Cocaine-induced intracellular signaling and gene expression are oppositely regulated by the dopamine D1 and D3 receptors

Abstract

Repeated exposure to cocaine can induce neuroadaptations in the brain. One mechanism by which persistent changes occur involves alterations in gene expression mediated by the dopamine receptors. Both the dopamine D1 and D3 receptors have been shown to mediate gene expression changes. Moreover, the D1 and D3 receptors are also coexpressed in the same neurons, particularly in the nucleus accumbens and also caudoputamen (CPu). Little is known however, whether these two receptors coordinately regulate gene expression after cocaine administration and the underlying mechanisms. We have used various gene mutant mice to address this issue. We show that extracellular signal-regulated kinase (ERK) activation and c-fos induction in the CPu in response to acute cocaine administration is mediated by the D1 receptor and inhibited by the D3 receptor. Moreover, ERK activation mediates acute cocaine-induced expression of Fos family genes, including c-fos, fosB and fra2. Interestingly, dynorphin, neogenin, and synaptotagmin VII, genes that possess cAMP-response element binding protein and AP-1 transcription complex-binding consensus sequences in their promoters, are also oppositely regulated by the D1 and D3 receptors after repeated exposure to cocaine. Furthermore, such regulation depends on proper ERK activation and c-fos function. These results suggest that the D1 and D3 receptors elicit opposite regulation of target gene expression by regulating ERK activation and c-fos induction after acute and chronic cocaine treatment.

Figure 1.

Opposite regulation of ERK activation in the CPu of D1 and D3 receptor mutant mice compared with wild-type mice after acute cocaine treatment. Time course (left) and quantification (right) of cocaine-induced ERK phosphorylation in the CPu in wild-type (A), D1 (B), and D3 receptor mutant (C) mice. Western blot analyses were performed 10 (n = 3 mice each), 20 (n = 8 mice each), 60 (n = 3 mice each), and 120 min (n = 3 mice each) after a cocaine injection at the 30 mg/kg dose. Antibodies against dually phosphorylated (Thr²⁰² and Tyr²⁰⁴) ERK (pERK) or total ERK were used. Data represent the mean + SEM pERK levels over the WT basal levels (n = 3 mice each). °p < 0.05 compared with basal levels. D, Comparison of the ERK phosphorylation in the CPu in D3 receptor mutant (D3–/–) and wild-type (WT) mice (n = 8 mice each) 20 min after a cocaine injection at the 30 mg/kg dose. *p < 0.05 compared with wild-type mice.

Figure 2.

Effects of D1 and D2 class receptor agonists on ERK activation in the CPu in D1, D3 receptor mutant, and wild-type mice. D1 (D1–/–), D3 (D3–/–) receptor mutant, and wild-type (WT) mice (n = 3 each) received injections of SKF81297 (A), PD128907 (B), or both (C). Western blot analyses were performed using CPu extracts prepared 20 min after the injections. Antibodies against dually phosphorylated ERK (pERK) or total ERK were used. Data represent the mean + SEM pERK levels over the WT basal levels (n = 3 mice each). *p < 0.05 treated groups versus WT basal levels. ^†p < 0.05 treated groups versus treated D1 receptor mutant mice. °p < 0.05 treated D3 receptor mutant mice versus treated WT mice.

Figure 3.

Phospho-ERK is induced by acute cocaine administration mostly in dynorphin-expressing neurons in the NAc in wild-type mice. Staining for dynorphin (A), phospho-ERK (B), and nuclear DNA (C, DAPI) show that phospho-ERK is expressed in dynorphin-expressing neurons (D). Double-labeled cells are indicated by white arrows. The results are representative of results of multiple sections from four wild-type mice. Sections were obtained 20 min after an intraperitoneal cocaine treatment. Scale bar, 10 μm.

Figure 4.

p38 and JNK phosphorylation in the CPu in D1, D3 receptor mutant, and wild-type mice is not obviously changed after acute cocaine treatment. Time course of cocaine-induced phosphorylation of p38 and JNK in the CPu in wild-type (A), D1 (B), and D3 receptor mutant (C) mice. The phosphorylation status of p38 and JNK was determined 10, 20, 60, and 120 min after a cocaine injection (30 mg/kg, i.p.). Total cell extracts were analyzed by Western blotting using antibodies against dually phosphorylated-p38 (Thr¹⁸⁰ and Tyr¹⁸²), dually phosphorylated-JNK (Thr¹⁸³ and Tyr¹⁸⁵), total p38, or JNK, respectively. The same set of mice used in probing ERK activation was used in analyzing p38 and JNK activation after cocaine injections.

