Abstracts

Rationale: Alterations to inhibitory interneurons within the hippocampus are thought to play a key role in the pathogenesis of mesial temporal lobe epilepsy (MTLE). Of particular importance, two distinct populations of interneurons, those expressing parvalbumin (PV) and the cannabinoid type 1 receptor (CB1R), exert powerful control over excitatory dentate granule neurons (DGN) through perisomatic innervation. Surprisingly little, however, is known about the organization of these inhibitory interneurons in the healthy human hippocampus. Furethermore, while PV cells appear disrupted in MTLE, we have no understanding of epilepsy-related changes in PV-containing basket cells (PVBC) and chandelier cells (PVChC) at the synaptic bouton level. In addition, a separate population of basket cells expressing CB1R has also been hypothesized to play a role in MTLE, although there are contradictory findings regarding CB1R-mediated anti-epileptic versus pro-epileptic effects. No study to date has simultaneously examined changes in CB1R+, PVBC, and PVChC inhibitory perisomatic bouton distribution, density and protein content in the normal human or epileptic DG. Methods: We obtained 12 hippocampal specimens from subjects with refractory MTLE who underwent en bloc hippocampectomy as part of standard anterior temporal lobectomy surgery (n=6 sclerotic, n=6 non-sclerotic), and 6 age- and sex-matched healthy postmortem controls. We modified a multi-label, semi-quantitative fluorescence confocal microscopy method and an advanced post-imaging analysis paradigm to analyze the densities and relative protein levels of PV+ and CB1R+ terminals in the granule cell layer of the human dentate gyrus (DG). Given that PVBC boutons in the human prefrontal cortex contain both GAD65 and GAD67, while PVChC contain GAD67 almost exclusively, differential GAD isoform expression was used to identify these two PV subpopulations. Results: PV and CB1R terminal density and distribution were significantly altered in MTLE. Specifically, PVBC bouton density was selectively and substantially increased in sclerotic samples, while PVChC bouton density was reduced. CB1R-BC bouton density was also modulated in sclerotic samples. In areas of preserved PV+ and CB1+ terminals, upregulation of PV, CB1, and GAD protein was observed. Conclusions: We demonstrate interneuron-subtype specific epilepsy-related changes at the synaptic bouton level in human MTLE for the first time. These data advance our understanding of the perisomatic innervation of principle neurons in the human dentate gyrus in both healthy and epileptic states. Future studies may determine the extent to which these findings are recapitulated in both the normal rodent hippocampus and in rodent models of MTLE, a knowledge gap that is critical to fill, given the importance of perisomatic innervation in determining the patterning of neural output from the hippocampus. Funding: Walter L. Copeland Fund of the Pittsburgh Foundation (RMR), Grant UL1 TR0000005 from the National Center for Advancing Translational Sciences (RMR and KNF).