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Distribution of melanin-concentrating hormone (MCH)-like immunoreactivity in neurons of the diencephalon of sheep
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Journal of Chemical Neuroanatomy 12 (1996) 135-145
Distribution of melanin-concentrating hormone (MCH)-like immunoreactivity in neurons of the diencephalon of sheep Y v e s T i l l e t a,*, M a r t i n e
B a t a i l l e r a, D o m i n i q u e
Fellmann b
aUnit~ de Neuroendocrinologie Sexuelle, INRA Station de Physiologie de la Reproduction des Mammifbres Domestiques, 37380 Nouzilly, France bLaboratoire d'Histologie, Embryologie, Cytogbn&ique, Facultk de MOdecine, 25030 Besan¢on, France
Received 21 September 1995; revised 3 May 1996; accepted 8 October 1996
Abstract
An immunohistochemical study with an antiserum raised against salmon melanin concentrating-hormone has demonstrated the presence of numerous melanin concentrating-hormone-immunoreactive neurons in the lateral hypothalamic areas of the sheep. The pattern of distribution of these perikarya is similar to that of rodents and primates. In sheep, however, melanin concentrating-hormone-immunoreactive neurons appeared to form two gatherings: the first is situated ventromedially to the internal capsule and the second in the dorsolateral hypothalamus. In these areas, numerous immunostained perikarya are observed. Compared to the rats, labelled neurons extended more caudally in the ventral tegmental area and more rostrally above the optic chiasma. Compared to primates, these neurons are less numerous in the periventricular area. In our study, dense networks of melanin concentrating-hormone-immunoreactive varicose fibers were observed in the supramamillary nucleus, the lateral hypothalamus, the nucleus medialis thalami and nucleus reuniens and in the bed nucleus of the stria terminalis. Copyright © 1996 Elsevier Science B.V. Keywords: Peptide; Immunohistochemistry; Hypothalamus; Ungulate; Mammal
I. Introduction
The melanin concentrating-hormone (MCH) is a 17 residue peptide which has been isolated from salmon pituitary and hypothalamus (Kawauchi et al., 1983). In fish, it is involved in the regulation of skin pigmentation (Baker and Rance, 1983). Specific antibodies have been raised against this molecule and immunohistochemical studies have demonstrated the presence of M C H in various classes of vertebrates (for review see Baker, 1991). In the rat, antibodies to salmon M C H stained a very abundant population of neurons mainly located in the lateral and dorsal parts of the hypothalamus (Skofitsch et al., 1985; Zamir et al., 1986b). It was then demonstrated (Naito et al., 1986; Fellmann et al.,
* Corresponding author. Tel.: + 33 474 27966; fax: + 33 474 27743.
1987; Pelletier et al., 1987) that these neurons corresponded to those previously described using antisera to c~-MSH (Watson and Akil, 1979) and to human growth hormone-releasing hormone 1-37 (Fellmann et al., 1985). A similar neuron population was observed in human and several other mammalian species (Sekiya et al., 1988; Bresson et al., 1989; Pelletier et al., 1987). After the identification of the rat peptide corresponding to salmon M C H (Vaughan et al., 1988) and the subsequent elucidation of the sequence of the m R N A encoding the precursor of this peptide (Nahon et al., 1989), immunohistochemical studies using antisera to rat M C H confirmed and completed the earlier results (Risold et al., 1992; Bittencourt et al., 1992). A complete mapping of perikarya and fibers has been realised in the rat (Bittencourt et al., 1992). The sheep has been used to study various neuroendocrine regulations involving the lateral hypothalamic area, such as reproduction (Goodman, 1994; Caraty et
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al., 1995) and feeding (Scallet et al., 1985; Della-Fera et al., 1981) and it constitutes an interesting model as an alternative to rodents and primates for studying the putative role of MCH. However, although the distribution of M C H immunoreactive neurons has been studied in various species, it has never been observed in sheep or in ungulates, and only one biochemical characterization has been conducted in pig (Sekiya et al., 1988). The aim of this study was therefore to investigate the distribution of MCH immunoreactive neurons in the diencephalon of the sheep using immunohistochemistry with antibodies raised against salmon MCH.
2. Materials and methods
1989; Risold et al., 1992). It was prefered to a rat MCH antiserum also prepared in the laboratory because of its higher titer and better specificity (Risold et al,, 1992), as verified in a side experiment performed on sheep sections (data not shown). For the present study, the specificity of the labelling was checked by preincubating the immune serum for 3 h with salmon or rat synthetic MCH (1 nmol//21 undiluted antiserum, synthetic MCH was kindly donated by Dr. J. Rivier, Salk Institute, La Jolla, Ca) and by substituting the specific antiserum with normal (nonimmune) rabbit serum. The distribution of MCH containing perikarya and fibers is shown on successive schematic drawings in the frontal plane and on representative photomicrographs.
