THE FUNCTION OF SYMPATHETIC INNERVATION IN THE SPLEEN AND THE ROLE OF ENDOGENOUS CB1/CB2 RECEPTOR SIGNALING By Tyrell Jonathan Simkins A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Neuroscience - Environmental Toxicology Ð Doctor of Philosophy 2014 ii ABSTRACT THE FUNCTION OF SYMPATHETIC INNERVATION IN THE SPLEEN AND THE ROLE OF ENDOGENOUS CB1/CB2 RECEPTOR SIGNALING By Tyr ell Jonathan Simkins The spleen is a multifunction organ that sits at a unique intersection between the circulatory, immune, and neurologic systems. The work in this dissertation endeavored to shed light on the interaction of the sympathetic nervous sys tem in the spleen with these other vital biologic systems. In addition, the role of signaling by the cannabinoid receptors 1 and 2 was explored as it relates the function of splenic sympathetic innervation. Specifically , it was found that splenic noradrene rgic neurons do not play a role in T cell independent humoral immunity, and that both norepinephrine and adenosine mediate spleen contraction. It was discovered that splenic sym pathetic noradrenergic neurons are likely not regulated by CB1 and that cannabi noid -mediated immunosuppression of humoral immunity is likely due solely to CB2 on immune cells. It was also found that CB1/CB2 play a permissive role in maintaining the relationship between NE release from splenic sympathetic neurons and spleen contracti on. These findings add to the knowledge base regarding both the spleen and extra -CNS cannabinoid effects and can be built upon for a more complete understanding of these systems. iii Dedicated to my D ad - ÒEducation is never a waste.Ó iv ACKNOWLEDGEMENTS The completion of a dissertation, much like raising children, takes Òa villageÓ. There are far too many people that have contributed in some respect to this dissertation, whether directly or indirectly , to list here . Therefo re, this w ill be short and broad. I have been directly surrounded by an amazing group of people during my life. At home this is my amazing partner, Jensie , without whom I would be lost. I also wish to thank my children, Lucie and Asher, for always making me smile and giving me perspective. My parents, Vione and Paul, raised me right. They are the origin for my own family ! s motto: Work hard, play hard. Th ank you, thank you. I also wish to thank my siblings for support, teasing, encouragement, and the perfect amount of brotherly competition. My research colleagues have been amazing. This could be a VERY long list, but I wish to specifically thank a few pe ople. This dissertation really is as much mine as is it Keith Lookingland ! s. Thank you for pushing me, guiding me, and being a great mentor. Barb Kaplan, this project was your whole idea in the beginning. Thank you for the guidance, laughs, and keeping m e in line . John Goudreau, thank you for showing me that it can be done and for being there when I needed it. There have been a host of Goud -Looking lab mates that have come and gone ov er the course of my time here: Dr. Bahareh Behrouz, Dr. Kelly v Janis, H ae-Young H awo ng, Joe Patterson, Teri Lansdell, Brittany Winner, Kelly McGregor, Ethan Edwin, and Kevin Parihk. But I need to especially thank Dr. Sam Pappas, Dr. Matthew Bensk ey, and Dr. Chelsea Tiernan. Whew! We all made it. Lastly I would like to broa dly thank and acknowledge the contribution of all the other people I have worked with over the years. Of particular note are Dr. Justin McCormick, Dr. Veronica Maher, Dr. Norbert Kaminski, Dr. James Galligan, Dr. Cheryl Sisk, Bob Crawford, Shawna D ! Ingill o, David Fried, Brian Jespersen, and Jim Stockmeyer. Thank you to the Stephanie Watts and the Watts Lab for their help with smooth muscle physiology and quantification. Other notable entities I need to acknowledge are the Neuroscience Program, the Center for Integrative Toxicology, the NIH NRSA program, the College of Osteopathic Medicine, and the DO/PhD program. And to everybody who I was unable to list here: Thank you! You are not forgotten. Thank you one and all. I did it. vi TABLE OF CONTENTS LIST OF TAB LESÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... xiii LIST OF FIGUR ESÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉ xiv KEY TO ABBREV IATIONSÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. ..xx Chapter 1: General Introduction ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.1 1.1 Statement of PurposeÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ1 1.2 Sple enÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉ..2 1.2.1 Splenic O rganizationÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ....2 1.2.1.1 Red Pulp..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...3 1.2.1.2 White Pulp..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ....4 1.2.1.2.1 White Pulp Structure..ÉÉÉÉÉÉÉÉ....4 1.2.1.2.2 White Pulp Function..ÉÉÉ.ÉÉÉÉÉ....4 1.2.1.3 Spl een Capsule..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ....9 1.2.2 Splenic Bl ood Flow...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..10 1.2.2.1 AnatomyÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..10 1.2.2.2 Regulation o f Splenic Blood Flow..ÉÉÉÉÉÉ...11 1.2.3 Splenic Innervation. ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..12 1.2.3.1 Sympathet ic Innervation..ÉÉÉÉÉÉÉÉÉÉ...12 1.2.3.1.1 Brain Nuclei Associated with Spleen Innervation .ÉÉÉÉÉÉÉÉÉÉÉÉÉ12 1.2.3.1.2 Post -Ganglionic Innervation of the Spleen É....ÉÉÉÉÉÉÉÉÉÉÉÉÉ15 1.2.3.1.3 Sympathet ic Neurotransmitters.ÉÉÉ.É16 1.2.3.1.3.1 NorepinephrineÉÉÉÉÉÉÉ16 1.2.3.1.3.1.1 Synthe sisÉÉÉÉÉÉ.. 16 1.2.3.1.3.1.2 MetabolismÉÉÉÉÉ.. 19 1.2.3.1.3.1.3 ReceptorsÉÉÉÉÉÉ .21 1.2.3.1.3.1.4 NE Function in Spleen.. .23 1.2.3.1.3.1.4.1 Spleen Blood Flow and Contraction É.23 1.2.3.1.3.1.4.2 NE Effec ts on the Immune SystemÉÉ ...23 1.2.3.1.3.2 ATP/Adenosine..ÉÉÉ..ÉÉ...25 1.2.3.1.3.2.1 SynthesisÉÉÉÉÉÉ.. 25 1.2.3.1.3.2.2 MetabolismÉÉÉÉÉ.. 27 1.2.3.1.3.2.3 ReceptorsÉÉÉÉÉÉ. 27 vii 1.2.3.1.3.2.4 ATP/Adensoine Function in SpleenÉÉÉÉÉÉÉ.. 28 1.2.3.1.3.2.4.1 Spleen Blood Flow and ContractionÉ 28 1.2.3.1.3.2.4.2 ATP/Adeno sine Effects on the Immune SystemÉÉÉ 29 1.2.3.1.3.3 NPY..ÉÉÉ..ÉÉÉÉÉÉÉ..29 1.2.3.1.3.3.1 SynthesisÉÉÉÉÉÉ.. 29 1.2.3.1.3.3.2 MetabolismÉÉÉÉÉ.. 30 1.2.3.1.3.3.3 ReceptorsÉÉÉÉÉÉ. 30 1.2.3.1.3.3.4 NPY Function in SpleenÉÉÉÉÉÉÉ.. 31 1.2.3.1.3.3.4.1 Spleen Blood Flow and ContractionÉ. 31 1.2.3.1.3.3.4.2 NPY Effects on the Imm une SystemÉÉ. ..32 1.2.3.2 Parasympathetic Innervation.ÉÉÉÉÉÉÉÉÉ 32 1.3 Cannabinoi dsÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..33 1.3.1 Cannabin oid Receptors..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...36 1.3.1.1 CB1..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..36 Structure and SignalingÉÉÉÉÉÉÉÉÉÉÉÉ.36 Location...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.37 LigandsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.37 1.3.1.2 CB2..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..38 Structure and SignalingÉÉÉÉÉÉÉÉÉÉÉÉ.38 Location...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.39 LigandsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.39 1.3.1.3 Non -CB1/ CB2 Receptors..ÉÉÉÉÉÉÉÉ...É..40 1.3.2 Cannabinoid Effect s..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.43 1.3.2.1 Neuronal Effe cts of CB1 Stimulation..ÉÉÉÉÉ..43 1.3.2.2 Immune Effec ts of CB2 Stimulatio n..ÉÉÉÉÉÉ43 1.4 Summ aryÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...45 1.5 Thesis Objec tiveÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ46 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É48 Chapter 2: General Materials and Methods ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..71 2.1 Mic eÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ71 viii 2.1.1 CB1/CB2 KO Mouse GenotypingÉÉÉÉÉ.ÉÉÉÉÉÉ71 2.2 General Material s and DrugsÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉ72 2.3 Isolation of the Spleen Capsule and Splenocytes ÉÉÉÉÉÉÉ..É78 2.4 Preparation of Brain Tissue for Neurochemical Analyses ÉÉÉÉÉ.78 2.5 Neurochemistr yÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ79 2.6 Western B lotÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ...84 2.7 Immunohistoc hemistryÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ85 2.7.1 Brain Tissue I mmunohistochemistryÉÉÉÉÉÉÉÉÉÉ85 2.7.2 Spleen I mmunohistochemistryÉÉÉÉÉÉÉÉÉÉÉÉ.85 2.8 Preparation and Cultu re o f SplenocytesÉÉÉÉÉÉÉÉÉÉÉÉ..86 2.9 Flow Cyto metryÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.87 2.9.1 Surface antibody labeling for flow cytometry ÉÉÉÉÉÉ..87 2.9.2 Intracellular antibody labeling for flow cytometry ÉÉÉÉ...88 2.9.3 Flow Cyto metry AnalysisÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...88 2.10 ELISA ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉ96 2.11 ELISPOTÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉ.97 2.12 Spleen Contra ction StudiesÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..97 2.12.1 Preparation of Spleen Tissue for Spleen Contraction Studies ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ..97 2.12.2 Spleen Contraction Measurement ÉÉÉÉÉÉÉÉ..ÉÉ98 2.13 Spleen Capsule Width Measurement ÉÉÉÉÉÉÉÉÉÉÉÉ...101 2.13.1 Hematoxylin and Eosin Staining ÉÉÉÉÉÉÉÉÉ..É.101 2.13.2 Quantification of Spleen Capsule ThicknessÉÉÉÉ É..101 2.14 Statistical A nalysisÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É103 2.14.1 Statis tic al ComparisonsÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.103 2.14.2 aMT ExperimentationÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.103 2.14.3 Flow Cytome try Data HandlingÉÉÉÉÉÉÉÉÉÉÉ.104 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..105 Chapter 3: Comparison of the Noradrenergic Innervation in the Murine Spleen and the Paraventricular Nucleus of the Hypothalamus ÉÉÉÉ...108 3.1 Introduc tionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.108 3.2 Materials and Methods... ÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉ.110 3.2.1 MiceÉ.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..110 3.2.2 Materials..ÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉ..110 3.2.3 Isolation of the Spleen Capsule and Splenocytes ÉÉÉ...112 3.2.4 Preparation of Brain Tissue for Neurochemical AnalysesÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.112 3.2.5 Neuroc hemistr y..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..113 3.2.6 West ern blot..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ114 3.2.7 Immunohis tochemistry..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..115 3.2.7.1 Brain Tissue ImmunohistochemistryÉÉÉÉÉ..115 3.2.7.2 Spleen Immunohistochemistry ÉÉÉÉÉÉ.......115 ix 3.2.8 Statistical Analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ116 3.2.8.1 Statistical Comparisons ..ÉÉ..ÉÉÉ..ÉÉÉÉ.116 3.2.8.2 aMT Experimentation ..ÉÉÉÉÉÉÉÉÉÉÉ.116 3.3 Results ÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ...ÉÉÉÉÉÉ.................117 3.3.1 Noradrenergic innervation of the PVN and the spleen i n miceÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ117 3.3.2 Noradrenergic neurochemical activity in the PVN a nd spleen of miceÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...1 22 3.4 Discu ssionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...135 3.4.1 Anatomic differences and consequences in noradrenergic innervation of th e spleen and PVNÉÉÉÉÉÉÉÉÉ....135 3.4.2 Noradrenergic neuron activity in the spleen capsule a nd PVNÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉ.É137 3.4.3 Conclusion.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...141 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..142 Chapter 4: The Contribution of Norepinephrine to Humoral Immune Responses in the Spleen ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ...148 4.1 Introduc tionÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉ.148 4.2 Materials and M ethods...ÉÉÉÉÉÉÉ.ÉÉ ÉÉÉÉÉÉÉÉÉ.153 4.2.1 MiceÉ.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..153 4.2.2 Mate rials..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..153 4.2.3 Isolation of the Spleen Capsule and Splenocytes ÉÉÉ...155 4.2.4 Preparation and Culture of SplenocytesÉÉÉÉÉÉÉ...15 5 4.2.5 Neuroc hemistry..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉ..156 4.2.6 West ern blot..ÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉ157 4.2.7 Flow CytometryÉÉÉ ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...158 4.2.7.1 Surface antibody labeling for flow cytometry .ÉÉ158 4.2.7.2 Intracellular antibody labeling for flow cytometry ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ159 4.2.7.3 Flow cytometry analysis .ÉÉÉÉÉÉÉÉÉÉ..159 4.2.8 ELISAÉÉÉ..ÉÉÉÉÉ.É ÉÉÉÉÉÉÉÉÉÉÉÉ..160 4.2.9 ELIS POTÉÉÉ..ÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉ..161 4.2.10 Statistical Analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ161 4.2.10.1 Statistical c omparisons ÉÉÉÉÉÉÉÉÉ.É..161 4.2.10.2 aMT e xperimentation ..ÉÉÉÉÉÉÉÉÉÉÉ162 4.2.10.3 Flow Cytometry Data Handling ..ÉÉÉÉÉÉÉ162 4.3 Results ÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ...ÉÉÉÉÉÉ.................163 4.3.1 Characterization of hu moral immune challenge modelsÉ164 4.3.2 Splenic sympathetic neuronal activity in response to humoral immune challenge models. ÉÉÉÉÉÉÉÉÉÉÉÉÉ.169 4.3.3 !2AR expression on splenic lymphocytesÉÉÉÉÉÉÉ176 4.3.4 The effect of !2AR stimulation on humoral immune responses in s plenic lymphocytesÉÉÉÉÉÉÉÉÉÉ.182 x 4.4 DiscussionÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉ...187 4.4.1 Activation of splenic sympathetic neurons following an immune challenge eliciting a humoral respo nse ÉÉÉÉ187 4.4.2 !2AR expression on splenic B cells. ÉÉÉÉÉÉÉ.ÉÉ191 4.4.3 The effect of !2AR on the humoral response of splenic B cellsÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉ192 4.4.4 Conclusion.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...194 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..195 Chapter 5: The Interac tion of Endogenous CB1/CB2 Receptor Signaling and Norepinephrine in Splenic Humoral Immune Responses ÉÉÉÉÉ..201 5.1 Introduc tionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.201 5.2 Materials and M ethods...ÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉ.206 5.2.1 MiceÉ.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..206 5.2.1.1 CB1/CB2 Mouse GenotypingÉ..ÉÉÉÉÉÉÉ206 5.2.2 Mate rials..ÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..207 5.2.3 Isolation of the Spleen Capsule and Splenocytes ÉÉÉ...208 5.2.4 Neuroc hemistry..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..209 5.2.5 West ern blot..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ210 5.2.6 Flow C ytometryÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...211 5.2.6.1 Surface antibody labeling for flow cytometry ÉÉ.211 5.2.6.2 Intracellular antibody labeling for flow cytometry ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ212 5.2.6.3 Flow cytometry analysis .ÉÉÉÉÉÉÉÉÉÉ..212 5.2.7 ELISAÉÉÉ..ÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉ..213 5.2.8 Statistical Analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ214 5.2.8.1 Statistical comparisons ÉÉÉÉÉÉÉÉÉ.É..214 5.2.8.2 aMT experimentation ..ÉÉÉÉÉÉÉÉÉÉÉ214 5.2.8.3 Flow Cytometry Data Handling ..ÉÉÉÉÉÉÉ215 5.3 Results ÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ...ÉÉÉÉÉÉ.................215 5.3.1 Enhanced humoral immunity in CB1/CB2 KO miceÉÉ É215 5.3.2 Elevated !2AR expression on B cells in CB1/CB2 KO miceÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉ225 5.3.3 Splenic sympathetic noradrenergic neuronal activity in CB1/ CB2 KO miceÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ232 5.3.4 Enhanced humoral immunity in CB1/CB2 KO mice is not due to increased stimulation of !2AR.. ÉÉÉÉÉÉÉÉÉÉ.238 5.4 Discu ssionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...243 5.4.1 Enhanced humoral immunity in CB1/CB2 KO mice ÉÉÉ243 5.4.2 !2AR expression in CB1/CB2 KO mice ..ÉÉÉÉÉÉÉ..245 5.4.3 Splenic sympathetic noradrenergic activity and signaling in CB1/CB2 KO mice ÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉ..247 5.4.4 Concl usion...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..248 xi REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..249 Chapter 6: Sympathetic nervous system control of spleen contraction and the role of CB1/CB2 signaling ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ256 6.1 Introduc tionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.256 6.2 Materials and Methods... ÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉ.261 6.2.1 MiceÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. .261 6.2.1.1 CB1/CB2 Mouse GenotypingÉ. .ÉÉÉÉÉÉ....262 6.2.2 Mate rials..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..262 6.2.3 Isolation of th e Spleen CapsuleÉÉÉÉÉÉÉ.. ÉÉÉ...264 6.2.4 West ern blot..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ264 6.2.5 Spleen Contraction StudiesÉ..ÉÉÉ..ÉÉÉÉ. ÉÉÉ...265 6.2.5.1 Preparation of Spleen Tissue for Spleen Contraction StudiesÉÉÉÉÉÉÉÉÉÉÉ É...265 6.2.5.2 Spleen Contraction Measurement ÉÉÉÉÉÉ..266 6.2.6 Spleen Capsul e Width MeasurementÉÉÉ..ÉÉÉÉÉ.266 6.2.6.1 Hematoxylin and Eosin Staining ÉÉÉÉÉÉÉ266 6.2.6.2 Quantification of Spleen Capsule Thickness É...267 6.2.7 Statistical Analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ267 6.2.7.1 Statistical comparisons ÉÉÉÉÉÉÉÉÉ.É..267 6.3 Results ÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ...ÉÉÉÉÉÉ.................268 6.3.1 Comparison of NE -induced spleen contraction in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ268 6.3.2 Comparison of EFS -induced spleen contraction in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ282 6.4 Discu ssionÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...294 6.4.1 NE-induced spleen contractility is decreased in CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ294 6.4.2 Normal spleen contraction in response to EFS is achieved in CB 1/CB2 KO mice by a compensatory mechanism É...296 6.4.3 Conclusion ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.298 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..299 Chapter 7 : General Discussion and Concluding Remarks ÉÉÉÉÉÉÉ.307 7.1 Anatomy and physiology of splenic noradrenergic post -ganglionic neurons ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É.307 7.2 Immunologic function of splenic noradrenergic post -ganglionic neurons ...ÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ...312 7.3 The role of CB1 and CB2 receptors on the immunologic role of noradrenergic splenic sympathetic innervation .ÉÉÉÉÉÉÉ..É.316 7.4 Sympathetic control of spleen contraction ÉÉÉÉÉÉÉÉÉÉÉ.321 7.5 The role of CB1 and CB2 receptors in spleen contr action ÉÉÉÉ..322 xii 7.6 Significance of cannabinoid use and abuseÉÉÉÉÉÉ. ÉÉÉÉ.326 7.7 Concluding RemarksÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉ.329 REFERENCES ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..330 xiii LIST OF TABLES Table 2.1. Summary of Drug Actions ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ77 xiv LIST OF FIGURES Figure 1.1. Synthetic pathway of NE ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ......17 Figure 1.2. Metabolic pathway of NE É.ÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉ20 Figure 1. 3. Schematic representation of the endocannabinoid system É...35 Figure 2. 1. Representative image from HPLC detection of biogenic amines in a standard solution ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.....81 Figure 2 .2. Representative image of NE detection by HPLC in the spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ82 Figure 2.3. Representative image of NE dectection by HPLC in the PVN É.......................................................................................................83 Figure 2.4. Representative gating of singlets in splenocytes prepared for flow cytometry ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..É90 Figure 2 .5. Representative gating of live cells in preparations of cultured splenocytes for flow cytometry ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ91 Figure 2 .6. Representative gating for spleen derived lymphocytes in preparations for flow cytometry ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ..É.92 Figure 2 .7. Representative gating for splenic B cells by flow cytometry ...93 Figure 2.8. Representative images from flow cytometric analysis of IgM producing splenic B cells ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...É..É94 Figure 2.9. Representative images from flow cytometric analysis of IgM producing splenic B cells expressing !2ARÉÉ.ÉÉÉÉÉÉÉÉÉÉ...É95 Figure 2. 10. Schematic of equipment setup used to record spleen contractile force ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉ........99 Figure 2.11. Representative image of the raw data obtained from spleen contraction experiments ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ..100 Figure 2. 12. Representative images comparing the spleen capsule thickness between WT and CB1/CB2 KO mice ÉÉ.ÉÉÉÉÉÉÉÉÉ....102 xv Figure 3.1. Immunohistochemical staining for TH in the PVN and spleen of mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉ...119 Figure 3.2. Western blot analysis of TH in the spleen of mice ÉÉÉÉ.É.120 Figure 3.3. Neurochemical analysis of NE in the murine spleen ÉÉ,,,,É.121 Figure 3.4. Comparison of NE concentrations in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..124 Figure 3.5. Comparison of NE content in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉ..125 Figure 3.6. Comparison of MHPG concentrations in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉ..126 Figure 3 .7. The ratio of MHPG to NE in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ127 Figure 3.8. Comparison of VMA concentrations in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É..128 Figure 3.9. The ratio of VMA to NE in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ129 Figure 3.10. Comparison of DOPA concentrations in the murine PVN and spleen capsule following treatment with NSD -1015ÉÉÉÉÉÉÉÉÉ.É131 Figure 3.11. Comparison of basal NE utilization rates in the murine PVN and spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉ..É133 Figure 3.12. Idazoxan i ncreases the rate of NE utilization and NE concentrations in the murine spleen capsule ÉÉÉÉÉÉÉÉÉÉÉÉ...134 Figure 4.1. The effect to experimental immune challenges on body weight in mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ164 Figure 4.2. The effect to experimental immune challenges on spleen weight in mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉ165 Figure 4.3. The effect to experimental immune challenges on the spleen:body weight ratio in mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ....166 xvi Figure 4 .4. IgM production from splenocytes from mice subject to experimental immune challenges ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ...É167 Figure 4.5. Analysis of IgM a ntibody production responses in splenic B cells from mice exposed to LPS in vitro ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..168 Figure 4.6. Spleen capsule NE concentrations in response to injection of sRBC ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..170 Figure 4.7. Spleen capsule TH content in response to injection of sRBC ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É..171 Figure 4.8. Spleen capsule noradr energic neuron activity in response to injection of sRBC ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É.172 Figure 4.9. Spleen capsule NE concentrations i n response to injection of LPS ................................................................................................173 Figure 4. 10. Spleen capsule TH content in response to injection of LPS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..174 Figure 4.11. Spleen capsule noradrenergic neuron activity in response to injection of LPS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ.175 Figure 4.12. Populations of splenic lymphocytes expressing !2AR ÉÉ...178 Figure 4 .13. Correlation between antigen presenting lymphocytes in the spleen and !2AR expression ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...ÉÉ..É.179 Figure 4.1 4. The response of !2AR expressing splenic B cells to in vitro LPS administration ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É180 Figure 4. 15. The IgM response of B2AR expressing splenic B cells to in vitro LPS administration ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..181 Figure 4. 16. The effect of NE on the IgM response of splenic B cells to in vitro LPS administration ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...É.É.183 Figure 4. 17. The effect of NE on the IgM response of !2AR expressing splenic B cells to in vitro LPS administration ...............................................184 Figure 4.18. The effect of !2AR antagonism on the IgM response of splenic B cells to in vivo LPS administration ÉÉÉÉÉÉÉÉÉÉ...É.É18 5 xvii Figure 4.19. The effect of !2AR antagonism on the IgM response of !2AR expressing splenic B to in vivo LPS administration ÉÉÉÉÉÉ.É186 Figure 5.1. Plasma IgM concentrations in immunologically naive WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ........217 Figure 5.2. Plasma IgG concentrations in immunologically naive WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ.218 Figure 5.3. Flow cytometric analysis of splenic IgM producing B cell populations from immunologically naŁve WT and CB1/CB2 KO mice É.É219 Figure 5.4. Flow cytometric analysis of splenic IgM producing B cells from immunologically naŁve WT and CB1/CB2 KO mice ÉÉÉÉÉÉ.É...220 Figure 5.5. Plasma IgM concentrations in WT and CB1/CB2 KO mice treated with LPS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉ221 Figure 5.6. Plasma IgG concentrations in WT and CB1/CB2 KO mice treated with LPS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉ...222 Figure 5.7. Flow cytometric analysis of splenic IgM producing B cell populations from LPS treated WT and CB1/CB2 KO mice ÉÉÉÉÉÉ.....223 Figure 5.8. Flow cytometric analysis of splenic IgM producing B cells from WT and CB1/CB2 KO mice treated with LPS ÉÉÉÉÉÉÉÉÉ...É.224 Figure 5.9. Flow cyto metric analysis of splenic B cells expressing !2AR from immunologically naive WT and CB1/CB2 KO mice ÉÉÉÉ.É227 Figure 5.10. Flow cytometric analysis of splenic IgM producing B cell populations expressing !2AR from immunologically naŁve WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..228 Figure 5.11. Flow cytometric analysis of IgM producing splenic B cells expressing !2AR from immunologically naive WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..229 Figure 5.12. Flow cytometric a nalysis of splenic IgM producing B cell populations expressing !2AR from LPS treated WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..230 xviii Figure 5.13. Flow cytometric analysis of IgM producing splenic B cells expressing !2AR from WT and CB1/CB2 KO mice treated with LPS ÉÉ..231 Figure 5.14. Spleen capsule NE concentrations in immunologically naŁve WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..234 Figure 5.15. Spleen capsule TH content in immunologically naŁve WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉ...235 Figure 5.16. Spleen capsule noradrenergic neuron activity in immunologically naŁve WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉ..ÉÉÉ236 Figure 5.17. Comparison of the effects of LPS on spleen capsule noradrenergic neuron activity in WT and CB1/CB2 KO mice ÉÉÉÉÉÉ237 Figure 5.18. Lack of effect of !2AR antagonism on plasma IgM concentrations in LPS exposed WT and CB1/CB2 KO mice ÉÉÉ..ÉÉÉ239 Figure 5.19. Lack of effect of !2AR antagonism on plasma IgG concentrations in LPS exposed WT and CB1/CB2 KO mice ÉÉÉÉ..ÉÉ240 Figure 5.20. Lack of effect of !2AR antagonism on the IgM producing B cell population in the spleen of LPS exposed WT and CB1/CB2 KO mice ÉÉ241 Figure 5.21. Lack of effect of !2AR antagonism on the IgM producing B cell population expressing !2AR in the spleen of LPS exposed WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ242 Figure 6.1. Dose response of NE -induced spleen contraction ÉÉÉÉÉ.269 Figure 6.2. Blockade of NE -induced spleen contraction by prazosin ÉÉ.270 Figure 6.3. Spleen contractions induced by repeated administration of 10, 100 or 500 nM NE ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.....271 Figure 6.4. Spleen contractions induced by repeated administration of 10-100 nM NE ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...272 Figure 6.5. Comparison of NE -induced spleen contraction force in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ274 Figure 6.6. Comparison of NE -induced weight -normalized spleen contr action force in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉ..ÉÉÉ..275 xix Figure 6.7. Spleen weight comparison in WT and CB1/CB2 KO mice É....276 Figure 6. 8. Representative histology of the spleen capsule from WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉ278 Figure 6 .9. Comparison of spleen capsule thickness in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É279 Figure 6. 10. Comparison of spleen capsule smooth muscle specific "-actin in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...280 Figure 6.11. Comparison of spleen capsule "1AR content in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ281 Figure 6. 12. Voltage response curve of EFS -induced spleen contraction ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.283 Figure 6.13. Frequency response of EFS -induced spleen contraction É..284 Figure 6 .14. Duration response of EFS -induced spleen contraction ÉÉ..285 Figure 6.15. EFS -induced spleen contraction is mediated by "1AR and adenosine A1 receptors ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...287 Figure 6. 16. Adenosine P2X receptors do not contribute to EFS -induced spleen contraction ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.288 Figure 6. 17. NPY Y1 receptors do not contribute to EFS -induced spleen contraction ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.289 Figure 6. 18. Comparison of EFS -induced spleen contraction force in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...291 Figure 6. 19. Comparison of EFS -induced weight -normalized spleen contraction force in WT and CB1/CB2 KO mice ÉÉÉÉÉÉÉÉÉÉÉÉ292 Figure 6 .20. The effect of prazosin on EFS -induced weight -normalized spleen contrac tion force in WT and CB1/CB2 KO miceÉÉÉÉÉÉÉÉ..293 xx KEY TO ABBREVIATIONS 2-AG 2-arachidonylglyceride ABTS 2,2'-Azino -di-[3-ethylbenzthiazoline sulfonate NSD -1015 3-hydroxybenzylhyrdazine MHPG 3-methoxy -3-hydroxyphenylglycol DOPAL 3,4-dihydroxyphenylacetaldehyde DHPG 3,4-dihydroxyphenylglycol 5HT 5-hydroxytryptamine DPCPX 8-Cyclopentyl -1,3-dipropylxanthine Ach Acetylcholine ADP Adenosine diphosphate AMP Adenosine monophosphate ATP Adenosine triphosphate AD Alcohol dehydrogenase ALD Aldehyde dehydrogenase ADR Aldehyde reductase AR Adrenergic receptor aMT "-Methyl -DL-tyrosine methyl ester hydrochloride A Amps AEA Arachidonoylethanolamide or anandamide BCR B cell receptor xxi 2-ME !-Mercaptoethanol BCA Bicinchoninic acid BCS Bovine calf serum BSA Bovine serum albumin BDNF Brain derived neurotrophic factor CBD Cannabidiol CB1 Cannabinoid receptor 1 CB2 Cannabinoid receptor 2 COMT Catechol -o-methyl transferase KO Knockout CNS Central nervous system cAMP Cyclic adenosine monophosphate CRH Corticotropin releasing hormone THC #9-Tetrahydrocannabinol DMSO Dimethyl sulfoxide DTT Dithiothreitol DA Dopamine DBH Dopamine -!-hydroxylase DRG Dorsal root ganglion EFS Electrical field stimulation ELISA Enzyme linked immunosorbant assay ELISPOT Enzyme Linked Immunosorbent Spot Assay xxii RBC Erythrocyte(s) EDTA Ethylenediaminetetraacetic acid FAAH Fatty acid amide hydrolase FACS Fluorescence activated cell sorting FDA Food and Drug Agency GABA $-Aminobutyric ac id GAPDH Glyceraldehyde 3 -phosphate dehydrogenase HBSS Hank's Buffered Saline Solution Hz Hertz HPLC -ED High performance liquid chromatography coupled with electrochemical detection HRP Gorseradish peroxidase HCl Hydrochloric acid HPA Hypothalamic -pituitary -adrenal Ig Immunoglobulin IP3 Inositol 1,4,5 -triphosphate IL Interleukin IML Intermediolateral cell column of the spinal cord i.p. Intraperitoneal L-AAAD L-aromatic amino acid decarboxylase DOPA L-dihyd roxyphenylalanine LPS Bacterial lipopolysaccharide xxiii MHC Major histocompatibility complex MFI Mean fluorescence intensity MAGL Monoacylglyceride lipase MAPK Mitogen activated protein kinase MAO Monoamine oxidase BIBP3226 N2-(Diphenyl acetyl) -N-[(4 -hydroxyphenyl)methyl] -D-arginine amide NPY Neuropeptide Y NE Norepinephrine PVN Paraventricular nucleus of the hypothalamus PALS Periarteriolar lymphatic sheath PNS Peripheral nervous system PMSF Phenylmethylsulfonyl fluoride PBS Phosphate buffered saline PLC Phospholipase C PCR Polymerase chain reaction KCl Potassium chloride KH2PO4 Postassium phosphate monobasic PPADS Pyridoxal phosphate -6-azo(benzene -2,4-disulfonic acid RCF Relative centrif ugal force RDU Relative density unit RT Room temperature xxiv Ser Serine sRBC Sheep erythrocytes NaHCO 3 Sodium bicarbonate NaCl Sodium chloride SDS -PAGE Sodium dodecyl sulfate poly -acrylamide gel electrophoresis NaOH Sodium hydroxide Na2HPO 4 Sodium phosphate dibasic sIPSCs Spontaneous inhibitory postsynaptic currents SEM Standard error of the mean TCR T cell receptor TH T helper cell TTX Tetrodotoxin TH Tyrosine hydroxylase VMA Vanillylmandel ic acid VMAT Vesicular monoamine transporter V Volts WT Wild-type !"!Chapter 1: General Introduction 1.1: Statement of Purpose The spleen is a visceral organ with both hematologic and immune functions. Hematologically the spleen serves as a site to remove foreign mate rial and dead, dying, or damaged erythrocytes from circulation. It also serves as a storage site for erythrocytes, which are released upon demand by contraction of the splenic capsule to increase hematocrit. Immunologically, the spleen is the largest secondary lymphoid organ and plays a significant role in the adaptive immune response by housing leukocytes and providing easy ac cess to the circulation and blood -borne antigens. Innervation of the spleen is via the sympathetic nervous system. The impact of sympathetic neuronal activity on the multifunctional role of the spleen is not entirely clear and is the major purpose of this work. The legalization and use of marijuana, as well as related cannabinoid compounds, for medicinal and a recreational purposes is increasing in the United States and worldwide. Despite this increasing approval and popularity, the biological ramifi cations of cannabinoid use are not completely understood. This is particularly true for the actions of cannabinoids outside the brain. Cannabinoid receptors are not only in the central nervous system, but also throughout the body of mammals. The presen ce of peripheral located cannabinoid receptors suggests a role for both exogenous (marij uana and cannabinoid based drugs) and endogenous cannabinoids (endocannabinoids) . Understanding the role of endocannabinoid signaling is significant !#!by itself, but can also inform s the scientific and medical community of t he possible ramifications of cannabinoid use and/or abuse. 1.2: Spleen The spleen is a visceral organ with both hematologic and immune functions. The major hematological purpose of the spleen is to remove foreign mate rial and dead, dying, or damaged erythrocytes from circulation. It also serves as a storage site for erythrocytes and iron as well as a site for limited hematopoiesis. Immunologically, the spleen is the largest secondary lymphoid organ a nd plays a significant role in the adaptive immune response by housing leukocytes and providing easy access to the bloodstream, and thus to blood -borne antigens. 1.2.1: Splenic Organization The spleen is divided into two major parenchymal compartments a nd one major structural component (Davies and Withrington, 1973; Cesta, 2006) . The parenchymal compartments of the spleen are the red and white pulp, which serve the hematologic and immunologic functions of the spleen, respectively. The major structural component of the spleen is the capsule, which gives rise to parenchyma penetrating trabeculae, which also serve s a structural function. !$!1.2.1.1: Red Pulp The red pulp is composed of a meshwork of reticular cells and fibers, in the midst of which are contained mostly erythrocytes ( RBC ) and macrophages. The r eticular cells in the red pulp are considered myofibroblasts as they contain contractile elements and likely contribute to the contraction of the spleen (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988) . Red pulp r eticular fibers are multi -comp onent structures composed of collagen fibers, elastic fibers, micofibrils, and unmyelinate d noradrenergic axons (Saito et al., 1988) , all of which is ensh eathed by reticular cells and their processes (Cesta, 2006) . Within this reticular meshwor k are largely RBC entering from branches of central arterioles in the red pulp , or through the white pulp marginal zone to red pulp (Schmidt et al., 1985; Satodate et al., 1986; Schmidt et al., 1993; Mebius and Kraal, 2005) . The concentration of RBC within the splenic red pulp, the likely site of RBC storage, is double that of the general circulation (Groom et al., 1991) . This area is also where macrophages actively phagocytiz e dead and damaged erythrocytes (Cesta, 2006). Macrophages within the red pulp are also constantly exposed to foreign particulate matter in the circulation , thus facilitating the speed and efficacy of immune responses (Cesta, 2006) . !%!1.2.1.2: White Pulp 1.2.1.2.1: Structure The white pulp is composed of T cells, B cells, and macrophages in a reticular framework similar to the red pulp (Saito et al., 1988) . As central arterioles enter the spleen they are surrounded the periarte riolar lymphatic sheath ( PALS ), which is composed of lymphocytes, T and B cells (Mebius and Kraal, 2005; Cesta, 2006) , and concentric layers of reticular fibers and cells , which have contractile machinery (Pinkus et al., 1986; Satodate et al., 1986; Cesta, 2006) . The PALS is roughly divided into the inner and outer zones . The inner PALS is largely composed of CD4 + T cells, also known as T helper cells (T H), whereas the outer PALS is a mixture of T cells, B cells, macrophages, and plasma cells (B cells differentiated into dedicated antibody producing cells) (Matsuno et al., 1989; Mebius and Kraal, 2005; Cesta, 2006) . Collectively, t he PALS is considered to be a site for plasma cell differentiation (Matsuno et al., 1989) . The marginal zone is a portion of the w hite pulp at the interface between the red and white pulp of the spleen. It is composed largely of macrophages and functions as a site where the blood is screened for systemic circulating antigens and pathogens (Mebius and Kraal, 2005; Cesta, 2006) 1.2.1.2.2: Function The spleen is the largest secondary lymphoid organ and the white pul p of the spleen mediates th e immune function of the spleen. The full complement of cells required for immune responses, particularly for humoral immunity, are found within the !&!splenic white pulp. The immune system produces highly complex, multi -cellular, coordinated responses that act to protect organisms from harmful infectious diseases and cancer. The presence of foreign partic les, pathogens, or tumor cells e llicit distinct actions with their own particular set of cells and signaling molecules. In gener al there are two broad divisions of the immune system: innate and adaptive. Innate immun ity is an intrinsic system that is ever present and provides an early and rapid defense against microbes (Oikonomopoulou et al., 2001; Quah and Parish, 2001). The innate immune s ystem does not confer long -term immunity to a pathogen, however it can help the adaptive immune system respond more efficaciously to microbes (Meager and Wadhwa, 2001) . The cells mediating the innate immune response include natural killer cells, neutrophils , monocytes/macrophages, and dendritic cells (Quah and Parish, 2001) . The adaptive immune system is directed by lymphocytes comprised of two major types: B cells and T cells (P Kane, 2001) , and is activated when an antigen is recognized by epitope specific receptors on lymphocytes (P Kane, 2001) . Both B cells and T cells exp ress epitope specific receptors composed of surface exp ressed immunoglobulin ( Ig), termed the B cell receptor (BCR) and T cell receptor (TCR), respectively (Ferrero et al., 2001; P Kane, 2001; Kurosaki and Hikida) . An important difference between these receptors is the types of antigens to which they respond . BCRs recognize a vast array of antigens such as proteins, polysaccharides, lipids, nucleic acids , and soluble antigens including small chemicals, whereas TCRs only recognize peptide fragments presented by host cells in the major histocompatibility !'!complex ( MHC ) (Ferrero et al., 2001; Saha, 2001; Kurosaki and Hikida) . The activation of these receptors results in highly specific responses centered about the ability of these cells to recognize the epitope on the inciting ant igen. Th ese response s are divided into cell -mediated and humoral immunity (Saha, 2001) . T cells implement cel l-mediated adaptive immune responses once an antigen has been recognized (Ademokun and Dunn -Walters, 2001) . Two major classes of T cells exist (Nath, 2001; Saha, 2001) . CD8 + cells, also known as cytotoxic T cells, aid in the removal of cells infected with intracellular pathogens such as viruses and intracellular bacteria (Gotch, 2001; Nath, 2001) . CD4 + T cells, or TH cell s, aid in the removal of extra cellular pathogens (Ademokun and Dunn -Walters, 2001; Ferrero et al., 2001). There are several T H cell subtypes; the T H1 subtype is involved in the stimulation of macrophage s to more effectively phagocytize and lyse bacteria, whereas the T H2 subtype is especially involved in the immune response against helminthes and parasites (Fer rero et al., 2001) . Another more recently recognized subtype of T H cell is the follicular T H cell, which are antigen experienced T H cells that reside within B -cell follicles of secondary lymphoid organs , such as the spleen, and assist in the production of antibodies, especially the IgG isotype (Fazilleau et al., 2009) . T cells also participate in humoral immune responses. Many of the T H subtypes (TH1, TH2, and follicular T H cells) assist in stimulating B cells to produce antibodies (Ademokun and Dunn -Walters, 2001; Ferrero et al., 2001) . Once activated by exposure to an extracellular antigen, T H cell types begin express ing CD40 -ligand, also known as CD154 , on their cell surface and release cytokines (i.e. interferon -! , interleukin -2, and !(!interleukin -4) in response to interaction with CD40 expressing B cells that recognize the same antigen (Ademokun and Dunn -Walters, 2001; N”ron et al., 2011) . The antigen specific interaction between B cells and T H cells, i ncluding CD40/CD40 -ligand interaction and cytokine stimulation, induces B cells to produce antibodies directed against the commonly recognized antigen (N”ron et al., 2011) . Antibodies are composed of two di stinct portions: the heavy chain and the light chain. Each heavy and light chain has a constant and variable region (Kenter, 2005) . The variable region of each chain confers the ability of the antibody to recognize a unique antigen ic site (Zouali, 2001a) . The constant portion of the heavy chain determines the subtype of antibody, of which there ar e 5 types: IgA, IgD, IgE, IgM, and IgG (Zouali, 2001a; Kenter, 2005) . The subtype of antibody plays a major role in determining its function as the heavy chain constant portion of the each antibody is the site recognized by receptors throughout the body, such as by F c receptors on macrophages or by complement (Da‚ron, 1997; Stavnez er et al., 2008) . IgM is a pentameric Ig with relatively low specificity, but high capacity, and is the first type of antibody produced in response to a novel antigen (Ademokun and Dunn -Walters, 2001; Zouali, 2001a; Czajkowsky and Shao, 2009) . Additionally, low specificity IgM is constitutively produced as a form of innate -type immunity (Zouali, 2001b; Czajkowsky and Shao, 2009; Baumgarth, 2013) . These antibodies are termed " natural antibodies # and are produced largely in the spleen and bon e marrow (Zouali, 2001b; Baumgart h, 2013) . The effector mechanism of IgM is complement activation (Czajkowsky and Shao, 2009; Baumgarth, 2013) . Over the course of a humoral immune !)!response the isotype of antibody changes from IgM to IgG (Ademokun and Dunn -Walters, 2001; Schroeder and Cavacini, 2010) . This process is termed class switching. IgG is a monomeric Ig with a high specificity, but relatively low capacity (Zouali, 2 001a; Schroeder and Cavacini, 2010) . IgG is the predominant antibody type produced several days (3 -4) following an initial immune exposure and during repeated exposure to the same antigen (Ademokun and Dunn -Walters, 2001) . Effector mechanisms of IgG inclu de complement activation, neutralization of toxins, and facilitation of phagocytosis (Da‚ron, 1997; Schroeder and Cavacini, 2010) . There are certain types of antigens that stimulate humoral response s that do not require T cells. These antigens are termed thymus -independent antigens, or T cell independent antigens, and stimulate B cells to produce antibodies by replacing the secondary stimulation provided by T H cells (Ademokun and Dunn -Walters, 2001) . These secondary signals come from either cross linking of BCRs or stimulation of toll -like receptors (TLR) that recognize specific pathogen associated molecules. A prime example of this type of antigen is bacterial lipopolysaccharide ( LPS ) (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . LPS is a component of the cell wall of many types of pathogenic bacteria (Miller et al., 2005) . Upon entering the circulation , LPS is recog nized by TLR4 rece ptors on the surface of B cells (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009). In addition, because of its large size and repeating subunits, LPS can be simultaneously be recognized by ma ny BCRs on the same lymphocyte, termed crosslinking (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and !*!Jego, 2009) . Together t hese two unique features of L PS are able to stimulate B cells to produce antibodies in the absence of T H cell involvement (Moller, 2001) . 1.2.1.3: Spleen Capsule The spleen is encapsulate d by a tissue layer with three major components: connective tissue, elastic tissue, and smooth muscle (Cesta, 2006) . Immunohistochemical detection of smooth muscle specific myosin, one of the core components required for muscle contraction (Squire, 2001) , is demonstrative of the large amount of smooth muscle contained within the spleen capsule (Pinkus et al., 1986) and confirms histologic evaluation (Davies and Withrington, 1973; Cesta, 2006) . However as mentioned above, contractile elements have also been observed in trabeculae of connective tissue that penetrate the sple en (Pinkus et al., 1986) . These trabeculae carry with them branches of the incoming arterioles with contractile components in their walls, and axons of noradrenergic sympathetic neurons (Davies and Withrington, 1973; Pinkus et al., 1986; Cesta, 2006) . Contraction of smooth muscle in the spleen serves two hematologic purposes . First, the spleen acts as a reservoir for RBC (Cesta, 2006) . Due to the reticular mesh network parenchyma surrounded by a fibromuscular spleen capsule (Davies and Withrington, 1973; Cesta, 2006) , contraction of the sp leen capsule reduces the overall size and volume of the spleen (Sandler et al., 1984; Richardson et al., 2009; Seifert et al., 2012). This contraction expels the cells contained within the spleen into the general circulation thereby increas ing the content of RBC, or he matocrit of the blood . !"+!Contraction of the spleen can increase the hematocrit of the blood by as much as 5% in humans (Sandler et al., 1984; Bakovic et al., 2005; Richardson et al., 2007; 2009), 10% in rats (Kuwahira et al., 1999) , and 16% in dogs (Sato et al., 1995) , thereby increasing its oxygen carrying capacity. This effect is considered the primary function of spleen contraction. In addition to being a reservoir for RBC, the spleen is the largest secondary lymphoid organ and contains a large num ber of immune cells (macrophages, leukocytes, and lymphocytes) (Mebius and Kraal, 2005 ; Cesta, 2006) . Thus, the immune cells of the spleen are also released during splenic contraction (Schaffner, 1985; Seifert et al., 20 12), but the consequences of this effect are essentially unknown. 1.2.2: Splenic Blood supply 1.2.2.1: Anatomy Blood enters the spleen via the splenic artery which branches into a number of trabecular arteries that follow connective tissue trabeculae into the spleen parenchyma (Cesta, 2006) . These vessels branch further and enter the red pulp of the spleen w here they are termed central arterioles and are surround ed by the PALS within the white pulp (Cesta, 2006) . Branches from central arterioles have several destinations including capillary beds within the white pulp (marginal zone) and the red pulp of the spleen (Schmidt et al., 1985; Satodate et al., 1986; Schmidt et al., 1993) . This distributi on of blood vessels leads to two defined pathways of blood flow through the spleen: the " fast # and " slow # pathways. Blood that flows through the white pulp marginal zone and directly into venous sinuses is considered the " fast # pathway (Cesta, 2006) . As much as 90% of !""!splenic blood flow travels through th is "fast # pathway (Schmidt et al., 1985; 1993; Cesta, 2006). Alternatively, the " slow # pathway is whe re blood enters the reticular meshwo rk of the red pulp (Schmid t et al., 1993) through terminal capillaries arising from central arterioles terminating within the reticular meshwork of the red pulp (Mebius and Kraal, 2005). Blood flow through the red pulp is collected into venous sinuses, which coalesce into venous collec ting veins and ultimately exit the spleen via the splenic vein to drain into the hepatic portal system , traveling in the same neurovascular bundle as the splenic artery (Mebius and Kraal, 2005; Cesta, 2006) . 1.2.2.2: Regulation of Splenic Blood F low Blood flow through the spleen is regulated by both arteriolar/capillary endothelial cells and red pulp reticular cell contraction (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988; Groom et al., 1991) . Contraction of endothelial cells in capillaries within the red pu lp serve to decrease blood flow to the reticular meshwork, thereby shu nting more blood flow through the " fast # pathway (Blue and Weiss, 1981; Groom et al., 1991). Reticular cells in the red pulp, which contain contractile elements, likely further prevent blood from entering by decreasing the size of passages through this area (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988) . Contraction of red pulp reticular cells also likely participates in ejecti on of the cells contained within this compar tment (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988) . Hypoxia is the most well research ed condition that causes spleen contraction and shuttling of blood to the " fast # pathway within the spleen (Sandler et al., 1984; Sato et al., 1997; Kuwahira !"#!et al., 1999; Bakovic et al., 2003; Richardson et al., 2007; 2009) . Spleen contraction has also been found to occur in response to both stroke and exercise (Schaffner, 1985; Seifert et al., 2012) . 1.2.3: Splenic Innervation 1.2.3.1: Sympathetic Innervation 1.2.3.1.1: Brain Nuclei Associated with Spleen Innervation Sympathetic neuron al activity in the periphery is controlled primarily by five brain nuclei. These nuclei are the A5 cell group, rostral ventrolateral medulla , ventromedial medulla, caudal raphe nuclei, and paraventricular nucleus of the hypothalamus ( PVN ) (Strack et al., 1989; Cano et al., 2001; Sved et al., 2001) . These nuclei all have direct descending connections with spinal cord pre -ganglionic neurons (Strack et al., 1989; Cano et al., 2001). Furthermore all of these brain nuclei are the first nuclei to become infected after injection of retrogradely transported reporter -viruses in to known sympathetic targets such as the spleen, kidney, adrenal gland, tail artery, and sympathet ic ganglia (Str ack et al., 1989; Li et al., 1992; Schramm et al., 1993; Smith et al., 1998; Weiss and Chowdhury, 1998; Cano et al., 2001) . Despite the common circuitry to all these peripheral targets , different subsets of nuclei are specifically involved in the control of each organ (Sved et al., 2001) . For example, both r etroviral tracing studies (Cano et al., 2001) and microinjection studies (Katafuchi et al., 1993) demonstrate that th e PVN is an upstream regulator of splenic sympathetic neuronal activity. !"$!The PVN is a bilateral nucleus located at the dorsal aspect of the 3 rd ventricle of the brain. The PVN has a multitude of functions which are aimed at maintaining the homeostasis of the body, particularly cardiovascular function (Pyner, 2009). The PVN is regarded as one of five canonical central pre -sympathetic nuclei , and has direct descending efferent projections to the intermediolateral cell column of the spinal cord (IML) where they synapse on pre -ganglionic sympathetic neurons (Strack et al., 1989; Schramm et al., 1993; Cano et al., 2001; Sved et al., 2001; Pyner, 2009) . These descending spinal projecting neurons specifically express vasopressin, oxytocin, or dopamine (DA) , and terminate at virtually every thoracic level (Saw chenko and Swanson, 1982; Strack et al., 1989; Sved et al., 2001; Pyner, 2009) . Both oxytocin and vasopressin are stimulatory to pre -ganglionic sympathetic neurons, whereas dopamine has mixed effects on pre -ganglionic sympathetic neurons , likely due to a combination of direct and indirect interactions (Pyner, 2009) . The PVN receives noradrenergic innervation from several brain nuclei, including the A1, A5, and A6 (locus coeruleus) noradrenergic cell groups (Byrum and Guyenet, 1987; Pacak et al., 1995; Samuels and Szabadi, 2008) . The PVN is divided int o three magnocellular and five parvocellular subdivisions (Pyner, 2009) . The majority of noradrenergic innervation to the PVN terminates in the parvocellular portio n, which is also the region where the cells bodies of pre -sympathetic spinal projecting neurons reside (Byrum and Guyenet, 1987; Cunningham and Sawchenko, 1988; Strack et al., 1989; Samuels and Szabadi, 2008; Pyner, 2009) . These descending pre -sympathetic neurons synapse upon the cell bodies of pre -ganglionic acetylcholine ( Ach ) expressing !"%!neurons in the IML of the spinal cord to control sympathetic outflow in the periphery (Kandel et al., 2000) . Stimulation of the PVN by direct intracerebral glutamate injection stimulates splenic sympathetic activity (Katafuchi et al., 1993) . Norepinephrine ( NE) release in the PVN, in turn, stimulates glutamate releasing neurons in the parvocellular regions of the PVN to contribute to the control of sympathetic outflow (Daftary et al., 2000). Noradrenergic signaling in the PVN is mediated by $ 1, $ 2, and %-adrene rgic receptors (AR) (Leibowitz and Hor, 1982; Goldman et al., 1985; Daftary et al., 2000; Chong et al., 2004) . Within the parvocellular regions, $ 1AR increase spontaneous inhibitory pos t-synaptic currents (sIPSC) in gamma -aminobutyric acid expressing (GABA) interneurons and stimulate the activity of glutamatergic interneurons, both of which synapse upon effector parvocellular neurons, such as directly descending pre -sympathetic neurons (Daftary et al., 2000; Chong et al., 2004) . Conversely, $ 2AR decrease sIPSCs in GABAergic interneurons (Chong et al., 2004) . Other parvocellular neurons are directly inhibited via %AR (Daftary et al., 2000) . Afferent PVN noradrenergic axon terminals are known to be regulated by pre -synaptic $ 2-adrenergic auto -receptors (Lookingland et al., 1991) . The rate of NE turnover (an estimate of noradrenergic neuronal activity) is generally higher in central nervous system ( CNS ) noradrenergic neurons compared to noradrenerg ic neurons in the peripheral nervous system ( PNS ). For instance, the half -life of NE turnover in the PVN and amygdala is <1.5 h, whereas NE turnover in the heart and pancreas is >3 h (Levin, 1995) . A well known and well studied cause for higher NE release in the PVN is !"&!stress, serving to activate the hypothalamic -pituitary -adrenal ( HPA ) axis by the inducing the production of stress -related corticosteroids, such a s cortisol, as well as increasing sympathetic outflow (Pacak et al., 1995; Itoi and Sugimoto, 2010; Inoue et al., 2013) . 1.2.3.1.2: Post -Ganglionic Innervation of the Spleen Sympathetic pre -ganglionic neurons innervating the spleen arise from the thoracic spinal cord, specifically T 3-T12 (Bellinger et al., 1987; Cano et al., 2001) . Upon exiting the spinal cord at these levels, axons of pre -ganglionic neurons regulating splenic sympathetic activity largely terminate in the celiac -mesenteric plexus (Bellinger et al., 1987; Nance and Sanders, 2007) . There is, however, a minor (<15%) sympathetic innervation contribution to the spleen from sources other than the celiac -mesenteric ganglion, perhaps from splanchnic nerves emanating directly from sympathetic chain ganglia (Bellinger et al., 1987; Nance and Burns, 1989) . Post -ganglionic s ympathetic neuron axons travel to the spleen via the splenic neurovascular bundle , which contains the splenic artery, vein, and nerve, to terminate diffusely in the spleen capsule and periarteriolar regions (Felten et al., 1987; Fe lten and Olschowka, 1987; Kohm and Sanders, 2001; Cesta, 2006) . Unmyelinated nerves are present in reticular fibers of the red and white pulp , presumably controlling the contraction of reticular cells in these areas (Pinkus et al., 1986; Satodate et al., 1986; Saito et al., 1988; Cesta, 2006) . Axon terminals have also been identified in lymphocyte -rich areas, such as the PALS, terminating in very close proximity (less than 8 microns) to immune cells (Felten et al., 1987; Felten and Olschowka, 1987) . !"'