The first experiment would be to use immunohistochemistry to investigate the overlapping/non-overlapping expression of Mc4rs and Leprs in the hippocampus. Both sets of neurons in the hypothalamus encode satiety signals and act to blunt feeding, of course with different signaling cascades and circuit mechanisms. Although neither of these receptors have good antibodies and in situs are nearly impossible given the low levels of expression, we can co-localized GFP-signaling from the Mc4r promoter-driven Cre line with STAT3 expression following leptin injection (see Myers et al 2012). Next, the obvious experiment is to transduce Lepr+ and/or Mc4r+ hippocampal neurons with ChR2 or eNpHR and assess the effects of photostimulation of photoinhibition on feeding (quantity as well as meal patterns including meal size, meal frequency, meal length) and body weight. If we see some convincing alteration in behavior, we are then in a great position again to dissect the circuit with regard to inputs and outputs using viral tools. Even more interesting is the ability to perform conditioned place preference and other memory/foraging tasks aimed at procuring food during optogenetic manipulation. We can perform these using a simple chamber or even design automated protocols using the specialized Phenotyper cages. As with the other projects listed above, we can (and definitely should) use a combination of in vitro and ex vivo slice recordings to analyze the firing patterns of these cell types in differential states (hungry versus sated) or specific tasks (memory assays, active eating ect). Tools Mice: Mc4r-t2a-Cre LepR-ires-Cre, Pacap-ires-Cre Virus: AAV-FLEX-ChR2-mCherry, AAV-FLEX-synapsin-mCherry, AAV-FLEX-eNpHR-YFP, AAV-FLEX-hM3Dq-mCherry, AAV-FLEX-hM4Di-mCherry, modified rabies virus Equipment: Phenotyper cage for measurements of food intake. Plexon Ominplex for in vivo recordings. Acute slice ephys rig.

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