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CREB Binding at the Zfp189 Promoter Within Medium Spiny Neuron Subtypes Differentially Regulates Behavioral and Physiological Adaptations Over the Course of Cocaine Use

Open AccessPublished:August 04, 2022DOI:https://doi.org/10.1016/j.biopsych.2022.07.022

      Abstract

      Background

      Over the course of chronic drug use, brain transcriptional neuroadaptation is thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB (cAMP response element binding protein) 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 the NAc of mice.

      Methods

      To investigate the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 constructs capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice.

      Results

      We observed that CREB binding to the Zfp189 promoter increased Zfp189 expression and diminished the reinforcing responses to cocaine. Furthermore, we showed that NAc Zfp189 expression increased within D1 medium spiny neurons in response to acute cocaine but increased in both D1- and D2-expressing medium spiny neurons in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiated electrophysiological activity of D1- and D2-expressing medium spiny neurons, recapitulating the known effect of CREB on these neurons. Finally, 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 the NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.

      Keywords

      Repeated drug use is correlated with persistent changes at the molecular, cellular, and circuit levels in the brain that are thought to give rise to the lasting behavioral maladaptations that define drug addiction (
      • Koob G.F.
      • Volkow N.D.
      Neurobiology of addiction: A neurocircuitry analysis.
      ,
      • Nestler E.J.
      • Aghajanian G.K.
      Molecular and cellular basis of addiction.
      ). The development of drug tolerance, a phenomenon describing the reduced sensitivity to a drug after repeated exposures, is an important component of the pathophysiology of drug addiction that contributes to the dangerous escalation of drug consumption over time (
      • Koob G.F.
      • Volkow N.D.
      Neurobiology of addiction: A neurocircuitry analysis.
      ). Understanding the molecular sequence of events that contribute to a reduction in the reinforcing effects of addictive drugs over the course of drug use will inform novel interventions with the capacity to prevent or reverse some of the most damaging consequences of chronic drug use.
      Previous research established that activation of CREB (cAMP response element binding protein) in the nucleus accumbens (NAc), a brain region involved in regulating reward and reinforcement, is a conserved mechanism capable of diminishing the reinforcing effects of addictive drugs across several drug classes (
      • Nestler E.J.
      • Aghajanian G.K.
      Molecular and cellular basis of addiction.
      ,
      • Brunzell D.H.
      • Mineur Y.S.
      • Neve R.L.
      • Picciotto M.R.
      Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference.
      ,
      • Pandey S.C.
      • Roy A.
      • Zhang H.
      • Xu T.
      Partial deletion of the cAMP response element-binding protein gene promotes alcohol-drinking behaviors.
      ,
      • Barrot M.
      • Olivier J.D.
      • Perrotti L.I.
      • DiLeone R.J.
      • Berton O.
      • Eisch A.J.
      • et al.
      CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli.
      ,
      • Terwilliger R.Z.
      • Beitner-Johnson D.
      • Sevarino K.A.
      • Crain S.M.
      • Nestler E.J.
      A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.
      ,
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • McClung C.A.
      • Nestler E.J.
      Regulation of gene expression and cocaine reward by CREB and DeltaFosB.
      ,
      • Teague C.D.
      • Nestler E.J.
      Key transcription factors mediating cocaine-induced plasticity in the nucleus accumbens.
      ). CREB is a ubiquitously expressed transcription factor involved in numerous nervous system functions including learning and memory, synaptic plasticity, and nervous system development (
      • Carlezon Jr., W.A.
      • Duman R.S.
      • Nestler E.J.
      The many faces of CREB.
      ,
      • Hong E.J.
      • West A.E.
      • Greenberg M.E.
      Transcriptional control of cognitive development.
      ). CREB regulates gene expression through binding to CRE motifs within the promoter or enhancer regions of target genes and recruiting CREB binding protein, a histone acetyltransferase, and the basal transcription complex to these genes (
      • McClung C.A.
      • Nestler E.J.
      Regulation of gene expression and cocaine reward by CREB and DeltaFosB.
      ,
      • Zhang X.
      • Odom D.T.
      • Koo S.H.
      • Conkright M.D.
      • Canettieri G.
      • Best J.
      • et al.
      Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
      ). CREB was implicated initially in drug addiction based on the empirical observation that cocaine and morphine elevate adenylyl cyclase and cAMP-dependent protein kinase A levels in the NAc (
      • Nestler E.J.
      • Aghajanian G.K.
      Molecular and cellular basis of addiction.
      ,
      • Terwilliger R.Z.
      • Beitner-Johnson D.
      • Sevarino K.A.
      • Crain S.M.
      • Nestler E.J.
      A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.
      ). Protein kinase A and other protein kinases phosphorylate CREB at serine residue 133 to activate CREB-dependent transcription (
      • Hong E.J.
      • West A.E.
      • Greenberg M.E.
      Transcriptional control of cognitive development.
      ,
      • Mattson B.J.
      • Bossert J.M.
      • Simmons D.E.
      • Nozaki N.
      • Nagarkar D.
      • Kreuter J.D.
      • Hope B.T.
      Cocaine-induced CREB phosphorylation in nucleus accumbens of cocaine-sensitized rats is enabled by enhanced activation of extracellular signal-related kinase, but not protein kinase A.
      ). In addition, subsequent studies confirmed that CREB is activated by many drugs of abuse including stimulants, morphine, and nicotine (
      • Shaw-Lutchman T.Z.
      • Barrot M.
      • Wallace T.
      • Gilden L.
      • Zachariou V.
      • Impey S.
      • et al.
      Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal.
      ,
      • Shaw-Lutchman T.Z.
      • Impey S.
      • Storm D.
      • Nestler E.J.
      Regulation of CRE-mediated transcription in mouse brain by amphetamine.
      ,
      • Walters C.L.
      • Cleck J.N.
      • Kuo Y.C.
      • Blendy J.A.
      Mu-opioid receptor and CREB activation are required for nicotine reward.
      ), suggesting that CREB-mediated transcriptional regulation is a common molecular response to different classes of drugs (
      • Nestler E.J.
      • Aghajanian G.K.
      Molecular and cellular basis of addiction.
      ).
      Viral overexpression of CREB in rodent NAc neurons reduces conditioned place preference (CPP) for cocaine and increases cocaine self-administration (
      • Barrot M.
      • Olivier J.D.
      • Perrotti L.I.
      • DiLeone R.J.
      • Berton O.
      • Eisch A.J.
      • et al.
      CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli.
      ,
      • McClung C.A.
      • Nestler E.J.
      Regulation of gene expression and cocaine reward by CREB and DeltaFosB.
      ,
      • Carlezon Jr., W.A.
      • Thome J.
      • Olson V.G.
      • Lane-Ladd S.B.
      • Brodkin E.S.
      • Hiroi N.
      • et al.
      Regulation of cocaine reward by CREB.
      ,
      • Larson E.B.
      • Graham D.L.
      • Arzaga R.R.
      • Buzin N.
      • Webb J.
      • Green T.A.
      • et al.
      Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.
      ). Both the reduction in cocaine conditioning and the seemingly paradoxical increase in cocaine self-administration are indicative of reduced cocaine reward and reinforcement in these animals. These data suggest that CREB function in the NAc represents a molecular mechanism that promotes a drug dependence–like behavioral state. However, studies using CREB overexpression and knockout approaches alter CREB regulation at all target genes, numbering in the hundreds to thousands (
      • Zhang X.
      • Odom D.T.
      • Koo S.H.
      • Conkright M.D.
      • Canettieri G.
      • Best J.
      • et al.
      Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
      ), making it difficult to determine which specific target genes causally mediate the effects of CREB activation on drug reward and volitional intake. Earlier studies using gene expression microarrays as well as chromatin immunoprecipitation paired with promoter microarrays identified numerous putative CREB target genes that may mediate its effects on drug tolerance, but most of these genes are yet to be studied (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • McClung C.A.
      • Nestler E.J.
      Regulation of gene expression and cocaine reward by CREB and DeltaFosB.
      ). In addition, it remains unclear whether the mechanisms downstream of CREB activation in the NAc differ between distinct neuronal cell types in this brain region.
      In this study, we set out to test the hypothesis that the cell type–specific action of CREB at key target genes differentially occurs over the course of drug use and drives the molecular neuroadaptations associated with chronic drug exposure. To address this hypothesis, we leveraged CRISPR (clustered regularly interspaced short palindromic repeats)-based locus-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 (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ). In this system, a nuclease-dead Cas9 protein is tethered to the phosphomimetic (constitutively active) form of CREB (dCas9-CREBS133D) and directed to the Zfp189 promoter using a DNA-targeting single guide RNA (sgRNA) (
      • Hamilton P.J.
      • Lim C.J.
      • Nestler E.J.
      • Heller E.A.
      Neuroepigenetic editing.
      ,
      • Yim Y.Y.
      • Teague C.D.
      • Nestler E.J.
      In vivo locus-specific editing of the neuroepigenome.
      ). Prior work suggests that cocaine experience elevates CREB binding at the Zfp189 promoter in the NAc (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ). By directing dCas9-CREBS133D specifically to the Zfp189 promoter, initially throughout the 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.

