Glycogen synthase kinase 3β in the nucleus accumbens core mediates cocaine-induced behavioral sensitization

Authors


Address correspondence and reprint requests to Dr. Jie Shi, National Institute on Drug Dependence, Peking University, Beijing, China. E-mail: shijie@bjmu.edu.cn

Abstract

Glycogen synthase kinase 3β (GSK-3β) is a ubiquitous serine/threonine protein kinase involved in a number of signaling pathways. Previous studies have demonstrated a role for GSK-3β in the synaptic plasticity underlying dopamine-associated behaviors and diseases. Drug sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. However, the role of GSK-3β in cocaine-induced behavior sensitization has not been examined. The present study investigated the effects of chronic cocaine exposure on GSK-3β activity in the nucleus accumbens (NAc) and determined whether changes in GSK-3β activity in the NAc are associated with cocaine-induced locomotor sensitization. We also explored whether blockade of GSK-3β activity in the NAc inhibits the initiation and expression of cocaine-induced locomotor sensitization in rats using systemic or brain region-specific administration of the GSK-3β inhibitors lithium chloride (LiCl) and SB216763. GSK-3β activity in the NAc core, but not NAc shell, increased after chronic cocaine (10 mg/kg, i.p.) administration. The initiation and expression of cocaine-induced locomotor sensitization was attenuated by systemic administration of LiCl (100 mg/kg, i.p.) or direct infusion of SB216763 (1 ng/side) into the NAc core, but not NAc shell. Collectively, these results indicate that GSK-3β activity in the NAc core, but not NAc shell, mediates the initiation and expression of cocaine-induced locomotor sensitization, suggesting that GSK-3β may be a potential target for the treatment of cocaine addiction.

Abbreviations used:
DMSO

dimethylsulfoxide

GSK3β

glycogen synthase kinase 3β

LTP

long-term potentiation

Repeated psychostimulant administration produces progressively enhanced and enduring behavioral sensitization (Robinson 1984; Kalivas and Stewart 1991; Carlezon and Nestler 2002), which has been suggested to model the neurobiological adaptations that contribute to compulsive drug craving (Robinson and Berridge 1993; White et al. 1997) and relapse (Bradberry 2007; Robinson and Berridge 2008). Chronic cocaine exposure increases extracellular dopamine levels in the nucleus accumbens (NAc) (Koob and Nestler 1997; Pierce and Kalivas 1997; Ikemoto and Wise 2004). Dopaminergic projections from the ventral tegmental area to the NAc and other forebrain nuclei play a crucial role in both the initiation and long-term expression of cocaine-induced behavioral sensitization (Robinson and Becker 1986; Kalivas and Stewart 1991; Robinson and Kolb 1999; Robinson et al. 2001; Kalivas 2004; Thomas et al. 2008). However, the precise neuronal processes underlying dopamine-dependent sensitization are still not clearly understood.

A current influential hypothesis is that cocaine addiction-related behaviors, including psychomotor sensitization, are caused by drug-induced neuroadaptations in the mesocorticolimbic dopamine system and glutamatergic corticolimbic circuitry in which dopamine projections are embedded. These neuroadaptations include the extracellular signal-regulated kinase (ERK) signaling pathway (Sweatt 2001), brain-derived neurotrophic factor (Poo 2001), glutamate transmission, and synaptic plasticity (Malenka 2003). Glycogen synthase kinase 3β (GSK-3β), a multifunctional serine/threonine (Ser/Thr) kinase (Embi et al. 1980), has been shown to be involved in synaptic plasticity, including the regulation of N-methyl-d-aspartate receptor-dependent long-term potentiation (LTP) and long-term depression (Hooper et al. 2007; Peineau et al. 2007). Once inhibitory control is impaired, GSK-3β activity becomes abnormally high, which has detrimental effects on neural plasticity and survival (Jope 2003; Jope and Johnson 2004; Beaulieu 2007; Koros and Dorner-Ciossek 2007; Lovestone et al. 2007). Furthermore, multiple lines of evidence support the involvement of the β-arrestin-2–Akt–GSK-3 pathway in the regulation of dopamine-associated behaviors. GSK-3β inhibitors can reduce locomotor hyperactivity in both dopamine transporter knockout mice and amphetamine-treated wildtype animals. GSK-3β heterozygote mice were less responsive to amphetamine-induced behavioral actions (Beaulieu et al. 2007, 2008a).

