Co-existence of leptin- and orexin-receptors in feeding-regulating neurons in the hypothalamic arcuate nucleus-a triple labeling study

The arcuate nucleus (ARC) of the hypothalamus has been identified as a prime feeding regulating center in the brain. Several feeding regulating peptides, such as neuropeptide Y (NPY) and proopiomelanocortin (POMC), are present in neurons of the ARC, which also serves as a primary targeting site for leptin, a feeding inhibiting hormone secreted predominantly by adipose tissues, and for orexin (OX)-containing neurons. OX is expressed exclusively around the lateral hypothalamus, an area also established as a feeding regulating center. Some recent physiological analyses have shown that NPY- and POMC-containing neurons are activated or inactivated by leptin and OX. Moreover, we have already shown, using double immunohistochemical staining techniques, that NPY- and POMC-containing neurons express leptin receptors (LR) and orexin type 1 receptors (OX-1R). However, no morphological study has yet described the possibility of whether or not these arcuate neurons are influenced by both leptin and OX simultaneously. In order to address this issue, we performed histochemistry on ARC neurons using a triple immunofluorescence method. We found that 77 out of 213 NPY- and 99 out of 165 POMC-immunoreactive neurons co-localized with both LR- and OX-1R-immunoreactivities. These findings strongly suggest that both NPY- and POMC-containing neurons are regulated simultaneously by both leptin and OX.