iii? In 'HESiS MICHIGAN STATEU uu vs I I IIII I IIIIIIIIIIIIIIIIIIIIIIIIII 3 12919395 II This is to certify that the dissertation entitled Olfactory Efferents to the Hypothalamic Paraventricular and Supraoptic Nuclei: An Anatomical and Electrophysiological Analysis in the Rat presented by Kenneth George Smithson II has been accepted towards fulfillment of the requirements for Ph.D- degree in Melange-Physiology ‘6 J Major professor Q Date 6 l/l‘fJfi'o MS U is an Affirmative Action/Equal Opportunity Institution 0.12771 _‘_——‘ _ A __—__ A__——___‘__ __.—.____ I LIBRARY 'Mlchlgan State 1 University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or betore date due. DATE DUE DATE DUE DATE DUE l I_—I—7I MSU Is An Affirmative ActlorVEqual Opportunity Institution cWMS-DJ AN! OLFACTORY EFFERENTS TO THE HYPOTHALAMIC PARAVENTRICULAR AND SUPRAOPTIC NUCLEI: AN ANATOMICAL AND ELECTROPHYSIOLOGICAL ANALYSIS IN THE RAT by Kenneth George Smithson II A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Neuroscience Program 1990 ANAT COunce 811me} using a Nellrop dendn‘ti SOmani. Ventroh architec microg] horserac into the ABSTRACT OLFACTORY EFFERENTS TO THE HYPOTHALAMIC PARAVENTRICULAR AND SUPRAOPTIC NUCLEI: AN ANATOMICAL AND ELECTROPHYSIOLOGICAL ANALYSIS IN THE RAT by Kenneth George Smithson II The morphological and physiological features of a putative connection between the main and accessory olfactory bulbs and the supraoptic (SON) and paraventricular nuclei (PVN) of the rat were studied using a combination of anatomical and electrophysiological techniques. Neurophysin immunocytochemistry revealed the supraoptic nucleus dendritic plexus which coursed anteroposteriorly ventral to supra0ptic somata. Additionally, a portion of this plexus also projected ventrolaterally into periamygdaloid areas, a feature of supraoptic nucleus architecture which is not generally appreciated. Injections of the anterogradely transported substances, wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) or Phaseolus vulgaris leucoagglutinin into the main accessory bulb and injections of WGA-HRP into the accessory bulb revealed a dense plexus of terminals and fibers ventrolateral to the fibers ventrt latter both the P Gold ipsila label: this c short. latera Cxcitz the at €0an medi: to the ipsilateral supraoptic nucleus. The pattern of anterogradely labeled fibers and terminals appeared to overlap with the distribution of ventrolaterally projecting neurophysin-containing processes. Since the latter consists of dendritic processes of supraoptic origin, this suggests that both olfactory bulbs project to the SON. No labeled fibers appeared near the PVN. Injections of rhodamine-labeled latex microspheres or Fluoro- Gold® resulted in retrogradely labeled mitral cells throughout the ipsilateral main and accessory bulbs. No mitral cells were retrogradely- labeled after an injection into the PVN. Electrophysiological analysis of this connection using an explant preparation confirmed the existence of a short, variable latency, excitatory response to electrical stimulation of the lateral olfactory tract. These responses were reversibly blocked by the excitatory amino acid receptor antagonist, kynurenic acid. Taken together the anatomical and physiological studies demonstrate a direct monosynaptic connection from the main and accessory bulbs which is excitatory and is mediated through an excitatory amino acid receptor on SON neurons. To n withou my fan DEDICATION To my mother Rosemary, who endowed me with an indomitable spirit, without which I would not have reached so high, nor traveled so far, and to my family and friends who have supported me through this long endeavor. ACKNOWLEDGMENTS In my ten short years in Glenn Hatton’s lab I have made many good friends and colleagues. I have reached the successful culmination of this journey only through your support and love. Thank you all! I wish I could recognize each of you individually, but that would require another volume. Thanks go to Drs. P. Cobbett, G. I. Hatton and C. D. Tweedle who reviewed earlier drafts of this document and offered many helpful suggestions. In this regard I’d also like to thank my committee members; Drs. K. Moore, B. Zipser, R. Pax , D. Kaufman and G. Hatton who also carefully evaluated the dissertation and offered many helpful criticisms and suggestions. Glenn Hatton, my mentor, has carefully guided me during this journey in many ways. His approach to research and insight into “our” system have substantially shaped my own science. He has taught me many skills (too many to list), of these, his careful attention to my writing I’ll cherish the most. He has generously supported my research providing me with all that I needed to pursue interesting projects. Need I say more. Thank you Glenn. Glenn and his wonderful wife Pat have also cared for me as if I were part of their family. This love and support has nurtured my growth. Thank you both. Dining the past ten years our lab has been a busy place with many graduate students, postdocs, and collaborators. Their keen minds provided a sea of ideas which has shaped my approach to science. In particular, I’d like to thank Drs. P. Cobbett, Q. Z. Yang, A. K. Salm, L. S. Pearlmutter, B. A. MacVicar, M. L. Weiss, A. A. Nunez, C. D. Tweedle and B. K. Modney for the many thou ght-provolcin g discussions I’ve had with each of you. The projects that entail this dissertation took several years and consumed much time, not only of myself but of Kevin Grant, Farshid Marzban, Louise Koran, Inge Smithson, Alisa Zapp, and Doug Hollowell. Thanks you all. The elecuophysiology portion of the dissertation was a real test of my patience; in the beginning nothing worked I received many helpful suggestions and encouragement from Drs. J. Krier, V. Gribkoff, P. Cobbett, Q. Z. Yang, R. Fax, and G. I. Hatton. Thank you, without this help this portion of the dissertation would not be done. Weiss’ much I thank T this Wt travele develo approa Complc convex approa acfivit) Tony! With hi Provide exam“; dissem live. Several portions of the dissertation were anatomical studies, and in this Dr. M. L. Weiss’s contributions have added immeasurably to the quality of the project. Thanks very much Mark, I’ve learned much from you and I am a better anatomist for it. I also like to thank Drs. L. Smith and B. Spann, fellow anatomists, who offered helpful suggestions on this work. I’d also like to thank my “lab sibling” B.K. Modney for her support. “We’ve traveled many roads together”; her company was wonderful and indispensable. Dr. P. Cobbett (also known as Swinemaster) has contributed much to my development as a scientist, electrophysiologist, and most of all as a debater. Peter’s unique approach of alternating points of view during a discussion (often taking a position completely opposite that originally poised) has provided us with hours of interesting conversation. Seriously, thanks much Peter for all your help. Dr. A. Nunez deserves a special thanks. He has become a friend and colleague. His perspectives on research have forced me to defend and often reevaluate my reductionist approach. Perhaps his most important contribution was as my squash partner. This activity has afforded me good physical and mental health during this long task. Thanks Tony! Dr. C. D. Tweedle also deserves much thanks. I’ve enjoyed many conversations with him regarding “orn' system”. His insights into the workings of the brain have provided much fodder for thought. As a collaborator we’ve stumbled onto some very exciting findings. These have maintained my interest in research while some projects of the dissertation were mired with difficulties. Thanks also go to Jan Harper, her bright shining face that greeted me each morning and her concern for me has contributed immeasurably to making the lab a homey place to live. I’d like to thank Drs. P. Gerhardt, J. McCormick, and V. Maher for the generous support I’ve received through the Medical Scientist Training Program This research was also support by two NIH grants to Glenn, NS 16942 and NS 09140. Many thanks go to my family, while they had little understanding of this undertaking they unconditionally supported me through this endeavor. A special thanks go to the Sloths Dave and Bob, true friends and confidants. Finally, but certainly not least I like to thank my wife Inge, her love and patience have supported me through these many months. She has also provided much help for the preparation of figures for this documents. Thanks much Wiffo! vi List of Figures List of Abbreviations .. The Hypothalamic-Neurohypophysial System 1.1. Olfactory Efferents to the l-INS? 1.2. Thesis Objectives 1.3. Experimental Approach 1.4. Organization of the Dissertation . . Anatomy of Supraoptico-Neurohypophysial System 2.1. Intrinsic Organization of the SON: somatic and dendritic 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2. 8. 29 2.10. Afferent to the Supraoptic Nucleus . Introduction to the Olfactory System TABLE OF CONTENTS x :21: domains Supraoptic Efferents Supraoptic Somatic Neurochernistry Oxytocin and Vasopressin Production Stimulus-Secretion Coupling Peripheral Effects of Oxytocin Peripheral Effects of Vasopressin Physiology of Oxytocin and Vasopressin Release Electrophysiology of Supraoptic Neurons NNNNHHI—IHr—Ii—i—IHH MMNNmm-FWNHHOOQWW “\l@M-§H 3.1. Receptor Systems in the Olfactory Mucosa 3.2. Olfactory bulb organization 3.3. Olfactory bulb intrinsic organization 3.4. Main Bulb Efferents 3.5. Accessory Bulb Efferents . . Exp.l. : Cytoarchitectural Organization of the SON Dendritic Zone 30 4.1. Introduction 30 4.2. Experimental Question 30 4.3. Methods Exps. 14: General Methods for Anatomical Studies 31 4.4. Methods Exp. 1: Neurophysin Immunocytochemistry 32 4.5. Results Exp. 1: Perikaryal labeling with Neurophysin Irnmunocytochemistry 32 4.6. Discussion Exp. 1. 46 vii 1 .Il ...1J]]1 plat-IIEISSQJ 99(11A111111 . . . . I 0 0 I 8 9 1 l 5. Exp. 2: Anterograde Tracing of Main Olfactory Bulb Efferents to the SON 5.1. Introduction 5.2. Experimental Question 5.3. Methods Exp. 2A: Anterograde studies with WGA- HRP 5.4. Methods Exp. 23: Anterograde studies with PHA-L 5.5. Results Exp. 2A: WGA-HRP labeling 5.6. Results Exp 2B: PHA-L labeling 5.7. Discussion Exp 2 6 . . Exp. 3: Anterograde tracing of Accessory Olfactory Bulb Efferents to SON 6.1. Introduction 6.2. Experimental Question 6.3. Methods Exp. 3: Anterograde studies with WGA-HRP 6.4. Results Exp. 3: WGA-HRP labeling 6.5. Discussion 7 . . . . Exp. 4: Retrograde tracing of Bulb efferents 7.1. Introduction 7.2 Experimental Question 7.3 Methods: Exp. 4: Retrograde studies with fluorescent tracers. 7.4 Results Exp. 4 7.5. Discussion 8 . Discussion of Anatomical Experiments 8.1. Summary of Anatomical Results 9 . .. Exp 5. :Electrophysiological analysis of connection 9.1. Introduction 9.2. Experimental Question 9.3. Methods Exp. 5: Methods for ElectrOphysiology experiments 9.4. Results 9.5. Discussion 10.. . General Conclusions 10.1 Functional Significance 10.2 Summary 1 l .. . Appendices 11.1 Buffers 11.2 Chromogens/Substrates 11.3 Fixatives 11.4 Slice Medium 11.5 Protocols 11.6 Equipment Sources viii 4 9 49 49 49 50 51 56 57 6 6 66 66 66 67 75 7 7 77 77 77 78 86 8 8 88 9 2 92 93 93 98 110 114 114 115 118 118 119 120 121 121 123 12... 11.7 List of Equipment 128 1 2.. . References l 47 Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 99° 9°“" >‘ 9‘ U‘PP‘N t" p—r $3 p—r p—a . . 12. 13. 14. 15. 16. 16 LIST OF FIGURES Schematic of the hypothalamo-neurohypophysial system. ................................................................. 2 Summary diagram of SON afferents ...................... 15 Chemoreceptor systems in the olfactory mucosa. 19 Atlas of the olfactory bulb ill the sagittal plane. ...... 23 Schematic drawing of the main olfactory bulb efferents. ............................................................ 26 Schematic drawing of accessory olfactory bulb efferents. ............................................................ 28 Distribution of neurophysin irmnunoreactivity in 100 pm thick coronal sections ............................... 33 (cont.): Plate 2 .................................................... 35 (cont.): Plate 3 .................................................... 37 Distribution of neurophysin irnmunoreactivity in 100 pm thick sagittal sections ................................ 39 (cont.) Plate 2 ..................................................... 41 Distribution of neurophysin irnmunoreactivity in 100 um thick sections in the horizontal plane .......... 43 Overlapping distributions of neurophysin stained processes and WGA-HRP labeling around the SON in the coronal plane ...................................... 52 The distribution of labeled cell bodies, fibers, and terminals, after an injection of WGA-HRP into the main bulb ................................................ 54 The distribution of labeled axons and terminals, after an injection of PHA-L into the main bulb. ...... 58 Diagram of the overlapping distributions of SON dendrites and main bulb axons in a parasagittal plane through the lateral margins of the SON. ........ 60 PHA-L labeled fibers in the perinuclear zone of the SON .............................................................. 62 Accessory olfactory bulb injection sites .................. 68 Distribution of labeled cell bodies and terminals after an injection of WGA-HRP into the ipsilateral accessory bulb ...................................... 71 (cont.) Plate 2 ..................................................... 73 Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 18. 19. 20. 21 22 23. 24. 25. 26 Line drawings illustrating injections sites of A) Fluoro-Gold and, B) rhodarnine-labeled microspheres, into the SON. ................................. 79 Distribution of labeled cells in the main bulb after an injection of Fluoro-Gold into the SON ....... 82 The distribution of labeled cells in the accessory bulb after an injection of rhodamine-labeled microspheres into the SON. .................................. 84 Photomicrographs of the explant preparation. ........ 96 Oscilloscope traces of evoked responses in SON neurons to electrical stimulation of the lateral olfactory tract (A & B) and neurohypophysial stalk (C) ............................................................. 99 Effects of bath application of 1 mM kynurenic acid on responses evoked in SON neurons by electrical stimulation of the lateral olfactory tract. ................................................................ 102 Effects of kynurenic acid on the threshold of the evoked responses. .............................................. 104 Reversiblity of kynurenate blockade of the excitatory responses in a SON neuron to lateral olfactory stimulation .......................................... 