Involvement of cAMP-dependent protein kinase in the nucleus accumbens in cocaine self-administration and relapse of cocaine-seeking behavior

Abstract

cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc) has been implicated in cocaine addiction because (1) cocaine reinforcement is mediated by dopamine receptors that modulate cAMP formation, and (2) repeated exposure to cocaine upregulates the cAMP system in NAc neurons. This study tested PKA involvement in cocaine self-administration and relapse of cocaine-seeking behavior by infusing cAMP analogs that activate or inhibit PKA into the NAc of rats. Bilateral intra-NAc infusions of the PKA inhibitor Rp-cAMPS reduced baseline cocaine self-administration, shifted the dose-response curve for cocaine self-administration to the left, and induced relapse of cocaine-seeking behavior after extinction from cocaine self-administration, consistent with an enhancement of cocaine effects in each paradigm. In contrast, pretreatment with intra-NAc infusions of a PKA activator, Sp-cAMPS or dibutyryl cAMP, increased baseline cocaine self-administration during the second hour of testing and shifted the dose-response curve to the right, consistent with an antagonist-like action. After extinction from cocaine self-administration, similar infusions of Sp-cAMPS induced generalized responding at both drug-paired and inactive levers. As an index of PKA activity in vivo, NAc infusions of Rp-cAMPS reduced basal levels of dopamine-regulated phosphoprotein-32 phosphorylation and blocked amphetamine-induced increases in cAMP response element-binding protein (CREB) phosphorylation. Conversely, NAc infusions of Sp-cAMPS increased phosphorylation of CREB. Together, these results suggest that sustained upregulation of the cAMP system in the NAc after repeated cocaine exposure could underlie tolerance to cocaine reinforcement, whereas acute inhibition of this system may contribute to drug craving and relapse in addicted subjects.

Fig. 1.

Event records from representative rats self-administering cocaine (0.5 mg/kg per injection, i.v.) during baseline (previous test session) and 30 min after bilateral NAc infusions of the PKA inhibitor R_p-cAMPS, the PKA activator S_p-cAMPS, and the PBS vehicle during daily, 2 hr test sessions. The vertical deflections mark the time of each self-injection response. The total number of self-injections (SA), number of lever press responses during the 15 sec time-out periods after each injection (TO), and responses at the inactive lever (IA) are listed to the right. NAc infusions of R_p-cAMPS decreased whereasS_p-cAMPS increased cocaine self-administration. Control infusions of vehicle had no effect.

Fig. 2.

Effects of bilateral infusions ofR_p-cAMPS,S_p-cAMPS, or vehicle (VHCL) in the NAc (top panel, n = 8–14) or in the caudate–putamen 2 mm dorsal to the NAc site (bottom panel, n = 5–7) on cocaine self-administration (0.5 mg/kg per injection, i.v.). Hourly self-administration rates (S.A.) are expressed as the mean ± SEM of each animal’s percent change from baseline self-administration rates from the previous test session.Asterisks indicate that values differ from baseline values by paired t test (*p < 0.05; **p < 0.01).

Fig. 3.

Effects of bilateral infusions of dibutyryl cAMP or vehicle (VHCL) in the NAc on cocaine self-administration (0.5 mg/kg per injection, i.v.). Hourly self-administration rates (S.A.) are expressed as the mean ± SEM (n = 7) of each animal’s percent change from baseline self-administration rates from the previous test session. The asterisk indicates that values differ from both baseline values (paired t test) and from rates after control infusions of vehicle (Dunnett’s test) in the same animals (*p < 0.05).

Fig. 4.

Autoradiograms showing the effects of NAc infusion of the PKA inhibitor R_p-cAMPS (Rp), the PKA activatorS_p-cAMPS (Sp), or vehicle (V) on phospho-CREB and phospho-DARPP-32 immunoreactivity in NAc homogenates. The cAMP analogs were both infused at a dose of 80 nmol in 1.0 μl, whereas the contralateral side received similar infusions of vehicle 1 hr before killing. SomeR_p-treated animals received a challenge injection of amphetamine (4.0 mg/kg, i.p.) 15 min before killing.R_p-cAMPS inhibited basal levels of phospho-DARPP-32 (first lane) and prevented amphetamine-stimulated phospho-CREB (fifth lane) immunoreactivity. In contrast, a NAc infusion ofS_p-cAMPS increased basal phospho-CREB immunoreactivity but had no effect on basal phospho-DARPP-32 immunoreactivity (third lane).

Fig. 5.

