Grant: Discovery Grants Program - Individual

Increasing evidence indicates that astrocytes play important roles in brain functions, including synaptic plasticity. Astrocytes demonstrate rapid remodeling of their distal processes over the course of minutes. Astrocytic remodeling can occur in response to homeostatic challenges, and these structural changes can alter synaptic efficacy and neuronal excitability. We hypothesize that astrocytes play a role in sleep/wake regulation through dynamic remodeling of their perisynaptic processes to regulate key sleep/wake-promoting neurons. We will use acute (6 h) sleep deprivation (SD) for prolonged wakefulness, along with undisturbed and recovery conditions, in conjunction with morphological (immunohistochemistry, confocal microscopy), electrophysiological (patch-clamp recording), and behavioural (EEG & EMG recording, inhibitors of astrocyte functions) techniques in rats. The first morphological study will use immunohistochemistry for glial glutamate transporter-1 (GLT-1) and confocal microscopy to analyze GLT-1 apposition with key sleep/wake-promoting neurons after 6 h of SD or control conditions. Preliminary results indicate that wake-promoting orexin (ORX)-containing neurons, and sleep-promoting melanin concentrating hormone (MCH)-containing neurons in the hypothalamus show reciprocal changes in the amount of astrocytic GLT-1 apposition with their cell bodies after 6 h of SD, relative to time-matched controls. These preliminary data suggest that astrocytic processes show dynamic changes in their apposition with sleep/wake-regulatory neurons depending on sleep/wake history in a cell type-specific manner. Such changes in astrocytic apposition can alter glutamate uptake efficiency, and thereby synaptic efficacy and neuronal excitability. To test this possibility, the second study will use patch-clamp recording from brain slices prepared from sleep-deprived or undisturbed rats. Basic membrane properties and synaptic efficacy will be investigated, and effects of SD as well as pharmacological blockade of GLT-1 using dihydorkainate (DHK) will be characterized. Our preliminary data with SD show depolarization of ORX, but not MCH, neurons, and occlusion of the DHK effects by SD in ORX neurons. These preliminary morphological and electrophysiological data are consistent with the notion that astrocytes can restructure to modulate sleep/wake regulatory neurons in a cell type-specific manner. Thus, the third study will examine the effect of brain region-specific inhibition of astrocytic remodeling (using siRNA to silence the expression of connexin 30, a negative regulator of astrocytic restructuring) on sleep/wake patterns, and on sleep rebound after SD, using EEG & EMG recordings in rats. The proposed research is expected to provide novel insights about how astrocytes interact with neurons to regulate sleep and wake states.