Purinergic Mechanisms in Homeostatic Sleep Control

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The broad objective of this proposal is to understand the cellular mechanisms which control sleepiness. Sleep deprivation (SD) and insomnia are experienced by ~40% of military personnel immediately after deployment and SD is a well-known risk factor that adversely affects the mental health of veterans. The basal forebrain (BF) is an important brain region for controlling the increased propensity for sleep after SD i.e. sleep homeostasis. Previous grant cycles investigated how sleep homeostasis is mediated by inhibition of BF wake-promoting neurons via increases in the extracellular concentration of adenosine (ADex), and investigated the role of extracellular adenosine triphosphate (ATPex) as a source of ADex. However, the circuit and neurotransmitter basis for these increases in ATPex and ADex has remained unresolved. At least three types of cortically-projecting, wake-active neurons coexist within BF: cholinergic (ChAT+), parvalbumin expressing GABAergic (PV+) and glutamatergic (vGluT2+). Selective lesion studies of BF ChAT+ neurons suggested that they are required for SD-induced increases in ADex and for sleep homeostasis. However, irreversible lesions may cause degenerative changes which go beyond loss of ChAT+ neurons. Thus, it is important to test the role of ChAT+ neurons using reversible stimulation or inactivation experiments. Furthermore, the need for intact ChAT+ neurons could reflect either ATPex/ADex release from ChAT+ neurons themselves and/or ChAT+ modulation of neighboring, non-cholinergic neurons. Thus, in this application we will use our novel methodology of optodialysis (Zant et al., 2016) to combine reversible, neuronal-subtype-specific optogenetic manipulations with simultaneous in vivo microdialysis to measure neurochemical changes in the presence and absence of selective antagonists for particular neurotransmitter receptors to delineate functional interactions between neuronal subtypes. Recent reports showed that optogenetic stimulation of vGluT2+ neurons excited ChAT+ neurons whereas PV+ neurons had only a weak influence on ChAT+ neurons. Furthermore, BF glutamate receptor activation enhanced acetylcholine (ACh) release in cortex and increased ADex levels in BF. These findings provide the basis for our hypothesis that local positive feedback between BF ChAT+ and vGluT2+ but not PV+ neurons leads to the ATPex/ADex increases which underlie the homeostatic sleep response. Three specific aims (SA) are proposed towards this goal: We will examine the role of cholinergic, parvalbumin expressing GABAergic, and glutamatergic neurons in sleep homeostasis using optogenetic stimulation and inhibition (SA1) and BF increases of ATPex and ADex using optodialysis (SA2). Using in vitro electrophysiology and in vivo optodialysis, SA3 will examine the hypothesis that a local positive feedback between cholinergic and glutamatergic neurons leads to BF ATPex/ADex increases during SD. Successful completion of these studies will provide insight into the cellular and neurochemical mechanisms underlying the homeostatic sleep response. Thereby, facilitating the development of therapeutic measures to reduce the deleterious effects of sleep loss in military personnel, veterans and people with sleep disorders.