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Light and electron microscopic immunocytochemistry of GRF-like immunoreactive neurons and terminals in the rat hypothalamic arcuate nuclues and median eminence
Brain Research, 370 (1986) 136-143
136
Elsevier BRE 11593
Light and Electron Microscopic Immunocytochemistry of GRF-Like Immunoreactive Neurons and Terminals in the Rat Hypothalamic Arcuate Nucleus and Median Eminence Y. IBATA j , H. OKAMURA 1, S. MAKINO 1, F. KAWAKAMI 2, N. MORIMOTO I and K. CHIHARA 3
1Department of Anatomy, Kyoto Prefectural University of Medicine, KawaramachiHirokofi; 2Departmentof Psychiatry, Kyoto Prefectural Universityof Medicine, KawaramachiHirokoji, Kyoto 602 and 3Third Division, Department of Medicine, Kobe UniversitySchool of Medicine, Kobe 650 (Japan) (Accepted August 13th, 1985)
Key words: arcuate nucleus - - median eminence - - immunocytochemistry - - growth hormone-releasing factor (GRF) - electron microscopy - - rat
Growth hormone-releasing factor (GRF) synthesizing neuronal perikarya and terminals were investigated by light and electron microscopic immunocytochemistry using rat hypothalamus. Immunoreactive neuronal perikarya were located mainly in the ventrolateral part of the arcuate nucleus. They contained well developed cell organella such as mitochondria and rough surfaced endoplasmic reticulum with some expansion. They also contained immunoreactive dense granules (80-120 nm in diameter). On the surface of the immunoreactive neuronal perikarya were frequently found non-immunoreactive axo-somatic synapses. Therefore, the GRF-like immunoreactive neurons were assumed to receive neuronal inputs from other neurons on their neuronal soma. In the external layer of the median eminence large numbers of immunoreactive terminals were distributed particularly around the capillaries of the portal vessel. Electron microscopic immunocytochemistry revealed large numbers of immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapillary space of the portal vessel. Therefore, we concluded that GRF-like immunoreactive substances are released into the portal capillaries from the nerve terminals, which originate from the neuronal perikarya in the ventrolateral part of the arcuate nucleus, and act on growth hormone release in the anterior pituitary. We also suggest that GRF-like immunoreactive neurons have abundant terminal arborization in the external layer of the median eminence.
INTRODUCTION G r o w t h h o r m o n e - r e l e a s i n g factor ( G R F ) has b e e n
Immunocytochemistry
using
specific
antisera
isolated f r o m h u m a n p a n c r e a t i c t u m o r s in patients with a c r o m e g a l y and its c h e m i c a l structure was de-
against h p G R F and rat G R F has d e m o n s t r a t e d G R F like i m m u n o r e a c t i v e n e u r o n a l p e r i k a r y a in the ar-
t e r m i n e d as a p e p t i d e with a 40 o r a n o t h e r 44 a m i n o acid residue9,17, 21. T h e s e p e p t i d e s are given the n a m e
cuate and v e n t r o m e d i a l nuclei and their n e r v e termi-
h u m a n p a n c r e a t i c g r o w t h h o r m o n e - r e l e a s i n g factor (hpGRF)9,17. T h e y are highly and specifically p o t e n t for releasing the g r o w t h h o r m o n e ( G H ) f r o m the anterior pituitary in v i v o and in vitro c o n d i t i o n s 2~6,13,19,
nals in the e x t e r n a l layer of the m e d i a n e m i n e n c e of the rat and o t h e r m a m m a l i a n species 3.18A9~23,25. H o w ever, their distributional p a t t e r n and density h a v e not b e e n e x a m i n e d in detail and no i m m u n o e l e c t r o n
24,26,28. T h e G R F s of 6 animal species h a v e n o w b e e n
microscopic o b s e r v a t i o n s a b o u t their n e u r o n a l perikarya and t e r m i n a l s have b e e n d e s c r i b e d until now.
identified and rat G R F has b e e n f o u n d to be a peptide with a 43 a m i n o acid residue 4,5,14. T h e effects of
W e a t t e m p t e d , t h e r e f o r e , to e x a m i n e their features in detail and fine structure by light and e l e c t r o n mi-
G R F including h p G R F on release of G H h a v e b e e n investigated physiologically14.24.27.29 and b i o c h e m i c ally1,15.
croscopic i m m u n o c y t o c h e m i s t r y and to o b t a i n m o r e i n f o r m a t i o n c o n c e r n i n g their functional significance from m o r p h o l o g i c a l points of view.
Correspondence: Y. Ibata, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kyoto Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
137 MATERIALS AND METHODS Fourteen male Wistar rats housed under a 14 h light-10 h dark schedule were given free access to food and water; 6 for light microscopic immunocytochemistry and 8 for electron microscopic immunocytochemistry. Under Nembutal anesthesia, colchicine (30 #g) was injected into the lateral ventricle of all the animals for light microscopic immunocytochemistry and 4 animals for examination of GRF-like immunoreactive neuronal perikarya by electron microscopic immunocytochemistry 2 days prior to sacrifice. This treatment was done to promote the storage of GRF-like immunoreactive substances in the neuronal perikarya. The 4 rats used for examination of the fine structures of GRF-like immunoreactive terminals were not given colchicine treatment. For light microscopic immunocytochemistry the animals were perfused via the left cardiac ventricle with a mixture of 4% paraformaldehyde, 0.2% picric acid and 0.35% glutaraldehyde buffered at pH 7.4 with 0.1 M phosphate buffer. The brain was removed after perfusion and further fixed with the same mixture but without glutaraldehyde, for 48 h at 4 °C. Thereafter, serial frontal frozen sections (30/~m thick) were made and stored in a phosphate-buffered saline (PBS) solution containing 0.3% Triton X for 4 days at 4 °C. Sections for immunocytochemistry were first incubated in bovine serum albumin (dilution x 100) for 1 h at room temperature, and then incubated in rat anti-GRF serum (dilution x 4000) for 48 h at 4 °C. They were next immersed in anti-rabbit IgG solution (dilution x 200, Miles Laboratory) for 2 h at room temperature followed by incubation in PAP solution (dilution × 200, D A K O immunoglobulins a/s) for 90 min at room temperature. Finally, they were exposed for 10 min at room temperature to 0.02% 3.3'-diaminobenzidine'4HCl (DAB) containing H202 (15 pl of 30% H202 solution into 100 ml DAB solution), mounted on glass slides and examined by light microscopy. As a control, the sections were treated with anti-GRF serum absorbed with synthetic rat G R F (0.1-10 /~g/ml). No positive immunoreactivity was detected in the control sections. The antiserum to rat G R F used in this present study was generated in rabbit using 1 mg synthetic rat GRF1_43-OH coupled to 2.5 mg bovine thyroglobulin by glutaraldehyde method.
