Regulation of extraysynaptic GABA-A receptors in health and disease

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This project is focused on the cellular and molecular changes caused by traumatic brain injury (TBI), specifically those affecting GABAergic signaling in the hippocampus. Maladaptive changes in cellular and molecular properties contribute to the cognitive and neuropsychiatric sequelae of TBI, and epileptic seizures. The chronic cellular and molecular alterations causing dysfunction after TBI, and the proximal factors triggered by TBI that induce these chronic changes, are incompletely understood. Microglia, the resident immune cells of the brain, serve beneficial functions in the healthy brain but in response to injury their actions can be maladaptive, injurious, and magnify the consequences of TBI. As such, microglia and their inflammatory signaling are candidate factors to trigger pathophysiological cellular and molecular changes after TBI. Prior work has established that downregulation of extrasynaptic GABA receptors is a consistent feature of TBI in the hippocampus. Experiments proposed here will test the hypotheses that: i) attenuation of microglia activation will limit TBI-induced changes in extrasynaptic GABA receptor expression and function and ii) attenuation of microglia activation will decrease the incidence of spontaneous seizures after TBI (epileptogenesis). Using a combination of patch-clamp electrophysiology and immunohistochemical techniques, the expression and function of extrasynaptic GABA receptors (both GABAA and GABAB) in hippocampal neurons will be assessed in a model of severe TBI (controlled cortical impact, CCI). To assess the role of microglia in these molecular alterations after TBI, a powerful pharmacological tool that dramatically depletes brain microglia will be used (PLX5622, a CSF1-R inhibitor). PLX5622-treated animals will be exposed to CCI, allowing the assessment of whether functional molecular alterations after TBI are dependent on microglia activation. Specifically, the proposed experiments will determine if changes in extasynaptic GABA receptors after TBI are dependent on microglia activation. As part of this aim, other areas affected by TBI (thalamus, cerebellum) will be examined for CCI-associated changes in extrasynaptic GABA receptor function. The cellular effects of TBI and outcomes will also be investigated to assess their dependence on microglia activation. Using the approach of depleting microglia with PLX5622 prior to CCI, the time course and magnitude of neuronal death after TBI (for all neurons and specific interneuronal subtypes) will be determined for control and PLX5622-treated animals. Mossy fiber sprouting (the pathophysiological arborizing response of DGGC axons to injury) is another cellular change that occurs as a consequence of TBI; mossy fiber sprouting will be quantified and compared between the two groups. The cellular and molecular changes proposed for study here are all believed to contribute to epileptogenesis and seizures, but irrespective of these specific cellular/molecular changes the recognition of significant microglia-dependent effects on epileptogenesis after CCI is of greatest importance. Video-EEG monitoring will be used to determine the incidence of spontaneous seizures (i.e. epilepsy) and seizure characteristics after CCI in control and PLX5622-treated animals. These data will provide evidence to support (or refute) a significant role of microglia in epileptogenesis after CCI. These insights are needed to inform areas of future investigation and therapeutic approaches.