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.     

Regulation of alpha-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus by serotonin and the interaction of serotonin with the dopaminergic system inhibiting peptide release

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from frontal slices of rat hypothalamus superfused with oxygenated artificial cerebrospinal fluid (ACSF) was quantified by radioimmunoassay. Control depolarisations with 50 mM KCl-containing ACSF produced significant increases in alpha-MSH release which were partially blocked by 10(-6) M cinanserin, a serotonin (5-HT) receptor antagonist. Superfusion of the tissues with varying concentrations of 5-HT (10(-7) M to 10(-4) M) resulted in an inverted U-shaped dose-response curve, maximum alpha-MSH release being obtained with 10(-6) M 5-HT. Addition of 10(-6) M cinanserin shifted the 5-HT dose-response curve to the right whilst the presence of 10(-8) M flupenthixol, a dopamine receptor antagonist, resulted in a sigmoidal 5-HT dose-response curve. Superfusion with ACSF containing either 10(-7) M fluoxetine, a 5-HT re-uptake inhibitor, or 10(-7) M p-chloroamphetamine, an agent releasing 5-HT, induced significant increases in alpha-MSH release which were abolished in the presence of 10(-6) M cinanserin. These data demonstrate the presence of an endogenous 5-HT system that exerts a biphasic effect on alpha-MSH release. A stimulatory effect caused by lower 5-HT concentrations appears to be a direct action whilst an inhibitory effect at higher concentrations is mediated through an inhibitory endogenous dopaminergic system. A significant proportion of K+-stimulated peptide release is 5-HT-mediated.     

Characterization of α-melanocyte-stimulating hormone (α-MSH)-like peptides in discrete regions of the rat brain. In vitro release of α-MSH from perifused hypothalamus and amygdala

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the α-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying α-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of α-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the syntheticdes-Nα-acetyl α-MSH standard. In contrast, 3 distinct forms of immunoreactive α-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl α-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated α-MSH (authentic α-MSH plus diacetyl α-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total α-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying α-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of α-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of α-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM). The effect of depolarizing concentrations of KCl was totally suppressed in the absence of Ca²⁺, indicating that high-K⁺ induced the opening of voltage-operated Ca²⁺ (VOC) channels. Veratridine (50 μM), a depolarizing agent which activates Na⁺ conductances, caused a robust stimulation of α-MSH release from hypothalamic slices but had virtually no effect on amygdala nuclei. ω-Conotoxin (1 μM), a peptide toxin which blocks L- and N-type VOC channels, caused a slight reduction of K⁺-evoked α-MSH release from hypothalamic slices but induced a dramatic decrease of α-MSH release from amygdala nuclei. These data suggest that acetylation of α-MSH to generate the biologically active forms of the peptide is a slow process which occurs gradually during axonal transport. Our results also indicate that release of α-MSH at the hypothalamic level mainly results from activation of T-type VOC channels whereas, in the amygdala nuclei, L- and (or) N-type VOC channels are involved in the regulation of α-MSH secretion.     

N-methyl-d-aspartate (NMDA) stimulates release of α-MSH from the rat hypothalamus through release of nitric oxide

Superfusion of rat hypothalamic slices with 10⁻⁴ MN-methyl-d-aspartic acid (NMDA) resulted in increased release of α-melanocyte-stimulating hormone (α-MSH). Peptide release was blocked by 10⁻⁶ MN^G-nitro-l-arginine methyl ester (l-NAME) a specific competitive inhibitor of nitric oxide synthase but not by the inactive enantiomerd-NAME at 10⁻⁶ M. The inhibition byl-NAME was reversed by the addition of 10⁻⁵ Ml-arginine, an excess of enzyme substrate. Release of nitric oxide products into tissue superfusates was stimulated by a 50 mM concentration of potassium ions and by 10⁻⁴ M NMDA. Potassium-stimulated release was blocked byl-NAME. Basal, potassium-stimulated and NMDA-stimulated release of nitric oxide products were significantly inhibited by the NMDA-receptor antagonistd-(−)-2-amino-5-phosphopentanoic acid (AP5) at 10⁻⁴ M and by the NMDA-channel blocker ketamine at 10⁻⁴ M. We conclude that nitric oxide mediates the stimulatory action of glutamic acid ont he release of α-MSH from the rat hypothalamus.     

