Developmental transcriptomic analysis of cultured primary mouse cortical neurons reveals sex-specific expression of neuropeptides

Primary neuronal cultures derived from mouse tissue serve as an essential model for investigating neuronal development and function. Despite this, comprehensive developmental and sex-specific transcriptomic profiles in primary neurons have not been defined. Here, we performed multiplexed RNA-sequencing of neurons derived from male and female cortices across time. We validated this approach by assessing established neuronal maturation genes and we identified highly stable genes to serve as controls across development. Next, using linear modeling and temporal regression, we defined developmentally regulated transcripts, longitudinal expression dynamics, and gene signatures associated with transition states throughout development. Unexpectedly, this also revealed sex-specific effects on autosomal genes that emerge only after neuronal maturation even in the absence of in vivo cues. Most notably, neuropeptide genes Cortistatin and Neurokinin A are more highly expressed in female neurons. Furthermore, exposure to these neuropeptides elicited distinct transcriptional responses in male-versus female-derived cultures. These findings provide a valuable resource and reveal sex-specific autosomal transcriptional signatures that emerge in neurons maintained ex vivo .