Analysis of the ACTH/β-End/α-MSH-Immunoreactive afferent input to the hypothalamic paraventricular nucleus of rat

Immunoreactive fibers and varicosities in the hypothalamic paraventricular nucleus (PVN) were examined by light- and electronmicroscopy, following treatment of brain slices with specific antibodies to adrenocorticotropin (ACTH), β-endorphin (β-End) and α-melanotropin (α-MSH) peptides. In an attempt to provide a more precise, quantitative definition of the densities of immunoreactive elements, sections were analyzed by computer based image-analysis techniques.Fibers and varicosities immunostained with the 3 different antibodies displayed an identical distribution pattern throughout the nucleus suggesting that they are parts of the same, arcuate pro-opiomelanocortin (POMC) neuron system. Although immunoreactive varicosities were found all over the PVN, it was possible to identify a characteristic, density distribution pattern. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites. The majority of immunoreactive elements were found in the dorsal parvo- and caudal magnocellular subdivisions which give rise to long projections to the lower brainstem. Moderate density of POMC neural elements was observed in the anterior and medial (ventral portion) parvocellular subdivisions which project to the external zone of the median eminence. Only a few, widely scattered immunostained varicosities are found in the medial and lateral magnocellular subdivisions which project to the neurohypophysis. A combined lesion and immunocytochemical approach has shown that the bulk of the afferent neuronal input from arcuate POMC cells enters the PVN from a ventral direction.     

α-Melanocyte stimulating hormone discloses a stimulatory effect of β-endorphin on somatostatin release

The influence of alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END) on the secretion of somatostatin (SRIF) from the median eminence (ME) was studied using an in vitro incubation system. The MEs from adult male rats were first preincubated at 37 degrees C for 30 min with constant shaking in 0.4 ml of Krebs-Ringer bicarbonate-glucose buffer (pH 7.4) containing bacitracin in an atmosphere of 95% O2/5% CO2. Medium was discarded and replaced by medium containing different doses of alpha-MSH, beta-END, or a fixed dose of alpha-MSH (10(-7) M or 10(-9) M) plus beta-END at various concentrations. By themselves alpha-MSH and beta-END did not alter basal SRIF release, but in the presence of alpha-MSH (10(-7) M) beta-END stimulated somatostatin release. This effect was significant at concentrations of beta-END of 10(-8) M and higher. The permissive effect of alpha-MSH was observed at a concentration as low as 10(-9) M, but in this case the stimulatory effect of beta-END became evident only at higher doses tested (10(-7) M). It is suggested that alpha-MSH and beta-END participate in the modulation of SRIF release. By themselves beta-END and alpha-MSH did not affect basal release of SRIF but in the presence of alpha-MSH, beta-END had a stimulatory effect on SRIF release. The mechanism for this interaction is unknown. The results are consistent with the possibility that beta-END neurons have stimulatory and inhibitory effects on SRIF release and that alpha-MSH, by blocking the inhibitory components, discloses the stimulatory effect of beta-END on SRIF release.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Characterization of the forms of β-endorphin and α-MSH in the caudal medulla of the rat and guinea pig

The post-translational processing of pro-opiomelanocortin (POMC) in brain remains controversial. Classically, there was thought to be a single cell group in the arcuate nucleus with long projections through limbic structures. More recently, a second cell group was discovered in the caudal medulla. This study addresses the question of POMC processing in this region. Steady-state analysis of acid extracts of dorsal caudal medulla from rat and guinea pig CNS by gel filtration chromatography and radioimmunoassay indicated that in both species the major POMC-related end products are α-MSH-sized material and ß-endorphin-sized. In this tissue ß-LPH and ACTH represent minor end products. Analysis of the α-MSH-sized material from both species by reverse-phase HPLC indicated that in the rat caudal medulla approximately 79% of the α-MSH-related material is acetylated, and in the guinea pig caudal medulla approximately 85% of the α-MSH-related material is acetylated. Analysis of the forms of ß-endorphin isolated from the rat caudal medulla by cation exchange chromatography revealed that acetylated and non-acetylated forms of ß-endorphin are present in this region of the rat CNS. Approximately 65% of the ß-endorphin in the rat caudal medulla is N-acetylated. Analysis of the forms of ß-endorphin isolated from the guinea pig caudal medulla indicated that approximately 63% of the ß-endorphin is N-acetylated in this region of the guinea pig CNS. These data indicate that the post-translational processing of POMC in the dorsal caudal medulla, the site of the nucleus tractus solitarius POMC cell group, is distinct from the processing patterns that have been reported for POMC systems in the mammalian anterior pituitary, intermediate pituitary and arcuate nucleus.     

