The role of lateral orbitofrontal cortex astrocytes in alcohol drinking

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PROJECT SUMMARY Alcohol use disorder (AUD) is characterized by the progression from recreational drinking to uncontrollable and excessive consumption resulting in a myriad of social and neurobiological complications. The mechanisms underlying the dependence-induced escalation in drinking are not completely understood. However, a key brain region disrupted in individuals with AUD is the orbitofrontal cortex (OFC). Studies from the Woodward laboratory show that acute ethanol inhibits action potential firing of lateral orbitofrontal (lOFC) cortex pyramidal neurons. This occurs via an astrocyte-dependent process involving activation of astrocytic D1/D5 dopamine receptors and the release of glycine via reversal of the GlyT1 glycine transporter. Following chronic intermittent exposure (CIE) to alcohol, lOFC neurons become hyperexcitable and are tolerant to acute ethanol. However, the effects of CIE exposure on lOFC astrocytes and how this affects lOFC neuronal excitability are not completely understood. The overarching hypothesis of this proposal is that CIE exposure impairs lOFC astrocyte function that contributes to hyperexcitability of lOFC pyramidal neurons and the resulting dependence-induced escalation in drinking. This hypothesis will be tested with two complementary aims. Aim 1 will test the hypothesis that CIE-induced increases in lOFC neuronal excitability and loss of acute ethanol inhibition involves astrocytic calcium signaling. To test this, male and female C57BL/6J mice will receive an intra-OFC infusion of an astrocyte-selective AAV encoding either a plasma membrane calcium ATPase (PMCA) or a reporter construct (tdTomato). Following repeated cycles of CIE exposure, slice electrophysiology will be used to measure current-evoked spiking of lOFC neurons and the membrane potential of lOFC astrocytes. Training in viral infusion surgeries and astrocyte and neuron slice electrophysiology will be achieved under this aim. Aim 2 will test the hypothesis that expressing PMCA in lOFC astrocytes prevents the increases in drinking following CIE exposure. In the first study, male and female C57BL/6J mice expressing either PMCA or tdTomato localized in lOFC astrocytes will undergo baseline sessions of two-bottle choice ethanol drinking. Weekly sessions of CIE exposure will then be interleaved with test weeks of drinking. The second study will follow the same CIE paradigm with the absence of homecage drinking and will use mice expressing GCaMP6f in lOFC astrocytes and the red-shifted opsin ChrimsonR in lOFC neurons. Training in astrocyte fiber photometry and optogenetics will be achieved under this aim. The proposed research studies will be complemented by career development activities including manuscript preparation, data presentation, networking, and training in the responsible conduct of research. These studies will provide novel insight into the role of lOFC astrocytes in AUD and position me to pursue a productive career in alcohol research.