The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.Since the discovery of the orexins (1) investigations of their functions have been guided by evidence for their hypothalamic distribution (1, 2), focusing on feeding, energy homeostasis (1, 3), and neurocrine functions (3). Our studies now show the presence of orexin A immunoreactive fibers and varicosities in extrahypothalamic areas, particularly the locus coeruleus, and demonstrate that the functions of orexin A extend beyond the hypothalamus.Orexin A and B are derived from a 130-aa precursor, prepro-orexin, which is encoded by a gene localized to human chromosome 17q21 (1). Prepro-orexin, or preprohypocretin (2), was identified in the rat hypothalamus by directional tag PCR subtractive hybridization (2) and has been shown by Northern blot analysis to be abundant in the brain and detectable at low levels in testes but not in a variety of other tissues (1, 2). Hypocretins had been identified as hypothalamic neuropeptides, but their biological role was not described (2). Nucleotide sequence alignment shows that hypocretins 1 and 2 have sequence in common with orexins A and B, respectively, but additional amino acids are present in both hypocretins. In situ hybridization maps confirm dense prepro-orexin mRNA expression in the hypothalamus (1, 2). Immunocytochemical mapping of orexin A has identified a population of mediumsized neurones within the hypothalamus, median eminence (3), and ventral thalamic nuclei of rat brain (1, 3). This distribution has been confirmed in human tissue (4).Orexin A binds with high affinity to the novel G proteincoupled receptors orexin 1 (OX 1 ) (IC 50 20 nM) and orexin 2 (OX 2 ) (IC 50 38 nM). Calcium mobilization assays in transfected HEK293 cells confirm that orexin A is a potent agonist at both OX 1 (EC 50 30 nM) and OX 2 (EC 50 34 nM) (1). Emerging evidence suggests the existence of an extensive extrahypothalamic projection of orexin-immunoreactive neurones. Peyron et al. (5), in addition to confirming the presence of immunoreactive cell somata within the hypothalamus, reported immunolabeled fibers throughout extrahypothalamic regions, including septal nuclei, substantia nigra, and raphe nucle...

No abstract

The underlying causes of obesity are poorly understood but probably involve complex interactions between many neurotransmitter and neuropeptide systems involved in the regulation of food intake and energy balance. Three pieces of evidence indicate that the neuropeptide melanin-concentrating hormone (MCH) is an important component of this system. First, MCH stimulates feeding when injected directly into rat brains; second, the messenger RNA for the MCH precursor is upregulated in the hypothalamus of genetically obese mice and in fasted animals; and third, mice lacking MCH eat less and are lean. MCH antagonists might, therefore, provide a treatment for obesity. However, the development of such molecules has been hampered because the identity of the MCH receptor has been unknown until now. Here we show that the 353-amino-acid human orphan G-protein-coupled receptor SLC-1 expressed in HEK293 cells binds MCH with sub-nanomolar affinity, and is stimulated by MCH to mobilize intracellular Ca2+ and reduce forskolin-elevated cyclic AMP levels. We also show that SLC-1 messenger RNA and protein is expressed in the ventromedial and dorsomedial nuclei of the hypothalamus, consistent with a role for SLC-1 in mediating the effects of MCH on feeding.

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.

No abstract