Dopamine D1 and D3 receptors are differentially involved in cue-elicited cocaine seeking

Abstract

Environmental cues previously associated with reinforcing drugs can play a key role in relapse to drug seeking behaviors in humans. The mesocorticolimbic dopamine system plays a critical role in cocaine-induced neurobiological changes. Dopamine D1 and D3 receptors modulate locomotor-stimulant and positive reinforcing effects of cocaine, and cue-induced reinstatement of cocaine-seeking. Moreover, activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is regulated by both D1 and D3 receptors. How D1 and D3 receptors modulate the acquisition and extinction of cue-elicited cocaine seeking behavior and associated changes in the MAPK signaling pathway in different brain regions, however, remains unclear. In the present study, we found that D1 receptor mutant mice failed to acquire conditioned place preference (CPP) while D3 receptor mutant mice show delayed CPP extinction compared with wild-type mice. Moreover, ERK, but not the c-jun N-terminal kinase and p38, is activated in wild-type and D3 receptor mutant mice but not in D1 receptor mutant mice following CPP acquisition. D3 receptor mutant mice also exhibit sustained ERK activation compared with wild-type mice following extinction training. Our results suggest that D1 and D3 receptors differentially contribute to learned association between cues and the rewarding properties of cocaine by regulating, at least in part, ERK activation in specific areas of the brain.

Fig. 1

The CPP behavioral paradigm and testing schedule. Preconditioning phase (Pre or Pre-test: day 1–2): mice were allowed to explore the CPP boxes for 20 minutes to determine the initial preference. Conditioning and testing phase (day 3–14): mice received once daily i.p. cocaine (C) or saline (S) injections alternatively and were confined in the white and black compartments respectively for 30 minutes. After every 2 days of training, mice were tested for place preference. Extinction and testing phase (day 15–33): mice received i.p. saline injections on both sides of the boxes and were confined for 30 minutes. The first extinction (Ext) test was given after 6 days of extinction training on day 21 and it was for 20 minutes. Then mice were tested after every 2 days of extinction training until CPP was extinguished. Reinstatement phase (Rein: day 34): mice received an i.p. cocaine injection before testing for CPP for 20 minutes.

Fig. 2

D1 receptor mutant mice do not acquire CPP at multiple cocaine doses. Five groups each of D1 receptor mutant (D1−/−) and wild-type (WT) mice received either cocaine (2.5, 5, 10, 20 mg/kg, n=12–16 per group) or saline (n=10 per group) injections alternatively and were confined to CPP chambers. Mice were then tested for place preference without injections at the indicated time points. Results represent mean ± SEM time spent on the drug-paired side minus the saline-paired side. Wild-type mice showed significant place preference at all 4 cocaine doses. In contrast, D1 receptor mutant mice did not. Saline-treated D1 receptor mutant and wild-type mice did not show significant preference. *p<0.05 compared with the same mouse group before drug administration (pre-test). #p<0.05 compared between two genotype at the same dose and test.

Fig. 3

D3 receptor mutant mice show similar CPP acquisition and reinstatement but delayed extinction compared to wild-type mice. a. CPP acquisition. Three groups each of D3 receptor mutant (D3−/−) and wild-type (WT) mice received either cocaine (10, 20 mg/kg, n=10–22 each) or saline (n=10 each) injections alternatively and were confined to specific compartments. These mice were tested for place preference without injections. Both D3 receptor mutant and wild-type mice showed equal CPP acquisition at 10 and 20 mg/kg cocaine doses but not following saline treatment. b. Extinction. Once acquired CPP, these mice (n=10–14 each) received saline injections and were confined to both sides alternatively. Extinction tests were performed at the indicated time points. Wild-type mice showed extinction of CPP on ext-test 1 and thereafter. D3 receptor mutant mice exhibited delayed extinction compared to wild-type mice. c. Reinstatement. One day after the last extinction test, mice (n=6 each) received an i.p. cocaine (20 mg/kg) injection and were tested for place preference. Both D3 receptor mutant and wild-type mice with prior exposure to cocaine but not to saline exhibited CPP following cocaine injections. Results represent mean ± SEM time spent on the drug-paired side minus the saline-paired side. *p<0.05 compared with the same mouse group before drug administration (pre-test). **p<0.05 compared with the last extinction test of the same mouse group. #p<0.05 compared between two genotype at the same cocaine dose and test.

