cAMP response element binding protein phosphorylation in nucleus accumbens underlies sustained recovery of sensorimotor gating following repeated D₂-like receptor agonist treatment in rats

Abstract

Background: Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating. PPI deficits are observed in humans and rats upon acute treatment with dopamine D₂-like receptor agonists and in patients with schizophrenia. Repeated treatment with a D₂-like agonist, however, reverses PPI deficits and increases cyclic adenosine monophosphate (cAMP) signaling in the nucleus accumbens (NAc). This study examined the short- and long-term effects on PPI of treatment with quinpirole and ropinirole, dopamine D₂/D₃ receptor agonists, and the molecular mechanism by which they occur.

Methods: PPI was assessed in adult male Sprague-Dawley rats following acute and chronic treatment with quinpirole or ropinirole and 1, 2, 3, and 4 weeks after termination of repeated ropinirole treatment. Finally, the effect of dominant negative mutant cAMP response element binding protein (CREB) overexpression in the NAc on PPI following chronic quinpirole treatment was assessed.

Results: Acute quinpirole produced dose-dependent PPI deficits, whereas ropinirole caused consistent PPI reduction at all but the highest dose. Repeated ropinirole treatment significantly increased PPI compared with acute treatment, and increased CREB phosphorylation in NAc neurons. Subsequent ropinirole challenge had no effect as long as 28 days later, at which time NAc CREB phosphorylation had normalized. Overexpression of dominant negative mutant CREB prevented PPI recovery induced by chronic quinpirole treatment.

Conclusions: Chronic quinpirole or ropinirole treatment produces sustained PPI recovery; CREB activity in the NAc is required to induce PPI recovery but not to maintain it. The results suggest that transcriptional regulation by CREB mediates long-lasting changes occurring within NAc circuits to promote recovery of sensorimotor gating.

Figure 1. PPI disruption by acute quinpirole or ropinirole

Animals were challenged with quinpirole (0.0, n=36; 0.05, n=12; 0.1, n=12; or 0.3 mg/kg, n=12) or ropinirole (0.0, n=36; 0.05, n=6; 0.1, n=10; 0.5, n=9; or 1.0 mg/kg, n=10) 10 min prior to PPI testing. Percent PPI (mean ± SEM) are mean values collapsed across three prepulse intensities. There was a significant effect of quinpirole dose (F_11,204 = 4.399, p ≤ 0.001) and ropinirole dose (F_14,297 = 2.383, p ≤ 0.005) using one-way ANOVA. * - p ≤ 0.05 compared to 0.0 mg/kg dose; † - p ≤ 0.05 compared to 0.05 mg/kg dose.

Figure 2. Recovery of PPI after 28 days of repeated ropinirole treatment

Animals were treated daily for 28 consecutive days with ropinirole (0.0, n=10; 0.05, n=11; 0.1, n=11; or 0.5 mg/kg, n=10). Following baseline testing, PPI was assessed 10 min after drug treatment on days 1 and 28. Percent PPI (mean ± SEM) are mean values collapsed across three prepulse intensities. There was no significant effect over time of saline vehicle (F_8,81 = 1.056, p > 0.05), however, there was a significant effect of ropinirole treatment (0.05 mg/kg: F_8,90 = 6.163, p ≤ 0.005; 0.1 mg/kg: F_8,90 = 6.787, p ≤ 0.005; 0.5 mg/kg: F_8,90 = 5.220, p ≤ 0.05) using repeated measures ANOVA. * - p ≤ 0.05 compared to baseline PPI within treatment group; † - p ≤ 0.05 compared to day 1 PPI within treatment group.

Figure 3. CREB phosphorylation increased after repeated ropinirole treatment in the NAc, but not in the caudatoputamen (CP)

Immunohistochemistry was performed on tissue obtained from rats treated for 28 days with ropinirole (0.0, n=10; 0.05, n=11; 0.1, n=11; or 0.5 mg/kg, n=10). Data are expressed as number of nuclear profiles (mean ± SEM) per mm². There was a significant effect in the NAc core (F_3,38 = 4.561, p ≤ 0.05) and shell (F_3,38 = 3.653, p ≤ 0.05), but not in the caudatoputamen (DL CP: F_3,38 = 1.837, p > 0.05; Med CP: F_3,38 = 0.727, p > 0.05) using repeated measures ANOVA. * - p ≤ 0.05 compared to 0.0 mg/kg dose within brain region.