2.1. Animals
3. Results
Five adult sheep (male and female) from the laboratory flock were used. They were killed by decapitation at the slaughterhouse by a licensed butcher (authorization no. A37801). The head was immediately perfused via both carotid arteries with 4 liters of a 4% paraformaldehyde solution in phosphate buffer pH 7.4, 0.1 M. The diencephalon was dissected and postfixed in the same fixative for 24 h and rinsed for at least 24 h in a 15% sucrose, 0.1% NaN3 solution in the same buffer. Sections (40 p m thick) were cut in the frontal plane throughout the diencephalon. 2.2. Immunohistochemistrv
The immunohistochemical procedure has been previously described (Tillet, 1987). Sections were successively incubated for 20 min with normal sheep serum diluted 1/15, for 5 days with the first antiserum (raised in rabbit) diluted 1/5000, overnight with a sheep serum anti rabbit gamma globulin diluted 1/400, and overnight with rabbit peroxidase antiperoxidase complex (PAP) diluted 1/2000. All incubations were performed in phosphate buffer saline (PBS) 0.1 M, pH 7.4, containing 0.3% Triton X100, 0.1% sodium azide, excepted the PAP complex which was diluted in PBS only. The peroxidase was visualized after incubation in a 0.02% diaminobenzidine hydrochloride, 0.5% nickel ammonium sulfate and 0.003% H20 2 solution in Tris HCI, 0.05 M, pH 7.9 for 5 15 min. 2.3, Antisera
The primary antiserum was raised against synthetic salmon MCH. The specificity of this antiserum have been thoroughly checked in previous studies by liquid and solid phase saturation with several peptides related to M C H or to its precursor and by competition experiments in a radioimmunoassay (Fellmann et al., 1987,
MCH like immunoreactivity was observed in the cytoplasm of neurons distributed in the diencephalon between the anterior and the posterior commissure (Fig. 1). These neurons were bipolar or multipolar, their diameter was comprised between 25 and 50 /~m for the smaller and larger value, respectively, and did not change according to the different studied areas, their nucleus was never stained. The peroxidase staining appeared homogeneous in most of the perikarya but was granular in some (Fig. 3B). No difference was observed between male and female. No immunoreactive neurons were observed on sections incubated with the first antiserum previously adsorbed with the specific antigen (Fig. 2). We therefore considered the labelling specific to MCH; MCH-containing neurons were referred to as MCH-immunoreactive (-IR). 3. I. Neurons
Within the diencephalon, MCH-IR neurons were observed in the lateral part of the hypothalamus. The most caudally labelled neurons were found in the rostral part of the ventral tegmental area (VTA), caudally to the mamillary bodies in the sagittal plane, and only one or two neurons were observed per section (Fig. 1L). In the following sections the number of labelled neurons increased around the medial mamillary nucleus. At this level, they are situated dorsally to this structure, near the sagittal plane, and laterally, in the VTA (Fig. l J, K and Fig. 3). In the caudal part of the third ventricle, M C H - I R neurons were observed between the ventral part of the ventricle and the fornix (Fig. 1H, I and Fig. 4). In the middle hypothalamus, labelled neurons were tbund around the fornix, between the third ventricle and the internal capsule, and under the mamillothalamic tract (Fig. 1E, F and G). At this level, two gather-
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Fig. I. Schematic draw!ngs of frontal sections through the diencephalon of sheep showing the distribution of MCH-IR perikarya (O: 1- 10 perkarya) and fibers (_ ~ ) from rostral to caudal levels. AHDM: area hypothalamica dorsomedialis; AHL: area hypothalamica lateralis; AHP: area hypothalamica posterioris; ATV: area tegmentalis ventralis; BNST: bed nucleus of the stria terminalis; CA: commissura anterioris; Cd: caudate nucleus; Ch capsula interna; CMm: medial mamillary nucleus; CP: commissura posterioris; Ent: nucleus entopeduncularis; Fx: fornix; FM: forarnen of Monro; FMT: fasciculus mamillothalamicus; Hb: habenular nucleus; IAM: nucleus interanteromedialis; IIIV: third ventricle; NM: nucleus medialis, pars ventralis and pars dorsalis (thalamus); NPV: nucleus paraventricularis hypothalami; NRT: nucleus reticularis thalami; NSO: nucleus supraopticus: OC: optic chiasma; OT: optic tract; Ped Cer: cerebral peduncle; PVT: nucleus paraventricularis thalami; Re: nucleus reuniens: SL: septum laterale: VL: ventriculus lateralis: VMN: nucleus ventromedialis hypothalami;
ings of labelled neurons appeared in the hypothalamus. The first is situated ventrally to the internal capsule, between the fornix and the optic tract, and the second is observed more dorsally, above the fornix in the dorsolateral hypothalamus. Severals M C H - I R neurons were observed between these two gatherings. Rostrally, the neurons of the ventral group extended in the lateral part of the hypothalamus, between the optic tract and the internal capsule (Fig. 1B-E). In this area, neurons were mainly bipolar and their long axes were parallel to the dorsal side of the optic tract (Fig. 5). M C H - I R neurons were never observed in the mediobasal hypothalamus, in the ventromedial nucleus nor in the arcuate nucleus but were observed laterally to these nuclei. In the anterior hypothalamus, few scattered M C H IR neurons were found in the lateral part, dorsal to the caudal part of the optic chiasma (Fig. 1A and B). They were not observed in the supraoptic nucleus. At
the level of the anterior commissure, only one or two immunoreactive neurons were observed per section. 3.2. Fibers
M C H - I R fibers were observed in almost all studied areas but their density varied in the different structures. Most of them presented varicosities. Caudally a weak density was observed in the rostral part of the ventral tegmental area and dorsally to the cerebral peduncles were they were oriented mediolaterally. The sagittal plane presented also a low density of vertically oriented fibers on each side of the third ventricle and laterally to the posterior commissure. The density of M C H - I R fibers became important in the caudal hypothalamus above the mamillary bodies (Fig. I I - K and Fig. 6A, a). This area presented one of the highest densities of labelled fibers observed in the sheep diencephalon. Few fibers were
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Y. Tiller et al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
Fig. 2. (A) MCH-IR neurons in the lateral diencephalonof the sheep; (B) Contralateral area immunostained with the anti-MCH preincubatedwith an excess of MCH. Scale bars: 50 pm.
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Fig. 3. MCH-IR neurons in the caudal diencephalonof the sheep observeddorsally (A) and laterally (B) to the mamillary bodies. Note that some neurons presented a granular labelling (arrowhead in B). Scale bars: 25/~m. observed in the mamillary bodies and in the habenula. Laterally, a low density of fibers was observed in an area corresponding to the nucleus reticularis thalami. In the medial part of the diencephalon, a low density of fibers was found between the optic tract and the internal capsule, and dorsally to the third ventricle where they were oriented horizontally and vertically, respectively. In the thalamus, the paraventricular thalamic nucleus and the area related to the nucleus medialis thalami and to the nucleus reuniens presented a noteworthy innervation whereas a lower density of labelled fibers was observed beneath the lateral ventricles. In the rostro-ventral part of the thalamus, dorsally to the third ventricle, a dense network of labelled fibers is observed in the area related to the interanteromedian nucleus (Fig. 1D and 6B, b) whereas a low density was found in the other thalamic nuclei. In the hypothalamus, the lateral and dorsomedial hypothalamic areas presented a dense network of M C H - I R fibers (Fig. 1 D - F ) . Only a few labelled fibers were observed close to the third ventricle and in the hypothalamic ventromedial nucleus. Small bundles of M C H - I R fibers were observed dorsally to the optic tract and to the internal capsule, running
towards the nucleus reticularis thalami. In the median eminence M C H - I R fibers were observed in the internal layer, and almost none in the external layer. In the rostral diencephalon, the bed nucleus of the stria terminalis presented a dense network of immunoreactive fibers (Fig. 1 A - C and Fig. 6C, c) and a bundle of vertically oriented labelled fibers ran along the medial edge of the caudate nucleus, in the stria terminalis. The periventricular area and the medial preoptic area presented a weak innervation and only a few small varicose M C H - I R fibers were observed in the paraventricular hypothalamic nucleus and supraoptic nucleus. Above the optic tract, a bundle of horizontal fibers ran toward more lateral areas such as the ,putamen and the ventral surface of the telencephalon, between the optic chiasma and the olfactory tract. The medial side of the internal capsule and the lateral preoptic area presented a moderate density of fibers (Fig. 1A and B). In the lateral preoptic area, some of them appeared to contact unlabelled perikarya and formed basket-like labellings. The lateral septum (Fig. 1A and Fig. 6D, d), the amygdala and the putamen received a noteworthy innervation of M C H - I R fibers but a lower density was observed in the caudate nucleus.
