PROJECT SUMMARY Understanding the neural mechanisms underlying narcolepsy with cataplexy (NC) is crucial, considering the debilitating and life-threatening nature of this sleep disorder and its high prevalence. Previous studies have established that the loss of orexins is the fundamental cause of NC in humans and animals. However, the role of melanin-concentrating hormone (MCH) neurons, which are reciprocally connected with orexin neurons and play a complementary role in sleep-wake regulation, in the pathophysiology of NC remains unclear. The proposed work is aimed to address this question and to define the neural circuitry through which MCH neurons may influence cataplexy. To understand if MCH neurons are active during cataplexy, in Aim1, we will image activity dynamics of MCH neurons in a mouse model of NC during spontaneous cataplexy as well as in the presence of positive emotional stimuli that enhance cataplexy. To demonstrate a causal role of MCH neurons in cataplexy, in Aim 2, we will selectively activate and silence the MCH neurons using chemogenetic methods and study the changes in spontaneous cataplexy and positive-emotion-triggered cataplexy (PES-cataplexy). As MCH neurons heavily project to the midbrain locomotor region (MLR; involved in facilitating muscle tone and locomotor activity), whose lesions in rats produced cataplexy, we then hypothesized that the MCH neurons might target the MLR to modulate cataplexy. We will test this hypothesis, in Aim 3, by studying the changes in cataplexy a) following in vivo optogenetic stimulation of the MCH terminals in the MLR with concurrent chemogenetic inhibition of MCH soma and b) following optogenetic inhibition of the MCH terminals in the MLR with concurrent chemogenetic activation of MCH soma. Finally, in Aim 4, to identify the neurochemically distinct subset(s) of MLR neurons involved in MCH control of cataplexy, we will chemogenetically activate or inhibit the glutamatergic and GABAergic neurons in the MLR in mice with or without orexins and study cataplexy behavior. Collectively, these experiments will identify the specific mechanisms and pathways by which MCH neurons influence cataplexy and thereby will improve our understanding of the neural basis of NC.