SEARCH

SEARCH BY CITATION

Keywords:

  • glycogen synthase kinase 3β;
  • locomotor activity;
  • methamphetamine;
  • nucleus accumbens;
  • sensitization

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07281.x

Abstract

As a ubiquitous serine/threonine protein kinase, glycogen synthase kinase 3β (GSK-3β) has been considered to be important in the synaptic plasticity that underlies dopamine-related behaviors and diseases. We recently found that GSK-3β activity in the nucleus accumbens (NAc) core is critically involved in cocaine-induced behavioral sensitization. The present study further explored the association between the changes in GSK-3β activity in the NAc and the chronic administration of methamphetamine. We also examined whether blocking GSK-3β activity in the NAc could alter the initiation and expression of methamphetamine (1 mg/kg, i.p.)-induced locomotor sensitization in rats using systemic administration of lithium chloride (LiCl, 100 mg/kg, i.p) and brain region-specific administration of the GSK-3β inhibitor SB216763 (1 ng/side). We found that GSK-3β activity increased in the NAc core, but not NAc shell, after chronic methamphetamine administration. The initiation and expression of methamphetamine-induced locomotor sensitization was attenuated by systemic administration of LiCl and direct infusion of SB216763 into the NAc core, but not NAc shell. These results indicate that GSK-3β activity in the NAc core mediates the initiation and expression of methamphetamine-induced locomotor sensitization, suggesting that GSK-3β may be a potential target for the treatment of psychostimulant addiction.

Abbreviations used
GSK-3β

glycogen synthase kinase 3β

LTP

long-term potentiation

NAc

nucleus accumbens

Glycogen synthase kinase 3β (GSK-3β) was discovered as the enzyme involved in glycogen biosynthesis that phosphorylates and inactivates glycogen synthase, but this evolutionarily conserved enzyme is also a multifunctional kinase that plays various roles in protein synthesis, cell proliferation, cell differentiation, microtubule dynamics, cell motility, and neuronal apoptosis (Beaulieu et al. 2004; Hooper et al. 2007; Koros and Dorner-Ciossek 2007). GSK-3β is highly enriched in the brain (Leroy and Brion 1999) and is involved in various neuronal functions, including synaptic plasticity (Hooper et al. 2007; Peineau et al. 2007, 2008) and reconsolidation of fear and spatial memory (Kimura et al. 2008). Experiments using GSK-3β inhibitors and genetic knockout mice have found that GSK-3β is involved in dopamine-mediated behavior and neuropsychiatric diseases, such as Parkinson’s disease, schizophrenia, obsessive compulsive disorder, and bipolar disorder (Anderton 1999; Beasley et al. 2001; Kozlovsky et al. 2002, 2004; Bauer et al. 2003; Beaulieu et al. 2004; Emamian et al. 2004; Takashima 2006; Hooper et al. 2007; O’Brien and Klein 2007). GSK-3β is highly regulated by phosphorylation at tyr216 or ser9 (Hughes et al. 1993; Wang et al. 1994). When the level of phosphorylation at Ser9 decreases, GSK-3β activity increases, and its substrates undergo phosphorylation to produce marked effects. Phosphorylation of Ser9 by various negative regulators regulates GSK-3β activity and most of its substrates, leading to GSK-3β inactivation (Bhat et al. 2000, 2003). The abnormal regulation of GSK-3β activity may have detrimental effects on neural plasticity, structure, and survival (Jope and Johnson 2004).

Behavioral sensitization is an enduring and progressively enhanced behavioral response to repeated administration of addictive drugs, such as cocaine, methamphetamine, opiates, nicotine, and ethanol (Robinson 1984; Stewart and Badiani 1993; Carlezon and Nestler 2002). Behavioral sensitization has been suggested to be a useful animal model of drug abuse that can model the neurobiological adaptations that contribute to synapse plasticity (Robinson and Berridge 1993; White and Kalivas 1998), which is hypothesized to result in drug relapse (Bradberry 2007; Robinson and Berridge 2008).

Long-lasting neurobiological changes occur in mesolimbic reward circuitry following chronic psychostimulant exposure. Most of these adaptive alterations are induced by increased extracellular dopamine levels in the nucleus accumbens. The initiation and expression of psychostimulant-induced behavioral sensitization is mediated by nucleus accumbens (NAc)-related circuitry (Robinson and Becker 1986; Kalivas and Stewart 1991). Experiments using behavioral sensitization have determined that dopamine transmission from the ventral tegmental area to the NAc and other forebrain nuclei plays a crucial role in 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, especially those downstream of dopamine D2 receptors, that underlie dopamine-mediated neuroadaptations during sensitization are still not clearly understood, despite numerous studies that have explored the molecular mechanisms downstream of dopamine receptors (Edwards et al. 2007; Masri et al. 2008). Recently, the role of the novel protein kinase B (Akt)-GSK-3 signaling pathway, located downstream of D2 receptors, in dopamine-associated behaviors and diseases has received much attention (Beaulieu et al. 2004; Gould and Manji 2005; Koros and Dorner-Ciossek 2007; Lei et al. 2008). For example, GSK-3β mutant mice have decreased locomotor activity compared with wildtype mice following amphetamine injection (Beaulieu et al. 2004). Our recent study also found that cocaine-induced locomotor sensitization was accompanied by enhanced GSK-3β activity in the NAc core, and inhibition of GSK-3β activity in the NAc core by systemically administered LiCl or microinjection of the GSK-3β inhibitor SB216763 in the NAc core attenuated the initiation and expression of cocaine-induced behavioral sensitization (Xu et al. 2009). Based on these findings, we hypothesized that GSK-3β in the NAc core mediates the initiation and expression of methamphetamine-induced sensitization.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects

Two hundred twenty 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 on a reverse 12 h/12 h light/dark cycle (lights off at 8 am) under temperature- (23 ± 2C°) and humidity- (50 ± 5%) controlled conditions with ad libitum access to food and water. All animal procedures were conducted according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Animal Care and Use Committee.

Drugs

Methamphetamine was obtained from the National Institute on Drug Dependence, Peking University, Beijing, China, and dissolved in 0.9% saline. SB216763 was obtained from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide at a concentration of 100% for intracranial injections according to our previous study (Xu et al. 2009). Drugs were freshly prepared before use, and the doses used in the present study were based on previous reports (Beaulieu et al. 2004; Parkitna et al. 2006).

Locomotor activity

Locomotor activity was measured by an automated video tracking system (DigBehv-LM4, Shanghai Jiliang Software Technology Co. Ltd, Shanghai, China) that contained eight identical clear Plexiglas chambers (40 × 40 × 65 cm). A monochrome video camera was mounted at the top of each chamber. All of the chambers were connected to a computer that recorded the locomotion of rats. The video files (stored on the computer) were analyzed by DigBehv analysis software. Locomotor activity is expressed as the total distance traveled during a pre-determined period of time (min).

Surgery and intracranial injections

Rats weighed 280–310 g when surgery began. Animals were first anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and permanent guide cannulae (23 gauge; Plastics One, Roanoke, VA, USA) were bilaterally implanted 1 mm above the NAc core or NAc shell. The cannulae were angled toward the midline at an angle of 16° to avoid penetration of the lateral ventricle. The brain coordinates were the following: NAc core (1.5 mm anterior to bregma, 3.8 mm lateral to midline, 6.0 mm ventral to flat skull surface, measured along the trajectory of the angled cannula), NAc shell (1.8 mm anterior to bregma, 3.2 mm lateral to midline, 6.6 ventral to flat skull surface) (Zangen et al. 2006; Xu et al. 2009). Following surgery, rats were allowed to recover for 5–7 days. Intracranial injections were performed with Hamilton microsyringes (Hamilton, Reno, NV, USA) connected to 30 gauge injectors (Plastics One, Roanoke, VA). SB216763 (1 ng/0.5 μL) was bilaterally infused into the NAc core or shell (0.5 μL per side) over 1 min.

Tissue sample preparation

Tissue sample preparation was based on previous reports from our laboratory (Lu et al. 2005; Li et al. 2008; Xu et al. 2009). After the experiment, rats were decapitated without anesthesia. Brains were then quickly removed, frozen in −60°C N-hexane, and transferred to a −80°C freezer. A freezing cryostat (−20°C; Reichert-Jung 2800 Frigocut E) was used to make 1 mm thick coronal sections located approximately 2.2 mm from bregma. Bilateral tissue punches (16 gauge) of the NAc core and NAc shell were then taken. After immersion for 30 min in RIPA lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3VO4, 0.5 μg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride), tissue punches were homogenized three times for 10–15 s at 5 s intervals with an electrical disperser (Wiggenhauser, Sdn Bhd). Tissue homogenates then underwent 10 000 g centrifugation at 4°C for 5 min. All of the above procedures were performed under low temperature (0–4°C). Protein sample concentrations were determined using the bichinconinic acid assay (Beyotime Biotechnology, Beijing, China), and samples were further diluted in RIPA lysis buffer to equalize protein concentrations.