This antiserum bound 50% of [125I]iodo rat GRFl_43-OH at a final dilution of 1:60,000, and the binding was inhibited in a dose-related manner by 5600 pg of unlabeled rat GRF1_a3-OH. There was no cross-reaction with human GRF1_44-NH2, human GRFI_a0-OH, human GRFI_26-NH2, peptide histidine isoleusine (PHI), vasoactive intestinal polypeptide (VIP), rat corticotropine releasing factor (CRF), human calcitonin, neurotensin, substance P, somatostatin~_14 or somatostatin1_28. For electron microscopic immunocytochemistry, the colchicine-treated animals were perfused via the left cardiac ventricle with the same fixative as for light microscopic immunocytochemistry. The animals not treated with colchicine were decapitated under Nembutal anesthesia, the brains immediately removed and thin tissue blocks including the hypothalamus were dissected out. They were immersed in the same fixative for 2 h and further fixed with the same mixture, from which glutaraldehyde had been excluded. Immersion fixation was used in order to avoid deformation of the fine structure of the median eminence caused by expansion of extracellular spaces during perfusion. Thin tissue blocks including the hypothalamus were also dissected from the brains of the perfused animals. They were further fixed with the same mixture from which glutaraldehyde had been excluded. Each sample was treated for electron microscopic immunocytochemistry as follows. Frontal sections, 30 ,um in thickness, were made within PBS by a microslicer (Dosaka EM) and incubated in bovine serum (dilution x 4000) for 96 h at 4 °C followed by incubation in anti-rabbit IgG solution (dilution x 200) for 2 h, at room temperature. Then they were incubated in PAP solution (dilution × 200) for 2 h at room temperature and finally allowed to react with 0.02% DAB solution containing H20 2 for 5 min at room temperature. Between each step of the reaction they were washed with PBS. Completion of the immunoreactive processes was judged by the appearance under light microscopy of a weak brown coloration of the neuronal perikarya, processes and terminals after treatment with DAB solution. The sections were fixed in chilled 1% OsO4 solution for 1 h and dehydration and embedding in Epon mixture were carried out in the usual manner. Semi-thin sections were examined by light microscopy to confirm the presence of immunoreactive perikarya, proces-
138 ses and terminals, following which ultrathin sections stained with uranyl acetate were examined in a JEM200CX electron microscope. RESULTS
Light microscopic immunocytochemistry G R F - l i k e immunoreactive neuronal perikarya were mainly located in the lateral and basolateral parts of the arcuate nucleus at the level of the central median eminence of the frontal section (Fig. l a ) . They were observed as small neurons (about 15 ~ m in diameter) with round, oval or fusiform shapes. Immunoreactive density was varied in each neuron and unipolar, bipolar or multipolar immunoreactive processes extended from some neuronal perikarya. However, immunoreactive dots or strands, which were considered to be G R F - l i k e dendritic branches, were rarely detected a r o u n d the immunoreactive neuronal perikarya. Few immunoreactive neuronal perikarya were found in the arcuate nucleus at the level of the rostral and caudal median eminence. W e also found some immunoreactive neuronal p e r i k a r y a in the mediobasal hypothalamus (Fig. l b ) . Their features were similar to those in the a b o v e - m e n t i o n e d
region, but we could not detect immunoreactive neuronal p e r i k a r y a in the ventromedial nucleus. Immunoreactive nerve terminals were distributed in the external layer of the rostral, central and caudal median eminence. Dense accumulation of immunoreactive dots were detected particularly a r o u n d the pericapillary space of the portal vessel (Fig. 2a). In the s u b e p e n d y m a l layer and internal layer of the median eminence fairly numbers of immunoreactive brown dots and b e a d e d strands were found particularly in the central median eminence and in the infundibular stalk (Fig. 2b). A r o u n d the pericapillary space of the portal vessel which p e n e t r a t e d into the internal layer, dense accumulation of immunoreactive terminals were also observed (Fig. 2c). M o d e r ate numbers of immunoreactive dots were also distributed in the region dorsal to the infundibular recessus at the level of the caudal median eminence. A t the more caudal level immunoreactive processes and terminals were found in the floor of the third ventricle near the p r e m a m m i l l a r y region.
Electron microscopic immunocytochemistry Immunoreactive neuronal p e r i k a r y a a p p e a r e d as brown neurons in the lateral and basolateral parts of
]
~i~~! iiliI ~i~ Fig. 1. a: GRF-like immunoreactive neurons with numerous shapes and immunoreactive density are distributed in the rat arcuate nucleus at the level of the central median eminence. V, third ventricle, x 240. b: in addition to the arcuate nucleus, immunoreactive neurons are also detected in the mediobasal hypothalamus, x450.
