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Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda
Peptides, Vol. 7, pp. 941-951, 1986.©AnkhoInternationalInc. Printedin the U.S.A.
0196-9781/86$3.00 + .00
Localization of Melanin-Concentrating Hormone-Like Immunoreactivity in the Brain and Pituitary of the Frog Rana ridibunda A N N C. A N D E R S E N , * G E O R G E S P E L L E T I E R , t A L E X N . E B E R L E , ~ PHILIPPE LEROUX,* SYLVIE JEGOU* AND HUBERT VAUDRY*
*Groupe de Recherche en Endocrinologie Mol~culaire, UA CNRS 650, Unitd Alli@e ~ I'INSERM Facult# des Sciences, Universit# de Rouen, 76130 Mont Saint Aignan, France t M R C Group in Molecular Endocrinology, Le Centre Hospitalier de l' Universit# Laval Qu#bec, G1V 4G2 Canada SKantonsspital Basel, Universitiitskliniken, Department Forschung Hebelstrasse 20 CH 4031 Basel, Switzerland R e c e i v e d 11 S e p t e m b e r 1986
ANDERSEN, A. C., G. PELLETIER, A. N. EBERLE, P. LEROUX, S. JEGOU AND H. VAUDRY. Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda. PEPTIDES 7(6) 941-951, 1986.--The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog
Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-iike containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area. Brightly immunofluorescent nerve bundles were found in the preoptic nucleus and in the ventral infundibular nucleus, coursing towards the internal zone of the median eminence and the pituitary stalk. An intense network of immunofluorescent fibers was localized in the neural lobe of the pituitary. The subcellular localization of MCH-like material was studied in the neurohypophysis using the immunogold technique. It was demonstrated that MCHlike material was contained in dense core vesicles (80-90 mm in diameter) within specific nerve terminals. The present findings indicate that, in amphibians, MCH-like peptide is located in specific hypothalamic neurons. Our data suggest that MCH may be released by neurohypophyseal nerve endings as a typical neurohormone. Melanin-concentrating hormone Neurosecretion Frog
Immunohistochemistry
THE intermediate lobe of the pituitary secretes a major peptide called alpha melanotropin (a-MSH) which plays an important role in skin color adaptation in cold blooded vertebrates [2]. ct-MSH is not only produced by endocrine cells of the intermediate lobe, but is also present in discrete neuronal systems of the brain of amphibians [5, 10, 16, 31, 36], reptiles [19,33] and mammals [6, 11, 13, 15, 17, 25, 27, 35, 38], supporting the view that most hormonal peptides are also potential neurotransmitters. Recently, a regulatory peptide called melanin-concentrating hormone (MCH) has been isolated from the fish pituitary and characterized [18]. Baker and Rance [3] have studied the distribution of MCH in the pituitary and brain of various species and have found that MCH-like bioactivity was detectable in the hypothalamus of fish, amphibians and mammals. Polyclonal antibodies have been raised against synthetic MCH and applied to the immunohistochemical identification of MCH in the brain and
Electron microscopy
Brain
Pituitary
pituitary gland of salmons [21] and rats [30,40]. In the present study we have examined the distribution of MCH in the brain and pituitary of the frog Rana ridibunda using the indirect immunofluorescence technique. METHOD
Tissue Preparation Adult male frogs (Rana ridibunda) of 30-40 g body weight, obtained from Cou~tard (St. Hilaire de Riez, France) were kept unfed in tanks continuously supplied with a trickle of water. The animals were submitted to a 12 hr-dark/light cycle, at a constant temperature (8-+0.5°C). The frogs were stunned and transcardially perfused with 30 ml of 0.1 M phosphate buffer (PBS, pH 7.4) containing 0.025% xylocaine. The perfusion was carded on with 50 ml of McLean's fixative as previously described [20]. The
941
942
ANDERSEN
TABLE
1
TABLE
LOCALIZATION AND RELATIVE ABUNDANCE OF N E U R O N A L PERIKARYA AND FIBERS EXHIBITING MCH-LIKE IMMUNOREACTIVITY IN THE FROG BRAIN Structure Telencephalon P a l l i u m d o r s a l i s (PD) P a l l i u m m e d i a l e (PM) Pallium laterale, pars dorsalis (PLd) Pallium iaterale, pars ventralis (PLv) Nucleus medialis septi (NMS) B e d n u c l e u s o f the pallial c o m m i s s u r e (NBPC) A m y g d a l a , p a r s l a t e r a l i s (A1) Amygdala, pars medialis (Am) Nucleus entopeduncularis (NEP) Pallial c o m m i s s u r e (PaC) A n t e r i o r c o m m i s s u r e (Ac) Diencephaion E p i p h y s i s (E) Habenular commissure (HC) Nucleus habenularis dorsalis (NHD) Nucleus habenularis ventralis (NHV) Area ventralis anterior thalami (AVA) Area ventrolateralis thalami (AVL) Area ventromedialis thalami (AVM) Nucleus dorsomedialis anterior thalami (NDMA) Nucleus dorsolateralis anterior thalami (NDLA) Corpus geniculatus laterale (CGL) Lateral forebrain bundle (LFB) N u c l e u s r o t u n d u s (NR) Posterocentralis thalami (NPC) Nucleus posterolateralis thalami (NPL) Nucleus preopticus, pars anterior (NPOa) Nucleus preopticus, pars posterior (NPOp) Nucleus infundibularis dorsalis (NID) Nucleus infundibularis ventralis (NIV)
Perikarya
ET AL.
1
(CONTINUED) Structure
Perikarya
Fibers
-
+ + + + -
-
-
-
-
-
-
-
-
-
-
-
-
Fibers
-
-
-
-
+ -
-
-
-
+ +
+ +
+ + +
++
+ + -
+ +
A r e a i n f u n d i b u l a r i s (AI) Median eminence (ME) P o s t e r i o r c o m m i s s u r e (PC) O p t i c c h i a s m a (OC) O p t i c t r a c t (OT) Optic nerve (ON) Mesencephalon Nucleus mesencephalicus nervitrigemini (NMNT) Stratum griseum superficiale tecti (SGS) Stratum griseum centrale tecti (SGC) S t r a t u m g r i s e u m p e r i v e n t r i c u l a r e tecti (SGP) N u c l e u s o f the film ( N F ) Nucleus profundus mesencephali (NPM) Nucleus anterodorsalis tegmenti mesencephali (NAD) Nucleus anteroventralis tegmenti mesencephali (NAV) N u c l e u s o f the o c u l o m o t o r n e r v e (NOM) T o r u s s e m i c i r c u l a r i s (TS) Nucleus reticularis isthmi (NRIS) N u c l e u s o f the t r o c h l e a r n e r v e ( N T R O ) Nucleus cerebelli (NCER) Oculomotor nerve (OMN)
-
-
-
-
Hypophysis P a r s n e r v o s a (PN) P a r s i n t e r m e d i a (PI) P a r s d i s t a l i s (PD)
-
+ + + -
+ , f e w ; + + , m e d i u m ; + + + , m a n y ; - , no i m m u n o r e a c t i v e cell b o d i e s or fibers. A b b r e v i a t i o n s a c c o r d i n g to W a d a et al. [37].