Western blot assays

Samples were treated according to previous studies from our laboratory (Lu et al. 2005; Li et al. 2008) with modification. 4× loading buffer (16% glycerol, 20% mercaptoethanol, 2% sodium dodecyl sulfate, 0.05% bromophenol blue) was added to each sample (3 : 1, sample : loading buffer) before boiling for 3 min. Samples were cooled and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% acrylamide/0.27%N,N′-methylenebisacryalamide resolving gel) for approximately 30 min at 80 V in stacking gel and approximately 90 min at 120 V in resolving gel. Increasing amounts of protein pooled from all samples were electrophoresed to produce a standard curve. Proteins were electrophoretically transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA, USA) at 0.25 A for 2.5 h. Membranes were washed with TBST (Tris-buffered saline plus 0.05% Tween-20, pH 7.4) and dipped in blocking buffer (5% bovine serum albumin in TBST) at room temperature (25°C) on an orbital shaker for 2 h. Membranes were then alternately incubated overnight with two different primary antibodies: anti-phospho-GSK-3β (1 : 1000; Cell Signaling Technology, Danvers, MA, USA) or anti-total-GSK (1 : 5000; Cell Signaling Technology) antibody in TBST plus 5% bovine serum albumin and 0.05% sodium azide at 4°C. The next day, after four 6 min washes in TBST buffer, the blots were incubated for 45 min at 25°C on a shaker with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG; goat anti-mouse IgG; Santa Cruz PI-1000; Vector Labs, Beijing, China) diluted 1 : 5000 in blocking buffer. The blots were then washed four times for 6 min each in TBST and incubated with a layer of Super Signal Enhanced Chemiluminescence (ECL) substrate (Detection Reagents 1 and 2 at a 1 : 1 ratio; Pierce Biotechnology, Rockford, IL, USA) for 1 min at 25°C. After dripping off the excess mixture, the blots were wrapped with a clean piece of plastic wrap (no bubbles between blot and wrap). The blots were then exposed for 30–300 s to X-ray film (Eastman Kodak Company, Rochester, NY, USA). Band intensities of GSK-3β and phosphorylated GSK-3β (pGSK-3β) were quantified using Quantity One software (v. 4.4.0; Bio-Rad Corporation, Hercules, CA, USA) by two observers who were blind to the experimental groups. Band intensities from each test sample were compared with the band intensities from the standard curves. The amount of the protein of interest in each sample was interpolated from the standard curve.

Experimental design

The behavioral sensitization experiments that was performed according to our previous study (Xu et al. 2009), consisted of four phases: (i) adaptation (day −2 to day 0), (ii) initiation (day 1 to day 14), (iii) withdrawal (day 15 to day 19), and (iv) expression (day 20). The 3-day adaptation procedure was used so that rats could adapt to the behavior chamber and the injection procedure, which consisted of placing the rats into the chambers for 2 h to monitor locomotor activity after daily injection of 0.9% saline (1 mL/kg, i.p.). The level of locomotor activity recorded on day 0 was defined as baseline. During the initiation training phase, locomotor activity was assessed for up to 2 h each day following methamphetamine injection (1 mg/kg, i.p.) or saline injection (1 mL/kg, i.p.). After 5 days of withdrawal in the home cage, rats were again monitored for 2 h following systemic challenge injection of methamphetamine (1 mg/kg, i.p.).

Experiment 1. GSK-3β expression in the NAc after chronic methamphetamine administration

To determine the effects of methamphetamine exposure on GSK-3β expression in the NAc, rats were randomly divided into saline and chronic methamphetamine groups (= 7–8 per group) and received either saline (1 mL/kg, i.p.) or methamphetamine (1 mg/kg, i.p.) once per day for 14 days in their home cages. Twenty-four hours after the last saline or methamphetamine administration, rats were decapitated, and brains were extracted to determine GSK-3β activity in the NAc core and shell by western blotting.

Experiment 2. Effect of LiCl on the initiation of methamphetamine-induced locomotor sensitization

To examine whether GSK-3β is involved in the initiation of methamphetamine-induced locomotor sensitization, four groups (= 7–8 per group) of rats received LiCl (100 mg/kg, i.p.) or saline (1 mL/kg, i.p.) 30 min before daily methamphetamine (1 mg/kg, i.p.) or saline (1 mg/mL, i.p.) administration for 14 days. Rats were then returned to the locomotor activity monitoring chamber for 2 h. The day after the last drug administration (day 15), rats were decapitated, and brains were extracted for subsequent determination of pGSK-3β in the NAc core and shell by western blotting. The LiCl dose was based on a previous study (Beaulieu et al. 2004).

Experiment 3. Effect of GSK-3β inhibition in the NAc on the initiation of methamphetamine-induced sensitization

To determine whether GSK-3β in the NAc plays a role in the initiation of methamphetamine-induced locomotor sensitization, we examined whether inhibiting GSK-3β activity in the NAc core with the selective antagonist SB216763 blocks the initiation of methamphetamine-induced behavioral sensitization. The experimental design was similar to experiment 2 above. We used four groups of rats (= 7 per group) that received bilateral SB216763 (0 and 1 ng/side) microinfusions into the NAc core 30 min before daily administration of methamphetamine every other day. We used another four groups of rats (= 7 per group) that received bilateral SB216763 (0 and 1 ng/side) microinfusion into the NAc shell 30 min before administration of methamphetamine every other day. SB216764 was not administered every day because excessive microinjections may cause brain destruction. The dependent measures were locomotor activity and pGSK-3β levels in the NAc core and shell. The day after the last monitoring of locomotor activity (day 15), rats were decapitated, and brains were extracted for subsequent determination of pGSK-3β.

Experiment 4. Effect of LiCl on the expression of methamphetamine-induced locomotor sensitization

To further examine whether GSK-3β contributes to the expression of methamphetamine-induced locomotor sensitization, four groups of rats (= 7–8 per group) received 14 daily systemic injections of methamphetamine (1 mg/kg, i.p.) or saline (1 mL/kg, i.p.) followed by 2 h of locomotor activity monitoring. After 5 days of withdrawal from methamphetamine in their home cage, animals were pre-treated with either saline (1 mL/kg, i.p.) or LiCl (100 mg/kg, i.p.) 30 min before receiving a systemic challenge injection of methamphetamine (1 mg/kg, i.p.). Two hours after locomotor activity monitoring, rats were decapitated, and brains were extracted for subsequent determination of pGSK-3β in the NAc.

