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O hormônio concentrador de melanina (MCH) foi inicialmente identificado em peixes teleósteos por regular a mudança de coloração, agregando os grânulos de pigmentos nas células pigmentares, levando ao clareamento dos animais. Possui dois receptores conhecidos: MCHR1, encontrado em todas as espécies, e o MCHR2, no SNC de alguns mamíferos e em outros tecidos de teleósteos. Devido a sua ampla distribuição, diversos estudos tem investigado o papel do MCH como um peptídeo que exerce diversas funções fisiológicas: regulação da ingestão de alimentos em mamíferos e peixes, regulação da resposta ao stress e à ansiedade, comportamento, locomoção, reprodução, sono, memória, inflamação. O objetivo desse trabalho foi investigar a possível participação do MCH na ativação das vias de sinalização para oxido nítrico em células de eritroforoma de Carassius auratus (GEM-81). Adicionalmente, incluímos o hormônio estimulador de melanócitos - ?-MSH no estudo, pois este é antagônico ao MCH nas mais diversas funções. Demonstramos que em GEM-81 ocorre a expressão da isoforma iNOS sem o estimulo de lipopolissacarídeos; e que com o tratamento de MCH e Soro Fetal Bovino (SFB) reduzido a 0,5%, sua expressão foi aumentada com o decorrer do tempo, com o máximo alcançado com 180 min e 360 min. Na produção de nitrito foi observado um aumento muito discreto nos mesmos tempos. O α-MSH não apresentou nenhuma diferença de expressão de iNOS e na produção de nitrito. Nossos dados apontam a existência da via do óxido nítrico em células de eritroforoma de Carassius auratus e sugerem que o MCH possa induzir a produção de oxido nítrico pelo aumento de expressão da iNOS / The melanin -concentrating hormone (MCH) was first identified in teleost fish by regulating the color change, aggregating the pigment granules in pigment cells, leading to bleaching of animals. It has two known receptors: MCHR1, found in all species, and MCHR2, in the CNS of mammals and some other tissues of teleosts. Because of their widespread distribution, various studies have investigated the role of MCH as a peptide that exerts various physiological functions: regulation of food intake in mammals and fish, regulation of stress responses and anxiety, behavior, locomotion, reproduction, sleep, memory and inflammation. The aim of this study was to investigate the possible involvement of MCH in the activation of signaling pathways for nitric oxide in cells of Carassius auratus eritroforoma (GEM -81). Additionally, we include the melanocyte stimulating hormone - α - MSH in the study, as this is antagonistic to MCH in various functions. We demonstrated that GEM -81 isoform expression of iNOS without lipopolysaccharide stimulation occurs, and the treatment with MCH and Fetal Bovine Serum (FBS) reduced to 0.5% increased the expression with the passage of time, the maximum reached at 180 min and 360 min. Regarding nitrite production, it was observed a very modest increase at the same time. The α-MSH showed no difference in the expression of iNOS or production of nitrite. Our data indicate the presence of the nitric oxide pathway in cells of eritroforoma Carassius auratus, suggesting that MCH could induce nitric oxide production by increasing the expression of iNOS .

A área incerto-hipotalâmica (IHy) é uma região diencefálica que atua em funções reprodutivas através da presença do hormônio concentrador de melanina (MCH). Nosso objetivo é estudar as conexões da IHy de fêmeas com áreas relacionadas ao controle neuroendócrino reprodutivo, usando traçador anterógrado dextrana amina biotinilada (BDA) e retrógrado Fluorogold (FG). Nesse sentido, destacamos que a IHy, em fêmeas, conecta-se reciprocamente com diversos núcleos hipotalâmicos da região periventricular e pré-óptica, além das áreas hipotalâmica anterior, posterior e lateral. Nosso resultados também sugerem um dimorfismo sexual das projeções da IHy, já que a área pré-óptica medial, o núcleo anteroventral periventrciular e o núcleo arqueado são mais densamente inervadas pelas IHy em fêmeas do que em machos. / The incerto-hypothalamic area (IHy) is a diencephalic region poorly studied which is characterized by two non-colocalized neurochemical groups composed by the A13 dopaminergic group intermingled with predominantly GABAergic cells co-expressing melanin-concentrating hormone (MCH) and/or cocaine and amphetamine regulated transcript (CART). Functional studies suggest that IHy is involved in the neuroendocrine control of female reproduction in specific metabolic states mediated by MCH. In this way, our aim is to study the IHy connections in female rats related to neuroendocrine control of female reproduction using neuronal anterograde Biotin Dextran Amine (BDA) and retrograde Flurogold (FG) tracers. As a result, we found that IHy projections are the lateral septal nucleus, bed nucleus of stria terminalis, paraventricular thalamic nucleus, reuniens thalamic nucleus, medial preoptic area, medial and lateral preoptic nucleus, median preoptic nucleus, periventricular hypothalamic nucleus, anteroventral periventricular nucleus, anterior, posterior and lateral hypothalamic area and precommissural nucleus. The main afferents to the IHy, in females, are the lateral septal nucleus, median preoptic nucleus and paraventricular thalamic nucleus. These results suggest sexually dimorphic projections from the IHy, since IHy more densely innervates neuroendocrine regions in female than in male rats.

