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Demonstration of serotoninergic axons terminating on luteinizing hormone-releasing hormone neurons in the preoptic area of the rat using a combination of immunocytochemistry and high resolution autoradiography
0306-4522/8S %3.00+0.00 Pergamon Press Ltd IBRO
~e~~u~eie~eeVol. 14, No. 1, pp. 69-78, 1985 Printed in Great Britain
DEMONSTRATION OF SEROTONINERGIC AXONS TERMINATING ON LUTEINIZING HORMONE-RELEASING HORMONE NEURONS IN THE PREOPTIC AREA OF THE RAT USING A COMBINATION OF IMMUNOCYTOCHEMISTRY AND HIGH RESOLUTION AUTORADIOGRAPHY J. KISS* and B. HAL&.Z Second Department of Anatomy, Semmelweis University, Medical School, H-1450 Budapest, Hungary Abstrac&--The synaptic relationship between serotoninergic terminals and luteinizing hormone-releasing ho~one~on~ning neurons was investiga~ in the medial preoptic area using a combined technique.
Axon terminals selectively taking up S-[~HJhydroxytryptaminewere labelled autoradiographi~lIy and luteinizing hormone-releasing hormone-containing neuronal elements were identified by means of immunocytochemistry. Synaptic contacts were observed between tritiated S-hydroxytryptamine-labelled boutons and luteinizing hormone-releasing hormone-immunoreactive dendrites. About 5% of the boutons which formed synapses with luteinizing hormone-releasing hormone-immunoreactive dendrites were found to be labelled by the tritiated indolamine. Luteinizing hormone-releasing hormone-immunoreactive axon terminals occurred as presynaptic elements in contact with unidenti~ed dendritic spines, shafts or perikarya. These observations provide morphological basis for the idea that S-hydroxyt~tamine-containing neurons can act directly on iuteinizing hormone-releasing hormone release. Further, they support the assumption that luteinizing hormone-releasing hormone is not only a neurohormone but may also function as a neurotransmitter or neuromodulator.
Luteinizing hormone-releasing hormone (LHRH, or gonadoliberin) which is synthetized by neurons situated in the rat brain mainly in the media1 preoptic area and septum,3s and liberated from axon terminals into the hypothalamohypophyseal portal vessels, stimulates the output of pituitary gonadotrophins, particularly of luteinizing hormone (LH).” It has been suggested4’ that serotoninergic neurons might be involved in the neural control of gonadotropin secretion. Serotonin (5-hydroxytryptamine; 5-HT) does not seem to act directly on pituitary LH secretion but exerts its influence probably through the LHRH neurons. This is suggested by the findings that when the medial basal hypothalamus and anterior pituitary of male rats were incubated together, addition of S-HT to the incubation medium had no effect on LH release. 33 Furthermore, there are serotoninergic fibers and presumably terminals in the medial preoptic area7,26,37where a significant number of LHRH neurons are located. The relationship between serotoninergic and LHRH-containing neuronal elements is unknown. Do serotoninergic fibers terminate directly on LHRH
neurons or do they end on neurons projecting to the LHRH cells? This question was investigated in the present studies examining the media1 preoptic area which contains both LHRH neurons projecting to the median eminence and serotoninergic fibers. A combination of immun~yt~hemistry and high resolution autoradiography was used. By this technique serotoninergic terminals selectively taking up [3H]5-HT can be labelled autoradiographically and the LHRH elements by the immunoreaction product. EXPERIME~AL
PROCEDURES
Trea~meR~of animals This study was carried out on eight adult male rats (body wt 210-230g). Each animal was pretreated with an intraventricular injection of colchicine (75p g/l 0 p 1 saline), administering the drug into the lateral ventricle of rats anesthetized with sodium hexobarbital (lOOmg/kg i.p.) and jmmobilized in a stereotaxic head frame. Colchicine injection was given 24 h prior to perfusion in order to increase the amount of LHRH in the perikarya.12 Each animal was pretreated with a monoamine oxidase inhibitor (pargyline/ N-methyl-N-benzyl-2_propynylamine,* 70 mg/kg i.p.) 18 and 2 h prior to microinfusion of t3H].5-HT to prevent the rapid metabolism of the tritiated monoamine. All animals pretreated with colchicine and with pargyline were reanestheti~ with hexobarbital, immobili~ in a stereotaxic head frame and submitted to intraventricuiar microinfusion of tritiated S-HT (5-[3H]hydroxytryptamine (G) creatinine sulfate, sp. act. 15 Ci/mmol, Amersham International) into the left lateral ventricle. In five animals 5 x lo-’ M [3H]5-HT was administered alone, in three others, 5 x 10m5 13H]5-HT was injected together with
*Please address all correspondence to: Dr. J. Kiss, Second Department of Anatomy, Semmelweis University, Medical School, Budapest, Tiizoltit II. 58, H-1450 Hungary. Abbreuiurions: 5-HT, 5-hydroxytryptamine; LH, luteinizing hormone; LHRH, luteinizing hormone-releasing hormone. 69
70
J. Kiss and B. Hal&
5 x 10m4 M non-radioactive noradrenaline (L-noradrenaline HCI). The [3H]5-HT was freshly diluted in cold saline containing 1% L-ascorbic acid and a total volume of 150 ~1 was infused over a period of 90 min as detailed elsewhere. ” Immediately after finishing the microinfusion of [jH]5-HT the animals were perfused through the heart first with Tyrode’s solution and then with a modified Zamboni’s fixative.