Experiment 5. Effect of GSK-3β inhibition in the NAc on the expression of methamphetamine-induced locomotor sensitization

To further determine whether GSK-3β in the NAc core plays a role in the expression of methamphetamine-induced locomotor sensitization, we inhibited GSK-3β activity in the NAc using the selective antagonist SB216763 (0 and 1 ng/side) 30 min before a systemic challenge injection of methamphetamine (1 mg/kg, i.p.) on day 20. The rats received 14 daily systemic injections of methamphetamine (1 mg/kg, i.p.) or saline (1 mL/kg, i.p.) followed by 2 h of locomotor activity monitoring. After 5 days of withdrawal from methamphetamine in their home cage, four groups of rats were injected with the selective GSK-3β antagonist SB216763 (0 and 1 ng/side) into the NAc core 30 min before a systemic challenge injection of methamphetamine (1 mg/kg, i.p.). Another set of four groups of rats received SB216763 (0 and 1 ng/side) microinfusion bilaterally into the NAc shell 30 min before a systemic challenge injection of methamphetamine (1 mg/kg, i.p.). Two hours after locomotor activity monitoring, all rats were decapitated, and their brains were extracted for subsequent determination of pGSK-3β in the NAc.

Statistical analysis

The results are expressed as mean ± SEM. Data were subjected to analysis of variance (anova), with treatment (psychostimulant injection and inhibitor administration) as the between-subjects factor and test day (day 1, day 7 and day 14) as the within-subjects factor for each of the experiments (see Results section). Significant main effects in the anova were followed by Tukey’s post hoc test. Values of < 0.05 were considered statistically significant. For clarity, significance in the post hoc analyses are indicated by asterisks in the figures but are not described in the Results section.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The alteration of GSK-3β expression in NAc after chronic methamphetamine exposure

One-way anova revealed that pGSK-3β at Ser9 in the NAc core (F1,13 = 9.46, < 0.05, Fig. 1b), but not NAc shell (F1,13 = 0.21, > 0.05) was decreased after14 days of chronic methamphetamine administration. Total GSK-3β protein levels were not affected by chronic methamphetamine exposure in the NAc core or shell (Fig. 1c).

image

Figure 1.  Effects of chronic methamphetamine administration on GSK-3β expression in the nucleus accumbens. (a) Behavioral procedure for experiment 1 (n = 7–8 per group). (b, c) Phosphorylated and total GSK-3β in the nucleus accumbens (NAc) core and NAc shell in rats after repeated methamphetamine (1 mg/kg, i.p.) exposure for 14 days in their home cages. Chronic methamphetamine exposure decreased GSK-3β phosphorylation at Ser9 in the NAc core, suggesting that GSK-3β activity was increased by methamphetamine administration. Data are expressed as a percentage (mean ± SEM) of saline control rats. *< 0.05, compared with saline group. METH, methamphetamine.

Download figure to PowerPoint

Effect of LiCl on the initiation of methamphetamine-induced locomotor sensitization and GSK-3β activity in the NAc

Rats were trained for the development of methamphetamine-induced locomotor sensitization (Fig. 2). Figure 2b showed that the development of methamphetamine-induced locomotor sensitization was inhibited by systemic LiCl administration (100 mg/kg, i.p.) 30 min before each daily methamphetamine injection (Fig. 2b).

image

Figure 2.  Lithium chloride inhibited the initiation of methamphetamine-induced sensitization and GSK-3β activity in the NAc core. (a) Behavioral procedure for experiment 2 (n = 7–8 per group). (b) Lithium chloride (LiCl) inhibited the initiation of methamphetamine-induced locomotor sensitization. Locomotor activity in rats was monitored for 2 h after daily injection of saline or methamphetamine (1 mg/kg, i.p.) for 14 days. Locomotor activity data on day 0 served as baseline. Data are expressed as mean ± SEM. *< 0.05, compared with locomotor activity on day 1 (within-group comparison); #< 0.05, compared with SAL + METH group on corresponding test day; *#< 0.05, compared with SAL + SAL group. (c, d) Systemic injection of LiCl (100 mg/kg, i.p.) blocked the decrease in pGSK-3β in the NAc core, but not NAc shell, whereas total GSK-3β protein levels in the NAc remained unchanged. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in the SAL + SAL control group; #< 0.05. Li100, LiCl 100 mg/kg, i.p; METH, methamphetamine; SAL, saline.

Download figure to PowerPoint

Locomotor activity was assessed using repeated-measures anova including between-subjects factors of methamphetamine (0 and 1 mg/kg) and LiCl (0 and 100 mg/kg), and within-subjects factor of test day (days 1, 7, and 14). The analysis revealed significant effects of methamphetamine (F1,26 = 117.52, < 0.05), LiCl (F1,26 = 27.26, < 0.05), and test day (F2,52 = 4.98, < 0.05), a significant methamphetamine × test day interaction (F2,52 = 9.87, < 0.05), and a significant methamphetamine × LiCl test day interaction (F2,52 = 10.24, < 0.05).

