Brain Research, 554 (1991) 293-298 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391168113
293
BRES 16811
The origin of somatostatin-containing nerve fibers innervating the hypothalamic supraoptic nucleus l~va Mezey 1, Csaba L6r~inth 2, Robert Eskay 3, S~indor Horv~ith I and Mikl6s Palkovits 1 1NIMH, LCB, Bethesda, MD (U.S.A.) and 1st Department of Anatomy, Semmelweis University Medical School, Budapest (Hungary), 2yale University, Department of Obstetrics and Gynaecology, New Haven (U.S.A.) and 3NIAAA, LCS, Bethesda, MD (U.S.A.) (Accepted 26 February 1991)
Key words: Somatostatin; Vasopressin; Supraoptic nucleus
Light and electron microscopic studies were performed to study the connections between somatostatin (SOS)-containing nerve terminals and vasopressin (VP)-containing neurons in the rat supraoptic nucleus (SON). SOS-positive fibers innervate the SON in both the oxytocinergic and vasopressinergic areas. Using double immunostaining symmetric synaptic contacts were visualized between SOS immunoreactive boutons and the soma of VP immunopositive neurons. Surgical transection deafferentating the SON from all possible directions do not effect the presence of SOS immunopositive fibers. These results suggest a local origin of the SOS fibers. Somatostatin-containing perikarya can indeed be found at the dorsal border of the SON at the rostral and caudal pole of the nucleus - we suggest that these cells innervate the SON. The presence of synaptic contacts between SOS fibers and VP neurons as well as the lack of these fibers in the VP deficient Brattleboro rats indicate a role for SOS in the synthesis and/or release of vasopressin in the SON.
INTRODUCTION
MATERIALS AND METHODS
S o m a t o s t a t i n ( S O S ) has b e e n d i s c o v e r e d a l m o s t 20 years ago 3"24 as the m a j o r i n h i b i t o r y n e u r o p e p t i d e in the
Adult (200-250 g) male Sprague-Dawley (NIH, Taconic Farm), Brattleboro and Long-Evans (Blue Spruce Farm) rats were used in all studies. The animals were kept under standard laboratory conditions.
h y p o t h a l a m u s . Since its d i s c o v e r y m a n y studies h a v e b e e n d o n e o n its b i o c h e m i c a l 6 and h i s t o c h e m i c a l 2'9' 13-15,25,26 d i s t r i b u t i o n d e m o n s t r a t i n g its w i d e p r e s e n c e in the rat c e n t r a l n e r v o u s system. T h e best k n o w n role for S O S is to inhibit the r e l e a s e of g r o w t h h o r m o n e f r o m the a n t e r i o r pituitary, but a bulk of e l e c t r o p h y s i o l o g i c a l d a t a indicates its n e u r o t r a n s m i t t e r role as well 2°. S o m a t o s t a t i n h a v e also b e e n s h o w n to i n f l u e n c e h o r m o n e r e l e a s e in the C N S i n c l u d i n g the r e l e a s e o f v a s o p r e s s i n 4,5,19,26. This effect m a y
be
indirect
(by affecting o t h e r
neuronal
systems which in t u r n affect v a s o p r e s s i n secretion) o r direct (by synaptic c o n t a c t s b e t w e e n s o m a t o s t a t i n axons and v a s o p r e s s i n - p o s i t i v e n e u r o n a l e l e m e n t s ) . T h e prese n c e of s o m a t o s t a t i n has b e e n p r o v e d in the h y p o t h a l a m i c s u p r a o p t i c n u c l e u s ( S O N ) with b o t h b i o c h e m i c a l 6 ,and h i s t o c h e m i c a l 8,13,22 m e t h o d s . In this w o r k we a i m e d to d e t e r m i n e the p r e s e n c e (or lack) of synaptic c o n t a c t s b e t w e e n S O S - and vasopressinp o s i t i v e n e u r o n a l e l e m e n t s in the rat S O N by e l e c t r o n m i c r o s c o p y . F u r t h e r m o r e , we u s e d surgical t e c h n i q u e s to localize the origin of t h e s o m a t o s t a t i n - i m m u n o p o s i t i v e fibers in the S O N .