‘6 Preparation of sections After perfusion the brains were removed, the preoptic and anterior hypothalamic regions were excised and immersed for an additional 1 h in an ice-cold glutaraldehyde-free Zamboni’s fixative. Frontal slices of 1-2 mm thickness were cut, postfixed for 1h in the same fixative then washed in 0.1 M phosphate buffer pH 7.4. Fifty micrometer thick sections were cut on a Vibratome (Oxford Instruments) and sections corresponding to levels between A7200 and A6500” containing media1 and lateral preoptic areas were selected for immunocytochemical procedure under microscopic control. Before immunostaining, the vials containing the sections in phosphate buffer were put into liquid nitrogen and then the slices thawed to room temperature. Antiserum An antiserum against LHRH [anti-LHRH (rabbit) No. H-141 was used. It was prepared and its specificity tested as described by Merchenthaler et al.24 Immunocytochemical procedure Incubation of sections was carried out according to Sternberger’s peroxidase-antiperoxidase technique3* in the following steps: after washing in phosphate buffer, sections were incubated in 207” normal goat serum (Human, Hungary), rinsed in phosphate-buffered saline containing 1:; normal roat serum. incubated at 4°C in the LHRH antiserum diluted to I : 10,000 for 24 h, rinsed in phosphatebuffered saline and then incubated overnight in the second (goat antirabbit immunoglobulin Cl) antiserum (Cappel) diluted I :40 at 4°C. After rinsing in phosphate-buffered saline, the sections were incubated for 6 h in rabbit complex (Sternberger-Meyer peroxidase-antiperoxidase Chemicals), diluted to 1: 100, then rinsed in phosphatebuffered saline, and subsequently in Tris-HCI. The antigen was located by incubation with O.OS’A 3,3_diaminobenzidine tetrahydrochloride (Sigma), dissolved in 0.05 M Tris-HCI buffer (pH 7.5) containing 0.01% hydrogen peroxide. The sections were washed in the Tris buffer, in phosphate buffer. postfixed in I”/, 0~0, dissolved in 0.1 M phosphate buffer, dehydrated and embedded in Durcupan as detailed elsewhere.2’ Procedure of autoradiography From the areas containing immunoreactive elements observed under the light microscope, four sections of 1 pm thickness were cut with a Reichert ultramicrotome for light microscopic autoradiography to check the intensity of the labelling. After 5, 10 or 20 days of exposure, when the light microscopic autoradiographic labelling was evident, blocks were retrimmed, and thin serial sections were cut by ultramicrotome, and dipped for electron microscope autoradiography as detailed previously.” Following 2, 3 or 4 months of exposure, the development was carried out using paraphenylenediamine.4 Sections were collected on Formvar-coated copper single-slot grids, and examined with a Jeol IOOC or Jeol 7C electron microscope. Earlier studies following intraventricular administration of [jH]5-HT’,9 suggested that the detection of serotoninergic fibers and axon terminals were possible by autoradiography. Specificity of the axonal labelling following uptake of [‘HIS-HT was confirmed by Descarries and Beaudet.’ Steinbusch” concluded later that the autoradiographic localization of serotoninergic neurons and terminals in the rat
hypothalamus coincided fairly accurately with the results obtained by the immunofluorescence technique.
RESULTS
Structure of 5-[3H]h,vdroxytryptamine-labelled axon terminals In light microscopic autoradiographs a diffuse pattern of scattered silver grains was evident particularly over the areas close to the ventricular surface; silver grain density diminished with increasing distance from the ventricle. In addition, there were clusters containing large numbers of silver grains. In electron microscopic autoradiographs small groups of silver grain aggregates were seen (Fig. 1A) in the neuropil. The structures overlaid by silver grain aggregates corresponded to roughly circular profiles of axon terminals (Figs IA,B, 2A,B, 4A-C and 5E). The labelled boutons measured 0.5-1.8 pm in diameter. They usually contained a heterogeneous population of vesicles: small, round, agranular vesicles of 35-55 nm in diameter as well as large, round granular vesicles, 80-120 nm in diameter (Figs lB, 2A,B, 4A,B and 5E). Approximately onethird of the labelled boutons showed the typical synaptic specialization at their surface membrane. Most of these labelled synaptic terminals clearly exhibited an asymmetrical junction (Fig. 2A). They were in synaptic contact with dendritic shafts (Fig. 2A) or dendritic spines (Fig. 2B). In most cases the postsynaptic dendrites did not contain immunoreactive reaction product (Fig. 2A,B). Luteinizing hormonePreleasing hormone -immune reactive neurons in postsynaptic contact with unlabelled boutons A small number of scattered LHRH-containing immunoreactive nerve cell bodies were found in the medial preoptic nucleus. They were of medium size (16-25 /*m in greatest dimension), had usually oval cell bodies and a centrally located round or oval nucleus with a smooth, not indented, nuclear envelope. Perikarya were filled with the reaction product associated with the surface of mitochondria, endoplasmic reticulum, the cell membrane and the outer membrane of vacuoles or vesicles. Synaptic contact onto one of the perikarya was only observed in one case. Immunoreactive dendritic elements were more numerous than immunostained perikarya and the peroxidase reaction product in them was located preferentially close to dendritic tubules (Figs 3A,B and 4A-C). Examination of serial sections of the immunostained dendrites revealed the presence of numerous synapses. In most cases the presynaptic boutons in these contacts were not radioactively labelled, and formed an asymmetrical synaptic contact with dendritic shafts (Fig. 3A,B). In these synapses the unlabelled presynaptic boutons contained small, round
Serotoninergic terminals on LHRH neurons