      Methods and Materials

      See the Supplement for methods relating to cocaine self-administration procedure, whole-cell patch-clamp electrophysiology, tissue collection, viral reagents, stereotaxic infusions, and RNAscope (RNAScope Multiplex Fluorescent V2 Assay; Advanced Cell Diagnostics).

      Animals

      C57BL/6J male and female mice ages 8–12 weeks were acquired from The Jackson Laboratory. D1-Cre and D2-Cre bacterial artificial chromosome transgenic mice (http://www.gensat.org/cre.jsp) were bred in-house. Animals were housed at 22–25 °C in a 12-hour light/dark cycle and provided food and water ad libitum. All tests were conducted during the light cycle. Animal procedures were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai or the Virginia Commonwealth University School of Medicine.

      Behavioral Paradigms

      For CPP, mice were placed in a 3-chambered CPP box for 20 minutes to assess pretest preferences. For the next 2 days, mice were injected in the morning (saline) and afternoon (drug) and then restricted to 1 chamber of the box for 30 minutes. During the posttest, mice were placed in the CPP box with free access to all chambers of the box for a 20-minute test session. Data are represented as the time spent in the cocaine-paired chamber minus the time spent in the saline-paired chamber during the posttest.

      Viral Reagents

      We used modified CRISPR constructs, which we have described previously (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ,
      • Lardner C.K.
      • van der Zee Y.
      • Estill M.S.
      • Kronman H.G.
      • Salery M.
      • Cunningham A.M.
      • et al.
      Gene-targeted, CREB-mediated induction of ΔFosB controls distinct downstream transcriptional patterns within D1 and D2 medium spiny neurons.
      ) and are described in more detail in the Supplement.

      RNAscope

      Tissue preparation is described in the Supplement. The following probes were used in this study: Zfp189 (catalog #: 569561-C3), Drd1 (catalog #: 461901), and Drd2 (catalog #: 406501-C2). Slides were imaged at 40× magnification using a Zeiss Laser Scanning Microscope (LSM) 900 confocal microscope, and images were quantified using CellProfiler 4.2.1 (
      • McQuin C.
      • Goodman A.
      • Chernyshev V.
      • Kamentsky L.
      • Cimini B.A.
      • Karhohs K.W.
      • et al.
      CellProfiler 3.0: Next-generation image processing for biology.
      ).

      RNA Isolation and Quantitative Polymerase Chain Reaction

      Total RNA was isolated from frozen dissected NAc tissue using the RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. Following isolation, RNA was quantified by Nano Drop (Thermo Fisher) and converted to complementary DNA with iScript (Bio-Rad). Quantitative polymerase chain reaction samples were analyzed in triplicate using the standard ΔΔCT method. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) was used as the reference gene for normalization in all experiments.

      Results

      NAc CREB-Zfp189 Interactions Regulate Behaviors Associated With Chronic Cocaine Use

