Abstracts

Rationale: The mechanisms of glutamatergic neurotransmission during status epilepticus (SE) are not well understood. We recently reported the expression of calcium permeable AMPA receptors (AMPARs) on hippocampal principal cells during SE. We further investigated the mechanisms of AMPAR plasticity in the hippocampus during SE.Methods: Adult rats experiencing pilocarpine-induced SE either 10 min (10 SE) or 60 min (60 SE) after the onset of the first Grade V behavioral seizure were used for electrophysiology and biochemical studies. Results: During prolonged SE, the frequency of AMPAR-mediated miniature EPSCs (mEPSCs) obtained from CA1 pyramidal neurons (PNs) was increased. The mean mEPSC frequency in CA1 PNs of the control group was 0.39 0.05 Hz (n=14 cells) whereas in the 60 SE group it was 1.59 0.4 Hz (n=12 cells, p<0.05). The mean mEPSC frequency in CA1 PNs of the 10 SE group was similar to controls (0.22 0.04 Hz, n = 8 cells). The amplitude of mEPSCs in CA1 PNs of 10 SE (11.74 0.8 pA) and 60 SE (11.97 0.8 pA) groups were similar to that in controls (11.40 0.6 pA); however, the net charge transfer of AMPAR-mediated mEPSCs was greater in the 60 SE group (40.25 6.1 pC vs. 138.9 29.9 pC; p<0.05). In contrast, SE was not associated with any change in the frequency and amplitude of mEPSCs in DGCs. Biotinylation and BS3 cross-linking assays revealed that compared to controls, the cell surface expression of GluA1 subunit in the hippocampi of 60 SE was increased (n = 9, p<0.05). Further, micro-dissection of hippocampal subfields revealed that increased surface expression of GluA1 subunit following 60 SE was observed in the CA1 (n=12) but not the DGC subfield (n=5). An increase in cell surface expression of GluA1 subunit during SE indicated potential alterations in the trafficking of AMPARs. Phosphorylation of AMPARs at the Ser831 and Ser845 residue on the C-tail of the GluA1 subunit can increase their surface expression and conductance. However, phosphorylation of neither Ser831 nor Ser845 residues was altered in the hippocampi (n=8) or CA1 subfield (n=5) during SE. The TARP, stargazin, is another potential modulator that enhances surface expression of AMPARs. Compared to controls, the cell surface expression of stargazin was greater in the hippocampi of animals experiencing prolonged SE (p<0.05, n =8). Since overexpression of stargazin can increase expression of extrasynaptic AMPARs, we tested the response of CA1 PN to bath applied AMPA (2.5 M). The capacitance normalized shift in holding current in response to AMPA was larger in CA1 PN from 60 SE group (20.6 3.8 pA, n=7) compared to control CA1 PNs (12.5 1.5 pA, n=10, p<0.05). Further, NMDAR activation during SE also likely increases GluA1 expression blockade with MK-801 (2 mg/kg) after 10 min of continuous seizure prevented increase in GluA1 surface expression in the CA1 region (n=5). Conclusions: The studies suggest strengthened AMPAR-mediated neurotransmission during prolonged SE is due to an increased surface expression of the GluA1 subunit.