Inducible CRISPR Epigenome Systems Mimic Cocaine Induced Bidirectional Regulation of Nab2 and Egr3.

Substance use disorder is a chronic disease and a leading cause of disability around the world. The 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 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-specific expressional changes of histone lysine demethylases Kdm1a, Kdm6a, and Kdm5c in NAc after repeated cocaine exposure in male mice. 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 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. Our study sheds light on the expression patterns of Nab2 and Egr3 in 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 that Egr3 and Nab2 are bidirectionally regulated in mouse 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 of Egr3 and Nab2 in Neuro2a cells.

Sex specific effects of dorsomedial striatal cannabinoid receptor-1 signaling on Pavlovian outcome devaluation.

Cannabinoid-1 receptor (CB1R) signaling in the dorsal striatum regulates the shift from flexible to habitual behavior in instrumental outcome devaluation. Based on prior work establishing individual, sex, and experience-dependent differences in Pavlovian behaviors, we predicted a role for dorsomedial striatum CB1R signaling in driving sign-tracking and rigid responding in Pavlovian outcome devaluation. We trained male and female rats in Pavlovian Lever Autoshaping to determine sign-, or goal- or intermediate tracking groups. After extended Pavlovian training, we gave intra-DMS infusions of the CB1R inverse agonist, rimonabant, before satiety-induced outcome devaluation test sessions, in which we sated rats on training pellets (devalued) or home cage chow (valued) and examined responding to cues in brief nonreinforced Pavlovian Lever Autoshaping sessions. Overall, DMS CB1R signaling inhibition blocked Pavlovian outcome devaluation. After extended training, male rats were sensitive to devaluation while female rats were not. Inhibition of DMS CB1R signaling impaired Pavlovian outcome devaluation in male sign-tracking rats making their behavior more rigid but had no effects in female sign-tracking rats. Intra-DMS rimonabant infusions before reinforced sessions had no effects on Pavlovian sign- or goal-tracking in either sex. The sex-specific and CB1R-dependent effects were specific to outcome devaluation and were consistent between sign- and goal-tracking groups. Our results demonstrate that DMS endocannabinoid receptor signaling regulates behavioral flexibility in a sex-specific manner, suggesting differences in CB1R signaling in DMS between male and female rats.

Gaining Wings to FLY: Using Drosophila Oogenesis as an Entry Point for Citizen Scientists in Laboratory Research.

Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.