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Demonstration of serotoninergic axon terminals on somatostatin-immunoreactive neurons of the anterior periventricular nucleus of the rat hypothalamus
Brain Research, 442 (1988) 23-32
23
Elsevier BRE 13300
Demonstration of serotoninergic axon terminals on somatostatinimmunoreactive neurons of the anterior periventricular nucleus of the rat hypothalamus J6zsef Kiss, Agnes Cs~iky and B61a Hal~isz Second Department of Anatomy. Semmelweis University Medical School, Budapest (Hungary)
(Accepted 4 August 1987) Key words: Periventricular nucleus: Somatostatin neuron: Serotoninergic terminal: Hypothalamus: Synaptic connection
Using a combination of electron microscopic autoradiography and immunocytochemistry, the connections between serotoninergic axons and somatostatin neurons of the anterior periventricular nucleus of the rat hypothalamus were examined. The serotoninergic elements were identified after selective uptake of tritiated serotonin and the somatostatin neurons with immunocytochemistry. Synaptic connections between labeled serotoninergic nerve endings and somatostatin-immunoreactive neurons were observed. This finding provides morphological evidence for a direct influence of serotoninergic elements on somatostatin neurons of the anterior periventricular nucleus projecting to the median eminence of the hypothalamus.
INTRODUCTION Secretion of growth hormone (GH) by the pituitary gland is believed to be controlled by the central nervous system through the release of two hypothalamic factors (hormones), growth hormone releasing hormone (GHRH)-" and somatostatin (SRIF) II. Rat G H R H was recently isolated from the hypothalamus, characterized and synthesized ~'~. It stimulates GH secretion. Specific antibody against rat G H R H suppresses spontaneous G H secretion in the conscious rat 39 Neurons producing G H R H and projecting to the median eminence are concentrated in the arcuate nucleus of the hypothalamus 9"31"34. SRIF, a tetradecapeptide, is present in the hypothalamus and pituitary portal blood ~'12'-''. In the hypothalamus the majority of the SRIF-immunoreactive cells are concentrated in the periventricular region forming 1-3 rows in the ventricular wall and extending from the middle of the optic chiasm to the rostral margin of the median eminence. Such cells have been also ob-
served in many other regions or nuclei of the hypothalamus 3'8'~7"t'~'-~'~. $RIF inhibits basal and stimulated release of GH from the pituitary cells both in vivo and in vitro (for details and refs. see Arimura and Culler4). The anterior periventricular region of the hypothalamus which comprises the majority of the SRIF neurons has a rich innervation by serotonin (5-HT)containing fibers 37. There is experimental evidence that the 5-HT system of the central nervous system can affect G H secretion. Most observations suggest that 5-HT stimulates G H secretion. Collu et al. t4 reported that intracerebroventricular injection of 5-HT raised plasma GH levels in rats. Smythe et al. 3"~and Kato et ai. :3 observed that 5-hydroxytryptophan increased serum GH concentrations. Martin et el. 3° found that parachlorophenylalanine, an inhibitor of 5-HT biosynthesis, suppressed GH secretion in conscious rats. An injection of metergoline, which presumably blocks 5-HT receptors in brain, suppressed episodic G H
Correspondence: B. Hal~isz, 2nd Department of Anatomy, Semmelweis University Medical School, Budapest, T0zolt6 u.58, H-145t)
Hungary. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
24 increases in plasma 5. The findings of Willoughby et al. 4° indicate that 5-HT may have both facilitatory and inhibitory effects on GH secretion. Alterations in GH secretion induced by 5-HT may be mediated by changes in GHRH release 32, SRIF release 6 or both. 5-HT does not stimulate GH release from the pituitary in vitro 23. To our knowledge nothing is known on the structural relationship.between 5-HT elements and SRIF neurons in the anterior periventricular region and 5HT elements and GHRH neurons of the arcuate nucleus of the hypothalamus. Observations on these relationships may provide useful information on the site of action of 5-HT neurons on CH secretion. Therefore, in the present investigations the relationship between 5-HT axon terminals and SRIF neurons in the anterior periventricular region of the rat hypothalamus was studied. A combination of electron microscopic autoradiography and immunocytochemistry was used. Serotoninergic elements taking up selectively [3H]5-HTI.2..16 were labeled autoradiographically. SRIF neurons were visualized immunocytochemically. MATERIALS AND METHODS A combination of high-resolution autoradiography and immunocytochemistry at electron microscopic level was performed on vibratome sections of the rat brain. Autoradiography and immunocytochemistry were first checked on semi-thin (1 ~m thick) sections at the light microscopic level. Selected areas of the hypothalamic periventricular nucleus showing immunopositivity for SRIF and autoradiographic labeling of the serotoninergic neuronal element within the same areas, were then processed for electron microscopy in order to study synaptic interconnections between SRIF-containing neuronal elements and serotoninergic axon terminals.
Antiserum A rabbit antiserum against SRIF (Antisomatostatin, Immunonuclear, Steelwater, MN) diluted in 0.1 M phosphate-buffered saline, pH 7.4 (PBS) containing 1% normal goat serum (NGS), was used in this study.
Tritiated 5-hydroxytryptamine [3H]5-Hydroxytryptamine (G) creatinine sulfate
([3H]5-HT), spec. act. 15 Ci/nmol (Amersham International), was evaporated under a nitrogen gas stream and re-dissolved to produce a concentration of 5 x 10-5 M in physiological saline containing 0.1% ascorbic acid and a mixture of 10-4 M non-radioactive noradrenaline (L-noradrenaline-HCl, Sigma) and 10-4 M non-radioactive dopamine (Sigma) to prevent non-specific incorporation of [3H]5-HT by catecholaminergic neuronal elements.