      To explore the possibility that Zfp189 expression within the NAc is sensitive to cocaine exposure, we first treated mice with daily intraperitoneal injections of either saline or 20 mg/kg cocaine (Figure 1A). Compared with saline-treated mice, mice treated with a single, acute cocaine injection exhibited a significant increase in Zfp189 messenger RNA expression within the NAc (Figure 1B). Mice treated with chronic cocaine did not display a significant increase in Zfp189 expression (Figure 1B), revealing dynamic regulation of NAc Zfp189 expression over the time course of cocaine exposure.
      Figure thumbnail gr1
      Figure 1Zfp189 expression in the NAc is increased in response to cocaine. (A) The experimental timeline to determine the effect of acute or chronic intraperitoneal injections of cocaine on NAc Zfp189 mRNA expression. Each bubble represents a day. Light blue bubbles correspond to intraperitoneal saline, whereas red bubbles correspond to intraperitoneal 20-mg/kg cocaine. (B) Bilateral NAc Zfp189 mRNA levels quantified via quantitative real time polymerase chain reaction from each treatment condition. An acute cocaine injection significantly increased Zfp189 mRNA levels relative to saline-treated animals. One-way analysis of variance followed by Holm-Sidak’s multiple comparison test; ∗p value < .05. n = 7 (saline; acute cocaine) or 9 (chronic cocaine). (C) The rate of IVSA infusions for mice self-administering either cocaine (0.5 mg/kg/infusion) or saline on a fixed ratio of 1 schedule of reinforcement (i.e., reinforcement given after every response) in 3-hour sessions. Cocaine IVSA mice self-administered more infusions relative to saline IVSA mice. Two-way repeated-measures analysis of variance drug effect; ∗p value < .05. n = 5–7 mice per condition. (D) Bilateral NAc Zfp189 mRNA levels quantified via quantitative real-time polymerase chain reaction from each treatment condition. A history of cocaine IVSA resulted in a trend toward increased NAc Zfp189 mRNA levels relative to saline IVSA animals. p = .066. Student t test. n = 5 mice (saline IVSA), 7 mice (cocaine IVSA). IVSA, intravenous self-administration; mRNA, messenger RNA; NAc, nucleus accumbens.
      Next, we explored the consequence of mouse cocaine intravenous self-administration (IVSA) on NAc Zfp189 expression levels. Mice were catheterized in their jugular vein and trained to respond on an active lever for either infusions of cocaine (0.5 mg/kg/infusion) or saline. As expected, cocaine IVSA mice self-administered significantly more infusions than saline IVSA mice (Figure 1C). Furthermore, there was a trend toward increased NAc Zfp189 expression in mice with a history of cocaine IVSA (Figure 1D). These data suggest that contingent and noncontingent cocaine exposure may similarly increase NAc Zfp189 messenger RNA expression.
      Because the promoter region of Zfp189 possesses a CRE motif (
      • Zhang X.
      • Odom D.T.
      • Koo S.H.
      • Conkright M.D.
      • Canettieri G.
      • Best J.
      • et al.
      Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
      ,
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ) and CREB has been shown to bind the Zfp189 promoter in the NAc following cocaine treatment (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ), we devised an approach to deliver active CREB specifically to the Zfp189 promoter in NAc neurons to determine the role of the CREB-Zfp189 interaction in behavioral responses to cocaine. We applied a modified CRISPR/dCas9 approach wherein dCas9-CREBS133D is targeted selectively to the promoter region of Zfp189 within the NAc through an sgRNA in proximity to the consensus CRE motif in the Zfp189 promoter (Zfp189-sgRNA) (Figure 2A). This approach has been validated and applied by our group to the Zfp189 locus in prefrontal cortex neurons to study stress-related behaviors (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ) and to the Fosb locus in the NAc to study MSN-specific transcriptional responses (
      • Lardner C.K.
      • van der Zee Y.
      • Estill M.S.
      • Kronman H.G.
      • Salery M.
      • Cunningham A.M.
      • et al.
      Gene-targeted, CREB-mediated induction of ΔFosB controls distinct downstream transcriptional patterns within D1 and D2 medium spiny neurons.
      ).
      Figure thumbnail gr2
      Figure 2CREB-mediated Zfp189 induction within the NAc diminishes the rewarding effects of cocaine. (A) Illustration of CRISPR-mediated localization of CREBS133D to the Zfp189 promoter within mouse NAc. (B) qRT-PCR quantification of NAc Zfp189 mRNA from mice virally manipulated with HSV-dCas9-CREBS133D and either NT or Zfp189-targeting sgRNA. Localizing dCas9-CREBS133D to the Zfp189 promoter resulted in elevated NAc Zfp189 expression. Two-tailed Student t test; ∗p value < .05. n = 11 (NT-sgRNA) and 17 (Zfp189-sgRNA) mice. (C) Experimental timeline for cocaine CPP. (D) Mice with induced NAc CREB-Zfp189 interaction spent less time on the 7.5 mg/kg intraperitoneal cocaine-paired side of the CPP chamber. Two-tailed Student t test; ∗p value < .05. n = 18 (NT-sgRNA) and 22 (Zfp189-sgRNA) mice. (E) Experimental timeline for mouse cocaine IVSA. (F) Mice were virally delivered CRISPR tools to localize either the transcriptionally activating CREBS133D or the transcriptionally repressive histone methyltransferase G9a to the Zfp189 promoter within the NAc. Mice with NAc Zfp189 levels elevated via the CREB-Zfp189 interaction elected to self-administer more cocaine infusions than mice in which G9a was targeted to the Zfp189 promoter. 0.5 mg/kg/infusion at a FR1 for a 3-hour session. Two-way repeated-measures ANOVA viral treatment effect within sessions post viral delivery; F1,17 = 6.686; ∗p value < .05. n = 10 (dCas9-CREBS133D) and 9 (dCas9-G9a) mice. ANOVA, analysis of variance; CPP, conditioned place preference; CREB, cAMP response element binding protein; CRISPR, clustered regularly interspaced short palindromic repeats; FR, fixed ratio; HSV, herpes simplex virus; IVSA, intravenous self-administration; JVC, jugular vein cannulation; mRNA, messenger RNA; NAc, nucleus accumbens; NT, nontargeting; qRT-PCR, quantitative real-time polymerase chain reaction; sgRNA, single guide RNA; TSS, transcription start site.
      We packaged dCas9-CREBS133D, Zfp189-sgRNA, and the control nontargeting sgRNA into separate herpes simplex virus (HSV) vectors to enable stereotaxic delivery and expression in the NAc of awake and behaving mice. Upon delivery to the NAc, we observed that targeting CREB to the Zfp189 promoter increased Zfp189 expression (Figure 2B). To investigate the contribution of NAc CREB-Zfp189 interaction to cocaine-related behaviors, we first delivered our CRISPR tools intra-NAc and tested the consequence of CREB-mediated Zfp189 activation on cocaine reward associative learning (Figure 2C, D). We found that animals with CREB-mediated Zfp189 activation throughout the NAc neurons exhibited decreased preference for the 7.5 mg/kg cocaine-paired side of the chamber relative to control animals (Figure 2D), which is consistent with a blunted response to the reinforcing effects of cocaine. This phenomenon of reduced cocaine CPP is also observed in female mice (Figure S1 in Supplement 1), suggesting a similar role for CREB-Zfp189 interaction in governing cocaine-related behaviors in both sexes.
      Interestingly, on repeating these experiments with morphine administration instead of cocaine, we observed no effect of CREB-Zfp189 interaction on morphine-induced locomotor activity or morphine CPP in male or female mice (Figure S2 in Supplement 1), signifying that the capacity of NAc Zfp189 to affect drug-related behaviors occurs in response to cocaine but may not extend to other classes of commonly used drugs such as opioids.
      We also tested cocaine IVSA in mice to further explore whether NAc CREB-Zfp189 interaction drives changes in reward-related behaviors to cocaine beyond investigator-administered paradigms. Mice were trained to self-administer 0.5 mg/kg/infusion of cocaine on a fixed ratio 1 schedule of reinforcement (Figure 2E). We elected to bidirectionally regulate NAc Zfp189 expression by coupling the delivery of Zfp189-sgRNA with either dCas9-CREBS133D or dCas9-G9a. The latter possesses the functional moiety of a transcriptionally repressive histone methyltransferase, which we have demonstrated previously suppresses Zfp189 expression in the brain (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ). While there was a general reduction of IVSA rates immediately following HSV delivery to the NAc, mice with CREB-mediated induction of Zfp189 in the NAc self-administered more cocaine infusions than mice with G9a targeted to the Zfp189 promoter (Figure 2F). These data further support the notion that NAc-localized Zfp189 expression mimics the hallmark of CREB activation itself (
      • Larson E.B.
      • Graham D.L.
      • Arzaga R.R.
      • Buzin N.
      • Webb J.
      • Green T.A.
      • et al.
      Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.
      ): 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 (
      • Neve R.L.
      • Neve K.A.
      • Nestler E.J.
      • Carlezon Jr., W.A.
      Use of herpes virus amplicon vectors to study brain disorders.
      ). 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.