Identification of a second category of α-melanocyte-stimulating hormone (α-MSH) neurons in the rathypothalamus

An immunocytochemical localization of alpha-melanocyte-stimulating hormone (alpha-MSH) as well as ACTH and a fragment (16K) of the common precursor of ACTH and beta-lipotropin (beta-LPH) was performed in rat brain. Two different groups of neuronal cell bodies showing alpha-MSH-like immunoreactivity (alpha-MSH-LI) were observed in the hypothalamus. One group of neurons located in the arcuate nucleus was shown to contain not only alpha-MSH-LI, but also ACTH and the 16K fragment. A second category of alpha-MSH-LI-containing neurons was characterized by the complete absence of staining for ACTH and 16K fragment. These neurons were mainly located in the dorsal-lateral portion of the hypothalamus. Immunoelectron microscopy showed that immunostaining for alpha-MSH was restricted to dense core vesicles in the positive perikarya. Nerve fibers staining for alpha-MSH (but not for ACTH and 16K fragment) were also observed outside the ACTH-beta-LPH pathway, especially in the cortex, caudate-putamen nucleus and hippocampus. These findings strongly suggest the presence of two different neuronal systems reacting with antibodies to alpha-MSH.     

Enhanced binocular interaction in the visual cortex of normal kittens subjected to intracortical norepinephrine perfusion

Immunoreactive fibers and varicosities in the hypothalamic paraventricular nucleus (PVN) were examined by light- and electronmicroscopy, following treatment of brain slices with specific antibodies to adrenocorticotropin (ACTH), beta-endorphin (beta-End) and alpha-melanotropin (alpha-MSH) peptides. In an attempt to provide a more precise, quantitative definition of the densities of immunoreactive elements, sections were analyzed by computer based image-analysis techniques. Fibers and varicosities immunostained with the 3 different antibodies displayed an identical distribution pattern throughout the nucleus suggesting that they are parts of the same, arcuate pro-opiomelanocortin (POMC) neuron system. Although immunoreactive varicosities were found all over the PVN, it was possible to identify a characteristic, density distribution pattern. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites. The majority of immunoreactive elements were found in the dorsal parvo- and caudal magnocellular subdivisions which give rise to long projections to the lower brainstem. Moderate density of POMC neural elements was observed in the anterior and medial (ventral portion) parvocellular subdivisions which project to the external zone of the median eminence. Only a few, widely scattered immunostained varicosities are found in the medial and lateral magnocellular subdivisions which project to the neurohypophysis. A combined lesion and immunocytochemical approach has shown that the bulk of the afferent neuronal input from arcuate POMC cells enters the PVN from a ventral direction.     

Beta-endorphin/ACTH immunocytochemistry in the CNS of the lizard Anolis carolinensis: evidence for a major mesencephalic cell group