Previous studies have demonstrated that neuroplasticity of NAc-related circuitry mediated the initiation and expression of psychostimulant-induced behavioral sensitization (Robinson and Becker 1986; Kalivas and Stewart 1991). We hypothesized here that GSK-3β in the NAc mediates cocaine-induced sensitization. The present study examined (i) the effects of chronic cocaine on GSK-3β activity in the NAc, (ii) whether GSK-3β activity in the NAc is altered during the initiation and expression of cocaine-induced behavioral sensitization, and (iii) the effects of GSK-3β inhibition in the NAc on the initiation and expression of cocaine-induced locomotor sensitization.

Materials and methods

Subjects

Three hundred male Sprague-Dawley rats (weighing 220–240 g upon arrival) were obtained from the Laboratory Animal Center, Peking University Health Science Center. Rats were housed in groups of five in a temperature (23 ± 2°C) and humidity (50 ± 5%) controlled room with food and water freely available in the home cage. Animals were maintained on a reverse 12 h/12 h light/dark cycle (lights off at 8:00 am). All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the procedures were approved by the local Animal Care and Use Committee.

Drugs

The drugs used were cocaine hydrochloride (Qinghai Pharmaceuticals, Xining, China), lithium chloride (LiCl, Beijing Chemical Plant, Beijing, China), and SB216763 (Sigma, St. Louis, MO, USA). Cocaine was dissolved in 0.9% saline. LiCl was dissolved in distilled water to reach a working concentration of 9 mg/mL. SB216763 was dissolved in a concentration of 100% dimethylsulfoxide (DMSO). Based on the manufacturer’s instructions, a 100% concentration of DMSO was used to dissolve SB216763 because it might be scarified if any other vehicle is added (e.g., normal saline or phosphate-buffered saline). Neuronal toxicity may be considered to be a potential problem with this concentration of DMSO, but during our experiments, we did not observe any adverse effects in rats, consistent with our previous experiments (Zhao et al. unpublished results), possibly because these rats received a very low volume (only 0.5 μL/side). All drugs were freshly prepared before use. Doses of drugs were based on previous reports (Beaulieu et al. 2004; Bilbao et al. 2008).

Locomotor activity

Locomotor activity was assessed using the Animal Locomotor Video Analysis System (JLsofttech Company, Shanghai, China) (see Appendix S1).

Surgery and intracranial injections

Rats (weighing 280–310 g when surgery began) were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and permanent guide cannulae (23 gauge; Plastics One, Roanoke, VA, USA) were implanted bilaterally 1 mm above the NAc core or NAc shell (see Appendix S1).

Tissue sample preparation and western blot assays

Tissue sample preparation and western blot assays were based on those previously used in our laboratory (Lu et al. 2005; Li et al. 2008) (see Appendix S1).

Experimental design

See Appendix S1.

Data analysis

Data are expressed as mean ± SEM. Data were analyzed with analysis of variance (anova) using appropriate between- and within-subjects factors for different experiments (see Results). All post hoc comparisons were made using Tukey’s test. Values of < 0.05 were considered statistically significant. For clarity, post hoc analyses are indicated by asterisks in the figures but are not described in Results.

Results

Effect of chronic cocaine administration on GSK-3β expression in the NAc

Figure 1b shows the level of phosphorylated GSK-3β in the NAc. One-way anova revealed that 14 days of chronic cocaine administration decreased GSK-3β phosphorylation at Ser9 in the NAc core (F1,14 = 12.95, < 0.01), but not NAc shell (> 0.05). Chronic cocaine exposure had no effect on total GSK-3β protein levels in the NAc core or shell (Fig. 1c).

Figure 1.

 Effects of chronic cocaine administration on GSK-3β expression in the nucleus accumbens. (a) Behavioral procedure for Experiment 1 (= 7–9 per group). (b and c) phosphorylated and total GSK-3β in the nucleus accumbens (NAc) core and NAc shell in rats after repeated cocaine (10 mg/kg, i.p.) exposure for 14 days in their home cages. In the NAc core, chronic cocaine exposure decreased GSK-3β phosphorylation at Ser9, suggesting that GSK-3β activity was increased. Data are expressed as percent (mean ± SEM) of saline control rats. *< 0.01, different from saline group. SAL, saline; COC, cocaine.

