Circuit-Wide Gene Network Analysis Reveals Sex-Specific Roles for Phosphodiesterase 1b in Cocaine Addiction

Abstract

Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b (Pde1b), a Ca²⁺/calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine.

Keywords: addiction; bioinformatics; cocaine; genes; phosphodiesterase; plasticity.

Figure 1.

MEGENA identifies gene networks associated with addiction-like behavior across the brain's reward circuitry in male mice. a, An overview of self-administration paradigm, RNA-seq across six brain regions (PFC, NAc, CPU, BLA, VTA, vHip), and MEGENA analysis to identify gene networks associated with addiction-like behavior. MEGENA modules were ranked by enrichment in genes associated with the AI or DEGs (Extended Data Tables 1-1, 1-2). b, Network structure of NAc M41. Each node within the network represents a gene, and the color of the node represents the gene's correlation with the AI (yellow, positive correlation; blue, negative correlation; gray, nonsignificant correlation). Pde1b, a NAc M41 hub gene, has a negative correlation with the AI (Extended Data Fig. 1-1). The lines connecting the nodes, commonly referred to as “edges,” indicate a statistically significant coexpression relationship. Hub genes (Extended Data Table 1-3) are scaled in size by node strength (see Materials and Methods, MEGENA). c, A dot plot depicting the normalized expression levels of the top 10 NAc M41 hub genes across all clusters within a single RNA-seq dataset (n = 15,631) from the NAc of adult male and female rats (Savell et al., 2020). The size of the dot represents the percentage of nuclei expressing a given gene. GO analysis reveals the known physiological functions of the genes in NAc M41 (Extended Data Table 1-4).

Figure 2.

Pde1b overexpression in the NAc reduces cocaine self-administration in female rats but elevates relapse-like behavior after prolonged abstinence in both sexes. a, Representative confocal microscopy image of viral targeting to the NAc (10× magnification). b, Experimental timeline of cocaine self-administration in male (n = 6 mCherry; n = 6 PDE1B) and female (n = 7 mCherry; n = 8 PDE1B) rats. We validated that Pde1b overexpression in the NAc significantly increases Pde1b expression (Extended Data Fig. 2-1). c–h, Combined across sexes, the overexpression of Pde1b in the NAc reduces cocaine intake [repeated measures (RM) ANOVA; virus, F_(1,23)= 4.774; p = 0.0393; sex, F_(1,23)= 0.1590; p = 0.6938] but has no significant effect on active (RM ANOVA; virus, F_(1,23)= 0.4654; p = 0.5019; sex, F_(1,23) = 0.02499; p = 0.8758) or inactive lever presses (mixed-effect model; virus, F_(1,23) = 0.5298; p = 0.4740; sex, F_(1,23) = 0.4213; p = 0.5227). Given the intrinsic sex differences in cocaine self-administration behavior and that the effect of Pde1b overexpression on infusions is driven primarily by female rats, we present the data and statistical analysis disaggregated by sex. In male rats, the overexpression of PDE1B in the NAc has no effect on active lever presses (RM ANOVA; virus, F_(1,10) = 0.2473; p = 0.6298), inactive lever presses (mixed-effect model; virus, p = 0.3427), or cocaine intake (RM ANOVA; virus, F_(1,10) = 0.5653; p = 0.4695) during the acquisition of cocaine self-administration behavior. In female rats, the overexpression of PDE1B in the NAc reduced cocaine intake (RM ANOVA; virus, F_(1,13) = 11.37; p = 0.0050) in female rats. No significant differences were observed in active (RM ANOVA; virus, F_(1,13) = 0.1907; p = 0.6695) or inactive (RM ANOVA; virus, F_(1,13) = 1.318; p = 0.2717) lever presses in female rats. i, j, Pde1b overexpression decreases active lever responses during a thresholding task in female rats (RM ANOVA; virus, F_(1,132) = 5.756; p = 0.0178) but has no effect in male rats (RM ANOVA; virus, F_(1,88) = 3.497 × 10⁻²; p = 0.8521). K, L, PDE1B overexpression in the NAc potentiates active lever responses in a drug-free–seeking test after a 30 d abstinence period in male and female rats [RM ANOVA; virus, F_(1,22) = 16.36; p = 0.0005; sex, F_(1,22) = 10.12; p = 0.0043; Sidak's post hoc test (mCherry males vs PDE1B males), p = 0.0171; Sidak's post hoc test (mCherry females vs PDE1B females), p = 0.0186]. No significant effects of Pde1b overexpression were observed after 24 h forced abstinence (RM ANOVA; virus, F_(1,22) = 8.922 × 10⁻⁴; p = 0.9764; sex, F_(1,22) = 5.183; p = 0.0329). Data are presented as the mean ± SEM.