! 1.2.3.1.3: Sympathetic Neurotransmitters 1.2.3.1.3.1: Norepinephrine 1.2.3.1.3.1.1: Synthesis NE is synthesized within the axon terminal of post -ganglionic sympathetic neurons through a multistep process (Figure 1.1 ) (Eisenhofer et al., 2004) . Dietary tyrosine is taken in to the cell via the L -neutral amino acid transporter (Oxender and Christensen, 1963) . Once inside the cell tyrosine is converted to L -3,4-dihydroxyphenylalanine (L -DOP A) by the rate -limited enzyme in catecholamine synthe sis, tyrosine hydroxylase (TH) (Levitt et al., 1965; Eisenhofer et al., 2004) . L-DOPA is then rapidly converted to DA by the acti ons of L -aromatic amino acid decarboxylase (L -AAAD), such that L -DOPA is normally undetectable within the neuron (Blaschko, 1942; Lookingland and Moore, 2005) . DA is then taken up into vesicles by the vesicular monoamine transporter (VMAT), where is it converted to NE by way of DA-!-hydroxylase (DBH) (Kaufman, 1974; Weinshilboum, 1977; Kumer and Vrana, 1996; Eisenhofer et al., 2004) !"(! Figure 1.1. Synthetic pathway of NE. Dietary tyrosine is hydroxylated to DOPA via TH, the rate -limiting enzyme in NE synthesis. DOPA is rapidly decarboxylated to DA by L-AAAD. DA is then hydroxylated by D !H to form NE. Figure adapted from Marino and Cosentino (Marino and Cosentino, 2013) . !")!The synthesis of NE is largely regulated by the activity of TH. Short -term regulation of TH is accomplished by phosphorylation (Kumer and Vrana, 1996) . Phosphorylation of serine (Ser) residues (Ser19, Ser 31, and Ser 40) located on the N -terminus of TH can increase enzyme efficiency (Haycock, 1990; Daubner et al., 2011) . Phosphorylation at Ser40, the most important site of phosphorylation, decreases e nd-product feedback inhibition (Daubner et al., 1992; Ribeiro et al., 1992; Ramsey and Fitzpatrick, 1998) . Phosphorylation of Ser19 and Ser 31 have modest direct effects on TH activity, but also increase the rate of Ser40 phosphorylation to indirectly potentiate TH activity (Bevilaqua et al., 2001; Lehmann et al., 2006) . Interestingly, the phosphorylation of TH to increase catalytic activity also reduces the stability of the phosphorylated enzyme, but not due to degrad ation of the entire protein (Lazar et al., 1981; Vrana et al ., 1981; Vrana and Roskoski, 1983) . Thus, while more catalytically active, the half -life of phosphorylated TH protein is decreased relative to non -phosphorylated TH (Gahn and Roskoski, 1995) . On the other hand, TH stability can be increased by both tyrosine and feedback inhibitors (i.e. , catecholamines) (Kumer and Vrana, 1996) . A number of Ca 2+-dependent pathways mediate TH phosphorylation including Ca 2+/phospholipid -dependent protein kinase C and Ca 2+/calmodulin -dependent multi -protein kinase (Yamauchi and Fujisawa, 1981; Albert et al., 1984; Vulliet et al., 1985) . Phosphorylation of TH is also a ctivity dependent , such that activated neurons increase the amount of phosphorylated enzyme (Kumer and Vrana, 1996). !"*!1.2.3.1.3.1.2: Metabolism NE is converted to 3-methoxy -4-hydroxyphenylglycol ( MHPG ), the major metabolite of NE in the brain, by successive processing of monoamine oxidase ( MAO), catechol -O-methyltransferase ( COMT ) and aldehyde reductase ( ADR) (Figure 1.2 ) (Lookingland et al., 1991; Hayley et al., 2001; Eisenhofer et al., 2004) . More specifically, in the brain NE is converted to 3,4-dihydroxyphenylglyco aldehyde ( DOP EGAL), by MAO, which diffuses out of the neuron to glia cells where it is converted to MHPG via COMT and ADR (Lookingland e t al., 1991; Eisenhofer et al., 2004) . In the spleen, MHPG is produced by MAO/A DR conversion of NE to 3,4-dihydroxyphenylglycol (DHPG ) in sympathetic neurons followed by the conversion of DHPG to MPHG by COMT in macrophages within the white pulp of the spleen (Karhunen et al., 1994; Eisenhofer et al., 2004; Myıh−nen et al., 2010) . MHPG is then released from the spleen into circulation and further metabolized to vanillylman delic acid ( VMA ) in the liver by alcohol dehydrogenase ( ADH) and aldehyde dehydrogenase ( AD) (Oh -hashi et al., 2001; Eisenhofer et al., 2004; Siraskar et al., 2011) . !#+!Figure 1.2. Metab olic pathway of NE . NE is converted to the major brain metabolite MHPG in neurons and glia/macrophages by successive deamination -hydroxylation, reduction, and methylation by MAO, A DR, and COMT, respectively. MHPG can the be converted in the liver to VMA, the major metabolite of NE found in the blood, by dehydr ogenation and hydroxylation via ADH and AD. Figure adapted from Eisenhofer, et al (Eisenhofer et al., 2004) . !#"!1.2.3.1.3.1.3: Receptors AR mediate the effects of NE released from sympathetic axon terminals . These receptors, which are also activated by epinephrine, are divided into two major types, " and ! (Malbon and Wang, 2001) . There are 2 identified subtypes of " AR and 3 ! subtypes (Marino and Cosentino, 2013) . The two " AR are designated "1 and "2. The "1AR is a G -protein coupled receptor that activates the G q pathway (Eltze, 1996; Aboud et al., 2012) . This pathway results in the activation of phospholipase C and generation of inositol 1,4,5 -triphosphate (IP3 ) by the hydrolysis of phospholipid precursors (Bootman et al., 2001; Malbon and Wang, 2001) . IP3 then acts upon the IP3 receptor found on intracellular Ca 2+ storag e sites, such as the endoplasmic or sarcoplasmic reticulum, leading to the release and elevation of intracellular Ca 2+ causing contraction of smooth muscle (Bootman et al., 2001; Malbon and Wang, 2001 ). Contraction of smooth muscle is the major physiologic action of "1AR at sites such as the urethra, vascular smooth muscle, and in the spleen . This specific subtype receptor has been demonstrated to undergo phosphorylation -mediated desensitization and internalization occurring as quickly as 5 min after NE binding (Leeb -Lundberg et al., 1987; Fonseca et al., 1995; Malbon and Wang, 2001) . The "2AR is also a G -pro tein coupled receptor, but this receptor activates the Gi/o pathway whose primary effects upon are to inhibit cyclic adenosine monophosphate (cAMP ) production via inhibition of adenylyl cyclase (Howlett and Mukhopadhyay, 2000; Marino and Cosentino, 2013) . Activation of these G -proteins also leads to the inhibition of Ca 2+ ion channels and increase s in K+ conductance (Bockaert, 2001; Khan et al., !##!2002). These effects result in a decrease in the firing of neurons and explains the physiological role of these receptor s, which is to inhibit the release of NE from the axon terminals of noradrenergic neurons as a form of autoregulatory feedback (Khan et al., 2002; Marino and Cosentino, 2013) . Thus , "2AR are largely expressed on pre -synaptic axon terminals of noradrenergic neurons in the brain and periphery, particularly in the spleen and kidney (Marino and Cosentino, 2013) . The three !AR are designated !1, !2, and !3. All these receptors are G -coupled proteins that activate the G s pathway (Malbon and Wang, 2001; Marino and Cosentino, 2013). Activation of the G s pathway stimulates adenylyl cyclase leading to an increase in the production intracellular cAMP , and therefore increased activity of protein kinase A (Bockaert, 2001; Malbon and Wang, 2001; Marino and Cosentino, 2013) . The most well known physiologic effect of !1AR is to increase cardiac output via increasing heart rate and contractility (Malbon and Wang, 2001; Marino and Cosentino, 2013) . However, this receptor is known to stimulate the release of renin from the juxtaglo merular apparatus of the kidney as well. !1, along with !3AR, also regulates the lipolysis adipose tissue (Marino and Cosentino, 2013) . The most well studied effect of !2AR is to induce the relaxation of smooth muscle (Malbon and Wang, 2001; Marino and Cosentino, 2011) . !2AR are also known to have an number of other actions including the regulation of glycogenolysis, aqueous humor production, and insulin secretion (Marino and Cosentino, 2013) . However, !2AR are also expressed by cells of the immune system, the highest expression being on B cells, !#$!suggesting an immunologic function role for these receptors (Kin and Sanders, 2006; Sanders, 2012) . 1.2.3.1.3.1.4: NE Function in Spleen: 1.2.3.1.3.1.4.1: Splenic Blood Flow and Contraction Spleen contraction is controlled by the sympathetic nervous system. NE was first found to bind to receptors located on smooth muscle cells within the spleen by Gillespie and Hamilton in 1966 (Gillespie and Hamilton, 1966) . These receptors were later identified as "1AR, more specifically the $ 1BAR subtype (Eltze, 1996; Aboud et al., 2012). In congruence with the ability of NE to stimulate splenic smooth muscle contraction, noradrenergic axon s densely innervate the spleen capsule, periarteriolar regi ons of the spleen , and in reticular fibers of the splenic white and red pulp (Davies and Withrington, 1973; Blue and Weiss, 1981; Pinkus et al., 1986; Felten et al., 1987; Felten and Olschowka, 1987; Saito et al., 1988; Elenkov and Vizi, 1991; Cesta, 2006) . 1.2.3.1.3.1.4.2: NE Effects on t he Immune System Interactions between the immune and nervous systems are coordinated within the spleen. The sympathetic nervous system is activated following the presentation of an immune challenge (Kohm et al., 2000; Meltzer et al., 2003; Hefco et al., 2004) , and sympathetic post -ganglionic noradrenergic axon terminal activity increases in the spleen following the injection of soluble protein antigens (Kohm et al., 2000; Sanders, 2012) . !#%!Pro -inflammatory cytokines are likely the activators of sympathetic neuronal activity during an immune challenge. The most studied of these early inflammatory factors a re interleukin (IL) -1% and IL-6. Both of these cytokines are produced by phagocytic monocytes early in inflammatory events (Dinarello, 2004) . The production of IL -1% and IL-6 in response to an immune challenge occurs within hours (Kakizaki et al., 1999) . This makes the se cytokines prime candidates to mediate rapid and early changes in sympathetic activity. In support of this, intraperitoneal (i.p.) injection of IL -1 % increases the release of NE in the spleen (Shimizu et al., 1994; Kohm and Sanders, 2001) . This suggests that IL -1% is the sympatho -stimulatory mediator in vivo . However, IL -1% is also able to stimulate the production of IL -6 from a myriad of cell types (Kauma et al., 1994; Spangelo et al., 1994; Parikh et al., 1997) . This leaves open the possibility that the effects of IL -1% on sympathe tic activity may be due to IL -6. Plasma and brain levels of IL-6 peak within 1 -3 h following an immune challenge (Kakizaki et al., 1999) . Intraventricular injection of IL -6 produces increased firing of spleen capsule sympathetic neurons (Helwig et al., 2008) , suggesting IL -6 may act centrally to increase NE release during an immune challenge. Taken together, these data strongly suggest that one, or both, of these cytokines are stimulating th e sympathetic nervous system during an immune challenge. Splenic B cells express functional % 2AR and respond to NE both in vitro and in vivo (Nance and Sanders, 2007; Sanders, 2012) . Engagement of NE with % 2AR on B cells within 24 h of an antigen exposure increases the amount of secreted IgM and IgG in response to a humoral immune challenge (Sanders, 2012) . Conversely, blockade of !#&!% 2AR on B cells during an immune challenge decreases the amount of secreted antibody (Sanders, 2012) . Transcriptional activity of the 3' -Ig heavy chain enhancer (3' -IgH) and the actions of the transcription factor OCA -B are critically important to antibody production (Stevens et al., 2000; Pinaud et al., 2001; Vincent -Fabert et al., 2010) . Activation of % 2AR on B cells, through a protei n-kinase A dependent mechanism, up -regulates OCA -B and OCA -B binding to the 3' -IgH, thereby connecting % 2AR stimulation to increased antibody production (Podojil et al., 2004) . Increased antibody production by B cells in response to NE is likely temporally dependent. Only exposure to NE at early time points (< 24 h) following antigen exposure has been shown to increase B cell proliferation and antibody production (Kohm and Sanders, 2000; 2001) . Exposure to NE prior to or 24 h after an immune challenge has no effect on the resulting antibody respo nse. However, there is also evidence that % 2AR stimulation may decrease proliferation in lymphocytes (Marino and Cosentino, 2011) . 1.2.3.1.3.2: ATP /Adenosine 1.2.3.1.3.2.1: Synthesis Adenosine 5 #-triphosphate (ATP) is the universal energy currency for biological systems. It is synthesized by the combination of three chemically distinct parts : the adenine ring, the ribose moiety , and the triphosphate chain (Sperl⁄gh and Vizi, 1996) . Each of these components are synthesized via independent pathways and assembled !#'!to form ATP. Adenine rings are formed during de novo purine synthesis by combining the carbon and nitrogen atoms derived from glycine, N 5, N10-methenyltetrahydrofolate, glu tamine, N 10-formyltetrahydrofolate, aspartate and respiratory CO 2 (Sperl⁄gh and Vizi, 1996). The ribose moiety, synthesized in the pentose phosphate pathway, is attached to the adenine ring and the ultimate end product of purine synthesis is inosine monophosphate, which can be converted to adenosine monophosphate (AMP) via a two -step amination process (Sperl⁄gh and Vizi, 1996) . AMP can also be scavenged through the purine salvage pathway , by the phosphorylation of scavenged adenosine by adenosine kinase (Sperl⁄gh and Vizi, 1996) . AMP is then again phosphorylated by nucleotide diphosphate kinase to create adenosine diphosphate (ADP). ADP is the major substrate for ATP synthase which is powered by the generation of a hydrogen ion concentration through the process of oxidative phosphorylation in mitoc hondria to generate ATP (Spe rl⁄gh and Vizi, 1996) . ATP is exp orted from the mitochondria by adenine nucleotide transporters, driven by the exchange of ATP for ADP (Sperl⁄gh and Vizi, 1996) . ATP is found in virtually all synaptic vesicles, but it is assumed there is a specific ADP/ATP translocase , believed to be structurally and functionally similar to the transporter used to export ATP from mitochondria, that allows for the accumulation of ATP in synaptic vesicles (Dowdall et al., 1974; Lagercrantz and Staj−rne, 1974; Winkler and Westhead, 1980; Sperl⁄gh and Vizi, 1996; Gualix et al., 1999; Pankr atov et al., 2006) . !#(!1.2.3.1.3.2.2: Metabolism After release, e xtracellular ATP is quickly degraded to AMP by extracellular phosphatases followed by conversion to adenosine by 5&-nucleos idase (Pearson et al., 1980; Welford et al., 1987; Juul et al., 1991) . Adenosine is rapidly cleared from the interstitium , at least in part , by re -uptake through pre -synaptic nucleoside transporters (Van Belle, 1993) . Recaptured a denosine can then be metabolized or recycled through the purine salvage pathway to generate new AMP, ADP, and ATP (Sperl⁄gh and Vizi, 1996). 1.2.3.1.3.2.3: Receptors Purinergic receptors are divided into two broad categories : P1 and P2 . P1 rece ptors are all G -coupled protein receptors that are agonized by adenosine (Ralevic, 2009). Of the four subtypes, the A1 and A3 activate the G i/o pathway, whereas the two isotypes of A2 both activate G s pathway (Ralevic, 2009) . The A1 receptor has been identified as being able to modulate the release of sympathetic neurotransmitters (Rongen et al., 1996; Ralevic and Kendall, 2002) . Specifically, A1 rece ptors are expressed on the pre -synaptic axon terminal of post -ganglionic sympathetic neurons and activation of these receptors inhibits the release of neurotransmitters (Rubino et al., 1991; 1993; Rongen et al., 1996) . Activation of A2 receptors has b een shown to induce relaxation of vascular smooth muscle (Ralevic, 2009) . There are two major categories of P2 receptors. P2X receptors are ligand -gated ion channels that when activated by ATP cause membrane depolarization and !#)!extracellular Ca 2+ entry into cells (Sedaa et al., 1990; Ren and Burnstock, 1997; North, 2002; Burnstock and Ralevic) . P2Y receptors are G -protein coupled re ceptors and depending upon which of the 8 subtypes activate either the G s or Gq pathway (Ralevic, 2009). All P2 receptors recognize ATP, as well other nucleotides such as ADP, uridine triphosphate, and uridine diphosphate (Ralevic, 2009) . P2X receptors are known to induce the contraction of vascular smooth muscle when released from sympathetic neurons (Ralevic and Kendall, 2002; Macarthur et al., 2011) . Activation of pre -synaptically expressed P2X receptors has been demonstrated to induce depolarization, whereas pre -synaptically expressed P2Y receptors are inhibitory to neu rotransmitter release (Ralevic, 2009) . P2Y and P 1-type A1 receptors have been shown to form a heterodimer, suggesting that the actions of one of these receptors may be assisted or mediated by the other (Yoshioka et al., 2002; Ralevic, 2009) . 1.2.3.1.3.2.4: ATP/Adenosine Function in Spleen: 1.2.3.1.3.2.4.1: Splenic Blood Flow and Contraction Purinergic receptors likely regulate two different aspects of splenic contraction. Adenosine has largely been know n to inhibit NE release from sympathetic neurons via acting on pre -synaptic A1 adenosine receptors (Kubo and Su, 1983; Wennmalm et al., 1988; Kgelgen et al., 1992; Rongen et al., 1996; Ralevic, 2009; Macarthur et al., 2011; Burnstock and Ralevic) . Interestingly, there is some preced ence that activation of A1 receptors can stimulate vascular smooth muscle contraction and contraction of the spleen (Fozard and Milavec -Krizman, 1993; Tawfik et al., 2005) . ATP released from !#*!sympathetic neurons is also able to induce the constriction of vascular smooth muscle in a variety of tissues (vas defe rens, aorta, splenic nerve) through P2X receptor s (Sedaa et al., 1990; Ren and Burnstock, 1997; Burnstock and Ralevic) , however, a specific role for P2X receptors in inducing splenic contraction has not been described. 1.2.3.1.3.2.4.2: ATP/Adenosine Effects on The Immune System The role of purine in the immune response is relatively unknown. The most well research ed aspect of purinergic effects on the immune system involved P2X receptor s. P2X receptors have been identified on near ly every cell type of the immune system (Jacob et al., 2013) . The role of these receptors is largely undefined, but i t appears to be mostly involved in chemotaxis of inflammatory cells, such as neutrophil s, macrophages, and eosinophils , as well as stimulating granular release from these latter cells (Jacob et al., 2013) . In addition to this direct effect, the role of purinergic receptor modulation of neurotransmitter release from sympathetic neu rons may also affect immune responses, such as those mediated by NE. 1.2.3.1.3.3: Neuropeptide Y 1.2.3.1.3.3.1: Synthesis Neuropeptide Y ( NPY ) is a 36 amino acid peptide expressed by many post -ganglionic sympathetic neurons, including those projecting to the spleen, kidney, and mesentery (Lundberg et al., 1990; Romano et al., 1991; Chevendra and Weaver, 1992) . NPY was name d as such due to the many tyrosine residues , designate d by the letter !$+!ÒYÓ, in its linear sequence (Tatemoto, 2004) . NPY is generated from a 97 amino acid that is cleaved at two sites producing NPY as well as a 28 amino acid peptide, termed Òsignal peptideÓ, and a 30 amino acid peptide, termed ÒCOOH -terminal peptideÓ (Tatemoto, 2004) . NPY distribution largely parallels that of TH and DBH and is considered the major signaling pept ide of sympathetic neurotransmission (Lundberg et al., 1990). 1.2.3.1.3.3.2: Metabolism After release, NPY signal is terminated by proteolytic cleavage. This proteolysis is accomplished by ectopeptidase s located on the cell surface of neurons, as has been described for other neuropeptides, such as enkephalins (Turner and Barnes, 1994) . Specifically , NPY is degraded predominantly by dipeptidyl peptidase IV, neutral endopeptidase -24.11, and by aminopeptidase P (Mentlein et al., 1993; Medeiros and Turner, 1996) . 1.2.3.1.3.3.3: Receptors To date, five different NPY receptors have been identified including Y1, Y2, Y4, Y5, and Y6 subtypes (Tatemoto, 2004) . All NPY receptors are G -protein coupled receptors that activate the G i/o pathway (Michel et al., 1998) . Y1 receptors are expressed b y vascular smooth muscle and can induce contraction and vasoconstriction (Westfall et al., 1987; 1990; Zukowska -Grojec et al., 1996; Michel et al., 1998; Wiest et !$"!al., 2006). Y2 receptors are expressed on the pre -synaptic terminal of post -ganglionic sympathetic neurons and inhibit the release of sympathetic neurotransmitter s (Westfall et al., 1987; 1990; Michel et al., 1998) . Y4 receptor s primarily bind a NPY related peptide called pancreatic polypeptide and only has significant affinity for a mutated form of NPY, [Leu31, Pro34]NPY (Michel et al., 1998) . Y5 receptor pharmacolog ical and physiological actions are very similar to that of Y1 receptors , and in fact may be considered a subtype of Y1 receptors (Michel et al., 1998) . While the Y6 receptor has been cloned and identified, a physiologic effect of the receptor has yet to be identified (Mi chel et al., 1998) . 1.2.3.1.3.3.4: NPY Function in Spleen 1.2.3.1.3.3.4.1: Splenic Blood Flow and Contraction Y1 receptors are expressed by vascular smooth muscle and can induce vasoconstriction , while Y2 receptors inhibit the release of sympathetic neurotransmitters (Westfall et al., 1987; 1990; Zukowska -Grojec et al., 1996; Michel et al., 1998; Wiest et al., 2006). Thus it is likely that NPY may act to regulate the flow o f arterial blood and thus modify blood flow through the spleen. Regarding spleen contraction, only one study has shown NPY capable of inducing spleen contraction, albeit very weakly in comparison to NE (Corder et al., 1987) . !$#! 1.2.3.1.3.3.4.2: NPY Effects on The Immune System NPY has been demonstrated to modulate a number of immune responses , and most cells in the immune system express NPY receptors , the predominant type being Y1 and Y2 (Bedoui et al., 2003; Wheway et al., 2005; Dimitrijevi ' and Stanojevi ', 2013) . While the consensus effect of NPY on immunity is not clear at this time, NPY acts generally to promote migration, inhibiting proliferation, and skew immune responses to a TH2-type (la Fuente et al., 1993; Levite, 1998; Puerto et al., 2005; Dimitrij evi ' and Stanojevi ', 2013) . In addition to these direct effects, the role of Y2 receptor modulation of neurotransmitter release from sympathetic neurons may also affect immune responses, such as those mediated by NE. 1.2.3.2: Parasympathetic Innervation Parasympathetic innervation of the spleen is somewhat controversial. A number of anatomical studies have failed to demonstrate cholinergic parasympathetic innervation of the spleen (Bellinger et al., 1993; Sch−fer et al., 1998; Cano et al., 2001; Nance and Sanders, 2007; Olofsson et al., 2012) , yet the spleen is known to contain acetylcholine (Ach) , and activation of muscarinic cholinergic receptor s induce s contraction of the spleen capsule through an action independent of "1AR (Davies and Withrington, 1973; Wong, 1990; Olofsson et al., 2012) . In addition, the va gus nerve plays a vital role in controlling the inflammatory response (i.e. , tumor necrosis factor !$$!production) of splenocytes to an immune challenge, an effect believed to be mediated by "7 nicotinic Ach receptors on macrophage immune cells (Olofsson et al., 2012) . The prevailing thought as to the source of splenic Ach is from T cells (Fujii et al., 2008). This finding, combined with the identification of vagus stimulation of spleen projecting sympathetic neurons of the celiac ganglion plexus provides a potential mechanism whereby vag al and cholinergic signaling can cause effects in the absence of direct parasympathetic innervation of the spleen (Vida et al., 2011; Olofsson et al., 2012). The final piece of this puzzle will then be to confirm the production of Ach from T cells in response to sympathetic activity in the spleen (Vida et al., 2011; Olofsson et al., 2012). 1.3: Cannabinoids Cannabinoids are a class of lipophilic compounds defined by their ability to bind to two identified cannabinoid receptors designated CB1 and CB2. The most widely known cannabinoid, #9-tetrahydrocannabinol (THC), is derived from plants of the genus cannabis , a.k.a. mariju ana. THC is known mainly for it s psychoactive properties when either ingested or inhaled (Pertwee, 2008) . Cannabinoid derivatives have been widely used throughout history for both medicinal and recreational purposes. In the U.S.A., dronabinol (synthetic THC) is approved by the Federal Drug Administration for the control of nausea and vomiting in cancer patients undergoing chemotherapy as well as an appetite stimulant in patients with AIDS (Galal et al., 2009) . In the United Kingdom, a drug named nabi ximols (Sativex ¨), which is a mixture of THC and another cannabis -!$%!derived cannabinoid called cannabidiol (CBD) , is approved for the control of spasticity in multiple sclerosis patients (Pharmaceuticals, 2011) . In the U.S.A. , 20 states and District of Columbia have approved measures allowing the possession and growing of marijuana for medicinal purposes (WhiteHouse.gov) . Furthermore, two of these states have legalized the recreat ional use of marijuana, which is an indicator of increasing popular support for the use of marijuana (drugpolicy.org; Wollner) . The effects of exogenous cannabinoids, s uch as THC, are mediated by modulation of the endocannabinoid system (Figure 1.3 ). The endocannabinoid system has largely been identified as modulating the release of neurotransmitters from axon terminals as a form of negative feed back. The two most well researched receptors of the endocannabinoid system are CB1 and CB2 (Mackie, 2008; Alger and Kim, 2011) . Endogenous ligands for CB1 and CB2, termed ÒendocannabinoidsÓ, were identified in the mid 1990's , of which t he two most widely studied endocannabinoids are N-arachidonoyl -ethanolamide (anandamide) and 2-arachidonoyl glycerol (2-AG) (Di Marzo, 2008; Alger and Kim, 2011) . !$&! Figure 1.3. Schemati c representation of the endocannabinoid system. The endogenous cannabinoid ligands AEA and 2 -AG can be synthesized from precursors in the p ost -synaptic membrane in response to binding of released neurotransmitters. CB1 activation by AEA or 2 -AG then resul ts in the inhibition of neurotransmitter release via through a variety of mechanisms, including a decrease in voltage -gated Ca 2+ channel conductance . THC, the major cannabinoid congener found in cannabis , is also CB1 agonist, and it through this action that its psychotropic actions are believed to be mediated. Figure adapted from Guzman (Guzm⁄n, 2003) . !$'!1.3.1: Cannabinoid Receptors 1.3.1.1: CB1 Structure and Signaling CB1 is considered to be the major neuronal cannabinoid receptor. CB1 is a G protein -couple d receptor consisting of seven -transmembrane domains, with an intracellular C terminus and an extracellular amino terminus (Mackie, 2008) . CB1 predominantly couple s with the G i/o subtype, whose primary effects upon activation are to inhibit cAMP production via inhibi tion of adenylyl cyclase (Howlett and Mukhopadhyay, 2000) . Inhibition of cAMP production leads to inhibition of protein kinase A which then leads to a number of downstream consequences, including a positive shift in the voltage -dependence of A -current K + channels (Childers and Deadwyler, 1996) . Activation of these G -proteins also leads to the inhibition of N -type (Felder et al., 1993; Mackie et al., 1993) , Q-type (Mackie et al., 1995) , and L -type Ca 2+ channels (Gebremedhin et al., 1999) . Calc ium influx through voltage -gated calcium channels, especially Q - and N -type, is essential for the release of neurotransmitters from axon terminals (Sheng et al., 1998) . CB1 receptor agonism also activates inwardly rectifying K + channels, which play a significant role in determining the resting membrane potential of neurons (Mackie et al., 1995) . It is by these mechanisms that CB1 inhibits neurotransmitter release from axon terminals. Additional effects of CB1 activation include s activation of the MAPK pathway, leading to increased glucose and fatty -acid oxidation, and well -characterized intracellular cal cium transients mediated by IP 3 (Howlett and Mukhopadhyay, 2000) . !$(!Location CB1 is localized to the pre -synaptic axon terminals of neurons (Ny™ri et al., 2005) . CB1 is found abundantly in CNS areas such as the cortex, basal ganglia, cerebellum, and brainstem emetic center, but expressed at very low levels in brainstem respiratory control centers and the thalamus (Herkenham et al., 1991; Mackie, 2008) . There is also evidence for functional CB1 on autonomic sympathetic axon terminals. Functional evidence for CB1 has been identified in sympathetic axon terminals of the atria, vas deferens, and mesenteric arteries (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . Interestingl y, CB1 mRNA has also been found in the superior celiac ganglion, vas deferens, and the spleen (Ishac et al., 1996; Schatz et al., 1997). In addition, CB1 is expressed by immune cells, including splenic lymphocytes, but t o a much lesser extent than CB2 (Galiegue et al., 1995; Kaplan, 2012) . The role of CB1 on immune cells remains somewhat controversial, but a handful of studies have shown CB1 to mediate, at least partially, some immune effects (Kaplan, 2012) . For example, CB1 transcription is in duced by IL -4, a TH2 cytokine, and once up -regulated CB1 stimulation inhibits cAMP formation and IL -2 production, a cytokine critical for T cell responses in vivo (Ferrero et al., 2001; Bırner et al., 2008) . Ligands CB1 is activated by both exogenous an d endogenous cannabinoids. THC is a partial agonist of the CB1 receptor (K i ~40 nM) compared to compounds such as WIN55212, a full agonist (K i ~2 nM) (Kuster et al., 1993; Pertwee, 2008) . The two most !$)!well characterized endocannabinoid compounds that bind to CB1 are AEA and 2 -AG. Neither of these compounds are stored in the neuron , but instead are made on demand from phospholipid components found in the membrane of post -synaptic neurons (Giuffrida and Mcmahon, 2010) . AEA is derived from the deacylation of N -arachidonoyl phosphatidylethanolamine, a phospholipid pr ecursor, in a Ca 2+-dependent process (Okamoto et al., 2004) . 2-AG is synthesized by sequential hydrolysis of inositol phospholipids containing arachidonic acid by PLC ! and diacylglycerol lipase (Giuffrida and Mcmahon, 2010). Following de novo synthesi s, endocannabinoids are released and travel retrogradely across the synapse to act on presynaptic CB1. Both AEA and 2 -AG are partial agonists of CB1, however AEA is more selective for CB1 than 2 -AG (Alger and Kim, 2011) . AEA and 2 -AG are rapidly degraded within minutes by the enzymes FAAH (primarily AEA) and MAGL (primarily 2 -AG), thereby terminating their actions (Gerra et al., 2010; Giuffrida and Mcmahon, 2010) . 1.3.1.2: CB2 Structure and Signaling CB2 is considered to be the major peripheral cannabinoid receptor. CB2 was first discovered in the HL60 cell line, a pro -myelocytic cell line (Munro et al., 1993) . It was subsequently discovered that CB2 mRNA is abundant in peripheral tissues, such as the sple en, but relatively absent from the brain (Shire et al., 1996; Griffin et al., 2000; Brown et al., 2002) . Like CB1, CB2 is a seven -transmembrane protein that couple s to Gi/o subtype G -proteins and inhibit adenylyl cyclase, preventing the formation of cAMP !$*!(Felder et al., 1995) . CB2 also activate s the MAPK/ERK pathway (modulating inflammation in microglia), the PI3K/AKT signaling pathway (promoting survival in oligodendrocyte precursors), and ceramide synthesis (inducing the apoptosis of tumor cells) (Molina -Holgado et al., 2002; Carracedo et al., 2006; Herrera et al., 2006; Romero -Sandoval et al., 2009) . Very different from CB1, CB2 agonism does not inhibit Q-type Ca 2+ channels, nor activate inwardly rectifying K + channels (Felder et al., 1995) . Location CB2 is expressed primarily on cells with an immunologic function. As mentione d above, the spleen was among the first organs shown to have abundant CB2 expression (Brown et al., 2002) . CB2 is found on T cells, B cells, macrophages, and natural killer cells of the immune system, with B -cells having the highest expression (Galiegue et al., 1995; Schatz et al., 1997; Cabral and Griffin -Thomas, 2009) . Microglia, the resident macrophage of the brain, also express CB2 (Cabral et al., 2008; Romero -Sandoval et al., 2009). Ligands Much like CB1, THC is a partial agonist of CB2 (K i ~20 nM) and WIN55212 is a full agonist (K i ~10 nM), although the difference in selectivity for these two ligands is less for CB2 as compared with CB1 (Shire et al., 1996; Griffin et al., 2000) . Both AEA and 2 -AG are a lso agonists for CB2, but 2 -AG is a more selective and potent agonist !%+!than AEA and is expressed in higher quantities in the spleen, bone marrow, and gut (Tanasescu and Constantinescu, 2010; Basu et al., 2011) . 1.3.1.3: Non -CB1/CB2 Receptors While CB1 and CB2 are the most well -known cannabinoid receptors, there are number of other receptors that bind cannabinoids with varying strength. These receptors can be divided broadly into two broad categories: G -protein coupled receptors and transient receptor potential (TRP) channels (De Petrocellis and Di Marzo, 2010) . GPR55 is a G -protein coupled receptors that binds 2 -AG, THC, and AEA, listed in order of affinity from highest to lowest (Ryberg et al., 2007) . Activation of GPR55 has been shown to activate the G 13 pathways which leads to the activation of rhoA, cdc42, and rac1 (Ryberg et al., 2007) . However, there is also evidence that GPR55 activation can increase intracellular Ca 2+ and increase neuronal excitability, via activation of IP3 receptors (Lauckner et al., 2008) . In humans th ese receptors are found in only in the striatum (Sawzdargo et al., 1999) , however in mice GPR55 has been found to be expressed in the spleen, adrenals, and distribution in the brain parallel s that of CB1, although at lower levels (Ryberg et al., 2007) . Functionally, GPR55 has been shown to be critical in inducing inflammatory hyperalgesia, as it failed to occur in a model in GPR55 knockout mice, and to induce an increase in intracellular Ca 2+ in dorsal root ganglion (DRG) cells (De Petrocellis and Di Marzo, 2010) . The other known G -protein coupled receptor to which cannabinoids bind is GPR119 , which is an orphan receptor expressed primarily in the pancreas and !%"!gastrointestinal tract that activates the G s pathway (De Petrocellis and Di Marzo, 2010) . GPR119 is activated by the endocannabinoid oleoylethanolamide and may enhance glucose -dependent insulin release (De Petrocellis and Di Marzo, 2010) . TRP channels are six -domain transmembrane channels that gate the passage of ion s, including Ca2+ (Latorre et al., 2007) . These channels are activated by physical stimuli, such as temperature or mechanical pressure, or chemical stimuli, such as acid, alkali, and lipids (Latorre et al., 2007) . TRP channels are divided into several subcategories: TRPC (canonical), TRPA (ankyrin type),TRPM (melastatin -type), TRPP (poly cystin -like), TRPML (mucolipin -like), and TRPV (vanilloid -type) channels (De Petrocellis and Di Marzo, 2010) . At least t wo TRPV channels are activated by cannabinoid compounds: TRPV1 and TRPV2. TRP V1 was originally described as the target for capsaicin and mediates the burning sensation of this compound (Juliu s et al., 1997) . This receptor is found in afferent nociceptive neurons of the DRG as well as in the brain , which interestingly corresponds with many of the same places as CB1 receptor s (Julius et al., 1997; Zygmunt et al., 1999; Mezey et al., 2000; Cristino et al., 2006) . However, unlike CB1, TRPV1 is typically located on the post -synaptic membranes (Maccarrone et al., 2008) . AEA and CBD are the known agonists of TRPV1 (Bisogno et al., 2001; de Petrocellis et al., 2001). The TRP V2 channel shares 50% homol ogy with TRPV1 and is activated by high temperature s (~52 ¡ C) and cell swelling (Caterina et al., 1999; De Petrocellis and Di Marzo, 2010) . TRPV2 is also found within DRG sensory ganglia (Qin et al., 2008) . !%#!TRPV2 channels are agonized by both AEA and CBD, but unlike TRPV1 channels, can be agonized by THC (De Petrocellis and Di Marzo, 2010) . Besides the TRPV1 and TRPV2 receptors, two other TRP channels are known to bind cannabinoids. The TRPM8 is gated by low temperatures (<25¡C) and natural or synthetic coo ling compounds , such as menthol, eucalyptol, spearmint (De Petrocellis and Di Marzo, 2010) . These receptors are e xpressed on nociceptors, trigeminal neurons , and a small proportion of DRG neurons (Peier et al., 2002; Nealen et al., 2003; Kobayashi et al., 2005; Xing et al., 2006; De Petrocellis and Di Marzo, 2010) . Contrary to the TRPV receptors, AEA and THC are antagonists of the TRPM8 receptor (De Petrocellis et al., 2007; 2008). The TRPA1 receptor is expressed in unmyelinated peptidergic polymodal nociceptors of the trigeminal ganglion and DRG , distinct from those expressing TRPM8, but overlapping with nociceptive neurons expressing TRPV1, substance P, CGRP, and bradykinin receptors (Story et al., 2003; Bandell et al., 2004; Bautista et al., 2005; Kobayashi et al., 2005) . These receptors are activated by a variety of stimuli including bradykinin, cold ( (17¡ C), formalin, aldehydes found in smoke, and plant oils, such as those from mustard, cinnamon, garlic, winterg reen, cloves, and ginger (Story et al., 2003; Bandell et al., 2004; De Petrocellis and Di Marzo, 2010) . Activation of TRPA1 induces burning, prickling, or aching pain in humans (Morin and Bushnell, 1998) . THC and AEA are agonist s of TRPA1, although the binding for AEA is very weak (EC 50 = ~5 µM) (Jordt et al., 2004; De Petrocellis and Di Marzo, 2009) . !%$!1.3.2: Cannabinoid Effects 1.3.2.1: Neuronal Effects of CB1 Stimulation As discussed above, CB1 is located on the pre -synaptic axon terminals (Ny™ri et al., 2005). The activation of CB1 induces a number of effects on ion channels that play critical rol es in the release of neurotransmitters. These effects include a positive shift in the voltage -dependence of A -current K + channels (Childers and Deadwyler, 1996) , inhibition of N -, Q-, and L -type Ca 2+ channels (Felder et al., 1993; Mackie et al., 1993; 1995; Gebremedhin et al., 1999) , and ac tivation of inwardly rectifying K + channels (Mackie et al., 1995) . These mechanisms result in the inhibition of neurotransmitter release from axon terminals. This effect was first described in CNS neurons (Mackie, 2008), however, CB1 expression and regulation of neurotransmitter release in post -ganglionic sympathetic neurons has also been described (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . 1.3.2.2: Immune effects of CB2 Stimulation Cannabinoids are known immunosuppressive compounds. This can be seen as a decrease in the immune profile, particularly of lymphocytes, obtained from humans who consume cannabis (Pacifici et al., 2003; Gohary and Eid, 2004) . Further, cannabinoids decrease serum antibody concentrations in humans consuming cannabinoids, in this case THC (Klein et al., 1998) . Interestingly, epidemiologic studies have yet to fully identify the consequences of cannabinoid -mediated immunosuppression, despit e the wealth of information in animal studies (Greineisen and Turner, 2010; Tashkin, 2013) . !%%!The most well researched cannabinoid compound in this regard is THC , which is kno wn to suppress both cell -mediated (Lu et al., 2009; Karmaus et al., 2012; 2013) and hum oral (Schatz et al., 1993; Jan et al., 2003) adaptive immune responses in animal models. For example, THC was recently shown to broadly inhibit the immune response of mice to influenza infection (Karmaus et al., 2013) . The results of this study strongly suggest a major site of action in this regard is inhibition of antigen presenting cell function. THC also inhibits the humoral response in mice, as demonstrated by decreasing the number of antibody producing cells in the s pleen in response to sheep RBC ( sRBC ) injection (Schatz et al., 1993) . THC is a known agonist of both C B1 and CB2. Therefore experiments using THC do not provide information regarding the specific immunosuppressive role of CB1 or CB2. However, t he expression of CB2 on immune cells makes it likely that at least part of THC -induced immunosuppression is media ted by CB2 (Schatz et al., 1997) . A number of in vitro studies summarized here have been used to assess the effect of CB2 receptor activation on a variety of immune cell functions. Stimulation of the CB2 receptor by 2 -AG induces the migration of immune cell types, including B cells, monocytes/macrophages, and microglia (Basu and Dittel, 2011) . However, migration of mouse macrophages in response to common chemotactants is inhibited by synthetic CB2 agonists (Ghosh et al., 2006; Montecucco et al., 2008; Raborn et al., 2008) . To date there is no consensus regarding the effect of CB2 on immune cell migration, but it is clear from these studies that CB2 can modula te immune ce ll chemotaxis. CB2 can also a ffect immune cell proliferation, however there is no clear consensus with studies !%&!showing stimulatory effects in microglia (Carrier et al., 2004) and suppressive effect in CD4+ T cells (Maresz et al., 2007) . More clear data is available regarding the ability of CB2 to inhibit cytokine production, especially pro -inflammatory cytokines (IL -10 and IL -23) (Basu et al. , 2011). In addition to studies using CB agonists, a number of studies have used mice gen etically manipulated to lack CB1 and CB2. These mice appear phenotypically normal and have normal immune cell profile s (Springs et al., 2008) . Yet, in accordance with the immunosuppression observed with CB receptor sti mulation, these mice demonstrate enhanced immunity. CB1/CB2 knockout ( KO) mice show increased numbers of antibody producing cells from the spleen in response to sRBC injection (Springs et al., 2008) . CB1/CB2 KO mice also demonstrate enhanced T cell immunity in response to influenza infection, which is associated with augmented antigen presenting cell function (Karmaus et al., 2011) . While these mice are useful in establishing the immunomodulatory role of the CB1 and/or CB2, they do not provide specific details regarding the contribution of the individual receptors. 1.4: Summary Sympathetic noradrenergic innervation of the spleen has multiple roles. It controls the contraction of the spleen , which serves the dual purposes of modulating the pathway of blood through this highly perfused organ as well as releasing the cellular contents of the splenic parenchyma , such as RBC and lymphocytes . Splenic !%'!innervation also serve s an immunologic purpose. NE released from sple en proje cting noradrenergic post -ganglionic sympathetic neurons can increase the production of antibodies in response to an immune challenge inducing a humoral immune response. Cannabinoids have multiple effects in the mammalian system. The most widely studied e ffect is the inhibition of neurotransmitter release caused by agonism of pre -synaptic CB1, which represents an emerging field in the immunologic effects of cannabinoids. It is assumed that most effects of cannabinoids are meditated by CB2 , which are expre ssed on immune cells. However, there has yet to be a consensus on the effect of CB2 on immunity, despite the consistent ability of cannabinoids to be immunosuppressive. Furthermore, the contribution of CB1 to this effect is unclear. The interaction betwe en sympathetic noradrenergic innervation and the effect of cannabinoid receptor signaling is relatively unknown. The experiments in this dissertation were designed to investigate the role of CB1 and CB2 on the function of splenic sympathetic innervation. 1.5: Thesis Objective The studies described in this dissertation were developed in order to test the central hypothesis that the genetic knockout of the CB1 and CB2 in mice will enhance splenic sympathetic neuronal function . The following specific aims and hypotheses were designed to test this central hypothesis: !%(!Specific Aim 1: Characterize the physiology and function of splenic sympathetic innervation Hypothesis: Splenic sympathetic innervation augments splenic humoral immune responses by NE agonism of the % 2AR on splenic B cells and controls spleen contraction through NE agonism of $ 1AR on capsular smooth muscle. Specific Aim 2 : Investigate the role of endogenous CB1/CB2 signaling in the interaction between NE and splenic humoral immune r esponses Hypothesis: Enhanced humoral immunity in mice lacking the CB1 and CB2 is due to increased splenic sympathetic NE release and activation of % 2AR on splenic B cells . Specific Aim 3 : Investigate the role of endogenous CB1/CB2 signaling in sympathetic nervous system control of spleen contraction Hypothesis: Spleen contraction in mice lacking the CB1 and CB2 is attenuated due to decreased $ 1AR expression secondary to chronically increased noradrenergic sympathetic neuron activity . The following chapters outlin e the research performed to address the above Specific Aims. The reader is referred to Chapter 2 for detailed methodology. Chapters 3-6 describe findings as they relate to the central hypothesis and the thesis objectives. Chapter 7 provides a general di scussion on the relevance and the importance of this research as it relates to the previous findings and information in the literature. !%)! REFERENCES !%*!REFERENCES Aboud R, Shafii M, Docherty JR. Investigation of the subtypes of $ 1-adrenoceptor mediating contractions of rat aorta, vas deferens and spleen. Br J Pharmacol. 2012 Jul 19;109(1):80 Ð7. Ademokun AA, Dunn -Walters D. Immune Responses: Primary and Secondary. e ls.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Albert KA, Helmer -Matyjek E, Nairn AC, Mller TH, Haycock JW, Greene LA, et al. Calcium/phospholipid -dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase. Proc Natl Acad Sci USA. 1984 Dec;81(24):7713 Ð7. PMCID: PMC392222 Alger B, Kim J. Supply and demand for endocannabinoids. Trends Neurosci. 2011 Jun 1;34(6):304 Ð15. Bakovic D, Eterovic D, Saratlija -Novakovi ' Z, Palada I, Valic Z, Bilopavlovi ' N, et al. Effect of human splenic contraction on variation in circulating blood cell counts. Clin Exp Pharmacol Physiol. 2005 Nov;32(11):944 Ð51. Bakovic D, Valic Z, Eterovic D, Vukovic I, Obad A, Marinovi '-Terzi ' I, et al. Spleen volume and blood flow response to repeated b reath -hold apneas. J. Appl. Physiol. 2003 Oct;95(4):1460 Ð6. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron. 2004 Mar 25;41(6):849 Ð57. Basu S, D ittel BN. Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunol. Res. 2011 Oct;51(1):26 Ð38. Basu S, Ray A, Dittel BN. Cannabinoid receptor 2 is critical for the homing and retention of marginal zone B lineage cells and for efficient T -independent immune responses. The Journal of Immunology. 2011 Dec 1;187(11):5720 Ð32. PMCID: PMC3226756 Baumgarth N. Innate -Like B Cells and Their Rules of Engagement. link.springer.com.proxy2.cl.msu.edu. New York, NY: Spr inger New York; 2013. p. 57Ð66. Bautista DM, Movahed P, Hinman A, Axelsson HE, Sterner O, Hıgest−tt ED, et al. Pungent products from garlic activate the sensory ion channel TRPA1. Proc Natl Acad Sci USA. 2005 Aug 23;102(34):12248 Ð52. PMCID: PMC1189336 !&+!Bed oui S, Miyake S, Lin Y, Miyamoto K, Oki S, Kawamura N, et al. Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor -specific inhibition of autoreactive Th1 responses in vivo. J Immunol. 2003 Oct 1;171(7):3451 Ð8. Bekeredj ian-Ding I, Jego G. Toll -like receptors --sentries in the B -cell response. Immunology. 2009 Nov;128(3):311 Ð23. PMCID: PMC2770679 Bellinger D, Felten S, Collier T, Felten D. Noradrenergic sympathetic innervation of the spleen: IV. Morphometric analysis in ad ult and aged F344 rats. J Neurosci Res. 1987;18(1):55 Ð63, 126Ð9. Bellinger DL, Lorton D, Hamill RW, Felten SY, Felten DL. Acetylcholinesterase staining and choline acetyltransferase activity in the young adult rat spleen: lack of evidence for cholinergic innervation. Brain Behav Immun. 1993 Sep;7(3):191 Ð204. Bevilaqua LR, Graham ME, Dunkley PR, Nagy -Felsobuki von EI, Dickson PW. Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40). J Biol Chem. 2001 Nov 2;276(44):40411 Ð6. Bisogno T, Hanus L, de Petrocellis L, Tchilibon S, Ponde DE, Brandi I, et al. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br J Pharmacol. 2001 Oct;134(4):845 Ð52. PMCID: PMC1573017 Blaschko H. The activity of l ( Ñ)-dopa decarboxylase. J. Physiol. (Lond.). 1942. Blue J, Weiss L. Electron microscopy of the red pulp of the dog spleen including vascular arrangements, periarterial macrophage sheaths (Ellipsoids), and the contractile, innervated reticular meshwork. Am. J. Anat. 1981 Jun;161(2):189 Ð218. Bockaert JL. G Protein -coupled Receptors. onlinelibrary.wiley.com. Chichester: John Wiley & Sons, Ltd; 2 001. Bootman MD, Rietdorf K, Hardy H, Dautova Y, Corps E, Pierro C, et al. Calcium Signalling and Regulation of Cell Function. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Bırner C, Bedini A, Hıllt V, Kraus J. Analysis of promoter regions regul ating basal and interleukin -4-inducible expression of the human CB1 receptor gene in T lymphocytes. Mol Pharmacol. 2008 Mar;73(3):1013 Ð9. Brown SM, Wager -Miller J, Mackie K. Cloning and molecular characterization of the rat CB2 cannabinoid receptor. Bioch im Biophys Acta. 2002 Jul 19;1576(3):255 Ð64. !&"!Burnstock G, Ralevic V. Purinergic Signaling and Blood Vessels in Health and Disease. Byrum CE, Guyenet PG. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J Comp Neurol. 1987 Jul 22;261(4):529 Ð42. Cabral GA, Griffin -Thomas L. Emerging role of the cannabinoid receptor CB2 in immune regulation: therapeutic prospects for neuroinflammation. Expert Rev Mol Med. 2009;11:e3. PMCID: PMC2768535 Cabral GA, Raborn ES, Griffin L, Dennis J, Marciano -Cabral F. CB 2receptors in the brain: role in central immune function. Br J Pharmacol. 2008 Jan;153(2):240 Ð51. PMCID: PMC2219530 Cano G, Sved AF, Rinaman L, Rabin BS, Card JP. Characterization of the central nervous system innervation of the ra t spleen using viral transneuronal tracing. J Comp Neurol. 2001 Oct 8;439(1):1 Ð18. Carracedo A, Gironella M, Lorente M, Garcia S, Guzm⁄n M, Velasco G, et al. Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress -related genes. Cancer Res. 2006 Jul 1;66(13):6748 Ð55. Carrier EJ, Kearn CS, Barkmeier AJ, Breese NM, Yang W, Nithipatikom K, et al. Cultured rat microglial cells synthesize the endocannabinoid 2 -arachidonylglycerol, which increases proliferation via a CB2 recepto r-dependent mechanism. Mol Pharmacol. 2004 Apr;65(4):999 Ð1007. Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin -receptor homologue with a high threshold for noxious heat. Nature. 1999 Apr 1;398(6726):436 Ð41. Cesta M. Normal Structure, F unction, and Histology of the Spleen. Toxicol Pathol. 2006;34(5):455 Ð65. Chevendra V, Weaver LC. Distributions of neuropeptide Y, vasoactive intestinal peptide and somatostatin in populations of postganglionic neurons innervating the rat kidney, spleen an d intestine. Neuroscience. 1992 Oct;50(3):727 Ð43. Childers SR, Deadwyler SA. Role of cyclic AMP in the actions of cannabinoid receptors. Biochem Pharmacol. 1996 Sep 27;52(6):819 Ð27. Chong W, Li LH, Lee K, Lee MH, Park JB, Ryu PD. Subtypes of alpha1 - and alpha2 -adrenoceptors mediating noradrenergic modulation of spontaneous inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus. J. Neuroendocrinol. 2004 May;16(5):450 Ð7. Corder R, Lowry PJ, Withrington PG. The actions of the peptides, neuropeptide Y and !&#!peptide YY, on the vascular and capsular smooth muscle of the isolated, blood -perfused spleen of the dog. Br J Pharmacol. 1987 Apr;90(4):785 Ð90. PMCID: PMC1917199 Cristino L, de Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo V. Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience. 2006;139(4):1405 Ð15. Cunningham ET, Sawchenko PE. Anatomical specificity of noradrenergic inp uts to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol. 1988 Aug 1;274(1):60 Ð76. Czajkowsky DM, Shao Z. The human IgM pentamer is a mushroom -shaped molecule with a flexural bias. Proc Natl Acad Sci USA. 2009 Sep 1;106(35): 14960Ð5. Da‚ron M. Fc RECEPTOR BIOLOGY - Annual Review of Immunology, 15(1):203. Annu. Rev. Immunol. 1997. Daftary SS, Boudaba C, Tasker JG. Noradrenergic regulation of parvocellular neurons in the rat hypothalamic paraventricular nucleus. Neuroscience. 2000 Mar;96(4):743 Ð51. Daubner SC, Lauriano C, Haycock JW, Fitzpatrick PF. Site -directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP -dependent phosphorylation on enzyme activity. J Biol Chem. 1992 Jun 25;267(18):1263 9Ð46. Daubner SC, Le T, Wang S. Tyrosine hydroxylase and regulation of dopamine synthesis. Arch. Biochem. Biophys. 2011 Apr 1;508(1):1 Ð12. PMCID: PMC3065393 Davies BN, Withrington PG. The actions of drugs on the smooth muscle of the capsule and blood vess els of the spleen. Pharmacol Rev. 1973 Sep;25(3):373 Ð413. de Petrocellis L, Bisogno T, Maccarrone M, Davis JB, Finazzi -Agro A, Di Marzo V. The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism. J Biol Chem. 2001 Apr 20;276(16):12856 Ð63. De Petrocellis L, Di Marzo V. Role of endocannabinoids and endovanilloids in Ca2+ signalling. Cell Calcium. 2009 Jun;45(6):611 Ð24. De Petrocellis L, Di Marzo V. Non -CB1, non -CB2 receptors for endocannabinoids, plant cannabinoids, and synthetic cannabimimetics: focus on G -protein -coupled receptors and transient receptor potential channels. J Neuroimmune Pharmacol. 2010 Mar;5(1):103 Ð21. !&$!De Petrocellis L, Starowicz K, Moriello AS, Vivese M, Orlando P, Di Marzo V. Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): Effect of cAMP, cannabinoid CB1 receptors and endovanilloids. Exp. Cell Res. 2007 May;313(9):1911 Ð20. De Petrocellis L, Vellani V, Schia no-Moriello A, Marini P, Magherini PC, Orlando P, et al. Plant -derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type -1 and melastatin type -8. Journal of Pharmacology and Experimental Therapeutics. 2008 Jun;325( 3):1007 Ð15. Di Marzo V. Endocannabinoids: synthesis and degradation. Rev. Physiol. Biochem. Pharmacol. 2008;160:1 Ð24. Dimitrijevi ' M, Stanojevi ' S. The intriguing mission of neuropeptide Y in the immune system. Amino Acids. 2013 Jul;45(1):41 Ð53. Dinarel lo C. Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. J Endotoxin Res. 2004;10(4):201 Ð22. Dowdall MJ, Boyne AF, Whittaker VP. Adenosine triphosphate. A constituent of cholinergic synaptic vesicles. Biochem J. 1974 Apr;140(1):1 Ð12. PMCID: PMC1167964 drugpolicy.org. Marijuana Legalization in Washington State and Colorado | Drug Policy Alliance [Internet]. drugpolicy.org. [cited 2013 Dec 31]. Retrieved from: http://www.drugpolicy.org/resource/marijuana -legalization -was hington -state -and-colorado Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. 2004 Sep 1;56(3):331 Ð49. Elenkov I, Vizi E. Presynaptic modulation of release of noradrenaline fr om the sympathetic nerve terminals in the rat spleen. Neuropharmacology. 1991 Dec 1;30(12A):1319 Ð24. Eltze M. Functional evidence for an alpha 1B -adrenoceptor mediating contraction of the mouse spleen. Eur J Pharmacol. 1996 Sep 12;311(2 -3):187 Ð98. Fazill eau N, Mark L, McHeyzer -Williams LJ, McHeyzer -Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009 Mar 20;30(3):324 Ð35. PMCID: PMC2731675 Felder CC, Briley EM, Axelrod J, Simpson JT, Mackie K, Devane WA. Anandamide, an endogenous ca nnabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor -mediated signal transduction. Proc Natl Acad Sci USA. 1993 Aug 15;90(16):7656 Ð60. PMCID: PMC47201 !&%!Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, et al. Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol. 1995 Sep;48(3):443 Ð50. Felten DL, Ackerman KD, Wiegand SJ, Felten SY. Noradrenergic sympathetic innervation of the spleen: I. Ner ve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neurosci Res. 1987;18(1):28 Ð36, 118Ð21. Felten SY, Olschowka J. Noradrenergic sympathetic innervation of the spleen: II. Tyrosine hydroxylase (TH) -positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp. J Neurosci Res. 1987;18(1):37 Ð48. Ferrero I, Michelin O, Luescher I. Antigen Recognition by T Lymphocytes. onlinelibrary.wiley.com.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Fonseca MI, Button DC, Brown RD. Agonist regulation of alpha 1B -adrenergic receptor subcellular distribution and function. J Biol Chem. 1995 Apr 14;270(15):8902 Ð9. Fozard JR, Milavec -Krizman M. Contraction of the rat iso lated spleen mediated by adenosine A1 receptor activation. Br J Pharmacol. 1993 Jul 19;109(4):1059 Ð63. PMCID: PMC2175713 Fujii T, Takada -Takatori Y, Kawashima K. Basic and clinical aspects of non -neuronal acetylcholine: expression of an independent, non -neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy. J. Pharmacol. Sci. 2008 Feb;106(2):186 Ð92. Gahn LG, Roskoski R. Thermal stability and CD analysis of rat tyrosine hydroxylase. Biochemistry. 1995 Jan 10;34(1):252 Ð6. Galal A, Slade D, Gul W, El -Alfy A, Ferreira D, Elsohly M. Naturally occurring and related synthetic cannabinoids and their potential therapeutic applications. Recent Pat CNS Drug Discov. 2009 Jun 1;4(2):112 Ð36. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, et al. Expression of Central and Peripheral Cannabinoid Receptors in Human Immune Tissues and Leukocyte Subpopulations. Eur J Biochem. 1995 Aug;232(1):54 Ð61. Gebremedhin D, Lange AR, Campbell WB, Hillard CJ, Harder DR. Cannabinoi d CB1 receptor of cat cerebral arterial muscle functions to inhibit L -type Ca2+ channel current. Am J Physiol. 1999 Jun;276(6 Pt 2):H2085 Ð93. Gerra G, Zaimovic A, Gerra ML, Ciccocioppo R, Cippitelli A, Serpelloni G, et al. Pharmacology and toxicology of C annabis derivatives and endocannabinoid !&&!agonists. Recent Pat CNS Drug Discov. 