Differential effect of ACTH1–24 and α-MSH induced grooming in the paraventricular nucleus of the hypothalamus

Injection of ACTH_1–24 as well as α-MSH in the paraventricular nucleus of the hypothalamus (PVH) induces intense grooming in the rat. While comparing the details of MSH, ACTH and control grooming, we found that the induction of grooming was highly site specific. Even injection of saline in that specific area produced some grooming, possibly due to the release of endogenous substances. To distinguish between effects caused by the peptides and the effects caused by the injection procedure, we compared the behavioural effects of saline and peptide injections in sites with exactly the same location in the PVH, in a post-hoc matched pairs design. Using this design we found that the grooming response induced by saline is of a limited. ACTH_1–24 and α-MSH prolong grooming beyond that period. Interestingly, rats receiving α-MSH continued to groom, while rats receiving ACTH_1–24 changed to scratching. This confirms earlier findings suggesting that grooming and scratching have a differential organization at the level of the PVH. Whether the peptides also have a role in the initiation of the grooming response, or just prolong a response caused by other local factors requires another experimental approach.     

Influence of centrally administered α- and γ2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat

The effect of intracerebroventricularly (i.c.v.) administered α-melanocyte-stimulating hormone (MSH) and γ₂-MSH on hypothalamic blood flow autoregulation was studied in anesthetized rats at different levels of standardized arterial hypotension. Autoregulation was impaired upon i.c.v. administration of 5 γ g/kg γ₂-MSH while α-MSH caused no change.. Since this effect of γ₂-MSH wa identical to that produced by i.c.v. naloxone in the same model, γ₂-MSH may be a functional antagonist of central opioid mechanisms participating in the control of cerebral blood flow autoregulation.     

Effect of opioid antagonism on β-endorphin processing and proopiomelanocortin-peptide release in the hypothalamus

Previous studies have shown that chronic opioid receptor blockade has significant effects on POMC gene expression and peptide levels in the hypothalamus. We have now examined the effects of the opioid antagonist naltrexone on β-EP processing in the hypothalamus and on the release of 2 POMC-derived peptides, β-EP andγ₃-MSH, from the perifused hypothalamus in vitro. The β-EP immunoactivity in the medial basal hypothalamus (MBH) of 7 rats infused for 1 week with naltrexone by osmotic minipump, was individually analyzed by HPLC and compared to 7 control rats. The mean ratio of β-EP_1–31 compared to β-EP_1–27 plus β-EP_1–26 was 2.34 ± 0.41 in the naltrexone treated rats, significantly higher than the ratio of 1.26 ± 0.09 in the control rats (P < 0.02). Thus in the setting of chronic opioid antagonism although β-EP content decreases, there is relatively more β-EP_1–31, the biologically active opioid form of the peptide, compared to the C-terminally cleaved forms of β-EP which have reduced biological activity. To study the effects of naltrexone on β-EP andγ₃-MSH release, hypothalami were perfused in vitro with 10⁻⁶M naltrexone. Basal release ofγ₃-MSH was significantly higher from the naltrexone treated brains compared to the controls (221 ± 20pg/60min vs.161 ± 6.7pg/60 min) (P < 0.01); KCl stimulatedγ₃-MSH was also significantly higher in the naltrexone group (951 ± 94 vs.543 ± 85pg/60 min) (P < 0.005). Basal release of β-EP was136 ± 45pg/60 min in the naltrexone treated brains compared to 93 ± 15pg in the controls, but this difference was not significant; KCl stimulated release of β-EP, however was significantly higher in the naltrexone group (558 ± 103 vs. 275 ± 49pg/60 min (P < 0.02). To study the acute and chronic effects of naltrexone in vivo on β-EP andγ₃-MSH release, rats were either injected with naltrexone and sacrificed 40–60 min later or were infused with naltrexone for 7 days. Baselineγ₃-MSH release was significantly higher in rats treated with naltrexone 40–60 min prior to the perifusion (P < 0.01). Baseline β-EP release was below the limit of assay detection. No differences were noted in the responses ofγ₃-MSH or β-EP to KCl in either group. In contrast after chronic treatment with naltrexone for 1 week, baseline peptide release was not different from the control animals despite a more than 50% fall in peptide content. Theγ₃-MSH and β-EP responses to KCl stimulation, however, were significantly less in the naltrexone treated animals. Thus there is an increase in POMC peptide release acutely after treatment with naltrexone in vitro and in vivo. After 1 week of naltrexone, baseline POMC peptide release continues unchanged despite the fall in peptide content, however, the response to KCl is blunted possibly reflecting the decrease in peptide content after chronic stimulation with naltrexone. We conclude that naltrexone has significant effects on POMC peptide release and on β-EP processing in the hypothalamus. These results further demonstrate that the brain POMC system can respond to opioid blockade at several levels and are consistent with inhibitory feedback mechanisms for the autoregulation of the POMC system by endogenous β-EP.     

