Cell Subtype Specific Investigation of Cocaine-induced Transcriptomic Adaptations in the Nucleus Accumbens

Abstract

Substance use disorder is a chronic disease and a leading cause of disability around the world. The nucleus accumbens (NAc) is a major brain hub mediating reward behavior. Studies demonstrate exposure to cocaine is associated with molecular imbalance in NAc medium spiny neurons (MSNs), dopamine receptor 1 and 2 enriched D1-MSNs and D2-MSNs. We previously reported repeated cocaine exposure induced transcription factor early growth response 3 (Egr3) mRNA in NAc D1-MSNs, and reduced it in D2-MSNs. Here, we report that repeated cocaine exposure in mice induces MSN subtype specific bidirectional expression of the Egr3 corepressor NGFI-A-binding protein 2 (Nab2). Using CRISPR activation and interference tools combined with Nab2 or Egr3 sgRNAs, we mimicked these bidirectional changes in Neuro2a cells. Furthermore, we investigated MSN subtype specific bidirectional expressional changes of histone lysine demethylases Kdm1a, Kdm6a and Kdm5c in mouse NAc after repeated cocaine exposure. Employing a light inducible Opto-CRISPR-KDM1a system, we were able to downregulate Egr3 and Nab2 transcripts in Neuro2A cells and cause similar bidirectional expression changes we observed in D1-MSNs and D2-MSNs of mouse repeated cocaine exposure model. Contrastingly, our Opto-CRISPR-p300 activation system induced the Egr3 and Nab2 transcripts and caused opposite bidirectional transcription regulations. We further investigated the role of Nab2 in mouse cocaine self-administration. Specifically, we report that NAc D2-MSN specific silencing of Nab2 attenuates cocaine seeking after 10 days of cocaine self-administration in mice. Furthermore, we observed that Nab2 KD in D2-MSNs caused lower cocaine intake in female mice. Notably, lower intake was not observed in male mice. Next, using single-nucleus RNA-sequencing (snRNA-seq), we investigated cocaine and D2-MSN specific Nab2 KD induced alterations in transcriptomic landscape in mouse NAc with single-cell resolution. We then performed cell population specific gene co-expression network analysis and unbiasedly identified modules of differentially expressed genes (DEGs). Notably, we observed that several D2-MSN specific modules of DEGs were up-regulated after cocaine self-administration, and this effect was blocked with Nab2 silencing. Together, the findings in this dissertation provide mechanistic insights into cocaine-induced MSN subtype specific adaptations in the NAc with a particular focus on the D2-MSN specific role of Nab2.