Neuropeptide Y release is elevated from the microdissected paraventricular nucleus of food-deprived rats: an in vitro study.

Abstract

Intracerebroventricular injection of neuropeptide Y (NPY) stimulates a robust dose-related feeding response in the rat. Experimental evidence attests to the view that the release of NPY in the paraventricular nucleus (PVN), a site richly innervated by NPY immunopositive fibers, is responsible for stimulation of feeding behavior. However, there is little information on the neuroendocrine factors involved in regulation of NPY release, in part due to the unavailability of reliable techniques to monitor PVN NPY release. In this study, we have validated an in vitro technique to assess NPY release from the PVN and other neighboring hypothalamic sites of the rat brain. In the first experiment, freshly dissected brains from male rats were processed for 300-microns thick sections with a vibratome. The PVNs were microdissected from the brain sections under a stereomicroscope and incubated in 250 microliters Krebs Ringer bicarbonate buffer at 37 C for basal and KCl-induced NPY release. The results showed that basal NPY efflux from the excised PVN was detectable and increased in relation to the number of PVNs in the incubation chambers. Addition of KCl at the end of the 60-min basal incubation period increased NPY release further, the increments were again closely related to the number of PVN punches in the incubation chambers. In the second experiment, the assumption that in vitro basal and KCl-evoked NPY release from the PVN reflected the in vivo pattern of PVN NPY secretion was validated. The effects of 4-day food deprivation (FD), an experimental paradigm known to augment in vivo PVN NPY secretion, on the in vitro NPY release from PVN and ventromedial nucleus were evaluated. The results showed that both basal and KCl-evoked NPY release was significantly higher from the PVN of food-deprived than control rats on ad libitum rat chow. This FD-induced incremental NPY response was site-specific because the basal and KCl-evoked NPY effluxes from the ventromedial nucleus of FD and control rats were similar. Thus, in agreement with previous in vivo findings, NPY release in vitro is also augmented selectively from the PVN in response to fasting. Cumulatively, these results demonstrate that NPY release in vitro from hypothalamic sites microdissected from fresh brains can be assessed in a reliable fashion and are in accord with the proposal that enhanced NPY action within the PVN is responsible for increased drive for food.