Immunocytochemically stained binding sites for oxytocin and α-melanocyte-stimulating hormone in rat brain following ventricular administration

Binding sites for oxytocin (OXT) and alpha-melanocyte-stimulating hormone (alpha-MSH) in brain of homozygous Brattleboro rats were immunocytochemically visualized after ventricular administration of the peptides by Accurel implants. Two patterns were found: 'ring type' staining in perineuronal structures was observed in CA1 and CA3 areas of ventral hippocampus and in subiculum for OXT implanted brains and a very weak staining in striatum for alpha-MSH-implanted brains; cytoplasmic staining of intracellular binding sites was observed in the bed nucleus of the stria terminalis (BST) in brains with OXT implants and in the anterodorsal thalamic nucleus (AD) and postcingulate cortex in brains with alpha-MSH implants. These localizations are different from those described for vasopressin binding sites in the same rat strain.     

α-Melanocyte-stimulating hormone (α-MSH) release from perifused rat hypothalamic slices

A perifusion system was developed to investigate the control of α-melanocyte-stimulating hormone (α-MSH) release from rat brain. Hypothalamic slices were perifused with Krebs-Ringer bicarbonate (KRB) medium supplemented with glucose, bacitracin and bovine serum albumine. Fractions were set apart every 3 min and α-MSH levels were measured by means of a specific and sensitive radioimmunoassay method. Hypothalamic tissue in normal KRB medium released α-MSH at a constant rate corresponding to 0.1% of the total hypothalamic content per 3 min. The basal release was not altered by Ca²⁺ omission in the medium or addition of the sodium channel blocker tetrodotoxine (TTX). Depolarizing agents such as potassium (50 mM) and veratridine (50 μM), which is known to increase Na⁺ conductance, significantly stimulated α-MSH release in a Ca²⁺-dependent manner. When Na⁺-channels were blocked by TTX (0.5 μM) the stimulatory effect of veratridine was completely abolished whereas the K⁺-evoked release was unaffected. These findings suggest that: (1) voltage-dependent sodium channels are present on α-MSH hypothalamic neurons; (2) depolarization by K⁺ induces a marked stimulation of α-MSH release; (3) K⁺- and veratridine-evoked releases are calcium-dependent. Altogether, these data provide evidence for a neurotransmitter or neuromodulator role for α-MSH in rat hypothalamus.     

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.     

Lack of effect of interleukins 1α and 1β, during in vitro perifusion,on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin-releasing factor (CRF)-induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations. The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF-induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10^?12 to 10^?9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley-Liss, Inc.     

Subcellular localization of immunoreactive α-melanocyte stimulating hormone in human brain

The regional and subcellular distribution of immunoreactive α-melanocyte stimulating hormone (α-MSH₁) in the post mortem adult human brain was investigated. α-MSH_i was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of α-MSH_i were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of α-MSH_i in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, α-MSH_i was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of α-MSH_i were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that α-MSH is a neuronal peptide in the human brain.     

The descending α-MSHergic (α-melanocyte-stimulating hormone-ergic) projections from the zona incerta and lateral hypothalamic area to the inferior colliculus and spinal cord in the rat

Neuronal pathways containing α-melanocyte-stimulating hormone (α-MSH) extending from the zona incerta and lateral hypothalamic area to the inferior colliculus and spinal cord were analyzed using both immunohistochemical localization and a retrograde tracer. Biotinized horseradish peroxidase injected into the inferior colliculus or the thoracic cord of the rat labeled a number of neurons in the zona incerta and lateral hypothalamic area. Simultaneous immunostaining of the same sections with α-MSH antiserum showed that some of these neurons are α-MSHergic.     

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.     

Microinjection of the α1-agonist methoxamine into the paraventricular hypothalamus induces anorexia in rats

Adrenergic receptors within the paraventricular hypothalamus (PVN) play a prominent role in the control of food intake: stimulation of α2-adrenoceptors induces food intake whereas stimulation of α1-adrenoceptors suppresses food intake. This study further examines the role of PVN α1-adrenoceptors hy examining the effects on food and water intake of the α1-adrenergic agonist methoxamine (100, 200, 400 nMol) microinjected into the rat paraventricular hypothalamus. Methoxamine suppressed food intake but not water intake. Doses of 100, 200, and 400 nMol methoxamine suppressed food intake by 47%. 64%, and 96%, respectively. These results further confirm the hypothesis that administration of α1-agonists into the PVN acts to significantly suppress food intake; an action that is in opposition to the facilitory effects of α2-adrenergic agonists on food intake.     