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Fig. 4. (A) Low power microphotograph of MCH-IR neurons in the caudal hypothalamus. Arrows showed the presence of labelled neurons between the ventricle and the fornix. (a) represents a successive Kluver-Barrera stained sections. (B) represents a higher magnification of the squared area situated above the internal capsule• Fx, fornix: CI, internal capsule: FMT, mamillothalamic tract: LHA, lateral hypothalamic area; IIIV, third ventricle• Scale bars, A, a, 500/~m; B, 50 /~m.
In all the areas studied, MCH-IR fibers appeared unmyelinated and they were sometimes found between myelinated bundles, never inside myelinated tracts such as the fornix, optic and mamillothalamic tracts.
4. Discussion
Using an antiserum raised against salmon MCH, a very large population of neurons was immunostained for the first time in the lateral hypothalamus of the sheep. This labelling appears specific because it disappears after preabsorption of the antiserum with the corresponding antigen• The general distribution of MCH-IR neurons observed in sheep is similar to that described in rats (Bittencourt et al., 1992; Fellmann et al., 1987; Naito et
al., 1986, 1988; Skofitsch et al., 1985; Zamir et al., 1986b) and humans (Pelletier et al., 1987; Bresson et al., 1989). However, each species presents specific characteristics and in sheep, the fine distribution of M C H - I R neurons is slightly different from that in rats and humans. In sheep, immunostained neurons extend more caudally than in rats. The most posterior labelled perikarya are found around the mamillary bodies, only few in the rostral part of the ventral tegmental area, but in rats they are not observed caudally to the mamillary bodies• Similarly, the most rostral M C H - I R neurons are found above the optic chiasma in sheep but in rats the most anterior immunostained neurons are located above the paraventricular nucleus in the dorsal hypothalamus• Whereas in sheep the M C H - I R neurons appear to form two dense gatherings of perikarya surrounded by scattered labelled neurons, in rats a
Y. Tillet et al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
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Fig. 5. Low power microphotograph of MCH-IR neurons in the rostral hypothalamus, at the level of Fig. 1G. CI, internal capsule; Fx, fornix; OT, optic tract; VMN, hypothalamic ventromedial nucleus; IIIV; third ventricle. Scale bars, A, 500/~m; B, 50/~m.
supplementary group of neurons is observed in the ventral zona incerta (Skofitsch et al., 1985). In both species, the lateral hypothalamic area presents numerous labelled neurons. The distribution of MCH-IR neurons observed in sheep is also different from that observed in humans. In this species, labelled neurons are abundant in the periventricular nucleus (Pelletier et al., 1987), an area where only few neurons are labelled in sheep. As in other species that have been studied, M C H containing fibers are widely distributed in the sheep diencephalon. However, as for the neuronal localization, this distribution presents some characteristics specific to sheep. As in the rat (Skofitsch et al., 1985; Bittencourt et al., 1992), the highest density of immunoreactive fibers is found in the lateral hypothalamic areas (where the MCH-IR neurons are observed), in the bed nucleus of the stria terminalis, and in the supramamillary nucleus, as observed in the present sutdy. However, the periventricular area of the third ventricle and the medial preoptic area contain very few MCH-IR fibers in sheep whereas in rats a moderate density of fibers is observed (Bittencourt et al., 1992). The arcuate nucleus and the medial septum are densely innervated in rats but receive few labelled fibers in sheep. Conversely, the lateral septum receives more MCH-IR fibers in sheep than in rats. The presence of MCH-IR terminals in basket-like structures in the hypothalamus of the sheep has been also observed in the diencephalon of human (Pelletier et al., 1987).
The presence of a dense network of labelled fibers in discrete areas of the diencephalon indicates putative interactions with other neuronal systems. In the lateral septum, somatostatin, cholecystokinin, neurotensin and neuropeptide Y containing neurons have been described in sheep (Tillet, 1995). These neurons, in addition to those containing substance P, GABA and methioninenkephalin described in the rat (Gall and Moore, 1984; K6hler et al., 1984; Onteniente et al., 1982) are the most abundant neuronal population and it could be hypothesized that these neurotransmitters present or putatively present in the lateral septum of the sheep received MCH afferents. Similarly, the presence of a dense network of MCH-IR fibers in the arcuate/premamillary area may indicate morphological interactions with the numerous different transmitter-containing neurons such as GABA, thyrotropin releasing hormone, met-enkephalin, galanin, substance P, neurotensin and histamine-containing neurons that have been described in this area in rodents and humans (Panula et al., 1984; Steinbusch, 1991; Airaksinen et al., 1992; Nieuwenhuys, 1985). In sheep, only the presence of somatostatin containing neurons was described in this area (Papadopoulos et al., 1986). The physiological role of MCH is well documented in fishes, but poorly understood in mammals. M C H is a peptide whose hormonal role in fish is not shared by mammals. However, it is present in larger amounts in the perikarya in the hypothalamus than many other peptides since the producing neurons are very abundant
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Fig. 6. (A-D} MCH-IR fibers in various areas of the diencephalon. (a d) are higher magnifications of the squared area in (A-D), respectivley. (A, a) dense network of labelled fibers observed dorsally to the mamillary bodies. (B, b) MCH containing fibers in the interanteromedial nucleus of the thalamus. (C, c) rostrally, numerous MCH-IR varicose fibers are found in the bed nucleus of the stria terminalis whereas the density is moderate in the lateral septum (D, d). BNST, bed nucleus of the stria terminalis; CMm, median mamillary bodies; FMT, mamillothalamic bundle; Fx, fornix; IAM, interanteromedial nucleus of the thalamus; LV, lateral ventricle; SL, lateral septum; stars on Figs. 6A, 6B and 6C indicate the third ventricle and on Fig. 6D. it indicates the sagittal plane. Scale bars, A -D. 1 mm; a-d, 25 /Lm.