agranular vesicles of 35-55 nm, and large granular vesicles of 80-120 nm in diameter. 5-[3H]Hydroxytryptamine-labelled boutons in synaptic contacts with luteinizing hormone-releasing hormoneimmunoreactive dendrites Immunoreactive dendrites and [3H]5-HT-labelled axons or terminals were often in direct apposition. In a few cases typical synaptic contacts between [3H]5-HT-labelled terminal and immunostained dendrite were observed (Fig. 4A-C). 5-[3H]Hydroxytryptamine-labelled axons contributed approximately 5% of the overall axon population synapsing on LHRH-immunostained dendrites. These [3H]5-HTlabelled axon terminals were very similar in appearance to the unlabelled boutons and contained a heterogenous population of vesicles (small, round agranular, as well as large, round granular vesicles) (Fig. 4A-C). Luteinizing hormone-releasing hormone-immunoreactive terminals making synapses with nonimmunostained neuronal elements A considerable number of LHRH-immunoreactive axonal varicosities was seen (Fig. 5A-E). These boutons were medium size (0.5-1.8 pm in diameter), contained small (30-50 nm in diameter) and large, round vesicles of 8Gl20 nm in diameter. They were filled with immunoreactive product associated with the membrane of vesicles, mitochondria and with the inner surface of the axonal membrane. The immunostained presynaptic boutons formed synaptic contacts with dendritic spines (Fig. 5A,B), dendritic shafts (Fig. 5E) and soma (Fig. 5C). In one case the immunoreactive bouton was found to be in synaptic contact with a profile containing large secretory granules of 120-180nm in diameter (Fig. 5D).
DISCUSSION
The present findings confirm, at the electron microscope level, the light microscopic observations that S-HT fibers terminate in the medial preoptic region.7.26”7 Further, they demonstrate that a portion of these fibers synapses with LHRH-immunoreactive neurons. This observation supports previous findings that suggest the involvement of 5-HT in pituitary gonadotrophin function. It has been reported that in rats blockade of 5-HT synthesis with pchlorophenylalanine, an inhibitor of tryptophan hydroxylase, prevents ovulation,2’ the proestrous rise in LH” as well as the daily surge of LH in estrogentreated ovariectomized rats5,6x”and causes persistent estrous syndrome.4’ Restoration of 5-HT synthesis by treatment with 5-hydroxytryptophan, its immediate precursor, reinstates surge.5,6,‘0 Intrathe cerebroventricular administration of 5-HT was found by Porter et a1.27to increase LH secretion of male rats. Others observed an inhibitory influence of 5-HT.
71
Infusion of the indolamine into the third ventricle caused a decrease in plasma LH levels of male ratsI and inhibited LH secretion in ovariectomized rats.34 Systemic administration of large amounts of 5-HT inhibited the ovulation in immature rats elicited by administration of pregnant mare serum gonadotropin.25 Tima et ~1.~~have reported that daily i.p. administration of 5-HT for 30 days counteracted the ovulation-inducing effect of pinealectomy in persistent estrous rats. Also pharmacological increase of serum 5-HT level prevented superovulation in immature rats.” According to Trentini et al.” a decrease in brain 5-HT level by prolongedp-chlorophenylalanine treatment induced luteinization in more than 50% of persistent estrous rats. Stimulation of the dorsal raphe nucleus, a cell group rich in 5-HT neurons, inhibited episodic LH release.2 Pharmacological destruction of S-HT neurons with 5,6-dihydroxytryptamine was reported to cause a marked increase in LH secretion in male rats.20 This neurotoxin was also shown to have the opposite effect.43 The significance of 5-HT in the neural control of pituitary gonadotropin function is also supported by the findings of Biegon et al.3 They found a clear and significant difference between the 5-HT binding on diestrus-1 and 2 and proestrus-estrus in the basal forebrain (including the hypothalamus, septum and preoptic area). The binding on proestrus and estrus was about 407, lower than on diestrus. There was no effect of the cycle on 5-HT binding in the cortex and caudate nucleus. The occurrence of 5-HT terminals on LHRHimmunoreactive neuronal elements provides the structural basis for the view that the action of 5-HT neurons on pituitary gonadotrophin secretion is mediated via the LHRH neurons as was hypothesized by Schneider and McCann.33 Furthermore, the existence of such synapses indicates that the 5-HT neurons can act directly on the LHRH neurons. This of course, does not exclude the possibility of an additional indirect influence. The present observation does not give any information about the origin of the 5-HT fibers terminating on LHRH neurons in the medial preoptic area. It is possible that the S-HT axons and terminals of the medial preoptic area arise from neurons of the nucleus raphe dorsalis and centralis superior.7,37 A hypothalamic origin ‘,I5of some of these axons cannot be excluded. Approximately 5% of the boutons in synaptic contact with immunoreactive LHRH dendrites were found to be 5-HT boutons. This indicates that the majority of the boutons terminating on LHRH neurons are not serotoninergic. This is consistent with the observations that, among others, catecholamines and various brain peptides influence pituitary gonadotroph hormone secretion.23,42 Many 5-HT-labelled axons formed synaptic contacts with non-immunoreactive neuronal elements suggesting that 5-HT fibers can influence not only
72
J. Kiss and 3. Hal&z
LHRH neurons, but they might also act on other neurons of the medial preoptic area. We observed LHRH-like immunoreactive terminals as presynaptic structures forming synaptic contacts with unidentifi~ dendrites and perikarya. Johansson et ~1.‘~ and Shioda and Nakai3* have reported thyrotropin-releasing hormoneimmunoreactive fibers to form axosomatic, axoaxonic and/or axodendritic synapses with unidentified neurons in the hypothalamic arcuate nucleus. The
existence of LHRH axon terminals as presynaptic structures is consistent with the view that LHRH is not only a neurohormone but it might also function as a neurotransmitter or neuromoduIator.2~3’ Acknowfedgemen~~-We are grateful to Dr. G. Semi6 (P&s, Hungary) for generous provision of antiserum to LHRH, to Dr. Cs. I_&nth for help in immunocytochemistry, to A. Werglesz and Mrs Es. Popp for excellent technical assistance, to Mrs E. Petranyi for excellent phototechnical work. We thank Mrs. E. Plesk6 for typing the manuscript.