LiCl inhibits the initiation of cocaine-induced locomotor sensitization and GSK-3β activity in the NAc core

As shown in Fig. 2a and b, rats were trained for the development of cocaine (10 mg/kg, i.p.)-induced locomotor sensitization. Locomotor activity of rats in the cocaine group progressively increased during the 14 days of initiation training (Fig. 2b). These effects were inhibited by systemic administration of LiCl (100 mg/kg, i.p.) 30 min before each daily injection of cocaine (Fig. 2b). Furthermore, the progressive increases in locomotor activity induced by cocaine were associated with decreased phosphorylated GSK-3β (pGSK-3β) in the NAc core but not NAc shell (Fig. 2c). LiCl administration blocked the decrease in pGSK-3β in the NAc core, but not NAc shell (Fig. 2c). Additionally, total GSK-3β protein levels in the NAc of all rats did not significantly change (Fig. 2d; statistical analyses not presented).

Figure 2.

 Lithium chloride (LiCl) inhibited the initiation of cocaine-induced sensitization and GSK-3β activity in the NAc core. (a) Behavioral procedure for Experiment 2 (n = 6–8 per group). (b) Lithium chloride (100 mg/kg, i.p.) inhibited the initiation of cocaine (10 mg/kg, i.p.)-induced locomotor sensitization. Locomotor activity in rats was monitored for 2 h after daily injection of saline or cocaine for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm). *< 0.05, different from locomotor activity on day 1 (within group). **< 0.05, different from Saline + Cocaine group on corresponding test day. ***< 0.05, different from Saline + Saline group. (c and d) Systemic injection of LiCl blocked the decrease in pGSK-3β in the NAc core, but not NAc shell, while total GSK-3β protein levels in the NAc remained stable. Data are expressed as percent (mean ± SEM) of phosphorylated and total GSK-3β in the Saline + Saline control group. *< 0.05, different from control group. **< 0.05, different from the control group. ***< 0.05, different from Saline + Cocaine group. Li100, LiCl 100 mg/kg, i.p.

We analyzed locomotor activity using repeated-measures anova, with cocaine (0 and 10 mg/kg) and LiCl (0 and 100 mg/kg) as the between-subjects factors and test day (day 1, 7, and 14) as the within-subjects factor. The analysis revealed significant effects of cocaine (F1,28 = 174.32, < 0.01), LiCl (F1,28 = 27.87, < 0.01), and test day (F2,56 = 4.98, < 0.01), a cocaine × test day interaction (F2,56 = 6.08, < 0.01), and a cocaine × LiCl × test day interaction (F2,56 = 5.82, < 0.01).

Mixed anova analyses of pGSK-3β levels were performed separately for the NAc core and NAc shell, with the between-subjects factors cocaine (0 and 10 mg/kg) and LiCl (0 and 100 mg/kg). For the NAc, the statistical analysis of pGSK-3β levels revealed significant effects of cocaine (F1,25 = 7.48, < 0.05) and LiCl (F1,25 = 9.49, < 0.05) and a cocaine × LiCl interaction (F1,25 = 6.28, < 0.05). For the NAc shell, the statistical analysis of pGSK-3β levels revealed no significant effects of cocaine (> 0.05) or LiCl (> 0.05) or a cocaine × LiCl interaction (> 0.05). Furthermore, LiCl increased pGSK-3β levels in the NAc core (Fig. 2c), but not NAc shell, in the saline group, although locomotor activity in the saline group was not affected, suggesting that brain function may not be affected when GSK-3β is inhibited under normal conditions (Kimura et al. 2008).

SB216763 inhibition of GSK-3β activity in the NAc core, but not NAc shell, attenuated the initiation of cocaine-induced sensitization

Inhibition of GSK-3β activity in the NAc core, but not NAc shell, attenuated the increase in cocaine-induced locomotor activity during the initiation phase (Figs 3b and 4a). The anova analysis of locomotor activity included the between-subjects factors SB216763 (0, 0.1, and 1 ng/side for the NAc core and 0 and 1 ng/side for the NAc shell) and cocaine (0 and 10 mg/kg) and the within-subjects factor test day (day 1, 7, and 14). This analysis of data from rats that received SB216763 in the NAc core revealed significant effects of cocaine (F1,38 = 195.16, < 0.05), SB216763 (F2,38 = 3.68, < 0.05), and test day (F2,76 = 3.42, < 0.05), a cocaine × test day interaction (F2,76 = 4.21, < 0.05), and a SB216763 × cocaine × test day interaction (F4,76 = 4.71, < 0.05) (Fig. 3b). The analysis of rats that received SB216763 in the NAc shell revealed significant effects of cocaine (F1,19 = 112.29, < 0.05) and test day (F2,38 = 7.32, < 0.05) and a cocaine × test day interaction (F2,38 = 6.16, < 0.01), but no effect of SB216763 (> 0.05) and no SB216763 × cocaine × test day interaction (> 0.05) (Fig. 4a). The results suggest that cocaine administration progressively increased locomotor activity, which may be inhibited by GSK-3β inhibition in the NAc core, but not NAc shell. Locomotor activity in the saline group was not affected by GSK-3β inhibition in the NAc core or NAc shell.

