Abstracts

Rationale: Early-life seizures (ELSs) can cause permanent cognitive deficits and network hyperexcitability. Using mice with targeted recombination of activated populations (TRAP) we genetically labeled neurons activated by kainate-induced ELSs in immature mice (P10). We have shown that only the labeled (tdT+) neurons in area CA1, but not surrounding neurons, exhibited persistent increases in glutamatergic receptor activity and dysregulated LTP and LTD at P14-16 and in later life at P30-35 (PMID: 38227384). However, cell type-specific molecular mechanisms driving these persistent deficits after ELS are unknown.

Methods: Given the electrophysiological changes observed only in TRAP-labeled neurons, we aimed to identify unique transcriptomic alterations in activated tdT+ compared to surrounding tdT- CA1 pyramidal neurons. Following ELS-TRAP at P10, we used fluorescence-activated cell sorting (FACS) to separate tdT+ and tdT- cells from microdissected hippocampal tissue at P30, followed by single-nucleus RNA-sequencing (snRNA-seq) analysis. Seurat and enrichR were used to identify cell types and analyze differential gene expression.

Results: The ELS-TRAPed neurons, marked with tdTomato (tdT) as a reporter gene under the immediate early gene cFos promoter, were highly enriched in the hippocampal CA1 region. These tdT+ neurons remained preferentially susceptible to reactivation by later-life seizures in adulthood (P60). Gene ontology analysis of differentially expressed genes in snRNA-seq of tdT-sorted cells at P30 revealed prominent dysregulation in several pathways related to chemical synaptic transmission in identified glutamatergic neurons. Genes that were most significantly dysregulated included an increase in Gria1 and Grin2b with a related decrease in Grin2d and Grid2. By contrast, GABAergic neurons and non-neuronal cells demonstrated more prominent changes in genes involved in mRNA processing.

Conclusions: These findings show that enduring transcriptomic modifications occur in a subpopulation of ELS-activated neurons, compared to the surrounding neurons. Consistent with the persistent hyperexcitability and synaptic dysplasticity we have previously reported, modifications were most prominent in glutamate receptor subunits. Determining the time course of such changes following ELS provides a basis for future studies to examine potential targetable pathways that may yield novel clinically relevant therapies to prevent long-lasting cognitive deficits and epilepsy following ELS.

Funding: This work was supported by the NIH grants R37NS115439 to FEJ, 1K08NS119797-01A1 and Harold Amos Medical Faculty Development Program to AGC.