Release of hypothalamic corticotropin-releasing hormone and arginine-vasopressin by interleukin 1β and αMSH: studies in rats with different susceptibility to inflammatory disease

The susceptibility of Lewis rats is related to blunted hypothalamic-pituitary-adrenal (HPA) axis responsiveness to a variety of inflammatory and neuroendocrine stimuli. In contrast resistance to inflammatory disease of histocompatible Fischer rats is associated with their intact HPA axis responses to the same stimuli. We have examined the contribution of IL-1β to in vitro corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) release from hypothalamic explants derived from LEW/N and F344/N rats. The same animal model has been used to investigate the regulatory effect of αMSH, an immunosuppressive neurohormone, on IL-1β stimulated CRH and AVP secretion. CRH basal release in both strains was similar. However, LEW/N hypothalamic AVP basal secretion was significantly elevated. CRH relative response of LEW/N hypothalamic explants to IL-1β stimulation was lower compared to Fischer, which is consistent with their hyporesponsiveness to inflammatory mediators. AVP secretion however, was significantly decreased in hypothalamic explants from both strains after 40 min exposure to IL-1β. αMSH suppressed basal CRH and AVP release in both LEW/N and F344/N rats and prevented IL-1β stimulated CRH secretion in these strains. AVP was further diminished in F344/N explants following incubation with αMSH+IL-1β, while LEW/N level was significantly elevated. However, AVP levels remained significantly below baseline in explants from both strains after final incubation with IL-1β. Although our findings indicate a modulatory action of αMSH in HPA axis regulation in vitro, the physiological importance of this phenomenon in Lewis and Fischer rats requires further investigation.     

β-Endorphin responses to different serotonin agonists: involvement of corticotropin-releasing hormone, vasopressin and direct pituitary action

Activation of serotonergic neurotransmission has been shown to increase plasma β-endorphin-like immunoreactivity (β-End-LI). To study the mechanism(s) of this action, we measured the effects of 3 potent serotonin (5-HT) agonists with different structures and 5-HT receptor binding profiles in conscious unrestrained Sprague-Dawley rats in vivo and in dispersed anterior pituicytes in vitro. The 5-HT_1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT_1C agonist, m-chlorophenylpiperazine(m-CCP), and the 5-HT₂ agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), all markedly increased β-End-LI in plasma in vivo. All 3 responses were blocked by dexamethasone pretreatment. Pituitary stalk transection (PST), as well as pretreatment with rabbit serum hyperimmune against rat corticotropin-releasing hormone (CRH, TS-6) completely abolished β-End-LI response to 8-OH-DPAT and attenuated the responses by about 60% to DOI. Responses to m-CPP were markedly attenuated in PST rats, but pretreatment with TS-6 had no significant effect. To examine whether vasopressin (AVP) might be involved in the CRH neutralizing antibody-resistant β-End-LI responses after m-CPP and DOI, we measured AVP concentrations after each agonist. m-CPP, but not DOI or 8-OH-DPAT, significantly elevated circulating AVP levels. As a proof of direct pituitary effectm DOI markedly stimulated β-End-LI release from the anterior pituitary cell culture preparation in vitro. It was approximately as potent as CRH in the picomolar range. m-CPP was much less effective than DOI, while 8-OH-DPAT did not stimulate β-End-LI release in vitro. Thus, the present findings suggest that the 5-HT_1A agonist 8-OH-DPAT causes plasma β-End-LI increases in vivo by stimulating hypothalamic CRH secretion. The mechanism of β-End-LI response to the 5-HT_1C agonist m-CPP is a complex phenomenon including mainly AVP-mediated and direct pituitary actions. The 5-HT₂ agonist DOI may elicit β-End-LI responses by CRH-mediated and direct pituitary effects.     

Differential projections of two immunoreactive α-melanocyte stimulating hormone (α-MSH) neuronal systems in the rat brain

Immunocytochemical localization of α-MSH was performed in the brain of rats of which the arcuate nucleus has been destroyed by treatment with monosodium glutamate in the neonatal period. In these animals, α-MSH cell bodies normally found in the arcuate nucleus were almost completely absent. The reactive fibers found in the ACTH-β-LPH pathway were also markedly decreased. On the other hand, α-MSH cell bodies located in the dorsolateral hypothalamus as well as fibers located outside the ACTH-β-LPH pathway were not decreased. These results strongly suggest that α-MSH cell bodies in dorsolateral hypothalamus have projections completely different from those located in the arcuate nucleus.