2010 Jan;5(1):46 Ð52. Ghosh S, Preet A, Groopman JE, Ganju RK. Cannabinoid receptor CB2 modulates the CXCL12/CXCR4 -mediated chemotaxis of T lymphocytes. Mol Immunol. 2006 Jul;43( 14):2169 Ð79. Gillespie JS, Hamilton DN. Binding of noradrenaline to smooth muscle cells in the spleen. Nature. 1966 Oct 29;212(5061):524 Ð5. Giuffrida A, Mcmahon LR. In vivo pharmacology of endocannabinoids and their metabolic inhibitors: therapeutic impl ications in Parkinson's disease and abuse liability. Prostaglandins and Other Lipid Mediators. 2010 Apr 1;91(3 -4):90 Ð103. Gohary ME, Eid MA. Effect of cannabinoid ingestion (in the form of bhang) on the immune system of high school and university students . Human & experimental toxicology. 2004. Goldman CK, Marino L, Leibowitz SF. Postsynaptic $ 2-noradrenergic receptors mediate feeding induced by paraventricular nucleus injection of norepinephrine and clonidine. Eur J Pharmacol. 1985 Sep;115(1):11 Ð9. Gotc h FM. T Lymphocytes: Cytotoxic. Chichester, UK: John Wiley & Sons, Ltd; 2001. Greineisen WE, Turner H. Immunoactive effects of cannabinoids: considerations for the therapeutic use of cannabinoid receptor agonists and antagonists. Int Immunopharmacol. 2010 May;10(5):547 Ð55. PMCID: PMC3804300 Griffin G, Tao Q, Abood ME. Cloning and pharmacological characterization of the rat CB(2) cannabinoid receptor. J Pharmacol Exp Ther. 2000 Mar;292(3):886 Ð94. Groom AC, Schmidt EE, MacDonald IC. Microcirculatory pathways and blood flow in spleen: new insights from washout kinetics, corrosion casts, and quantitative intravital videomicroscopy. Scanning Microsc. 1991 Mar;5(1):159 Ð73Ðdiscussion173 Ð4. Gualix J, Pin tor J, Miras -Portugal MT. Characterization of nucleotide transport into rat brain synaptic vesicles. J Neurochem. 1999 Sep;73(3):1098 Ð104. Guzm⁄n M. Cannabinoids: potential anticancer agents. Nat Rev Cancer. Nature Publishing Group; 2003 Oct;3(10):745 Ð55. Haycock JW. Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40. J Biol Chem. 1990 Jul 15;265(20):11682 Ð91. Hayley S, Lacosta S, Merali Z, van Rooijen N, Anisman H. Central monoamine and plasma corticosterone changes induced by a bacterial endotoxin: sensitization and cross -sensitization effects. Eur J Neurosci. 2001 Mar;13(6):1155 Ð65. !&'!Hefco V, Olariu A, Hefco A, Nabeshima T. The modulator role of the hypothalamic paraventricular nucleus on immune responsiveness. Brain Behav Immu n. 2004 Mar;18(2):158 Ð65. Helwig BG, Craig RA, Fels RJ, Blecha F, Kenney MJ. Central nervous system administration of interleukin -6 produces splenic sympathoexcitation. Autonomic Neuroscience. 2008 Aug;141(1 -2):104 Ð11. Herkenham M, Lynn AB, Johnson MR, M elvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. 1991 Feb 1;11(2):563 Ð83. Herrera B, Carracedo A, Diez -Zaera M, GŠmez del Pulgar T, Guzm⁄n M, Velasco G . The CB2 cannabinoid receptor signals apoptosis via ceramide -dependent activation of the mitochondrial intrinsic pathway. Exp. Cell Res. 2006 Jul 1;312(11):2121 Ð31. Howlett AC, Mukhopadhyay S. Cellular signal transduction by anandamide and 2 -arachidonoyl glycerol. Chemistry and Physics of Lipids. 2000 Nov;108(1 -2):53 Ð70. Inoue W, Baimoukhametova DV, Fzesi T, Cusulin JIW, Koblinger K, Whelan PJ, et al. Noradrenaline is a stress -associated metaplastic signal at GABA synapses. Nat. Neurosci. 2013 May;16(5): 605Ð12. Ishac E, Jiang L, Lake K, Varga K, Abood M, Kunos G. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves. Br J Pharmacol. 1996 Aug 1;118(8):2023 Ð8. PMCID: PMC1909901 Itoi K, Sugi moto N. The brainstem noradrenergic systems in stress, anxiety and depression. J. Neuroendocrinol. 2010 May;22(5):355 Ð61. Jacob F, Novo CP, Bachert C, Van Crombruggen K. Purinergic signaling in inflammatory cells: P2 receptor expression, functional effect s, and modulation of inflammatory responses. Purinergic Signalling. Springer; 2013 Sep 1;9(3):285. Jan T -R, Farraj AK, Harkema JR, Kaminski NE. Attenuation of the ovalbumin -induced allergic airway response by cannabinoid treatment in A/J mice. Toxicol App l Pharmacol. 2003 Apr 1;188(1):24 Ð35. Jordt S -E, Bautista DM, Chuang H -H, McKemy DD, Zygmunt PM, Hıgest−tt ED, et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature. 2004 Jan 15;427(6971):260 Ð5. Julius D, Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD. The capsaicin receptor: a heat -activated ion channel in the pain pathway. Nature. Nature Publishing Group; 1997 Oct 23;389(6653):816 Ð24. !&(!Juul B, Lscher ME, Aalkjaer C, Plesner L. Nucleotide hy drolytic activity of isolated intact rat mesenteric small arteries. Biochim Biophys Acta. 1991 Aug 26;1067(2):201 Ð7. Kakizaki Y, Watanobe H, Kohsaka A, Suda T. Temporal profiles of interleukin -1beta, interleukin -6, and tumor necrosis factor -alpha in the p lasma and hypothalamic paraventricular nucleus after intravenous or intraperitoneal administration of lipopolysaccharide in the rat: estimation by push -pull perfusion. Endocr J. 1999 Aug 1;46(4):487 Ð96. Kandel E, Schwartz J, Jessell T. Principles of neura l science. 2000. Kaplan BLF. The Role of CB(1) in Immune Modulation by Cannabinoids. Pharmacol. Ther. 2012 Dec 19. Karhunen T, Tilgmann C, Ulmanen I, Julkunen I, Panula P. Distribution of catechol -O-methyltransferase enzyme in rat tissues. É of Histochem istry & É. 1994. Karmaus PWF, Chen W, (null), Kaplan BLF, Kaminski NE. ) 9-Tetrahydrocannabinol Impairs the Inflammatory Response to Influenza Infection: Role of Antigen -Presenting Cells and the Cannabinoid Receptors 1 and 2. Toxicological Sciences. 2013. PMCID: PMC3551428 Karmaus PWF, Chen W, Crawford RB, Harkema JR, Kaplan BLF, Kaminski NE. Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection. J Leukoc Biol. 2011 Nov;9 0(5):983 Ð95. PMCID: PMC3206470 Karmaus PWF, Chen W, Kaplan BLF, Kaminski NE. ) 9-tetrahydrocannabinol suppresses cytotoxic T lymphocyte function independent of CB1 and CB 2, disrupting early activation events. J Neuroimmune Pharmacol. 2012 Dec;7(4):843 Ð55. PMCID: PMC3266990 Katafuchi T, Ichijo T, Take S, Hori T. Hypothalamic modulation of splenic natural killer cell activity in rats. J. Physiol. (Lond.). 1993 Nov;471:209 Ð21. PMCID: PMC1143959 Kaufman S. Dopamine -beta -hydroxylase. J Psychiatr Res. 1974;11:303 Ð16. Kauma SW, Turner TT, Harty JR. Interleukin -1 beta stimulates interleukin -6 production in placental villous core mesenchymal cells. Endocrinology. 1994;134(1):457 Ð60. Kenter AL. Class Switch Recombination: An Emerging Mechanism - Springer. Molecular Analysis of B Lymphocyte Development and É. 2005. Khan ZP, Ferguson CN, Jones RM. Alpha -2 and imidazoline receptor agonistsTheir pharmacology and therapeutic role. Anaesthesia. 2002 Apr 6;5 4(2):146 Ð65. !&)!Kin NW, Sanders VM. It takes nerve to tell T and B cells what to do. J Leukoc Biol. 2006 Jun;79(6):1093 Ð104. Klein TW, Friedman H, Specter S. Marijuana, immunity and infection. J Neuroimmunol. 1998 Mar 15;83(1 -2):102 Ð15. Kobayashi K, Fukuok a T, Obata K, Yamanaka H, Dai Y, Tokunaga A, et al. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c -fibers and colocalization with trk receptors. J Comp Neurol. 2005 Dec 26;493(4):596 Ð606. Kohm AP, Sander s VM. Norepinephrine: a messenger from the brain to the immune system. Immunology Today. 2000 Nov;21(11):539 Ð42. Kohm AP, Sanders VM. Norepinephrine and beta 2 -adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev. 2001 Dec;53(4):487 Ð525. Kohm AP, Tang Y, Sanders VM, Jones SB. Activation of antigen -specific CD4+ Th2 cells and B cells in vivo increases norepinephrine release in the spleen and bone marrow. 2000 Jul 15;165(2):725 Ð33. Kubo T, Su C. Effe cts of adenosine on [3H]norepinephrine release from perfused mesenteric arteries of SHR and renal hypertensive rats. Eur J Pharmacol. 1983 Feb 18;87(2 -3):349 Ð52. Kumer SC, Vrana KE. Intricate regulation of tyrosine hydroxylase activity and gene expression . J Neurochem [Internet]. 1996 Aug 1;67(2):443 Ð62. Retrieved from: https://mail.google.com/mail/ca/u/0/?ui=2&view=bsp&ver=ohhl4rw8mbn4 Kurosaki T, Hikida M. B Lymphocytes: Receptors. eLS. Kuster JE, Stevenson JI, Ward SJ, D'Ambra TE, Haycock DA. Aminoalky lindole binding in rat cerebellum: selective displacement by natural and synthetic cannabinoids. J Pharmacol Exp Ther. 1993 Mar;264(3):1352 Ð63. Kuwahira I, Kamiya U, Iwamoto T, Moue Y, Urano T, Ohta Y, et al. Splenic contraction -induced reversible increas e in hemoglobin concentration in intermittent hypoxia. J. Appl. Physiol. 1999 Jan;86(1):181 Ð7. Kgelgen von I, Sp−th L, Starke K. Stable adenine nucleotides inhibit [3H] -noradrenaline release in rabbit brain cortex slices by direct action at presynaptic a denosine A1 -receptors. Naunyn -Schmied Arch Pharmacol. 1992 Aug;346(2):187 Ð96. la Fuente De M, Bernaez I, Del Rio M, Hernanz A. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein !&*!kinase C. Immunol ogy. 1993 Oct;80(2):259 Ð65. PMCID: PMC1422192 Lagercrantz H, Staj−rne L. Evidence that most noradrenaline is stored without ATP in sympathetic large dense core nerve vesicles. Nature. 1974 Jun 28;249(460):843 Ð5. Lanzavecchia A, Sallusto F. Toll -like recep tors and innate immunity in B -cell activation and antibody responses. Curr Opin Immunol. 2007 Jun;19(3):268 Ð74. Latorre R, Brauchi S, Orta G, Zaelzer C, Vargas G. ThermoTRP channels as modular proteins with allosteric gating. Cell Calcium. 2007 Oct;42(4 -5):427 Ð38. Lauckner JE, Jensen JB, Chen H -Y, Lu H -C, Hille B, Mackie K. GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proc Natl Acad Sci USA. 2008 Feb 19;105(7):2699 Ð704. PMCID: PMC2268199 Lazar MA, Truscott R JW, Raese JD, Barchas JD. Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme. J Neurochem. 1981 Feb;36(2):677 Ð82. Leeb-Lundberg LM, Cotecchia S, DeBlasi A, Caron MG, Lefkowitz RJ . Regulation of adrenergic receptor function by phosphorylation. I. Agonist -promoted desensitization and phosphorylation of alpha 1 -adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF -2 smooth muscle cells. J Biol Chem. 1987 Mar 5;262(7):3098 Ð105. Lehmann IT, Bobrovskaya L, Gordon SL, Dunkley PR, Dickson PW. Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation. J Biol Chem. 2006 Jun 30;281(26):17644 Ð51. Leibowitz SF, Hor L. Endorphiner gic and $-noradrenergic systems in the paraventricular nucleus: Effects on eating behavior. Peptides. 1982 May;3(3):421 Ð8. Levin BE. Reduced norepinephrine turnover in organs and brains of obesity -prone rats | Regulatory, Integrative and Comparative Physiology. É Journal of Physiology -Regulatory. 1995. Levite M. Neuropeptides, by direct interaction with T cells, induce cytoki ne secretion and break the commitment to a distinct T helper phenotype. Proc Natl Acad Sci USA. 1998 Oct 13;95(21):12544 Ð9. PMCID: PMC22867 Levitt M, Spector S, Sjoerdsma A, Udenfriend S. Elucidation of the rate -limiting step in norepinephrine biosynthesis in the perfused guinea -pig heart. J Pharmacol Exp Ther. 1965 Apr;148:1 Ð8. Li YW, Ding ZQ, Wesselingh SL, Blessing WW. Renal and adrenal sympathetic preganglionic neurons in rabbit spinal cord: tracing with herpes simplex virus. Brain !'+!Res. 1992 Feb 21;573 (1):147 Ð52. Lookingland KJ, Ireland LM, Gunnet JW, Manzanares J, Tian Y, Moore KE. 3 -Methoxy -4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons. Brain Res. 1991 Sep 13;559(1):82 Ð8. Lookingland KJ, Moore KE. Chapter VIII Functional neuroanatomy of hypothalamic dopaminergic neuroendocrine systems. Handbook of chemical neuroanatomy. 2005. Lu H, Kaplan BLF, Ngaotepprutaram T, Kaminski NE. Suppression of T cell costimulator ICOS by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2009 Feb;85(2):322 Ð9. PMCID: PMC2631366 Lundberg JM, Franco -Cereceda A, Lacroix JS, Pernow J. Neuropeptide Y and sympathetic neurotransmission. Ann N Y Acad Sci. 1990;611:166 Ð74. Macarthur H, Wilken GH, Westfall TC, K olo LL. Neuronal and non -neuronal modulation of sympathetic neurovascular transmission. Acta Physiol (Oxf). 2011 Sep;203(1):37 Ð45. PMCID: PMC3139802 Maccarrone M, Rossi S, Bari M, De Chiara V, Fezza F, Musella A, et al. Anandamide inhibits metabolism and p hysiological actions of 2 -arachidonoylglycerol in the striatum. Nat. Neurosci. 2008 Feb;11(2):152 Ð9. Mackie K. Signaling via CNS cannabinoid receptors. Mol. Cell. Endocrinol. 2008 Apr 16;286(1 -2 Suppl 1):S60 Ð5. PMCID: PMC2435200 Mackie K, Devane WA, Hille B. Anandamide, an endogenous cannabinoid, inhibits calcium currents as a partial agonist in N18 neuroblastoma cells. Mol Pharmacol. 1993 Sep;44(3):498 Ð503. Mackie K, Lai Y, Westenbroek R, Mitchell R. Cannabinoids activate an inwardly rectifying potassium conductance and inhibit Q -type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor. J Neurosci. 1995 Oct;15(10):6552 Ð61. Malbon CC, Wang H -Y. Adrenergic Receptors. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Lt d; 2001. Maresz K, Pryce G, Ponomarev ED, Marsicano G, Croxford JL, Shriver LP, et al. Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells. Nat. Med. 2007 Apr;13(4):492 Ð7. Marino F , Cosentino M. Adrenergic modulation of immune cells: an update. Amino Acids. 2011 Dec 8. Marino F, Cosentino M. Adrenergic modulation of immune cells: an update. Amino !'"!Acids. 2013 Jul;45(1):55 Ð71. Matsuno K, Ezaki T, Kotani M. Splenic outer periarterial lymphoid sheath (PALS): an immunoproliferative microenvironment constituted by antigen -laden marginal metallophils and ED2 -positive macrophages in the rat. Cell Tissue Res. 1989 Sep;257(3):459 Ð70. Meager A, Wadhwa M. An Overview of Cytokine Regulation of Inflammation and Immunity. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol. Nature Publishing Group; 2005 Aug;5(8):606 Ð16. Medeiros MDS, Turner AJ. Metaboli sm and functions of neuropeptide Y. Neurochem Res. 1996 Sep;21(9):1125 Ð32. Meltzer JC, MacNeil BJ, Sanders V, Pylypas S, Jansen AH, Greenberg AH, et al. Contribution of the adrenal glands and splenic nerve to LPS -induced splenic cytokine production in the rat. Brain Behav Immun. 2003 Dec;17(6):482 Ð97. Mentlein R, Dahms P, Grandt D, Kger R. Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul. Pept. 1993 Dec 10;49(2):133 Ð44. Mezey E, TŠth ZE, Cortright DN, Arzubi MK, Krause JE, Elde R, et al. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1 -like immunoreactivity, in the central nervous system of the rat and human. Proc Natl Acad Sci USA. 2000 Mar 28;97(7):3655 Ð60. PMCID: PMC16295 Michel MC, Beck -Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, et al. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev. 1998 Mar;50(1):143 Ð50. Miller SI, E rnst RK, Bader MW. LPS, TLR4 and infectious disease diversity. Nat. Rev. Microbiol. 2005 Jan;3(1):36 Ð46. Molina -Holgado E, Vela JM, Ar”valo -Mart™n A, Almaz⁄n G, Molina -Holgado F, Borrell J, et al. Cannabinoids promote oligodendrocyte progenitor survival: involvement of cannabinoid receptors and phosphatidylinositol -3 kinase/Akt signaling. Journal of Neuroscie nce. 2002 Nov 15;22(22):9742 Ð53. Moller G. Antigens: Thymus Independent. els.net.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Montecucco F, Burger F, Mach F, Steffens S. CB2 cannabinoid receptor agonist JWH -015 modulates human monocyt e migration through defined intracellular signaling !'#!pathways. Am J Physiol Heart Circ Physiol. 2008 Mar;294(3):H1145 Ð55. Morin C, Bushnell MC. Temporal and qualitative properties of cold pain and heat pain: a psychophysical study. Pain. 1998 Jan;74(1):67 Ð73. Munro S, Thomas KL, Abu -Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. Nature Publishing Group; 1993 Sep 2;365(6441):61 Ð5. Myıh−nen TT, Schendzielorz N, M−nnistı PT. Distribution of catechol -O-methyltransferase (COMT) proteins and enzymatic activities in wild -type and soluble COMT deficient mice. J Neurochem. 2010 Mar 31;:no Ðno. Nance DM, Burns J. Innervation of the spleen in the rat: Evidence for absence of afferent innervation. Brain Behav Immun. 1989 Dec;3(4 ):281 Ð90. Nance DM, Sanders VM. Autonomic innervation and regulation of the immune system (1987 -2007). Brain Behav Immun. 2007 Aug;21(6):736 Ð45. PMCID: PMC1986730 Nath I. Immune Mechanisms against Intracellular Pathogens. onlinelibrary.wiley.com.proxy2.cl .msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Nealen ML, Gold MS, Thut PD, Caterina MJ. TRPM8 mRNA is expressed in a subset of cold -responsive trigeminal neurons from rat. J Neurophysiol. 2003 Jul;90(1):515 Ð20. N”ron S, Nadeau PJ, Darveau A, Le blanc J -F. Tuning of CD40 -CD154 interactions in human B -lymphocyte activation: a broad array of in vitro models for a complex in vivo situation. Arch. Immunol. Ther. Exp. (Warsz.). 2011 Feb;59(1):25 Ð40. Niederhoffer N, Szabo B. Effect of the cannabinoid r eceptor agonist WIN55212 -2 on sympathetic cardiovascular regulation. Br J Pharmacol. 1999 Jan;126(2):457 Ð66. North RA. Molecular physiology of P2X receptors. Physiol. Rev. 2002 Oct;82(4):1013 Ð67. Ny™ri G, Cser”p C, Szabadits E, Mackie K, Freund TF. CB1 c annabinoid receptors are enriched in the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons. Neuroscience. 2005;136(3):811 Ð22. Oh-hashi Y, Shindo T, Kurihara Y, Imai T, Wang Y, Morita H, et al. Elevated Sympathetic Nervous Act ivity in Mice Deficient in CGRP. Circ. Res. 2001 Nov 23;89(11):983 Ð90. Oikonomopoulou K, Reis ES, Lambris JD. Complement System and Its Role in Immune Responses. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N. Molecular characterization of a !'$!phospholipase D generating anandamide and its congeners. J Biol Chem. 2004 Feb 13;279(7):5298 Ð305. Olofsson PS, Rosas -Ballina M, Levine YA, Tracey KJ. Rethinking inflammation: neural circuits in the regulation of immunity. Immunol. Rev. 2012 Jul;248(1):188 Ð204. Oxender D, Christensen H. Distinct Mediating Systems for the Transport of Neurtral Amino Acids by the Ehrlich Cell. J Biol Chem. 1963 Nov;238:3686 Ð99. P Kane L. Lymphocyte Activation: Signal Transduction. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. Pacak K, Palkovits M, Kopin IJ, Goldstein DS. Stress -Induced Norepinephrine Release in the Hypothalamic Paraventricular Nucleus and Pituitary -Adrenocortical and Sympathoadrenal Activity: In Vivo Microdialysis Studies. Frontiers in É. 1995. Pacifici R, Zuccaro P, Pichini S, Roset PN, Poudevida S, Farr” M, et al. Modulation of the immune system in cannabis users. JAMA. 2003 Apr 16;289(15):1929 Ð31. Pankratov Y, Lalo U, Verkhratsky A, North RA. Vesicular release of ATP at central synapses. Pflgers Archiv. 2006. Parikh AA, Salzman AL, Kane CD, Fischer JE, Hasselgren PO. IL -6 production in human intestinal epithelial cells following stimulation with IL -1 beta is associ ated with activation of the transcription factor NF -kappa B. J Surg Res. 1997 Apr 1;69(1):139 Ð44. Pearson JD, Carleton JS, Gordon JL. Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth -muscle cells in culture. Biochem J. 1980 Aug 15;190(2):421 Ð9. PMCID: PMC1162107 Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, et al. A TRP channel that senses cold stimuli and menthol. Cell. 2002 Mar 8;108(5):705 Ð15. Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: ) 9-tetrahydrocannabinol, cannabidiol and ) 9-tetrahydrocannabivarin. Br J Pharmacol [Internet]. 2008 Jan;153(2):199 Ð215. Retrieved from: http://onlinelibrary.wiley.com.proxy2.cl.msu.edu/doi/10.1038/sj.bjp.0707442/fu ll. PMCID: PMC2219532 Pharmaceuticals G. Sativex. 2011;(3/8/2012). Pinaud E, Khamlichi A, Le Morvan C, Drouet M, Nalesso V, Le Bert M, et al. Localization of the 3' IgH locus elements that effect long -distance regulation of class switch recombination. Imm unity. 2001 Aug 1;15(2):187 Ð99. Pinkus GS, Warhol MJ, O'Connor EM, Etheridge CL, Fujiwara K. Immunohistochemical !'%!localization of smooth muscle myosin in human spleen, lymph node, and other lymphoid tissues. Unique staining patterns in splenic white pulp a nd sinuses, lymphoid follicles, and certain vasculature, with ultrastructural correlations. Am. J. Pathol. 1986 Jun;123(3):440 Ð53. PMCID: PMC1888274 Podojil JR, Kin NW, Sanders VM. CD86 and beta2 -adrenergic receptor signaling pathways, respectively, increa se Oct -2 and OCA -B Expression and binding to the 3' -IgH enhancer in B cells. 2004 May 28;279(22):23394 Ð404. Puerto M, Guayerbas N, Alvarez P, la Fuente De M. Modulation of neuropeptide Y and norepinephrine on several leucocyte functions in adult, old and very old mice. J Neuroimmunol. 2005 Aug;165(1 -2):33 Ð40. Pyner S. Neurochemistry of the paraventricular nucleus of the hypothalamus: Implications for cardiovascular regulation. J. Chem. Neuroanat. 2009. Qin N, Neeper MP, Liu Y, Hutchinson TL, Lubin ML, Fl ores CM. TRPV2 is activated by cannabidiol and mediates CGRP release in cultured rat dorsal root ganglion neurons. Journal of Neuroscience. 2008 Jun 11;28(24):6231 Ð8. Quah BJ, Parish CR. Innate Immune Mechanisms: Nonself Recognition. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Raborn ES, Marciano -Cabral F, Buckley NE, Martin BR, Cabral GA. The cannabinoid delta -9-tetrahydrocannabinol mediates inhibition of macrophage chemotaxis to RANTES/CCL5: linkage to the CB2 receptor. J Neuroimmune Pharmaco l. 2008 Jun;3(2):117 Ð29. PMCID: PMC2677557 Ralevic V. Purines as Neurotransmitters and Neuromodulators in Blood Vessels. CVP. 2009 Jan 1;7(1):3 Ð14. Ralevic V, Kendall DA. Cannabinoids inhibit pre - and postjunctionally sympathetic neurotransmission in rat mesenteric arteries. Eur J Pharmacol. 2002 May 31;444(3):171 Ð81. Ramsey AJ, Fitzpatrick PF. Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of catecholamines: evidence for a novel regulatory mechanism. Biochemistry. 1998 Jun 23; 37(25):8980 Ð6. Ren LM, Burnstock G. Prominent sympathetic purinergic vasoconstriction in the rabbit splenic artery: potentiation by 2,2 &-pyridylisatogen tosylate - Ren - 2009 - British Journal of Pharmacology - Wiley Online Library. Br J Pharmacol. 1997. Ribeiro P, Wang Y, Citron BA, Kaufman S. Regulation of recombinant rat tyrosine hydroxylase by dopamine. Proc Natl Acad Sci USA. 1992 Oct 15;89(20):9593 Ð7. PMCID: PMC50178 !'&!Richardson MX, de Bruijn R, Schagatay E. Hypoxia augments apnea -induced increase in hemoglobin concentration and hematocrit. Eur. J. Appl. Physiol. 2009 Jan;105(1):63 Ð8. Richardson MX, Lodin A, Reimers J, Schagatay E. Short -term effects of normobaric hypoxia on the human spleen. Eur. J. Appl. Physiol. Springer -Verlag; 2007 Nov 28;104(2) :395Ð9. Romano TA, Felten SY, Felten DL, Olschowka JA. Neuropeptide -Y innervation of the rat spleen: another potential immunomodulatory neuropeptide. Brain Behav Immun. 1991 Mar;5(1):116 Ð31. Romero -Sandoval EA, Horvath R, Landry RP, DeLeo JA. Cannabinoid receptor type 2 activation induces a microglial anti -inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation. Mol Pain. 2009;5:25. PMCID: PMC2704199 Rongen GA, Lenders JW, Lambrou J, Willemsen JJ, Van Belle H, Thien T, et al. Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension. 1996 Apr;27(4):933 Ð8. Rubino A, Amerini S, Mantelli L, Ledda F. Adenosine receptors involved in the inhibitory control of non -adrene rgic non -cholinergic neurotransmission in guinea -pig atria belong to the A1 subtype. É -Schmiedeberg's archives of É. 1991. Rubino A, Ralevic V, Burnstock G. The P1 -purinoceptors that mediate the prejunctional inhibitory effect of adenosine on capsaicin -sensitive nonadrenergic noncholinergic neurotransmission in the rat mesenteric arterial bed are of the A1 subtype. J Pharmacol Exp Ther. 1993 Dec;267(3):1100 Ð4. Ryberg E, Larsson N, Sjıgren S, Hjorth S, Hermansson N -O, Leonova J, et al. The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol. 2007 Dec;152(7):1092 Ð101. PMCID: PMC2095107 Saha B. Antigens. els.net.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Saito H, Yokoi Y, Watanabe S, Tajima J, Kuroda H, Namihisa T. Re ticular meshwork of the spleen in rats studied by electron microscopy. Am. J. Anat. 1988 Mar;181(3):235 Ð52. Samuels ER, Szabadi E. Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation. Curr Neuropharmacol. 2008 Sep;6(3):235 Ð53. PMCID: PMC2687936 !''!Sanders VM. The beta2 -adrenergic receptor on T and B lymphocytes: Do we understand it yet? Brain Behav Immun. 2012 Feb;26(2):195 Ð200. PMCID: PMC3243812 Sandler MP, Kronenberg MW, Forman MB, Wolfe OH, Clanton JA, Partain CL. Dynamic fluctuations in blood and spleen radioactivity: splenic contraction and relation to clinical radionuclide volume calculations. J. Am. Coll. Cardiol. 1984 May;3(5):12 05Ð11. Sato N, Shen YT, Kiuchi K, Shannon RP, Vatner SF. Splenic contraction -induced increases in arterial O2 reduce requirement for CBF in conscious dogs. American Journal of Physiology - Heart and Circulatory Physiology. American Physiological Society; 1995 Aug 1;269(2):H491 ÐH503. Sato S, Steeber DA, Jansen PJ, Tedder TF. CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19. J Immunol. 1997 May 15;158(10):4662 Ð9. Satodate R, Tanak a H, Sasou S, Sakuma T, Kaizuka H. Scanning electron microscopical studies of the arterial terminals in the red pulp of the rat spleen. Anat. Rec. 1986 Jul;215(3):214 Ð6. Sawchenko PE, Swanson LW. Immunohistochemical identification of neurons in the parave ntricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol. 1982 Mar 1;205(3):260 Ð72. Sawzdargo M, Nguyen T, Lee DK, Lynch KR, Cheng R, Heng HH, et al. Identification and cloning of three novel human G protein -coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain. Brain Res Mol Brain Res. 1999 Feb 5;64(2):193 Ð8. Schaffner A. The Hypersplenic SpleenA Contractile Reservoir of Granulocytes and Platelets. Arch Int ern Med. 1985 Apr 1;145(4):651. Schatz A, Lee M, Condie R, Pulaski J, Kaminski N. Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Pharmacol. 1997 Feb 1;142(2):278 Ð87. Schatz AR, Koh WS, Kaminski NE. Delta 9 -tetrahydrocannabinol selectively inhibits T -cell dependent humoral immune responses through direct inhibition of accessory T -cell function. Immunopharmacology. 1993 Sep;26(2):129 Ð37. Sch−fer MK, Eiden LE, Weihe E. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience. 1998 May;84(2):361 Ð76. !'(!Schmidt EE, MacDonald IC, Groom AC. Microcirculation in mouse spleen (nonsinusal) studied by means of corrosion casts. Journal of morphology. 1985. Schmidt EE, MacDonald IC, Groom AC. Comparative aspects of splenic microcirculatory pathways in mammals: the region bordering the white pulp. Scanning Microsc. 1993 Jun ;7(2):613 Ð28. Schramm LP, Strack AM, Platt KB, Loewy AD. Peripheral and central pathways regulating the kidney: a study using pseudorabies virus. Brain Res. 1993 Jul 9;616(1 -2):251 Ð62. Schroeder HW Jr., Cavacini L. Structure and function of immunoglobuli ns. Journal of Allergy and Clinical Immunology. Elsevier; 2010 Feb;125(2):S41 ÐS52. Sedaa KO, Bjur RA, Shinozuka K, Westfall DP. Nerve and drug -induced release of adenine nucleosides and nucleotides from rabbit aorta. J Pharmacol Exp Ther. 1990 Mar;252(3): 1060Ð7. Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR. A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation. J Neuroimmune Pharmacol. 2012 Dec;7(4):1017 Ð24. PMCID: PMC35 18577 Sheng ZH, Westenbroek RE, Catterall WA. Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery. J. Bioenerg. Biomembr. 1998 Aug;30(4):335 Ð45. Shimizu N, Hori T, Nakane H. An interleuki n-1 beta -induced noradrenaline release in the spleen is mediated by brain corticotropin -releasing factor: an in vivo microdialysis study in conscious rats. Brain Behav Immun. 1994 Mar 1;8(1):14 Ð23. Shire D, Calandra B, Rinaldi -Carmona M, Oustric D, Pess‘g ue B, Bonnin -Cabanne O, et al. Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor. Biochim Biophys Acta. 1996 Jun 7;1307(2):132 Ð6. Siraskar B, Vılkl J, Ahmed MSE, Hierlmeier M, Gu S, Schmid E, et al. Enhanced cate cholamine release in mice expressing PKB/SGK -resistant GSK3. Pflugers Arch. 2011 Dec;462(6):811 Ð9. Smith JE, Jansen AS, Gilbey MP, Loewy AD. CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat . Brain Res. 1998 Mar 9;786(1 -2):153 Ð64. Spangelo BL, deHoll PD, Kalabay L, Bond BR, Arnaud P. Neurointermediate pituitary lobe cells synthesize and release interleukin -6 in vitro: effects of lipopolysaccharide and interleukin -1 beta. Endocrinology. 1994 Aug 1;135(2):556 Ð63. !')!Sperl⁄gh B, Vizi SE. Neuronal synthesis, storage and release of ATP. Seminars in Neuroscience. 1996 Aug;8(4):175 Ð86. Springs AEB, Karmaus PWF, Crawford RB, Kaplan BLF, Kaminski NE. Effects of targeted deletion of cannabinoid receptor s CB1 and CB2 on immune competence and sensitivity to immune modulation by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2008 Dec;84(6):1574 Ð84. PMCID: PMC2614598 Squire JM. Muscle Contraction: Regulation. els.net. Chichester, UK: John Wiley & Sons, Ltd; 200 1. Stavnezer J, Guikema JEJ, Schrader CE. Mechanism and Regulation of Class Switch Recombination. Annu. Rev. Immunol. 2008 Apr;26(1):261 Ð92. Stevens S, Ong J, Kim U, Eckhardt L, Roeder R. Role of OCA -B in 3' -IgH enhancer function. J Immunol. 2000 May 15;164(10):5306 Ð12. Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP -like channel expressed in nociceptive neur ons, is activated by cold temperatures. Cell. 2003 Mar 21;112(6):819 Ð29. Strack AM, Sawyer WB, Hughes JH, Platt KB, Loewy AD. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brai n Res. 1989 Jul 3;491(1):156 Ð62. Sved AF, Cano G, Card JP. Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin Exp Pharmacol Physiol. 2001;28(1 -2):115 Ð9. Tanasescu R, Constantinescu C. Cannabinoids and t he immune system: an overview. Immunobiology. 2010 Aug 1;215(8):588 Ð97. Tashkin DP. Effects of marijuana smoking on the lung. Ann Am Thorac Soc. 2013 Jun;10(3):239 Ð47. Tatemoto K. Neuropeptide Y and related peptides. Alfalah M, Michel MC, editors. 2004. Tawfik HE, Schnermann J, Oldenburg PJ, Mustafa SJ. Role of A1 adenosine receptors in regulation of vascular tone. Am J Physiol Heart Circ Physiol. 2005 Mar;288(3):H1411 Ð6. Turner AJ, Barnes K. Neuropeptidases: candidate enzymes and techniques for study. Biochem. Soc. Trans. 1994 Feb;22(1):122 Ð7. Van Belle H. Nucleoside transport inhibition: a therapeutic approach to cardioprotection via adenosine? Cardiovasc. Res. 1993 Jan;27(1):68 Ð76. !'*!Vida G, Pena G, Deitch EA, Ulloa L. $ 7-cholinergic receptor mediates vagal induction of splenic norepinephrine. The Journal of Immunology. 2011 Apr 1;186(7):4340 Ð6. PMCID: PMC3083451 Vincent -Fabert C, Fiancette R, Cogn” M, Pinaud E, Denizot Y. The IgH 3' regulatory region and its implication in lymphomagenesis. Eur J Immun ol. 2010 Dec 1;40(12):3306 Ð11. Vrana KE, Allhiser CL, Roskoski R. Tyrosine hydroxylase activation and inactivation by protein phosphorylation conditions. J Neurochem. 1981 Jan;36(1):92 Ð100. Vrana KE, Roskoski R. Tyrosine hydroxylase inactivation followin g cAMP -dependent phosphorylation activation. J Neurochem. 1983 Jun;40(6):1692 Ð700. Vulliet PR, Woodgett JR, Ferrari S, Hardie DG. Characterization of the sites phosphorylated on tyrosine hydroxylase by Ca2+ and phospholipid -dependent protein kinase, calmo dulin -dependent multiprotein kinase and cyclic AMP -dependent protein kinase. FEBS Lett. 1985 Mar 25;182(2):335 Ð9. Weinshilboum RM. Serum dopamine -beta -hydroxylase activity and blood pressure. Mayo Clin. Proc. 1977 Jun;52(6):374 Ð8. Weiss ML, Chowdhury SI. The renal afferent pathways in the rat: a pseudorabies virus study. Brain Res. 1998 Nov 23;812(1 -2):227 Ð41. Welford LA, Cusack NJ, Hourani SM. The structure -activity relationships of ectonucleotidases and of excitatory P2 -purinoceptors: evidence that dep hosphorylation of ATP analogues reduces pharmacological potency. Eur J Pharmacol. 1987 Sep 2;141(1):123 Ð30. Wennmalm M, Fredholm BB, Hedqvist P. Adenosine as a modulator of sympathetic nerve -stimulation -induced release of noradrenaline from the isolated r abbit heart. Acta Physiol. Scand. 1988 Apr;132(4):487 Ð94. Westfall TC, Carpentier S, Chen X, Beinfeld MC, Naes L, Meldrum MJ. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat. Journal of Cardiovascular Pharmacology. 1987 Dec;10(6):716 Ð22. Westfall TC, Chen XL, Ciarleglio A, Henderson K, Del Valle K, Curfman -Falvey M, et al. In vitro effects of neuropeptide Y at the vascular neuroeffector junction. Ann N Y Acad Sci. 1990;611:145 Ð55. Wheway J, Mackay CR, Newton RA, Sainsbury A, Boey D, Herzog H, et al. A fundamental bimodal role for neuropeptide Y1 receptor in the immune system. J Exp Med. 2005 Dec 5;202(11):1527 Ð38. PMCID: PMC2213323 !(+!WhiteHouse.gov. Mar ijuana Resource Center: State Laws Related to Marijuana | The White House [Internet]. whitehouse.gov. [cited 2013 Dec 31]. Retrieved from: http://www.whitehouse.gov/ondcp/state -laws -related -to-marijuana Wiest R, Jurzik L, Moleda L, Froh M, Schnabl B, Hırst en SV, et al. Enhanced Y1 -receptor -mediated vasoconstrictive action of neuropeptide Y (NPY) in superior mesenteric arteries in portal hypertension. Journal of Hepatology. 2006 Mar;44(3):512 Ð9. Winkler H, Westhead E. The molecular organization of adrenal c hromaffin granules. Neuroscience. 1980;5(11):1803 Ð23. Wollner A. Public Support For Marijuana Legalization Hits Record High : It's All Politics : NPR [Internet]. npr.org. [cited 2014 Jan 4]. Retrieved from: http://www.npr.org/blogs/itsallpolitics/2013/10/22/239847084/public -support -for -marijuana -legalization -hits -record -high Wong KK. Bethanechol induced contraction in mouse spleen. Chin J Physiol. 1990;33(2):161 Ð7. Xing H, Ling J, Chen M, Gu JG. Chemical and cold sensitivity of two distinct populations of TRPM8 -expressing somatosensory neurons. J Neurophysiol. 2006 Feb;95(2):1221 Ð30. Yamauchi T, Fujisawa H. Tyrosine 3 -monoxygenase is phosphorylated by Ca2+ -, calmodulin -dependent protein kinase, followed by a ctivation by activator protein. Biochemical and Biophysical Research Communications. 1981 May 29;100(2):807 Ð13. Yoshioka K, Hosoda R, Kuroda Y, Nakata H. Hetero -oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains. FEBS Lett. 2002 N ov 6;531(2):299 Ð303. Zouali M. Antibodies. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001a. Zouali M. Natural Antibodies. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001b. Zukowska -Grojec Z, Dayao EK, Karwatowska -Prokopczuk E, Hauser GJ, Do ods HN. Stress -induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y Y1 receptors. Am J Physiol. 1996 Feb;270(2 Pt 2):H796 Ð800. Zygmunt PM, Petersson J, Andersson DA, Chuang H, S¿rg„rd M, Di Marzo V, et al. Vanilloid receptors on senso ry nerves mediate the vasodilator action of anandamide. Nature. 1999 Jul 29;400(6743):452 Ð7. 71 Chapter 2: General Materials and Methods 2.1: Mice C57BL/6 WT female mice (NCI/Charles River, Portage, MI) and female CB1/CB2 KO mice were used in all experiment unless otherwise indicated. CB1/CB2 KO mice, on a C57BL/6 background, were created by Dr. Andreas Zimmer at the University of Bonn, Germany as previously described (Jarai et al., 1999; Zimmer et al., 1999; Buckley et al., 2000; Gerald et al., 2006) . CB1/CB2 KO m ice for these studies were obtained from Drs. Norbert Kaminski and Barbara Kaplan who maintain a breeding colony of CB1/CB2 KO mice at Michigan State University. All animals were housed two to five per cage and maintained in a sterile, temperature (22 ± 1 ¡C) and light controlled (12L:12D) room, and provided with irradiated food and bottled tap water ad libitum. All experiments used the minimal number of animals required for statistical analyses, minimized suffering, and followed the guidelines of the Natio nal Institutes of Health Guide for the Care and Use of Laboratory Animals. The Michigan State Institutional Animal Care and Use Committee approved all drug administrations and methods of euthanasia (AUF# 03/12!060!00). 2.1.1: CB1/CB2 KO Mouse Genotyping PCR was used to confirm knockout of CB1 and CB2 receptor genes. Genomic DNA was isolated from ~0.5 cm tail snips using 100 µl DirectPCR Lysis Reagent 72 (Viagen Biotech, Los Angeles, CA) plus 0.1 mg/ml proteinase K. Tails were incubated overnight at 55¡C foll owed by a 45 min incubation at 85¡C. Crude DNA extract was obtained following centrifugation at 300 RCF for 1 min. One µl of extract was used in a Taqman PCR reaction using Cnr1 stock primers (CB1 receptor gene) or Cnr2 (CB2 receptor gene) custom primers ( Life Technologies/Applied Biosystems, Foster City, CA) (Kaplan et al., 2010) . PCR primers for CB1 receptors were forward 5"-AGGAGCAAGGACCTGAGACA -3", reverse 5"-GGTCACCTTGGCGATCTTAA -3" , for CB2 receptor were forward 5 "-CCTGATAGGCTGGAAGAAGTATCTAC -3 " , reverse 5 "-ACATCAGCCTCTGTTTCTGTAACC -3 " , neomycin cassette primers were forward 5"-ACCGCTGTTGACCGCTACCTATGTCT -3 " , and reverse 5 "-TAAAGCGCATGCTCCAGACTGCCTT -3 " . The average ± standard deviatio n Ct values for Cnr1 and Cnr2 in WT mice were 25.0 ± 0.54 and 26.0 ± 1.22, respectively. All samples obtained from CB1/CB2 KO tail snips resulted in an ÒundeterminedÓ Ct value, indicating lack of expression of both Cnr1 and Cnr2 . 2.2: General Materials and Drugs All drug concentrations were calculated as free -base. All chemicals were prepared fresh for each experiment. Table 2.1 summarizes the pharmacologic action of drugs listed in this section. 73 3-hydroxybenzylhyrdazine ( NSD -1015): NSD -1015 (Molekula) was dissolved in 0.9% isotonic saline to a final concentration of 10 mg/ml and administered at dose of 100 mg/kg. 8-Cyclopentyl -1,3-dipropylxanthine ( DPCPX ): DPCPX (Sigma) was dissolved in DMSO to a concentration of 100 # M and used at a final concentration of 100 nM. !-Methyl -DL-tyrosine methyl ester hydrochloride ( aMT ): aMT ester (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline to a final concentration of 30 mg/ml and administered at dose of 300 mg/kg. Butoxamine: Butoxamine (B1385, Sigma) was dissolved in sterile isotonic saline at a concentration of 5 mg/ml and administered at doses ranging from 1-10 mg/kg (i.p.). Hank's Buffered Saline Solution ( HBSS ): 10x HBSS powder (Gibco) was diluted with ultra -pure H 2O (NaCl 138 mM, KCl 5.3 mM, Na 2HPO 4 0.3 mM, NaHCO 3 4.2 mM, KH 2PO4 0.4 mM, and glucose 5.6 mM), autoclaved, and stored at 4 ¡ C. Idazoxan: Idazoxan (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline to a final concentration of 0.4 mg/ml and administered at dose o f 4 mg/kg. 74 Isotonic Saline: 1 L of 0 .9% saline was prepared using ultra -pure H2O and 9 grams of NaCl. The solution was autoclaved and kept closed at room temperature. Krebs bicarbonate buffer: NaCl 120.0 mM, KCl 5.5 mM, CaCI 2 2.5 mM, NaH 2PO4 1.2 mM, MgCl 2 1.2 mM, NaHCO 3 20.0 mM, and glucose 11.0 mM in ultra -pure H2O. LPS: LPS ( E. coli 055:B5 catalog L2880, lot 066K4096, 5 EU/ng (Limulus lysate assay) and 10 EU/ng (chromogenic assay) , Sigma , St. Louis, MO) was dissolved in RPMI -1640 at used a t a final concentration of 10 #g/ml for in vitro studies. For in vivo experim ents, LPS was dissolved in HBSS to a concentration of 50 #g/ml and injected at 25 # g per mouse (i.p.) . N2-(Diphenylacetyl) -N-[(4 -hydroxyphenyl)methyl] -D-arginine amide ( BIBP3226 ): BIBP3226 (Tocris) was dissolved in DMSO to a concentration of 1 mM and used at a final concentration of 1 # M. Norepinephrine (NE): NE (Cat# A7257, Sigma) was dissolved in RPMI media to a concentration of 1 mM and used at a final concentration between 20 -80 #M. For spleen contraction studies, NE was dissolved in less than 20 ml of Krebs 75 bicarbonate buffer that was acidified by a single drop of concentrated (18 M) HCl to a concentration of 1 mM . Paraformaldehyde: 4% Paraformaldehyde, buffered with 0.1 M phosphate at pH 7.4, was made by combining 1:1 an 8% paraformaldehyde stock solution prepared from prills (Sigma) and a 0.2 M phosphate buffer (pH 7.4) followed by adjustment with either sodium hyd roxide or concentrated phosphoric acid. Phosphate Buffere Saline ( PBS ): NaCl 137 mM, KCl 2.7 mM, Na 2HPO 4 10 mM, and KH 2PO4 1.8 mM in ultra -pure H2O. Prazosin: Prazosin (Sigma) was dissolved in DMSO to concentration of 1 mM and used at a final concentrati on of 1 # M. Pyridoxal phosphate -6-azo(benzene -2,4-disulfonic acid ( PPADS ): PPADS (Sigma) was dissolved in H2O to a concentration of 10 mM and used at a final concentration of 10 # M. RPMI -1640: RPMI -1640 stock solution (Life Technologies) was supplemented with 15 ml of 1 M HEPES per 0.5 L prior to sterile filtration. Sterile bottles were stored under sterile conditions at 4¡ C. 76 sRBC: An aliquot of sRBC was placed in a 50 -ml conical t ube. 25 ml of HBSS was added to the sRBC and centrifuged at 300 RCF for 5 min. The supernatant was removed from the concentrated sRBC pellet. This process was performed 3 additional times. sRBC were then counted and adjusted to 2 x 10 9 cells/ml in HBSS . In experiments using sRBC, mice received 1x 10 9 cells via a single i.p. injection. Tetrodotoxin ( TTX): TTX (Sigma) was dissolved in H2O at a concentration of 0.3 mM and used at a final concentration of 0.3 # M. 77 Table 2.1. Summary of Drug Actions. Drug Action Reference 3-Hydroxybenzylhydrazine dihydrochloride (NSD -1015) L-aromatic amino acid decarboxylase inhibitor (Carlsson et al., 1972) 8-Cyclopentyl -1,3-dipropylxanthine (DPCPX) Adenosine A1 receptor antagonist (Fozard and Milavec -Krizman, 1993) $-Methyl -DL-tyrosine (aMT) Tyrosine hydroxylase inhibitor (Brodie et al., 1966) Butoxamine %2-adrenergic receptor antagonist (Burns et al., 1967) Idazoxan $2-adrenergic receptor antagonist (Doxey et al., 2012) N2-(Diphenylacetyl) -N-[(4 -hydroxyphenyl)methyl] -D-arginine amide (BIBP3226) Neuropeptide Y Y1 receptor antagonist (Rudolf et al., 1994) Norepinephrine (NE) Adrenergic receptor agonist ( $ 1, $ 2, and %2) (Furchgott, 1967) Prazosin $1AR antagonist (Cambridge and Davey, 1977) Pyridoxal phosphate -6-azo(benzene -2,4-disulfonic acid) (PPADS) Adenosine P2X receptor antagonist (Lambrecht et al., 1992) Tetrodotoxin (TTX) Voltage -gated sodium channel blocker (Hille, 19 75) 78 2.3: Isolation of the Spleen Capsule and Splenocytes After euthanasia spleens were removed by an incision in the left lateral abdomen under sterile conditions , which entails spraying the area of removal with 70% ethanol and using ethanol cleaned scissors and forceps to cut through the skin, and underlying muscle and connective tissue . The spleen was placed in a 6-well plate and mechanically crushed with the blu nt end of a 10 ml syringe in 2 ml of HBSS to separate the spleen capsule (insoluble tissue) from the splenocytes (contained in the disruption supernatant). The spleen capsule was removed from the supernatant using forceps and taken whole or divid ed into t wo parts using ethanol -cleaned scissors depending on the needs of the experiment . Splenocytes were separated from the disruption supernatant by centrifugation at 300 RCF for 5 min and the supernatant was decanted . The separated splenocytes were then re-suspended in differing buffers and taken whole or divided into two parts depending on the needs of the experiment . 2.4: Preparation of Brain Tissue for Neurochemical Analys es Following sacrifice by decapitation , the brains were rapidly removed and quick -frozen on dry ice and stored at -80¡ C until sectioning. Coronal sections of the brain (500 #m) were prepared by cryostat ( -10¡ C) for mic ropunching according to the method of Palkovits (Palkovits, 1973) . For the PVN a 21 g oval shaped tool was used to take a single midline punch. 79 2.5: Neurochemistry All samples were placed in ice -cold tissue buffer following isolation or dissection and kept frozen at -80¡C until analysis. Samples were thawed on the day of analysis and sonicated with 3 one -sec bursts (Sonicator Cell Disruptor, Heat Systems -Ultrasonic, Plainview, NY, USA) and centrifuged at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R cent rifuge. The supernatant of brain samples was removed and brought up to 65 # l (q.s.) with fresh cold tissue buffer. The supernatant from the first centrifugation of the spleen capsule was removed and spun again at 18,000 RCF for 5 min in a Beckman -Coulter M icrofuge 22R centrifuge before being brought up to 100 # l (q.s.) with fresh cold tissue buffer. Spleen samples were then filtered using a 0.2 # M syringe driven Millex -LG filter (Millipore, Billerica, MA). All samples were anal yzed for NE , MHPG, VMA , and/or DOPA content using high performance liquid chromatography coupled with electrochemical detection ( HPLC -ED) (Lindley et al., 1990; Eaton et al., 1994) using C18 reverse phase columns (ESA Inc., Sunnyvale, CA ) combined with a low pH buffered mobile phase (0 .05 M Sodium Phosphate, 0.03 M Citrate, 0.1 mM EDTA at a pH of 2.65) composed of 5 -15% methanol and 0.03 -0.05% sodium octyl sulfate . Oxidation of monoamines was measured at a constant potential of -0.4 V by coulometric detection using a Coulochem Electrochemical Detector (Thermo Scientific). The amount of each substance in the samples was determined by comp aring peak height values (as determined by a Hewlett Packard Integrator, 80 Model 3395) with those obtained from known standards run on the same day. Representative HPLC tracings from a standard solution ( Figure 2.1 ), the spleen capsule ( Figure 2.2) and PVN ( Figure 2.3) are provided as a reference. Tissue pellets remaining from preparation were dissolved in 1 N NaOH and assayed for protein using the BCA method (Noble and Bailey, 2009) . 81 Figure 2.1. Representative image from HPLC detection of biogenic amines in a standard solution . HPLC -ED can be used to separate and quantify the amount of biogenic amines in prepared solutions . The biogenic amines NE, DA, and 5HT can be separated and identified based upon their retention time. The values obtained from known stand ards , such as this, run on the same day were used to quantify the amount of in experimental samples from various tissues. 82 Figure 2. 2. Representative image of NE detection by HPLC in the spleen capsule. HPLC -ED was used to separate and quantify th e amount of NE from spleen capsule samples prepared as described. The two biogenic amines found in the spleen capsule, NE and 5HT, can be separated and identified based upon their retention time. The values obtained from HPLC standards run on the same day were used to quantify the amount of NE and normalized to the amount of protein per sample. 83 Figure 2. 3. Representative image of NE dectection by HPLC in the PVN. HPLC -ED was used to separate and quantify the amount of NE from PVN samples prepared as described. The values obtained from HPLC standards run on the same day were used to quantify the amount of NE and normalized to the amount of protein per sample. 84 2.6: Western Blot All samples were placed in ice -cold lysis buffer (water containing 1% Triton -x 100, 250 mM sucrose, 50 mM NaCl, 20 mM tris -HCl, 1 mM EDTA, 1 mM PMSF protease inhibitor cocktail, 1 mM DTT) immediately following isolation and kept frozen at -80¡ C until analysis. On the day of analysis samples were thawed, heated for 30 min at 100¡ C, sonicated for 8 sec, and spun at 12,000 RCF for 5 min. The supernatant was collected and a BCA protein assay performed (Noble and Bailey, 2009) . Equal amounts of protein were separated using SDS -PAGE and transferred to PVDF -FL membranes (Millipore, Billeri ca, MA). The resulting membranes were reacted against antibodies for TH (AB152 1:2000, Millipore, Billerica, MA) , smooth muscle $-actin (CP47 1:5000, Millipore, Billerica, MA) , or $1AR (A270 1:400, Sigma) whose intensities were normalized to "-Actin (8H10D10 1:8000, Cell Signaling, Danvers, MA) or GAPDH (G8795, 1:2000, Sigma) to account for loading variability. Each PVDF -FL membrane contained samples representing all experimental conditions to avoid variability due to run, transfer, or antibod y exposure conditions. Blots were visualized and quantified using an Odyssey Fc Infrared Imaging system (Li -Cor, Lincoln, NE) by utilization of IRDye conjugated secondary antibodies ( goat an ti-Mouse 800CW (1:20,000) or goat anti -rabbit 680LT (1:20,000) ) a nd/or HRP -conjugated anti -rabbit antibodies (1:5000; Cell Signaling) visualized using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific, Rockford, IL). 85 2.7: Immunohistochemistry 2.7.1: Brain Tissue Immunohistochemistry Mice were an esthetized with a lethal dose of ketamine:xylazine (244 mg/kg:36 mg/kg; i.p.) and transcardially perfused first with ~5 ml of 0.9% isotonic saline immediately followed by ~100 ml of 4% paraformaldehyde prepared fresh that day. Perfused brains were removed and placed in vials of 4% paraformaldehyde and transferred to 20% sucr ose in PBS after 24 h in paraformaldehyde and stored at 4 ¡ C until processing. Sections of the rostral 3rd ventricle containing the PVN (20 #m) were prepared in a cryostat at &20 C ¡. Section s were immunostained for TH (1:2,000 AB152, Millipore) using free -floating sections. Biotin conjugated goat anti -rabbit secondary antibodies (1:500) (Vector Laboratories, Burlingame, CA) were reacted with an avidin -biotin complex using an ABC Vectast ain kit (Vector Laboratories), followed by 3,3 '-diaminobenzidine (Sigma -Aldrich) to visual immuno specific staining for TH . 2.7.2: Spleen Immunohistochemistry Freshly dissected spleen s were quick frozen on dry ice and then stored at -80 C ¡ for not more that 1 week. For sectioning s pleens were embedded in Tissue -Tek OCT compound (VWR) and horizontal sections (8 # m) prepared in a cryostat at -20 C ¡ and directly mounted onto gelatin -coated slides. The sections were then allowed to dry at room temperature for one h. Sections were soaked in 86 50% ethanol for 5 min to remove residual OCT compound and then fixed in 4 C ¡ acetone prior to immunostaining. The sections were then immunostained for TH (1:250 AB152, Millipore) . Biotin conjugated goat anti -rabbit secon dary antibodies (1:500) (Vector Laboratories, Burlingame, CA) were reacted with an avidin -biotin complex using an ABC Vectastain kit (Vector Laboratories), followed by 3,3 '-diaminobenzidine (Sigma -Aldrich) to visual ize immuno specific staining for TH . 2.8: Preparation and Culture of Splenocytes Two mice were killed by decapitation and their spleens removed aseptically via a single ethanol incision in the left flank, which was wetted with 70% ethanol, using ethanol -cleaned scissors . Single -cell suspensions were prepared by disrupting the spleen capsule with the blunt end of a sterile disposable 5 -ml syringe in a 6 -well plate in ~2 ml of RPMI -1640 media. The isolated splenocytes were then cultured in RPM I-1640 media supplemented with BCS (percentage of BCS de pendent on length of culture and assay; Hyclone, Logan, UT, USA), 100 units penicillin/ml (Gibco), 100 #g streptomycin/ml (Gibco), and 50 #M 2 -ME (Gibco). Splenocytes were cultured in a humidified atmosphere at 37 ¡C and 5% CO 2. 87 2.9: Flow Cytometry 2.9.1: Surface antibody labeling for flow cytometry All staining protocols were performed in 96 -well round bottom plates (BD Falcon, Franklin Lakes, NJ). Splenocytes were washed 3x with HBSS by centrifugation at 1000 RCF for 5 min, the supernatant was decant ed, and the cells re -suspen ded in HBSS . Cultured s plenocytes were then incubated for 30 min on ice in the dark in a 1x solution of near IR (APY -Cy7) live/dead stain (#L10119, Invitrogen, Grand Island, NY) , a step which was omitted for splenocytes obtained directly from spleens. Following a wash in HBSS (as described above) , splenocytes were then washed with FACS buffer ( HBSS, 1% bovine serum albumin, 0.1% sodium azide, pH 7.6) as was done with HBSS . Surface Fc receptors were then blocked with anti -mouse C D16/CD32 [0.5 mg/ml] (#553142, BD Biosciences, Franklin Lakes, NJ) at 0.5 # l/well, IgM was blocked with anti -IgM [0.5 mg/ml] (#553425, BD Biosciences) at 1 # l/well, and IgG was blocked with anti -IgG [1.3 mg/ml] (#115 -006-071, Jackson Immunoresearch, West G rove, PA) at 0.5 # l/well for 15 min each at RT. Cells were stained for 30 min at RT with the following antibody clones: CD19 (clone 6D5) [0.2 mg/ml] (Biolegend, San Diego, CA) at 1.25 # l/well and % 2AR [0.25 mg/ml] (#AP7263d, Abgent, San Diego, CA) at 2 # l/ well. Cells were then washed 3x with FACS buffer. A secondary antibody for % 2AR, donkey anti -rabbit DyLight 649 (clone Poly4064) [0.5 mg/ml] (Biolegend), at 0.5 # l/well was incubated for 30 min at RT. Subsequently cells were washed with FACS buffer, fixe d with Cytofix (BD 88 Biosciences) for 15 min at RT, washed 3x with FACS buffer, and finally suspended in FACS buffer for intracellular staining. Stained and fixed cells were stored in the dark at 4 ¡C for up to 2 weeks. 2.9.2: Intracellular antibody labeling for flow cytometry Within 2 weeks of surface staining (described above), cells were washed 2x with Perm/Wash (BD) and incubated with Perm/Wash for 30 min at RT. Fluorescently labeled antibodies for IgM (Clone II/41) [0.5 mg/mL] (Biolegend) and/or IgG [0.5 mg/mL] (#115 -606-071, Jackson Immunoresearch, West Grove, PA) were added at 1 # l/well (IgM) or 0.5 µl/well (IgG) for 30 min. Cells were washed 2x with Perm/Wash and suspended in FACS buffer. After intracellular staining , cells were analyzed the s ame day. 2.9.3: Flow Cytometry Analysis Fluorescent staining was analyzed using a BD Biosciences FACSCanto II flow cytometer. Data were analyzed using Kaluza (Beckman Coulter Inc., Brea, CA) or Flo wJo software ( Tree Star Inc., Ashland, OR) . Compensatio n and voltage settings of fluorescent parameters were performed using fluorescence -minus -one controls. Cells were gated on singlets (forward scatter height versus area) (Figure 2.4) followed by determination of live cells (low APC -Cy7 signal) only in samples obtained from splenocyte culture (Figure 2.5). Cells were then gated to select lymphocytes using forward versus side scatter (Figure 2.6). For 89 some analyses , an additional gate was created for CD19 expression to select for B cells (Figure 2.7). These sequential gates were used to identify IgM producing B cells ( Figure 2.8) and IgM producing B cells that express % 2AR ( Figure 2.9). The percentage of cells gated to individual populations relative to the entire population were collected and analyzed. Additionally, the numerical intensity of the fluorescent signal , termed the mean fluorescence intensity (MFI), was also quantified and analyzed. 90 NOTE: For interpretation of the references to color in this and all other figures, the reader is referre d to the electr onic version of this dissertation . Figure 2.4. Representative gating of singlets in splenocytes prepared for flow cytometry . The cells with a linear relationship when comparing the forward scatter area and height is encircled in black. This is the population single cells in solution, termed singlets. 91 Figure 2. 5. Representative gating of live cell s in preparations of cultured splenocytes for flow cytometry. Within the si nglet population, cells having low APC -Cy7 are encircled in black. This is the population singlets that are alive following isolation and preparation. 92 Figure 2. 6. Representative gating for spleen derived lymphocytes in preparations for flow cytometry. Within the singlet/live cells , the population of lymphocytes in the spleen (encircled in black) was identified as cells having a relatively high forward scatter and low side scatter profile. 93 Figure 2. 7. Representati ve gating for splenic B cell s by flow cytometry. Among splenic lymphocytes, the population of B cells was identified by high CD19 expression (indicated by the brackets). 94 Figure 2. 8. Representative images from flow cytometric analysis of IgM producing splenic B cells. Among splenic lymphocytes, I gM prod ucing B cells were identified as cells with high IgM and CD19 expression (upper right quadrant). An image from both immunologically naŁve and LPS treated splenocytes is provided for comparison. 95 Figure 2. 9. Representative images from flow cytometric analysis of IgM producing splenic B cells expressing "2AR. Among gated splenic B cells , IgM producing cells expressing "2AR were identified (upper right quadrant). An image from both immunologically naŁve and LPS treated splenocytes is provided for comparison . 96 2.10: Enzyme Linked Immunosorbant Assay ( ELISA ) Serum IgM and IgG were detected by sandwich ELISA. In preparation, 100 #l of 1 #g/ml anti -mouse IgM (Sigma -Aldrich, St. Louis, MO) or 1 # g/ml anti -mouse IgG (Sigma -Aldrich) was added to wells of a 96 -well microtiter plate and stored at 4 ¡C overnight. After the pre -coating step, the plate was washed twice with 0.05% Tween -20 in PBS and three times with H 2O. Following this, 200 #l of 3% BSA -PBS was added to the wells and incubated at RT for 1.5 h to block nonspecific binding followed by the same washing steps described above. Serum samples were diluted and added to the coated plate (100 # l) for 1.5 h at RT. After the incubation, the plate was w ashed again, followed by addition of 100 #l of HRP -conjugated goat anti -mouse IgM (A8786, Sigma -Aldrich) or HRP -conjugated goat IgG (A3673, Sigma -Aldrich). Following the HRP incubation for 1.5 h at RT, any unbound detection antibody was washed away from th e plate, and 100 # l ABTS (Roche Applied Science, Indianapolis, IN) added. The detection of the HRP substrate reaction was conducted over a 1 h period using a plate reader with a 405 -nm filter (Bio -Tek). The KC4 computer analysis program (Bio -Tek) calculate d the concentration of IgM or IgG in each sample based on a standard curve generated from the absorbance readings of known concentrations of IgM (range 6 -1600 pg/ml, clone TEPC 183, Sigma -Aldrich) or IgG (range 3 -800 pg/ml, Sigma -Aldrich). 97 2.11: Enzyme L inked Immunosorbent Spot Assay ( ELISPOT ) ELISPOT was performed as described previously (Lu et al., 2009) . ELISPOT wells were coated with purified anti -mouse (Sigma -Aldrich) IgM antibody and blocked with 5% BSA. Splenocytes from freshly disrupted spleens were washed , via centrifugation as described for flow cytometry, and incubated in the ELISPOT wells for 16 Ð20 h. Biotin -conjugated anti -mouse IgM antibody (Sigma -Aldrich, St. Louis, MO) and streptavidin -horseradish peroxidase (Sigma -Aldrich, St. Louis, MO) were sequentially added to the wel ls. The spots were developed with the aminoethylcarbazole staining kit (Sigma -Aldrich, St. Louis, MO). Data were collected and analyzed using the CTL ImmunoSpot system (Cellular Technology Ltd, Shaker Heights, OH). 