In vivo analysis of astroctye-neuron dynamics in circuit formation, function, and maintenance

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PROJECT SUMMARY The mammalian brain is formed by billions of neurons which communicate at specialized chemical junctions called synapses. Individual neurons connect to form functional circuits which are required for proper learning and memory, and disruption of neuronal circuitry underlies the debilitating symptoms experienced by patients suffering from neurological disorders such as epilepsy and schizophrenia. Although proper formation and maintenance of neuronal circuits is essential for a high quality of human life, the process by which a given neuron finds the correct synaptic pair, and how these synapses are maintained and modified over time is poorly understood. Recent work from our labs and others have identified astrocytes, the most abundant CNS glial cell type, as a major regulator of synaptic development. Astrocytes are both pro-synaptogenic (e.g. loss of astrocytes results in decreased synaptogenesis) as well as anti-synaptogenic (e.g. astrocytes engulf and prune synapses). These important functions of astrocytes in regulating synapse number suggest that astrocytes may regulate broader circuit formation, though this hypothesis has not been fully investigated. Characterization of astrocyte-neuron dynamics within a behaviorally-relevant circuit has not been performed, probably because it requires in vivo manipulation of a defined pair of synaptically-coupled neurons and the associated astrocytes. Given the enormous complexity of the mammalian nervous system, these types of experiments are not yet feasible in mammals. Excitingly, it is now possible to perform these studies in the Drosophila nervous system due to the recent development of tools for astrocyte manipulation from the Freeman lab, and identification of neural circuits governing larval locomotion in the Doe lab. As a co-mentored postdoctoral fellow within the Doe and Freeman laboratories, I will merge these new tools to have the unique ability to visualize and genetically manipulate individual central synapses, which I will couple with targeted manipulation of the associated astrocytes to define the role of astrocytes in synapse formation, maintenance, and function. For all studies, I will use recently identified transgenic lines that label defined synaptic pairs: the excitatory cholinergic synapses between E2 and SA1 interneurons, and the inhibitory, GABAergic synapses between A31k interneuron and RP2 motor neuron. Astrocytes will be visualized using anti-Gat immunofluorescence or expression of UAS-myr::Cerulean under alrm-GAL4. In my first aim, I will couple astrocyte ablation experiments with mutant analyses to test the necessity of functional astrocytes in the development (formation) of excitatory and inhibitory synapses. In my second aim, I will use an optogenetic strategy to measure the activity (function) of excitatory and inhibitory synapses in response to changes in astrocyte function. Finally, in my third aim, I will manipulate neuronal activity (through constitutive activation or silencing of defined pre-synaptic neurons) and test the hypothesis that neuronal activity influences both astrocyte morphology and function. In sum, these experiments will define the in vivo role of astrocytes in the formation, function, and maintenances of excitatory and inhibitory synapses within a behaviorally-relevant, sensorimotor circuit.