331
J. Anat. (1979), 128, 2, pp. 331-347 With 14 figures Printed in Great Britain
Simultaneous localization of LHRH and catecholamines in rat hypothalamus KATSUYA AJIKA
Department of Obstetrics and Gynaecology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo, Japan
(Accepted 4 April 1978) INTRODUCTION
Luteinizing hormone releasing hormone (LHRH) has recently been demonstrated by immunofluorescence and immunohistochemistry in the hypothalamus of various species of mammals including the guinea-pig (Barry, Dubois & Poulain, 1973; Silverman, 1976), rat (SetAlo et al. 1975; Naik, 1975) and monkey (Barry & Carette, 1975; Silverman, Antunes, Ferin & Zimmerman, 1977) (see Dubois, 1976 for detailed list of publications). There is general agreement that substantial amounts of LHRH reside in the neuronal processes in the median eminence and the organum vasculosum of the lamina terminalis (OVLT) of these animals. In the mouse, however, LHRH was localized in the tanycytes rather than in the neuronal processes (Silverman, 1976; Zimmerman, Hsu, Ferin & Kozlowski, 1974). With respect to the localization of the immunoreactive LHRH cell bodies, reports differ according to the species and technique used. Thus the immunoreactive LHRH perikarya were rather widely localized, extending from the parolfactory and precommissural area to the caudal end of the tuber cinereum in the guinea-pig (Barry, Dubois & Carette, 1974), and monkey (Barry & Carette, 1975; Silverman et al. 1977). In immunohistochemical studies, however, some investigators were unable to demonstrate LHRH in the perikarya of the rat hypothalamus (King, 1974; Baker, Dermody & Reel, 1975; Gross, 1976), while others claimed that LHRH was demonstrable in the nerve cell bodies of several hypothalamic nuclei (Naik, 1975; Setalo et al. 1976). An array of evidence indicates that catecholamines participate in the regulatory mechanism of the release of gonadotropin secretion (McCann et al. 1971; Hokfelt and Fuxe, 1971). McCann's group suggested that dopamine was involved in stimulating LHRH release (McCann et al. 1971), while Fuxe's group showed that dopamine inhibited the release of gonadotropin secretion (Fuxe et al. 1976). In both cases it was inferred that catecholamine acted in the median eminence, dopamine being released from the nerve terminals here to influence the release of LHRH. However, the precise mechanism and the histological background have not received much attention (see Hokfelt & Fuxe, 1971; Ajika & Hokfelt, 1975). In fact clear evidence is lacking that LHRH and catecholamine are contained in different neuronal systems. The present study was concerned with the demonstration of LHRH-positive perikarya in the pregnant rat, and the differentiation of LHRH and catecholamine neuronal systems with an immunohistochemical double staining technique.
3'3 2
K. AJIKA MATERIALS AND METHODS
Animals and tissue preparations Adult male and pregnant female rats (15 to 20 days of gestation) of the Wistar strain were used. They were maintained in air-conditioned quarters with a controlled lighting schedule and were fed ad libitum. For light microscopy the animals were perfused with Zamboni's picric acid paraformaldehyde fixative (PAF) via the ascending aorta, under Nembutal anaesthesia. The brain was dissected out and immersed in the same fixative for 4 to 10 hours. In another group of rats the brain was dissected out under Nembutal anaesthesia and immersed in PAF for 48 hours. Specimens containing the hypothalamus were embedded in paraffin and sectioned serially at 5 to 7 ,tm in a coronal or sagittal plane. For electron microscopy, hypothalamic fragments, fixed (either by cardiac perfusion or simple immersion) in either PAF or a mixture of 2 % paraformaldehyde and 2 0 glutaraldehyde, were dehydrated in ethanol and embedded in Epon. They were sectioned on a Reichert Om U3 ultramicrotome equipped with a diamond knife. The ultrathin sections were collected on nickel grids and served for immunohistochemical staining. In another experiment the hypothalamic tissue fixed with PAF or with the mixture of 2 % paraformaldehyde and 2 % glutaraldehyde was sectioned with a Vibratome (Oxford Instruments, Pasadena, California, U.S.A.) at 40 to 50 ,um, in phosphate buffer. These sections were subjected to immunohistochemical staining procedures. After staining, they were osmified, dehydrated and embedded in Epon. Ultrathin sections were obtained from the surface zone of these immunohistochemically stained sections and examined in a Hitachi 1 ID electron microscope. Immunohistochemical staining Light microscopy Following deparaffinization and hydration the sections were stained for LHRH and/or tyrosine hydroxylase (TH), catecholamine synthesizing enzyme, by the peroxidase-antiperoxidase (PAP) method of Sternberger, Hardy, Cuculis & Meyer (1970). Prior to immunohistochemical staining the sections were incubated with 1 % normal goat serum in PBS for 10 minutes to reduce non-specific protein binding. The sections were then incubated sequentially in 1 hour steps with LHRH antiserum (1:200), goat anti-rabbit IgG (1:30; Miles Laboratories, U.S.A.) and PAP (1:70). All staining procedures were carried out at room temperature in a moist chamber, and two 5 minute washes in PBS followed each step. Peroxidase activity was revealed by brown products of 3,3'-diaminobenzidine-H202 (DAB-H202) as in the case of LHRH, and by blue products of 4-Cl-l-naphtol as in TH. In the case of double staining of LHRH and TH, the procedure was fundamentally similar to that of Nakane (1968) except for using the PAP complex as a marker for the antibody in the present study. The sections were reacted first with LHRH antiserum and stained for peroxidase, using DAB as substrate. They were immersed and washed in 0-1 M-glycine-HCl (Ph 2 2) for 1 hour to remove the first antibody and conjugate. The DAB reaction product remained on the tissue. The sections were then serially incubated with TH antiserum, IgG and PAP complex. Peroxidase activity was revealed by incubation in 4-Cl-l-naphtol-H20, for 7 to 10 minutes. Specificity control included substitution of normal rabbit serum for the rabbit
LHRH and dopamine