Fig. 1. Electron microscope autoradiographs of [3H]5-HT-labelled boutons in the rat preoptic area. (A) Two labelled boutons (t) can be seen on this low-power electron micrograph. Aggregates of silver grains cover the labelled boutons. Labelled axon terminal marked with asterisk in (A) is shown in (B) at higher power (asterisk). It contains small, round, agranular synaptic vesicles and a large granular vesicle (arrowhead). Scale bars: (A) 1 pm; (B) 0.4pm. Fig. 2. Labelled axon terminal making synaptic contacts with non-immunoreactive dendrites. (A) Asymmetrical synaptic contact (arrow) between a labelled bouton containing small, round or flattened agranular and large granular (arrowhead) vesicles and a dendritic shaft (d). (B) A labelled bouton forms symmet~~l synaptic contact (arrow) with a dendritic spine (s). The bouton contains small, round, agranular synaptic vesicles and large granular vesicles (arrowhead). Scale bars: (A) 0.7 pm; (B) 0.7 pm. Fig. 3. Luteinizing hormone-releasing hormone-immunoreactive dendrites can be seen in synaptic contact with non-labelled axon terminals. (A) A presynaptic bouton containing small, round, agranular synaptic vesicles and a number of large granular vesicles (arrowheads) makes an asymmetrical synaptic contact (arrow) with an immunostained dendrite (d). The presynaptic bouton in (B) making asymmetrical synapse (arrow) with the immunostained dendrite (d) contains mainly small, round, agranular synaptic vesicles. Scale bars: (A) 0.7 pm; (B) 0.8 pm. Fig. 4. S-[3HJHydroxytr~tamine-labelled terminals (T) make synaptic contacts (arrows in A, B and C) with LHRH-immunoreactive dendrites. (A) Two radiolabelled boutons, one of them forming a synapse with an immunostained dendrite (dl) containing a large amount of reaction product, that is associated with all dendritic organelles. The other labelled terminal is in synaptic contact with a dendrite (d2) where the reaction product is concentrated to a smaller site of the dendritic cytoplasm. The labelled bouton forming synaptic contact with dl dendrite is shown at a higher magnification in (B). (B) Large, round, granular vesicles (arrowheads) are visible in the labelled axon terminal. (C) A labelled bouton containing a great number of small, round, agranufar synaptic vesicles makes synaptic contact at two sites (arrows) with an immunoreactive dendrite (d3). Open arrows indicate areas of a higher concentration of reaction product in the postsynaptic dendrite region. Scale bars: (A) 0.7 pm; (B) 0.2 pm; (C) 0.3 pm. Fig. 5(A). An LHRH-immunoreactive terminal (t) forming an axodendritic synapse. The profile marked with asterisk in (A) is shown in (B) at a higher power. The immunostained bouton (t) contains a great number of small, round vesicles and makes symmetrical synaptic contact (arrow) with a dendritic spine (d). (C) An LHRH-immunostained bouton (t) can be seen forming a symmetrical axosomatic synapse (arrow) @k = ~~karyon). (0) An imm~oreactive bouton makes synaptic contact (arrow) with a neuronal profile containing neurosecretory granules (arrowheads). (E) Two immunostain~ boutons are seen (t). One of them makes synaptic contact (arrow) with a large dendrite (d). A [3H].5-HT-labelled terminal (T) is in close membrane contact with the same dendrite (d). Scale bars: (A) 0.4 pm; (B) 0.2 pm; (C) 0.2 pm; (D) 0.4 pm; (E) 0.4 pm.
