Abstracts

Rationale: Traumatic brain injury is one of the most common causes of acquired epilepsy and the underlying mechanisms are not fully understood. Brain injury leads to loss of interneurons in the dentate hilus, enhanced dentate excitability and activation of sterile inflammatory responses. We previously reported that concussive brain injury increases expression of toll-like receptor 4 (TLR4), an innate immune receptor, in dentate hilar neurons. In glutamatergic granule cells and mossy cells, TLR4 signaling enhanced non-NMDA currents selectively after brain injury. The cell-type specific expression of TLR4 in interneurons is currently unknown. This study examined TLR4 expression and physiological effect of TLR4 signaling on dentate hilar interneurons after brain injury.   Methods: Young Wistar rats (25-27 day old) were subject to sham- or moderate (2 atm) lateral fluid percussion injury (FPI) (Li et al., 2015). Immunostaining was used to determine the cell-type specific expression of TLR4. Afferent-evoked granule cell field responses were recorded in acute hippocampal slices prepared 3-7 days after FPI. Whole cell recording were performed on granule cells and dentate hilar neurons in hippocampal slices from rats 5-7 days after FPI or sham injury. Slices were incubated in control ACSF or in the specific TLR4 antagonist LPS-RS-Ultrapure (LPS-RS-U) for 2 hours. AMPA currents were evoked by performant path stimulation and recorded in NMDA and GABA receptor blockers. AMPA rectification index was calculated as the ratio of non-NMDA current amplitude measured from holding potentials of +40mV and -60mV.  Single cell PCR was used to identify interneuron subtypes. Results: In the presence of GABA_A receptor antagonist, the TLR4 antagonist mediated reduction of injury-induced dentate hyperexcitability was decreased suggesting that TLR4 signaling modifies dentate inhibition. Brain injury resulted in an increase in afferent-evoked IPSC amplitude in granule cells.  TLR4 antagonist reversed the post-FPI reduction of granule cell eIPSC while decreasing eIPSCs in sham. Immunostaining revealed that somatostatin and not parvalbumin interneurons co-localized with TLR4. In whole cell recordings, somatostatin neurons had increased sEPSC frequency and reduced AMPA current rectification after FPI which were reversed by LPS-RS-U (Sham: aCSF: 0.62 ± 0.14, LPS-RS-U: 0.71 ± 0.15 p>0.05; FPI: aCSF: 0.11 ± 0.12, LPS-RS-U: 0.53 ± 0.16, n=6 each, p<0.05). Rectification index of parvalbumin neurons was not affected by LPS-RS-U. Blocking TLR4 selectively reduced the loss of somatostatin neurons during a high-K challenge after FPI. Conclusions: TLR4 signaling leads to differential modulation of GABA currents in granule cells from control and FPI rats. Cell specific expression of TLR4 may underlie loss of somatostatin interneurons early after brain injury. TLR4 antagonist treatment early after brain injury has the potential to limit neuronal loss, limit excitotoxicity and promote GABAergic inhibition in the dentate gyrus and prevent post-traumatic epileptogenesis. Funding: CURE Foundation CF 259051, NJCBIR CBIR14RG024 and NIH R01NS097750 to V.S. and and NJCBIR CBIR15FEL011 to A.K.