320 - Sex differences in the hippocampal tyrosine kinase B receptor gene (Ntrk2) expression profile within the interneurons after neonatal hypoxia ischemia

Poster Number: 320
Publication Number: 320.2856

Background: The hippocampus plays a critical role in learning and memory. Neonatal hypoxia ischemia (HI) related brain injury is a known risk factor for learning and memory deficits in children. Sex differences in response to neonatal HI has been described. We previously showed that there is a sexually differential pathway in tyrosine kinase B receptor (TrkB) in the hippocampus post-HI. However, the impact of HI on the individual hippocampal cells and their gene expressions have not been explored.

Objective: In this study, we aimed to determine the sex differences in gene expression profiles of the individual cell clusters following HI by using single nuclear RNA sequencing and identify the cell types associated with TrkB signaling in a sex specific way.

Design/Methods: Neonatal HI was induced in postnatal day (P) 9 mice using Vannucci’s HI model. Ipsilateral hippocampi were extracted 3 days post-HI. Variability of the HI injury was controlled by only using the moderately injured hippocampi. Following hippocampal extraction and nuclear isolation, quality control of sequencing was done by FastQC (v0.11.5). Reads were aligned with mouse genome using cell ranger (v6.1.2). Approximately 13,000 nuclei per sequenced sample, with approximately 20,000 reads per nuclei, associated to approximately 1,300 genes per nuclei. For statistical analysis, R environment (v4.1.2), R package SingleR (v1.8.1), R package Seurat (v4.0.6) used to identify sex-specific differences for inter-group comparisons for each cluster. An adjusted P-value cutoff of 0.1 was used to identify differentially expressed genes.

Results: We identified 44 different hippocampal cell clusters which consisted 12 glutaminergic and 6 GABAergic neuron, 7 astrocyte, 4 oligodendrocyte precursor, 3 microglia, 2 oligodendrocyte cluster subtypes and 11 other uncategorized clusters. The majority of the nuclei were coming from the excitatory neuron clusters of the dentate gyrus. We identified sexually differential response to HI in terms of hippocampal gene expression profiles in inhibitory neurons. Particularly the TrkB gene (Ntrk2) expression found to be increased in female hippocampal inhibitory interneurons compared to males post-HI. Detailed analysis of this large dataset is undergoing.

Conclusion(s): Single nuclear RNA sequencing is a promising method to determine the sexually differential pathways activated on different neonatal hippocampal cell clusters. Identifying the cell- and sex-specific pathways associated with gene of interest in our case Ntrk2 gene is important to mitigate the negative effects of HI on the learning and memory.