Serotoninergic terminals on LHRH neurons
77
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35. S&al6 G., Flerko B., Arimura A. and Schally A. V. (1978) Brain cells as producers of releasing and inhibiting hormones Rev. Cytol., Suppl. 7, l-52. 36. Somogyi P. and Takagi H. (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7, 1779-1783. 37. Steinbusch H. W. M. (1981) Distribution of serotonin-immunoreactivity in the central nervous system of the rat+ell bodies and terminals. Neuroscience 6, 557-618. 38. Stemberger L. A., Hardy P. H., Cuculis J. J. and Meyer H. G. (1970) The unlabelled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen-antibody complex (horseradish-peroxidaseantiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-337. 39. Tima L., Trentini G. P. and Mess B. (1973) Effect of serotonin on ovulation induced by pinealectomy in anovulatory frontal-deafferented rats. Neuroendocrinology 12, 149-152. 40. Trentini G. P., Tima L., De Gaetani C. F. and Mess B. (1974) Luteinization induced by p-chlorophenylalanine treatment in constant oestrous anovulatory rats. Steroids Lipids Res. 5, 262-267. 41. Walker R. F., Cooper R. L. and Timiras P. S. (1980) Constant estrous: role of rostra1 hypothalamic monoamines in development of reproductive disfunction in aging rats. Endocrinology 107, 249-255. 42. Weiner R. I. and Ganong W. F. (1978) Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol. Rev. 58, 905-976. 43. Wuttke W., Bjorklund A., Baumgarten H.-G., Lachenmayer L., Fenske M. and Klemm H. P. (1977) De- and regeneration of brain serotonin neurons following 5,7-dihydroxytryptamine treatment: effects on serum LH, FSH and prolactin levels in male rats. Brain Res. 134, 317-331. (Accepted 25 July 1984)
~e~~u~eie~eeVol. 14, No. 1, pp. 69-78, 1985 Printed in Great Britain
DEMONSTRATION OF SEROTONINERGIC AXONS TERMINATING ON LUTEINIZING HORMONE-RELEASING HORMONE NEURONS IN THE PREOPTIC AREA OF THE RAT USING A COMBINATION OF IMMUNOCYTOCHEMISTRY AND HIGH RESOLUTION AUTORADIOGRAPHY J. KISS* and B. HAL&.Z Second Department of Anatomy, Semmelweis University, Medical School, H-1450 Budapest, Hungary Abstrac&--The synaptic relationship between serotoninergic terminals and luteinizing hormone-releasing ho~one~on~ning neurons was investiga~ in the medial preoptic area using a combined technique.
Axon terminals selectively taking up S-[~HJhydroxytryptaminewere labelled autoradiographi~lIy and luteinizing hormone-releasing hormone-containing neuronal elements were identified by means of immunocytochemistry. Synaptic contacts were observed between tritiated S-hydroxytryptamine-labelled boutons and luteinizing hormone-releasing hormone-immunoreactive dendrites. About 5% of the boutons which formed synapses with luteinizing hormone-releasing hormone-immunoreactive dendrites were found to be labelled by the tritiated indolamine. Luteinizing hormone-releasing hormone-immunoreactive axon terminals occurred as presynaptic elements in contact with unidenti~ed dendritic spines, shafts or perikarya. These observations provide morphological basis for the idea that S-hydroxyt~tamine-containing neurons can act directly on iuteinizing hormone-releasing hormone release. Further, they support the assumption that luteinizing hormone-releasing hormone is not only a neurohormone but may also function as a neurotransmitter or neuromodulator.
Luteinizing hormone-releasing hormone (LHRH, or gonadoliberin) which is synthetized by neurons situated in the rat brain mainly in the media1 preoptic area and septum,3s and liberated from axon terminals into the hypothalamohypophyseal portal vessels, stimulates the output of pituitary gonadotrophins, particularly of luteinizing hormone (LH).” It has been suggested4’ that serotoninergic neurons might be involved in the neural control of gonadotropin secretion. Serotonin (5-hydroxytryptamine; 5-HT) does not seem to act directly on pituitary LH secretion but exerts its influence probably through the LHRH neurons. This is suggested by the findings that when the medial basal hypothalamus and anterior pituitary of male rats were incubated together, addition of S-HT to the incubation medium had no effect on LH release. 33 Furthermore, there are serotoninergic fibers and presumably terminals in the medial preoptic area7,26,37where a significant number of LHRH neurons are located. The relationship between serotoninergic and LHRH-containing neuronal elements is unknown. Do serotoninergic fibers terminate directly on LHRH
neurons or do they end on neurons projecting to the LHRH cells? This question was investigated in the present studies examining the media1 preoptic area which contains both LHRH neurons projecting to the median eminence and serotoninergic fibers. A combination of immun~yt~hemistry and high resolution autoradiography was used. By this technique serotoninergic terminals selectively taking up [3H]5-HT can be labelled autoradiographically and the LHRH elements by the immunoreaction product. EXPERIME~AL
PROCEDURES