Figure 3.

 Inhibition of GSK-3β activity in the NAc core attenuated the initiation of cocaine-induced sensitization. (a) Behavioral procedure for Experiment 3 (n = 7–8 per group). (b) NAc core microinjection of SB216763 attenuated the initiation of cocaine-induced locomotor sensitization. Locomotor activity in rats was monitored for 2 h after daily injection of saline or cocaine (10 mg/kg, i.p.) for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm). *< 0.05, different from locomotor activity on day 1 (within group). **< 0.05, different from Vehicle + Cocaine group on corresponding test day. ***< 0.05, different from Vehicle + Saline group. (c and d) NAc core injections of SB216763 decreased GSK-3β activity induced by cocaine, while total GSK-3β protein levels in the NAc core remained unchanged. Data are expressed as percent (mean ± SEM) of phosphorylated and total GSK-3β in the Vehicle + Saline control group. *< 0.05, different from control group. **< 0.05, different from control group. ***< 0.05, different from Vehicle + Cocaine group. VEH, vehicle; SB0.1, SB216763 0.1 ng/0.5 μl per side; SB1, SB216763 1 ng/0.5 μl per side.

Figure 4.

 NAc shell microinjection of GSK-3β inhibitor SB216763 did not attenuate the initiation of cocaine-induced sensitization. (a) Microinjections of SB216763 into the NAc shell did not attenuate the initiation of cocaine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or cocaine (10 mg/kg, i.p.) for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm). *< 0.05, different from locomotor activity on day 1 (within group). **< 0.05, different from locomotor activity on day 1 (within group). ***< 0.05, different from Vehicle + Saline group. (b and c) NAc shell injections of SB216763 did not affect the activity or total protein levels of GSK-3β. Data are expressed as percent (mean ± SEM) of phosphorylated and total GSK-3β in vehicle/saline control rats (= 6 per group).

The mixed anova analysis of pGSK-3β levels in the NAc core, with the between-subjects factors SB216763 (0, 0.1, and 1 ng/side) and cocaine (0 and 10 mg/kg), revealed significant effects of cocaine (F1,34 = 5.16, < 0.05) and SB216763 (F2,34 = 10.69, < 0.05) and a cocaine × SB216763 interaction (F2,34 = 8.38, < 0.05) (Fig. 3c). In contrast, neither cocaine administration (> 0.05) nor NAc shell infusion of SB216763 (> 0.05) had a significant effect on pGSK-3β levels in the NAc shell (Fig. 4b).

The inhibitory effects of SB216763 on cocaine-induced increases in GSK-3β activity in the NAc core were observed 24 h after SB216763 administration in the present study, consistent with a previous report (Cross et al. 2001), suggesting that inhibition of GSK-3β activity by SB216763 could be detected long after inhibitor administration only when GSK-3β was abnormally activated (Cross et al. 2001). Additionally, total GSK-3β protein levels in the NAc of all rats were not significantly affected (Figs 3d and 4c; statistical analyses not presented).

LiCl inhibited the expression of cocaine-induced locomotor sensitization and GSK-3β activity in the NAc core

As shown in Fig. 5b, locomotor activity in the cocaine (10 mg/kg, i.p.) group progressively increased during the 14 daily injections. Furthermore, locomotor activity in the cocaine group was higher than the saline group following systemic cocaine (10 mg/kg, i.p.) challenge on day 20 after withdrawal. The expression of cocaine-induced locomotor sensitization was inhibited by LiCl (100 mg/kg, i.p., injected 30 min before the cocaine challenge injection). Additionally, locomotor activity in the saline group was also inhibited by LiCl (100 mg/kg, i.p.) after a cocaine challenge injection (Fig. 5c). Furthermore, the expression of cocaine-induced locomotor sensitization after cocaine challenge was associated with decreased pGSK-3β levels in the NAc core, but not NAc shell. Inhibition of this expression by LiCl was associated with increased pGSK-3β levels in the NAc core, but not NAc shell (Fig. 5d and e). The experimental manipulations had no effect on total GSK-3β protein levels in the NAc core or NAc shell (Fig. 5f and g; statistical analyses not presented).

