Abstracts

Rationale:
Neuroimmune interactions following trauma may play a critical role in the development of post traumatic epilepsy (PTE). We recently demonstrated that the expression of Toll-Like Receptor 4 (TLR4), an innate immune receptor, is enhanced after brain injury and contributes to epileptogenesis (Korgaonkar et al., 2020). While pharmacological suppression of TLR4 after brain injury reduces seizure susceptibility and memory deficits, TLR4 antagonism resulted in a paradoxical increase in seizure susceptibility in the uninjured brain. These data suggest mechanistic differences between TLR4 signaling and its effects on neurophysiology in the naïve and injured brain which are not fully characterized. This study examines the cellular and molecular mechanisms downstream of TLR4 which contribute to differential modulation of synaptic excitation and inhibition in the uninjured and injured brain. 
Method:
Young male Wistar rats or C57Bl6 mice of both sexes (25-27 day old) were subject to moderate (2 atm in rats, or 1.5atm in mice) lateral fluid percussion injury (FPI) or sham injury (Li et al., 2015). Whole cell recordings were obtained from granule cells in acute hippocampal slices prepared 5-7 days after FPI or sham injury. Synaptic responses evoked by perforant path stimulation were recorded at holding potentials of -70mV or 0mV using a cesium-methansulfonate based to isolate evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) respectively. A potassium-gluconate based solution was used to record EPSCs in some experiments. A subset of sham and FPI mice were treated in vivo with the TLR4 antagonist CLI-095 (3 mg/ml) and sacrificed 3 days after injury to obtain sections for immunostaining.
Results:
We previously demonstrated that TLR4 signaling contributes to increase in perforant path evoked granule cell AMPA currents after FPI (Korgaonkar et al., 2020). In slices from brain injured rats, granule cell eEPSC amplitude and charge transfer in were not altered by incubation in glial metabolic inhibitors (GMI: 1 mM fluoroacetate and 50mM minocycline) indicating lack of glial involvement. Moreover, HMGB1 (10 ng/ml), a TLR4 agonist, enhanced AMPA currents even in the presence of GMI. However, blocking TNF-α reduced the peak amplitude of granule cell eEPSCs recorded in HMGB1, indicating that TNFα may be downstream of neuronal TLR4 signaling. One week after FPI, granule cell eIPSC amplitude was reduced in both mice (Sham: 1186pA±131 FPI: 718pA±168, n=5, p< 0.05) and rat. TLR4 antagonist reduced eIPSC amplitude in granule cells from sham-injured mice while increasing eIPSC amplitude in granule cells from FPI mice, demonstrating  an opposing effect of TLR4 signaling on dentate inhibition in sham and FPI mice. Immunostaining for GABAAR α1 subunit in sham and FPI mice treated with CLI-095 revealed layer specific increase in GABAAR α1 subunit expression in the dentate molecular layer in FPI mice treated with CLI-095 compared to vehicle treated FPI mice.
Conclusion:
TLR4 signaling leads to differential modulation of glutamatergic and GABAergic currents in granule cells in the control and FPI mice. While TLR4 signaling, acting acutely, appears to enhance GABA currents without altering excitatory currents in controls, it enhances excitatory currents and reduces inhibitory currents in dentate granule cells contributing to enhanced network excitability early after injury. Targeting TLR4 signaling after brain injury could mitigate excitation-inhibition imbalance brain injury by combined effects on glutamate and GABA synapses.
Funding:
:AES Predoctoral Fellowship #695548 to SN; NIH/NINDS R01NS069861 and R01NS097750 to VS