      Cell Type–Specific Effects of the CREB-Zfp189 Interaction in the NAc

      Approximately 95% of the neurons within the NAc are MSNs, which are differentiated into 2 primary subtypes based on their predominant expression of dopamine receptor genes, Drd1 versus Drd2, which exist in roughly equal numbers in this brain region (
      • Lobo M.K.
      • Karsten S.L.
      • Gray M.
      • Geschwind D.H.
      • Yang X.W.
      FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.
      ,
      • Kronman H.
      • Richter F.
      • Labonté B.
      • Chandra R.
      • Zhao S.
      • Hoffman G.
      • et al.
      Biology and bias in cell type-specific RNAseq of nucleus accumbens medium spiny neurons.
      ). Emerging evidence implicates D1-MSN function in promoting reward and motivated behaviors, whereas D2-MSNs are often associated with decreased reward and aversive behaviors (
      • Tai L.H.
      • Lee A.M.
      • Benavidez N.
      • Bonci A.
      • Wilbrecht L.
      Transient stimulation of distinct subpopulations of striatal neurons mimics changes in action value.
      ,
      • Calipari E.S.
      • Bagot R.C.
      • Purushothaman I.
      • Davidson T.J.
      • Yorgason J.T.
      • Peña C.J.
      • et al.
      In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward.
      ,
      • Heinsbroek J.A.
      • Neuhofer D.N.
      • Griffin W.C.
      • Siegel G.S.
      • Bobadilla A.C.
      • Kupchik Y.M.
      • Kalivas P.W.
      Loss of plasticity in the D2-accumbens pallidal pathway promotes cocaine seeking.
      ,
      • Cole S.L.
      • Robinson M.J.F.
      • Berridge K.C.
      Optogenetic self-stimulation in the nucleus accumbens: D1 reward versus D2 ambivalence.
      ,
      • Hikida T.
      • Kimura K.
      • Wada N.
      • Funabiki K.
      • Nakanishi S.
      Distinct roles of synaptic transmission in direct and indirect striatal pathways to reward and aversive behavior.
      ,
      • Lobo M.K.
      • Covington H.E.
      • Chaudhury D.
      • Friedman A.K.
      • Sun H.
      • Damez-Werno D.
      • et al.
      Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward.
      ,
      • van Zessen R.
      • Li Y.
      • Marion-Poll L.
      • Hulo N.
      • Flakowski J.
      • Lüscher C.
      Dynamic dichotomy of accumbal population activity underlies cocaine sensitization.
      ). In particular, D1- and D2-MSNs have been shown to drive distinct and often opposing behavioral responses to drugs such as cocaine. Therefore, we investigated MSN subtype–specific expression of Zfp189 in the NAc in response to either acute or chronic cocaine exposure (Figure 3). In performing RNAscope in situ hybridization for Zfp189, Drd1, and Drd2 in fixed NAc sections from mice exposed to varying cocaine treatment regimens, we observed that following acute cocaine exposure, solely the D1-MSN population exhibited a robust increase in the number of Zfp189+ cells (Figure 3A–C). This observation is consistent with rodent NAc single-cell RNA sequencing data demonstrating that acute cocaine upregulates Zfp189 specifically in the Drd1+ cell cluster (
      • Savell K.E.
      • Tuscher J.J.
      • Zipperly M.E.
      • Duke C.G.
      • Phillips R.A.
      • Bauman A.J.
      • et al.
      A dopamine-induced gene expression signature regulates neuronal function and cocaine response.
      ). After chronic cocaine, by contrast, we observed an increased number of Zfp189+ cells in both D1- and D2-MSNs (Figure 3D–F). Thus, over the course of cocaine exposure, Zfp189 was activated within D1-MSNs acutely and in both D1- and D2-MSNs at chronic time points. This analysis does not quantify the expression level of Zfp189 within MSNs, but rather the percentage of Zfp189+ MSNs by subtype, which may explain the difference in results at the chronic cocaine time point between Figure 1, Figure 3. Therefore, while acute cocaine induced a significant increase in total NAc Zfp189 expression levels (localized primarily within D1-MSNs), chronic cocaine increased the number of Zfp189-expressing D2-MSNs.
      Figure thumbnail gr3
      Figure 3Acute cocaine exposure selectively induces Zfp189 expression within D1-MSNs, whereas chronic cocaine exposure induces Zfp189 expression within D1- and D2-MSNs. (A) The experimental timeline of acute 20 mg/kg IP cocaine injection. (B) Representative images for Drd1, Drd2, and Zfp189 mRNA probes in mouse NAc from animals acutely exposed to cocaine. Scale bar = 30 μm. Merged image is magnified 2×, labeled with DAPI nuclear labeling, and Zfp189+ regions are denoted with white arrows. (C) Quantification of the percentage of Drd1- or Drd2-positive MSNs that expressed Zfp189 in each treatment condition. Two-way analysis of variance followed by Holm-Sidak’s multiple comparison test; ∗∗∗∗p value < .0001. n = 4–5 mice per condition. (D) The experimental timeline of chronic 20 mg/kg IP cocaine injection. (E) Representative images for Drd1, Drd2, and Zfp189 mRNA probes in mouse NAc from animals chronically exposed to cocaine. Scale bar = 30 μm. Merged image is magnified 2×, labeled with DAPI nuclear labeling, and Zfp189+ regions are denoted with white arrows. (F) Quantification of the percentage of Drd1- or Drd2-positive MSNs that expressed Zfp189 in each treatment condition. Two-way analysis of variance followed by Holm-Sidak’s multiple comparison test; ∗p < .05; ∗∗∗p < .001. n = 5 mice per condition. IP, intraperitoneal; MSN, medium spiny neuron; mRNA, messenger RNA; NAc, nucleus accumbens; ns, nonsignificant.