139
V
Fig. 2. a: concentrations of GRF-like immunoreactive terminals are observed in the external layer of the central median eminence. Large numbers of immunoreactive dots are accumulated particularly around the pericapillary space of the portal vessel (arrows). V, third ventricle, x 180. b: in the subependymal and internal layer some immunoreactive fibers are also detected in the central median eminence. V, third ventricle, x880. c: dense accumulation of immunoreactive terminals are observed around the pericapillary regions of the portal vessel which penetrate into the internal layer through the external layer. C, capillary, x 1600.
the arcuate nucleus in the semi-thin sections of Epon embedding materials by light microscopy. By electron microscopy, dense osmiophilic immunoreactive substances were disseminated throughout the cytoplasma of moderately immunoreactive neuronal perikarya, though the entire cytoplasmic matrix and cell organella showed a diffuse high electron density in strong immunoreactive neurons. GRF-like immunoreactive neurons were characterized by oval nuclei with diffusely distributed D N A particle, well developed mitochondria, rough surfaced endoplasmic reticulum (rER) with some expansion, polysomes and Golgi complex. Invagination of the nuclear envelope was also frequently observed. Lysosomes in the cytoplasma were hard to detect. In addition, dense granules (80-120 nm in diameter)
with immunoreactivity were scattered throughout the cytoplasma (Fig. 3a, b). On the surface of immunoreactive neuronal perikarya non-immunoreactive preterminal axons containing synaptic vesicles (40-60 nm in diameter), occasionally cored vesicles (80-100 nm in d i a m e t e r ) a n d mitochondria terminated as axo-somatic synapses (Fig. 3a, b). There were only a few immunoreactive processes around the immunoreactive neuronal perikarya by electron microscopy as shown by light microscopic immunocytochemistry. In the median eminence large numbers of immunoreactive dots were found particularly in the lateral part of the external layer in the semithin sections from the Epon embedding materials, although light microscopic immunocytochemistry of the sections of
140 DISCUSSION
Fig. 3. Fine structures of GRF-like immunoreactive neuronal perikarya in the rat arcuate nucleus. Immunoreactiveneuronal perikarya contain well developed mitochondria, rER, polysomes and Golgi complex (G). Besides the cell organellae, immunoreactive dense granules (arrows) are also distributed throughout the cytoplasma. Invaginationof nuclear envelope is also observed. On the surface of immunoreactive neuronal perikarya non-immunoreactive preterminal axons (double arrows) containing synaptic vesicles and mitochondria terminate as axo-somaticsynapses, a: x4000; b: x5800. 30/~m in thickness showed even distributional density in immunoreactive terminals throughout the entire external layer. We could easily detect GRF-like immunoreactive terminals containing dense granules (80-120 nm in diameter), synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapiUary space of the portal vessel (Fig. 4a, b). Immunoreactive preterminal axons intimately contacting with immunoreactive or non-immunoreactive processes were also observed in the external layer of the median eminence, although the synaptic membrane specialization between them was not clear (Fig. 4c).
In this present study GRF-like immunoreactive neurons were distributed in the basolateral and lateral parts of the arcuate nucleus and in the mediobasai hypothalamus. We could not find immunoreactive neuronal perikarya in the ventromedial nucleus in which Merchenthaler et a1.18 reported the presence of immunoreactive neuronal perikarya. We also failed to detect GRF-like immunoreactive neuronal perikarya around the anterior commissure where Smith et al. 25 demonstrated immunoreactive neuronal perikarya using antibodies against hpGRF. Most recently, Sawchenko et al. mentioned the existence of small GRF-like immunoreactive neuronal perikarya in the paraventricular nucleus and broad expansion of their terminal fields in the hypothalamus as well as main location of immunoreactive neuronal perikarya in the arcuate nucleus and of terminals in the external layer of the median eminence by immunofluorescent and immunoperoxidase methods 23. We failed to detect any immunoreactive neuronal perikarya in the paraventricular nucleus and such expansion of immunoreaction terminal fields in the present study. Immunoreactive neurons appeared as relatively small round, oval or fusiform shapes and some of them extended immunoreactive processes from the neuronal perikarya. In the median eminence massive immunoreactive dots were accumulated in the external layer particularly around pericapillary spaces of the portal vessel. Therefore, we assumed that the main terminal portions of the immunoreactive processes around the capillaries originated from the neuronal perikarya in the arcuate nucleus and that GRF-like substances are released into the portal vessel and act on activation of the growth hormone release from the anterior pituitary. This assumption was confirmed by immunoelectron microscopy, which showed many GRF-like immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria around the basement membrane of the pericapillary space of the portal vessel. The fine structure of the GRF-like immunoreactive neurons have well developed cell organellae such as mitochondria and rER. They also contain sparsely distributed immunoreactive dense granules. These immunoreactive neurons are considered to re-
141
Fig. 4. GRF-like immunoreactive endings in the external layer of rat median eminence. Many immunoreactive endings containing immunoreactive dense granules, synaptic vesicles and mitochondria are distributed in the vicinity of pericapillary region of the portal vessels (a and b). Immunoreactive endings are in intimately contact with the non-immunoreactive endings in the external layer. L, capillary lumen of the portal vessel, a: x 11,500; b: x 12,000; c: x 14,000.
