Optogenetics: A tool to probe mechanism and an agent to block TBI-induced epileptogenesis.

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Over two million people are treated medically each year in the United States after sustaining a traumatic brain injury (TBI). Posttraumatic epilepsy (PTE) develops in up to 39% of patients with moderate to severe, non-penetrating TBI. As with other acquired epilepsies, spontaneous recurrent seizures associated with PTE develop with a latency (>1 week and up to many years) after the initial injury. This seizure-free period after TBI represents the period of epileptogenesis, during which the brain undergoes physiological, anatomical, cellular, and molecular changes leading to a state of chronically increased seizure susceptibility. This delay between the TBI and development of PTE also represents a period during which strategies might be employed to inhibit the reactive plasticity in the brain that leads to PTE, but the molecular mechanisms underlying the epileptogenic process leading to acquired epilepsy are largely unknown and no anti-epileptogenic therapies have been successfully developed to date. Animal models of posttraumatic epileptogenesis (PTEgenesis) point to reactive plasticity of hippocampal networks, with alteration in the balance of excitation/inhibition as a driver of permanent brain changes and the epileptic state. However, the prime molecular and electrophysiological transformations remain murky. The hypothesis to be tested: Post-injury activity and network changes in the hippocampus, induced in part by alterations in the vesicular neurotransmitter release machinery, are primary drivers of PTEgenesis. The Specific Aims are to: 1) Use channelrhodopsin-2 (ChR2) to optogenetically drive neural activity and the process of PTEgenesis by depolarizing specific primary neuronal populations in dentate gyrus (DG). 2) Use halorhodopsin (NpHR) to retard PTEgenesis, induced using a standard method, by optogenetically inhibiting neural activity in DG. Proven techniques will be integrated into a new and unique model to detect network and molecular drivers of PTE and PTEgenesis. Using the controlled cortical impact (CCI) model of TBI, our proposed studies combine 1) unique microelectrode array electrochemistry (MEA) to monitor real-time glutamate release and oxygen change as a metric of epileptiform activity; 2) immunohistochemistry to define changes in specific cell phenotypes; and 3) slice electrophysiology with custom Western blot quantitation of neurotransmitter release machinery on a novel, optogenetically-modified hippocampal platform. Aim 1: Studies will be accomplished by AAV2/5 viral transfection of a ChR2-promotor construct into hippocampal DG of rats, utilizing optogenetic activation of DG neurons of free-roaming rats after CCI-induced TBI to enhance PTEgenesis. Extra-cellular glutamate, electrophysiological, immunohistochemical, and vesicular release biochemical measures will be made on animals at discrete behavioral stages during the progression of epileptogenesis. Aim 2: Studies will be accomplished by AAV2/5 viral transfection of an NpHR- promoter construct into hippocampal DG of rats prior to CCI injury and optogenetic inhibition of DG circuits after CCI to inhibit PTEgenesis. MEA, electrophysiological, immunohistochemical, and biochemical measures will be made. As one component of these studies, transient glutamate surges and changes in oxygen in DG detected by MEAs will trigger real-time optogenetic inhibition as a potential means to abort PTEgenesis. Our approach should allow network, neuronal, and presynaptic release changes to be clearly tied to biochemical, anatomical, electrophysiological, and behavioral outcomes associated with epileptogenesis. The studies in this proposal will pave the way to development of comprehensive, novel analyses of the progression of PTE and will provide preliminary data to support studies identifying causative effects of activity-dependent synaptogenesis in the development of PTE. In addition, these studies will test a potentially novel therapy for PTEgenesis. Thus, this research is directly relevant to the care of a large proportion of service men and women, veterans, and the general population.