106 Long depolarizations in response to lateral olfactory stimulation .......................................... 108 Schematic of olfactory connections with the SON .. 116 xi IIn 3V ACo AH AOB AOD AOE AOL AON AOV BAOT bv DAB EAA(s) EPl eWINS) Fluoro-Gold fx G1 Gr GrA HDB HNS OVLT LIST OF ABBREVIATIONS optic nerve, third cerebral ventricle; anterior cortical amygdaloid nucleus; anterior hypothalamic area; accessory olfactory bulb; anterior olfactory nucleus, dorsal subdivision; anterior olfactory nucleus ,extemal portion; anterior olfactory nucleus, lateral subdivision; anterior olfactory nucleus; anterior olfactory nucleus, ventral subdivision; bed nucleus of the accessory olfactory tract; blood vessel; 3,3' diaminobenzidine tetrahydrochloride excitatory amino acid(s); external plexiforrn layer of the main olfactory bulb; excitatory postsynaptic potential(s); Fluoro-Gold®; fomix; glomerular layer of the main olfactory bulb; granule cell layer of main bulb; granule cell layer of accessory bulb; nucleus of the horizontal limb of the diagonal band; hypothalamo-neurohypophysial system; horseradish peroxidase; lateral hypothalarnic area; lateral olfactory tract; medial nucleus of amygdala; mitral cell layer of the main olfactory bulb; mitral cell layer of accessory olfactory bulb; main olfactory bulb; nucleus of the lateral olfactory tract; N-acetyl-L-aspartylglutamate N-methyl-D-aspartate; optic chiasm; optic tract; organum vasculosum of the lamina terminalis xii OX PBS Pe PHA- ' Pir PLCo PMCc PSMS) PVN SCN SON SONt TBS Tu VP WGA. OX PBS Pe PHA-L Pir PLCo PMCo 9813(8) PVN SCN SON SONt TBS TTl Tu WGA-HRP oxytocin; phosphate-buffered saline; periventricular hypothalamic nucleus; Phaseolus vulgaris leucoagglutinin; piriforrn cortex; posterolateral cortical amygdaloid nucleus; posteromedial cortical amygdaloid nucleus; postsynaptic potential(s); paraventricular nucleus, hypothalamus; suprachiasmatic nucleus; supraoptic nucleus-main (or anterior) portion; supraoptic nucleus, tuberal subdivision; Tris-buffered saline; ventral tenia tecta olfactory tubercle; ventral medial nucleus hypothalamus; vasopressin; wheatgerm agglutinin conjugated to horseradish peroxidase. xiii W The hypothalamo-neurohypophysial system (HN S) is composed of several prominent aggregations of cells located in the anterior hypothalamus. These include the supraoptic (SON), paraventricular (PVN), and anterior commissural nuclei. Several additional smaller cell groups have also been described (Fisher et al.,1979; Peterson,1966), and are collectively referred to as accessory nuclei. A common feature of these cells is their prominent efferent projection to the neurohypophysis (Fig. l). The major secretory products of the HNS are the neurohonnones oxytocin and vasopressin which are released from the terminals located within the posterior pituitary. Unlike many other neuronal systems, the output of this system (i.e. vasopressin and oxytocin) has well defined functional consequences. The most notable function of vasopressin is to increase reabsorption of water at the collecting duct of the kidney. Oxytocin plays an equally prominent role in the processes of parturition and milk ejection by stimulating contraction of the smooth muscles of the uterus and of the myoepithelia of the breast. Understanding the function of oxytocin and vasopressin has permitted the design of experimental paradigms which manipulate (i. e. stimulate or inhibit) the quantity of these hormones released; an approach which has been profitably employed by many investigators to explore both central and peripheral mechanisms which underlie changing patterns of hormone release. Under this scrutiny, much has been learned about the HNS, which has emerged as a model system for the study of many facets of neurosecretion. Fig.1. Sch Schematic prominent neurohypo 2 Fig.1. Schematic of the hypothalamo-neurohypophysial system. Schematic drawing of the HNS in the sagittal plane illustrating the prominent nuclei within this system and their primary efferent to the neurohypophysis. 06200050502 .8an .9. 30.32 0: ofiaa IF In; 5.8.8.. + 8.15 128 52.00800232068 .0 3953.8 .m 33:05 320:2 .< 6.32 203003 ‘V 89. 0:8 I //IIIIII\\.9=8 5.2.2 , 320:2 028905 _ < e 3232 5:32:50 .E< 3232 8323393 C0] One important aspect in our understanding of the control of oxytocin and vasopressin secretion is the description of afferent input to the HNS. Such information provides insights into the pathways required to transduce and/or relay the changing demands placed upon the animal and may also lead to a clearer understanding of the integrative mechanisms which transform the seemingly stochastic volley of incoming signals into a meaningful physiological response. 1.1. Olfactory Efferents to the HNS? Suggestions that the main olfactory bulb (main bulb or MOB) may project directly to the region surrounding the SON have appeared several times in the literature over the past decade or so. Using degeneration methods, after main bulb ablation, Scalia and Winans (1975) described terminal fields in the area immediately ventral and lateral to the more posterior portions of the SON. These authors interpreted the projections as terminating in the region bordered medially by the SON and laterally by the anteroventromedial portion of the medial amygdaloid nucleus. A similar pattern of terminal degeneration was found by Heirner, using the same method (Heirner,1978). Precluded by the limitations inherent in the Fink- Heirner method was fine resolution of the terminal distribution in the region. For example, it is difficult to distinguish fine diameter terminals from background and nonspecific staining of the pial membrane. The latter is of particular importance for terminals contacting SON dendrites which run in the ventral glial lamina subjacent to the pial surface. This problem was further exacerbated by the lack of detailed information regarding the SON dendritic architecture. In a review, Brooks et al.(1980) made the most positive statement concerning the anatomical connections of the olfactory bulb with the SON. 01 go 19 SE dir he: ho the ant Spe org in r Cfft of l eva Pro: and Writ moc' Prof ‘7er llIXr; They stated that horseradish peroxidase tracing had shown that “the olfactory bulb seems to project monosynaptically upon SON as seen in the guinea pig...”, and then cited “J.R. DeOlmos, personal communication, 1978.” No experimental paper has appeared confirming this claim. This statement is intriguing in that such a connection could provide the HNS with direct sensory information. This is a relationship in the hypothalamus which heretofore has been exclusively reserved to the suprachiasmatic nucleus (see, however, Youngstrom et al.,1987). 1.2. Thesis Objectives The experiments in this thesis are designed to test the hypothesis that the main and/or accessory olfactory bulbs project to nuclei within the HNS and, more specifically, the SON. In order to evaluate this hypothesis, specific experiments were designed to: 1) delineate the somatic and dendritic organization of the SON, 2) determine the distribution of main bulb efferents in relationship to the SON and PVN; 3) similarly investigate accessory bulb efferents to the SON and PVN, 4) ascertain the type and regional distribution of projection neurons contributing to this putative HNS afferent and 5) evaluate some of the neurophysiological and neuropharrnacological properties of this putative connection. Much of the data presented is of special relevance to only the SON, and not the PVN or accessory nuclei of the HNS. Hence, much of what is written will be limited to the SON. Nevertheless, since the SON contains almost twice as many neurosecretory cells as other nuclei within the HNS, modulation of SON activity alone could play a prominent role in altering the profile of oxytocin and vasopressin secretion. Furthermore, the SON lies very close to the subarachnoid space, and nearby the cistema chiasma; this juxtaposition suggests that the SON may also secrete oxytocin and vasopressin into the cerebral spinal fluid. This would greatly extend the influence of the SON to include many central targets as well as the well- known peripheral effects. 1.3. Experimental Approach In the years following these earlier studies, techniques have been developed allowing a much finer resolution of axon terminals at the light microscopic level. Other new methods also permit high resolution retrograde transport studies to be done in confirmation of the data obtained by anterograde methods. Furthermore, with the development of brain slices and explant preparations, much more of the mammalian brain is now accessible to study using intracellular electrophysiological techniques. In order to characterize the anatomical and physiological properties of a putative connection, a variety of techniques are required. Each method presents a somewhat biased view of the structure under study. Therefore the use of several techniques to illustrate the features of a pathway permits the investigator to identify incongruent findings. This reduces the interpretational errors that may result when only one approach is employed. Accordingly, the possible existence of direct projections from the main and accessory olfactory bulbs to the SON was investigated using: 1) neurophysin irnmunocytochemistry to delineate the extent of the SON dendritic zone, 2) anterograde transport of wheatgerm agglutinin-horseradish peroxidase conjugate (WGA-HRP) after injection into either the main or accessory bulb, or Phaseolus vulgaris leucoagglutinin (PHA-L) after injection into the main bulb, to label bulb efferents, 3) retrograde transport of rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro- Gold®(F1uoro-Gold) after injection into the SON and PVN to determine projection neurons and, 4) intracellular electrophysiological analysis of “olfactory”-evoked responses from in vitro incubated explants to identify neurophysiological and pharmacological properties of this connection. 1.4. Organization of the Dissertation The dissertation is organized into 12 sections. Section two provides a brief overview of the anatomy and physiology of the SON. No attempt has been made to completely review this large literature. For this the reader is referred to several recent reviews by Hatton (1990), Morris (1987), Poulain (1982), Silverrnan (1983), Swanson and Sawchenko (1983), and Wakerley (1987). Rather it provides a framework of essential material for those less familiar with this system. Likewise section three provides a brief introduction to the anatomy of the olfactory system. More complete descriptions may be found in reviews by Scott (1986), Doty (1986), Mair (1986), Wysocki (1986), and Switzer (1985). Sections four to nine contain the experiments comprising this dissertation. Section ten summarizes the results and further discusses the implication of these observations. Section eleven is a group of appendices providing additional details of the materials and methods employed in these experiments.and, finally, section twelve provides the references cited / Beacon cote—.2 _ E:__o£_ne2:e b20m=0 Ems. 128 t‘\ . £:m >803 0< :3: E2936 5e: 21 have access to only volatile chemicals which are freely diffusible in inspired air. 3.1.2. Septal Organ The septal organ is ventral to the main olfactory receptors at the entrance to the nasopharyngeal passage and isolated from other sensory epithelia by respiratory mucosa (Rodolfo-Masera,l943). Receptors in the septal organ project as a separate fascicle through the cribriforrn plate to terminate within glomeruli of the main bulb (Bojsen-Moller,l975). Unlike the main olfactory receptors, the septal organ projects to a restricted area within the posteroventral area of the main bulb (Astic and Saucier,l988). The function of the septal organ remains somewhat obscure. Its position at the entrance to the nasopharyngeal passage suggests that it serves an “alerting” function, especially at low tidal volumes (e.g., during quiet respiration) when chemotactic agents may not reach other receptors (Bojsen- Moller,1975; Rodolfo-Masera,l943). This view is consistent with electrofactograrn studies of the septal organ which demonstrate that septal organ receptors show greater sensitivity to certain odors than the main olfactory neuroepithelium (Marshall and Maruniak,l986). Also by virtue of its position, the septal organ should have access to both volatile and non- volatile stimuli, suggesting it may play a more comprehensive role in sensing the environment. 3.1.3. Vomeronasal Organ The vomeronasal organ is a cigar-shaped patch of neuroepithelium located rostrally within the nasal septum and overlies the nasopalatine duct. Thin unmyelinated fibers coalesce to form the vomeronasal nerve which, after passing through the cribriforrn plate, terminates within the glomeruli of the accessory olfactory bulb. Unlike either the septal organ or the main 011 8C (ii: 131 v0 wi 011 the the ple bu? Fu. ple Pill Ple rev den thic late 22 olfactory neuroepithelium, the vomeronasal organ innervates only the accessory olfactory bulb (Barber and Raisman,l974; Bojsen-Moller,l975). The vomeronasal organ senses non-volatile compounds which, when dissolved or suspended in saliva (or respiratory mucus), reach these receptors through the nasopalatine duct (W ysocki et al.,1980). The vomeronasal organ is thought to be responsible for guiding many “pre- wired” (Keveme et al.,1986) social behaviors (e.g. maternal behavior, sexual behavior). 3.2. Olfactory bulb organization The olfactory bulb in the rat is a six-layer laminated structure. It can be subdivided into a rostromedial main olfactory bulb and an accessory olfactory bulb, a crescent-shaped inclusion within dorsolateral margins of the posterior main bulb (Fig. 4). In the main bulb, from superficial to deep, the six layers are: 1) olfactory nerve layer; 2) glomerular; 3) external plexiform; 4) mitral cell; 5) internal plexiform; and 6) granule cell layer. The accessory olfactory formation is organized similar to the main bulb, but, the mitral cells are more pleomorphic than those of the main bulb. Further, mitral cells are organized into a thick band within the external plexiform layer, hence no real mitral layer exists. 