2.12: Spleen Contraction Studies 2.12.1: Preparation of Spleen Tissue for Spleen Contraction Studies Spleens were obtained from mice euthanized via isoflurane (primary death by cervical dislocation results in less contractile responses and necessitates prolonged equilibration of the tissue (Eltze, 1996) . Spleens were removed and connected via loops of nylon string to an isometric force transducer (Radnoti, Monrovia, CA) and placed in a 20 -ml organ bath (Norman D. Erway Glass Blowing) under a resting tension of 0.8 g (7.84 mN) for recording isometric contractile responses in Krebs bicarbonate buffer maintained at 37¡C and gassed with 95% 0 2 - 5% CO 2 (Figure 2.10 ). 98 2.12.2: Spleen Contraction Measurement Isometric contractions were recorded in response to EFS or direct injection of NE into the bath medium. EFS was produced by two ~1 cm platinum ring electrodes (custom built), above and below the spleen, connected to a Grass S48 Stimulator (Grass Technologies, Warwi ck, RI). All spleens were simultaneously stimulated using a Med -Lab Stimu -Splitter II (Med -Lab Instruments). The EFS consisted of square wave pulses 0.2 -0.25 ms in duration with the following characteristics: 30 V, 25 hertz (Hz), and 3 s train duration. Prior to any testing, each spleen was given at least 45 min of undisturbed time to acclimate to the ex vivo environment. Increased tension on the nylon string from spleen contraction resulted in deflection of a post on the isometric force transducer , the deflection of which is read as a change in voltage ( Figure 2.11). This change in voltage is converted to grams at a rate of 0.28 volts equaling 1 gram. All data was then con verted to and expressed as mN by multiplying the gram data output by 9.8 m/s 2 and then multiplying by 1000. 99 Figure 2. 10. Schematic of equipment setup used to record spleen contractile force. Isolated spleens connected by nylon string to an isometric force transducer while residing in a physiological bath aerated with 95% oxygen and 5% carbon dioxide maintained at 37 ¡ C. Spleen contractile force was recorded by the tension generated on the nylon string. Figure adapted from Evora, et al (Evora et al ., 2007). 100 Figure 2. 11. Representative image of the raw data obtained from spleen contraction experiments. The force of spleen contraction was measured by the tension generated on nylon strings connected from the spleen to the post of an isometric force transduction unit. The force of contraction was defined as the maximum voltage change, as indicated by the br acketed arrow, in response to a contractile stimulus. 101 2.13: Spleen Capsule Width Measurement 2.13.1: Hematoxylin and Eosin Staining Mice were given a lethal dose of ketamine:xylazine (244 mg/kg:36 mg/kg; i.p.) and the spleen removed and dropped fixed i n 4% paraformaldehyde. Fixed spleens were then taken to the Michigan State University Department of Pathology Histology Laboratory where they were paraffin embedded, sectioned at 4 # m, and stained with hematoxylin and eosin using standard accepted methodol ogy. 2.13.2: Quantification of Spleen Capsule Thickness Stained spleen sections were imaged at 40x magnification using a Nikon TE2000 -S Inverted Microscope (Nikon, Melville, NY) with a Spot Insight QE camera (SPOT Imaging, Sterling Heights, MI) with SPOT 3.5.8 Imaging software (SPOT Imaging) ( Figure 2.12). Each spleen was imaged, focused on the spleen capsule, two times at random locations on both the internal and external side of the spleen. ImageJ software (National Institutes of Health, USA) was used to measure the width of the spleen capsule, in pixels, at four evenly spaced locations on each image. All 12 values from each spleen were then averaged and this number used a s a single n for comparison between genotypes . 102 Figure 2. 12. Representative images comparing the spleen capsule thickness between WT and CB1/CB2 KO mice. Spleens from WT and CB1/CB2 KO mice were fixed, stained with hematoxylin and eosin, and imaged at 40x magnificati on. Representative images from each genotype are presented. The black bar in the images is 50 µm in length. 103 2.14: Statistical Analysis 2.14.1: Statistical Comparisons Prism software version 4.0a was used to make statistical comparisons between groups using the appropriate statistical test. Differences with a probability of error of less than 5% (p<0.05) were considered statistically significant. Two group comparisons were done using the S tudent " s t-test. Two group comparisons where in one group had more than one degree or factor were done using a One -way ANOVA followed by a Bonferroni or Tukey " s post -test for multiple comparisons. Experiments in which there were two groups with more than one degree or factor in each group, such as a 2x2 design, were analyzed using a Two -way ANOVA followed b y Bonferroni post -test for multiple comparisons. Consideration for repeated measurements, as in spleen contraction studies, was given in analysis where appropriate. 2.14.2: aMT Experimentation In experiments in which aMT was used to assess neuronal activ ity, NE concentrations from aMT treated and non -aMT (saline) treated mice were used for a regression analysis with saline animals acting as the 0 time control and aMT animals a 4 h time point. The rate constant was determined using this formula: =(Log 10[B] -Log10[A])/( -0.434*t). Where B is the concentration of NE after aMT, A is the concentration of A in saline treated animals, and t is the time of aMT treatment (Brodie et al., 1966) . The slopes were compared via t -test using the 104 mean slope, SEM, and an n equal to the total number of data points used in the analysis to account for the total number of independent measurements used to generate the regression equations. Differences with a probability of error of less than 5% were considered statistically significant . 2.14.3: Flow Cytometry Data Handling Population percentage data was transformed in Excel (Microsoft Corporation, Redmond, WA) to a parametric form before ANOVA analysis using the formula: =arcsin(sqrt(DAT A/100)) (Ahrens et al., 1990) . Raw percentage data was used for visual representation s, while statistical significance indicated on these figures was performed on the transformed data. 105 REFERENCES 106 REFERENCES Ahrens WH, Cox DJ, Budhwar G. Use of the arcsine and square root transformations for subjectively determined percentage data. Weed Science. 1990. Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines. J Pharmacol Exp Ther. 1966 Dec;154(3):493 Ð8. Buckley N, McCoy K, Mezey E, Bonner T, Zimmer A, Felder C, et al. Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB( 2) receptor. Eur J Pharmacol. 2000 May 19;396(2 -3):141 Ð9. Burns JJ, Salvador RA, Lemberger L. METABOLIC BLOCKADE BY METHOXAMINE AND ITS ANALOGS. Ann NY Acad Sci. 1967 Feb;139(3 New Adrenergi):833 Ð40. Cambridge D, Davey MJ. Prazosin, a selective antagonis t of post -synaptic alpha -adrenoceptors [proceedings]. British journal of É. 1977. Carlsson A, Davis JN, Kehr W, Lindqvist M, Atack CV. Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in vivo using an inhibitor of the ar omatic amino acid decarboxylase. Naunyn -Schmied Arch Pharmacol. 1972;275(2):153 Ð68. Doxey JC, Roach AG, Smith CFC. Studies on RX 781094: a selective, potent and specific antagonist of $ 2-adrenoceptors. Br J Pharmacol. 2012 Jul 19;78(3):489 Ð505. Eaton MJ, Lookingland KJ, Moore KE. Effects of the selective dopaminergic D2 agonist quinelorane on the activity of dopaminergic and noradrenergic neurons projecting to the diencephalon of the rat. 1994 Feb 1;268(2):645 Ð52. Eltze M. Functional evidence for an alph a 1B-adrenoceptor mediating contraction of the mouse spleen. Eur J Pharmacol. 1996 Sep 12;311(2 -3):187 Ð98. Evora PRB, Cable DG, Chua YL, Rodrigues AJ, Pearson PJ, Schaff HV. Nitric oxide and prostacyclin -dependent pathways involvement on in vitro induced hypothermia. Cryobiology. 2007 Feb;54(1):106 Ð13. Fozard JR, Milavec -Krizman M. Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation. Br J Pharmacol. 1993 Jul 19;109(4):1059 Ð63. PMCID: PMC2175713 107 Furchgott RF. The Pharmacolog ical Differentiation of Adrenergic Receptors. Ann NY Acad Sci. 1967. Gerald T, Ward G, Howlett A, Franklin S. CB1 knockout mice display significant changes in striatal opioid peptide and D4 dopamine receptor gene expression. Brain Res. 2006 Jun 6;1093(1): 20Ð4. Hille B. The receptor for tetrodotoxin and saxitoxin. A structural hypothesis. Biophysical Journal. The Biophysical Society; 1975 Jun 1;15(6):615. Jarai Z, Wagner J, Varga K, Lake K, Compton D, Martin B, et al. Cannabinoid -induced mesenteric vasodi lation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14136 Ð41. Kaplan BLF, Lawver JE, Karmaus PWF, Ngaotepprutaram T, Birmingham NP, Harkema JR, et al. The effects of targeted deletion of cann abinoid receptors CB1 and CB2 on intranasal sensitization and challenge with adjuvant -free ovalbumin. Toxicol Pathol. 2010 Apr;38(3):382 Ð92. PMCID: PMC2941344 Lambrecht G, Friebe T, Grimm U, Windscheif U, Bungardt E, Hildebrandt C, et al. PPADS, a novel functionally selective antagonist of P2 purinoceptor -mediated responses. Eur J Pharmacol. 1992 Jul 7;217(2 -3):217 Ð9. Lindley SE, Gunnet JW, Lookingland KJ, Moor e KE. 3,4 -Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity. Brain Res. 1990 Jan 1;506(1):133 Ð8. Lu H, Kaplan BLF, Ngaoteppru taram T, Kaminski NE. Suppression of T cell costimulator ICOS by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2009 Feb;85(2):322 Ð9. PMCID: PMC2631366 Noble J, Bailey M. Quantitation of protein. Methods Enzymol. 2009;463:73 Ð95. Palkovits M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 1973 Sep 14;59:449 Ð50. Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD, Entzeroth M, et al. The first highly potent and selective non -peptide neuropeptide Y Y1 receptor antagonist: BIBP322 6. Eur J Pharmacol. 1994 Dec 27;271(2 -3):R11 Ð3. Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci USA. 1999 May 11;96(10):5780 Ð5. PMCID : PMC21937 108 Chapter 3 : Comparison of the Noradrenergic Innervation in the Murine Spleen and the Par aventricular Nucleus of the Hypothalamus 3.1: Introduction The PVN is a bilateral nucleus in the hypothalamus that has a multitude of functions that are aimed at maintaining the homeostasis of the body , particularly cardiovascular function . It is also regarded as one of five canonical central pre -sympathetic nuclei (Strack et al., 1989; Sved et al., 2001; Pyner, 20 09). Like all central pre -sympathetic nuclei, the PVN has directly descending e fferent projections to the IML of the spinal cord where they synapse on pre -ganglionic sympathetic neurons (Strack et al., 1989; Schramm et al., 1993; Cano et al., 2001; Sved et al., 2001; Pyner, 2009) . The directly descending spinal projecting neuron s of the PVN express vasopressin and oxytocin , which are stimulatory, or dopamine , which has mixed effects on pre -ganglionic sympathetic neurons (Sawc henko and Swanson, 1982; Strack et al., 1989; Sved et al., 2001; Pyner, 2009) . The PVN receives noradrenergic innervation from several brain nuclei , including the A1, A5, and A6 (locus coeruleus) noradrenergic syste ms (Byrum and Guyenet, 1987; Pacak et al., 1995; Samuels and Szabadi, 2008) . Most noradrenergic innervation of the PVN is to the parvocellular portion, which is where the cells bodies of pre -autonomic spinal projecting neurons reside (Byrum and Guyenet, 1987; Cunningham and Sawchenko, 1988; Strack et al., 1989; Samuels and Szabadi, 2008; Pyner, 2009) . Retroviral tracing and microinjection studies of the PVN demonstrate that this nucleus is 109 an upstream regulator of splenic sympathetic activity (Katafuchi et al., 1993; Cano et al., 2001). Stimulation of the PVN by direct intracerebral glutamate injection stimulate s splenic sympathetic act ivity (Katafuchi et al., 1993) . Noradrenergic signaling in the PVN is mediated largel y by !1 and !2AR (Leibowitz and Hor, 1982; Goldman et al., 1985; Daftary et al., 2000; Chong et al., 2004) . Stimulation of !1AR on parvocellular neurons increase sIPSCs in GABAergic inte rneurons and stimulate s the activity of glutamatergic interneurons, both of which synapse upon effector parvocellular neurons, including directly descending pre -autonomic neurons (Daftary et al., 2000; Chong et al., 2004) . Conversely, ! 2AR decrease sIPSCs in GABAergic interneurons (Chong et al., 2004) . Afferent PVN noradrenergic axon terminals have also known to be regulated by ! 2AR auto -receptors (Lookingland et al., 1991) . The rate of NE turnover (an estimate of noradrenergic neuronal activity ) is general ly higher in CNS noradrenergic neurons compared to noradrenergic neurons in the PNS . For instance, the half-life of NE turnover in the PVN is <1.5 h, whereas NE turnover in the heart and pancreas is >3 h (Levin, 1995) . Stress is a well known cause for NE release in the PVN to activate the HPA -axis and induc e the production of stres s-related corticosteroids as well as increasing sympathetic outflow (Pacak et al., 1995; Itoi and Sugimoto, 2010; Inoue et al., 2013) . In comparison to the PVN, relatively little is known about the physiology of the splenic sympathetic neurons. The purpose of this chapter is to use the PVN, a central noradrenergic nucleus, as a comparator in evaluating the spleen projecting 110 noradrenergic sympathetic neurons. Not only are the noradrenergic neurons in the PVN well studied, but also are likely upstream in controlling the activity of splenic sympathetic neurons. There fore, the ir activity is likely coupled with splenic sympathetic neurons and also likely share a number of physiological properties in common. Thus, identification of the similarities and differences between these two regions will be of use in identifying ways to stud y splen ic noradrenergic neurons, the focus of this dissertation. Furthermore many of the tools used to study PVN noradrenergic neurons will likely be useful for studying splenic noradrenergic neurons: including immunostaining, neurochemical analysis, and pharmacologic manipulation. The goal of the chapter is , therefore, to provide a basis of understanding and methodology necessary to interpret the findings in subsequent experimental paradigms with regard to the activity and function of splenic sympathetic neurons. 3.2: Materials and Methods 3.2.1:Mice C57BL/6 WT female mice were h oused two to five per cage and maintained in a temperature (22 ± 1 ¡C) and light controlled (12L:12D) room, and provided with food and water ad libitum. 3.2.2: Materials NSD -1015: NSD -1015 (Molekula) was dissolved in 0.9% isotonic saline to a final concen tration of 10 mg/ml and administered at dose of 100 mg/kg. 111 aMT: aMT ester (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline to a final concentration of 30 mg/ml and administered at dose of 300 mg/kg. HBSS: 10x HBSS powder (Gibco) was diluted with ultra -pure H 2O (NaCl 138 mM, KCl 5.3 mM, Na 2HPO 4 0.3 mM, NaHCO 3 4.2 mM, KH 2PO4 0.4 mM, and glucose 5.6 mM), autoclaved, and stored at 4 ¡ C. Idazoxan: Idazoxan (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline to a final concentration of 0.4 mg/ml and administered at dose of 4 mg/kg. Isotonic Saline: 1 L of 0.9% saline was prepared using ultra -pure H 2O and 9 grams of NaCl. The so lution was autoclaved and kept closed at room temperature. Paraformaldehyde: 4% Paraformaldehyde, buffered with 0.1 M phosphate at pH 7.4, was made by combining 1:1 an 8% paraformaldehyde stock solution prepared from prills (Sigma) and a 0.2 M phosphate b uffer (pH 7.4) followed by adjustment with either sodium hydroxide or concentrated phosphoric acid. Phosphate Buffered Saline (PBS): NaCl 137 mM, KCl 2.7 mM, Na 2HPO 4 10 mM, and KH2PO4 1.8 mM in ultra -pure H2O. 112 3.2.3: Isolation of the Spleen Capsule and S plenocytes After euthanasia , spleens were removed by an incision in the left lateral abdomen under sterile conditions, which entails spraying the area of removal with 70% ethanol and using ethanol cleaned scissors and forceps to cut through the skin, and underlying muscle and connecti ve tissue. The spleen was placed in a 6 -well plate and mechanically crushed with the blunt end of a 10 ml syringe in 2 ml of HBSS to separate the spleen capsule (insoluble tissue) from the splenocytes (contained in the disruption supernatant). The spleen capsule was removed from the supernatant using forceps and taken whole or divided into two parts using ethanol -cleaned scissors depending on the needs of the experiment . Splenocytes were separated from the disruption supernatant by centrifugation at 300 R CF for 5 min and the supernatant was decanted. The separated splenocytes were then re -suspended in differing buffers and taken whole or divided into two parts depending on the needs of the experiment. 3.2.4: Preparation of Brain Tissue for Neurochemical Analys es Following sacrifice by decapitation, the brains were rapidly removed and quick -frozen on dry ice and stored at -80¡ C until sectioning. Coronal sections of the brain (500 "m) were prepared by cryostat ( -10¡ C) for micropunching according to the me thod of Palkovits (Palkovits, 1973) . For the PVN a 21 g oval shaped tool was used to take a single midline punch. 113 3.2.5: Neurochemistry All samples were placed in ice -cold tissue buffer following isolation or dissection and kept frozen at -80¡C until analysis. Samples were thawed on the day of analysis and sonicated with 3 one -sec bursts (Sonicator Cell Disruptor, Heat Systems -Ultrasonic, Plainview, N Y, USA) and centrifuged at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge. The supernatant of brain samples was removed and brought up to 65 " l (q.s.) with fresh cold tissue buffer. The supernatant from the first centrifugation of the s pleen capsule was removed and spun again at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge before being brought up to 100 " l (q.s.) with fresh cold tissue buffer. Spleen samples were then filtered using a 0.2 " M syringe driven Millex -LG filter (Millipore, Billerica, MA). All samples were analyzed for NE, MHPG, VMA, and/or DOPA content using HPLC -ED (Lindley et al., 1990; Eaton et al., 1994) using C18 reverse phase columns (ESA) combined with a low pH buffered mobile phase (0 .05 M Sodium Phosphate, 0.03 M Citrate, 0.1 mM EDTA at a pH of 2.65) composed of 5 -15% methanol and 0.03 -0.05% sodium octyl sulfate. Oxidation of monoamines was measured at a constant potential of -0.4 V by coulometric detection using a Coulochem Electrochemical Detector (Thermo Scientific). The amount of each substance in the samples was determined by comparing peak height values (as determined by a Hewlett Packard Integrator, Model 3395) with those obtained from known standards run on the same day. Tissue pellets remaining from preparation were dissolved in 1 N NaOH and a ssayed for protein using the BCA method (Noble and Bailey, 2009) . 114 3.2.6: Western Blot All samples were placed in ice -cold lysis buffer (water containing 1% Triton -x 100, 250 mM sucrose, 50 mM NaCl, 20 mM tris -HCl, 1 mM EDTA, 1 mM PMSF protease inhibitor cocktail, 1 mM DTT) immediately following isolation and kept frozen at -80¡C until analysis. On the day of analysis samples were thawed, heated for 30 min at 100¡ C, sonicated for 8 sec, and spun at 12,000 RCF for 5 min. The supernatant was collected and a BCA protein assay performed (Noble and Bailey, 2009) . Equal amounts of protein were separated using SDS -PAGE and transferred to PVDF -FL membranes (Millipore, Billerica, MA). The resulting membranes were reacted against antibodies for TH (AB152 1:2000, Millipore, Billerica, MA), s mooth muscle !-actin (CP47 1:5000, Millipore, Billerica, MA), or ! 1AR (A270 1:400, Sigma) whose intensities were normalized to Beta -Actin (8H10D10 1:8000, Cell Signaling, Danvers, MA) or GAPDH (G8795, 1:2000, Sigma) to account for loading variability. Eac h PVDF -FL membrane contained samples representing all experimental conditions to avoid variability due to run, transfer, or antibody exposure conditions. Blots were visualized and quantified using an Odyssey Fc Infrared Imaging system (Li -Cor, Lincoln, NE ) by utilization of IRDye conjugated secondary antibodies, goat anti -Mouse 800CW (1:20,000) and/or goat anti -rabbit 680LT (1:20,000). 115 3.2.7: Immunohistochemistry 3.2.7.1: Brain Tissue Immunohistochemistry Mice were anesthetized with a lethal dose of ketamine :xylazine (244 mg/kg : 36 mg/kg; i.p.) and transcardially perfused first with ~5 ml of 0.9% isotonic saline immediately followed by ~100 ml of 4% paraformaldehyde prepared fresh that day. Perfused brains were removed and placed in vials of 4% parafo rmaldehyde and transferred to 20% sucrose in PBS after 24 h in paraformaldehyde and stored at 4 ¡ C until processing. Sections of the rostral 3rd ventricle containing the PVN (20 "m) were prepared in a cryostat at #20 C¡ . Sections were immunostained for TH (1:2,000 AB152, Millipore) using free -floating sections. Biotin conjugated goat anti -rabbit secondary antibodies (1:500) (Vector Laboratories, Burlingame, CA) were reacted with an avidin -biotin complex using an ABC Vectastain kit (Vector Laboratories), fol lowed by 3,3 $-diaminobenzidine (Sigma -Aldrich) to visual immunospecific staining for TH. 3.2.7.2: Spleen Immunohistochemistry Freshly dissected spleens were quick frozen on dry ice and then stored at -80 C¡ for not more that 1 week. For sectioning spl eens were embedded in Tissue -Tek OCT compound (VWR) and horizontal sections (8 " m) prepared in a cryostat at -20 C¡ and directly mounted onto gelatin -coated slides. The sections were then allowed to dry at room temperature for one hour. Sections were soak ed in 50% ethanol for 5 min to remove residual OCT compound and then fixed in 4 C¡ acetone prior to immunostaining. The sections were then immunostained for TH (1:250 AB152, Millipore) . Biotin 116 conjugated goat anti -rabbit secondary antibodies (1:500) (Vect or Laboratories, Burlingame, CA) were reacted with an avidin -biotin complex using an ABC Vectastain kit (Vector Laboratories), followed by 3,3 $-diaminobenzidine (Sigma -Aldrich) to visual immunospecific staining for TH. 3.2.8: Statistical Analysis: 3.2.8.1: Statistical Comparisons Prism software version 4.0a was used to make statistical comparisons between groups using the appropriate statistical test. Differences with a probability of error of less than 5% (p< 0.05) were considered statistically significant. Two group comparisons were done using the Student % s t-test. Two group comparisons where in one group had more than one degree or factor were done using a One -way ANOVA followed by a Bonferroni or Tukey % s p ost -test for multiple comparisons. Experiments in which there were two groups with more than one degree or factor in each group, such as a 2x2 design, were analyzed using a Two -way ANOVA followed by Bonferroni post -test for multiple comparisons. Considerat ion for repeated measurements, as in spleen contraction studies, was given in analysis where appropriate. 3.2.8.2: aMT Experimentation In experiments in which aMT was used to assess neuronal activity, NE concentrations from aMT treated and non -aMT (salin e) treated mice were used for a regression analysis with saline animals acting as the 0 time control and aMT animals a 117 4 h time point. The rate constant was determined using this formula: =(Log 10[B] -Log10[A])/( -0.434*t). Where B is the concentration of NE after aMT, A is the concentration of A in saline treated animals, and t is the time of aMT treatment (Brodie et al., 1966) . The slopes were compared via t -test using the mean slope, SEM, and an n equal to the total number of data points used in the analysis to account for the total number of independent measurements used to generate the regression equations. Differences with a probability of error of less than 5% were considered statistically significant. 3.3: Resu lts 3.3.1: Noradrenergic innervation of the PVN and the spleen in mice The noradrenergic innervation of the PVN and spleen can be visualized using immunohistochemical techniques. Im munostaining for TH demonstrates that the den sity of noradrenergic terminals differs greatly b etween these two areas. In the PVN, immunostaining for TH reveals dense noradrenergic axon terminal innervation largely in the dorsolateral portion of the PVN , with some axon terminals reaching periventricul ar areas of the PVN (Figure 3.1 ). Thes e area s of the PVN are considered to be the lateral and dorsal parvocellular region s, respectively (Strack et al., 1989; Pacak et al., 1995; Daftary et al., 2000; Pyner, 2009) . Neurons in this area are known to project to brain stem/ spinal cord autonomic targets to control the activation of peripheral sympathetic neurons , as well as controlling CRH expressing neurons leading to HPA axis activation (Chong et al., 2004; Pyner, 2009) . NE is reported to participate in the control of 118 sympathetic stimulation from this region, but interestingly only to certain types of stressors (Pacak et al., 1995) . Much like the PVN, TH staining in the spleen is heterogeneous and is localized to two regions within the spleen : the spleen capsule a nd plexus innervation of arterioles their surrounding areas, known as the PALS (Figure 3. 1). This distribution is suggestive of the function of splenic sympathetic innervation , which is to control spleen contraction and the flow of blood through the spleen (Groom et al., 1991; Su et al., 1991; Eltze, 1996). Noradrenergic innervation o f the PALS area immediately adjacent to splenic central arterioles is also supported by the staining demonstrated here (Felten et al., 1987; Felten and Olschowka, 1987) . Western blot an alysis for TH is congruent with immunohistochemical staining in showing that the majority of TH in the spleen is contained in the spleen capsule rather than the cellular parenchyma of the spleen, splenocytes ( Figure 3. 2). This distribution is further conf irmed in that NE in the spleen is almost exclusively found in the spleen capsule as compared with splenocytes (Figure 3.3). 119 Figure 3.1 . Immunohistochemical staining for TH in the PVN and spleen of mice. Fixed tissue from the PVN and spleen were stained for TH using standard immunohistochemical techniques (described in detail in Chapter 2). TH specific staining is shown as brown. Arrowheads denote TH immunostaining in the spleen capsule. Arrows denote TH immunostaining surrounding blood vessels. A scale bar for s ize reference is provided and represents the size denoted in the figure. 120 Figur e 3.2 . Western blot analysis of TH in the spleen of mice. Freshly removed mouse spleens were disrupted to separat e the spleen capsule from the splenocytes. The tis sue was then homogenized and lys ed. Lysates were separated by SDS -PAGE then probed for TH and &-actin via Western blot. A) Labeled picture of the resulting image used for quantification. Red bands are T H and green bands are &-actin. B) Columns represent that mean TH RDU normalized to &-actin in the same sample + one SEM (n=6). * Differs from Spleen Capsule (p<0.05). 121 Figure 3.3 . Neurochemical analysis of NE in the murine spleen. Freshly removed mouse spleens were disrupted to separate the spleen capsule from the splenocytes. The resulting samples were then prepared and analyzed via HPLC -ED for the amount of NE, which was normalized to the amount of protein per sample as determi ned by a BCA assay. Columns represent the mean c oncentration of NE + one SEM (n=7 -8). * Differs from Spleen Capsule (p<0.05). 122 3.3.2: Noradrenergic neurochemi cal activity in the PVN and spleen of mice Neurochemical analysis of noradrenergic axon terminals in the PVN and spleen capsule reveals a number of significant differences. First, there is a large difference in the concentration of NE, with the PVN having a significantly higher NE concentration than the spleen capsule ( Fig ure 3.4 ). However, the total amount of NE in the spleen capsule is much greater ( Figure 3.5 ). These findings are cons istent with what was observed with TH immunostaining showing a higher density of TH fibers in t he PVN versus the spleen . The relative pr oportion of axon terminals in re lation to the size of the PVN appears to be much greater than the spleen capsule , but the spleen capsule is much larger than the PVN. Therefore, it is not surprising that the PVN has a higher concentration of NE, but less overall NE content than the spleen capsule . The two major metabolites of NE, VMA and MHPG, were also measured in the PVN and spleen capsule. Commensurate with higher concentrations of NE in the PVN, the concentration of MHPG is higher in the PVN as compared with the spleen capsule (Figure 3.6 ). This difference is likely due to the relatively small size of the PVN compared to the spleen capsule, but may also be reflective of a higher metabolic rate in the PVN compared to the spleen capsule. The r atio of MHPG to NE can be used as an indirect index of the rate of NE release, re -uptake and metabolism (Lookingland et al., 1991). Successive processing of NE by MAO, COMT, and AR produces MHPG, which is considered to be the major metabolite of NE in the brain (Lookingland et al., 1991; Hayley et a l., 2001; Eisenhofer et al., 2004) . In the brain this metabolism take s place in both the neuron and glia cells , whereas in the spleen this conversion takes place in 123 neurons and macrophages (Karhunen et al., 1994; Eisenhofer et al., 2004; Myıh−nen et al., 2010). The MHPG/NE ratio is higher in the PVN ( Figure 3. 7). These data, calculated using the content of each neurochemical, suggest that the PVN has a higher rate of metabolic activity. NE is converted to MHPG locally and can then be further metabolized to VMA by AD and ALD (Eisenhofer, Kopin et al. 2004). In the periphery this occurs largely in the liver and VMA is considered the major end metabolite of NE outside the brain (Oh -hashi et al., 2001; Eisenhofer et al., 2004; Siraskar et al., 2011) . Similar to MPHG, the concentration of VMA was observed to be higher in the PVN ( Figure 3 .8). However, t he VMA/ NE ratio was equivalent between the PVN and spleen capsule ( Figure 3. 9). These data suggests that the differences in VMA concentrations may be due largely to size dif ference, rather than increased metabolic activity. But since VMA is largely generated in non -neuronal cells it may not be a truly accurate indicator of neuronal metabolic activity. 124 Figure 3.4 . Comparison of NE concentration s in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen capsule was isolated and brains were sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of NE which was normalized to the amount of protein per sample as determined by a BCA assay. Columns represent the mean conc entration of NE + one SEM (n=7 ). * Differs from PVN (p<0.05). 125 Figure 3.5 . Comparison of NE content in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen cap sule was isolated and brains were sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of NE. Columns represent the mean amount of NE per sample + one SEM (n=7 ). * Differs from PVN (p<0.05). 126 Figure 3. 6. Comparison of MHPG concentration s in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen capsule was isolated and brains were sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule s amples were then prepared and analyzed via HPLC -ED for the amount of MHPG which was normalized to the amount of protein per sample as determined by a BCA assay. Columns represent the mean concentration of MHPG + one SEM (n=4 -7). * Differs from PVN (p<0.05 ). 127 Figure 3.7. The ratio of MHPG to NE in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen capsule was isolated and brains were sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of MHPG and NE, which was normalized to the amount of protein per sample as determined by a BCA assay. Col umns represent the mean ration of MHPG to NE + one SEM (n=4 -7). * Differs from PVN (p<0.05). 128 Figure 3. 8. Comparison of VMA concentration s in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen capsule was isolated and brains were sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of VMA whi ch was normalized to the amount of protein per sample as determined by a BCA assay. Columns represent the mean concentration of VMA + one SEM (n=4 -7). * Differs from PVN (p<0.05). 129 Figure 3. 9. The ratio of VMA to NE in the murine PVN and spleen capsule. Immediately following sacrifice by decapitation, the spleens and brains of mice were rapidly removed and frozen on dry ice. The spleen capsule was isolated and brains were sliced at 500 " M and the PVN microdissected . The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of VMA and NE, which was normalized to the amount of protein per sample as determined by a BCA assa y. Columns represent the mean ration of VMA to NE + one SEM (n=4 -7). 130 The synthetic capacity of TH, the rate -limiting enzyme in NE synthesis, can be assessed utilizing an inhibitor of L -AAAD, the enzyme that catalyzes the conversion of DOPA to DA in noradrenergic axon terminals . Normally DOPA is undetectable due to its rapid conversion to DA following its synthesis (Lookingland and Moore, 2005) . Therefore, during inhibiti on of L -AAAD the accumulation of DOPA is a way to assess the in vivo activity of TH. Using this methodology, it was shown that DOPA accumulation in the PVN occurs at a much higher rate than in the spleen capsule during the same time period ( Figure 3.10). This reveals that NE synthe sis in central noradrenergic axon terminals in the PVN is higher than in peripheral autonomic noradrenergic axon terminals in the spleen capsule. This data is in full agreement with the observed MPHG/NE ratio by indicating that PVN noradrenergic neurons terminating in the PVN have a higher metabolic rate than those in the spleen capsule. 131 Figure 3.10. Comparison of DOPA concentration s in the murine PVN and spleen capsule following treatment with NSD -1015. Mice received a single i.p. injection of NSD -1015 (100 mg/kg) and sacrificed 30 min later. Freshly removed mouse spleens were disrupted to isolate the spleen capsule. In the same mice, the brain was rapidly removed and frozen on dry ice. Frozen brains were then sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of DOPA which was normalized to the amount of protein per sample as determined by a BCA assay. Columns represent the mean concen tration of DOPA + one SEM (n=4 ). * Differs from PVN (p<0.05). 132 The rate of NE utilization was also assessed in the PVN and spleen capsule , as an indirect index of NE release from axon terminals. Under normal conditions th ere is a tight coupling between catecholamine synthesis and release whereby newly synthesized NE is preferentially released from neurons before utilization of NE stores (Kopin et al., 1968) . By blocking de novo synthesis of NE using an inhibitor of TH, aMT, and measuring the rate of decline of NE after a specified amount of time , the NE utilization rate, which is directly related to the release of NE, can be measured . Utilizing this methodology th e rate of NE decline following aMT in the PVN was found to be much higher than the spleen under normal, quiescent conditions ( Figure 3.11). The rate of spleen capsule NE utilization was also assessed following the administration of idazoxan, an ! 2AR antagonist. Antagonism of pre -junctional ! 2AR increases periphera l sympathetic neuron activity (Cheng et al., 2000; Khan et al., 2002; Doxey et al., 2012) . In congruence, i dazoxan significantly increased the rate of NE utilization in the spleen capsule ( Figure 3.12). Increased NE utilization in the spleen capsule was accompanied by an increase in the concentration of NE in the spleen capsule (Figure 3.12). 133 Figure 3.11. Comparison of basal NE utilization rate s in the murine PVN and spleen capsule. Mice were injected with sa line/aMT (300 mg/kg, i.p.) and sacrificed 4 h later . Freshly removed mouse spleens were disrupted to isolate the spleen capsule. In the same mice, the brain was rapidly removed and frozen on dry ice. Frozen brains were then sliced at 500 " M and the PVN microdissected. The resulting PVN and spleen capsule samples were then prepared and analyzed via HPLC -ED for the amount of NE which was n ormalized to the amount of protein per sample as determined by a BCA assay. Saline -treated mice were used for the 0 h time point and a linear regression analysis performed to determine the rate of NE utilization (the slope of the line generated). Columns depict the rate of NE utilization (absolute valu e of the slo pe of NE decline) + one SEM (n=14 ). * Differs from PVN (p<0.05). 134 Fig ure 3.12. Idazoxan increases t he rate of NE utilization and NE concentrations in the murine spleen capsule . Mice received a single injection saline/aMT (300 mg/kg, i.p.) immediately followed by saline/idazoxan (4 mg/kg, i.p.) and sacrificed 4 h later . HPLC -ED was used to determine amount of NE in the spleen capsule (A) normalized to the protein per sample. Non -aMT treated mice were used for the 0 h time point and a linear regression analysis performed to determine the rate of NE utilization (the slope of the line generated). Columns depict the rate of NE utilization or NE concentrations + one SEM (n= 15-16). * Differs from saline (p<0.05). 135 3.4: Discussion The data presented here demonstrate differences in noradrenergic innervation between the spleen, a peripheral organ receiving PNS sympathetic efferent axon termina ls, with that of the PVN, a brain nucleus receiving CNS efferent axon terminals . Noradrenergic innervation of the PVN is heterogeneous. In agreement with other investigators, t he data presented in this chapter demonstrate that only dorsal and periventricu lar portions of the parvocellular division receive noradrenergic innervation (Pacak et al., 1995; Chong et al., 2004) . Innervation of the spleen is also heterogeneous , the targets of innervation being the spleen capsule and surroundin g parenchymal arteriolar areas. While the spleen capsule contains more NE, due to it % s relatively large size, the concentration of NE in the spleen capsule is less than that of the PVN. Noradrenergic activity in the spleen capsule is measurable and regulated, at least in part, by !2AR. Interestingly, by comparison, the PVN has much a higher intrinsic rate of metabolism and activity compared to NE axon terminals in the spleen capsule. 3.4.1: Anatomi c differences and consequences i n noradrenergic innervation of the spleen and PVN Noradrenergic terminals in the spleen and PVN can be visualized by TH immunostaining and are confirmed as being noradrenergic by a lack of finding significant amounts of either DA (see Figure s 2.2 and 2.3) or epinephrine (data not shown) in the spleen and PVN by HPLC -ED, despite known adrenergic input to the PVN 136 from the C1, C2, and C3 nuclei (Cunningham et al., 1990) . A major purpose of noradrenergic innervation of the spleen is to contr ol smooth muscle contraction (Eltze, 1996). Thus, it is not surprising to find noradrenergic nerve terminals localized to the spleen capsule. The spleen capsule contains s mooth muscle and contracts in response to a number of stimuli (Eltze, 1996; Cesta, 2006) . It is also not surprising to find noradrenergic nerve terminals near arterioles in the spleen, as it is presumed that sympathetic innervation of these vessels is able to regulate blood flow through the spleen (Groom et al., 1991; Su et al., 1991) . The PALS is a spleen specific tissue surround ing incoming arterioles as they enter the spleen compo sed largely of lymphocytes, T and B cells (Cesta, 2006) . Finding noradrenergic nerve terminals in this lymphocyte rich tissue, as well as reports of NE containing terminals in very close approxima tion to lymphocytes (Felten and Olschowka, 1987), supports the hypothesis that noradrenergic innervation of the spleen has a neuroimmune function. The paucity of noradrenergic nerve terminals , cate cho lamine synthetic machinery (TH) , and NE in the remainder of the spleen parenchyma , including splenocytes, is suggestive of a major ro le for noradrenergic innervation in this compartment of the spleen. Differences between the noradrenergic innervation of the spleen and PVN are reflective of differences in function. Noradrenergic innervation of the PVN has the purpose of regulating other neurons to cause down stream effects, such as participation in the activation of the HPA -axis during stress, hypoglycemia, and inflammation (Pyner, 2009). The PVN is also an upstream nucleus for sympathetic control of the spleen , with 137 the dorsal parvocellular region being one of the major areas in the brain labeled following retrograde tracers injection into the spleen (Cano et al., 2001) . Stimulation of the PVN by glutamate injection is able to stimula te splenic sympathetic activity (Katafuchi et al., 1993) . NE release d in the PVN can stimulate glutamate releasing neurons in the parvocellular regions of the PVN to control sympathetic outflow (Daftary et al., 2000) , thus establishing t he link between NE release in the PVN and splenic sympathetic activity. Noradrenergic innervation of the spleen, on the other hand, is aimed at eliciting end -motor effects in smooth muscle and lymphocytes. Th erefore, innervation distribution is locali zed specifically to those areas, as it is required that axon terminal s end in direct opposition to t he post -neuron target cell. 3.4.2: Noradrenergic neuron activity in the spleen capsule and PVN The basal activity of noradrenergic neurons in the spleen is lower than the PVN. The differences observed between the spleen and PVN in the concentration of NE metabolites suggest NE axon terminals in the PVN are more metabolically active than those in the spleen . Differences in the concentration of these metab olites may simply be due to the size difference between the two regions. The ratio of total metabolite content to total pr imary catecholamine content is more suited for conclusions with regards to metabolic activity (Lookingland et al., 1991) . It is interesting that the VMA/NE ratio was not different between these tissue s, whereas the MHPG/NE rati o was. This difference may be due to differences in the site of NE metabolism . Briefly, NE is converted to DOP EGAL, by MAO, which diffuses out of the neuron to glia cells where is 138 it converted to MHPG via COMT and A DR (Lookingland et al., 1991; Eisenhofer et al., 2004). In the spleen, MHPG is produced by MAO and A DR conversion of NE to DHPG in sympathetic neurons followed by the conversion of DHPG to MPHG in macrophages within the wh ite pulp of the spleen by COMT (Karhunen et al., 1994; Eisenhofer et al., 2004; Myıh− nen et al., 2010) . MHPG is then released from the spleen into the circulation and further metabo lized to VMA , by AD H and AD, in the liver (Oh -hashi et al., 2001; Eisenhofer et al., 2004; Siraskar et al., 2011) . It is interesting that despite the obvious ability of VMA to be produced in the brain, as demonstrated above, MHPG is considered the major metabolite in the brain (Lookingland et al., 1991; Eisenhofer et al., 2004). However , VMA synthesis is mostly dependent upon extra -neuronal conversion of MHPG, whereas MHPG synthesis is largely dependent upon neuronal MAO activity. Therefore, MHPG is likely a better indicator of the metabolic capacity of the neurons. Thus, the large difference in the MHPG/NE ratio is more telling of a reduce d metabolic rate in splenic sympathetic neurons. In agreement with the assessment of the metabolic activity in the PVN and spleen using endogenous neurochemical concentrations, the higher accumulation of DOPA following blockade of NE synthesis with NDS -1015 suggests a faster rate of NE synthesis in axon terminals of the PVN versus the spleen capsule under basal conditions . The rate of NE release was also indirectly assessed in the PVN and spleen . The most direct way to assess the firing activity of any s et of neurons is using in vivo 139 electrophysiological techniques , such as patch -clamp and whole -cell recording (Kandel et al., 2000) . While these techniques provide a great amount of detail (Katafuchi et al., 1993), they have several drawbacks. One of the most salient drawback s is the inability to measuring the activity of two different sites in the body in the same animal (Lee et al., 1992; Helwig et al., 2008) . Another significant drawback is that they are time intensive , typically only one to two specimens can be assessed in a single day . Lastly, the endpoint of electrophysiological studies is the neuronal activity itself, leaving out the possibility to measure physio logic endpoints related to the activity of the released neurotransmitters , especially with single -cell or slice preparations . Alternative to electrophysiological methods is the use of site -specific microdialysis to measure released neurochemicals (Chefer et al., 2009) . It is more plausible to assess multiple sites within a single animal with this technique , however this would require extensive animal surgery and manipulation. Additionally , the size of available probes presents a problem relative to the s ize of the areas being assessed. This is particularly true with reference to the dorsal parvocellular portion of t he PVN , which is on the order of 100 x 50 microns in two dimensional area ( Figure 3.1 ), and commercially available dialysis probes , on the order of 200+ microns in diameter (Olive et al ., 2000; Ortega et al., 2012; CMA Microdialysis) . In this work , pharmacologic technique s were utilized to estimate the rate of neuronal activity. Th e activity rate o f neurons in the PVN and spleen were measured using aMT , an inhibitor of tyrosi ne hydro xylase that prevents de novo NE synthesis (Brodie et al., 1966) . Using this drug , and comparing treated mice with non -treated 140 mice after a set time period , the rate of NE utilization can be assessed. This technique has several advantages to those just mentioned. First, it easy to assess rate s of neuronal NE utilization in multiple tissues within each animal. Second, many animals can be treated and assessed simultaneously. Lastly, the rate of NE uti lization, indicated by the rate c onstant, is generated by comparing values within the same site of sampling , which leaves tissue endpoints in the rest of the body intact . This also allows for more valid comparison between areas with differences in size an d innervation density, such as PVN and spleen. Using this method, the rate of noradrenergic activity in the PVN was found to be much faster tha n that of the spleen . Further, it also demonstrated that increases in activity in the spleen could be induced and measured using the drug Idazoxan , an !2AR antagonist . Much like D2 -dopamine receptors, ! 2AR act on presynaptic neuronal terminals to inhibit the synthesis and release of neurotransmitters (Khan et al., 2002) . Therefore, antagonism of this receptor was expected to increase the synthesis and release of NE , measured as an increase in the rate constant of NE utilization. Interestingly, the concentration of NE in the spleen capsule significantly increased in c onjunction with increased neuronal activity. Normally , the coupling of NE synthesis and release maintains a steady -state amount of stored NE in the axon terminal. However, it appears that in splenic noradrenergic neurons the synthetic capacity of these n eurons out -paces the release o f NE leading to accumulation during activation. Activity dependent increases in the activity of TH is a known phenomenon in catecholaminergic neurons (Kumer and Vrana, 1996) . These changes occur in 141 response to phosphorylation -induced increases in the catalytic -activity of TH (Kumer and Vrana, 1996) . Thus, if the synthetic capacity of TH increases non -proportionally to incr eases in the rate of NE release this will lea d to an accumulation of NE in the spleen capsule . This hypothesis needs further validation, but if true may provide an easily measured , albeit indirect , index of splenic noradrenergic activity. 3.4.3: Conclusion The purpose of this chapter is aimed at understanding the noradrenergic innervation of the spleen as compared to a well -known CNS noradrenergic innervated nucleus. A number of important differences were observed between the PVN and spleen , including differences in the pattern of innerv ation, density of innervation, and rate of activity. The data presented here provide a comprehensive understanding of noradrenergic innervation of the spleen as compared to the PVN . PVN innervation by noradrenergic neurons is heterogeneous and site speci fic, indicating specific functional roles for NE in this nucleus. These observations in the PVN are paralleled in the spleen by finding NE containing terminals almost exclusively reside in the capsule and periarteriolar regions , indicating site -specific f unctions of these neurons , as well . This focuses further investigation to the impact of NE on the function carried out in the se specific regions of the spleen . In addition , reliable methods of measuring splenic noradrenergic neuronal activity , such as aMT methodology, were identified and will be essential in ongoing pursuits to investigate the role of these neurons in the various aspects of spleen function. 142 REFERENCES 143 REFERENCES Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines. J Pharmacol Exp Ther. 1966 Dec;154(3):493 Ð8. Byrum CE, Guyenet PG. Afferent and effere nt connections of the A5 noradrenergic cell group in the rat. J Comp Neurol. 1987 Jul 22;261(4):529 Ð42. Cano G, Sved AF, Rinaman L, Rabin BS, Card JP. Characterization of the central nervous system innervation of the rat spleen using viral transneuronal t racing. J Comp Neurol. 2001 Oct 8;439(1):1 Ð18. Cesta M. Normal Structure, Function, and Histology of the Spleen. Toxicol Pathol. 2006;34(5):455 Ð65. Chefer VI, Thompson AC, Zapata A, Shippenberg TS. Overview of brain microdialysis. Curr Protoc Neurosci. 2 009 Apr;Chapter 7:Unit7.1. PMCID: PMC2953244 Cheng Y, Planta F, Ladure P, Julien C, Barres C. Acute cardiovascular effects of the alpha2 -adrenoceptor antagonist, idazoxan, in rats: influence of the basal sympathetic tone. Journal of Cardiovascular Pharmaco logy. 2000 Jan;35(1):156 Ð63. Chong W, Li LH, Lee K, Lee MH, Park JB, Ryu PD. Subtypes of alpha1 - and alpha2 -adrenoceptors mediating noradrenergic modulation of spontaneous inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus. J. Neuroendocrinol. 2004 May;16(5):450 Ð7. CMA Microdialysis. Microdialysis Probes [Internet]. CMA Microdialysis AB. [cited 2013 Dec 18]. Retrieved from: http://www.microdialysis.com/probe_brochure.pdf?cms_fileid=2772532331977349a4 a942f6d40fafca Cunningham ET, Bohn MC, Sawchenko PE. Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol. 1990 Feb 22;292(4):651 Ð67. Cunningham ET, Sawchenko PE. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol. 1988 Aug 1;274(1):60 Ð76. Daftary SS, Boudaba C, Tasker JG. Noradrenergic regulation of parvocellular neurons in the rat hypothalamic paraventricular nucleus. Neuroscience. 2 000 Mar;96(4):743 Ð51. 144 Doxey JC, Roach AG, Smith CFC. Studies on RX 781094: a selective, potent and specific antagonist of ! 2-adrenoceptors. Br J Pharmacol. 2012 Jul 19;78(3):489 Ð505. Eaton MJ, Lookingland KJ, Moore KE. Effects of the selective dopaminerg ic D2 agonist quinelorane on the activity of dopaminergic and noradrenergic neurons projecting to the diencephalon of the rat. 1994 Feb 1;268(2):645 Ð52. Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. 2004 Sep 1;56(3):331 Ð49. Eltze M. Functional evidence for an alpha 1B -adrenoceptor mediating contraction of the mouse spleen. Eur J Pharmacol. 1996 Sep 12;311(2 -3):187 Ð98. Felten DL, Ackerman KD, Wiegand SJ, Felten SY. Noradr energic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neurosci Res. 1987;18(1):28 Ð36, 118Ð21. Felten SY, Olschowka J. Noradrenergic sympathetic inner vation of the spleen: II. Tyrosine hydroxylase (TH) -positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp. J Neurosci Res. 1987;18(1):37 Ð48. Goldman CK, Marino L, Leibowitz SF. Postsynaptic ! 2-noradrenergic receptors mediate feeding induced by paraventricular nucleus injection of norepinephrine and clonidine. Eur J Pharmacol. 1985 Sep;115(1):11 Ð9. Groom AC, Schmidt EE, MacDonald IC. Microcirculatory pathways and blood flow in spleen: new insights from washout kinetic s, corrosion casts, and quantitative intravital videomicroscopy. Scanning Microsc. 1991 Mar;5(1):159 Ð73Ðdiscussion173 Ð4. Hayley S, Lacosta S, Merali Z, van Rooijen N, Anisman H. Central monoamine and plasma corticosterone changes induced by a bacterial en dotoxin: sensitization and cross -sensitization effects. Eur J Neurosci. 2001 Mar;13(6):1155 Ð65. Helwig BG, Craig RA, Fels RJ, Blecha F, Kenney MJ. Central nervous system administration of interleukin -6 produces splenic sympathoexcitation. Autonomic Neuroscience. 2008 Aug;141(1 -2):104 Ð11. Inoue W, Baimoukhametova DV, Fzesi T, Cusulin JIW, Koblinger K, Whelan PJ, et al. Noradrenaline is a stress -associated metaplastic signal at GABA synapses. Nat. Neurosci. 2013 May;16(5):605 Ð12. Itoi K, Sugimoto N. The brainstem noradrenergic systems in stress, anxiety and depression. J. Neuroendocrinol. 2010 May;22 (5):355 Ð61. 145 Kandel E, Schwartz J, Jessell T. Principles of neural science. 2000. Karhunen T, Tilgmann C, Ulmanen I, Julkunen I, Panula P. Distribution of catechol -O-methyltransferase enzyme in rat tissues. J. Histochem. Cytochem. 1994 Aug;42(8):1079 Ð90. Katafuchi T, Ichijo T, Take S, Hori T. Hypothalamic modulation of splenic natural killer cell activity in rats. J. Physiol. (Lond.). 1993 Nov;471:209 Ð21. PMCID: PMC1143959 Khan ZP, Ferguson CN, Jones RM. Alpha -2 and imidazoline receptor agonistsTheir phar macology and therapeutic role. Anaesthesia. 2002 Apr 6;54(2):146 Ð65. Kopin IJ, Breese GR, Krauss KR, Weise VK. Selective release of newly synthesized norepinephrine from the cat spleen during sympathetic nerve stimulation. J Pharmacol Exp Ther. 1968 Jun;1 61(2):271 Ð8. Kumer SC, Vrana KE. Intricate regulation of tyrosine hydroxylase activity and gene expression. J Neurochem. 1996 Aug 1;67(2):443 Ð62. Lee TH, Ellinwood EH Jr., Einstein G. Intracellular recording from dopamine neurons in the substantia nigra: double labelling for identification of projection site and morphological features. Journal of Neuroscience Methods. 1992 Jul;43(2 -3):119 Ð27. Leibowitz SF, Hor L. Endorphinergic and !-noradrenergic systems in the paraventricular nucleus: Effects on eating behavior. Peptides. 1982 May;3(3):421 Ð8. Levin BE. Reduced norepinephrine turnover in organs and brains of obesity -prone rats | Regulatory, Integrative and Comparative Physiology. É Journal of Physiology -Regulatory. 1995. Lindley SE, Gunnet JW, Lookingl and KJ, Moore KE. 3,4 -Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity. Brain Res. 1990 Jan 1;506(1):133 Ð8. Lookingland KJ, Ireland LM, Gunnet JW, Manzanares J, Tian Y, Moore KE. 3 -Methoxy -4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons. Brain Res. 1991 Sep 13;559(1):82 Ð8. Lookingland KJ, Moore KE. Chap ter VIII Functional neuroanatomy of hypothalamic dopaminergic neuroendocrine systems. Handbook of chemical neuroanatomy. 2005. Myıh−nen TT, Schendzielorz N, M−nnistı PT. Distribution of catechol -O-methyltransferase (COMT) proteins and enzymatic activities in wild -type and soluble COMT deficient mice. J Neurochem. 2010 Mar 31. Noble J, Bailey M. Quantitation of protein. Methods Enzymol. 2009;463:73 Ð95. 146 Oh-hashi Y, Shindo T, Kurihara Y, Imai T, Wang Y, Morita H, et al. Elevated Sympathetic Nervous Activity in Mice Deficient in CGRP. Circ. Res. 2001 Nov 23;89(11):983 Ð90. Olive MF, Mehmert KK, Hodge CW. Microdialysis in the mouse nucleus accumbens: a method for detection of monoamine and amino acid neurotransmitters with simultaneous assessment of locomotor activity. Brain Res. Brain Res. Protoc. 2000 Feb;5(1):16 Ð24. Ortega JE, Katner J, Davis R, Wade M, Nisenbaum L, Nomikos GG, et al. Modulation of neurotransmitter release in orexin/hypocretin -2 receptor knockout mice: a microdialysis study. J Neurosci Res . 2012 Mar;90(3):588 Ð96. Pacak K, Palkovits M, Kopin IJ, Goldstein DS. Stress -Induced Norepinephrine Release in the Hypothalamic Paraventricular Nucleus and Pituitary -Adrenocortical and Sympathoadrenal Activity: In Vivo Microdialysis Studies. Frontiers in É. 1995. Palkovits M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 1973 Sep 14;59:449 Ð50. Pyner S. Neurochemistry of the paraventricular nucleus of the hypothalamus: Implications for cardiovascular regulation. J. Chem. N euroanat. 2009. Samuels ER, Szabadi E. Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation. Curr Neuropharmacol. 2008 Sep;6(3):235 Ð53. PMCID: PMC2687936 Sawchenko PE, Swanson LW. Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol. 1982 Mar 1;205(3):260 Ð72. Schramm LP, S track AM, Platt KB, Loewy AD. Peripheral and central pathways regulating the kidney: a study using pseudorabies virus. Brain Res. 1993 Jul 9;616(1 -2):251 Ð62. Siraskar B, Vılkl J, Ahmed MSE, Hierlmeier M, Gu S, Schmid E, et al. Enhanced catecholamine release in mice expressing PKB/SGK -resistant GSK3. Pflugers Arch. 2011 Dec;462(6):811 Ð9. Strack AM, Sawyer WB, Hughes JH, Platt KB, Loewy AD. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res. 1989 Jul 3;491(1):156 Ð62. Su CY, Tsai AI, Liu HP, Chen LT, Chien S. Ultrastructural studies on splenic microcirculation of the rat. Chin J P hysiol. 1991;34(2):223 Ð34. 147 Sved AF, Cano G, Card JP. Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin Exp Pharmacol Physiol. 2001;28(1 -2):115 Ð9. 