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 and functional imbalance in NAc medium spiny neuron subtypes (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 our findings of repeated cocaine exposure in male mice inducing MSN subtype specific bidirectional expression of theEgr3corepressor NGFI-A-binding protein 2 (Nab2). Using CRISPR activation and interference (CRISPRa and CRISPRi) tools combined withNab2orEgr3targeted sgRNAs, we mimicked these bidirectional changes in Neuro2a cells. Furthermore, we investigated D1-MSN and D2-MSN specific expressional changes of histone lysine demethylasesKdm1a,Kdm6aandKdm5cin NAc after repeated cocaine exposure in male mice. SinceKdm1ashowed bidirectional expression patterns in D1-MSNs and D2-MSNs, likeEgr3, we developed a light inducible Opto-CRISPR-KDM1a system. We were able to downregulateEgr3andNab2transcripts 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 theEgr3andNab2transcripts and caused opposite bidirectional transcription regulations. Our study sheds light on the expression patterns ofNab2andEgr3in specific NAc MSNs in cocaine action and uses CRISPR tools to further mimic these expression patterns.Significance Statement:Substance use disorder is a major societal issue. The lack of medication to treat cocaine addiction desperately calls for a treatment development based on precise understanding of molecular mechanisms underlying cocaine addiction. In this study, we show thatEgr3andNab2are bidirectionally regulated in mouse nucleus accumbens (NAc) D1-MSNs and D2-MSNs after repeated exposure to cocaine. Furthermore, histone lysine demethylations enzymes with putative EGR3 binding sites showed bidirectional regulation in D1- and D2-MSNs after repeated exposure to cocaine. Using Cre and light inducible CRISPR tools, we show that we can mimic this bidirectional regulation ofEgr3andNab2in Neuro2a cells.

No abstract

Sign-tracking rats show enhanced cue sensitivity before drug experience that predicts greater discrete cue-induced drug-seeking compared to goal-tracking or intermediate- rats. Cue-evoked dopamine in the nucleus Accumbens (NAc) is a neurobiological signature of sign-tracking behaviors. Here, we examine a critical regulator of the dopamine system; endocannabinoids, which bind the cannabinoid receptor-1 (CB1R) in the Ventral Tegmental Area (VTA) to control cue-evoked striatal dopamine levels. We use cell-type specific optogenetics, intra-VTA pharmacology and fiber photometry to test the hypothesis that VTA CB1R receptor signaling regulates NAc dopamine levels to control sign-tracking. We trained rats in a Pavlovian lever autoshaping task (PLA) to determine their tracking groups before testing the effect of VTA to NAc dopamine inhibition. We found this circuit is critical for mediating the vigor of the ST response. Upstream of this circuit, intra-VTA infusions of rimonabant, a CB1R inverse agonist, during PLA decrease lever and increase foodcup approach in sign-tracking rats. Using fiber photometry to measure fluorescent signals from dopamine sensor, GRABDA, we tested the effects of intra-VTA rimonabant on NAc dopamine dynamics during autoshaping. We found that intra-VTA rimonabant-induced decreases in sign-tracking behaviors are not associated with changes to phasic cue(CS)-evoked NAc dopamine activity. Instead, intra-VTA rimonabant increases NAc shell, but not core, dopamine levels during reward delivery (US). Our results suggest that CB1R signaling in the VTA influences the balance between the CS- and US-evoked dopamine responses in the NAc shell and biases behavioral responding to cues in sign-tracking rats.

Substance use disorder is a debilitating chronic disease and a leading cause of disability around the world. The nucleus accumbens (NAc) is a major brain hub that mediates reward behavior. Studies demonstrate exposure to cocaine is associated with molecular and functional imbalance in two NAc medium spiny neuron subtypes (MSNs), dopamine receptor 1 and 2 enriched D1-MSNs and D2-MSNs. Our previous reports showed that repeated cocaine exposure induced transcription factor early growth response 3 (Egr3) mRNA in NAc D1-MSNs, while reducing it in D2-MSNs. Here, we report our findings of repeated cocaine exposure inducing cell subtype specific bidirectional expression of the Egr3 corepressor NGFI-A-binding protein 2 (Nab2). Using CRISPR activation and interference (CRISPRa and CRISPRi) tools combined with Nab2 or Egr3 targeted sgRNAs, we mimicked these bidirectional changes in Neuro2a cells. Furthermore, we investigated D1-MSN and D2-MSN subtype specific expressional changes of histone lysine demethylases Kdm1a, Kdm6a and Kdm5c in NAc after repeated cocaine exposure. Since Kdm1a showed bidirectional expression patterns in D1-MSNs and D2-MSNs, like Egr3, we developed a light inducible Opto-CRISPR-KDM1a system. We were able to downregulate Egr3 and Nab2 transcripts and cause bidirectional expression changes in D1-MSNs and D2-MSNs similar to cocaine exposure in Neuro2A cells. In contrast, our Opto-CRISPR-p300 activation system induced the Egr3 and Nab2 transcripts and caused bidirectional transcription regulations in D1-MSNs and D2-MSNs. Our study sheds light on the expression patterns of Nab2 and Egr3 in specific NAc MSN subtypes in cocaine action and uses CRISPR tools to further mimic these expression patterns.