y . 9": LC... :3» gig. fin: C. 'l’kn"" I I. It!!! ~r Iv'llt roofi- vrrzi.i. z... it!!! ’glr’4’t vl‘ ’4‘. vtvovflrfihukw cl: I!» 1.. 2 l. . . u i!!!- vEFfilg-‘OBI‘LHCEA 3 in... . 1:33;!!! II?» ‘1‘.. i I flrlfaiinK 2 Din-11"; "l V ' :J‘L “q.” n "i .i! :v .. ‘ , , . A. . , «352$ 333$. 33% . .. ‘ ; . ‘ I .. ...s~ . .....+¢..Ia..v4..f .1! .ll‘l-d' 9" I!!! .ll lbxvlor". III 1 |||||‘I.ll'lll|'¢| NIVERSITY LIBRARIES Illllllllllllllu lll l ll l l 3 1293 00914 2757 This is to certify that the dissertation entitled STUDYING THE PLASTICITY OF THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM IN DEHYDRATED RATS USING POSTEMBEDDING IMMUNOLOGY CYTOCHEMISTRY AT THE ELECTRON MICROSCOPIC LEVEL presented by Farshid Marzban has been accepted towards fulfillment of the requirements for Ph . D . degree in Anatomy / ka 1 -x 2V 0 (\/’ my, V Major professor Date i/ZIB / Cl? MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771 T LIBRARY Michigan State University L A PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE I fir“ “=7 +=_T_| MSU Is An Affirmative Action/Equal Opportunity Inaitution cWeInS-m STUDYING THE PLASTICITY OF THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM IN DEHYDRATED RATS USING POSTEMBEDDING IMMUNOLOGY CYTOCHEMISTRY AT THE ELECTRON MICROSCOPIC LEVEL. By Farshid Marzban A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy and Neuroscience Program 1992 ABSTRACT STUDYING THE PLASTICITY OF THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM IN DEHYDRATED RATS USING POSTEMBEDDING IMMUNOGOLD CYTOCHEMISTRY AT THE EM LEVEL. BY Farshid Marzban Oxytocin (OT), involved in uterine contraction, milk ejection, and vasopressin (VP), an antidiuretic hormone, are the main neurohormones of the magnocellular neuroendocrine cells (MNC) of the paraventricular (PVN) and supraoptic (SON) nuclei. The OT and VP with their respective neurophysins (I and II) are synthesized in separate populations of neurons (De Mey et al., 74; van Leeuwen & Swaab, 77; Vandesande & Dierickx, 79) and conveyed intraaxonally to the posterior lobe of the pituitary (PP) gland to be stored in the Herring bodies or released from the terminals. The hypothalamo- neurohypophysial system (HNS) undergoes dramatic morphological changes during various physiological states. However, little is known about how much each type of cell, OT or VP, contributes to this plasticity during dehydration. To address these questions post-embedding immunogold cytochemistry for both OT and VP hormones was used at the electron microscopic level. In the first experiment VP and OT neurons in SON were studied in rats dehydrated for 10 days. The results were compared to control animals which had free access to water. Both VP and OT somata showed an enlargement in size in dehydrated animals. The percentage of soma-somatic/dendritic membrane contacts increased significantly in both VP and OT neurons, whereas percentage of coverage of the cells by astrocytic membrane reduced. Both the VP and OT cells had a lesser amount of axo- somatic membrane contact while the number axons contacting 100nm of OT or VP cell membrane did not change after dehydration. This may suggest that during dehydration, smaller size terminals may form new axo-capillary apposition. Multiple synapses (MSs, i.e. single axonal terminals which form more than one synapse with adjacent somata and/or dendrites) occurred only between OT neurons or VP neurons, but not between adjacent OT and VP cells. The number of M85 per 100 pm of OT somatic membrane or per 100 OT cells was significantly higher in dehydrated rats but was unchanged with regards to VP neurons in dehydrated vs. control rats. In the second experiment axonal and terminal VP and OT neurons in dehydrated rats were studied. VP and OT axons engulfed by pituicytes (i.e. completely surrounded by pituicyte cytoplasm) decreased in number. Both VP and OT axe-capillary apposition per unit length of basal lamina increased, whereas pituicyte-capillary apposition per unit of basal lamina length decreased. These results indicate that both VP and OT neurons undergo morphological changes during chronic dehydration. This dissertation is dedicated to my father Dr. Farrokh Marzban whose strength and capacity for love was truly remarkable and to my wife Shabnam and my Mom Ozra for their unlimited unconditional love and understanding. ACKNOWLEDGMENTS l have many people to thank for various things they have done for me since I came to Michigan State. I would like to express my sincere appreciation and thanks to my advisor, Dr. Charles D. Tweedle, who not only provided generous moral support throughout my entire graduate training, but, more importantly, provided a role model for what a neuroscientist and an adviser should be. To Glenn Hatton, who provided generous educational and financial support. To my dissertation committee members - Drs. R. Bowker, P. Cobbett, W. Falls and l. Grotova, Your advice and encouragement were truly appreciated and will never be forgotten. To B. Modney, T. Nunez, K. Smithson, M. Weiss - Who have been great supporters and friends in my entire Ph. D. training. To the many able technicians who have run the lab and have helped me enormously: L. Koran, P. Rusch & I. Smithson. The following people have been helpful in many ways: G. Beagley, L. Bergland, A, Elliot, J. Harper, J. Smith, C. Sisk, B. Smisch & Q. Yang. Last but not least, To Farzad, Farshad, Farshad, Golchehreh, Ferydoun, Farhad, Golii and Shahroukh Marzban, Javiid, Nader, Parviz, Debbie & Mark -Thanks for constant support. TABLE OF CONTENTS PAGE ListofFiguresandTables......................Vll| Abbreviation............................Xl Introduction............................1 General Methods and Statementotproblems . . . . . . . . . . . . . . .21 Experiment l-RE-EVALUATION OF PLASTICITY IN THE RAT SUPRAOPTIC NUCLEUS AFTER CHRONIC DEHYDRATION USING IMMUNOGOLD FOR OXYTOCIN AND VASOPRESSIN AT THE ULTRASTRUCTURAL LEVEL. lntroduction..........................25 MaterialandMethods......................26 Results............................28 Discussion..........................29 Figures............................33 EXPERIMENT ll-MORPHOLOGICAL STUDY OF POSTERIOR PITUITARY IN CHRONICALLY DEHYDRATED RATS USING AN IMMUNOGOLD CYTOCHEMICAL LABEL FOR VASOPRESSIN. Introduction..........................54 MaterialandMethods......................55 Results . Technical Considerations Discussion Figures . Concluding Remarks List of References vii .56 .57 .59 .64 . 76 .81 LIST OF FIGURES AND TABLES EXPERIMENT I: PAGE Table Figure Figure Figure Figure Figure Figure Figure 1: Effects of chronic dehydration on magnocellular neurons in the rat supraoptic nucleus. 33 Accumulation of gold particles on the granules (arrows) on either side, cis (forming face) and trans (maturing face), of or within Golgi apparatus (*) in a vasopressin immunostained positive cell. 35 VP positive inclusions (arrows) larger than the typical vasopressin granules with an unknown function in a somatic proximal dendrite (D). N=nucleus. 37 Two pairs of adjacent sections where A and C are not immunostained and B and D are processed for immunocytochemistry. Note the presynaptic (filled arrows) and postsynaptic (empty arrows) thickenings and membranes in A and C are not as clearly visible in the B and D. 39 Effects of chronic dehydration on the oxytocin and vasopressin somatic size in the SON. Values shown represent the means iS.E.M.. * p<0.03, ** p<0.01 significantly different from the control. 41 (A) Some-somatic contact (arrowheads) between vasopressin positively labelled cells (arrows) showing a multiple synapse (*) contacting two vasopressin positive somata. (B) A higher magnification of 5A showing a multiple synapse (‘) without clear thickenings. vasopressin labelled granules are shown with arrows. 43 (A) Some-somatic contact (arrowheads) between vasopressin positive and negative somata. Note a vaSOpressin positive dendrite (*) contacting the two cells. Positively and negatively vasopressin labelled granules are shown with small and large arrows respectively. (B) A higher micrograph of 6A (inset) showing positive labelled granules (arrows) on either side (cis and trans) of Golgi apparatus (*). 46 A multiple synapse (*) contacting a vasopressin positive cell and an adjacent undetermined dendrite (D). Positive granules are shown with arrows. 48 viii Figure 8: Shows an oxytocin positively labeled cell (P) making some-somatic and dendro-somatic appositions (arrow heads). Notice the dendrite (d) branching from the P cell apposes with adjacent oxytocin cell and a multiple synapse (*) contacts both of them. 50 Figure 9: Diagrammatic sketch of vasopressin and oxytocin somata (circles) contacting neighboring vasopressin (VP) and oxytocin (OT) magnocellular neuroendocrine cells (semicircles) and afferent single synapsing (open ovals) and multiple synapses (shaded ovals) terminals in the control and chronically dehydrated supraoptic nucleus. VP and OT somatic size and the amount of soma-somatlc/dendritic contact increase in dehydrated animals. The number of axons per unit of somatic area in either OT and VP neurons increases with the same proportion as the cell sizes are enlarged. Only OT neurons contact more multiple synapses, whereas lesser axonal contact apposes both VP and OT somatic membrane in chronically dehydrated animals. Note there are different types of terminal and every terminal may carry different types of neurotransmitter secreted differently under various physiological stimuli. MNC=magnocellular neuroendocrine cells (including both somata and dendrites). ~ 52 EXPERIMENT II: Table 2: Effects of chronic dehydration on the oxytocin and vasopressin terminal and pituicyte apposition in the PP. Values shown represent the means iS.E.M.. 1] p<0.05, * p<0.03, “ p<0.01 significantly different from the control. 62 Figure 10: A. Low power electron micrograph of the neurohypophysis. A pituicyte (Pit) can be seen to be surrounded by many VP-positive (p) and negative (n) neurosecretory processes. Note that around the capillary (Cap), there are VP-positive (arrow heads) and negative (arrows) terminals apposing the perivascular space. Pituicyte-capillary apposition is shown with dashed lines. B. Higher micrograph of 1A (inset) showing the specificity of the labeling. Bar = tum 64 Figure 11: A. Area of the neurohypophysis of a control rat. B. High power electron micrograph of a pituicyte (P) (2A inset), has two VP-positive neurosecretory axons (p) enclosed within its cytoplasm. C. High power electron micrograph of 2A (inset) showing a VP (-) terminal (n) apposing a capillary. Bar = 1pm 67 Figure 12: Micrograph of two terminals one stained positively for OT (P) apposing (arrows) a capillary and one did not stain for CT (n) and also apposing (arrow heads) the capillary. Pituicyte-capillary apposition is shown with dashed line. Bar = 1pm 71 ix Figure 13: Micrographs of 10 days dehydrated animal. A. Stained for VP. Note that the labelled granules are clear in the center. B. Stained for OT. Note that the labelled granules are pale in color. Also note the empty terminals (*) around the capillary (Cap). Bar = 1pm 73 ABBREVIATIONS Agll - Angiotensin II Ab - Antibody ACh - Acetylcholine Ag - Antigen AV3V - Antroventral Third Ventricle B-End - B-endorphin BBB - Blood brain barrier CSF - Cerebral spinal fluid DA - Dopamine DYN - Dynorphin GA - Golgi apparatus GABA - Gamma-aminobutyric acid HNS - Hypothalamo-neurohypophysial system Leu-Enk - Leu-enkephalin Met-Enk - Met-enkephalin MNC - Magnocellular neuroendocrine cells MS - Multiple synapse NG - Neurosecretory granules NT - Neurotransmitter OT - Oxytocin OVLT - Organum vasculum lamina terminalis PP - Posterior pituitary PVN - Paraventricular nucleus SFO - Subfornical organ SON - Supraoptic nucleus xi TBS - Tris buffer solution TX-100 - Triton X-100 VP - Vasopressin xii Introduction A) Mammy. Paraventricular (PVN) and supraoptic (SON) nuclei are parts of the hypothalamo-neurohypophysial system (HNS) of the diencephalon. PVN is located dorso-laterally at either side of the third ventricle and SON is located at the lateral portion of the optic tract. PVN is composed of two subgroups, the magnocellular neurons, located laterally and posteriolaterally, and parvocellular neurons, located medially (Armstrong et al., 80; Van den Pol, 82). Fibers from PVN magnocellular neuroendocrine cells (MNC) run laterally around and through the fornix and then turn ventrally to pass over and join the axons from SON which are directed dorsally and medially to form a tract converging in the zona interna of the median eminence. Sometimes these fibers send off collateral branches to the hypothalamic area (Hatton, 1985; Mason et al., 1984; Sofroniew & Glasmann, 81; Van den Pol, 82) on their way to the internal zone of the median eminence. The pathway then continues through the pituitary