NEURONAL FIRING PROPERTIES IN SYMPATHETIC GANGLION NEURONS IN HYPERTENSION By Xiaohong Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Physiology -Doctor of Philosophy 2014 ABSTRACT NEURONAL FIRING PROPERTIES IN SYMPATHETIC GANGLION NEURONS IN HYPERTENSION By Xiaohong Wang The activity of both sympathetic neurons in the central nervous system and peripheral sympathetic nerves that innervate the heart and blood vessels is elevated in hypertension. However it is unclear about the roles of sympathetic ganglia, which integrate central sympathetic signals and send their signals to cardiovascular end organs via sympathetic nerve. The celiac ganglion (CG) plays an important function in regulating blood pressure by the controlling s planchnic circulation containing 30% of total blood volume . Reactive oxygen species (ROS) are elevated in CG in hypertension. Ion channels, essential factors for neu ronal firing, are possible targets for ROS. The purp ose of this study is to find if neuronal firing and ion channel s are modulated in hypertension and if ROS are related to changes in neuronal firing and ion channels in hypertension. Neuronal firing patterns were explored in dissociated CG neurons from DOCA-salt hypertensive rats. In response to sustained su prathreshold current injection, more phasic neurons, fewer adaptive neurons and tonic neurons were shown in dissociated CG neurons from hypertensi ve rats. Moreover, t he firing frequencies of tonic neurons were significantly lower in hypertensive rats compared to matched normotensive rats. Furthermore several K + currents were found different in CG from the same animal model. Delay rectifier K + current (IKv), big conductance Ca2+-activated K + current (IBK) and A -type K + current (IA) were significantly lower in hypertensive rats compared to normotensive rats. The contribution of the decreased K+ currents to neuronal firing changes in hypertension is confirmed by using specific K+ channel blockers. H2O2, one type of ROS, was applied to dissociated CG neurons. H2O2 caused more accommodating firing by converting tonic neurons to phasic /adaptive and adaptive neurons to phasic in response to sustained suprathreshold current injection . Neuronal firing frequency was also decreased by H2O2. All the changes in neuronal firing by H2O2 are similar to those differences between normotensive and hypertensive rats. Moreover H2O2 mimicked some of the reductions of K currents in hypertension by decreasing IA and IBK. In conclusion, the changes in distribution of different neuronal firing pattern s, associated with attenuated K+ currents, are contributed by elevated ROS in CG neurons from hypertensive rats. The electrophysiological changes in CG might contribute to the increased sympathetic activity in DOCA-salt hypertensive rats . iv ACKNOWLEDGEMENT S I am sincerely thankful to my advisor, David Kreulen, who gives me enormous help s , enthusiasm and broad knowledge. Whenever I felt I could not achieve a goal , patient and motivational guidance I overcame many difficulties and finished my dissertation writing. I could not have imagined finishing my Ph.D study without his mentoring. Besides my advisor, I would also like to thank the rest of my committee members: Drs . Bruce Uhal, James Galligan, Stephen Schneider, Steven Heidemann. Thank you for all your encouragement, suggestions and continuous supports. Furthermore, I would like to thank the director of Physiology department, Dr. Arthur Weber for offering me the opportunity to study in the program and your many supports. I am indebted to my many fellow labmates for their helps and camaraderie : Xiaoling Dai, Jolene Zheng, Erica Wehrwein, Xian Cao, Anna Wright, Lindsay Parker, Mohammad Esfahanian , Casey Henley, Timothy Houchin, Amit Shah. I would also like to show my gratitude and thanks to my friends in the neighborhood laboratories. I could not finish my project without their helps: Xiaochun Bian, Hui Xu, We i Li, Tracy Walker, Wen - hsin Ku and a ll the researchers working in like to thank specially to Xiaochun Bian who taught me patch clamp which is the major technique for my research . v Last but not the least, I would like to give thanks to my fami ly: my parents Zhenying Wang and Xiulan Chen , my husband Hui Wang and my two angels Timothy and Angelina . Thank my parents for giving birth to me and supporting me throughout my life. Thank my husband for the love and supports. Thank my angels for growing healthily and smiling to me everyday . vi TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ .......... i x LIST OF FIGURES ................................ ................................ ................................ .......... x LIST OF ABBREVIATIONS ................................ ................................ .......................... x i i CHAPTER 1: GENERAL INTRODUCTION ................................ ................................ .... 1 Hypertension ................................ ................................ ................................ ................. 2 Introduction of hypertension ................................ ................................ .................... 2 Hypertension animal models ................................ ................................ .................... 3 Characteristics of the DOCA - salt hypertensive rat ................................ ................ 6 Sympathetic nervous system and hypertension ................................ ........................ 9 SNS and BP regulation ................................ ................................ .............................. 9 Role of SNS in hypertension ................................ ................................ .................. 1 0 Structure of SNS ................................ ................................ ................................ ...... 11 Sympathetic neurons in the CNS and hypertension ................................ ............ 1 1 Sympathetic nerve acti vity and hypertension ................................ ....................... 1 3 Sympathetic ganglia and hypertension ................................ ................................ ..... 14 The structure of sympathetic ganglia ................................ ................................ .. 1 4 Signal transmission in sympathetic ganglia ................................ ......................... 1 5 Neurotransmission of sympathetic ganglia in hypertension ............................... 16 Neuronal membrane firing properties of sympathetic postganglionic neurons 17 Celiac ganglion and hyper tension ................................ ................................ ......... 19 Ion channels and hypertension ................................ ................................ .................. 20 I on channels and neuronal firing ................................ ................................ ........... 20 K + and Ca 2+ channel currents in CG neurons ................................ ....................... 2 1 K + channels and neuronal firing properties ................................ ......................... .2 2 Reactive Oxygen Species (ROS) and hypertension ................................ ................. 2 4 Introduction ................................ ................................ ................................ .............. 2 4 Elevated ROS in hypertension ................................ ................................ ............... 2 5 Oxidation function of ROS on regulation of ion channels ................................ ... 2 6 ROS and neuronal firing properties in hypertension ................................ ........... 2 7 Summary ................................ ................................ ................................ ...................... 2 8 BIBLIOGRAP HY ................................ ................................ ................................ ........... 31 CHAPTER 2: NEURONAL MEMBRANE FIRING PROPERTIES OF DISSOCIATED CELIAC GANGLION NEURONS IN DOCA - SALT HYPERTENSIVE RATS ................ 43 Abstract ................................ ................................ ................................ .................... 44 Introduction ................................ ................................ ................................ .............. 4 5 vii M aterials and Methods ................................ ................................ ............................ 4 6 Animals ................................ ................................ ................................ ................. 46 Tissure collection ................................ ................................ ................................ 47 Cell culture ................................ ................................ ................................ ........... 48 Whole - cell patch clamp ................................ ................................ ....................... 49 Data acquisition ................................ ................................ ................................ ... 50 Statistical analysis ................................ ................................ ............................... 50 Results ................................ ................................ ................................ ...................... 51 Discussion ................................ ................................ ................................ ............... 56 APP ENDIX ................................ ................................ ................................ ................ 59 BIB L IOGRAPHY ................................ ................................ ................................ ....... 80 CHAPTER 3: CALCIUM AND POTASSIUM CHANNEL CURRENTS OF DISSOCIATED CELIAC GANGLION NEURONS IN DOCA - SALT HYPERTENSIVE RATS ................................ ................................ ................................ ............................ 83 Abstract ................................ ................................ ................................ .................... 84 Introduction ................................ ................................ ................................ .............. 85 Materials and Methods ................................ ................................ ............................ 87 Animals ................................ ................................ ................................ ................. 87 Tissure collection ................................ ................................ ................................ 87 Cell culture ................................ ................................ ................................ ........... 87 Whole - cell patch clamp ................................ ................................ ....................... 87 Data acquisition ................................ ................................ ................................ ... 88 Statistical analysis ................................ ................................ ............................... 89 Results ................................ ................................ ................................ ...................... 8 9 Discussion ................................ ................................ ................................ ............... 9 2 APPENDIX ................................ ................................ ................................ ................ 96 BIB L IOGRAPHY ................................ ................................ ................................ ..... 1 11 CHAPTER 4: EFFECTS OF HYDROGEN PEROXIDE ON NEURONAL FIRING AND POTASSIUM CHANNEL CURRENTS OF DISSOCIATED RAT C ELIAC GANGLION NEURONS ................................ ................................ ................................ .................. 1 1 5 Abstract ................................ ................................ ................................ .................. 1 1 6 Introduction ................................ ................................ ................................ ............ 1 1 7 Materials and Methods ................................ ................................ .......................... 1 1 9 Animals ................................ ................................ ................................ ............... 119 Tissure collection ................................ ................................ .............................. 119 Cell culture ................................ ................................ ................................ ......... 119 Whole - cell patch clamp ................................ ................................ ..................... 119 Statistical analysis ................................ ................................ ............................. 120 Results ................................ ................................ ................................ .................... 1 2 0 Discussion ................................ ................................ ................................ ............. 1 27 APPENDIX ................................ ................................ ................................ .............. 131 BIB L IOGRAPHY ................................ ................................ ................................ ..... 1 57 viii CHAPTER 5 : CONCLUSION AND DISCUSSION ................................ ...................... 160 The Sympath etic nervous system in hypertension ................................ ................ 162 Increased sympathetic activity in hypertension ................................ ................. 1 62 The s ympathetic nervous system in DOCA - salt hypertensive rats .................. 1 63 Predicting the possible roles of altered firing properties in CG neurons from hypertensive rats ................................ ................................ ................................ ... 1 64 Neuronal firing pattern is a changeable firing property ................................ ... 165 Neuro transmission in sympath etic ganglia ................................ ....................... 