Intracerebroventricular administration of alpha-melanocyte stimulating hormone increases phosphorylation of CREB in TRH- and CRH-producing neurons of the hypothalamic paraventricular nucleus.

Changes in circulating leptin levels, as determined by nutritional status, are important for the central regulation of neuroendocrine axes. Among these effects, fasting reduces TRH gene expression selectively in the hypothalamic paraventricular nucleus (PVN), which can be reversed by leptin administration. Intracerebroventricular (i.c.v.) infusion of alpha-MSH recapitulates the effects of leptin on hypophysiotropic TRH neurons, completely restoring proTRH mRNA to levels in fed animals despite continuation of the fast, making alpha-MSH a candidate for mediating the central effects of leptin. As alpha-MSH binds to a G-protein coupled receptor that activates cAMP and alpha-MSH stimulates the TRH promoter through the phosphorylation of the transcription factor CREB in vitro, we determined whether i.c.v. injection of alpha-MSH to rats regulates phosphorylation of CREB, specifically in hypophysiotropic TRH neurons of PVN. As alpha-MSH also induces the activation of CRH gene expression in the PVN, we further determined whether i.c.v. injection of alpha-MSH regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. In vehicle-treated animals, only rare neurons contained nuclear phospho-CREB (PCREB) immunoreactivity in the parvocellular PVN. I.c.v. injection of 10 microg alpha-MSH dramatically increased the number of PCREB-immunolabeled cell nuclei in the PVN in fasted groups at 10 min postinjection, particularly in the medial, periventricular, anterior and ventral parvocellular subdivisions, whereas a moderate increase of PCREB immunoreactivity was observed at 30 min and PCREB immunoreactivity was lowest at 1 h postinfusion. Double immunolabeling with proTRH antiserum revealed that following i.c.v. alpha-MSH infusion at 10 min, the majority of TRH neurons contained PCREB in the anterior (71%), medial (83%) and periventricular (63%) parvocellular subdivisions. The percentage of double-labeled TRH neurons declined at 30 min and 1 h post alpha-MSH infusion. Similarly, only 16% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle treated animals, whereas 10 min following alpha-MSH infusion the percentage of CRH neurons colocalizing with the PCREB rose to 54%, then fell to 37 and 17% at 30 and 60 min postinfusion, respectively. These data demonstrate that i.c.v. alpha-MSH administration increases the phosphorylation of CREB in several subdivisions of the PVN including TRH and CRH neurons in the medial and periventricular parvocellular subdivisions, suggesting that phosphorylation of CREB may be necessary for alpha-MSH-induced activation of the TRH and CRH genes. The increase in PCREB in the anterior and ventral parvocellular subdivisions of the PVN, regions linked to nonhypophysiotropic functions such as autonomic regulation, would also imply a role for these neurons in anorectic and energy wasting responses of melanocortin signaling.