Treatment of animals Ten adult male Sprague-Dawley rats (body weight 230-250 g) were pretreated with a monoamine oxidase inhibitor (pargyline/N-methyl.N-ben. zyl-2-propynyl amine, 70 mg/kg i.p.) at 18 h and 2 h prior to microinfusion of monoamines to prevent their rapid metabolism. Under sodium hexobarbital anesthesia (100 mg/kg i.p.) coichicine was administered into the right lateral ventricle (70 #g colchicine, BDH Chemicals, Poole, U.K.) diluted in 201d saline. After an interval of 24-48 h, the animals were reanesthetized, immobilized in a stereotaxic headframe and submitted to a microinfusion of tritiated monoamine, by means of a micro-infusion pump. A total volume of 120 ~d of [3H]5-HT was administered over a period of 6(]-9(] rain into the left lateral ventricle. The animals were perfused immediately thereafter through the heart with 50 ml of oxygenized Tyrode's solution followed by 3(]0 ml of a fixative containing 3.6% of paraformaldehyde, 0.1% glutaraldehyde and 2% saturated picric acid in PBS, pH 7.4. After perfusion, the brains were removed, 1 mm thick slices from the rostral edge of the optic chiasma up to the median eminence were cut at the frontal plane and postfixed for an additional hour in the same fixative but without glutaraldehyde.
Preparation of sections After postfixation, brain slices were washed in PBS, then 501tm thick sections were cut on the Vibratome (Oxford Instruments) and kept overnight in PBS at 4 °C. Sections corresponding to levels between A8200 ~m and A6000/~m 28 containing the periventricular nucleus were selected for immunocytochemistry under microscopic control. Before immunostaining, the sections were frozen in liquid nitrogen then thawed to room temperature to enhance penetration of the immunoreagent.
25
Irnmunocytochemical procedure Immunostaining for SRIF was performed on sections of 50 F~m thickness using the peroxidase-antiperoxidase (PAP) technique of Sternberger 3s according to the protocol described in detail previously -~4"-'6. Briefly, sections were incubated in PBS containing 20% normal goat serum (Human, Hungary), washed in PBS containing 1% NGS, then incubated in the antiserum against SRIF diluted 1:1000, for 24 h at 4 °C. After washing in PBS containing 1% NGS, the sections were incubated in goat anti-rabbit IgG (Human, Hungary) diluted 1:40 for 6 h at 4 °C, washed and incubated overnight in rabbit peroxidase-antiperoxidase complex (Miles) diluted 1:100. The bound peroxidase was then reacted for 6-10 min with 0.06% 3,3'-diaminobenzidine tetrahydrochioride (Sigma) and 0.01% hydrogen peroxide in 0.05 M Tris buffer,
Procedure of autoradiography After washing, sections were postfixed for 30 min in 1% OsO4 dissolved in 0.1 M phosphate buffer, dehydrated and stained with uranyi acetate (Analar), then embedded in Durcupan by flat-embedding between a glass microscope slide and a glass coverslip. SRIF immunoreactivity was checked on the Durcupan-embedded sections. Selected areas of sections, showing immunopositivity for SRIF were re-embedded in a Durcupan block and 4 semithin sections of 1/~m were cut and processed for light microscopic autoradiography as detailed elsewhere 27 to check the intensity of radiolabeling. Blocks showing radiolabeling suitable for electron microscopic autoradiography were retrimmed, thin serial sections were cut by an ultramicrotome (Reichert) and dipped for electron microscope autoradiography as detailed previously24.27.
Controls To identify immunocytochemicai specificity, randomly selected sections were incubated in normal rabbit serum which replaced the specific antibody in the immuno-staining procedure. To ascertain the specific uptake of [3H]5-HT by serotoninergic but not by other monoamine-containing axon terminals, tritiated 5-HT was substituted with [3H]L-noradrenaline (L-[7,8-3H]noradrenaline, spec. act. 313-50 Ci/nmol, Amersham International) on a group of 4 experimental animals.
Evaluation of electron micrographs for analysis of immuno- or radiolabeled profi'les and synapses Electron micrographs of areas containing either SRIF-immunolabeled or radiolabeled neuronal profiles, or both, were taken at random at an initial magnification of 10,000x and finally printed at 26,000x. SRIF-immunolabeled or 5-HT-radiolabeled neuronal profiles were identified cn micrographs and characterized on the basis of their contacts. Ten single uitrathin sections originating from each of the 6 animals were analyzed so that the proportion of SRIF-immunolabeled perikarya, dendritic processes and axonal elements could be ascertained. Further analysis was carried out of all the sections examined under the electron microscope and particular note taken of contacts made with SRIF-immunolabeled profiles. Estimations were also made in these sections of the occurrence of radiolabeled or autoradiographically non-labeled axonai varicosities and terminals adjacent to the SRIF-immunoreactive neuronal elements. Connections between immunoreactive dendrites and radiolabeled axonai elements were classified as a synapse or as a close membrane contact. The proportion of radiolabeled and autoradiographically non-labeled axonal connections made with SRIF-immunopositive dendrites was estimated in 10 single ultrathin sections from each experimental animal. RESULTS In Durcupan-embedded 50 l~m thick coronal sections, SRIF-immunoreactive neuronal structures including cell bodies and dendrites, part of a complex network of immunoreactive processes, as well as a few punctate elements were observed throughout the area of the periventricular nucleus. Most of the immunolabeled perikarya were restricted to areas in the immediate vicinity of the ependymal layer of the third ventricle. Perikarya of labeled SRIF neurons were frequently located in a few layers under the ependymal wall. In 2 l~m thick semi-thin sections processed for light-microscopic autoradiography, numerous SRIF-immunoreactive nerve cell bodies and dendrites were detected. Superimposed over the inamunolabeled and unlabeled neuronal structures a diffuse autoradiographic labeling was also seen and aggregates of silver grains were found to be present
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Fig. 1. A radiolabeled varicosity filled with small round and oblong vesicles is seen emerging from a thin unmyelinated intervaricose axonal segment (AxS). Dense-cored vesicles are evident in the varicosity and in the axonal segment. The labeled varicosity is directly opposed (arrows) to a neuronal element but no synaptic differentiation is visible at the site of the contact. Note the presence of a small immunolabeled dendritic branchlet (asterisk) close to the radiolabeled varicosity. Scale bar: 0.3011m. Fig. 2. Radiolabeled axon terminal encounters unlabeled dendritic profiles (D), but as in Fig. 1, no synaptic contact is seen between the two elements. Scale bar: 0.40,um.