Methamphetamine induced locomotor sensitization accompanied decreased pGSK-3β in the NAc core, but not NAc shell (Fig. 2c). LiCl blocked the decrease in pGSK-3β in the NAc core (Fig. 2c). pGSK-3β levels were analyzed separately for the NAc core and shell using mixed anova including the between-subjects factors of methamphetamine (0 and 1 mg/kg) and LiCl (0 and 100 mg/kg). The analysis of pGSK-3β levels in the NAc core revealed significant effects of methamphetamine (F1,26 = 12.54, < 0.05) and LiCl (F1,26 = 17.78, < 0.05) and a significant methamphetamine × LiCl interaction (F1,26 = 6.43, < 0.05). The anova of pGSK-3β levels in the NAc shell revealed no significant interaction between the two factors(p > 0.05). Moreover, LiCl increased pGSK-3β levels in the NAc core (Fig. 2c), but not shell, in the saline group, although locomotor activity in the saline group was unaffected. Total GSK-3β protein levels in the NAc did not significantly change in any of the groups (Fig. 2d; statistical analyses not presented).

Effect of GSK-3β inhibition in the NAc on the initiation of methamphetamine-induced sensitization

SB216763 injections into the NAc core, but not NAc shell, attenuated the increase in methamphetamine-induced locomotor activity during the initiation of locomotor sensitization. Locomotor activity in the saline group was not affected by GSK-3β inhibition in the NAc core or shell (Figs 3b and 4a). For the anova of locomotor activity, SB216763 (0 and 1 ng/side) and methamphetamine (0 and 1 mg/kg) served as the between-subjects factors, and test day (days 1, 7, and 14) served as the within-subjects factor. For administration of SB216763 into the NAc core, the anova revealed significant effects of methamphetamine (F1,24 = 320.65, < 0.05), SB216763 (F1,24 = 7.28, < 0.05), and test day (F2,48 = 3.71, < 0.05), a significant methamphetamine × test day interaction (F2,48 = 4.68, < 0.05), and a significant methamphetamine × SB216763 × test day interaction (F2,48 = 5.37, < 0.05) (Fig. 3b). For administration of SB216763 into the NAc shell, the anova revealed significant effects of methamphetamine (F1,24 = 72.25, < 0.05) and test day (F2,48 = 6.79, < 0.05) and a significant methamphetamine × test day interaction (F2,48 = 9.97, < 0.05), but no main effect of SB216763 (> 0.05) and no methamphetamine × SB216763 × test day interaction (> 0.05) (Fig. 4a).

image

Figure 3.  Inhibition of GSK-3β activity in NAc core attenuated the initiation of methamphetamine-induced sensitization. (a) Behavioral procedure for experiment 3 (n = 7 per group). (b) NAc core microinjection of SB216763 attenuated the initiation of methamphetamine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or methamphetamine (1 mg/kg, i.p.) for 14 days. Locomotor activity on day 0 served as baseline. Data are expressed as mean ± SEM locomotor activity counts. *< 0.05, compared with locomotor activity on day 1 (within-group comparison); #< 0.05, compared with VEH + METH group; *#< 0.05, compared with VEH + SAL group. (c, d) NAc core injections of SB216763 decreased GSK-3β activity induced by methamphetamine in the NAc core, whereas total GSK-3β protein levels in the NAc remained unchanged. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in the VEH + SAL control group. *< 0.05. VEH, vehicle; METH, methamphetamine; SB, SB216763, 1 ng/0.5 μL per side.

Download figure to PowerPoint

image

Figure 4.  NAc shell microinjection of GSK-3β antagonist SB216763 did not alter the initiation of methamphetamine-induced sensitization. (a) Microinjection of SB216763 into the NAc shell did not attenuate the initiation of methamphetamine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or methamphetamine (1 mg/kg, i.p.) for 14 days. Locomotor activity on day 0 served as baseline. Data are expressed as mean ± SEM of locomotor activity. *p <0.05, compared with locomotor activity on day 1 (within-group comparison); *#p <0.05, compared with VEH + SAL group (= 7 per group). (b, c) Nucleus accumbens shell microinjections of SB216763 did not affect locomotor activity or total GSK-3β protein levels. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in vehicle/saline control rats. VEH, vehicle; METH, methamphetamine; SB1, SB216763, 1 ng/0.5 μL per side.

Download figure to PowerPoint

pGSK-3β levels determined by western blot assay were analyzed with mixed anova. SB216763 (0 and 1 ng/side) and methamphetamine (0 and 1 mg/kg) served as the between-subjects factors. For SB216763 administration into the NAc core, the anova revealed significant effects of methamphetamine (F1,24 = 11.19, < 0.05) and SB216763 (F1,24 = 28.42, < 0.01) and a significant methamphetamine × SB216763 interaction (F1,24 = 12.16, < 0.05) (Fig. 3c). The anova of pGSK-3β levels in the NAc shell revealed no significant effects of the two main factors (> 0.05) or interaction between the two factors(p > 0.05). (Fig. 4b). Total GSK-3β protein levels in the NAc were not significantly affected in any of the rats (Figs 3d and 4c; statistical analyses not presented).