stalk to terminate on the perivascular spaces around the fenestrated capillaries in the posterior pituitary (PP). The anatomical aspects of this pathway have been studied using such as different techniques as antidromic recordings (Barker et al, 71; Yamashita et al., 70; Pittman et al., 81; Bourque & Renaud, 84), anterograde labeling (Ju et al., 1986; Poulain & Wakerley, 1982) and retrograde labeling (Onto et al., 78; Sherlock et al., 75; Dyball et al., 88b; Alonso and Assenmacher, 78). The 2 somata of magnocellular vasopressin (VP) and oxytocin (OT) neurons are round to oval and multipolar with a long axis of 20-35um, usually with one axon and 1-3 dendritic processes (Dyball & Kemplay, 82; Morris et al, 87; Sofroniew & Glasmann, 81). The SON dendritic trees extend ventrally into the ventral glial lamina toward the ventral pia matter surface of the brain (Armstrong et al., 82). They are characterized as thick, unbeaded processes with some spines and varicosities (Dyball & Kemplay, 82; Morris et al., 87; LuOul & Fox, 76). On the other hand, PVN magnocellular dendritic processes extend medially and abut the ependyma of the third ventricle although, in the caudal portion of the PVN MNC may also send dendritic branches laterally (Sofroniew & Glasmann, 81; Van den Pol, 82; Armstrong et al., 80). lmmunocytochemically and electrophysiologically, it has been shown that the OT and VP perikarya in the SON and PVN and their fibers in the PP are not distributed randomly (Rhodes et al., 81; van Leeuwen et al, 79; Bourque & Renaud 84). The OT neurons in the SON are located mainly in the rostral and dorsal regions, while the majority of the VPergic somata are in at the caudal and ventral portion of SON (Rhodes et al., 81). In the PVN, OT neurons are primarily found in the posterior and medial part of the nucleus and VP neurons are located preferentially laterally with a rim of OT cells (Rhodes et al., 81). In the PP, the OTergic and VPergic sites are preferentially situated at the peripheral and central part, respectively (van Leeuwen & Swaab, 1977). Beth at the somatic (SON and PVN) and at the terminal (PP) levels the MNC are neighbored with astrocytes and astrocyte-like glial cells (pituicytes), respectively. Astrocytes separate the somata of MNC and decrease the soma-somatic/dendritic apposition in SON and PVN. ln PP, the roles of pituicytes have been hypothesized to morphologically wrap around some of the axons and reduce the axonal contact with the fenestrated capillaries (Tweedle and Hatton, 1980b). 8) WSW Both VP and CT peptides at the terminals originate from larger prohormones, synthesized in the cell bodies of the MNC. VP prohormone (19,000 MW in rat) contains a nonapeptide VP at its N-terminus, a neurophysin II in the middle, and a 39 amino acid glycopeptide at the C-terminus. OT prohormone (15,000 MW in rat) includes a nonapeptide OT at the N-terminus and a neurophysin l at the C-terminus where glycopeptide is absent (Gainer 83). The post-translation events occur in different organelles in the MNC (Gainer et al., 77, 83; Brownstein & at al. 80; Liston et al., 84; Hook et al. 90). Signal peptide cleavage, disulfide bond formation and the first stages of glycosylation take place in association with the rough endoplasmic reticulum. After the prohormones are packaged and transported to the Golgi apparatus (GA), where the glycosylation is completed, they are loaded into the neurosecretory vesicles (or granules) and freed in the cytoplasmic space. Enzymatic cleavages and ' amidation of the prohormones occur while the prohormones are within the granules and transported to the terminals. Anderson and Keiding (1988) reported three different sizes of granules in the SON and PP. Based on the results, they speculated that OT and VP are stored in large and medium size granules, respectively, and small granules may contain some other substances. These hormones were hypothesized to be independently released from the terminal during different physiological states (Kasting, 88). Therefore, release of the hormones at the terminals would depend upon the frequency and the pattern of electrical activity invading PP terminals (Gainer et al., 1986; Nordman & Stuenkel, 1986), their external and/or internal concentration of Ca++ (Sladek & Joynt, 1978), sodium, chloride, magnesium and potassium (Dayanithi & Nordmann, 1989; Bony et al., 1987; Cazalis et al., 1987a,b). These ions might, thus, play a major role in action potential conduction along the axons and vesicle fusion with the terminal membrane. C) .-..IZAT.L.FN '0 'AL A Ti L A In. ‘IF’RAA'I'_LI_::O,-L."I:L QELIXQBAIIQN; Some neurotransmitters, either co-localized within OT or VP fibers or separately stored in different terminals, are found in the PP. These NTs may modulate hormonal release and possibly would cause morphological changes at the terminal in the PP. Dynorphin (DYN), an inhibitory neurotransmitter to OT and VP, is co-released with VP (Bondy et al., 89; Gayman & Martin, 87; Martin et al., 81, 83). Cholecystokinin and corticotropin releasing hormone are co-released with OT in the PP and the intermediate lobe, respectively. Both of these NTs stimulate PP secretion of OT and VP (Bondy et al., 1989; Miaskiewicz et al. 1989). It is speculated that corticotropin releasing hormone stimulates OT release via the release of melanocyte stimulating hormone in the intermediate lobe, since there are no corticotropin releasing hormone receptors in the PP. Corticotropin releasing hormone has no effect on CT release in the PP free of the intermediate lobe. A high number of corticotropin releasing hormone receptors are present in the intermediate lobe and melanocyte stimulating hormone increases OT release in the PP (Bondy et al. 89). Galanin, which is found in the SON, PVN and the PP, coexists with VP (Skofitsch et al. 1989; Arai et al. 1990) and, during osmotic stimuli and in Brattleboro rats (which have a lack of VP synthesis), galanin synthesis in the SON and galanin secretion in the PP increase (Rokaeus et al. 88, Skafitisch 89; Wilkin et al. 89; Meister et al. 90). In addition, Leu-enkephalin (Leu- Enk), a VP inhibitor (Iversen et al., 80; Lightman et al., 82; Summy-Long et al. 84, Zhao et al., 88), and peptide histidine-isoleucine co-localized with VP (Meister et al., 1990) and Met-enkephalin (Met-Enk) co-exist with OT. Calcitonin, Iuteinizing hormone releasing hormone, vasoactive intestinal polypeptide, somatostatin, gamma- aminobutyric acid (GABA), dopamine (DA) and acetylcholine have also been reported to be present in the PP. Meister et. al. (90) reevaluated the coexistence of these NTs 5 with OT and VP in the SON and PVN and reported that the level of these chemical messengers in the MNC varies under various physiological conditions. Beside in the SON and PVN, opioid peptides are found in the neurons of the arcuate nucleus, the preoptic area and the brain stem, which all send efferent fibers to the MNC and the neurointermediate lobe (Finely et al., 81; Khachaturin et al., 85; Millan et al., 83; Weber et al., 82). Generally, there are 3 types of opiate receptors in the central nervous system (CNS): mu (u), delta (3) and kappa (k) and activation of each one of the receptors may cause a postsynaptic inhibition. Activation of the k receptors causes a reduction in the voltage-dependent Ca++ conductance to block the secretion whereas activation of the u- or 3- receptors causes an increase in the K+ conductance to shorten the postsynaptic action potential duration (North, 86; Bicknell, 88). Met-enkephalin (Met-ENK), Leu-enkephalin (Leu-ENK) and B-endorphin (Ii-End) are the main endogenous ligands for the u- and a- binding sites. Lightman and Young (87) reported a progressive increase in OT, VP, DYN mRNAs in the MNC of the SON and PVN in 2% NaCl treated rats. Ten day lactating female rats showed a very marked increase in OT mRNA with a small increase in VP and DYN mRNAs in the nuclei (Lightman 8 Young, 87). DYN is the most k-selective endogenous opioid peptide (Chavkin et al., 82; Corbett et al., 82; Gerstberger & Barden, 86) and heavily concentrated in the PP (Khachaturin et al., 85, Lightman et al., 83). Kappa receptors are found both on the neurosecretosomes, of which a majority are on the OT endings (Falke & Martin, 85,88b, Herkenham et al., 86) and the pituicytes in the PP with no or a fewer number of u and 3 receptors (Bicknell et al., 89; Bunn et al., 85; Falke & Martin, 85; Lightman et al., 83; Pesce et al., 87). Opioid peptides can affect MNC hormone secretion both directly by acting on MNC cell bodies or the terminals and indirectly by acting on astrocytes. The hypothalamus contains all three types of opioid receptors (Wakerley et al., 83; Mansour et al., 86). At the SON, lnenaga et al. (90) reported that k receptor activation 6 inhibits both the OT and VP neurons, whereas activation of a and u receptors inhibits primarily OT neurons. Also intracerebroventricular administration of opioids inhibits both OT and VP release (van den Heijning et al., 90; Summy-Long et al., 81 ). In vitro, at the PP level, opioids inhibit OT secretion but the VP secretion is not affected by opioid administratiOn (van de Heijning et al., 90; Falke, 1988a). These peptides suppress OT secretion at the neural lobe during NaCl treatment, electrical stimulation or suckling (Bicknell & Leng, 82p; Bicknell et al., 83,85; Bondy et al., 88 Carter et al., 87, Clarke et al., 79; Clarke & Patrick, 83; Shibuki et al., 88; Summy-Long et al., 84; Wammack & Racke, 88; Zhao et al., 88). However, VP secretion was not affected during electrical stimulation, lactation, hemorrhage or dehydration by naloxone (an opiate antagonist) (Bicknell & Leng, 82; Clarke & Patrick, 83; Summy-Long et al., 84). Based on these findings, one could speculate that during dehydration, when both OT and VP cells are stimulated, more DYN is secreted at the PP (Racke et al.,1986). In order to conserve the stored CT in the PP, DYN binds to the k-receptors on the OT terminals and reduces OT secretion (North, 86; Gross et al., 90). Brady et al. (88) suggested that k-receptor down-regulation occurs as a result of the elevation of opiate release (Brady & Herkenham, 87; Brady et al., 88) and this may reduce the inhibitory effects of DYN on the VP terminals. Therefore more VP would be secreted. On the other hand, during lactation, when OT MNC are activated, some Met-ENK is co- released with CT in the PP. Since Met-Enk has low affinity binding capacity to k receptors, and there are not as many ll and 3 receptors in the PP, this neuromodulator can impose only small inhibitory effect on both VP and CT secretion. In addition, during lactation, VP secretion is not elevated as much to need opioid peptides to be around to inhibit its release. B-End has been found in high concentrations in the intermediate lobe (Pesce et al., 87; Rossier et al., 80) and there is a possibility that there is enough diffusion of substances from the intermediate lobe to the PP to expect that B-End can modulate the 7 HNS at the PP such as by elevating VP release (Weitzman et al., 77). The arcuate nucleus is also rich in enkephalin and, with its direct projection to PVN, may modulate the MNC activities at the hypothalamic level. Pituicytes (the specialized astrocytes of the PP) have receptors for opiate peptides, receive axonal apposition stained for Enk (van Leeuwen, 82; van Leeuwen et al., 83) and may express proenkephalin mRNA (Schafer et al., 90). Stiene-Martin and Hauser (90) noted an inhibition of DNA synthesis in cultured astrocytes by Enk. As of now, the role of ENK on pituicytes is not clear, but pituicytes may mediate the inhibition of VP and CT release from PP by opiates. D) YTE F I Astrocytes are star-shaped specialized cells with a large and usually round nucleus and some cytoplasmic matrix. Astrocytes and pituicytes cover most of the lining of intersomatic spaces in SON and PVN and the perivascular spaces in PP respectively. Pituicytes may be capable of blocking the neurohormones from reaching the periphery via reducing direct membrane contact of the terminals with perivascular spaces. Under physiological changes (for instance during high hormonal demand during dehydration and/or lactation) it has been hypothesized that pituicytes retract and permit either more terminals and/or a bigger axonal area to make contact with the perivascular space (Tweedle, 83,87; Tweedle & Hatton, 1987). Beside acting as a insulator wrapping around somata and their branches and terminals, astrocytes may also act as modulators. Astrocytes are capable of doing many different functions which may change the neighboring cellular activities. Astrocytes may alter the extracellular ion concentration by uptaking K+ ions as well as externalizing Ca“ ions (Bambaure et al., 1984; Wuttke, 90). Recently, Canady et al. (1990) reported that high [K+]° facilitates glial morphological plasticity in culture. Therefore, when the HNS is stimulated, neural excitation elevates [K+]° which, in turn, facilitates astrocytic morphological changes. This results in retraction of glial processes from the 8 interneuronal space and leads to elevation of cell-cell apposition. In return, the astrocytic K+ uptake mechanism accelerates to buffer extracellular K+ to nermal levels. Astrocytes also respond to NTs. Ahmad et al. (90) reported that glutamate stimulates cultured spinal cord astrocytes to release Ca++ from internal stores. Pituicytes stain for GABA and are capable of taking up GABA from the extracellular fluid (Didier-Bazes et al., 89). This mechanism might serve to eliminate GABA inhibitory effects on neighboring nerve processes. On the other hand, if locally, pituicytes are able to release GABA into the interstitial spaces, this secretion may inhibit axonal activity. E). “CT FF_ 0N ,_ 3. '.P Y A Y ll' OT and VP hormones are synthesized and released at different rates in response to various stimuli (Cunningham et al., 1991 ). This may be due to the fact that the neurons and their respective terminals are probably controlled by different mechanisms based on individual physiological conditions. 