27 o
Fig. 3. Ultrastructural immunocytochemistry of a portion of an SRIF-positive perikaryon (Pk). The reaction product is confined to the cytoplasm, in which large immunoreactive neurosecretory granules (arrowheads) are also present. An unlabeled axon terminal containing small pleomorphic vesicles forms a asymmetrical axosomatic synaptic contact (arrow). Scale bar: 0.24 !¢m. Fig. 4. SRIF-immunopositive proximal dendritic portion (pD) containing immunolabeled microtubules and large, intensely stained dense granules is synapticaUy contacted by an unlabeled varicosity which shows several small granular vesicles in addition to the small clear ones. The contacts between the two elements are asymmetrical synapses (arrows). Scale bar: 0.30,um. Fig. 5. lmmunoreactive axon terminal filled with intensely labeled small, electron-lucent vesicles and several large, intensely stained dense granules forms symmetrical synaptic contact (thick arrow) with an unlabeled nerve cell body (Pk). Note that within the labeled terminal dense granules are localized immediately under the presynaptic membrane (white arrows). Scale bar: 0.30 !¢m.
28 in the entire periventricular nuclear region. t~H]5-HT radiolabeled axonal elements in the periventricular nuclear area Electron microscopic autoradiographs showed a considerable number of radiolabeled axonal elements corresponding to thin unmyelinated axons, axonal varicosities and axon terminals (Figs. 1, 2). The ultrastructure of these radiolabeled axonal elements was similar to serotoninergic terminals previously identified in other hypothalamic nuclei 1°'15"24--'6and in other regions of the brain 1'16"33"37. The radiolabeled boutons in the periventricular nucleus measured 0.5-1.8~m in diameter and usually contained a heterogeneous population of vesicles, the majority exhibiting mainly round or oblong, clear synaptic vesicles 35-55 nm in diameter, and a few large granular vesicles measuring 80-120 nm across. Radiolabeling of these axonal profiles varied in intensity and in the accumulation of silver grains. The radiolabeled profiles were frequently found to be in close contact with neuronal processes or perikarya, and in some cases these contacts exhibited the classic morphological features of synapses. In a great number of the serotoninergic boutons, however, the contacts between 5-HT-labeled terminals and immunopositive or immunocytochemically non-labeled neuronal elements were formed without synaptic-like membrane specialization. SRIF-immunoreactive neuronal elements The reaction product indicating SRIF-immunoreactivity occurs throughout the cytoplasm of immunostained perkarya (Fig. 3). It appears to be associated with membranes of the rough endoplasmic reticulum, mitochondria, Golgi membranes, microtubules and the inner surface of the cell membrane. Also, SRIF-immunoreactive neuronal elements, including perikarya, contain neurosecretory granules associated with the diaminobenzidine reaction product (Fig. 3, 4). Dendrites immunolabeled for SRIF varied in diameter, and the reaction product was restricted to the proximal part and extent of the distal branches. The proximal dendritic parts contain a great number of microtubules, fewer endoplasmic reticulum cisternae than perikarya, and neurosecretory granules in variable number, associated with the reaction product, lmmunoreactive perikarya may re-
ceive contacts from non-radiolabeled axonai afferents. Most often, these unlabeled axon terminals containing small round clear vesicles and a few small or medium-sized granular vesicles, form asymmetrical synaptic contacts with SRIF-immunoreactive perikarya, as seen in Fig. 3. Similar asymmetrical synaptic connections between unlabeled ierminals and immunostained profiles occur on proximal dendrites (Fig. 4) and distal dendritic branches. SRIF-immunolabeling also occurred in axon terminal~ in the periventricular nucleus. These axonal elements contained round, clear vesicles of about 35-50 nm in diameter and a varying number of neurosecretory granules, 80-120 in diameter, but no dense-cored vesicles. In some cases, immunoreactive terminals made contact with non-immunolabeled perikarya and occasionally formed symmetrical synapses, as seen in Fig. 5. As a rough estimate, the large majority of the immunoreactive elements (about 80%) were dendritic profiles, about 10% perikarya and less than 10% axonal profiles. Synaptic connections between serotoninergic nerve terminals and SRIF.immunopositive neuronal elements Autoradiographically labeled serotoninergic terminals were occasionally seen to be in contact with SRIF-immunopositive dendritic elements in the perivenrricular nucleus of the rat. These immunostained dendritic profiles included distal shafts, medium-tosmall distal branches and occasionally more-distal branchlets. Most contained neurosecretory granules too, and all were filled with the diaminobenzidine reaction product (Figs. 6-10). In a number of cases, labeled terminals in these contacts appeared to be in close proximity to the immunostained dendritic profile (about 10% of the immunoreactive elements was in contact with radiolabeled axon profiles) without there being any synaptic relationship (Fig. 6), perhaps because of disruption of membranes due to our 'freezing-thawing' treatments, the synaptic membrane specialization could not be clearly revealed (Fig. 8). However, in about one-third of the contacts between radiolabeled terminals and immunopositive dendrites, definitive synaptic structures were found which exhibited asymmetrical synapses, as seen in Figs. 7, 9 and 10. No SRIF-immunoreactive cell body
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Fig. 6. A radiolabeled varicosity containing large (80-100 nm in diameter) dense-cored vesicles (arrows) is seen in contact with an SRIF.immunopositive small dendritic profile (dD) without synaptic differentiation. In the dendrite, intense immunostaining is associated with cisternal membrane.2,, microtubules and large neurosecretory granules. Scale bar: 0.24 ~m. Fig. 7. A radiolabeled 5-HT varicosity filled with small pleomorphic vesicles, makes an asymmetrical synaptic contact (arrow) with an immunopositive distal dendritic shaft (dD). Scale bar: 0.301~m. Fig. 8. Immunolabeled distal dendritic branch is in contact with two axon terminals, one of which is radiolabeled and nnakes a synapse (arrow) on the dendrite shaft. The unlabeled axonal profile (asterisk) containing dense-cored vesicles is not synaptically in contact with the immunostained dendrite. Scale bar: 0.40F~m. Fig. 9. Radiolabeled axonal varicosity emerging from an axonal segment (Ax) containing dense-cored vesicles makes asymmetrical synaptic contact (arrow) with an immunopositive dendrite. Scale bar: 0.30 F~m. Fig. 10. Immunopositive small distal dendritic branchlet (dD) receives three axon terminals (t I, t2, t3) each of which is in synaptic contact (arrows) with the dendritic profile. The connection of the radiolabeled bouton (t L) seems to be symmetrical synaptic differentiation. Scale bar: 0.24!~m.
