PROJECT SUMMARY/ABSTRACT Neurons in the lateral hypothalamic area (LHA) that express the neuropeptide hypocretin/orexin (H/OX) are critical regulators of sleep-wake architecture, arousal, and motivated behavior. The progressive destruction of these neurons, and/or disrupted H/OX signaling, in humans causes a devastating, chronic neurological disease—narcolepsy with cataplexy—which results in disordered wakefulness, loss of muscle control, and late- onset metabolic effects. Experimental disruption of H/OX signaling in vertebrate animal models recapitulate many of these symptoms including aberrant sleep-wake states. Despite the clear and conserved role of the H/OX system in behavioral state control, the molecular and cellular underpinnings of H/OX synaptic function are not well understood. Thus, the long-term objective of this proposal is to fill this knowledge gap by defining the molecular machinery underlying the structure and function of H/OX synapses throughout the central nervous system and illuminating the molecular pathways that drive their behavioral effects. To begin addressing this gap, this proposal will build off the discovery that mRNA encoding the synaptic organizing protein complement C1q- like 3 (C1QL3) is uniquely and robustly expressed in H/OX neurons, as identified in a recent transcriptomic analysis of the LHA. Work in other circuits demonstrated that C1QL3 is important for establishing and maintaining excitatory synapse density, and it was further shown that C1QL3 global deletion results in deficits in sleep-wake behavior, reminiscent of features of human narcolepsy. C1QL3 was also shown to bind pre- and postsynaptic proteins, one of which is also robustly expressed in H/OX neurons. Based on these strong preliminary data, the hypothesis underlying this proposal is that C1QL3, together with its pre- and postsynaptic binding partners, forms a novel trans-synaptic adhesion complex, which is necessary for both H/OX synaptic function and its role in regulating behavioral state. This will be systematically investigated in the following three Specific Aims: 1) In Aim 1, C1QL3 will be visualized in H/OX cell bodies and axons at the light and ultrastructural level using a newly developed epitope-tagged knockin mouse line, and effects of C1QL3 global and conditional knockout (cKO) and overexpression on H/OX neuron fiber density and synaptic ultrastructure will be examined. 2) In Aim 2, the impact of C1QL3 cKO and overexpression on excitatory synaptic function in targets of the H/OX system will be examined using both in vitro slice electrophysiology and optogenetics, and on sleep-wake behavior using electroencephalographic recording of vigilance states. 3) In Aim 3, the molecular mechanisms through which C1QL3 contributes to a trans-synaptic adhesion complex will be dissected using biochemical assays, viral manipulations, the novel epitope-tagged knockin mouse line, and super-resolution microscopy. This investigation will not only provide critical mechanistic insight into the basic biology of excitatory synaptic transmission at H/OX synapses, but also identify a novel molecular pathway through which H/OX signaling may be manipulated as a therapeutic intervention in chronic neurological and sleep disorders.

PROJECT NARRATIVE Hypocretin/orexin (H/OX) neurons in the mammalian hypothalamus are critical for coordinating fundamental behavioral states such as arousal, stress, and motivation, and their dysfunction is implicated in devastating neurological and sleep disorders, notably narcolepsy with cataplexy. This proposal will use new and innovative molecular, cellular, and behavioral techniques to dissect the synaptic signaling pathways through which H/OX neurons shape physiology and behavior. In so doing, we hope to identify targets for therapeutic intervention in treating and improving the quality of life of patients suffering from neurological and sleep disorders.