Instructor/Research University of Texas Health San Antonio San Antonio, Texas
Rationale: The overall goal of this study is to identify molecular mechanisms resulting from traumatic brain injury (TBI) that precede epileptogenesis. Approximately 20% of symptomatic epilepsies and 5-6% of all cases of epilepsy result from TBI. After a TBI, but before any seizures begin, there are pathophysiological changes in the brain that produce neuronal hyperexcitability and epileptogenesis. Cascades of pathophysiological changes affect neuronal signaling within the cortex and the hippocampus. Adaptations to cholinergic signaling after TBI suggest muscarinic receptor-mediated changes to TRPC1/4/5 cation channels. However, there have been no investigations of TRPC channel activity in the context of TBI-induced epileptogenesis. Our overall hypothesis is that TRPC channels are up-regulated after TBI thus promoting neuronal hyperexcitability in the cortex and hippocampus. Therefore, we investigated whether TRPC1/4/5 cation channels contribute to seizure susceptibility and epileptogenesis following TBI-induced hyperexcitability. Methods: We performed closed-cortical impact to the right parietal cortex (5 mm tip, 4.5m/s, 0.5s dwell time) or sham surgery on two- to three-month-old C57BL/6 mice. Real-time PCR and western immunoblot were performed for TRPC1, C4, and C5 from microdissected regions of the hippocampus and parietal cortex after seven days. Ipsilateral and contralateral hemispheres were compared among each mouse, and sham and TBI brains were compared for changes in TRPC1/4/5 expression. Acetylcholinesterase (AChE) activity was measured with the AChE–Amplex Red assay. We investigated neuronal activity from brain slices of TBI mice using GCaMP6f mice in measurement of Ca2+ influx and action potential firing. Whole-cell patch clamp electrophysiology was performed in neurons to test the hypothesis that TRPC channels promote hyperexcitability after injury. To evaluate seizure susceptibility after TBI, mice were given pentylenetetrazol (PTZ, 35 mg/kg s.c.) once per day in development of kindling epileptogenesis. Results: Seven days after TBI, CA3 exhibited significant upregulation of TRPC1, C4, and C5 mRNA, and CA1 exhibited upregulation of TRPC1 mRNA in ipsilateral tissue compared to contralateral side of injury. Furthermore, all examined regions (cortex, DG, CA3, and CA1) of TBI mice from both ipsilateral and contralateral side of injury displayed significantly increased TRPC4 and C5 mRNA compared to sham controls. TRPC4 and C5 protein were significantly greater in TBI ipsilateral cortex, DG, and CA3, but not in CA1. Sham mice did not exhibit inter-hemispheric differences in TRPC channels. AChE activity was significantly decreased seven days after TBI in cortex and hippocampus. In brain slice, TRPC4/5-mediated Ca2+ influx resulted in greater neuronal hyperexcitability in TBI mice in hippocampus. We observed that TBI mice were more susceptible to sub-threshold dose of PTZ (35 mg/kg) and more rapidly progressed to forelimb clonus and tonic-clonic seizures. The TRPC4/5 antagonist M_084 impeded the rate of PTZ kindling in TBI mice, suggesting a role of TRPC channels in the modulation of hyperexcitability after injury. Conclusions: These findings demonstrate that TRPC channels are upregulated in compensatory modulation of neuronal excitability after TBI, facilitated by increased cholinergic activity. The cholinergic-TRPC1/4/5 axis of neuronal signaling in the hippocampus and cortex contributes to hyperexcitability in transformation of network activity after injury. Our data suggest that TRPC channel plasticity may maladaptively contribute to greater seizure susceptibility and sensitivity within the hippocampus and cortex. Funding: Please list any funding that was received in support of this abstract.: Department of Defense Idea Development Award W81XWH-19-1-0400 to C.M.C.