Figure 5.

 Lithium chloride inhibited the expression of cocaine-induced locomotor sensitization and GSK-3β in the NAc core. (a) Behavioral procedure for Experiment 4 (n = 6–8 per group). (b) Initiation of cocaine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or cocaine (10 mg/kg, i.p.) for 14 days. Locomotor activity on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm). *< 0.05, different from day 1 (within group). (c) LiCl (100 mg/kg, i.p.) inhibited the expression of cocaine-induced locomotor sensitization. After cocaine challenge, locomotor activity was recorded every 15 min for a total of 2 h. Distances during the first 15 min served as baseline. Data are expressed as mean ± SEM of locomotor distances. #< 0.05, different from LiCl (100 mg/kg) + Cocaine group. ##< 0.05, different from Saline + Saline group. (d–g) Systemic injection of LiCl (100 mg/kg, i.p.) 30 min before cocaine challenge injection increased pGSK-3β activity which was decreased by cocaine in the NAc core, but not NAc shell, while total GSK-3β protein levels in the NAc remained stable. Data are expressed as percent (mean ± SEM) of phosphorylated and total GSK-3β in the Saline + Saline control group. *< 0.05, different from control group. **< 0.05, different from control group. ***< 0.05, different from Saline + Cocaine group. Li30, LiCl 30 mg/kg, i.p.

We analyzed locomotor activity during the 14-day development of sensitization using repeated-measures anova, with cocaine (0 and 10 mg/kg) as the between-subjects factor and test day (day 1, 7, and 14) as the within-subjects factor. The analysis revealed significant effects of cocaine (F1,36 = 372.56, < 0.01) and test day (F2, 72 = 40.21, < 0.01) and a cocaine × test day interaction (F2,72 = 44.01, < 0.01), suggesting that 14 daily injections of cocaine progressively increased locomotor activity.

Locomotor activity during the expression phase was analyzed by mixed anova, with the between-subjects factors cocaine (0 and 10 mg/kg, i.p.) and LiCl (0, 30, and 100 mg/kg) and the within-subjects factor test interval (every 15 min). This analysis revealed significant effects of cocaine (F1,36 = 7.63, < 0.01), LiCl (F2,36 = 8.58, < 0.01), and test interval (F7,252 = 105.32, < 0.01) and a cocaine × LiCl × test interval interaction (F14,252 = 1.89, < 0.05) (Fig. 5b). This interaction was attributable to the fact that LiCl inhibited the increased locomotor activity induced by a cocaine challenge injection on the expression day.

Analyses were further performed separately for pGSK-3β levels in the NAc core and NAc shell using mixed anova, with the between-subjects factors cocaine (0 and 10 mg/kg) and LiCl (0, 30, and 100 mg/kg). The analysis of pGSK-3β levels in the NAc core revealed significant effects of cocaine (F1,39 = 25.26, < 0.01) and LiCl (F2,39 = 7.15, < 0.01) and a cocaine × LiCl interaction (F2,39 = 6.89, < 0.01) (Fig. 5d). The statistical analysis of pGSK-3β levels in the NAc shell revealed no significant effects of cocaine (> 0.05) or LiCl (> 0.05) and no cocaine × LiCl interaction (> 0.05) (Fig. 5e).

SB216763 inhibition of GSK-3β in the NAc core, but not NAc shell, attenuated the expression of cocaine-induced locomotor sensitization

Locomotor activity in the cocaine (10 mg/kg, i.p.) group progressively increased during the 14 daily injections (Figs 6b and 7a). Repeated-measures anova, with the between-subjects factor cocaine (0 and 10 mg/kg, i.p.) and the within-subjects factor test day (day 1, 7, and 14), was used to analyze locomotor activity during the initiation phase. The analysis of locomotor activity in rats with NAc core infusion of SB216763 revealed significant effects of cocaine (F1,34 = 60.20, < 0.01) and test day (F2,68 = 13.37, < 0.05) and a cocaine × test day interaction (F2,68 = 7.84, < 0.01) (Fig. 6b). As shown in Fig. 7a, the analysis of locomotor activity in rats with NAc shell infusion of SB216763 also revealed significant effects of cocaine (F1,24 = 314.14, < 0.01) and test day (F2,48 = 7.89, < 0.01) and a cocaine × test day interaction (F2,48 = 13.09, < 0.01).