Surgical procedures Rats were anaesthetized with ether and their heads were f'Lxedin a stereotaxic device in a 5° nose-down position. Four different types of surgical cuts were performed in different groups of animals (n = 4 per group). Knife cuts were performed with a variety of 'glass knives' with 2.5-4 mm cutting edge made of histological coverslips. The stereotaxic parameters were measured on the skull where a thin line shaped cut was drilled to fit the size of the knife. The glass knives were then lowered into the brain far enough to touch the base of the skull. All cuts were unilateral. The knife cuts isolated the supraoptic nucleus from all possible directions of potential neural inputs (Fig. 1). Cut no. 1. A 4 mm wide mediolaterally-oriented oblique cut was made between the level of the bregma and 4 mm caudal to it. This cut that runs dorsal and lateral from the SON separates the nucleus from ipsilateral fibers coming from the direction of the forebrain (Fig. 1B and C). Cut no. 2. This is a 2.5 mm wide rostral coronal cut at the level of the bregma. Fibers coming from all brain areas rostral to the SON are disconnected by this transection (Fig. 1A and C). Cut no. 3. Parasagittal cut placed along the third ventricle at 0.5 mm from the midline. The 4 mm long knife cut runs caudalwards from the bregma level. The surgery separates the major periventricular hypothalamic somatostatin cell group from the SON (Fig. 1B and C). Cut no. 4. Coronal plane section at the caudal hypothalamic level (between 3.6 and 4.0 mm caudal to the bregma). This 2.5 mm wide
Correspondence: E. Mezey, NIMH, LCB bldg. 36, 3A17, Bethesda, MD 20892, U.S.A.
294 cut - starting at the midline - separates the SON from ipsilateral fibers ascending from the lower brainstem (Fig. 1A and C). Only rats where the cut reached the base of the brain were used for further evaluation. The animals were sacrificed on the 14th postoperative day.
lmmunohistochemistry Control, surgically transected and colchicine-treated (90/~g/rat, administered intraventricularly, 48 h survival time) animals were anaesthetized with ether and perfused through the ascending aorta with 4% paraformaldehyde and 0.75% picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Coronal 40/~m thick sections were cut with a vibratome and immunostained using a rabbit anti-somatostatin antibody raised against SOS14 (R85/722 - a gift from T. G6rcs, Budapest) that have been used earlier ~°. The antibody recognizes both the hypothalamic and the brainstem somatostatin systems. Absorption of the working (1:2000) dilution of the antibody with 5 /~g/ml somatostatin peptide eliminated the staining. The primary antibody was visualized using the avidin-biotin-peroxidase technique ~2 on free floating sections.
Electron microscopy Tissue preparation. Rats were sacrificed under ether anesthesia by transcardial perfusion of 10 ml physiological saline followed by first 80 ml of the following fixative: 3.75% acrolein and 2% paraformaldehyde in 0.1 M (pH 7.35) phosphate buffer (PB). To improve the uitrastructure the rats were then perfused with 200 ml of 2% paraformaldehyde alone and the dissected hypothalami were postfixed in the last fixative for an additional 2 h at 4 °C. Coronal plane 40 ~m thick sections were cut with a vibratome and the sections were rinsed in PB for 2 h. Then the sections were placed in 10% sucrose (in PB) for cryoprotection and frozen in liquid nitrogen then thawed at 4 °C - to improve antibody penetration. The sections were then soaked in 1% sodium borohydride 16 and rinsed in PB.
B
Double immunostaining. The procedure used has been reported previously 17'18. Briefly the sections were first incubated with a 1:4000 dilution of a monoclonal antibody raised against SOSIJ. At the end of the incubation the antibody was visualized using the avidin-biotin-peroxidase technique 12 with the ABC kit of Vector Laboratories (Burlingame, CA). After several rinses in PB the sections were then incubated with a 1:1500 dilution of a rabbit polyclonal antiserum against vasopressin. The specificity of this antiserum - called VA4 ~ - have been reported. At the end of the primary incubation the antibody was visualized with a 1:15 dilution of goat anti-rabbit IgG conjugated to 5 nm gold particles (Polysciences, Warrington, PA). After rinsing the sections were postfixed in 1% OsO 4 (in PB), dehydrated (the 70% ethanol contained 1% uranyl acetate for 30 min) and embedded in resin. The method is rather specific, usually - unlike postembedding methods - there is no background staining with the gold particles TM. To control the vasopressin immunostaining at the light microscopic level a few sections were removed after the primary incubation and incubated with rabbit PAP (1:80 dilution for 2 hours at room temperature) and developed using diaminobenzidine as a substrate.
RESULTS In c o n t r o l S p r a g u e - D a w l e y
r a t s t h e r e is a m o d e r a t e l y
d e n s e S O S i n n e r v a t i o n o f t h e s u p r a o p t i c n u c l e u s (Fig. 2). T h e n e t w o r k o f f i b e r s a r e l o c a l i z e d m o s t l y in t h e v e n t r a l portion of the nucleus where the vasopressin producing neurons
are
l o c a t e d 11'21'23.