Effect of LiCl on the expression of methamphetamine-induced locomotor sensitization and GSK-3β activity in the NAc

Locomotor activity in the methamphetamine (1 mg/kg, i.p.) group progressively increased during the 14 daily injections (Fig. 5b), which indicate that all rats have developed methamphetamine induced locomotor sensitization. The anova revealed significant effects of methamphetamine (F1,28 = 407.74, < 0.05) and test day (F2,56 = 60.47, < 0.05) and a significant methamphetamine × test day interaction (F2,56 = 37.03, < 0.05).

image

Figure 5.  Lithium chloride inhibited the expression of methamphetamine-induced locomotor sensitization and GSK-3β in the NAc core. (a) Behavioral procedure for experiment 4 (n = 7–8 per group). (b) Initiation of methamphetamine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injection of saline or methamphetamine (1 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, compared with day 1 (within-group comparison). (c) LiCl (100 mg/kg, i.p.) inhibited the expression of methamphetamine-induced locomotor sensitization on day 20 after a 5 day drug-free period. After methamphetamine challenge, locomotor activity was recorded every 15 min for a total of 2 h. Distances traveled during the first 15 min served as baseline. #< 0.05, compared with LiCl + METH group; #*< 0.05, compared with SAL + SAL group. (d–g) Systemic injection of LiCl (100 mg/kg, i.p.) 30 min before a methamphetamine challenge injection increased pGSK-3β activity, which was decreased by methamphetamine in the NAc core, but not NAc shell, whereas total GSK-3β protein levels in the NAc remained unchanged. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in the SAL + SAL control group. #< 0.05. Li100, LiCl 100 mg/kg, i.p; SAL, saline; METH, methamphetamine.

Download figure to PowerPoint

Figure 5c showed that following systemic methamphetamine (1 mg/kg, i.p.) challenge on day 20 after withdrawal, the locomotor activity was inhibited by LiCl (100 mg/kg, i.p.). The locomotor activity was analyzed using mixed anova. Methamphetamine (0 and 1 mg/kg, i.p.) and LiCl (0 and 100 mg/kg) served as the between-subjects factors, and test interval (every 15 min) served as the within-subjects factor. The anova revealed significant effects of methamphetamine (F1,28 = 7.23, < 0.05), LiCl (F1,28 = 21.54, < 0.05), and test interval (F7,196 = 53.56, < 0.05) and a significant methamphetamine × LiCl × test interval interaction (F7,196 = 2.07, < 0.05) (Fig. 5c).

The expression of locomotor sensitization after methamphetamine challenge accompanied decreased pGSK-3β levels in the NAc core, but not shell. and systemic administration of LiCl could restore the decreased pGSK-3β levels in the NAc core (Fig. 5d and e). Analyses were performed separately for pGSK-3β levels in the NAc core and NAc shell using mixed anova. The anova of pGSK-3β levels in the NAc core revealed significant effects of methamphetamine (F1,23 = 10.02, < 0.05) and LiCl (F1,23 = 8.20, < 0.05) and a significant methamphetamine × LiCl interaction (F1,23 = 5.27, < 0.05) (Fig. 5d). The anova of pGSK-3β levels in the NAc shell revealed no significant effects of methamphetamine (F1,23 = 0.002, > 0.05) or LiCl (F1,23 = 0.003, > 0.05) or the interaction of the two factors (F1,23 = 0.001, > 0.05) (Fig. 5e). The experimental manipulations had no effect on total GSK-3β protein levels in the NAc core or shell (Fig. 5f and g; statistical analyses not presented).

Effect of inhibition of GSK-3β in the NAc on the expression of methamphetamine-induced locomotor sensitization

Locomotor activity in the methamphetamine (1 mg/kg, i.p.) group progressively increased during the 14 daily injections (Fig. 6b and 7a). Repeated-measures anova was used to analyze locomotor activity during the initiation phase. The analysis revealed significant effects of methamphetamine (F1,26 = 382.59, < 0.05 for core group; F1,22 = 85.79, < 0.05 for shell group) and test day (F2,52 = 3.69, < 0.05 for core group; F1,22 = 85.79, < 0.05 for shell group) and a significant methamphetamine × test day interaction (F2,52 = 9.19, < 0.05 for core infusions group; F2,44 = 17.75, < 0.05 for shell group) (Fig. 6b and 7a).

image

Figure 6.  Inhibition of GSK-3β in the NAc core attenuated the expression of methamphetamine-induced locomotor sensitization. (a) Behavioral procedure for experiment 5 (n = 6–7 per group). (b) Initiation of methamphetamine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injections of saline or methamphetamine (1 mg/kg, i.p.) for 14 days. Locomotor activity on day 0 served as baseline. Data are expressed as mean ± SEM of distances traveled (mm). *< 0.05, compared with day 1 (within-group comparison). (c) Infusions of SB216763 (1 ng/0.5 μL per side) into the NAc core inhibited the expression of methamphetamine-induced locomotor sensitization. SB216763 (1 ng/0.5 μL per side) or vehicle was microinjected bilaterally into the NAc core 30 min before methamphetamine challenge (1 mg/kg, i.p.) on day 20. After the methamphetamine challenge injection, distances traveled at 15 min intervals were recorded for a total of 2 h. #< 0.05, compared with SB + METH; #*< 0.05, compared with VEH + SAL. (d, e) Infusions of SB216763 into the NAc core 30 min before the methamphetamine challenge injection decreased GSK-3β activity in the NAc core. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in the VEH + SAL control group. #< 0.05. VEH, vehicle; SB, SB216763; METH, methamphetamine.