A área incerto-hipotalâmica (IHy) é uma região diencefálica que atua em funções reprodutivas através da presença do hormônio concentrador de melanina (MCH). Nosso objetivo é estudar as conexões da IHy de fêmeas com áreas relacionadas ao controle neuroendócrino reprodutivo, usando traçador anterógrado dextrana amina biotinilada (BDA) e retrógrado Fluorogold (FG). Nesse sentido, destacamos que a IHy, em fêmeas, conecta-se reciprocamente com diversos núcleos hipotalâmicos da região periventricular e pré-óptica, além das áreas hipotalâmica anterior, posterior e lateral. Nosso resultados também sugerem um dimorfismo sexual das projeções da IHy, já que a área pré-óptica medial, o núcleo anteroventral periventrciular e o núcleo arqueado são mais densamente inervadas pelas IHy em fêmeas do que em machos. / The incerto-hypothalamic area (IHy) is a diencephalic region poorly studied which is characterized by two non-colocalized neurochemical groups composed by the A13 dopaminergic group intermingled with predominantly GABAergic cells co-expressing melanin-concentrating hormone (MCH) and/or cocaine and amphetamine regulated transcript (CART). Functional studies suggest that IHy is involved in the neuroendocrine control of female reproduction in specific metabolic states mediated by MCH. In this way, our aim is to study the IHy connections in female rats related to neuroendocrine control of female reproduction using neuronal anterograde Biotin Dextran Amine (BDA) and retrograde Flurogold (FG) tracers. As a result, we found that IHy projections are the lateral septal nucleus, bed nucleus of stria terminalis, paraventricular thalamic nucleus, reuniens thalamic nucleus, medial preoptic area, medial and lateral preoptic nucleus, median preoptic nucleus, periventricular hypothalamic nucleus, anteroventral periventricular nucleus, anterior, posterior and lateral hypothalamic area and precommissural nucleus. The main afferents to the IHy, in females, are the lateral septal nucleus, median preoptic nucleus and paraventricular thalamic nucleus. These results suggest sexually dimorphic projections from the IHy, since IHy more densely innervates neuroendocrine regions in female than in male rats.