Figure 4. Ropinirole challenge 28 days after repeated ropinirole treatment does not disrupt PPI

Rats were treated daily for 28 consecutive days with ropinirole (0.0, n=7 or 0.1 mg/kg, n=7). Thereafter, PPI testing was accomplished 28 days after termination of daily treatment following challenge with the same dose received previously. Percent PPI (mean ± SEM) are mean values collapsed across three prepulse intensities. There was a significant effect on Day 1 (F_1,40 = 4.442, p ≤ 0.05), but not on any other day (Baseline: F_1,40 = 0.002, p > 0.05; Day 28: F_1,40 = 0.002, p > 0.05; Day 28 after treatment: F_1,40 = 0.268, p > 0.05) using repeated measures ANOVA. There was no significant effect over time of saline (F_3,200 = 2.207, p > 0.05) or ropinirole treatment (F_3,200 = 1.896, p > 0.05). * - p ≤ 0.05 compared to 0.0 mg/kg dose.

Figure 5. CREB phosphorylation returned to basal levels 28 days after the termination of repeated ropinirole treatment

Immunohistochemistry was performed on tissue obtained from rats following a ropinirole challenge 29 days after termination of daily treatment. Rats that previously received vehicle treatment received either 0.0 or 0.1 mg/kg of ropinirole, while rats that previously received 0.1 mg/kg ropinirole received the same challenge dose (n=9 in each group). Data are expressed as number of nuclear profiles (mean ± SEM) per mm². There was no significant effect in any brain region (NAc Core: F_2,24 = 1.548, p > 0.05; NAc Shell: F_2,24 = 1.150, p > 0.05; DL CP: F_2,24 = 0.704, p > 0.05; Med CP: F_2,24 = 1.006, p > 0.05) using repeated measures ANOVA.

Figure 6. CREB phosphorylation increased in the NAc immediately following repeated ropinirole, but not four weeks after termination of treatment

Image of phosphoCREB labeling in the NAc core from (A) saline-, (B) acute ropinirole-, or (C) repeated ropinirole-treated rats, or (D) repeated ropinirole-treated rats 29 days after termination of treatment. Anterior commissure is shown at lower left in each illustration. Scale bar: 100 µm.

Figure 7. mCREB prevents PPI recovery induced by chronic quinpirole treatment

PPI was determined before and 24 hr after intracranial infusion of adeno-associated virus for gene transfer of eGFP alone, mCREB-eGFP (mCREB) or CREB-eGFP (CREB). Rats were challenged with quinpirole (0.0 or 0.1 mg/kg) 3 weeks later. Pellets were then implanted which released quinpirole (0.0 or 0.1 mg/kg/day, n=9 per virus group). PPI was tested 1 and 28 days after pellet implantation, then following quinpirole challenge on day 29 (0.0 or 0.1 mg/kg). Percent PPI (mean ± SEM) are mean values collapsed across three prepulse intensities. There was no significant effect in the control group (F_8,207 = 1.669, p > 0.05), however there was a significant effect of quinpirole treatment (GFP/quinpirole: F_8,207 = 3.211, p < 0.005; mCREB/quinpirole: F_8,207 = 5.337, p < 0.005; CREB/quinpirole: F_8,207 = 4.894, p < 0.005) using repeated measures ANOVA. * - p ≤ 0.05 compared to post-virus surgery within treatment group; † - p ≤ 0.05 compared to challenge 1 within treatment group.

Figure 8. CREB phosphorylation increased after chronic quinpirole treatment in the NAc, but not in the caudatoputamen (CP)

Immunohistochemistry was performed on tissue obtained from rats treated for 28 days with blank or quinpirole pellets (0.0 or 0.1 mg/kg/day, n=9 per virus group) after intracranial infusion of adeno-associated virus for gene transfer of eGFP alone, mCREB-eGFP (mCREB) or CREB-eGFP (CREB). Data are expressed as number of nuclear profiles (mean ± SEM) per mm². There was a significant effect of quinpirole treatment in the NAc core (F_5,22 = 15.83, p ≤ 0.01) and shell (F_5,22 = 7.116, p ≤ 0.01), but not in the CP (DL CP: F_5,22 = 0.9656, p > 0.05; Med CP: F_5,22 = 0.39, p > 0.05) using repeated measures ANOVA. * - p ≤ 0.05 compared to blank pellet in the same viral treatment group.