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.     

Release of immunoreactive alpha-MSH by synaptosome-enriched fractions of homogenates of hypothalami.

Immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and alpha-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound alpha-MSH, recovered by extraction with acid ethanol, and alpha-MSH released from the synaptosome preparation, were immunologically similar to synthetic alpha-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound alpha-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca2+, alpha-MSH release increased with increasing concentrations (30-150 mM) of K+. The stimulatory effect of 60 mM K+ was complete within 2 min and was potentiated by increasing Ca2+ concentrations over the range of 0 to 2 mM. K+-induced release of alpha-MSH was independent of temperature from 1 to 30 degrees C, and neither glucose (10 mM) nor dopamine (10(-10)-10(-2) M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive alpha-MSH that is mobilized by depolarizing concentrations of K+ in a Ca2+-dependent manner.     

Alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) containing neurons and the molecular forms of alpha-MSH-related peptides exhibit substantial differences in the brains of fish and amphibians. Lungfishes, which share similarities with both fishes and tetrapods, represent a valuable group in which to investigate the neuroanatomical and neurochemical facets of evolution. In the present study, we have localized and characterized alpha-MSH-immunoreactive peptides in the central nervous system of the African lungfish Protopterus annectens. Perikarya exhibiting alpha-MSH-like immunoreactivity were observed in two distinct regions of the hypothalamus: the rostral part of the preoptic nucleus and the caudal part of the hypothalamus. In the caudal hypothalamus most alpha-MSH-immunopositive perikarya were located in both the subependymal and deepest layers of the ventral periventricular region. Scattered alpha-MSH-immunopositive cells were occasionally detected in the dorsal side of the caudal hypothalamus. The alpha-MSH-immunoreactive material localized in the brain was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The displacement curves obtained with synthetic alpha-MSH and serial dilutions of brain and pituitary extracts were parallel. HPLC analysis of lungfish hypothalamic extracts showed that the major immunoreactive peak coeluted with synthetic desacetyl alpha-MSH and its sulfoxide derivative. An additional peak coeluted with synthetic sulfoxide alpha-MSH. In contrast, in the pituitary, the predominant form of alpha-MSH-like material coeluted with the N,O-diacetyl alpha-MSH standard. These results provide the first evidence for the presence of alpha-MSH-related peptides in the brain of a lungfish. The distribution of alpha-MSH neuronal systems in the lungfish is very similar to that reported in amphibians, supporting the existence of phylogenetic convergences between these two vertebrate groups.     

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) is not under dopaminergic control

A possible dopaminergic regulation of hypothalamic proopiomelanocortin (POMC)-containing neurons has been investigated in rats by means of in vivo and in vitro approaches. Acute or 3-weeks chronic in vivo treatments with the dopaminergic agonists apomorphine (1 mg/kg: s.c.) and 2-Br-alpha-ergocriptine (2.5 mg/kg; s.c.) or the dopaminergic antagonist haloperidol (0.15-3 mg/kg; i.p.) had no significant effect on the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) in two hypothalamic regions: arcuate nucleus (AN) and dorsolateral area (DLH). In the same way, chronic administration of the dopaminergic agonists or antagonist did not induce any change in hypothalamic contents of beta-endorphin, another peptide derived from POMC. Reverse-phase high-performance liquid chromatographic analysis revealed that acetic acid extracts of AN and DLH both contained two major forms of alpha-MSH-like peptides: deacetylated alpha-MSH and authentic alpha-MSH. The ratio between these two forms was not altered after acute haloperidol treatment (3 mg/kg, i.p.). The possible effect of dopamine on the release of hypothalamic alpha-MSH was studied in vitro using perifused rat hypothalamic slices. Infusion of dopamine (10(-7)-10(-5)M) or its antagonist haloperidol (10(-5)M) had no effect on spontaneous alpha-MSH release from hypothalamic tissue. In addition, none of these drugs had any effect on potassium (50 mM)-induced alpha-MSH release. It is concluded that dopaminergic neurons are not involved in the regulation of synthesis, post-translational processing (acetylation) or release of hypothalamic alpha-MSH.     