      To explore the consequence of Zfp189 induction within NAc MSNs on cocaine-induced physiological function, we first virally overexpressed Zfp189 through a conventional overexpression vector in the NAc, which has been validated in the brain previously (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ). Mice were given an acute cocaine treatment regimen, and 12 hours after the final injection, we performed whole-cell patch-clamp recording of virally infected or uninfected MSNs. While we observed that neither cocaine treatment nor Zfp189 overexpression had any discernable effect on the spontaneous excitatory postsynaptic current (sEPSC) amplitudes (Figure 4A), this acute cocaine procedure increased the frequency of sEPSCs in both nontransduced and green fluorescent protein–expressing MSNs (Figure 4B). HSV-Zfp189 delivery increased the sEPSC frequency of saline-treated mice to the level of cocaine-treated mice, with the effect of cocaine and Zfp189 overexpression being nonadditive (Figure 4B). These results indicate that acute cocaine treatment specifically enhances sEPSC frequency in NAc MSNs and that Zfp189 overexpression is sufficient to induce this effect in saline-treated mice.
      Figure thumbnail gr4
      Figure 4Cocaine exposure and CRISPR-mediated CREB-Zfp189 interactions differentially regulate excitatory synaptic transmission to D1- and D2-MSNs. (A) Whole-cell patch-clamp recording of sEPSC amplitudes from NAc MSNs in acutely prepared brain slices from mice with intra-NAc injection of either HSV-GFP or HSV-Zfp189 immediately after acute injections of either saline or cocaine (10 mg/kg). Neither viral treatment nor drug experience affected MSN sEPSC amplitudes. Two-way analysis of variance: viral effect F2,82 = 0.89; drug effect, F1,82 = 0.01; n = 10 to 13 recordings from 7 to 8 mice (nontransduced), 18 to 19 recordings from 5 mice (HSV-GFP), and 14 recordings from 4 mice (HSV-Zfp189). (B) Acute cocaine IP injection increased the sEPSC frequency in all treatment groups, and HSV-Zfp189 expression was sufficient to enhance sEPSC frequency in saline mice to levels observed in cocaine mice. Two-way analysis of variance viral or drug treatment effect; F2,84 = 6.504 (viral effect); F1,84 = 18.23 (drug effect); Holm-Sidak’s multiple comparison test; ∗p value < .05. n = 12 to 13 recordings from 7 to 8 mice (HSV-Zfp189), 18 to 19 recordings from 5 mice (HSV-GFP), and 14 recordings from 4 mice (nontransduced). (C) To assess the MSN-type–specific consequences of CREB-mediated induction of Zfp189 on cocaine-induced physiological function, we combined viral delivery of Cre-dependent CRISPR expression vectors and transgenic mice, which express Cre-recombinase under the Drd1 or Drd2 promoter. (Top panel) Representative sEPSCs in transduced D1- and D2-MSNs. (Bottom panel) In catalyzing CREB-Zfp189 interactions within D1-MSNs and exposing mice to the acute cocaine regimen, either an acute cocaine injection or CRISPR-mediated CREB-Zfp189 interactions was sufficient to increase sEPSC frequency in D1-MSNs. Two-way analysis of variance followed by Holm-Sidak’s multiple comparison test; ∗∗p < .001, ∗∗∗p < .0005. n = 13 to 16 recordings from 5 or 6 mice (NT-sgRNA) and 12 recordings from 4 or 5 mice (Zfp189-sgRNA). (D) The experiment was repeated in Drd2-Cre mice. (Top panel) Representative sEPSCs. (Bottom panel) In delivering CREB-Zfp189 interactions within D2-MSNs and exposing mice to the treatment regimen described above, we observed that only CRISPR-mediated CREB-Zfp189 interactions, and not an acute cocaine treatment, increased sEPSC frequency in D2-MSNs. Two-way analysis of variance followed by Holm-Sidak’s multiple comparison test; ∗p value < .05. n = 16 to 17 recordings from 4 or 5 mice (NT-sgRNA) and 15 to 18 recordings from 5 mice (Zfp189-sgRNA). CREB, cAMP response element binding protein; CRISPR, clustered regularly interspaced short palindromic repeats; HSV-GFP, herpes simplex virus–green fluorescent protein; IP, intraperitoneal; MSN, medium spiny neuron; NAc, nucleus accumbens; ns, nonsignificant; NT, nontargeting; sEPSC, spontaneous excitatory postsynaptic current; sgRNA, single guide RNA.
      To understand the MSN subtypes responsible for this phenomenon, we adapted our viral delivery strategy to use Cre-dependent (loxP-STOP-loxP) expression vectors to facilitate CRISPR-mediated CREB-Zfp189 interaction in an MSN subtype–dependent manner. This approach has been applied by our group previously (
      • Lardner C.K.
      • van der Zee Y.
      • Estill M.S.
      • Kronman H.G.
      • Salery M.
      • Cunningham A.M.
      • et al.
      Gene-targeted, CREB-mediated induction of ΔFosB controls distinct downstream transcriptional patterns within D1 and D2 medium spiny neurons.
      ), and as in this earlier work, we saw Zfp189 activation preferentially in the NAc neurons that are conditionally expressing our CRISPR constructs (Figure S3 in Supplement 1). In Drd1-Cre+ mice in the loxP-STOP-loxP-dCas9-CREBS133D+NT-sgRNA control condition, acute cocaine treatment potentiated sEPSC frequency (Figure 4C), consistent with our observations in wild-type mice (Figure 4B). In addition, targeting CREB to the Zfp189 promoter within D1-MSNs was sufficient to potentiate sEPSC frequency, recapitulating the effect of acute cocaine (Figure 4C). In Drd2-Cre+ mice, acute cocaine had no effect on sEPSC frequency in the control viral treatment condition (Figure 4D). This result indicates that only the D1-MSN population is sensitive to acute cocaine treatment by this metric. However, targeting CREB binding to the Zfp189 promoter within D2-MSNs increased the sEPSC frequency in both the saline and cocaine treatment conditions (Figure 4D). Collectively, these data reveal that only D1-MSNs were sensitive to acute cocaine exposure, yet CRISPR-mediated delivery of CREB to the Zfp189 promoter was sufficient to potentiate the function of both D1- and D2-MSN subtypes. Furthermore, none of our manipulations affected basal MSN electrophysiological metrics such as membrane capacitance or membrane resistance (Figure S4 in Supplement 1).