The immunocytochemical distribution of beta-endorphin and other proopiomelanocortin (POMC) peptides in the central nervous system of the lizard Anolis carolinensis was determined. Colchicine pretreatment was used to enhance perikaryal immunoreactivity. A major finding of this study is the localization of a previously undetected mesencephalic cell group which exhibits immunoreactivity to beta-endorphin, ACTH, and alpha-MSH. The perikarya of these neurons are large, bipolar, and situated in the mesencephalic tegmental area. They appear to project to the mesencephalic central gray and other brainstem structures. In contrast, the immunoreactive parvicellular perikarya of the medial-basal hypothalamus, corresponding to the POMC perikarya of the rodent arcuate nucleus, exhibit major rostral projections to various telencephalic and diencephalic structures. The exact extent of fiber projections and innervation patterns arising from either of these two groups is not clear at this time and will require further analyses. Scattered fiber immunoreactivity was also seen in the medial cerebral cortex and the striatal complex, regions which apparently are not innervated by beta-endorphin fibers in the rodent brain. Also, no immunoreactivity was seen to an antiserum to the 16K peptide of POMC. Other similarities and differences in the brain distribution of POMC in reptiles and mammals are discussed.     

Immunocytochemical localization of pro-opiomelanocortin-derived peptides in the adult rat spinal cord

A dispersed descending pro-opiomelanocortin (POMC) fiber system has been demonstrated by peroxidase-antiperoxidase (PAP) immunocytochemistry in the adult rat spinal cord. beta-endorphin, adrenocorticotrophic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and 16K immunoreactive fibers exist in the spinal cord from cervical down to sacral level. Descending fibers running parallel in the dorsolateral and lateral funiculus send collaterals ventromedially or medially to terminate in the gray matter surrounding the central canal, where nociceptive neurons have recently been located, in addition to those nociceptive cells in the dorsal horn. After spinal transection at lower thoracic level, POMC peptide immunoreactivities disappeared below the lesion. Moreover, no POMC cell bodies were found in the spinal cord. Therefore, the descending fibers are most likely of supraspinal origin.     

Injection of alpha-MSH, but not beta-endorphin, into the PVN decreases POMC gene expression in the ARC

beta-Endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH), neuropeptides derived from proopiomelanocortin (POMC), have opposite effects on eating behavior. We injected rats with alpha-MSH (0.6 nmol) or beta-END (1 nmol) into the PVN (three times in a 26 h period). These doses of alpha-MSH and beta-END decreased and increased feeding respectively. Following alpha-MSH administration into the PVN, mRNA levels of POMC decreased by 17%, whereas there was no significant change in gene expression of either proDynorphin or proEnkephalin. PVN injection of beta-END failed to alter gene expression of POMC, proDynorphin or proEnkephalin. These data suggest that a feedback pathway exists between the PVN and ARC for alpha-MSH and POMC, but not for beta-END and POMC.     

Distribution of β-lipotropin (β-LPH), adrenocorticotropin (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the rat brain. I. Origin of the extrahypothalamic fibers

By immunocytochemical techniques, the neuronal cell bodies containing ACTH, β-LPH and α-MSH have only been found in the area of the arcuate nucleus of the hypothalamus, whereas positive nerve fibers have been observed in many hypothalamic and extra-hypothalamic areas. The possible contribution of neurons which could be located in other brain areas has been studied in the rat by experiments involving hypothalamic deafferentation or destruction of the acurate nucleus. Fourteen days after deafferentation, no immunoreactive fibers could be detected in extrahypothalamic areas whereas the concentration of positive cell bodies and fibers remained unchanged within the hypothalamic island. In rats which had been injected in the neonatal period with monosodium glutamate (MSG), which selectively destroys the arcuate nucleus, only a few immunostained cell bodies were observed in hypothalamic region lateral to the arcuate nucleus. As compared to control animals, the concentration of immunostained fibers in both hypothalamic and extrahypothalamic regions was markedly decreased. These results strongly suggest that neuronal cell bodies producing ACTH, β-LPH and α-MSH are located in the region of the arcuate nucleus and send axonal projections into many brain areas.     

Distribution of immunoreactive substance P neurons in the hypothalamus and pituitary of the rhesus monkey

The distribution of immunoreactive substance P (IR-SP) neurons was examined in the hypothalamus and pituitary gland of the rhesus monkey by using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactive SP cell bodies were observed in the arcuate nucleus, in the region lateral to the arcuate nucleus, and in the median eminence (ME). Immunoreactive SP cells were also seen in the periventricular area of the dorsal tuberal region. A rich network of SP fibers was concentrated in the arcuate region, and the fiber stain was particularly dense in the external zone of the median eminence and in the external layer of the infundibular stalk. Also, substance P fibers were seen in the internal layer of the pituitary stalk and in the neural lobe of the pituitary gland. Outside the hypothalamus a dense network of IR-SP fibers was observed in the globus pallidus.     

