To pinpoint the downstream projection targets of POMC neurons that mediate satiety. Hypothesis: Hypothalamic POMCARC neuron projections modulate MC4R+ neurons in the paraventricular nucleus of the hypothalamus (PVN) to signal satiety. Rationale: Studies have demonstrated that the selective restoration of MC4R activity in the PVN and a subpopulation of amygdaloid neurons of MC4R-null knockout mice rescued the MC4R knockout-induced obesity (Balthasar 2005). Our lab has access to a MC4R-T2a-Cre mouse line, which expresses Cre recombinase under the endogenous promoter of the Mc4r gene, allowing precise access to mark, manipulate and record activity from this specific subset. Our lab also has access to the POMC-Cre mouse line for the purpose of probing neural connections and modulation. This experiment is designed to elucidate the anatomical structures in which MC4Rs are expressed to mediate satiety, as well as the connections and modulatory roles of POMCARC neurons. Approach: We will identify MC4R-expressing neurons by crossing MC4R-T2a-Cre with a GFPflox/flox reporter mouse and in combination with anterograde tracing (via a Cre-dependent adeno-associated virus (AAV) expressing synaptophysin injected into the arcuate (ARC) nucleus of POMC-Cre mice) and immunohistochemistry, map POMCARC projections in this complex network. Based on these tracing studies, we will selectively target the light-activated ion channel, channelrhodopsin2 (ChR2) specifically to POMCARC neurons (via stereotaxic injection of a Cre-dependent AAV-ChR2), followed by selective photo-stimulation of distinct POMC terminal fields and assess the resulting behavioral output. These in vivo optogenetic studies will tease apart the functionally relevant downstream sites of POMCARC neurons that decrease food intake and body weight, and ultimately lead to a clearer idea of the neural circuits controlling satiety. We will also generate double transgenic POMC-Cre;MC4R-T2a-Cre mice, which allows for bimodal regulation of two separate neural populations, to verify sequential neural networks. For instance, we can acutely activate the POMC terminal field to the PVN, which we hypothesize will result in reduced feeding, and simultaneously inhibit the relevant downstream MC4RPVN neurons (via Cre-dependent viruses expressing inhibitory GPCRs or ion pumps), which we hypothesize will reverse this fall in food intake. These occlusion studies are extremely elegant and grant the experimenter unparalleled control of these specific circuits. Predictions, interpretations &Future Experiments: We do not anticipate any problems with these experiments. Preliminary data has already been generated labeling MC4R+ neurons and we will subsequently assess POMCARC projections to this cell type both within and outside the hypothalamus. In addition, the viruses proposed here have been used successfully by a number of laboratories. We predict that activation of the POMCARC terminal field in the PVN will reduce feeding behavior and ultimately reduce body weight. Furthermore, we have preliminary data showing that DREADD-mediated acute inhibition (a pharmaco-genetic approach used to acutely manipulate neural activity via specific expression of an exogenous receptor and its subsequent binding and activation through a pharmacologically inert ligand;Krashes et al., 2011) of MC4RPVN neurons can drive feeding behavior, and that MC4RPVN neurons receive direct monosynaptic inputs from AGRP neurons (utilizing AgRP-ires-Cre mice), both strong lines of evidence that suggest an opposing role for POMCARC neurons on these downstream targets.