SAT-605 Characterization of Dual Projection Patterns of Refeeding-Activated Neurons in the Parasubthalamic Nucleus

We have observed that following a fast, animals terminate their food intake within 2h after refeeding accompanied by a pattern of neuronal activation as identified by c-fos immunostaining that involves a number of brain regions associated with the regulation of food intake including the nucleus tractus solitarius (NTS), parabrachial nucleus (PBN), central nucleus of the amygdala (CEA), hypothalamic arcuate and paraventricular nuclei, and bed nucleus of the stria terminalis. We also observed striking c-fos activation in the posterior-lateral hypothalamus called the parasubthalamic nucleus or PSTN, raising the possibility that it may also be an important anorectic center in the brain. To establish how the PSTN is integrated into the CNS, we performed dual-label retrograde tract tracing studies to characterize whether refeeding-activated PSTN neurons project to one, or more than one target area in the CNS. Adult, Sprague-Dawley rats received dual stereotaxic injections of Alexa Fluor 488- and Alexa Fluor 555-conjugated cholera toxin β subunit (CTB; 0.1%, 0.5–1 µl volume) into the 1) PBN and NTS, 2) PBN and CEA and 3) NTS and CEA. After 7–12 days, the animals were fasted for 24 h and then given free access to food for 2 h before euthanasia by transcardial perfusion with 4% paraformaldehyde. Brains with successful dual injections were further processed for c-fos immunohistochemistry. The results showed that 26.5±3.8% of PSTN neurons projecting to the PBN also project to the CEA, and 34.6±7.6% of PSTN neurons that project to the CEA also project to the PBN. In addition, 20.2±2.7% of PSTN neurons that project to the PBN also project to the NTS, and 38.1±9.7% of PSTN neurons that project to the NTS also project to the PBN. Furthermore, 35.0±12.5% of PSTN neurons that project to the CEA project to the NTS and 37.1±4.0% of PSTN neurons that project to the NTS project to the CEA. Finally, up to 15% of the neurons with dual projections to the PBN and CEA contained c-fos after refeeding; up to 18% of the neurons with dual projections to the PBN and NTS contained c-fos; and up to 30% of neurons with dual projections to the NTS and CEA contained c-fos. We conclude that a large number of PSTN neurons have more than one projection site within the brain, thus the PSTN appears to have the capability of simultaneously communicating information about appetite to several, major feeding-related sites within the brain, presumably to terminate feeding.