A
somewhat
less
dense
n e t w o r k c a n b e o b s e r v e d in t h e d o r s a l p o r t i o n o f t h e
C
Fig. 1. Surgical knife cuts transecting neuronal inputs to the supraoptic nucleus. A: midsagittal; B: coronal; and C: horizontal schematic drawings of the rat forebrain indicating the surgeries performed. Transections are labelled with numbers: 1 = lateral (oblique) cut; 2 = rostral preoptic-hypothalamic; 3 = paramedian sagittal cut between the periventricular area and supraoptic nucleus; 4 = caudal hypothalamic (medial forebrain bundle) cut. Abbreviations: AA, anterior amygdaloid area; AL, lateral amygdaloid nucleus; AM, medial amygdaloid nucleus; C, claustrum; EC, entorhinal cortex; F, fornix; HI, hippocampus; M, midbrain; MB, mammillary body; OC, optic chiasm; OT, optic tract; P, preoptic area; S, supraoptic nucleus (solid area in Fig. 1C); ST, stria terminalis; Th, thalamus.
295 nucleus corresponding to the oxytocin cells. In addition to the network that is present there are also occasional very long beaded SOS-immunopositive axons present in the nucleus (Fig. 2A). Our electron microscopical results show that these somatostatin-containing fibers make axo-somatic symmetrical synapses in the SON with the vasopressin neurons (Fig. 3). Although we did not do immunostaining for oxytocin, we presumed that the non-stained magnocellular neurons are oxytocinergic in character. Somatostatin-immunopositive terminals also make synaptic contacts with these non-labelled neurons (not shown). The presence of the somatostatin fibers could not be influenced by surgical transections that disconnected the supraoptic nucleus from the main hypothalamic periventricular or the paraventricular nuclei somatostatin system (Fig. 1, Cut no. 3); somatostatin neurons in the lateral forebrain (Fig. 1, Cut no. 2); the somatostatin cells in the
A
amygdala (by transecting the stria terminalis) (Fig. 1, Cut nos. 1 and 3); the brainstem somatostatin (by a coronal cut at the caudal hypothalamic level (Fig. 1, Cut no. 4). Two weeks after all the above surgical procedures the SOS innervation of the supraoptic nucleus have remained practically unchanged. In colchicine-treated rats we saw a small number (2-3/section) of somatostatin-positive cell bodies around the rostral pole and dorsal to the SON (Fig. 4). These cells are medium sized, multipolar and are not part of the magnocellular system. In a few sections we could follow long beaded-like axons from these neurons entering the SON. We did not observe any SOS-containing magnocellular neurons in the SON itself. In homozygous Brattleboro rats (a strain that genetically is unable to make vasopressin and develops diabetes insipidus) we could hardly detect somatostatin-immunopositive nerve fibers and terminals in the SON (Fig. 5). In the control Long-Evans rats the innervation was similar to that seen in Sprague-Dawley rats (not shown). DISCUSSION Somatostatin has been shown to be present in the rat
hypothalamic SON 6,s,13,22. The most likely source of this
B
Fig. 2. Somatostatin-immunoreactive network of fibers and terminals in the rat hypothalamic supraoptic nucleus in a 40/~m thick coronal vibratome section. OC, optic chiasm. Bars = 200/zm.
is the main hypothalamic somatostatin periventricular system. Transecting the fibers from the periventricular nucleus (medial Cut no. 2), however, left the SON SOS innervation unchanged. Similarly, knife cuts lateral to the SON did not have an effect on the SON SOS content either - excluding the somatostatin - containing cells in the limbic system (Cut. no. 1) and cerebral cortex (Cut nos. 1 and 2) as a positive source. Brainstem somatostatin-containing neurons project to the SON through pathways cut by the caudal coronal cut (no. 4). This cut did not have an effect on the SON SOS fiber density either, the brainstem can also be excluded as the major source towards the SON (although these cells do project to the PVN22). The resistence of the innervation to change following depletion of outside inputs indicated a local source. Since - like earlier data suggested s,13,22 - we could not demonstrate somatostatin-positive perikarya inside the SON itself we concluded that the only possible source must be the few somatostatin cells that are consistently present around the dorsal border of the rostral and caudal pole of the nucleus (Fig. 4). The double immunostaining at the EM level showed that there are synaptic contacts between the somatostatin nerve terminals and vasopressin neurons and dendrites in the SON. These synapses would supply the morphological basis for a direct effect of SOS on vasopressin synthesis and/or release. Sawchenko et al.22 demonstrated the presence of SOS-immunopositive fibers in the
296 SON, but mostly in the dorsal portion that corresponds to oxytocin containing neurons rather than vasopressincontaining ones. In our studies we have seen those fibers as well as synaptic contacts between the SOS terminals and the oxytocin cells. A l t h o u g h the present study was designed to study the effect of SOS on vasopressin we do not exclude the effect on oxytocin neurons. Most likely both populations are effected by somatostatin. The different distribution of SOS fibers in the B r a t t l e b o r o versus the L o n g - E v a n s (control) rat also indicates that
there is an interaction between the somatostatin and the vasopressin systems in the SON. Sawchenko et al. 22 in their study also mention the brainstem as the most likely source of the SON somatostatin innervation. However, their tract tracing studies were c o n c e n t r a t e d on the PVN magnocellular cells - that do receive somatostatin from the brainstem. They only suggested that the SON magnocellular neurons would also be likely to be innervated by the same cells. O u r present results do not support that hypothesis, though.
