Determining the role of dysregulated GABA uptake by reactive astrocytes in thalamic circuit hyperexcitability and seizures

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PROJECT SUMMARY & ABSTRACT Acquired epilepsies can occur following brain lesions such as stroke or traumatic brain injury, and particularly affects elderly people children. However, there is no effective treatment or prevention strategy for post-traumatic epilepsy (PTE). Key to finding therapeutic targets is understanding epileptogenesis, the latent period between the initial injury and the development of epilepsy. Reactive astrocytes, or astrogliosis, form in response to neurological insults, and are strongly associated with epileptogenesis and with intractable, drug-resistant, epilepsies. In order to understand how reactive astrocytes contribute to diseases characterized by circuit abnormalities such as seizures, there is an urgent need to understand how they affect complex neuronal activity. The thalamus has been implicated in PTE—following cortical injuries and stroke, the thalamus becomes hyperexcitable, and develops chronic astrogliosis preceding the onset of seizures. Using a viral model of selectively induced astrogliosis that I previously characterized, I will investigate the cellular and circuit mechanisms by which reactive astrocytes drive circuit hyperexcitability in the thalamus and enable seizures. My preliminary electrophysiological and transcriptomic studies have suggested a direct, mechanistic link between astrocyte dysfunction and neural circuit hyperexcitability, in the context of inflammation. In this proposal, I will test the working hypothesis that thalamic reactive astrocytes downregulate GABA uptake via reduction of GABA transporters, which leads to enhanced tonic GABA currents in thalamocortical neurons, ultimately resulting in thalamic circuit hyperexcitability and seizures. To test this hypothesis, I will characterize the effects of bidirectional manipulation of astrocytic GABA uptake on GABAergic signaling in thalamocortical neurons, rhythmogenesis of the intrathalamic circuit in vitro, and seizure susceptibility in awake, behaving mice. I will use a dual adeno-associated virus (AAV) CRISPR- Cas9 approach to decrease astrocytic GABA transporter expression in wild-type mice (Aim 1), and an AAV- mediated overexpression approach to selectively enhance astrocytic GABA transporter expression in wild-type mice that have thalamic astrogliosis and abnormal thalamocortical hyperexcitability (Aim 2). The proposed work will harness the selectivity of the viral astrogliosis approach in combination with slice and in vivo electrophysiological assessments of neuronal circuits. These results will elucidate our basic understanding of astrocytic GABA uptake and neural circuit plasticity. Furthermore, by investigating the functional changes in reactive astrocytes that enable pathological circuit activity and mediate epileptogenesis, the proposed work will identify potential therapeutic targets to intervene and prevent PTE.