12mm The HNS can sense plasma osmotic and blood pressure changes via different channels. The system by itself may act as an osmoreceptor. First of all, the MNC terminals in the PP may sense osmolality levels through direct contact with the perivascular space surrounding the fenestrated capillaries and these capillaries are, thus, able to exchange substances freely either way. Secondly, since the MNC dendritic processes contact the vascular and/or the ependymal cells covering the internal surface of the third ventricle and ventral surface of brain, the neurons may monitor the CSF content as well as plasma constituent at the hypothalamic level. Leng et al. (1982) reported that SON neurons can respond to the osmotic changes in their immediate environment and SON lesions eliminate VP release in response to osmotic stimuli. Most likely, HNS indirectly adjusts its own activity based on the incoming information from central sensory sites. HNS receives afferent fibers from 9 baroreceptor and chemoreceptor nuclei in the medulla and circumventricular structures (OVLT, SFO) where each directly plays a major role as the sensory sites to evaluate the plasma osmolality or blood pressure in the CNS. These HNS afferent fibers are discussed in detail in the HNS afferent connections. VP is an antiduretic hormone via acting on V2 receptors located on the renal collecting tubules. OT can positively modulate VP effects on V2 receptors. It seems reasonable to expect a larger release of VP and OT hormones during dehydration in order to increase water reabsorption to bring down the plasma osmolality back to the basal level. During dehydration, the amount of VP and OT mRNA increases in the MNC (McCabe et al., 90), MNC become more active (Walters & Hatton, 1974) and CT and VP hormone synthesis becomes faster. In order to MNC reach to the capability of producing a larger amount of the OT and VP hormones during the needs, the MNC may change morphologically. At the somatic level these morphological changes are as such: the number of nucleoli, the rough endoplasmic reticulum is enlarged and distended, somatic size increases and the number of the NGs and lysosomes decreases in the SON and PVN (Hatton & Walters, 73; Morris & Dyball, 74; Krisch, 76; Wakerly et al., 78; Whitnall & Gainer, 85; Anderson, 86). As the size of the MNC increases and astrocytic processes may retract, the soma-somatic/dendritic membrane apposition and the number of multiple synapse (MS, one presynaptic terminal contacting more than one postsynaptic some/dendrite) increase (Tweedle & Hatton, 1977; Gregory et al., 80; Tweedle & Hatton, 84; Modney & Hatton, 89). At the terminals, the microvesicles are more often seen at the contact zone with the perivascular space, the number of NGs decreases, hormone secretion (which is Ca++ dependent (Sladek & Joynt, 78)) elevates and partial depletion of VP and OT content can be detected. Plasma VP and CT both increase (Kasting, 88; Robertson & Athar, 76; Sladek & Knigge, 77; Anderson, 87, Jones & Pickering, 69; Januszewicz et al., 86; Hattori et al., 88) and the content of OT and VP in the CSF increases (Januszewicz et 10 al., 86; Morris et al., 84) during dehydration. Some of the terminals, normally wrapped by the pituicytic processes, become free and, therefore, glia-capillary apposition decrease, whereas the axo-capillary and neuro-glial synaptoid contact increase. These physiological and morphological changes gradually return back to normal after a period of rehydration (2-5 weeks) (Wittkowski & Brinkmann, 74; Tweedle & Hatton, 80a, 80b; Tweedle, 87a; Tweedle & Hatton, 87b; Perlmutter et al., 84). At the dendritic zone, dendritic bundling and dendro-dendritic apposition significantly increase. This is also a reversible event following rehydration (Perlmutter et al., 85). F) QT AND VP ACT AS NT§ AND HQRMQNES: Hypothalamo—neurohypophysial VP and OT act both as NTs and play a major role within the CNS and as neurohormones released into the hypophysial portal system at the PP. These hormones induce such changes in behavior and homeostasis such as memory, respiratory, heart rate, body temperature and fluid balance, milk ejection and vascular smooth muscle and uterine contraction (Meisenberg & Simmons, 1983). Luppi et al. (88) and Caverson et al. (87) reported that neurophysin- and VP- and OT-labeled perikarya in the SON, PVN, the diagonal band of Broca, the periventricular area, the nucleus circularis, the anterior hypothalamic preoptic area, the peritornical area of the lateral hypothalamus, the accessory SON, the area of the tuber cinerum and the medial region of the amygdala may project to the PP. The suprachiasmatic nucleus, the caudal part of the PVN and the ventral globus pallidus which are also immunolabeled for CT and/or VP have not been shown to project to the PP. Peripherally, VP and CT reach the target organs via the circulatory system. As mentioned previously, VP increases the permeability of the kidney distal tubules and collecting ducts to water reabsorption. VP can also be a factor for long-lasting release of neurotransmitter at the neuromuscular junction (Abdul-ghain et al. 90). On the other hand, OT causes uterine contraction and milk ejection. OT may also be involved in 11 elevation of renal Na+ and CI' excretion and urine flow during dehydration (Dyball & Leng, 86; Balment et al., 80). VP released from PP elevates melanocyte stimulating hormone release after reaching the intermediate lobe through the neurohypophysial portal system (Howe & Ray, 85). Buma and Niewenhuys (87) found the release sites for OT and VP in the median eminence along the pituitary stalk and speculated that their release could affect anterior pituitary hormone excretion. Johnson et al. (90) reported that lobectomy of PP alters the amount of the CT in the PVN as well as the adrenocorticotropic hormone, luteinizing hormone and prolactin release in the anterior pituitary. Centrally, the VP fibres have been reported to feed back to the nuclei of origin (Berlove & Piekut, 90; Mason et al., 84; Hatton, 85; Ray & Choudhury, 90) and act as a positive feedback on its own release after binding to V2 receptors (Ramirez et al., 90). Moos et al. (89) reported that CT may also be released within the SON and act as a neurotransmitter or modulator. The sources of the OT released in the SON are most probably local, either neurons within the SON (Theodosis, 85) which may participate as a feedback factor and/or the afferent projections from hypothalamic accessory neurons, which also stain for OT. An increase of OT release has been reported when the SON, PVN and neural lobe are incubated separately in OT media both in male and lactating rats (Moos et al., 84; Yamashita et al., 87; Falke, 1989). It has been reported that OT locally induces steroid dependent morphological changes in SON neurons during milk ejection (Montagnese et al., 90; Theodosis et al., 1986). OT and VP concentrations in both the CSF and plasma vary during suckling or hypertonic stimulation (Morris et al., 84; Kendrik et al., 86; Kasting, 88; Szczepanska- Sadowska et al., 83). Direct secretion of OT and VP into CSF has been suggested since OT and VP content increased in CSF in lobectomized animals (Dogterom et al., 77). One of the candidates for the source of the CSF OT and VP would be the PVN 12 dendritic processes extended to the third ventricle. Upon stimulation, they may release their content into the CSF. The NTs in the CSF may affect on the juxtaventricular or juxtasubarachnoidal neurons possibility via crossing the ependymal cells covering the internal layer of the ventricles where no barrier exists (Luiten et al., 1980). The stimulatory effect of OT content in CSF on OT release has been reported (Moos et al., 89). It was thought that OT and VP can not cross the blood brain barrier (BBB) and the content of these peptides in the blood stream and the CSF are independent of each other (Varma et al., 69). However, Banks & Kastin, (1990) suggested that some peptides such as CT and VP can cross BBB bidirectionally and the presence of peptides on one side of the barrier can be effective on the other side of the wall as well. If this finding might be true, a direct contact of the perikarya with capillaries in the SON (Krisch 1977) could allow the magnocellular neurons to detect the plasma osmolality directly at the somata level. One especially can not disregard the possibility of the free passage of plasma OT and VP peptides and other substances across the fenestrated capillary wall found in the regions which are free of BBB (e.g. PP, SFO and OVLT) and having a direct affect on the neighboring neurons in the central nervous system. G) F ENT ND EFFE ENT N T NEUBQTBANSMITTEBS: G-I-I MW Besides the PP, Castel and Morris (1988) reported that PVN and SON efferent fibers project to the median eminence, preoptic area, septum, hypothalamus, subthalamus, habenula, amygdala and ventral hippocampus. The PVN and SON also project to brain stem nuclei such as the dorsal vagus nucleus, the nucleus of the solitary tract and the locus ceruleus (Camier et al., 1985; Sinding et al., 1982; Voorn and Buijs, 1983) as well as to lamina l of the spinal dorsal horn and the sympathetic 13 and parasympathetic pregangllonic columns (Swanson, 77; Swanson & McKellar, 79). The SON MNC projecting to the septal and the hypothalamic areas are VP-, DYN-, and a-norepinephrin (NE)-immunocytochemically labeled (Millan et al., 83). In addition, PVN MNC which contribute to the afferent PP, septal, medulla, pons, and spinal cord fibers are also immunolabeled for OT, VP, DYN and a-NE (Millan et al., 84; Cechetto et al., 88). There is some evidence for hyperpolarization of the caudal medulla by OT and activation of NE neurons in the locus ceruleus by VP (Morris et al., 80; Olpe & Baltzer, 81). Buijs et al. (1990) suggested that stimulation of the parvocellular OT synapsing NE neurons at the dorsolateral medulla (A1 area) excites VPergic cells in SON and PVN and, as the result, VP release in the nuclei elevates by a feedback mechanism. (32-) mm It has been shown that MNC’s receive afferent fibers from many different areas of the CNS (Tribollet & Dreifuss, 81 ; Tribollet et al., 85) carrying many different types of NTs and each one may modulate and control the function of the MNC's differently. The MNC's in SON and PVN receive afferent fibres synapsing on both the somata and the dendritic processes. Van den Pol (82) reported that the area where both parvocellular somata and magnocellular dendrites are present contain the highest axon density in the PVN. This indicates that any afferent fibres coming to the parvocellular area may also make synapses on the dendritic processes of the magnocellular neurons of the PVN. However SON somata and dendrites alSo receive a fair amount of axonal projections. DAergic terminals are present in the PVN, SON and PP (Buijs et al., 84; Bjorklund et al., 73). Neurons in the DA anterior hypothalamic periventricular areas such as the arcuate nucleus and rostral periventricular region project to the PVN, SON and neurointermediate lobe (Kawano et al., 87; Bjorklund et al., 73; Luppi et al., 1986, Buijs et al., 84, Lookingland et al., 85). DA injection intraventricularly and into 14 hypothalamic slice preparations has shown that DA stimulates OT and VP neuronal activity and secretion (Bridges et al., 76. Mason, 83). On the other hand, DA terminals in the isolated PP inhibit both the OT and VP release (Vizi & Volbekas, 80; Racke et al., 82). Since DA agonists and antagonists do not change the DA turnover in the PP, it has been suggested that the DA terminals lack