Fig. 3. Electron micrographs show a double-immunostaining for somatostatin and vasopressin in the rat supraoptic nucleus. Immunoreactivity for somatostatin was visualized by an immunoperoxidase reaction, while vasopressin-containing elements were labelled with 5-nm diameter gold particles. Symmetric synaptic connections (arrowheads) formed between immunoperoxidase labelled somatostatin immunoreactive boutons with the soma of gold particles (small arrows) containing vasopressin immunoreactive neurons. OC, optic chiasm. Bars = 1 /~m.
297
OC
m
Fig. 5. Somatostatin-containing nerve terminals in the supraoptic nucleus of a Brattleboro rat. OC, optic chiasm. The solid arrows indicate the dorsal portion of the supraoptic nucleus with somatostatin-immunoreactive fibers. The empty arrows point to the ventral region with no immunostaining. Bars = 50/~m.
OC
matostatin in sheep. Intracerebroventricular injection of SOS also blocks VP release 4'5. Recent data indicate that in isolated nerve endings of the rat n e u r o n a l lobe
Fig. 4. Somatostatin-containing cells in the rostral pole of the supraoptic nucleus of rats with surgical transection. A: midsagittal cut; B" rostral cut. OC, optic chiasm. Bars = 100/zm.
somatostatin inhibits high potassium-induced secretion of both oxytocin and vasopressin 19. The somatostatin fibers in the SON may have a similar function and may thus participate in the fine regulation of the release of magnocellular hormones.
The exact physiological role for somatostatin in vasopressin release is not yet clear. Wang et al. 26 demonstrated that haemorrhage-induced vasopressin release can be blocked by intraventricularly administered so-
Acknowledgements. Cs.L. is being supported by the following Grants: NIH - NF26068 and NIH - HD23830.
REFERENCES
8 Dierickx, K. and Vandesande, F., Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus, Cell Tissue Res., 201 (1979) 349-359• 9 Finley, J.C.W., Maderdrut, J.L., Roger, L.J. and Petrusz, P., The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system, Neuroscience, 6 (1981) 2173-2192. 10 Horv~lth,S., Palkovits, M., G6rcs, T. and Arimura, A., Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormonereleasing hormone- and somatostatin-containing neurons in the hypothalamus of the rat, Brain Research, 481 (1989) 8-15. 11 Hou-Yu, A., Lamme, A.T., Zimmerman, E.A. and Silverman, A.J., Comparative distribution of vasopressin and oxytocin neurons in the rat brain and spinal cord, Neuroendocrinology, 44 (1986) 235-246. 12 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 13 Johansson, O., H6kfelt, T. and Elde, R.P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat, Neuroscience, 13 (1984) 265-339• 14 Kawano, H., Daikoku, S. and Saito, S., Immunohistochemical
1 Altstein, M., Whitnall, M.H., House, S., Key, S. and Gainer, H., An immunocytochemical analysis of oxytocin and vasopressin prohormone processing in vivo, Peptides, 9 (1988) 87-105• 2 Bennett-Clarke, C., Romagnano, M.A. and Joseph, S.A., Distribution of somatostatin in the rat brain: telencephaion and diencephalon, Brain Research, 188 (1980) 473-486• 3 Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. and Guillemin, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 4 Brown, M.R., Somatostatin-28 effects on central nervous system regulation of vasopressin secretion and blood pressure, Neuroendocrinology, 47 (1988) 556-562. 5 Brown, M.R., Mortrud, M., Crum, R. and Sawchenko, P., Role of somatostatin in the regulation of vasopressin secretion, Brain Research, 452 (1988) 212-218. 6 Brownstein, M.J., Arimura, A., Sato, H., Schally, A.V. and Kizer, J.S., The regional distribution of somatostatin in the rat brain, Endocrinology, 96 (1975) 1456-1461. 7 Buchan, A.M.J., Sikora, L.K.J., Levy, J.G., McIntosh, C.H.S., Dyck, I. and Brown, J.C., An immunocytochemical investigation with monoclonal antibodies to somatostatin, Histochemistry, 83 (1985) 175-180•
