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The rat nucleus incertus (NI) contains GABA/peptide-projection neurons responsive to orexin (hypocretin)/orexin receptor-2 (OX2) signalling. Melanin-concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor-1 (MCH1) expression, and neurophysiological and behavioural effects of MCH c.f. orexin-A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub-zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH-containing fibres in NI. Single-cell RT-PCR detected MCH1 and OX2 mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co-expression patterns of MCH1 and OX2 mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch-clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1μM), representing a distinct population from OXA-sensitive NI neurons (35%). Intra-NI OXA infusion (600pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra-NI MCH infusion (600pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre-fasted rats tested during the dark/active phase, intra-NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide-immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours.Nucleus incertus (NI) receives an innervation from MCH and orexin neurons.GABAergic and non-GABAergic NI neurons express MCH1 and/or OX2 receptor mRNA.NI neurons display differential responses to MCH (inhibition) and OXA (excitation).OXA injection into NI enhanced light/inactive phase locomotor activity and feeding.MCH injection into NI did not alter light or dark phase activity or food intake.