Y. Tiller et.al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
and colchicine treatment of animals was not required before immunohistochemical treatment (Risold et al., 1991; Bittencourt et al., 1992). In sheep, similar observations were recorded, and excepted for hypothalamic releasing hormones, most of the other neuropeptide immunohistochemistry required colchicine treatment. Alternatively, axonal transport in this neurons may be very low, inducing a noteworthy accumulation of the peptide in perikarya, easily detected by immunohistochemistry in these conditions. The absence of fibers observed in the external zone of the median eminence led us to hypothesize that M C H does not act directly on the anterior pituitary secretion. Zamir et al. (1986a) observed a 2-fold increase of MCH level in the pituitary gland and lateral hypothalamus of rats maintained on 2% saline drinking solution for 5 days, and in addition, variations in the electric activity of neurons of the lateral hypothalamus have been observed in response to osmotic changes (Tanaka and Seto, 1988). As in rats, a putative role of this peptide in the regulation of water-electrolyte balance could be possible in sheep, but physiological experiments are needed to test this hypothesis. The distribution of MCH-IR perikarya in the lateral hypothalamus also points to a possible involvement of this peptide in functions involving neurons of the lateral hypothalamus, such as control of eating behaviour (Oomura et al., 1969, 1974; Oomura, 1980). Electrophysiological data show that neurons of this area are sensitive to glucose level (Himmi et al., 1988; Oomura et al., 1969, 1974). Immunohistochemical study of fos expression in the lateral hypothalamus of rats shows that most neurons expressing fos immunoreactivity after insulin treatment expressed prolactin immunoreactivity, but only few of them were MCH-IR (Bahjaoui-Bouhaddi et al., 1994) suggesting that M C H neurons are not glucose sensitive. However, their interaction with the ventromedial nucleus, strongly implicated in control of behaviour associated with hunger, was demonstrated by lesion experiments (Griffond et al., 1995). Moreover, their role in the regulation of feeding behaviour was recently shown in rat intracerebroventricularly injected with M C H and in genetically obese mice (Presse et al., 1996; Qu et al., 1996). It has also been shown in the rat that a moderate MCH projection to the posterior pituitary is associated with oxytocin terminals, Since M C H stimulated in vitro oxytocin secretion (Parkes and Vale, 1993), and M C H neuron population displayed changes in lactating rats, a role for this peptide in the regulation of reproductive and maternal functions has been suggested. Thus, our observation of few fibers in the internal zone of the median eminence, probably projecting to the neural lobe, is likely related to an organization of the posterior pituitary M C H terminals similar in sheep and rat.
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Changes in this pituitary projection during the reproductive cycle of the ewe should be investigated. The involvement of M C H in the control of hypothalamic-pituitary-adrenal function was also studied in the rat. It has been shown that chronic stress, but also adrenalectomy induced a decrease in M C H mRNA, providing evidence for a negative regulation of M C H gene by stress and for a positive feedback of glucocorticoids on M C H gene activity (Presse et al., 1992). The projections of MCH-IR neurons have not been studied in sheep. However, after injections of a retrograde tracer, fluorogold, in the medial preoptic area (Tillet et al., 1993) several neurons were retrogradely labelled in the lateral hypothalamus, but double labelling with M C H antibodies revealed only few MCHIR fluorogold containing neurons in the rostral part of the area (unpublished data). This is confirmed by the low density of labelled fibers observed in the medial preoptic area of the sheep. In conclusion, the general distribution of M C H containing neurons in the diencephalon of sheep is very similar to that described in rats and humans. It is not known if these morphological differences are related to different functional characteristics, but according to the role of M C H in feeding, we may hypothesize that MCH is involved in the difference of 'feeding strategy' between sheep (ruminant) and rat (rodent). Although the role of this peptide is poorly understood in mammals and unknown in sheep, this study may constitute a first step to initiate physiological investigations in a new species whose endocrine characteristics are different from those of rats or humans.
Acknowledgements We thank Christine Young of the INRA translation service and Dr. Graeme B. Martin for revising the English.
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Oomura. Y. (1980) Input-output organization in the hypothalamus relating to food intake behavior. In Handbook of the Hypothalamus, (eds Morgane, P.J. and Panksepp, J.), Vot. 2, Marcel Dekker, New York, pp. 557 576. Oomura, Y., Ono, T., Ooyama, H. and Wayner, M.J. (1969) Glucose and osmosensitive neurons of the rat hypothalamus. Nature 222, 282 284. Oomura, Y., Ooyama, H., Sugimori, M., Nakamura, T. and Yamada, Y. (1974) Glucose inhibition of the glucose-sensitive neurons in the lateral hypothalamus. Nature 247, 284 286. Panula, P., Yang, H.Y.T. and Costa, E. (19841 Histamine-containing neurons in the rat hypothalamus. Proc. Natl. Acad. Sei. LiSA 81. 2572-2576. Papadopoulos, G.C, Karamanlidis, A.N., Dinopoulos, A. and Antonopoulos, J. (1986) Somatostatin like immunoreactive neurons in the hedgehog (Erinaeeus europaeus) and the sheep (O~is aries) central nervous system. J. Comp. Neurol. 244, 174 192. Parkes, D. and Vale, W. (1993) Contrasting actions of melanin-concentrating hormone and neuropeptide-Eq on posterior pituitary function. In Melanotropie Peptides, (eds Vaudry, H. and Eberle, A.I, NY Acad. Sci. Ser. 680, New York Academy of Sciences, New York, pp. 588. Pelletier, G., Guy, J., D6sy, L., Li, S., Eberle, A.N. and Vaudry, H. (19871 Melanin-concentrating hormone (MCH) is colocalized with alpha-melanocyte-stimulating hormone (alpha-MSH) in the rat but not in the human hypothalamus. Brain Res. 423, 247 253. Presse, F., Hervieu, G., lmaki, T., Sawchenko, P., Vale, W. and Nahon. J.L. (1992) Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli. Endocrinology 131, 1241- 1250. Presse. F., Sorokovsky, 1., Max, J.-P., Nicolaidis, S. and Nahon, J.L. (1996) Melanin-concentrating hormone is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat, Neuroseience 71, 735 745. Qu, D., Ludwig, D.S., Gammeltoft, S., Piper, M., Pelleymounter, M.A., Cullen, M.J., Foulds Mathes, W., Przypek, J., Kanarek, R. and Maratos-Flier, E. (19961 A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380, 243 247. Risold, P.Y., Fellmann, D. and Bugnon, C. (1991) Effet de la colchicine sur les neurones "a vasopressine et "a I'hormone de melano-concentration de l'hypothalamus du rat: 6tudes hybridocytochimique et immunocytochimique. C.R. Acad. Sei. 313, 311 317. Risold, P.Y., Fellmann, D., Rivier, J., Vale, W. and Bugnon, C. (1992) lmmunoreactivities for antisera to three putative neuropeptides of the rat melanin-concentrating hormone precursor are coexpressed in neurons of the rat lateral dorsal hypothalamus. Neurosci. Lett. 136, 145 149. Scallet, A.C., Della-Fera, M.A. and Baile, C. (t985) Satiety, hunger and regional brain content of cholecystokinin/gastrin and metenkephalin immunoreactivity in sheep. Peptides 6, 937 -943. Sekiya, K., Ghatel, M.A., Lacoumenta, S., Burner, P.W.J., Zamir, N.. Burrin, J.M., Polak, J.M. and Bloom, S.R. (1988) The distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of rat, guinea pig, pig and man. Neuroscienee 25, 925 930. Skofitsch, G., Jacobowitz, D.M. and Zamir, N. (1985) Immunohistochemical localization of a melanin-concentrating hormone-like peptide in the rat brain. Brain Res. Bull. 15, 635-649. Steinbusch, H.W.M. (1991) Distribution of histaminergic neurons and fibers in rat brain. Acta Otolaryngol 479 (Stockh) (Suppl.), 12 23.