Download figure to PowerPoint

image

Figure 7.  Inhibition of GSK-3β in the NAc shell did not attenuate the expression of methamphetamine-induced locomotor sensitization. (a) Initiation of methamphetamine-induced locomotor sensitization. Locomotor activity was monitored for 2 h after daily injections of saline or methamphetamine (1 mg/kg, i.p.) for 14 days. Locomotor activity on day 0 served as baseline. Data are expressed as mean ± SEM of distance traveled (mm). *< 0.05, compared with day 1 (within-group comparison). (b) Infusions of SB216763 into the NAc shell did not inhibit the expression of methamphetamine-induced locomotor sensitization. SB216763 (1 ng/0.5 μL per side) or vehicle was microinjected bilaterally into the NAc shell 30 min before methamphetamine challenge (1 mg/kg, i.p.) on day 20. After the methamphetamine challenge injection, distances traveled at 15 min intervals were recorded for a total of 2 h. Data are expressed as mean ± SEM of distances traveled. #< 0.05, compared with VEH + SAL control group (= 6 per group). (c, d) Infusions of SB216763 into the NAc shell 30 min before methamphetamine challenge injection had no effect on phosphorylated or total GSK-3β protein levels in the NAc shell. Data are expressed as a percentage (mean ± SEM) of phosphorylated and total GSK-3β in the VEH + SAL control group (= 6 per group). VEH, vehicle; SB, SB216763; METH, methamphetamine.

Download figure to PowerPoint

Figure 6c showed that following systemic methamphetamine (1 mg/kg, i.p.) challenge on day 20 after withdrawal, the locomotor activity in the group with daily methamphetamine injection was higher than the group with daily saline injection, but this effect was inhibited by NAc core microinjection of SB216763 30 min before the methamphetamine challenge injection. Repeated-measures anova was used to analyze locomotor activity in the NAc core and NAc shell microinjection groups on day 20. Methamphetamine (0 and 1 mg/kg, i.p.) and SB216763 (0 and 1 ng/side) served as the between-subjects factors, and test interval (every 15 min) served as the within-subjects factor. The anova of NAc core microinjection of SB216763 revealed significant effects of methamphetamine (F1,23 = 4.22, < 0.05), SB216763 (F1,23 = 34.65, < 0.05), and test interval (F7,161 = 15.63, < 0.05) and a significant methamphetamine × SB216763 × test interval interaction (F7,161 = 2.64, < 0.05) (Fig. 6c). NAc shell microinjection of SB216763 30 min before the methamphetamine challenge injection had no effect on the expression of locomotor sensitization (Fig. 7b). The anova of locomotor activity in rats that received NAc shell microinfusion of SB216763 revealed significant effects of methamphetamine (F1,20 = 20.28, < 0.05) and test interval (F7,140 = 42.80, < 0.05) but no significant effect of SB216763 (F1,20 = 0.34, > 0.05) and no methamphetamine × SB216763 × test interval interaction (F7,140 = 2.96, > 0.05) (Fig. 7b).

The expression of locomotor sensitization after methamphetamine challenge accompanied decreased pGSK-3β levels in the NAc core, and intra-NAc core injections of SB216763 could restore the decreased pGSK-3β levels in the NAc core (Figs 6d, e and 7c). The anova analysis of pGSK-3β levels in the NAc core revealed significant effects of methamphetamine (F1,23 = 8.63, < 0.05) and SB216763 (F1,23 = 11.50, < 0.05) and a significant methamphetamine × SB216763 interaction (F1,23 = 14.98, < 0.05). Intra-NAc shell injections of SB216763 had no effect on the pGSK-3β levels in the NAc shell. The anova of pGSK-3β levels in the NAc shell of rats that received SB216763 microinjection into the NAc shell revealed no significant effects of methamphetamine (F1,23 = 0.001, > 0.05) or SB216763 (F1,23 = 0.001, > 0.05) (Fig. 7c). As shown above, the experimental manipulations also had no effects on total GSK-3β levels in either the NAc core or shell (Figs 6e and 7d; statistical analyses not presented).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The main findings of the present study were: (i) chronic methamphetamine exposure increased GSK-3β activity in the NAc core but not shell, (ii) locomotor activity progressively increased during 14 days of methamphetamine administration and was accompanied by increased GSK-3β activity in the NAc core, but not NAc shell, and (iii) inhibition of GSK-3β activity within systemic administration of LiCl or intra-NAc core injection of SB216763 attenuated the initiation of methamphetamine-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 methamphetamine-induced sensitization.