auto-receptors (Lookingland et al., 85). However, opioids abolish DA secretion in the PP (Garris & Benjonathan, 1990; Vizi & Volbekas, 80). Therefore; during a high demand of hormone secretion, when OT and VP are secreted with their co-Iocalized Opioids, inhibition of DA release by opioids would indirectly stimulate the release of the neurohypophysial hormone. OTergic and VPergic neurons in SON and PVN and their terminals in the PP also receive GABAergic fibers (Theodosis et al., 1986; Rabhi et al., 1987; Van Den Pol, 1985, and Buijs et al., 1987) and activation of the GABAa receptors in the SON decreases neural activity (Randle & Renaud, 1987; Mason et al., 1987) via opening Cl' channels. The diagonal band of Broca and the septum are two main candidates for sending the GABAergic afferent fibers to the SON and/or PVN (Jhamandas et al., 1989; Poulain et al., 1980). Acetylcholine (ACh) stimulates and inhibits VP release at the SON level depending on if it is bound to the nicotinic or muscarinic receptors, respectively. ACh- muscarine receptor coupling also increases the release of VP at the PP (Gregg, 1985; Bioulac et al., 1978). Hosli et al., (88) noted that the spinal cord and brain stem astrocytes also exibit both types of ACh receptors and stimulating them hyperpolarizes the majority of the pituicytes. Glutamate fibers form synapses on the SON neurons and excite the NHS (Meeker et al., 1989). Glutamate may also activate Ca++ influx into the astrocytes, changing the extracellular Ca++ concentration. A close apposition between the adrenocorticotropic hormone fibres with the OT-IR MNC of the PVN as well as with parvocellular neurons was reported (Pieknut, 85). Adrenal cortical glucocorticoid 15 receptors are also found on the MNC of the SON and their activation has been reported to inhibit VP release (Ahmed et al., 1967; and Streeten et al., 1981). Serotonergic projections to the PVN and SON originating from raphe nuclei have also been demonstrated (Sawchenko et al., 83). It has been shown that the limbic system (the septum, the olfactory bulb, the prepiriform cortex and the amygdaloid nuclei) projects directly to the SON and PVN (Poulain et al., 80, 81 ; Silverman et al., 81; Smithson et al., 89; Ferreyra et al., 83). The electrically stimulatedlimbic system inhibits MNC activity (Ferreyra et al., 1983; Gray et al., 1980). Smithson et al., (1989) and Hatton and Yang (89) reported a direct projection from the main olfactory bulb to the,SON and they speculated that this contact could be one of the main factors controlling neuronal secretions. In addition, Grosvenor et al. (1986) found a greater level of plasma OT in lactating mothers with 8 pups compared to mothers caged with 6 pups. This may occur either via the main olfactory bulb being more stimulated by odor (Slotnick et al., 1989) from pups in the cage with 8 pups and/or the higher possibility of suckling 8 pups compared to 6 pups. lwahara et al. (73) noted elevated olfactory bulb electrical activity in postnatal rats. Yang and Hatton (87) reported that the incidence of dye coupling in the SON is enhanced by lateral olfactory tract stimulation in lactating mothers. This event did not occur in virgin or male rats. Modney et. al. (90) also reported an increased incidence of dye coupling in maternally behaving virgin rats. They suggested that sniffing and anogenital licking of the pups stimulate SON neurons via stimulation of olfactory bulb. Baroreceptor adrenergic sensory relay medullary nuclei project to the SON and PVN (Weiss & Hatton 1990a; Cunningham et al., 1990; McAllen &Blessing, 1987; Wilkin et al., 89) and PP (Garten et al., 1989). The SON neurons can be inhibited by stimulating baroreceptors (carotid sinus and vagus sensory neurons) (Mc Allen & Blessing, 87; Yamashita & Koizumi, 79) while activation of the chemoreceptors excites the neurons (Yamashita, 77; Yamashita & Koizumi, 79). Activation of the 16 baroreceptors inhibits neurons in the caudal ventral medulla (A1 area) neurons which project to the SON and PVN and causes excitation of A1 neurons when atrial pressure decreases (McAllen & Blessing, 1987). Day and Sibbald (1990a) reported that activation of A1 neurons could modulate inhibition of the VP neurons in the SON. Based on what type of adrenergic receptors is activated (a2 or a1), the MNC in the SON and PVN become inhibited or excited, respectively (Bush at al., 1990; Song et al., 1988; Armstrong et al., 1986b; Day et al., 1985). On the other hand, activation of the B-adrenergic receptors causes inhibition of OT release and/or enhancing of VP release. The other two routes by which adrenalin could reach the PP are from a direct superior cervical ganglia projection (Saavedra, 1985) and a free access of some of the terminals and the pituicyte processes to peripheral adrenalin released into circulatory system by the adrenal medulla. Sladek and Yagil (1990) demonstrated that not only is the type of the receptor important, but also the effects of NE on the receptors are based on both NE concentration and the physiological state of the individuals. These baroreceptor neurons could also indirectly inhibit the MNC through activation of the GABA containing diagonal band of Broca (Jhamandas & Renaud, 1986). Astroglial cells have adrenergic receptors on their cytoplasmic membrane (Bicknell et al., 1989; Lerea &McCarthy, 1989) It has been shown under B-adrenergic stimulation, maybe adenylate-cyclase dependent (Stone et al., 1990), the pituicytes retract and the percentage of terminal contact with the perivascular space increases (Smithson et al., 1990; Luckman & Bicknell, 1990). Hatton et. al. (91) reported that activation of 82 receptors causes plasticity of cultured astrocytes whereas 81 antagonists did not reduce this effect. As was mentioned before, activation of the pituicytes may-lead to the extracellular ionic changes and, consequently, the alterations of terminal activity (Bicknell, 88). 17 Substance P coexists with A1 adrenergic neurons and such neurons may project to the SON, PVN and PP (Bittencourt et al., 1991; Van den Pol). Holzbauer et al. (84) reported that SP-immunoreactivity in the PP is decreased with dehydration, where substance P increases both OTergic and VPergic neuronal activity (Sladek 8 Johnson, 1983). Neuropeptide Y, also co-localized in the A1 catecholamine area projecting to both PVN and SON, causes VP release in rats (Hooi et al., 1989; Levine 8 Morley, 84; Willoughby 8 Blessing, 87; Sawchenko et al., 85). Neuropeptide Y - containing fibers synaptically contact the VP-containing somata and their dendrites in the SON (Beroukas et al., 1989) and Morley and Flood (1989) reported that intracerebroventricular injection of Neuropeptide Y inhibits drinking behavior in mice. This peptide was also found in the arcuate nucleus which sends out some of their efferent fibers to the SON and PVN as well as the PP. Galanin is another neuropeptide originating from the brain stem catecholaminergic area that projects to magno- and parvocellular neurons of the PVN (Sawchenko 8 Pfeiffer, 88b). What effects galanin might have on the magnocellular neurons is not clear. Histaminergic terminals (originating from the mammilary nucleus) are found in the PVN, SON (Weiss 91 al., 1989) and many other parts of the brain. Histamine mainly causes excitation by acting on H1 receptors, but some depression by acting on H2 receptors of the MNC in the hypothalamus (Hass 8 Wolf 77; Yang 8 Hatton 89). Some investigators have reported that VP release is stimulated at the PP by histamine (Bennett 8 Pert, 1974; Hass 8 Wolf, 1977; Tuoristo 1984; Yang 8 Hatton, 1989). Angiotensin II (Agll) receptors are found in the PVN, SON, AV3V and SFO somata (Castern 8 Saavedra, 1989; Plunkett et al., 87) and on hypothalamic glial cells (Raizoda et al., 87). When plasma volume decreases, in order to conserve body water, plasma All increases. Allstimulates neurons in SON, SFO and Antroventral Third Ventricle (AV3V) slice preparations (Okuya et al., 87). All microelectrophoreticaly injected into the PVN or in the ventricles stimulates MNC 18 activity (Akaishi et al., 81) and VP release (Shoji et al., 89). The somata in the SON, PVN, SFO, medial preoptic area, medial nucleus of the amygdala, nucleus of the solitary tract (near the margin of the area postrema), hypothalamic perifornical area and fibres of the SFO are stained for Agll (Lind et al., 1985) and most of these nuclei project to the SON, PVN and/or PP. In addition, it has been suggested that the MNC are capable of angiotensin synthesis. Its function is not clear (Aronsson et al., 1988) but its release in the PP may act on the receptors and modulate hypophysial hormone release. Most possibly, this would elevate VP secretion (Sladek 8 Joynt, 79). H.) 3 DE 3 A: 0' A AL. l-‘ I L O ll-TL.L:.L-. ntlix-|-|:; .L There are reports of impairing the sensitivity of SON and PVN to. systemic osmotic stimulation by local anesthesia of or lesions of the SFO or AV3V (Tanaka et al., 1989; Mangiapane et al., 84; Hosutt et al., 1981; Blackburn et al., 1987). SFO (located medially at the rostral end of the third ventricle) is a neurosecretory structure free of a BBB. SFO neurons directly project to the MNC, NL and AV3V region (Weiss 8 Hatton, 90b, Lind et al., 82, 85, Miselis, 81 ; Ju et al., 86). Agll may indirectly affect OT and VP secretion via excitation of the osmotic sensory circumventricular SFO which has receptors for the peptide (Okuya et al., 87; Castern 8 Saavedra, 89, Wilkin et al., 89). Activation of the SFO stimulates OT and VP secretion directly (Knepel et al., 1982; Sladek 8 Johnson, 1983; Ferguson 8 Kasting, 1987) or, indirectly, SFO inhibits VP secretion by activation of the septum (Tanaka et al., 1988a). Szcepanska-S‘adowska et al. (1979) and Moran and Blass (1976) reported inhibition of osmotic thirst by electrical stimulation of the septum and recently Staiger reported that septal efferents directly project to the VP neurons in the SON and PVN. On the other hand VP terminals are found in the lateral septum (Shaw et al., 1987) and activation of these neurons by VP was reported (Raggenbass et al., 1988). These VP neurons likely originate from the SON and PVN to modulate their own activity using septal neurons 19 as the mediator. In addition, the solitary tract nucleus , a baroreceptor, besides its input to the MNC (Weiss 8 Hatton 1990a; Sawchenko et al., 1988a; Horst et al., 1989), projects to the SFO and inhibits its activity (Shioyva 8 Tanaka, 1989). This indicates another route of controling the MNC activities and secretion by the baroreceptor neurons in the medulla. AV3V includes OVLT and median preoptic area. OVLT, a circumventricular organ, receives afferent fibres from the SFO, median preoptic and SON and projects to the SON and median preoptic. OVLT neurons are also stimulated by both All and high sodium concentration (Okuya et al., 87; Knowles 8 Phillips, 80; Vivas et al., 90). The median preoptic nucleus, which receives afferent fibres from both the SFO and OVLT, projects to and stimulates both the VP and OT neurons in the SON and PVN (Tanaka et al., 1988b; Leng et al., 1989; Wilkin et al., 89, Honda et al., 90). With regard to PVN, SON, SFO, AV3V and baro- and chemoreceptors, the question is where the central osmotic sensory center is located. Destruction of either AV3V or SFO partially impairs the response of the magnocellular neurons to systemic osmotic stimuli (Blackburn et al., 87), whereas lesions of AV3V do not affect OT release in response to suckling (Blackburn et al., 87). Leng et al. (89) believe in the necessity of excitatory postsynaptic input provided by the SFO and the AV3V nuclei for the MNC to be responsive to the osmotic changes. Baro- and chemoreceptor nuclei can sense the blood pressure and plasma osmolarity, respectively, and manipulate MNC function. In in vitro studies, the MNC in the SON respond to osmotic changes which indicates that these neurons are able to sense their extracellular osmotic changes as well (Yagil and Sladek, 1990). Overall, it seems like that neither the SON, PVN, AV3V, OVLT nor solitary tract nucleus is the primary source of the somatic sensory system and most probably serves as a relay among all of these regions to give a final order to the VP and CT neurons to act under various osmotic stimuli. 