333 LHRH or TH antisera, and the absorption of each antisera with 10 to 1000 ng of synthetic LHRH (Takeda Chemical Industries, Osaka, Japan) or bovine adrenal TH. The LHRH antiserum was tested not to cross-react with vasopressin, oxytocin, TRH or BSA, and the TH antisera was tested not to cross-react with dopamine-,fhydroxylase or phenylethanolamine-N-methyltransferase. The PAP complex was a gift from Dr L. A. Sternberger. LHRH antisera no. 185 raised in a rabbit immunized with LHRH conjugated to BSA was generously supplied by Dr W. C. Dermody, and TH antisera raised in a rabbit immunized with bovine adrenal tyrosine hydroxylase by Dr I. Nagatsu. Electron microscopy The electron microscopical staining procedures were fundamentally similar to those previously described (Moriarty & Halmi, 1972; Sternberger, 1972). Prior to immunohistochemical staining the ultrathin sections mounted on Formvar-coated nickel grids were etched by flotation on a drop of distilled water containing 0-01 % benzene, 5 00 methanol and 5 % ethanol. To reduce non-specific protein binding the grids were first floated on normal goat serum (1:30 dilution with PBS containing 0.1 % gelatin). The grids were then floated sequentially in 10 minute steps on a drop of LHRH antiserum (1:1000 to 1: 5000 dilution with PBS containing 04 % gelatin), goat anti-rabbit IgG (1: 30) and PAP (1: 50). After exposure to each solution they were rinsed in PBS containing 0-1 00 gelatin. DAB-H202 was used as substrate. In the case of TH simple staining, and the double staining for LHRH and TH, the hypothalamic vibratome sections were used for immunohistochemical staining. For the double staining of LHRH and TH, these sections were sequentially immersed in a drop of normal goat serum (1:100), LHRH antisera (1:100 to 1:400), IgG (1 :30) and PAP complex (1 :70). They were immersed and washed in 01 Mglycine-HCl for 10 minutes to remove the first antibody and conjugate. They were then immersed in a drop of TH antisera, IgG and PAP complex. For the simple staining of TH, only the latter procedures were carried out. Each solution was diluted either with PBS or with PBS containing 0-1 00 gelatin. All staining procedures were carried out at room temperature in a moist chamber, and washing in three consecutive changes of PBS followed each step. DAB-H202 was used as peroxidase substrate. After application of DAB-H202 the grids were washed in distilled water, stained on a drop of buffered 2 % OS04 for 30 minutes and washed in distilled water. -
RESULTS
LHRH-positive cell bodies
LHRH-positive cell bodies were observed in the medial preoptic area and in the medial prechiasmatic area of the pregnant rat (Fig. 1). Not more than three LHRHpositive perikarya were seen in any one sagittal section. LHRH-positive cell bodies were rarely encountered in coronal sections. Even in mid-sagittal sections more than half the specimens failed to show any LHRH-positive cell bodies. The LHRHpositive perikarya were ovoid in shape, and ranged from 10 to 13 ,m in diameter. They appeared most frequently in the medial preoptic area, with beaded string-like processes directing ventrally to the organum vasculosum of the lamina terminalis (OVLT), and less often in the median eminence. LHRH-positive cell bodies were not observed in other areas, including the arcuate nucleus, ventromedial nucleus
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Electron microscopy Electron microscopy in the tissue routinely processed without immunohistochemical staining revealed that a number of nerve terminals were densely arranged in the perivascular region of the median eminence of normal male and pregnant female rats (Fig. 6). Many nerve terminals were in direct contact with each other, and with the basement membrane of a portal vessel, without the intervention of glial elements. These terminals contained large dense vesicles ranging from 80 to 110 nm in diameter, and small lucent vesicles with a diameter of 50 nm. Some nerve terminals contained even larger dense vesicles, the diameters of which ranged from 130 to 150 nm. The immunohistochemical PAP staining which was carried out on ultrathin sections on the nickel grids labelled exclusively the dense vesicles, 80 to 110 nm diameter, within the LHRH-positive terminals in the lateral perivascular region of the median eminence. No other micro-organelles were stained in the LHRHpositive axon terminals. The LHRH-positive terminals appeared in close proximity to portal vessels (Fig. 8).
Dopamine cell bodies Light microscopy In the pregnant rat dopamine-positive cell bodies were consistently revealed in the arcuate nucleus (A12 cell group, according to Dahlstrom & Fuxe, 1964) using tyrosine hydroxylase (TH) as marker. The cytoplasm of TH-positive cells was strongly stained blue when naphtol was used as substrate (Fig. 3). The cells ranged from 10 to 13 ,tm in diameter. Other TH-positive cell groups appeared in the rostral periventricular area (A14 cell group), in the dorsal hypothalamic area (Al3) and in the caudal hypothalamic area (Al1). In the male rat, TH-positive cell groups (except A12) were consistently stained. However, the stainability of A12 cells was considerably less than that of the pregnant rat. In some cases, TH-positive cells were not seen in the arcuate nucleus of the male rat. No TH-positive cell body was stained in the control sections from the pregnant rat treated with normal rabbit serum or the TH antiserum absorbed in advance with TH in substitution of TH antiserum. Fig. 3. A frontal section of the median eminence. The TH-positive terminals are densely localized in the lateral perivascular region of the median eminence (me), as indicated by arrowheads. A considerable number of TH-positive fibres are also seen in the medial part of the median eminence (arrow). In the arcuate nucleus (ac), a number of TH-positive perikarya are seen. v, third ventricle. x 135. Fig. 4. A frontal section is stained with LHRH antiserum using 3,3'-diaminobenzidine as substrate. The LHRH-positive terminals are densely localized in the lateral perivascular region of the median eminence, while in the medial part there are a few LHRH-positive processes (arrow). In the arcuate nucleus (ac), no LHRH-positive perikarya are seen. v, third ventricle. x 160. Fig. 5. The median eminence (me) is simultaneously stained with TH and LHRH antiserum. TH-positive terminals are densely localized in the lateral and medial part of the perivascular region of the median eminence, while LHRH-positive terminals are mostly localized in the lateral part. In the arcuate nucleus, the stained perikarya are exclusively TH-positive. Figs. 4 and 5 are serial sections. x 160.
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Electron microscopy Electron microscopy of the tissue immunohistochemically stained with TH antiserum and PAP revealed the presence of TH-positive cell bodies in the arcuate nucleus of the pregnant rat. Within the cytoplasm the endoplasmic reticulum, membranes of the Golgi apparatus and the neurotubules were strongly and evenly stained, while the nucleus was less heavily stained (Fig. 7). Dopamine terminals in the median eminence Light microscopy In both male and pregnant female rats, TH-positive terminals were densely stained in the perivascular region of the median eminence with TH antiserum and PAP. The TH-positive nerve fibres appeared as delicate beaded strings and were blue when naphtol was used as substrate (Figs. 3, 5). A number of TH-positive terminals were densely arranged along the portal vessels.
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In coronal view, TH-positive fibres were most concentrated in the lateral penivascular region of the median eminence. In contrast with LHRH-positive terminals, however, considerable numbers of TH-positive fibres were also observed in the medial perivascular region. No TH-positive terminals were stained in the control section which was treated with normal rabbit serum or with the TH antiserum absorbed in advance with TH in substitution of TH antiserum.