30 or terminal was found to receive radiolabeled synapse, and no symmetrical synapses were detected between serotoninergic boutons and any immunopositive neuronal elements. DISCUSSION The present findings indicate that serotoninergic axons are in synaptic contacts with other neuronal elements in the anterior periventricular nucleus of the hypothalamus. Further. they show that some of the serotoninergic endings form synaptic junctions with SRIF-immunoreactive neurons, mainly with their dendrites. This is the first demonstration of the existence of synaptic contacts between the mentioned elements in this hypothalamic region. The finding can be interpreted as morphological evidence for the assumption that serotoninergic elements may act directly on SRIF neurons and thus, may influence the release of SRIF from these cells. As mentioned in the Introduction, SRIF inhibits GH release at the pituitary level and 5-HT appears to affect release of the hormone at tne central nervous system level. Direct connection between these two elements suggests that serotoninergic axons terminating on SRIF neurons might influence, stimulate or inhibit, the release of SRIF. Most investigators found a stimulatory effect of 5-HT on GH secretion 5'~4'''a' .a..3.~. From the recent years there is one reporP ° suggesting that besides stimulation, 5-HT may have also an inhibitory effect on GH. Willoughby et al. 4° have found that GH secretion was inhibited by parachlorophenylalanine, an inhibitor of 5-HT biosynthesis, and for three days following 5,7-dihydroxytryptamine, a neurotoxin selectively destroying 5-HT structures. Acute 5-HT release stimulated by fenfluramine inhibited GH in intact and 5-HT-depleted rats. If we accept that 5-HT primarily stimulates GH secretion, then it may be assumed that the 5-1tT terminals on SRIF neurons in the anterior periventricular
REFERENCES 1 Aghajanian, G.K. and Bloom, F.E., Localization of tritiated serotonin in rat brain by electron microscopicautoradiography,J. Pharmacol. Exp. Ther.. 156 (1967) 23-30.
nucleus probably inhibit SRIF release. Such an assumption is in line with the observation that administration of anti-somatostatin serum to rats reverses the inhibition of pulsatile GH secretion produced by injection of metergoline, which presumably blocks 5HT receptors 6. The findings of Kato et al. 23 suggest that endogenous SRIF levels may affect GH secretion induced by serotoninergic mechanisms. These authc~rs have demonstrated that pretreatment with anti-somatostatin antibody exaggerated GH secretion induced by 5-hydroxytryptophan in rats. It should be mentioned that Chihara et ai. 13 have reported that 5-HT injected into the third ventricle had no effect on hypophyseal portal blood. The exact projection site of the SRIF cells being in synaptic contact with serotoninergic axons is not known. Most probably they project, at least some of them, to the median eminence. It could also be that some others terminate on other SRIF neurons. Very recently, Epelbaum et al. ~s have observed classical synapses between SRIF containing axonal processes and SRIF perikarya and dendrites. We did not find such synapses. SRIF-immunoreactive axon terminal forming synaptic contact with an unlabeled nerve cell body was seen also in our material. The anatomical origin of the serotoninergic fibers in the anterior periventricular nucleus is not clear, it is most probable that these fibers arise from 5-HT neurons of the raphe nuclei 7, but a hypothalamic origin, at least for some of them, cannot be excluded I.~.20. We observed many terminations of unidentified axons on SRIF neurons. This clearly shows that not only serotoninergic axons but also other nerve cells, operating with unidentified neurotransmitters, terminate on SRIF cells of the anterior periventricular nucleus. Our investigations are in progress studying the relationship between 5-HT axon terminals and G H R H neurons of the arcuate nucleus.
