Abstracts

Rationale: Brain injury leads to early activation of immune responses including increasing dentate hilar neuronal expression of toll-like receptor4 (TLR4), a class of innate immune receptors implicated in enhanced hippocampal excitability in epilepsy. Here we examine if activation of TLR4 signaling modulates dentate excitability in vitro and seizure thresholds in vivo. Methods: Wistar rats (25-27 day old) were subject to moderate (2 atm) lateral fluid percussion injury (FPI) or used as sham-controls (Gupta et al., 2012). The effect of TLR4 ligands (agonists: LPS-RS and TLR4 antibody; and agonist: HMGB1) on afferent-evoked granule cell population spike amplitude, and granule cell and mossy cell EPSC amplitude and charge transfer were examined in hippocampal slices 1 week after FPI. The effect of in vivo hippocampal injection of LPS-RS (5μg) or vehicle, administered 24 hours after FPI, on latency to kainic acid induced seizures was examined 30 days after FPI. Results: The TLR4 antagonist LPS-RS increased and agonist HMGB1 reduced afferent evoked dentate population spike amplitude in slices from control rats indicating a constitutive TLR4 mediated suppression of dentate excitability. In contrast, TLR4 antagonists decreased (amplitude in mV, FPI-aCSF: 1.61±0.24, FPI-LPS-RS: 0.55±0.09, n=15 slices each, p≤0.05) and HMGB1 increased dentate population spike amplitude after FPI (amplitude in mV, FPI-aCSF: 1.65±0.20, FPI-HMGB1: 2.89±0.29, n=11 slices each, p≤0.05) revealing a post-injury reversal of TLR4 effect on network excitability. Afferent-evoked granule cell NMDA EPSCs were not modulated by LPS-RS in both sham and FPI rats. However, granule cell and mossy cell non-NMDA EPSC amplitude and charge transfer were increased after FPI. LPS-RS incubation in vitro selectively decreased non-NMDA EPSC amplitude in granule cells (in pA, FPI-aCSF: 139.69±13.64, FPI LPS-RS: 92.44±10.92 pA, n=10 each, p≤0.05) and mossy cells (in pA, FPI-aCSF: 189.49±11.88 FPI-LPS-RS: 135.20±5.89, n=6 each, p≤0.05) form FPI rats. Crucially, a single bolus treatment with LPS-RS 24 hours after FPI prolonged the latency for kainic acid induced seizures 30 days after FPI (in sec, FPI-vehicle: 16.2±2.09, FPI-LPS-RS: 180±0, p≤0.05) but reduced seizure latency in age matched controls (in sec, sham-vehicle: 52.2±5.48, sham-LPS-RS: 15.6±1.38, p≤0.05). Conclusions: Our findings demonstrate a constitutive modulation of dentate excitability by TLR4 signaling and a novel TLR4-dependent enhancement of non-NMDA glutamatergic synaptic currents in dentate mossy cells and granule cells after brain injury. The mechanisms and physiological role of the novel constitutive and bi-directional effects of TLR4 on dentate excitability need further investigation. Although in vivo studies suggest that TLR4 signaling may provide a valuable target for therapeutic interventions to prevent neurological sequelae of brain injury, the opposing physiological effects of constitutive TLR4 signaling, need to be considered before manipulating TLR4 signaling. Support: CURE Foundation and NJCBIR CBIR11PJT003 to V.S.