Figure 6.

 Inhibition of GSK-3β in the NAc core attenuated the expression of cocaine-induced locomotor sensitization. (a) Behavioral procedure for Experiment 5 (= 7–8 per group). (b) Initiation of cocaine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or cocaine (10 mg/kg, i.p.) for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm). *< 0.05, different form day 1 within group. (c) Infusions of SB216763 (1 ng/0.5 μL per side) into the NAc core inhibited the expression of cocaine-induced locomotor sensitization. SB216763 (0.1 and 1 ng/0.5 μL per side) or vehicle (DMSO, 0.5 μL per side) was microinjected bilaterally into the NAc core 30 min before cocaine challenge (10 mg/kg, i.p.) on day 20. After the challenge injection of cocaine, distances were recorded every 15 min for a total of 2 h. #< 0.05, different from SB216763 (1 ng/side) + Cocaine group. ##< 0.05, different from Vehicle + Saline. (d and e) Infusions of SB216763 (1 ng/0.5 μL per side) into the NAc core 30 min before cocaine challenge decreased GSK-3β activity in the NAc core. Data are expressed as percent (mean ± SEM) of phosphorylated and total GSK-3β in the Vehicle + Saline control group. *< 0.05, different from control group. **< 0.05, different from control group. ***< 0.05, different from Vehicle + Cocaine group.

Figure 7.

 NAc shell microinjection of GSK-3β inhibitor SB216763 did not attenuate the expression of cocaine-induced locomotor sensitization. (a) Initiation of cocaine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or cocaine (10 mg/kg, i.p.) for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor distance (mm) (= 7 per group). *< 0.05, different form day 1 within group. (b) Infusions of SB216763 (1 ng/0.5 μL per side) into the NAc shell did not inhibit the expression of cocaine-induced locomotor sensitization. SB216763 (1 ng/0.5 μL per side) or vehicle (DMSO, 0.5 μL per side) was microinjected bilaterally into the NAc shell 30 min before cocaine challenge (10 mg/kg, i.p.) on day 20. After the challenge injection of cocaine, distances were recorded every 15 min for a total of 2 h. #< 0.05, different from Vehicle + Saline group.

As shown in Figs 6c and 7b, similar to Experiment 4, the expression of locomotor sensitization in the cocaine (10 mg/kg, i.p.) group on day 20 was attenuated by NAc core, but not NAc shell, microinjection of SB216763 30 min before the cocaine challenge injection (10 mg/kg, i.p.). Repeated-measures anova, with the between-subjects factors cocaine (0 and 10 mg/kg, i.p.) and SB216763 (0, 0.1, and 1 ng/side for the NAc core, and 0 and 1 ng/side for the NAc shell) and the within-subjects factor test interval (every 15 min), was used to analyze locomotor activity in the NAc core and NAc shell microinjection groups on day 20. The analysis of NAc core infusion of SB216763 revealed significant effects of cocaine (F1,30 = 11.12, < 0.01), SB216763 (F2,30 = 10.87, < 0.05) and test interval (F7,210 = 102.82, < 0.05) and a cocaine × SB216763 × test interval interaction (F14,210 = 3.27, < 0.05) (Fig. 6c). As shown in Fig. 7b, the analysis of locomotor activity in rats with NAc shell infusion of SB216763 also revealed significant effects of cocaine (F1,23 = 8.66, < 0.01) and test interval (F7,161 = 107.41, < 0.01), but no effect of SB216763 (> 0.05) and no cocaine × test interval × SB216763 interaction (> 0.05). This effect was attributable to the fact that NAc shell infusion of SB216763 did not inhibit the increase in locomotor activity induced by a cocaine challenge injection on the expression day.

The analysis of western blot data indicated that the reduction of locomotor activity by SB216763 microinjection into the NAc core was associated with increased pGSK-3β in the NAc core (Fig. 6d). SB216763 microinjection into the NAc shell, however, did not increase pGSK-3β in the NAc shell. The anova of pGSK-3β levels in the NAc core, with cocaine and SB216763 as between-subjects factors, revealed significant effects of cocaine (F1,36 = 46.37, < 0.05) and SB216763 (F2,36 = 10.60, < 0.05) and a cocaine × SB216763 interaction (F2,36 = 12.76, < 0.05). In contrast, the anova of pGSK-3β levels in the NAc shell revealed no significant effects of cocaine or SB216763 (> 0.05). As shown above, the experimental manipulations also had no effects on total GSK-3β levels in the NAc core or NAc shell (statistical analyses not presented).