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studies of intrahypothalamic somatostatin-containing neurons in rat, Brain Research, 242 (1982) 227-232. Kiyama, H. and Emson, P.C., Distribution of somatostatin mRNA in the rat nervous system as visualized by a novelnon-radioactive in situ hybridization histochemistry procedure, Neuroscience, 38 (1990) 223-244. Kosaka, T., Nagatsu, I., Wu, J.Y. and Hama, K., Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter synthesizing enzymes in the central nervous system, Neuroscience, 18 (1986) 975-990. LEr~nth, C. and Frotscher, M., Organization of the septal region in the rat brain: cholinergic-GABA-ergic interconnections and the termination of hippocampo-septal fibers, J. Cornp. Neurol., 289 (1989) 304-414. L6r~inth, C., Maclusky, N.J., Shanabrough, M. and Naftolin, E, Catecholaminergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic area, Neuroendocrinology, 48 (1988) 591-602. Payza, K. and Russel, J., Presynaptic modulation of oxytocin and vasopressin secretion from isolated nerve terminals of the rat neural lobe, Ann. N.Y. Acad. Sci., 604 (1990) 632-634. Reichlin, S., Somatostatin. In D.T. Krieger, M.J. Brownstein and J.B. Martin (Eds.), Brain Peptides, Wiley, New York/
Chichester/Brisbane/Toronto/Singapore, 1983, pp. 711-752. 21 Rhodes, C.H., MoreU, J.l. and Pfaff, D.W., lmmunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and number of cells containing neurophysin, oxytocin and vasopressin, J. Comp. Neurol., 223 (1981) 556-582. 22 Sawchenko, P.E., Benoit, R. and Brown, M.R., Somatostatin 28-immunoreactive inputs to the paraventricular and supraoptic nuclei: principal origin from non-aminergic neurons in the nucleus of the solitary tract, J. Chem. Neuroanat.. 1 (1988) 81-94. 23 Sofroniew, M.V., Vasopressin, oxytocin and their related ncurophysins. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatorny. GABA and Neuropeptides in the CNS. Part 1, Elsevier, Amsterdam, 1985, pp. 93-165. 24 Vale, W., Brazeau, P., Rivier, C., Grant, G., Burgus, R. and Guillemin, R., Inhibitory hypophysiotropic activities of hypothalamic somatostatin, Fed. Proc., 32 (1973) 211. 25 Vincent, S.R., Mclntosh, C.H.S., Buchan, A.M.J. and Brown, J.C., Central somatostatin systems revealed with monoclonal antibodies, J. Cornp. Neurol., 238 (1985) 169-186. 26 Wang, X., Tresham, J.J., Congiv, M. and Goghlan, J.P., Somatostatin centrally inhibits vasopressin secretion during haemorrhage, Brain Research, 436 (1987) 199-203.
293
BRES 16811
The origin of somatostatin-containing nerve fibers innervating the hypothalamic supraoptic nucleus l~va Mezey 1, Csaba L6r~inth 2, Robert Eskay 3, S~indor Horv~ith I and Mikl6s Palkovits 1 1NIMH, LCB, Bethesda, MD (U.S.A.) and 1st Department of Anatomy, Semmelweis University Medical School, Budapest (Hungary), 2yale University, Department of Obstetrics and Gynaecology, New Haven (U.S.A.) and 3NIAAA, LCS, Bethesda, MD (U.S.A.) (Accepted 26 February 1991)
Key words: Somatostatin; Vasopressin; Supraoptic nucleus
Light and electron microscopic studies were performed to study the connections between somatostatin (SOS)-containing nerve terminals and vasopressin (VP)-containing neurons in the rat supraoptic nucleus (SON). SOS-positive fibers innervate the SON in both the oxytocinergic and vasopressinergic areas. Using double immunostaining symmetric synaptic contacts were visualized between SOS immunoreactive boutons and the soma of VP immunopositive neurons. Surgical transection deafferentating the SON from all possible directions do not effect the presence of SOS immunopositive fibers. These results suggest a local origin of the SOS fibers. Somatostatin-containing perikarya can indeed be found at the dorsal border of the SON at the rostral and caudal pole of the nucleus - we suggest that these cells innervate the SON. The presence of synaptic contacts between SOS fibers and VP neurons as well as the lack of these fibers in the VP deficient Brattleboro rats indicate a role for SOS in the synthesis and/or release of vasopressin in the SON.