Figure 3.

Chronic cocaine increases Pde1b expression in D2 MSNs of NAc in male mice. a, Experimental timeline of saline or cocaine injections in WT male mice (saline, n = 6; acute cocaine, n = 7; chronic cocaine, n = 9). b, Acute or chronic injections of cocaine (20 mg/kg) had no effect on Pde1b expression in bulk NAc tissue (one-way ANOVA; Dunnett's multiple-comparison test, saline vs acute cocaine, adj. p = 0.7445; saline vs chronic cocaine, adj. p = 0.9992). Pde1b expression values were normalized to Hprt1. Similarly, cocaine self-administration has no effect on NAc Pde1b expression (Extended Data Fig. 3-1). c, Experimental timeline of chronic saline or cocaine injections in WT mice (n = 4 per group). d, Representative confocal microscopy (40× objective) images representing DAPI (blue), Pde1b (green), Drd1 (red), and Drd2 (yellow) RNAscope in situ hybridization staining in the NAc. The fluorescence intensity of Pde1b was measured in Drd1+ and Drd2+ nuclei from male (saline Drd1+ nuclei, n = 291; cocaine Drd1+ nuclei, n = 435; saline Drd2+ nuclei, n = 304; cocaine Drd2+ nuclei, n = 409) and female (saline Drd1+ nuclei, n = 492; cocaine Drd1+ nuclei, n = 481; saline Drd2+ nuclei, n = 545; cocaine Drd2+ nuclei, n = 628) mice treated with chronic saline or cocaine (n = 4 mice per group). We present the data and statistical analysis disaggregated by sex due to observed sex differences in Pde1b intensity measurements (ANOVA; sex, F_(1,24) = 29.00; p < 0.0001). e, In male mice, chronic cocaine has no effect on Pde1b intensity in Drd1+ and Drd2+ nuclei (two-way ANOVA; cocaine, F_(1,12) = 1.509; p = 0.2428; cell type, F_(1,12) = 0.9592; p = 0.3467). f, Chronic cocaine increases the percentage of Drd2+ nuclei expressing Pde1b (two-way ANOVA; cocaine, F_(1,12) = 11.21; p = 0.0058; Drd1+ nuclei Sidak's post hoc test; p = 0.1546; Drd2+ nuclei Sidak's post hoc test, p = 0.0303). g–h, In female mice, chronic cocaine has no effect on Pde1b intensity (two-way ANOVA; cocaine, F_(1,12) = 1.044; p = 0.3270) or the percentage of nuclei expressing Pde1b (two-way ANOVA; cocaine, F_(1,12) = 0.1656; p = 0.6912). Data are presented as the mean ± SEM.

Figure 4.