148 Chapter 4: The Contribution of Norepinephrine to Humoral Immune Responses in the Spleen 4.1: Introduction The immune system produces highly complex, multi -cellular, coordinated responses that act to protect organisms from harmful infectious disease s and cancer. The presence of foreign particles, pathogens, or tumor cells illicit distinct actions with their ow n particular set of cells and signaling molecules. In general there are two broad divisions of the immune system: innate and adaptive. The innate immune response is an intrinsic system that is ever present and provides an early and rapid defense against microbes (Oikono mopoulou et al., 2001; Quah and Parish, 2001) . The innate immune system does not confer long -term immunity to a pathogen , but can facilitate adaptive immune system respon ses (Meager and Wadhwa, 2001) . The adaptive immune system is activated when an anti gen is recognized by epitope specific receptors on B cells and T cells (P Kane, 2001) . BCRs recognize a vast array of antigens such as proteins, polysaccharides, lipids, nucleic acids, and soluble antigens including small chemicals, whereas TCRs only recognize peptide fragments presented by host cells in the MHC (Ferrero et al., 2001; Saha, 2001; Kurosaki and Hikida) . The activation of these receptors results in highly specific responses centered around the rec ogni tion of specific epitope s on the inciting antigen. 149 T cells implement cell -mediated adaptive immune responses. CD8 + cells, or cytotoxic T cells, aid in the removal of cells infected with intracellular pathogens, such as viruses and intracellular bacteria (Gotch, 2001; Nath, 2001) . CD4 + T cells, or TH cells , aid in the removal of extra -cellular pathogens by macrophages (Ademokun and Dunn -Walters, 2001; Ferrero et al., 2001) . Important TH cell subtypes include the T H1 subtype , (involved in the sti mulation of macrophages to more effectively phagocytize and lyse bacteria ), the TH2 subtype (involved in the immune response against helminthes and parasites ), and follicular TH cell s ( antigen experienced TH cells that reside within B -cell follicles of sec ondary lymphoid organs and assist in the production of antibodies ) (Ferrero et al., 2001; Fazil leau et al., 2009) . T cells also participate in humoral immune responses. Many T H subtypes assist B cells to produce antibodies (Ademokun and Dunn -Walters, 2001; Ferrero et al., 2001) . Once activated by exposure to an extracellular antigen, TH cell s express CD154 on their cell surface and release cytokines (i.e. interferon -!, IL-2, and IL-4) in response to interaction with CD40 expressing B cells recogniz ing the same antigen (Ademokun and Dunn -Walters, 2001; N”ron et al., 2011) . Antigen specific interaction between B cells and T H cells, including CD40 -CD154 interaction and cytokine stimulation, induce B cells to produce antibodies directed against the commonly recognized antigen (N”ron et al., 2011). 150 The isotype of antibody , determined by the constant portion of the heavy chain, plays a major role in determining the function of the ant ibody . This portion is the site recognized by receptors throughout the body, such as by F c receptors on macrophages or by complement (Da‚ron, 1997; Kenter, 2005; Stavnezer et al., 2008) . IgM is the first type of antibody produced in response to a novel antigen (Ademokun and Dunn -Walters, 2001; Czajkowsky and Shao, 2009) . Additionally, low specificity IgM , " natural antibodies #, are constitutively produce d in the spleen and bone marrow as a form of innate -type immunity (Czajkowsky and Shao, 2009; Baumgarth, 2013) . The effector mechanism of IgM is through complement activa tion (Czajkowsky and Shao, 2009; Baumgarth, 2013) . Over the co urse of an immune response the isotype of antibody changes from IgM to IgG (Ademokun and Dunn -Walte rs, 2001; Schroeder and Cavacini, 2010) . IgG is a monomeric Ig with a high specificity, but relatively low capacity and is the predominant antibody type produced several days (3 -4) following an initial immune exposure and during repeated exposure to the same antigen (Ademokun and Dunn -Walters, 2001; Schroeder and Cavacini, 2010) . Effector mechani sms of IgG include complement activation, neutralization of toxins, and facilitation of phagocytosis (Da‚ron, 1997; Schroeder and Cavacini, 2010) . Certain types of antigens stimulate a humoral response, but do not require T cells. These T cell independent antigens stimulate B cells to produce antibodies by replacing the secondary stimul ation provided by T H cells (Ademokun and Dunn -Walters, 2001) . These secondary signals come from either cross linking of BCRs or stimulation of TLR 151 that recognize specific pathogen associated molecules. A prime example of this type of antigen is bacterial LP S (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . Upon release , LPS can be recognized by TLR4 receptors on the surface of B cells (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . In addition, because of its large size and repeating subunits, LPS is simultaneously recognized by many BCRs on the same lymphocyte (Moller, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . The combination of these features serve to stimulate B cell antibody produc tion in the absence of T H cell involvement (Moller, 2001) . Interactions between the immune and nervous systems are coordinated with in the spleen. Sympathetic post -ganglionic axon terminal NE activit y increases in the spleen following the injection of soluble protein antigens (Kohm et al., 2000; Sanders, 2012). These neurons have synaptic -like terminations in very close proximity (less than 8 microns) to immune cells in the spleen (Felten and O lschowka, 1987) . Sympathetic axon terminals, visualized by immunostaining for the rate -limiting enzyme tyrosine hydroxylase (TH), are found in the PALS , which is composed of T cells, B cells, and macrophages (Felten et al., 1987; Felten and Olschowka, 1987; Cesta, 2006) . This distribution is confirmed in Chapter 3 by demonstrating that NE and TH exist in high concentratio ns within the splenic capsule , but are almost absent in splenocyte preparations . Splenic B cells express functional $ 2AR and respond to NE both in vitro and in vivo (Nance and Sanders, 2007; Sanders, 2012) . Engagement of NE with $ 2AR on B cells within 24 h of an antigen exposure increase s the amount of secreted 152 antibodies in response to a humoral immune challenge (Sanders, 2012) . This effect is mediated by regulating the t ranscriptional activity of the 3' -IgH (Stevens et al., 2000; Pinaud et al., 2001; Vincent -Fabert et al., 2010) . Activation of $ 2AR on B cells, through a protein -kinase A dependent mechanism, up -regulates 3' -IgH transcription leading to increased antibody production (Podojil et al., 2004) . Interestingly , o nly exposure to NE at early time points (<24 h) following antigen exposure increase s B cell antibody production (Kohm and Sanders, 2000; 2001) . The c lose proximity of sympathetic neurons to immune cells in the spleen along with the research showing NE can increase antibody production has led to the hypothesis that splenic sympathetic neurons are a source of immunomodulatory NE. Extended, it is believed that sympathetic nervous system activation during immune responses causes the release of NE from axon terminals in the spleen , which acts upon B cells expressing $2AR resulting in enhanced antibody production. The purpose of the research described in this chapter is to test this hypothesis. First, the correlation between splenic sympathetic noradrenergic neuronal activity and humoral immunity will be established. The expression of !2AR on splenic B cells will then be confirmed, thereby providing a mechan ism by which these lymphocytes can sense NE. Finally, the effect of NE and !2AR stimulation will be tested with specific regards to the humoral immune response of splenic B cells. 153 4.2: Materials and Methods 4.2.1: Mice C57BL/6 WT female mice (NCI/Charles River, Portage, MI) obtained from were used in all experiment unless otherwise indicated. All animals were housed two to five per cage and maintained in a sterile, temperature (22 ± 1 ¡C) and light controlled (12L:12D) room, and provided with irradiated food and bottled tap water ad libitum. All experiments used the minimal number of animals required for statistical analyses, minimized suffering, and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Labo ratory Animals. The Michigan State Institutional Animal Care and Use Committee approved all drug administrations and methods of euthanasia (AUF# 03/12%060%00). 4.2.2: Materials aMT: aMT ester (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline t o a final concentration of 30 mg/ml and administered at dose of 300 mg/kg. Butoxamine: Butoxamine (B1385, Sigma) was dissolved in sterile isotonic saline at a concentration of 5 mg/ml and administered at doses ranging from 1 -10 mg/kg (i.p.). HBSS: 10x HBSS powder (Gibco) was diluted with ultra -pure H 2O (NaCl 138 mM, KCl 5.3 mM, Na 2HPO 4 0.3 mM, NaHCO 3 4.2 mM, KH 2PO4 0.4 mM, and glucose 5.6 mM), autoclaved, and stored at 4 ¡ C. 154 Isotonic Saline: one L of 0.9% saline was prepared using ultra -pure H 2O and 9 grams of NaCl. The solution was autoclaved and kept closed at room temperature. LPS: LPS ( E. coli 055:B5 catalog L2880, lot 066K4096, 5 EU/ng (Limulus lysate assay) and 10 EU/ng (chromogenic assay) , Sigma , St. Louis, MO) was dissolved in RPMI -1640 at us ed at a final concentration of 10 &g/ml for in vitro studies. For in vivo experiments, LPS was dissolved in HBSS to a concentration of 50 &g/ml and injected at 25 & g per mouse (i.p.) . PBS: NaCl 137 mM, KCl 2.7 mM, Na 2HPO 4 10 mM, and KH 2PO4 1.8 mM in ultra -pure H2O. sRBC: An aliquot of sRBC was placed in a 50 -ml conical tube. 25 ml of HBSS was added to the sRBC and centrifuged at 300 RCF for 5 min. The supernatant was removed from the concentrated sRBC pellet. This process was performed 3 additional times. sRBC were then counted and adjusted to 2 x 10 9 cells/ml in HBSS. In experiments using sRBC, mice received 1x 10 9 cells via a single i.p. injection. 155 4.2.3: Isolation of the Spleen Capsule and Splenocytes After euthanasia spleens were removed by an incision in the left lateral abdomen under sterile conditions, which entails spraying the area of removal with 70% ethanol and using ethanol cleaned scissors and forceps to cut through the skin, and underlying muscle and connect ive tissue . The spleen was placed in a 6 -well plate and mechanically crushed with the blunt end of a 10 ml syringe in 2 ml of HBSS to separate the spleen capsule (insoluble tissue) from the splenocytes (contained in the disruption supernatant). The splee n capsule was removed from the supernatant using forceps and taken whole or divided into two parts using ethanol -cleaned scissors depending on the needs of the experiment Splenocytes were separated from the disruption supernatant by centrifugation at 300 R CF for 5 min and the supernatant was decanted . The separated splenocytes were then re-suspended in differing buffers and taken whole or divided into two parts depending on the needs of the experiment . 4.2.4: Preparation and Culture of Splenocytes Two mice were killed by decapitation and their spleens removed aseptically via a single ethanol incision in the left flank, which was wetted with 70% ethanol, using ethanol -cleaned scissors . Single -cell suspensions were prepared by disrupting the spleen capsul e with the blunt end of a sterile disposable 5 -ml syringe in a 6 -well plate in ~2 ml of RPMI -1640 media. The isolated splenocytes were then cultured in RPM I-1640 media supplemented with BCS (percentage of BCS dependent on length of culture and assay; Hyclo ne, Logan, UT, USA), 100 units penicillin/ml (Gibco), 100 &g 156 streptomycin/ml (Gibco), and 50 &M 2 -ME (Gibco). Splenocytes were cultured in a humidified atmosphere at 37 ¡C and 5% CO 2. 4.2.5: Neurochemistry All samples were placed in ice -cold tissue buffer following isolation or dissection and kept frozen at -80¡C until analysis. Samples were thawed on the day of analysis and sonicated with 3 one -sec bursts (Sonicator Cell Disruptor, Heat Systems -Ultrasonic, Plainview, NY, USA) and centrifuged at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge. The supernatant from the first centrifugation of the spleen capsule was removed and spun again at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge before being brought up to 100 & l (q.s.) with fresh cold tissue buffer. Spleen samples were then filtered using a 0.2 & M syringe driven Millex -LG filter (Millipore, Billerica, MA). All samples were analyzed for NE , MHPG, VMA, and/or DOPA content using HPLC -ED (Lindley et al., 1990; Eaton et al., 1994) using C18 reverse phase columns (ESA Inc., Sunnyvale, CA ) combined with a low pH buffered mobi le phase (0 .05 M Sodium Phosphate, 0.03 M Citrate, 0.1 mM EDTA at a pH of 2.65) composed of 5 -15% methanol and 0.03 -0.05% sodium octyl sulfate. Oxidation of monoamines was measured at a constant potential of -0.4 V by coulometric detection using a Couloch em Electrochemical Detector (Thermo Scientific). The amount of each substance in the samples was determined by comparing peak height values (as determined by a Hewlett Packard Integrator, Model 3395) with those obtained from known standards run on the 157 same day. Tissue pellets remaining from preparation were dissolved in 1 N NaOH and assayed for protein using the BCA method (Noble and Bailey, 2009) . 4.2.6: Western Blot All samples were placed in ice -cold lysis buffer (water containing 1% Triton -x 100, 250 mM sucrose, 50 mM NaCl, 20 mM tris -HCl, 1 mM EDTA, 1 mM PMSF protease inhibitor cocktail, 1 mM DTT) immediately following isolation and kept frozen at -80¡C until analysis. On the day of analysis samples were thawed, heated for 30 min at 100¡ C, sonicated for 8 sec, a nd spun at 12,000 RCF for 5 min. The supernatant was collected and a BCA protein assay performed (Noble and Bailey, 2009) . Equal amounts of protein were separated using SDS -PAGE and transferred to PVDF -FL membranes (Millipore, Billerica, MA). The resulting membranes were reacted against antibodies for TH (AB152 1:2000, Millipore, Billerica, MA) , whose intensities were normalized to !-Actin (8H10D10 1:8000, Cell Signaling, Danvers, MA) to account for loading variability. Each PVDF -FL membrane contained samples represe nting all experimental conditions to avoid variability due to run, transfer, or antibody exposure conditions. Blots were visualized and quantified using an Odyssey Fc Infrared Imaging system (Li -Cor, Lincoln, NE) by utilization of IRDye conjugated seconda ry antibodies, goat anti -Mouse 800CW (1:20,000) and/or goat anti -rabbit 680LT (1:20,000). 158 4.2.7: Flow Cytometry 4.2.7.1: Surface antibody labeling for flow cytometry All staining protocols were performed in 96 -well round bottom plates (BD Falcon, Franklin Lakes, NJ). Splenocytes were washed 3x with HBSS by centrifugation at 1000 RCF for 5 min, the supernatant was decanted , and the cells re -suspen ded in HBSS. Cultured splenocytes were then incubated for 30 min on ice in the dark in a 1x solution of near IR (APY -Cy7) live/dead stain (#L10119, Invitrogen, Grand Island, NY), a step which was omitted for splenocytes obtained directly from spleens. Following a wash in HBSS (as described above), splenocytes were then washed with FACS buffer (HBSS, 1% bov ine serum albumin, 0.1% sodium azide, pH 7.6) as was done with HBSS. Surface Fc receptors were then blocked with anti -mouse CD16/CD32 [0.5 mg/ml] (#553142, BD Biosciences, Franklin Lakes, NJ) at 0.5 & l/well, IgM was blocked with anti -IgM [0.5 mg/ml] (#5534 25, BD Biosciences) at 1 & l/well, and IgG was blocked with anti -IgG [1.3 mg/ml] (#115 -006-071, Jackson Immunoresearch, West Grove, PA) at 0.5 & l/well for 15 min each at RT. Cells were stained for 30 min at RT with the following antibody clones: CD19 (clone 6D5) [0.2 mg/ml] (Biolegend, San Diego, CA) at 1.25 & l/well and $ 2AR [0.25 mg/ml] (#AP7263d, Abgent, San Diego, CA) at 2 & l/ well. Cells were then washed 3x with FACS buffer. A secondary antibody for $ 2AR, donkey anti -rabbit DyLight 649 (clone Poly4064) [0.5 mg/ml] (Biolegend), at 0.5 & l/well was incubated for 30 min at RT. Subsequently cells were washed with FACS buffer, fixed with Cytofix (BD Biosciences) for 15 min at RT, washed 3x with FACS buffer, and finally 159 suspended in FACS buffer for intracellula r staining. Stained and fixed cells were stored in the dark at 4 ¡C for up to 2 weeks. 4.2.7.2: Intracellular antibody labeling for flow cytometry Within 2 weeks of surface staining (described above), cells were washed 2x with Perm/Wash (BD) and incubated with Perm/Wash for 30 min at RT. Fluorescently labeled antibodies for IgM (Clone II/41) [0.5 mg/mL] (Biolegend) were added at 1 & l/well for 30 min. Cells were washed 2x with Perm/Wash and suspended in FACS buffer. After intracellular staining, cells were analyzed the same day. 4.2.7.3: Flow Cytometry Analysis Fluorescent staining was analyzed using a BD Biosciences FACSCanto II flow cytometer. Data were analyzed using Kaluza (Beckman Coulter Inc., Brea, CA) or FlowJo software (Tree Star Inc., Ashland, OR). Compensation and voltage settings of fluorescent parameters were performed using fluorescence -minus -one controls. Cells were gated on singlets (forward scatter height versus area) followed by determination of live cells (low APC -Cy7 signal) only in samples obtained from splenocyte culture. Cells were then gated to select lymphocytes using forward versus side scatter. For some analyses, an addit ional gate was created for CD19 or $2AR expression to select for B cells or $2AR expressing cells, respectively . These sequential gates were used to identify IgM producing B cells and IgM producing B cells that express $ 2AR. The percentage of cells gated to individual populations relative to the entire population were 160 collected and analyzed. Additionally, the numerical intensity of the fluorescent signal, termed the MFI, was also quant ified and analyzed. 4.2.8: ELISA Serum IgM was detected by sandwich ELISA. In preparation, 100 & l of 1 & g/ml anti -mouse IgM (Sigma -Aldrich, St. Louis, MO) was added to wells of a 96 -well microtiter plate and stored at 4 ¡C overnight. After the pre -coating step, the plate was washed twice with 0.05% Tween -20 in PBS and three times with H 2O. Following this, 200 &l of 3% BSA -PBS was added to the wells and incubated at RT for 1.5 h to block nonspecific binding followed by the same washing steps described above. Serum samples were diluted and added to the coated plate (100 & l) for 1.5 h at RT. After the incubation, the plate was washed again, followed by addition of 100 & l of HRP -conjugated goat anti -mouse IgM (A8786, Sigma -Aldrich). Following the HRP incubation for 1.5 h at RT, any unbound detection antibody was washed away from the plate, and 100 & l ABTS (Roche Applied Science, Indianapolis, IN) added. The detection of the HRP substrate reaction was conducted over a one h period using a plate reader with a 405-nm filter (Bio -Tek). The KC4 computer analysis program (Bio -Tek) calculated the concentration of IgM in each sample based on a standard curve generated from the absorbance readings of known concentrations of IgM (range 6 -1600 pg/ml, clone TEPC 183, Sigma -Aldrich) . 161 4.2.9: ELISPOT ELISPOT was performed as described previously (Lu et al., 2009) . ELISPOT wells were coated with purified anti -mouse (Sigma -Aldrich) IgM antibody and blocked with 5% BSA. Splenocytes from freshly disrupted spleens were washed, via centrifugation as described for flo w cytometry, and incubated in the ELISPOT wells for 16 Ð20 h. Biotin -conjugated anti -mouse IgM antibody (Sigma -Aldrich, St. Louis, MO) and streptavidin -horseradish peroxidase (Sigma -Aldrich, St. Louis, MO) were sequentially added to the wells. The spots wer e developed with the aminoethylcarbazole staining kit (Sigma -Aldrich, St. Louis, MO). Data were collected and analyzed using the CTL ImmunoSpot system (Cellular Technology Ltd, Shaker Heights, OH). 4.2.10: Statistical Analysis 4.2.10.1: Statistical Compar isons Prism software version 4.0a was used to make statistical comparisons between groups using the appropriate statistical test. Differences with a probability of error of less than 5% (p<0.05) were considered statistically significant. Two group compari sons were done using the Student # s t-test. Two group comparisons where in one group had more than one degree or factor were done using a One -way ANOVA followed by a Bonferroni or Tukey # s post -test for multiple comparisons. Experiments in which there were two groups with more than one degree or factor in each group, such as a 2x2 design, were analyzed using a Two -way ANOVA followed by Bonferroni post -test for multiple comparisons. 162 4.2.10.2: aMT Experimentation In experiments in which aMT was used to assess neuronal activity, NE concentrations from aMT treated and non -aMT (saline) treated mice were used for a regression analysis with saline animals acting as the 0 time control and aMT animals a 4 h time point. Th e rate constant was determined using this formula: =(Log 10[B] -Log10[A])/( -0.434*t). Where B is the concentration of NE after aMT, A is the concentration of A in saline treated animals, and t is the time of aMT treatment (Brodie et al., 1966) . The slopes were compared via t -test using the mean slope, SEM, and an n equal to the total number of data points used in the analysis to account for the total number of independent measurements used to generate the regression eq uations. Differences with a probability of error of less than 5% were considered statistically significant . 4.2.10.3: Flow Cytometry Data Handling Population percentage data was transformed in Excel (Microsoft Corporation, Redmond, WA) to a parametric fo rm before ANOVA analysis using the formula: =arcsin(sqrt(DATA/100)) (Ahrens et al., 1990) . Raw percentage data was used for visual representations, while statistical significance indicated on these figures was performed on the transformed data. 163 4.3: Results 4.3.1: Characterization of humoral immune challenge models Initial experiments sought to determine the efficacy of sRBC and LPS to induce a humoral immune response in vivo. While neither sRBC nor LPS significantly changed the body weight of mice 4 days after injection ( Figure 4.1 ), both immunogens were able to i ncrease the weight of the spleen and the spleen:body weight ratio ( Figures 4.2 and 4.3). S pleens from LPS treated mice were found to be heavier than those from sRBC treated mice ( Figures 4.2 and 4.3 ). The humoral immune response of lymphocytes contained within the spleen was assessed using ELISPOT specific for IgM. Injection of either immunogen significantly increased the number of splenic IgM producing lymphocytes (Figure 4.4 ). Next the ability of LPS to induce a humoral immune response in vitro was ev aluated using s plenocytes obtained from untreated mice. Addition of LPS to the culture medium for 3 days increase d the number of IgM producing B cells, indicated by surface expression of CD19 (CD19 +) as measured by flow cytometry ( Figure 4.5A ). The signi ficant increase in IgM producing B cells in response to LPS in vitro was accompanied by a decrease in the MFI of IgM suggesting an overall decrease in the amount of IgM being produced per B cell ( Figure 4.5B ). These data establish both sRBC and LPS as immu nogens able to induce a humor al immune response in vivo (sRBC and LPS) and in vitro (LPS). 164 Figure 4.1. The effect to experimental immune challenges on body weight in mice. Female mice were injected with HBSS, sRBC (1x10 9 cells; i.p.), or LPS (25 µg; i.p .) and weighed 4 days later. Columns represent the average weight (g) + one SEM (n=3). 165 Figure 4.2. The effect to experimental immune challenges on spleen weight in mice. Female mice were injected with HBSS, sRBC (1x10 9 cells; i.p.), or LPS (25 µg; i.p.) and sacrificed 4 days later. The spleen was rapidly and aseptically removed and weighed. Columns represent the average weight (g) + one SEM (n=3). * Differs from HBSS (p<0.05). # Differs from sRBC (p<0.05). 166 Figure 4.3. The effect to experi mental immune challenges on the spleen:body weight ratio in mice. Female mice were injected with HBSS, sRBC (1x10 9 cells; i.p.), or LPS (25 µg; i.p.). Four days later the mice were sacrificed, weighed, and the spleen was weighed following rapid aseptic rem oval. Columns represent the average spleen:body weight ratio + one SEM (n=3). * Differs from HBSS (p<0.05). # Differs from sRBC (p<0.05). 167 Figure 4.4. IgM production from splenocytes from mice subject to experimental immune challenges. Female mice were injected with sRBC (1x10 9 cells; i.p.), LPS (25 µg; i.p.), or vehicle (HBSS). Four days later the mice were sacrificed and the spleen was rapidly and aseptically removed. The splenocytes were isolated and subject to ELISPOT detection of IgM productio n. The data presented in this graph are derived from separate experiment s: one in which sRBC or its vehicle was injected into mice , and another using LPS or its vehicle . Columns represent the spots generated per 10 6 cells + one SEM (n=3). * Differs from H BSS (p<0.05). 168 Figure 4.5. Analysis of IgM a ntibody production responses in splenic B cells from mice exposed to LPS in vitro. Isolated splenocytes were cultured in the presence of LPS (10 & g/ml), or vehicle (RPMI) , for 3 days. On the 3rd day flow cytometry was used to detect CD19 (PE -Cy7) and IgM (FITC) on splenocytes . Cells were gated sequentially on singlet live lymphocytes, prior to plotting CD19 and IgM for analysis. Uncultured, freshly isolated splenocytes are designated as Day 0. All columns represent means + 1 SEM (n=3). Columns depict the percent of lymphocytes (A) or MFI of IgM (B) in lymphocytes expressing CD19 and IgM. Results are representative of at least 2 separate experiments. * Significantly differs from Day 0 (p<0.05). # Significant ly differs from Day 3 NA (p<0.05). 169 4.3.2: Splenic sympathetic neuronal activity in response to humoral immune challenge models Next, the activity of the sympathetic noradrenergic neurons in the spleen was assessed following T cell dependent (sRBC) and T cell independent (LPS) immune challenges in vivo . Neuroimmune interactions were hypothesized to occur within hours following an immune challenge; therefore the activity of splenic sympathetic noradrenergic neurons was evaluated between 90 min and 2.5 h fol lowing the injection of immunogens. Injection of sRBC increase d the concentration of spleen capsule NE and splenic TH content within 90 min ( Figures 4.6 and 4.7 ). The measured rate constant of NE utilization in the spleen following aMT administration wa s significantly elevated following the injection of sRBC ( Figure 4.8 ). Injectio n of LPS was also able to increase the activity of splenic sympathetic noradrenergic neurons. Within 2.5 h of LPS injection spleen capsule NE concentrations and splenic TH cont ent were increased (Figures 4.9 and 4.10 ). The rate of NE utilization was also increased in response to LPS ( Figure 4.11 ). T he data presented here demonstrate a correlation between splenic sympathetic neuronal activity and the initiation of a humoral imm une response. The data here also suggest that increases in sympathetic neuronal activity in response to immunogens eliciting a humoral immune response are not dependent on the involvement of T cells; the activity of splenic sympathetic noradrenergic innervation increased in response to both sRBC and LPS. 170 Figure 4.6. Spleen capsule NE concentrations in response to injection of sRBC. Female mice were injected with HBSS or sRBC (1x10 9 cells; i.p.). Ninety minutes later the mice were sacrificed a nd the spleen capsule was collected. Spleen capsule samples were prepared for and analyzed by HPLC -ED for NE as described. Columns represent average concentration of NE + one SEM (n=6 -8). * Differs from HBSS (p<0.05). 171 Figure 4.7. Spleen capsule TH content in response to injection of sRBC. Female mice were injected with HBSS or sRBC (1x10 9 cells; i.p.). Ninety minutes later the mice were sacrificed and the spleen capsule was collected. Spleen capsule samples were prepa red for and analyzed by Western blot for TH as described in the Methods (Section 4.2.6). Columns represent the average amount of TH , normalized to !-actin, in the spleen + one SEM (n=6 -8). * Differs from HBSS (p<0.05). 172 Figure 4.8. Spleen capsule n oradrenergic neuron activity in response to injection of sRBC. Female mice received a single injection saline/aMT (300 mg/kg, i.p.) followed 2.5 hours later by single injection of HBSS or sRBC (1x10 9 cells; i.p.). Mice were sacrificed 90 min after the s RBC injection and the spleen capsule collected and prepared analysis of NE by HPLC -ED. Non -aMT treated mice were used for the 0 h time point and a linear regression analysis performed to determine the rate of NE utilization. Columns depict the average rat e constant of NE utilization + one SEM (n=15 -16). * Differs from HBSS (p<0.05). 173 Figure 4.9. Spleen capsule NE concentrations in response to injection of LPS. Female mice were injected with HBSS or LPS (25 µg; i.p.). After 2.5 h the mice were sacrifi ced and the spleen capsule was collected. Spleen capsule samples were prepared for and analyzed by HPLC -ED for NE as described. Columns represent average concentration of NE + one SEM (n=6 -8). * Differs from HBSS (p<0.05). 174 Figure 4.10. Spleen capsule TH content in response to injection of LPS. Female mice were injected with HBSS or LPS (25 µg; i.p.). After 2.5 h the mice were sacrificed and the spleen capsule was collected. Spleen capsule samples were prepared for and analyzed by Western blot for TH as described in the Methods (Section 4.2.6) . Columns represent the average amount of TH , normalized to !-actin, in the spleen + one SEM (n=6 -8). * Differs from HBSS (p<0.05). 175 Figure 4.11. Spleen capsule noradrenergic neuron activity in response to injection of LPS. Female mice received a single injection saline/aMT (300 mg/kg, i.p.) immediately followed by single injection of HBSS or LPS (25 µg; i.p.). Mice were sacrificed 4 h after the LPS injection and the spleen capsule collected and prepared analysis of NE by HPLC -ED. Non -aMT treated mice were used for the 0 h time point and a linear regression analysis performed to determine the rate of NE utilization. Columns depict th e average rate constant of NE utilization + one SEM (n=15 -16). * Differs from HBSS (p<0.05). 176 4.3.3: !2AR expression on splenic lymphocytes The correlation between humoral immune responses and splenic sympathetic noradrenergic activation suggests these two physiologic processes are dependent upon one another . For such a link to exist, it was hypothesized that components of the humoral immune response would be able to recognize signals from the sympathetic nervous system, including activation of adrenergic receptors. This hypothesis was tested by evaluation of !2AR expression on lymphocytes, as there is precedence for this in the literature (Sanders, 2012) . The surface expression of !2AR was assessed among lymphocytes, T cells (CD3 +) and B cells (CD19 +). Both T cells and B cells were found to express !2AR (Figure 4.12 ), however the expression of !2AR in B cells (~20%) is nearly double than that of T cells (~10%) . Within the B cells , nea rly all of the cells expressing !2AR also express ed MHC type II , which is involved in the antigen presenting abilities of B cells (Figure 4.13 ). These data suggest B cell s are the likely targets for NE -mediated effects of the sympathetic nervous system on humor al immunity. Next , the expression of !2AR on B cells was assessed in response to LPS in vitro . In vitro LPS stimulation of splenocytes not only increased the percentage of B cells expressing !2AR over a 3 day period, but also increased the number of receptors expressed per cell as indicated by an elevation of the !2AR MFI in B cells (Figure 4.14 ). It was hypothesized that if !2AR stimulation were to have a direct effect on humoral immunity , then B cells expressing !2AR should be able to produce immunoglobulins in response to an immune challenge. In congruence with this hypothesis, !2AR expressing B cells were able to produce IgM 177 (Figure 4.15A ), expand in response to in vitro LPS over 3 days (Figure 4.15A ), and incre ase the number of !2AR expressed per cell ( Figure 4.15 B). 178 Figure 4.12 . Populations of splenic lymphocytes expressing ! 2AR. Lymphocytes were isolated from spleens of untreated mice and analyzed by flow cytometry as described . Cells were gated on singlet lymphocytes prior to plotting the depicted markers. Numbers in the plot are the percent of events occurring in the respective quadrant. (A) B cell s ( $ 2AR -APC versus CD19 -PE-Cy7); (B) T cell s ( $ 2AR -APC versus CD3 -APC -Cy7). 176 179 Figure 4.13 . Correlation between antigen presenting lymphocytes in the spleen and !2AR expression . Lymphocytes were isolated from spleens of untreated mice and analyzed by flow cytometry as described . Cells were gated on singlet lymphocytes prior to plottin g the depicted markers. Numbers in the plot are the percent of events occurring in the respective quadrant. (A) antigen -presenting B cell s (MH CII -FITC versus CD19 -PE-Cy7); (B ) $2AR expressing antigen -presenting cells ( $ 2AR -APC versus MHCII -FITC) 177 180 Figure 4.14. The response of !2AR expressing splenic B cells to in vitro LPS administration. Isolated splenocytes were cultured with LPS or its vehicle (RPMI) 3 days. Flow cytometry was used to assess surface $ 2AR and CD19 expression on lymphocytes on days 2 and 3 of culture as well as immediately after isolation (Day 0). Bars represent the average percentage of B cells expressing $ 2AR (A) or the MFI of $ 2AR on B cells (B) + one SEM. * Differs from Day 0 of the same treatment group (p<0.05); # Differs from the same time point in the Vehicle treated group (p<0.05); ^ Differs from Day 2 in the same treatment group (p<0.05). 181 Figure 4.15. The IgM response of B2AR expressing splenic B cells to in vitro LPS administration. Isolated splenocytes were cultured wit h LPS or its vehicle (RPMI) 3 days. Flow cytometry was used to assess surface $ 2AR and CD19 expression and intracellular IgM in lymphocytes on days 2 and 3 of culture as well as immediately after isolation (Day 0). Bars represent the average percentage of IgM producing B cells expressing $ 2AR (A) or the MFI of IgM in the same cells (B) + one SEM. * Differs from Day 0 of the same treatment group (p<0.05); # Differs from the same time point in the Vehicle treated group (p<0.05); ^ Differs from Day 2 in the sa me treatment group (p<0.05). 182 4.3.4: The effect of !2AR stimulation on humoral immune responses in splenic lymphocytes The presence of !2AR on B cells that produce IgM , coupled with the expansion of !2AR expressing cells in response to LPS , suggest NE i s able to exert a direct effect in these cells. Work in this arena suggest s NE stimulation of !2AR increase s antibody production in response to immune challenges (Sanders, 2012) . Therefore it was hypothesized NE would enhance the humoral immune response of cultured splenocytes to LPS. However, NE was not found to alter the proportion of IgM producing B cells , nor the amount of IgM per cell, at the concentrations tested ( Figure 4.16 ). Furthermore, NE did not change the proportion of IgM producing B cells expressing !2AR, nor the MFI for IgM in these cells ( Figure 4.17 ). To further test the effect of NE stimulation of !2AR on humoral immunity, immune challenged mice were treated with butoxamine, a !2AR antagonist, for 24 h before and after LPS. LPS significantly increase d the proportion of splenic IgM producing B cell s and the amount of IgM per B cell ( Figure 4.18 ). This is congruent with the effect s observed during in vitro administration of LPS. Butoxamine did not change the proportion of IgM producing B cells nor the amount of IgM per B cell (Figure 4.18 ). Interestingly, LPS d id not expand the population of IgM producing B cells expressing !2AR ( Figure 4.19A ), but increase d the amount of IgM per cell of this population ( Figure 4.19B ). Butoxamine also ha d no effect on the population of IgM producing B cells expressing !2AR ( Figure 4.19 ). 183 Figure 4.16. The effect of NE on the IgM response of splenic B cells to in vitro LPS administration. Isolated splenocytes were cultured with LPS or its vehicle (Veh) for 3 days. Flow cytometry was used to assess surface CD19 expression and intracellular IgM in lymphocytes after 3 days of culture. Bars represent the average percentage of IgM producing B ce lls (A) or the MFI of IgM in the same cells (B) + one SEM. * Differs from Veh (p<0.05). 184 Figure 4.17. The effect of NE on the IgM response of !2AR expressing splenic B cells to in vitro LPS administration. Isolated splenocytes were cultured with LPS or its vehicle (Veh) for 3 days. Flow cytometry was used to assess surface $ 2AR and CD19 expression and intracellular IgM in lymphocytes after 3 days of culture. Bars represent the average percentage of IgM producing B cells expressing $ 2AR (A) or the MFI of IgM in the same cells (B) + one SEM. * Differs from Veh (p<0.05). 185 Figure 4.18. The effect of !2AR antagonism on the IgM response of splenic B cells to in vivo LPS administration. Female mice were injected 8 times with butoxamine (various doses; i.p.), or vehicle, every 6 h for 48 h. Mice received a single injection of LPS (25 µg; i.p.), or vehicle (Veh), one h prior to the 5 th injection. Mice were sacrificed 4 days after LPS administration and their splenocytes isolated. Flow cytometry was used to assess s urface CD19 expression and intracellular IgM in splenic lymphocytes. Bars represent the average percentage of IgM producing B cells (A) or the MFI of IgM in the same cells (B) + one SEM. * Differs from Veh (p<0.05). 186 Figure 4.19. The effect of !2AR anta gonism on the IgM response of !2AR expressing splenic B to in vivo LPS administration. Female mice were injected 8 times with butoxamine (various doses; i.p.), or vehicle, every 6 h for 48 h. Mice received a single injection of LPS (25 µg; i.p.), or vehicl e (Veh), one h prior to the 5 th injection. Mice were sacrificed 4 days after LPS administration and their splenocytes isolated. Flow cytometry was used to assess surface $ 2AR and CD19 expression and intracellular IgM in lymphocytes after 3 days of culture. Bars represent the average percentage of IgM producing B cells expressing $ 2AR (A) or the MFI of IgM in the same cells (B) + one SEM. 187 4.4: Discussion The results from experiments presented in this chapter illustrate that the sympathetic nervous system is activated during an immune challenge, but noradrenergic signaling does not modulate humoral immune responses. Splenic sympathetic neurons increase their activity within hours of an immune c hallenge. The activation of these neurons was hypothesized to release NE to act on !2AR on lymphocytes. However, stimulation of !2AR was not found to affect the humoral response of splenic B cell s. 4.4.1: Activation of splenic sympathetic neurons foll owing an immune challenge eliciting a humoral response Both sRBC and LPS induce humoral immune responses. Both immunogens increase the weight of the spleen. This increase is due t o a combination of recruitment of immune cells to the spleen and the prolife ration of immune cells within the spleen (Rasmussen et al., 2012) . Interestingly, the increase in spleen weight was greater in LPS treated mice. LPS is a polyclonal immune stimulator, activating all B cells expressing TLR4 (Ademokun and Dunn -Walters, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) , while sRBC will only activate B cells with unique BCR that can recognize epitopes from sRBC (Ademokun and Dunn -Walters, 2001). Thus, the difference may be due to a relatively larger population of expanding cells within the spleen due to the nature of immune stimulation unique to these immunogens. This difference, however, does not translate into LPS producing more 188 significant responses later on as b oth immunogen increased the number of B cells producing IgM with relatively similar magnitudes. Despite the similarities in the humoral immune response to both of these immunogen s, there is a significant difference in their actio n of immune stimulation. sRBC s are a traditional soluble protein antigen requiring processing by macrophages, interactions with T cell s, and eventually T cell to B cell interaction s to stimulation antibody production (Ademokun and Dunn -Walters, 2001) . LPS, on the other hand, is a direct stimulator of B cells. LPS bind TLR 4 receptors and cross -links BCR on B cells directly causing proliferation, activation, and ultimately IgM production in the same cell (Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . Therefore, while sRBC require B cells that selectively recognize sRBC epitopes, LPS is a polyclonal B cell activator in that it activates any B cell expressing TLR 4. This fact makes LPS a suitable and preferable immunogen for in vitro work , as demonstrated by the ab ility of LPS to induce and increase in the number of IgM producing cells in cultured splenocytes. Activation of splenic sympathetic noradrenergic neurons in response to an immune challenge is T -cell independent. The i njection of sRBC and LPS induce simi lar changes in splenic noradrenergic neurons , and b oth im munogens significantly increase the concentration of NE in the spleen capsule. As discussed in Chapter 3 , increased spleen capsule NE is correlated with increased noradrenergic activity. This corre lation holds true with regards to immune challenge activation i n that both immunogens 189 increase the rate of NE utilization in the spleen. Interestingly, the amount of TH also increased in response to both immunogens. Activity dependent increases in the content TH of catecholaminergic neurons is not a new finding (Kumer and Vrana, 1996) , but the time frame of TH increases is of interest. TH within the spleen is found exclusively within the axon terminals of sympathetic neurons , the cell bodies of which lie in the pre -vertebral celiac -mesenteric ganglion plexus. Thus, due to the distance between the cell bodies and axon terminals in the spleen there is not sufficient time for increased translation and transport of TH to splenic axon terminals within 2.5 h (the longest time point of the observed effects). Feedback inhibitor stabilization of TH is more likely the cause for increased splenic TH content seen following an immune challenge (Kumer and Vrana , 1996). Thus, if no significant changes occur in the synthetic rate of TH, but there is decrease in the degradation of the enzyme this can account for the accumulation of splenic TH observed. Phosphorylation of TH is known to occur during times of activation . This phosphorylation increase s the catalytic activity of TH and leads to an increase in TH stabilizing feedback inhibitors, such as dopamine (Kumer and Vrana, 1996) . Therefore, increased amounts of TH with increased catalytic activity are the potential cause for increased NE concentrations in the spleen during times of activation. Yet, the question as to how increased feedback inhibitors of TH can stabilize the protein while still allowing sufficient synthesis for the accumulation of NE. This phenomenon occurs in response to sRBC, a T -cell dependent immunogen, and LPS, a T -cell independent 190 immunogen. Therefore, it can be conc luded that T -cell involvement is not necessary in the activation of splenic sympathetic neurons. Accordingly, LPS was used in subsequent experiments since this is a clinically relevant immunogen (Miller et al., 2005; Lanzavecchia and Sallusto, 2007) , and is an effective in vitro model (Lee et al., 1995; Gururajan et al., 2007) . Pro-inflammatory cytokines are likely the activators of sympathetic neuronal activity during an immune challenge. The finding that T -cells are not involved in splenic sympathetic i nnervation raises the question: what induce s sympathetic neuronal activa tion dur ing an immune challenge? Due to the rapid onset of sympathetic activation following immun ogen administration, the leading hypothesis is that sympathetic activity is induced by early pro -inflammatory cytokines. The most studied of these early inflammatory f actors are interleukin -1 $ (IL -1 $ ) and IL -6. Both of these cytokines are produced by phagocytic monocytes early in inflammatory events (Dinarello, 2004) . The temporal profile of IL -1$ and IL -6 production in response to an immune challenge is within hours (Kakizaki et al., 1999) . This makes them prime candidates to media te rapid and early changes in sympathetic activity. In support of this, intraperitoneal injection of IL -1 $ increases the release of NE in the spleen (Shimizu et al., 1994; Kohm and Sanders, 2001) . This suggests that IL -1$ is the sympatho -stimulatory mediator in vivo . However, IL -1$ is also able to stimulate the production of IL -6 from a myriad of cell types (Kauma et al., 1994; Spangelo et al., 1994; Parikh e t al., 1997) . This leaves open the possibility that the effects of IL -1$ on sympathetic activity may be due to IL -6. Plasma and brain levels of IL -6 peak within 1 -3 191 h following an immune challenge (Kakizaki et al., 1999) , correlating with increased splenic noradrenergic activity seen in this work. Intraventricular injection of IL -6 produces increased firing of spleen capsule sympathetic neurons (Helwig et al., 2008) , suggesting IL -6 may act centrally to increase NE release during an immune challenge. Taken together, these data strongly suggest that one, or both, of these cytokines are stimulating the sympathetic nervous system during an immune challenge. 4.4.2: !2AR expression on splenic B cells Splenic B cells express !2AR and are immunologically functional. The demonstration of increased splenic sympathetic activity during an immune challenge in conjunction with NE containing axon terminals being in very close proximity to immune cells (Felten et al., 1987; Felten and Olschowka, 1987) suggest s a potential interaction between sympathetic neurons and immune cells . The present study confirms observations by other investigators that $ 2AR are expressed by both B cells and T cells, with B cell expression being highe st (Kin an d Sanders, 2006) . Therefore, $ 2AR expression on B cells was further pursued, especially with regards to humoral immunity in which the B cell plays a central role. Co-localization of $ 2AR with other B cell surface markers has not been previously repor ted. B cells constitutively express MHCII and are considered professional antigen presenting cells (Painter and Stern, 2012) . The finding that the majority of B cells expressing $ 2AR are also MHCII positive suggests $ 2AR might play a role in antigen presentation , in addition to the effects on antibody production. The data 192 presented here also suggest that B cells become more sensitive to NE following LPS stimulation by increasing the number of cells expressing $ 2AR and the amount of $ 2AR per B cell. This occurs within the general population of B cells as well as B cells producing IgM. The expression of $ 2AR on IgM producing cells further supports the hypothesis that NE stimulation of $ 2AR can have a direct impact on IgM production. 4.4.3: The effect of !2AR on the humoral response of spleni c B cells Stimulation of !2AR on splenic B cells does not augment IgM production in response to LPS , although !2AR expression on B cells producing IgM allows for direct interaction s. NE is an endogenous agonist of !2AR and the catecholamine released by sympathetic neuron axon terminals in the spleen. Therefore, it was hypothesized that addition of NE to LPS stimulated cultured splenocytes would increase IgM production. This turned out not be the case. Despite the inability of NE to increase IgM product ion in vitro , it was postulated that perhaps the simplified conditions of splenocyte culture were not suitable to reveal the augmenting effect of !2AR stimulation. Therefore, !2AR were blocked in vivo during an immune challenge by LPS. LPS had previously been shown to increase splenic sympathetic activity , and therefore NE release , to augment the humoral response in the spleen. It was expected that blockade of !2AR under these conditions would decreas e the humoral response of splenic B cells by blocking the effect of NE released in response to LPS. B lockade of !2AR did not significantly affect the humoral response of splenic B cells. Taken together, th ese data lead to the 193 conclusion that NE stimulatio n of !2AR does not augment the humoral response of splenic B cells in response to LPS. These findings argue against a direct role for !2AR stimulation in antibody production. The question then remains as to the function of !2AR on B cells. It is demonst rated here that B cells express !2AR and these cells are immunologically functional . Furthermore, t he data show that !2AR stimulation is not necessary for a normal antibody response to LPS, a T cell independent antigen. This raises the question of why the data obtained in this chapter is incongruent with the reported effect of !2AR on antibody production. A potential explanation of this discrepancy has to do with the nature of the immune challenge used. The correlation between !2AR and antibody production was explored here using LPS, a T cell independent immunogen, which induces a humoral immune response through activation of TLR4 and crosslinking of the BCR (Ademokun and Dunn -Walters, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . However, the published research on the correlation between antibody production and !2AR has been accomplished using either T cell dependent immunogens or through direct stimulation of B cells by CD40 (Kohm et al., 2000; Kohm and Sanders, 2001; Pongratz et al., 2006; Padro et al., 2013) . Therefore, it may be the case that !2AR augmentation of antibody production is specific to B cell responses in which a CD40 -CD154 interaction has occurred. If this is the case, the lack of !2AR augmentation of the humoral immune response to LPS is due to the absence of CD40 being involved in this response. This hypothesis has not been directly tested and is an avenue for future research. 194 4.4.4: Conclusion The purpose of this chapter was to test the hypothesis that !2AR stimulation on B cells by NE released in the spleen augments humoral responses. The data here demonstrate that splenic sympathetic noradrenergic activity is increased during immune challenges. Splenic B cells are shown to express !2AR and are able to produce IgM, which allows for a direct effect of NE on the effector cells of the humoral immune response. However, stimulation of the !2AR was not found to enhance the humoral immune response of splenic B cells to LPS in vitro or in vivo . Thus, the roles of !2AR on B cells and neuroimmune interactions in humoral immunity remain unclear, but the data here demonstrate that they do not directly modulate the production of antibodies in response to LPS, a T cell independent immunogen . 195 REFERENCES 196 REFERENCES Ademokun AA, Dunn -Walters D. Immune Responses: Primary and Secondary. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Ahrens WH, Cox DJ, Budhwar G. Use of the arcsine and square root trans formations for subjectively determined percentage data. Weed Science. 1990. Baumgarth N. Innate -Like B Cells and Their Rules of Engagement. link.springer.com.proxy2.cl.msu.edu. New York, NY: Springer New York; 2013. p. 57Ð66. Bekeredjian -Ding I, Jego G. Toll -like receptors --sentries in the B -cell response. Immunology. 2009 Nov;128(3):311 Ð23. PMCID: PMC2770679 Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of steady state kinetics to the estimation of synthesis rate and turnover time of ti ssue catecholamines. J Pharmacol Exp Ther. 1966 Dec;154(3):493 Ð8. Cesta M. Normal Structure, Function, and Histology of the Spleen. Toxicol Pathol. 2006;34(5):455 Ð65. Czajkowsky DM, Shao Z. The human IgM pentamer is a mushroom -shaped molecule with a flex ural bias. Proc Natl Acad Sci USA. 2009 Sep 1;106(35):14960 Ð5. Da‚ron M. Fc RECEPTOR BIOLOGY - Annual Review of Immunology, 15(1):203. Annu. Rev. Immunol. 1997. Dinarello C. Infection, fever, and exogenous and endogenous pyrogens: some concepts have chan ged. J Endotoxin Res. 2004;10(4):201 Ð22. Eaton MJ, Lookingland KJ, Moore KE. Effects of the selective dopaminergic D2 agonist quinelorane on the activity of dopaminergic and noradrenergic neurons projecting to the diencephalon of the rat. 1994 Feb 1;268(2 ):645 Ð52. Fazilleau N, Mark L, McHeyzer -Williams LJ, McHeyzer -Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009 Mar 20;30(3):324 Ð35. PMCID: PMC2731675 Felten DL, Ackerman KD, Wiegand SJ, Felten SY. Noradrenergic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neurosci Res. 197 1987;18(1):28 Ð36, 118Ð21. Felten SY, Olschowka J. Noradrenergic sympathetic innervation of the spleen: II . Tyrosine hydroxylase (TH) -positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp. J Neurosci Res. 1987;18(1):37 Ð48. Ferrero I, Michelin O, Luescher I. Antigen Recognition by T Lymphocytes. onlinelibrary.wiley.com.pr oxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Gotch FM. T Lymphocytes: Cytotoxic. Chichester, UK: John Wiley & Sons, Ltd; 2001. Gururajan M, Jacob J, Pulendran B. Toll -Like Receptor Expression and Responsiveness of Distinct Murine Spleni c and Mucosal B -Cell Subsets. PLoS ONE. Public Library of Science; 2007 Sep 12;2(9):e863. PMCID: PMC1955832 Helwig BG, Craig RA, Fels RJ, Blecha F, Kenney MJ. Central nervous system administration of interleukin -6 produces splenic sympathoexcitation. Auton omic Neuroscience. 2008 Aug;141(1 -2):104 Ð11. Kakizaki Y, Watanobe H, Kohsaka A, Suda T. Temporal profiles of interleukin -1beta, interleukin -6, and tumor necrosis factor -alpha in the plasma and hypothalamic paraventricular nucleus after intravenous or intr aperitoneal administration of lipopolysaccharide in the rat: estimation by push -pull perfusion. Endocr J. 1999 Aug 1;46(4):487 Ð96. Kauma SW, Turner TT, Harty JR. Interleukin -1 beta stimulates interleukin -6 production in placental villous core mesenchymal cells. Endocrinology. 1994;134(1):457 Ð60. Kenter AL. Class Switch Recombination: An Emerging Mechanism - Springer. Molecular Analysis of B Lymphocyte Development and É. 2005. Kin NW, Sanders VM. It takes nerve to tell T and B cells what to do. J Leukoc B iol. 2006 Jun;79(6):1093 Ð104. Kohm AP, Sanders VM. Norepinephrine: a messenger from the brain to the immune system. Immunology Today. 2000 Nov;21(11):539 Ð42. Kohm AP, Sanders VM. Norepinephrine and beta 2 -adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev. 2001 Dec;53(4):487 Ð525. Kohm AP, Tang Y, Sanders VM, Jones SB. Activation of antigen -specific CD4 + Th2 cells and B cells in vivo increases norepinephrine release in the spleen and bone marrow. 2000 Jul 15;165(2):725 Ð33. Kumer SC, Vrana KE. Intricate regulation of tyrosine hydroxylase activity and gene 198 expression. J Neurochem. 1996 Aug 1;67(2):443 Ð62. Kurosaki T, Hikida M. B Lymphocytes: Receptors. eLS. Lanzavecchia A, Sallusto F. Toll -like receptors and innate immunity in B -cell activation and antibody responses. Curr Opin Immunol. 2007 Jun;19(3):268 Ð74. Lee M, Yang KH, Kaminski NE. Effects of puta tive cannabinoid receptor ligands, anandamide and 2 -arachidonyl -glycerol, on immune function in B6C3F1 mouse splenocytes. Journal of Pharmacology and Experimental É. 1995. Lindley SE, Gunnet JW, Lookingland KJ, Moore KE. 3,4 -Dihydroxyphenylacetic acid con centrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity. Brain Res. 1990 Jan 1;506(1):133 Ð8. Lu H, Kaplan BLF, Ngaotepprutaram T, Kaminski NE. Suppression of T c ell costimulator ICOS by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2009 Feb;85(2):322 Ð9. PMCID: PMC2631366 Meager A, Wadhwa M. An Overview of Cytokine Regulation of Inflammation and Immunity. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Lt d; 2001. Miller SI, Ernst RK, Bader MW. LPS, TLR4 and infectious disease diversity. Nat. Rev. Microbiol. 2005 Jan;3(1):36 Ð46. Moller G. Antigens: Thymus Independent. els.net.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Nance DM, Sand ers VM. Autonomic innervation and regulation of the immune system (1987 -2007). Brain Behav Immun. 2007 Aug;21(6):736 Ð45. PMCID: PMC1986730 Nath I. Immune Mechanisms against Intracellular Pathogens. onlinelibrary.wiley.com.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. N”ron S, Nadeau PJ, Darveau A, Leblanc J -F. Tuning of CD40 -CD154 interactions in human B -lymphocyte activation: a broad array of in vitro models for a complex in vivo situation. Arch. Immunol. Ther. Exp. (Warsz.). 2011 Feb;5 9(1):25 Ð40. Noble J, Bailey M. Quantitation of protein. Methods Enzymol. 2009;463:73 Ð95. Oikonomopoulou K, Reis ES, Lambris JD. Complement System and Its Role in Immune Responses. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. P Kane L. Lymphocyte Activation: Signal Transduction. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. 199 Padro CJ, Shawler TM, Gormley MG, Sanders VM. Adrenergic Regulation of IgE Involves Modulation of CD23 and ADAM10 Expression on Exos omes. The Journal of Immunology. 2013 Oct 18. PMCID: PMC3842235 Painter CA, Stern LJ. Conformational variation in structures of classical and non -classical MHCII proteins and functional implications. Immunol. Rev. 2012 Nov;250(1):144 Ð57. PMCID: PMC3471379 Parikh AA, Salzman AL, Kane CD, Fischer JE, Hasselgren PO. IL -6 production in human intestinal epithelial cells following stimulation with IL -1 beta is associated with activation of the transcription factor NF -kappa B. J Surg Res. 1997 Apr 1;69(1):139 Ð44. Pinaud E, Khamlichi A, Le Morvan C, Drouet M, Nalesso V, Le Bert M, et al. Localization of the 3' IgH locus elements that effect long -distance regulation of class switch recombination. Immunity. 2001 Aug 1;15(2):187 Ð99. Podojil JR, Kin NW, Sanders VM. CD 86 and beta2 -adrenergic receptor signaling pathways, respectively, increase Oct -2 and OCA -B Expression and binding to the 3' -IgH enhancer in B cells. 2004 May 28;279(22):23394 Ð404. Pongratz G, McAlees JW, Conrad DH, Erbe RS, Haas KM, Sanders VM. The Level of IgE Produced by a B Cell Is Regulated by Norepinephrine in a p38 MAPK - and CD23 -Dependent Manner. J Immunol. 2006 Sep 1;177(5):2926 Ð38. Quah BJ, Parish CR. Innate Immune Mechanisms: Nonself Recognition. