predicted that the POMC gene should be expressed in ARC neurons that do not contain Agrp mRNA. To determine if NPY mRNA is coexpressed with either Agrp or POMC mRNA in ARC neurons of fed and fasted mice and rats, we colocalized nature neuroscience • volume 1 no 4 • Neuropeptide Y (NPY) stimulates food intake and promotes weight gain, whereas melanocortins have the opposite effect. Yet both peptides are synthesized in the arcuate nucleus, a hypothalamic area involved in energy homeostasis. We report here that mRNA encoding NPY and the melanocortin precursor, proopiomelanocortin (POMC) are expressed in adjacent, but distinct, subpopulations of arcuate nucleus neurons. Moreover, these NPY neurons coexpress mRNA encoding Agouti-related protein (Agrp), an endogenous melanocortin receptor antagonist, and fasting increases the expression of both of these mRNA species. Our findings suggest that hypothalamic NPY/Agrp neurons constitute a unique cell type that is activated by fasting to stimulate food intake via a simultaneous increase of NPY and decrease of melanocortin.The ability to recover weight lost during periods of limited access to food is important for survival. Accordingly, a highly integrated central nervous system response to starvation and weight loss has evolved that includes changes in the activity of discrete hypothalamic pathways involved in energy homeostasis 1,2 . Components of this response include the activation of hypothalamic neurons that contain NPY (which stimulates food intake) coupled with reduced signaling by neurons that contain melanocortins (which inhibit food intake). Melanocortin-containing cell bodies in the forebrain have been detected only in the hypothalamic arcuate nucleus (ARC), the same nucleus that contains a population of NPY neurons that are implicated as critical in energy homeostasis 3 . Similarly, hypothalamic expression of Agrp 4,5 (also known as Agouti-related transcript, ART), a newly discovered antagonist of melanocortin receptors, has also been found only in neurons of the ARC.These anatomical considerations, as well as the ability of NPY and Agrp to promote increased food intake and weight gain 1,4 , led us to propose that these two gene products are expressed by the same ARC neurons, as proposed previously 5 . We hypothesized further that if NPY and Agrp are coexpressed by ARC neurons, their expression should be regulated in parallel during fasting. In contrast, because melanocortins reduce food intake 6 , we Fig. 1. Fluorescent in situ hybridization of mRNA encoding Agrp in the ARC of mice fed ad libitum (a) or fasted (b). Adult male mice (25 g) were fasted for 48 h. Brains were removed after decapitation, frozen on dry ice and sectioned at 14 µm. In situ hybridization used antisense Agrp cRNA probe. Sections were viewed with a Zeis Axioplan fluorescence microscope and quantified using MCID M2 imaging software (Imaging Research, Ontario, Canada). The expression of Agrp mRNA increases with fasting. All animals were treated in accordance with University of Washington gui...

The decline of leptin (Ob protein) concentrations during fasting is implicated as a signal for increasing the expression of the orexigenic peptide neuropeptide Y (NPY) in the hypothalamus. To test the hypothesis that the effects of food intake on arcuate nucleus NPY activation are mediated by leptin, we performed simultaneous triple in situ hybridization colocalization studies to determine whether the subset of NPY neurons that are activated by fasting preferentially expresses the long form of the leptin receptor (Ob-Rb). Thus, mRNAs encoding NPY and pro-opiomelanocortin (POMC) were colocalized in the arcuate nucleus of fed and fasted rats by fluorescence in situ hybridization in combination with isotopic in situ hybridization for Ob-Rb mRNA. In fed animals, 47% of arcuate nucleus neurons containing NPY mRNA also contained Ob-Rb mRNA, compared with 79% of POMC neurons (P < 0.01). After a 2-day fast, the number of arcuate nucleus neurons with NPY mRNA increased 50% (P < 0.05); the number of these that coexpressed Ob-Rb increased twofold (P = 0.013). Furthermore, Ob-Rb mRNA hybridization in individual NPY neurons increased by 64% (P < 0.02). In contrast, the number of POMC neurons that coexpressed Ob-Rb was unchanged. A significant interpretation of these findings is that the NPY neurons that do not express detectable levels of Ob-Rb mRNA are not activated by fasting, whereas the NPY neurons that are activated by fasting are the ones that express Ob-Rb. These data demonstrate a significant physiological difference between NPY neurons that express Ob-Rb and those that do not. The results support the conclusion that the effect of food intake on NPY neurons is mediated by the direct action of leptin via Ob-Rb receptors expressed by these NPY cells. The results also indicate that expression of Ob-Rb is a defining phenotypic characteristic of the subset of arcuate nucleus NPY neurons that are activated by fasting and play a central role in the adaptive response to negative energy balance.

Three experiments were performed to investigate the hypothesis that leptin action within the caudal brain stem (CBS) contributes to its intake inhibitory effects. The first experiment evaluated the anatomical distribution of leptin receptor mRNA in rat CBS using a sensitive fluorescence in situ hybridization method with a riboprobe specific for the long form of the leptin receptor (Ob-Rb). An Ob-Rb mRNA hybridization signal was detected in neurons of several CBS nuclei involved in the control of food intake, including the dorsal vagal complex and parabrachial nucleus. A strong hybridization signal was also obtained from neuronal cell bodies of a number of other structures including the hypoglossal, trigeminal, lateral reticular, and cochlear nuclei; locus ceruleus; and inferior olive. The anatomical profile revealed by fluorescence in situ hybridization was in good agreement with immunocytochemical analysis with an antibody specific to Ob-Rb. In a second experiment, exploring the relevance of CBS Ob-Rb to feeding behavior, rats were given a fourth intracerebroventricular (i.c.v.) injection of leptin (0.1, 0.83, or 5.0 microg; n = 9-11/group) or vehicle 30 min before lights-out on three consecutive days. The two higher doses reduced food intake significantly at 2, 4, and 24 h after injection and caused significant reductions of body weight. The dose-response profiles for fourth i.c.v. administration were indistinguishable from those obtained from separate groups of rats that received leptin via a lateral i.c.v. cannula. In the last experiment, a ventricle-subthreshold dose of leptin (0.1 microg) microinjected unilaterally into the dorsal vagal complex suppressed food intake at 2, 4, and 24 h. The results indicate that the CBS contains neurons that are potentially direct targets for the action of leptin in the control of energy homeostasis.