Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine

Abstract

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

Fig. 1.

Targeted inactivation of Camk4 and Creb1 genes. Immunostaining for CaMKIV (A and B) or CREB (C and D) was performed following the protocol described in Methods. Striatum from control animal (A), Camk4^D1Cre mouse (B), Creb1^loxP/loxP, Crem^−/− mouse (C), and Creb1^Camkcre4, Crem^+/− mouse (D).

Fig. 2.

Loss of CaMKIV does not impair induction of IEGs. (A) Profiling of gene transcription in the striatum 1 h after cocaine treatment. Gene expression profiling was performed using Affymetrix 420A 2.0 arrays on RNA samples derived from striatum from the Camk4^D1Cre animals and littermate controls 1 h after i.p. injection with 25 mg/kg cocaine or saline. On the heat map shown in the figure, each column represents the average (3 for saline treated groups, 6 for cocaine treated groups) log₂ expression values corresponding to significantly induced transcripts as indicated on the right. The color intensity is proportional to the normalized expression value as shown in the legend below. Transcripts are ordered by fold of induction in the controls treated with cocaine vs. saline. (B) Induction of Fos protein in the striatum 2 h after cocaine injection. Coronal sections from cocaine-injected Camk4^D1Cre and control mice were immunostained for Fos. The upper four panels correspond to representative fragments of the dorsal striatum, and the lower four panels show a fragment of the NAc on the border of the shell and core divisions. The corresponding genotypes and treatments are indicate above and left of the images. (C) Expression of Fos, Fosb, and Pdyn after a saline or cocaine (10 mg/kg) challenge 7 days after a drug-free period in cocaine-sensitized mice. The bars represent transcript abundance normalized to the levels observed in saline-treated control animals with the SEM shown (n = 4–7). Empty bars correspond to control animals, and black bars correspond to Camk4^D1Cre mice treated as indicated below the graphs. A significant difference (P < 0.05) between cocaine-treated Camk4^D1Cre vs. controls is indicated by an asterisk.

Fig. 3.

Locomotor effects of cocaine in Camk4^D1Cre and Creb1^Camkcre4 mice. Initial locomotor response (A–C) and development of cocaine sensitization (d–f) (10 mg/kg, i.p.) in Camk4^D1Cre (n = 9); Creb1^Camkcre4, Crem^+/− (n = 6); Creb1^Camkcre4, Crem^−/− (n = 4); and control mice for each genotype (n = 8, n = 7, and n = 8, respectively). (a–c) Cocaine induced a higher increase in locomotor activity during the first 10 min in Camk4^D1Cre mutant mice (t (15) = −3.33, P = 0.004) but in none of the Creb1^Camkcre4 genotypes or control groups (Creb1^Camkcre4, Crem^+/−: t (11) = −0.4, P = 0.6; Creb1^Camkcre4, Crem^−/−: t (10) = −1.12, P = 0.3). (D–F) Control and both Creb1^Camkcre4 genotypes showed intact development and expression of cocaine sensitization (Creb1^Camkcre4, Crem^+/− [two-way ANOVA cocaine effect: F (3, 30) = 21.33, P < 0.001; Creb1^Camkcre4, Crem^−/− F (2, 18) = 13.76, P < 0.001]. Development of sensitization was absent in Camk4^D1Cre mutant mice, but they expressed a significantly higher response to cocaine after drug-free intervals than their control littermates [two-way ANOVA day × genotype effect for Camk4^D1Cre: F (3, 45) = 10.52, P < 0.001; Newman-Keuls posthoc test: CamKIV^D1Cre vs. control, *P < 0.01 for coc-12 and coc-19]. Data represent the mean increase in percentage in activity in respect to saline over a 10-min (A–C) or 30 min (D–F) recording period after injection of cocaine. # represents P < 0.05 compared with day 1 and *P < 0.01 compared with control group. Because of the progressive neurodegeneration (25), Creb1^Camkcre4, Crem^−/− mice were not tested on day 19.

Fig. 4.

Cocaine-induced reinforcement and drug-seeking behavior in Camk4^D1Cre and Creb1^Camkcre4 mice. Cocaine-induced CPP (A–C), extinction and reinstatement (D and E) in Camk4^D1Cre (n = 8); Creb1^Camkcre4, Crem^+/− (n = 8); Creb1^Camkcre4, Crem^−/− (n = 4); and control mice for each genotype (n = 10, n = 13, and n = 13, respectively). (A–C) Camk4^D1Cre mutant mice showed higher preference for the cocaine-paired compartment [t (16) = −3.96. P = 0.001], whereas both Creb1^Camkcre4 genotypes and control mice showed similar scores [Creb1^Camkcre4, Crem^+/−: t (19) = −0.23, P = 0.6; Creb1^Camkcre4, Crem^−/−: t (15) = −0.3, P = 0.7]. (D and F) Camk4^D1Cre mutant mice showed a more robust CPP at a dose of 5 mg/kg when compared with the control group (n = 5 per genotype). After extinction, a challenge injection of cocaine (3 mg/kg, i.p.) induced a similar reinstatement of the CPP in Creb1^Camkcre4, Crem^+/− and control mice (n = 8 and n = 13, respectively) [two-way ANOVA conditioning × genotype effect: F (2, 18) = 0.3, P = 0.7], except for the Camk4^D1Cre animals, which displayed stronger preference than controls [two-way ANOVA conditioning × genotype effect: Camk4^D1Cre: F (2, 30) = 2.91, P < 0.05; post hoc for CPP, P < 0.05]. Results are presented as the means + SEM. CPP scores shown correspond to induction, followed by extinction and reinstatement of CPP. Statistical significance of P < 0.05 compared with control group is indicated by an asterisk.

Fig. 5.

Cocaine-induced reinforcement in heterozygous and virus-treated mice. Cocaine-induced CPP (10 mg/kg) in Camk4^D1Cre heterozygous (n = 6) and recombinant adenoassociated virus (rAAV)–dnCaMKIV (n = 9) mice and their respective control littermates (controls [n = 10] or empty virus-treated mice [n = 8]). Both heterozygous mice for Camk4 (A) and rAAV-dnCaMKIV mice (B) showed a more robust CPP compared with controls [for Camk4 ^D1Cre heterozygous mice: t (14) = −2.06, P = 0.05; for rAAV-dnCaMKIV mice: t (15) = −2.1, P = 0.05].