      Catalyzing the CREB-Zfp189 Interaction Specifically Within NAc Drd2+ Neurons Reduces CPP for Cocaine

      Next, we tested the consequence of delivering the CREB-Zfp189 interaction within individual neuron subtypes on cocaine reward–related behaviors. In Drd1+ neurons, recruitment of CREB-Zfp189 interaction through CRISPR tools had no effect on cocaine CPP (Figure 5B). By contrast, inducing the CREB-Zfp189 interaction in Drd2+ neurons significantly decreased cocaine CPP (Figure 5C). This latter effect is consistent with the effect of CREB-Zfp189 interaction in all neurons in the NAc (Figure 2D), consistent with the interpretation that this latter effect is driven by the CREB-mediated induction of Zfp189 in Drd2+ neurons selectively.
      Figure thumbnail gr5
      Figure 5CREB-mediated Zfp189 induction within NAc Drd2+ neurons drives reduced sensitivity to the reinforcing properties of cocaine. (A) Experimental timeline for cocaine conditioned place preference in Drd1- or Drd2-Cre+ mice. (B) Drd1-Cre mice in which the NAc CREB-Zfp189 interaction was induced within Drd1+ neurons show no effect relative to NT-sgRNA controls; cocaine 7.5 mg/kg. Two-tailed Student t test; p > .05. n = 15 (NT-sgRNA) and 13 (Zfp189-sgRNA) mice. (C) Drd2-Cre mice in which the NAc CREB-Zfp189 interaction is induced within Drd2+ neurons showed reduced cocaine conditioned place preference relative to NT-sgRNA controls; cocaine 15 mg/kg. Two-tailed Student t test; ∗p < .05. n = 8 (NT-sgRNA) and 6 (Zfp189-sgRNA) mice. CREB, cAMP response element binding protein; NAc, nucleus accumbens; NT, nontargeting; sgRNA, single guide RNA.