20 W Present studies focus on studing HNS plasticity to examine specifically which type of neuron (OT and /or VP) morphologically changes in SON and PP when the system is stimulated by chronic dehydration. Theodosis et al. (1984, 1986) and Chapman et al. (1986) reported an increase in total direct apposition and that in the number of M85 associated only OTergic cells in the SON in dehydrated and lactating animals. The results relating to dehydrated animals surprised us since electrophysiologically both types of neurons (OT and VP) are stimulated and both OT and VP content elevates in plasma in dehydrated animals. Therefore, one would expect that both OT and VP neurons (if either) would morphologically change. It has been shown that during dehydration (10 days of saline treatment) the percentage axo- capillary membrane contact increases wheras glia-capillary membrane contact decreases (Tweedle 1987). In this set of experiments I am trying to determine the type of the terminals showing the plasticity during chronic dehydration stimulation and, in addition, I would like to reevaluate Chapman et al. (86) findings in respect to the effects of dehydration on SON neurons in rats. This question will be addressed using electron microscopic immunogold cytochemistry. 21 ,'A ILTHOP -_ 0 ‘ __I .FP:.7-- u lmmunogold is a reliable technique to label hormones and NTs at different sites of a cell in tissues of interest, including the CNS. Theodosis et al. (1984, 86), Chapman et al. (86), and Castel et al. (1986) previously have labeled OT and VP neurons in the SON and the terminals in the PP applying a similar immunogold technique. Because of its electron dense properties, colloidal gold is easily detected by transmission electron microscopy. Colloidal gold carries a negative charge in water and is able to noncovalently bind to macromolecules such as antibodies (Abs), enzymes, lectin and polysaccharide. A uniform colloidal gold particle can be made ranging from 3 nm to 150 nm in diameter. Based on their size, colloidal gold particles show reversible binding affinity, which means that the smaller sized particles give a higher chance of Antibody-Antigen (Ab-Ag) coupling (Kent et al., 78) and better resolution (Bendyan, 84). Since different sizes of gold are available, multiple marking is also possible. Different antigens are labelled with different sizes of particles and the labeled sites are easily identifiable in the EM and quantitative studies are possible. The Abs for OT neurophysin and VP glyCOpeptide were kindly provided by J.P.H. Burbach and F. van Leeuwen, respectively. The OT antiserum is against the unique C-terminal part (the last 9 amino acids of neurophysin exclusive Argenin), and VP antiserum is against the glycopeptide fragment (22-39) which is absent in the OT prohormone gene sequences and both are raised in the rabbit. Since the neurophysins (I and II) and glycopeptide are co-stored and released with their respective hormones, for simplicity, I will consider these Abs specific as for the hormones (OT and VP). To accomplish our goal, the following sequence of steps should be considered: 1) The antigenicity should not be destroyed so as to preserve a 22 highly Ag-Ab coupling afinity, 2) the Ag binding sites should be freely available to the Abs, 3) the Ab should bind specifically to the Ag (OT or VP), 4) the gold particles conjugated to a macromolecular should bind to the primary Ab with a high affinity, 5) the background should be so low that will not interfere with interpretation and 6) the labeled granules of the positive cells should have a good number of particles that would be easily identified. Post-embedding immunocytochemistry was used in these studies for two reasons. 1) I am trying to use two different Abs to label the somata and terminals of the same tissue. Post-embedding staining would let us stain several ultrathin sections of a block for one Ab, while the adjacent sections of the same tissue can be used to stain for the other Ab; 2) using post-embedding technique allowed us conservatively to use tissues in the preliminary studies while for each try only few nanometers of tissue would be used. Using a high concentration of glutaraldehyde has been shown to be detrimental to recognition of Ag by Ab. Therefore, I used a low concentration of glutaraldehyde (0.5-1 %) to fix the tissues. In the preliminary studies different concentrations of glutaraldehyde (0-2%) and paraformaldehyde (0-4%) were used for making fixatives. pH of fixatives were ranged from 68 Both perfusion and immersion fixation were tried to prepare the tissues. Pre- embedding osmication and osmicated free tissues were tried. Either of the above combinations with Spurrs embedding plastic did not give an optimal staining and the background was high. Using Epon-Araldite embedding plastic greatly increased the labelling, even when the tissue was preosmicated. The best result was achieved when acetone instead of propylene oxide was used as the step in swiching alcohol to embedding medium. But when acetone was used in preparing PP, lipid droplets lose their attachments to the surrounding matrix, therefore, I used propylene oxide in PP studies 23 Since our procedure includes post-fixation with osmium tetroxide, which may cover the protein antigenic sites, it has been suggested that strong oxidizing agents such as sodium metaperiodate, hydrogen peroxide and periodic acid can unmask the antigenic sites (Bendyan 8 Zollinger, 83). In our case, using sodium metaperiodate did not improve the labeling. Triton X-100 (TX—100), a detergent solution, was used in the following studies for two reasons, 1) TX-100, for its low viscosity, used in the rinsing solution to improve washing out Abs and gold particles from the sections being treated for labelling and 2) TX-100 was used in blocking agent and diluting Abs in order to reduce the non-specificity binding sites on sections. The other important and crucial point in immunocytochemical studies at the EM level is using nickel or gold grids to prevent oxidation of the metal and contamination of sections (Bendayan, 84). The pH of blocking agent, diluted Abs, rinsing solution and gold solution is a very important factor to achieve better labelling with low background. The suggested pH for the Abs and immunogold is 8.6 and 8.2, respectively. At the beginning, gold particles conjugated with protein A was made in our laboratory. The protocol is as follow: 1) Add freshly prepared 4 ml 1% sodium citrate to boiled 100 ml 0.01 chloroauric acid. This step is light sensitive and this step should be done in dark. 2) Centrifuge the solution at 10,000 rpm for 15 minutes. 3) Adjust the pH of the suspension with 0.2M K2Co3 and 0.1 M HCI to reach pH 6.0-6.9. 4) Add 10 ml of the suspension to 0.3 mg of protein A solved in 0.2 ml distilled water. 5) Centrifuge the solution at 25,000 rpm for 30 minutes. 6) Carefully remove the top clear protein A solution layer and middle black metallic gold layer and then transfer the dark red sediment protein A gold to 1.5 ml of 0.01M phosphate buffered saline with 0.02% polyethylene glycol, pH 7.3. 7) Store Protein A gold at 4 degree centigrade (the stock solution is good for 1 month). 8) At the time of use, dilute Protein A gold 5-10X in 0.01 M phosphate buffered saline with 0.02% polyethylene glycol. 24 Either protein A gold made in the lab or purchased from manufacture did not specifically bind to the Abs. Since the Abs were raised in rabbits, we tried lgG goat anti rabbit gold which drastically improved the labelling with low background The procedure of tissue preparation and immunocytochemistry is discussed in the following experiments. 25 A. Ell A 'I ' A A .A . . A A I =A .:-.- L . 3 ni'k ' I '3. 0L L IIII L. 0 U 0: .A . AAA. A .-= k. I :A = :A W The neurohypophysial hormones, OT and VP, are synthesized in different populations of MNC in the SON and PVN of the HNS (De Mey et. al., 1974; van Leeuwen and Swaab, 1977; Vandesande and Dierickx 1977). Upon neural activation, these hormones are secreted into the perivascular spaces around fenestrated capillaries in the PP. In response to various physiological stimuli such .as chronic lactation or dehydration, the MNC in the SON undergo dramatic reversible morphological changes (Gregory et. al., 1980; Hatton, 1985; Hatton and Walter, 1973; Modney and Hatton, 1989; Tweedle and Hatton, 1977; Tweedle and Hatton 1984) such as increases in cell size, number of nucleoli, total soma-somatic/dendritic membrane apposition and number of M85 (one presynaptic terminal contacting more than one postsynaptic soma/dendrite). Physiologically, during dehydration and osmotic stimulation, the amount of VP and CT mRNA increases (McCabe et. al. 1990), the VP and CT neurons become more active (Poulain and Wakerley, 1989; Walter and Hatton 1974) and the VP and OT plasma content increases (Kasting, 1988). Based on physiological findings, it seems likely that the neural plasticity observed in dehydrated animals is associated with both OT and VP cell types. Chapman et al. (1986) reported that the high incidence of M83 and soma- somatic/dendritic apposition is associated with only OT neurons in the SON of chronically dehydrated rats. ln‘the present study, our aim is to re-evaluate the neural plasticity in the SON of chronically dehydrated rats using a post-embedding 26 immunocytochemical technique for CT and VP. A preliminary report of these findings has appeared (Marzban et. al. 1991). MATERIALS AND METHODS: W Sixteen (8 male and 8 female) rats, 100-110 days old, were divided into two groups. The first group of eight animals (4 male and 4 female) was dehydrated (given 2% saline instead of water for 10 days) and a second group of eight animals (4 male and 4 female) given tap water to drink was used as the control. All were housed in a 12:12 h light-dark cycle. Rats were decapitated without anesthesia during the light cycle. The brains were taken out and left in fixative (3% paraformaldehyde and 0.5-1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) for 4-5 hours. The anterior, middle and posterior portions of the SON were taken and coronally cut into 500 pm slices using a tissue chopper while the brains were incubated in the fixative for another 2 hours. The tissues were osmicated for one hour and stained en bloc in 4% uranyl acetate at 4°C overnight. The tissues were dehydrated and embedded in an Epon-Araldite resin mixture. The middle portions of the SONs were thin sectioned (silver) and picked up on 300 mesh nickel grids. W Two sets of sections from each animal were incubated in blocking agent (0.1% bovine serum albumin, 0.1% TX100, 0.5M NaCl in 0.05M tris buffer solution (TBS) pH 8.6) for 15 minutes. Without rinsing, the first set of sections was incubated in 1:1000 VP Ab and the second set in 1:1000 OT Ab (both Abs were diluted in 0.1% TX100 and 0.5M NaCl in 0.05M tris buffer pH 8.6) for 3 days in a refrigerator (4°C). (The OT and VP Abs were kindly provided by J.P.H. Burbach and F. van Leeuwen.) Following a jet wash with a rinsing solution (0.5M NaCl, 0.5% TX100 in 0.05M tris buffer pH 8.6), the grids were incubated in 1:10 lgG goat anti rabbit Gold (20 nm) (Polyscience, 27 Warington, PA) diluted in TBS (1% BSA, 0.05% sodium azide in 0.02M tris buffer pH 8.2) for 1 hour at room temperature. The grids were jet washed first with rinsing solution and then with distilled water. To control for non-specific staining: 1) adjacent sections were incubated in lgG-gold complex omitting the Ab step and 2) sections were incubated in nonimmune rabbit serum in place of the Ab followed by the lgG- gold. WW For viewing under the electron microscope, the sections were stained in lead citrate for 3-5 seconds. 