The overexpression of Pde1b in D1 or D2 MSNs in NAc of male and female mice oppositely regulates the acute locomotor response to cocaine, but not CPP. a, A timeline of CPP in D1-Cre (n = 11 mCherry males; n = 12 mCherry females; n = 12 PDE1B males; n = 13 PDE1B females) and D2-Cre (n = 9 mCherry males; n = 13 mCherry females; n = 12 PDE1B males; n = 10 PDE1B females) mice. b, Cre-dependent mCherry and Pde1b overexpression plasmids. c, d, The overexpression of Pde1b has no effect on CPP for cocaine in D1 (two-way ANOVA; virus, F_(1,44) = 1.118; p = 0.2960; sex, F_(1,44) = 2.038; p = 0.1605) or D2 (two-way ANOVA; virus, F_(1,38) = 1.163; p = 0.2877; sex, F_(1,38) = 0.6587; p = 0.4221) MSNs. e, f, During Day 1 of conditioning, Pde1b overexpression reduces the cocaine-induced locomotor response in D1 MSNs (ANOVA; virus, F_(1,45) = 5.092; p = 0.0289; sex, F_(1,45) = 5.108; p = 0.0287; virus:sex interaction, F_(1,45) = 0.7589; p = 0.3883; virus:cocaine interaction, F_(1,45) = 3.654; p = 0.0623) but increases the cocaine-induced locomotor response in D2 MSNs (ANOVA; virus, F_(1,38) = 7.288; p = 0.0103; sex, F_(1,38) = 0.3286; p = 0.5699; virus:sex interaction, F_(1,38) = 2.240; p = 0.1428; virus:cocaine interaction, F_(1,38) = 5.705; p = 0.0220). The overexpression of Pde1b had no effect on the locomotor response to saline administration in D1 or D2 MSNs. Data are presented as the mean ± SEM collapsed across sexes.

Figure 5.

Pde1b overexpression oppositely regulates cocaine-induced locomotor responses in NAc D1 and D2 MSNs female mice, but not male mice. a, A timeline for locomotor activity measures in D1-cre male mice (n = 9 mCherry; n = 9 Pde1b), D1-cre female mice (n = 6 mCherry; n = 6 Pde1b), D2-cre male mice (n = 6 mCherry; n = 7 Pde1b), and D2-cre female mice (n = 9 mCherry; n = 8 Pde1b). b, Cre-dependent mCherry and Pde1b overexpression plasmids. c–f, Combined across sexes, PDE1B overexpression reduces the locomotor response to chronic cocaine in D1 MSNs (ANOVA; virus, F_(1,26) = 5.165; p = 0.0316; sex, F_(1,26) = 4.734; p = 0.0388; virus:sex interaction, F_(1,26) = 2.354; p = 0.1371), with the opposite effect observed in D2 MSNs (ANOVA; virus, F_(1,26) = 5.021; p = 0.0338; sex, F_(1,26) = 1.775, p = 0.1943; virus:sex interaction, F_(1,26) = 8.137; p = 0.0084). Given the observed sex differences in locomotor response following Pde1b overexpression and cocaine administration, we present the data and statistical analysis disaggregated by sex. In D1 MSNs, Pde1b overexpression decreased cocaine-induced locomotor responses in female mice (RM ANOVA; virus, F_(1,10) = 6.663; p = 0.0274), but not male mice (RM ANOVA; virus, F_(1,16) = 0.3009; p = 0.5909). In D2 MSNs, Pde1b overexpression increased cocaine-induced locomotor responses in female mice (RM ANOVA; main effect of virus, F_(1,15) = 20.54; p = 0.0004), with no effect in male mice (RM ANOVA; main effect of virus, F_(1,11) = 0.2514; p = 0.6260). Data are presented as the mean ± SEM.

Figure 6.