α-Melanotropin, β-endorphin and adrenocorticotropin-like immunoreactivities are colocalized within duodenal myenteric plexus perikarya

Adrenocorticotropin (ACTH) immunoreactivity was localized at the ultrastructural level as positive 'cores' within large dense-cored vesicles (LDVs) of axons and dendrites of the rat duodenum. The immunostained vesicle 'cores' were 35-50 nm in mean diameter, corresponding to 'cores' of LDVs with a mean diameter of 80-90 nm. alpha-melanotropin (alpha-MSH) was detected also within LDVs, expressing the same mean diameter as ACTH-stained vesicles. alpha-MSH and ACTH were localized only within structures belonging to the enteric nervous system of the rat duodenum. alpha-MSH and ACTH, as detected by immunostaining, were absent in endocrine cells of the rat duodenum. These findings suggest the possibility that these peptides may have important physiological roles in the rat duodenum.     

Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein.

The arcuate nucleus of the hypothalamus contains various types of peptidergic neurons. In particular, two distinct populations of neurosecretory neurons containing neuropeptide Y (NPY)- and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity have been identified in the arcuate nucleus. Double-labeling immunocytochemical data have recently shown that NPY-containing fibers make synaptic contacts with proopiomelanocortin (POMC) immunoreactive neurons. We have thus investigated the possible effect of NPY on the release of alpha-MSH from rat hypothalamic slices in vitro, using the perifusion technique. NPY significantly inhibited KCl-stimulated alpha-MSH release in a dose-dependent manner. The inhibitory effect of NPY was mimicked by the Y2 agonist, NPY-(13-36), while the Y1 agonist, [Leu31,Pro34]NPY, was devoid of effect. Pretreatment of hypothalamic slices with pertussis toxin (PTX) blocked the inhibitory effect of NPY, suggesting that the action of NPY on POMC neurons is mediated through a PTX-sensitive G protein. These results support the notion that NPY may play a physiological role in the regulation of alpha-MSH release from hypothalamic neurons.     

Presence of strong glucocorticoid receptor immunoreactivity within hypothalamic and hypophyseal cells containing pro-opiomelanocortic peptides.

The presence of nuclear glucocorticoid receptor immunoreactivity (GR IR) was studied in the adrenocorticotropin (ACTH), beta-Endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) IR neuronal populations of the rat hypothalamus and hypophysis using double immunolabelling techniques. All the nuclei of the ACTH/beta-END/alpha-MSH IR neurons of the arcuate and periarcuate nuclei were strongly GR IR in the 48 h colchicine treated animal, but very few alpha-MSH-like IR perikarya located in the dorsal and lateral hypothalamus displayed nuclear GR IR. GR IR was present in the ACTH/beta-END corticotrophs and absent in the intermediate lobe of the hypophysis. The data provide morphological evidence for a glucocorticoid action through a nuclear GR in the arcuate ACTH/beta-END/alpha-MSH IR neurons and the ACTH/beta-END corticotrophs, whereas the alpha-MSH-like IR neurons of the lateral hypothalamus and the melanotropes of the intermediate lobe may not be directly affected by glucocorticoids under normal conditions.     

