The identity of higher-order neurons and circuits playing an associative role to control feeding is unknown. We injected pseudorabies virus, a retrograde tracer, into masseter muscle, salivary gland, and tongue of BAC-transgenic mice expressing GFP in specific neural populations and identified several CNS regions that project multisynaptically to the periphery. MCH and orexin neurons were identified in the lateral hypothalamus, and Nurr1 and Cnr1 in the amygdala and insular/rhinal cortices. Cholera toxin beta tracing showed that insular Nurr1(+) and Cnr1(+) neurons project to the amygdala or lateral hypothalamus, respectively. Finally, we show that cortical Cnr1(+) neurons show increased Cnr1 mRNA and c-Fos expression after fasting, consistent with a possible role for Cnr1(+) neurons in feeding. Overall, these studies define a general approach for identifying specific molecular markers for neurons in complex neural circuits. These markers now provide a means for functional studies of specific neuronal populations in feeding or other complex behaviors.
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