3.3. Olfactory bulb intrinsic organization The important feature of the intrinsic organization of the bulb (for our purposes) is that the mitral cells, and tufted cells within the external plexiform area, are the output neurons of the bulb. The reader is referred to reviews by Scott (1986), Shepard (1972) and Switzer (1985) for a more detailed discussion. Their axons join within the granule cell layer to form thick fascicles which, at the caudal end of the bulb, converge to form the lateral olfactory tract. Similarly, mitral cells of the accessory bulb also 23 Fig. 4. Atlas of the olfactory bulb in the sagittal plane. Sections are 100 um thick and stained with thionine. A line drawing on the right delineates the various structures within each bulb. Mitlal cell layers for each bulb are shaded in the drawings. A. The lateral margins of the bulb, here both the main and accessory bulb are visible. In the main bulb the laminated organization is readily apparent. The internal plexiform layer which lies between the mitral cell and granule cell layers has been omitted for the sake of clarity. B. Sagittal section through the main and accessory bulb 300 pm medial to section in A. C. Sagittal section through the main olfactory bulb 300 um medial to B. Here the accessory bulb is no longer visible. Abbreviations: AOB accessory olfactory bulb; AOD anterior olfactory nucleus, dorsal subdivision; AOE anterior olfactory nucleus, external portion; AOL anterior olfactory nucleus, lateral subdivision; AON anterior olfactory nucleus; AOV anterior olfactory nucleus, ventral subdivision; EPl external plexiform layer of the main olfactory bulb; Gl glomerular layer of the main olfactory bulb; Gr granule cell layer of main bulb; GrA granule cell layer of accessory bulb; LOT lateral olfactory tract; Mi mitral cell layer of the main olfactory bulb; MiA mitral cell layer of accessory olfactory bulb; MOB main olfactory bulb. 24 1&3 . \ Rhin 25 contribute axons to the lateral olfactory tract, joining the tract along its dorsal margins 3.4. Main Bulb Efferents The mitral and tufted cells of the main bulb project ipsilaterally through the lateral olfactory tract to several caudal structures (Fig. 5), including the anterior olfactory nucleus, primary olfactory cortex, olfactory tubercle, several subnuclei within the arnygdala (anterior area, anterior cortical, posterolateral cortical and medial), and the entorhinal cortex (Broadwell,l975; Devour,l976; Heimer,l968; Price and Powell,l97l; Scalia and Winans,l975; Shipley and Adamek,1984). It is generally believed that the tufted cells project to only rostral targets while mitral cells project to all target areas (Haberly and Price,1977; Macrides and Schneider,1982; Scott,198l). 3.5. Accessory Bulb Efferents The mitral cells of the accessory bulb have a somewhat different set of target nuclei (Fig. 6). This relationship, first demonstrated by Scalia and Winans (1975) and subsequently confirmed by others (Broadwell,1975; Devour,l976; Price and Powell,l971; Shipley and Adamek,1984), is the anatomical underpinning of the notion that the accessory and main bulb (hence, the vomeronasal organ and main bulb neuroepithelia) subserve different chemosensory functions. These mitral cells project to the nucleus of the accessory olfactory tract, medial and posteromedial cortical amygdaloid nuclei, and the bed nucleus of the stria terminalis. Fig. Arrc mitr tlIiCI l‘OStl Abb 26 Fig. 5 Schematic drawing of the main olfactory bulb efferents. Arrows represent an input to that structure. As illustrated, axons from mitral cells in the main olfactory bulb project through the lateral olfactory tract to terminate in many caudal targets. Tufted cells which also project to rostral targets are not drawn(e.g., Tu). Abbreviations: 3V third cereme ventricle; ACo anterior cortical amygdaloid nucleus; AON anterior olfactory nucleus; LOT lateral olfactory tract; Me medial nucleus of amygdala; MOB main olfactory bulb; Pir piriform cortex; PLCo posterolateral cortical amygdaloid nucleus; SON supraoptic nucleus-main (or anterior) portion; Tu olfactory tubercle; NLOT nucleus of the lateral olfactory tract IIn optic nerve. 27 Main Olfactory Bulb Efferents OB PCo U 28 Fig. 6. Schematic drawing of accessory olfactory bulb efferents. Mitral cells from the accessory olfactory bulb also project through the lateral olfactory tract. There is little overlap between the main and accessory bulb efferents except within the medial amygdala and possibly the SON. Abbreviations 3V third cerebral ventricle; AOB accessory olfactory bulb; BAOT bed nucleus of the accessory olfactory tract; LOT lateral olfactory tract; Me medial nucleus of amygdala; PMCo posteromedial cortical amygdaloid nucleus; SON supraoptic nucleus-main (or anterior) portion; Tu olfactory tubercle; Iln optic nerve. 29 Accessory Olfactory Bulb Efferents ‘ . o ' 01 A 0 2. 1 Zone 4.1. Introduction An essential first step in an anatomical study of putative projections is to accurately define the extent of the target nuclei. Since both somata and dendrites are post synaptic elements for afferents, their morphology needs to be understood. The use of Nissl stains amply illustrates the cell bodies within many neuronal systems. Indeed, much of the present nomenclature and organization of cell aggregates into nuclei are based on these stains. The distribution of PVN and SON somata has likewise been described for many years (Bleier et al.,1979). There is little ambiguity as to their organization. The morphology and distribution of dendritic processes in many systems, however, are less well understood. In some areas (e.g. cerebellum, cerebral cortex) Golgi methods have revealed much of the detail of these dendritic trees. Unfortunately, the SON and PVN do not stain well with this technique. The SON and/or PVN dendritic architectures have now been analyzed using irnmunocytochemistry (Armstrong et al.,1982; Sofroniew and Glasmann,1981), Golgi (Armstrong et al.,1982; Dyball and Kemplay,1982), retrogradely transported HRP (Ju et al.,1986) and intracellular injection of Lucifer Yellow CH (Hatton,l990; Randle et al.,1986). Unfortunately, these techniques have different sensitivities, consequently disagreements have arisen. 4.2. Experimental Question One such disagreement particularly pertinent to these studies is the issue of whether SON dendrites project into periamygdaloid areas. 30 31 The neurophysins, oxytocin- and vasopressin-associated neurophysins are found in the soma, dendrites, and axons of oxytocin- and vasopressin- producing cells in the PVN and SON and other neurons which produce these peptides. Since neurophysins are widely distributed within these neurons they may be used as markers to study the morphology of these cells. 4.3. Methods Exps. 14: General Methods for Anatomical Studies Male and female Sprague-Dawley rats 90-150 days old either obtained from the Holtzrnan Co. or raised in our colony were used. Surgeries were performed on animals which were deeply anesthetized with equithesin. Micropipettes containing one of several tracers were placed, with the aid of a stereotaxic holder, either by visual guidance or with coordinates from the atlas of Paxinos and Watson (1986). Glass micropipettes used for injections were manufactured on a Kopf vertical electrode puller from microfilarnent glass (1.0 mm O.D., 0.58 mm 1. D.) after which a final tip diameter of 5-15 pm for the iontophoretic injection or 25-50 pm for pressure injections was obtained by breaking the tip with a Kirnwipe®. After appropriate survival times, animals were transcardially perfused, the brains removed from the skull and stored in buffer prior to further histological processing. Unless otherwise noted, all reagents were diluted in one of two buffers: 1) 0.05 M Tris, 0.15 M NaCl, pH 7.4 (TBS) or 2) 0.01 M Na phosphate, 0.14 M NaCl, pH 7.4 (PBS). Sections were examined with a Zeiss standard microscope using brightfield, polarized darkfield, and/or epifluorescence (50 W Hg) illumination. The epifluorescence was configured with a “green” filter set consisting of a BP 546 supplemental exciter, FT 580 dichroic mirror, or a LP 590 barrier; and a “UV” set consisting of a G 365 supplemental exciter, FT 395 mirror, and LP 420 barrier. 32 4.4. Methods Exp. 1: Neurophysin Immunocytochemistry Male rats were fixed with Bouin's fixative, brains were removed and stored in the same fixative at 5°C for several days. In some cases, 24 h prior to fixation the animal was given an intracerebral ventricular injection of colchicine (0.1 ug/ kg). Brains were sectioned in either the coronal, sagittal, or horizontal plane with a Vibratome® at a thickness of 100 um. Tissue sections were rinsed in TBS over a period of several days to completely remove the fixative. Neurophysin-containing processes were detected by an irnmunocytochemical method (Hsu et al.,1981). This was accomplished by the successive application of the following reagents: rabbit anti-neurophysin (48 h at 4°C; Chemicon), biotinylated goat anti-rabbit (24 h at 4°C), and then avidin-biotinylated HRP complex (24 h at 4°C; Vector). The tissue was rinsed for 2 h with TBS at room temperature after each reagent incubation. The HRP was visualized with a glucose oxidase, irnidazole, diaminobenzidine solution (Smithson eta1.,1984). Labeled perikarya and their processes were identified by their dark brown coloration, as seen in brightfield illumination. 4.5. Results Exp. 1: Perikaryal labeling with Neurophysin Immunocytochemistry Dense irnmunocytochemical labeling around the SON revealed three distinct patterns; these corresponded to somatic, axonal, and dendritic labeling of SON neurons as previously reported (Armstrong et al.,1982). Features of neurophysin labeled material are illustrated in the coronal (Fig. 7), sagittal (Fig. 8) and horizontal planes (Fig. 9). 33 Fig. 7. Distribution of neurophysin immunoreactivity in 100 um thick coronal sections. Panels A-I, covering 3 consecutive plates, illustrates the course of SON dendrites in the periamygdaloid cortex. In each panel the photomicrograph on the right illustrates the SON dendritic zone at higher magnification. The irnmunocytochenrical reaction product appears dark gray to black while the unstained tissue is light gray. In all panels the SON dendrites are delineated with arrowheads. Bar== 375 & 250 pm for the photomicrographs on the left and light respectively. A-C. A dark black aggregation of irnmunoreactive cell bodies lateral to the optic tract is the somatic portion of the SON. The wavy arrow (in A) denotes the prominent efferent to the neurohypophysis. Arrows in the higher power photomicrographs illustrate axons projecting into the perinuclear zone/lateral hypothalamus. In each plate note the SON dendrites which project into the periamygdaloid cortex. Abbreviations: SON supraoptic nucleus-main (or anterior) portion; OT optic tract. 34 35 Fig. 7 (cont.): Plate 2 DE. Note the long dendritic projections (arrowheads) in D that extent several hundred microns into the periamygdaloid cortex. Also note the neurophysin-irnmunoreactive fibers (arrows) which appear juxtaposed to the nearby lateral olfactory tract. Abbreviations: SON supraoptic nucleus-main (or anterior) portion; OT optic tract. 36 37 Fig. 7 (cont.): Plate 3 G-I. Note that at these posterior levels of the nucleus, few SON cell bodies remain. However, SON dendrites are still observed in the cortex. Abbreviations: SON supraoptic nucleus-main (or anterior) portion; OT optic tract. 38 39 Fig. 8. Distribution of neurophysin immunoreactivity in 100 um thick sagittal sections. Panels A-F proceed from lateral to medial to further illustrate the course of SON dendrites. Anterior is positioned left, while dorsal is up. SON dendrites are delineated with arrowheads. Bar = 375 & 150 pm for photomicrographs on the left and right, respectively. A. SON somata at the posterolateral margins of the nucleus are positioned dorsal to the medial amygdala (Me in A). B & C. More medially, the region subjacent to the optic tract is filled with SON dendrites mnning in an anteroposterior direction. Also seen at these lateral levels of the SON are efferents which project dorsally (arrows) into the lateral hypothalamus. Abbreviations: Me medial nucleus of amygdala; OT optic tract; SON supraoptic nucleus-main (or anterior) portion. 40 Fig. after the not (ant Abb: 41 Fig. 8. (cont.) Plate 2 D-F. Progressing medially the periamygdaloid cortex disappears (see B), after which SON dendrites lie immediately subjacent to SON somata. An efferent to the lateral hypothalamus (upper arrow in D) and another projecting anteriorly towards the olfactory tubercle/lateral preoptic area (arrows in E & F) are observed at these levels. Abbreviations: OT optic tract; SON supraoptic nucleus—main (or anterior) portion. 42 43 Fig. 9. Distribution of neurophysin immunoreactivity in 100 pm thick sections in the horizontal plane. Panels A-C are arranged ventral to dorsal, anterior is left and medial is up. As before, SON dendrites are marked with arrowheads. Bar = 375 & 150 pm for panels on the left and right, respectively. A-B. Note the thick plexus of SON dendrites at the caudal margins of the SON extending beyond the confines of the nucleus. C. At more dorsal levels neurophysin-irnmunoreactive somata (wavy arrow) in the preoptic area appear as a rostral extension of the SON. Many thin neurophysin processes (arrow) were also observed in the perinuclear zone of the SON. Abbreviations: OT optic tract; SON supraoptic nucleus-main (or anterior) portion. 44 45 4.5.1. Somatic Labeling SON somata appeared as an aggregation of densely stained ovoid cells located lateral to the optic tract (Figs 7) and dorsal to a plexus of thick well- stained processes (i.e. SON dendrites). The distribution of SON somata is well documented and deserves little further comment. One important observation, however, was that the SONa extends further rostrally than generally appreciated as is readily apparent in the horizontal plane (Fig.9 C). Here SON somata appear at levels as rostral as the medial preoptic area and are positioned ventromedial to the anteroventral preoptic nucleus. 4.5.2. Axonal Labeling Axonal processes were observed as a plexus of small caliber varicose fibers radiating dorsomedially from the dorsal and dorsolateral margins of the somatic portion of the nucleus. Additionally, another group of fibers was also observed at this level of the SON. These fibers projected laterally into the adjacent nucleus of the horizontal limb of the diagonal band and lateral SON perinuclear zone (Fig. 7 B & C). Within the amygdala at this level were also thin fascicles of discontinuous fibers which circumscribed the dorsolateral margins of the nucleus of the lateral olfactory tract, ending abruptly within the lateral olfactory tract itself (Fig.7 D-F). In the sagittal plane (Figs. 9) a dense projection was observed which arched dorsally over the optic tract representing the well-known SON efferent to the neurohypophysis. Additionally, at the lateral margins of the nucleus two other efferent pathways appeared to project from the nuclear region. One projected dorsally into the lateral hypothalamus and horizontal limb of the diagonal band as several groups of fibers (Fig. 8 A-D). The other projected anteriorly into the olfactory tubercle and ramified diffusely within this structure (Fig. 8 DP). Unlike the neurohypophysial and lateral hypothalamic 46 projections in which fibers could be traced from the nuclear borders for a considerable distance, this olfactory tubercle projection appeared as a collection of discontinuous fibers. Consequently their origin is undetermined. 