Y. Tillet et al. / Journal of Chemical Neuroanatomy 12 (1996) 135-145 Tanaka, J. and Seto, K. (1988) Neurons in the lateral hypothalamus area and zona incerta with ascending projections to the subfornical organ area in the rat. Brain Res. 456, 397-400. Tillet, Y. (1987) Immunocytochemical localization of serotonin-conraining neurons in the myelencephalon, brainstem and diencephalon of the sheep. Neuroscience 23, 501-527. Tillet, Y. (1995) Distribution of neurotransmitters in the sheep brain. J. Reprod. Fertil. 49 (Suppl.), 199-220. Tillet, Y., Batailler, M. and Thibault, J. (1993) Neuronal projections to the medial preoptic area of the sheep, with special reference to monoaminergic afferents, lmmunohistochemical and retrograde tract tracing studies. J. Comp. Neurol. 330, 195220.
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Vaughan, J.M., Fischer, W.H., Hoeger, C., Rivier, J. and Vale, W. (1988) Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125, 1660-1665. Watson, S.J. and Akil, H. (1979) The presence of two ~-MSH positive cell groups in rat hypothalamus. Europ. J. Pharmacol. 58, 101 103. Zamir, N., Skofitsch, G. and Jacobowitz, D.M. (1986a) Distribution of immunoreactive melanin-concentrating hormone in the central nervous system of the rat. Brain Res. 373, 240-245. Zamir, N., Skofitsch, G., Bannon, M.J. and Jacobowitz, D.M. (1986b) Melanin-concentrating hormone: unique peptide neuronal system in the rat brain and pituitary gland. Proe. Natl. Acad. Sci. USA 83, 1528 1531.
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ELSEVIER
Journal of Chemical Neuroanatomy 12 (1996) 135-145
Distribution of melanin-concentrating hormone (MCH)-like immunoreactivity in neurons of the diencephalon of sheep Y v e s T i l l e t a,*, M a r t i n e
B a t a i l l e r a, D o m i n i q u e
Fellmann b
aUnit~ de Neuroendocrinologie Sexuelle, INRA Station de Physiologie de la Reproduction des Mammifbres Domestiques, 37380 Nouzilly, France bLaboratoire d'Histologie, Embryologie, Cytogbn&ique, Facultk de MOdecine, 25030 Besan¢on, France
Received 21 September 1995; revised 3 May 1996; accepted 8 October 1996
Abstract
An immunohistochemical study with an antiserum raised against salmon melanin concentrating-hormone has demonstrated the presence of numerous melanin concentrating-hormone-immunoreactive neurons in the lateral hypothalamic areas of the sheep. The pattern of distribution of these perikarya is similar to that of rodents and primates. In sheep, however, melanin concentrating-hormone-immunoreactive neurons appeared to form two gatherings: the first is situated ventromedially to the internal capsule and the second in the dorsolateral hypothalamus. In these areas, numerous immunostained perikarya are observed. Compared to the rats, labelled neurons extended more caudally in the ventral tegmental area and more rostrally above the optic chiasma. Compared to primates, these neurons are less numerous in the periventricular area. In our study, dense networks of melanin concentrating-hormone-immunoreactive varicose fibers were observed in the supramamillary nucleus, the lateral hypothalamus, the nucleus medialis thalami and nucleus reuniens and in the bed nucleus of the stria terminalis. Copyright © 1996 Elsevier Science B.V. Keywords: Peptide; Immunohistochemistry; Hypothalamus; Ungulate; Mammal
I. Introduction
The melanin concentrating-hormone (MCH) is a 17 residue peptide which has been isolated from salmon pituitary and hypothalamus (Kawauchi et al., 1983). In fish, it is involved in the regulation of skin pigmentation (Baker and Rance, 1983). Specific antibodies have been raised against this molecule and immunohistochemical studies have demonstrated the presence of M C H in various classes of vertebrates (for review see Baker, 1991). In the rat, antibodies to salmon M C H stained a very abundant population of neurons mainly located in the lateral and dorsal parts of the hypothalamus (Skofitsch et al., 1985; Zamir et al., 1986b). It was then demonstrated (Naito et al., 1986; Fellmann et al.,
* Corresponding author. Tel.: + 33 474 27966; fax: + 33 474 27743.
1987; Pelletier et al., 1987) that these neurons corresponded to those previously described using antisera to c~-MSH (Watson and Akil, 1979) and to human growth hormone-releasing hormone 1-37 (Fellmann et al., 1985). A similar neuron population was observed in human and several other mammalian species (Sekiya et al., 1988; Bresson et al., 1989; Pelletier et al., 1987). After the identification of the rat peptide corresponding to salmon M C H (Vaughan et al., 1988) and the subsequent elucidation of the sequence of the m R N A encoding the precursor of this peptide (Nahon et al., 1989), immunohistochemical studies using antisera to rat M C H confirmed and completed the earlier results (Risold et al., 1992; Bittencourt et al., 1992). A complete mapping of perikarya and fibers has been realised in the rat (Bittencourt et al., 1992). The sheep has been used to study various neuroendocrine regulations involving the lateral hypothalamic area, such as reproduction (Goodman, 1994; Caraty et
0891-0618/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S0891-0618(96)00195-0
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al., 1995) and feeding (Scallet et al., 1985; Della-Fera et al., 1981) and it constitutes an interesting model as an alternative to rodents and primates for studying the putative role of MCH. However, although the distribution of M C H immunoreactive neurons has been studied in various species, it has never been observed in sheep or in ungulates, and only one biochemical characterization has been conducted in pig (Sekiya et al., 1988). The aim of this study was therefore to investigate the distribution of MCH immunoreactive neurons in the diencephalon of the sheep using immunohistochemistry with antibodies raised against salmon MCH.