166 Predicting the significance of alterations in firing properties of CG neurons for hypertension ................................ ................................ ................................ .......... 16 7 Change in K + currents and neurotransmitter release in hypertension ............. 1 6 9 Changes in the neuronal firing properties of SCG neurons in other animal models of hypertension ................................ ................................ ........................ 172 Neuronal firing properties and ROS ................................ ................................ ........ 1 73 K + currents and neur onal firing ................................ ................................ ........... 1 73 ROS and neuronal firing in the SNS ................................ ................................ ..... 1 75 H 2 O 2 in sympathetic ganglia and hypertension ................................ ................. 1 77 Factors that may contribute the observed changes in n euronal firing properties and elevated ROS levels in CGs from DOCA - salt hypertensive rats .................. 1 79 Increased central sympathetic drive ................................ ................................ .... 1 79 Upregulation of ET - B receptor ................................ ................................ ............. 1 8 1 Other possible factors ................................ ................................ ........................... 1 82 Vascular neurons in the CG ................................ ................................ ..................... 1 83 Substructure of the CG ................................ ................................ ......................... 1 83 Identification of vas c ular neurons ................................ ................................ ....... 1 84 Conclusion and perspectives ................................ ................................ .................. 1 85 APPENDIX ................................ ................................ ................................ .................. 188 BIB L IOGRAPHY ................................ ................................ ................................ ......... 1 9 1 ix LIST OF TABLES Table 2 - 1 : Electrical membrane properties of dissociated CG neurons from NT and HT rats ................................ ................................ ................................ ................... 55 Table 2 - 2 : Single AP properties in response to sustained current injection of CG neurons in NT and HT rats ................................ ................................ ............. 60 Table 2 - 3: Afterhyperpolarizaion of single AP of CG neurons in NT and HT rats ........ 60 Table 4 - 1 : The effects of H 2 O 2 on electrical parameters of the first AP in the neuronal firing by a sustained depolarizing current injection ........................ 1 25 Table 4 - 2 : The effects of H 2 O 2 on membrane electrical parameters of a single AP .. 1 25 Table 5 - 1 : Summaries of the differences between HT and NT rats and the changes by H 2 O 2 in CG neuronal firing properties and K + channel current amplitudes of CG neurons ................................ ................................ ................................ .......... 1 73 x LIST OF FIGURES Figure 2 - 1 : Neuronal firing patterns of celiac neurons from NT and HT rats .............. 5 6 Figure 2 - 2 : The characteristics of firing frequency change in response to a sustained current injection in CG neurons ................................ ................................ ........ 5 8 Figure 2 - 3: The effect of TEA on outward K + currents ................................ ............... 58 Figure 2 - 4 : Effect of TE A on neuronal firing of dissociated CG neurons .................... 6 1 Figure 2 - 5 : Effect of paxilline on outward K + currents ................................ ................ 6 5 Figure 2 - 6 : Effect of paxilline on neuronal firing of dissociated CG neurons .............. 6 6 Figure 2 - 7 : Effect of 4 - AP on neuronal firing of dissociated CG neurons ..................... 6 7 Figure 3 - 1 : Sustained outward K + currents of CG neurons in HT and NT rats ............. 84 Figure 3 - 2 : I Kv and I BK of CG neurons in HT and NT rats ................................ ............ 87 Figure 3 - 3 : I A of CG neurons in HT and NT rats ................................ .......................... 90 Figure 3 - 4 : I m of CG neurons in HT and NT rats ................................ .......................... 92 Figure 3 - 5 : I Ca of celiac neurons in HT and NT rats ................................ ..................... 94 Figure 4 - 1 : Effects of H 2 O 2 on firing patterns of dissociated CG neurons .................. 118 Figure 4 - 2 : Effects of H 2 O 2 on firing frequency in dissociated CG neurons ............... 1 22 Figure 4 - 3 : Effects of H 2 O 2 on sustained outward K + currents in dissociated CG neurons ................................ ................................ ................................ .......... 126 Figure 4 - 4 : The effects of H 2 O 2 on transient outward K + current ............................... 129 Figure 4 - 5 : The effects of H 2 O 2 on activation and inactivation transient outward K + current ................................ ................................ ................................ ............ 1 30 xi F igure 4 - 6 : The effects of H 2 O 2 on activation and inactivation transient outward K + current in presence of intracellular catalase ................................ ................... 132 F igure 5 - 1 : Prediction of the role of altered firing properties of CG neurons in HT ..... 15 6 F igure 5 - 2 : A diagram illustrating the possible role of CG neurons in sympathetic neurotransmission in DOCA - salt hypertensive rats ................................ ........ 1 7 4 xii LIST OF ABBREVIATION S 4 - AP 4 - Aminopyridine AHP afterhyperpolarization AP action potential APs action potentials BK K + channel large - conductance calcium - activated K + channel BP blood pressure CG celiac ganglion DOCA deoxycorticosterone acetate DOCA - salt deoxycorticosterone acetate salt f i instantaneous firing frequency f 1st first instantaneous firing frequency f last last instantaneous firing frequency H 2 O 2 hydrogen peroxide I A A - type K + channel current I BK large - conductance calcium - activated K + channel current I KCa calcium - activated K + channel current I Kv delayed rectifier K + channel current I m M - type K + channel current I r rheobasic current I SK small - conductance calcium - activated K + channel current xiii NADPH nicotinamide adenine dinucleotide phosphate NE norepinephrine O 2 superoxide anion hydroxyl radical R a access resistance R in input resistance RMP resting membrane potential ROS reactive oxygen species SCG Superior cervical ganglia SK K + channel small - conductance calcium - activated K + channel SNA sympathetic nerve activity SNS sympathetic nervous system TEA Tetraethylammonium TTX tetrodotoxin SHR spontaneous hypertensive rat CAP compound action potential CHAPTER 1 GENERAL INTRODUCTION - - - - - . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CHAPTER 2 NEURONAL MEMBRANE FI RING PROPERTIES OF D ISSOCIATED CELIAC GANGLION NEURONS IN DOCA - SALT HYPERTENSIVE RA TS The activity of both sympathetic neurons in the central nervous system and peripheral sympathetic nerves that innervate the heart and blood vessels is elevated in hypertension. However, it is unclear about the roles of sympathetic ganglia, which integrate central sympathetic signals and send their signals to cardiovascular end organs via sympathetic nerve. The celiac ganglion (CG) plays an important function in regulating blood pressure by the controlling splanchnic circulation containing 30% of total blood volume. The purpose of this study is to find if the electrophysiological properties of CG neurons are modulated in DOCA - salt hypertensive rats. Using whole - cell patch clamp, dissociated CG neurons from hypertensive and matched normotensive rats were divi ded into three groups based on their action potential (AP) firing patterns to a sustained suprathreshold depolarizing current (100pA; 5s). Phasic neurons fired several APs and adapted quickly; adaptive neurons fired several APs and adapted slowly; tonic ne urons fired continuously. When compared to CG neurons from normotensive rats, the distribution of these three groups was significantly different in neurons from hypertensive rats. There were more phasic neurons, fewer adaptive neurons and tonic neurons in hypertensive rats. Moreover, the firing frequencies of tonic neurons were significantly lower in hypertensive rats compared to normotensive rats. Furthermore, the electrophysiological properties of a single AP were compared between normotensive and hyperte nsive rats. TEA), a delay rectifier K + channel blocker, the firing frequencies of phasic, adaptive and tonic neurons were all significantly atte nuated. When Paxilline, a big - conductance Ca 2+ - activated K + channel blocker, was used, both tonic and adaptive neurons were converted to phasic neurons. After application of 4 - Aminopyridine ( 4 - AP ), - This study demonstrates that the CG neuronal firing patterns and firing frequencies are modulated in DOCA - salt hypertensive rats, which may be contributed by some changes of K + channel currents including delay rectifier K + current, big - conductance Ca 2+ - activated K + current and A - type K + current. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CHAPTER 3 CALCIUM AND POTASSIU M CHAN N EL CURRENTS OF DISSOCIA TED CELIAC GANGLIO N N EURONS IN DOCA - SALT HYPERTENSIVE RA TS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 115 CHAPTER 4 EFFECTS OF HYDROGEN PEROXIDE ON NEURONAL FIRING AND POTASSIUM CHANNEL CURRENTS OF DISSOCIATED RAT CELIAC GANGLION NEURONS 116 Abstract: Elevated reactive oxygen species (ROS) have been found in celiac ganglia from DOCA - salt hypertensive rats. In chapters 2 and 3, altered neuronal firing patterns associated with reduced K + currents were detected in dissociated CG neurons from hypertensive rats. In this study, one type of ROS, H 2 O 2 was used in CG neurons to explore if the changes in neuronal firing properties are related to elevated ROS and whether the changes by H 2 O 2 mimic differences in neuronal firing properties found in hypertensive rats . Using whole - cell patch clamp, dissociated CG neurons from normal SD rats were divided into three groups based on their action potential (AP) firing patterns in response to a sustained suprathreshold depola rizing current step (100pA; 5s ). Phasic neurons fired several APs and adapted quickly; adapt ive neurons fired several APs and adapted slowly; tonic neurons fired continuously. Neurons were exposed to 3mM H 2 O 2 for 1 min. After treatment , t he phasic neurons remained phasic but fired fewer APs and t he adaptive neurons became phasic neurons. 21 % of tonic neurons w ere changed to phasic neurons, 14 % were changed to adaptive neurons, and 65 % tonic remained tonic but with lower firing frequency. Further effects on K + currents were explored by applying 3mM H 2 O 2 for 1min. The amplitude of the sustained out ward potassium current was increased at potentials positive to - 20mv in Ca 2+ - free extracellular solution but not in regular extracellular solut ion with Ca 2+ . The peak amplitude and mid - width of A - type K + current were decreased by H 2 O 2 . The 117 inactivation curve of A - type K + current was shifted to left by H 2 O 2 . The effects of H 2 O 2 on K + currents were partially blocked by c atalase (100 kU/ml) applied intracellularly. These findings demonstrate that H 2 O 2 acts intracellularly in CG neurons to reduc e neuronal firing frequency and alter firing patterns. The changes in firing properties by H 2 O 2 may be associated with changes by H 2 O 2 in sustained outward K + current s and decreased A - type K + current . Introduction: ROS are reactive derivatives of oxygen metabolism and include superoxide anion (O 2 ) , hydrogen peroxide (H 2 O 2 ) and hydroxyl radical . This study targeted H 2 O 2 as a potential candidate for neuronal firing properties changes in CG neurons from hypertensive rats. One reason is that H 2 O 2 is a potent and stable molecule. It is also a source of in the presence of ferrous - containing low - molecular - weight proteins or free cytoplasmic Fe 2+ . is one of the most potent oxidants that oxidizes proteins and lipids and breaks apart DNA strands (Stadtman & Levine, 2000) . The other reason is that H 2 O 2 is lipid soluble , and it can diffuse out into the cytoplasm (Halliwell, 1992) . H 2 O 2 applied extracellularly would dif fuse into the neurons and function intracellularly. The production of H 2 O 2 is checked by enzymes such as catalase and glutathione peroxidase that convert H 2 O 2 to water and molecular oxygen . 118 ROS are produced during normal metabolism and might be associated with physiological or pathological functions, even though an e xcessive amount of ROS can result in tissue damage (Halliw ell, 1992) . Elevated ROS is found in many neuronal (Barkats et al. , 2006) (Behl et al. , 1994) and multiple sclerosis (Ferretti et al. , 2006) . It also has been found that ROS is associated with hypertension. ROS are elevated in blood and tissues in hyp ertension (Somers et al. , 2000) .P rolonged antioxidant administration prevents superoxide accumulation and attenuate systolic BP elevation in DOCA - salt hypertensive rats (Beswick et al. , 2001) . Interestingly, e levated ROS are also shown in celiac g anglia from hypertensive rats (Dai et al. , 2006) . However it is unknown about the pathological role of elevated ROS in sympathetic ganglia for hypertension Different CG neuronal firing pattern exist in response to a sustained depolarizing current. The firing pattern is generated by activation and inactivation of a series of i on channels . M embrane ion channels are the possible targets of RO S (Kourie, 1998) . It has been demonstrated that H 2 O 2 can modulate ICC pacemaker activity and this occurs by the activation of K ATP channels (Choi et al. , 2008) . Voltage - gated K + currents are also altered by H 2 O 2 in cultured hippocampal neurons (Mul ler & Bittner, 2002) . In chapter 2 and 3, It was found that the neuronal firing patterns and K + currents were modulated in CG neurons from DOCA - salt hypertensive rats. It is not known if the elevated the ROS level in sympathetic ganglia wound contribute to the neuronal firing property changes of CG neurons in this hypertensive animal model. In this study, I explored the effects of H 2 O 2 on neuronal firing properties of dissoci ated CG neurons , 119 which would illustrate if there is a relationship between ROS and neuronal firing properties in sympathetic ganglion neurons . Materials and Methods: Animals The animals and number used in the experiments were conformed t National Institutes of Health Guide for the Care and Use of Laboratory Animals 3 - 4 week male Sprague Dawley (SD) rats ( 10 0 - 150 g) were housed and used in accordance with guidel ines established by the Animal Use and Care Committee of Michigan State University. Tissue collection CG was removed from the animals (details seen in Chapter 2). Cell culture The harvested celiac ganglia were dissociated enzymatically and plated in glass bottom culture dish es for electrophysiological studies (details seen in Chapter 2). Whole - cell patch clamp The dissociated CG neurons were tested under whole - cell patch clamp (details seen in Chapter 2). Access resistance (R a ) was monitored at regu lar intervals. The ruptured neurons with R a higher than 20M were not accepted for data acquisition in voltage clamp mode. The neurons with resting membrane potential more depolarized than - 45mv were not included in current clamp mode . The extracellular so lution contained 120 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.2 MgCl 2 , 1.2 NaHPO 4 , 25 NaHCO 3 , and 11 Glucose in mM which was equilibrated with a 95% O 2 - 120 5%CO 2 mixture. The intracellular solution contained 150 K Acetate, 3 MgCl 2 , 40 H EPES, 10 ATP, and 2.5 GTP in mM . In n o Ca 2+ extracellular solution, CaCl 2 was replaced by NaCl. 3mM H 2 O 2 was applied through drug administration capillary tube by gravity which was positioned 500 - system was still running at the rate of 20 - 30 drops/min during H 2 O 2 application. 3x10 - 7 M tetrodotoxin (TTX) was applied to neurons during the experiments for measuring the K + currents through dru g administration capillary tube. Some recording were made with intracellular solution containing catalase (100kU/ml). Catalase did not alter the p H of the intracellular solution at the concentration s used. Statistical analysis: Al l values were presented as mean ± SEM and n refers to the number of neurons Less than 4 neurons were recorded per animal . Statistical significance between means was determined using two - tailed paired t - test for two independent variables or one - way ANOVA for more than two independent variables . The neuronal firing pattern change was an alyzed by 2 - test. The statistical significance was set for P <0.05 . Results: Effects of H 2 O 2 on firing patterns of dissociated CG neurons Using whole - cell patch clamp, dissociated rat CG neurons (n=54) were tested for neuronal firing in current clamp mode by injecting a sustained suprathreshold 121 depolarizing current (100pA; 5 s) . The neurons were divided into three groups , phasic, adaptive and tonic neurons based on their AP firing pattern ( Figure 4 - 1, A ) . The same classification standard was used as in Chapter 2 . Some phasic neurons did not fire after 100pA current injection , but did fire after injection of a larger current ( 100 to 300pA ) . In these 54 neurons, 13% of them were phasic neurons, 9% were adapting neurons and 78% were tonic neurons. After 1min H 2 O 2 (3mM) application, the neurons were tested again for neuronal firing. The phasic neurons (n=7) remained phasic ( Figure 4 - 1, B ) . All adapting neurons (n=4) changed to phasic ( Figure 4 - 1, C ) . As for tonic n eurons , 2 1 % of them (n=9) changed to phasic neurons, 14 % (n=6) changed to adapting neurons, and 65 % (n=27) remained tonic ( Figure 4 - 1, D ) . In summary there were 39% phasic, 11% adapting and 50% tonic neurons a fter H 2 O 2 application, which was statistically different ( 2 - test, p<0.05) from the firing pattern distribution before H 2 O 2 application ( Figure 4 - 1, E ) . These data indicate that H 2 O 2 increased the accommodating capacity of CG neurons to sustained depolarizing current injection. The effec ts of H 2 O 2 were reversible in 50% patched neurons by 4min wash with extracellular solution . The effects of H 2 O 2 on neuronal firing pattern were not blocked by applying 100kU/ml catalase intracellularly . The changes by H 2 O 2 in neuronal firing pattern with catalase were similar to those without catalase shown before. In the presence of intracellular catalase, one adaptive neuron changed to phasic neuron, 25% of tonic neurons (n=2) changed to phasic neurons, 12% of tonic (n=1 ) changed to adaptive neuron and 62% of tonic (n=5) remained tonic after H 2 O 2 tre atment. 122 Effects of H 2 O 2 on neuronal firing frequency The neuronal firing frequency was not constant when depolarizing current injection applied . It declined gradually and stop ped firing in phasic and adapting neurons or went to a stable plateau in tonic neurons. To examine CG neuronal firing frequency, the time to first AP ( t 1 ), the time from the first AP to the second AP ( t 2 ) and the time from the penultimate AP and the last AP ( t 3 ) in the plateau region were measured before and after 1 min 3mM H 2 O 2 treatment ( Figure 4 - 2, A ). The t 3 value was not tested in phasic and adapting neurons because they did not have a stable plateau. t 2 and t 1 were measured when they occurred. t 2 was measured when there were more than 2 APs and t 1 when there was an AP in 100pA current injection. 3mM H 2 O 2 significantly increased t 1 ( t - test, p<0.05, n=52) in all measurable neurons including phasic, adapting and tonic neurons ( Figure 4 - 2, B ). The first firing frequency (1/ t 2 ) was significantly decreased by H 2 O 2 in all accessible neurons ( t - test, p<0.05, n=44) ( Figure 4 - 2, C ). The last firing frequency (1/ t 3 ) was only tested in tonic neurons that remained tonic after H 2 O 2 treatment. It was significantly decreased by H 2 O 2 ( t - test, p<0.05, n=27) ( Figure 4 - 2, D ). The phasic, adaptive and tonic neurons were tested for the action potential firing frequency during the entire 5 s of depolarization (f 5s ) as number of APs during current injecting . The f 5s was statistically decreased by H 2 O 2 in all three types of neurons ( Figure 4 - 2, E ). In summary, t hese data showed that H 2 O 2 delayed the time 123 to generate the first AP and decreased firing frequencies under a sustained depolarizing current injection. Effects of H 2 O 2 on electrical membrane parameters of a single AP Neuronal firing is composed of a series of APs. Altered AP shape may reflect altered neuronal firing pattern and firing frequency. AP electrical membrane properties were measured before and after H 2 O 2 treatment. The capacitance of these dissociated neurons was 53±10pF , which is not changed by H 2 O 2 treatment . The input resistance (R in ) was measured by applying a constant 20pA hyperpolarizing current for 5 s . R in was lowered by H 2 O 2 from n =42, paired t - test p<0.05). The resting membrane potential (RMP) was hyperpolarized by H 2 O 2 from - 56. 