INTRODUCTION
MATERIALS AND METHODS
S o m a t o s t a t i n ( S O S ) has b e e n d i s c o v e r e d a l m o s t 20 years ago 3"24 as the m a j o r i n h i b i t o r y n e u r o p e p t i d e in the
Adult (200-250 g) male Sprague-Dawley (NIH, Taconic Farm), Brattleboro and Long-Evans (Blue Spruce Farm) rats were used in all studies. The animals were kept under standard laboratory conditions.
h y p o t h a l a m u s . Since its d i s c o v e r y m a n y studies h a v e b e e n d o n e o n its b i o c h e m i c a l 6 and h i s t o c h e m i c a l 2'9' 13-15,25,26 d i s t r i b u t i o n d e m o n s t r a t i n g its w i d e p r e s e n c e in the rat c e n t r a l n e r v o u s system. T h e best k n o w n role for S O S is to inhibit the r e l e a s e of g r o w t h h o r m o n e f r o m the a n t e r i o r pituitary, but a bulk of e l e c t r o p h y s i o l o g i c a l d a t a indicates its n e u r o t r a n s m i t t e r role as well 2°. S o m a t o s t a t i n h a v e also b e e n s h o w n to i n f l u e n c e h o r m o n e r e l e a s e in the C N S i n c l u d i n g the r e l e a s e o f v a s o p r e s s i n 4,5,19,26. This effect m a y
be
indirect
(by affecting o t h e r
neuronal
systems which in t u r n affect v a s o p r e s s i n secretion) o r direct (by synaptic c o n t a c t s b e t w e e n s o m a t o s t a t i n axons and v a s o p r e s s i n - p o s i t i v e n e u r o n a l e l e m e n t s ) . T h e prese n c e of s o m a t o s t a t i n has b e e n p r o v e d in the h y p o t h a l a m i c s u p r a o p t i c n u c l e u s ( S O N ) with b o t h b i o c h e m i c a l 6 ,and h i s t o c h e m i c a l 8,13,22 m e t h o d s . In this w o r k we a i m e d to d e t e r m i n e the p r e s e n c e (or lack) of synaptic c o n t a c t s b e t w e e n S O S - and vasopressinp o s i t i v e n e u r o n a l e l e m e n t s in the rat S O N by e l e c t r o n m i c r o s c o p y . F u r t h e r m o r e , we u s e d surgical t e c h n i q u e s to localize the origin of t h e s o m a t o s t a t i n - i m m u n o p o s i t i v e fibers in the S O N .
Surgical procedures Rats were anaesthetized with ether and their heads were f'Lxedin a stereotaxic device in a 5° nose-down position. Four different types of surgical cuts were performed in different groups of animals (n = 4 per group). Knife cuts were performed with a variety of 'glass knives' with 2.5-4 mm cutting edge made of histological coverslips. The stereotaxic parameters were measured on the skull where a thin line shaped cut was drilled to fit the size of the knife. The glass knives were then lowered into the brain far enough to touch the base of the skull. All cuts were unilateral. The knife cuts isolated the supraoptic nucleus from all possible directions of potential neural inputs (Fig. 1). Cut no. 1. A 4 mm wide mediolaterally-oriented oblique cut was made between the level of the bregma and 4 mm caudal to it. This cut that runs dorsal and lateral from the SON separates the nucleus from ipsilateral fibers coming from the direction of the forebrain (Fig. 1B and C). Cut no. 2. This is a 2.5 mm wide rostral coronal cut at the level of the bregma. Fibers coming from all brain areas rostral to the SON are disconnected by this transection (Fig. 1A and C). Cut no. 3. Parasagittal cut placed along the third ventricle at 0.5 mm from the midline. The 4 mm long knife cut runs caudalwards from the bregma level. The surgery separates the major periventricular hypothalamic somatostatin cell group from the SON (Fig. 1B and C). Cut no. 4. Coronal plane section at the caudal hypothalamic level (between 3.6 and 4.0 mm caudal to the bregma). This 2.5 mm wide
Correspondence: E. Mezey, NIMH, LCB bldg. 36, 3A17, Bethesda, MD 20892, U.S.A.
294 cut - starting at the midline - separates the SON from ipsilateral fibers ascending from the lower brainstem (Fig. 1A and C). Only rats where the cut reached the base of the brain were used for further evaluation. The animals were sacrificed on the 14th postoperative day.
lmmunohistochemistry Control, surgically transected and colchicine-treated (90/~g/rat, administered intraventricularly, 48 h survival time) animals were anaesthetized with ether and perfused through the ascending aorta with 4% paraformaldehyde and 0.75% picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Coronal 40/~m thick sections were cut with a vibratome and immunostained using a rabbit anti-somatostatin antibody raised against SOS14 (R85/722 - a gift from T. G6rcs, Budapest) that have been used earlier ~°. The antibody recognizes both the hypothalamic and the brainstem somatostatin systems. Absorption of the working (1:2000) dilution of the antibody with 5 /~g/ml somatostatin peptide eliminated the staining. The primary antibody was visualized using the avidin-biotin-peroxidase technique ~2 on free floating sections.