Electron microscopy In both male and pregnant female rats, electron micrographs of sections through the median eminence incubated with TH antiserum and PAP showed a number of dopamine neurons stained with electron-dense material (Figs. 9, 10, 11, 12, 13). The peroxidase reaction products were preferentially associated with endoplasmic reticulum in the perikarya or in the dendrites, with neurotubules of 22 nm diameter in the axons and with small vesicles of 50 nm in the terminals (Figs. 9, 10, 11, 12). In the lateral perivascular region of the median eminence some of the terminals 22-2
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LHRH and dopamine 341 stained with TH antiserum were in direct contact with each other, and with the basement membrane of the portal vessels, without the intervention of glial elements (Fig. 13). Simultaneous staining of LHRH and dopamine terminals Light microscopy In the tissue which was reacted consecutively with LHRH and TH antiserum, both LHRH and TH-positive terminals were revealed as brown precipitates with DAB and as blue precipitates with naphtol, respectively (Fig. 5). In the lateral perivascular region of the median eminence these two kinds of terminal overlapped in places because of their high density, but were clearly differentiated from one another by their colour. The medial part of the perivascular region, on the other hand, was almost exclusively occupied with the blue precipitate of the TH-positive terminals and few brown precipitates of LHRH terminals were observed. In the arcuate nucleus of the pregnant rat the cell bodies appeared exclusively blue. These findings were clearly verified when sections stained for either TH or LHRH were compared with the double stained sections (Figs. 3, 4 and 5). Electron microscopy Electron micrographs of the lateral perivascular region of the median eminence incubated consecutively with LHRH and TH antiserum showed two kinds of terminal stained with electron-dense materials (Fig. 14). One type, judged as LHRH, contained many lar-ge electron-dense vesicles with a diameter of 100 nm. The other type, judged as dopamine, was more densely stained and contained many small vesicles with a diameter of 50 nm. These two kinds of terminal were in direct contact with each other without the intervention of glial elements. A number of other terminals, and the processes of ependymal cells and tanycytes, were negative. DISC USSION
LHRH in the 0 VLT The localization of LHRH in the OVLT was confirmed in the male and the pregnant female rat, as previously reported in the mouse (Zimmerman et al. 1974; Baker, 1975) and the rat (Weiner, Pattou, Kerdelhue & Kordon, 1975). In the present study, the dense meshwork of LHRH-positive axonal processes terminated Fig. 9. Electron micrograph of the median eminence, immunohistochemically stained with TH antiserum by PAP method. The peroxidase reaction products are preferentially associated with endoplasmic reticulum (double arrows) in the dendrites or in the perikarya, with neurotubules (short arrows) in the axons and with small vesicles (long arrows) in the terminals. x 5200. The inset is a high power view of the marked area in Fig. 9. In the axonal process (ax) neurotubules with a diameter of 22 nm (short arrow) are preferentially stained, while in the terminal (t) the small vesicles with a diameter of 50 nm are specifically stained. x 10000. Fig. 10. Electron micrograph of the median eminence, immunohistochemically stained with TH antiserum by the PAP method. TH is specifically localized in the small vesicles (arrows) in the DA terminal (t). x 28000. Figs. 11 and 12. Higher power electron micrographs of the median eminence, immunohistochemically stained with TH antiserum by the PAP method. The reaction product peroxidase is selectively associated with neurotubules (arrows) in the transverse profile (Fig. 11) and in the longitudinal profile (Fig. 12). x 27000.
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in close proximity to the blood vessels of the rostral part of the OVLT, while more caudally these LHRH terminals were localized in the ependymal layer close to the third ventricle. Neither tanycytes nor ependymal process were stained. These findings may indicate that the neuronal system is the major pathway for the transport of LHRH in the OVLT, although the tanycyte route cannot be excluded, as previously suggested for the mouse (Zimmerman et al. 1974; Gross, 1976). Although the source of these LHRH terminals has not been clarified, the LHRH-positive perikarya observed in the preoptic area could be a candidate, since Weiner et al. (1975) have reported that complete deafferentation caused severe reduction of LHRH in the median eminence without affecting the LHRH in the OVLT.
LHRH in the median eminence The present study confirmed the dense localization of LHRH-positive neuronal processes in the lateral perivascular region of the median eminence of the normal male and the pregnant female rat as previously reported in several species of mammals (Barry et al. 1973; Silverman, 1976; Setalo et al. 1975; Naik, 1975; King, 1974; Baker et al. 1975; Gross, 1976). Two routes of LHRH fibre tracts were present, namely the cephalocaudal and the dorsoventral projections. This finding may mean the presence of more that one source of LHRH-positive cells from which fibres originate, corresponding to the preoptico-infundibular and the tuberoinfundibular pathways, respectively (Szentagothai, 1962). However, only the presence of the former pathway was suggested, since the LHRH-positive perikarya
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chemically stained with both LHRH and TH antiserum. In the LHRH-positive neurons (LRH) a number of electron-dense vesicles with a diameter of about 100 nm are strongly stained. In the dopamine terminals (da) small vesicles with a diameter of 50 nm are strongly stained. Both stained terminals are clearly differentiated from the surrounding unstained terminals (*). LHRH- and TH-positive terminals are in axo-axonic contact (arrows). x 15000.
were observed in the preoptic area but not in the arcuate nucleus region. Compared with the profusion of LHRH-positive terminals, the paucity of LHRH-positive perikarya has been a major problem in many studies reported to date (King, 1974; Gross, 1976; Setalo et al. 1976). This might be due to the low concentration of LHRH in the perikarya below the threshold of the immunohistochemical technique. To cope with this, the intraventricular administration of various drugs including colchicine, melatonin, methanol and dopamine (Barry & Carette, 1975; Barry et al. 1974) or the neuronal deafferentation (Setalo et al. 1976) has been used to demonstrate LHRH-positive cell bodies. In the present study, LHRH-positive perikarya were demonstrable in the pregnant rat. In this context, species differences are apparent, for preliminary observations in the present study revealed that LHRHpositive perikarya were readily demonstrable in the periventricular nucleus of the guinea-pig, but not of the rat. In the light microscope, as mentioned above, LHRHpositive fibres appeared in the form of beaded strings, and this finding was one of the bases for the assumption that these fibres belonged to the neuronal processes rather than the tanycytes or the ependymal processes. The electron microscopical observations proved beyond doubt that these fibres were neuronal processes, and that the bead-like bodies belonged to the boutons or the terminals of the axon. These boutons contained many LHRH-positive granules with diameters of about 100 nm, as
344 K. AJIKA previously reported (Pelletier et al. 1974; Goldsmith & Ganong, 1975). Some of these terminals were observed to terminate directly on the basement membrane of the pericapillary space, suggesting that LHRH is released directly into portal vessels. Dopaminergic system In the present study, TH, the catecholamine synthesizing enzyme, was immunohistochemically localized in tubero-infundibular dopamine neurons. TH-positive terminals were densely localized in the perivascular region of the median eminence. The cell bodies from which these terminals originated were consistently demonstrable in the region of the arcuate nucleus of the pregnant rat. These TH-positive neurons were diagnosed as dopaminergic for the following reasons: (1) previous fluorescence microscopical studies by the Falck-Hillarp technique (Hokfelt & Fuxe, 1971; Fuxe & Hokfelt, 1969) and electron microscopical studies (Ajika & Hokfelt, 1973) revealed the existence of a very prominent dopaminergic system in this region; (2) a preliminary test in the present study confirmed that anti-DA-,/-hydroxylase (DBH) did not stain these neurons suggesting that they do not belong to NA; (3) the present technique stained cell groups not only in the arcuate nucleus (A1 2) but also in other regions of the brain (A9, Al 1, A1 3, A14) where the existence of DA cell groups has been previously reported (Dahlstrom & Fuxe, 1964; Bjorklund & Nobin, 1973); (4) recent immunofluorescence investigations reached similar conclusions (Hokfelt et al. 1975). As previously reported (Fuxe & Hokfelt, 1970), DA cell groups in the arcuate nucleus were markedly increased in number and intensity in the pregnant rat. It seemed, therefore, that the dense accumulation of TH in the arcuate cell group of the pregnant rat in the present study reflected well the DA concentration level in the perikarya. Pickel, Joh & Reis (1976), in another region of the brain, mention that TH is not evenly distributed in the cell; this was the experience of the present author also. TH was localized in the endoplasmic reticulum and Golgi apparatus in the perikaryon, in microtubules in the axons, and in small vesicles (50 nm in diameter) in the terminals. These small vesicles were identical with those previously reported as dopamine vesicles in a study using the pseudotransmitter, 5-hydroxydopamine, as a marker (Ajika & Hokfelt, 1973). Some of the DA terminals were shown to terminate on the basement membrane of the pericapillary space, suggesting the possible release of DA into portal vessels. Simultaneous staining of LHRH and DA neurons In the present study two distinct neuronal systems of LHRH and DA were differentiated by the immunohistochemical double staining technique. In the arcuate nucleus and the median eminence, simultaneous staining of LHRH and DA simply added the results when each was stained separately. The present study showed that no TH was discernible in LHRH neurons and vice versa. This is in agreement with a previous report that 6-hydroxydopamine causes severe depletion of DA without affecting LHRH (Kizer, Arimura, Schally & Brownstein, 1975). The present ultrastructural study clearly demonstrated that LHRH and DA terminals were in direct contact without the intervention of glial elements in the perivascular region of the median eminence. This suggests that DA reJeased from the DA terminals controls LHRH secretion via axo-axonic influences in the median eminence. This interpretation is supported by many previous investigations suggesting the