Proopiomelanocortin peptide immunocytochemistry in rhesus monkey brain

The immunocytochemical distribution of proopiomelanocortin (POMC) peptides (beta-endorphin, ACTH, alpha-MSH, 16K fragment) was studied in the brain of the rhesus monkey (Macaca mulatta). Some animals were administered colchicine intracerebroventricularly prior to sacrifice to enhance the visualization of perikaryal immunoreactivity. Immunoreactive perikarya are localized to hypothalamic infundibular nucleus, giving rise to several distinct projections. Rostral projections extend through midline diencephalic and preoptic areas, and enter the telencephalon. Along this course, immunoreactive fibers are seen in midline hypothalamic and preoptic nuclei, nucleus of the diagonal band, olfactory tubercle, nucleus accumbens, bed nucleus of stria terminalis, septum, and other limbic structures in telencephalon. Caudal to the anterior commissure, some fibers ascend dorsally to enter the midline thalamus, which they innervate. Lateral projections of the infundibular perikarya course through the medial-basal hypothalamus, dorsal to the optic tracts, and enter the amygdala region where they innervate more medially situated amygdaloid nuclei. Caudal projections of the POMC neurons also extend through midline diencephalon, some coursing along a periventricular path to innervate midline hypothalamic and thalamic nuclei. This projection extends into the mesencephalic substantia grisea centralis and may also contribute to the innervation of more dorsally situated nuclei in the pons and medulla, such as the parabrachial nuclei and nucleus tractus solitarius. Other caudal projections originating in the hypothalamus course through the ventral tegmentum of mesencephalon and pons and may contribute to the innervation of midline raphe and other ventrally situated nuclei in the pons and medulla. The distribution of immunoreactive perikarya and fibers in the brain of rhesus monkey is strikingly similar to that found in the rat brain. However, subtle differences appear to exist in the innervation patterns of particular brain regions.     

Biological and immunological characterization of α-melanocyte-stimulating hormone (α-MSH) in two neuronal systems of the rat brain

Recent immunocytochemical studies have demonstrated the existence of two different neuronal systems containing α-MSH-like material in the brain: one originating from the arcuate nucleus and the other one from the dorsolateral hypothalamus. The aim of the present study was to further characterize α-MSH in these two discrete regions of the rat diencephalon. Intracerebroventricular administration of colchicine resulted in a marked decrease in the number of ACTH and β-endorphin nerve fibers and a significant reduction in ACTH and β-endorphin content in the dorsolateral hypothalamus. Conversely, colchicine treatment did not alter α-MSH, ACTH or β-endorphin content in the arcuate nucleus and did not significantly affect α-MSH concentration in the dorsal region. Reverse-phase high-performance liquid chromatography showed that the major α-MSH-like compound localized in the dorsal hypothalamus co-migrated exactly with synthetic α-MSH, whereas the arcuate nucleus contained 5 peptides cross-reacting with α-MSH antibodies, 4 of them being different from standard α-MSH. Significant amounts of biologically active melanotropin, which migrated on Sephadex G-25 columns like synthetic α-MSH, were also detected in both the arcuate nucleus and dorsolateral hypothalamus. Taken together, these data demonstrate that the α-MSH cell bodies located in the dorsolateral hypothalamus specifically produce authentic α-MSH, whereas the α-MSH cell bodies in the arcuate nucleus also contain ACTH, β-endorphin and several peptides immunologically related but not identical to α-MSH.     

Lateral hypothalamic innervation of the cerebral cortex: Immunoreactive staining for a peptide resembling but immunochemically distinct from pituitary/arcuate α-melanocyte stimulating hormone

The combination of retrograde transport of fluorescent dyes and indirect immunofluorescence has been used to study the putative neurotransmitter specificity of the tuberal lateral hypothalamic projection to the cerebral cortex. Injections of either fast blue or diamidino yellow dye into the cerebral cortex or hippocampus retrogradely labeled large, multipolar neurons scattered through the lateral hypothalamic area and zona incerta at the level of the ventromedial nucleus of the hypothalamus. Approximately 80% of these neurons stained immunohistochemically with an antiserum against α-melanocyte stimulating hormone (α-MSH). A second population of smaller, predominantly bipolar α-MSH-like immunoreactive neurons was seen in the arcuate nucleus and retrochiasmatic area, but none of these projected to the cerebral cortex. Immunohistochemical staining for ACTH (18–24), another proopiomelanocortin series peptide, or with an antiserum against α-MSH (4–10) demonstrated only the second of these cell groups. Our results indicate that the tuberal lateral hypothalamic projection to the cerebral cortex contains a substance similar but not identical to α-MSH, and that this material is probably not derived from the same proopiomelanocortin precursor as true α-MSH.     

