Circadian rhythms are critical for human health and are highly conserved across species. Disruptions in these rhythms contribute to many diseases, including psychiatric disorders. Previous results suggest that circadian genes modulate behavior through specific cell types in the nucleus accumbens (NAc), particularly dopamine D1-expressing medium spiny neurons (MSNs). However, diurnal rhythms in transcript expression have not been investigated in NAc MSNs. In this study we identified and characterized rhythmic transcripts in D1- and D2-expressing neurons and compared rhythmicity results to homogenate as well as astrocyte samples taken from the NAc of male and female mice. We find that all cell types have transcripts with diurnal rhythms and that top rhythmic transcripts are largely core clock genes, which peak at approximately the same time of day in each cell type and sex. While clock-controlled rhythmic transcripts are enriched for protein regulation pathways across cell type, cell signaling and signal transduction related processes are most commonly enriched in MSNs. In contrast to core clock genes, these clock-controlled rhythmic transcripts tend to reach their peak in expression about 2 hours later in females than males, suggesting diurnal rhythms in reward may be delayed in females. We also find sex differences in pathway enrichment for rhythmic transcripts peaking at different times of day. Protein folding and immune responses are enriched in transcripts that peak in the dark phase, while metabolic processes are primarily enriched in transcripts that peak in the light phase. Importantly, we also find that several classic markers used to categorize MSNs are rhythmic in the NAc. This is critical since the use of rhythmic markers could lead to over- or under-enrichment of targeted cell types depending on the time at which they are sampled. This study greatly expands our knowledge of how individual cell types contribute to rhythms in the NAc.
Overall design: To sequence actively translated mRNAs, male and female Drd1- and A2a-Cre mice were crossed to RiboTag mice31, expressing a Cre-inducible HA-Rpl22. C57BL/6J mice were used to generate homogenate samples. Previously generated data was used for astrocyte analysis28 wherein the GFP-inducible alternative to HA-Rpl22, Rpl10a, was crossed to Aldh1l1-eGFP mice. Mice were over 10 weeks old and maintained on a 12:12 light-dark (LD) schedule with lights on (Zeitgeber Time (ZT0)) at 0700. Food and water were provided ad libitum and procedures were approved by the University of Pittsburgh IACUC. NAc tissue was collected from male and female mice pseudo-randomly across 6 times of day (ZT2,6,10,14,18,22). As described previously28,31, co-immunoprecipitation was used to isolate cell-type specific ribosome-associated mRNAs. Final eluted homogenate and D1 or D2 specific RNA was used for sequencing. Following quality/integrity assessment, libraries were prepared and sequenced (blinded samples). mRNA (D1/D2) or total (homogenate) RNA sequencing was performed (n>3) using the NextSeq 500 platform (Illumina) at the University of Pittsburgh Health Sciences Sequencing Core. After preprocessing and filtering of sequencing data, a parametric cosinor model32 assuming a sinusoidal relationship between the gene expression level and ZT was used to detect rhythmicity (Supp.Table.1). Six total samples were excluded, due to insufficient RNA, library preparation or raw counts.
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