2 Aghajanian, G.K., Bloom, F.E., Lovell, R., Sheard, M. and Freedman, D.X., The uptake of 5-hydroxy-tryptamine-3H from the cerebral ventricle: autoradiographic localization, Biochem. Pharmacol., 15 (1966) 1401-1403. 3 AIpert, L.C., Brawer, J.R., Patel, Y.C. and Reichlin, S.,
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histochemistry preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes, J. Histochem. Cytochern., 18 (1970) 315-337. 39 Wehrenberg, W.B., Brazeau, P., Luben, R., BOhlen, P. and Guillemin, R., Inhibition of the pulsatile secretion of growth hormone by monoclonai antibodies to the hypothaiamic growth hormone releasing factor, GRF, Endocrinology. 111 11982) 2147-2148. 40 Willoughby, J.O., Menadue, M. and Jervois, P., Function of serotonin in physiologic secretion of growth hormone and prolactin: action of 5,7-dihydroxytryptamine, fenfluramine and p-chlorophenylalanine, Brain Research, 249 (1982) 291-299.
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Elsevier BRE 13300
Demonstration of serotoninergic axon terminals on somatostatinimmunoreactive neurons of the anterior periventricular nucleus of the rat hypothalamus J6zsef Kiss, Agnes Cs~iky and B61a Hal~isz Second Department of Anatomy. Semmelweis University Medical School, Budapest (Hungary)
(Accepted 4 August 1987) Key words: Periventricular nucleus: Somatostatin neuron: Serotoninergic terminal: Hypothalamus: Synaptic connection
Using a combination of electron microscopic autoradiography and immunocytochemistry, the connections between serotoninergic axons and somatostatin neurons of the anterior periventricular nucleus of the rat hypothalamus were examined. The serotoninergic elements were identified after selective uptake of tritiated serotonin and the somatostatin neurons with immunocytochemistry. Synaptic connections between labeled serotoninergic nerve endings and somatostatin-immunoreactive neurons were observed. This finding provides morphological evidence for a direct influence of serotoninergic elements on somatostatin neurons of the anterior periventricular nucleus projecting to the median eminence of the hypothalamus.
INTRODUCTION Secretion of growth hormone (GH) by the pituitary gland is believed to be controlled by the central nervous system through the release of two hypothalamic factors (hormones), growth hormone releasing hormone (GHRH)-" and somatostatin (SRIF) II. Rat G H R H was recently isolated from the hypothalamus, characterized and synthesized ~'~. It stimulates GH secretion. Specific antibody against rat G H R H suppresses spontaneous G H secretion in the conscious rat 39 Neurons producing G H R H and projecting to the median eminence are concentrated in the arcuate nucleus of the hypothalamus 9"31"34. SRIF, a tetradecapeptide, is present in the hypothalamus and pituitary portal blood ~'12'-''. In the hypothalamus the majority of the SRIF-immunoreactive cells are concentrated in the periventricular region forming 1-3 rows in the ventricular wall and extending from the middle of the optic chiasm to the rostral margin of the median eminence. Such cells have been also ob-
served in many other regions or nuclei of the hypothalamus 3'8'~7"t'~'-~'~. $RIF inhibits basal and stimulated release of GH from the pituitary cells both in vivo and in vitro (for details and refs. see Arimura and Culler4). The anterior periventricular region of the hypothalamus which comprises the majority of the SRIF neurons has a rich innervation by serotonin (5-HT)containing fibers 37. There is experimental evidence that the 5-HT system of the central nervous system can affect G H secretion. Most observations suggest that 5-HT stimulates G H secretion. Collu et al. t4 reported that intracerebroventricular injection of 5-HT raised plasma GH levels in rats. Smythe et al. 3"~and Kato et ai. :3 observed that 5-hydroxytryptophan increased serum GH concentrations. Martin et el. 3° found that parachlorophenylalanine, an inhibitor of 5-HT biosynthesis, suppressed GH secretion in conscious rats. An injection of metergoline, which presumably blocks 5-HT receptors in brain, suppressed episodic G H
Correspondence: B. Hal~isz, 2nd Department of Anatomy, Semmelweis University Medical School, Budapest, T0zolt6 u.58, H-145t)
Hungary. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
24 increases in plasma 5. The findings of Willoughby et al. 4° indicate that 5-HT may have both facilitatory and inhibitory effects on GH secretion. Alterations in GH secretion induced by 5-HT may be mediated by changes in GHRH release 32, SRIF release 6 or both. 5-HT does not stimulate GH release from the pituitary in vitro 23. To our knowledge nothing is known on the structural relationship.between 5-HT elements and SRIF neurons in the anterior periventricular region and 5HT elements and GHRH neurons of the arcuate nucleus of the hypothalamus. Observations on these relationships may provide useful information on the site of action of 5-HT neurons on CH secretion. Therefore, in the present investigations the relationship between 5-HT axon terminals and SRIF neurons in the anterior periventricular region of the rat hypothalamus was studied. A combination of electron microscopic autoradiography and immunocytochemistry was used. Serotoninergic elements taking up selectively [3H]5-HTI.2..16 were labeled autoradiographically. SRIF neurons were visualized immunocytochemically. MATERIALS AND METHODS A combination of high-resolution autoradiography and immunocytochemistry at electron microscopic level was performed on vibratome sections of the rat brain. Autoradiography and immunocytochemistry were first checked on semi-thin (1 ~m thick) sections at the light microscopic level. Selected areas of the hypothalamic periventricular nucleus showing immunopositivity for SRIF and autoradiographic labeling of the serotoninergic neuronal element within the same areas, were then processed for electron microscopy in order to study synaptic interconnections between SRIF-containing neuronal elements and serotoninergic axon terminals.
Antiserum A rabbit antiserum against SRIF (Antisomatostatin, Immunonuclear, Steelwater, MN) diluted in 0.1 M phosphate-buffered saline, pH 7.4 (PBS) containing 1% normal goat serum (NGS), was used in this study.