10-15 micrographs of one section from each animal were taken randomly (of neurons in the two corners opposing each other of each grid square bar) at 3,000x with a Philips 201 electron microscope. Prints were made at a final magnification of 12,000x. Labelled and unlabelled cells were identified. Labelled cells were characterized as having 70% of their granules labeled with at least two gold particles and having not fewer than three granules. Unlabelled cells were identified as having at least six unlabeled NGs and not more than 10% of the total NGs may include, due to the background, at most two gold particles. The membrane length was measured using the SummaSketch ll. The total soma- somatic/dendritic apposition, axo-somatic apposition and soma-glial apposition membrane of each animal for both the positively labelled H and unlabelled (+) magnocellular neurons were measured. The percentage of each measurement for every animal was calculated with: total cell membrane (pm) of + (or-) cells The total number of M85 (a presynaptic terminal containing microvesicles contacting one or more somata and/or one or more dendrites while it is apposing the labelled and unlabelled cells with or without pre- or post-synaptic thickening) contacting cells 28 was counted. Number of M85 per 100nm of cell membrane and per 100 cells was calculated for both positively labelled and unlabelled cells from each animal with: total cell membrane (pm) of + (or -) cells and r f ' r- total number of + (or -) cells The total number of axons contacting 100um of + (or-) cell membrane was calculated with: l r i - total cell membrane (pm) of + (or -) cells The treated and control animals for each Ab were compared using the non-parametric one tailed Mann-Whitney U test. Sizes of each type of cells which contained more than three-fourth of their nucleus in the micrographs of dehydrated and control animals were measured using a point intersection analysis method [21]. The mean cell size of each animal was calculated and the treated group was compared with the control group using an unpaired one tailed t test. RESULTS: Without etching the embedding plastic, I obtained good labelling in the sections using the OT and VP Abs (Fig.1). This indicates the compatibility of the postembedding immunocytochemistry technique and highly specific antigen (CT or VP hormone-antibody binding) in our study. Among the cytoplasmic organelles, immunogold labeling was found mainly on the granules of positively labeled neurons. The labelled granules were concentrated around the nucleus and on either side (cis and trans) of the Golgi apparatus (Figs.1,6). Thus, the hormones are labelled even at the stage of being synthesized in rough endoplasmic reticulum before reaching the Golgi apparatus for further hormonal processing. The granules at the peripheral regions of the perikarya were not heavily labelled. The maximum sizes of the VP and 29 OT granules in the cell bodies were different (175 nm and 215 nm, respectively). At the dendritic level, occasional inclusions, bigger than the sizes of NG, stained for VP were seen in the treated animals (Fig.2). Usually the nucleoli were also labelled in either type of neuron whereas the cytoplasmic rRNA are not labelled. Background was very light and did not interfere with data evaluation. The quality of membrane appearance and pre- and post-synaptic thickenings predictably was not as good as seen in the non-immunostained sections (Fig.3). Dehydrated animals showed morphological changes in both VP and OT magnocellular neurons in the SON (Table 1). VP and CT somatic size increased significantly in the dehydrated animals (Fig.4). The soma-somatic/dendritic membrane contact increased significantly in both VP (Figs.5,6) and OT (Fig.6,8) cells. The percentage of astrocytic coverage reduced in both cell type. The percentage of axo- somatic contact decreased significantly with VP and CT neurons, while the total number of axons contacting each type of cell per unit of area of somatic membrane did not change significantly. Finally, contact by MSs (Fig.7,8) was also studied morphologically and was found only between labelled cells (Fig.5) or between unlabeled cells but not between a labeled and unlabelled cell. The number of contacts by MSs increased only between the OT neurons with no such change between the VP neurons. DISCUSSION: SON and PVN magnocellular neurons previously have shown morphological plasticity during lactation and dehydration (for review see Hatton 1990). In this study, I found that both VP and OT cells undergo increased cell/cell apposition after 10 days of 2% saline drinking. This is in contrast to the report of Chapman et al. (1986) who stated that such changes occurred exclusively in OT neurons in the SON of animals undergoing chronic dehydration. There are two main differences in how our experiments were done compared to theirs. First of all, for consistency, I used only the 30 middle portion of the SON which contains the most densely packed cells in the SON and which contains both OT and VP cells. They studied sections sampled from the whole SON. Secondly, their study included the somatic as well as the dendritic portion of the SON. In our experiment, since not many dendrites in the SON contain NGs and the population ratio of VP and OT varies along the SON, I studied only the somatic portion of the SON to reduce variability in the results. Our laboratory, using serial sections, previously reported that over 90% of apposed terminals (i.e. not having two definite synaptic thickenings) in the SON will eventually have postsynaptic thickenings along their length while still apposing adjacent cell bodies and/or dendrite(s) (Modney and Hatton, 1989; IL Smithson, unpublished results). Since, in this study, synaptic thickenings were not well preserved after the sections were immunostained, I included into our data all apposed terminals regardless of having any thickenings. Abs were very specific to labelled granules and the labelled granules were found on both cis (forming face where transporting vesicles fuse with GA) and trans (maturing face where secretory granules are formed) sides of GA. This indicates that glycosylation, phosphorylation and sulfation of hormones which may occur in GA do not alter the binding sides of the hormones to the Abs. It is hard to be certain why usually the nucleoli of neurons were labeled for the hormones. So far there is no report of any portion of VP and CT prohormone transport across the nuclear membrane, but it seems like that sequences of amino acid similar to the hormonal amino acid sequences are present in the MNC nucleoli. This finding may suggest that at least the neurophysin portion of OT prohormone and glycopeptide portion of VP prohormone may have a major input on the neural synthetic processes. Here I report that in the SON, the soma-somatic/dendritic contact elevated in both VP and CT neurons. Yang and Hatton (1988) reported that the MNC in the SON are electrically coupled. This possibly could be to synchronize MNC activity when 31 greater hormone secretion is required such as during lactation and dehydration. One possible way to enhance the likelihood of communication between cells is by increasing the length of direct membrane contact. At this point in time, with an ever- increasing list of possibilities, it is hard to say what types of information may be passing among the cells. In this study, both OT and VP cells share soma-somatic apposition. This means that if soma-somatic/dendritic membrane contact is the only requirement for electronic coupling then electronic coupling should be detected not only between homotypical, but also between heterotypical directly apposed MNC. Based on Cobbett et al. (1985) and Hatton et al. (1987) reports, dye couplings are observed only between homotypic MNC. This suggests that there should be other factor(s) involved in the interneuronal electrotonic coupling. In addition, both the VP and CT neurons had a lesser axo-somatic and glio- somatic contact and the number of MSs associated with CT but not VP cells increased in chronically dehydrated animals (as was shown by Chapman et al. (1986)). Previously, it has been shown that SON somatic size increases in water deprived rats (Bandaranayake, 1974; Hatton and Walter, 1973). It has also been suggested that increases in the M85 and increased cell-cell apposition could be because of astrocytic process retraction (Tweedle and Hatton, 1976) and somatic enlargement (Chapman and Theodosis, 1986; Modney and Hatton, 1889). Now it is observed that both OT and VP somatic size was enlarged in chronically dehydrated animals possibly, larger somatic surface area are avalable, to allow newly axonal contact to form on both OT and VP neurons. The magnocellular neurons receive both stimulatory and inhibitory afferent fibers from different areas of the CNS (for review see Hatton, 1990) and each may play a major role in controlling the hypothalamo-neurohypophysial activity differently while the neurons are under influence of various physiological stimuli, (e.g. lactation and dehydration). The axonal membrane contacting VP and OT neurons decreased 32 significantly. On the other hand, the number of axonal contacts per unit somatic membrane did not change in either CT or VP cells. This suggests that the number of axonal contacts increases as the size of VP and OT cells enlarges and axonal contacts, apposing on VP and OT neurons, occupy a lesser axe-somatic apposition when compared to control rats. Possibly, based on the terminal size, these newly formed terminals may bring in additional and different types of information to the MNC during dehydration. In addition, it was only OT neurons that demonstrated an increase in the number of M83. This difference between OT and VP axonal contact may indicate that some of the axons innervating VP neurons may be functionally different from those axons contacting OT neurons. It is hard to ascertain how these changes in axe-somatic and multiple synaptic contact may affect neuronal performance since these terminals were not lmmunocytochemically studied in this experiment and the types of receptors on the SON somatic membrane are not fully understood. Nonetheless, rules to guide neuronal activity show specificity because multiple synaptic terminals were found only contacting similar cells. This could serve to orchestrate neuronal synthesis and secretion of the two hormones differentially and M85 might be the main causal factor for electrical coupling to occur only among homotypical somata via adjusting VP or CT membrane physiological activities. Since using either type of Ab leads to the same result, one could conclude that both types of neuron in the SON of rats can change morphologically following chronic dehydration (Fig. 9) and the rules that guide the neuronal activity show specificity. These alterations could result in differential synchronization of OT and VP neuronal activities. lmmunocytochemical double staining studies labelling the neurons and the terminals following dehydration will help to better understand the HNS. 33 Table 1. Effects of chronic dehydration on magnocellular neurons in the rat supraoptic nucleus. Values shown represent the means iS.E.M.. 1] p< 0.05, * p<0.03, ** p<0.01 significantly different from the control. 34 8QO53 mazszumz .89 3 3523.28 .688 .2, 5.9:: .888. 8.9% zzmmw :38 85 mm 838:8 9m 38 {.989 9.98.3 3.99; 8. E :9 $3.98.: .399: .828 3 New 99.8 3.98.: 9.93.0 3.93.8 2.98.? 8.938 .88 xo ._.0 :3 3+3 on o F. 98.9 3.988 1.8.98.8 3.93.8 5.3.93.3 .938 3 3.0 98.2 3.99.: 3.93.0 3.09:3 3.93.9 3.93; .28 8 3.93.8 3.99.: 9.9 5.0 $393.2 8.9k I 5.3.98.3 3.38 3 3.93.9 N a 9.8.2 3.0.2 I 3. 99.8 9.93.2 8.939 .88 :> d> 80393.3 3.92.: .383: 9.98.9:3.093.588.9992 .938 3 9.93.: 3.93 3.99.0 8.98.3 3. 93.? 898.9 3:8 :> £8 .nEmE 859:8 83:8 83:8 83:8 83:8 3 + 09. 8a 8 88 Lo 3:: o_8E0m “.0 EB 833:8 :mn\mE0m 0.5.822 mm: 8 a :8 83:8 8:09 :3 3:8 3 .93 £3 .88 8 oz: _m:oxm 8 3 mm: .8 a mDmJUDZ UrEoImo 920qu do whomutm F 39; 35 Fig. 1. Accumulation of gold particles on the granules (arrows) of the cis and trans face of the Golgi apparatus (*) in a vasopressin immunostained positive cell. 36 37 Fig. 2. VP positive inclusions (arrows), which are larger than the typical vasopressin granules with an unknown function in a somatic proximal dendrite (D) in the dehydrated animals. N=nucleus. 38 s997' 39 Fig. 3. Two pairs of adjacent sections where A and C are not immunostained and B and D are processed for immunocytochemistry. Note the presynaptic (filled arrows) and postsynaptic (empty arrows) thickenings and membranes in A and C are not so clearly visible in the B and D. 4O 1954.} :1. . no. .( . . .vfl/ . _ , \1... . ,, u 4 ..., filth”: a e...1.za.% . A. . . . .A‘. x 4%. = A a A h. .1 i. 5.1.. I. . x ~ me... A A: . z .. t ,. .....1r I w .1. < . 177.... r fag}. .Hi quiltinq , S i ‘31! i. ' \\__,.4~ . 41 Fig. 4. Effects of chronic dehydration on the oxytocin and vasopressin somatic size in the SON. Values shown represent the means iS.E.M.. * p<0.03, ** p<0.01 significantly different from the control. CELL SIZE (umZ) 400q 300- 200‘ tooJ II VR(+) D VP(-) 969(- q CONTROL TREATMENT 42 CELL SIZE (urn?) 400‘ 300- 200‘ 100‘ g OT(+) I OT(-) as CONTROL TREATMENT 43 Fig. 5. (A) Soma-somatic contact (arrowheads) between vasopressin positively labelled cells (arrows) showing a multiple synapse (*) contacting two vasopressin positive somata. (B) A higher magnification of 5A showing a multiple synapse (*) without clear thickenings. vasopressin labelled granules are shown with arrows. 44 .5 a ‘ a . ‘ , 34 , .. .L J #4.: . . _ .w a: .. .4 I. . . . 3»; E h : . . v . . a . u: . . .. . . . g. , . .v .‘ . r n. a A . :. . .1. _ t..,.4.n,...d._.m..n,.v ... . .. . at», VA..;...MW 6.. . r 4 . . .. . Ruth. 1x , a. . 46 Fig. 6. (A) Soma-somatic contact (arrowheads) between vasopressin positive and negative somata. Note a vasopressin positive dendrite (*) contacting the two cells. Positively and negatively vasopressin labelled granules are shown with small and large arrows respectively. (B) A higher micrograph of 6A (inset) showing positive labelled granules (arrows) on either side (cis and trans) of Golgi apparatus (*). 48 Fig. 7. A multiple synapse (*) contacting a vasopressin positive cell and an adjacent undetermined dendrite (D). Positive granules are shown with arrows. 