FACING PAGE F I G . 1. F r o n t a l s e c t i o n s o f t h e frog b r a i n at the l e v e l o f t h e p r e o p t i c n u c l e u s ( N P O ) ( c o r r e s p o n d i n g to c o o r d i n a t e s + 2 . 6 o n Fig. 7A). T h i s photomicrograph shows MCH-like immunoreactive perikarya and fibers (arrows) forming a triangular-shaped network. The arrows indicate t h e v e n t r o l a t e r a l p r o j e c t i o n s o f t h e f i b e r s a b o v e the o p t i c n e r v e (ON). P R = P r e o p t i c R e c e s s , ×368. F I G . 2. H i g h m a g n i f i c a t i o n o f a f r o n t a l s e c t i o n o f t h e p r e o p t i c n u c l e u s ( N P O ) ( c o o r d i n a t e s + 2 . 6 o n Fig. 7A) s h o w i n g M C H i m m u n o r e a c t i v e p e r i k a r y a a n d fibers. I n t e n s e i m m u n o f l u o r e s e n c e l a b e l l i n g is o b s e r v e d in the c y t o p l a s m w h e r e a s the n u c l e u s d o e s n o t s h o w i m m u n o r e a c t i v e m a t e r i a l . T h e cell p r o c e s s e s a n d f i b e r s f o r m a d e n s e b u n d l e ( a r r o w s o n t h e r i g h t side o f the figure) p r o j e c t i n g v e n t r o l a t e r a l l y , x 5 8 0 . F I G . 3. F r o n t a l s e c t i o n o f the b r a i n ( c o o r d i n a t e s + 1.4 o n Fig. 7B) s h o w i n g the M C H - l i k e c o n t a i n i n g p e r i k a r y a g a t h e r e d in the d o r s a l i n f u n d i b u l a r n u c l e u s ( N I D ) c l o s e to the t h i r d v e n t r i c l e (III). T h e a r r o w s i n d i c a t e fine i m m u n o r e a c t i v e f i b e r s c o u r s i n g in a l a t e r a l d i r e c t i o n . x 193. F I G . 4. F r o n t a l s e c t i o n o f t h e frog b r a i n in the c a u d a l r e g i o n o f the p r e o p t i c n u c e l u s ( N P O ) ( c o o r d i n a t e s × 2 . 4 Fig. 7A). I m m u n o r e a c t i v e p e r i k a r y a s h o w a l i n e a r d i s t r i b u t i o n o n the s u r f a c e o f the e p i t h e l i a l b o r d e r i n g c e l l s o f the t h i r d v e n t r i c l e (III). B e a d e d h o r i z o n t a l l y - o r i e n t e d i m m u n o f l u o r e s c e n t f i b e r s are s h o w n b y a r r o w s , x 589. F I G . 5. P a r a s a g i t t a l s e c t i o n ( c o r r e s p o n d i n g to t h e p a r a s a g i t t a l d r a w i n g p r e s e n t e d in Fig. 6) o f the i n f u n d i b u l a r a r e a (AI) a n d p i t u i t a r y . D o r s a l side to t h e right, a n t e r i o r s i d e to the t o p o f the figure. I m m u n o r e a c t i v e p e r i k a r y a ( t h i c k a r r o w s ) are g a t h e r e d in t h e d o r s a l i n f u n d i b u l a r n u c l e u s ( N I D ) . I m m u n o r e a c t i v e f i b e r s (thin a r r o w s ) are p r e s e n t t h r o u g h o u t t h e i n f u n d i b u l a r a r e a (AI): d o r s a l ( N I D ) a n d v e n t r a l ( N I V ) i n f u n d i b u l a r nuclei. I m m u n o r e a c t i v e f i b e r s o r i g i n a t i n g f r o m the p r e o p t i c n u c l e u s c o u r s e v e n t r a l l y in t h e N I V t o w a r d s t h e p i t u i t a r y s t a l k a n d the i n t e r n a l z o n e o f the m e d i a n e m i n e n c e ( M E ) . I n t e n s e i m m u n o f l u o r e s c e n c e l a b e l l i n g is r e v e a l e d in t h e p a r s n e r v o s a (PN), w h i l e the p a r s i n t e r m e d i a (PI) a n d the p a r s d i s t a l i s (PD) are d e v o i d o f i m m u n o r e s p o n s e . × 184.
MCH IN THE FROG BRAIN AND PITUITARY
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FIG. 6. Schematic parasagittal section of the frog brain depicting the distribution of MCH-containing perikarya and neuronal processes. See list of abbreviations in Table 1. The dorsal reference zero point of the scale was defined by Wada et al. [37] as the apex of the angle formed by the junction of the dorsal midline meeting of the two optic lobes.
-2.0
.su.ou~L
PERIKARYA:
KEY:
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M C H IN T H E F R O G BR AIN A N D P I T U I T A R Y
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FIG. 7. A, B and C. Shematic drawings of serial frontal sections of the frog brain. Only the parts of the brain exhibiting a positive immunoreaction with the MCH antiserum are shown. The series start 3.2 mm anterior to the zero reference point of Wada et al. [37] and end 0.2 mm posterior to this point; the tracings are from sections 0.2 mm apart. The MCH-like containing perikarya are represented by triangles and the fibers by dots; the density of these symbols is proportional to the density of immunoreactive elements. See list of abbreviations in Table 1. FIG. 7 B AND C FOLLOWING PAGES.
P
~m
CO
Z
Z
MCH IN THE FROG BRAIN A N D PITUITARY
B
Ea FIG. 7 C
947
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A N D E R S E N ET AL.
948
FIG. 8. Immunoelectron microscopic localization of MCH using the immunogold technique. Several positive nerve terminals are decorated by gold particles located in dense core vesicles (arrows). Other nerve terminals appear non-immunoreactive (ni). ×47,000. brains were dissected and postfixed in the same fixative solution for 3 hr. The tissues were rinsed overnight in 0.1 M PBS containing 15% sucrose and transferred into 0.1 M PBS containing 30% sucrose for at least 24 hr. Then the brains were frozen in liquid nitrogen, embedded in O.C.T. Tissue Teck and sliced at 8 /zm in frontal or sagittal planes on a cryostat (Frigocut 2700, Reichert Jung).
Preparation of Antisera Synthetic MCH [12] was coupled to thyroglobulin using glutaraldehyde. The conjugate was dialyzed extensively and diluted with saline to a concentration of 1 mg/ml (with respect to conjugate concentration) and stored at -70°C. The complex was mixed with complete Freund's adjuvant (1:1) and injected intradermally into rabbits at multiple sites (100 or 200 /zg per animal). Booster injections were done every 4-5 weeks using 20/zg of complex per injection and incomplete Freund's adjuvant. The titer as measured by RIA ranged from 1:30,000 to 1:180,000 after the first booster injection. The antigenic determinant read by the antibodies was not determined. There was however full cross-reaction between iodinated MCH and tritiated (Nva3.6]-MCH which
means that the two methionine residues did not play a crucial role in the recognition.
