Previous studies have shown that the ventrolateral preoptic nucleus (VLPO) is a critical structure for promoting sleep. The VLPO is reciprocally connected with major wake-promoting centers in the caudal hypothalamus and the brainstem, and the loss of VLPO neurons (chemical lesions) produces profound insomnia and sleep fragmentation. Neurochemical studies identified galanin as a specific marker for the sleep-active VLPO neurons. However, past VLPO lesion studies also knocked out many other cell types (other than galaninergic neurons) in and around the VLPO region, and thus the effect of selectively lesioning the galaninergic VLPO neurons on sleep-wakefulness is not known. In this project, we will selectively destroy the galaninergic VLPO neurons by stereotaxically injecting adeno-associated viral vectors (AAV) containing transcriptionally blocked pro-apoptotic transgene, truncated BH3-interacting domain death agonist (tBID), into the preoptic hypothalamus of transgenic mice that express Cre-recombinase (Cre) specifically in the galaninergic neurons (Gal-Cre knockin mice). Injection of AAV-tBID into the preoptic hypothalamus acts selectively on the Cre- containing galaninergic VLPO neurons and triggers apoptotic cell death. We will study the changes in sleep- wake amounts, architecture and state transitions in these mice that lack galaninergic VLPO neurons (Specific Aim 1). We anticipate that the loss of these neurons will result in severe insomnia and sleep fragmentation. Interestingly, all the galaninergic neurons in the VLPO contain GABA. The VLPO-GABA has been hypothesized to mediate the inhibition of wake-promoting cell groups during sleep, although it has never been definitively determined. Hence, we will focally and selectively eliminate GABA release from the VLPO neurons and evaluate sleep-wakefulness changes that are secondary to the loss of VLPO GABA neurotransmission (Specific Aim 2). Specific elimination of GABA release will be achieved -1) by delivering AAV-Cre into the VLPO of conditional vesicular GABA transporter knockout (VGAT) mice that contain loxP sites flanking exon 2 of the VGAT gene and 2) by crossing the conditional VGAT mice with Gal-cre mice. Addition of Cre excises VGAT exon 2 and makes the entire gene non-functional, which results in the absence of functional VGAT protein in the VLPO neurons. As VGAT is critical for transporting GABA into the synaptic vesicles, the loss of VGAT will result in selective loss of GABA neurotransmission from the VLPO neurons. Studying sleep- wakefulness in these animals will provide critical insight into the specific contribution of VLPO-GABA in sleep regulation and in sleep-wake transitions. Collectively, the outcomes of this proposal will help us better understand the hypothalamic control mechanisms controlling sleep generation, maintenance and state transitions. This information will ultimately be important in designing pharmacological treatment for insomnia and arousal disorders. PUBLIC HEALTH RELEVANCE: Understanding the neural mechanisms controlling sleep-wakefulness is critical considering the high prevalence of insomnia in the general population and its social and economic consequences. The objective of the proposal is to study the neuronal mechanisms by which the sleep promoting area in the brain, known as ventrolateral preoptic area (VLPO), initiates and maintains sleep. Outcomes of this proposal will help us better understand the specific role of VLPO neurotransmitters in sleep generation, maintenance and state transitions. This information will ultimately be important in designing pharmacological treatment for insomnia and arousal disorders.