Effects of NAc infusions ofR_p-cAMPS (Rp) andS_p-cAMPS (Sp) at a dose of 80 nmol in 1.0 μl and systemic amphetamine (AMPH, 4.0 mg/kg, i.p.) on phospho-CREB (n = 6–8) and phospho-DARPP-32 (n = 3–6) immunoreactivity in NAc homogenates. Separate groups of animals were given unilateral infusions of the cAMP analogs concurrent with vehicle infusions into the contralateral side and killed 1–2 hr later (see Results). Some animals were challenged with amphetamine 15 min before killing. Data are expressed as percent change in immunoreactivity from the contralateral side for Rp and Sp(**p < 0.01, Student’s pairedt-test). Values for amphetamine in vehicle-treated (VHCL) and R_p-treated sides are expressed as a percentage of the vehicle-infused side ofR_p-treated animals not receiving amphetamine (*p < 0.05; ***p < 0.001, Fisher’s LSD test).

Fig. 6.

Effects of bilateral NAc infusions ofR_p-cAMPS (A) or S_p-cAMPS (B) on the dose–response relationship of cocaine self-administration. Self-administration rates are shown for the first hour of the test session in experiments with R_p-cAMPS and during the second hour in experiments withS_p-cAMPS, when the cAMP analogs produced their maximal behavioral effects (see Fig. 2). The data are expressed as the mean number of self-injections (n = 10–12). Baseline values represent pooled data from tests preceding both the 40 and 80 nmol/1.0 μl per side doses of each cAMP analog.Asterisks indicate that values differ from baseline values by paired t test for the 40 or 80 nmol/side dose (*p < 0.05; **p < 0.01; ***p = 0.001).

Fig. 7.

Effects of bilateral NAc infusions ofR_p-cAMPS orS_p-cAMPS (40 nmol/μl per side) or vehicle on food-reinforced responding. Each food-reinforced response was followed by a 2 min time-out period when subsequent responding was not reinforced. Under these conditions, food reinforcement rates approached a maximum of 60 food pellets in 2 hr. Open bars show baseline responding; solid bars show the effect of NAc infusions. Data are expressed as the mean ± SEM (n = 8) for the rate of food reinforcement (A), for nonreinforced lever press responding during the time-out periods (B), and for responding at the inactive lever (C). Asterisksindicate that values differ from baseline values by pairedt test (*p < 0.05).

Fig. 8.

Event records from a representative rat showing reinstatement of nonreinforced lever press responding at the drug-paired lever after bilateral NAc-priming infusions of the PKA inhibitor R_p-cAMPS (80 nmol/μl per side) or vehicle during the reinstatement paradigm. NAc-priming infusions were given after extinction from 2 hr of intravenous cocaine self-administration (0.5 mg/kg per injection), when only intravenous saline injections were available. The vertical deflections mark the times of each self-injection of cocaine in the cocaine phase and of saline in the saline phase.

Fig. 9.

Effects of bilateral priming infusions ofR_p-cAMPS,S_p-cAMPS, or vehicle (VHCL) in the NAc (n = 10–12) or caudate–putamen (n = 8) on nonreinforced lever press responding. Data are expressed as the mean ± SEM of total lever press responses during the final hour of the saline phase, 30 min after priming infusions with the cAMP analogs at doses of 40 and 80 nmol/1.0 μl per side, or vehicle. Responding at the drug-paired or inactive lever differs from the vehicle condition by Dunnett’s test (*p < 0.05; **p < 0.01) or by the Wilcoxon signed ranks test of related subjects (⁺p < 0.05;⁺⁺p < 0.01).

Fig. 10.

Event records from a representative rat showing the effects of pretreatment with bilateral NAc infusions ofR_p-cAMPS orS_p-cAMPS (40 nmol/μl per side) or vehicle on reinstatement of nonreinforced lever press responding induced by an intravenous priming injection of cocaine (2.0 mg/kg). Priming injections of cocaine were given 30 min after the NAc pretreatments during the saline phase of the reinstatement paradigm. Thevertical deflections mark the times of each self-injection of cocaine in the cocaine phase and saline in the saline phase.

Fig. 11.

Effects of pretreatment with bilateral infusions of R_p-cAMPS orS_p-cAMPS (40 nmol/μl per side) or vehicle (VHCL) on nonreinforced lever press responding induced by priming injections of cocaine (n = 13–15). Data are expressed as the mean ± SEM of total lever press responses during the final hour of the saline phase, 30 min after the NAc pretreatments with cAMP analogs, and immediately after an intravenous priming injection of cocaine (0.5 and 2.0 mg/kg). Responding at the drug-paired or inactive lever differs from the baseline condition (<5 responses at either lever) by the Wilcoxon signed ranks test of related subjects (⁺p < 0.05;⁺⁺p < 0.01).

Fig. 12.

Localization of infusion sites in the NAc and caudate–putamen experiments. CPu, caudate putamen, Acb, nucleus accumbens, aca, anterior commissure, (from Paxinos and Watson, 1982).