Abstracts

Rationale: Seizures in neonates increase the risks for autistic disorders and intellectual disability. We have previously shown that a kainate induced early life seizure (ELS) administered to post-natal (P) 7 day old rats has been shown to result in abnormal working memory and autistic features. These are associated with loss of LTP and enhanced mGluR-LTD. How ELS initiates and propagates these changes is unknown. AMPAR desensitization plays a role in the time course of excitatory post synaptic currents and may influence the induction of LTP. Changes in AMPAR desensitization can critically affect summation of synaptic currents to in-turn affect overall network properties, potentially triggering later life changes; thus we investigated AMPAR desensitization. Methods: ELS is induced by kainate injection (subcutaneous 2 mg/kg) in P7 male Sprague-Dawley rats. Brains are harvested and sagittal slices are prepared 2 and 7 days following injections. Whole-cell patch-clamp recordings of isolated AMPAR synaptic currents were recorded from CA1 hippocampal neurons to measure desensitization, and performed in ACSF with 100uM picrotoxin (to block GABAR), 50uM 4-aminopyridine, and 5mM CaCl2 (to increase release probability), at -70mV membrane holding potential (to minimize contribution from NMDAR) while stimulating Schaffer-Collateral axons with 5 pulses (20us) 20ms apart every 20 seconds. AMPAR desensitization was determined by comparing the amplitude of each successive response to the first response of the 5 pulses. Total protein and crosslinked protein was isolated from CA1 region of the hippocampal slices, then analyzed by Western blot. RNA was extracted from CA1 region of the hippocampal slices and reverse transcribed, then used for PCR and Sanger sequencing with AMPAR GluA2 subunit specific primers.Results: We find that a single kainate induced ELS at P7 results in decreased desensitization of CA1 synaptic AMPARs (compared to controls) in response to synaptic glutamate release. (Response 2: p = 0.055, Response 3: p = 0.040, Response 4: p = 0.043, Response: p = 0.054). AMPAR desensitization properties are determined in part by mRNA editing and alternative splicing of the individual subunits. We find that Adenosine Deaminase Acting on RNA 2 (ADAR2) expression is increased at P14 in CA1 7 days after seizure induction (p = 0.02). ADAR2 is responsible for editing of AMPAR GluA2 mRNA, causing the resulting protein to contain an amino acid switch from R to G at position 764. GluA2 R/G site editing results in fewer AMPARs maximally desensitized in response to glutamate stimulation and faster recovery from desensitization, thus the data suggests that editing of the R/G site may be increased by ELS. ADAR2 editing of the R/G site ensures accurate splicing of GluA2, which also effects desensitization; thus it is possible that ELS alters editing of the R/G site and the splice variant of GluA2 expressed.Conclusions: ELS leads to early changes in AMPAR desensitization that may underlie the progression to later-life abnormalities in behavior and plasticity.