A dynamic gene regulatory code drives synaptic development of hippocampal granule cells [SS2]
Connecting neurons into functional circuits requires the formation, maturation, and plasticity of synapses. While advances have been made in identifying individual genes regulating synapse development, the molecular programs orchestrating their action during circuit integration of neurons remain poorly understood. Here, we have employed a single-cell RNA sequencing (scRNAseq) method (Smart-seq2) to study the development of hippocampal GCs. GCs represent the first relay in the hippocampal tri-synaptic loop and play an essential role in processing spatial representations and memory acquisition. This study provides a high temporal resolution analysis of the transcriptome of GCs during early postnatal development (P5, P7, P10, P15, and P28). Together, these findings highlight the networks of key TFs and target genes orchestrating GC synapse development. Overall design: We performed scRNAseq on GCs from Rbp4-Cre:Ai9 mice at key timepoints during synapse development. Brain slices from Rbp4-Cre:Ai9 mice were made using a 500 µm brain matrix, and the dentate gyrus (DG) was dissected. Next, the DG was chopped in smaller pieces and digested using Pronase. Following the digestion, the tissue pieces were homogenized using two-size Pasteur pipettes and the mix was loaded in a 20% Percoll solution to remove debris. Following centrifugation, the pellet was resuspended in OptiMEM-trehalose, and DAPI was added. Fluorescence Activated Cell Sorting (FACS) was done based on DAPI and tdTomato signal, into 96-well plates containing lysis buffer