Immunofluorescence of MCH The brain sections were incubated with normal goat serum (diluted 1:25) for one hour at room temperature. PBS supplemented with 1% human serum albumin, and 0.03% triton was used to dilute all the antisera. The sections were rinsed in two 5-rain baths of PBS and incubated overnight in a moistened atmosphere at 4°C with MCH-antiserum (No. 526) at the working dilution of 1:200. Thereafter the sections were thoroughly rinsed in several baths of PBS and incubated in a dark moistened chamber at 20°C for one hour with fluorescein-isothiocyanate conjugated antirabbit y-globulin (GAR/FITC, Nordic Immunology) diluted 1:60. Finally the sections were rinsed again in PBS, counterstained for 3 rain with Evan's blue (diluted 1:10,000 in distilled water) and mounted with PBS-glycerol (1:1). The mapping and the photomicrographs of the MCHimmunoreactive structures were performed with a Leitz Orthoplan microscope fitted with a Vario-Orthomat photographic system. The controls were performed on sections
MCH IN THE FROG BRAIN AND PITUITARY incubated either with non-immunized rabbit serum or with anti-MCH preabsorbed overnight, at room temperature, with 10-6 M synthetic MCH (Peninsula Laboratories). Electron Microscopy The frogs used for electron microscopy were transcardially perfused first with 30 ml of 0.1 M PBS then with 50 ml of 4% paraformaldehyde in PBS without any osmic postfixation. The neurointermediate lobes of the pituitary were dissected, postfixed for 3 hr in the same solution, rinsed overnight in PBS containing 5% sucrose and embedded in araldite. Ultrathin sections of the pars nervosa were processed for the immunogold technique as described by Roth et al. [28]. The MCH antiserum was used at a final dilution 1:4,000. RESULTS Localization of MCH-Containing Elements by Immunofluorescence The results of this study are presented in the form of a series of line drawings and photomicrographs. The line drawings are shematic drawings of the frog diencephalon and mesencephalon, in the parasagittal and frontal planes, depicting the distribution of MCH-like immunoreactivity in cells and fibers. Cell bodies positive for MCH are shown as filled triangles in the drawings; fine fibers and terminals containing MCH-like material are shown as dots. Nomenclature for this study was based on the work of Wada et al. [37] and Netchitailo et al. [22]. A list of identified structures and the relative frequencies of immunoreactive perikarya and fibers is presented in Table 1. Cell bodies containing MCH-like immunoreactivity were located exclusively in the diencephalon. Perikarya were found primarily in the preoptic nucleus (NPO), mainly in the mid-part of the NPO (Figs. l, 4 and 6). On frontal brain sections, the perikarya appeared to be gathered within a triangular framework. Other immunoreactive perikarya were observed in a more caudal area of the NPO. These latter neurons showed a linear distribution on the basal surface of the epithelial bordering cells of the third ventricle (Figs. 4 and 7A). A second group of MCH-positive perikarya, although less abundant than in the NPO, was observed in the dorsal infundibular nucleus (NID), close to the third ventricle (Figs. 3, 6 and 7B). In contrast, no cell body was localized in the ventral infundibular nucleus (NIV). At the level of the ventral thalamic area (AVA) scattered neurons were visualized (Figs. 6 and 7A). Immunoreactive neurites originating from the NPO formed a dense bundle which projected ventro-laterally and coursed caudally just above the optic nerve (Figs. 1 and 2). Discrete fibers were occasionally seen in the zone between the optic chiasma and the infundibular area, suggesting that preoptic MCH-positive perikarya may project caudally towards the median eminence, as schematically shown on Fig. 6. A system of coarse, thick MCH-positive fibers observed in the NIV also coursed ventrally towards the pituitary stalk and the internal zone of the median eminence (ME) (Fig. 5). A high density of immunoreactive nerve terminals was found in the neural lobe of the pituitary. No immunoreactivity was found in the intermediate and distal lobes. Preincubation of the MCH antiserum with l0 -6 M of the corresponding peptide resulted in complete loss of staining. Replacement of the MCH antiserum by non immunized rabbit serum also resulted in loss of staining.
949 Electron Microscopy Staining of ultrathin sections with the MCH antiserum, using the immunogold technique made it possible to visualize the immunoreactive material in specific nerve terminals of the frog neurohypophysis (Fig. 8). The gold particles appeared concentrated in dense core vesicles of about 80-90 nm in diameter. Beside these positive nerve endings other nerve terminals did not show any immunoreactivity to MCH (Fig. 8). DISCUSSION The results of the present study show for the first time that specific neurons of the frog hypothalamus contain a peptide immunologically similar to salmon MCH. The highest concentration of MCH perikarya was observed in the preoptic region where numerous peptidergic neurons are located in amphibians: a-MSH [5,10]; corticotropin-releasing factor (CRF) [26,32]; thyrotropin-releasing hormone (TRH) [29]; neuropeptide Y [9]; gonadotropin-releasing hormone (LH-RH) [8,24]; and somatostatin (SRIF) [14]. A second group of cell bodies was detected in the dorsal infundibular nucleus which also contains a variety of neurosecretory elements such as: NPY [9], mesotocin and vasotocin [32,34], TRH [29] and SRIF [39]. The distribtuion of MCH has recently been described in the brain of the rat [30, 40, 41] and teleost fishes [21]. In all species studied, MCH-containing neurons are exclusively located in the diencephalon. In the rat, no immunoreactive perikarya were visualized in the preoptic periventricular nucleus (homologous to the frog NPO) which only contained moderate numbers of fibers. In fishes, most MCH-immunostained perikarya were observed in the ventral floor of the basal hypothalamus [21] a region named pars lateralis of the nucleus lateralis tuberalis, which is homologous to the amphibian ventral infundibular nucleus. MCH bioactivity was also detected in the nucleus lateralis tuberis and the dorsal hypothalamus of fish [3]. Thus, although MCH neurons are consistently restricted to the hypothalamic area, the distribution of MCH-containing cell bodies shows marked differences among vertebrate species. In contrast, a similar distribution of nerve processes was found in the ventral hypothalamic area and median eminence in fishes [21] and amphibians. In addition, both salmons and frogs possess a remarkably dense network of MCH nerve terminals in the neurohypophysis while, in the rat, the posterior pituitary contains few MCH-like fibers [30]. In teleost fishes, Naito et al. [21] have shown that MCH projections extend into the intermediate lobe and distal lobe of the pituitary. In contrast, the intermediate and distal lobes of the frog and rat pituitary are devoid of MCH-like innervation. Taken together, these studies indicate that the distribution of MCH-containing neurons and fibers are distinct in fishes, amphibians and mammals. Most peptidergic hypophysiotropic neuronal systems are found not only in the hypothalamus but also in various extrahypothalamic brain region: CRF [32]; NPY [9]; SRIF [14]; TRH [29]; L H R H [1,24]. In the frog, the MCH-containing neurons represent an unique neurosecretory system which appears to be specifically located in the hypothalamo-neurohypophyseal complex. Using the highly sensitive immunogold technique, we have been able to study the distribution of the MCH-like peptide in the frog neurohypophysis. The immunoreaction was concentrated in specific nerve endings while other terminals were devoid of MCH-like immunoreactivity. The gold particles appeared mainly located in dense core vesicles suggesting that MCH is an authentic neurosecretory product
950
ANDERSEN ET AL.