O hormônio concentrador de melanina (MCH) foi inicialmente identificado em peixes teleósteos por regular a mudança de coloração, agregando os grânulos de pigmentos nas células pigmentares, levando ao clareamento dos animais. Possui dois receptores conhecidos: MCHR1, encontrado em todas as espécies, e o MCHR2, no SNC de alguns mamíferos e em outros tecidos de teleósteos. Devido a sua ampla distribuição, diversos estudos tem investigado o papel do MCH como um peptídeo que exerce diversas funções fisiológicas: regulação da ingestão de alimentos em mamíferos e peixes, regulação da resposta ao stress e à ansiedade, comportamento, locomoção, reprodução, sono, memória, inflamação. O objetivo desse trabalho foi investigar a possível participação do MCH na ativação das vias de sinalização para oxido nítrico em células de eritroforoma de Carassius auratus (GEM-81). Adicionalmente, incluímos o hormônio estimulador de melanócitos - ?-MSH no estudo, pois este é antagônico ao MCH nas mais diversas funções. Demonstramos que em GEM-81 ocorre a expressão da isoforma iNOS sem o estimulo de lipopolissacarídeos; e que com o tratamento de MCH e Soro Fetal Bovino (SFB) reduzido a 0,5%, sua expressão foi aumentada com o decorrer do tempo, com o máximo alcançado com 180 min e 360 min. Na produção de nitrito foi observado um aumento muito discreto nos mesmos tempos. O α-MSH não apresentou nenhuma diferença de expressão de iNOS e na produção de nitrito. Nossos dados apontam a existência da via do óxido nítrico em células de eritroforoma de Carassius auratus e sugerem que o MCH possa induzir a produção de oxido nítrico pelo aumento de expressão da iNOS / The melanin -concentrating hormone (MCH) was first identified in teleost fish by regulating the color change, aggregating the pigment granules in pigment cells, leading to bleaching of animals. It has two known receptors: MCHR1, found in all species, and MCHR2, in the CNS of mammals and some other tissues of teleosts. Because of their widespread distribution, various studies have investigated the role of MCH as a peptide that exerts various physiological functions: regulation of food intake in mammals and fish, regulation of stress responses and anxiety, behavior, locomotion, reproduction, sleep, memory and inflammation. The aim of this study was to investigate the possible involvement of MCH in the activation of signaling pathways for nitric oxide in cells of Carassius auratus eritroforoma (GEM -81). Additionally, we include the melanocyte stimulating hormone - α - MSH in the study, as this is antagonistic to MCH in various functions. We demonstrated that GEM -81 isoform expression of iNOS without lipopolysaccharide stimulation occurs, and the treatment with MCH and Fetal Bovine Serum (FBS) reduced to 0.5% increased the expression with the passage of time, the maximum reached at 180 min and 360 min. Regarding nitrite production, it was observed a very modest increase at the same time. The α-MSH showed no difference in the expression of iNOS or production of nitrite. Our data indicate the presence of the nitric oxide pathway in cells of eritroforoma Carassius auratus, suggesting that MCH could induce nitric oxide production by increasing the expression of iNOS .

Les neurones exprimant l'hormone de mélano-concentration (MCH) sont observés dans l'hypothalamus postérieur de tous les vertébrés, de la lamproie à l'Homme. Ces neurones sont impliqués dans diverses fonctions comme le cycle veille/sommeil ou la prise alimentaire. Ils forment une population non homogène et au moins deux sous-populations sont reconnues, chez le rat. La première sous-population est composée de neurones nés au 11ème jour de vie embryonnaire (E11) qui projettent massivement sur les régions les plus postérieures du système nerveux central. La seconde est générée à E12/E13 et les neurones la caractérisant projettent sur les régions les plus antérieures du cerveau et expriment le peptide CART (cocaine and amphetamine regulated transcript) et le récepteur NK3 (neurokinine). L'objectif de notre travail était de comprendre l'origine de ces deux sous-populations. Pour cela, nous avons utilisé des approches histologiques, moléculaires et in vitro. Les neurones à MCH sont parmi les premiers neurones à naître et à différencier leur phénotype chimique le long d'une région longitudinale définie par une prolifération intense, appelée " cell cords " par Keyser en 1972. Cette bande longitudinale est caractérisée par l'expression de gènes comme Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 et a été récemment renommée " diagonale intrahypothalamica " ou ID. La différenciation des neurones à MCH dépend de l'expression du facteur morphogène Shh et ces neurones expriment Nkx2.1 et Nkx2.2, facteurs de transcription régulés positivement par Shh. Les neurones de la première sous-population envoient des projections le long du premier tractus longitudinal à se mettre en place, le tractus postopticus (tpoc). Ceux issus de la deuxième sous-population se différencient concomitamment au développement des régions télencéphaliques et leurs projections changent de direction pour innerver les régions antérieures du cerveau sous la dépendance de protéines de guidage axonal, Nétrine1 et Slit2. Nétrine1 permet d'attirer les axones MCH exprimant le récepteur DCC précocement