4.5.3. Dendritic Labeling The dendritic labeling was observed as a dense collection of thick varicose processes immediately ventral to SON somata, including a fascicle of processes which also projected ventrolaterally into periamygdaloid regions along the ventromedial surface of the brain. In the coronal plane, the periamygdaloid projecting dendrites were first observed at the mid rostrocaudal levels of the nucleus (Fig. 7 A). Here the dendrites appeared to project directly ventral, extending more than several hundred micrometers (Fig. 7 D) into the cortex. Caudally, the number of ventrally projecting dendrites diminished rapidly. At these levels, only punctate neurophysin labeling (i.e. SON dendrites cut in cross-section) was observed within the cortex (Fig. 7 F-I). That this labeling was of dendritic processes was more easily observed in the sagittal plane (Fig. 8) at the posterolateral boundaries of the SON. Here many thick processes were observed coursing in an anteroposterior orientation ventral to the optic tract within the periamygdaloid cortex (Fig. 8 B-E). Progressing medially this cortical region diminished rapidly along with this group of SON dendrites. It also appeared that dendrites extended beyond the caudal boundaries of the somatic region, as observed in the horizontal plane (Fig. 9 A & B). 4.6. Discussion Exp. 1. 4.6.1. Somatic Labeling The distribution of neurophysin-labeled somata are consistent with previous reports employing irnmunocytochemical techniques (Armstrong et 47 al.,1982; Sofroniew and Glasmann,198l; Yulis et al.,1984). However, only a passing mention (Rhodes et al.,1981; Sofroniew,l985; Yulis et al.,1984) has been made of the rostral extension of SON somata into the medial preoptic area. Irnmunocytochemical analysis of the distribution of oxytocin and vasopressin cells (Rhodes et al.,1981) suggest these cells may be oxytocinergic. The medial preoptic area has been implicated in the control of maternal behavior (Numan,1990). Furthermore, oxytocin seems to play an important role in promoting this behavior (Insel,1990; Pedersen,1982). Whether the nearby SON sends collateral branches of axons into the medial preoptic area is an important question awaiting further experimental evaluation. 4.6.2. Dendritic Ramifications That SON dendrites do rarnify ventrolaterally into periamygdaloid regions is not generally appreciated. Rather they are believed to traverse ventral to the cell bodies within the mediolateral confines of the nucleus (Dyball and Kemplay,1982; Felten and Cashner,l979; Randle et al.,1986). However, irnmunocytochemical studies (Armstrong et al.,1982, and the present study) as well as an analysis of retrogradely-filled SON neurons from posterior pituitary injections (Ju et al.,1986) clearly illustrate that many SON dendrites do traverse out of the confines of the nucleus. This fact is easily appreciated in the coronal plane where several prominent fascicles of dendrites project ventrally into the periamygdaloid cortex. However, since the predominant orientation of this dendritic projection is anteroposterior, the prominence of this projection disappears rapidly in this plane of section. It is replaced with small punctate profiles of dendrites cut in cross section, an appearance which may easily be mistaken for non-specific staining. Consequently, while the coronal plane clearly illustrates this dendritic 48 projection, it also obfuscates the magnitude of the projection. The frequent and almost exclusive use of the coronal plane by many investigators has, in all likelihood, perpetuated this misunderstanding. 5.1 . Introduction It seems clear after evaluation of the SON dendritic zone in the previous experiment and reviewing published line drawings of anterograde labeling of main bulb efferents from the rat (Heimer,l968; Scalia and Winans,l975) and rabbit (Broadwell,1975; Scalia and Winans,l975) that the main bulb probably projects to the SON. Yet no specific comment on this putative connection was made in these papers, and has only since appeared in a review by Heirner (1978). 5.2. Experimental Question Does the main bulb project to the SON? If so, what are the distribution of inputs within the SON? Does the main bulb also send projections to other nuclei within the HNS? Armed with more sensitive and specific anatomical tracing tools, this reevaluation of main bulb efferents should provide a more definitive answer to these questions. 5.3. Methods Exp. 2A: Anterograde studies with WGA-HRP Pressure injections (6 bilateral and 12 unilateral) of 100-150 nl of 0.5 - 0.7% WGA-HRP were stereotaxically placed into the main bulb of 18 female rats. The animals were allowed to survive 24-72 h after which they were perfusion-fixed with 500ml of 1% paraformaldehyde, 2% glutaraldehyde in 0.1 M Na phosphate, pH 7.4, followed by equal volumes of 10% sucrose in PBS. The brains were stored in this solution for up to 3 days after which they were frozen sectioned at 40-50 pm in either the sagittal, horizontal or coronal plane. After sectioning, the tissue was processed immediately or stored overnight at 4°C in PBS. The WGA-HRP was visualized with tetrarnethyl benzidine (Mesulam,1982). In order to 49 50 facilitate precise localization of staining, tissue sections were arranged into three adjacent sets consisting of every third section. Two sets were counterstained with bisbenzirnide (Schmued et al.,1982) while the third was counterstained with thionine. All slides were coverslipped with DPX, then stored at 4°C to prevent deterioration of the labeling. Labeled fibers and terminals were identified by their bright granular appearance in darkfield illumination. 5.4. Methods Exp. 28: Anterograde studies with PHA-L The PHA-L method of Gerfen and Sawchenko (Gerfen and Sawchenko,l984) was employed with some modifications in the histological processing to permit thinner tissue sections. Briefly, bilateral iontophoretic injections of PHA-L (2.0 mg/ml solution; Vector Labs) were placed into the main bulbs of 22 female rats and 2 male rats. After survival times ranging from 7-21 days, animals were fixed with 4% parafonnaldehyde in a 0.1M Na acetate pH 6.5, followed by 4% paraformaldehyde, 0.5% glutaraldehyde in a 0.1M Na borate, pH 9.5 (Berod et al.,1981). The brains were removed and stored in TBS for 1-3 days, then embedded in polyethylene glycol (Smithson et al.,1983) for sectioning in the horizontal, sagittal or coronal plane. Ribbons of tissue sections of various thicknesses between 10-25 pm were cut on a rotary microtome and collected onto an acetate film strip as they came off the blade. Sections were then rinsed in TBS to remove the polyethylene glycol and stored in TBS for further processing. The PHA-L was detected by an irnmunocytochemical method similar to that previously described. This was accomplished by the successive application of the following reagents: goat anti-phaseolus (24-48 h at 4°C; Vector), biotinylated rabbit anti-goat (1-2 h at room temperature), and then avidin- biotinylated HRP complex (1-2 h at room temperature). After rinsing, the 51 HRP was visualized with a glucose oxidase, irnidazole, diaminobenzidine solution (Smithson et al.,1984). After mounting, tissue sections were counterstained in either thionine or bisbenzirnide. Labeled processes and terminals were readily identified in brightfield illumination by their dark brown coloration which appeared bright in darkfield and polarized-darkfield illumination. 5.5. Results Exp. 2A: WGA-HRP labeling 5.5.1. injection Sites All injections of WGA-HRP were confined to the main bulb with no involvement of the accessory bulb or anterior olfactory nuclei, except in one case in which there was limited involvement with both these structures. 5.5.2. Anterograde Labeling The WGA-HRP technique consistently revealed more anterograde labeling around SON than the PHA-L technique. Anterograde labeling with WGA-HRP, however, did not reveal the same quality of morphology as did PHA-L, in that individual terminal morphology could not be readily discerned at the light microscopic level. In the coronal plane, dense labeling was observed throughout the ipsilateral lateral olfactory tract. Immediately anterior to the nucleus of the lateral olfactory tract, anterograde label was observed outside of this tract along the ventromedial surface of the brain. Further caudally, at the mid-rostrocaudal extent of the SON, punctate labeling was observed along the ventromedial edge of the brain spreading dorsally to the ventrolateral margins of the SON (Fig. 10 C & D). In sagittally cut material dense anterograde label was seen throughout the lateral olfactory tract, while a less dense more punctate pattern of labeling was observed deeper within the la layer of the piriform cortex (Fig. 11). Caudal to the nucleus of the lateral olfactory tract, and at the lateral borders 52 Fig. 10. Overlapping distributions of neurophysin stained processes and WGA-HRP labeling around the SON in the coronal plane. A & B. Neurophysin staining in a 100 pm thick section midway through the rostrocaudal extent of the SON. A. Immunocytochemistry illustrating a dense somatic region immediately lateral to the OT, a prominent efferent projection (closed arrow) dorsally, and a ventrally located dendritic projection which courses outside the confines of the nucleus (open arrow). B. At higher power, the efferent projections are illustrated by a dense plexus of varicose fibers projecting dorsally, and dorsolaterally (closed arrows). The dendritic region of the nucleus lies irnmediately ventral (open white arrows) to SON cell bodies and also send a prominent projection ventrolaterally (black open arrows) into periamygdaloid regions. A blood vessel interrupts the dendritic projection in this section (clear space). C & D. Distribution of labeled terminals and fibers in the coronal plane after an injection of Wheatgerm-HRP into the main bulb, as seen in polarized darkfield. This injection encroached marginally on the accessory bulb and rostral anterior olfactory nucleus. These micrographs are taken at approximately the same rostral-caudal level of the SON as those shown A & B. C. The WGA-HRP labeling is seen in darkfield as a bright punctate stippling on the dark background of surrounding unlabeled tissue. In this section a prominent aggregation of labeled terminal (arrows) appears immediately ventrolateral to the somatic portion of SON. The OT is located medial to the nucleus and appears bright in polarized darkfield. A few retrogradely labeled cells are also seen scattered throughout the adjacent neuropil. D. At higher power the darkfield illumination was combined with epifluorescence to reveal bisbenzirnide counterstained somata; conspicuous is the aggregation of cells in the SON (arrow). The WGA-HRP label (open arrows) is seen ventrolateral to the somatic region of SON within the field of the ventrolaterally projecting dendrites. Bar = 178 pm in A and C and, 100 pm in B and D. Abbreviations: OT optic tract; SON supraoptic nucleus-main (or anterior) portion. 53 54 Fig. 11. The distribution of labeled cell bodies, fibers, and terminals, after an injection of WGA-HRP into the main bulb. A. A montage of low power darkfield photomicrographs in a parasagittal plane (50 pm thick) through the lateral margins of SON, just medial to nucleus of the lateral olfactory tract. Rostrally (left), the anterogradely- labeled fibers present as a thin band within the LOT tract (arrows), while deeper within the la layer of the Pir punctate terminal labeling is observed. Many retrogradely-labeled cell bodies are also seen within HDB. At the level of the OT, another band of terminals (closed arrowhead) circumscribes the ventral margins of the OT, dorsal to the Me. B. At higher power, the labeled fibers and terminals (closed arrows) are immediately ventral to the OT. This area encompasses the perinuclear area of the SON as well as the lateral aspects of the SON dendritic zone. C. Another section, 150 tun medial to that seen in B illustrates many tenninals/aXons (closed arrows) lateral and ventral to SON. At this level SON cell bodies are positioned along the anterior margins of the OT, while the dendritic field is distributed ventrally within the area of labeling. D. Site of injection within the main bulb at its largest extent; note that the accessory bulb remains unstained. Bar = 190 pm in A, 100 pm in B and C, and 400 um in D. Abbreviations: AOB accessory olfactory bulb; LOT lateral olfactory tract; OT optic tract; MOB main olfactory bulb; HDB nucleus of the horizontal limb of the diagonal band; Me medial nucleus of amygdala; Pir piriform cortex; NLOT nucleus of the lateral olfactory tract; SON supraoptic nucleus-main (or anterior) portion. 55 56 of the SON, a small crescent of labeling was observed immediately ventral to the optic tract and dorsal to the medial amygdala (Fig. 11 B). More medially, within the lateral margins the SON, this labeling increased in both density and size to virtually fill the area subjacent to the optic tract (Fig. 11 C compare with neurophysin labeling in Fig. 8 A-C) No anterograde labeling was observed around the contralateral SON, nor was any labeling seen around the SONt, PVN or the accessory magnocellular nuclei. 5.5.3. Retrograde Labeling Retrograde labeling was routinely observed in the nucleus of the horizontal limb of the diagonal band and piriform cortex ipsilaterally, and bilaterally in the nucleus of the lateral olfactory tract. 