Astrocytes remove the excitatory neurotransmitter glutamate from the extracellular space following neuronal activity via sodium-driven, voltage-dependent excitatory amino acid transporters (EAATs). Robust glutamate uptake by EAATs ensures the temporal and spatial fidelity of glutamate signaling. Interestingly, we recently found that neuronal activity rapidly (within milliseconds) and reversibly slows glutamate uptake in the adult cerebral cortex. This slowing prolongs neuronal NMDA responses, consistent with prolonged extracellular glutamate dynamics, and is highly dependent on the frequency and duration of stimulation. Additionally, glutamate clearance can be modulated by neuronal activity with synapse specificity, even within a single astrocyte. We believe this may have important consequences on neurotransmission, extrasynaptic receptor activation, and synaptic plasticity. Based on that exciting finding, we hypothesized that neuronal activity induces microdomain-level changes in astrocyte membrane potential (Vm) that locally modulate EAAT function. GLT1 is the predominant astrocytic EAAT in the adult forebrain, is abundantly expressed, and ensures that glutamate in the extracellular space is rapidly sequestered by EAAT binding. Once bound to EAATs, the transport of glutamate into the astrocyte is both sodium-driven and voltage-dependent. Under normal conditions, astrocytes are hyperpolarized (-80 mV) due to their high permeability to potassium. However, neuronal activity increases extracellular potassium, [K ]e, and astrocyte Vm is especially sensitive to [K ]e changes. Therefore, it stands to reason that neuronal activity can alter EAAT function by depolarizing astrocytes. Changes in astrocytic Vm may be especially relevant in fine astrocytic processes, where EAATs are concentrated, and where small intracellular volumes may amplify changes in Vm, as compared to soma. A major challenge to testing our hypothesis, however, is an inability to monitor astrocyte Vm at distal processes due to low membrane resistance and process morphology. Overcoming this challenge is important because astrocyte distal processes are the site of synaptic interaction and EAATs localization. In order to detect distal changes in astrocyte Vm, we have begun to develop an approach to image Vm in astrocyte processes using QuasAr, a genetically-encoded voltage sensor. Dr. Adam Cohen, who developed QuasArs, is a collaborator on this proposal. Utilizing astrocyte and neuron electrophysiological recording, optogenetic manipulation of astrocyte Vm, and QuasAr imaging of astrocyte membrane potential we have generated preliminary data that supports our hypothesis that EAAT function can be modulated by activity-induced changes in astrocyte Vm.
Neuronal activity modulates astrocyte glutamate uptake. We hypothesize that this occurs via activity-induced modulation of membrane potential (Vm) in astrocyte distal processes. We will determine if modulating astrocyte Vm, independent of neuronal activity, alters glutamate uptake and utilize a genetically encoded voltage sensor to quantify Vm in astrocyte distal processes.