      Discussion

      In this study, we investigated a cell type–specific molecular mechanism by which CREB regulates cocaine-induced neuroadaptations in the NAc through the induction of Zfp189. Our findings show that in response to a single dose of cocaine, Zfp189 was rapidly and selectively induced in D1-MSNs. This expression persisted in D1-MSNs following repeated doses of cocaine. However, at these chronic time points, Zfp189 expression became induced in D2-MSNs as well. We further found that this increased Zfp189 expression in D2-MSNs promoted excitatory inputs upon this cell type, which drives heightened physiological activity and generates behaviors associated with chronic cocaine exposure, including behaviors indicative of a reduction in the reinforcing effects of cocaine. We have summarized this proposed neurobiological mechanism as a graphic in Figure S5 in Supplement 1. This work points to an NAc Drd2+ neuron–specific transcriptional cascade from CREB to Zfp189 as a novel mechanism that drives some of the damaging neuroadaptations associated with chronic cocaine use.
      The CRISPR-mediated recruitment of CREB to the Zfp189 promoter, depicted in Figure 2, models a single molecular interaction that occurs within the NAc neurons in response to cocaine, including both D1- and D2-MSNs. Given that Zfp189 is naturally activated by a single cocaine dose within D1-MSNs only (Figure 3C) and that each of our behavioral paradigms involves pretreatment of cocaine (CPP, IVSA), it is likely that the endogenous mechanism of activating Zfp189 within D1-MSNs occurs in parallel with our CRISPR manipulation. However, because observed that Zfp189 is induced within Drd2+ NAc neurons only after chronic cocaine treatment (Figure 3F), our NAc-wide CREB-Zfp189 interaction may be preferentially affecting behavioral responses to cocaine by regulating the function of Drd2+ neurons. Thus, we propose that the reduced reinforcing properties of cocaine observed in Figure 2 occur as a result of CREB-Zfp189 interaction within NAc Drd2+ neurons. This is corroborated by the selective delivery of CRISPR tools that drive CREB-Zfp189 interaction within Drd2+ neurons and the recapitulation of the behavioral effects on CPP (Figure 5C). In sum, our findings support the hypothesis that CREB-Zfp189 interaction, particularly within NAc Drd2+ neurons, drives the animal into a chronic cocaine-exposed state, including a decreased sensitivity to the reinforcing effects of cocaine.
      The cell type–specific features of Zfp189 regulation are consistent with published reports that D1- and D2-MSNs display distinct patterns of activity and gene profiles (
      • Kronman H.
      • Richter F.
      • Labonté B.
      • Chandra R.
      • Zhao S.
      • Hoffman G.
      • et al.
      Biology and bias in cell type-specific RNAseq of nucleus accumbens medium spiny neurons.
      ,
      • Calipari E.S.
      • Bagot R.C.
      • Purushothaman I.
      • Davidson T.J.
      • Yorgason J.T.
      • Peña C.J.
      • et al.
      In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward.
      ). Furthermore, considerable evidence points to the function of D1-MSNs regulating the function of D2-MSNs through the recruitment of cholinergic interneurons (
      • Francis T.C.
      • Yano H.
      • Demarest T.G.
      • Shen H.
      • Bonci A.
      High-frequency activation of nucleus accumbens D1-MSNs drives excitatory potentiation on D2-MSNs.
      ), to cholinergic interneurons contributing to cocaine self-administration behaviors (
      • Lee J.H.
      • Ribeiro E.A.
      • Kim J.
      • Ko B.
      • Kronman H.
      • Jeong Y.H.
      • et al.
      Dopaminergic regulation of nucleus accumbens cholinergic interneurons demarcates susceptibility to cocaine addiction.
      ), to cholinergic interneurons regulating glutamatergic synaptic strength upon NAc MSNs (
      • Lee J.
      • Finkelstein J.
      • Choi J.Y.
      • Witten I.B.
      Linking cholinergic interneurons, synaptic plasticity, and behavior during the extinction of a cocaine-context association.
      ), and to the dramatic effects of cocaine exposure on gene expression within cholinergic interneurons (
      • Lee J.H.
      • Ribeiro E.A.
      • Kim J.
      • Ko B.
      • Kronman H.
      • Jeong Y.H.
      • et al.
      Dopaminergic regulation of nucleus accumbens cholinergic interneurons demarcates susceptibility to cocaine addiction.
      ). Importantly, for the experiments reported within this article, we used Drd2-Cre+ mice, which drive the expression of Cre-recombinase in all Drd2+ brain cells. Given that there is a sparse population of cholinergic interneurons that are Drd2+, we cannot exclude the possibility that our conditional manipulations affected Zfp189 function within NAc Drd2+ cholinergic interneurons, which might have contributed to our results, specifically those shown in Figure 5C.
      The exact brain mechanisms responsible for the diminished behavioral sensitivity to cocaine seen in response to Zfp189 induction in the NAc must be further elucidated. It is possible that Zfp189 expression in NAc Drd2+ neurons singularly drives drug reward tolerance, which is a hallmark of chronic drug use and refers to a decreased sensitivity to a drug following repeated exposures. Conversely, it is possible that the Zfp189-driven function of D1-MSNs opposes the Zfp189-driven function of D2-MSNs in an opponent process that is differentially balanced over the course of cocaine experience, with increased weight dedicated to the D2-MSN–driven aversive properties as a function of chronic cocaine exposure. Either of these possibilities could explain the behavioral results observed in this work and warrant future investigation.
      Another major area for future research is investigating the molecular mechanisms responsible for the differential time course of CREB-mediated Zfp189 induction within D1- versus D2-MSNs. We currently possess an incomplete picture of what distinguishes the sensitivity of the Zfp189 locus in these two closely related neuronal subtypes. It is possible that the expression of transcription factor regulatory cofactors, Zfp189 promoter chromatin state and CRE accessibility, as well as transcript processing, among many other possibilities, are differentially sensitive to cocaine exposure and responsible for the time course of Zfp189 induction across MSN subtypes. This work also points to the possibility that CREB function is differentially engaged to regulate distinct gene targets over the course of drug use in a cell type–specific manner. This possibility is supported by evidence that CRE motif accessibility and CREB-mediated gene regulation vary widely by cell type (
      • Cha-Molstad H.
      • Keller D.M.
      • Yochum G.S.
      • Impey S.
      • Goodman R.H.
      Cell-type-specific binding of the transcription factor CREB to the cAMP-response element.
      ).
      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 initiated the endogenous, drug-course–dependent mechanism of CREB regulation and precipitated the behavioral and physiological consequences of CREB-mediated Zfp189 induction in the NAc. While our cocaine treatment regimen was acute in our electrophysiological experiments, our CRISPR-mediated induction of CREB-Zfp189 interaction within D2-MSNs modeled the transcriptional regulation that would occur with 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 or 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 MSNs (
      • Dong Y.
      • Green T.
      • Saal D.
      • Marie H.
      • Neve R.
      • Nestler E.J.
      • Malenka R.C.
      CREB modulates excitability of nucleus accumbens neurons.
      ), that cocaine exposure results in increased frequency of glutamate-mediated evoked excitatory postsynaptic currents upon NAc MSNs (
      • Dobi A.
      • Seabold G.K.
      • Christensen C.H.
      • Bock R.
      • Alvarez V.A.
      Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal.
      ), and that altered function of MSNs can regulate synaptic transmission upon distinct MSN subtypes through multineuronal circuit regulation (
      • Francis T.C.
      • Yano H.
      • Demarest T.G.
      • Shen H.
      • Bonci A.
      High-frequency activation of nucleus accumbens D1-MSNs drives excitatory potentiation on D2-MSNs.
      ). Furthermore, the fact that the increase in sEPSC frequency was not additive upon the combination of cocaine treatment and Zfp189 induction supports the notion of a conserved mechanism between cocaine treatment and Zfp189 induction rather than two independent mechanisms for potentiating MSN excitability. These data further indicate that even a relatively modest yet physiologically relevant increase in Zfp189 expression within MSNs, as is achieved through CREB-mediated activation, is sufficient to manifest a subset of the neuroadaptations associated with chronic cocaine exposure.
      Despite the multiple lines of evidence presented herein that causally link the NAc CREB-Zfp189 interaction to regulating cocaine-induced behaviors, we found no evidence that CREB-mediated regulation of Zfp189 influences morphine-elicited behaviors (Figure S2 in Supplement 1). While NAc CREB function is well documented in being sensitive to and regulating the rewarding properties of morphine (
      • Barrot M.
      • Olivier J.D.
      • Perrotti L.I.
      • DiLeone R.J.
      • Berton O.
      • Eisch A.J.
      • et al.
      CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli.
      ,
      • Terwilliger R.Z.
      • Beitner-Johnson D.
      • Sevarino K.A.
      • Crain S.M.
      • Nestler E.J.
      A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.
      ), the observed lack of an effect supports the conclusion that CREB achieves these effects independently of its regulation at Zfp189. This points to the possibility that while NAc CREB function regulates the rewarding properties of many drug classes (
      • Brunzell D.H.
      • Mineur Y.S.
      • Neve R.L.
      • Picciotto M.R.
      Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference.
      ,
      • Misra K.
      • Pandey S.C.
      The decreased cyclic-AMP dependent-protein kinase A function in the nucleus accumbens: A role in alcohol drinking but not in anxiety-like behaviors in rats [published correction appears in Neuropsychopharmacology 2011;36:2149].
      ), CREB achieves this outcome by regulation of partly distinct downstream transcriptional networks. Here, CREB’s activity at Zfp189 appears causal in governing cocaine-related outcomes specifically. The degree to which other classes of stimulants, such as amphetamines, share this CREB to Zfp189 mechanism is an interesting area for future research.
      Given the possibility that CREB functions as a negative feedback mechanism to oppose the rewarding properties of classes of drugs via engagement of distinct transcriptional cascades, novel and highly specific targets for drug addiction pharmacotherapies may be identified within these transcriptional networks. Therefore, it is worthwhile to explore transcriptional regulation downstream of the CREB-Zfp189 interaction to identify possible interventions for cocaine use disorder. The Zfp189 gene product is a Krüppel-associated box domain containing zinc finger protein, which suggests that ZFP189 acts as a transcription factor, but its function remains poorly understood (
      • Najafabadi H.S.
      • Mnaimneh S.
      • Schmitges F.W.
      • Garton M.
      • Lam K.N.
      • Yang A.
      • et al.
      C2H2 zinc finger proteins greatly expand the human regulatory lexicon.
      ). Zfp189 induction in prefrontal cortex neurons has been demonstrated to regulate the expression of genes in response to chronic stress (
      • Lorsch Z.S.
      • Hamilton P.J.
      • Ramakrishnan A.
      • Parise E.M.
      • Salery M.
      • Wright W.J.
      • et al.
      Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
      ), but there is no evidence that this is the result of direct ZFP189-gene interactions. Moreover, it is probable that ZFP189 target genes vary by brain region and cell type. A thorough analysis of NAc MSN-specific ZFP189 gene targets, particularly in the context of cocaine exposure, would be an important future direction.
      Collectively, this work points to the CREB-mediated induction of Zfp189 within the NAc Drd2+ neurons as a key molecular event that drives the transition into phenotypes associated with chronic cocaine use and may be a promising molecular target for the development of interventions to combat the pathophysiology of cocaine addiction.