ceive neuronal input from other neurons on the surface of their soma mainly since we could clearly demonstrate non-immunoreactive axo-somatic synapses on their surface. The GRF-like immunoreactive neurons have very few dendritic arborizations in the arcuate nucleus as compared with neurotensin-like and fl-endorphin-like immunoreactive neurons around which immunoreactive processes have been found by light and electron microscopic immunocytochemistry11,12. On the other hand, we speculate that abundant terminal arborization occurs in the external layer of the median eminence since a large number of immunoreactive nerve endings were detected by both light and electron microscopic immunocytochemistry in spite of the relatively small number of immunoreactive neuronal perikarya in the arcuate nucleus. Recently, the coexistence of peptides and classical
neurotransmitters in the neurons in the arcuate nucleus have been demonstrated; neurotensin and dopamine 1°, gamma-aminobutyric acid ( G A B A ) and dopamine in the same neurons 7. We have demonstrated GRF-like immunoreactive substance and tyrosine hydroxylase-like immunoreactive substance in the same neurons in the ventrolateral arcuate nucleus 20. Large numbers of neurotensin-like immunoreactive neurons and almost all the GRF-like immunoreactive neurons are located in the basolateral part of the arcuate nucleus. Therefore, it is also interesting to examine the colocalization of the neurotensin-like substance and GRF-like substance in the same neurons. We have obtained the evidence suggesting coexistence of both peptides in the same neurons by light microscopic immunocytochemistry (in preparation). Most recently, Sawchenko et al. have reported
142 the coexistence of GRF-like and neurotensin-like immunoreactive substances in the same neurons in the basolateral part of the arcuate nucleusz3. The size of dense granules immunoreactive to G R F in GRF-like immunoreactive neurons is similar to that of them with immunoreactivity to neurotensin in neurotensin-like immunoreactive neurons by immunoelectron microscopy. It is necessary to ascertain whether both peptides are localized in the same secretory granules by immunoelectron microscopy on the
neurons for functional control of G R F neurons has also been suggested by the fact that administration of a-methyltyrosine, an inhibitor of tyrosine hydroxylase, or reserpine abolish G H secretion z0. Our finding of axo-somatic preterminal axons containing synaptic vesicles and cored vesicles on GRF-like immunoreactive neuronal perikarya may be a feature of direct neuronal input of m o n o a m i n e n e u r o n on G R F like immunoreactive neuron.
same ultrathin sections. Many monoaminergic innervations originating from m o n o a m i n e neurons located in the brainstem have been found in the arcuate nu-
ACKNOWLEDGEMENTS
cleus 8. Therefore, it would also be interesting to examine their influence upon GRF-like immunoreactive neurons since interference' with the a-adrenergic pathway completely abolishes the G H secretion from
Institute,
the anterior pituitary 19. Participation of m o n o a m i n e
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The authors wish to thank Dr. Nicholas Ling (Salk San
Diego,
CA)
for
supplying
rat
GRFl_43-OH. This work was supported in part by Grants (nos. 57440021, 57370002 and 59225023) from the Ministry of Education, Science and Culture, Japan.
8 Fuxe, K., Cellular localization of monoamines in the median eminence and the infundibular system of some mammals, Z. Zellforsch., 61 (1964) 710-724. 9 Guillemin, R., Brazeau, P., B6hlen, P., Esch, F.M., Ling, N. and Wehrenberg, W.B., Growth hormone-releasing factor from a human pancreatic tumor that caused aeromegaly, Science, 218 (1982) 585-587. 10 Ibata, Y., Fukui, K., Okamura, H., Kawakami, F., Tanaka, M., Obata, H.L., Tsuto, T., Terubayashi, H., Yanaihara, C. and Yanaihara, N., Coexistence of dopamine and neurotensin in hypothalamic arcuate and periventricular neurons, Brain Research, 269 (1983) 177-179. 11 Ibata, Y., Kawakami, F., Fukui, K., Obata-Tsuto, H.L., Tanaka, M., Kubo, T., Okamura, H., Morimoto, N., Yanaihara, C. and Yanaihara, N., Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus, Brain Research, 302 (1984) 221-230. 12 Ibata, Y., Kawakami, F., Okamura, H., Obata-Tsuto, H.L., Morimoto, N. and Zimmerman, E.A., Light and electron microscopic immunocytochemistry of fl-endorphin//3-LPH-like immunoreactive neurons in the arcuate nucleus and surrounding areas of the rat hypothalamus, Brain Research, 341 (1985) 233-242. 13 Jansson, J.-O., Carlsson, L. and Isaksson, O.G.P., Growth hormone (GH)-releasing factor (GRF) pretreatment enhances the GRF-induced GH secretion in rats with the pituitary autotransplanted to the kidney capsule, Endocrinology, 116 (1985) 95-98. 14 Kita, T., Chihara, K., Abe, H., Minamitani, N., Kaji, H., Kodama, H., Kashio, Y., Okimura, Y., Fujita, T. and Ling, N., Regional distribution of rat growth hormone releasing factor-like immunoreactivity in rat hypothalamus, Endocrinology, 116 (1985) 259-262. 15 Law, G.J., Ray, K.P. and Wallis, M., Effects of growth hormone-releasing factor and somatostatin on growth hor-
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mone secretion and cellular cyclic AMP levels. Cultured ovine and rat anterior pituitary cells show markedly different responses, Fed. Eur. Biochem. Soc., 179 (1985) 12-16. Lechan, R.M., Lin, H.D., Ling, N., Jackson, I.M.D., Jacobson, S. and Reichlin, S., Distribution of immunoreactive growth hormone releasing factor (1-44) NH 2 in the tuberoinfundibular system of the rhesus monkey, Brain Research, 309 (1984) 55-61. Ling, N., Esch, F., B6hlen, P., Brazeau, P., Wehrenberg, W.B. and Guillemin, R., Isolation, primary structure and synthesis of human hypothalamic somatocrinin: growth hormone-releasing factor, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4302-4306. Marchenthaler, I., Vigh, S., Schally, A.V. and Petrusz, P., Immunocytochemical localization of growth hormone releasing factor in the rat hypothalamus, Endocrinology, 114 (1984) 1082-1085. Martin, J., Discovery of a new peptide with growth hormone releasing activity, Trends Neurosci., 6 (1983) 1-4. Durand, D., Martin, J.B. and Brazeau, P., Evidence for a role of a-adrenergic mechanism in regulation of episodic growth hormone secretion in the rat, Endocrinology, 100 (1977) 722-728. Okamura, H., Murakami, S., Chihara, K., Nagatsu, I. and Ibata, Y., Coexistence of growth hormone releasing factor (GRF)-like and tyrosine hydroxylase-like immunoreactives in neurons of the rat arcuate nucleus, Neuroendoerinology, 41 (1985) 177-179. Rivier, J., Spiess, J., Thorner, M. and Vale, W., Characterization of growth hormone-releasing factor from a human pancreatic islet tumor, Nature (London), 300 (1982) 276-278.