Trea~meR~of animals This study was carried out on eight adult male rats (body wt 210-230g). Each animal was pretreated with an intraventricular injection of colchicine (75p g/l 0 p 1 saline), administering the drug into the lateral ventricle of rats anesthetized with sodium hexobarbital (lOOmg/kg i.p.) and jmmobilized in a stereotaxic head frame. Colchicine injection was given 24 h prior to perfusion in order to increase the amount of LHRH in the perikarya.12 Each animal was pretreated with a monoamine oxidase inhibitor (pargyline/ N-methyl-N-benzyl-2_propynylamine,* 70 mg/kg i.p.) 18 and 2 h prior to microinfusion of t3H].5-HT to prevent the rapid metabolism of the tritiated monoamine. All animals pretreated with colchicine and with pargyline were reanestheti~ with hexobarbital, immobili~ in a stereotaxic head frame and submitted to intraventricuiar microinfusion of tritiated S-HT (5-[3H]hydroxytryptamine (G) creatinine sulfate, sp. act. 15 Ci/mmol, Amersham International) into the left lateral ventricle. In five animals 5 x lo-’ M [3H]5-HT was administered alone, in three others, 5 x 10m5 13H]5-HT was injected together with
*Please address all correspondence to: Dr. J. Kiss, Second Department of Anatomy, Semmelweis University, Medical School, Budapest, Tiizoltit II. 58, H-1450 Hungary. Abbreuiurions: 5-HT, 5-hydroxytryptamine; LH, luteinizing hormone; LHRH, luteinizing hormone-releasing hormone. 69
70
J. Kiss and B. Hal&
5 x 10m4 M non-radioactive noradrenaline (L-noradrenaline HCI). The [3H]5-HT was freshly diluted in cold saline containing 1% L-ascorbic acid and a total volume of 150 ~1 was infused over a period of 90 min as detailed elsewhere. ” Immediately after finishing the microinfusion of [jH]5-HT the animals were perfused through the heart first with Tyrode’s solution and then with a modified Zamboni’s fixative.‘6 Preparation of sections After perfusion the brains were removed, the preoptic and anterior hypothalamic regions were excised and immersed for an additional 1 h in an ice-cold glutaraldehyde-free Zamboni’s fixative. Frontal slices of 1-2 mm thickness were cut, postfixed for 1h in the same fixative then washed in 0.1 M phosphate buffer pH 7.4. Fifty micrometer thick sections were cut on a Vibratome (Oxford Instruments) and sections corresponding to levels between A7200 and A6500” containing media1 and lateral preoptic areas were selected for immunocytochemical procedure under microscopic control. Before immunostaining, the vials containing the sections in phosphate buffer were put into liquid nitrogen and then the slices thawed to room temperature. Antiserum An antiserum against LHRH [anti-LHRH (rabbit) No. H-141 was used. It was prepared and its specificity tested as described by Merchenthaler et al.24 Immunocytochemical procedure Incubation of sections was carried out according to Sternberger’s peroxidase-antiperoxidase technique3* in the following steps: after washing in phosphate buffer, sections were incubated in 207” normal goat serum (Human, Hungary), rinsed in phosphate-buffered saline containing 1:; normal roat serum. incubated at 4°C in the LHRH antiserum diluted to I : 10,000 for 24 h, rinsed in phosphatebuffered saline and then incubated overnight in the second (goat antirabbit immunoglobulin Cl) antiserum (Cappel) diluted I :40 at 4°C. After rinsing in phosphate-buffered saline, the sections were incubated for 6 h in rabbit complex (Sternberger-Meyer peroxidase-antiperoxidase Chemicals), diluted to 1: 100, then rinsed in phosphatebuffered saline, and subsequently in Tris-HCI. The antigen was located by incubation with O.OS’A 3,3_diaminobenzidine tetrahydrochloride (Sigma), dissolved in 0.05 M Tris-HCI buffer (pH 7.5) containing 0.01% hydrogen peroxide. The sections were washed in the Tris buffer, in phosphate buffer. postfixed in I”/, 0~0, dissolved in 0.1 M phosphate buffer, dehydrated and embedded in Durcupan as detailed elsewhere.2’ Procedure of autoradiography From the areas containing immunoreactive elements observed under the light microscope, four sections of 1 pm thickness were cut with a Reichert ultramicrotome for light microscopic autoradiography to check the intensity of the labelling. After 5, 10 or 20 days of exposure, when the light microscopic autoradiographic labelling was evident, blocks were retrimmed, and thin serial sections were cut by ultramicrotome, and dipped for electron microscope autoradiography as detailed previously.” Following 2, 3 or 4 months of exposure, the development was carried out using paraphenylenediamine.4 Sections were collected on Formvar-coated copper single-slot grids, and examined with a Jeol IOOC or Jeol 7C electron microscope. Earlier studies following intraventricular administration of [jH]5-HT’,9 suggested that the detection of serotoninergic fibers and axon terminals were possible by autoradiography. Specificity of the axonal labelling following uptake of [‘HIS-HT was confirmed by Descarries and Beaudet.’ Steinbusch” concluded later that the autoradiographic localization of serotoninergic neurons and terminals in the rat
hypothalamus coincided fairly accurately with the results obtained by the immunofluorescence technique.