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.     

Characteristics of NMDA receptors that stimulate release of hypothalamic alpha-MSH.

N-methyl-D-aspartic acid (NMDA) 10(-4) M stimulated release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus through receptors which shared common features with other central NMDA-type glutamate receptors. The receptors possessed inhibitory sites for both Mg2+ and ketamine; basal and NMDA-stimulated alpha-MSH release was reduced by high (5 mM) Mg2+ ion concentrations and by 10(-4) M ketamine, whilst use of Mg(2+)-free media led to a prolongation of the NMDA-stimulated response. The receptors were also shown to possess an allosteric glycine site. The glycine site agonist D-serine 10(-4) M potentiated basal and NMDA-stimulated alpha-MSH release whilst the antagonist, 7-chlorokynurenic acid 10(-4) M, reduced NMDA-stimulated release, an effect which was partially reversed by 10(-4) M D-serine.     

Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus

The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta-endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta-endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1-26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.     

Involvement of voltage-operated calcium channels in alpha-melanocyte-stimulating hormone (alpha-MSH) release from perifused rat hypothalamic slices

The contribution of voltage-operated calcium (VOC) channels in the mechanism of release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated using perifused rat hypothalamic slices. The stimulatory effect of potassium (50 mM) on alpha-MSH release was completely blocked by cadmium (1 mM) a calcium competitor which indifferently blocks T-, L-and N-type VOC channels. To determine the nature of calcium conductances involved in K+-evoked alpha-MSH release, we have investigated the effect of a VOC channel agonist and 3 antagonists on the secretion of the neuropeptide. Administration of synthetic omega-conotoxin fraction GVIA (1 microM), a peptide toxin which blocks both N- and L-type VOC channels, reduced by 33% K+-induced alpha-MSH release. In contrast, the 1,4-dihydropyridine (DHP) antagonist nifedipine, at concentrations up to 100 microM, did not affect the response of hypothalamic alpha-MSH neurons to depolarizing concentrations of KCl. In addition, the secretion of alpha-MSH induced by high K+ concentrations was not reduced by nifedipine (10 microM) in the presence of diltiazem (1 microM), a benzothiazepine derivative which increases the affinity of the DHP antagonist for L-type VOC channels. The DHP agonist BAY K 8644 (0.1-10 microM) did not modify the early phase of the response of alpha-MSH neurons to K+-induced depolarization. In contrast BAY K 8644 (1 or 10 microM) significantly prolonged the duration of K+-induced alpha-MSH release. This sustained release of alpha-MSH induced by BAY K 8644 (10 microM) was totally suppressed by nifedipine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral hypothalamic innervation of the cerebral cortex: immunoreactive staining for a peptide resembling but immunochemically distinct from pituitary/arcuate alpha-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 alpha-melanocyte stimulating hormone (alpha-MSH). A second population of smaller, predominantly bipolar alpha-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 alpha-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 alpha-MSH, and that this material is probably not derived from the same proopiomelanocortin precursor as true alpha-MSH.     

Localization of vasopressin in the rat brain

The distribution of arginine vasopressin (AVP) in the rat brain was studied using a sensitive radioimmunoassay. The highest concentration of AVP was found in the hypothalamus. Individually, the supraoptic, paraventricular and suprachiasmatic nuclei contained in the order of 10% of the total hypothalamic content. Vasopressin was also found in the thalamus, medulla, cerebellum, amygdala, substantia nigra and hippocampus. Much lower levels were detected in the pons, spinal cord, frontal and occipital lobes and caudate putamen. No AVP could be detected in any other regions of the cortex or corpus callosum. Chromatographically the vasopressin found outside the hypothalamus is of a similar nature to that of hypothalamo-hypophysial origin. This study supports previous reports of extrahypothalamic localization of vasopressin by immuno-histochemical methods. It is clear that AVP is not confined to the hypothalamo-hypophysial axis, and the possibilities that this may reflect an involvement in brain function are discussed.     