50 Fig. 8. Shows an oxytocin positively labeled cell (P) making soma-somatic and denciro-somatic appositions (arrow heads). Notice the dendrite (d) branching from the P cell apposes with adjacent oxytocin cell and a multiple synapse (*) contacts both of them. 51 52 Fig. 9. Diagrammatic sketch of vasopressin and oxytocin somata (circles) contacting neighboring vasopressin (VP) and oxytocin (OT) magnocellular neuroendocrine cells (semicircles) and afferent single synapsing (open ovals) and multiple synapses (shaded ovals) terminals in the control and chronically dehydrated supraoptic nucleus. VP and CT somatic size and the amount of soma-somatic/dendrltic contact increase in dehydrated animals. The number of axons per unit of somatic area in either OT and VP neurons increases with the same proportion as the cell sizes are enlarged. Only OT neurons contact more multiple synapses, whereas lesser axonal contact apposes both VP and CT somatic membrane in chronically dehydrated animals. Note there are different types of terminal and which may carry different types of neurotransmitter secreted differently under various physiological stimuli. MNC=magnocellular neuroendocrine cells (including both somata and dendrites). 53 MNC ‘ \ OT CONTROL TREHTMENT 54 A' in l -u.:'n... - D 0 '0 303' A3 l .301 z D u '3; 0 3;. L- AL nu L.-. D INTRODUCTION: The PP is a gathering region of the end terminals originating largely (but not exclusively) from neurons in two hypothalamic magnocellular nuclei, the SON and PVN. The MNC synthesize either OT or VP hormones with their respective neurophysins (I or II) in the somata. After being transported axonally, the hormones can be either stored in axonal swellings, called Herring bodies, or secreted mainly from terminals onto the basal lamina adjacent to the perivascular spaces around the fenestrated hypophysial portal system. Hormonal synthesis and secretion in this system are under the influence of physiological stimuli such as dehydration and lactation (McCabe et al., 90; Hattori et al., 88; Kasting, 88; Sladek & Joynt, 1978; Sladek & Knigge, 77; Robertson & Athar, 76; Jones & Pickering, 69). Previously, I have reported that chronic dehydration alters both OT and VP MNC morphologically but semi-differentially (Marzban et. al. 92). It has been reported that these neurons also show morphological plasticity at their terminals in the PP (Anderson, 87,Poulain & Tasker, 85; Tweedle, 83,85; Hatton & Tweedle, 82; Hatton, 1988; Wittkowski & Brinkmann, 1974). So far there is not a solid answer as to whether chronic dehydration preferentially affects the morphology of either OT or VP terminals. To answer this question I have adapted an immunogold cytochemical technique at the 55 electron microsc0pic level to study quantitatively certain measures in the PP of control vs. chronically dehydrated rats. MATERIALS AND METHODS: W Ten female rats, 100-110 days old, were divided into two groups. The first group of five animals was given 2% saline Instead of normal drinking water for 8 days prior to 4 [days rehydration. The second group of five control animals was given tap water to drink. After decapitation, the PPs were taken out and fixed (3% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) for 4-5 hours at room temperature. The tissue was a) osmicated, b) block stained with 4% uranyl acetate, 3) dehydrated and 4) embedded in iEpon-Araldite resin mixture. Mid- horizontal ultrathin sections (silver) were cut and collected on 300 mesh nickel grids. LMMQNQQQLQ Q YTQQflEM/fi TB Y: Two sets of a section from each animal were incubated in a blocking agent (0.1% bovine serum albumin , 1% TX100 and 0.5M NaCl in TBS, pH 8.6) for 15 minutes. Without rinsing, the first set of sections was incubated in 1:500 VP Ab and the second set in 1:500 OT Ab (both Abs were diluted in 0.1% TX100 and 0.5M NaCI in 0.05M tris buffer pH 8.6). The sections were left in Abs for 3 days in a refrigerator (4°C). Following a jet wash with a rinsing solution (0.5M NaCl, 0.5% TX100 and 0.5M NaCI in TBS, pH8.6), the grids were incubated in 1:10 lgG goat anti rabbit gbld (20 nm) diluted in TBS (1% bovine serum albumin and 0.05% sodium azide in 0.02M TBS, pH 8.2) for 1 hour at the room temperature. Finally, the grids were jet washed first with rinsing solution and then with distilled water. To control for non-specific staining: 1) adjacent sections were incubated in lgG-gold complex omitting the Ab step and 2) sections were incubated in nonimmune rabbit serum in place of the Ab followed by the lgG-gold. 56 ELEQIBQALMIQBQEQQBX: For viewing under the electron microscope, the sections were stained in lead citrate for 3-5 sec. 12-15 pictures of the capillaries and the pituicytes of one section of each animal were taken randomly at 4500x and 3000x respectively with a Philips 201 electron microscope. From the photographic negative, positive and negative terminals and axons were identified. Labelled VP and OT axons and terminals were characterized as having at least 50% and 30% of their NGs labelled respectively and each containing not less than five granules. Unlabelled terminals and axons were identified as having at least five granules, of these not more than 10% of the total NGs , due to background, may contain some gold particles. The total pituicyte-capillary membrane apposition and the membrane apposition of positive and negative terminals with capillary apposition membranes were measured from the negatives using a point intersection analysis method (Williams, 1981). The percentage of each measurement for every animal was calculated with: x 100 total cell membrane apposing capillaries The number of axons enclosed by pituicytes was counted and the number of the enclosed axons per 100 pituicytes for every animal was calculated with: tetal numeer ef eneleeeg ME lil er H labelled exene x 100 total number of pituicytes The treated and control animals were compared using the non-parametric one tailed Mann-Whitney U test. RESULTS : Reflecting the plasticity of the hypothalamo-neurohypophysial system to chronic dehydration, both VP and OT axonal terminals were affected (Table 1). The VP and OT terminal-capillary appositions (Fig. 1,2D,3) increased in the treated animals compared to the control, whereas the pituicyte-capillary apposition (Fig. 1) decreased 57 significantly after treatment. Based on Livingston's findings (1975), a significant increase in blood vessel cross sectional area in four day 2% saline treated rats PP, our measurements of terminal-capillary apposition may be underestimated. The labelling of OT granules was not as dense as for VP granules either at the axonal or the terminal levels. This could be the reason for the average number of OT (+) and VP (+) axons is less than the average number of VP (-) and OT (-) axons, respectively. Furthermore, the number of VP and OT axons enclosed by pituicytes (Fig. 2) decreased significantly in dehydrated animals. TECHNICAL CONSIDERATIONS: Nordmann (1985) reported that at earlier stages of dehydration granules at the nerve terminals preferentially are released whereas it is at the later stage of dehydration that the stored granules in the Herring bodies will be used. During rehydration, the granule density elevates first in the terminals and then gradually fill up the swellings. From our unpublished results and the report of Luckman and Bicknell (1990), most of the terminals in the PP of rats were seen to be either depleted of or have fewer granules with a higher number of microvesicles after drinking 2% saline instead of tap water for 10 days (Fig. 4) or 4 days. After labelling for OT or VP in these earlier experiments, there was a large number of undetermined terminals. This explains that, even though the hormonal synthetic activity increases during dehydration, it is not long after the granules reach the terminals that these hormones are released into the perivascular spaces. To overcome the statistical problem of having a large number of empty terminals in this study I rehydrated the animals for 4 days following 8 days dehydration to allow reaccumulation of granules, but not enough time for the morphological changes to return to the basal level (Tweedle 8 Hatton 87b) Previously, I have reported that the maximal VP and OT granule size are 175 nm and 215 nm respectively in the SON somata (Marzban et. al., 1992). In the 10 58 days dehydrated study of PP, the OT positive granules were also relatively larger in size and paler in appearance, whereas the VP granules were smaller size and stained more darkly peripherally and have a lighter central appearance (Fig.4). These samples were not taken from a defined plane of PP but rather the sections were taken randomly from a portion of the entire PP. In this study the sections were taken only at the mid-coronal plane in an effort to collect both preferentially medic-ventrally located VP neurons and dorsally located OT neurons (Vandesande, F. and Dierickx, K., 1975; van Leeuwen et. al., 1979; Redecker, P. and Hoffman, K.,1988). VP axons and terminals contained smaller size granules. The number of axons and terminals with larger granules did not seem to be as frequent as in our previous PP 10-day dehydrated study and did not stain for OT. However, in both the control and dehydrated tissue, the identified OT terminals contained smaller sized granules (Fig.3) whereas the larger granules did not usually stain for either OT or VP. Castel and her colleagues (1986) reported that altering treatments in tissue preparation (e.g, osmication) may change the diameters and the electron density of the granules. They reported larger granules stained in rat and mice PP. They used free-handcutting of the PP and embedded the tissues in a different resin than that used in the present study. There are also other reports of sizes of granular diameters which do not match the earlier Castel et. al. report. Finally the labelling of neither VP nor OT was as dense as in the 10 days of dehydration. Table 2 shows the similarity of the results collected for both VP and OT staining. There are three possible answers. 1) The VP and OT neurons may share VP and OT granules in the central region of PP. Mezey (1991) reported that in lactating rats, SON neurons coexpress for VP and OT and neurosecretory granules in the nerve endings in the PP stain for both hormones. 2) Midcoronally located small sized OT granules in PP may originate from PVN or different areas than the middle region of SON where OT hormones may be housed in the smaller granules. Krisch (1974) reported that the 59 median diameters of the granules in the secretory cells of PVN are significantly smaller than those of SON. 3) The techniques used in the two studies of PP may be different, resulting in change the sizes of the granules holding OT hormones. The only alterations in the techniques between this study and our previous 10 days dehydrated studies of SON and PP was that acetone, instead of propylene oxide, was used prior to embedding in Epon and the osmication period was increased from one hour to one and half hours. Using acetone in the embedding processing of tissues in 10 days dehydrated animals made lipid droplets more susceptible to the incubating solutions. lncubating the tissues for several days in the Ab solutions damaged the attachment of lipid droplets to the surrounding cytoplasmic matrix. This could cause the tissue to become more sensitive to the electron beam and the holes made by separation of the lipid droplets from the surrounding cytoplasm become larger and the sections become stretched out and damaged. Therefore collecting correct measurements could become more difficult. It is hard to be certain what difference using acetone makes since both acetone and propylene oxide are strong oxidizing agents. Probably,leither using propylene oxide or longer osmication changes the granule size, These chemicals may also alter the conformation of some percentage of the hormonal structure, somehow to block the binding sites of the hormones to the Abs since the labelling density of the granules was not as high as the 10 days dehydration study. DISCUSSION: During dehydration plasma VP increases (Kasting, 88, Robertson & Athar, 76; Sladek & Knigge, 77; Anderson, 87, Jones & Pickering, 69; Januszewicz et al., 86; Hattori et al., 88) to accomodate water loss by conserving water excretion at the level of the kidney collecting tubules. Plasma OT also increases during water loss (Kasting, 88). There are reports that OT produces natriuresis (Balment et al. 1980; Verbalis, 91 ), lowers sodium appetite (Stricker, 87a, 87b) and also mediates VP effects on V2 60 receptors in renal medullary collecting tubules (Teitelbaum, 1991). It is likely that the HNS changes morphologically in order to reach the maximal hormonal secretion at the time of high demand as during dehydration. Tweedle and Hatton (1987) reported an increase in axo-capillary contact, a decrease in the pituicyte-capillary contact and fewer axons engulfment by pituicytes in 10-day dehydrated rats PP. Such enlarged terminal