2. Materials and methods
1989; Risold et al., 1992). It was prefered to a rat MCH antiserum also prepared in the laboratory because of its higher titer and better specificity (Risold et al,, 1992), as verified in a side experiment performed on sheep sections (data not shown). For the present study, the specificity of the labelling was checked by preincubating the immune serum for 3 h with salmon or rat synthetic MCH (1 nmol//21 undiluted antiserum, synthetic MCH was kindly donated by Dr. J. Rivier, Salk Institute, La Jolla, Ca) and by substituting the specific antiserum with normal (nonimmune) rabbit serum. The distribution of MCH containing perikarya and fibers is shown on successive schematic drawings in the frontal plane and on representative photomicrographs.
2.1. Animals
3. Results
Five adult sheep (male and female) from the laboratory flock were used. They were killed by decapitation at the slaughterhouse by a licensed butcher (authorization no. A37801). The head was immediately perfused via both carotid arteries with 4 liters of a 4% paraformaldehyde solution in phosphate buffer pH 7.4, 0.1 M. The diencephalon was dissected and postfixed in the same fixative for 24 h and rinsed for at least 24 h in a 15% sucrose, 0.1% NaN3 solution in the same buffer. Sections (40 p m thick) were cut in the frontal plane throughout the diencephalon. 2.2. Immunohistochemistrv
The immunohistochemical procedure has been previously described (Tillet, 1987). Sections were successively incubated for 20 min with normal sheep serum diluted 1/15, for 5 days with the first antiserum (raised in rabbit) diluted 1/5000, overnight with a sheep serum anti rabbit gamma globulin diluted 1/400, and overnight with rabbit peroxidase antiperoxidase complex (PAP) diluted 1/2000. All incubations were performed in phosphate buffer saline (PBS) 0.1 M, pH 7.4, containing 0.3% Triton X100, 0.1% sodium azide, excepted the PAP complex which was diluted in PBS only. The peroxidase was visualized after incubation in a 0.02% diaminobenzidine hydrochloride, 0.5% nickel ammonium sulfate and 0.003% H20 2 solution in Tris HCI, 0.05 M, pH 7.9 for 5 15 min. 2.3, Antisera
The primary antiserum was raised against synthetic salmon MCH. The specificity of this antiserum have been thoroughly checked in previous studies by liquid and solid phase saturation with several peptides related to M C H or to its precursor and by competition experiments in a radioimmunoassay (Fellmann et al., 1987,
MCH like immunoreactivity was observed in the cytoplasm of neurons distributed in the diencephalon between the anterior and the posterior commissure (Fig. 1). These neurons were bipolar or multipolar, their diameter was comprised between 25 and 50 /~m for the smaller and larger value, respectively, and did not change according to the different studied areas, their nucleus was never stained. The peroxidase staining appeared homogeneous in most of the perikarya but was granular in some (Fig. 3B). No difference was observed between male and female. No immunoreactive neurons were observed on sections incubated with the first antiserum previously adsorbed with the specific antigen (Fig. 2). We therefore considered the labelling specific to MCH; MCH-containing neurons were referred to as MCH-immunoreactive (-IR). 3. I. Neurons
Within the diencephalon, MCH-IR neurons were observed in the lateral part of the hypothalamus. The most caudally labelled neurons were found in the rostral part of the ventral tegmental area (VTA), caudally to the mamillary bodies in the sagittal plane, and only one or two neurons were observed per section (Fig. 1L). In the following sections the number of labelled neurons increased around the medial mamillary nucleus. At this level, they are situated dorsally to this structure, near the sagittal plane, and laterally, in the VTA (Fig. l J, K and Fig. 3). In the caudal part of the third ventricle, M C H - I R neurons were observed between the ventral part of the ventricle and the fornix (Fig. 1H, I and Fig. 4). In the middle hypothalamus, labelled neurons were tbund around the fornix, between the third ventricle and the internal capsule, and under the mamillothalamic tract (Fig. 1E, F and G). At this level, two gather-
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Fig. I. Schematic draw!ngs of frontal sections through the diencephalon of sheep showing the distribution of MCH-IR perikarya (O: 1- 10 perkarya) and fibers (_ ~ ) from rostral to caudal levels. AHDM: area hypothalamica dorsomedialis; AHL: area hypothalamica lateralis; AHP: area hypothalamica posterioris; ATV: area tegmentalis ventralis; BNST: bed nucleus of the stria terminalis; CA: commissura anterioris; Cd: caudate nucleus; Ch capsula interna; CMm: medial mamillary nucleus; CP: commissura posterioris; Ent: nucleus entopeduncularis; Fx: fornix; FM: forarnen of Monro; FMT: fasciculus mamillothalamicus; Hb: habenular nucleus; IAM: nucleus interanteromedialis; IIIV: third ventricle; NM: nucleus medialis, pars ventralis and pars dorsalis (thalamus); NPV: nucleus paraventricularis hypothalami; NRT: nucleus reticularis thalami; NSO: nucleus supraopticus: OC: optic chiasma; OT: optic tract; Ped Cer: cerebral peduncle; PVT: nucleus paraventricularis thalami; Re: nucleus reuniens: SL: septum laterale: VL: ventriculus lateralis: VMN: nucleus ventromedialis hypothalami;
ings of labelled neurons appeared in the hypothalamus. The first is situated ventrally to the internal capsule, between the fornix and the optic tract, and the second is observed more dorsally, above the fornix in the dorsolateral hypothalamus. Severals M C H - I R neurons were observed between these two gatherings. Rostrally, the neurons of the ventral group extended in the lateral part of the hypothalamus, between the optic tract and the internal capsule (Fig. 1B-E). In this area, neurons were mainly bipolar and their long axes were parallel to the dorsal side of the optic tract (Fig. 5). M C H - I R neurons were never observed in the mediobasal hypothalamus, in the ventromedial nucleus nor in the arcuate nucleus but were observed laterally to these nuclei. In the anterior hypothalamus, few scattered M C H IR neurons were found in the lateral part, dorsal to the caudal part of the optic chiasma (Fig. 1A and B). They were not observed in the supraoptic nucleus. At
the level of the anterior commissure, only one or two immunoreactive neurons were observed per section. 3.2. Fibers
M C H - I R fibers were observed in almost all studied areas but their density varied in the different structures. Most of them presented varicosities. Caudally a weak density was observed in the rostral part of the ventral tegmental area and dorsally to the cerebral peduncles were they were oriented mediolaterally. The sagittal plane presented also a low density of vertically oriented fibers on each side of the third ventricle and laterally to the posterior commissure. The density of M C H - I R fibers became important in the caudal hypothalamus above the mamillary bodies (Fig. I I - K and Fig. 6A, a). This area presented one of the highest densities of labelled fibers observed in the sheep diencephalon. Few fibers were
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Y. Tiller et al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
Fig. 2. (A) MCH-IR neurons in the lateral diencephalonof the sheep; (B) Contralateral area immunostained with the anti-MCH preincubatedwith an excess of MCH. Scale bars: 50 pm.