LHRH and dopamine
345
presence of adrenergic synapses responsible for the release of LHRH (McCann et al. 1971; Hokfelt & Fuxe, 1971; Fuxe et al. 1976). In some instances, DA terminals were observed to terminate directly in the pericapillary space of a portal vessel. This perhaps suggests that DA may be released directly into portal vessels to affect secretion in the anterior pituitary gland. Previous data seem to be incompatible with the view that LH secretion is stimulated in this way (McCann et al. 1971), but it is possible that direct release of DA into portal vessels affects the secretion of some other pituitary hormone, perhaps prolactin (McCann & Ojeda, 1976). SUMMARY
The PAP unlabelled antibody enzyme method of Sternberger was used for the histochemical demonstration of LHRH and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) in the hypothalamus of the adult male and pregnant female rat. The sections for light and electron microscopy were serially treated with normal goat serum, LHRH antiserum and/or TH antiserum, goat anti-rabbit IgG, PAP complex and 3,3'-diaminobenzidine (DAB) or 4-Cl-l-naphtol. LHRH-positive cell bodies were discernible in the medial preoptic area. The LHRH-positive terminals were densely localized in the organum vasculosum of the lamina terminalis and in the perivascular region of the median eminence (PVME). Dopamine (DA)-positive cell groups (TH-positive perikarya) were discernible in the arcuate nucleus, and its terminals were densely localized in the PVME. The simultaneous identification of LHRH and DA in the distinctive neuronal system of the median eminence was possible with the PAP double staining technique, in which LHRH is revealed as a brown precipitate with DAB, and TH is revealed as a blue reaction product with naphtol. The LHRH neuronal system did not contain TH and vice versa. The ultrastructural study revealed that LHRH was localized in large vesicles with a diameter of 100 nm within the axon terminals, while TH was localized in the endoplasmic reticulum, the neurotubules and small vesicles with a diameter of 50 nm within the DA neuron. The axo-axonic contact of LHRH and DA terminals was demonstrated in close proximity to portal vessels, suggesting the synaptic influence of DA on the release of LHRH into these vessels. The author wishes to extend his sincere appreciation to Dr L. A. Sternberger, Immunology Branch, Edgewood Arsenal, Maryland, for PAP complex, to Dr W. C. Dermody, Endocrine Section, Parke-Davis Research Laboratories, Ann Arbor, Michigan, for LHRH antiserum no. 185, to Dr I. Nagatsu, Department of Anatomy, Fujita-Gakuen University, Toyoake, Japan, for TH antiserum and to Dr M. Fujino, Medicinal Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd Osaka, Japan, for the synthetic LHRH. The author is also indebted to Ms E. Kikuchi and Mr J. K6taki for their skilful technical assistance. REFERENCES
AJIKA, K. & HOKFELT, T. (1973). Ultrastructural identification of catecholamine neurons in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects. Brain Research 57, 97-117. AJIKA, K. & HOKFELT, T. (1975). Projections to the median eminence and the arcuate nucleus with special reference to the monoamine systems: Effects of lesions. Cell and Tissue Research 158, 15-35.
346 K. AJIKA BAKER, B. L., DERMODY, W. C. & REEL, J. R. (1975). Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocytochemistry. Endocrinology 97, 125-135. BARRY, J. & CARETTE, B. (1975). Immunofluorescence study of LRF neurons in primates. Cell and Tissue Research 164, 163-178. BARRY, J., DUBOIS, M. P. & CARETTE, B. (1974). Immunofluorescence study of the preopticoinfundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male guinea pig. Endocrinology 95, 1416-1423. BARRY, J., DUBOIS, M. P. & POULAIN, P. (1973). LRF producing cells of the mammalian hypothalamus: a fluorescent antibody study. Zeitschrift fur Zellforschung und mikroskopische Anatomie 146, 351-366. BJORKLUND, A. & NOBIN, A. (1973). Fluorescence histochemical and microspectrofluorometric mapping of dopamine and noradrenaline cell groups in the rat diencephalon. Brain Research 51, 193-205. DAHLSTROM, A. & FUXE, K. (1964). Evidence for the existence of monoamine-containing neurons in the central nervous system. Acta physiologica scandinavica 62, 1-55. DUBOIS, M. P. (1976). Immunocytological evidence of LH-RF in hypothalamus and median eminence: a review. Annales de Biologie animale, biochimie et biophysique 16, 177-194. FUXE, K. & HOKFELT, T. (1969). Catecholamines in the hypothalamus and the pituitary gland. In Frontiers in Neuroendocrinology (ed. W. F. Ganong & L. Martini), pp. 47-96. New York: Oxford University Press. FUXE, K. & HOKFELT, T. (1970). Central monoaminergic systems and hypothalamic function. In The Hypothalamus (ed. L. Martini, M. Motta & F. Fraschini), pp. 123-138. New York: Academic Press. FUXE, K., LOFSTROM, A., ENEROTH, P., GUSTAFSSON, J. A., HOKFELT, T., SCETT, P., WuTTKE, W., FRASER, H. & JEFFCOATE, S. (1976). Interactions between hypothalamic catecholamine nerve terminals and LRF containing neurons. Further evidence for an inhibitory dopaminergic and a facilitatory noradrenergic influence. Excerpta Medica International Congress Series 374, p. 165. Madrid: Proceedings of the First International Symposium on Basic Applications and Clinical Uses of Hypothalamic Hormones. GOLDSMITH, P. C. & GANONG, W. F. (1975). Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat. Brain Research 97, 181-193. GROSS, D. S. (1976). Distribution of gonadotropin-releasing hormone in the mouse brain as revealed by immunohistochemistry. Endocrinology 98, 1408-1417. HOKFELT, T. & FUXE, K. (1971). On the morphology and the neuroendocrine role of the hypothalamus catecholamine neurons. In Brain-Endocrine Interaction. Median Eminence: Structure and Function (ed. K. M. Knigge, D. E. Scott & A. Weindl), pp. 181-223, Basel: Karger. HOKFELT, T., FUXE, K., GOLDSTEIN, M., JOHANSSON, O., PARK, D., FRASER, H. & JEFFCOATE, S. L. (1975). Immunofluorescence mapping of central monoamine and releasing hormone (LRH) system. In Anatomical Neuroendocrinology (ed. W. E. Stumpf & L. D. Grant), pp. 381-392, Basel: Karger. KING, J. C. (1974). Luteinizing hormone-releasing hormone (LH-RH) pathway of the rat hypothalamus revealed by the unlabelled antibody peroxidase-antiperoxidase method. Cell and Tissue Research 153, 211-218. KIZER, J. S., ARIMURA, A., SCHALLY, A. V. & BROWNSTEIN, M. J. (1975). Absence of luteinizing hormonereleasing hormone (LH-RH) from catecholaminergic neurons. Endocrinology 96, 523-525. MCCANN, S. M., KALRA, P. S., DONOSO, A. 0., BISHOP, W., SCHNIDER, H. P. G., FAWCETT, C. P. & KRULICH, L. (1971). The role of monoamines in the control of gonadotropin and prolactin secretion. In Brain-Endocrine Interaction. Median Eminence: Structure and Function (ed. K. M. Knigge, D. E. Scott & A. Weindl), pp. 221-235. Basel: Karger. MCCANN, S. M. & OJEDA, S. R. (1976). Synaptic transmitters involved in the release of hypothalamic releasing and inhibitory hormones. In Reviews of Neuroscience, vol. 11 (ed. S. Ehrenpreis & I. J. Kopin), pp. 91-110. New York: Raven Press. MORIARTY, G. C. & HALMI, N. S. (1972). Electron microscopic study of the adrenocorticotropin-producing cell with the use of unlabelled antibody and the soluble peroxidase-antiperoxidase complex. Journal of Histochemistry and Cytochemistry 20, 590-603. NAIK, D. V. (1975). Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescence microscopy. Cell and Tissue Research 157, 423-436. NAKANE, P. K. (1968). Simultaneous localization of multiple tissue antigens using the peroxidase-labelled antibody method: a study on pituitary glands of the rat. Journal of Histochemistry and Cytochemistry 16, 557-560. PELLETIER, G., LABRIE, F., PUVIANI, R., ARIMuRA, A. & SCHALLY, A. V. (1974). Immunohistochemical localization of luteinizing hormone-releasing hormone in the rat median eminence. Endocrinology 95, 314-317. PICKEL,V. M., JOH, T. H. & REIS, D. J. (1976). Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy. Journal of Histochemistry and Cytochemistry 24, 792-806. SETALO, G., VIGH, S., SCHALLY, A. V., ARIMURA, A. & FLERK6, B. (1975). LHRH-containing neuronal elements in the rat hypothalamus. Endocrinology 96, 135-142.