released by n e r v e terminals of the frog neurohypophysis. Besides the n e u r o h y p o p h y s e a l nona-peptides m e s o t o c i n and vasotocin, several o t h e r neuropeptides h a v e been visualized in the neural lobe of the amphibian pituitary: T R H [7,29], C R F [32] and atrium natriuretic factor ( A N F ) [23]. W h e t h e r these regulatory peptides co-exist within the same nerve terminals or whether they are located in distinct fibers remains to be examined. S o m e of these neuropeptides---such as T R H [29] or A N F (unpublished observations)---are clearly involved in the control of the secretion of intermediate lobe hormones. Bearing in mind that M C H antagonizes a - M S H
action on fish melanophores [4] a possible i n v o l v e m e n t of M C H in the mediation of color change via the control of M S H secretion by the frog pars intermedia d e s e r v e s further investigations.
ACKNOWLEDGEMENTS This research was supported in part by research grants from INSERM (84-6020 and 86--4016), CNRS and France-Qu6bec exchange program. The authors gratefully acknowledge Mrs. R. Bensaadoune for typing the manuscript.
REFERENCES 1. Alpert, L. C., J. R. Brawer, I. M. D. Jackson and S. Reichlin. Localization of LHRH in neurons in frog brain Rana pipiens and Rana eatesbeiana). Endocrinology 98: 910-921, 1976. 2. Bagnara, J. T. and M. E. Hadley. Chromatophores and Color Change. The Comparative Physiology o f Animal Pigmentation. Englewood Cliffs, NJ: Prentice-Hall, 1973. 3. Baker, B. I. and T. A. Rance. Further observations on the distribution and properties of teleost melanin concentrating hormone. Gen Comp Endocrinol 50: 423-431, 1983. 4. Baker, B. I., A. N. Eberle, J. B. Bauman, W. Siegrist and J. Girard. Effect of malanin concentrating hormone on pigment and adrenal cells in vitro. Peptides 6:1125-1130, 1985. 5. Benyamina, M., C. Delbende, S. J6gou, P. Leroux, F. Leboulenger, M. C. Tonon, J. Guy, G. Pelletier and H. Vaudry. Localization and identification of a-melanocyte-stimulating hormone (a-MSH) in the frog brain. Brain Res 366: 230-237, 1986. 6. Bloch, B., C. Bugnon, D. Fellmann, D. Lenys and A. Gouget. Neurons of the rat hypothalamus reactive with antisera against endorphins, ACTH, MSH and fl-LPH. Cell Tissue Res 204: 1-15, 1979. 7. Bolaffi, J. L. Immunohistochemical localization of thyrotropinreleasing hormone in amphibian brain. 65th Annu Meet Endocrinol Soc Abstr No. 274, p. 149, 1983. 8. Crim, J. W. Immunocytochemistry of luteinizing hormone releasing hormone and sexual maturation of the frog brain: comparisons of juvenile and adult bullfrogs (Rana catesbeiana). Gen Comp Endocrinol 59: 424-433, 1985. 9. Danger, J. M., J. Guy, M. Benyamina, S. Jrgou, F. Leboulenger, J. C6tr, M. C. Tonon, G. Pelletier and H. Vaudry. Localization and identification of neuropeptide Y (NPY)-like immunoreactivity in the frog brain. Peptides 6: 1225-1236, 1985. 10. Delbende, C., S. J6gou, G. Pelletier, D. Tranchand-Bunel, F. Leboulenger, J. Guy, M. Benyamina and H. Vaudry. L'alphamelanotropine dans l'encephale de grenouille: rrgionalisation, quantification et distribution subcellulaire. C R Acad Sci [D] (Paris) 302: 359-363, 1986. I 1. D6sy, L. and G. Pelletier. Immunohistochemical localization of a-melanocyte hormone (a-MSH) in the human hypothalamus. Brain Res 154: 377-381, 1978. 12. Eberle, A. N. Peptide synthesis. Part 9. Solid-phase synthesis of melanin-concentrating hormone using a continuous-flow polyamide method. J Chem Soc Perkin Trans h 361-367, 1986. 13. Guy, J., R. Leclerc, H. Vaudry and G. Pelletier. Identification of a second category of c~-melanocyte-stimulating hormone (a-MSH) neurons in the rat hypothalamus. Brain Res 199: 135146, 1980. 14. Inagaki, S., S. Shiosaka, K. Takatsuki, M. Sakanaka, H. Takagi, E. Senba, T. Matsuzaki and M. Tohyama. Distribution of somatostatin in the frog brain, Rana catesbeiana, in relation to location of catecholamine containing neuron system. J Comp Neurol 202: 89-101, 1981. 15. Jacobowitz, D. M. and T. L. O'Donohue. a-Melanocytestimulating hormone: immunohistochemical identification and mapping in neurons of the rat brain. Proc Natl Acad Sci USA 75: 6300-6304, 1978.
16. J6gou, S., M. C. Tonon, P. Leroux, F. Leboulenger, C. Delbende, P. Netchitailo, G. Pelletier, A. Dupont and H. Vaudry. Effect of hypophysectomy and pituitary stalk transection on a-melanocyte-stimulating hormone-like immunoreactivity in the brain of frog Rana ridibunda Pallas. Brain Res 220: 287-298, 1981. 17. J6gou, S., M. C. Tonon, J. Guy, H. Vaudry and G. Pelletier. Biological and immunological characterization of a-melanocytestimulating hormone (a-MSH) in two neuronal systems of the rat brain. Brain Res 260: 91-98, 1983. 18. Kawauchi, H., I. Kawazoe, M. Tsubokawa, M. Kishida and B. I. Baker. Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 305: 321-333, 1983. 19. Khachaturian, H., R. M. Dores, S. J. Watson and H. Akil. /3-Endorphin/ACTH immunocytochemistry in the CNS of the lizard Anolis carolinensis: evidence for a major mesencephalic cell group. J Comp Neurol 229: 576--684, 1984. 20. Leboulenger, F., P. Leroux, C. Delarue, M. C. Tonon, Y. Charnay, P. M. Dubois, D. H. Coy and H. Vaudry. Colocalization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP. Life Sci 32: 375-383, 1983. 21. Naito, N., Y. Nakai, H. Kawauchi and Y. Hayashi. lmmunocytochemical identification of melanin-concentrating hormone in the brain and pituitary gland of the teleost fishes Oncorhyncus keta and Salmo gairdneri. Cell Tissue Res 242: 41-48, 1985. 22. Netchitailo, P., M. Feuilloley, G. Pelletier, F. Leboulenger, M. Cantin, J. Gutkowska and H. Vaudry. Atrial natriuretic factorlike immunoreactivity in the central nervous system of the frog. Neuroscience, in press, 1986. 23. Netchitailo, P., M. Feuilloley, G. Pelletier, M. Cantin, F. Leboulenger, A. C. Andersen and H. Vaudry. Localization of atrial natriuretic factor (ANF)-immunoreactive material in the hypothalamo-pituitary complex of the frog. Neurosci Lett. in press, 1986. 24. Nozaki M. and H. Kobayashi. Distribution of LH-RH-like substance in the vertebrate brain as revealed by immunohistochemistry. Arch Histol Jpn 42: 201-219, 1979. 25. O'Donohue, T. L., R. L. Miller and D. M. Jacobowitz. Identification, characterization of intraneural c~-melanocyte-stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain. Brain Res 176: 101-124, 1979. 26. Olivereau, M., F. Vandesance, E. Boucique, F. Ollevier and J. M. Olivereau. Mise en 6vidence immunocytochimique d'un systrme peptidergique de type CRF (corticotropin-releasing factor) dans le cerveau des amphibiens. Comparaison avec la rrpartition du systrme h somatostatine. C R Acad Sci [D] (Paris) III: 871-876, 1984. 27. Pelletier, G. and D. Dub6. Electron microscopic immunohistochemical localization of a-MSH in the rat brain. Am J Anat 150: 201-205, 1977.