Fig. 4

ERK is activated in the NAc and PFC in wild-type mice and not in D1 receptor mutant mice following CPP expression. Mice were given cocaine (COC, 20 mg/kg) or saline (SAL) injections and were sacrificed immediately after the last test. Western blotting were performed using brain samples from D1 receptor mutant (D1−/−, n=6 each) and wild-type (WT, n=7 each) mice after CPP acquisition test. Ratios of phospho-ERK relative to total ERK protein levels in the NAc, AMG and PFC were analyzed. Data were expressed as mean± SEM relative to saline controls that were set as 1. Actin levels were used as a loading control. *p<0.05 compared with the saline control group of the same genotype. #p<0.05 compared with cocaine-treated D1 receptor mutant mice.

Fig. 5

JNK and p38 are not obviously activated in wild-type and D1 receptor mutant mice following CPP expression. Western blotting for a JNK and b p38 were performed using samples from D1 receptor mutant (D1−/−, n=6 each) and wild-type (WT, n=7 each) mice after CPP acquisition test as before. Ratios of phospho-JNK and phosphor-p38 relative to total JNK and p38 protein levels in the NAc, AMG and PFC were analyzed. Data represent mean± SEM relative to saline controls that were set as 1. Actin levels were used as a loading control.

Fig. 6

ERK is similarly activated in the NAc and PFC in D3 receptor mutant and wild-type mice following CPP expression. Mice were given cocaine (COC, 20 mg/kg) or saline and were sacrificed immediately after the last CPP test. Western blotting were performed using brain samples from D3 receptor mutant (D3−/−, n=8 each) and wild-type (WT, n=8 each) mice after CPP acquisition test. Ratios of phospho-ERK relative to total ERK protein levels in the NAc, AMG and PFC were analyzed. Data were expressed as mean± SEM relative to saline controls that were set as 1. Actin levels were used as a loading control. *p<0.05 compared with the saline control mouse group of the same genotype.

Fig. 7

JNK and p38 are not obviously activated in D3 receptor mutant and wild-type mice following CPP expression. Western blotting for a JNK and b p38 were performed using brain samples from D3 receptor mutant (D3−/−, n=8 each) and wild-type (WT, n=8 each) mice after CPP acquisition test as before. Ratios of phospho-JNK and phospho-p38 over total JNK and p38 protein levels in the NAc, AMG and PFC were analyzed. Data represent mean± SEM relative to saline controls and were set as 1. Actin levels were used as a loading control.

Fig. 8

Sustained ERK activation in D3 receptor mutant mice compared to wild-type mice following 6 days of extinction training and extinction test. Mice were given saline for 6 days of extinction training and were sacrificed immediately after extinction test which was 20 minutes. At the same time, the No-CPP (No) control groups were sacrificed 24 hours after the last saline injection in the behavioral room. Western blotting were performed using brain samples from D3 receptor mutant (D3−/−) and wild-type (WT) mice from the extinction groups (n=7 each) and No-CPP groups (D3−/− and WT: n=6 each). Ratios of phospho-ERK relative to total ERK levels in the NAc, AMG and PFC were analyzed. Data were expressed as mean± SEM relative to No-CPP controls that were set as 1. Actin levels were used as a loading control. *p<0.05 compared with the No-CPP control mouse group of the same genotype. #p<0.05 compared with cocaine-treated wild-type mice with CPP training.

Fig. 9

JNK and p38 are not changed in wild-type and D3 receptor mutant mice following 6 days of CPP extinction training and test. Western blotting for a JNK and b p38 were performed using brain samples from D3 receptor mutant (D3−/−) and wild-type (WT) mice from the extinction groups (n=7 each) and the No-CPP groups (D3−/− and WT: n=6 each) as before. Ratios of phospho-JNK and phospho-p38 relative to total JNK and p38 protein levels in the NAc, AMG and PFC were analyzed. Data were expressed as mean± SEM relative to No-CPP controls that were set as 1. Actin levels were used as a loading control.