0 ±1.4mv to - 6 0.1±2. 2 mv ( n =42, paired t - test p<0.05) . The peak amplitude and the mid - width of the first AP of neuronal firing were measured under the sustained 100pA depolarizing current injection. The peak amplitude was not changed by H 2 O 2 . However the mid - width was statistically increased by H 2 O 2 from 2.8 ±0.2ms to 3.2±0.2ms ( n =50, paired t - test p<0.05) (Table 4 - 1 ) . To characterize the AP in more detail, a short depolarizing current injection (400pA, 2ms) was applied to the neurons to induce just one AP. It was confirmed that the effects of H 2 O 2 on RMP an d AP peak amplitude under this short current injection were consistent with those found under 5 s depolarizing current injection. The RMP was 124 hyperpolarized significantly from - 49.8±2.7 mv to - 5 2.7 ± 2 .9mv (n=13, paired t - test p<0.05) . The amplitude of AP was not changed by H 2 O 2 application. However, H 2 O 2 did not alter t he mid - width of the single AP despite being significantly increased in the first AP under 5 - s depolarizing current injection. Furthermore it has been found that the repolarizing rate of the sin gle AP was significantly decreased by H 2 O 2 from 29.4 .0± 6.7 mv/ms to 25.8 ± 5.5 mv/ms (n=13, paired t - test p<0.05) ; the amplitude of afterhyperpolarization (AHP) was not changed by H 2 O 2 , but the area of AHP was significantly decreased from 1400 ± , paired t - test p<0.05) (Table 4 - 2 ) . In summary, H 2 O 2 treatment lead to hyperpolarized RMP, decreased R in , slower AP repolarization and attenuated AHP in dissociated CG neurons. Effects of H 2 O 2 on sustained outward potassium currents Previous findings in electrical membrane firing properties indicate that the voltage - gated K + channels might be modulated by H 2 O 2 . To explore the mechanisms of changed neuronal firing by H 2 O 2 , the voltage - gated K + currents were tested before and after H 2 O 2 treatment. Current - voltage (I - V) relationships were generated in voltage - clamped neurons held at a membrane potential of - 70mV and then stepped in 10mV increments from - 120 to +50 mV. Each Voltage step was 0.2s duration. 0.19s int ervals were allowed betw een steps. Furthermore, a single - step depolarization protocol , stepping from - 70mv to +50mv for 0.2 s was also applied to neurons before and after 125 H 2 O 2 treatment . The amplitudes of K + currents were normalized to cell capacitance to obtain the current densities. The outward K + currents included a transient outward K + current ( I A ) and a sustained outward K + current ( I so ) (Figure. 4 - 3, A). I so was composed of a delayed rectifier potassium current ( I Kv ) and a big - conductance calcium - activated potassium channel (BK) current ( I BK ). 3mM H 2 O 2 applied for 1 min, did not significantly change the amplitude of I so under protocol for I - V relationships (n=17, two - way ANOVA p>0.05) ( Figure 4 - 3, B). Ho wever, it significantly increased the amplitude of I so under single - step depolarization protocol (n=5, paired t - test p<0.05) ( Figure 4 - 3, C). There would be no Ca 2+ influx in single step depolarization from - 70mv to +50mv because the equilibrium potential for Ca 2+ current is around +45 ( for details see C hapter 3). Therefore, t he BK channel current would be less activated under single - step depolarization compared to the last step of the series depolarization steps. The I - V relationships of I so were further tested in Ca 2+ - free extracellular solution. The amplitudes of I so were significantly enhanced 5 to 6 pA/pF at the voltage steps positive to - 20mv in the Ca 2+ - free extracellular solution (n=7, two - way ANOVA p<0.05) (Figure 4 - 3, D). This effect was blocked by addition of the H 2 O 2 catalyzing enzyme, catalase. When 100kU/ml catalase was applied i n the intracellular solution, I so was not significantly changed by H 2 O 2 in Ca 2+ - free extracellular solution (Figure 4 - 3, E). Effects of H 2 O 2 on A - type potassium current 126 In the beginning of the outward K + currents generated by depolarization steps, there was transient outward A - type potassium current ( I A ) exhibiting quick inactivation. Two depolarization steps (from - 40mv to 30mv and f rom - 70mv to 30mv) were used to separate I A from I so in the whole outward K + current. I A was obtained by subtracting current under - 40mv to 30mv step from one under - 70mv to 30mv step. It was compared between before and after H 2 O 2 application (Figure 4 - 4, A). The H 2 O 2 significantly decreased the peak amplitude of I A by 50% - 60% and the mid - width from 7.3 1.ms to 5.8 0.6ms (n=9, paired t - test p<0.05) (Figure 4 - 4, B). The activation and inactivation of I A were further studied before and after H 2 O 2 treatment. Voltage - dependent I A activation was generated using two protocols. One protocol is to depolarize neurons from - 70mv to +50mv in 10mv. Extra - 20mv depolarization for 0.5 s was applied right before depolarization from - 70mv to +50mv in the other protocol. I A was achieved by subtracting currents from these two protocols (Figure 4 - 5, A). I A inactivated was studied by depolarizing neurons to +50mv proceeded by prepulse, lasting 0.5s, from - 120mv to - 20mv (Figure 4 - 5, B). The neurons were held at a membrane potential of - 70mV. I A activation curves and inactivation curves were compared before and after H 2 O 2 treatment using the conductance - voltage plot. The conductance was calculated by peak current during the test pulse divided by maximum peak current . H 2 O 2 shifted voltage dependence of steady state inactivation to the left but did not affect the voltage dependence of 127 activation (Figure 4 - 5, C). When c atalase (100kU/ml) was applied intracellularly , the effects H 2 O 2 on I A were partially checked. Peak amplitude was not significantly changed by H 2 O 2 , but the mid - width remained decreased by H 2 O 2 in the presence of catalase (Figure 4 - 6, A). With catalase, H 2 O 2 still decreased the mid - width of I A from 7.0 1.6ms to 6.1 1.6ms (n= 8, paired t - test p<0.05). There were no shifts of either voltage dependence activation curve or voltage dependence steady - state inactivation curve by H 2 O 2 with intracellular c atalase application ( Figure 4 - 6, B ) . The effects of H 2 O 2 on K + currents were not reversible after a 4 min wash with control solution . Di scussion: The major findings of this study are that, in dissociated CG neurons 1) H 2 O 2 converted neuronal firing pattern to more accommodating type from tonic to phasic or adaptive neurons and from adaptive to phasic neurons; 2) i t decreased neuronal firing frequency in all three types of neurons; 3) it enhanced I so in no Ca 2+ extracellular solution and inhibited I A in regu lar extracellular solution. 