23 Sawchenko, P.E., Swanson, L.W., Rivier, J. and Vale, W.W., The distribution of growth-hormone-releasing factor (GRF) immunoreactivity in the central nervous system of the rat. An immunohistochemical study using antisera directed against rat hypothalamic GRF, J. Comp. Neurol., 273 (1985) 100-115. 24 Schriock, E.A., Lustig, R.H., Rosenthal, S.M., Kaplan, S.L. and Grumbach, M.V., Effect of growth hormone (GH)-releasing hormone (GRH) on plasma GH in relation to magnitude and duration of GH deficiency in 26 children and adults with isolated GH deficiency or multiple pituitary hormone deficiencies: evidence for hypothalamic GRH deficiency, J. Clin. Endocrinol. Meta. , 58 (1984) 1043-1049. 25 Smith, R.M., Howe, P.R.C., Oliver, J.R. and Willoughby, J.O., Growth hormone releasing factor immunoreactivity in rat hypothalamus, Neuropeptides, 4 (1984) 109-115. 26 Spiess, J., Rivier, J. and Vale, W., Characterization of rat hypothalamic growth hormone-releasing factor, Nature (London), 303 (1983) 532-535. 27 Tannenbaum, G.S., Growth hormone-releasing factor: direct effects on growth hormone, glucose and behavior via the brain, Science, 226 (1984) 464-466. 28 Thorner, M.O., River, J., Spiess, J., Borges, J.L., Vance, M.L., Bloom, F.E., Rogol, A.D., Cronin, M.J., Kaiser, D.L., Evans, W.S., Webster, J.D., MacLeod, R.M. and Vale, W., Human pancreatic growth-hormone-releasing factor selectively stimulates growth-hormone secretion in man, Lancet, 1/8 (1983) 24-28. 29 Wahrenberg, W.B., Brazeau, P., Luben, R., Ling, N. and Guillemin, R., A non-invasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormonereleasing factor, Neuroendocrinology, 36 (1983) 489-491.
136
Elsevier BRE 11593
Light and Electron Microscopic Immunocytochemistry of GRF-Like Immunoreactive Neurons and Terminals in the Rat Hypothalamic Arcuate Nucleus and Median Eminence Y. IBATA j , H. OKAMURA 1, S. MAKINO 1, F. KAWAKAMI 2, N. MORIMOTO I and K. CHIHARA 3
1Department of Anatomy, Kyoto Prefectural University of Medicine, KawaramachiHirokofi; 2Departmentof Psychiatry, Kyoto Prefectural Universityof Medicine, KawaramachiHirokoji, Kyoto 602 and 3Third Division, Department of Medicine, Kobe UniversitySchool of Medicine, Kobe 650 (Japan) (Accepted August 13th, 1985)
Key words: arcuate nucleus - - median eminence - - immunocytochemistry - - growth hormone-releasing factor (GRF) - electron microscopy - - rat
Growth hormone-releasing factor (GRF) synthesizing neuronal perikarya and terminals were investigated by light and electron microscopic immunocytochemistry using rat hypothalamus. Immunoreactive neuronal perikarya were located mainly in the ventrolateral part of the arcuate nucleus. They contained well developed cell organella such as mitochondria and rough surfaced endoplasmic reticulum with some expansion. They also contained immunoreactive dense granules (80-120 nm in diameter). On the surface of the immunoreactive neuronal perikarya were frequently found non-immunoreactive axo-somatic synapses. Therefore, the GRF-like immunoreactive neurons were assumed to receive neuronal inputs from other neurons on their neuronal soma. In the external layer of the median eminence large numbers of immunoreactive terminals were distributed particularly around the capillaries of the portal vessel. Electron microscopic immunocytochemistry revealed large numbers of immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapillary space of the portal vessel. Therefore, we concluded that GRF-like immunoreactive substances are released into the portal capillaries from the nerve terminals, which originate from the neuronal perikarya in the ventrolateral part of the arcuate nucleus, and act on growth hormone release in the anterior pituitary. We also suggest that GRF-like immunoreactive neurons have abundant terminal arborization in the external layer of the median eminence.
