Abstracts

Rationale: Impaired cortical inhibition occurs in many difficult-to-treat epilepsies and can originate from developmental disruptions of inhibitory circuit maturation. ~60% of cortical interneurons (IN) express parvalbumin- (PV) or somatostatin (SST) and provide powerful constraint of cortical activity. Our preliminary data suggests that PV-INs in mice are tonically depolarized by ambient glutamate acting at GluN2C/D-containing NMDA receptors during early development. Recently, human GluN2D mutations were shown to cause severe epileptic encephalopathies. Based on our data and these clinical studies, I hypothesize that disturbing early-life GluN2D signaling leads to dysfunctional cortical inhibition and epilepsy. Methods: To test my hypothesis, I will use electrophysiological, pharmacological, and genetic approaches to investigate tonic NMDAR activation of PV and SST cortical INs during normal development and in GluN2D deficient mice. Lhx6-GFP/SSTCre/Flx-td-Tomato mice enable us to identify SST- and PV-INs in the neonatal brain allowing us to study the role of GluN2D in developing SST- and PV-INs. Next, I will use genetic tools to determine how GluN2D contributes to the maturation of excitatory and inhibitory neurotransmission. Finally, I will determine if pharmacologic or genetic disruption of developmental GluN2D activation causes epilepsy and/or lowers seizure threshold. Results: Previous work and current preliminary data from our lab supports that GluN2D activation during early postnatal life (P7-9) contributes to IN maturation necessary to form functioning adult cortical inhibitory networks. We found that ambient glutamate is elevated during the first two postnatal weeks activating GluN2C/GluN2D subunit containing NMDA receptors on PV-INs in early cortical development. Moreover, preliminary FISH results in the cortex show that GluN2D is expressed by developing INs, but not excitatory neurons, and GluN2D expression in INs is developmentally regulated and peaks around P7-9. Consistent with these findings, our recordings suggest that cortical PV- and SST-INs, but not excitatory neurons, are tonically depolarized by GluN2D in the first postnatal week in mice. We believe this tonic depolarization contributes to PV+ IN maturation, as pharmacologically blocking GluN2C/D activation in vivo from P7-9 leads to functional and morphological alterations of PV-INs and impaired cortical inhibition at P28. My current preliminary results suggest that the depolarizing GluN2D mediated tonic current is also present in developing SST-INs at P7-9. In addition, first experiments investigating the developmental timeline of excitatory input onto PVs and SST-INs indicate that excitation onto INs intensifies after tonic depolarization between P7-9. Conclusions: These findings suggest that GluN2D-mediated tonic activation of INs at P7-9 might serve as a crucial developmental cue necessary for proper development of cortical inhibition. Early-life interruptions of GluN2D activation thus may lead to epilepsy or a lower seizure threshold. I will directly test the hypothesis whether attenuating GluN2D activation between P7-9 causes epilepsy as one of the first experiments after the AES fellowship start and will report on results at the annual AES meeting. Funding: 2019 American Epilepsy Society & Lennox Gastaut Syndrome Foundation Postdoctoral Fellowship