5.6. Results Exp 28: PHA-L labeling 5.6.1. Injections sites All injections were confined to relatively small regions of the main bulb (Fig. 12C) with no involvement of either the anterior olfactory nucleus or the accessory bulb. 5.6.2. Anterograde Labeling The number of labeled cell bodies within the main bulb varied considerably among injections (here only the labeled cells are those which contribute to the PHA-L-labeled fibers). However, each injection consistently revealed labeled fibers and terminals adjacent to the SON. PHA-L labeling was in good agreement with that observed with WGA-HRP; however, the density of labeled fibers around the SON was considerably less with PHA-L. In the sagittal plane (Fig. 12), the full course of fibers from the bulb to the SON was observed. Rostrally at the main bulb, separate fiber bundles within the bulb converged to form a single thick fascicle at the anterior margin of the lateral olfactory tract. Proceeding caudally, the 57 number of labeled fibers within the tract diminished progressively. Caudal to the piriform cortex, only a few fibers remained in a thin fascicle on the ventral surface of the brain. Posterior to the nucleus of the lateral olfactory tract, another aggregation of fibers appeared immediately anterior and ventral to the SON. The labeled axons (Fig. 12 B) had many varicosities, and a few appeared to have tenninal-like endings. Similar to that observed in the WGA-HRP studies, labeled fibers and terminals were first observed lateral to the SON and superior and medial to the margins of the medial amygdala. Progressing medially toward the lateral margins of the SON, the amount of labeling increased dramatically. Reconstruction of this projection from adjacent sections (Fig. 13 B) revealed that it approached the SON anteriorly along its ventrolateral margin as a sheet of fibers approximately 250 pm thick. Furthermore, this distribution of axons seemed to overlap considerably with the arrangement of neurophysin stained processes ( i.e. dendrites; Fig. 13 A) as observed along the lateral margins of the nucleus. In material prepared in the horizontal plane, many fibers gave rise to small collateral branches whose bulbous terminal-like structures ended along the posterolateral margins of the nucleus. Additionally, fibers and terminals were also observed more anteriorly in the perinuclear zone of the nucleus (Fig. 14). While not a prominent feature of this projection, terminals were occasionally found within the somatic portion of the SON. 5.7. Discussion Exp 2 The pattern of anterograde labeling observed here from injections limited to the main bulb are in agreement with previous studies of main bulb efferents in the rat (Heimer,l968; Scalia and Winans,l975), and those from other mammals (Broadwell,1975; Davis and Macrides,l981; Devour,l976; Scalia and Winans,l975; Shipley and Adamek,1984). Retrograde labeling, 58 Fig. 12. The distribution of labeled axons and terminals, after an injection of PHA-L into the main bulb. A. A montage of low magnification darkfield photomicrographs through a parasagittal plane (15 um thick) along the lateral margins of the SON. Rostrally (left) many thick axonal process may be seen within the LOT. Throughout the full extent of the Pir the stained axons remained tightly organized within the LOT. Immediately anterior to the SON an aggregation of processes (delineated with lines) fans out immediately anterior to the SON. B. This area (delineated at lower power in A) contains many fibers (two of which are indicated by open arrows) with varicosities, and terminal profiles. C. Injection site at its largest extent through a parasagittal plane of the main bulb illustrates that the PHA-L was completely confined to the anterior portions of the main bulb. Many mitral cells (open arrow) appeared darkly stained by the DAB chromogen. Bar = 253 pm in A, 138 pm in B and 800 pm in C. Abbreviations: DAB 3,3' diaminobenzidine LOT lateral olfactory tract; MOB main olfactory bulb; OT optic tract; Pir piriform cortex; SO supraoptic nucleus-main (or anterior) portion. 59 . 55,5555»; 60 Fig. 13. Diagram of the overlapping distributions of SON dendrites and main bulb axons in a parasagittal plane through the lateral margins of the SON. A. Tracing from a 100 pm thick section irnmunocytochenrically stained for neurophysin. Open circles represent SON somata. Due to the thickness of the section it appears as if SON somata are embedded within the OT, this however, is not the case. Thick lines represent neurophysin stained processes (i.e. dendrites). A prominent feature of these processes at this mediolateral level is that they extend anterior to the nucleus, sweeping ventrolaterally and posteriorly outside the boundaries of the nucleus. The most anteriorly extending processes probably originate from cells placed more medially within the nucleus. B. A composite line drawing of PHA-L labeled fibers (same animal as seen in Fig. 3) from four altemate 15 um thick sections at approximately the same mediolateral level as that drawn in A. Similar to Fig. 12 B many PHA-L-labeled fibers are seen anterior and ventrolateral to the somatic (open circles) portion of the nucleus. Additionally, these processes seem to sweep from lateral to medial towards the nucleus. A few processes appear overlying SON somata; occasionally a process was observed within the nucleus. This however, is not a general feature of these afferents. A and B are at the same magnification. Abbreviations: MOB main olfactory bulb; OT optic tract; SON supraoptic nucleus-main (or anterior) portion. SON Dendrites 61 62 Fig. 14. PHA-L labeled fibers in the perinuclear zone of the SON. A. A photomicrograph illustrating the approach of fibers to the supraoptic nucleus (SON) in the horizontal plane. Rostral is positioned up and medial is to the left. Fibers are seen running along the lateral border of the SON. In some cases these fibers (arrow) give off bulbous-like terminal profiles. A thionine counterstain appears light gray in cell bodies unstained by the irnmunocytochemical procedure. B. Features of several bulbous profiles (arrow) at higher power. Bar: 75 um and 30 um in A & B, respectively. 63 64 while not the focus of this study, was also in agreement with that previously reported (DeOlmos et al.,1978; Shipley and Adamek,1984). 5.7.1. Exp 2A: WGA-HRP Labeling The WGA-HRP experiments, like the PHA-L experiments, also demonstrated a pattem of densely labeled fibers and terminals along the ventrolateral margin of the nucleus. The punctate character of the label, particularly that seen in the coronal plane (Fig. 10 C & D), is similar to that seen in the piriform cortex. Since the latter is thought to represent terminal labeling (DeOlmos eta1.,1978), a similar interpretation for the staining observed ventrolateral to SON is plausible. The WGA-HRP labeling was considerably denser than that seen with the PHA-L. This is probably due in large measure to the differences in the respective sizes of the injection sites. Iontophoretic injections of PHA-L were generally small compared to the pressure injections of WGA-HRP which often filled over one-half of the main bulb. Given these differences, WGA-HRP experiments probably produced a more accurate estimate of the size of the terminal field in and around SON. 5.7.2. Exp 28: PHA—L Labeling Many PHA-L fibers and terminal profiles were observed lateral and anteroventral to the SON. This was interpreted to suggest that the main bulb projects, at least in part, monosynaptically onto SON cell dendrites which are present in this area (see section 4.5.3). That these terminal-like profiles are indeed terminals is given credence by the electron microscopic analysis by Wouterlood et al. ( 1985) of PHA-L labeled material. In the sagittal plane, approaching fibers had less terminal profiles than that observed in the horizontal plane. The reasons for this difference are unclear 65 but may represent a mediolateral orientation of terminals as they rarnify over SON dendrites. Many labeled fibers had a varicose appearance, suggesting a possible en passant synaptic arrangement. Results from the PHA-L experiments also illustrate fibers and terminals in the perinuclear region of the SON. Cells in this area around the SON are known to project into the nucleus (Hatton et al.,1985; Hatton et al.,1983; Mason et al.,1984; Tribollet et al.,1985) and have been postulated to function as intermediaries in modulating a divergent set of efferents (e.g. from locus coeruleus, parabrachial, raphe, subiculum) which terminate within this region (Tribollet et al.,1985). These data are consistent with the view that the main bulb may also be connected to SON in a polysynaptic manner. . A. -;.-..,;.-;...; -~~.‘e; ..:,. W 6.1 . Introduction Similar to our observations concerning main bulb efferents to the SON, it also seems clear after evaluation of the SON dendritic zone and reviewing published line drawings of anterograde labeling of accessory bulb efferents from the rat (Heimer,l968; Scalia and Winans,l975) and rabbit (Broadwell,l975; Scalia and Winans,l975) that the accessory bulb also probably projects to the SON. No other reports or inferences of such a connection have been published. 6.2. Experimental Question Simply stated, does the accessory olfactory bulb also project to SON? It is clear from the previous experiments that the PHA-L labeling while revealing the morphology of the bulb efferents greatly underestimates input to the SON. The WGA-HRP labeling on the other hand better represents the magnitude of the input to the SON. Since the accessory bulb efferent is likely less robust because of the fewer mitral cells involved, WGA-HRP labeling is more likely to reveal a connection if one exists. 6.3. Methods Exp. 3: Anterograde studies with WGA-HRP Pressure injections of 60-160 ill of 0.3% WGA-HRP were stereotaxically placed, under visual guidance, unilaterally into the accessory bulbs of 3 female and 2 male rats. Iontophoretic injections of 0.7 % WGA- HRP were made in a similar fashion in the accessory bulbs of 20 additional rats (3 male and 17 female). Two of these injections were into both accessory bulbs. All iontophoretic injections were made at an angle approximately 23° from the vertical and roughly parallel to the long axis of the accessory bulb mitral cell layer. 66 67 Animals were allowed to survive 24-72 h; then they were perfusion- fixed with one of two perfusion protocols: l) 500 ml of 1% paraformaldehyde, 2% glutaraldehyde in 0.1 M Na phosphate—pH 7.4, followed by equal volumes of 10% sucrose in 0.1 M Na phosphate, 0.14 M NaCl, pH 7.4 or 2) 500 ml of 0.75% paraformaldehyde, 1.5% glutaraldehyde in 0.05 M Na phosphate—pH 7.4, followed by equal volumes of 20% sucrose in 0.05 M Na phosphate, 0.14 M NaCl, pH 7.4. The brains were then stored in their final perfusion solution at 4° C for up to 3 days, after which they were frozen sectioned at 50 pm in either the coronal or sagittal planes. After sectioning, the tissue was processed immediately or stored overnight at 4°C in PBS. The WGA-HRP was visualized with tetramethyl benzidine (Mesulam,1982). In order to facilitate precise localization of staining, tissue sections were arranged into 3 sets of altemate sections. Two sets were counterstained with bisbenzirnide (Schmued et al.,1982) while the third was counterstained with thionine. All slides were coverslipped with DPX, and stored at 4°C to prevent deterioration of the anterograde labeling. Labeled fibers and terminals were identified by their bright granular appearance in darkfield illumination. 6.4. Results Exp. 3: WGA-HRP labeling 6.4.1. Injection Sites In previous work in the main bulb (Smithson et al.,1989b) injections of anterograde tracers were easily confined to this structure. The accessory bulb, however, is much smaller. Consequently, these injections required closer scrutiny. We have taken the dense core of the tetramethyl benzidine reaction product, which surrounds the injection site, to be the effective site of uptake as suggested by Mesulam (Mesulam,1982). On this basis, injections were classified into five groups (Fig. 15). These injection sites 68 Fig. 15. Accessory olfactory bulb injection sites. A. Line drawing of the main and accessory bulbs. Injections sites included the following structures. B. The AOB only, N=2. C. The AOB and rostral AOD, N=4. D. The AOD only, N=l. E. The frontal cortex, N=8, or the rostral AOB and MOB, N=1. F. The AOB and the MOB, N=ll. Abbreviations: AOB accessory olfactory bulb AOD anterior olfactory nucleus, dorsal subdivision; AOE anterior olfactory nucleus, external portion; AOV anterior olfactory nucleus, ventral subdivision; EPl external plexiform layer of the main olfactory bulb; Gl glomerular layer of the main olfactory bulb; Gr granule cell layer of main bulb; GrA granule cell layer of accessory bulb; LOT lateral olfactory tract; Mi mitral cell layer of the main olfactory bulb; MiA mitral cell layer of accessory olfactory bulb; MOB main olfactory bulb. 70 included those structures involving portions of: the accessory bulb only (2), the accessory bulb and a small portion of the dorsal subdivision of the anterior olfactory nuclei, the rostral anterior olfactory nucleus (4), the accessory bulb and a small portion of the main bulb (l), the overlying frontal cortex (8) and injections which encroached extensively into the adjacent main bulb (11). 6.4.2. Anterograde labeling No anterograde labeling was observed within the lateral olfactory tract after an injection into the anterior olfactory nucleus (i.e.AOD and AOL). Labeled fibers were observed within the anterior commisure, an observation consistent with previously demonstrated projections of the anterior olfactory nucleus to the contralateral main bulb (Davis and Macrides,l98l , see also Switzer et al.,1985). Injections limited to the accessory bulb and those that also encroached upon the anterior olfactory nucleus had similar pattems of anterograde labeling within the lateral olfactory tract and amygdaloid areas. Illustrated and described is anterograde labeling from a unilateral injection into the caudal accessory bulb which spread slightly into the anterior olfactory nucleus (i.e. Fig. 15 C). Anterograde labeling appeared at rostral levels within the dorsomedial margins of the lateral olfactory tract, and remained in this position until the caudal termination of the tract near the nucleus of the lateral olfactory tract. Ventral to this nucleus, the projection turned dorsally while remaining within the superficial layers of the ventromedial surface of the amygdala. Caudally, posterior to the nucleus (i.e. NLOT), retrogradely labeled cells within the bed nucleus of the accessory olfactory tract appeared, capping the anterogradely labeled fibers in their dorsal projection (Fig. 16). As the projection progressed posteriorly (Fig. 16 A-D), both anterogradely labeled fibers and retrogradely labeled 71 Fig. 16. Distribution of labeled cell bodies and terminals after an injection of WGA-HRP into the ipsilateral accessory bulb. Polarized darkfield photomicrographs of coronal sections, proceeding from rostral (A) to most caudal (E) illustrating retrogradely labeled cell bodies and anterogradely labeled axons/tenninals. Sections are 50 um thick and 100 um apart. These levels of the SON correspond approximately to those levels of the SON illustrated in Fig. 7. D-I. Bar = 150 um & 75 for photomicrographs, left & right, respectively A. At this rostrocaudal level, retrogradely labeled cells in the BAOT and anterogradely labeled fibers ventrolateral to these cells are observed. Photomicrograph on th right illustrates feature of the labeled BAOT neurons and fibers/terminals at higher power. B. More caudally, labeled cell bodies and fibers (arrows) are immediately subjacent to SON. At higher power terminals decorate the ventrolateral margins of SON. Abbreviations: BAOT bed nucleus of the accessory olfactory tract OT optic tract; SON supraoptic nucleus-main (or anterior) portion. 