LHRH and dopamine
347
SETAL6, G., VIGH, S., SCHALLY, A. V., ARIMURA, A. & FLERKO, B. (1976). Immunohistological study of the origin of LH-RH-containing nerve fibres of the rat hypothalamus. Brain Research 103, 597-602. SILVERMAN, A. J. (1976). Distribution of luteinizing hormone-releasing hormone (LHRH) in the guinea pig brain, Endocrinology 99, 30-46. SILVERMAN, A. J., ANTUNES, J. L., FERIN, M. & ZIMMERMAN, E. A. (1977). The distribution of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of the rhesus monkey. Light microscopic studies using immunoperoxidase technique. Endocrinology 101, 134-142. STERNBERGER, L. A. (1972). The unlabelled antibody-peroxidase and the quantitative-immunouranium methods in light and electron-immunohistochemistry. In Techniques of Biochemical and Biophysical Morphology, vol. I (ed. D. Glick & R. M. Rosenbaum), pp. 67-88. New York: Wiley. STERNBERGER, L. A., HARDY, P. H., CUCULIS, J. J. & MEYER, H. G. (1970). The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. Journal of Histochemistry and Cytochemistry 18, 315-333. SZENTAGOTHAI, J. (1962). Hypothalamic Control of the Anterior Pituitary (ed. J. Szentigothai, B. Flerk6, B. Mess & B. Halasz), p. 95. Budapest: Akademiai Kiado. WEINER, R. I., PATTOU, E., KERDELHUE, B. & KORDON, C. (1975). Differential effects of hypothalamic deafferentation upon luteinizing hormone-releasing hormone in the median eminence and organum vasculosum of the lamina terminalis. Endocrinology 97, 1597-1600. ZIMMERMAN, E. A., Hsu, K. C., FERIN, M. & KoZLOWSKI, G. P. (1974). Localization of gonadotropinreleasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique. Endocrinology 95, 1-8.
J. Anat. (1979), 128, 2, pp. 331-347 With 14 figures Printed in Great Britain
Simultaneous localization of LHRH and catecholamines in rat hypothalamus KATSUYA AJIKA
Department of Obstetrics and Gynaecology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo, Japan
(Accepted 4 April 1978) INTRODUCTION
Luteinizing hormone releasing hormone (LHRH) has recently been demonstrated by immunofluorescence and immunohistochemistry in the hypothalamus of various species of mammals including the guinea-pig (Barry, Dubois & Poulain, 1973; Silverman, 1976), rat (SetAlo et al. 1975; Naik, 1975) and monkey (Barry & Carette, 1975; Silverman, Antunes, Ferin & Zimmerman, 1977) (see Dubois, 1976 for detailed list of publications). There is general agreement that substantial amounts of LHRH reside in the neuronal processes in the median eminence and the organum vasculosum of the lamina terminalis (OVLT) of these animals. In the mouse, however, LHRH was localized in the tanycytes rather than in the neuronal processes (Silverman, 1976; Zimmerman, Hsu, Ferin & Kozlowski, 1974). With respect to the localization of the immunoreactive LHRH cell bodies, reports differ according to the species and technique used. Thus the immunoreactive LHRH perikarya were rather widely localized, extending from the parolfactory and precommissural area to the caudal end of the tuber cinereum in the guinea-pig (Barry, Dubois & Carette, 1974), and monkey (Barry & Carette, 1975; Silverman et al. 1977). In immunohistochemical studies, however, some investigators were unable to demonstrate LHRH in the perikarya of the rat hypothalamus (King, 1974; Baker, Dermody & Reel, 1975; Gross, 1976), while others claimed that LHRH was demonstrable in the nerve cell bodies of several hypothalamic nuclei (Naik, 1975; Setalo et al. 1976). An array of evidence indicates that catecholamines participate in the regulatory mechanism of the release of gonadotropin secretion (McCann et al. 1971; Hokfelt and Fuxe, 1971). McCann's group suggested that dopamine was involved in stimulating LHRH release (McCann et al. 1971), while Fuxe's group showed that dopamine inhibited the release of gonadotropin secretion (Fuxe et al. 1976). In both cases it was inferred that catecholamine acted in the median eminence, dopamine being released from the nerve terminals here to influence the release of LHRH. However, the precise mechanism and the histological background have not received much attention (see Hokfelt & Fuxe, 1971; Ajika & Hokfelt, 1975). In fact clear evidence is lacking that LHRH and catecholamine are contained in different neuronal systems. The present study was concerned with the demonstration of LHRH-positive perikarya in the pregnant rat, and the differentiation of LHRH and catecholamine neuronal systems with an immunohistochemical double staining technique.
