Abstracts

Rationale: Traumatic brain injury is a major risk factor for acquired epilepsies. We recently identified that brain injury enhances neuronal expression of the innate immune receptor, Toll-like receptor 4 (TLR4) in the dentate gyrus (Li et al., 2015). TLR4 was found to augment post-traumatic dentate excitability by a cell-type specific increase in non-NMDA synaptic excitation. Here, we examine the molecular mechanisms by which TLR4 modulates granule cell excitability after brain injury.Methods: Wistar rats (25-27 day old) were subject to sham or moderate (2 atm) lateral fluid percussion injury (FPI) (Li et al., 2015). Whole cell recordings were obtained from granule cells and dentate hilar neurons in hippocampal slices obtained 5-7 days after FPI. Incubation in LPS-RS ultrapure (LPS-RS-U) was used to acutely and selectively block TLR4 signaling. Afferent-evoked granule cell AMPA currents were recorded in the presence of NMDA and GABA receptor antagonists. Rectification index was measured as the ratio of currents recorded from holding potentials of +40mV and -60mV. Biotinylation assay was used to quantify the effect of FPI and acute TLR4 antagonism on surface expression of AMPA receptor subunits in slices from rats 24 hours after FPI.Results: In agreement with earlier studies, there was an increase in the number of afferent-evoked action potentials in dentate granule cells one week after FPI. LPS-RS-U treatment selectively reversed the post-FPI increase in granule cell evoked firing. In whole cell recordings, granule cell AMPA current amplitude recorded at -60 mV was higher and rectification index significantly lower after FPI (Sham: 0.90 ± 0.06; FPI: 0.12 ± 0.02, P<0.05, n=6 each), indicting an increase in calcium-permeable AMPA (CP-AMPA) currents after FPI. LPS-RS-U treatment selectively enhanced AMPA rectification index in slices from FPI rats but not in controls. Consistent with the physiology data, surface biotinylation assay identified an increase in surface expression of the calcium-permeable GluR1 AMPA subunit expression 24 hours after FPI compared to sham (% GluR1 expression in FPI normalized to sham: 134.41±9.91, n=6 each, ANOVA, P<0.05). The post-FPI increase in GluR1 surface expression was abolished following acute incubation of the slices in LPS-RS-U (% GluR1 expression in LPS-RS-U treated slices normalized to untreated sham: Sham: 102.86±3.54, FPI: 103.87±5.69%, n=6 each, ANOVA, P>0.05). Surface expression of the calcium-impermeable GluR2 AMPA subunit showed no differences between slices from sham and FPI rats and no alteration following TLR4 antagonist treatment.Conclusions: The early increase in surface expression of the CP-AMPA in hippocampus after FPI could contribute to excitotoxic damage following brain injury. Since TLR4 actively modulates CP-AMPA currents early after brain injury, TLR4 antagonism early after brain injury could, by limiting early increases in excitability, reduce subsequent risk for epilepsy after brain injury.