Corticotropin-releasing factor immunoreactivity is widely distributed within the central nervous system of the rat: an immunohistochemical study

The discovery of a 41-amino acid peptide with potent corticotropin-releasing factor properties has prompted a search for neurons that contain this substance and potentially utilize it in intercellular communication. The present study utilized immunohistochemical methods and an antiserum directed against a synthetic replica of ovine corticotropin-releasing factor. The rat hypothalamus was found to contain striking immunoreactive groups of neuronal perikarya within the paraventricular, periventricular, and anterior hypothalamic nuclei, some of which are likely to project to the external layer of the median eminence and thereby comprise a hypophysiotropic system. Certain other hypothalamic nuclei, as well as many other regions of the central nervous system, were found to contain corticotropin-releasing factor-immunoreactive neurons. Among the most prominent of these were neurons in the bed nucleus of stria terminalis, the central nucleus of the amygdala, the region of the dorsal raphe, locus ceruleus, the external cuneate nucleus, and the medullary reticular formation. Thus, corticotropin-releasing factor, like many other neurohormones and peptides, may participate in neuroendocrine regulation as well as play a role as a neurotransmitter-like substance in numerous extrahypothalamic circuits.     

Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.     

Identification, characterization and stereotaxic mapping of intraneuronal alpha-melanocyte stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain.

A highly specific antibody to alpha-melanocyte stimulating hormone (alpha-MSH) was used to histochemically localize and biochemically identify and quantitate alpha-MSH immunoreactivity in nerve fibers and cell bodies of the rat brain. alpha-MSH-like immunoreactivity was contained in fibers throughout the brain. The distribution of alpha-MSH was determined by immunocytochemistry as well as by radioimmunoassay combined with microdissection techniques. High concentrations of alpha-MSH were contained in the nucleus interstitialis stria terminalis, the median eminence and the medial preoptic, anterior hypothalamic, periventricular, paraventricular, arcuate, dorsomedial, and posterior hypothalamic nuclei. Moderate alpha-MSH concentrations were noted in the amygdala, septum, central gray, dorsal raphe, and the nucleus tractus solitarius. Cell bodies containing alpha-MSH were observed only in the arcuate nucleus. The alpha-MSH-like compound in brain had similar immunochemical and electrophoretic properties of standard alpha-MSH but high pressure liquid chromatographic analysis demonstrated that the alpha-MSH-like immunoreactivity was comprised of one major and two minor components. The major immunoreactive peak had an identical retention time as alpha-MSH and therefore may be chemically identical to alpha-MSH. The similar retention times and immunoreactivity of the other two compounds suggest a similarity in size and structure to alpha-MSH. These observations demonstrate that fibers containing alpha-MSH emanate from the arcuate nucleus to innervate many other regions of the rat brain.     

Characterization of alpha-MSH-related peptides released from rat hypothalamic neurons in vitro

Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for alpha-melanocyte-stimulating hormone (alpha-MSH), was used to characterize the alpha-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of alpha-MSH-like immunoreactivity (alpha-MSH-LI) co-eluting with synthetic des-N alpha-acetyl alpha-MSH, alpha-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl alpha-MSH which was the predominant peptide represented 94.4% of total alpha-MSH-LI content, while the relative amount of alpha-MSH was only 5.6%. Analysis of alpha-MSH-related peptides contained in effluent perifusates showed that deacetyl alpha-MSH and its oxidized form were the major peptides released from neurons in basal conditions or under KCl-induced depolarization (50 mM KCl for 75 min). However, the proportion of acetylated peptide was 3-4 times higher in the perifusion medium than in hypothalamic extracts. Our data indicate that acetylation of des-N alpha-acetyl alpha-MSH may occur during the process of exocytosis. Since acetylation of alpha-MSH markedly increases the behavioural potency of the peptide, these results suggest that regulation of the acetyltransferase activity could be a key mechanism to modulate the bioactivity of alpha-MSH-related peptides in the brain.     

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.     

