Journal Pre-proof CREB binding at the Zfp189 promoter within medium spiny neuron subtypes differentially regulates behavioral and physiological adaptations over the course of cocaine use regulates

CREB binding at the Zfp189 promoter within medium spiny neuron subtypes differentially behavioral and physiological adaptations over the course of Abstract: 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. specific epigenome editing to study the downstream effects of CREB binding to a single CREB target gene – zinc finger protein 189 ( Zfp189 ), which was identified in our earlier work as a key driver of a gene network associated with responses to social stress (19). In this system, a nuclease-dead Cas9 protein is tethered to the phosphomimetic (constitutively active) form of CREB (dCas9-CREB S133D ) and directed to the Zfp189 promoter using a DNA-targeting single guide RNA (sgRNA) (20,21). Prior work suggests that cocaine experience elevates CREB binding at the Zfp189 promoter in the NAc(7). By directing dCas9-CREB S133D specifically to the Zfp189 promoter, initially throughout NAc neuronal populations and subsequently in a medium spiny neuron (MSN) subtype-dependent manner, we show that increased CREB binding at Zfp189 causally increases Zfp189 expression and controls the behavioral and physiological responses to cocaine. These data suggest a cell-type-specific mechanism by which CREB acts through Zfp189 in the NAc to regulate the physiological and behavioral adaptations to cocaine exposure. These data further support the notion that NAc-localized Zfp189 expression mimics the hallmark of CREB activation itself it diminishes the reinforcing effects of cocaine which is manifested here as heightened rates of cocaine self-administration. This effect of Zfp189 induction was maximal at the time of peak transgene expression of HSV vectors(24). These results suggest that Zfp189 is a CREB target gene whose activation is singularly sufficient to recapitulate the increased cocaine self-administration effect of general CREB overexpression. Despite the difference in time course, our findings suggest that CREB-mediated induction of Zfp189 might be a molecular mechanism through which both D1 and D2 MSNs respond to cocaine use to alter their physiological function. The application of our novel CRISPR approach for cell-type-specific recruitment of CREB- Zfp189 interaction initiates the endogenous, drug-course-dependent mechanism of CREB regulation, and precipitates the behavioral and physiological consequences of CREB-mediated Zfp189 induction in NAc. While our cocaine treatment regimen was acute in our electrophysiological experiments, our CRISPR-mediated induction of CREB- Zfp189 interaction within D2 MSNs modelled the transcriptional regulation that would occur upon chronic cocaine exposure. Shifting the population of NAc D2 MSNs to a chronic cocaine-like state would have circuit-wide consequences emanating from the cell types in which the CREB- Zfp189 interaction occurred. This may explain our observed increase in sEPSC frequency, which can be mediated by increases in presynaptic release probability, number of presynaptic inputs, number of synapses/release sites, or the general activity state of presynaptic terminals, all suggesting a strengthening of excitatory synaptic input on these cell types. Indeed, there is evidence that increased CREB activity enhances the intrinsic membrane excitability of NAc exposure results in increased frequency of glutamate-J