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Rasmussen JW, Tam JW, Okan NA, Mena P, Furie MB, Thanassi DG, et al. Phenotypic, morphological, and functional heterogeneity of splenic immature myeloid cells in the host response to tularemia. Infect. Immun. 2012 Jul;80(7):2371 Ð81. PMCID: PMC34164 75 Saha B. Antigens. els.net.proxy2.cl.msu.edu. Chichester, UK: John Wiley & Sons, Ltd; 2001. Sanders VM. The beta2 -adrenergic receptor on T and B lymphocytes: Do we understand it yet? Brain Behav Immun. 2012 Feb;26(2):195 Ð200. PMCID: PMC3243812 Schroeder HW Jr., Cavacini L. Structure and function of immunoglobulins. Journal of Allergy and Clinical Immunology. Elsevier; 2010 Feb;125(2):S41 ÐS52. Shimizu N, Hori T, Nakane H. An interleukin -1 beta -induced noradrenaline release in the spleen is media ted by brain corticotropin -releasing factor: an in vivo microdialysis study in conscious rats. Brain Behav Immun. 1994 Mar 1;8(1):14 Ð23. 200 Spangelo BL, deHoll PD, Kalabay L, Bond BR, Arnaud P. Neurointermediate pituitary lobe cells synthesize and release in terleukin -6 in vitro: effects of lipopolysaccharide and interleukin -1 beta. Endocrinology. 1994 Aug 1;135(2):556 Ð63. Stavnezer J, Guikema JEJ, Schrader CE. Mechanism and Regulation of Class Switch Recombination. Annu. Rev. Immunol. 2008 Apr;26(1):261 Ð92. Stevens S, Ong J, Kim U, Eckhardt L, Roeder R. Role of OCA -B in 3' -IgH enhancer function. J Immunol. 2000 May 15;164(10):5306 Ð12. Vincent -Fabert C, Fiancette R, Cogn” M, Pinaud E, Denizot Y. The IgH 3' regulatory region and its implication in lymphomagen esis. Eur J Immunol. 2010 Dec 1;40(12):3306 Ð11. 201 Chapter 5: The Interaction of Endogenous CB1/CB2 Receptor Signaling and Norepinephrine in Splenic Humoral Immune Responses 5.1: Introduction Cannabinoids are known immunosuppressive compounds. In humans, t his can be seen as a decrease in serum lymphocytes and serum antibody concentrations in cannabis users (Klein et al., 1998; Pacifici et al., 2003; Gohary and Eid, 2004) . Interestingly, epidemiologic studies have yet to fully identify the consequences of cannabinoid -mediated immunosuppression, despite the wealth of information in animal studies (Greineisen and Turner, 2010; Tashkin, 2013) . The most well researched cannabinoid compound in this regard is THC . This compound is known to suppress both cell -mediated (Lu et al., 2009; Karmaus et al., 2012; 2013) and humoral (Schatz et al., 1993; Jan et al., 2003) adaptive immune responses in animal models. The effects of exogenous cannabinoids occur by pharmacologically mimicking the actions of the endocannabinoid s. The endocannabinoid system has largely been identified as inhibit ory to the release of neurotransmitters from axon terminals as a form of retrograde tran s-synaptic negative feedback . The two most well researched receptors of the endocannabinoid system are CB1 and CB2 (Mackie, 2008; Alger and Kim, 2011) . CB1 is considered to be the major n euronal cannabinoid receptor. CB1 is a G-protein coupled receptor s that activate the G i/o pathway leading to decreased cAMP production via inhibition of adenylyl cyclase (Howlett and Mukhopadhyay, 2000; Mackie, 2008). Inhibition of cAMP production decreases protein kinase A activity resulting in a 202 number of downstream consequences, including a positive shift in the voltage -dependence of A -current K + channels (Childers and Deadwyler, 1996) , inhibition of voltage -gate Ca 2+ channels (Felder et al., 1993; Mackie et al., 1993; 1995; Gebremedhin et al., 1999) , and a ctivat ion of inwardly rectifying K + channels (Mackie et al., 1995). It is by these mechanisms that CB1 activation inhibits neurotransmitter release from axon terminals. CB1 is predominantly localized to the pre -synaptic axon terminals of neurons and found abundantly in the brain (Herkenham et al., 1991; Ny™ri et al., 2005; Mackie, 2008). There is also functional evidence for CB1 regulatory process es in sympathetic axon terminals of the atria, vas deferens, and mesenteric arteries (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . CB1 mRNA is found in the superior celiac ganglion, vas deferens, and the spleen (Ishac e t al., 1996; Schatz et al., 1997). Interestingly, CB1 is also expressed by immune cells, including sple nic lymphocytes, but to a much lesser extent than CB2 (Galiegue et al., 1995; Kaplan, 2012). The role of CB1 on immune cells remains somewhat controv ersial, but a handful of studies have shown the CB1 to mediate, at least partially, some immune effects (Kaplan, 2012) . For example (Bırner et al., 2008) , CB1 transcription is induced by IL -4, a TH2 cytokine, and once up -regulated CB1 stimulation inhibits cAMP formation and IL -2 production, a cytokine critical for T cell responses in vivo (Ferrero et al., 2001) . CB1 is activated by both exogenous and endogenous cannabinoids (See Figure 1.3). THC is a partial agonist of the CB1 receptor (Kuster et al., 1993; Pertwee, 2008) . 203 The two most well characterized endocannabinoid compounds that bind to CB1 are AEA and 2 -AG. Neither of these compounds are stored, but rather made on demand from phospholipid components found in the membrane of post -synaptic neurons (Giuffrida and Mcmahon, 2010) . Once synthesized endocannabinoids travel retrogradely across the synapse to act on presynaptic CB1 receptors. Both AEA and 2 -AG are partial agonists of CB1, however AEA is more selective for CB1 than 2 -AG (Alger and Kim, 2011) . AEA and 2 -AG are rapidly degraded within minutes by the enzymes FAAH (primarily AEA) and MAGL (primarily 2 -AG), thereby terminating their actions (Gerra et al., 2010; Giuffrida and Mcmahon, 201 0). CB2 is considered to be the major peripheral cannabinoid receptor. CB2 mRNA is abundant in peripheral tissues, such as the spleen, but almost completely absent from the brain (Shire et al., 1996; Griffin et al., 2000; Brown et al., 2002) . Like CB1, CB2 is coupled to Gi/o subtype G -proteins and inhibit s adenylyl cyclase, preventing the formation of cAMP (Felder et al., 1995) . Very different from CB1, CB2 agonism does not inhibit Q -type Ca 2+ channels, nor activate inwardly rectifying K + channels (Felder et al., 1995). CB2 is expressed primarily on cells w ith an immunologic function, and the spleen was among the first organs shown to have abundant CB2 expression (Brown et al., 2002). CB2 is found on T -cells, B -cells, macrophages, and natural killer cells of the immune system, with B -cells having the highest expression (Galiegue et al., 1995; Schatz et al., 1997; Cabral and Griffin -Thomas, 2009) . 204 CB2 is also activated by both exogenous and endogenous cannabinoids . THC is a partial agonist of CB2 (K i ~20 nM) (Shire et al., 1996; Griffin et al., 2000) . Both AEA and 2 -AG are also agonists for CB2, but 2 -AG is a mor e selective and potent agonist than AEA and is expressed in higher quantities in the spleen, bone marrow, and gut (Tanasescu and Constantinescu, 2010; Basu et al., 2011) . The expression of CB2 on immune cells suggests a role for these receptors. A numbe r of in vitro studies have been used to assess the effect of CB2 activation on a variety of immune cell functions. Stimulation of CB2 with 2 -AG induces the migration of immune cell types, including B cells, monocytes/macrophages, and microglia (Basu and Dittel, 2011) . However, migration of mouse macrophages in response to common chemoattractants is inhibited by synthe tic CB2 agonists (Ghosh et al., 2006; Montecucco et al., 2008; Raborn et al., 2008) . To date, there is no consensus regarding the effect of CB2 on immune cell migration, but it is clear from these studies that they can modulate immune cell chem otaxis. CB2 can also affect immune cell proliferation, however there is no clear consensus with studies showing both stimulatory effects on microglia (Carrier et al., 2004) and suppressive effects on CD4 + T cells (Maresz et al., 2007). In contrast , the ability of CB2 receptors to inhibit cytokine production, especially pro -inflammatory cytokines (IL -10 and IL -23) has been well established (Basu et al., 2011). The use of in vitro assays, while useful, cannot replicate the complexity of the immune system in vivo . Thus, the role of cannabinoid receptors in vivo has received a good deal of attention, and a wealth of studies have shown the immunosuppressive 205 ability of cannabis or cannabinoids (Klein et al., 1998) . For example, THC was recently shown to broadly inhibit the immune response of mice to influenza infection (Karmaus et al., 2013). These results strongly suggest the site of action in this regard is inhibition of antigen presenting cell function. THC also inhibits the humoral response in mice, as demonstrated by decr easing the number of antibody producing cells in the spleen in response to sRBC administration (Schatz et al ., 1993). In addition to pharmacological experiments using CB receptor agonists, a number of studies have used mice genetically manipulated to lack the CB1 and CB2 receptors. These mice appear phenotypically normal and have a normal immune cell profil e (Springs et al., 2008) . Yet, in accordance with the immun osuppression observed with CB receptor stimulation, these mice demonstrate enhanced immunity. CB1/CB2 KO mice show increased numbers of antibody producing cells from the spleen in response to sRBC administration (Springs et al., 2008) . Accordingly, these mice are useful in establishing the immunomodulatory role of the CB1 and/or CB2 receptors. The purpose of this chapter is to test the hypothesis that enhanced humoral immunity in CB1/CB2 KO mice is due to increased stimulation of !2AR secondary to increased release NE from splenic sympathetic neurons. First, enhanced humoral immune responses will be evaluated in CB1/CB2 KO mice. Next, the expression of !2AR on splenic B cells will be assessed to confirm the adrenergic sensing ability of B cells in the absence of CB1/CB2. Lastly, the effect of CB1/CB2 KO on the activity of splenic sympathetic noradrenergic neurons, and subsequent !2AR stimulation, will be determined. 206 5.2: Materials and Methods 5.2.1: Mice C57BL/6 WT female mice (NCI/Charles River, Portage, MI) and female CB1/CB2 KO mice were used in all experiment unless otherwise indicated. CB1/CB2 KO mice, on a C57BL/6 background, were created by Dr. And reas Zimmer at the University of Bonn, Germany as previously described (Jarai et al., 1999; Zimmer et al., 1999; Buckley et al., 2000; Gerald et al., 2006) . CB1/CB2 KO mice for these studies were obtained from Drs. Norbert Kaminski and Barbara Kaplan who maintain a breeding colony of CB1/CB2 KO mice at Michigan State University. All animals were housed two to five per cage and maintained in a sterile, temperature (22 ± 1 ¡C) and light controlled (12L:12D) room, and provided with irradiated food and bottled tap water ad libitum. All experiments used the minimal number of animals required for statistical analyses, minimized suffering, and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Michigan State Institutional Animal Care and Use C ommittee approved all drug administrations and methods of euthanasia (AUF# 03/12!060!00). 5.2.1.1: CB1/CB2 KO Mouse Genotyping Polymerase chain reaction (PCR) was used to confirm knockout of CB1 and CB2 receptor genes. Genomic DNA was isolated from ~0.5 cm tail snips using 100 µl DirectPCR Lysis Reagent (Viagen Biotech, Los Angeles, CA) plus 0.1 mg/ml proteinase K. Samples were in cubated overnight at 55¡C followed by a 45 min incubation at 85¡C. 207 Crude DNA extract was obtained following centrifugation at 300 RCF for 1 min. One µl of extract was used in a Taqman PCR reaction using Cnr1 stock primers (CB1 receptor gene) or Cnr2 (CB2 receptor gene) custom primers (Life Technologies/Applied Biosystems, Foster City, CA) (Kaplan et al., 2010) . PCR primers for CB1 receptors were forward 5"-AGGAGCAAGGACCTGAGACA -3" , reverse 5"-GGTCACCTTGGCGATCTTAA -3" , for CB2 receptor were forward 5 "-CCTGATAGGCTGGAAGAAGTATCTAC -3 " , reverse 5 "-ACATCAGCCTCTGTTTCTGTAACC -3 " , neomycin cassette primers were forward 5 "-ACCGCTGTTGACCGCTACCTATGTCT -3 " , and reverse 5 "-TAAAGCGCATGCTCCAGACTGCCTT -3 " . The average ± standard deviatio n Ct values for Cnr1 and Cnr2 in WT mice were 25.0 ± 0.54 and 26.0 ± 1.22, respectively. All samples obtained from CB1/CB2 KO tail snips resulted in an ÒundeterminedÓ Ct value, indicating lack of expression of both Cnr1 and Cnr2 . 5.2.2: Materials aMT: aMT ester (Sigma, St. Louis, MO) was dissolved in 0.9% isotonic saline to a final concentration of 30 mg/ml and administered at dose of 300 mg/kg. Butoxamine: Butoxamine (B1385, Sigma) was dissolved in sterile isotonic saline at a concentration of 5 mg/ml and administered at doses ranging from 1 -10 mg/kg (i.p.). 208 HBSS: 10x HBSS powder (Gibco) was diluted with ultra -pure H 2O (NaCl 138 mM, KCl 5.3 mM, Na 2HPO 4 0.3 mM, NaHCO 3 4.2 mM, KH 2PO4 0.4 mM, and glucose 5.6 mM), autoclaved, and stored at 4 ¡ C. Isotonic Saline: 1 L of 0.9% saline was prepared using ultra -pure H 2O and 9 grams of NaCl. The solution was autoclaved and kept closed at room temperature. LPS: LPS ( E. coli 055:B5 catalog L2880, lot 066K4096, 5 EU/ng (Limulus lysate assay) and 10 EU/ng (chromog enic assay) , Sigma , St. Louis, MO) was dissolved in RPMI -1640 at used at a final concentration of 10 #g/ml for in vitro studies. For in vivo experiments, LPS was dissolved in HBSS to a concentration of 50 #g/ml and injected at 25 # g per mouse (i.p.) . PBS: NaCl 137 mM, KCl 2.7 mM, Na 2HPO 4 10 mM, and KH 2PO4 1.8 mM in ultra -pure H2O. 5.2.3: Isolation of the Spleen Capsule and Splenocytes After euthanasia spleens were removed by an incision in the left lateral abdomen under sterile conditions, which entails spraying the area of removal with 70% ethanol and using ethanol cleaned scissors and forceps to cut through the skin, and underlying 209 muscle and connect ive tissue . The spleen was placed in a 6 -well plate and mechanically crushed with the blunt end of a 10 ml syringe in 2 ml of HBSS to separate the spleen capsule (insoluble tissue) from the splenocytes (contained in the disruption supernatant). The splee n capsule was removed from the supernatant using forceps and taken whole or divided into two parts using ethanol -cleaned scissors depending on the needs of the experiment. Splenocytes were separated from the disruption supernatant by centrifugation at 300 RCF for 5 min and the supernatant was decanted . The separated splenocytes were then re-suspended in differing buffers and taken whole or divided into two parts depending on the needs of the experiment . 5.2.4: Neurochemistry All samples were placed in ice -cold tissue buffer following isolation or dissection and kept frozen at -80¡C until analysis. Samples were thawed on the day of analysis and sonicated with 3 one -sec bursts (Sonicator Cell Disruptor, Heat Systems -Ultrasonic, Plainview, NY, USA) and cen trifuged at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge. The supernatant from the first centrifugation of the spleen capsule was removed and spun again at 18,000 RCF for 5 min in a Beckman -Coulter Microfuge 22R centrifuge before bein g brought up to 100 # l (q.s.) with fresh cold tissue buffer. Spleen samples were then filtered using a 0.2 # M syringe driven Millex -LG filter (Millipore, Billerica, MA). All samples were analyzed for NE content using HPLC -ED (Lindley et al., 1990; Eaton et al., 1994) using C18 reverse phase columns (ESA Inc., Sunnyvale, CA ) 210 combined with a low pH buffered mobile phase (0 .05 M Sodium Phosphate, 0.03 M Citrate, 0.1 mM EDTA at a pH of 2.65) composed of 5 -15% methanol and 0.03 -0.05% sodium octyl sulfate. Oxidation of NE was measured at a constant potential of -0.4 V by coulometric detection using a Coulochem Electro chemical Detector (Thermo Scientific). The amount of NE in the samples was determined by comparing peak height values (as determined by a Hewlett Packard Integrator, Model 3395) with those obtained from known standards run on the same day. Tissue pellets w ere dissolved in 1 N NaOH and assayed for protein using the BCA method (Noble and Bailey, 2009) . 5.2.5: Western Blot All samples were placed in ice -cold lysis buffer (water containing 1% Triton -x 100, 250 mM sucrose, 50 mM NaCl, 20 mM tris -HCl, 1 mM EDTA, 1 mM PMSF p rotease inhibitor cocktail, 1 mM DTT) immediately following isolation and kept frozen at -80¡C until analysis. On the day of analysis samples were thawed, heated for 30 min at 100¡ C, sonicated for 8 sec, and spun at 12,000 RCF for 5 min. The supernatant was collected and a BCA protein assay performed (Noble and Bailey, 2009) . Equal amounts of protein were separated using SDS -PAGE and transferred to PVDF -FL membranes (Millipore, Billerica, MA). The resulting membranes were reacted against antibodies for TH (AB152 1:20 00, Millipore, Billerica, MA) , whose intensities were normalized to !-Actin (8H10D10 1:8000, Cell Signaling, Danvers, MA) to account for loading variability. Each PVDF -FL membrane contained samples representing all experimental conditions to avoid variability due to run, transfer, or antibody exposure conditions. Blots we re 211 visualized and quantified using an Odyssey Fc Infrared Imaging system (Li -Cor, Lincoln, NE) by utilization of IRDye conjugated secondary antibodies, goat anti -Mouse 800CW (1:20,000) and/or goat anti -rabbit 680LT (1:20,000). 5.2.6: Flow Cytometry 5.2.6.1: Surface antibody labeling for flow cytometry All staining protocols were performed in 96 -well round bottom plates (BD Falcon, Franklin Lakes, NJ). Splenocytes were washed 3x with HBSS by centrifugation at 1000 RCF for 5 min, the supernatant was decante d, and the cells re -suspen ded in HBSS. Cultured splenocytes were then incubated for 30 min on ice in the dark in a 1x solution of near IR (APY -Cy7) live/dead stain (#L10119, Invitrogen, Grand Island, NY), a step which was omitted for splenocytes obtained directly from spleens. Following a wash in HBSS (as described above), splenocytes were then washed with FACS buffer (HBSS, 1% bovine serum albumin, 0.1% sodium azide, pH 7.6) as was done with HBSS. Surface Fc receptors were blocked with anti -mouse CD16/CD 32 [0.5 mg/ml] (#553142, BD Biosciences, Franklin Lakes, NJ) at 0.5 # l/well, IgM was blocked with anti -IgM [0.5 mg/ml] (#553425, BD Biosciences) at 1 # l/well, and IgG was blocked with anti -IgG [1.3 mg/ml] (#115 -006-071, Jackson Immunoresearch, West Grove, PA) at 0.5 # l/well for 15 min each at RT. Cells were stained for 30 min at RT with the following antibody clones: CD19 (clone 6D5) [0.2 mg/ml] (Biolegend, San Diego, CA) at 1.25 # l/well and $ 2AR [0.25 mg/ml] (#AP7263d, Abgent, San Diego, CA) at 2 # l/ well. Cells were then washed 3x with FACS buffer. A secondary antibody for $ 2AR, donkey anti -rabbit DyLight 649 212 (clone Poly4064) [0.5 mg/ml] (Biolegend), at 0.5 # l/well was incubated for 30 min at RT. Subsequently cells were washed with FACS buffer, fixed with Cytofix (BD Biosciences) for 15 min at RT, washed 3x with FACS buffer, and finally suspended in FACS buffer for intracellular staining. Stained and fixed cells were stored in the dark at 4 ¡C for up to 2 weeks. 5.2.6.2: Intracellular antibody labeling for flow cytometry Within 2 weeks of surface staining, cells were washed 2x with Perm/Wash (BD) and incubated with Perm/Wash for 30 min at RT. Fluorescently labeled antibodies for IgM (Clone II/41) [0.5 mg/mL] (Biolegend) were added at 1 # l/well for 30 min. Cells were washed 2x with Perm/Wash and suspended in FACS buffer. After intracellular staining, cells were analyzed the same day. 5.2.6.3: Flow Cytometry Analysis Fluorescent staining was analyzed using a BD Biosciences FACSCanto II flow cytometer. Data were analyzed using Kaluza (Beckman Coulter Inc., Brea, CA) or FlowJo software (Tree Star Inc., Ashland, OR). Compensation and voltage settings of fluorescent parameters were performed using fluorescence -minus -one controls. Cells were gated on singl ets (forward scatter height versus area) followed by determination of live cells (low APC -Cy7 signal) only in samples obtained from splenocyte culture. Cells were then gated to select lymphocytes using forward versus side scatter. For some analyses, an a dditional gate was created for CD19 or $2AR expression to select for B 213 cells or $2AR expressing cells, respectively . These sequential gates were used to identify IgM producing B cells and IgM producing B cells that express $ 2AR. The percentage of cells gat ed to individual populations relative to the entire population were collected and analyzed. Additionally, the numerical intensity of the fluorescent signal, termed the MFI, was also quantified and analyzed. 5.2.7: ELISA Serum IgM and IgG were detected by sandwich ELISA. In preparation, 100 # l of 1 #g/ml anti -mouse IgM (Sigma -Aldrich, St. Louis, MO) or 1 # g/ml anti -mouse IgG (Sigma -Aldrich) was added to wells of a 96 -well microtiter plate and stored at 4 ¡C overnight. After the pre -coating step, the plate w as washed twice with 0.05% Tween -20 in PBS and three times with H 2O. Following this, 200 #l of 3% BSA -PBS was added to the wells and incubated at RT for 1.5 h to block nonspecific binding followed by the same washing steps described above. Serum samples we re diluted and added to the coated plate (100 # l) for 1.5 h at RT. After the incubation, the plate was washed again, followed by addition of 100 #l of HRP -conjugated goat anti -mouse IgM (A8786, Sigma -Aldrich) or HRP -conjugated goat IgG (A3673, Sigma -Aldri ch). Following the HRP incubation for 1.5 h at RT, any unbound detection antibody was washed away from the plate, and 100 # l ABTS (Roche Applied Science, Indianapolis, IN) added. The detection of the HRP substrate reaction was conducted over a 1 h period u sing a plate reader with a 405-nm filter (Bio -Tek). The KC4 computer analysis program (Bio -Tek) calculated the concentration of IgM or IgG in each sample based on a standard curve generated from 214 the absorbance readings of known concentrations of IgM (range 6-1600 pg/ml, clone TEPC 183, Sigma -Aldrich) or IgG (range 3 -800 pg/ml, Sigma -Aldrich). 5.2.8: Statistical Analysis 5.2.8.1: Statistical Comparisons Prism software version 4.0a was used to make statistical comparisons between groups using the appropriate statistical test. Differences with a probability of error of less than 5% (p<0.05) were considered statistically significant. Two group comparisons were done using the Student " s t-test. Two group comparisons where in one group had more than one degree or factor were done using a One -way ANOVA followed by a Bonferroni or Tukey " s post -test for multiple comparisons. Experiments in which there were two groups with more than one degre e or factor in each group, such as a 2x2 design, were analyzed using a Two -way ANOVA followed by Bonferroni post -test for multiple comparisons. 5.2.8.2: aMT Experimentation In experiments in which aMT was used to assess neuronal activity, NE concentratio ns from aMT treated and non -aMT (saline) treated mice were used for a regression analysis with saline animals acting as the 0 time control and aMT animals a 4 h time point. The rate constant was determined using this formula: =(Log 10[B] -Log10[A])/( -0.434*t). Where B is the concentration of NE after aMT, A is the concentration of A in saline treated animals, and t is the time of aMT treatment (Brodie 215 et al., 1966) . The slopes were compared via t -test using the mean sl ope, SEM, and an n equal to the total number of data points used in the analysis to account for the total number of independent measurements used to generate the regression equations. Differences with a probability of error of less than 5% were considered statistically significant . 5.2.8.3: Flow Cytometry Data Handling Population percentage data was transformed in Excel (Microsoft Corporation, Redmond, WA) to a parametric form before ANOVA analysis using the formula: =arcsin(sqrt(DATA/100)) (Ahrens et al., 1990) . Raw percentage data was used for visual representation s, while statistical significance indicated on these figures was performed on the transformed data. 5.3: Results 5.3.1: Enhanced humoral immunity In CB1/CB2 KO mice Humoral immunity in CB1/CB2 KO mice was compared with that of WT mice using antibody production from B cells and the number of B cells as indices. Here it is shown that CB1/CB2 KO have elevated serum IgM and IgG concentrations compared to WT mice, even whe n immunologically naŁve ( Figures 5.1 and 5.2 ). One potential cause for increased serum antibodies is an increase in the number of antibody producing B cells. To test this possibility the percentage of IgM producing B cells was assessed in the spleen by fl ow cytometry. It was discovered that CB1/CB2 KO have 216 more splenic IgM producing B cells than WT mice ( Figures 5.3 and 5.4 ). Based upon these results, it was hypothesized that CB1/CB2 KO mice would also have enhanced humoral immunity in response to an im mune challenge by LPS. Congruent with this hypothesis, serum IgM and IgG levels are elevated in LPS treated CB1/CB2 KO mice (Figures 5.5 and 5.6 ), which was associated with an increase in the population of splenic antibody producing B cells in CB1/CB2 KO mice ( Figures 5.7 and 5.8 ). These data support the hypothesis that CB1/CB2 KO mice have enhanced humoral immunity, which is due, at least in part, to an increase in the number of splenic antibody producing B cells. 217 Figure 5.1. Serum IgM concentrations in immunologically naive WT and CB1/CB2 KO mice . The serum from f emale WT and CB1/CB2 KO mice collected and assayed for IgM using ELISA . Columns represent the concentration of IgM (pg/ml) + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 218 Figure 5.2. Serum IgG concentrations in immunologically naive WT and CB1/CB2 KO mice . The serum from f emale WT and CB1/CB2 KO mice collected and assayed for IgG using ELISA . Columns represent the concentration of IgG (pg/ml) + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 219 Figure 5.3 . Flow cytometric analysis of splenic IgM producing B cell populations from immunologically naŁve WT and CB1/CB2 KO mice . Lymphocytes were isolated from the spleens of untreated WT and CB 1/CB2 KO mice and analyzed by flow cytometry as described . IgM (FITC) producing B cells (CD19 -PE-Cy7) reside in the upper right quadrant of the displayed plots . Each plot represents the concatenated data from 3 -5 mice. 216 220 Figure 5.4. Flow cytometric analysis of splenic IgM producing B cells from immunologically naŁve WT and CB1/CB2 KO mice . Flow cytometry was used to detect B cells (CD19 -PE-Cy7) and IgM (FITC) on splenocytes freshly isolated from mice . Cells were gated sequentially on singlet live lym phocytes, prior to plotting CD19 and IgM for analysis. Columns represent the percentage of IgM + CD19 + cells + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 221 Figure 5.5. Serum IgM concentrations in WT and CB1/CB2 KO mice treated with LPS . Four days following an injection with LPS (25 µg; i.p.), the serum from f emale WT and CB1/CB2 KO mice was collected and assayed for IgM using ELISA . Columns represent the concentration of IgM (ng/ml) + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 222 Figure 5.6. Serum IgG concentrations in WT and CB1/CB2 KO mice treated with LPS . Four days following an injection with LPS (25 µg; i.p.), the serum from f emale WT and CB1/CB2 KO mice was collected and assayed for IgG using ELISA . Columns represent t he concentration of IgG (ng/ml) + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 223 Figure 5.7 . Flow cytometric analysis of splenic IgM producing B cell populations from LPS treated WT and CB1/CB2 KO mice . Lymphocytes were isolated from the spleens of WT and CB1/CB2 KO mice treated with LPS (25 µg; i.p.) 4 days prior and analyzed by flow cytometry as described . IgM (FITC) producing B cells (CD19 -PE-Cy7) reside in the upper right quadrant of the displayed plo ts. Each plot represents the concatenated data from 3 -5 mice. 220 224 Figure 5.8. Flow cytometric analysis of splenic IgM producing B cells from WT and CB1/CB2 KO mice treated with LPS . Flow cytometry was used to detect B cells (CD19 -PE-Cy7) and IgM (FITC) on splenocytes isolated from mice treated with LPS (25 µg; i.p.) 4 days prior . Cells were gated sequentially on singlet live lymphocytes, prior to plotting CD19 and IgM for analysis. Columns represent the percentage of IgM + CD19 + cells + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 225 5.3.2: Elevated !2AR expression on B cells in CB1/CB2 KO mice The underlying hypothesis of this chapter is that CB1/CB2 KO exhibit enhanced humoral immunity due to enhanced activity of splenic sympathetic noradrenergic neurons. CB1/CB2 KO mice were confirmed to have enhanced humoral immunity. Following this, it was hypothesized that B cells from CB1/CB2 KO mice would also express !2AR, as was seen in WT mice ( Chapter 4 ). It was expected that !2AR expression in CB1/CB2 KO mice would be reduced due to activity dependent down -regulation (Zastrow and Kobilka, 1992; Hudson et al., 2010) secondary to an increased rate of NE rele ase and binding to these receptors. Flow cytometry was used to measure the expression of !2AR on B cells and it was discovered that CB1/CB2 KO have a greater population of splenic B cells expressing !2AR ( Figure 5.9 ). These data suggest that either there is an interaction between cannabinoid receptors and !2AR such that the lack of CB1/CB2 allows for increased expression of !2AR on lymphocytes, or CB1/CB2 KO mice have a reduced rate of NE release from splenic sympathetic neurons resulting in up -regulation of !2AR. It was also discovered that CB1/CB2 KO mice possess a larger percentage of !2AR expressing B cells producing IgM when immunologically naŁve ( Figures 5.10 and 5.11) and when immune challenged by LPS ( Figures 5.12 and 5.13 ). Thus, the B cell popula tion in CB1/CB2 KO mice possess an enhanced ability to sense NE, namely increased !2AR expression. Furthermore, this population with enhanced adrenergic sensing capacity is able to produce antibodies. These data, in addition to the 226 hypothesized effect of cannabinoid receptor knockout on NE release, support a role for !2AR activation as the cause for enhanced humoral immunity in CB1/CB2 KO mice. 227 Figure 5.9. Flow cytometric analysis of splenic B cells expressing !2AR from immunologically naive WT and CB1/CB2 KO mice . Flow cytometry was used to detect B cells (CD19 -PE-Cy7) expressing !2AR (APC ) on splenocytes isolated from untreated mice . Cells were gated sequentially on singlet lymphocytes, prior to plotting CD19 and !2AR for analysis. Columns represen t the percentage of !2AR + CD19 + cells + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 228 Figure 5.10 . Flow cytometric analysis of splenic IgM producing B cell populations expressing !2AR from immunologically naŁve WT and CB1/CB2 KO mice . Lymphocytes were isolated from the spleens of untreated WT and CB1/CB2 KO mice and analyzed by flow cytometry as described . Cells were gated sequentially on singlet lymphocytes expressing !2AR (APC) prior to plotting CD19 and IgM for analysis . IgM (F ITC) producing B cells (CD19 -PE-Cy7) reside in the upper right quadrant of the displayed plots . Each plot represents the concatenated data from 3 -5 mice. 225 229 Figure 5.11. Flow cytometric analysis of IgM producing splenic B cells expressing !2AR from immunologically naive WT and CB1/CB2 KO mice . Flow cytometry was used to detect IgM (FITC) producing B cells (CD19 -PE-Cy7) expressing !2AR (APC ) on splenocytes isolated from untreated mice . Cells were gated sequentially on singlet lymphocytes expressing !2AR, prior to plotting CD19 and IgM for analysis. Columns represent the percentage of IgM + !2AR + CD19 + cells + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 230 Figure 5.12 . Flow cytometric analysis of splenic IgM producing B cell populations expressing !2AR from LPS treated WT and CB1/CB2 KO mice . Lymphocytes were isolated from the spleens of WT and CB1/CB2 KO mice treated with LPS (25 µg; i.p.) 4 days prior and analyzed by flow cytometry as described . Cells were gated sequentially on singlet lymphocytes expressing !2AR (APC) prior to plotting CD19 and IgM for analysis . IgM (FITC) producing B cells (CD19 -PE-Cy7) reside in the upper right quadrant of the displayed plots . Each plot represents the concatenated data from 3 -5 mice. 227 231 Figure 5.13. Flow cytometric analysis of IgM producing splenic B cells expressing !2AR from WT and CB1/CB2 KO mice treated with LPS . Flow cytometry was used to detect IgM (FITC) producing B cells (CD19 -PE-Cy7) expressing !2AR (APC ) on splenocytes isolated from WT and CB1/CB2 KO mice treated with LPS (25 µg; i.p.) 4 days prior . Cells were gated sequentially on singlet lymphocytes expressing !2AR , prior to plotting CD19 and IgM for analysis. Columns represent the percentage of IgM + !2AR + CD19 + cells + 1 SEM (n=5 ). * Significantly differs from WT (p<0.05 ). 232 5.3.3: Splenic sympathetic noradrenergic neuronal activity in CB1/CB2 KO mice Following the discovery of enhanced adrenergic sensing capacity of B cells in CB1/CB2 KO mice, the effect of cannabinoid receptor knockout on splenic sympathetic activity was tested. It was hypothesized that these mice will have enhanced splenic noradrenergic neuronal activity due to a lack of the inh ibitory effects of CB1 on sympathetic axon terminals. First, it was observed that CB1/CB2 KO mice have an increased concentration of NE in the spleen capsule compared to WT ( Figure 5.14 ). This data is suggestive of increased noradrenergic neuronal activit y, as increased splenic NE concentrations were previously positively correlated with increased activity (Chapters 3 and 4 ). Despite elevated NE concentrations in the spleen capsule of CB1/CB2 KO mice, TH is not elevated in these mice ( Figure 5.15 ). Previo usly it was observed that increased noradrenergic activity in the spleen capsule was correlated with increased NE concentrations and TH expression ( Chapter 4 ). Therefore, the discordance of these data, in the light of the current hypothesis, was further i nvestigated. To this end, the rate of NE release in CB1/CB2 KO mice was assessed using the aMT method. The results revealed that under immunologically naŁve conditions CB1/CB2 KO mice have a decrease in the rate constant of NE utilization in the splee n capsule, associated with increased NE concentrations ( Figure 5.16 ). When challenged with LPS, there is no significant difference between WT and CB1/CB2 KO with regards to the rate of spleen capsule NE utilization ( Figure 5.17 ). These data argue against a role for CB1 inhibition of NE release in the spleen, and suggest that CB1/CB2 receptors 233 are permissive to a regular rate of NE release as a decrease in NE utilization was observed in the spleen capsule under immunologically naŁve conditions. These data d o not support the hypothesis that increased NE release is the cause for enhance humoral immunity in CB1/CB2 KO mice. 234 Figure 5.14. Spleen capsule NE concentrations in immunologically naŁve WT and CB1/CB2 KO mice . The spleen capsule was collected from untreated female WT and CB1/CB2 KO mice . Spleen capsule samples were prepared for and analyzed by HPLC -ED for NE as described. Columns represent average concentration of NE + 1 SEM (n=6 -8). * Differs from WT (p<0.05). 235 Figure 5.15. Spleen capsu le TH content in immunologically naŁve WT and CB1/CB2 KO mice . The spleen capsule was collected from untreated female WT and CB1/CB2 KO mice . Spleen capsule samples were prepared for and analyzed by Western blot for TH as described. Columns represent av erage normalized amount of TH in the spleen + 1 SEM (n=6 -8). * Differs from WT (p<0.05). 236 Figure 5.16. Spleen capsule noradrenergic neuron activity in immunologically naŁve WT and CB1/CB2 KO mice . Female WT and CB1/CB2 KO mice received a single injection of saline/aMT (300 mg/kg, i.p.) and were sacrificed by decapitation 4 h later. T he spleen capsule was collected and prepared for analysis of NE by HPLC -ED. Non -aMT treated mice were used for the 0 h time point and a regress ion analysis performed to determine the rate of NE utilization. Columns depict the average rate constant of NE utilization or the concentration of NE (ng/mg protein) in non -aMT treated mice + one SEM . * Differs from WT (p<0.05). 237 Figure 5.17. Compar ison of the effects of LPS on s pleen capsule noradrenergic neuron activity in WT and CB1/CB2 KO mice . Female WT and CB1/CB2 KO mice received a single injection of saline/aMT (300 mg/kg, i.p.) immediately followed by single injection of HBSS or LPS (25 µg; i.p.). Mice were sacrificed 4 h after the LPS injection and the spleen capsule collected and prepared analysis of NE by HPLC -ED. Non -aMT treated mice were used for the 0 h time point and a linear regression analysis performed to determine the rate of NE utilization. Columns depict the average rate constant of NE utilization or the concentration of NE (ng/mg protein) in non -aMT treated mice + one SEM . * Differs from WT (p<0.05). 238 5.3.4: Enhanced humoral immunity in CB1/CB2 KO mice is not due to increas ed stimulation of !2AR Despite the finding that CB1/CB2 receptor deficiency does not affect NE release in the spleen, it was still hypothesized that !2AR may play a role in enhanced humoral immunity, based upon the finding of increased !2AR expression by splenic B cells. It was hypothesized that enhanced !2AR stimulation, due to an increased presence of these receptors on B cells, may be the cause of enhanced humoral immunity following knockout of the CB1/CB2 receptors. To test this hypot hesis !2AR were blocked by butoxamine, a !2AR specific antagonist, during an immune challenge by LPS. Serial b utoxamine injections were used to antagonize !2AR for 24 h before and after an immune challenge by LPS. This time frame chosen because it ha d bee n previously published that adrenergic effect s on immunity likely happen within 24 h before or after an immune challenge (Kohm and Sanders, 2001; Sanders, 2012) . Consistent with data presented above, enhancement of humoral immunity in CB1/CB2 KO , as compared with WT mice , was observed in all endpoints ( Figures 5.18 -21). Butoxamine failed to alter serum concentrations of IgM (Figure 5.18 ) or IgG ( Figure 5.19 ) in WT or CB1/CB2 KO mice. Moreover, antagonism of !2AR with butoxamine did not alter the percentage of B cells expressing !2AR (Figure 5.20 ), or the subset of these cells producing IgM ( Figure 5.21) in WT or CB1/CB2 KO mice. These data are inconsistent with the hypothesis that increased !2AR expression and stimulation is responsible for enhanced humoral immunity in CB1/CB2 KO mice. 239 Figure 5.18. Lack of effect of !2AR antagonism on serum IgM concentrations in LPS exposed WT and CB1/CB2 KO mice . Female WT and CB1/CB2 KO mice were injected 8 times with butoxamine (10 mg/kg ; i.p.), or vehicle, every 6 h for 48 h. Mice received a single injection of LPS (25 µg; i.p.) one h prior to the 5 th injecti on and were sacrificed 4 days later . Serum was collected and assayed for IgM using ELISA . Columns represent the concentration of IgM (ng/ml) + 1 SEM (n=5 ). * Significantly differs from WT of the same treatment group (p<0.05 ). 240 Figure 5.19. Lack of effect of !2AR antagonism on serum IgG concentrations in LPS exposed WT and CB1/CB2 KO mice . Female WT and CB1/CB2 KO mice were injected 8 times with butoxamine (10 mg/kg ; i.p.), or vehicle, every 6 h for 48 h. Mice received a single injection of L PS (25 µg; i.p.) one h prior to the 5 th injection and were sacrificed 4 days later . Serum was collected and assayed for IgG using ELISA . Columns represent the concentration of IgG (ng/ml) + 1 SEM (n=5 ). * Significantly differs from WT of the same treatment group (p<0.05 ). 241 Figure 5.20. Lack of effect of !2AR antagonism on the IgM producing B cell population in the spleen of LPS exposed WT and CB1/CB2 KO mice . Female WT and CB1/CB2 KO mice were injected 8 times with butoxamine (10 mg/kg ; i.p.), or vehicl e, every 6 h for 48 h. Mice received a single injection of LPS (25 µg; i.p.) one h prior to the 5 th injection and were sacrificed 4 days later . The splenocytes were isolated and flow cytometry was used to identify B cells (CD19 -PE-Cy7) producing IgM (FITC) . Cells were gated sequentially on singlet lymphocytes, prior to plotting CD19 and IgM for analysis. Columns represent the average percentage of IgM + CD19 + cells + 1 SEM (n=5). * Significantly differs from WT of the same treatment group (p<0.05 ). 242 Figure 5.21. Lack of effect of !2AR antagonism on the IgM producing B cell population expressing !2AR in the spleen of LPS exposed WT and CB1/CB2 KO mice. Female WT and CB1/CB2 KO mice were injected 8 times with butoxamine (10 mg/kg ; i.p.), or vehicle, every 6 h for 48 h. Mice received a single injection of LPS (25 µg; i.p.) one h prior to the 5 th injection and were sacrificed 4 days later . The splenocytes were isolated and flow cytometry was used to identify !2AR (APC) expressing B cells (CD19 -PE-Cy7) producing IgM (FITC) . Cells were ga ted sequentially on singlet lymphocytes expressing !2AR prior to plotting CD19 and IgM for analysis. Columns represent the average percentage of IgM + !2AR + B cells + 1 SEM (n=5). * Significantly differs from WT of the sam e treatment group (p<0.05 ). 243 5.4: Discussion In the present chapter the hypothesis was tested that enhanced humoral immunity in mice lacking the CB1 and CB2 receptor is due to increased stimulation of !2AR secondary to increased release NE from splenic sympathetic neurons. The data presented confirm that CB1/CB2 KO mice do exhibit enhanced humoral immunity. Interestingly, B cells from CB1/CB2 KO mice possess an enhanced adrenergic sensing capacity; they have an increase in B cells expressing !2AR . Ho wever, lack of CB1/CB2 does not result in increased NE release from splenic sympathetic neurons. Increased !2AR stimulation, due to increased !2AR expression, was also ruled out as a potential cause for enhanced humoral immunity in CB1/CB2 KO mice. 5.4.1: Enhanced humoral immunity in CB1/CB2 KO mice Congruent with a previously published report (Springs et al., 2008) , CB1/CB2 KO mice demonstrate enhanced humoral immunity. Increased serum IgM and IgG concentrations suggest several things. IgM is constitutively secreted without antigen stimu lation as a form of innate immunity by innate -like B cells (Baumgarth, 2013) . These IgM antibodies are termed % natural antibodies " . Thus, elevated serum IgM in immunologically naŁve CB1/CB2 KO mice suggests these receptors may be inhibitor y to this specific type of immunity, a finding that is unexplored in scientific literature. However, natural antibodies are reported to be almost exclusively IgM, where as IgG is produced in response to antigenic stimulation (Zouali, 2001) . Therefore, the production 244 of natural antibodies does not explain the elevated serum IgG in immunologically naŁve CB1/CB2 KO mice. Elevated IgG in im munologically naŁve CB1/CB2 KO could have two likely sources. First, it could be due to antigenic stimulation. The only way that this is likely to be the true is if the lack of CB1 and CB2 increases the sensitivity of B cells to foreign antigens. This po ssibility seems unlikely to be the cause for increased IgG, though, as the elevation in IgG is approximately 4 -fold higher compared to WT, whereas under true antigenic stimulation the elevation of antibodies is on the order of log -fold changes (Ademokun and Dunn -Walters, 2001) . The second possible explanation for increased serum IgG is due to an increased presence of long -lived plasma cells. Plasma cells are professional antibody producing B cells produced in response to antigenic stimulation (Ademokun and Dunn -Walters, 2001; Tangye, 2011) . Some plasma cells are short -lived, lasting few days to weeks, but others persist for years thereby conferring long -term immunity (Tangye, 2011) . These long -lived p lasma cells constitutively produce antigen specific IgG designed to speed the removal of antigens upon re -exposure (Tangye, 2011). It may be the case that lack of CB1/CB2 results in an increase in the survival or generation of these cells. This possibility seems likely to be the cause of increased IgG in immunologically naŁve cells as it would likely increase IgG serum concentrations, but not at the level seen with antigenic stimulation. This suggests that CB1 and/or CB2 play a role in the formation of these specialized B cells. The effect of cannabinoids on long -term immunity is also virtually unexplored in the literature with only one report in CB2 245 knockout mice showing no significant effect of plasma cell generation in response to a T-cell dependent antigen (Basu et al., 2013) . Overall, however, it seems clear that CB1 and/or CB2 receptors play a role in the development and maintenance of B cells as CB1/CB2 KO mice have more splenic B cells and B cells that produce antibodies. These effects are also observed when CB1/CB2 KO mice are immune challenged by LPS, likely due to the increased number of B cells at rest resulting in more B cells that prod uce antibodies. This effect may be more pronounced following exposure to LPS, a polyclonal B cell stimulator, as opposed to T cell dependent immunogen requiring specific antigen recognizing T and B cell populations (Ademokun and Dunn -Walters, 2001) . 5.4.2: !2AR expression in CB1/CB2 KO mice Considering the hypothesis that enhanced sympathetic noradrenergic stimulation may be the underlying cause for enhanced humoral responses, it was expected that $ 2AR expression in CB1/CB2 KO mice would be reduced due to ligand/ activity dependent down -regulation (Zastrow and Kobilka, 1992; Hudson et al., 2010) . However, as demonstrated, this is not the case. The observed up -regulation of $2AR in CB1/CB2 KO mice could be due to the absence of cannabinoid receptors on B cells, most likely CB2 given it " s higher expression in B cells compared to CB1 (Schatz et al., 1997) . In support of t his possibility, a recent study demonstrated a physical interaction between CB1 and $2AR , and this interaction was able modulate the surface expression of these receptors (Hudson et al., 2010). Thus, although CB1 and CB2 do not have 100% homology, it might be hypothesized that CB2 may also be able to alter $2AR expression 246 as was shown for CB1. Another potential explanation of increased $2AR is that CB1/CB2 KO mice release less NE than WT in the spleen, resulting in receptor up -regulation due to a lack of ligand -binding (Zastrow and Kobilka, 1992; Hudson et al., 2010). While obviously in direct contradiction to the original underlying hypothesis of this chapter, this explanation for increased $2AR expression turns out to be the most likely explanation given decreased NE utilization in CB1/CB2 KO mice under immunologically naŁve conditions. The increase of $2AR expressing B cells producing IgM also suggests an interaction between CB1 and/or CB2 receptors with $2AR . These data were obtained by gating for cells expressing $2AR then assessing the number of IgM producing B cells. Therefore, if $2AR expression were globally increased on all $2AR -expressing lymphocytes, the relative ratio of each cell type would not change. However, this is not the case. It appears that knockout of CB1/CB2 increases the relative proportion of these IgM producing B cells. These data cannot specifically identify the cause of this shift as both NE signaling and CB signaling are simultaneously changed in these mice . However, at least two possibilities can be considered. First, despite the reported effect of $2AR on the stimulation of antibody production, there is also evidence that $2AR stimulation may decrease proliferation in lymphocytes (Marino and Cosentino, 2011) . Thus, decreased NE release in CB1/CB2 KO mice might preferentially allow for the survival of IgM producing B cells. The second possibility to be considered is a direct effect of C B2 on B cells themselves, which have been show to modulate lymphocyte proliferation (Basu and Dittel, 2011) . Thus, it is possible that CB2 receptor mediated 247 preferential survival/proliferation of IgM producing B cells accounts for these differences. 5.4.3: Splenic sympathetic noradrenergic activity and signaling in CB1/CB2 KO mice The lack of the CB1 and CB2 recep tors does not result in increased NE release from splenic sympathetic neurons. This strongly argues against the existence of CB1 receptors on the axon terminals of spleen projection post -ganglionic sympathetic neurons, although CB1 receptor expression and regulation of neurotransmitter release in post -ganglionic sympathetic neurons is not without precedence (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . Interestingly, the present study revealed that CB1/CB2 KO mice have lower splenic sympathetic noradrenergic activity. The explanation for this effect is unclear and may involve CNS -mediated CB1 receptor effects upstream of post -ganglionic neurons. How ever, the decreased release of NE from sympathetic neurons in the spleen may explain the accumulation of NE in the spleen capsule without a concomitant increase in TH enzyme content. Regardless, the lack of increased NE release in the spleen of CB1/CB2 KO leads to the acceptance of the null hypothesis regarding this explanation of enhanced humoral immunity in CB1/CB2 KO mice. Next, due to the increased $2AR expression by B cells in CB1/CB2 KO mice, it was hypothesized that $2AR activity may still the ca use of enhanced humoral immunity in these mice. It was expected that if increased $2AR stimulation was, at least in part, responsible for increased humoral immune responses in CB1/CB2 KO mice, then blockade of these receptors would significantly attenuate antibody production or reduce 248 the population of antibody producing cells. This hypothesis was rejected following the observation that blockade of $2AR had no observable effect in CB1/CB2 KO mice. These data also confirm what was observed in Chapter 4 , namely that $2AR activity does not play a significant role in the humoral response to LPS. 5.4.4: Conclusion The data in this chapter rule out the involvement of splenic NE release and !2AR in conferring enhanced humoral immunity in CB1/CB2 KO mice. Intere stingly, B cells from CB1/CB2 KO mice possess an enhanced adrenergic sensing capacity. These data, however, do give some clues as to the underlying mechanism of enhanced humoral immunity in CB1/CB2 KO mice, namely increased % natural antibody " production an d increased long -lived plasma cell survival. Future investigation into the immunologic effects of cannabinoi d receptors should be focused towards the effect of CB2 receptors on immune cells themselves, rather than the effect of CB1 modulation of local neur otransmitter release in secondary lymphoid organs, such as the spleen. 249 REFERENCES 250 REFERENCES Ademokun AA, Dunn -Walters D. Immune Responses: Primary and Secondary. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Ahrens WH, Cox DJ, Budhwar G. Use of the arcsine and square root transformations for subjectively determined percentage data. Weed Science. 1990. Alger B, Kim J. Supply and demand for endocannabinoids. Trends Neurosci. 2011 Jun 1;34(6) :304Ð15. Basu S, Dittel BN. Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunol. Res. 2011 Oct;51(1):26 Ð38. Basu S, Ray A, Dittel BN. Cannabinoid receptor 2 is critical for the homing and retention of marginal zone B lineage cells and for efficient T -independent immune responses. The Journal of Immunology. 2011 Dec 1;187(11):5720 Ð32. PMCID: PMC3226756 Basu S, Ray A, Dittel BN. Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen -Specific IgG and IgM, in Response to T -dependent Antigens. PLoS ONE. 2013;8(6):e67587. PMCID: PMC3695093 Baumgarth N. Innate -Like B Cells and Their Rules of Engagement. link.springer.com.proxy 2.cl.msu.edu. New York, NY: Springer New York; 2013. p. 57Ð66. Bırner C, Bedini A, Hıllt V, Kraus J. Analysis of promoter regions regulating basal and interleukin -4-inducible expression of the human CB1 receptor gene in T lymphocytes. Mol Pharmacol. 2008 Mar;73(3):1013 Ð9. Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines. J Pharmacol Exp Ther. 1966 Dec;154(3):493 Ð8. Brown SM, Wager -Miller J, Mackie K. Cloning and molecular characterization of the rat CB2 cannabinoid receptor. Biochim Biophys Acta. 2002 Jul 19;1576(3):255 Ð64. Buckley N, McCoy K, Mezey E, Bonner T, Zimmer A, Felder C, et al. Immunomodulation by cannabinoids is absent in mic e deficient for the cannabinoid CB(2) receptor. Eur J Pharmacol. 2000 May 19;396(2 -3):141 Ð9. Cabral GA, Griffin -Thomas L. Emerging role of the cannabinoid receptor CB2 in immune regulation: therapeutic prospects for neuroinflammation. Expert Rev Mol Med. 2009;11:e3. PMCID: PMC2768535 251 Carrier EJ, Kearn CS, Barkmeier AJ, Breese NM, Yang W, Nithipatikom K, et al. Cultured rat microglial cells synthesize the endocannabinoid 2 -arachidonylglycerol, which increases proliferation via a CB2 receptor -dependent mecha nism. Mol Pharmacol. 2004 Apr;65(4):999 Ð1007. Childers SR, Deadwyler SA. Role of cyclic AMP in the actions of cannabinoid receptors. Biochem Pharmacol. 1996 Sep 27;52(6):819 Ð27. Eaton MJ, Lookingland KJ, Moore KE. Effects of the selective dopaminergic D2 agonist quinelorane on the activity of dopaminergic and noradrenergic neurons projecting to the diencephalon of the rat. 1994 Feb 1;268(2):645 Ð52. Felder CC, Briley EM, Axelrod J, Simpson JT, Mackie K, Devane WA. Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor -mediated signal transduction. Proc Natl Acad Sci USA. 1993 Aug 15;90(16):7656 Ð60. PMCID: PMC47201 Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, et al. Compa rison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol. 1995 Sep;48(3):443 Ð50. Ferrero I, Michelin O, Luescher I. Antigen Recognition by T Lymphocytes. onlinelibrary.wiley.com.proxy2.cl.msu.edu. Chi chester, UK: John Wiley & Sons, Ltd; 2001. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, et al. Expression of Central and Peripheral Cannabinoid Receptors in Human Immune Tissues and Leukocyte Subpopulations. Eur J Biochem. 1995 Aug; 232(1):54 Ð61. Gebremedhin D, Lange AR, Campbell WB, Hillard CJ, Harder DR. Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L -type Ca2+ channel current. Am J Physiol. 1999 Jun;276(6 Pt 2):H2085 Ð93. Gerald T, Ward G, Howlett A , Franklin S. CB1 knockout mice display significant changes in striatal opioid peptide and D4 dopamine receptor gene expression. Brain Res. 2006 Jun 6;1093(1):20 Ð4. Gerra G, Zaimovic A, Gerra ML, Ciccocioppo R, Cippitelli A, Serpelloni G, et al. Pharmacol ogy and toxicology of Cannabis derivatives and endocannabinoid agonists. Recent Pat CNS Drug Discov. 2010 Jan;5(1):46 Ð52. Ghosh S, Preet A, Groopman JE, Ganju RK. Cannabinoid receptor CB2 modulates the CXCL12/CXCR4 -mediated chemotaxis of T lymphocytes. Mo l Immunol. 2006 Jul;43(14):2169 Ð79. Giuffrida A, Mcmahon LR. In vivo pharmacology of endocannabinoids and their 252 metabolic inhibitors: therapeutic implications in Parkinson's disease and abuse liability. Prostaglandins and Other Lipid Mediators. 2010 Apr 1 ;91(3 -4):90 Ð103. Gohary ME, Eid MA. Effect of cannabinoid ingestion (in the form of bhang) on the immune system of high school and university students. Human & experimental toxicology. 2004. Greineisen WE, Turner H. Immunoactive effects of cannabinoids: considerations for the therapeutic use of cannabinoid receptor agonists and antagonists. Int Immunopharmacol. 2010 May;10(5):547 Ð55. PMCID: PMC3804300 Griffin G, Tao Q, Abood ME. Cloning and pharmacological characterization of the rat CB(2) cannabinoid rec eptor. J Pharmacol Exp Ther. 2000 Mar;292(3):886 Ð94. Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. 1991 Feb 1;11( 2):563 Ð83. Howlett AC, Mukhopadhyay S. Cellular signal transduction by anandamide and 2 -arachidonoylglycerol. Chemistry and Physics of Lipids. 2000 Nov;108(1 -2):53 Ð70. Hudson BD, H”bert TE, Kelly MEM. Physical and functional interaction between CB1 canna binoid receptors and beta2 -adrenoceptors. Br J Pharmacol. 2010 Jun;160(3):627 Ð42. PMCID: PMC2931563 Ishac E, Jiang L, Lake K, Varga K, Abood M, Kunos G. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves. Br J Pharmacol. 1996 Aug 1;118(8):2023 Ð8. PMCID: PMC1909901 Jan T -R, Farraj AK, Harkema JR, Kaminski NE. Attenuation of the ovalbumin -induced allergic airway response by cannabinoid treatment in A/J mice. Toxicol Appl Pharmacol. 2003 Apr 1;188(1):24 Ð35. Jarai Z, Wagner J, Varga K, Lake K, Compton D, Martin B, et a l. Cannabinoid -induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14136 Ð41. Kaplan BLF. The Role of CB(1) in Immune Modulation by Cannabinoids. Pharmacol. Ther. 2012 Dec 19. Kaplan BLF, Lawver JE, Karmaus PWF, Ngaotepprutaram T, Birmingham NP, Harkema JR, et al. The effects of targeted deletion of cannabinoid receptors CB1 and CB2 on intranasal sensitization and challenge with adjuvant -free ovalbumin. Toxicol Pathol. 2010 Apr;38(3):382 Ð92. PMCID: PMC2941344 253 Karmaus PWF, Chen W, (null), Kaplan BLF, Kaminski NE. & 9-Tetrahydrocannabinol Impairs the Inflammatory Response to Influenza Infection: Role of Antigen -Presenting Cells and the Cannabinoid Receptors 1 and 2. Toxico logical Sciences. 2013. PMCID: PMC3551428 Karmaus PWF, Chen W, Kaplan BLF, Kaminski NE. & 9-tetrahydrocannabinol suppresses cytotoxic T lymphocyte function independent of CB1 and CB 2, disrupting early activation events. J Neuroimmune Pharmacol. 2012 Dec;7( 4):843 Ð55. PMCID: PMC3266990 Klein TW, Friedman H, Specter S. Marijuana, immunity and infection. J Neuroimmunol. 1998 Mar 15;83(1 -2):102 Ð15. Kohm AP, Sanders VM. Norepinephrine and beta 2 -adrenergic receptor stimulation regulate CD4+ T and B lymphocyte fu nction in vitro and in vivo. Pharmacol Rev. 2001 Dec;53(4):487 Ð525. Kuster JE, Stevenson JI, Ward SJ, D'Ambra TE, Haycock DA. Aminoalkylindole binding in rat cerebellum: selective displacement by natural and synthetic cannabinoids. J Pharmacol Exp Ther. 1 993 Mar;264(3):1352 Ð63. Lindley SE, Gunnet JW, Lookingland KJ, Moore KE. 3,4 -Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activit y. Brain Res. 1990 Jan 1;506(1):133 Ð8. Lu H, Kaplan BLF, Ngaotepprutaram T, Kaminski NE. Suppression of T cell costimulator ICOS by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2009 Feb;85(2):322 Ð9. PMCID: PMC2631366 Mackie K. Signaling via CNS cannabinoid receptors. Mol. Cell. Endocrinol. 