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Fig. 3. MCH-IR neurons in the caudal diencephalonof the sheep observeddorsally (A) and laterally (B) to the mamillary bodies. Note that some neurons presented a granular labelling (arrowhead in B). Scale bars: 25/~m. observed in the mamillary bodies and in the habenula. Laterally, a low density of fibers was observed in an area corresponding to the nucleus reticularis thalami. In the medial part of the diencephalon, a low density of fibers was found between the optic tract and the internal capsule, and dorsally to the third ventricle where they were oriented horizontally and vertically, respectively. In the thalamus, the paraventricular thalamic nucleus and the area related to the nucleus medialis thalami and to the nucleus reuniens presented a noteworthy innervation whereas a lower density of labelled fibers was observed beneath the lateral ventricles. In the rostro-ventral part of the thalamus, dorsally to the third ventricle, a dense network of labelled fibers is observed in the area related to the interanteromedian nucleus (Fig. 1D and 6B, b) whereas a low density was found in the other thalamic nuclei. In the hypothalamus, the lateral and dorsomedial hypothalamic areas presented a dense network of M C H - I R fibers (Fig. 1 D - F ) . Only a few labelled fibers were observed close to the third ventricle and in the hypothalamic ventromedial nucleus. Small bundles of M C H - I R fibers were observed dorsally to the optic tract and to the internal capsule, running
towards the nucleus reticularis thalami. In the median eminence M C H - I R fibers were observed in the internal layer, and almost none in the external layer. In the rostral diencephalon, the bed nucleus of the stria terminalis presented a dense network of immunoreactive fibers (Fig. 1 A - C and Fig. 6C, c) and a bundle of vertically oriented labelled fibers ran along the medial edge of the caudate nucleus, in the stria terminalis. The periventricular area and the medial preoptic area presented a weak innervation and only a few small varicose M C H - I R fibers were observed in the paraventricular hypothalamic nucleus and supraoptic nucleus. Above the optic tract, a bundle of horizontal fibers ran toward more lateral areas such as the ,putamen and the ventral surface of the telencephalon, between the optic chiasma and the olfactory tract. The medial side of the internal capsule and the lateral preoptic area presented a moderate density of fibers (Fig. 1A and B). In the lateral preoptic area, some of them appeared to contact unlabelled perikarya and formed basket-like labellings. The lateral septum (Fig. 1A and Fig. 6D, d), the amygdala and the putamen received a noteworthy innervation of M C H - I R fibers but a lower density was observed in the caudate nucleus.
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Y. Tiller et al. / Journal qf Chemical Neuroanatomy 12 (1996) 135 145
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Fig. 4. (A) Low power microphotograph of MCH-IR neurons in the caudal hypothalamus. Arrows showed the presence of labelled neurons between the ventricle and the fornix. (a) represents a successive Kluver-Barrera stained sections. (B) represents a higher magnification of the squared area situated above the internal capsule• Fx, fornix: CI, internal capsule: FMT, mamillothalamic tract: LHA, lateral hypothalamic area; IIIV, third ventricle• Scale bars, A, a, 500/~m; B, 50 /~m.
In all the areas studied, MCH-IR fibers appeared unmyelinated and they were sometimes found between myelinated bundles, never inside myelinated tracts such as the fornix, optic and mamillothalamic tracts.
4. Discussion
Using an antiserum raised against salmon MCH, a very large population of neurons was immunostained for the first time in the lateral hypothalamus of the sheep. This labelling appears specific because it disappears after preabsorption of the antiserum with the corresponding antigen• The general distribution of MCH-IR neurons observed in sheep is similar to that described in rats (Bittencourt et al., 1992; Fellmann et al., 1987; Naito et
al., 1986, 1988; Skofitsch et al., 1985; Zamir et al., 1986b) and humans (Pelletier et al., 1987; Bresson et al., 1989). However, each species presents specific characteristics and in sheep, the fine distribution of M C H - I R neurons is slightly different from that in rats and humans. In sheep, immunostained neurons extend more caudally than in rats. The most posterior labelled perikarya are found around the mamillary bodies, only few in the rostral part of the ventral tegmental area, but in rats they are not observed caudally to the mamillary bodies• Similarly, the most rostral M C H - I R neurons are found above the optic chiasma in sheep but in rats the most anterior immunostained neurons are located above the paraventricular nucleus in the dorsal hypothalamus• Whereas in sheep the M C H - I R neurons appear to form two dense gatherings of perikarya surrounded by scattered labelled neurons, in rats a
Y. Tillet et al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
141
Fig. 5. Low power microphotograph of MCH-IR neurons in the rostral hypothalamus, at the level of Fig. 1G. CI, internal capsule; Fx, fornix; OT, optic tract; VMN, hypothalamic ventromedial nucleus; IIIV; third ventricle. Scale bars, A, 500/~m; B, 50/~m.
supplementary group of neurons is observed in the ventral zona incerta (Skofitsch et al., 1985). In both species, the lateral hypothalamic area presents numerous labelled neurons. The distribution of MCH-IR neurons observed in sheep is also different from that observed in humans. In this species, labelled neurons are abundant in the periventricular nucleus (Pelletier et al., 1987), an area where only few neurons are labelled in sheep. As in other species that have been studied, M C H containing fibers are widely distributed in the sheep diencephalon. However, as for the neuronal localization, this distribution presents some characteristics specific to sheep. As in the rat (Skofitsch et al., 1985; Bittencourt et al., 1992), the highest density of immunoreactive fibers is found in the lateral hypothalamic areas (where the MCH-IR neurons are observed), in the bed nucleus of the stria terminalis, and in the supramamillary nucleus, as observed in the present sutdy. However, the periventricular area of the third ventricle and the medial preoptic area contain very few MCH-IR fibers in sheep whereas in rats a moderate density of fibers is observed (Bittencourt et al., 1992). The arcuate nucleus and the medial septum are densely innervated in rats but receive few labelled fibers in sheep. Conversely, the lateral septum receives more MCH-IR fibers in sheep than in rats. The presence of MCH-IR terminals in basket-like structures in the hypothalamus of the sheep has been also observed in the diencephalon of human (Pelletier et al., 1987).