INTRODUCTION G r o w t h h o r m o n e - r e l e a s i n g factor ( G R F ) has b e e n
Immunocytochemistry
using
specific
antisera
isolated f r o m h u m a n p a n c r e a t i c t u m o r s in patients with a c r o m e g a l y and its c h e m i c a l structure was de-
against h p G R F and rat G R F has d e m o n s t r a t e d G R F like i m m u n o r e a c t i v e n e u r o n a l p e r i k a r y a in the ar-
t e r m i n e d as a p e p t i d e with a 40 o r a n o t h e r 44 a m i n o acid residue9,17, 21. T h e s e p e p t i d e s are given the n a m e
cuate and v e n t r o m e d i a l nuclei and their n e r v e termi-
h u m a n p a n c r e a t i c g r o w t h h o r m o n e - r e l e a s i n g factor (hpGRF)9,17. T h e y are highly and specifically p o t e n t for releasing the g r o w t h h o r m o n e ( G H ) f r o m the anterior pituitary in v i v o and in vitro c o n d i t i o n s 2~6,13,19,
nals in the e x t e r n a l layer of the m e d i a n e m i n e n c e of the rat and o t h e r m a m m a l i a n species 3.18A9~23,25. H o w ever, their distributional p a t t e r n and density h a v e not b e e n e x a m i n e d in detail and no i m m u n o e l e c t r o n
24,26,28. T h e G R F s of 6 animal species h a v e n o w b e e n
microscopic o b s e r v a t i o n s a b o u t their n e u r o n a l perikarya and t e r m i n a l s have b e e n d e s c r i b e d until now.
identified and rat G R F has b e e n f o u n d to be a peptide with a 43 a m i n o acid residue 4,5,14. T h e effects of
W e a t t e m p t e d , t h e r e f o r e , to e x a m i n e their features in detail and fine structure by light and e l e c t r o n mi-
G R F including h p G R F on release of G H h a v e b e e n investigated physiologically14.24.27.29 and b i o c h e m i c ally1,15.
croscopic i m m u n o c y t o c h e m i s t r y and to o b t a i n m o r e i n f o r m a t i o n c o n c e r n i n g their functional significance from m o r p h o l o g i c a l points of view.
Correspondence: Y. Ibata, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kyoto Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
137 MATERIALS AND METHODS Fourteen male Wistar rats housed under a 14 h light-10 h dark schedule were given free access to food and water; 6 for light microscopic immunocytochemistry and 8 for electron microscopic immunocytochemistry. Under Nembutal anesthesia, colchicine (30 #g) was injected into the lateral ventricle of all the animals for light microscopic immunocytochemistry and 4 animals for examination of GRF-like immunoreactive neuronal perikarya by electron microscopic immunocytochemistry 2 days prior to sacrifice. This treatment was done to promote the storage of GRF-like immunoreactive substances in the neuronal perikarya. The 4 rats used for examination of the fine structures of GRF-like immunoreactive terminals were not given colchicine treatment. For light microscopic immunocytochemistry the animals were perfused via the left cardiac ventricle with a mixture of 4% paraformaldehyde, 0.2% picric acid and 0.35% glutaraldehyde buffered at pH 7.4 with 0.1 M phosphate buffer. The brain was removed after perfusion and further fixed with the same mixture but without glutaraldehyde, for 48 h at 4 °C. Thereafter, serial frontal frozen sections (30/~m thick) were made and stored in a phosphate-buffered saline (PBS) solution containing 0.3% Triton X for 4 days at 4 °C. Sections for immunocytochemistry were first incubated in bovine serum albumin (dilution x 100) for 1 h at room temperature, and then incubated in rat anti-GRF serum (dilution x 4000) for 48 h at 4 °C. They were next immersed in anti-rabbit IgG solution (dilution x 200, Miles Laboratory) for 2 h at room temperature followed by incubation in PAP solution (dilution × 200, D A K O immunoglobulins a/s) for 90 min at room temperature. Finally, they were exposed for 10 min at room temperature to 0.02% 3.3'-diaminobenzidine'4HCl (DAB) containing H202 (15 pl of 30% H202 solution into 100 ml DAB solution), mounted on glass slides and examined by light microscopy. As a control, the sections were treated with anti-GRF serum absorbed with synthetic rat G R F (0.1-10 /~g/ml). No positive immunoreactivity was detected in the control sections. The antiserum to rat G R F used in this present study was generated in rabbit using 1 mg synthetic rat GRF1_43-OH coupled to 2.5 mg bovine thyroglobulin by glutaraldehyde method.