Tritiated 5-hydroxytryptamine [3H]5-Hydroxytryptamine (G) creatinine sulfate
([3H]5-HT), spec. act. 15 Ci/nmol (Amersham International), was evaporated under a nitrogen gas stream and re-dissolved to produce a concentration of 5 x 10-5 M in physiological saline containing 0.1% ascorbic acid and a mixture of 10-4 M non-radioactive noradrenaline (L-noradrenaline-HCl, Sigma) and 10-4 M non-radioactive dopamine (Sigma) to prevent non-specific incorporation of [3H]5-HT by catecholaminergic neuronal elements.
Treatment of animals Ten adult male Sprague-Dawley rats (body weight 230-250 g) were pretreated with a monoamine oxidase inhibitor (pargyline/N-methyl.N-ben. zyl-2-propynyl amine, 70 mg/kg i.p.) at 18 h and 2 h prior to microinfusion of monoamines to prevent their rapid metabolism. Under sodium hexobarbital anesthesia (100 mg/kg i.p.) coichicine was administered into the right lateral ventricle (70 #g colchicine, BDH Chemicals, Poole, U.K.) diluted in 201d saline. After an interval of 24-48 h, the animals were reanesthetized, immobilized in a stereotaxic headframe and submitted to a microinfusion of tritiated monoamine, by means of a micro-infusion pump. A total volume of 120 ~d of [3H]5-HT was administered over a period of 6(]-9(] rain into the left lateral ventricle. The animals were perfused immediately thereafter through the heart with 50 ml of oxygenized Tyrode's solution followed by 3(]0 ml of a fixative containing 3.6% of paraformaldehyde, 0.1% glutaraldehyde and 2% saturated picric acid in PBS, pH 7.4. After perfusion, the brains were removed, 1 mm thick slices from the rostral edge of the optic chiasma up to the median eminence were cut at the frontal plane and postfixed for an additional hour in the same fixative but without glutaraldehyde.
Preparation of sections After postfixation, brain slices were washed in PBS, then 501tm thick sections were cut on the Vibratome (Oxford Instruments) and kept overnight in PBS at 4 °C. Sections corresponding to levels between A8200 ~m and A6000/~m 28 containing the periventricular nucleus were selected for immunocytochemistry under microscopic control. Before immunostaining, the sections were frozen in liquid nitrogen then thawed to room temperature to enhance penetration of the immunoreagent.
25
Irnmunocytochemical procedure Immunostaining for SRIF was performed on sections of 50 F~m thickness using the peroxidase-antiperoxidase (PAP) technique of Sternberger 3s according to the protocol described in detail previously -~4"-'6. Briefly, sections were incubated in PBS containing 20% normal goat serum (Human, Hungary), washed in PBS containing 1% NGS, then incubated in the antiserum against SRIF diluted 1:1000, for 24 h at 4 °C. After washing in PBS containing 1% NGS, the sections were incubated in goat anti-rabbit IgG (Human, Hungary) diluted 1:40 for 6 h at 4 °C, washed and incubated overnight in rabbit peroxidase-antiperoxidase complex (Miles) diluted 1:100. The bound peroxidase was then reacted for 6-10 min with 0.06% 3,3'-diaminobenzidine tetrahydrochioride (Sigma) and 0.01% hydrogen peroxide in 0.05 M Tris buffer,
Procedure of autoradiography After washing, sections were postfixed for 30 min in 1% OsO4 dissolved in 0.1 M phosphate buffer, dehydrated and stained with uranyi acetate (Analar), then embedded in Durcupan by flat-embedding between a glass microscope slide and a glass coverslip. SRIF immunoreactivity was checked on the Durcupan-embedded sections. Selected areas of sections, showing immunopositivity for SRIF were re-embedded in a Durcupan block and 4 semithin sections of 1/~m were cut and processed for light microscopic autoradiography as detailed elsewhere 27 to check the intensity of radiolabeling. Blocks showing radiolabeling suitable for electron microscopic autoradiography were retrimmed, thin serial sections were cut by an ultramicrotome (Reichert) and dipped for electron microscope autoradiography as detailed previously24.27.
Controls To identify immunocytochemicai specificity, randomly selected sections were incubated in normal rabbit serum which replaced the specific antibody in the immuno-staining procedure. To ascertain the specific uptake of [3H]5-HT by serotoninergic but not by other monoamine-containing axon terminals, tritiated 5-HT was substituted with [3H]L-noradrenaline (L-[7,8-3H]noradrenaline, spec. act. 313-50 Ci/nmol, Amersham International) on a group of 4 experimental animals.
Evaluation of electron micrographs for analysis of immuno- or radiolabeled profi'les and synapses Electron micrographs of areas containing either SRIF-immunolabeled or radiolabeled neuronal profiles, or both, were taken at random at an initial magnification of 10,000x and finally printed at 26,000x. SRIF-immunolabeled or 5-HT-radiolabeled neuronal profiles were identified cn micrographs and characterized on the basis of their contacts. Ten single uitrathin sections originating from each of the 6 animals were analyzed so that the proportion of SRIF-immunolabeled perikarya, dendritic processes and axonal elements could be ascertained. Further analysis was carried out of all the sections examined under the electron microscope and particular note taken of contacts made with SRIF-immunolabeled profiles. Estimations were also made in these sections of the occurrence of radiolabeled or autoradiographically non-labeled axonai varicosities and terminals adjacent to the SRIF-immunoreactive neuronal elements. Connections between immunoreactive dendrites and radiolabeled axonai elements were classified as a synapse or as a close membrane contact. The proportion of radiolabeled and autoradiographically non-labeled axonal connections made with SRIF-immunopositive dendrites was estimated in 10 single ultrathin sections from each experimental animal. RESULTS In Durcupan-embedded 50 l~m thick coronal sections, SRIF-immunoreactive neuronal structures including cell bodies and dendrites, part of a complex network of immunoreactive processes, as well as a few punctate elements were observed throughout the area of the periventricular nucleus. Most of the immunolabeled perikarya were restricted to areas in the immediate vicinity of the ependymal layer of the third ventricle. Perikarya of labeled SRIF neurons were frequently located in a few layers under the ependymal wall. In 2 l~m thick semi-thin sections processed for light-microscopic autoradiography, numerous SRIF-immunoreactive nerve cell bodies and dendrites were detected. Superimposed over the inamunolabeled and unlabeled neuronal structures a diffuse autoradiographic labeling was also seen and aggregates of silver grains were found to be present
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Fig. 1. A radiolabeled varicosity filled with small round and oblong vesicles is seen emerging from a thin unmyelinated intervaricose axonal segment (AxS). Dense-cored vesicles are evident in the varicosity and in the axonal segment. The labeled varicosity is directly opposed (arrows) to a neuronal element but no synaptic differentiation is visible at the site of the contact. Note the presence of a small immunolabeled dendritic branchlet (asterisk) close to the radiolabeled varicosity. Scale bar: 0.3011m. Fig. 2. Radiolabeled axon terminal encounters unlabeled dendritic profiles (D), but as in Fig. 1, no synaptic contact is seen between the two elements. Scale bar: 0.40,um.