3'3 2
K. AJIKA MATERIALS AND METHODS
Animals and tissue preparations Adult male and pregnant female rats (15 to 20 days of gestation) of the Wistar strain were used. They were maintained in air-conditioned quarters with a controlled lighting schedule and were fed ad libitum. For light microscopy the animals were perfused with Zamboni's picric acid paraformaldehyde fixative (PAF) via the ascending aorta, under Nembutal anaesthesia. The brain was dissected out and immersed in the same fixative for 4 to 10 hours. In another group of rats the brain was dissected out under Nembutal anaesthesia and immersed in PAF for 48 hours. Specimens containing the hypothalamus were embedded in paraffin and sectioned serially at 5 to 7 ,tm in a coronal or sagittal plane. For electron microscopy, hypothalamic fragments, fixed (either by cardiac perfusion or simple immersion) in either PAF or a mixture of 2 % paraformaldehyde and 2 0 glutaraldehyde, were dehydrated in ethanol and embedded in Epon. They were sectioned on a Reichert Om U3 ultramicrotome equipped with a diamond knife. The ultrathin sections were collected on nickel grids and served for immunohistochemical staining. In another experiment the hypothalamic tissue fixed with PAF or with the mixture of 2 % paraformaldehyde and 2 % glutaraldehyde was sectioned with a Vibratome (Oxford Instruments, Pasadena, California, U.S.A.) at 40 to 50 ,um, in phosphate buffer. These sections were subjected to immunohistochemical staining procedures. After staining, they were osmified, dehydrated and embedded in Epon. Ultrathin sections were obtained from the surface zone of these immunohistochemically stained sections and examined in a Hitachi 1 ID electron microscope. Immunohistochemical staining Light microscopy Following deparaffinization and hydration the sections were stained for LHRH and/or tyrosine hydroxylase (TH), catecholamine synthesizing enzyme, by the peroxidase-antiperoxidase (PAP) method of Sternberger, Hardy, Cuculis & Meyer (1970). Prior to immunohistochemical staining the sections were incubated with 1 % normal goat serum in PBS for 10 minutes to reduce non-specific protein binding. The sections were then incubated sequentially in 1 hour steps with LHRH antiserum (1:200), goat anti-rabbit IgG (1:30; Miles Laboratories, U.S.A.) and PAP (1:70). All staining procedures were carried out at room temperature in a moist chamber, and two 5 minute washes in PBS followed each step. Peroxidase activity was revealed by brown products of 3,3'-diaminobenzidine-H202 (DAB-H202) as in the case of LHRH, and by blue products of 4-Cl-l-naphtol as in TH. In the case of double staining of LHRH and TH, the procedure was fundamentally similar to that of Nakane (1968) except for using the PAP complex as a marker for the antibody in the present study. The sections were reacted first with LHRH antiserum and stained for peroxidase, using DAB as substrate. They were immersed and washed in 0-1 M-glycine-HCl (Ph 2 2) for 1 hour to remove the first antibody and conjugate. The DAB reaction product remained on the tissue. The sections were then serially incubated with TH antiserum, IgG and PAP complex. Peroxidase activity was revealed by incubation in 4-Cl-l-naphtol-H20, for 7 to 10 minutes. Specificity control included substitution of normal rabbit serum for the rabbit
LHRH and dopamine
333 LHRH or TH antisera, and the absorption of each antisera with 10 to 1000 ng of synthetic LHRH (Takeda Chemical Industries, Osaka, Japan) or bovine adrenal TH. The LHRH antiserum was tested not to cross-react with vasopressin, oxytocin, TRH or BSA, and the TH antisera was tested not to cross-react with dopamine-,fhydroxylase or phenylethanolamine-N-methyltransferase. The PAP complex was a gift from Dr L. A. Sternberger. LHRH antisera no. 185 raised in a rabbit immunized with LHRH conjugated to BSA was generously supplied by Dr W. C. Dermody, and TH antisera raised in a rabbit immunized with bovine adrenal tyrosine hydroxylase by Dr I. Nagatsu. Electron microscopy The electron microscopical staining procedures were fundamentally similar to those previously described (Moriarty & Halmi, 1972; Sternberger, 1972). Prior to immunohistochemical staining the ultrathin sections mounted on Formvar-coated nickel grids were etched by flotation on a drop of distilled water containing 0-01 % benzene, 5 00 methanol and 5 % ethanol. To reduce non-specific protein binding the grids were first floated on normal goat serum (1:30 dilution with PBS containing 0.1 % gelatin). The grids were then floated sequentially in 10 minute steps on a drop of LHRH antiserum (1:1000 to 1: 5000 dilution with PBS containing 04 % gelatin), goat anti-rabbit IgG (1: 30) and PAP (1: 50). After exposure to each solution they were rinsed in PBS containing 0-1 00 gelatin. DAB-H202 was used as substrate. In the case of TH simple staining, and the double staining for LHRH and TH, the hypothalamic vibratome sections were used for immunohistochemical staining. For the double staining of LHRH and TH, these sections were sequentially immersed in a drop of normal goat serum (1:100), LHRH antisera (1:100 to 1:400), IgG (1 :30) and PAP complex (1 :70). They were immersed and washed in 01 Mglycine-HCl for 10 minutes to remove the first antibody and conjugate. They were then immersed in a drop of TH antisera, IgG and PAP complex. For the simple staining of TH, only the latter procedures were carried out. Each solution was diluted either with PBS or with PBS containing 0-1 00 gelatin. All staining procedures were carried out at room temperature in a moist chamber, and washing in three consecutive changes of PBS followed each step. DAB-H202 was used as peroxidase substrate. After application of DAB-H202 the grids were washed in distilled water, stained on a drop of buffered 2 % OS04 for 30 minutes and washed in distilled water. -
RESULTS
LHRH-positive cell bodies
LHRH-positive cell bodies were observed in the medial preoptic area and in the medial prechiasmatic area of the pregnant rat (Fig. 1). Not more than three LHRHpositive perikarya were seen in any one sagittal section. LHRH-positive cell bodies were rarely encountered in coronal sections. Even in mid-sagittal sections more than half the specimens failed to show any LHRH-positive cell bodies. The LHRHpositive perikarya were ovoid in shape, and ranged from 10 to 13 ,m in diameter. They appeared most frequently in the medial preoptic area, with beaded string-like processes directing ventrally to the organum vasculosum of the lamina terminalis (OVLT), and less often in the median eminence. LHRH-positive cell bodies were not observed in other areas, including the arcuate nucleus, ventromedial nucleus
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Electron microscopy Electron microscopy in the tissue routinely processed without immunohistochemical staining revealed that a number of nerve terminals were densely arranged in the perivascular region of the median eminence of normal male and pregnant female rats (Fig. 6). Many nerve terminals were in direct contact with each other, and with the basement membrane of a portal vessel, without the intervention of glial elements. These terminals contained large dense vesicles ranging from 80 to 110 nm in diameter, and small lucent vesicles with a diameter of 50 nm. Some nerve terminals contained even larger dense vesicles, the diameters of which ranged from 130 to 150 nm. The immunohistochemical PAP staining which was carried out on ultrathin sections on the nickel grids labelled exclusively the dense vesicles, 80 to 110 nm diameter, within the LHRH-positive terminals in the lateral perivascular region of the median eminence. No other micro-organelles were stained in the LHRHpositive axon terminals. The LHRH-positive terminals appeared in close proximity to portal vessels (Fig. 8).