This antiserum bound 50% of [125I]iodo rat GRFl_43-OH at a final dilution of 1:60,000, and the binding was inhibited in a dose-related manner by 5600 pg of unlabeled rat GRF1_a3-OH. There was no cross-reaction with human GRF1_44-NH2, human GRFI_a0-OH, human GRFI_26-NH2, peptide histidine isoleusine (PHI), vasoactive intestinal polypeptide (VIP), rat corticotropine releasing factor (CRF), human calcitonin, neurotensin, substance P, somatostatin~_14 or somatostatin1_28. For electron microscopic immunocytochemistry, the colchicine-treated animals were perfused via the left cardiac ventricle with the same fixative as for light microscopic immunocytochemistry. The animals not treated with colchicine were decapitated under Nembutal anesthesia, the brains immediately removed and thin tissue blocks including the hypothalamus were dissected out. They were immersed in the same fixative for 2 h and further fixed with the same mixture, from which glutaraldehyde had been excluded. Immersion fixation was used in order to avoid deformation of the fine structure of the median eminence caused by expansion of extracellular spaces during perfusion. Thin tissue blocks including the hypothalamus were also dissected from the brains of the perfused animals. They were further fixed with the same mixture from which glutaraldehyde had been excluded. Each sample was treated for electron microscopic immunocytochemistry as follows. Frontal sections, 30 ,um in thickness, were made within PBS by a microslicer (Dosaka EM) and incubated in bovine serum (dilution x 4000) for 96 h at 4 °C followed by incubation in anti-rabbit IgG solution (dilution x 200) for 2 h, at room temperature. Then they were incubated in PAP solution (dilution × 200) for 2 h at room temperature and finally allowed to react with 0.02% DAB solution containing H20 2 for 5 min at room temperature. Between each step of the reaction they were washed with PBS. Completion of the immunoreactive processes was judged by the appearance under light microscopy of a weak brown coloration of the neuronal perikarya, processes and terminals after treatment with DAB solution. The sections were fixed in chilled 1% OsO4 solution for 1 h and dehydration and embedding in Epon mixture were carried out in the usual manner. Semi-thin sections were examined by light microscopy to confirm the presence of immunoreactive perikarya, proces-
138 ses and terminals, following which ultrathin sections stained with uranyl acetate were examined in a JEM200CX electron microscope. RESULTS
Light microscopic immunocytochemistry G R F - l i k e immunoreactive neuronal perikarya were mainly located in the lateral and basolateral parts of the arcuate nucleus at the level of the central median eminence of the frontal section (Fig. l a ) . They were observed as small neurons (about 15 ~ m in diameter) with round, oval or fusiform shapes. Immunoreactive density was varied in each neuron and unipolar, bipolar or multipolar immunoreactive processes extended from some neuronal perikarya. However, immunoreactive dots or strands, which were considered to be G R F - l i k e dendritic branches, were rarely detected a r o u n d the immunoreactive neuronal perikarya. Few immunoreactive neuronal perikarya were found in the arcuate nucleus at the level of the rostral and caudal median eminence. W e also found some immunoreactive neuronal p e r i k a r y a in the mediobasal hypothalamus (Fig. l b ) . Their features were similar to those in the a b o v e - m e n t i o n e d
region, but we could not detect immunoreactive neuronal p e r i k a r y a in the ventromedial nucleus. Immunoreactive nerve terminals were distributed in the external layer of the rostral, central and caudal median eminence. Dense accumulation of immunoreactive dots were detected particularly a r o u n d the pericapillary space of the portal vessel (Fig. 2a). In the s u b e p e n d y m a l layer and internal layer of the median eminence fairly numbers of immunoreactive brown dots and b e a d e d strands were found particularly in the central median eminence and in the infundibular stalk (Fig. 2b). A r o u n d the pericapillary space of the portal vessel which p e n e t r a t e d into the internal layer, dense accumulation of immunoreactive terminals were also observed (Fig. 2c). M o d e r ate numbers of immunoreactive dots were also distributed in the region dorsal to the infundibular recessus at the level of the caudal median eminence. A t the more caudal level immunoreactive processes and terminals were found in the floor of the third ventricle near the p r e m a m m i l l a r y region.
Electron microscopic immunocytochemistry Immunoreactive neuronal p e r i k a r y a a p p e a r e d as brown neurons in the lateral and basolateral parts of
]
~i~~! iiliI ~i~ Fig. 1. a: GRF-like immunoreactive neurons with numerous shapes and immunoreactive density are distributed in the rat arcuate nucleus at the level of the central median eminence. V, third ventricle, x 240. b: in addition to the arcuate nucleus, immunoreactive neurons are also detected in the mediobasal hypothalamus, x450.
139
V
Fig. 2. a: concentrations of GRF-like immunoreactive terminals are observed in the external layer of the central median eminence. Large numbers of immunoreactive dots are accumulated particularly around the pericapillary space of the portal vessel (arrows). V, third ventricle, x 180. b: in the subependymal and internal layer some immunoreactive fibers are also detected in the central median eminence. V, third ventricle, x880. c: dense accumulation of immunoreactive terminals are observed around the pericapillary regions of the portal vessel which penetrate into the internal layer through the external layer. C, capillary, x 1600.
the arcuate nucleus in the semi-thin sections of Epon embedding materials by light microscopy. By electron microscopy, dense osmiophilic immunoreactive substances were disseminated throughout the cytoplasma of moderately immunoreactive neuronal perikarya, though the entire cytoplasmic matrix and cell organella showed a diffuse high electron density in strong immunoreactive neurons. GRF-like immunoreactive neurons were characterized by oval nuclei with diffusely distributed D N A particle, well developed mitochondria, rough surfaced endoplasmic reticulum (rER) with some expansion, polysomes and Golgi complex. Invagination of the nuclear envelope was also frequently observed. Lysosomes in the cytoplasma were hard to detect. In addition, dense granules (80-120 nm in diameter)
with immunoreactivity were scattered throughout the cytoplasma (Fig. 3a, b). On the surface of immunoreactive neuronal perikarya non-immunoreactive preterminal axons containing synaptic vesicles (40-60 nm in diameter), occasionally cored vesicles (80-100 nm in d i a m e t e r ) a n d mitochondria terminated as axo-somatic synapses (Fig. 3a, b). There were only a few immunoreactive processes around the immunoreactive neuronal perikarya by electron microscopy as shown by light microscopic immunocytochemistry. In the median eminence large numbers of immunoreactive dots were found particularly in the lateral part of the external layer in the semithin sections from the Epon embedding materials, although light microscopic immunocytochemistry of the sections of
140 DISCUSSION
Fig. 3. Fine structures of GRF-like immunoreactive neuronal perikarya in the rat arcuate nucleus. Immunoreactiveneuronal perikarya contain well developed mitochondria, rER, polysomes and Golgi complex (G). Besides the cell organellae, immunoreactive dense granules (arrows) are also distributed throughout the cytoplasma. Invaginationof nuclear envelope is also observed. On the surface of immunoreactive neuronal perikarya non-immunoreactive preterminal axons (double arrows) containing synaptic vesicles and mitochondria terminate as axo-somaticsynapses, a: x4000; b: x5800. 30/~m in thickness showed even distributional density in immunoreactive terminals throughout the entire external layer. We could easily detect GRF-like immunoreactive terminals containing dense granules (80-120 nm in diameter), synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapiUary space of the portal vessel (Fig. 4a, b). Immunoreactive preterminal axons intimately contacting with immunoreactive or non-immunoreactive processes were also observed in the external layer of the median eminence, although the synaptic membrane specialization between them was not clear (Fig. 4c).