The presence of a dense network of labelled fibers in discrete areas of the diencephalon indicates putative interactions with other neuronal systems. In the lateral septum, somatostatin, cholecystokinin, neurotensin and neuropeptide Y containing neurons have been described in sheep (Tillet, 1995). These neurons, in addition to those containing substance P, GABA and methioninenkephalin described in the rat (Gall and Moore, 1984; K6hler et al., 1984; Onteniente et al., 1982) are the most abundant neuronal population and it could be hypothesized that these neurotransmitters present or putatively present in the lateral septum of the sheep received MCH afferents. Similarly, the presence of a dense network of MCH-IR fibers in the arcuate/premamillary area may indicate morphological interactions with the numerous different transmitter-containing neurons such as GABA, thyrotropin releasing hormone, met-enkephalin, galanin, substance P, neurotensin and histamine-containing neurons that have been described in this area in rodents and humans (Panula et al., 1984; Steinbusch, 1991; Airaksinen et al., 1992; Nieuwenhuys, 1985). In sheep, only the presence of somatostatin containing neurons was described in this area (Papadopoulos et al., 1986). The physiological role of MCH is well documented in fishes, but poorly understood in mammals. M C H is a peptide whose hormonal role in fish is not shared by mammals. However, it is present in larger amounts in the perikarya in the hypothalamus than many other peptides since the producing neurons are very abundant
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Y. Tillet et al. , Journal q[ Chemical Neuroanatomy 12 (1996) 135 145
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Fig. 6. (A-D} MCH-IR fibers in various areas of the diencephalon. (a d) are higher magnifications of the squared area in (A-D), respectivley. (A, a) dense network of labelled fibers observed dorsally to the mamillary bodies. (B, b) MCH containing fibers in the interanteromedial nucleus of the thalamus. (C, c) rostrally, numerous MCH-IR varicose fibers are found in the bed nucleus of the stria terminalis whereas the density is moderate in the lateral septum (D, d). BNST, bed nucleus of the stria terminalis; CMm, median mamillary bodies; FMT, mamillothalamic bundle; Fx, fornix; IAM, interanteromedial nucleus of the thalamus; LV, lateral ventricle; SL, lateral septum; stars on Figs. 6A, 6B and 6C indicate the third ventricle and on Fig. 6D. it indicates the sagittal plane. Scale bars, A -D. 1 mm; a-d, 25 /Lm.
Y. Tiller et.al. /Journal of Chemical Neuroanatomy 12 (1996) 135-145
and colchicine treatment of animals was not required before immunohistochemical treatment (Risold et al., 1991; Bittencourt et al., 1992). In sheep, similar observations were recorded, and excepted for hypothalamic releasing hormones, most of the other neuropeptide immunohistochemistry required colchicine treatment. Alternatively, axonal transport in this neurons may be very low, inducing a noteworthy accumulation of the peptide in perikarya, easily detected by immunohistochemistry in these conditions. The absence of fibers observed in the external zone of the median eminence led us to hypothesize that M C H does not act directly on the anterior pituitary secretion. Zamir et al. (1986a) observed a 2-fold increase of MCH level in the pituitary gland and lateral hypothalamus of rats maintained on 2% saline drinking solution for 5 days, and in addition, variations in the electric activity of neurons of the lateral hypothalamus have been observed in response to osmotic changes (Tanaka and Seto, 1988). As in rats, a putative role of this peptide in the regulation of water-electrolyte balance could be possible in sheep, but physiological experiments are needed to test this hypothesis. The distribution of MCH-IR perikarya in the lateral hypothalamus also points to a possible involvement of this peptide in functions involving neurons of the lateral hypothalamus, such as control of eating behaviour (Oomura et al., 1969, 1974; Oomura, 1980). Electrophysiological data show that neurons of this area are sensitive to glucose level (Himmi et al., 1988; Oomura et al., 1969, 1974). Immunohistochemical study of fos expression in the lateral hypothalamus of rats shows that most neurons expressing fos immunoreactivity after insulin treatment expressed prolactin immunoreactivity, but only few of them were MCH-IR (Bahjaoui-Bouhaddi et al., 1994) suggesting that M C H neurons are not glucose sensitive. However, their interaction with the ventromedial nucleus, strongly implicated in control of behaviour associated with hunger, was demonstrated by lesion experiments (Griffond et al., 1995). Moreover, their role in the regulation of feeding behaviour was recently shown in rat intracerebroventricularly injected with M C H and in genetically obese mice (Presse et al., 1996; Qu et al., 1996). It has also been shown in the rat that a moderate MCH projection to the posterior pituitary is associated with oxytocin terminals, Since M C H stimulated in vitro oxytocin secretion (Parkes and Vale, 1993), and M C H neuron population displayed changes in lactating rats, a role for this peptide in the regulation of reproductive and maternal functions has been suggested. Thus, our observation of few fibers in the internal zone of the median eminence, probably projecting to the neural lobe, is likely related to an organization of the posterior pituitary M C H terminals similar in sheep and rat.
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Changes in this pituitary projection during the reproductive cycle of the ewe should be investigated. The involvement of M C H in the control of hypothalamic-pituitary-adrenal function was also studied in the rat. It has been shown that chronic stress, but also adrenalectomy induced a decrease in M C H mRNA, providing evidence for a negative regulation of M C H gene by stress and for a positive feedback of glucocorticoids on M C H gene activity (Presse et al., 1992). The projections of MCH-IR neurons have not been studied in sheep. However, after injections of a retrograde tracer, fluorogold, in the medial preoptic area (Tillet et al., 1993) several neurons were retrogradely labelled in the lateral hypothalamus, but double labelling with M C H antibodies revealed only few MCHIR fluorogold containing neurons in the rostral part of the area (unpublished data). This is confirmed by the low density of labelled fibers observed in the medial preoptic area of the sheep. In conclusion, the general distribution of M C H containing neurons in the diencephalon of sheep is very similar to that described in rats and humans. It is not known if these morphological differences are related to different functional characteristics, but according to the role of M C H in feeding, we may hypothesize that MCH is involved in the difference of 'feeding strategy' between sheep (ruminant) and rat (rodent). Although the role of this peptide is poorly understood in mammals and unknown in sheep, this study may constitute a first step to initiate physiological investigations in a new species whose endocrine characteristics are different from those of rats or humans.
Acknowledgements We thank Christine Young of the INRA translation service and Dr. Graeme B. Martin for revising the English.
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