MCH IN THE FROG BRAIN AND PITUITARY 28. Roth, J., M. Ravazolla, M. Bendayan and L. Orci. Application of the protein A-gold technique for electron microscopic demonstration of polypeptide hormones. Endocrinology 108: 247-253, 1981. 29. Seki, T., Y. Nakai, S. Shioda, T. Mitsuma and S. Kikuyama. Distribution of immunoreactive thyrotropin-releasing hormone in the forebrain and hypophysis of the bullfrog, Rana catesbeiana. Cell Tissue Res 233: 507-516, 1983. 30. Skofitsch, G., D. M. Jacobowitz and N. Zamir. Immunohistochemical localization of a melanin concentrating hormone-like peptide in the rat brain. Brain Res Bull 15: 635-649, 1985. 31. Tonon, M. C., P. Leroux, M. E. Stoeckel, S. Jtgou, G. PeUetier and H. Vaudry. Catecholamine control of amelanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro: Evidence for direct stimulation of a-MSH release by thyrotropin-releasing hormone. Endocrinology 112: 133-141, 1983. 32. Tonon, M. C., A. Burlet, M. Lanber, P. Cuet, S. Jtgou, L. Gouteux, N. Ling and H. Vaudry. Immunohistochemical localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda. Neuroendocrinology 40:109-119, 1985. 33. Vallarino, M. Immunocytochemical localization of a-melanocytestimulating hormone in the brain of the lizard Lacerta muralis. Cell Tissue Res 237: 521-524, 1984. 34. Vandesande, F. and K. Dierick. Immunocytochemical demonstration of separate vasotocinergic and mesotocinergic neurons in the amphibian hypothalamic magnocellular neurosecretory system. Cell Tissure Res 175: 289-296, 1976.
951 35. Vaudry, H., M. C. Tonon, C. Delarue, R. Vaillant and J. Kraicer. Biological and radioimmunological evidence for melanocyte-stimulating hormones (a-MSH) of extra-pituitary origin in the rat brain. Neuroendocrinology 27: 9-24, 1978. 36. Vaudry, H., C. Oliver, S. Jtgou, F. Leboulenger, M. C. Tonon, C. Delarue, J. P. Morin and R. Vaillant. Immunoreactive a-melanocyte-stimulating hormone (a-MSH) in the brain of the frog Rana esculenta L. Gen Comp Endocrinol 36: 327-338, 1978. 37. Wada, M., A. Urano and A. Gorbman. A stereotaxic atlas for diencephalic nuclei of the frog, Rana pipiens. Arch Histol Jpn 43: 157-173, 1980. 38. Watson, S. J. and H. Akil. The presence of two a-MSH positive cell groups in the rat hypothalamus. Eur J Pharmacol 48: 101103, 1979. 39. Yui, R. Immunohistochemical studies on peptide neurons in the hypothalamus of the bullfrog Rana catesbeiana. Gen Comp Endocrinol 49: 195--209, 1983. 40. Zamir, N., G. Skofitsch, M. J. Bannon and D. M. Jacobowitz. Melanin-concentrating hormone: Unique peptide neuronal system in the rat brain and pituitary gland. Proc Natl Acad Sci USA 83: 1528-1531, 1986. 41. Zamir, N., G. Skotfitsch and D. M. Jacobowitz. Distribution of immunoreactive melanin-concentrating hormone in the central nervous system of the rat. Brain Res 373: 240-245, 1986.