Dopamine cell bodies Light microscopy In the pregnant rat dopamine-positive cell bodies were consistently revealed in the arcuate nucleus (A12 cell group, according to Dahlstrom & Fuxe, 1964) using tyrosine hydroxylase (TH) as marker. The cytoplasm of TH-positive cells was strongly stained blue when naphtol was used as substrate (Fig. 3). The cells ranged from 10 to 13 ,tm in diameter. Other TH-positive cell groups appeared in the rostral periventricular area (A14 cell group), in the dorsal hypothalamic area (Al3) and in the caudal hypothalamic area (Al1). In the male rat, TH-positive cell groups (except A12) were consistently stained. However, the stainability of A12 cells was considerably less than that of the pregnant rat. In some cases, TH-positive cells were not seen in the arcuate nucleus of the male rat. No TH-positive cell body was stained in the control sections from the pregnant rat treated with normal rabbit serum or the TH antiserum absorbed in advance with TH in substitution of TH antiserum. Fig. 3. A frontal section of the median eminence. The TH-positive terminals are densely localized in the lateral perivascular region of the median eminence (me), as indicated by arrowheads. A considerable number of TH-positive fibres are also seen in the medial part of the median eminence (arrow). In the arcuate nucleus (ac), a number of TH-positive perikarya are seen. v, third ventricle. x 135. Fig. 4. A frontal section is stained with LHRH antiserum using 3,3'-diaminobenzidine as substrate. The LHRH-positive terminals are densely localized in the lateral perivascular region of the median eminence, while in the medial part there are a few LHRH-positive processes (arrow). In the arcuate nucleus (ac), no LHRH-positive perikarya are seen. v, third ventricle. x 160. Fig. 5. The median eminence (me) is simultaneously stained with TH and LHRH antiserum. TH-positive terminals are densely localized in the lateral and medial part of the perivascular region of the median eminence, while LHRH-positive terminals are mostly localized in the lateral part. In the arcuate nucleus, the stained perikarya are exclusively TH-positive. Figs. 4 and 5 are serial sections. x 160.
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Electron microscopy Electron microscopy of the tissue immunohistochemically stained with TH antiserum and PAP revealed the presence of TH-positive cell bodies in the arcuate nucleus of the pregnant rat. Within the cytoplasm the endoplasmic reticulum, membranes of the Golgi apparatus and the neurotubules were strongly and evenly stained, while the nucleus was less heavily stained (Fig. 7). Dopamine terminals in the median eminence Light microscopy In both male and pregnant female rats, TH-positive terminals were densely stained in the perivascular region of the median eminence with TH antiserum and PAP. The TH-positive nerve fibres appeared as delicate beaded strings and were blue when naphtol was used as substrate (Figs. 3, 5). A number of TH-positive terminals were densely arranged along the portal vessels.
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In coronal view, TH-positive fibres were most concentrated in the lateral penivascular region of the median eminence. In contrast with LHRH-positive terminals, however, considerable numbers of TH-positive fibres were also observed in the medial perivascular region. No TH-positive terminals were stained in the control section which was treated with normal rabbit serum or with the TH antiserum absorbed in advance with TH in substitution of TH antiserum.
Electron microscopy In both male and pregnant female rats, electron micrographs of sections through the median eminence incubated with TH antiserum and PAP showed a number of dopamine neurons stained with electron-dense material (Figs. 9, 10, 11, 12, 13). The peroxidase reaction products were preferentially associated with endoplasmic reticulum in the perikarya or in the dendrites, with neurotubules of 22 nm diameter in the axons and with small vesicles of 50 nm in the terminals (Figs. 9, 10, 11, 12). In the lateral perivascular region of the median eminence some of the terminals 22-2
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LHRH and dopamine 341 stained with TH antiserum were in direct contact with each other, and with the basement membrane of the portal vessels, without the intervention of glial elements (Fig. 13). Simultaneous staining of LHRH and dopamine terminals Light microscopy In the tissue which was reacted consecutively with LHRH and TH antiserum, both LHRH and TH-positive terminals were revealed as brown precipitates with DAB and as blue precipitates with naphtol, respectively (Fig. 5). In the lateral perivascular region of the median eminence these two kinds of terminal overlapped in places because of their high density, but were clearly differentiated from one another by their colour. The medial part of the perivascular region, on the other hand, was almost exclusively occupied with the blue precipitate of the TH-positive terminals and few brown precipitates of LHRH terminals were observed. In the arcuate nucleus of the pregnant rat the cell bodies appeared exclusively blue. These findings were clearly verified when sections stained for either TH or LHRH were compared with the double stained sections (Figs. 3, 4 and 5). Electron microscopy Electron micrographs of the lateral perivascular region of the median eminence incubated consecutively with LHRH and TH antiserum showed two kinds of terminal stained with electron-dense materials (Fig. 14). One type, judged as LHRH, contained many lar-ge electron-dense vesicles with a diameter of 100 nm. The other type, judged as dopamine, was more densely stained and contained many small vesicles with a diameter of 50 nm. These two kinds of terminal were in direct contact with each other without the intervention of glial elements. A number of other terminals, and the processes of ependymal cells and tanycytes, were negative. DISC USSION
LHRH in the 0 VLT The localization of LHRH in the OVLT was confirmed in the male and the pregnant female rat, as previously reported in the mouse (Zimmerman et al. 1974; Baker, 1975) and the rat (Weiner, Pattou, Kerdelhue & Kordon, 1975). In the present study, the dense meshwork of LHRH-positive axonal processes terminated Fig. 9. Electron micrograph of the median eminence, immunohistochemically stained with TH antiserum by PAP method. The peroxidase reaction products are preferentially associated with endoplasmic reticulum (double arrows) in the dendrites or in the perikarya, with neurotubules (short arrows) in the axons and with small vesicles (long arrows) in the terminals. x 5200. The inset is a high power view of the marked area in Fig. 9. In the axonal process (ax) neurotubules with a diameter of 22 nm (short arrow) are preferentially stained, while in the terminal (t) the small vesicles with a diameter of 50 nm are specifically stained. x 10000. Fig. 10. Electron micrograph of the median eminence, immunohistochemically stained with TH antiserum by the PAP method. TH is specifically localized in the small vesicles (arrows) in the DA terminal (t). x 28000. Figs. 11 and 12. Higher power electron micrographs of the median eminence, immunohistochemically stained with TH antiserum by the PAP method. The reaction product peroxidase is selectively associated with neurotubules (arrows) in the transverse profile (Fig. 11) and in the longitudinal profile (Fig. 12). x 27000.
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in close proximity to the blood vessels of the rostral part of the OVLT, while more caudally these LHRH terminals were localized in the ependymal layer close to the third ventricle. Neither tanycytes nor ependymal process were stained. These findings may indicate that the neuronal system is the major pathway for the transport of LHRH in the OVLT, although the tanycyte route cannot be excluded, as previously suggested for the mouse (Zimmerman et al. 1974; Gross, 1976). Although the source of these LHRH terminals has not been clarified, the LHRH-positive perikarya observed in the preoptic area could be a candidate, since Weiner et al. (1975) have reported that complete deafferentation caused severe reduction of LHRH in the median eminence without affecting the LHRH in the OVLT.