Pde1b overexpression regulates the electrophysiological activity of D1 and D2 MSNs in the NAc of male and female mice. a, b, Pde1b overexpression reduces sEPSC frequency in D1 MSNs (nested t test, t₍₉₎ = 2.453; p = 0.0366; mCherry group, n = 22 cells; n = 6 mice; Pde1b group, n = 15 cells; n = 6 mice) but has no effect on sEPSC amplitude in D1 MSNs (nested t test, t₍₉₎ = 0.8943; p = 0.3945). Sex differences in D1 MSN sEPSC frequency (two-way ANOVA; sex, F_(1,33) = 1.310; p = 0.2606; sex:virus interaction, F_(1,33) = 0.9009; p = 0.3494) and amplitude (two-way ANOVA; sex, F_(1,35) = 1.434; p = 0.2392; sex:virus interaction, F_(1,35) = 0.7673; p = 0.3870) were not observed. c, d, Pde1b overexpression has no effect on sEPSC frequency (nested t test, t₍₁₀₎ = 0.1824; p = 0.8589; mCherry group, n = 25 cells; n = 6 mice; Pde1b group, n = 20 cells; n = 6 mice) or amplitude (unpaired t test, t₍₄₃₎ = 1.177; p = 0.2458) in D2 MSNs. Sex differences in D2 MSN sEPSC frequency (two-way ANOVA; sex, F_(1,41)= 3.486; p = 0.0691; sex:virus interaction, F_(1,41)= 1.866; p = 0.1794) and amplitude (two-way ANOVA; sex, F_(1,41)= 0.5838; p = 0.4492; sex:virus interaction, F_(1,41)= 0.4599; p = 0.5015) were not observed. e, f, Representative sEPSC traces from D1 (left) and D2 (right) MSNs. g, h, Pde1b overexpression has no effect on passive membrane properties in D1 (ANOVA; virus, F_(1,35)= 0.7135; p = 0.4040; sex, F_(1,35)= 4.508; p = 0.0409; sex:virus interaction, F_(1,35)= 2.802; p = 0.1030) or D2 (ANOVA; virus, F_(1,40)= 1.048; p = 0.3121; sex, F_(1,40)= 3.113 × 10⁻³; p = 0.9558; virus:sex interaction, F_(1,40)= 1.990; p = 0.1661) MSNs. i, j, We observed sex differences in the effect of Pde1b overexpression on the excitability of D1 (ANOVA; virus, F_(1,28)= 4.446; p = 0.0441; sex, F_(1,28)= 4.987; p = 0.0337; virus:sex interaction, F_(1,28)= 13.58; p = 0.0010) and D2 (ANOVA; virus, F_(1,32)= 170.2; p < 0.0001; sex, F_(1,32)= 1.084; p = 0.3056; virus:sex interaction, F_(1,32)= 15.97; p = 0.0004) MSNs. Pde1b overexpression reduces excitability in D1 MSNs of female mice (two-way ANOVA; virus, F_(1,12)= 24.23; p = 0.0004), but not male mice (two-way ANOVA; virus, F_(1,16)= 1.090; p = 0.3120). k, l, Pde1b overexpression increases excitability in D2 MSNs of male mice (two-way ANOVA; virus, F_(1,16)= 22.69; p = 0.0002), but not female mice (two-way ANOVA; virus, F_(1,16)= 3.530; p = 0.0786). m, n, Representative action potentials evoked by 200 pA injection currents. Data are presented as the mean ± SEM.

Figure 7.

Pde1b overexpression regulates the transcriptomic response to cocaine in the NAc of male and female mice. a, Experimental timeline (mCherry males, n = 5; mCherry females, n = 5; PDE1B males, n = 5; PDE1B females, n = 5). b, Validation of PDE1B overexpression (two-way ANOVA, F_(1,36)= 59.75; p < 0.0001) in mice treated with saline (Sidak's post hoc test, p < 0.0001) or cocaine (Sidak's post hoc test, p < 0.0001). These data were analyzed collapsed across sexes, given that sex differences in Pde1b expression levels following Pde1b overexpression were not observed (ANOVA; virus, F_(1,32)= 54.89, p < 0.0001; sex, F_(1,32)= 0.1875; p = 0.6679; virus:sex interaction, F_(1,32)= 0.2082; p = 0.6521). c, Heatmaps comparing DEGs (Extended Data Tables 7-1, 7-2, 7-3, 7-4) after chronic cocaine across viral treatment groups and sexes [p value <0.05; log₂(fold change) >0.3]. d–e, RRHO plots comparing the overlap of cocaine-induced changes in gene expression between sexes in mCherry and PDE1B mice (Extended Data Tables 7-5, 7-6). Data are presented as the mean ± SEM.