4) The effects of H 2 O 2 on CG neuronal firing and K + current were rarely reversible by simple wash. The effects were partially eliminated by adding catalase in the intracellular solution. Taken together, 128 these finding s suggest that H 2 O 2 exert different effects on the various K + curre nts which may contribute the effects of H 2 O 2 on neuronal firing. In the previous study, firing properties change s were seen in dissociated CG neurons from normotensive and DOCA - salt hypertensive rats. The changes in neuronal firing pattern from NT rats to HT rats are similar to those before and after H 2 O 2 treat ment. There were more phasic neurons and less adaptive and tonic neurons in HT rats; the firing frequency was lower in tonic neurons from HT rats. It is shown that superoxide level is increased in sympathetic ganglia from this hypertensive animal model (Dai et al. , 2004) . Elevated superoxide would lead to increased H 2 O 2 , which may contribute CG neuronal firing pattern change in hypertension. The effect of H 2 O 2 on voltage - dependent K + currents was examined in this study. There is evidence indicating that chemical oxidat ion and reduction may modulate the activity of ion channels (Gulbis, 2002;Ruppersberg et al. , 1991) . I have shown that the sustained potassium channel currents ( I so ) were increased by H 2 O 2 in calcium free extracellular solution but unchanged in normal calcium extracellular solution. I so include mainly I Kv and I BK . Since BK channel are activated by calcium, the increased effect by H 2 O 2 is probably through delay rectifier potassium channel currents. The unchanged effect in normal calcium solution, which is also shown in colonic smooth muscle cell s (Prasad & Goyal, 2004) , may be contributed to decreased BK Ca channel currents which cover the increased K v current. The increased AP duration by H 2 O 2 may also 129 contributed by decreased BK Ca channel current. Decreased peak amplitude of afterhyperpolarization (AHP) is also consistent with decreased BK Ca channel current. It also shown that the neuronal firing was more phasic when BK Ca channel was blocked in Chapter 2. It has been show that I A was reduced by H 2 O 2 in rat CA1 pyramidal neurons (Angelova & Muller, 2006) . The I A contributes to the RMP by t he so - called window current. B ecause of its fast activation, it is primarily res ponsible for repolarization, the AP duration, and repetitive firing (Connor & Stevens, 1971a;Connor & St evens, 1971b) . It would increase AP duration, which was consistent with the finding in the membrane electrical properties of single AP. The role of A - type K + channel on neuronal firing was discussed in Chapter 3. Via action potential duration, I A chan nels would also strongly affect presynaptic Ca 2+ influx and transmitter release (Pongs, 1999) . A large range of H 2 O 2 conc entrations from several 10mM (Prasad & Goyal, 2004) are used in the research work. It has been estimated that local H 2 O 2 c oncentration can be higher than 1 - 2mM (Bychkov et al. , 1999) . 3mM H 2 O 2 applied for 1 min give me the consistent effect and also the effect can be parti ally washable. I used high concentration due to my drug administration system. My drug perfusion tube is 600 - away from patched cell. The extracellular solution perfusion system is still on during the drug perfusion to ensure the neuron gets enough O 2 . The actual H 2 O 2 130 conc entration working on the neurons will be much less than 3mM. It has been shown that the effect of H 2 O 2 is hardly washed away , partly because it is related to covalent chemical modification (Prasad & Goyal, 2004) . The effects of H 2 O 2 on neuronal firing pattern are partially washed away, whereas the changes on K + currents by H 2 O 2 are not washed away in this study. It should be mentioned in this study that I focus ed only on short - term effects of H 2 O 2 , which occurred at the first 1 min after application of H 2 O 2 to the bath solution. Further research could be done to elucidate the long - term effects of H 2 O 2 . In conclusion, H 2 O 2 converted neurons to more accommodating neuronal firing pattern and decreased firing frequency in response to sustained suprathreshold depolarizing currents, which could be related to its inhibitory effect on BK channels, and A - type K + channel. Interestin gly all these effects by H 2 O 2 are similar to the changes found in CG neurons from HT rats. Therefore, the changes in neuronal firing and K + currents are possibly related to elevated ROS in hypertension. 131 APPENDIX 132 A. Figure 4 - 1 : Effects of H 2 O 2 on firing patterns of dissociated CG neurons. 133 Figure 4 - 1 B. 134 Figure 4 - 1 C. 135 Figure 4 - 1 D. 136 Figure 4 - 1 E. P (7) A (4) T (42) P (7) A (6) T (27) P (4) P (9) control H 2 O 2 13% 9% 78% 39% 11% 50% 137 Figure 4 - 1 Three categorical firing patterns including phasic, adapting, or tonic were found in dissociated CG neurons in response to sustained suprathreshold 100pA depolarizing current injection in current clamp mode. Firing patterns from three representative cells (A). H 2 O 2 (3mM) was applied on 54 neurons for 1 min. The neuronal firing pattern was tested before and after H 2 O 2 application. Phasic neurons (n=7) remained phasic after H 2 O 2 treatment (B). A representative cell is shown. Adapting neurons (n=4) changed to be phasic by H 2 O 2 (C). A representative cell is show n. Out of 42 tonic neurons, nine changed to phasic, six changed to adapting, and the remaining twenty - seven remained tonic (D). Representative cells are shown. The schematic summary diagram was shown c neurons, respectively. The firing pattern was statistically different between before and after H 2 O 2 2 - test, p<0.05). The percentages of specific neuronal firing pattern before and after H 2 O 2 treatment were shown adjacent to the representative schematic boxes. 138 A. Figure 4 - 2 : Effects of H 2 O 2 on firing frequency in dissociated CG neurons. 139 Figure 4 - 2 B. C. 140 Figure 4 - 2 D. E. 141 Figure 4 - 2 CG neuronal firing frequency declined gradually and stopped firing in phasic and adapting neurons or went to a stable plateau in tonic neurons . T he time to first AP ( t 1 ), the time from the first AP to the second AP ( t 2 ) and the time from the penultimate AP and the last AP ( t 3 ) in plateau region were measured before and after 1 - min 3mM H 2 O 2 treatment . A schematic graph was shown to illustrate how to measure t 1 , t 2 and t 3 (A). H 2 O 2 significantly increased t 1 ( n=52, paired t - test, p<0.05) in measurable neurons (B). H 2 O 2 significantly decreased the first firing frequency ( n=44, paired t - test, p<0.05) (C) and significantly decr eased the last firing frequency (1/ t 3 ) ( n=27, paired t - test, p<0.05) (D). H 2 O 2 also significantly decreased the action potential firing frequency during the entire 5 s of depolarization (number of APs during 5 - s current injection) ( n=7 in phasic group, n=5 in adapting group , n=41 in tonic group ; paired t - test, p<0.05) (E) 142 RMP ( - mv) R in (M ) p eak amplitude (mv) m id - width (ms) control 56.0 1.4 1039 68 121.7 2.6 2.8 0.2 H 2 O 2 60.1 2.2 * 821 69 * 120.0 3.0 3.2 0.2 * Table 4 - 1 . The effects of H 2 O 2 on electrical parameters of CG neurons in response to a sustained depolarizing current injection The electrical parameters of dissociated CG neurons in response to a sustained 100pA depolarizing current were measured in before and after H 2 O 2 treatment. R in was lowered by H 2 O 2 from n =42, paired t - test p<0.05). The resting membrane potential (RMP) was hyperpolarized by H 2 O 2 from - 56. 