RESULTS
Structure of 5-[3H]h,vdroxytryptamine-labelled axon terminals In light microscopic autoradiographs a diffuse pattern of scattered silver grains was evident particularly over the areas close to the ventricular surface; silver grain density diminished with increasing distance from the ventricle. In addition, there were clusters containing large numbers of silver grains. In electron microscopic autoradiographs small groups of silver grain aggregates were seen (Fig. 1A) in the neuropil. The structures overlaid by silver grain aggregates corresponded to roughly circular profiles of axon terminals (Figs IA,B, 2A,B, 4A-C and 5E). The labelled boutons measured 0.5-1.8 pm in diameter. They usually contained a heterogeneous population of vesicles: small, round, agranular vesicles of 35-55 nm in diameter as well as large, round granular vesicles, 80-120 nm in diameter (Figs lB, 2A,B, 4A,B and 5E). Approximately onethird of the labelled boutons showed the typical synaptic specialization at their surface membrane. Most of these labelled synaptic terminals clearly exhibited an asymmetrical junction (Fig. 2A). They were in synaptic contact with dendritic shafts (Fig. 2A) or dendritic spines (Fig. 2B). In most cases the postsynaptic dendrites did not contain immunoreactive reaction product (Fig. 2A,B). Luteinizing hormonePreleasing hormone -immune reactive neurons in postsynaptic contact with unlabelled boutons A small number of scattered LHRH-containing immunoreactive nerve cell bodies were found in the medial preoptic nucleus. They were of medium size (16-25 /*m in greatest dimension), had usually oval cell bodies and a centrally located round or oval nucleus with a smooth, not indented, nuclear envelope. Perikarya were filled with the reaction product associated with the surface of mitochondria, endoplasmic reticulum, the cell membrane and the outer membrane of vacuoles or vesicles. Synaptic contact onto one of the perikarya was only observed in one case. Immunoreactive dendritic elements were more numerous than immunostained perikarya and the peroxidase reaction product in them was located preferentially close to dendritic tubules (Figs 3A,B and 4A-C). Examination of serial sections of the immunostained dendrites revealed the presence of numerous synapses. In most cases the presynaptic boutons in these contacts were not radioactively labelled, and formed an asymmetrical synaptic contact with dendritic shafts (Fig. 3A,B). In these synapses the unlabelled presynaptic boutons contained small, round
Serotoninergic terminals on LHRH neurons
agranular vesicles of 35-55 nm, and large granular vesicles of 80-120 nm in diameter. 5-[3H]Hydroxytryptamine-labelled boutons in synaptic contacts with luteinizing hormone-releasing hormoneimmunoreactive dendrites Immunoreactive dendrites and [3H]5-HT-labelled axons or terminals were often in direct apposition. In a few cases typical synaptic contacts between [3H]5-HT-labelled terminal and immunostained dendrite were observed (Fig. 4A-C). 5-[3H]Hydroxytryptamine-labelled axons contributed approximately 5% of the overall axon population synapsing on LHRH-immunostained dendrites. These [3H]5-HTlabelled axon terminals were very similar in appearance to the unlabelled boutons and contained a heterogenous population of vesicles (small, round agranular, as well as large, round granular vesicles) (Fig. 4A-C). Luteinizing hormone-releasing hormone-immunoreactive terminals making synapses with nonimmunostained neuronal elements A considerable number of LHRH-immunoreactive axonal varicosities was seen (Fig. 5A-E). These boutons were medium size (0.5-1.8 pm in diameter), contained small (30-50 nm in diameter) and large, round vesicles of 8Gl20 nm in diameter. They were filled with immunoreactive product associated with the membrane of vesicles, mitochondria and with the inner surface of the axonal membrane. The immunostained presynaptic boutons formed synaptic contacts with dendritic spines (Fig. 5A,B), dendritic shafts (Fig. 5E) and soma (Fig. 5C). In one case the immunoreactive bouton was found to be in synaptic contact with a profile containing large secretory granules of 120-180nm in diameter (Fig. 5D).
DISCUSSION
The present findings confirm, at the electron microscope level, the light microscopic observations that S-HT fibers terminate in the medial preoptic region.7.26”7 Further, they demonstrate that a portion of these fibers synapses with LHRH-immunoreactive neurons. This observation supports previous findings that suggest the involvement of 5-HT in pituitary gonadotrophin function. It has been reported that in rats blockade of 5-HT synthesis with pchlorophenylalanine, an inhibitor of tryptophan hydroxylase, prevents ovulation,2’ the proestrous rise in LH” as well as the daily surge of LH in estrogentreated ovariectomized rats5,6x”and causes persistent estrous syndrome.4’ Restoration of 5-HT synthesis by treatment with 5-hydroxytryptophan, its immediate precursor, reinstates surge.5,6,‘0 Intrathe cerebroventricular administration of 5-HT was found by Porter et a1.27to increase LH secretion of male rats. Others observed an inhibitory influence of 5-HT.
71
Infusion of the indolamine into the third ventricle caused a decrease in plasma LH levels of male ratsI and inhibited LH secretion in ovariectomized rats.34 Systemic administration of large amounts of 5-HT inhibited the ovulation in immature rats elicited by administration of pregnant mare serum gonadotropin.25 Tima et ~1.