Discussion

Our findings provide the first demonstration that GSK-3β activity contributes to behavioral changes after repeated cocaine administration. The main findings of the present study were (i) GSK-3β activity in the NAc core, but not NAc shell, increased following chronic cocaine exposure, (ii) cocaine-induced locomotor sensitization was accompanied by enhanced GSK-3β activity in the NAc core, but not NAc shell, (iii) systemically administered LiCl prevented the initiation and expression of cocaine-induced behavioral sensitization and increased pGSK-3β levels in the NAc core, but not NAc shell, and (iv) inhibition of GSK-3β activity by SB216763 in the NAc core, but not NAc shell, attenuated the initiation of cocaine-induced behavioral sensitization and decreased locomotor activity during the expression phase. Altogether, our findings suggest a novel role for NAc core GSK-3β activity in the initiation and expression of cocaine-induced sensitization.

Repeated drug exposure could make the brain reward system highly sensitive to drugs and drug-related stimuli, a phenomenon referred to as sensitization. During the behavioral sensitization process, neuroadaptive and sensitized molecular changes occur in the central nervous system, especially in NAc-related reward circuitry (Robinson and Berridge 2003; Vezina 2004). GSK-3β, a multifunctional serine/threonine (Ser/Thr) kinase (Embi et al. 1980), has been shown to be involved in synaptic plasticity. For example, GSK-3β activation is required for long-term depression and was inhibited during LTP (Peineau et al. 2007). Activation of GSK-3β in mouse brain or rat hippocampus inhibited LTP (Hooper et al. 2007; Zhu et al. 2007) and caused spatial memory deficits in rats (Hernandez et al. 2002; Peineau et al. 2007; Kimura et al. 2008). These neuroadaptations induced by GSK-3β may be one of the molecular mechanisms underlying dopamine-mediated behavior changes (Kimura et al. 2008; Noh et al. 2008). Indeed, multiple lines of evidence support the involvement of GSK-3β in the regulation of dopamine-related behaviors (Beaulieu et al. 2004, 2005; Gould and Manji 2005; Sotnikova et al. 2005; Beaulieu et al. 2006; Prickaerts et al. 2006). Persistent activation of GSK-3β substrates occur in the striatum in dopamine transporter knockout mice and wild-type mice with administration of amphetamine or the non-selective dopamine receptor agonist apomorphine (Beaulieu et al. 2004, 2005, 2006). GSK-3β knockout animals exhibit dopamine-dependent locomotor behavior (Beaulieu et al. 2004), whereas GSK3β-over-expressing transgenic mice display increased general locomotor activity and reduced immobility in the forced swim test (i.e., antidepressant-like effect) (Prickaerts et al. 2006). Additionally, GSK-3β inhibitors have been shown to inhibit locomotor hyperactivity induced by acute administration of amphetamine or cocaine (De Sarno et al. 2002; Beaulieu et al. 2004; Gould and Manji 2005). Consistent with these findings, we found that GSK-3β activity in the NAc core was elevated by chronic cocaine exposure. More importantly, our results suggest that inhibiting GSK-3β activity with LiCl could suppress the initiation and expression of cocaine-induced behavioral sensitization. However, Experiment 2 of the present study showed that LiCl increased GSK-3β phosphorylation in the NAc core, but not NAc shell, in the saline group, while locomotor activity in this group was unaffected. The explanation for these contradictory results may be that brain function might not be affected when GSK-3β is inhibited under normal conditions (Kimura et al. 2008). Much evidence supports this hypothesis; knockout of GSK-3β in the striatum had no effect on locomotor activity in mice during 1 h monitoring (Beaulieu et al. 2004). The pharmacological or genetic inhibition of GSK-3β significantly reduced dopamine-dependent locomotor activity only after administration of psychostimulants or knockout of the dopamine transporter (Beaulieu et al. 2004, 2005, 2007, 2008a). Therefore, inhibition of GSK-3β by LiCl in drug-free conditions had no effect on locomotor activity. However, LiCl administration was reported to decrease locomotor activity in mice during 30 min of monitoring (Beaulieu et al. 2008b). The discrepancy with our present results might be attributable to the different durations of monitoring and different animal sensitization models. The present study monitored locomotor sensitization for 2 h in rats, while Beaulieu et al. (2008b) monitored spontaneous activity for 30 min after LiCl administration.

