Opposing role for Egr3 in nucleus accumbens cell subtypes in cocaine action

Abstract

An imbalance in molecular signaling cascades and transcriptional regulation in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes, those enriched in dopamine D1 versus D2 receptors, is implicated in the behavioral responses to psychostimulants. To provide further insight into the molecular mechanisms occurring in MSN subtypes by cocaine, we examined the transcription factor early growth response 3 (Egr3). We evaluated Egr3 because it is a target of critical cocaine-mediated signaling pathways and because Egr3-binding sites are found on promoters of key cocaine-associated molecules. We first used a RiboTag approach to obtain ribosome-associated transcriptomes from each MSN subtype and found that repeated cocaine administration induced Egr3 ribosome-associated mRNA in NAc D1-MSNs while reducing Egr3 in D2-MSNs. Using Cre-inducible adeno-associated viruses combined with D1-Cre and D2-Cre mouse lines, we observed that Egr3 overexpression in D1-MSNs enhances rewarding and locomotor responses to cocaine, whereas overexpression in D2-MSNs blunts these behaviors. miRNA knock-down of Egr3 in MSN subtypes produced opposite behavioral responses from those observed with overexpression. Finally, we found that repeated cocaine administration altered Egr3 binding to promoters of genes that are important for cocaine-mediated cellular and behavioral plasticity. Genes with increased Egr3 binding to promoters, Camk2α, CREB, FosB, Nr4a2, and Sirt1, displayed increased mRNA in D1-MSNs and, in some cases, a reduction in D2-MSNs. Histone and the DNA methylation enzymes G9a and Dnmt3a displayed reduced Egr3 binding to their promoters and reduced mRNA in D1-MSNs. Our study provides novel insight into an opposing role of Egr3 in select NAc MSN subtypes in cocaine action.

Keywords: Egr3; RiboTag; cocaine; medium spiny neurons; nucleus accumbens; transcription.

Figure 1.

Egr3 expression after repeated cocaine exposure. A, qRT-PCR and Western blot analysis of Egr3 demonstrate that mRNA and protein levels are decreased in NAc after repeated cocaine exposure (7 d, 20 mg/kg) followed by 24 h of withdrawal in mice. B, Rats that self-administer cocaine display a similar reduction of Egr3 mRNA and protein. All values were normalized to GAPDH. Error bars indicate SEM.

Figure 2.

D1-Cre-RT and D2-Cre-RT validation and Egr3 ribosome-associated mRNA levels in MSN subtypes after cocaine exposure. A, Illustration of HA-tagged Rpl22 in D1-MSNs or D2-MSNs by crossing RT mice to D1-Cre or D2-Cre mice. Polyribosomes are immunoprecipitated from D1-MSNs or D2-MSNs with HA-tagged magnetic beads and then cell-specific ribosome-associated mRNA is isolated. B, C, MSN subtypes display enrichment of ribosome-associated mRNA for known MSN subtype genes. D, qRT-PCR analysis of Egr3 ribosome-associated mRNA in D1-Cre-RT and D2-Cre-RT NAc tissue after repeated cocaine (7 d, 20 mg/kg) or saline exposure. Repeated cocaine exposure induces Egr3 ribosome-associated mRNA in D1-MSNs while decreasing Egr3 in D2-MSNs. Error bars indicate SEM.

Figure 3.

Cre-inducible AAV overexpression of Egr3 in D1-MSN and D2-MSN subtypes. A, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Egr3-EYFP. B, Image of a D2-Cre NAc demonstrating expression of Egr3-EYFP in NAc but no expression is visible in wild-type (WT) mice. Scale bar, 100 μm. C, D, AAV-DIO-Egr3-EYP overexpression D1-Cre and D2-Cre NAc results in the upregulation of Egr3 mRNA (C) and expression of 70 kDa Egr3-EYFP protein in NAc (D) compared with mice receiving the AAV-DIO-EYFP control virus. Error bars indicate SEM.