0 ±1.4mv to - 6 0.1±2. 2 mv ( n =42, paired t - test p<0.05) . The peak amplitude of the first AP was not changed by H 2 O 2 . However the mid - width of the first AP was statistically increased by H 2 O 2 from 2.8 ±0.2ms to 3.2±0.2ms ( n =50, paired t - test p<0.05) . 143 RMP ( - mv) p eak amplitude (mv) m id - width (ms) control 49.8 2.7 117.4 4.0 2.7 0.3 H 2 O 2 52.7 2.9 * 110.2 5.5 2.8 0.3 Repolarizing rate (mv/ms) AHP amplitude (mv) AHP area (mv ) control 29.4 6.7 13.3 1.8 1400 193.8 H 2 O 2 25.8 5.5 * 13.4 2.4 996 192.2 * Table 4 - 2 . The effects of H 2 O 2 on membrane electrical parameters of a single AP M embrane electrical parameters was measured in response to a short depolarizing current injection (400pA, 2ms) . The RMP was hyperpolarized significantly from - 49.8±2.7 mv to - 5 2.7 ± 2 .9mv (n=13, paired t - test p<0.05) . The amplitude of AP was not changed by H 2 O 2 application. However, H 2 O 2 did not alter t he mid - width of the single AP. Furthermore it has been found that the repolarizing rate of the single AP was significantly decreased by H 2 O 2 from 29.4 .0± 6.7 mv/ms to 25.8 ± 5.5 mv/ms (n=13, paired t - test p<0.05) ; the amplitude of afterhyperpolarization (AHP) was not changed by H 2 O 2 , but the area of AHP was significantly decreased from 1400 ± paired t - test p<0.05) 144 A. Figure 4 - 3. Effects of H 2 O 2 on sustained outward K + currents in dissociated CG neurons. - - 145 Figure 4 - 3 B. 146 Figure 4 - 3 C. 147 Figure 4 - 3 D 148 Figure 4 - 3 E. 20 100 80 60 149 Figure 4 - 3 The CG neurons were held at - 70mv in voltage clamp mode. Outward K + currents were generated by an I - V relationship protocol ( from - 120 to +50 mV at 10mv step, 0.2s) (A). H 2 O 2 (3mM) applied for 1 min did not change the amplitude of I so under I - V relationshi p protocol (n=17, two - way ANOVA p>0.05) (B) , but s ignificantly increased the amplitude of I so under one - step depolarization protocol from - 70mv to +50mv, 0.2s) (n=5, paired t - test p<0.05) (C). I n the Ca 2 + - free extracellular solution , H 2 O 2 significantly enhanced I so by 5 to 6 pA/pF at the voltage steps positive to - 20mv (n=7, two - way ANOVA p<0.05) (D) , However H 2 O 2 did not change the amp litude of I so in the Ca 2+ - free extracellular solution when 100kU/ml catalase applied intracellularly (n=5, two - way ANOVA p> 0.05) (E). 150 A. B. Figure 4 - 4 . The effects of H 2 O 2 on transient outward K + current control H 2 O 2 500pA 10msec 151 Figure 4 - 4 I A obtained by subtracting current under - 40mv to 30mv step from one under - 70mv to 30mv step was compared between before and after H 2 O 2 application. The subtracting traces were shown before and after H 2 O 2 treatment (A). The dark line represe nts the control; the red line presents the H 2 O 2 treatment . Both the peak amplitude and mid - width of were I A significantly decreased by H 2 O 2 (t - test, p<0.05, n=9) (B). ( For interpretation of the references to color in this figure , the reader is referred to the electronic version of this dissertation ) 152 A . B. Figure 4 - 5. The effects of H 2 O 2 on activation and inactivation transient outward K + current - - - 153 Figure 4 - 5 C. 154 Figure 4 - 5 Voltage - dependent I A activation was generated using two protocols. One protocol is to depolarize neurons from - 70mv to +50mv in 10mv. Extra - 20mv depolarization for 0.5 s was applied right before depolarization from - 70mv to +50mv in the other protocol. I A was achieved by subtracting currents from these two protocols (A). I A inactivated was studied by depolarizing neurons to +50mv proceeded by a prepulse, lasting 0.5s, from - 120mv to - 20mv (B). I A activation curves and inactivation curves were compared bef ore and after H 2 O 2 treatment using the conductance - voltage plot. H 2 O 2 shifted voltage dependence of steady state inactivation to the left but did not affect the voltage dependence of activation (C) 155 A. B . Figure 4 - 6 . The effects of H 2 O 2 on activation and inactivation transient outward K + current in presence of intracellular catalase 156 Figure 4 - 6 In presence of intracellular catalase (100kU/ml), I A obtained by subtracting current under step from - 40mv to 30mv from one under step from - 70mv to 30mv wa s compared between before and after H 2 O 2 application. Peak amplitude was not significantly changed by H 2 O 2 (t - test, p>0.05, n=8), but the mid - width was significantly decreased by H 2 O 2 (t - test, p<0.05, n=8) (A). 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Specifically, hypertensive rats had more phasic neurons, fewer adaptive neurons and tonic neurons than normotensive rats . The firing frequencies of the tonic neurons from the hypertensive rats were also significantly lower than the firing frequencies of th e tonic neurons from the normotensive rats. 2) - - - Following more phasic neurons were observed, and there appeared to be fewer adaptive neurons and tonic neurons , similar to what was observed in neurons from hypertensive rats . Moreover, exposure to - - - - - - - The results showed that Previous research on neuronal firing patterns and signal transmission in sympathetic ganglia explain this prediction - - - - - - - - - - - - - considered dissociated CG neurons from DOCA - salt hypertensive rats more neurons with phasic firing patterns and fewer with tonic firing patterns in response to sustained suprathreshold current injection. This inconsistency may be related to inherent differences in the types of hypertensive models used, namely differences between genetic and non - genetic models. The observed discrepancy might also be caused by differences between paravertebral and prevertebral sympathetic ganglia. My study indicates that the observed changes in the distribution of neuronal firing patterns could be related to the altered voltage - gated K + currents and elevated ROS levels that occur in the CG of DOCA - salt hypertensive rats. The ROS levels in SCG from SHR animals have not been determined, but in contrast to my finding, the densities of A - type K + currents ( - elevated ROS concentration in CG neurons might reduce the - . Similarly, the firing pattern changes observed in SCG neurons from SHR might be related to an elevated . K + currents and neuronal firing - - - - - - - - - - - - - - - - - dihydroethidium) - is not strictly specific to O . 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