2008 Apr 16;286(1 -2 Suppl 1):S60 Ð5. PMCID: PMC2435200 Mackie K, Devane WA, Hille B. Anandamide, an endogenous cannabinoid, inhibits calcium currents as a partial agonist in N18 neuroblastoma cells. Mol Pharmacol. 1993 Sep ;44(3):498 Ð503. Mackie K, Lai Y, Westenbroek R, Mitchell R. Cannabinoids activate an inwardly rectifying potassium conductance and inhibit Q -type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor. J Neurosci. 1995 Oct;15(10): 6552Ð61. Maresz K, Pryce G, Ponomarev ED, Marsicano G, Croxford JL, Shriver LP, et al. Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells. Nat. Med. 2007 Apr;13(4):492 Ð7. 254 Marino F , Cosentino M. Adrenergic modulation of immune cells: an update. Amino Acids. 2011 Dec 8. Montecucco F, Burger F, Mach F, Steffens S. CB2 cannabinoid receptor agonist JWH -015 modulates human monocyte migration through defined intracellular signaling pathw ays. Am J Physiol Heart Circ Physiol. 2008 Mar;294(3):H1145 Ð55. Niederhoffer N, Szabo B. Effect of the cannabinoid receptor agonist WIN55212 -2 on sympathetic cardiovascular regulation. Br J Pharmacol. 1999 Jan;126(2):457 Ð66. Noble J, Bailey M. Quantitati on of protein. Methods Enzymol. 2009;463:73 Ð95. Ny™ri G, Cser”p C, Szabadits E, Mackie K, Freund TF. CB1 cannabinoid receptors are enriched in the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons. Neuroscience. 2005;136(3):8 11Ð22. Pacifici R, Zuccaro P, Pichini S, Roset PN, Poudevida S, Farr” M, et al. Modulation of the immune system in cannabis users. JAMA. 2003 Apr 16;289(15):1929 Ð31. Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: &9-tetrahydrocannabinol, cannabidiol and & 9-tetrahydrocannabivarin. Br J Pharmacol [Internet]. 2008 Jan;153(2):199 Ð215. Retrieved from: http://onlinelibrary.wiley.com.proxy2.cl.msu.edu/doi/10.1038/sj.bjp.0707442/full. PMCID: PMC2219532 Raborn ES, Marciano -Cabral F, Buckley NE, Martin BR, Cabral GA. The cannabinoid delta -9-tetrahydrocannabinol mediates inhibition of macrophage chemotaxis to RANTES/CCL5: linkage to the CB2 receptor. J Neuroimmune Pharmacol. 2008 Jun;3(2):117 Ð29. PMCID: PMC2677557 Ralevic V, Ke ndall DA. Cannabinoids inhibit pre - and postjunctionally sympathetic neurotransmission in rat mesenteric arteries. Eur J Pharmacol. 2002 May 31;444(3):171 Ð81. Sanders VM. The beta2 -adrenergic receptor on T and B lymphocytes: Do we understand it yet? Brain Behav Immun. 2012 Feb;26(2):195 Ð200. PMCID: PMC3243812 Schatz A, Lee M, Condie R, Pulaski J, Kaminski N. Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Pharmacol. 1997 Feb 1;142(2):278 Ð87. Schatz AR, Koh WS, Kaminski NE. Delta 9 -tetrahydrocannabinol selectively inhibits T -cell dependent humoral immune responses through direct inhibition of accessory T -cell function. Immunopharmacology. 1993 Sep;26(2):129 Ð37. 255 Shire D, Calandra B, Rinaldi -Carmona M, Oustric D, Pess‘gue B, Bonnin -Cabanne O, et al. Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor. Biochim Biophys Acta. 1996 Jun 7;1307(2):132 Ð6. Springs AEB, Karmaus PWF, Crawford RB, Kaplan BLF, Kaminski NE. Effects of targeted deletion of cannabinoid receptors CB1 and CB2 on immune competence and sensitivity to immune modulation by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2008 Dec;84(6):1574 Ð84. PMCID: PMC2614598 Tana sescu R, Constantinescu C. Cannabinoids and the immune system: an overview. Immunobiology. 2010 Aug 1;215(8):588 Ð97. Tangye SG. Staying alive: regulation of plasma cell survival. Trends Immunol. 2011 Dec;32(12):595 Ð602. Tashkin DP. Effects of marijuana s moking on the lung. Ann Am Thorac Soc. 2013 Jun;10(3):239 Ð47. Zastrow von M, Kobilka BK. Ligand -regulated internalization and recycling of human beta 2 -adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem. 1992 Feb 15;267(5):3530 Ð8. Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci USA. 1999 May 11;96(10):5780 Ð5. PMCID: PMC21937 Zou ali M. Natural Antibodies. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. 256 Chapter 6: Sympathetic nervous system control of spleen contraction and the role of CB1/CB2 signaling 6.1: Introduction Contraction of smooth muscle in the spleen serves two hematologic purposes. First, the spleen acts as a reservoir for RBC (Cesta, 2006) . Due to the organization of the spleen , contraction of the spleen capsule reduces its overall size and volume (Davies and Withrington, 1973; Sandler et al., 1984; Cesta, 2006; Richardson et al., 2009; Seifert et al., 2012) . This contraction expels the cellular co ntents of the spleen into the general circulation leading to an increased hematocrit of the blood . Contraction of the spleen can increase the blood hematocrit by as much as 5% in humans (Sandler et al., 1984; Bakovic et al., 2005; Richardson et al., 2007; 2009) , 10% in rats (Kuwahira et al., 1999) , and 16% in dogs (Sato et al., 1995) , thereby increasing the oxygen carrying capacity of the blood. This is considered the primary function of spleen contraction and has been primarily studied in the context of hypoxia (Sandler et al., 1984; Sato et al., 1997; Kuwahira et al., 1999; Bakovic et al., 2003; Richardson et al., 2007; 2009). In addition to being a reservoir for RBC, the spleen is the largest secondary lymphoid organ and contains a large number of immune cells (macrophages, leukocytes, and lymphocytes) (Cesta, 2006) . These cells are also released during spleen contraction (Seifert et al., 2012) , but the consequences of this inc reased leukocyte/lymphocyte content in the blood are essentially unknown. 257 The second role for spleen contraction is to regulate blood flow through the spleen. Blood enters the spleen via the splenic artery which the n branches into a number of trabecular arteries which eventually enter the red pulp of the spleen where they are termed central arterioles and are surrounded by lymphoid tissue, PALS (Cesta, 2006). Branches from central arterioles have several destinations including capillary beds within the white pulp (marginal zone) and the red pulp of the spleen (Schmidt et al., 1985; Satodate et al., 1986; Schmidt et al., 1993) . Blood that flows through the white pulp marginal zone and directly into venous sinuses is considered the ! fast " pathway (Cesta, 2006) . As much as 90% of splenic blood flow travels through the ! fast " pathway (Schmidt et al., 1985; 1993; Cesta, 2006) . Alternatively , the !slow " pathway is blood that enters the reticular meshwork of the red pulp (Schmidt et al., 1993; Mebius and Kraal, 2005) . The reticular meshwork of the red pulp is composed of reticular fibers, reticular cells, and macrophages and is the area where macrophages actively phagocytize dead and damaged erythrocytes (Saito et al., 1988; Cesta, 2006) . Macrophages within the red pulp of the spleen are also constantly exposed to foreign particulate s in the blood (Cesta, 2006) . Blood flow through the spleen is regulated by both arteriolar/capillary endothelial cells and red pulp reticular cell contraction (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988; Groom et al., 1991) . Contraction of reticular cells surrounding capillaries and capillary endothelial cells in the red pulp serve to decrease blood flow to the reticular meshwork of the red pulp and shuttl e more blood through the ! fast " pathway (Blue and Weiss, 1981; Groom et al., 1991) . Contraction of r eticular cells in the red pulp 258 likely further prevent blood from entering this area (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988) . Contraction of red pulp reticular cells also participates in ejecting the cells contai ned within this compartment (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988) . The spleen capsule is composed of three major tissues : connective tissue, elastic tissue, and smooth muscle (Cesta, 2006) . Immunohistochemical detection of smooth muscle specific myosin is demonstrative of the large amount of smooth muscle contained within the spleen capsule (Davies and Withrington, 1973; Pinkus et al., 1986; Cesta, 2006) . Cont ractile elements are also observed in trabeculae of connective tissue that penetrate the spleen , in the periarteriolar meshwork of the white pulp in a circumferential pattern around incoming vessels , and in the reticular meshwork of the red pulp arranged i n an orderly fashion parallel to the long -axis of the spleen (Blue and Weiss, 1981; Pinkus et al., 1986) . Therefore, contraction of the spleen include s not only the contraction of spleen capsule s mooth muscle, but also a network of contractile component s throughout the spleen. Spleen contraction is controlled by the sympathetic nervous system. NE activation of !1AR, specifically the # 1BAR subtype , mediate s much of sympathetically induce d spleen co ntraction (Gillespie and Hamilton, 1966; Eltze, 1996; Aboud et al., 2012). Activation of !1AR results in the activation of phospholipase C and generation of IP3 which then lead s to the release and elevation of intracellular Ca 2+ and contraction of smooth muscle (Bootman et al., 2001; Malbon and Wang, 2001) . In congruence with the ability of NE to stimulate splenic smooth muscle contraction, noradrenergic fibers 259 and axon terminals densely innervate the spleen capsule and periarteriolar regions of the spleen ( Chapter 3 )(Davies and Withrington, 1973; Blue and Weiss, 1981; Pinkus et al., 1986; Felten et al., 1987; Felten and Olschowka, 1987; Saito et al., 1988; Elenkov and Vizi, 1991) . Co-localized neurotransmitters in post -ganglionic sympathetic neurons other than NE also contribute to spleen contraction. NPY and ATP are commonly recognized neurotransmitters that are co -released from sympathetic neurons (Macarthur et al., 2011). NPY is a 36 amino acid peptide expressed by many post -ganglionic sympathetic neurons, including those projecting to the spleen, kidney, and mesentery (Lu ndberg et al., 1990; Romano et al., 1991; Chevendra and Weaver, 1992) . NPY is able to induce the contraction of vascular smooth muscle through Y1 NPY receptors (Westfall et al., 1987; 1990; Zukowska -Grojec et al., 1996; Michel et al., 1998; Wiest et al., 2006) . NPY also has pre -junctional effects, mediated by Y2 receptors, whereby it can autoregulate sympathetic neurotransmitter release (Westfall et al., 1987; 1990; Michel et al., 1998) . Only one study has reported NPY capable of inducing spleen contraction, albeit very weakly in comparison to NE (Corder et al., 1987) . ATP released from sympathetic neurons is also able to induce the contraction of vascular smooth muscl e in a variety of tissues (i.e., vas deferens, aorta, splenic nerve) through the P2X receptor (Sedaa et al., 1990; Ren and Burnstock, 1997; Burnstock and Ralevic) . Extracellular ATP is quickly converted to adenosine which is then know n to inhibit NE release from sympathetic neurons via an action on pre -synaptic A1 adenosine receptors, a G-coupled protein that signals through the G i/o pathway (Kubo and Su, 260 1983; Wennmalm et al., 1988; Kgelgen et al., 1992; Rongen et al., 1996; Ralevic, 2009; Macarthur et al., 2011; B urnstock and Ralevic) . Interestingly, there is some precedence that activation of A1 receptors can stimulate vascular smooth muscle contraction in the spleen (Fozard and Milavec -Krizman, 1993; Tawfik et al., 2005) . To date, the effect of cannabinoids on spleen contraction has not been reported in the scie ntific literature. Yet, the ability of CB1 receptors to modulate neurotransmitter release from other peripheral sympathetic neurons suggests a potential effect (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . It was originally hypothesized that NE releas e from sympathetic neurons in the spleens of CB1/CB2 KO mice would be increased due to the absence of pre -synaptic CB1 receptors. However, this hypothesis was not supported by the data presented in Chapter 5 which showed that NE release in the spleen of C B1/CB2 KO mice is not elevated as compared with WT mice. With this discovery it was then hypothesized that spleen contraction would be enhanced in CB1/CB2 KO mice secondary to an increase in # 1AR expression from decreased NE release. A decrease in NE rel ease, and therefore NE binding to # 1AR mediating spleen contraction, would result in increased expression of this receptor due to a lack of ligand -binding/activity dependent receptor internalization (Leeb -Lundberg et al., 1987; Malbon and Wang, 2001) . The purpose of this chapter is to test the hypothesis that spleen contraction is enhanced in CB1/CB2 KO in response to exogenously applied NE and electrically evoked neurotransmitter release. To this end, an experimental apparatus was employed in which the ability of isolated spleen to pull on a nylon string attached to an isometric 261 force transducer ex vivo . This method was used to test the contractile response of spleens from both WT and CB1/CB2 KO mice to exogenous NE and EFS -induced neurotransmitter release from s plenic sympathetic axon terminals. 6.2: Materials and Methods 6.2.1: Mice C57BL/6 WT female mice (NCI/Charles River, Portage, MI) and female CB1/CB2 KO mice were used in all experiment s unless otherwise indicated. CB1/CB2 KO mice, on a C57BL/6 background, were created by Dr. Andreas Zimmer at the University of Bonn, Germany as previously described (Jarai et al., 1999; Zimmer et al., 1999; Buckley et al., 2000; Gerald et al., 2006) . CB1/CB2 KO mice for these studies were obtained from Drs. Norbert Ka minski and Barbara Kaplan who maintain a breeding colony of CB1/CB2 KO mice at Michigan State University. All animals were housed two to five per cage and maintained in a sterile, temperature (22 ± 1 ¡C) and light controlled (12L:12D) room, and provided wi th irradiated food and bottled tap water ad libitum. All experiments used the minimal number of animals required for statistical analyses, minimized suffering, and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Michigan State Institutional Animal Care and Use Committee approved all drug administrations and methods of euthanasia (AUF# 03/12$060$00). 262 6.2.1.1: CB1/CB2 KO Mouse Genotyping PCR was used to confirm knockout of CB1 and CB2 rece ptor genes. Genomic DNA was isolated from ~0.5 cm tail snips using 100 µl DirectPCR Lysis Reagent (Viagen Biotech, Los Angeles, CA) plus 0.1 mg/ml proteinase K. Samples were incubated overnight at 55¡C followed by a 45 min incubation at 85¡C. Crude DNA ext ract was obtained following centrifugation at 300 RCF for 1 min. One µl of extract was used in a Taqman PCR reaction using Cnr1 stock primers (CB1 receptor gene) or Cnr2 (CB2 receptor gene) custom primers (Life Technologies/Applied Biosystems, Foster City, CA) (Kaplan et al., 2010) . PCR primers for CB1 receptors were forward 5"-AGGAGCAAGGACCTGAGACA -3" , reverse 5"-GGTCACCTTGGCGATCTTAA -3" , for CB2 receptor were forward 5 "-CCTGATAGGCTGGAAGAAGTATCTAC -3 " , reverse 5 "-ACATCAGCCTCTGTTTCTGTAACC -3 " , neomycin cassette primers were forward 5 "-ACCGCTGTTGACCGCTACCTATGTCT -3 " , and reverse 5 "-TAAAGCGCATGCTCCAGACTGCCTT -3 " . The average ± standard deviatio n Ct values for Cnr1 and Cnr2 in WT mice were 25.0 ± 0.54 and 26.0 ± 1.22, respectively. All samples obtained from CB1/CB2 KO tail snips resulted in an ÒundeterminedÓ Ct value, indicating lack of expression of both Cnr1 and Cnr2 . 6.2.2: Materials DPCPX: D PCPX (Sigma) was dissolved in DMSO to a concentration of 100 % M and used at a final concentration of 100 nM. 263 Krebs bicarbonate buffer: NaCl 120.0 mM, KCl 5.5 mM, CaCI 2 2.5 mM, NaH 2PO4 1.2 mM, MgCl 2 1.2 mM, NaHCO 3 20.0 mM, and glucose 11.0 mM in ultra -pure H2O. BIBP3226: BIBP3226 (Tocris) was dissolved in DMSO to a concentration of 1 mM and used at a final concentration of 1 % M. NE: NE (Cat# A7257, Sigma) was dissolved in RPMI media to a concentration of 1 mM and used at a final concentration between 20 -80 %M. For spleen contraction studies, NE was dissolved in less than 20 ml of Krebs bicarbonate buffer that was acidified by a single drop of concentrated (18 M) HCl to a concentration of 1 mM. Paraformaldehyde: 4 % Paraformaldehyde, buffered with 0.1 M phosphate at pH 7.4, was made by combining 1:1 an 8% paraformaldehyde stock solution prepared from prills (Sigma) and a 0.2 M phosphate buffer (pH 7.4) followed by adjustment with either sodium hydroxide or concentra ted phosphoric acid. Prazosin: Prazosin (Sigma) was dissolved in DMSO to concentration of 1 mM and used at a final concentration of 1 % M. PPADS: PPADS (Sigma) was dissolved in H2O to a concentration of 10 mM and used at a final concentration of 10 % M. 264 TTX: TTX ( Sigma) was dissolved in H2O at a concentration of 0.3 mM and used at a final concentration of 0.3 % M. 6.2.3: Isolation of the Spleen Capsule After euthanasia spleens were removed by an incision in the left lateral abdomen under sterile conditions, which entails spraying the area of removal with 70% ethanol and using ethanol cleaned scissors and forceps to cut through the skin, and underlying muscle and connect ive tissue . The spleen was placed in a 6 -well plate and mechanically crushed with the blunt end of a 10 ml syringe in 2 ml of HBSS to separate the spleen capsule (insoluble tissue) from the splenocytes (contained in the disruption supernatant). The spleen capsule was removed from the supernatant using forceps and taken whole or divided into two parts using ethanol -cleaned scissors depending on the needs of the experiment. 6.2.4: Western Blot All samples were placed in ice -cold lysis buffer (water contain ing 1% Triton -x 100, 250 mM sucrose, 50 mM NaCl, 20 mM tris -HCl, 1 mM EDTA, 1 mM PMSF protease inhibitor cocktail, 1 mM DTT) immediately following isolation and kept frozen at -80¡C until analysis. On the day of analysis samples were thawed, heated for 30 min at 100¡ C, sonicated for 8 sec, and spun at 12,000 RCF for 5 min. The supernatant was collected and a BCA protein assay performed (Noble and Bailey, 2009) . Equal amounts of protein were separated using SDS -PAGE and transferred to PVDF -FL membranes (Millipore, 265 Biller ica, MA). The resulting membranes were reacted against antibodies for smooth muscle #-actin (CP47 1:5000, Millipore, Billerica, MA), or # 1AR (A270 1:400, Sigma) whose intensities were normalized to GAPDH (G8795, 1:2000, Sigma) to account for loading variability. Each PVDF -FL membrane contained samples representing all experimental conditions to avoid variability due to run, transfer, or antibo dy exposure conditions. Blots were visualized and quantified using an Odyssey Fc Infrared Imaging system (Li -Cor, Lincoln, NE) by utilization of IRDye conjugated secondary antibodies (goat an ti-Mouse 800CW (1:20,000) or goat anti -rabbit 680LT (1:20,000) ) a nd/or HRP -conjugated anti -rabbit antibodies (1:5000; Cell Signaling) visualized using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific, Rockford, IL). 6.2.5: Spleen Contraction Studies 6.2.5.1: Preparation of Spleen Tissue for Splee n Contraction Studies Spleens were obtained from mice euthanized by placing them in a chamber with an isoflurane soaked pad until respiration ceased as death by cervical dislocation results in less contractile responses and necessitates prolonged equilibr ation of the tissue (Ignarro and Titus, 1968; Wong, 1990; Eltze, 1996) . Spleens were removed and connect ed via loops of nylon string to an isometric force transducer (Radnoti, Monrovia, CA) and placed in a 20 -ml organ bath (Norman D. Erway Glass Blowing) under a resting tension of 0.8 g (7.84 millinewtons [mN] ) for recording isometric contractile responses i n Krebs bicarbonate buffer maintained at 37¡C and gassed with 95% 0 2 - 5% CO 2. 266 6.2.5.2: Spleen Contraction Measurement Isometric contractions were recorded in response to EFS or direct injection of NE into the bath medium. EFS was produced by two ~1 cm custom built platinum ring electrodes placed above and below the spleen connected to a Grass S48 Stimulator (Grass Technologies, Warwick, RI). All spleens were simultaneously stimulated using a Med -Lab Stimu -Splitter II (Med -Lab Instruments). The EFS consi sted of square wave pulses 0.2 -0.25 ms in duration with the following characteristics: 30 V, 25 hertz (Hz), and 3 s train duration. Prior to any testing, each spleen was given at least 45 min of undisturbed time to acclimate to the ex vivo environment. Increased tension on the nylon string from spleen contraction resulted in deflection of a post on the isometric force transducer, the deflection of which is read as a change in voltage. This change in voltage is converted to grams at a rate of 0.28 V equaling 1 gram. All data was then converted to and expressed as mN by multiplying the gram data output by 9.8 m/s 2 and then multiplying by 1000. 6.2.6: Spleen Capsule Width Measurement 6.2.6.1: Hematoxylin and Eosin Staining Mice were given a lethal dose of ketamine:xylazine (244 mg/kg:36 mg/kg; i.p.) and the spleen removed and dropped fixed in 4% paraformaldehyde. Fixed spleens were taken to the Michigan State University Department of Pathology Histology Laboratory where they were paraffin embedded, sectioned at 4 % m, and stained with hematoxylin and eosin using standard histological methodology. 267 6.2.6.2: Quantification of Spleen Capsule Thickness Stained spleen sections were imaged at 40x magnification using a Nikon TE2000 -S Inverted Microscope (Nikon, Melville, NY) with a Spot Insight QE camera (SPOT Imaging, Sterling Heights, MI) with SPOT 3.5.8 Imaging software (SPOT Imaging). Each spleen was imaged, focused on the spleen capsule, two times at random locations on both the internal and external side of the spleen. ImageJ software (National Institutes of Health, USA) was used to measure the width of the spleen capsule, in pixels, at four evenly spaced locations on each image. All 12 values from each spleen were then averaged and this number used as a single n for comparison between genotypes. 6.2.7: Statistical Analysis 6.2.7.1: Statistical Comparisons Prism software version 4.0a was used to make statistical comparisons between groups using the appropriate statistical test. Differences with a probability of error of less than 5% (p<0.05) were considered statistically significant. Two group comparisons were done using the Student " s t-test. Two group comparisons where in one group had more than one degree or factor were done usi ng a One -way ANOVA followed by a Bonferroni or Tukey " s post -test for multiple comparisons. Experiments in which there were two groups with more than one degree or factor in each group, such as a 2x2 design, were analyzed using a Two -way ANOVA followed by B onferroni post -test for multiple 268 comparisons. Consideration for repeated measurements, as in spleen contraction studies, was given in analysis where appropriate. 6.3: Results 6.3.1: Comparison of NE -induced spleen contraction in WT and CB1/CB2 KO mice Spleen contraction can be induced ex vivo in a dose dependent manner by the exogenous application of NE ( Figure 6.1 ). The effective NE concentrations ranged from minimal induction of spleen contraction at 1 nM to a large induction at 1 µM, which is in cohe rence with previous published reports (Ignarro and Titus, 1968; Takano, 1969; Eltze, 1996; Aboud et al., 2012 ). Also in agreement with previous published reports, NE -induced spleen contraction was blocked by the !1AR antagonist prazosin ( Figure 6.2 ) (Cambridge and Davey, 1977; Eltze, 1996; Aboud et al., 2012) . An initial experiment testing the effect of repeated NE -induced stimulation of spleen capsule contraction found rapid develop ing desensitization at concentrations & 100 nM ( Figure 6.3 ). In a follow -up experiment, this phenomenon was found to occur at concentrations >25 nM NE ( Figure 6.4 ). This desensitizing response to high -level stimulation of !1AR is post -synaptic in origin, an d likely due to receptor desensitization by phosphorylation and internalization within smooth muscle (Garcõ !a-S⁄inz et al., 2000) . Thus, subsequent expe riments used '25 nM NE to avoid agonist -induced desensitization. 269 Figure 6.1. Concentrations response of NE -induced spleen contraction. A single spleen was removed, hung in a physiological bath, and connected to an isometric force transducer. NE was added directly to the bath medium and the resulting force of contraction was recorded to incremental NE concentrations. Columns represent the force of contra ction in a single spleen to increasing concentrations of NE. 270 Figure 6. 2. Blockade of NE-induced spleen contraction by prazosin . A single spleen was removed, hung in a physiological bath, and connected to an isometric force transducer. Prazosin (1.0 µM) was added 10 min before the direct addition of NE (1 % M) to the bath medium, and the resulting force of contraction was recorded. Columns represent the force of contraction in a single spleen to NE. 271 Figure 6.3. Spleen contraction s induced by repeate d administration of 10, 100 or 500 nM NE. Spleens were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction in response to repeated administration of increasing NE concentrations was assessed. NE w as added 5 times in a row, with a 3 -min wash between, for each concentration. The order of testing was from low [NE] to high [NE]. Data points represent the average force (mN) of each NE addition (in consecutive order by trial) ± one SEM (n=2). 272 Figure 6.4. Spleen contraction s induced by repeated administration of 10 -100 nM NE. Spleens were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction in response to repeated administrations of incr easing NE concentrations was assessed. NE was added 5 times in a row, with a 3 -min wash between, for each concentration. The order of testing was from low [NE] to high [NE]. Data points represent the average force (mN) of each NE addition (in consecutive o rder by trial) ± one SEM (n=2). 273 Once it was established that NE could produce effective and reproducible spleen contractions ex vivo, NE-induced spleen contraction was assessed in CB1/CB2 KO mice. The working hypothesis was that s pleen contraction in CB1/CB2 KO mice would be enhanced because of increased # 1-adrenergic receptor expression secondary to chronically decreased spleen capsule noradrenergic sympathetic neuron activity (Chapter 5 ). Contrary to prediction, spleens from CB1/CB2 KO mice produced less forceful contractions in response to exogenous NE than those obtained from WT mice (Figure 6.5 ). The force of contraction was normalized to the weight of the spleen to control for potential differences in the absolute size and muscle mass. The spleens from CB1/CB2 KO mice also produce less force per contraction after weight normalization (Figure 6.6 ). It should be noted, too, that there is not a significant difference in spleen weight between WT and CB1/CB2 KO mice ( Figure 6.7 ). These data suggest that the congenital lack of CB1 and/or CB2 receptors produces a deficit in the ability of the spleen to contract in response to NE. 274 Figure 6.5. Comparison of NE -induced spleen contraction force in WT and CB1/CB2 KO mice. Spleens were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens in response to 25 nM NE was measured in WT and CB1/CB2 KO mice. Columns represent the average force (mN) of contraction + one SEM (n=3). * Dif fers from WT (p<0.05). 275 Figure 6.6. Comparison of NE -induced weight -normalized spleen contraction force in WT and CB1/CB2 KO mice. Spleens were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contract ion generated by spleens in response to 25 nM NE was measured and normalized to the weight of the spleen in WT and CB1/CB2 KO mice. Columns represent the average weight -normalized force (mN) of contraction + one SEM (n=3). * Differs from WT (p<0.05). 276 Figure 6.7. Spleen weight comparison in WT and CB1/CB2 KO mice. Spleens were removed and weighed. Columns represent the average weight (mg) + one SEM (n=6). 277 Reduced spleen contractibility in CB1/CB2 KO mice was not hypothesized to be due to a differen ce in the amount of smooth muscle in the spleen, as this would be expected to decrease the overall weight of the spleen. However, upon histological analysis, the thickness of the spleen capsule was significantly smaller in CB1/CB2 KO mice ( Figures 6.8 and 6.9). A possible explanation for reduced spleen capsule thickness is a reduction in smooth muscle content. Therefore, the amount of smooth muscle in the spleen capsule was evaluated by Western blot determination of the smooth muscle specific form of !-acti n. Smooth muscle !-actin content in the spleen capsule of CB1/CB2 KO mice was not different than that of WT mice ( Figure 6.10 ). With this data it was concluded that differences in the smooth muscle content of the spleen capsule was not a contributory fact or to reduced spleen contractility in CB1/CB2 KO mice. It was hypothesized that reduced !1AR expression might contribute to less forceful spleen contraction in mice lacking CB1/CB2. Since the contractile response of the spleen to NE is dose dependent, redu ced !1AR activity (secondary to reduced expression) would produce a reduced spleen contraction. In agreement, there is a lower amount !1AR in the spleen capsule of CB1/CB2 KO mice as compared with WT controls ( Figure 6.11 ). Collectively, these data reveal that reduced force of NE -induced spleen contractility in the absence of CB1/CB2 is due to reduced expression of !1AR on smooth muscle in the spleen capsule. 278 Figure 6.8. Representative histology of the spleen capsule from WT and CB1/CB2 KO mice. Spleens from WT and CB1/CB2 KO mice were fixed, stained with hematoxylin and eosin, and imaged at 40x magnification. Representative images of the spleen capsule from each genotype are displayed. 274 279 Figure 6.9. Comparison of s pleen capsule thickness in WT and CB1/CB2 KO mice. Spleens from WT and CB1/CB2 KO mice were fixed, stained with hematoxylin and eosin, and imaged at 40x magnification. The pixel width of the spleen capsule was sampled at 12 random locations for each spleen, and the mean of these values calculated and compared statistically. Columns represent the average spleen capsule thickness (pixels) + one SEM (n=3). * Differs from WT (p<0.05). 280 Figure 6.10. Comparison of s pleen capsule smooth muscle specific !-actin in WT and CB1/CB2 KO mice . Spleen capsule samples from both WT and CB1/CB2 KO were prepared for analy sis by Western blot for #-actin as described in the Methods (Section 6.2.4). Top : Columns represent the average amount of #-actin, normalized to GAPDH, in the spleen capsule + 1 SEM (n=5 ). * Differs from WT (p<0.05). Bottom: Representative Western blot image used for quantification. 281 Figure 6.11. Comparison of s pleen capsule !1AR content in WT and CB1/CB2 KO mice. Spleen capsule samples from both WT and CB1/CB2 KO were prepared for analysis by Western blot for #1AR as described in the Methods (Section 6.2.4). Top: Columns represent the average amount of # 1AR, normalized to GAPDH, in the spleen capsule + 1 SEM (n=7 ). * Differs from WT (p< 0.05). Bottom: Representative Western blot image used for quantification. 282 6.3.2: Comparison of EFS -induced spleen contraction in WT and CB1/CB2 KO mice Following experiments utilizing exogenously applied NE, the contractile response of the spleen to EFS was investigated. The intent of electrically stimulating the spleen is to induce the release of endogenous NE and co -released neurotransmitters from sympathetic post -ganglionic axon terminals remaining in the spleen following excision. The optimization of EFS parameters was undertaken prior to experimental testing. The voltage response of spleen contraction following EFS ranged from little/no response at 10 V to a maximal response at 30 V ( Figure 6.12 ). Next, it was determined that individual pulses of 0.2 ms duration induced spleen contraction through the release of neurotransmitters (as opposed to direct stimulation of voltage -gated calcium channel dependent smooth muscle contraction) since spleen contraction to EFS was blocked (zero or minimal measure able response) by TTX ( Figure 6.12 ), a voltage -gated sodium channel blocker (Hille, 1975) . A frequency of 25 Hz ( Figure 6.13 ) and 3 sec of stimulation ( Figure 6.14 ) produced optimal contraction of the spleen roughly equivalent in magnitude to that produced by 25 nM NE ( Figure 6.5 ). It should be noted , however, that Hz modulation did not induce any statistically s ignificant changes in the force of contraction (Figure 6.13 ). 283 Figure 6.12. Voltage response curve of EFS -induced spleen contraction . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens was measured in response to increasing voltage and in the absence and presence of TTX (0.3 % M). EFS was done using circular platinum electrodes located above and below the spleen. Stimulation consisted of 0.2 m s square wave pulses at 20 -28 mA and 8 Hz for 10 s. All data was performed in the same spleens with the control stimulations done prior to incubation with TTX and subsequent stimulation. Each point represents the average force (nM) of contraction generated ± one SEM (n=2). * Significantly differs from TTX treatment (p<0.05). 284 Figure 6.13. Frequency response of EFS -induced spleen contraction . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens was measured in response to increasing frequency electric pulses. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses at 20 -28 mA and 40 V for 1 s. Columns represent the average force of contraction generated + one SEM (n=5). 285 Figure 6.14. Duration response of EFS -induced spleen contraction . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens was measured in response to differing durations of stimulation. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0. 2 ms square wave pulses at 20 -28 mA, 40 V, and 25 Hz. Columns represent the average force of contraction generated + one SEM (n=5). * Significantly differs from 1 s stimulation (p<0.05). # Significantly differs from 2 s stimulation (p<0.05). 286 After EF S parameters were established, the neurotransmitters responsible for EFS -induced spleen contraction were assessed. NE is considered to be the major neurotransmitter of sympathetic neurons (Macarthur et al., 2011) , however, both ATP and NPY are well -known neurotransmitters co -released with NE from sympathetic neurons (Macarthur et al., 2011) . The contribution of NE to EFS -induced spleen contraction was determined by the addition of the # 1AR antagonist prazosin. Prazosin pre -treatment blocked approximatel y 2/3 of EFS -induced spleen contraction ( Figure 6.15). Essentially all prazosin -insensitive spleen contraction was blocked by DPCPX (Figure 6.15 ), an adenosine A1 receptor antagonist (Fozard and Milavec -Krizman, 1993), demonstrating the role of purinergic neurotransmiss ion in EFS spleen contraction. The contribution of NPY Y1 receptors and ATP P2X receptors, both commonly known mediators of sympathetically released NPY and ATP (Macarthur et al., 2011) , were also investigated. Neither BIBP3226 ( Figure 6.16 ), a Y1 receptor antagonist (Rudolf et al., 1994), nor PPADS ( Figure 6.17 ), a P2X receptor antagonist (Lambrecht et al., 1992) , had a significant effect on EF S-induced spleen contraction. 287 Figure 6.15. EFS -induced spleen contraction is mediated by !1AR and adenosine A1 receptors . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens was measured in response to EFS. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V and 25 Hz for 3 s. The additi on of drugs was done in the order demonstrated on the X -axis. Each drug was allowed to incubate for at least 20 min prior to EFS. The final concentration s of the individual drugs w ere as follows : Prazosin (P) Ð 1.0 µM, DPCPX (D) Ð 100 nM, and TTX - 0.3 %M. Columns represent the average force (mN) of contraction generated + one SEM (n=5). * Differs from Control (p>0.05). # Differs from Prazosin (p>0 .05). " No response. 288 Figure 6.16. Adenosine P2X receptors do not contribute to EFS -induced spleen contraction . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction generated by spleens was measured in response to EFS. EFS was done using circular platinum electrodes loc ated above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V and 25 Hz for 3 s. PPADS (10 µM) was allowed to incubate for at least 20 min prior to EFS. Columns represent the average force (mN) of contraction generated + one SEM (n=3). 289 Figure 6.17. NPY Y1 receptors do not contribute to EFS -induced spleen contraction . Spleens from WT mice were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction g enerated by spleens was measured in response to EFS. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V and 25 Hz for 3 s. BIBP3226 (1 µM) was allowed to incubate for at least 20 min prior to EFS. Columns represent the average force (mN) of contraction generated + one SEM (n=4). 290 Once the characteristics of EFS -induced spleen contraction were determined in WT mice, CB1/CB2 KO mice were investiga ted. It was expected that EFS -induced spleen contraction would be reduced in CB1/CB2 KO mice based on the results obtained from exogenous NE application. However, EFS -induced spleen contraction force did not differ between WT and CB1/CB2 KO mice ( Figure 6.18). Weight -normalized EFS -induced spleen contraction also did not differ ( Figure 6.19 ). These data suggest the possibility that compensatory mechanisms in CB1/CB2 KO mice act to produce normal strength spleen contraction despite reduced # 1AR. This co uld be due to compensation by an increased contribution of adenosine Ð A1 receptor signaling. To determine if this is the case, the spleen contractile response to EFS was tested in WT and CB1/CB2 mice in the presence of prazosin. It was reasoned that if adenosine signaling compensates for reduced noradrenergic signaling, then in the absence of # 1AR signaling the contraction of the spleen would be greater in CB1/CB2 KO than WT mice. Contrary to this hypothesis, EFS -induced spleen contraction was reduced t o an equivalent extent by prazosin in both WT and CB1/CB2 KO mice ( Figure 6.20 ). Thus, the non -adrenergic components of EFS -induced spleen contraction do not compensate for decreased # 1AR signaling in CB1/CB2 KO mice. This may suggest that changes in the release of NE are responsible for the absence of decreased spleen contraction in response to EFS in CB1/CB2 KO mice. 291 Figure 6.18. Comparison of EFS -induced spleen contraction force in WT and CB1/CB2 KO mice. Spleens were removed, hung in a physiolog ical bath, and connected to an isometric force transducer. The force of contraction induced by EFS was measured in spleens from both WT and CB1/CB2 mice. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V for 3 s at a frequency of 25 Hz. Columns represent the average force of contraction + one SEM (n=6). 292 Figure 6.19. Comparison of EFS -induced weight -normalized spleen contraction force in WT and CB1/CB2 KO mice. Spleens were removed, hung in a physiological bath, and connected to an isometric force transducer. The force of contraction induced by EFS was mea sured and normalized to spleen weight for both WT and CB1/CB2 mice. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V for 3 s at a frequency of 2 5 Hz. Columns represent the average weight -normalized force of contraction + one SEM (n=6). 293 Figure 6.20. The effect of prazosin on EFS -induced weight -normalized spleen contraction force in WT and CB1/CB2 KO mice. Spleens were removed, hung in a physiol ogical bath, and connected to an isometric force transducer. Prazosin (1 µM) was added to the bath medium and allowed to incubate for 20 before testing. The force of contraction induced by EFS was measured and normalized to spleen weight for both WT and CB 1/CB2 mice. EFS was done using circular platinum electrodes located above and below the spleen. The stimulation consisted of 0.2 ms square wave pulses with 20 -28 mA at 30 V for 3 s at a frequency of 35 Hz. Columns represent the average weight -normalized fo rce of contraction + one SEM (n=6). 294 6.4: Discussion The data presented in this chapter suggest that the CB1 and/or CB2 receptors are involved in the development of noradrenergic control of splenic contraction. CB1/CB2 KO mice were shown to have a de creased contractile response to exogenously applied NE that is due to a decreased expression of the # 1AR in the spleen capsule. This effect, however, is absent when splenic contraction is induced by EFS. The mechanism compensating for a decrease in # 1AR is not due to changes in non -adrenergic neurotransmitters. 6.4.1: NE -induced spleen contractility is decreased in CB1/CB2 KO mice The data set forth here demonstrate that NE is able to contract the spleen. Furthermore the interaction between NE and sple en contraction is concentration -dependent. Spleen contraction is mediated by # 1AR, as demonstrated by the ability of the pan # 1AR antagonist prazosin to block this response. More specifically, other investigators have narrowed the type of # 1AR to be the #1BAR subtype (El tze, 1996; Aboud et al., 2012) . This specific subtype of the # 1AR has previously been demonstrated to undergo phosphorylation mediated desensitization and internalization in response to NE binding (Leeb -Lundberg et al., 1987; Malbon and Wang, 2001) . Internalization occurs as quickly as 5 min, which is within the time scale observed in this work (Fonseca et al., 1995) . Thus, receptor phosphorylation and internalization is the likely cause for the NE -induced attenuation of spleen contraction obser ved at repeated high doses. 295 The mechanism for decreased spleen contraction force in CB1/CB2 KO mice is decreased # 1AR expression on smooth muscle in the spleen capsule. This conclusion is supported by the concentration dependent increase in spleen contr action force in response to NE, and demonstrates that increasing activation of # 1AR receptors results in an increased force of contraction. Thus, decreased activation of # 1AR, due to decreased expression, may account for the decreased contraction observed in CB1/CB2 KO mice. This is in the face of similar weights and amounts of smooth muscle specific #-actin (Skalli et al., 1986; Johnson et al., 2010) , both of which suggest an equivalent amount of smooth muscle in the spleen capsule. However, the reason for decreased # 1AR expression remains unclear. It was hypothesized that # 1AR would be increased in CB1/CB2 KO mice secondary to decreased receptor stimulation, and thus decreased internalization (Chapter 5 ). However the data suggests a different interaction between # 1AR expression and CB1/CB2 receptors. Wh ether this interaction is direct or indirect cannot be determined by the data obtained in these experiments. It remains a possibility that CB receptor expression may directly alter # 1AR expression, just as was described for CB1 and (2AR (Hudson et al., 2010) . While this supposition is without precedence and speculative, increasing evidence for protein -protein interactive effects between CB receptors and other G -protein coupled rec eptors are known (Kearn, 2005; Hudson et al., 2010; Ward et al., 2011) . This hypothesis requires further investigation and likely should start with determining which receptor, CB1 or CB2, is the major mediator of decreased # 1AR expression. 296 Interestingly, the capsule layer of the spleen in CB1/CB2 KO mice is thinner than in WT mice. The explanation for this is unclear, but does not appear to be due to differences in smooth muscle content. The other two major components of the spleen capsule are connectiv e tissue and elastic tissue (Cesta, 2006) . CB1 and CB2 have been found to have effects on the production of extracellular matrix components from fibroblast. CB1 are stimulatory to fibroblasts (Marquart et al., 2010; Lazzerini et al., 2012), whereas CB2 are inhibitory (Akhmetshina et al., 2009; Defer et al., 2009) . Despite these opposing effects, these reports demonstrate that CB receptors can modulate the act ivity of fibroblasts. It may be the case that these effects during development account for reduced spleen capsule thickness in CB1/CB2 KO mice. Thus, an interesting line of experimentation would be to evaluate the relative contribution of CB1 and CB2 to c onnective tissue development in the spleen. 6.4.2: Normal spleen contraction in response to EFS is achieved in CB1/CB2 KO mice by a compensatory mechanism EFS -induced spleen contraction is mediated by two different neurotransmitters. The major neurotran smitter of the sympathetic nervous system (Macarthur et al., 2011) , NE, mediates approximately 2/3 of EFS -induced spleen contr action. The receptor responsible for this is the # 1BAR (Eltze, 1996; Aboud et al., 2012) . The remaining component mediating EFS -induced spleen contraction is release of adenosine derived from the sympathetic neurotransmitter ATP (Rongen et al., 1996) . Adenosine activation of A1 receptors is responsible for this action, which in agreement with other 297 investigators (Fozard and Milavec -Krizman, 1993) . These receptors are reported to mediate a relatively small portion (18 -33%) of vascular smooth muscle contraction (Tawfik et al., 2005) , in agreement with the data obtained in the present study. The compensatory mechanism whereby EFS -induced s pleen contraction in CB1/CB2 KO mice is equivalent to that of WT mice does not involve changes in non -adrenergic transmission. This was demonstrated by an equivalent reduction of spleen contractile force following prazosin blockade of noradrenergic transm ission through # 1AR. Thus, the contribution of NE to EFS -induced spleen contraction in CB1/CB2 KO mice is equivalent to WT despite a reduction in # 1AR. A possible explanation for this phenomenon is an increase in the quantal release of NE in CB1/CB2 KO m ice. An increase in the amount of NE released per action potential would hypothetically stimulate more # 1AR in CB1/CB2 KO mice, thus compensating for decreased # 1AR expression. CB1/CB2 KO mice may indeed be primed for this scenario as splenic noradrenerg ic axon terminals in these mice contain more NE than WT mice ( Chapter 5 ). Additionally, increased release of neurotransmitter is not without precedence as it has been shown that BDNF can increase the quantal release of glutamate in the hippocampus (Tyler and Pozzo -Miller, 2001; Amaral and Pozzo -Miller, 2012) . However, while this hypothesis may explain the observed data, it is untested and requires further validation. 298 6.4.3: Conclusion The purpose of this chapter was to test the hypothesis that spleen contraction is enhanced in CB1/CB2 KO mice presumably do to an up -regulation of # 1AR expression compensating for decreased NE engagement of these receptors. First, it was demonstrated that NE can cause a concentration -dependent induction of spleen contraction via # 1AR activation, but the force of contraction is reduced in CB1/CB2 KO mice. Reduced NE -induced spleen contraction in CB1/CB2 KO mice is due to decreased # 1AR expression. However, EFS -induced spleen contraction in CB1/CB2 KO is not different than WT. Thus, compensatory mechanisms are able to overcome the deficit of # 1AR expression in these mice. This co mpensation is not due to changes in adenosine stimulation of A1 adenosine receptors of EFS -induced spleen contraction. 299 REFERENCES 300 REFERENCES Aboud R, Shafii M, Docherty JR. Investigation of the subtypes of # 1-adrenoceptor mediating contractions of rat aorta, vas deferens and spleen. Br J Pharmacol. 2012 Jul 19;109(1):80 Ð7. Akhmetshina A, Dees C, Busch N, Beer J, Sarter K, Zwerina J, et al. The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis. Arthritis Rheum. 2009 Apr;60(4):1129 Ð36. Amaral MD, Pozzo -Miller L. Intracellular Ca2+ stores and Ca2+ influx are both required for BDNF to rapidly increase quantal ves icular transmitter release. Neural Plast. 2012;2012:203536. PMCID: PMC3397209 Bakovic D, Eterovic D, Saratlija -Novakovi ) Z, Palada I, Valic Z, Bilopavlovi ) N, et al. Effect of human splenic contraction on variation in circulating blood cell counts. Clin Exp Pharmacol Physiol. 2005 Nov;32(11):944 Ð51. Bakovic D, Valic Z, Eterovic D, Vukovic I, Obad A, Marinovi )-Terzi ) I, et al. Spleen volume and blood flow response to repeated breath -hold apneas. J. Appl. Physiol. 2003 Oct;95(4):1460 Ð6. Blue J, Weiss L. Ele ctron microscopy of the red pulp of the dog spleen including vascular arrangements, periarterial macrophage sheaths (Ellipsoids), and the contractile, innervated reticular meshwork. Am. J. Anat. 1981 Jun;161(2):189 Ð218. Bootman MD, Rietdorf K, Hardy H, Da utova Y, Corps E, Pierro C, et al. Calcium Signalling and Regulation of Cell Function. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Buckley N, McCoy K, Mezey E, Bonner T, Zimmer A, Felder C, et al. Immunomodulation by cannabinoids is absent in m ice deficient for the cannabinoid CB(2) receptor. Eur J Pharmacol. 2000 May 19;396(2 -3):141 Ð9. Burnstock G, Ralevic V. Purinergic Signaling and Blood Vessels in Health and Disease. Cambridge D, Davey MJ. Prazosin, a selective antagonist of post -synaptic alpha -adrenoceptors [proceedings]. British journal of É. 1977. Cesta M. Normal Structure, Function, and Histology of the Spleen. Toxicol Pathol. 2006;34(5):455 Ð65. Chevendra V, Weaver LC. Distributions of neuropeptide Y, vasoactive intestinal peptide and somatostatin in populations of postganglionic neurons innervating the rat kidney, spleen and intestine. Neuroscience. 1992 Oct;50(3):727 Ð43. 301 Corder R, Lowry PJ, Withrington PG. The actions of the peptides, neuropeptide Y and peptide YY, on the vascular a nd capsular smooth muscle of the isolated, blood -perfused spleen of the dog. Br J Pharmacol. 1987 Apr;90(4):785 Ð90. PMCID: PMC1917199 Davies BN, Withrington PG. The actions of drugs on the smooth muscle of the capsule and blood vessels of the spleen. Pharmacol Rev. 1973 Sep;25(3):373 Ð413. Defer N, Wan J, Souktani R, Escoubet B, Perier M, Caramelle P, et al. The cannabinoid receptor type 2 promotes cardiac myocyte and fibroblast survival and protects against ischemia/reperfusion -induced cardiomyopathy. FASEB J. 2009 Jul;23(7):2120 Ð30. Elenkov I, Vizi E. Presynaptic modulation of release of noradrenaline from the sympathetic nerve termina ls in the rat spleen. Neuropharmacology. 1991 Dec 1;30(12A):1319 Ð24. Eltze M. Functional evidence for an alpha 1B -adrenoceptor mediating contraction of the mouse spleen. Eur J Pharmacol. 1996 Sep 12;311(2 -3):187 Ð98. Felten DL, Ackerman KD, Wiegand SJ, Fe lten SY. Noradrenergic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neurosci Res. 1987;18(1):28 Ð36, 118Ð21. Felten SY, Olschowka J. Noradrenergic sy mpathetic innervation of the spleen: II. Tyrosine hydroxylase (TH) -positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp. J Neurosci Res. 1987;18(1):37 Ð48. Fonseca MI, Button DC, Brown RD. Agonist regulation of alpha 1B-adrenergic receptor subcellular distribution and function. J Biol Chem. 1995 Apr 14;270(15):8902 Ð9. Fozard JR, Milavec -Krizman M. Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation. Br J Pharmacol. 1993 Jul 19;109(4):1 059Ð63. PMCID: PMC2175713 Garcõ !a-S⁄inz JA, V⁄zquez -Prado J, del Carmen Medina L. # 1-Adrenoceptors: function and phosphorylation. Eur J Pharmacol. 2000 Feb;389(1):1 Ð12. Gerald T, Ward G, Howlett A, Franklin S. CB1 knockout mice display significant changes in striatal opioid peptide and D4 dopamine receptor gene expression. Brain Res. 2006 Jun 6;1093(1):20 Ð4. Gillespie JS, Hamilton DN. Binding of noradrenaline to smooth muscle cells in the spleen. Nature. 1966 Oct 29;212(5061):524 Ð5. 302 Groom AC, Schmidt EE, MacDonald IC. Microcirculatory pathways and blood flow in spleen: new insights from washout kinetics, corrosion casts, and quantitative intravital videomicroscopy. Scanning Microsc. 1991 Mar;5(1):159 Ð73Ðdiscussion173 Ð4. Hille B. The receptor for tetrodot oxin and saxitoxin. A structural hypothesis. Biophysical Journal. The Biophysical Society; 1975 Jun 1;15(6):615. Hudson BD, H”bert TE, Kelly MEM. Physical and functional interaction between CB1 cannabinoid receptors and beta2 -adrenoceptors. Br J Pharmacol . 2010 Jun;160(3):627 Ð42. PMCID: PMC2931563 Ignarro LJ, Titus E. The presence of antagonistically acting alpha and beta adrenergic receptors in the mouse spleen. J Pharmacol Exp Ther. 1968 Mar;160(1):72 Ð80. Ishac E, Jiang L, Lake K, Varga K, Abood M, Kuno s G. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves. Br J Pharmacol. 1996 Aug 1;118(8):2023 Ð8. PMCID: PMC1909901 Jarai Z, Wagner J, Varga K, Lake K, Compton D, Martin B, et al. Canna binoid -induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14136 Ð41. Johnson KB, Thompson JM, Watts SW. Modification of proteins by norepinephrine is important for va scular contraction. Front Physiol. 2010;1:131. PMCID: PMC3059971 Kaplan BLF, Lawver JE, Karmaus PWF, Ngaotepprutaram T, Birmingham NP, Harkema JR, et al. The effects of targeted deletion of cannabinoid receptors CB1 and CB2 on intranasal sensitization and challenge with adjuvant -free ovalbumin. Toxicol Pathol. 2010 Apr;38(3):382 Ð92. PMCID: PMC2941344 Kearn CS. Concurrent Stimulation of Cannabinoid CB1 and Dopamine D2 Receptors Enhances Heterodimer Formation: A Mechanism for Receptor Cross -Talk? Molecular Pharmacology. 2005 Feb 9;67(5):1697 Ð704. Kubo T, Su C. Effects of adenosine on [3H]norepinephrine release from perfused mesenteric arteries of SHR and renal hypertensive rats. Eur J Pharmacol. 1983 Feb 18;87(2 -3):349 Ð52. Kuwahira I, Kamiya U, Iwamoto T, Mo ue Y, Urano T, Ohta Y, et al. Splenic contraction -induced reversible increase in hemoglobin concentration in intermittent hypoxia. J. Appl. Physiol. 1999 Jan;86(1):181 Ð7. Kgelgen von I, Sp−th L, Starke K. Stable adenine nucleotides inhibit [3H] -noradrena line release in rabbit brain cortex slices by direct action at presynaptic adenosine A1 - 303 receptors. Naunyn -Schmied Arch Pharmacol. 1992 Aug;346(2):187 Ð96. Lambrecht G, Friebe T, Grimm U, Windscheif U, Bungardt E, Hildebrandt C, et al. PPADS, a novel functi onally selective antagonist of P2 purinoceptor -mediated responses. Eur J Pharmacol. 1992 Jul 7;217(2 -3):217 Ð9. Lazzerini PE, Natale M, Gianchecchi E, Capecchi PL, Montilli C, Zimbone S, et al. Adenosine A2A receptor activation stimulates collagen producti on in sclerodermic dermal fibroblasts either directly and through a cross -talk with the cannabinoid system. J. Mol. Med. 2012 Mar;90(3):331 Ð42. Leeb-Lundberg LM, Cotecchia S, DeBlasi A, Caron MG, Lefkowitz RJ. Regulation of adrenergic receptor function by phosphorylation. I. Agonist -promoted desensitization and phosphorylation of alpha 1 -adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF -2 smooth muscle cells. J Biol Chem. 1987 Mar 5;262(7):3098 Ð105. Lundberg JM, Franco -Cereceda A, Lacroix JS, Pernow J. Neuropeptide Y and sympathetic neurotransmission. Ann N Y Acad Sci. 1990;611:166 Ð74. Macarthur H, Wilken GH, Westfall TC, Kolo LL. Neuronal and non -neuronal modulation of sympathetic neurovascular tran smission. Acta Physiol (Oxf). 2011 Sep;203(1):37 Ð45. PMCID: PMC3139802 Malbon CC, Wang H -Y. Adrenergic Receptors. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. Marquart S, Zerr P, Akhmetshina A, Palumbo K, Reich N, Tomcik M, et al . Inactivation of the cannabinoid receptor CB1 prevents leukocyte infiltration and experimental fibrosis. Arthritis Rheum. 2010 Nov;62(11):3467 Ð76. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol. Nature Publishing Group; 2005 Au g;5(8):606 Ð16. Michel MC, Beck -Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, et al. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev. 1998 Mar;50(1):143 Ð50. Niederhoffer N, Szabo B. Effect of the cannabinoid receptor agonist WIN55212 -2 on sympathetic cardiovascular regulation. Br J Pharmacol. 1999 Jan;126(2):457 Ð66. Noble J, Bailey M. Quantitation of protein. Methods Enzymol. 2009;463:73 Ð95. Pinkus GS, Warhol MJ, O'Connor EM, Etheridge CL, Fujiwara K. Immunohistochemical localization of smooth muscle myosin in human spleen, lymph node, and other 304 lymphoid tissues. Unique staining patterns in splenic white pulp and sinuses, lymphoid follic les, and certain vasculature, with ultrastructural correlations. Am. J. Pathol. 1986 Jun;123(3):440 Ð53. PMCID: PMC1888274 Ralevic V. Purines as Neurotransmitters and Neuromodulators in Blood Vessels. CVP. 2009 Jan 1;7(1):3 Ð14. Ralevic V, Kendall DA. Canna binoids inhibit pre - and postjunctionally sympathetic neurotransmission in rat mesenteric arteries. Eur J Pharmacol. 2002 May 31;444(3):171 Ð81. Ren LM, Burnstock G. Prominent sympathetic purinergic vasoconstriction in the rabbit splenic artery: potentiati on by 2,2 *-pyridylisatogen tosylate - Ren - 2009 - British Journal of Pharmacology - Wiley Online Library. Br J Pharmacol. 1997. Richardson MX, de Bruijn R, Schagatay E. Hypoxia augments apnea -induced increase in hemoglobin concentration and hematocrit. E ur. J. Appl. Physiol. 2009 Jan;105(1):63 Ð8. Richardson MX, Lodin A, Reimers J, Schagatay E. Short -term effects of normobaric hypoxia on the human spleen. Eur. J. Appl. Physiol. Springer -Verlag; 2007 Nov 28;104(2):395 Ð9. Romano TA, Felten SY, Felten DL, O lschowka JA. Neuropeptide -Y innervation of the rat spleen: another potential immunomodulatory neuropeptide. Brain Behav Immun. 1991 Mar;5(1):116 Ð31. Rongen GA, Lenders JW, Lambrou J, Willemsen JJ, Van Belle H, Thien T, et al. Presynaptic inhibition of nor epinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension. 1996 Apr;27(4):933 Ð8. Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD, Entzeroth M, et al. The first highly potent and selective non -peptide neuropeptide Y Y1 r eceptor antagonist: BIBP3226. Eur J Pharmacol. 1994 Dec 27;271(2 -3):R11 Ð3. Saito H, Yokoi Y, Watanabe S, Tajima J, Kuroda H, Namihisa T. Reticular meshwork of the spleen in rats studied by electron microscopy. Am. J. Anat. 1988 Mar;181(3):235 Ð52. Sandler MP, Kronenberg MW, Forman MB, Wolfe OH, Clanton JA, Partain CL. Dynamic fluctuations in blood and spleen radioactivity: splenic contraction and relation to clinical radionuclide volume calculations. J. Am. Coll. Cardiol. 