Effects of mediobasal hypothalamic lesion on immunoreactive ACTH/beta-endorphin levels in cerebrospinal fluid, in discrete brain regions, in plasma, and in pituitary of the rat.

One week after complete destruction of the mediobasal hypothalamus, immunoreactive adrenocorticotropin (ACTH) and beta-endorphin levels were determined in cerebrospinal fluid, trunk blood, as well as in brain and pituitary tissue samples collected from anaesthetized and cisternally cannulated rats. Control rats were sham operated. In lesioned rats we observed: (a) 60% decrease in the immunoreactive beta-endorphin concentrations in the cerebrospinal fluid, (b) decreased immunoreactive ACTH and beta-endorphin levels in the hypothalamus, in the thalamus and in the amygdala, (c) unaffected immunoreactive ACTH/beta-endorphin levels in the septum and in the hippocampus, (d) decreased immunoreactive beta-endorphin levels both in the anterior and neurointermediate pituitary but unchanged immunoreactive ACTH contents in the anterior lobe, and (e) unaffected immunoreactive ACTH and beta-endorphin levels in the plasma under stressful conditions. From these findings the following conclusions can be drawn: (1) more than 50% of the beta-endorphin-like peptide content of the cerebrospinal fluid originates from the periventricular nuclei of the hypothalamus and thalamus in the rat; (2) the loss of the hypothalamic control probably enhances the intracellular proteolytic degradation of beta-endorphin both in the anterior and neurointermediate pituitary lobe; (3) rats with mediobasal hypothalamic lesion cannot react to the stressful stimuli of ether anaesthesia or cisternal cannulation with elevated plasma immunoreactive ACTH and beta-endorphin levels.     

Role of alpha-MSH and related peptides in the central nervous system

Alpha-melanocyte stimulating hormone (alpha-MSH) is a tridecapeptide secreted by intermediate lobe cells and synthesized in the brain as well. As a hormonal peptide, the physiological function of alpha-MSH consists mainly in the control of pigment movements within dermal melanophores. At the pituitary level, alpha-MSH secretion is under multifactorial control: it is inhibited by dopamine and GABA and stimulated by corticoliberin (CRF), thyroliberin (TRH), beta-adrenergic agonists and (or) serotonin. Identification of alpha-MSH containing neurons in the hypothalamus and other brain regions (septum, thalamus, mid-brain, striatum, hippocampus, cerebral cortex and spinal cord) has been carried out by means of immunological and biochemical techniques combined with bioassays. In the central nervous system (CNS) as in the hypophysis, alpha-MSH is synthesized from a high molecular weight precursor, pro-opiomelanocortin (POMC). Maturation of this protein yield similar end products in the hypothalamus and the intermediate lobe. Several peptides chemically related to alpha-MSH are generated including the desacetyl and monoacetyl (authentic alpha-MSH) forms; the latter has the greatest behavioral activity. The demonstration that alpha-MSH has numerous central nervous system effects has raised the possibility that this neuropeptide acts as a neuromodulator or a neurotransmitter. In the rat, intra-cerebroventricular administration of ACTH/MSH peptides induces the stretching-yawning syndrome (SYS) which is frequently preceded by excessive grooming. This excessive grooming is blocked by neuroleptics indicating that the central dopaminergic neurons are implicated in this behavioral effect of the peptide. alpha-MSH is involved in memory, arousal and attention; in hypophysectomized animals, the learning ability is restored after administration of MSH or related peptides. Injection of alpha-MSH delays also extinction of passive avoidance behavior and affects performances motivated by hunger as well as aggressive behavior. Recent studies concerning the role of alpha-MSH have been undertaken in human beings. The effects of MSH-related peptides favour a role of these peptides in arousal: they maintain a high level of vigilance and improve visual discrimination. These behavioral changes were accompanied by marked changes in CNS electrophysiology. Current studies, which aim at establishing a neurotransmitter function for alpha-MSH, concern the distribution and characterization of alpha-MSH receptors in the central nervous system and the mechanism controlling the release of neuronal alpha-MSH.     