Glucocorticoids potentiate the adenylyl cyclase—cAMP system mediated immunoreactive β-endorphin production and secretion from hypothalamic neurons in culture

Beta-endorphin(βEP)₁₋₃₁, a potent opioid peptide of proopiomelanocortin (POMC) derivatives, is produced and released from neurons at arcuate nuclei of the rat hypothalamus. Although dexamethasone (DM) suppresses the production and secretion of POMC related peptides from rat pituitary corticotrophs, the effect of glucocorticoids on the function of hypothalamic βEP neurons remains unclear. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 day treatment with 10 μM of forskolin increased ir-βEP levels in cell content and culture media by approximately1.7 (P < 0.05) and 4.1 times (P < 0.01) above vehicle treated control cultures (mean ±S.E.M., 47.3 ± 2.6 pg/well 40.4 ± 3.0 pg/well; n = 3) respectively. Although 4 day treatment with DM alone had little effect on the release and the cell content of ir-βEP, it significantly enhanced forskolin-induced elevation of ir-βEP levels in cell content and in culture media. The effect of DM was dose-related and time-dependent, with an EC₅₀ of about 1 nM; at this concentration DM enhanced ir-βEP secretion about2.1 times (P < 0.01) above that induced by 10 μM of forskolin alone. Furthermore, the potentiating effect of DM was specifically suppressed by 100 nM of RU38486 (P < 0.01), a glucocorticoid receptor antagonist, but not by an equivalent dose of RU28318, a mineralocorticoid receptor antagonist. In addition, Northern blot analysis showed that forskolin (10 μM) increased the abundance of POMC mRNA 1.4 fold above that of vehicle treated control cultures. Whereas by itself, DM (10 nM) had little effect on the level of POMC mRNA, it enhanced forskolin-stimulated increase of the abundance of POMC mRNA approximately 2.6 times. Moreover, DM also augmented1.6 times (P < 0.05) forskolin-induced but not 3-isobutyl-1-methylxanthine (IBMX)-induced increase of cAMP production (5.5 ± 0.4 pmol/well; mean ± S.E.M., n = 3) in the cultures. Taken together, our findings suggest that in contrast to the inhibitory effect on pituitary corticotrophs, glucocorticoids enhance the production and secretion of βEP from rat hypothalamic neurons by facilitating the stimulatory effect mediated, in part, through the adenylyl cyclase-cAMP system.     

Melanin-concentrating hormone (MCH) is colocalized with α-melanocyte-stimulating hormone (α-MSH) in the rat but not in the human hypothalamus

Melanin-concentrating hormone (MCH)-containing neurons have recently been localized in the dorsolateral region of the rat hypothalamus, an area where the second α-MSH system is found which contains only α-MSH and none of the pro-opiomelanocortin (POMC)-related peptides. In order to study the morphological relationships between the MCH and α-MSH neuronal systems, we have studied the immunocytochemical localization of both MCH and α-MSH in the rat hypothalamus. The same study was also performed in the human hypothalamus where there is only one α-MSH system which contains α-MSH as well as the other POMC-related peptides (first α-MSH system). In the rat dorsolateral hypothalamus, we could demonstrate that most neuronal cell bodies stained for MCH also contained immunoreactive α-MSH. In the human hypothalamus, neuronal cell bodies stained for MCH were observed only in the periventricular area whereas cell bodies containing α-MSH were exclusively located in the infundibular (arcuate) nucleus. In the rat, immunoelectron microscopy showed labelling for MCH in the dense core vesicles of positive neurons and double-staining techniques clearly demonstrated that both immunoreactive MCH and α-MSH could be consistently detected in the same dense core vesicles. These ultrastructural studies then suggest that these two peptides should be released simultaneously from neurons located in the rat dorsolateral hypothalamus.