vers la moelle épinière et plus tardivement vers le télencéphale alors que Slit2 contraint les axones MCH exprimant Robo2 à sortir de l'hypothalamus. L'étude du modèle " MCH " permet de mettre en lumière un changement d'organisation précocement au cours du développement dans l'axe longitudinal du prosencéphale. La bande longitudinale d'expression des facteurs de transcription Shh, Nkx2.2 peut être perçue comme une extension rostrale de la colonne neurogénique médiane déjà décrite chez des espèces d'invertébrés possédant une symétrie bilatérale. Les neurones générés le long de cette colonne le sont très tôt au cours du développement. / Neurons expressing melanin-concentrating hormone (MCH) are observed in the vertebrate posterior hypothalamus, from lampreys to humans. These neurons are involved in various functions such as sleep/wake cycle or food intake. They form a non-homogeneous population and at least two sub-populations are indentified in the rat. The first sub-population is composed of neurons born on the 11th embryonic day (E11) that project heavily on posterior regions of the central nervous system. The second is characterized by neurons born at E12/E13, projecting in anterior regions of the brain and expressing the peptide CART (cocaine and amphetamine Regulated Transcript) and the NK 3 receptor (neurokinin). The aim of this study was to understand the origin of these two sub-populations. For this, we used histological, molecular and in vitro approaches. MCH neurons are among the first neurons to be born and to differentiate their chemical phenotype along a longitudinal region defined by intense proliferation and called " cell cord " by Keyser in 1972. This longitudinal band is characterized by the expression of genes such as Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 and was recently named " diagonal intrahypothalamica " or ID. Differenciation of MCH neurons depends on expression of the morphogenetic factor Shh and these neurons express Nkx2.1 and Nkx2.2, transcription factors upregulated by Shh. The neurons of the first sub-population send projections along the tractus postopticus (tpoc), which is the first longitudinal tract to develop. Neurons of the second sub-population differentiate concomitantly to the development of the basal forebrain and their projections innervate anterior brain regions. Our results obtained in vitro showed that Netrin1 attracts MCH axons and that this reponse is mediated by DCC. Slit2 repulses MCH axons and this reponse is mediated by the Robo2 receptor. Overall, our study of the development of the MCH system shed light on an organizational change in the longitudinal axis of the forebrain during early development : a primary longitudinal organization characterized by the longitudinal expression of Shh and Nkx2.2 and the path of the tractus postopticus in the diencephalon and mesencephalon. MCH neurons of the first sub-population develop during this stage. Then, as the basal telencephalon extends and expresses Netrin1, the medial forebrain bundle differentiates, inducing a change in the main axis of the forebrain ; meanwhile MCH neurons of the second sub-population appear. MCH sub-populations reflect distinct developmental stages of the forebrain.

A área incerto-hipotalâmica (IHy) está envolvida no controle neuroendócrino de fêmeas, com associação de suas células MCHérgicas (hormônio concentrador de melanina). Em ratos, a área pré-óptica medial (MPA), o núcleo periventricular anteroventral (AVPe) e o núcleo arqueado (Arc) são mais densamente inervadas pela IHy em fêmeas do que em machos, sugerindo um dimorfismo sexual das projeções da IHy relevantes para o controle reprodutivo. Nosso objetivo é caracterizar as áreas relacionadas ao controle reprodutivo inervadas pela IHy em camundongos fêmeas, utilizando traçador neuronal anterógrado, analisar a inervação das células GnRH (hormônio liberador de gonadotrofinas) da MPA pela IHy e analisar a inervação do AVPe e Arc pela IHy, usando camundongos Kiss1-hrGFP. Como resultado, observamos que MPA, AVPe e Arc recebem projeções da IHy, assim como áreas relacionadas ao circuito de defesa. Nos animais Kiss1-hrGFP, não encontramos inervação de células KiSS-1 pela IHy. Problemas metodológicos impossibilitaram a análise das projeções da IHy para os neurônios GnRH. / The incerto-hypothalamic area (IHy) is related to the neuroendocrine control of females, involving their MCHergic cells (melanin-concentrating hormone). In rats, the medial preoptic area (MPA), the anteroventral periventricular nucleus (AVPe) and the arcuate nucleus (Arc) are more densely innervated by IHy in females than in males, suggesting a sexual dimorphism of IHy projections relevant to reproductive control. Our aim is to characterize the areas related to reproductive control innervated by IHy in female mice using anterograde neuronal tracer, analyze the innervation of GnRH cells (gonadotropin-releasing hormone) of MPA by IHy and analyze the innervation of AVPe and Arc by IHy using Kiss1-hrGFP mice. As a result, we observed that MPA, AVPe and Arc are innervated by IHy, as well as areas related to the defensive circuit. In Kiss1-hrGFP animals, KiSS-1 cells were not innervated by IHy cells. Methodological problems made it impossible to analyze the projections of IHy to the GnRH neurons.