contact onto the basal lamina is probably the main site of hormone release (Tweedle & Hatton, 1987) while other possibilities certainly exist for non-terminal release (Pow & Morris, 89; Morris 8 Pow, 91). These morphological changes gradually return to normal after a period of rehydration (Wittkowski & Brinkmann, 74; Tweedle & Hatton, 80a, 80b; Tweedle, 87a; Tweedle & Hatton, 87b; Perlmutter et al., 84). In these studies, it is not clear if either OT and/or VP nerve terminals participate in such neural plasticity. Previously, we reported that these changes in the SON include both OT and VP neurons in the SON (Marzban et. al., 1992). Now the present study addresses that the morphological changes at the PP during dehydration also includes both OT and VP terminals. It suggests that during dehydration when both OT and VP secretion increases there are a greater length of axo-capillary apposition and a lesser percentage of glia-capillary apposition to allow the terminals to have a free access, without any barriers (pituicytes), to release their hormones to the circulatory system. This increase in axe-capillary apposition could be formed by either astrocytic retraction, where astrocytic process move away from inter-axo-capillary space, and allow a greater contact of axons with capillary and/or 2) axonal enlargement and sprouting, while capillaries are increasing in size, more chance of axo-capillary contact can be furnished. It is not clearly understood how freeing some axons from pituicytic engulfment might have any effect on the hormonal secretion during dehydration while such larger number of axons are not wrapped around by astrocytes. Close apposition release of neuropeptides from axons along pituicytes has been reported (Morris & Pow, 1991) 61 and it has been also suggested that pituicytes can alter the extracellular ionic concentrations (Wuttke, 1990). These might suggest two ways communication between axons and pituicytes. The axons engulfed by pituicytes may directly control pituicyte activities via releasing opioid peptides and/or maybe those axons are the prime axons, from which other axonal branches are originated, and pituicytes may adjust their activities via changing extracellular ionic concentrations. These results suggest that, with greater demands for hormonal secretion during dehydration and recovery, both VP and OT terminals make greater contact with perivascular spaces and fewer VP and OT axons are engulfed by pituicytes in dehydrated animals. We therefore conclude that these morphological changes are not exclusive to one type of terminal but rather these changes allow both VP and OT terminals to have a greater access to capillaries to release their hormones into circulatory system. 62 Table 2. Effects of chronic dehydration on the oxytocin and vasopressin terminal and pituicyte apposition in the PP. Values shown represent the means iS.E.M.. 1] p<0.05, * p<0.03, ** p<0.01 significantly different from the control. 63 .226ch S 886% 302332 3 68:63 23:78.. A+v $8 3 $522,252 .6580 .m> deaI .modva. .modvaF .zmmw :88 9: mm 889qu 2m 33 8.38.0 .5833 .38; At 8.08 S 8.98 E .28 5 Imm. 32.3 .28; 8.33.0 ImOMHemée 8mm; 3 HO 323.8 .28 m 2.3.3.0 0380.8 .28 B .8388 3688.2 .22., 3 8.03 to Ne. 33.... .28 5 8.38.0 3033 .222. g 8.88.0 N P. .83 .28 a> .mmxfl 5.3V .28; Immodueod .. 533.3 88:. A+V m> $2302 .28 $8320 2830.2 .28 a> 88.0.... ho $838 .28; 3 9.0.83.0 mm. 33.2 .28 .3 83:8 8.8553298 58:8 .2832 as 3528 28-92 x. m._.mImo U_zOmIU no 953% N 59: 64 Fig. 10. A. Low power electron micrograph of the neurohypophysis. A pituicyte (Pit) can be seen to be surrounded by many VP-positive (p) and negative (n) neurosecretory processes. Note that around the capillary (Cap), there are VP-positive (arrow heads) and negative (arrows) terminals apposing the perivascular space. Pituicyte-capillary apposition is shown with dashed lines. B. Higher micrograph of 1A (inset) showing the specificity of the labeling. Bar = tum 65 66 67 Fig.11. A. Area of the neurohypophysis of a control rat. B. High power electron micrograph of a pituicyte (P) (2A inset), has two VP-positive neurosecretory axons (p) enclosed within its cytoplasm. C. High power electron micrograph of 2A (inset) showing a VP (-) terminal (n) apposing a capillary. Bar = tum 68 i»... e . 69 fi 70 71 Fig. 12. Micrograph of two terminals one stained positively for OT (P) apposing (arrows) a capillary and one did not stain for OT (n) and also apposing (arrow heads) the capillary. Pituicyte-capillary apposition is shown with dashed line. Bar = tum 72 73 Fig. 13. Micrographs of 10 days dehydrated animal. A. Stained for VP. Note that the labelled granules are clear in the center. B. Stained for OT. Note that the labelled granules are pale in color. Also note the. empty terminals (*) around the capillary (Cap.). Bar = tum 74 75 76 CONCLUDING REMARKS VP ,an antidiuretic hormone, helps to conserve water in case of water loss and increasing of plasma osmolality. OT, in addition to being involved in uterine contraction and milk ejection, is an antidiuretic hormone (Balment et.al., 1980,1982) and a mediator to VP2 receptors in renal medullary collecting ducts (Teitelbaum, 1991). VP and OT hormones are synthesized in separate magnocellur somata in the SON and PVN of HNS. These hormones are transported intra-axonally to PP where either stored in the Herring bodies or move to readily secreted sites in the terminals. HNS morphologically adjusts itself, at somatic, dendritic and terminal sites, to become more efficient in synthesizing and secreting VP and OT hormones during different physiological stimuli such as dehydration. These morphological adjustments are called plasticity. MNC plasticity in SON has been studied in depth in dehydrated rats (Hatton & Walters, 1973; Tweedle & Hatton, 1977; Tweedle & Hatton, 1984; Modney, Hatton, 1989) and are as such: 1) the somatic size increases, 2) soma- somatic/dendritic contact increases 3) axo-somatic contact increases 4) number and percentage of M83 contact increases, 5) number of cells with dilated rough endoplasmic reticulum increases, 6) number of multiple nucleoli increases and 7) number of lysosomes per cell decreases. Tweedle (1983) reviewed the ultrastructural plasticity of PP of dehydrated rats. During dehydration, 1) axo-capillary apposition increases, 2) pituicyte-capillary apposition decreases and 3) number of axons enclosed by pituicytes decreases. 77 It is clear that both plasma VP and OT increases during dehydration (Kasting, 88; Robertson & Athar, 76; Sladek & Knigge, 77; Anderson, 87, Jones & Pickering, 69; Januszewicz et al., 86; Hattori et al., 88). However it is not apparent that if only OT and/or VP magnocellular neurons and their terminals in the SON and PP respectively are involved in the ultrastructural plasticity in dehydrated rats. A post-embedding immunogold cytochemistry technique was used to label VP and OT magnocellular neurons in the SON and their terminals in the PP using Abs for glycopeptide of VP prohormone and neurophysin of OT prohormone. To study SON MNC, rats were given 2% saline to drink for 10 days. Whereas PP plasticity was studied on rats given 2% saline for 8 days followed by 4 days of rehydration to accumulate granules dense enough in the terminals to be detected while the morphological plasticity does not return back to base line completely. The results were compared to control animals which had free access to water. It is also clearly shown that magnocellular neurons in SON are electrically coupled (Yang 8 Hatton, 1988). This electrical communication may occur either through direct membrane apposition found between the neurons and maybe initiated by multiple synaptic terminals apposing more than one soma. Hatton et al. (87,88) reported that lactation, a strong stimulation to HNS, increases the number of the electrically coupled MNC in the SON parallel with an increase in soma- somatic/dendritic membrane apposition and number of M85. Hatton et al. (1987) and Cobbet et al. 1985) also reported that electrical coupling are exclusively homotypic (only between OTs or VPs neurons). Experiment I suggests that either three possible combinations between OT and VP neuronal membrane apposition can happen. Therefore, membrane apposition may not seem to be the causal factor of electrical coupling between one or the other type of neurons. On the other hand, MSs are exclusively homotypic which means MSs apposition can be found only onto two OT neurons or tonP neurons. These results strongly suggest that MSs may be the main 78 factor to cause synchronization between the homotypic magnocellular neurons. Therefore, soma-somatic/dendritic contact may also play a role in synchronizing the magnocellular cells of SON as a whole. HNS receives axonal inputs from different sources of sensory centers of the brain (Cunningham et al. 1991). AV3V is the best candidate to detect plasma osmotic changes and relay the information to HNS. It is important to emphasis that changing plasma osmolality either via water deprivation or drinking salt water may also indirectly change the cardiac output and stimulate baroreceptor, chemoreceptor, the release of renin from kidney and increase plasma Agll. Since HNS receives afferent fibers from baro- and chemosensory catecholaminergic medullary nuclei (A1 and A2) and from Agll sensory SFO, one can not exclude these important sources of afferent fibers to HNS during dehydration. It is not fully understood all the interneuronal pathways that connect AV3V, SFO and A1 and A2 medullary nuclei to HNS and how many types of neurotransmitter these nuclei and the interneurons might have on HNS. At least two types of M83 might be involved in apposing the magnocellular neurons are NE (Tweedle and Hatton, 1984a) and GABAergic terminals (Theodosis et al. 1986a). During dehydration the number of M85 only increases between OT neurons. Since the contents and the sources of these MSs are unknown it is hard to ascertain what physiological effects these MSs might have postsynaptically. But it suggests that these newly formed MSs either stimulate, inhibit or act as a modulator control on only OT neuronal activity. It is important for HNS to have this type of mechanism to adjust VP and OT secretion independently during different types of stimuli such as lactation, dehydration and hypotension. The increase in the percentage of soma-somatic/dendritic membrane apposition is inversely related to a decrease in percentage of glia-somatic membrane apposition of both VP and OT neurons in the dehydrated rats. Since the VP and OT neural size increases during dehydration, one can not conclude that there is a 79 retraction of interneuronal astrocytic processes from decreasing in glia-somatic membrane contact. But newly formed M83 and axonal contact may be the result of both enlargement of cellular size ( Modney and Hatton, 1989) and/or retraction of the astrocytic processes from certain areas of the neural surface (Hatton, 1985) to allow certain contacts to form. At the terminals, during dehydration when there is a higher demand of both OT and VP hormonal secretion, experiment ll found that both OT and VP terminal membranes have a larger apposition to the perivascular space, whereas percentage of pituicyte-capillary contact was reduced. The increase of the contact between terminals and the capillaries allows a higher quantity of the VP and OT secreted hormones reach the neurohypophysial portal system easily. The increase of the axo- capillary apposition in PP may be formed as the result of pituicytes retraction (Tweedle, 1983). Also experiment It suggest that number of both VP and OT axons enclosed by pituicytes decreases in dehydrated rats. It is not clear enough why pituicytes engulf only few number of axons while the majority of swellings and axons are free. Two possible answers to this are that the axons engulfed by pituicytes might be 1) those major branches from which peripheral branches are originated and their activities are being controlled by the pituicytes or 2) those axons having a direct input on the pituicytes engulfing the axons to manipulate the movements of the pituicytic processes. Based on 1) double synaptic contact only occurring between similar neurons in SON 2) differential OT and VP hormonal release under various physiological stimuli, 3) topographical arrangement of VP and OT magnocellular neurons in SON and PVN and their terminals in PP, respectively and 4) differential organization of thefafferent inputs to VP and OT neurons in SON and PVN, we suggest that even though the VP and OT neural activity seems to be in some synchrony synthesizing and secreting hormones, the VP and OT neuronal systems are controlled by two different neural 80 pathways in order to secrete their hormones differently under various physiological stimuli. 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