Figure 4.

Egr3 overexpression in MSN subtypes alters cocaine CPP and cocaine-induced locomotor activity. A, Cocaine (7.5 mg/kg) CPP is enhanced with D1-Cre mice that received AAV-DIO-Egr3-EYFP into NAc, whereas cocaine CPP is decreased in D2-Cre mice that received AAV-DIO-Egr3-EYFP into NAc compared with AAV-DIO-EYFP controls. B, Repeated cocaine (10 mg/kg)-induced locomotor activity is increased in D1-Cre mice (days 2–5), whereas it is reduced in D2-Cre mice (day 2–3) expressing AAV-DIO-Egr3-EYFP in NAc compared with controls expressing AAV-DIO-EYFP. Error bars indicate SEM.

Figure 5.

Cre-inducible AAV miRNA knock-down of Egr3 in D1-MSN and D2-MSN subtypes. A, Mouse Egr3 shRNA sequences and their targeting of the Egr3 gene. The position is relative to the ATG start site of Egr3 cDNA. B, qRT-PCR analysis of Egr3 demonstrates Egr3shRNA knock-down in Neuro2a cells. C, Illustration of the miRNA engineering sequence. The first 21 bp of the Egr3 target sequence (red) of shRNA is used for miRNA engineering. D, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Egr3miR and mCitrine. E, Image of a D2-Cre NAc demonstrating expression of mCitrine in NAc, but no visible expression is observed in wild-type (WT) NAc. Scale bar, 100 μm. F, G, D1-Cre and D2-Cre mice expressing AAV-DIO-Egr3miR-mCitrine in NAc display reduction of Egr3 mRNA (F) and protein (G) compared with AAV-DIO-Scramble mCitrine controls. Error bars indicate SEM.

Figure 6.

Egr3miR knock-down in MSN subtypes alters cocaine CPP and cocaine-induced locomotor activity. A, Cocaine (7.5 mg/kg) CPP is decreased in D1-Cre mice that received AAV-DIO-Egr3miR-mCitrine into NAc, whereas it is increased in D2-Cre mice that received AAV-DIO-Egr3miR-mCitrine compared with AAV-DIO-scramble-mCitrine controls. Error bars indicate SEM. B, Cocaine (10 mg/kg)-induced locomotor activity is decreased in D1-Cre mice (days 1, 2, 3, and 5), whereas it is increased in D2-Cre mice expressing AAV-DIO-Egr3miR-mCitrine in NAc compared with controls expressing AAV-DIO-Scramble.

Figure 7.

Egr3 transcriptional regulation of cocaine-associated genes after repeated cocaine exposure. ChIP using an Egr3 antibody to immunoprecipitate Egr3 bound to promoters of cocaine-associated genes after 7 d of cocaine (20 mg/kg) or saline exposure followed by 24 h withdrawal. Egr3 binding to the Camk2α, CREB, FosB, Nr4a2, and Sirt1 promoters is increased, whereas binding to the Dnmt3a and G9a promoters is decreased in the cocaine group. Error bars indicate SEM.

Figure 8.

Egr3-regulated genes are differentially expressed in MSN subtypes after repeated cocaine exposure. Cell-type-specific ribosome-associated mRNA analysis from D1-Cre-RT and D2-Cre-RT NAc mice after repeated cocaine (7 d, 20 mg/kg) exposure followed by 24 h withdrawal demonstrates altered induction of Egr3-regulated genes. Ribosome-associated mRNA for Camk2α and CREB is increased in D1-MSNs, whereas it is decreased in D2-MSNs in the cocaine group. FosB, Nr4a2, and Sirt1 are increased in D1-MSNs, with no change in D2-MSNs in the cocaine group. Dnmt3a and G9a are decreased in D1-MSNs, with no change in D2-MSNs in the cocaine group. Error bars indicate SEM.