72 73 Fig. 16 (cont.) Plate 2 D-E. Anterograde labeling at more caudal levels of the SON after an accessory bulb injection. Here, anterogradely labeled terminals continue to interdigitate with the SON dendritic zone. 74 75 neurons within the nucleus of the accessory olfactory tract migrated dorsally towards the ventral margins of the SON. Here, at these posterior levels of the SON, anterograde labeling was distributed within the region occupied by ventrolaterally projecting SON dendrites (Fig. 7 D-I). The accessory bulb projection continued posteriorly in its dorsal path along the optic tract sweeping adjacent to the medial amygdala and posteromedial cortical amygdaloid nucleus. No anterograde labeling was observed near the contralateral SONa. Furthermore no anterograde labeling was observed near the SONt, PVN or accessory magnocellular nuclei. 6.4.3. Retrograde labeling Many retrogradely-labeled cells within the ipsilateral bed nucleus of the accessory olfactory tract and posteromedial cortical amygdaloid nucleus were consistently observed. A few retrogradely cells were also observed within the medial amygdala, the bed nucleus of the stria medularis, and infrequently, the caudal portion of the horizontal limb of the diagonal band. 6.5. Discussion The pattern of anterograde labeling observed here, from injections limited to the accessory bulb as well as those which encroached slightly upon the rostral anterior olfactory nucleus is in agreement with previous reports of accessory bulb efferents in the rat (Heimer,l968; Scalia and Winans,l975), and those from other mammals (Broadwell,l975; Davis and Macrides,1981; Devour,l976; Scalia and Winans,l975; Shipley and Adamek,1984). Retrograde labeling, while not the focus of this study, was also in agreement with that previously reported (DeOlmos et al.,1978; Shipley and Adamek,1984). 76 Similar to those reported in previous experiments of main bulb efferents (in this thesis , and Smithson et al.,1989b), accessory bulb efferents approach the ventrolateral margins of the SON, terminating within the area occupied by SON dendrites. The accessory bulb projection approaches the nucleus more posteriorly than main bulb efferents, terminating near the caudal borders of the nucleus. While this was not a quantitative study, the magnitude of the terminal labeling after an accessory bulb injection appeared smaller than after main bulb injections. In part, this is because the accessory bulb projection approaches the nucleus more caudally where only a small portion of the SON somata and dendritic region remain. W 7.1 . Introduction The literature suggests that there is an approximate rostrocaudal topography of main bulb projection neurons to their target nuclei (i.e. anterior positioned mitral cells project to more anterior targets; Haberly and Price,1977). This notion, however, was recently disputed (Ojirna et al.,1984). Another important feature of the main bulb is that it contains two types of projection neurons, mitral cells and tufted cells. Mitral cells project to all target nuclei. The efferents of tufted cells, on the other hand, are limited to primarily rostral targets (Haberly and Price,1977). The accessory bulb seems to have a different organization. Projection neurons are exclusively mitral cells with little topography described for its projections. 7.2. Experimental Question The previous experiment employing anterograde tracers suggests strongly that both the accessory and main bulb project to the SON. This conclusion could be strengthened considerably by a complementary retrograde tracing study. The prediction from the anterograde studies is that mitral (or tufted) cells would be retrogradely labeled after confined injections into the SON. Furthermore, since these projections are virtually unknown, no other published information is available, consequently this experiment could also determine the projection neuron involved (i .e whether mitral or tufted), as well as any preferential distribution within bulbar structures. 7.3. Methods: Exp. 4: Retrograde studies with fluorescent tracers. Pressure injections of 20-70 n] of Fluoro-Gold (Schmued and Fallon,l986; 1-2% in distilled water; courtesy of L. Schmued) or 70-150 n1 of rhodamine-label latex microspheres (rhodamine beads; LUMA-FLUOR 77 78 Inc.) were stereotaxically placed into the SON of 17 male rats. Injections were aimed at either the SON or tuberal portion of the supraoptic nucleus (SONt). In most cases, injections were also placed in the ipsilateral PVN, with the tracer that was not injected into the SON or SONt (e.g. Fluoro-Gold into PVN and rhodamine beads into SON). After a 3-7 day survival time, the animals were fixed with neutral buffered parafonnaldehyde (4%) or formalin (10%). The brains were blocked into diencephalic and telencephalic portions, left in fixative ovemight, then transferred to a solution containing 10-30% sucrose, 2% parafonnaldehyde in a 0.1 M phosphate buffer (pH 7.3-7.4) and stored at 5°C prior to sectioning. Blocks were frozen sectioned at 50 pm in the coronal plane through the preoptic/ diencephalic block and sagittally through the olfactory bulb/ telencephalic block. Sections were collected into either PBS or TBS and arranged into three adjacent sets consisting of every third section and stored refrigerated until they were mounted. After mounting, two sets were often counterstained with either ethidium bromide (Schmued et al.,1982) or bisbenzirnide (for rhodamine bead injections) and the remaining set with thionine, after which they were coverslipped in DPX or Entellan. Cells were identified by their bright fluorescence when exposed to the appropriate filter combinations. We only considered an area to definitely project to the SON if retrogradely labeled cells were observed with both tracers. 7.4. Results Exp. 4 7.4.1. Injection Sites Fluoro-Gold injection sites have been described (Schmued and Fallon,l986) as containing essentially three zones: a) a central zone which usually shows some necrosis, b) a zone of brilliant fluorescence where all tissue elements are stained, and c) a region of very diffuse fluorescence in 79 Fig. 17. Line drawings illustrating injections sites of A) Fluoro-Gold and, B) rhodamine-labeled microspheres, into the SON. A. The effective site of uptake from this Fluoro-Gold injection is demarcated by the inner dashed line, and includes the SON and some of the perinuclear zone surrounding the nucleus. This region did not, however, extend laterally into amygdaloid areas. Retrogradely labeled cells within the main bulb from this injection are presented in Fig. 18. As with all our Fluoro-Gold injections diffuse perikaryal staining (outer dashed line) could be observed surrounding the site of tracer uptake. B. Injections of rhodamine-labeled microspheres were typically more confined (dashed line), limited to the somatic and dendritic portions of the SON, however, not completely filling its anteroposterior dimensions. Retrogradely labeled cells within the accessory bulb from this injection are shown in Fig. 19. A and B are at the same magnification. Abbreviations 3V third cereme ventricle; AH anterior hypothalamic area; fx fornix; LH lateral hypothalamus NLOT nucleus of the lateral olfactory tract; 0C optic chiasm; Pe periventricular hypothalamic nucleus; SCN suprachiasmatic nucleus; SON supraoptic nucleus-main (or anterior) portion. 8O LH AH Site of SON 1mm 81 which occasional cellular elements are stained. The injection sites here also showed a similar organization. Accordingly, we too have considered the site of uptake for the Fluoro-Gold as encompassing only the first two zones of the injection site. The Fluoro-Gold injections used in the present study were largely limited to the SON, nearly filling the complete anteroposterior dimension of the SON. In most cases the region of brilliant fluorescence was confined to the SON and surrounding perinuclear area (Fig. 17 A). These injections were further subdivided into those which spread dorsally into the perinuclear area or lateral hypothalamus, and those spreading ventrally along the ventromedial surface of the periamygdaloid cortex. In the latter case, the adjacent medial amygdala was never involved. In all cases the third region (the area of diffuse fluorescence) spread beyond the confines of the SON into the lateral hypothalamus and occasionally ventrally into the periamygdaloid region. Uptake from Fluoro-Gold injections into the PVN frequently extended beyond the confines of the nucleus into adjacent areas including the zona incerta, perifomical region, and the lateral hypothalamus. In the case of the rhodamine bead injections, the uptake site of the tracer was very localized and taken to be the area of intense fluorescence surrounding the end of the injection tract. Rhodamirre beads were virtually limited to either the SON (Fig. 17 B) or SONt and adjacent optic tract. SON injections of rhodamine beads did not fill the complete anteroposterior dimensions of the nucleus, but were centered in its posterior portions. 7.4.2. Distribution of retrogradely labeled cells Qualitatively, the two fluorescent tracers yielded the same results with respect to retrograde filling of mitral cells within the main and accessory bulbs. Unilateral injections into the SON revealed only ipsilateral labeling within the main (Fig. 18) and accessory bulbs (Fig. 19). Retrograde labeling 82 Fig. 18. Distribution of labeled cells in the main bulb after an injection of Fluoro-Gold into the SON. A. A montage of low power epifluorescence micrographs through a parasagittal plane of the main bulb. Many labeled cells within the mitral cell layer of the main bulb are observed. No retrogradely labeled cells were seen in the EPl or G] of the main bulb and no regional distribution of labeled cells was noted. Open arrow points to a reference blood vessel. B. At higher power (arrow points to the same blood vessel) many large and small nritral cells are retrogradely labeled through the full thickness of the mitral cell layer. C. Brightfield micrograph of a section adjacent to that seen in A through the main bulb illustrating the relationship of the Mi, EPl and G] in the sagittal plane. Bar= 190 pm in A, 45 pm in B and 600 pm in C. Abbreviations Mi mitral cell layer of the main olfactory bulb; EPl external plexiform layer of the main olfactory bulb; Gl glomerular layer of the main olfactory bulb. 83 84 Fig. 19. The distribution of labeled cells in the accessory bulb after an injection of rhodamine-labeled nricrospheres into the SON. A. Montage of epifluorescence micrographs in a parasagittal plane through the MOB and AOB illustrating many labeled cells within the mitral cell layer of the accessory bulb. B. Low power bright field micrograph of a thionine stained section adjacent to that seen in A. Bag 100 pm in A and 500 pm in B. Abbreviations AOB accessory olfactory bulb; MiA mitral cell layer of accessory olfactory bulb; MOB main olfactory bulb. 85 86 of mitral cells throughout the main and accessory bulbs was observed without apparent regional distribution. Additionally, Fluoro-Gold injections revealed two populations of cells within the mitral cell layer of the main bulb: large mitral cells, and a much smaller cell type, possibly petite mitral cells (Fig. 18 B; Cajal,1911). No tufted cells were retrogradely labeled. Our quantitative impressions were that the small rhodamine bead injections had fewer retrogradely filled cells than did the larger Fluoro-Gold injections which seemed to label virtually all mitral cells within the main and accessory bulbs. Injections of Fluoro-Gold which spread dorsally consistently labeled cells within the ipsilateral ventral tenia tecta (i.e. TI‘1 , or medial transition zone), and bilaterally within the caudal horizontal limb of the diagonal band. In one case, cells within the contralateral anterior olfactory nucleus were labeled. However, in no cases were retrogradely labeled cells observed in the portion of the anterior olfactory nucleus bordering the accessory bulb (i.e. dorsal subdivision; AOD; see Fig. 4) In ventrally spreading injections, retrogradely-labeled cells were virtually limited to mitral cells within the main and accessory bulbs, with a rare retrogradely-labeled cell in the tinea tecta. Injections into the PVN (even those that spread considerably into surrounding areas) or the SONt failed to reveal any retrogradely labeled cells in either the main or accessory bulbs. Retrogradely labeled cells within the frontal cortex were routinely observed from these injections. 7.5. Discussion We have presented data from a Fluoro-Gold injection, because only in these injections did we fill the complete anteroposterior dimensions of the SON, and thus more accurately represents the input to the nucleus. This is particularly true for the main bulb efferents which terminate more anteriorly in the SON. That leakage of Fluoro-Gold into the subarachnoid space may 87 in part account for this labeling is highly unlikely. First, in those cases in which we have missed the SON and injected this space (data not presented) mitral cells were never labeled. Secondly if Fluoro-Gold were to be distributed in this space by diffusion one would expect that mitral cells would be labeled bilaterally, this was also never observed. The rhodamine bead injections, on the other hand, were smaller and confined to the more posterior portions of the SON, and thus underestimates the main bulb projection. While these injections labeled fewer mitral cells in the main bulb, all mitral cells within the accessory bulb were labeled by this injection. This finding is congruent with the fact that the main bulb efferent terminates more anteriorly than accessory bulb efferents. Warm 8.1 . Summary of Anatomical Results Irnmunocytochenrical staining for neurophysin revealed irnmunoreactive processes (i.e. dendrites) of probable SON origin extending ventrolaterally outside the boundaries of the nucleus into periamygdaloid areas. Injections of WGA-HRP into the main or accessory bulbs or injections of PHA-L into the main bulb revealed anterogradely-labeled fibers and terminals ventrolateral to ipsilateral SON somata; the same area occupied by ventrolaterally projecting SON processes. Injections of rhodamine beads or Fluoro-Gold into the SON resulted in many retrogradely labeled mitral cells throughout the main and accessory bulbs. We have approached the demonstration of this pathway through the use of several different techniques in order that their individual biases may be overcome. That the interpretation of the observations from the, PHA-L, WGA-HRP, Fluoro-Gold, and rhodamine beads are in good agreement with each other argues strongly against the possibility of artifactual staining in each case. Furthermore, many problems associated with interpreting “tract tracing” experiments center around two chief issues; a) uptake of the tracers by damaged fibers of passage, and b) the size of the effective site of uptake. The location and relatively large size of the main bulb within the