LHRH in the median eminence The present study confirmed the dense localization of LHRH-positive neuronal processes in the lateral perivascular region of the median eminence of the normal male and the pregnant female rat as previously reported in several species of mammals (Barry et al. 1973; Silverman, 1976; Setalo et al. 1975; Naik, 1975; King, 1974; Baker et al. 1975; Gross, 1976). Two routes of LHRH fibre tracts were present, namely the cephalocaudal and the dorsoventral projections. This finding may mean the presence of more that one source of LHRH-positive cells from which fibres originate, corresponding to the preoptico-infundibular and the tuberoinfundibular pathways, respectively (Szentagothai, 1962). However, only the presence of the former pathway was suggested, since the LHRH-positive perikarya
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chemically stained with both LHRH and TH antiserum. In the LHRH-positive neurons (LRH) a number of electron-dense vesicles with a diameter of about 100 nm are strongly stained. In the dopamine terminals (da) small vesicles with a diameter of 50 nm are strongly stained. Both stained terminals are clearly differentiated from the surrounding unstained terminals (*). LHRH- and TH-positive terminals are in axo-axonic contact (arrows). x 15000.
were observed in the preoptic area but not in the arcuate nucleus region. Compared with the profusion of LHRH-positive terminals, the paucity of LHRH-positive perikarya has been a major problem in many studies reported to date (King, 1974; Gross, 1976; Setalo et al. 1976). This might be due to the low concentration of LHRH in the perikarya below the threshold of the immunohistochemical technique. To cope with this, the intraventricular administration of various drugs including colchicine, melatonin, methanol and dopamine (Barry & Carette, 1975; Barry et al. 1974) or the neuronal deafferentation (Setalo et al. 1976) has been used to demonstrate LHRH-positive cell bodies. In the present study, LHRH-positive perikarya were demonstrable in the pregnant rat. In this context, species differences are apparent, for preliminary observations in the present study revealed that LHRHpositive perikarya were readily demonstrable in the periventricular nucleus of the guinea-pig, but not of the rat. In the light microscope, as mentioned above, LHRHpositive fibres appeared in the form of beaded strings, and this finding was one of the bases for the assumption that these fibres belonged to the neuronal processes rather than the tanycytes or the ependymal processes. The electron microscopical observations proved beyond doubt that these fibres were neuronal processes, and that the bead-like bodies belonged to the boutons or the terminals of the axon. These boutons contained many LHRH-positive granules with diameters of about 100 nm, as
344 K. AJIKA previously reported (Pelletier et al. 1974; Goldsmith & Ganong, 1975). Some of these terminals were observed to terminate directly on the basement membrane of the pericapillary space, suggesting that LHRH is released directly into portal vessels. Dopaminergic system In the present study, TH, the catecholamine synthesizing enzyme, was immunohistochemically localized in tubero-infundibular dopamine neurons. TH-positive terminals were densely localized in the perivascular region of the median eminence. The cell bodies from which these terminals originated were consistently demonstrable in the region of the arcuate nucleus of the pregnant rat. These TH-positive neurons were diagnosed as dopaminergic for the following reasons: (1) previous fluorescence microscopical studies by the Falck-Hillarp technique (Hokfelt & Fuxe, 1971; Fuxe & Hokfelt, 1969) and electron microscopical studies (Ajika & Hokfelt, 1973) revealed the existence of a very prominent dopaminergic system in this region; (2) a preliminary test in the present study confirmed that anti-DA-,/-hydroxylase (DBH) did not stain these neurons suggesting that they do not belong to NA; (3) the present technique stained cell groups not only in the arcuate nucleus (A1 2) but also in other regions of the brain (A9, Al 1, A1 3, A14) where the existence of DA cell groups has been previously reported (Dahlstrom & Fuxe, 1964; Bjorklund & Nobin, 1973); (4) recent immunofluorescence investigations reached similar conclusions (Hokfelt et al. 1975). As previously reported (Fuxe & Hokfelt, 1970), DA cell groups in the arcuate nucleus were markedly increased in number and intensity in the pregnant rat. It seemed, therefore, that the dense accumulation of TH in the arcuate cell group of the pregnant rat in the present study reflected well the DA concentration level in the perikarya. Pickel, Joh & Reis (1976), in another region of the brain, mention that TH is not evenly distributed in the cell; this was the experience of the present author also. TH was localized in the endoplasmic reticulum and Golgi apparatus in the perikaryon, in microtubules in the axons, and in small vesicles (50 nm in diameter) in the terminals. These small vesicles were identical with those previously reported as dopamine vesicles in a study using the pseudotransmitter, 5-hydroxydopamine, as a marker (Ajika & Hokfelt, 1973). Some of the DA terminals were shown to terminate on the basement membrane of the pericapillary space, suggesting the possible release of DA into portal vessels. Simultaneous staining of LHRH and DA neurons In the present study two distinct neuronal systems of LHRH and DA were differentiated by the immunohistochemical double staining technique. In the arcuate nucleus and the median eminence, simultaneous staining of LHRH and DA simply added the results when each was stained separately. The present study showed that no TH was discernible in LHRH neurons and vice versa. This is in agreement with a previous report that 6-hydroxydopamine causes severe depletion of DA without affecting LHRH (Kizer, Arimura, Schally & Brownstein, 1975). The present ultrastructural study clearly demonstrated that LHRH and DA terminals were in direct contact without the intervention of glial elements in the perivascular region of the median eminence. This suggests that DA reJeased from the DA terminals controls LHRH secretion via axo-axonic influences in the median eminence. This interpretation is supported by many previous investigations suggesting the
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presence of adrenergic synapses responsible for the release of LHRH (McCann et al. 1971; Hokfelt & Fuxe, 1971; Fuxe et al. 1976). In some instances, DA terminals were observed to terminate directly in the pericapillary space of a portal vessel. This perhaps suggests that DA may be released directly into portal vessels to affect secretion in the anterior pituitary gland. Previous data seem to be incompatible with the view that LH secretion is stimulated in this way (McCann et al. 1971), but it is possible that direct release of DA into portal vessels affects the secretion of some other pituitary hormone, perhaps prolactin (McCann & Ojeda, 1976). SUMMARY
The PAP unlabelled antibody enzyme method of Sternberger was used for the histochemical demonstration of LHRH and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) in the hypothalamus of the adult male and pregnant female rat. The sections for light and electron microscopy were serially treated with normal goat serum, LHRH antiserum and/or TH antiserum, goat anti-rabbit IgG, PAP complex and 3,3'-diaminobenzidine (DAB) or 4-Cl-l-naphtol. LHRH-positive cell bodies were discernible in the medial preoptic area. The LHRH-positive terminals were densely localized in the organum vasculosum of the lamina terminalis and in the perivascular region of the median eminence (PVME). Dopamine (DA)-positive cell groups (TH-positive perikarya) were discernible in the arcuate nucleus, and its terminals were densely localized in the PVME. The simultaneous identification of LHRH and DA in the distinctive neuronal system of the median eminence was possible with the PAP double staining technique, in which LHRH is revealed as a brown precipitate with DAB, and TH is revealed as a blue reaction product with naphtol. The LHRH neuronal system did not contain TH and vice versa. The ultrastructural study revealed that LHRH was localized in large vesicles with a diameter of 100 nm within the axon terminals, while TH was localized in the endoplasmic reticulum, the neurotubules and small vesicles with a diameter of 50 nm within the DA neuron. The axo-axonic contact of LHRH and DA terminals was demonstrated in close proximity to portal vessels, suggesting the synaptic influence of DA on the release of LHRH into these vessels. The author wishes to extend his sincere appreciation to Dr L. A. Sternberger, Immunology Branch, Edgewood Arsenal, Maryland, for PAP complex, to Dr W. C. Dermody, Endocrine Section, Parke-Davis Research Laboratories, Ann Arbor, Michigan, for LHRH antiserum no. 185, to Dr I. Nagatsu, Department of Anatomy, Fujita-Gakuen University, Toyoake, Japan, for TH antiserum and to Dr M. Fujino, Medicinal Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd Osaka, Japan, for the synthetic LHRH. The author is also indebted to Ms E. Kikuchi and Mr J. K6taki for their skilful technical assistance. REFERENCES
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