Les neurones exprimant l'hormone de mélano-concentration (MCH) sont observés dans l'hypothalamus postérieur de tous les vertébrés, de la lamproie à l'Homme. Ces neurones sont impliqués dans diverses fonctions comme le cycle veille/sommeil ou la prise alimentaire. Ils forment une population non homogène et au moins deux sous-populations sont reconnues, chez le rat. La première sous-population est composée de neurones nés au 11ème jour de vie embryonnaire (E11) qui projettent massivement sur les régions les plus postérieures du système nerveux central. La seconde est générée à E12/E13 et les neurones la caractérisant projettent sur les régions les plus antérieures du cerveau et expriment le peptide CART (cocaine and amphetamine regulated transcript) et le récepteur NK3 (neurokinine). L'objectif de notre travail était de comprendre l'origine de ces deux sous-populations. Pour cela, nous avons utilisé des approches histologiques, moléculaires et in vitro. Les neurones à MCH sont parmi les premiers neurones à naître et à différencier leur phénotype chimique le long d'une région longitudinale définie par une prolifération intense, appelée " cell cords " par Keyser en 1972. Cette bande longitudinale est caractérisée par l'expression de gènes comme Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 et a été récemment renommée " diagonale intrahypothalamica " ou ID. La différenciation des neurones à MCH dépend de l'expression du facteur morphogène Shh et ces neurones expriment Nkx2.1 et Nkx2.2, facteurs de transcription régulés positivement par Shh. Les neurones de la première sous-population envoient des projections le long du premier tractus longitudinal à se mettre en place, le tractus postopticus (tpoc). Ceux issus de la deuxième sous-population se différencient concomitamment au développement des régions télencéphaliques et leurs projections changent de direction pour innerver les régions antérieures du cerveau sous la dépendance de protéines de guidage axonal, Nétrine1 et Slit2. Nétrine1 permet d'attirer les axones MCH exprimant le récepteur DCC précocement vers la moelle épinière et plus tardivement vers le télencéphale alors que Slit2 contraint les axones MCH exprimant Robo2 à sortir de l'hypothalamus. L'étude du modèle " MCH " permet de mettre en lumière un changement d'organisation précocement au cours du développement dans l'axe longitudinal du prosencéphale. La bande longitudinale d'expression des facteurs de transcription Shh, Nkx2.2 peut être perçue comme une extension rostrale de la colonne neurogénique médiane déjà décrite chez des espèces d'invertébrés possédant une symétrie bilatérale. Les neurones générés le long de cette colonne le sont très tôt au cours du développement. / Neurons expressing melanin-concentrating hormone (MCH) are observed in the vertebrate posterior hypothalamus, from lampreys to humans. These neurons are involved in various functions such as sleep/wake cycle or food intake. They form a non-homogeneous population and at least two sub-populations are indentified in the rat. The first sub-population is composed of neurons born on the 11th embryonic day (E11) that project heavily on posterior regions of the central nervous system. The second is characterized by neurons born at E12/E13, projecting in anterior regions of the brain and expressing the peptide CART (cocaine and amphetamine Regulated Transcript) and the NK 3 receptor (neurokinin). The aim of this study was to understand the origin of these two sub-populations. For this, we used histological, molecular and in vitro approaches. MCH neurons are among the first neurons to be born and to differentiate their chemical phenotype along a longitudinal region defined by intense proliferation and called " cell cord " by Keyser in 1972. This longitudinal band is characterized by the expression of genes such as Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 and was recently named " diagonal intrahypothalamica " or ID. Differenciation of MCH neurons depends on expression of the morphogenetic factor Shh and these neurons express Nkx2.1 and Nkx2.2, transcription factors upregulated by Shh. The neurons of the first sub-population send projections along the tractus postopticus (tpoc), which is the first longitudinal tract to develop. Neurons of the second sub-population differentiate concomitantly to the development of the basal forebrain and their projections innervate anterior brain regions. Our results obtained in vitro showed that Netrin1 attracts MCH axons and that this reponse is mediated by DCC. Slit2 repulses MCH axons and this reponse is mediated by the Robo2 receptor. Overall, our study of the development of the MCH system shed light on an organizational change in the longitudinal axis of the forebrain during early development : a primary longitudinal organization characterized by the longitudinal expression of Shh and Nkx2.2 and the path of the tractus postopticus in the diencephalon and mesencephalon. MCH neurons of the first sub-population develop during this stage. Then, as the basal telencephalon extends and expresses Netrin1, the medial forebrain bundle differentiates, inducing a change in the main axis of the forebrain ; meanwhile MCH neurons of the second sub-population appear. MCH sub-populations reflect distinct developmental stages of the forebrain.