Figure 5.

Opposite regulation of c-fos induction in the CPu by the D1 and D3 receptors after cocaine treatment is dependent on ERK activation. A, Opposite regulation of c-fos induction by cocaine in the CPu in D1 and D3 receptor mutant mice. D1 (D1–/–), D3 (D3–/–) receptor mutant, and wild-type (WT) mice (n = 3 mice each) were treated with cocaine at the 30 mg/kg dose for 2 hr. Protein extracts were isolated from the CPu of individual mouse, and Western blotting was performed. c-Fos levels in the wild-type mice were set at 1 for quantifications. B, The basal c-fos expression in the CPu of D1, D3 receptor mutant, and wild-type mice. c-Fos levels in the wild-type mice were set at 1 for quantitative comparisons. C, The induction of several Fos family proteins, including c-Fos, FosB, and Fra2, depends on ERK activation. Western blot analyses were performed in the absence or presence of SL327. SL327 was injected 15 min before cocaine treatment, and protein extracts from the CPu (n = 4 mice each) were isolated 2 hr after cocaine injections. Equal amounts of protein were loaded in each lane. Data represent mean + SEM of c-Fos, FosB, and Fra2 expression in the CPu of D3 receptor mutant and wild-type mice. Saline-injected WT levels were set at 1 for quantifications. Treated mice versus cocaine-treated WT mice *p < 0.05; **p < 0.01.

Figure 6.

Dynorphin expression is oppositely regulated in the NAc and CPu of D1, D3 receptor mutant mice after repeated cocaine exposure. D1, D3 receptor mutant, and wild-type mice (n = 4 each) were treated with cocaine (20 mg/kg) twice daily for 7 consecutive days. Coronal sections from wild-type (A, D, G), D1 (B, E, H), and D3 receptor mutant (C, F, I) mice were stained with an anti-dynorphin antibody. Scale bar, 15 μm.

Figure 7.

Neogenin and synaptotagmin VII are oppositely regulated in the NAc and CPu in D1 and D3 receptor mutant mice after repeated cocaine administration. D1 (D1–/–), D3 (D3–/–) receptor mutant, and wild-type (WT) mice were treated with 20 mg/kg of cocaine twice daily for 7 consecutive days or with saline (n = 8 mice each). Whole-cell extracts were isolated from both the NAc and CPu of individual mouse, and Western blotting was performed for neogenin (A) and synaptotagmin VII (B). Equal amounts of protein were loaded in each lane. Data represent mean + SEM of the expression of neogenin and synaptotagmin VII in the NAc and CPu of D1 and D3 receptor mutant and wild-type mice. Basal levels in WT mice were set at 1 for quantitative comparisons. °p < 0.05 treated WT mice versus WT basal levels. ^†p < 0.05 treated D3 receptor mutant mice versus treated WT mice. *p < 0.05 treated D1 receptor mutant mice versus treated WT mice.

Figure 8.

Changes in the expression of neogenin and synaptotagmin VII in the NAc and CPu of wild-type mice induced by repeated cocaine exposure depend on ERK activation and c-Fos function. A, Wild-type (WT) mice were treated with cocaine twice daily for 7 consecutive days in the absence or presence of SL327 or with saline (n = 4 mice each). SL327 were injected 15 min before cocaine treatment. Whole-cell extracts from the NAc and CPu were isolated, and Western blotting was performed for neogenin and synaptotagmin VII. B, Wild-type and D1 receptor-expressing neuron-specific c-fos mutant (DC) mice (n = 4 mice each) were treated with cocaine at the 20 mg/kg dose twice daily for 7 consecutive days or with saline (n = 4 mice each). Whole-cell extracts were isolated from both the NAc and CPu, and Western blotting was performed for neogenin and synaptotagmin VII. Equal amounts of protein were loaded in each lane. Results using extracts from two different mice are shown. Data represent the mean + SEM percent change over the basal levels in wild-type mice. B indicates basal levels. °p < 0.05 cocaine-treated WT versus WT basal. *p < 0.05 cocaine and SL327-treated WT versus cocaine-treated WT. ^†p < 0.05, cocaine-treated DC mice versus cocaine-treated WT mice.

Figure 9.

Promoter sequence comparisons of genes encoding dynorphin, neogenin, and synaptotagmin VII. Promoter sequences were obtained from GenBank. TGAGTAA and TGA(C/G)TCA were used for the AP-1 binding site, and CGTCA was used for the core CRE binding site, respectively, to search for sequences upstream from the transcription initiation sites of the three genes.