      Acknowledgments and Disclosures

      This work was supported by the National Institute on Drug Abuse grants (Grant Nos. P50DA047233 and R37DA007359 [to EJN]; R00DA045795 and P30DA033934 [to PJH]). Microscopy was performed at the Virginia Commonwealth University Microscopy Facility, supported, in part, by funding from National Institutes of Health-National Cancer Institute (NIH-NCI) Cancer Center Support grant (Grant No. P30CA016059 [to RAW]). Services in support of the research project were provided by the VCU Massey Cancer Center Transgenic or Knockout Mouse Core , supported, in part, with funding from NIH-NCI Cancer Center Support grant (Grant No. P30CA016059 [to RAW]).
      The authors report no biomedical financial interests or potential conflicts of interest.

      References

        • Koob G.F.
        • Volkow N.D.
        Neurobiology of addiction: A neurocircuitry analysis.
        Lancet Psychiatry. 2016; 3: 760-773
        • Nestler E.J.
        • Aghajanian G.K.
        Molecular and cellular basis of addiction.
        Science. 1997; 278: 58-63
        • Brunzell D.H.
        • Mineur Y.S.
        • Neve R.L.
        • Picciotto M.R.
        Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference.
        Neuropsychopharmacology. 2009; 34: 1993-2001
        • Pandey S.C.
        • Roy A.
        • Zhang H.
        • Xu T.
        Partial deletion of the cAMP response element-binding protein gene promotes alcohol-drinking behaviors.
        J Neurosci. 2004; 24: 5022-5030
        • Barrot M.
        • Olivier J.D.
        • Perrotti L.I.
        • DiLeone R.J.
        • Berton O.
        • Eisch A.J.
        • et al.
        CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli.
        Proc Natl Acad Sci U S A. 2002; 99: 11435-11440
        • Terwilliger R.Z.
        • Beitner-Johnson D.
        • Sevarino K.A.
        • Crain S.M.
        • Nestler E.J.
        A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.
        Brain Res. 1991; 548: 100-110
        • Renthal W.
        • Kumar A.
        • Xiao G.
        • Wilkinson M.
        • Covington H.E.
        • Maze I.
        • et al.
        Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
        Neuron. 2009; 62: 335-348
        • McClung C.A.
        • Nestler E.J.
        Regulation of gene expression and cocaine reward by CREB and DeltaFosB.
        Nat Neurosci. 2003; 6: 1208-1215
        • Teague C.D.
        • Nestler E.J.
        Key transcription factors mediating cocaine-induced plasticity in the nucleus accumbens.
        Mol Psychiatry. 2022; 27: 687-709
        • Carlezon Jr., W.A.
        • Duman R.S.
        • Nestler E.J.
        The many faces of CREB.
        Trends Neurosci. 2005; 28: 436-445
        • Hong E.J.
        • West A.E.
        • Greenberg M.E.
        Transcriptional control of cognitive development.
        Curr Opin Neurobiol. 2005; 15: 21-28
        • Zhang X.
        • Odom D.T.
        • Koo S.H.
        • Conkright M.D.
        • Canettieri G.
        • Best J.
        • et al.
        Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
        Proc Natl Acad Sci U S A. 2005; 102: 4459-4464
        • Mattson B.J.
        • Bossert J.M.
        • Simmons D.E.
        • Nozaki N.
        • Nagarkar D.
        • Kreuter J.D.
        • Hope B.T.
        Cocaine-induced CREB phosphorylation in nucleus accumbens of cocaine-sensitized rats is enabled by enhanced activation of extracellular signal-related kinase, but not protein kinase A.
        J Neurochem. 2005; 95: 1481-1494
        • Shaw-Lutchman T.Z.
        • Barrot M.
        • Wallace T.
        • Gilden L.
        • Zachariou V.
        • Impey S.
        • et al.
        Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal.
        J Neurosci. 2002; 22: 3663-3672
        • Shaw-Lutchman T.Z.
        • Impey S.
        • Storm D.
        • Nestler E.J.
        Regulation of CRE-mediated transcription in mouse brain by amphetamine.
        Synapse. 2003; 48: 10-17
        • Walters C.L.
        • Cleck J.N.
        • Kuo Y.C.
        • Blendy J.A.
        Mu-opioid receptor and CREB activation are required for nicotine reward.
        Neuron. 2005; 46: 933-943
        • Carlezon Jr., W.A.
        • Thome J.
        • Olson V.G.
        • Lane-Ladd S.B.
        • Brodkin E.S.
        • Hiroi N.
        • et al.
        Regulation of cocaine reward by CREB.
        Science. 1998; 282: 2272-2275
        • Larson E.B.
        • Graham D.L.
        • Arzaga R.R.
        • Buzin N.
        • Webb J.
        • Green T.A.
        • et al.
        Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.
        J Neurosci. 2011; 31: 16447-16457
        • Lorsch Z.S.
        • Hamilton P.J.
        • Ramakrishnan A.
        • Parise E.M.
        • Salery M.
        • Wright W.J.
        • et al.
        Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
        Nat Neurosci. 2019; 22: 1413-1423
        • Hamilton P.J.
        • Lim C.J.
        • Nestler E.J.
        • Heller E.A.
        Neuroepigenetic editing.
        Methods Mol Biol. 2018; 1767: 113-136
        • Yim Y.Y.
        • Teague C.D.
        • Nestler E.J.
        In vivo locus-specific editing of the neuroepigenome.
        Nat Rev Neurosci. 2020; 21: 471-484
        • Lardner C.K.
        • van der Zee Y.
        • Estill M.S.
        • Kronman H.G.
        • Salery M.
        • Cunningham A.M.
        • et al.
        Gene-targeted, CREB-mediated induction of ΔFosB controls distinct downstream transcriptional patterns within D1 and D2 medium spiny neurons.
        Biol Psychiatry. 2021; 90: 540-549
        • McQuin C.
        • Goodman A.
        • Chernyshev V.
        • Kamentsky L.
        • Cimini B.A.
        • Karhohs K.W.
        • et al.
        CellProfiler 3.0: Next-generation image processing for biology.
        PLoS Biol. 2018; 16e2005970
        • Neve R.L.
        • Neve K.A.
        • Nestler E.J.
        • Carlezon Jr., W.A.
        Use of herpes virus amplicon vectors to study brain disorders.
        Biotechniques. 2005; 39: 381-391
        • Lobo M.K.
        • Karsten S.L.
        • Gray M.
        • Geschwind D.H.
        • Yang X.W.
        FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.
        Nat Neurosci. 2006; 9: 443-452
        • Kronman H.
        • Richter F.
        • Labonté B.
        • Chandra R.
        • Zhao S.
        • Hoffman G.
        • et al.
        Biology and bias in cell type-specific RNAseq of nucleus accumbens medium spiny neurons.
        Sci Rep. 2019; 9: 8350
        • Tai L.H.
        • Lee A.M.
        • Benavidez N.
        • Bonci A.
        • Wilbrecht L.
        Transient stimulation of distinct subpopulations of striatal neurons mimics changes in action value.
        Nat Neurosci. 2012; 15: 1281-1289
        • Calipari E.S.
        • Bagot R.C.
        • Purushothaman I.
        • Davidson T.J.
        • Yorgason J.T.
        • Peña C.J.
        • et al.
        In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward.
        Proc Natl Acad Sci U S A. 2016; 113: 2726-2731
        • Heinsbroek J.A.
        • Neuhofer D.N.
        • Griffin W.C.
        • Siegel G.S.
        • Bobadilla A.C.
        • Kupchik Y.M.
        • Kalivas P.W.
        Loss of plasticity in the D2-accumbens pallidal pathway promotes cocaine seeking.
        J Neurosci. 2017; 37: 757-767
        • Cole S.L.
        • Robinson M.J.F.
        • Berridge K.C.
        Optogenetic self-stimulation in the nucleus accumbens: D1 reward versus D2 ambivalence.
        PLoS One. 2018; 13e0207694
        • Hikida T.
        • Kimura K.
        • Wada N.
        • Funabiki K.
        • Nakanishi S.
        Distinct roles of synaptic transmission in direct and indirect striatal pathways to reward and aversive behavior.
        Neuron. 2010; 66: 896-907
        • Lobo M.K.
        • Covington H.E.
        • Chaudhury D.
        • Friedman A.K.
        • Sun H.
        • Damez-Werno D.
        • et al.
        Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward.
        Science. 2010; 330: 385-390
        • van Zessen R.
        • Li Y.
        • Marion-Poll L.
        • Hulo N.
        • Flakowski J.
        • Lüscher C.
        Dynamic dichotomy of accumbal population activity underlies cocaine sensitization.
        Elife. 2021; 10e66048
        • Savell K.E.
        • Tuscher J.J.
        • Zipperly M.E.
        • Duke C.G.
        • Phillips R.A.
        • Bauman A.J.
        • et al.
        A dopamine-induced gene expression signature regulates neuronal function and cocaine response.
        Sci Adv. 2020; 6eaba4221
        • Francis T.C.
        • Yano H.
        • Demarest T.G.
        • Shen H.
        • Bonci A.
        High-frequency activation of nucleus accumbens D1-MSNs drives excitatory potentiation on D2-MSNs.
        Neuron. 2019; 103: 432-444.e3
        • Lee J.H.
        • Ribeiro E.A.
        • Kim J.
        • Ko B.
        • Kronman H.
        • Jeong Y.H.
        • et al.
        Dopaminergic regulation of nucleus accumbens cholinergic interneurons demarcates susceptibility to cocaine addiction.
        Biol Psychiatry. 2020; 88: 746-757
        • Lee J.
        • Finkelstein J.
        • Choi J.Y.
        • Witten I.B.
        Linking cholinergic interneurons, synaptic plasticity, and behavior during the extinction of a cocaine-context association.
        Neuron. 2016; 90: 1071-1085
        • Cha-Molstad H.
        • Keller D.M.
        • Yochum G.S.
        • Impey S.
        • Goodman R.H.
        Cell-type-specific binding of the transcription factor CREB to the cAMP-response element.
        Proc Natl Acad Sci U S A. 2004; 101: 13572-13577
        • Dong Y.
        • Green T.
        • Saal D.
        • Marie H.
        • Neve R.
        • Nestler E.J.
        • Malenka R.C.
        CREB modulates excitability of nucleus accumbens neurons.
        Nat Neurosci. 2006; 9: 475-477
        • Dobi A.
        • Seabold G.K.
        • Christensen C.H.
        • Bock R.
        • Alvarez V.A.
        Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal.
        J Neurosci. 2011; 31: 1895-1904
        • Misra K.
        • Pandey S.C.
        The decreased cyclic-AMP dependent-protein kinase A function in the nucleus accumbens: A role in alcohol drinking but not in anxiety-like behaviors in rats [published correction appears in Neuropsychopharmacology 2011;36:2149].
        Neuropsychopharmacology. 2006; 31: 1406-1419
        • Najafabadi H.S.
        • Mnaimneh S.
        • Schmitges F.W.
        • Garton M.
        • Lam K.N.
        • Yang A.
        • et al.
        C2H2 zinc finger proteins greatly expand the human regulatory lexicon.
        Nat Biotechnol. 2015; 33: 555-562