0196-9781/86$3.00 + .00
Localization of Melanin-Concentrating Hormone-Like Immunoreactivity in the Brain and Pituitary of the Frog Rana ridibunda A N N C. A N D E R S E N , * G E O R G E S P E L L E T I E R , t A L E X N . E B E R L E , ~ PHILIPPE LEROUX,* SYLVIE JEGOU* AND HUBERT VAUDRY*
*Groupe de Recherche en Endocrinologie Mol~culaire, UA CNRS 650, Unitd Alli@e ~ I'INSERM Facult# des Sciences, Universit# de Rouen, 76130 Mont Saint Aignan, France t M R C Group in Molecular Endocrinology, Le Centre Hospitalier de l' Universit# Laval Qu#bec, G1V 4G2 Canada SKantonsspital Basel, Universitiitskliniken, Department Forschung Hebelstrasse 20 CH 4031 Basel, Switzerland R e c e i v e d 11 S e p t e m b e r 1986
ANDERSEN, A. C., G. PELLETIER, A. N. EBERLE, P. LEROUX, S. JEGOU AND H. VAUDRY. Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda. PEPTIDES 7(6) 941-951, 1986.--The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog
Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-iike containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area. Brightly immunofluorescent nerve bundles were found in the preoptic nucleus and in the ventral infundibular nucleus, coursing towards the internal zone of the median eminence and the pituitary stalk. An intense network of immunofluorescent fibers was localized in the neural lobe of the pituitary. The subcellular localization of MCH-like material was studied in the neurohypophysis using the immunogold technique. It was demonstrated that MCHlike material was contained in dense core vesicles (80-90 mm in diameter) within specific nerve terminals. The present findings indicate that, in amphibians, MCH-like peptide is located in specific hypothalamic neurons. Our data suggest that MCH may be released by neurohypophyseal nerve endings as a typical neurohormone. Melanin-concentrating hormone Neurosecretion Frog
Immunohistochemistry
THE intermediate lobe of the pituitary secretes a major peptide called alpha melanotropin (a-MSH) which plays an important role in skin color adaptation in cold blooded vertebrates [2]. ct-MSH is not only produced by endocrine cells of the intermediate lobe, but is also present in discrete neuronal systems of the brain of amphibians [5, 10, 16, 31, 36], reptiles [19,33] and mammals [6, 11, 13, 15, 17, 25, 27, 35, 38], supporting the view that most hormonal peptides are also potential neurotransmitters. Recently, a regulatory peptide called melanin-concentrating hormone (MCH) has been isolated from the fish pituitary and characterized [18]. Baker and Rance [3] have studied the distribution of MCH in the pituitary and brain of various species and have found that MCH-like bioactivity was detectable in the hypothalamus of fish, amphibians and mammals. Polyclonal antibodies have been raised against synthetic MCH and applied to the immunohistochemical identification of MCH in the brain and
Electron microscopy
Brain
Pituitary
pituitary gland of salmons [21] and rats [30,40]. In the present study we have examined the distribution of MCH in the brain and pituitary of the frog Rana ridibunda using the indirect immunofluorescence technique. METHOD
Tissue Preparation Adult male frogs (Rana ridibunda) of 30-40 g body weight, obtained from Cou~tard (St. Hilaire de Riez, France) were kept unfed in tanks continuously supplied with a trickle of water. The animals were submitted to a 12 hr-dark/light cycle, at a constant temperature (8-+0.5°C). The frogs were stunned and transcardially perfused with 30 ml of 0.1 M phosphate buffer (PBS, pH 7.4) containing 0.025% xylocaine. The perfusion was carded on with 50 ml of McLean's fixative as previously described [20]. The
941
942
ANDERSEN
TABLE
1
TABLE
LOCALIZATION AND RELATIVE ABUNDANCE OF N E U R O N A L PERIKARYA AND FIBERS EXHIBITING MCH-LIKE IMMUNOREACTIVITY IN THE FROG BRAIN Structure Telencephalon P a l l i u m d o r s a l i s (PD) P a l l i u m m e d i a l e (PM) Pallium laterale, pars dorsalis (PLd) Pallium iaterale, pars ventralis (PLv) Nucleus medialis septi (NMS) B e d n u c l e u s o f the pallial c o m m i s s u r e (NBPC) A m y g d a l a , p a r s l a t e r a l i s (A1) Amygdala, pars medialis (Am) Nucleus entopeduncularis (NEP) Pallial c o m m i s s u r e (PaC) A n t e r i o r c o m m i s s u r e (Ac) Diencephaion E p i p h y s i s (E) Habenular commissure (HC) Nucleus habenularis dorsalis (NHD) Nucleus habenularis ventralis (NHV) Area ventralis anterior thalami (AVA) Area ventrolateralis thalami (AVL) Area ventromedialis thalami (AVM) Nucleus dorsomedialis anterior thalami (NDMA) Nucleus dorsolateralis anterior thalami (NDLA) Corpus geniculatus laterale (CGL) Lateral forebrain bundle (LFB) N u c l e u s r o t u n d u s (NR) Posterocentralis thalami (NPC) Nucleus posterolateralis thalami (NPL) Nucleus preopticus, pars anterior (NPOa) Nucleus preopticus, pars posterior (NPOp) Nucleus infundibularis dorsalis (NID) Nucleus infundibularis ventralis (NIV)
Perikarya
ET AL.
1
(CONTINUED) Structure
Perikarya
Fibers
-
+ + + + -
-
-
-
-
-
-
-
-
-
-
-
-
Fibers
-
-
-
-
+ -
-
-
-
+ +
+ +
+ + +
++
+ + -
+ +
A r e a i n f u n d i b u l a r i s (AI) Median eminence (ME) P o s t e r i o r c o m m i s s u r e (PC) O p t i c c h i a s m a (OC) O p t i c t r a c t (OT) Optic nerve (ON) Mesencephalon Nucleus mesencephalicus nervitrigemini (NMNT) Stratum griseum superficiale tecti (SGS) Stratum griseum centrale tecti (SGC) S t r a t u m g r i s e u m p e r i v e n t r i c u l a r e tecti (SGP) N u c l e u s o f the film ( N F ) Nucleus profundus mesencephali (NPM) Nucleus anterodorsalis tegmenti mesencephali (NAD) Nucleus anteroventralis tegmenti mesencephali (NAV) N u c l e u s o f the o c u l o m o t o r n e r v e (NOM) T o r u s s e m i c i r c u l a r i s (TS) Nucleus reticularis isthmi (NRIS) N u c l e u s o f the t r o c h l e a r n e r v e ( N T R O ) Nucleus cerebelli (NCER) Oculomotor nerve (OMN)
-
-
-
-
Hypophysis P a r s n e r v o s a (PN) P a r s i n t e r m e d i a (PI) P a r s d i s t a l i s (PD)
-
+ + + -
+ , f e w ; + + , m e d i u m ; + + + , m a n y ; - , no i m m u n o r e a c t i v e cell b o d i e s or fibers. A b b r e v i a t i o n s a c c o r d i n g to W a d a et al. [37].