1984 May;3(5):1205 Ð11. Sato N, Sh en YT, Kiuchi K, Shannon RP, Vatner SF. Splenic contraction -induced increases in arterial O2 reduce requirement for CBF in conscious dogs. American 305 Journal of Physiology - Heart and Circulatory Physiology. American Physiological Society; 1995 Aug 1;269(2): H491 ÐH503. Sato S, Steeber DA, Jansen PJ, Tedder TF. CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19. J Immunol. 1997 May 15;158(10):4662 Ð9. Satodate R, Tanaka H, Sasou S, Saku ma T, Kaizuka H. Scanning electron microscopical studies of the arterial terminals in the red pulp of the rat spleen. Anat. Rec. 1986 Jul;215(3):214 Ð6. Schmidt EE, MacDonald IC, Groom AC. Microcirculation in mouse spleen (nonsinusal) studied by means of c orrosion casts. Journal of morphology. 1985. Schmidt EE, MacDonald IC, Groom AC. Comparative aspects of splenic microcirculatory pathways in mammals: the region bordering the white pulp. Scanning Microsc. 1993 Jun;7(2):613 Ð28. Sedaa KO, Bjur RA, Shinozuk a K, Westfall DP. Nerve and drug -induced release of adenine nucleosides and nucleotides from rabbit aorta. J Pharmacol Exp Ther. 1990 Mar;252(3):1060 Ð7. Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR. A transient decrease in splee n size following stroke corresponds to splenocyte release into systemic circulation. J Neuroimmune Pharmacol. 2012 Dec;7(4):1017 Ð24. PMCID: PMC3518577 Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha -smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 1986 Dec;103(6 Pt 2):2787 Ð96. PMCID: PMC2114627 Takano S. A study on the contraction of spleen strips in kids and dogs, with special reference to the cholinergic and ad renergic receptors. Jpn. J. Pharmacol. 1969 Dec;19(4):563 Ð8. Tawfik HE, Schnermann J, Oldenburg PJ, Mustafa SJ. Role of A1 adenosine receptors in regulation of vascular tone. Am J Physiol Heart Circ Physiol. 2005 Mar;288(3):H1411 Ð6. Tyler WJ, Pozzo -Mille r LD. BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. Journal of Neuroscience. 2001 Jun 15;21(12):4249 Ð58. PMCID: PMC2806848 Ward RJ, Pediani JD, Milligan G. Heteromultimerization of cannabinoid CB(1) receptor and orexin OX(1) receptor generates a unique complex in which both protomers are 306 regulated by orexin A. Journal of Biological Chemistry. 2011 Oct 28;286(43):37414 Ð28. PMCID: PMC3199489 Wennmalm M, Fredho lm BB, Hedqvist P. Adenosine as a modulator of sympathetic nerve -stimulation -induced release of noradrenaline from the isolated rabbit heart. Acta Physiol. Scand. 1988 Apr;132(4):487 Ð94. Westfall TC, Carpentier S, Chen X, Beinfeld MC, Naes L, Meldrum MJ. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat. Journal of Cardiovascular Pharmacology. 1987 Dec;10(6):716 Ð22. Westfall TC, Chen XL, Ciarleglio A, H enderson K, Del Valle K, Curfman -Falvey M, et al. In vitro effects of neuropeptide Y at the vascular neuroeffector junction. Ann N Y Acad Sci. 1990;611:145 Ð55. Wiest R, Jurzik L, Moleda L, Froh M, Schnabl B, Hırsten SV, et al. Enhanced Y1 -receptor -mediate d vasoconstrictive action of neuropeptide Y (NPY) in superior mesenteric arteries in portal hypertension. Journal of Hepatology. 2006 Mar;44(3):512 Ð9. Wong KK. Bethanechol induced contraction in mouse spleen. Chin J Physiol. 1990;33(2):161 Ð7. Zimmer A, Z immer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci USA. 1999 May 11;96(10):5780 Ð5. PMCID: PMC21937 Zukowska -Grojec Z, Dayao EK, Karwatowska -Prokopcz uk E, Hauser GJ, Doods HN. Stress -induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y Y1 receptors. Am J Physiol. 1996 Feb;270(2 Pt 2):H796 Ð800. 307 Chapter 7: General Discussion and Concluding Remarks The spleen is a visceral abdominal organ with both h ematologic and immune functions that is innervated by the sympathetic nervous system. Cannabinoid receptors are present throughout the body of mammals, including in the spleen. The multi -functional role of splenic sympathetic innervation is not completely understood, and the effect s of cannabinoids on the function of splenic sympath etic innervation are not known. The present dissertation characterizes the physiology of splenic noradrenergic sympatheti c innervation and explores its role in both immunologic and hematologic functions. The effects of CB1 and CB2 deficiency on both the immunologic and hematologic functions of these neurons were also examined . Understanding the role of endocannabinoid signaling is significant considering the increase in medicinal use of cannabinoids and can inform the scientific and medical community of the possible ramific ations of cannabinoid use and abuse. 7.1: Anatomy and physiology of splenic noradrenergic post -ganglionic neurons Innervation of the spleen is heterogeneous, the targets of innervation being the spleen capsule and parenchymal tissue surrounding arte riole s. This innervation is sympathetic in origin and noradrenergic in nature . This was confirmed by visualization with TH immunostaining and a lack of finding signif icant amounts of either DA or epinephrine in the spleen by HPLC -ED, 308 confirming the findings of other investigators (Felten et al., 1987; Felten and Olschowka, 1987; Nance and Sanders, 2007; Olofsson et al., 2012) . A major function of noradrenergic innervation to the spleen is control of smooth muscle contraction , which expels the cellular content of the sple en and regulat es blood flow through the spleen (Blue and Weiss, 1981; Pinkus et al., 1986; Saito et al., 1988; Groom et al., 1991) . Therefore the distribution of noradre nergic terminals in the spleen is not surprising. Finding noradrenergic nerve terminals in the lymphocyte -rich PALS (Felten and Olschowka, 1987; Cesta, 2006) supports the hypothesis of an immunologic function for noradrenergic innervation of the spleen (Sanders, 2012) . The paucity of noradrenergic axon terminals, catecholamine synthetic machinery (TH), and NE in the remainder of the spleen parenc hyma suggest s no direct role for noradrenergic innervation in the se regions . The basal metabolic and synthetic activity of noradrenergic neurons in the spleen capsule is lower than the PVN. This was illustrated by differences in the concentration of NE metabolites and suggests NE axon terminals in the PVN are more metabolically active than those in the spleen. However, differences in the concentration of these metabolites may simply be due to the size difference. The ratio of total metabolite content to total primary catecholamine content is more suited for conc lusions with regards to metabolic activity (Lookingland et al., 1991) . Interestingly, the VMA/NE ratio was not different between these tissu es, whereas the MHPG/NE rat io wa s lower in the spleen . This difference may be due to the tissue location where VMA and MHPG are respectively created . VMA synthesis 309 is mostly dependent upon extra -neuronal conversion of MHPG, whereas MHPG synthesis is largely dependent upon neuronal MAO activity (Lookingland et al., 1991; Karhunen et al., 1994; Hayley et al., 2001; Oh -hashi et al., 2001; Eisenhofer et al., 2004; Myıh−nen et al., 2010; Siraskar et al., 2011) making it a better indicator of the metabolic c apacity of neurons. Thus, the large difference in the MHPG/NE ratio is more telling of a reduced metabolic rate in splenic sympathetic neurons. Furthermore , increased accumulation of DOPA following blockade of NE synthesis with NDS -1015 suggests a faster r ate of NE synthesis in axon terminals of the PVN versus the spleen capsule under basal conditions. The rate of NE release in the spleen is lower compared to the PVN. This was determined using the aMT method , which has a number of advantages compared to electrophysiology or microdialysis, such as be ing able to measure the activity of two different sites in the body in the same animal (Lee et al., 1992; Helwig et al., 2008) , the amount of time required, and not requiring the insertion of large microdialysis probes (Olive et al., 2000; Ortega et al., 2012; CMA Microdialysis) . Using this method, the rate of noradrenergic activity in the PVN was found to be much faster than that of the spleen. The rate of NE release from spleni c noradrenergic sympathetic neurons is regulated by ! 2AR autoreceptors. This was demonstrated by measuring the effect of the !2AR antagonist idazoxan on the activity of spleen capsule noradrenergic neurons. These receptors are act much like D2 -autoreceptor s in that their stimulation on pre -synaptic terminals decreases the synthesis and 310 release of neurotransmitters (Khan et al., 2002; Marino and Cosentino, 2013) . Thus a ntagonism of these receptors leads to an increase d release of NE and demonstrates the ability of these neurons to self -regulate their activity. Interestingly , the activity of splenic noradrenergic neurons is coupled to increases in the concentrat ion of NE and TH in the spleen. Normally under basal conditions , the coupling of NE synt hesis and release maintains a steady -state amount of stored NE in the axon terminal s (Brodie et al., 1966; Lookingland et al., 1991). For example , during times of activation the amount of NE does not change in the PVN , demonstrating the maintenance of steady -state NE concentrations in a central noradrenergic nucleus (Lookingland et al., 1991) . Yet during increased neuronal activity the amount of NE s ignificantly increases in the spleen capsule . Thus , it appears that in splenic noradrenergic neurons the synthetic capacity out -paces the release of NE leading to accumulation during activation. Activity dependent increases in the activity of TH are a kno wn phenomenon in catecholaminergic neurons (Kumer and Vrana, 1996) . The enzyme activity of TH can be increased in response to neuronal activation via phosphorylation (Kumer and Vrana, 1996) . However, the phosphorylation of TH also reduces the stability o f the phosphorylated enzyme (not due to degradation of the entire protein ) (Lazar et al., 1981; Vrana et al., 1981; Vrana and Roskoski, 1983). On the other hand, TH stability is increased by feedback inhibitors, such as DA (Kumer and Vrana, 1996) . The refore, it may be the case that two different 311 mechanisms are occurring simultaneously to cause bot h increased TH activity and increased stability of this enzyme. During times of activation the rate of NE production is out -pacing the rate of NE release due to increased enz yme activity by phosphorylation, t hus explaining the increase in spleen capsule NE during activation. During the times of increased production, however, there should also be an increase in the synthesis of feedback inhibitors of TH, such as DA, which stabilize TH proteins. This occurs by DA forming a complex with ferric iron within t he active site of the enzyme . The binding of this complex serves to not only decrease the catalytic activity of TH, but also yields a very stable form of the enzyme that can be reversibly activated (Okuno and Fujisawa, 1991) . This may explain the increase in splenic TH during times of activation. What is unclear, however, is how increase d synthesis of NE can occur in the presence of increas ed TH -stabilizing feedback inhibitors that should shut down the production of NE due to phosphorylation induced increases in TH catalytic activity . This paradoxical aspect of splenic noradrenergic neuron physiology requires further investigation and pre sents a number of future directions to explore. For instance, c hanges in the rate of TH synthesis could be measured in spleen capsule during times of activity by measuring the accumulation of DOPA following blockade of L -AAAD (Lookingland and Moore, 2005) . If phosphorylation -dependent increases the catalytic activity are occurring, then it would be expected that DOPA accumulation would occur quicker during 312 activation. However, other causes for NE accumulation in the spleen are also possible. For instance, increased NE storage , due to increased vesicular number and size , or displacement of co -released transmitters , effectively increasing the storage available for NE , could also account for this phenomenon . Ultrastructural analysis by electron microscopy of NE containing terminals in the spleen capsule would reveal differences due to these fact ors . In addition to these experiments, differences in the ability of feedback inhibitors, such as DA, to inhibit and stabilize isolated TH from the brain spleen might be assessed. This may reveal differences in the sensitivity of TH from different tissues to feedback inhibitors, or even reveal differences in DA -mediated stabilization. 7.2: Immunologic function of splenic noradrenergic post -ganglionic neurons The activity of splenic noradrenergic neurons is increased in respon se to humoral immune challenges regardless of T -cell involvement. Both sRBC and LPS induce humoral immune responses. sRBC are a traditional soluble protein antigen requiring processing by macrophages, interactions with T cells, and eventually T cell to B cell interactions to s timulate antibody production (Ademokun and Dunn -Walters, 2001) . LPS, on the other hand, is a direct stimulator of B cells. LPS binds TLR4 receptors and cross -links BCR on B cells to directly caus e proliferation, activation, and IgM production in the same cel l (Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . Therefore, while sRBC require both T and B cells that selectively recognize 313 sRBC epitopes, LPS is a polyclonal B cell activator by activa ting any B cell expressing TLR4. Both sRBC and LPS were found to significantly increase the rate of NE release in the spleen capsule. This demonstrates that humoral immunogen induced increases in the activity of splenic sympatheti c noradrenergic neurons does not depend on the involvement of T cells. Pro -inflammatory cytokines , such as IL-1" and IL -6, are the likely activators of sympathetic neuronal activity during an immune challenge. Peripheral injection of IL -1 " increases the release of NE in the spleen (Shimizu et al., 1994; Kohm and Sanders, 2001) . However, IL -1" also stimulate s the production of IL -6 from a myriad of cell types (Kauma et al., 1994; Spangelo et al., 1994; Parikh et al., 1997) leaving open the possibility that the effects of IL -1" on sympathetic activity may be due to IL -6. In support of this hypothesis, central injection of IL -6 produces increased firing of spleen capsule sympathetic neurons (Helwig et al., 2008) . The role for each of these specific cytokines could be tested using biological inhibitors. For instance, the administration of MP161 -1, an IL-6 receptor neutralizing antibody (Uchiyama et al., 2008; Tomiyama -Hanayama et al., 2009), prior to an immune challenge would be expected to inhibit sympathetic activation in the periphery if IL-6 is the sympatho -stimulatory factor. If MR161 -1 is unable to block sympathetic nervous system stimulation then it might reasonably be assumed that IL -1" is the mediating factor, leading to a similar experiment using a blockade of IL -1" effects. 314 Splenic B cells express !2AR and are immunologically functional. The observations by other investigators were confirmed in that " 2AR are expressed by both B cells and T cells, with B cell expression being highest (Kin and Sanders, 2006) . The data presented here also suggest that B cells become more sensitive to NE following LPS stimulation by increasing the number of cells expressing " 2AR and th e amount of " 2AR per B cell. This also occurs within the population of B cells producing IgM. Thus, changes in the expression of " 2AR on B cells, and on IgM producing cells , supports the hypothesis that NE stimulation of " 2AR can have a di rect impact on IgM production. Unfortunately, s timulation of !2AR on splenic B cells does not augment Ig M production in response to LPS . The question then remains as to the function of !2AR on B cells. It wa s demonstrated here that B cells express !2AR and are immunologically functional. However , the data show that !2AR stimulation is not necessary for normal antibody response s to LPS, a T cell independent antigen. This raises the question of why the data obtained is seemingly incongruent with the repor ted effect of !2AR on antibody production (Kohm and Sanders, 2001; Nance and Sanders, 2007; Sanders, 2012) . The explanation may lie in the nature of the immune challenge used. The correlation between !2AR and antibody production was explored in this dissertation using LPS, a T cell independent immunogen, which induces a humoral immune response through activation of TLR4 and crosslinking of the BCR in the absence of other co -stimulators, such as CD40 315 (Ademokun and Dunn -Walters, 2001; Lanzavecchia and Sallusto, 2007; Bekeredjian -Ding and Jego, 2009) . On the other hand , the published research correlating antibody production and !2AR utilized either T cell dependent immunogens or direct stimulation of B cells by CD40 (Kohm et al., 2000; Kohm and Sanders, 2001; Pongratz et al., 2006; Padro et al., 2013) . Therefore, it may be the case that !2AR augmentation of antibody production is specific to B cell responses in which a CD40 -CD154 interaction is required , such as during immune responses requiring T cells involvement (Ademokun and Dunn -Walters, 2001; N”ron et al., 2011) . If this is the case, the lack of !2AR augmentation of the humoral immune response to LPS is due to the absence of CD40 being involved in this response. This hypothesis has not been directly tested and is an avenue for future research. To test this hypothesis, in vitro stimulation of !2AR could be compared in the face of immunologic stimulation by both LPS and CD40. It would be expected that !2AR stimulation would augment humoral immune responses to CD40, but not to LPS. This could t hen be further tested in vivo by blocking !2AR in mice immunized with sRBC and LPS. It would be expected in this case that blockade of !2AR signaling would decrease antibody production in sRBC, but not LPS, mice. 316 7.3: The role of CB1 and CB2 on the immunologic role of noradrenergic splenic sympathetic innervation Congruent with a previously published report (Springs et al., 2008) , CB1/CB2 KO mice demonstrate enhanced humoral immunity. This is a partially manifest ed in the present studies by higher plasma IgM and IgG in immunologically naŁve CB1/CB2 KO mice. IgM is constitutively secreted without antigen stimulation , termed # natural antibodies $ , as a form of innate immunity by innate -like B cells (Baumgarth, 2013) . Thus, elevated plasma IgM in immunologically naŁve CB1/CB2 KO mice sugges ts these receptors are inhibitory to this specific type of immunity, a finding that is unexplored in scientific literature. This hypothesis could be tested by chronically treating mice with THC, a non -selective CB1/CB2 agonist, under immunologically naŁve conditions and then testing for the presence of natural antibodies and natural antibody producing cells in the spleen. These innate -type B cells are known as Marginal Zone B cells, identified by an IgM high IgD low CD21 high CD23 low phenotype, and B -1a cel ls, identified by expressi ng CD19 and CD5 (Zouali, 2001) . If this hypothesis is correct, then it is expected that THC treatment would cause a decrease in plasm a IgM as well as a decrease in marginal z one B cells and B -1a cells. If this is found to be the case, then the contribution of CB1 versus CB2 on this effect could be explored using individual CB receptor KO mi ce, or receptor specific pharmacologic agents. 317 IgG , on the other hand, is produced in response to antigenic stimulation (Zouali, 2001) . Therefore, # natural antibody $ production does not explain elevated plasma IgG in immuno logically naŁve CB1/CB2 KO mice. The most likely explanation for increased plasma IgG is an increased presence of long -lived plasma cells. Plasma cells can be short -lived (days to weeks) or persist for years (conferring long -term immunity ) (Ademokun and Dunn -Walters, 2001; Tangye, 2011). These long -lived plasma cells constitutively produce antigen specific IgG to speed the removal of antigens upon re -exposure (Tangye, 2011) . It may be the case that CB1/CB2 deficiency results in an increase in the survival or generation of these cells. This possibility seems very likely to be the cause of increased IgG i n immunologically naŁve mice as this would be expected to increase IgG plasma concentrations, but not at the level seen with antigenic stimulation. This suggests that CB1 and/or CB2 play a n inhibitory role in the function of formation of these specialized B cells. The effect of canna binoids on long -term immunity are also virtually unexplored in the literature with only one report in CB2 knockout mice showing no significant effect of plasma cell generation in response to a T -cell dependent antigen (Basu et al., 2013) . A simple experiment to test this hypothesis would be to measure and compare the number of plasma cells in the sp leen and bone marrow of WT and CB1/CB2 KO mice. Thi s could be accomplished using CD138, a marker for plasma cells (O'Connell et al., 2004) . CB1/CB2 KO mice are expected to have an increased number of CD138 positive lymphocytes if this 318 hypothesis is correct . If KO of CB1/CB2 results in an increased presence of plasma cells, then a role for each specific receptor could be explored using CB1 or CB2 KO mice using a similar approach. Alternatively, the use of CB1 and CB2 specific antagonists could be utilized. To do this, antagonists of CB1 or CB2 would be administered prior to an immune challenge. After sufficient time has passed for the differentiation of plasma cells (4 days) , the effect of the individual receptor antagonists would be tested by comparing the size of the CD138 lymphocyte population in untreated and antagonist treated mice. It would be expected in this case that mice treated with the antagonist of the CB receptor res ponsible for inhibiting plasma cell differentiation would have an increased population of plasma cells. Considering the hypothesis that enhanced sympathetic noradrenergic stimulation may be the underlying cause for enhanced humoral responses, it was expe cted that " 2AR expression in CB1/CB2 KO mice would be reduced due to ligand/activity dependent down -regulation (Zastrow and Kobilka, 1992; Hudson et al., 2010). Contrary to expectation, " 2AR expression is elevated on B cells in CB1/CB2 KO mice . The most likely explanation for this phenomenon is the release of less splenic NE than in WT mice . This is hypothesized to result in "2AR receptor up -regulation due to a lack of ligand -binding , or rather decreased ligand -dependent internalization (Zastrow and Kobilka, 1992; Hudson et al., 2010). This hypothesis requires further experimentation and could be tested several ways. For instance, the administration of an "2AR antagonist, which 319 increases the release of NE from splenic sympathetic neurons, would be expected to result in a de crease of "2AR expression on B cells. Alternatively, an "2AR agonist , which is expected to decrease the release of NE from splenic sympathetic neurons, would be expected to increase the expression of "2AR on B cells. These experiments would demonstrate the direct correlation between splenic NE release and the expression of "2AR on B cells, providing sup port for the above hypothesis. The results obtained in this dissertation argue against the regulation of splenic post -ganglionic sympathetic neurons by CB1. CB1 are pre -synaptically expressed receptors known to inhibit the release of neurotransmitters from peripheral sympathetic neurons (Ishac et al., 1996; Niederhoffer and Szabo, 1999; Ralevic and Kendall, 2002) . It was expected that the absence of CB1 inhibitory action s on splenic sympathetic neurons would result in increased NE release. This was not found to be true and theref ore argues against the ability of CB1 to regulate splenic sympathetic neurons. Interestingly , it was found that CB1/CB2 KO mice actually have lower splenic sympathetic noradrenergic activity . The explanation for this effect is unclear and may involve CNS -mediated CB1 receptor effects upstream of post -ganglionic neurons. However, d ecreased release of NE from sympathetic neurons in the spleen may explain the accumulation of NE in the spleen capsu le without a concomitant increase in TH enzyme content. Regardless, the results regarding the rate of NE in the spleen of CB1/CB2 KO mice leads to the 320 acceptance of the null hypothesis regarding the absence of CB1 inhibition on NE release in the spleen as a mechanism for enhanced humoral immunity in CB1/CB2 KO mice. Due to the increased " 2AR expression of B cells in CB1/CB2 KO mice, it was hypothesized that " 2AR activity may still be the cause for enhanced humoral immunity in these mice. It was expecte d that if increased " 2AR stimulation were at least part ly responsible for increased humoral immune responses in CB1/CB2 KO mice, then blockade of these receptors would significantly attenuate antibody production in response to LPS . This hypothesis was reje cted following the observation that blockade of " 2AR had no observable effect in LPS -treated CB1/CB2 KO mice. Yet, as discussed above, "2AR stimulation is likely not involved in the humoral response to LPS. Therefore, this hypothesis needs to be revisited in response to an immunogen for which "2AR plays a role in modulating humoral responses, such as sRBC or CD40 stimulation . Collectively , the data presented in this dissertation are consistent with the conclusion that enhanced humoral immunity in the absence of CB1/CB2 is not due to dis -inhibition of splenic sympathetic noradrenergic neurons . Given that CB1 is the predominant neuronal receptors, this also suggests that enhanced humoral immunity in CB1/CB2 KO mice is not due to the absence of this receptor (Mackie, 2008) . Therefore, CB2 signaling directly on cells o f the immune system (Schatz et al., 1997; Tanasescu and Constantinescu, 2010) is the likely immunosuppressive mediator of the endocannabinoid system . 321 Future work should be directed towards this possibility by utilizing tools such as CB2 KO mice or CB2 specific agonists and antagonists. Assessing the activity of splenic sympathetic neurons in CB2 KO mice could rule out any contributing effect of this receptor to the function of these neurons. Of particular interest is the interaction between B2AR and CB2 receptors. The expression of "2AR on B cells should be evaluated in CB2 KO mice to determine if the lack of CB2 modulates the its expression. Furthermore, the effect of "2AR stimulation in response to appropriate immunogens can be evaluated in the absence of CB2 or during pharmacologic activation/ inhibition of CB2. Beyond these stu dies, an interesting point of investigation is the effect of CB2 on the transcription of the 3 $-IgH enhancer. It is unknown if the actions of CB2 are able to directly inhibit 3 $-IgH enhancer activity to mediate cannabinoid -mediated suppression of antibody production. 7.4: Sympathetic control of spleen contraction Spleen contraction is mediated by two different neurotransmitters. NE can induce spleen contraction and is responsible for approximately 2/3 of EFS -induced spleen contraction . NE -induced splee n contraction is mediated by ! 1AR, as demonstrated by the ability of the pan ! 1AR antagonist prazosin to block this response. More specifically, the type of ! 1AR mediating this effect is the ! 1BAR subtype (Eltze, 1996; Aboud et al., 2012) . This specific subty pe of the ! 1AR has previously been demonstrated to undergo phosphorylation mediated 322 desensitization and internalization in response to NE binding , occurring as quickly as 5 min after binding (Leeb -Lundberg et al., 1987; Fonseca et al., 1995; Malbon and Wang, 2001) . Thus , receptor internalization is the likely cause for the NE -induced attenuation of spleen contraction observed at repeated high concentrations of exogenous NE . The other neurotransmitter mediating spleen contraction is adenosine derived from ATP (Rongen et al., 1996) . Results from the present studies reveal that a denosine activation of A1 receptors is responsible for this action, which is in agreement with other investigators (Fozard and Milavec -Krizman, 1993) . A1 receptors are also known to mediate a relatively small portion (18 -33%) of vascular smooth muscle contraction (Tawfik et al., 2005) . 7.5: The role of CB1 and CB2 in spleen contraction The spleens from CB1/CB2 KO mice produce less force during contraction tha n WT in response to NE. The mechanism for this phenomenon is d ecreased ! 1AR expression on smooth muscle in the spleen capsule of CB1/CB2 KO mice . In support of this conclusion, NE induces a concentration -dependent increase in spleen cont raction force, which demons trates that increasing activation of ! 1AR receptors re sults in increased force of contraction. Thus, decreased activation of ! 1AR, due to decreased expression, likely account s for the decreased contraction observed in CB1/CB2 KO mice. This is despite similar weights and similar amounts of smooth muscle specific !-actin (Skalli et al., 1 986; Johnson et 323 al., 2010), both of which suggest an equivalent amount of smooth muscle in the spleen capsule of WT and CB1/CB2 KO mice . The reason for decreased spleen capsule !1AR expression in CB1/CB2 KO mice remains unclear. It was hypothesized th at ! 1AR would be increased in CB1/CB2 KO mice secondary to decreased receptor stimulation from decreased NE release . However, this does not appear to be the case since ! 1AR are decreased, not increased , in CB1/CB2 deficient mice . Thus, mechanisms other than ligand -mediated receptor internalization are likely at play . Whether this effect is direct or indirect cannot be determined by the data obtained in these experiments. It is possible that CB receptors directly alter ! 1AR expression, just as has been recently described for CB1 and " 2AR (Hudson et al., 2010) . While this supposition is without precedence and speculative, increasing evidence for protein -protein interactive effect s between CB receptors and other G -protein coupled receptors are known (Kearn, 2005; Hudson et al., 2010; Ward et al., 2011). This hypothesis requires further investigation and likely should start with determining which receptor, CB1 or CB2, is the major mediator of decreased ! 1AR expression through the use of selective CB1 and CB2 antagonists or mouse models employing specific knockout of CB1 or CB2 . The compensatory mechanism whereby EFS -induced spleen contraction in CB1/CB2 KO mice is equivalent to th at of WT mice does not involve changes in non -adrenergic transmission. Prazosin blockade of noradrenergic transmission reduces EFS -induced spleen contraction to an equivalent extent in both WT and 324 CB1/CB2 KO mice. Thus, the contribution of NE to EFS -induc ed spleen contraction in CB1/CB2 KO mice is equivalent to WT, despite the reduction in ! 1AR. This phenomenon may be due to an increase in the quantal release of NE in CB1/CB2 KO mice. Increased NE released per action potential would hypothetically stimula te more ! 1AR in CB1/CB2 KO mice, thus compensating for lower ! 1AR expression. In fact, CB1/CB2 KO mice may be primed for this scenario as the axon terminals of splenic sympathetic neurons in these mice contain more NE than WT mice. Increased release of ne urotransmitter s is not without precedence. For instance, BDNF can increase the quantal release of glutamate in the hippocampus (Tyler and Pozzo -Miller, 2001; Amaral and Pozzo -Miller, 2012) . This hypothesis may explain the observed data and could be further validat ed using microdialysis. It would be expected if this were true the concentration of captured NE in the dialysate from spleens of CB1/CB2 KO mice would be higher than those from WT spleen in response to sympathetic neuron excitation . Alternatively, a n electrochemical method utilizing boron -doped diamond nanoelect rodes, which can quantify the amount of NE released from in situ neurons, could be utilized to compared the quantal relea se of NE in the spleens of WT and CB1/CB2 KO mice (Dong et al., 2011) . The mechanism underlying this hypothesis could then be studied by ultrastructure analysis of splenic sympathetic neuron axon te rminals using electron microscopy. If this hypothesis is true then the size of synaptic vesicles or the number of docking 325 synaptic vesicles should be increased in splenic sympathetic axon terminals of CB1/CB2 KO mice compared to WT. Interestingly, the capsule of the spleen in CB 1/CB2 KO mice is thinner than WT mice, but not due to differences in smooth muscle content. The other two major tissues of the spleen capsule are connective tissue and elastic tissue (Cesta, 2006) . CB1 and CB2 have demonstrated effects on the production of extracellular matrix components from fibroblast. CB1 are stimulatory to fibroblasts (Marquart et al., 2010; Lazzerini et al., 2012) , whereas CB2 are inh ibitory (Akhmetshina et al., 2009; Defer et al., 2009) . Despite t hese opposing effects, these reports demonstrate that CB receptors can modulate the activity of fibroblasts. It may be the case that CB1 and/or CB2 receptors allow for an appropriate amount , or structure , of extracellular material and these effects during development account for reduced spleen capsule thickness in CB1/CB2 KO mice. Alternative to changes in the amount or structure of extracellular material, changes in the number, or cytoarchitecture, of cells in the spleen capsule could account for a decreas ed th ickness of the spleen capsule of CB1/CB2 KO mice . However, any changes in the cytoarchitecture would likely not affect smooth muscle cells, a s they are able to contract and have a no minal amount of !-actin. A n interesting line of experimentation woul d be to evaluate the relative contribution of CB1 and CB2 to connective tissue development in the spleen. This line of investigation could initiated by histologically evaluating the 326 structure and development of the spleen capsule in the individual CB1 and CB2 KO mice. 7.6: Significance of cannabinoid use and abuse Cannabis, or marijuana, has been used by humans for over 5000 years, the earliest recorded use of cannabis fibers occurred circa 4,000 B.C. (Zuardi, 2008). The use of marijuana for medicinal and possibly s piritual purposes arose in China and the Himalay as as far back as 2000 B.C. (Kalant, 2001; Zuardi, 2008). Over time marijuana has been used by a multitude of cultures for spiritual, religious, medicinal, and recreation al purposes. Even today cannabis is still widely used. In fact, in the U.S., whe re marijuana is illegal according to federal law, it is the most widely used illicit drug with an estimated ~17.4 million people using marijuana every month (National Institute on Drug Abuse, 2013) . Additionally, studies show that approxim ately 1/3 of 12 th grade students ha ve tried marijuana (National Institute on Drug Abuse, 2013) . Desp ite the illegal status given to cannabis by multiple national governments (Hall and Diehm, 2014) , the use of marijuana persists and may be gaining mainstream acceptance (Swift, 2014) . In support of this, 20 U.S. states and the Distr ict of Columbia have approved measures allowing for the possession and growing of marijuana for medicinal purposes (WhiteHouse.gov, 2014). Two of these states, Colorado and Washington, have legalized the recreational use of marijuana, which i s an indicator of increasing popular support 327 for the use of marijuana (Wollner, 2014; 2014) . In South America , Paraguay recently legaliz ed the production and sale of marijuana (Watts, 2014) . In addition to the accept ance of marijuana in a modern social context, cannabinoid -based compounds are finding acceptance in modern Western medicine. T he U.S. F ood and Drug Agency (FDA) has approved several cannabinoid -based compounds for medicinal use and prescription. Dronabinol is synthetic ally created THC and is approved by the FDA for the control of nausea and vomiting in cancer patients undergoing chemotherapy as well as an appetite stimulant in patients with AIDS (Galal et al., 20 09). In the United Kingdom, a drug named nabiximols (Sativex¨), which is a mixture of THC and another cannabis -derived cannabinoid called cannabidiol (CBD), is approved for the control of spasticity in multiple sclerosis patients (Pharm aceuticals, 2011) . Despite th e increasing acceptance and popularity of marijuana , the biological ramifications of cannabinoid use are not completely understood. This is particularly true for the actions of cannabinoids outside the brain. As has been discussed, c annabinoid receptors are found not only in the CNS , but als o throughout the body (Schatz et al., 1997; Ralevic and Kendall, 2002; Cabral et al., 2008; Mackie, 2008; Cabral and Griffin -Thomas, 2009; Atwood and Mackie, 2010; Kaplan, 2012) . The presen ce of peripheral ly located cannabinoid receptors suggests a role for both exogenous and endogenous cannabinoids at these sites . Understanding the role of endocannabinoid signaling is significant by itself, but is also able to inform the scientific and med ical community of the 328 possible ramifications of cannabinoid use and/or abuse , which remains a persistent world -wide issue (Degenhardt et al., 2013) . With particular reference to work in this dissertation , the immunologic function of cannabinoids is of clinical interest. The use of potentially immunosuppressive cannabinoids need s to be evaluated and understood as these compounds are already potentially being give n to immunocompromised patients with AIDS or undergoing chemotherapy (Galal et al., 2009) . However, what may be even more relevant is the potential impact of cannabinoids in new and chronic users of cannabis as it become increasingly popular and available . The effect of cannabinoids on the contraction of the spleen is wholly untouched by the scientific community. The work in this dissertation, while of scientific interest, is difficult as yet to relate to a clinic or biologic effect. This may be simply due to the lack of understanding regarding the function of spleen contraction as a whole. Of particular interest are the consequences of spleen contraction on immune function . It has been demonstrated that i mmune cells from the spleen are released in response to spleen contraction (Seifert et al., 2012). Yet it is unknown if this release of immu ne cells is beneficial or detrimental to immunity. It can be hypothesized that increasing the number of circulating macrophages and lymphocytes can increase the probability of these cells to interact with an immunogen thus initiating immune responses more quickly to infection. On the other, the dispersion of immune cells from their close packed environment of the spleen may impede the requirement for these cells to 329 interact with each other in the process of an immune response. Which of these hypothesized effects are true or predominant is unknown and presents an interesting avenue for future research. Regardless , cannabinoid receptors definitely play a role in regulating the contraction of the spleen and warrants further investigation. 7.7: Concluding Remarks The spleen is a multifunction organ that sits at a unique intersection between the circulatory, immune, and neurologic systems. The work in this dissertation endeavored to shed light on the interaction of the sympathetic nervous system in the spl een with these other vital biologic systems. In addition , the role of CB1/CB2 signaling was explored as it relates the function of splenic sympathetic noradrenergic innervation . Specifically it was found that splenic noradrenergic neurons do not play a rol e in T cell independent humoral immunity, and that both NE and adenosine mediate spleen contraction. It was concluded that splenic sympathetic noradrenergic neurons likely are not regulated by CB1 and that cannabinoid -mediated immunosuppression of humoral immunity is likely due solely to CB2 on immune cells. It was also found that CB1/CB2 play a permissive role in maintaining the relationship between NE release from splenic sympathetic neurons and spleen contraction. These findings add to the knowledge ba se regarding both the spleen and extra -CNS cannabinoid effects and can be built upon for a more complete understanding of these systems. 330 REFERENCES 331 REFERENCES Aboud R, Shafii M, Docherty JR. Investigati on of the subtypes of ! 1-adrenoceptor mediating contractions of rat aorta, vas deferens and spleen. Br J Pharmacol. 2012 Jul 19;109(1):80 Ð7. Ademokun AA, Dunn -Walters D. Immune Responses: Primary and Secondary. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. Akhmetshina A, Dees C, Busch N, Beer J, Sarter K, Zwerina J, et al. The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis. Arthritis Rheum. 2009 Apr;60(4):1129 Ð36. Amaral MD, Pozzo -Miller L. Intracellular Ca2 + stores and Ca2+ influx are both required for BDNF to rapidly increase quantal vesicular transmitter release. Neural Plast. 2012;2012:203536. PMCID: PMC3397209 Atwood BK, Mackie K. CB2: a cannabinoid receptor with an identity crisis. Br J Pharmacol. 2010 Mar 4;160(3):467 Ð79. Basu S, Ray A, Dittel BN. Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen -Specific IgG and IgM, in Response to T -dependent Antigens. PLoS ONE. 201 3;8(6):e67587. PMCID: PMC3695093 Baumgarth N. Innate -Like B Cells and Their Rules of Engagement. link.springer.com.proxy2.cl.msu.edu. New York, NY: Springer New York; 2013. p. 57 Ð66. Bekeredjian -Ding I, Jego G. Toll -like receptors --sentries in the B -cell response. Immunology. 2009 Nov;128(3):311 Ð23. PMCID: PMC2770679 Blue J, Weiss L. Electron microscopy of the red pulp of the dog spleen including vascular arrangements, periarterial macrophage sheaths (Ellipsoids), and the contractile, innervated reticular meshwork. Am. J. Anat. 1981 Jun;161(2):189 Ð218. Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines. J Pharmacol Exp Ther. 1966 Dec;154(3): 493Ð8. Cabral GA, Griffin -Thomas L. Emerging role of the cannabinoid receptor CB2 in 332 immune regulation: therapeutic prospects for neuroinflammation. Expert Rev Mol Med. 2009;11:e3. PMCID: PMC2768535 Cabral GA, Raborn ES, Griffin L, Dennis J, Marciano -Cabral F. CB 2receptors in the brain: role in central immune function. Br J Pharmacol. 2008 Jan;153(2):240 Ð51. PMCID: PMC2219530 Cesta M. Normal Structure, Function, and Histology of the Spleen. Toxicol Pathol. 2006;34(5):455 Ð65. CMA Microdialysi s. Microdialysis Probes [Internet]. CMA Microdialysis AB. [cited 2013 Dec 18]. Retrieved from: http://www.microdialysis.com/probe_brochure.pdf?cms_fileid=277253 2331977349a4a942f6d40fafca Defer N, Wan J, Souktani R, Escoubet B, Perier M, Caramelle P, et al. The cannabinoid receptor type 2 promotes cardiac myocyte and fibroblast survival and protects against ischemia/reperfusion -induced cardiomyopathy. FASEB J. 2009 Jul;23(7):2120 Ð30. Degenhardt L, Whiteford HA, Ferrari AJ, Baxter AJ, Charlson FJ, Hall WD, e t al. Global burden of disease attributable to illicit drug use and dependence: findings from the Global Burden of Disease Study 2010. Lancet. 2013 Nov 9;382(9904):1564 Ð74. Dong H, Wang S, Galligan JJ, Swain GM. Boron -doped diamond nano/microelectrodes fo r biosensing and in vitro measurements. Front Biosci (Schol Ed). 2011;3:518 Ð40. Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. 2004 Sep 1;56(3):331 Ð49. Eltze M. Functiona l evidence for an alpha 1B -adrenoceptor mediating contraction of the mouse spleen. Eur J Pharmacol. 1996 Sep 12;311(2 -3):187 Ð98. Felten DL, Ackerman KD, Wiegand SJ, Felten SY. Noradrenergic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neurosci Res. 1987;18(1):28 Ð36, 118Ð21. Felten SY, Olschowka J. Noradrenergic sympathetic innervation of the spleen: II. Tyrosine hydroxylase (TH) -positive nerve termin als form synapticlike contacts on lymphocytes in the splenic white pulp. J Neurosci Res. 1987;18(1):37 Ð48. 333 Fonseca MI, Button DC, Brown RD. Agonist regulation of alpha 1B -adrenergic receptor subcellular distribution and function. J Biol Chem. 1995 Apr 14;270(15):8902 Ð9. Fozard JR, Milavec -Krizman M. Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation. Br J Pharmacol. 1993 Jul 19;109(4):1059 Ð63. PMCID: PMC2175713 Galal A, Slade D, Gul W, El -Alfy A, Ferreira D, Elsohly M. Nat urally occurring and related synthetic cannabinoids and their potential therapeutic applications. Recent Pat CNS Drug Discov. 2009 Jun 1;4(2):112 Ð36. Groom AC, Schmidt EE, MacDonald IC. Microcirculatory pathways and blood flow in spleen: new insights from washout kinetics, corrosion casts, and quantitative intravital videomicroscopy. Scanning Microsc. 1991 Mar;5(1):159 Ð73Ðdiscussion173 Ð4. Hall K, Diehm J. The World's Most Marijuana -Friendly Countries (INFOGRAPHIC) [Internet]. huffingtonpost.com. 2014 [cit ed 2014 Jan 10]. Retrieved from: http://www.huffingtonpost.com/2013/08/27/marijuana -world -map-_n_3805800.html Hayley S, Lacosta S, Merali Z, van Rooijen N, Anisman H. Central monoamine and plasma corticosterone changes induced by a bacterial endotoxin: sen sitization and cross -sensitization effects. Eur J Neurosci. 2001 Mar;13(6):1155 Ð65. Helwig BG, Craig RA, Fels RJ, Blecha F, Kenney MJ. Central nervous system administration of interleukin -6 produces splenic sympathoexcitation. Autonomic Neuroscience. 2008 Aug;141(1 -2):104 Ð11. Hudson BD, H”bert TE, Kelly MEM. Physical and functional interaction between CB1 cannabinoid receptors and beta2 -adrenoceptors. Br J Pharmacol. 2010 Jun;160(3):627 Ð42. PMCID: PMC2931563 Ishac E, Jiang L, Lake K, Varga K, Abood M, Kun os G. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves. Br J Pharmacol. 1996 Aug 1;118(8):2023 Ð8. PMCID: PMC1909901 Johnson KB, Thompson JM, Watts SW. Modification of proteins by norep inephrine is important for vascular contraction. Front Physiol. 2010;1:131. PMCID: PMC3059971 Kalant H. Medicinal use of cannabis: History and current status. Pain Res Manag. 334 2001;6(2):80 Ð91. Kaplan BLF. The Role of CB(1) in Immune Modulation by Cannabino ids. Pharmacol. Ther. 2012 Dec 19. Karhunen T, Tilgmann C, Ulmanen I, Julkunen I, Panula P. Distribution of catechol -O-methyltransferase enzyme in rat tissues. J. Histochem. Cytochem. 1994 Aug;42(8):1079 Ð90. Kauma SW, Turner TT, Harty JR. Interleukin -1 beta stimulates interleukin -6 production in placental villous core mesenchymal cells. Endocrinology. 1994;134(1):457 Ð60. Kearn CS. Concurrent Stimulation of Cannabinoid CB1 and Dopamine D2 Receptors Enhances Heterodimer Formation: A Mechanism for Receptor Cross -Talk? Molecular Pharmacology. 2005 Feb 9;67(5):1697 Ð704. Khan ZP, Ferguson CN, Jones RM. Alpha -2 and imidazoline receptor agonistsTheir pharmacology and therapeutic role. Anaesthesia. 2002 Apr 6;54(2):146 Ð65. Kin NW, Sanders VM. It takes nerve to tell T and B cells what to do. J Leukoc Biol. 2006 Jun;79(6):1093 Ð104. Kohm AP, Sanders VM. Norepinephrine and beta 2 -adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pha rmacol Rev. 2001 Dec;53(4):487 Ð525. Kohm AP, Tang Y, Sanders VM, Jones SB. Activation of antigen -specific CD4+ Th2 cells and B cells in vivo increases norepinephrine release in the spleen and bone marrow. 2000 Jul 15;165(2):725 Ð33. Kumer SC, Vrana KE. In tricate regulation of tyrosine hydroxylase activity and gene expression. J Neurochem. 1996 Aug 1;67(2):443 Ð62. Lanzavecchia A, Sallusto F. Toll -like receptors and innate immunity in B -cell activation and antibody responses. Curr Opin Immunol. 2007 Jun;19( 3):268 Ð74. Lazar MA, Truscott RJW, Raese JD, Barchas JD. Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme. J Neurochem. 1981 Feb;36(2):677 Ð82. Lazzerini PE, Natale M, Gianchec chi E, Capecchi PL, Montilli C, Zimbone S, et al. 335 Adenosine A2A receptor activation stimulates collagen production in sclerodermic dermal fibroblasts either directly and through a cross -talk with the cannabinoid system. J. Mol. Med. 2012 Mar;90(3):331 Ð42. Lee TH, Ellinwood EH Jr., Einstein G. Intracellular recording from dopamine neurons in the substantia nigra: double labelling for identification of projection site and morphological features. Journal of Neuroscience Methods. 1992 Jul;43(2 -3):119 Ð27. Leeb-Lundberg LM, Cotecchia S, DeBlasi A, Caron MG, Lefkowitz RJ. Regulation of adrenergic receptor function by phosphorylation. I. Agonist -promoted desensitization and phosphorylation of alpha 1 -adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF -2 smooth muscle cells. J Biol Chem. 1987 Mar 5;262(7):3098 Ð105. Lookingland KJ, Ireland LM, Gunnet JW, Manzanares J, Tian Y, Moore KE. 3 -Methoxy -4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons. Brain Res. 1991 Sep 13;559(1):82 Ð8. Lookingland KJ, Moore KE. Chapter VIII Functional neuroanatomy of hypothalamic dopaminergic neuroendocrine systems. Handbook of chemical neuroanatomy. 2005. Mackie K. Signaling via CNS cannabi noid receptors. Mol. Cell. Endocrinol. 2008 Apr 16;286(1 -2 Suppl 1):S60 Ð5. PMCID: PMC2435200 Malbon CC, Wang H -Y. Adrenergic Receptors. onlinelibrary.wiley.com. Chichester, UK: John Wiley & Sons, Ltd; 2001. Marino F, Cosentino M. Adrenergic modulation of immune cells: an update. Amino Acids. 2013 Jul;45(1):55 Ð71. Marquart S, Zerr P, Akhmetshina A, Palumbo K, Reich N, Tomcik M, et al. Inactivation of the cannabinoid receptor CB1 prevents leukocyte infiltration and experimental fibrosis. Arthritis Rheum. 20 10 Nov;62(11):3467 Ð76. Myıh−nen TT, Schendzielorz N, M−nnistı PT. Distribution of catechol -O-methyltransferase (COMT) proteins and enzymatic activities in wild -type and soluble COMT deficient mice. J Neurochem. 2010 Mar 31. Nance DM, Sanders VM. Autonomi c innervation and regulation of the immune system (1987 -2007). Brain Behav Immun. 2007 Aug;21(6):736 Ð45. PMCID: PMC1986730 336 National Institute on Drug Abuse. Marijuana Abuse. 2013 Jul 24;:1 Ð12. Retrieved from: http://www.drugabuse.gov/sites/default/files/rr marijuana.pdf N”ron S, Nadeau PJ, Darveau A, Leblanc J -F. Tuning of CD40 -CD154 interactions in human B -lymphocyte activation: a broad array of in vitro models for a complex in vivo situation. Arch. Immunol. Ther. Exp. (Warsz.). 2011 Feb;59(1):25 Ð40. Niede rhoffer N, Szabo B. Effect of the cannabinoid receptor agonist WIN55212 -2 on sympathetic cardiovascular regulation. Br J Pharmacol. 1999 Jan;126(2):457 Ð66. O'Connell FP, Pinkus JL, Pinkus GS. CD138 (syndecan -1), a plasma cell marker immunohistochemical pr ofile in hematopoietic and nonhematopoietic neoplasms. Am. J. Clin. Pathol. 2004 Feb;121(2):254 Ð63. Oh-hashi Y, Shindo T, Kurihara Y, Imai T, Wang Y, Morita H, et al. Elevated Sympathetic Nervous Activity in Mice Deficient in CGRP. Circ. Res. 2001 Nov 23 ;89(11):983 Ð90. Okuno S, Fujisawa H. Conversion of tyrosine hydroxylase to stable and inactive form by the end products. J Neurochem. 1991 Jul;57(1):53 Ð60. Olive MF, Mehmert KK, Hodge CW. Microdialysis in the mouse nucleus accumbens: a method for detecti on of monoamine and amino acid neurotransmitters with simultaneous assessment of locomotor activity. Brain Res. Brain Res. Protoc. 2000 Feb;5(1):16 Ð24. Olofsson PS, Rosas -Ballina M, Levine YA, Tracey KJ. Rethinking inflammation: neural circuits in the reg ulation of immunity. Immunol. Rev. 2012 Jul;248(1):188 Ð204. org D, editor. Marijuana Legalization in Washington State and Colorado | Drug Policy Alliance [Internet]. drugpolicy.org. 2014 [cited 2014 Jan 10]. Retrieved from: http://www.drugpolicy.org/resource/marijuana -legalization -washington -state -and-colorado Ortega JE, Katner J, Davis R, Wade M, Nisenbaum L, Nomikos GG, et al. Modulation of neurotransmitter release in orexin/hypocretin -2 receptor knockout mice: a microdialy sis study. J Neurosci Res. 2012 Mar;90(3):588 Ð96. Padro CJ, Shawler TM, Gormley MG, Sanders VM. Adrenergic Regulation of IgE Involves Modulation of CD23 and ADAM10 Expression on Exosomes. The Journal of Immunology. 2013 Oct 18. PMCID: PMC3842235 337 Parikh AA , Salzman AL, Kane CD, Fischer JE, Hasselgren PO. IL -6 production in human intestinal epithelial cells following stimulation with IL -1 beta is associated with activation of the transcription factor NF -kappa B. J Surg Res. 1997 Apr 1;69(1):139 Ð44. Pharmace uticals G. Sativex. 2011;(3/8/2012). Pinkus GS, Warhol MJ, O'Connor EM, Etheridge CL, Fujiwara K. Immunohistochemical localization of smooth muscle myosin in human spleen, lymph node, and other lymphoid tissues. Unique staining patterns in splenic white p ulp and sinuses, lymphoid follicles, and certain vasculature, with ultrastructural correlations. Am. J. Pathol. 1986 Jun;123(3):440 Ð53. PMCID: PMC1888274 Pongratz G, McAlees JW, Conrad DH, Erbe RS, Haas KM, Sanders VM. The Level of IgE Produced by a B Cell Is Regulated by Norepinephrine in a p38 MAPK - and CD23 -Dependent Manner. J Immunol. 2006 Sep 1;177(5):2926 Ð38. Ralevic V, Kendall DA. Cannabinoids inhibit pre - and postjunctionally sympathetic neurotransmission in rat mesenteric arteries. Eur J Pharmacol . 2002 May 31;444(3):171 Ð81. Rongen GA, Lenders JW, Lambrou J, Willemsen JJ, Van Belle H, Thien T, et al. Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension. 1996 Apr;27(4):933 Ð8. Saito H , Yokoi Y, Watanabe S, Tajima J, Kuroda H, Namihisa T. Reticular meshwork of the spleen in rats studied by electron microscopy. Am. J. Anat. 1988 Mar;181(3):235 Ð52. Sanders VM. The beta2 -adrenergic receptor on T and B lymphocytes: Do we understand it yet? Brain Behav Immun. 2012 Feb;26(2):195 Ð200. PMCID: PMC3243812 Schatz A, Lee M, Condie R, Pulaski J, Kaminski N. Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Phar macol. 1997 Feb 1;142(2):278 Ð87. Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR. A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation. J Neuroimmune Pharmacol. 2012 Dec;7 (4):1017 Ð24. PMCID: PMC3518577 338 Shimizu N, Hori T, Nakane H. An interleukin -1 beta -induced noradrenaline release in the spleen is mediated by brain corticotropin -releasing factor: an in vivo microdialysis study in conscious rats. Brain Behav Immun. 1994 Mar 1;8(1):14 Ð23. Siraskar B, Vılkl J, Ahmed MSE, Hierlmeier M, Gu S, Schmid E, et al. Enhanced catecholamine release in mice expressing PKB/SGK -resistant GSK3. Pflugers Arch. 2011 Dec;462(6):811 Ð9. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha -smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 1986 Dec;103(6 Pt 2):2787 Ð96. PMCID: PMC2114627 Spangelo BL, deHoll PD, Kalabay L, Bond BR, Arnaud P. Neurointermediate pituit ary lobe cells synthesize and release interleukin -6 in vitro: effects of lipopolysaccharide and interleukin -1 beta. Endocrinology. 1994 Aug 1;135(2):556 Ð63. Springs AEB, Karmaus PWF, Crawford RB, Kaplan BLF, Kaminski NE. Effects of targeted deletion of ca nnabinoid receptors CB1 and CB2 on immune competence and sensitivity to immune modulation by Delta9 -tetrahydrocannabinol. J Leukoc Biol. 2008 Dec;84(6):1574 Ð84. PMCID: PMC2614598 Swift A. For First Time, Americans Favor Legalizing Marijuana [Internet]. gal lup.com. 2014 [cited 2014 Jan 10]. Retrieved from: http://www.gallup.com/poll/165539/first -time -americans -favor -legalizing -marijuana.aspx Tanasescu R, Constantinescu C. Cannabinoids and the immune system: an overview. Immunobiology. 2010 Aug 1;215(8):588 Ð97. Tangye SG. Staying alive: regulation of plasma cell survival. Trends Immunol. 2011 Dec;32(12):595 Ð602. Tawfik HE, Schnermann J, Oldenburg PJ, Mustafa SJ. Role of A1 adenosine receptors in regulation of vascular tone. Am J Physiol Heart Circ Physiol. 2 005 Mar;288(3):H1411 Ð6. Tomiyama -Hanayama M, Rakugi H, Kohara M, Mima T, Adachi Y, Ohishi M, et al. Effect of interleukin -6 receptor blockage on renal injury in apolipoprotein E -deficient mice. AJP: Renal Physiology. 2009 Sep 1;297(3):F679 Ð84. Tyler WJ, Pozzo -Miller LD. BDNF enhances quantal neurotransmitter release 339 and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. Journal of Neuroscience. 2001 Jun 15;21(12):4249 Ð58. PMCID: PMC2806848 Uchiyama Y, Yoshida H , Koike N, Hayakawa N, Sugita A, Nishimura T, et al. Anti -IL-6 receptor antibody increases blood IL -6 level via the blockade of IL -6 clearance, but not via the induction of IL -6 production. Int Immunopharmacol. 2008 Nov 1;8(11):1595 Ð601. Vrana KE, Allhise r CL, Roskoski R. Tyrosine hydroxylase activation and inactivation by protein phosphorylation conditions. J Neurochem. 1981 Jan;36(1):92 Ð100. Vrana KE, Roskoski R. Tyrosine hydroxylase inactivation following cAMP -dependent phosphorylation activation. J Neurochem. 1983 Jun;40(6):1692 Ð700. Ward RJ, Pediani JD, Milligan G. Heteromultimerization of cannabinoid CB(1) receptor and orexin OX(1) recept or generates a unique complex in which both protomers are regulated by orexin A. Journal of Biological Chemistry. 2011 Oct 28;286(43):37414 Ð28. PMCID: PMC3199489 Watts J. Uruguay legalises production and sale of cannabis | World news | The Guardian [Intern et]. theguardian.com. 2014 [cited 2014 Jan 4]. Retrieved from: http://www.theguardian.com/world/2013/dec/11/uruguay -cannabis -marijuana -production -sale -law WhiteHouse.gov. Marijuana Resource Center: State Laws Related to Marijuana | The White House [Interne t]. whitehouse.gov. 2014 [cited 2013 Dec 31]. Retrieved from: http://www.whitehouse.gov/ondcp/state -laws -related -to-marijuana Wollner A. Public Support For Marijuana Legalization Hits Record High : It's All Politics : NPR [Internet]. npr.org. 2014 [cited 2 014 Jan 10]. Retrieved from: http://www.npr.org/blogs/itsallpolitics/2013/10/22/239847084/public -support -for -marijuana -legalization -hits -record -high Zastrow von M, Kobilka BK. Ligand -regulated internalization and recycling of human beta 2 -adrenergic recept ors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem. 1992 Feb 15;267(5):3530 Ð8. Zouali M. Natural Antibodies. els.net. Chichester, UK: John Wiley & Sons, Ltd; 2001. 340 Zuardi AW. Cannabidiol: from an inactive cannabin oid to a drug with wide spectrum of action. Rev Bras Psiquiatr [Internet]. 2008 Sep;30(3):271 Ð80. Retrieved from: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&i d=18833429&retmode=ref&cmd=prlinks npr.org [Internet]. [cited 2013 Dec 31]. Retrieved from: http://www.npr.org