Abstracts

Rationale: Brain injury is a leading cause of acquired temporal lobe epilepsy. We recently reported that brain injury enhances neuronal expression of the innate immune receptor Toll-Like receptor 4 (TLR4) in the hippocampal dentate gyrus and showed that TLR4 signaling promotes post-injury increase in dentate excitability (Li et al., 2015). We demonstrated that TLR4 signaling augments non-NMDA synaptic currents in dentate excitatory neurons after brain injury. Interestingly, TLR4 was expressed in a majority of dentate hilar neurons suggesting that TLR4 could modulate inhibitory circuits. Here we examine TLR4 regulation of granule cell inhibition and GABAergic neurons in the normal and injured brain. Methods: Wistar rats (25-27 day old) were subject to sham- or moderate (2 atm) lateral fluid percussion injury (FPI). Whole cell recording were obtained from granule cells and dentate hilar neurons in hippocampal slices prepared 5-7 days after FPI. Data from slices incubated in the selective TLR4 antagonist LPS-RS ?"ultrapure (LPS-RS-U) for 2 hours were compared to vehicle-treated controls. Cell types were identified based on single cell PCR. Non-NMDA currents were isolated in NMDA and GABA antagonists. Rectification index, a measure of calcium permeability, was defined as the ratio of peak Non-NMDA currents measured at +40mV and -60mV. As subset of live slices were incubated with 10mM potassium along with LPS-RS-U or vehicle, fixed and immunostained to assess excitotoxic cell death. Results: The amplitude of perforant-path evoked inhibitory postsynaptic currents (eIPSCs) in granule cells was decreased after FPI. LPS-RS-U pretreatment reversed the decrease in granule cell eIPSC amplitude after FPI (in pA, vehicle: 71.19 13.98, LPS-RS-U: 125.97 13.85, n=5, p < 0.05) yet decreased it in sham controls (in pA, vehicle: 141.63 15.65, LPS-RS-U 100.98 14.24, n=5, p < 0.05). Immunostaining revealed that somatostatin (SOM) and not parvalbumin (PV) neurons expressed TLR4. Voltage clamp recordings identified a post-FPI increase in spontaneous EPSC frequency in SOM neurons which was reversed by LPS-RS-U treatment. There was a post-FPI decrease in the rectification index of non-NMDA eEPSCs in SOM neurons, indicating an increase in calcium permeability, which was reversed by LPS-RS-U incubation. LPS-RS-U reduced SOM neuron death in response to a 10 mM K+ challenge after FPI but not sham. Rectification index of non-NMDA eEPSCs in PV neurons was not modulated by LPS-RS-U. Conclusions: TLR4 signaling in the injured brain contributes to enhanced calcium-permeable AMPA currents and excitotoxic death of hilar SOM neurons. TLR4 antagonist treatment has the potential to limit neuronal loss and excitotoxicity after brain injury and reduce the risk for post-traumatic epilepsy. Funding: CURE Foundation CF259051 and NJCBIR CBIR14RG024 to VS