rat brain, permits injection in such a fashion as to virtually eliminate these problems for the WGA-HRP and PHA-L experiments. Injections into SON with Fluoro-Gold or rhodamine beads, however, must be more closely scrutinized because of these problems. This is especially true because the nearby amygdala is known to receive main bulb efferents. Some of the Fluoro-Gold injections did spread into the lateral hypothalamus and possibly into the amygdaloid areas making our interpretation more tentative. However, the 88 89 rhodamine bead injections which were strictly confined to the SON also resulted in retrogradely labeled mitral cells throughout the main and accessory bulbs, thus supporting our interpretation of these results. Likewise injections into the accessory bulb in many cases also spread to the adjacent anterior olfactory nucleus. However, several facts argue against this nucleus, rather than the accessory bulb, as the source of afferent to the SON: 1) no retrogradely labeled cells were observed in this portion of the anterior olfactory nucleus (i.e. adjacent to the accessory bulb) after injections of either Fluoro-Gold or rhodamine beads into the SON, 2) the pattern of anterograde labeling within the lateral olfactory tract and caudal targets was similar to injections which were confined to the accessory bulb, and 3) the pattem of anterograde labeling after injections into only the anterior olfactory nucleus was limited to very rostral structures and did not involve the lateral olfactory tract. Our data suggest that the SON receives a relatively large input from the main bulb and a somewhat smaller input from the accessory bulb. This interpretation is supported by both, the dense terminal labeling from WGA- HRP injections into the main and accessory bulbs, and the extensive mitral cell labeling after injections of Fluoro-Gold into the SON. The implications of these findings are that mitral cells of the main bulb which presumably project to the anterior olfactory nucleus and piriform cortex also project to the SON. That individual mitral cells may project to both these structures is supported by electrophysiological (Scott,1981) as well as anatomical evidence (Luskin and Price,1982; Ojirna et al.,1984). While these authors have not investigated these relationships in the caudal piriform cortex or the SON, Ojirna (Ojirna eta1.,1984) noted that the trajectory of HRP-stained fibers within the lateral olfactory tract suggested that mitral cells could 90 project to the anterior olfactory nucleus and anterior as well as the caudal piriform cortex. It appears from our work that the main bulb projection to the SON is a continuation of that to the caudal piriform cortex, and is thus likely that individual mitral cells may project to the anterior olfactory nucleus, divergent areas of the piriform cortex, and the SON. Additional strong support for a monosynaptic projection comes from the observations of retrogradely filled mitral cells after injections of Fluoro- Gold or rhodamine beads into the SON. In particular, the small injections of rhodamine beads which were completely confined to SON somata and dendrites (e.g. Fig. 17 B) consistently revealed labeled mitral cells within the main and accessory bulbs, while injections into the SONt or PVN never labeled mitral cells. Many anatomical studies have described the efferent projection from the main and accessory bulbs (for a concise description see Switzer et al. 1985) and other mammals. Indeed, the pattems of anterograde labeling and retrogradely labeled cell are in agreement with these previous reports. Nonetheless, no anatomical study to date has described the connection between either the main or accessory bulb and the SON. This is probably in large part because the dendritic projections of SON neurons have only recently begun to be understood (Annstrong et al.,1982; Ju et al.,1986, and the present study). In all likelihood, some labeling formerly interpreted as evidence for a medial amygdaloid afferent was in reality a SON afferent. Many have also looked at the afferents to the SON, (Iijirna and Ogawa,1981; Tribollet et al.,1985; Wilkin et al.,1989) but again no mention was made of retrogradely filled cells in either the main or accessory bulbs. Data from both the anterograde and retrograde studies fail to show any apparent monosynaptic bulbar connections with the PVN. This distinguishes 91 these efferents to the SON by the fact that virtually all other known major inputs to the SON also go to the PVN (Tribollet et al.,1985). The functional significance of this difference is presently unclear. Retrogradely-labeled cells were consistently observed within the ventral tenia tecta suggesting that this area may also project to the SON. .0 ' ' Hi 000::12"- 'll‘ or 9.1 . Introduction The mitral cell, the major output neuron of the main and accessory olfactory bulbs, provides excitatory input to the pyramidal cells of the piriform cortex. Extracellular analysis of diencephalic neurons also reveals a predominantly excitatory response to olfactory stimuli (Kogure and Onoda,l983; Komisaruk and Beyer,l972; Scott and Leonard,197l; Scott and Pfaffmann,1967). Indeed, a recent anatomical study employing intracellular injection of HRP demonstrates that mitral cells send collateral projections to divergent areas within the piriform cortex (Ojirna et al.,1984). It is not unlikely that these diencephalic connections are collaterals of the same mitral cells rather than a special subset of pyramidal neurons. The previous anatomical experiments (section 4-7) suggest that both the main and accessory bulbs project to the SON monosynaptically, and possibly polysynaptically. Intracellular analysis of this newly-discovered connection in the in vitro brain slice demonstrated a predominantly short- latency excitatory response to stimulation of the lateral olfactory tract (Hatton and Yang,1989). This finding supports the hypothesis that the main and accessory bulb are monosynaptically connected to the SON. This study also offers further evidence that mitral cells provide direct excitatory input to diencephalic neurons. The identity of the chemical(s) which mediate mitral cell neurotransmission is controversial. Several lines of evidence suggest that the transmitter may be glutamate (Bradford and Richards,l976; Yamamoto and Matsui,l976), aspartate (Collins,1979; Collins et al.,1981; Collins and Probett,l981), or the related dipeptide N-acetyl-L-aspartyl-L-glutamate (N AAG; Anderson et al.,1986; Blakely et al.,1987; ffrench-Mullen et 92 93 al.,1985; Zollinger et al.,1988). Regardless of this disagreement, it is clear that the excitatory postsynaptic effects observed are mediated through excitatory amino acid (EAA) receptors systems (see Mayer and Westbrook,l987 for a review of these receptor systems) because the excitatory responses are blocked by known antagonists of these receptors (Collins,1982; Collins and Howlett,l988; ffrench-Mullen et al.,1985; Hori et al.,1982). Recently, Gribkoff and Dudek (1988; 1990) have demonstrated electrically-evoked excitatory postsynaptic potentials (epsp) in SON neurons which were reversibly blocked by the EAA antagonist, kynurenic acid (Cotrnan et al.,1986; Mayer and Westbrook,l987; Perkins and Stone,1982; Watkins and Evans,l981). This demonstration of an EAA input to the SON provides the first evidence that these compounds may mediate fast excitatory responses in this system. Unfortunately, the afferent(s) mediating these SON responses are unknown. Two likely candidates are the main and accessory bulb efferents to the SON (Smithson et al.,1989b; Smithson et al.,1988) which, when electrically stimulated, elicit a fast, short-latency excitatory response (Hatton and Yang,1989). 9.2. Experimental Question Here the possibility that these short-latency responses were mediated through an EAA receptor was investigated in an in vino-incubated explant which contain virtually the complete olfactory projection from the rostral end of the lateral olfactory tract to the SON. 9.3. Methods Exp. 5: Methods for Electrophysiology experiments Male and female Sprague-Dawley rats 36-58 days old were used. All animals were maintained in a 12:12 h light-dark cycle and given food and water ad libitum. Artificial cerebral spinal fluid used to prepare and 94 maintain the explant contained the following ingredients ( in mM) NaCl (126), NaH2PO4 (1.3), NaHCO3 (26), KC1(5), CaC12 (2.4), MgSO4 (1.3), glucose (10) and MOPS (3-[N-Morpholino]pr0panesulfonic acid) (5), pH 7.4. 9.3.1. Explant Preparation Animals, while freely exploring a guillotine, were quickly decapitated. The parietal and frontal bones were removed thus exposing the cortex and the olfactory bulbs. The lateral olfactory tracts were carefully transected immediately caudal to the olfactory bulbs. The brain was then gently coaxed from the skull while cutting the neurohypophysial stalk and the optic tracts, and then immediately placed into medium (5 °C) which had been gassed with 95% 02, 5% C02. Explants were hand-cut with a razor blade by first blocking the tissue behind the infundibulum, then by cutting the tissue 2 mm dorsal to an imaginary line between the SON and the most rostral portion of the lateral olfactory tract. In older animals with larger brains, the piriform cortex lateral to the tract was also removed. The SON-containing tissue block was then transferred to a ramp-type recording chamber (Haas et al.,1979) and laid ventral-side up on a bed of filter paper affixed to the ramp floor with bone wax (Ethicon). The chamber was maintained at 32-35°C by a heated water-jacket. Strands of surgical gauze were carefully draped over the tissue to direct the flow of medium as it was superfused over the explant. The gassed medium was delivered to the chamber by a 4-channel peristaltic pump (Gilson) at 1.0 - 1.5 ml/ min, and warmed to chamber temperature as it passed through tubing within the water jacket. The dura and blood vessels overlying the SON were carefully dissected away with Dumont #5 forceps and iris scissors (Robaz), under microscopic observation with direct 95 illumination. The explant was allowed to incubate for two or more hours prior to intracellular recording. 9.3.2. Electrical Stimulation A stimulating electrode was placed in the most rostral portion of the lateral olfactory tract, approximately 6 mm anterior to the SON (Fig. 20). In some cases, a stimulating electrode was also placed in the cut end of the pituitary stalk to antidromically activate SON neurons. In a few cases, a third electrode was also placed in the olfactory tubercle about midway between the SON and the stimulating electrode in the lateral olfactory tract. Either concentric or twisted bipolar electrodes were used. In all cases when multiple electrodes were employed, they were of the same design. Constant current stimulus pulses, 0.10 -0.15 ms duration, at 2-4 Hz with currents between 2.0 -100 uA were typically employed to evoke responses. 9.3.3. Electrophysiology Glass microelectrodes, manufactured on a Brown and Flaming-type puller (Sutter Instruments) and filled with 3 M K+ acetate (80-150 m9), were used for these recordings. Electrodes, visually guided under microscopic observation and direct illumination, were positioned in the SON which was easily located by its position in relation to the optic tract (Fig. 20 B). Irnpalements were obtained by advancing the electrode in 4-8 pm increments using a microdrive (Burleigh Instruments) while concomitantly ejecting brief pulses of positive or altemating current. Signals were amplified with a conventional intracellular preamplifier (Neurodata). After further amplification with an oscilloscope, signals were recorded onto magnetic tape (Hewlett Packard) for later analysis. 96 Fig. 20. Photomicrographs of the explant preparation. A. Low power photomicro graph of the explant illustrating the uncut ventral surface of the brain. Two stimulating electrode (S) as well as an intracellular recording electrode (R) have been placed in the tissue. B. At higher magnification the position of the stimulating electrodes in the rostral lateral olfactory tract (LOT), and the pituitary stalk (PS) is observed. The intracellular recording electrode is positioned in the SON. 97 l roar-rd. ’ en ‘- ; . . v 98 9.3.4. Application of EAA antagonist kynurenic acid The focus of the present study was to determine if the short-latency excitatory responses from lateral olfactory stimulation, previously reported (Hatton and Yang,1989). are mediated through an EAA receptor. This was accomplished, after obtaining a stable irnpalement in a SON neuron, by first determining the nature (i.e. whether excitatory or inhibitory) and threshold of the lateral olfactory tract evoked response, then exchanging the superfusion medium with medium containing 1 mM kynurenic acid (Sigma) while monitoring the effects of this EAA antagonist on the evoked responses. Finally, irnpalement permitting, the explant was retumed to control medium. 9.4. Results Recordings were obtained from a total of 66 neurons from 22 animals. These neurons all had overshooting action potentials initially following irnpalement, with a mean transmembrane potential of -57 mV 2 1 mV (mean :t standard error) and input resistances which ranged from 70-217 MO (108 i 5 MO). 9.4.1. Synaptic Responses In fifty of the recorded neurons, stimulation of the lateral olfactory tract resulted in only excitatory responses. No inhibitory responses were observed. The remaining neurons were unresponsive to lateral olfactory tract stimulation. These responses displayed variable latencies (Fig. 21) and, at threshold, often revealed small depolarizing postsynaptic potentials (psp; see Fig. 23 A), or action potentials preceded by a small epsps . In contrast, antidromic activation via stalk stimulation, when successful, produced a short latency response with a virtually constant latency (Fig. 21 C). Stimulation of the olfactory tubercle never resulted in an evoked 99 Fig. 21. Oscilloscope traces of evoked responses in SON neurons to electrical stimulation of the lateral olfactory tract (A & B) and neurohypophysial stalk (C) In all cases arrows denote onset of stimulation. A. Four consecutive traces illustrating a variable latency response to 3 uA of + polarity current at 2 Hz. The shortest latency response observed is spontaneous activity of the cell. B. Variable latency responses of the same cell as in A to 6 11A of - polarity current; five consecutive traces are shown. C. Seven consecutive traces illustrating a constant latency response of another cell to antidromic activation following stalk stimulation. 