~~have reported that daily i.p. administration of 5-HT for 30 days counteracted the ovulation-inducing effect of pinealectomy in persistent estrous rats. Also pharmacological increase of serum 5-HT level prevented superovulation in immature rats.” According to Trentini et al.” a decrease in brain 5-HT level by prolongedp-chlorophenylalanine treatment induced luteinization in more than 50% of persistent estrous rats. Stimulation of the dorsal raphe nucleus, a cell group rich in 5-HT neurons, inhibited episodic LH release.2 Pharmacological destruction of S-HT neurons with 5,6-dihydroxytryptamine was reported to cause a marked increase in LH secretion in male rats.20 This neurotoxin was also shown to have the opposite effect.43 The significance of 5-HT in the neural control of pituitary gonadotropin function is also supported by the findings of Biegon et al.3 They found a clear and significant difference between the 5-HT binding on diestrus-1 and 2 and proestrus-estrus in the basal forebrain (including the hypothalamus, septum and preoptic area). The binding on proestrus and estrus was about 407, lower than on diestrus. There was no effect of the cycle on 5-HT binding in the cortex and caudate nucleus. The occurrence of 5-HT terminals on LHRHimmunoreactive neuronal elements provides the structural basis for the view that the action of 5-HT neurons on pituitary gonadotrophin secretion is mediated via the LHRH neurons as was hypothesized by Schneider and McCann.33 Furthermore, the existence of such synapses indicates that the 5-HT neurons can act directly on the LHRH neurons. This of course, does not exclude the possibility of an additional indirect influence. The present observation does not give any information about the origin of the 5-HT fibers terminating on LHRH neurons in the medial preoptic area. It is possible that the S-HT axons and terminals of the medial preoptic area arise from neurons of the nucleus raphe dorsalis and centralis superior.7,37 A hypothalamic origin ‘,I5of some of these axons cannot be excluded. Approximately 5% of the boutons in synaptic contact with immunoreactive LHRH dendrites were found to be 5-HT boutons. This indicates that the majority of the boutons terminating on LHRH neurons are not serotoninergic. This is consistent with the observations that, among others, catecholamines and various brain peptides influence pituitary gonadotroph hormone secretion.23,42 Many 5-HT-labelled axons formed synaptic contacts with non-immunoreactive neuronal elements suggesting that 5-HT fibers can influence not only
72
J. Kiss and 3. Hal&z
LHRH neurons, but they might also act on other neurons of the medial preoptic area. We observed LHRH-like immunoreactive terminals as presynaptic structures forming synaptic contacts with unidentifi~ dendrites and perikarya. Johansson et ~1.‘~ and Shioda and Nakai3* have reported thyrotropin-releasing hormoneimmunoreactive fibers to form axosomatic, axoaxonic and/or axodendritic synapses with unidentified neurons in the hypothalamic arcuate nucleus. The
existence of LHRH axon terminals as presynaptic structures is consistent with the view that LHRH is not only a neurohormone but it might also function as a neurotransmitter or neuromoduIator.2~3’ Acknowfedgemen~~-We are grateful to Dr. G. Semi6 (P&s, Hungary) for generous provision of antiserum to LHRH, to Dr. Cs. I_&nth for help in immunocytochemistry, to A. Werglesz and Mrs Es. Popp for excellent technical assistance, to Mrs E. Petranyi for excellent phototechnical work. We thank Mrs. E. Plesk6 for typing the manuscript.
Fig. 1. Electron microscope autoradiographs of [3H]5-HT-labelled boutons in the rat preoptic area. (A) Two labelled boutons (t) can be seen on this low-power electron micrograph. Aggregates of silver grains cover the labelled boutons. Labelled axon terminal marked with asterisk in (A) is shown in (B) at higher power (asterisk). It contains small, round, agranular synaptic vesicles and a large granular vesicle (arrowhead). Scale bars: (A) 1 pm; (B) 0.4pm. Fig. 2. Labelled axon terminal making synaptic contacts with non-immunoreactive dendrites. (A) Asymmetrical synaptic contact (arrow) between a labelled bouton containing small, round or flattened agranular and large granular (arrowhead) vesicles and a dendritic shaft (d). (B) A labelled bouton forms symmet~~l synaptic contact (arrow) with a dendritic spine (s). The bouton contains small, round, agranular synaptic vesicles and large granular vesicles (arrowhead). Scale bars: (A) 0.7 pm; (B) 0.7 pm. Fig. 3. Luteinizing hormone-releasing hormone-immunoreactive dendrites can be seen in synaptic contact with non-labelled axon terminals. (A) A presynaptic bouton containing small, round, agranular synaptic vesicles and a number of large granular vesicles (arrowheads) makes an asymmetrical synaptic contact (arrow) with an immunostained dendrite (d). The presynaptic bouton in (B) making asymmetrical synapse (arrow) with the immunostained dendrite (d) contains mainly small, round, agranular synaptic vesicles. Scale bars: (A) 0.7 pm; (B) 0.8 pm. Fig. 4. S-[3HJHydroxytr~tamine-labelled terminals (T) make synaptic contacts (arrows in A, B and C) with LHRH-immunoreactive dendrites. (A) Two radiolabelled boutons, one of them forming a synapse with an immunostained dendrite (dl) containing a large amount of reaction product, that is associated with all dendritic organelles. The other labelled terminal is in synaptic contact with a dendrite (d2) where the reaction product is concentrated to a smaller site of the dendritic cytoplasm. The labelled bouton forming synaptic contact with dl dendrite is shown at a higher magnification in (B). (B) Large, round, granular vesicles (arrowheads) are visible in the labelled axon terminal. (C) A labelled bouton containing a great number of small, round, agranufar synaptic vesicles makes synaptic contact at two sites (arrows) with an immunoreactive dendrite (d3). Open arrows indicate areas of a higher concentration of reaction product in the postsynaptic dendrite region. Scale bars: (A) 0.7 pm; (B) 0.2 pm; (C) 0.3 pm. Fig. 5(A). An LHRH-immunoreactive terminal (t) forming an axodendritic synapse. The profile marked with asterisk in (A) is shown in (B) at a higher power. The immunostained bouton (t) contains a great number of small, round vesicles and makes symmetrical synaptic contact (arrow) with a dendritic spine (d). (C) An LHRH-immunostained bouton (t) can be seen forming a symmetrical axosomatic synapse (arrow) @k = ~~karyon). (0) An imm~oreactive bouton makes synaptic contact (arrow) with a neuronal profile containing neurosecretory granules (arrowheads). (E) Two immunostain~ boutons are seen (t). One of them makes synaptic contact (arrow) with a large dendrite (d). A [3H].5-HT-labelled terminal (T) is in close membrane contact with the same dendrite (d). Scale bars: (A) 0.4 pm; (B) 0.2 pm; (C) 0.2 pm; (D) 0.4 pm; (E) 0.4 pm.
Serotoninergic terminals on LHRH neurons
77
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