Neurons commonly have a primary cilium, which is a non-motile organelle extending from the centrosome into the extracellular space. In most brain regions, neuronal cilia are enriched in either somatostatin receptor type 3 (SstR3) or melanin concentrating hormone receptor type 1 (MCHR1), or both. The present immunohistochemical study provides novel evidence that primary cilia regulate neuronal excitability via G-protein coupled receptors (GPCRs), and that their identity is governed by brain region and by competition, both in adulthood and in postnatal development. The hippocampus, which is particularly vulnerable to seizures, has opposing gradients of SstR3(+) and MCHR1(+) ciliary GPCRs. We hypothesized that there is a competition between these two ciliary GPCRs, which might take place on any level from gene expression to presence in the cilium. We examined whether receptor colocalization occurs transiently in development before ciliary GPCR dominance is established in neurons in the CNS. In postnatal CA1 and CA3, the first GPCR to appear in cilia was the one that will dominate in adults: MCHR1 in CA1 and SstR3 in CA3. Some days later, the second GPCR was expressed along with the first; dual-receptor cilia were the exclusive type until single-receptor cilia emerged again around P14. Single-receptor cilia then increased in numbers through adulthood. By identifying ciliary receptors that modulate seizure activity in mice, the present study lays a foundation for therapeutic approaches to reduce neuronal excitotoxicity underlying cell death in epilepsy, CNS injury, and neurodegenerative diseases.

O MCH é um neuropeptídeo sintetizado preferencialmente no hipotálamo que exibe projeções para todo o neuroeixo, podendo modular vários processos fisiológicos, como a ingestão aguda de alimento. Um alvo de suas projeções é o núcleo accumbens (Acb), componente do estriado ventral envolvido na recompensa de estímulos hedônicos. Nossa contribuição visa mapear as células imunorreativas (ir) ao MCH responsáveis pelas aferências do Acb, onde também investigamos aposições entre os sistemas MCH, GABA e ChAT. Utilizaram-se os métodos de simples e duplas imunoperoxidases e combinação de imunofluorescência e captação de traçadores retrógrados. A maior densidade de fibras ir-MCH ocupa a parte shell do Acb (AcbSh); as aferências ir-MCH partem, por exemplo, da área hipotalâmica lateral e área perifornicial. Controles anterógrados foram obtidos a partir da LHA. Observaram-se aposições entre terminais ir-MCH e células ir-GABA e ir-ChAT, no AcbSh. Assim, sugere-se que os territórios hipotalâmicos que contém MCH inervem o AcbSh e contatem outros sistemas neuroquímicos no Acb. / MCH is a neuropeptide synthesized preferentially in the hypothalamus that shows projections throughout neuraxis, and may modulate various physiological processes such as food intake. One target of its projections is the nucleus accumbens (Acb), a component of the striatum involved in reward of hedonic stimuli. Our contribution aims to map immunoreactive (ir) cells responsible for the MCH inputs of the Acb, and to investigate appositions between MCH fibers, GABA and ChAT cells. We used the single and double immunoperoxidase methods and immunofluorescence combined with retrograde tracer. The highest density of fiber MCH-ir occupies the shell part of the Acb (AcbSh); MCH-ir inputs are mainly from lateral hypothalamic and perifornical areas. Anterograde controls were obtained from the LHA. We have founded appositions between MCH-ir terminals and GABA- and ChAT-ir cells at AcbSh. Thus, is suggested that hypothalamic areas containing MCH innervate the AcbSh and contact other neurochemical systems systems in the Acb.