Probes for INS

Background A salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type-II receptor (D2R) availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs). Methods Cre-conditional, Translating Ribosome Affinity Purification (TRAP) was used to purify and analyze the translatome (ribosome-bound mRNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed. Results Innate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA and cAMP signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 mRNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. Conclusion This study provides strong molecular evidence that in addiction inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.

Opioid exposure is known to cause transcriptomic changes in the nucleus accumbens (NAc). However, no studies to date have investigated cell type-specific transcriptomic changes associated with volitional opioid taking. Here, we use single nucleus RNA sequencing (snRNAseq) to comprehensively characterize cell type-specific alterations of the NAc transcriptome in rats self-administering morphine. One cohort of male Brown Norway rats was injected with acute morphine (10 mg/kg, i.p.) or saline. A second cohort of rats was allowed to self-administer intravenous morphine (1.0 mg/kg/infusion) for 10 consecutive days. Each morphine-experienced rat was paired with a yoked saline control rat. snRNAseq libraries were generated from NAc punches and used to identify cell type-specific gene expression changes associated with volitional morphine taking. We identified 1106 differentially expressed genes (DEGs) in the acute morphine group, compared to 2453 DEGs in the morphine self-administration group, across 27 distinct cell clusters. Importantly, we identified 1329 DEGs that were specific to morphine self-administration. DEGs were identified in novel clusters of astrocytes, oligodendrocytes, and D1R- and D2R-expressing medium spiny neurons in the NAc. Cell type-specific DEGs included Rgs9, Celf5, Oprm1, and Pde10a. Upregulation of Rgs9 and Celf5 in D2R-expressing neurons was validated by RNAscope. Approximately 85% of all oligodendrocyte DEGs, nearly all of which were associated with morphine taking, were identified in two subtypes. Bioinformatic analyses identified cell type-specific upstream regulatory mechanisms of the observed transcriptome alterations and downstream signaling pathways, including both novel and previously identified molecular pathways. These findings show that volitional morphine taking is associated with distinct cell type-specific transcriptomic changes in the rat NAc and highlight specific striatal cell populations and novel molecular substrates that could be targeted to reduce compulsive opioid taking.

Background Over the course of chronic drug use, brain transcriptional neuroadaptation are thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuroadaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within mouse NAc. Methods To query the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified CRISPR/dCas9 constructs, capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice. Results We observe that CREB binding to the Zfp189 promoter increases Zfp189 expression and diminishes the reinforcing responses to cocaine. We show further that NAc Zfp189 expression is increased within D1 medium spiny neurons (MSNs) in response to acute cocaine, but increased in both D1 and D2 MSNs in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiates electrophysiological activity of D1 and D2 MSNs - recapitulating the known effect of CREB on these neurons. Lastly, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors. Conclusions Together, these findings point to the CREB-Zfp189 interaction within NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.