A ballistic pendulum impulse generator was used to impact networks in primary culture growing on microelectrode arrays. This approach has the advantage of imparting pure tangential acceleration insults (50 to 300 g) with simultaneous morphological and electrophysiological multichannel monitoring for days before and after the impact. Action potential (AP) production, network activity patterns, and cell electrode coupling of individual units using AP waveshape templates were quantified. Network adhesion was maintained after tangential impacts up to 300g with minimal loss of pre-selected active units. Time lapse phase contrast microscopy revealed stable nuclei pre-impact, but post impact nuclear rotation in 95% of observations (n= 30). All recording experiments (n=31) showed a repeatable two-phase spike production response profile: recovery to near reference in 1-2 hrs, followed by a slow activity decay to a stable, level plateau approximately 30-40% below reference. Phase 1 consisted of a complex two-step recovery: rapid activity increase to an average 23.6% (range: 11-34%) below reference, forming a level plateau lasting from 5 to 20 min, followed by a climb to within 20% of reference where a second plateau was established for 1 to 2 hrs. Cross correlation profiles showed changes in firing hierarchy after impact, and in spontaneous network oscillatory activity. Native oscillations were found in the Delta band (2 to 3 Hz), and decreased by approximately 20% after impact. Under network disinhibition with bicuculline, oscillations were slower (0.8-1Hz) and decreased 40% after impact. These data link network performance deficits with microscopically observable subcellular changes.

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Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE) following traumatic brain injury, accounts for 20% of symptomatic epilepsy. Reorganization of mossy fibers within the hippocampus is a common pathological finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus where they form synapses with pyramidal cells. During TLE, mossy fibers are observed to innervate the inner molecular layer where they synapse onto the dendrites of other dentate granule cells, leading to the formation of recurrent excitatory circuits. To date, the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown. Recent focus has centered on the involvement of tropomycin-related kinase receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β) inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2 (CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation of GSK3β should therefore increase CRMP2 activity. To determine the involvement of CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it was demonstrated that increased neurite outgrowth following GSK3β inactivation is CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the transience of GSK3β-inactivation. Based on previous work, it was hypothesized that activity-dependent mechanisms may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI. While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it did not differ significantly from vehicle-treated animals. Therefore, the necessity of CRMP2 in mossy fiber sprouting following TBI remains unknown.