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Fig. 3. Ultrastructural immunocytochemistry of a portion of an SRIF-positive perikaryon (Pk). The reaction product is confined to the cytoplasm, in which large immunoreactive neurosecretory granules (arrowheads) are also present. An unlabeled axon terminal containing small pleomorphic vesicles forms a asymmetrical axosomatic synaptic contact (arrow). Scale bar: 0.24 !¢m. Fig. 4. SRIF-immunopositive proximal dendritic portion (pD) containing immunolabeled microtubules and large, intensely stained dense granules is synapticaUy contacted by an unlabeled varicosity which shows several small granular vesicles in addition to the small clear ones. The contacts between the two elements are asymmetrical synapses (arrows). Scale bar: 0.30,um. Fig. 5. lmmunoreactive axon terminal filled with intensely labeled small, electron-lucent vesicles and several large, intensely stained dense granules forms symmetrical synaptic contact (thick arrow) with an unlabeled nerve cell body (Pk). Note that within the labeled terminal dense granules are localized immediately under the presynaptic membrane (white arrows). Scale bar: 0.30 !¢m.
28 in the entire periventricular nuclear region. t~H]5-HT radiolabeled axonal elements in the periventricular nuclear area Electron microscopic autoradiographs showed a considerable number of radiolabeled axonal elements corresponding to thin unmyelinated axons, axonal varicosities and axon terminals (Figs. 1, 2). The ultrastructure of these radiolabeled axonal elements was similar to serotoninergic terminals previously identified in other hypothalamic nuclei 1°'15"24--'6and in other regions of the brain 1'16"33"37. The radiolabeled boutons in the periventricular nucleus measured 0.5-1.8~m in diameter and usually contained a heterogeneous population of vesicles, the majority exhibiting mainly round or oblong, clear synaptic vesicles 35-55 nm in diameter, and a few large granular vesicles measuring 80-120 nm across. Radiolabeling of these axonal profiles varied in intensity and in the accumulation of silver grains. The radiolabeled profiles were frequently found to be in close contact with neuronal processes or perikarya, and in some cases these contacts exhibited the classic morphological features of synapses. In a great number of the serotoninergic boutons, however, the contacts between 5-HT-labeled terminals and immunopositive or immunocytochemically non-labeled neuronal elements were formed without synaptic-like membrane specialization. SRIF-immunoreactive neuronal elements The reaction product indicating SRIF-immunoreactivity occurs throughout the cytoplasm of immunostained perkarya (Fig. 3). It appears to be associated with membranes of the rough endoplasmic reticulum, mitochondria, Golgi membranes, microtubules and the inner surface of the cell membrane. Also, SRIF-immunoreactive neuronal elements, including perikarya, contain neurosecretory granules associated with the diaminobenzidine reaction product (Fig. 3, 4). Dendrites immunolabeled for SRIF varied in diameter, and the reaction product was restricted to the proximal part and extent of the distal branches. The proximal dendritic parts contain a great number of microtubules, fewer endoplasmic reticulum cisternae than perikarya, and neurosecretory granules in variable number, associated with the reaction product, lmmunoreactive perikarya may re-
ceive contacts from non-radiolabeled axonai afferents. Most often, these unlabeled axon terminals containing small round clear vesicles and a few small or medium-sized granular vesicles, form asymmetrical synaptic contacts with SRIF-immunoreactive perikarya, as seen in Fig. 3. Similar asymmetrical synaptic connections between unlabeled ierminals and immunostained profiles occur on proximal dendrites (Fig. 4) and distal dendritic branches. SRIF-immunolabeling also occurred in axon terminal~ in the periventricular nucleus. These axonal elements contained round, clear vesicles of about 35-50 nm in diameter and a varying number of neurosecretory granules, 80-120 in diameter, but no dense-cored vesicles. In some cases, immunoreactive terminals made contact with non-immunolabeled perikarya and occasionally formed symmetrical synapses, as seen in Fig. 5. As a rough estimate, the large majority of the immunoreactive elements (about 80%) were dendritic profiles, about 10% perikarya and less than 10% axonal profiles. Synaptic connections between serotoninergic nerve terminals and SRIF.immunopositive neuronal elements Autoradiographically labeled serotoninergic terminals were occasionally seen to be in contact with SRIF-immunopositive dendritic elements in the perivenrricular nucleus of the rat. These immunostained dendritic profiles included distal shafts, medium-tosmall distal branches and occasionally more-distal branchlets. Most contained neurosecretory granules too, and all were filled with the diaminobenzidine reaction product (Figs. 6-10). In a number of cases, labeled terminals in these contacts appeared to be in close proximity to the immunostained dendritic profile (about 10% of the immunoreactive elements was in contact with radiolabeled axon profiles) without there being any synaptic relationship (Fig. 6), perhaps because of disruption of membranes due to our 'freezing-thawing' treatments, the synaptic membrane specialization could not be clearly revealed (Fig. 8). However, in about one-third of the contacts between radiolabeled terminals and immunopositive dendrites, definitive synaptic structures were found which exhibited asymmetrical synapses, as seen in Figs. 7, 9 and 10. No SRIF-immunoreactive cell body
