The ontogeny of rat diencephalic melanin-concentrating hormone (MCH) neurons has been analysed, using the bromodeoxyuridine method to determine the period of birth of these neurons, and using in situ hybridization and immunohistochemistry to study their chemical differentiation. The spatiotemporal pattern of MCH neuron generation is complex, although it is broadly lateromedial with a peak between embryonic days (E) 12 and E13. The first expression of the MCH gene has been detected on E13 in neurons in the presumptive lateral hypothalamic area. But the adult-like pattern was observed from E18. Medial-most MCH neurons express the peptide CART (cocaine-amphetamine-regulated transcript) from E18, and the receptor neurokinin 3 (NK3) from between postnatal day (P) 0 and P5. These results are discussed and compared with data from the literature to better understand the organization of the 'MCH-containing area'.

Neurons producing melanin-concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the 'cocaine-and amphetamine-regulated transcript' (CART) peptide, while another expresses neither one of these two molecules. MCH+/ CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/ CART-axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART-projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions. Keywords: calcium-binding proteins, cocaine-and amphetamine-regulated transcript, hypothalamus, neurokinin 3. The peptide melanin-concentrating hormone (MCH) has been implicated in reproductive and feeding behaviors as well as in arousal and cognitive processes (Griffond and Baker 2002). Its mechanisms of action are unknown, but the diencephalic MCH neurons (Risold et al. 1992;Broberger et al. 1998) are obviously largely involved in these responses.It has been known since the mid 1980s that all MCH neurons express the enzyme acetylcholinesterase (Risold et al. 1989). During the last few years it has also been shown that most, if not all, MCH neurons express the enzyme glutamic acid decarboxylase (Elias et al. 2001; DallvechiaAdams et al. 2002; unpublished observations). However, the MCH neuron population is not homogeneous. A previous study combining BrdU and retrograde tract tracing protocols (Brischoux et al. 2002) has demonstrated that at least two types of MCH neurons can be distinguished: (i) those that show a peak of birth between E12 and E13, that are retrogradely labeled after fastblue injections in the cerebral cortex, and that express high levels of the tachykininergic receptor neurokinin 3 (NK3); and (ii) those born earlier (peak of birth at E11), retrogradely labeled after fastblue injections in the spinal cord, and that do not express NK3. Meanwhile, other authors showed the expression of the cocaine-and amphetamine-regulated transcript (CART) in many MCH neurons (Broberger 1999;Vrang et al. 1999). As MCH neurons are involved in many responses and as specific subpopulations might subserve different functions, it appeared critical to us to better characterize putative subpopulations, and to verify if CART is coexpressed with NK3 in MCH neurons.In many parts of the brain, calcium-binding proteins...

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