100 101 response.The current required to elicit a response varied with the stimulus polarity employed, as well as between preparations. That is, response thresholds varied less within a single explant than between preparations. The median threshold current for positive polarity stimulation was 9.5 uA and, when employed, negative polarity stimulation required approximately twice as much current to evoke a synaptic response. Synaptic latencies also varied with stimulus polarity. The mean synaptic latency was 3 ms and 10 ms for positive and negative polarity stimulation, respectively (Fig. 22). In either case, synaptic latencies decreased as the stimulation cunent was increased above that which produced a threshold response. 9.4.2. Kynurenate Blocking Out of the 50 cells; demonstrating synaptic responses from electrical stimulation of the lateral olfactory tract, long-tenn stable irnpalements were maintained during the medium exchange in 15 neurons. In each case, the evoked response at threshold currents was blocked in kynurenate-containing (1 mM kynurenate) medium (Figs. 22-24). This was observed approximately 3-4 min into the exchange. Higher currents, however, often produced an evoked response (Fig. 23 D). Further superfusion of the explant with kynurenate-containing medium resulted in blocking the evoked response at increasingly higher currents. After approximately 7 min in kynurenate-containing medium, currents 50% larger than threshold cunents failed to evoke a response. Indeed in most cases, currents several times threshold also failed to evoke a response. Throughout the superfusion in kynurenate-containing medium, action potentials could be evoked by current injection through the intracellular amplifier or, when previously successful, through antidromic activation via the pituitary stalk (Fig. 22). The kynurenate-containing medium had no apparent effect on either the spike 102 Fig. 22. Effects of bath application of 1 mM kynurenic acid on responses evoked in SON neurons by electrical stimulation of the lateral olfactory tract. A. Variable latency responses evoked by electrical stimulation of the lateral olfactory tract. B. This evoked response is no longer observed after approximately 3 min in kynurenate-containing medium. C. Action potentials, however, may be elicited with current injection (0.1 nA . ' D. Action potential may also be produced by antidromic activation via stalk stimulation. Arrows indicate onset of stimulus, or visible portion of the stimulus artifact in all panels. 104 Fig. 23. Effects of kynurenic acid on the threshold of the evoked responses. A & B. Oscilloscope traces of multiple consecutive records of synaptic responses ill SON neurons to stimulation of the lateral olfactory tract at threshold cunents, 10 MA positive polarity and 20 uA negative polarity, A & B respectively. Note the small depolarizing event in A (arrowhead) and the variable latencies of the responses in both records. C. After 3 min in kynurenate-containing medium electrically evoked responses are blocked at 30 uA (polarity), but as before may be elicited by current injection through the intracellular amplifier. D. However, immediately following trace in C responses from lateral olfactory stimulation may be evoked with 60 uA of current. This response failed altogether with longer treatment in this medium. Arrows as in figures 1 & 2. 106 Fig. 24. Reversiblity of kynurenate blockade of the excitatory responses in a SON neuron to lateral olfactory stimulation. A. SON action potential evoked by intracellular current injection. B. Responses to lateral olfactory stimulation with 50 11A of positive polarity current. C. After 3 minutes in kynurenate-containing medium stimulation of the lateral olfactory tract with up to 150 uA no longer produced an effect. D. After a 4 min wash-out in control medium a lateral olfactory response is once again observed with 50 uA of current. In B-D 3 consecutive responses are illustrated, here the stimulus artifact is indicated by an arrow in only the first trace. 107 10 mVn 8 II 108 Fig. 25. Long depolarizations in response to lateral olfactory stimulation. A. Shown at increased amplification to that in B. action potentials in A are truncated. Stimulation of the lateral olfactory tract in this SON neuron appeared to result in a prolonged depolarization (onset of depolarization marked with an asterisk), which upon cessation of the stimulation (double- headed arrow) resulted in repolarization of the cell's membrane. Arrows with bar denote the resultant hyperpolarization. A few calibration pulses (10 mV, 5 ms) are labeled with arrowheads at the beginning of each trace. 109 110 threshold or transmembrane potential of the cell. Inconsistently, both increases and decreases in input resistance were observed in the presence of kynurenic acid. Also observed in several cases were bursts of action potentials apparently triggered by stimulation of the lateral olfactory tract (Fig. 25). In five cells, the explant was retumed to control medium in an attempt to restore the blocked response. In two cases, the explant was retumed to control medium after the synaptic response was blocked at threshold currents, approximately 4 min into the exchange (Fig. 24 C). Following a 4 min wash in control medium, the response was once again observed at threshold currents (Fig. 24 D). In another case the blockage was partially reversed. That is, evoked response could be elicited at currents below that which did not previously evoke a response. However, these currents were higher than the original threshold current. In two other cases, the irnpalements were lost before the response returned. In these last three cases the initial blockades were virtually complete with cunents 4-10 X above the threshold stimulus required to evoke a response. 9.5. Discussion A short latency excitatory response in SON neurons to lateral olfactory tract stimulation has been demonstrated which was reversibly blocked by the excitatory amino acid (BAA) antagonist kynurenic acid. The short latency excitatory responses observed here in the explant are similar to those previously reported in the brain slice (Hatton and Yang,1989; Hatton and Yang,1990). The range of stimulus currents required to elicit threshold responses was also similar. Synaptic latencies, while marginally longer here, probably reflect the differences in placement of stimulating electrodes. In the explant, the lateral olfactory tract is intact rostrally to the anterior 111 olfactory nucleus, i.e. approximately 4-6 mm anterior to the SON. In the slice this tract is intact for approximately 2-3 mm. In this regard, the explant has an advantage over the slice in that it is easier to resolve these fast events. The similarities observed between the two, different, preparations further strengthen the conclusion that the SON receives an excitatory input from the olfactory bulbs. These fmdings are also consistent with previous anatomical data (presented in this thesis) which suggest that the SON receives monosynaptic efferents for both the main (Smithson et al.,1989b) and accessory (Smithson et al.,1988) bulbs. An additional finding in the present electrophysiological studies is that these excitatory responses are blocked by kynurenic acid, a result in agreement with previous anatomical (Anderson et al.,1986; Blakely et al.,1987), biochemical (Collins,1984) and electrophysiological (Collins,1982; Collins and Howlett,l988; ffrench- Mullen et al.,1985; Hori et al.,1982)studies, suggesting that mitral cell neurotransmission is mediated, at least in part, via EAAs. Kynurenate blocked the evoked responses without noticeably altering either the cells' input resistance, spike threshold, or membrane potential; features of kynurenate pharmacology which have been previously reported for SON neurons (Gribkoff and Dudek,1990). Taken together, this evidence suggests that the kynurenate blockage of these evoked responses was not due to the indiscriminate alterations of the cells' membrane properties which might have rendered it unresponsive. Recently, Gribkoff and Dudek (1990), employing a SON-containing hypothalamic brain slice demonstrated spontaneous and electrically-evoked epsps which were kynurenate sensitive. A plausible interpretation of this experiment is that kynurenic acid is acting postsynaptically on SON EAA receptor similar to that observed in other systems (Cotrnan et al.,1986). 112 Using the same concentration of kynurenic acid, we have confumed and extended these findings: one source of the EAA input is from the olfactory bulbs. Interestingly, our time-course of antagonist blockade was very similar to that reported by Gribkoff and Dudek (1990), with complete inhibition of the evoked events occurring around 7 min. Infrequently, a stimulus dependent slow depolarization, which resulted in bursts of action potentials was observed. The prominent feature of this afferent under our experimental conditions were fast epsps which often resulted in action potentials. This suggests that this input was activating either a quisqualate or kainate receptor (for reviews on these different receptors see Colman and Iversen,l987; Johnson and Koemer,l988; Mayer and Westbrook,l987). Indeed, the fast epsps observed by Gribkoff and Dudek (1990) were not diminished by DL-2-amino-5-phosphonopentanoic acid, an antagonist of the N-methyl-D-aspartate (NMDA) EAA receptor, suggesting these events were not mediated through this receptor. SON neurons, however, are likely to have NMDA receptors since application of NMDA does produce effects which are reversible and blocked by appropriate antagonists (Gribkoff and Dudek,1990). Furthermore, since Mg2+, a known (non-competitive) antagonist of the NMDA receptor was included in the medium these experimental conditions probably artificially masked the interaction of this receptor with incoming olfactory information. In fact, it is quite plausible that during conditions in which hormone release is enhanced (e.g. during dehydration, parturition, or lactation) this receptor becomes more efficacious. In these activated states, the SON dendritic zone undergoes a dramatic reorganization of neuronal-glial relationships (see Hatton (1988a; 1990) for a review) resulting in increased dendritic bundling; that is increased direct apposition of dendritic 113 membranes with each other. This could lead to decreased spatial buffering of [Kt]0 by glia in the area of increased apposition resulting in activity dependent increase in [K+]o, subsequent depolarization of the membrane, and release of the Mg2+ block of the NMDA channel. In conclusion, electrical stimulation of the lateral olfactory tract produced a short, variable latency excitatory response in the SON. On the basis of previous anatomical investigations ill this thesis, this represents the electrophysiological properties of a monosynaptic projection from the main and accessory bulbs to SON dendrites. These excitatory responses are mediated through an EAA receptor which is blocked by kynurenic acid. Further investigations with more specific antagonists should reveal which receptor sub-type is involved in these responses. SIEMENS 10.1 Functional Significance The functional significance of a connection from the main bulb or accessory bulb to the SON is presently unclear. Unfortunately, there are few well-designed behavior studies which offer any insight into the function of this connection. The few studies that have been published suggest that this connection may participate in fluid homeostasis. Studies in the rat (Novakova and Dlouha,l960) and in the sheep (Bell et al.,1979) demonstrate changes in fluid balance resulting in an increase ill urine volume after bilateral bulb ablation. These changes could reasonably be caused by a reduction in circulating vasopressin. That such is the case, is supported by observations in the rat, that bulb ablation decreases circulating vasopressin (as judged by a plasma bioassay; Novakova and Dlouha,1960) Together these observations suggest that the olfactory bulbs play an excitatory role in vasopressin secretion. This view is consistent with the previous report that this connection is excitatory to vasopressin neurons (Hatton and Yang,1989). Additionally, a recent report has demonstrated that olfactory bulbs have receptors for atrial natriuretic peptide (Gibson et al.,1988), further implicating a role in fluid homeostasis for this connection. These olfactory efferents also provides an excitatory input to oxytocin cells (Hatton and Yang,1989) suggesting a function other than fluid homeostasis for this pathway. Indeed, in two recent reports it was found that electrical stimulation of the lateral olfactory tract in brain slices from either lactating, or matemally-behaving, animals dramatically increased the amount of dye-coupling that was observed (Hatton and Yang,1990; Modney et al.,1990). This observation suggests that in animals which presumably have increased dendritic bundling, that is dendritic membranes directly 114 115 apposed with one another without an intervening glial process, (Perlrnutter et al.,1984; Salrn et al.,1988; Taubitz et al.,1987) this input can selectively increases cell-cell communication, presumably to promote release of oxytocin. It is likely that increases in dye-coupling will also occur in other “activated” states (i.e. dehydration). That activation of a connection can specifically alter junctional conductances between cells is an important observation that deserves further experimentation. 10.2 Summary In summary results from anterograde tracing studies with PHA-L and WGA-HRP as well as retrograde tracing experiments, provide strong evidence that the main and accessory bulbs are connected to the SON in a monosynaptic manner. The anatomical data also suggests that this connection may be polysynaptic as well. Additionally, the use of an in vitro explant preparation, has confirmed one finding in earlier reports (Hatton and Yang,1989; Hatton and Yang,1990; Modney et al.,1990); that this connection is excitatory. Furthermore, results from these electrophysiological studies suggest that this excitatory response is mediated through (an) excitatory amino acid receptor on SON neurons (Fig. 26). 116 Fig. 26 Schematic of olfactory connections with the SON. Diagram on left illustrates full course of olfactory projection from mitral cells in the MOB and AOB through the LOT. Inset is drawn at higher magnification on the right . Inset: MOB and AOB efferents project to dendrites of SON neurons which lie outside the nucleus proper. Electrophysiological evidence is consistent with and BAA as a transmitter (e.g. aspartate, glutamate, or NAAG), and a non-NMDA EAA receptor on these SON dendrites. Abbreviations Iln optic nerve. 3V third cerebml ventricle; AOB accessory olfactory bulb; BAOT bed nucleus of the accessory olfactory tract; EAA(s) excitatory amino acid(s); LOT lateral olfactory tract; MOB main olfactory bulb NAAG N-acetyl-L-aspartylglutamate NLOT nucleus of the lateral olfactory tract; SON supraoptic nucleus-main (or anterior) portion. 117 00:6:8 20m :0 029.0003. <