FACING PAGE F I G . 1. F r o n t a l s e c t i o n s o f t h e frog b r a i n at the l e v e l o f t h e p r e o p t i c n u c l e u s ( N P O ) ( c o r r e s p o n d i n g to c o o r d i n a t e s + 2 . 6 o n Fig. 7A). T h i s photomicrograph shows MCH-like immunoreactive perikarya and fibers (arrows) forming a triangular-shaped network. The arrows indicate t h e v e n t r o l a t e r a l p r o j e c t i o n s o f t h e f i b e r s a b o v e the o p t i c n e r v e (ON). P R = P r e o p t i c R e c e s s , ×368. F I G . 2. H i g h m a g n i f i c a t i o n o f a f r o n t a l s e c t i o n o f t h e p r e o p t i c n u c l e u s ( N P O ) ( c o o r d i n a t e s + 2 . 6 o n Fig. 7A) s h o w i n g M C H i m m u n o r e a c t i v e p e r i k a r y a a n d fibers. I n t e n s e i m m u n o f l u o r e s e n c e l a b e l l i n g is o b s e r v e d in the c y t o p l a s m w h e r e a s the n u c l e u s d o e s n o t s h o w i m m u n o r e a c t i v e m a t e r i a l . T h e cell p r o c e s s e s a n d f i b e r s f o r m a d e n s e b u n d l e ( a r r o w s o n t h e r i g h t side o f the figure) p r o j e c t i n g v e n t r o l a t e r a l l y , x 5 8 0 . F I G . 3. F r o n t a l s e c t i o n o f the b r a i n ( c o o r d i n a t e s + 1.4 o n Fig. 7B) s h o w i n g the M C H - l i k e c o n t a i n i n g p e r i k a r y a g a t h e r e d in the d o r s a l i n f u n d i b u l a r n u c l e u s ( N I D ) c l o s e to the t h i r d v e n t r i c l e (III). T h e a r r o w s i n d i c a t e fine i m m u n o r e a c t i v e f i b e r s c o u r s i n g in a l a t e r a l d i r e c t i o n . x 193. F I G . 4. F r o n t a l s e c t i o n o f t h e frog b r a i n in the c a u d a l r e g i o n o f the p r e o p t i c n u c e l u s ( N P O ) ( c o o r d i n a t e s × 2 . 4 Fig. 7A). I m m u n o r e a c t i v e p e r i k a r y a s h o w a l i n e a r d i s t r i b u t i o n o n the s u r f a c e o f the e p i t h e l i a l b o r d e r i n g c e l l s o f the t h i r d v e n t r i c l e (III). B e a d e d h o r i z o n t a l l y - o r i e n t e d i m m u n o f l u o r e s c e n t f i b e r s are s h o w n b y a r r o w s , x 589. F I G . 5. P a r a s a g i t t a l s e c t i o n ( c o r r e s p o n d i n g to t h e p a r a s a g i t t a l d r a w i n g p r e s e n t e d in Fig. 6) o f the i n f u n d i b u l a r a r e a (AI) a n d p i t u i t a r y . D o r s a l side to t h e right, a n t e r i o r s i d e to the t o p o f the figure. I m m u n o r e a c t i v e p e r i k a r y a ( t h i c k a r r o w s ) are g a t h e r e d in t h e d o r s a l i n f u n d i b u l a r n u c l e u s ( N I D ) . I m m u n o r e a c t i v e f i b e r s (thin a r r o w s ) are p r e s e n t t h r o u g h o u t t h e i n f u n d i b u l a r a r e a (AI): d o r s a l ( N I D ) a n d v e n t r a l ( N I V ) i n f u n d i b u l a r nuclei. I m m u n o r e a c t i v e f i b e r s o r i g i n a t i n g f r o m the p r e o p t i c n u c l e u s c o u r s e v e n t r a l l y in t h e N I V t o w a r d s t h e p i t u i t a r y s t a l k a n d the i n t e r n a l z o n e o f the m e d i a n e m i n e n c e ( M E ) . I n t e n s e i m m u n o f l u o r e s c e n c e l a b e l l i n g is r e v e a l e d in t h e p a r s n e r v o s a (PN), w h i l e the p a r s i n t e r m e d i a (PI) a n d the p a r s d i s t a l i s (PD) are d e v o i d o f i m m u n o r e s p o n s e . × 184.
MCH IN THE FROG BRAIN AND PITUITARY
943
I
N~[)
't
Ill
.....
NID
t
3
5
-tO
~
I
• &•
ON
I
.1.O
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N~ . .'::::~-~*.:. ~,. ~-::: :.........-.. ~
*4.0
~
k
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FIG. 6. Schematic parasagittal section of the frog brain depicting the distribution of MCH-containing perikarya and neuronal processes. See list of abbreviations in Table 1. The dorsal reference zero point of the scale was defined by Wada et al. [37] as the apex of the angle formed by the junction of the dorsal midline meeting of the two optic lobes.
-2.0
.su.ou~L
PERIKARYA:
KEY:
-
r-,
Z
~
M C H IN T H E F R O G BR AIN A N D P I T U I T A R Y
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A
"*'
"_
//
",-_r-'/
rn;
E!
El
\~.t<,..o ..L " / : , '...", / X j ~ .../ ¥_ ,, , k-.;/
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--
/,.-~o,,~ ~ O _ ' ~ " - ~ - ' ' J
~ NR ~" AVN \
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.
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FIG. 7. A, B and C. Shematic drawings of serial frontal sections of the frog brain. Only the parts of the brain exhibiting a positive immunoreaction with the MCH antiserum are shown. The series start 3.2 mm anterior to the zero reference point of Wada et al. [37] and end 0.2 mm posterior to this point; the tracings are from sections 0.2 mm apart. The MCH-like containing perikarya are represented by triangles and the fibers by dots; the density of these symbols is proportional to the density of immunoreactive elements. See list of abbreviations in Table 1. FIG. 7 B AND C FOLLOWING PAGES.
P
~m
CO
Z
Z
MCH IN THE FROG BRAIN A N D PITUITARY
B
Ea FIG. 7 C
947
N
A N D E R S E N ET AL.
948
FIG. 8. Immunoelectron microscopic localization of MCH using the immunogold technique. Several positive nerve terminals are decorated by gold particles located in dense core vesicles (arrows). Other nerve terminals appear non-immunoreactive (ni). ×47,000. brains were dissected and postfixed in the same fixative solution for 3 hr. The tissues were rinsed overnight in 0.1 M PBS containing 15% sucrose and transferred into 0.1 M PBS containing 30% sucrose for at least 24 hr. Then the brains were frozen in liquid nitrogen, embedded in O.C.T. Tissue Teck and sliced at 8 /zm in frontal or sagittal planes on a cryostat (Frigocut 2700, Reichert Jung).
Preparation of Antisera Synthetic MCH [12] was coupled to thyroglobulin using glutaraldehyde. The conjugate was dialyzed extensively and diluted with saline to a concentration of 1 mg/ml (with respect to conjugate concentration) and stored at -70°C. The complex was mixed with complete Freund's adjuvant (1:1) and injected intradermally into rabbits at multiple sites (100 or 200 /zg per animal). Booster injections were done every 4-5 weeks using 20/zg of complex per injection and incomplete Freund's adjuvant. The titer as measured by RIA ranged from 1:30,000 to 1:180,000 after the first booster injection. The antigenic determinant read by the antibodies was not determined. There was however full cross-reaction between iodinated MCH and tritiated (Nva3.6]-MCH which
means that the two methionine residues did not play a crucial role in the recognition.