Electron microscopy Tissue preparation. Rats were sacrificed under ether anesthesia by transcardial perfusion of 10 ml physiological saline followed by first 80 ml of the following fixative: 3.75% acrolein and 2% paraformaldehyde in 0.1 M (pH 7.35) phosphate buffer (PB). To improve the uitrastructure the rats were then perfused with 200 ml of 2% paraformaldehyde alone and the dissected hypothalami were postfixed in the last fixative for an additional 2 h at 4 °C. Coronal plane 40 ~m thick sections were cut with a vibratome and the sections were rinsed in PB for 2 h. Then the sections were placed in 10% sucrose (in PB) for cryoprotection and frozen in liquid nitrogen then thawed at 4 °C - to improve antibody penetration. The sections were then soaked in 1% sodium borohydride 16 and rinsed in PB.
B
Double immunostaining. The procedure used has been reported previously 17'18. Briefly the sections were first incubated with a 1:4000 dilution of a monoclonal antibody raised against SOSIJ. At the end of the incubation the antibody was visualized using the avidin-biotin-peroxidase technique 12 with the ABC kit of Vector Laboratories (Burlingame, CA). After several rinses in PB the sections were then incubated with a 1:1500 dilution of a rabbit polyclonal antiserum against vasopressin. The specificity of this antiserum - called VA4 ~ - have been reported. At the end of the primary incubation the antibody was visualized with a 1:15 dilution of goat anti-rabbit IgG conjugated to 5 nm gold particles (Polysciences, Warrington, PA). After rinsing the sections were postfixed in 1% OsO 4 (in PB), dehydrated (the 70% ethanol contained 1% uranyl acetate for 30 min) and embedded in resin. The method is rather specific, usually - unlike postembedding methods - there is no background staining with the gold particles TM. To control the vasopressin immunostaining at the light microscopic level a few sections were removed after the primary incubation and incubated with rabbit PAP (1:80 dilution for 2 hours at room temperature) and developed using diaminobenzidine as a substrate.
RESULTS In c o n t r o l S p r a g u e - D a w l e y
r a t s t h e r e is a m o d e r a t e l y
d e n s e S O S i n n e r v a t i o n o f t h e s u p r a o p t i c n u c l e u s (Fig. 2). T h e n e t w o r k o f f i b e r s a r e l o c a l i z e d m o s t l y in t h e v e n t r a l portion of the nucleus where the vasopressin producing neurons
are
l o c a t e d 11'21'23.
A
somewhat
less
dense
n e t w o r k c a n b e o b s e r v e d in t h e d o r s a l p o r t i o n o f t h e
C
Fig. 1. Surgical knife cuts transecting neuronal inputs to the supraoptic nucleus. A: midsagittal; B: coronal; and C: horizontal schematic drawings of the rat forebrain indicating the surgeries performed. Transections are labelled with numbers: 1 = lateral (oblique) cut; 2 = rostral preoptic-hypothalamic; 3 = paramedian sagittal cut between the periventricular area and supraoptic nucleus; 4 = caudal hypothalamic (medial forebrain bundle) cut. Abbreviations: AA, anterior amygdaloid area; AL, lateral amygdaloid nucleus; AM, medial amygdaloid nucleus; C, claustrum; EC, entorhinal cortex; F, fornix; HI, hippocampus; M, midbrain; MB, mammillary body; OC, optic chiasm; OT, optic tract; P, preoptic area; S, supraoptic nucleus (solid area in Fig. 1C); ST, stria terminalis; Th, thalamus.
295 nucleus corresponding to the oxytocin cells. In addition to the network that is present there are also occasional very long beaded SOS-immunopositive axons present in the nucleus (Fig. 2A). Our electron microscopical results show that these somatostatin-containing fibers make axo-somatic symmetrical synapses in the SON with the vasopressin neurons (Fig. 3). Although we did not do immunostaining for oxytocin, we presumed that the non-stained magnocellular neurons are oxytocinergic in character. Somatostatin-immunopositive terminals also make synaptic contacts with these non-labelled neurons (not shown). The presence of the somatostatin fibers could not be influenced by surgical transections that disconnected the supraoptic nucleus from the main hypothalamic periventricular or the paraventricular nuclei somatostatin system (Fig. 1, Cut no. 3); somatostatin neurons in the lateral forebrain (Fig. 1, Cut no. 2); the somatostatin cells in the
A
amygdala (by transecting the stria terminalis) (Fig. 1, Cut nos. 1 and 3); the brainstem somatostatin (by a coronal cut at the caudal hypothalamic level (Fig. 1, Cut no. 4). Two weeks after all the above surgical procedures the SOS innervation of the supraoptic nucleus have remained practically unchanged. In colchicine-treated rats we saw a small number (2-3/section) of somatostatin-positive cell bodies around the rostral pole and dorsal to the SON (Fig. 4). These cells are medium sized, multipolar and are not part of the magnocellular system. In a few sections we could follow long beaded-like axons from these neurons entering the SON. We did not observe any SOS-containing magnocellular neurons in the SON itself. In homozygous Brattleboro rats (a strain that genetically is unable to make vasopressin and develops diabetes insipidus) we could hardly detect somatostatin-immunopositive nerve fibers and terminals in the SON (Fig. 5). In the control Long-Evans rats the innervation was similar to that seen in Sprague-Dawley rats (not shown). DISCUSSION Somatostatin has been shown to be present in the rat
hypothalamic SON 6,s,13,22. The most likely source of this
B
Fig. 2. Somatostatin-immunoreactive network of fibers and terminals in the rat hypothalamic supraoptic nucleus in a 40/~m thick coronal vibratome section. OC, optic chiasm. Bars = 200/zm.