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Fig. 6. A radiolabeled varicosity containing large (80-100 nm in diameter) dense-cored vesicles (arrows) is seen in contact with an SRIF.immunopositive small dendritic profile (dD) without synaptic differentiation. In the dendrite, intense immunostaining is associated with cisternal membrane.2,, microtubules and large neurosecretory granules. Scale bar: 0.24 ~m. Fig. 7. A radiolabeled 5-HT varicosity filled with small pleomorphic vesicles, makes an asymmetrical synaptic contact (arrow) with an immunopositive distal dendritic shaft (dD). Scale bar: 0.301~m. Fig. 8. Immunolabeled distal dendritic branch is in contact with two axon terminals, one of which is radiolabeled and nnakes a synapse (arrow) on the dendrite shaft. The unlabeled axonal profile (asterisk) containing dense-cored vesicles is not synaptically in contact with the immunostained dendrite. Scale bar: 0.40F~m. Fig. 9. Radiolabeled axonal varicosity emerging from an axonal segment (Ax) containing dense-cored vesicles makes asymmetrical synaptic contact (arrow) with an immunopositive dendrite. Scale bar: 0.30 F~m. Fig. 10. Immunopositive small distal dendritic branchlet (dD) receives three axon terminals (t I, t2, t3) each of which is in synaptic contact (arrows) with the dendritic profile. The connection of the radiolabeled bouton (t L) seems to be symmetrical synaptic differentiation. Scale bar: 0.24!~m.
30 or terminal was found to receive radiolabeled synapse, and no symmetrical synapses were detected between serotoninergic boutons and any immunopositive neuronal elements. DISCUSSION The present findings indicate that serotoninergic axons are in synaptic contacts with other neuronal elements in the anterior periventricular nucleus of the hypothalamus. Further. they show that some of the serotoninergic endings form synaptic junctions with SRIF-immunoreactive neurons, mainly with their dendrites. This is the first demonstration of the existence of synaptic contacts between the mentioned elements in this hypothalamic region. The finding can be interpreted as morphological evidence for the assumption that serotoninergic elements may act directly on SRIF neurons and thus, may influence the release of SRIF from these cells. As mentioned in the Introduction, SRIF inhibits GH release at the pituitary level and 5-HT appears to affect release of the hormone at tne central nervous system level. Direct connection between these two elements suggests that serotoninergic axons terminating on SRIF neurons might influence, stimulate or inhibit, the release of SRIF. Most investigators found a stimulatory effect of 5-HT on GH secretion 5'~4'''a' .a..3.~. From the recent years there is one reporP ° suggesting that besides stimulation, 5-HT may have also an inhibitory effect on GH. Willoughby et al. 4° have found that GH secretion was inhibited by parachlorophenylalanine, an inhibitor of 5-HT biosynthesis, and for three days following 5,7-dihydroxytryptamine, a neurotoxin selectively destroying 5-HT structures. Acute 5-HT release stimulated by fenfluramine inhibited GH in intact and 5-HT-depleted rats. If we accept that 5-HT primarily stimulates GH secretion, then it may be assumed that the 5-1tT terminals on SRIF neurons in the anterior periventricular
REFERENCES 1 Aghajanian, G.K. and Bloom, F.E., Localization of tritiated serotonin in rat brain by electron microscopicautoradiography,J. Pharmacol. Exp. Ther.. 156 (1967) 23-30.
nucleus probably inhibit SRIF release. Such an assumption is in line with the observation that administration of anti-somatostatin serum to rats reverses the inhibition of pulsatile GH secretion produced by injection of metergoline, which presumably blocks 5HT receptors 6. The findings of Kato et al. 23 suggest that endogenous SRIF levels may affect GH secretion induced by serotoninergic mechanisms. These authc~rs have demonstrated that pretreatment with anti-somatostatin antibody exaggerated GH secretion induced by 5-hydroxytryptophan in rats. It should be mentioned that Chihara et ai. 13 have reported that 5-HT injected into the third ventricle had no effect on hypophyseal portal blood. The exact projection site of the SRIF cells being in synaptic contact with serotoninergic axons is not known. Most probably they project, at least some of them, to the median eminence. It could also be that some others terminate on other SRIF neurons. Very recently, Epelbaum et al. ~s have observed classical synapses between SRIF containing axonal processes and SRIF perikarya and dendrites. We did not find such synapses. SRIF-immunoreactive axon terminal forming synaptic contact with an unlabeled nerve cell body was seen also in our material. The anatomical origin of the serotoninergic fibers in the anterior periventricular nucleus is not clear, it is most probable that these fibers arise from 5-HT neurons of the raphe nuclei 7, but a hypothalamic origin, at least for some of them, cannot be excluded I.~.20. We observed many terminations of unidentified axons on SRIF neurons. This clearly shows that not only serotoninergic axons but also other nerve cells, operating with unidentified neurotransmitters, terminate on SRIF cells of the anterior periventricular nucleus. Our investigations are in progress studying the relationship between 5-HT axon terminals and G H R H neurons of the arcuate nucleus.
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