Immunofluorescence of MCH The brain sections were incubated with normal goat serum (diluted 1:25) for one hour at room temperature. PBS supplemented with 1% human serum albumin, and 0.03% triton was used to dilute all the antisera. The sections were rinsed in two 5-rain baths of PBS and incubated overnight in a moistened atmosphere at 4°C with MCH-antiserum (No. 526) at the working dilution of 1:200. Thereafter the sections were thoroughly rinsed in several baths of PBS and incubated in a dark moistened chamber at 20°C for one hour with fluorescein-isothiocyanate conjugated antirabbit y-globulin (GAR/FITC, Nordic Immunology) diluted 1:60. Finally the sections were rinsed again in PBS, counterstained for 3 rain with Evan's blue (diluted 1:10,000 in distilled water) and mounted with PBS-glycerol (1:1). The mapping and the photomicrographs of the MCHimmunoreactive structures were performed with a Leitz Orthoplan microscope fitted with a Vario-Orthomat photographic system. The controls were performed on sections
MCH IN THE FROG BRAIN AND PITUITARY incubated either with non-immunized rabbit serum or with anti-MCH preabsorbed overnight, at room temperature, with 10-6 M synthetic MCH (Peninsula Laboratories). Electron Microscopy The frogs used for electron microscopy were transcardially perfused first with 30 ml of 0.1 M PBS then with 50 ml of 4% paraformaldehyde in PBS without any osmic postfixation. The neurointermediate lobes of the pituitary were dissected, postfixed for 3 hr in the same solution, rinsed overnight in PBS containing 5% sucrose and embedded in araldite. Ultrathin sections of the pars nervosa were processed for the immunogold technique as described by Roth et al. [28]. The MCH antiserum was used at a final dilution 1:4,000. RESULTS Localization of MCH-Containing Elements by Immunofluorescence The results of this study are presented in the form of a series of line drawings and photomicrographs. The line drawings are shematic drawings of the frog diencephalon and mesencephalon, in the parasagittal and frontal planes, depicting the distribution of MCH-like immunoreactivity in cells and fibers. Cell bodies positive for MCH are shown as filled triangles in the drawings; fine fibers and terminals containing MCH-like material are shown as dots. Nomenclature for this study was based on the work of Wada et al. [37] and Netchitailo et al. [22]. A list of identified structures and the relative frequencies of immunoreactive perikarya and fibers is presented in Table 1. Cell bodies containing MCH-like immunoreactivity were located exclusively in the diencephalon. Perikarya were found primarily in the preoptic nucleus (NPO), mainly in the mid-part of the NPO (Figs. l, 4 and 6). On frontal brain sections, the perikarya appeared to be gathered within a triangular framework. Other immunoreactive perikarya were observed in a more caudal area of the NPO. These latter neurons showed a linear distribution on the basal surface of the epithelial bordering cells of the third ventricle (Figs. 4 and 7A). A second group of MCH-positive perikarya, although less abundant than in the NPO, was observed in the dorsal infundibular nucleus (NID), close to the third ventricle (Figs. 3, 6 and 7B). In contrast, no cell body was localized in the ventral infundibular nucleus (NIV). At the level of the ventral thalamic area (AVA) scattered neurons were visualized (Figs. 6 and 7A). Immunoreactive neurites originating from the NPO formed a dense bundle which projected ventro-laterally and coursed caudally just above the optic nerve (Figs. 1 and 2). Discrete fibers were occasionally seen in the zone between the optic chiasma and the infundibular area, suggesting that preoptic MCH-positive perikarya may project caudally towards the median eminence, as schematically shown on Fig. 6. A system of coarse, thick MCH-positive fibers observed in the NIV also coursed ventrally towards the pituitary stalk and the internal zone of the median eminence (ME) (Fig. 5). A high density of immunoreactive nerve terminals was found in the neural lobe of the pituitary. No immunoreactivity was found in the intermediate and distal lobes. Preincubation of the MCH antiserum with l0 -6 M of the corresponding peptide resulted in complete loss of staining. Replacement of the MCH antiserum by non immunized rabbit serum also resulted in loss of staining.
949 Electron Microscopy Staining of ultrathin sections with the MCH antiserum, using the immunogold technique made it possible to visualize the immunoreactive material in specific nerve terminals of the frog neurohypophysis (Fig. 8). The gold particles appeared concentrated in dense core vesicles of about 80-90 nm in diameter. Beside these positive nerve endings other nerve terminals did not show any immunoreactivity to MCH (Fig. 8). DISCUSSION The results of the present study show for the first time that specific neurons of the frog hypothalamus contain a peptide immunologically similar to salmon MCH. The highest concentration of MCH perikarya was observed in the preoptic region where numerous peptidergic neurons are located in amphibians: a-MSH [5,10]; corticotropin-releasing factor (CRF) [26,32]; thyrotropin-releasing hormone (TRH) [29]; neuropeptide Y [9]; gonadotropin-releasing hormone (LH-RH) [8,24]; and somatostatin (SRIF) [14]. A second group of cell bodies was detected in the dorsal infundibular nucleus which also contains a variety of neurosecretory elements such as: NPY [9], mesotocin and vasotocin [32,34], TRH [29] and SRIF [39]. The distribtuion of MCH has recently been described in the brain of the rat [30, 40, 41] and teleost fishes [21]. In all species studied, MCH-containing neurons are exclusively located in the diencephalon. In the rat, no immunoreactive perikarya were visualized in the preoptic periventricular nucleus (homologous to the frog NPO) which only contained moderate numbers of fibers. In fishes, most MCH-immunostained perikarya were observed in the ventral floor of the basal hypothalamus [21] a region named pars lateralis of the nucleus lateralis tuberalis, which is homologous to the amphibian ventral infundibular nucleus. MCH bioactivity was also detected in the nucleus lateralis tuberis and the dorsal hypothalamus of fish [3]. Thus, although MCH neurons are consistently restricted to the hypothalamic area, the distribution of MCH-containing cell bodies shows marked differences among vertebrate species. In contrast, a similar distribution of nerve processes was found in the ventral hypothalamic area and median eminence in fishes [21] and amphibians. In addition, both salmons and frogs possess a remarkably dense network of MCH nerve terminals in the neurohypophysis while, in the rat, the posterior pituitary contains few MCH-like fibers [30]. In teleost fishes, Naito et al. [21] have shown that MCH projections extend into the intermediate lobe and distal lobe of the pituitary. In contrast, the intermediate and distal lobes of the frog and rat pituitary are devoid of MCH-like innervation. Taken together, these studies indicate that the distribution of MCH-containing neurons and fibers are distinct in fishes, amphibians and mammals. Most peptidergic hypophysiotropic neuronal systems are found not only in the hypothalamus but also in various extrahypothalamic brain region: CRF [32]; NPY [9]; SRIF [14]; TRH [29]; L H R H [1,24]. In the frog, the MCH-containing neurons represent an unique neurosecretory system which appears to be specifically located in the hypothalamo-neurohypophyseal complex. Using the highly sensitive immunogold technique, we have been able to study the distribution of the MCH-like peptide in the frog neurohypophysis. The immunoreaction was concentrated in specific nerve endings while other terminals were devoid of MCH-like immunoreactivity. The gold particles appeared mainly located in dense core vesicles suggesting that MCH is an authentic neurosecretory product
950
ANDERSEN ET AL.
released by n e r v e terminals of the frog neurohypophysis. Besides the n e u r o h y p o p h y s e a l nona-peptides m e s o t o c i n and vasotocin, several o t h e r neuropeptides h a v e been visualized in the neural lobe of the amphibian pituitary: T R H [7,29], C R F [32] and atrium natriuretic factor ( A N F ) [23]. W h e t h e r these regulatory peptides co-exist within the same nerve terminals or whether they are located in distinct fibers remains to be examined. S o m e of these neuropeptides---such as T R H [29] or A N F (unpublished observations)---are clearly involved in the control of the secretion of intermediate lobe hormones. Bearing in mind that M C H antagonizes a - M S H
action on fish melanophores [4] a possible i n v o l v e m e n t of M C H in the mediation of color change via the control of M S H secretion by the frog pars intermedia d e s e r v e s further investigations.
ACKNOWLEDGEMENTS This research was supported in part by research grants from INSERM (84-6020 and 86--4016), CNRS and France-Qu6bec exchange program. The authors gratefully acknowledge Mrs. R. Bensaadoune for typing the manuscript.
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