In this present study GRF-like immunoreactive neurons were distributed in the basolateral and lateral parts of the arcuate nucleus and in the mediobasai hypothalamus. We could not find immunoreactive neuronal perikarya in the ventromedial nucleus in which Merchenthaler et a1.18 reported the presence of immunoreactive neuronal perikarya. We also failed to detect GRF-like immunoreactive neuronal perikarya around the anterior commissure where Smith et al. 25 demonstrated immunoreactive neuronal perikarya using antibodies against hpGRF. Most recently, Sawchenko et al. mentioned the existence of small GRF-like immunoreactive neuronal perikarya in the paraventricular nucleus and broad expansion of their terminal fields in the hypothalamus as well as main location of immunoreactive neuronal perikarya in the arcuate nucleus and of terminals in the external layer of the median eminence by immunofluorescent and immunoperoxidase methods 23. We failed to detect any immunoreactive neuronal perikarya in the paraventricular nucleus and such expansion of immunoreaction terminal fields in the present study. Immunoreactive neurons appeared as relatively small round, oval or fusiform shapes and some of them extended immunoreactive processes from the neuronal perikarya. In the median eminence massive immunoreactive dots were accumulated in the external layer particularly around pericapillary spaces of the portal vessel. Therefore, we assumed that the main terminal portions of the immunoreactive processes around the capillaries originated from the neuronal perikarya in the arcuate nucleus and that GRF-like substances are released into the portal vessel and act on activation of the growth hormone release from the anterior pituitary. This assumption was confirmed by immunoelectron microscopy, which showed many GRF-like immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria around the basement membrane of the pericapillary space of the portal vessel. The fine structure of the GRF-like immunoreactive neurons have well developed cell organellae such as mitochondria and rER. They also contain sparsely distributed immunoreactive dense granules. These immunoreactive neurons are considered to re-
141
Fig. 4. GRF-like immunoreactive endings in the external layer of rat median eminence. Many immunoreactive endings containing immunoreactive dense granules, synaptic vesicles and mitochondria are distributed in the vicinity of pericapillary region of the portal vessels (a and b). Immunoreactive endings are in intimately contact with the non-immunoreactive endings in the external layer. L, capillary lumen of the portal vessel, a: x 11,500; b: x 12,000; c: x 14,000.
ceive neuronal input from other neurons on the surface of their soma mainly since we could clearly demonstrate non-immunoreactive axo-somatic synapses on their surface. The GRF-like immunoreactive neurons have very few dendritic arborizations in the arcuate nucleus as compared with neurotensin-like and fl-endorphin-like immunoreactive neurons around which immunoreactive processes have been found by light and electron microscopic immunocytochemistry11,12. On the other hand, we speculate that abundant terminal arborization occurs in the external layer of the median eminence since a large number of immunoreactive nerve endings were detected by both light and electron microscopic immunocytochemistry in spite of the relatively small number of immunoreactive neuronal perikarya in the arcuate nucleus. Recently, the coexistence of peptides and classical
neurotransmitters in the neurons in the arcuate nucleus have been demonstrated; neurotensin and dopamine 1°, gamma-aminobutyric acid ( G A B A ) and dopamine in the same neurons 7. We have demonstrated GRF-like immunoreactive substance and tyrosine hydroxylase-like immunoreactive substance in the same neurons in the ventrolateral arcuate nucleus 20. Large numbers of neurotensin-like immunoreactive neurons and almost all the GRF-like immunoreactive neurons are located in the basolateral part of the arcuate nucleus. Therefore, it is also interesting to examine the colocalization of the neurotensin-like substance and GRF-like substance in the same neurons. We have obtained the evidence suggesting coexistence of both peptides in the same neurons by light microscopic immunocytochemistry (in preparation). Most recently, Sawchenko et al. have reported
142 the coexistence of GRF-like and neurotensin-like immunoreactive substances in the same neurons in the basolateral part of the arcuate nucleusz3. The size of dense granules immunoreactive to G R F in GRF-like immunoreactive neurons is similar to that of them with immunoreactivity to neurotensin in neurotensin-like immunoreactive neurons by immunoelectron microscopy. It is necessary to ascertain whether both peptides are localized in the same secretory granules by immunoelectron microscopy on the
neurons for functional control of G R F neurons has also been suggested by the fact that administration of a-methyltyrosine, an inhibitor of tyrosine hydroxylase, or reserpine abolish G H secretion z0. Our finding of axo-somatic preterminal axons containing synaptic vesicles and cored vesicles on GRF-like immunoreactive neuronal perikarya may be a feature of direct neuronal input of m o n o a m i n e n e u r o n on G R F like immunoreactive neuron.
same ultrathin sections. Many monoaminergic innervations originating from m o n o a m i n e neurons located in the brainstem have been found in the arcuate nu-
ACKNOWLEDGEMENTS
cleus 8. Therefore, it would also be interesting to examine their influence upon GRF-like immunoreactive neurons since interference' with the a-adrenergic pathway completely abolishes the G H secretion from
Institute,
the anterior pituitary 19. Participation of m o n o a m i n e
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The authors wish to thank Dr. Nicholas Ling (Salk San
Diego,
CA)
for
supplying
rat
GRFl_43-OH. This work was supported in part by Grants (nos. 57440021, 57370002 and 59225023) from the Ministry of Education, Science and Culture, Japan.
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