One of the molecular mechanisms underlying the involvement of GSK-3β in the initiation and expression of behavioral sensitization may be the dopamine D2 receptor–Akt–GSK-3 pathway. A previous study reported that morphine-induced sensitization increased D2 receptor mRNA expression (Heidari et al. 2006). A D2-like receptor antagonist also reduced apomorphine- or nornicotine-induced behavioral sensitization (Chausmer and Katz 2001; Green et al. 2002), whereas microinfusion of the D2 receptor agonist quinpirole into the NAc increased locomotor activity (Gong et al. 1999). Persistently elevated extracellular dopamine levels were associated with a reduction of Akt phosphorylation and activity in the striatum of dopamine transporter knockout mice (Beaulieu et al. 2004, 2006). The inactivation of Akt in these mice resulted in concomitant activation of GSK-3α and GSK-3β substrates (Beaulieu et al. 2004) which could be reversed by Akt. Studies with dopamine depletion (Beaulieu et al. 2004; Sotnikova et al. 2005) or dopamine receptor antagonists in dopamine transporter knockout mice demonstrate that Akt, GSK-3α, and GSK-3β are regulated by D2-class receptors (Gould and Manji 2005). Administration of amphetamine or the nonselective dopamine receptor agonist apomorphine to non-transgenic mice also results in inhibition of Akt activity, thus confirming the regulation of the Akt–GSK-3 pathway by dopamine (Beaulieu et al. 2004, 2005).

The NAc core and shell are heterogeneous structures with distinct immunohistochemical characteristics and afferent and efferent connections (Jongen-Relo et al. 1994; Nordquist et al. 2008). Numerous studies have examined the differential roles of the NAc core and shell in motivated behavior and the actions of drugs of abuse (Di Chiara et al. 2004). Cadoni et al. (2000) found that rats with chronic administration of 1 mg/kg amphetamine or 5 mg/kg cocaine exhibited sensitization of dopamine transmission in the NAc core, but not NAc shell. Increased dopamine is also seen in the NAc core, but not NAc shell, in yoked controls in cocaine self-administration studies (Lecca et al. 2007). Additionally, the c-fos response to amphetamine in the NAc core was augmented in amphetamine-pretreated animals, while no effect of sensitization was seen in the NAc shell (Nordquist et al. 2008). Consistent with these studies, our results showed that GSK-3β activity in cocaine-sensitized rats increased in the NAc core, but not NAc shell. Moreover, the initiation and expression of cocaine-induced sensitization was attenuated by inhibition of GSK-3β activity only in the NAc core. Our findings are consistent with previous studies in which microinjection of a D2 receptor antagonist into the NAc core blocked nicotine- and methamphetamine-induced sensitization, and microinjection into the NAc shell had no effect (Boye et al. 2001). However, a number of studies also suggest that the NAc shell plays an important role in the expression of locomotor sensitization and dopamine sensitization (Pierce and Kalivas 1995; Pierce et al. 1997, 1998). Therefore, GSK-3β in the NAc core, but not NAc shell, may have a crucial role in the neuroplasticity induced by conditioned cocaine administration (i.e., the development and expression of locomotor sensitization). This possibility also supports the hypothesis that the NAc core is a component of the neural circuitry involved in the storage of reward-related information derived from conditioned reinforcers, and NAc shell dopamine is essential for the invigorating effect of stimulant drugs (Ito et al. 2004).

In summary, we demonstrated that GSK-3β activity in the NAc core contributed to the development and expression of cocaine-induced locomotor sensitization, highlighting the need for additional studies to further explore the molecular influence of GSK-3β in the NAc. Behavioral sensitization has been suggested to model the neurobiological adaptations that result in compulsive drug craving, and our findings provide support for the potential therapeutic value of GSK-3β inhibitors in the treatment of cocaine addiction.

Acknowledgments

This work was supported in part by the National Basic Research Program of China (No. 2007CB512302 and 2009CB522004) and the Natural Science Foundation of China (No. 30670713 and 30725016). The authors would like to thank Dr Yavin Shaham for his comments on the manuscript. The authors declare that they do not have any conflicts of interest (financial or otherwise) related to the data presented in this manuscript.

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