Melanin-concentrating hormone (MCH) is colocalized with alpha-melanocyte-stimulating hormone (alpha-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 alpha-MSH system is found which contains only alpha-MSH and none of the pro-opiomelanocortin (POMC)-related peptides. In order to study the morphological relationships between the MCH and alpha-MSH neuronal systems, we have studied the immunocytochemical localization of both MCH and alpha-MSH in the rat hypothalamus. The same study was also performed in the human hypothalamus where there is only one alpha-MSH system which contains alpha-MSH as well as the other POMC-related peptides (first alpha-MSH system). In the rat dorsolateral hypothalamus, we could demonstrate that most neuronal cell bodies stained for MCH also contained immunoreactive alpha-MSH. In the human hypothalamus, neuronal cell bodies stained for MCH were observed only in the periventricular area whereas cell bodies containing alpha-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 alpha-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.     

Corticotropin releasing factor-like immunoreactivity in the rat brain as revealed by a modified cobalt-glucose oxidase-diaminobenzidine method

A cobalt-glucose-oxidase diaminobenzidine (Co-GOD) method, employing a specific antiserum against rat corticotropin releasing factor (CRF), was applied to determine immunohistochemically a widespread and detailed localization of corticotropin releasing factor-like immunoreactivity (CRFI) in the rat brain. Besides the CRFI cells in the paraventricular hypothalamic nucleus that project to the median eminence, CRFI cells were demonstrated in many brain regions, including the olfactory bulb, cerebral cortex, septal nuclei, hippocampus, amygdala, thalamic nuclei, medial hypothalamic nuclei, lateral hypothalamic area, perifornical area, central gray, cuneiform nucleus, inferior colliculus, raphe nuclei, mesencephalic reticular formation, laterodorsal tegmental nucleus, locus coeruleus, parabrachial nuclei, mesencephalic tract of the trigeminal nerve, pontine reticular formation, lateral superior olive, vestibular nuclei, prepositus hypoglossal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, lateral reticular nucleus, nucleus of the spinal tract of the trigeminal nerve, external cuneate nucleus, inferior olive, and medullary reticular formation. CRFI-reacting neural processes were also detected in these same areas. In particular, the median eminence, lateral septum, bed nucleus of the stria terminalis, mesencephalic reticular formation, parabrachial nuclei, and nucleus of the solitary tract contained large numbers of CRFI fibres. The widespread localization of CRFI demonstrated in the present study strongly suggests that CRF, like many other neurohormones and peptides, may act as a neurotransmitter and/or neuromodulator in numerous extrahypothalamic circuits, as well as participate in neuroendocrine regulation.     

Regional distribution of gastrin-releasing peptide- and somatostatin-like immunoreactivity in the rabbit hypothalamus

Regional distribution of gastrin-releasing peptide- (GRP) and somatostatin (SRIF)-like immunoreactivity in the discrete nuclei of the hypothalamus was examined in the rabbit according to Palkovits' microdissection method. GRP-like immunoreactivity (LI) was detected abundantly in the hypothalamus as compared with the cerebral cortex when measured by radioimmunoassay using the antiserum recognizing the C-terminal portion of synthetic porcine GRP. On gel-filtration chromatography of the hypothalamic extracts, two major peaks of GRP-LI were eluted; the peak with larger molecular size corresponded to synthetic porcine GRP1-27 and the smaller size to porcine GRP14-27. A concentration of GRP-LI was highest in the infundibular nuclei (IFN) as well as the ventromedial nuclei (VMN), and next high in the paraventricular nuclei (PVN), suprachiasmatic nuclei (SCN) and periventricular nuclei (PEV). The content of GRP-LI in the median eminence was not so much when compared with them. On the other hand, SRIF was localized in the highest concentration in the ME, followed by the VMN and IFN, as well as the PEV. The findings indicate that porcine GRP-LI exists in the hypothalamus of rabbits with characteristic regional distribution. Concurrent localization of GRP-LI and SRIF in some parts of the hypothalamus may suggest the interaction of both peptides in these areas under various physiological and pathological status.