is the main hypothalamic somatostatin periventricular system. Transecting the fibers from the periventricular nucleus (medial Cut no. 2), however, left the SON SOS innervation unchanged. Similarly, knife cuts lateral to the SON did not have an effect on the SON SOS content either - excluding the somatostatin - containing cells in the limbic system (Cut. no. 1) and cerebral cortex (Cut nos. 1 and 2) as a positive source. Brainstem somatostatin-containing neurons project to the SON through pathways cut by the caudal coronal cut (no. 4). This cut did not have an effect on the SON SOS fiber density either, the brainstem can also be excluded as the major source towards the SON (although these cells do project to the PVN22). The resistence of the innervation to change following depletion of outside inputs indicated a local source. Since - like earlier data suggested s,13,22 - we could not demonstrate somatostatin-positive perikarya inside the SON itself we concluded that the only possible source must be the few somatostatin cells that are consistently present around the dorsal border of the rostral and caudal pole of the nucleus (Fig. 4). The double immunostaining at the EM level showed that there are synaptic contacts between the somatostatin nerve terminals and vasopressin neurons and dendrites in the SON. These synapses would supply the morphological basis for a direct effect of SOS on vasopressin synthesis and/or release. Sawchenko et al.22 demonstrated the presence of SOS-immunopositive fibers in the
296 SON, but mostly in the dorsal portion that corresponds to oxytocin containing neurons rather than vasopressincontaining ones. In our studies we have seen those fibers as well as synaptic contacts between the SOS terminals and the oxytocin cells. A l t h o u g h the present study was designed to study the effect of SOS on vasopressin we do not exclude the effect on oxytocin neurons. Most likely both populations are effected by somatostatin. The different distribution of SOS fibers in the B r a t t l e b o r o versus the L o n g - E v a n s (control) rat also indicates that
there is an interaction between the somatostatin and the vasopressin systems in the SON. Sawchenko et al. 22 in their study also mention the brainstem as the most likely source of the SON somatostatin innervation. However, their tract tracing studies were c o n c e n t r a t e d on the PVN magnocellular cells - that do receive somatostatin from the brainstem. They only suggested that the SON magnocellular neurons would also be likely to be innervated by the same cells. O u r present results do not support that hypothesis, though.
Fig. 3. Electron micrographs show a double-immunostaining for somatostatin and vasopressin in the rat supraoptic nucleus. Immunoreactivity for somatostatin was visualized by an immunoperoxidase reaction, while vasopressin-containing elements were labelled with 5-nm diameter gold particles. Symmetric synaptic connections (arrowheads) formed between immunoperoxidase labelled somatostatin immunoreactive boutons with the soma of gold particles (small arrows) containing vasopressin immunoreactive neurons. OC, optic chiasm. Bars = 1 /~m.
297
OC
m
Fig. 5. Somatostatin-containing nerve terminals in the supraoptic nucleus of a Brattleboro rat. OC, optic chiasm. The solid arrows indicate the dorsal portion of the supraoptic nucleus with somatostatin-immunoreactive fibers. The empty arrows point to the ventral region with no immunostaining. Bars = 50/~m.
OC
matostatin in sheep. Intracerebroventricular injection of SOS also blocks VP release 4'5. Recent data indicate that in isolated nerve endings of the rat n e u r o n a l lobe
Fig. 4. Somatostatin-containing cells in the rostral pole of the supraoptic nucleus of rats with surgical transection. A: midsagittal cut; B" rostral cut. OC, optic chiasm. Bars = 100/zm.
somatostatin inhibits high potassium-induced secretion of both oxytocin and vasopressin 19. The somatostatin fibers in the SON may have a similar function and may thus participate in the fine regulation of the release of magnocellular hormones.
The exact physiological role for somatostatin in vasopressin release is not yet clear. Wang et al. 26 demonstrated that haemorrhage-induced vasopressin release can be blocked by intraventricularly administered so-
Acknowledgements. Cs.L. is being supported by the following Grants: NIH - NF26068 and NIH - HD23830.
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