One of the molecular mechanisms underlying dopamine-mediated behavior changes is the involvement of GSK-3β in synaptic plasticity. Activation of GSK-3β is required for long-term depression, and GSK-3β was inhibited during long-term potentiation (LTP) (Peineau et al. 2007), consistent with recent research showing that conditional expression of GSK-3 in mouse brain or rat hippocampus inhibited LTP (Hooper et al. 2007; Zhu et al. 2007). Inhibition of LTP by activation of GSK-3β further impaired learning and memory, including memory recall or reconsolidation (Hernandez et al. 2002; Kimura et al. 2008).

Much evidence at different levels of analysis suggests that drug addiction represents a pathological usurpation of the neural mechanisms of learning and memory that under normal circumstances serve to shape survival behaviors related to the pursuit of rewards and the cues that predict them (White et al. 1997; Hyman 2005; Hyman et al. 2006). Long-term drug use is hypothesized to result in molecular changes in NAc-related reward circuitry (Robinson and Berridge 2003; Vezina 2004), which make the reward system highly sensitive to drugs and drug-related stimuli. This phenomenon is referred to as sensitization, which has been further hypothesized to underlie drug craving (Robinson and Berridge 1993; White and Kalivas 1998) and relapse (Bradberry 2007; Robinson and Berridge 2008). GSK-3β is associated with synaptic plasticity and learning and memory and may therefore be involved in the neuronal processes of drug addiction.

Nucleus accumbens-related circuitry, including the dopamine projection from the ventral tegmental area to NAc and striatum, plays a critical role in the control of sensitization (Bradberry and Roth 1989; Kalivas and Duffy 1990; Koob and Le Moal 2008). Psychostimulants such as cocaine, methamphetamine, and amphetamine can cause increased extracellular dopamine levels and increased activation of both dopamine D1 and D2 receptors through inhibiting the function of the dopamine transporter and the uptake of synaptic dopamine or acting as a reversal of transport by the dopamine transporter. Dopamine-related functions are mediated by an intricate signaling network, including cyclic adenosine monophosphate (cAMP)-independent dopamine receptor signaling, such as the protein kinase B (Akt)/GSK-3 signaling cascade, which involves D2-class receptors (Beaulieu et al. 2004). GSK-3 activity is negatively regulated by Akt activity; therefore, as p-Akt levels decrease, pGSK3 levels decrease, resulting in increased GSK-3 enzymatic activity. GSK-3 can regulate dopaminergic signaling and behavior. Increased striatal GSK-3α-β activity (i.e., decreased pGSK3) accompanies decreased pAkt levels in dopamine transporter-knockout mice and animals exposed to amphetamine (Beaulieu et al. 2004, 2007a). Studies using dopamine depletion or dopamine receptor antagonists in dopamine transporter knockout mice showed that protein kinase B (Akt)/GSK-3 pathway is regulated by D2-class receptors (Beaulieu et al. 2005, 2007b). GSK-3 knockout mice exhibited decreased spontaneous activity compared with wildtype mice following amphetamine injection (Beaulieu et al. 2004), whereas GSK-3β-over-expressing transgenic mice displayed increased general locomotor activity, reduced immobility in the forced swim test (i.e. an antidepressant-like effect), and most notably up-regulated Akt expression, suggesting a compensatory mechanism (Prickaerts et al. 2006).

LiCl has been used for its anti-manic and antidepressant effects (Sachs 1996; Kleindienst and Greil 2003). Recently, multiple lines of evidence have shown that the effects of LiCl on mood stabilization and neurogenesis are attributable to LiCl-induced GSK-3β inhibition (Wada 2009). Our previous study (Xu et al. 2009) and current findings confirmed that GSK-3β may be a possible molecular mechanism of the therapeutic effect of LiCl, and indicate that GSK-3β is involved in neuroadaptation and behavioral adaptation after repeated psychostimulant exposure. Previous study also demonstrated that LiCl, a GSK-3β inhibitor, may be useful in the treatment of drug addiction (Flemenbaum et al. 1979; Abrahamson 1983).

We did not find a significant difference between the LiCl or SB216763 plus methamphetamine group compared with the vehicle plus methamphetamine group on day 1 during the initiation period. However, on the expression day, LiCl or SB216763 attenuated locomotor activity after a challenge injection in the LiCl or SB216763 plus saline group compared with the vehicle plus saline group. These inconsistent results might be attributable to the different treatments. Rats that received saline pre-treatment may have adapted to the training environment and were different from those that received chronic methamphetamine injection on their first treatment day. Unfortunately, we did not assess the acute methamphetamine-induced changes in pGSK levels. However, previous studies have shown that acute injection of psychostimulants, including cocaine and amphetamine, can regulate GSK-3 activity (Beaulieu et al. 2004; Perrine et al. 2008; Miller et al. 2009). A recent study by Miller showed that an acute cocaine injection decreased pGSK-3β levels, and cocaine-induced hyperlocomotor activity was dose-dependently attenuated by SB216763, a GSK-3β inhibitor, which also inhibited the development of cocaine-induced locomotor sensitization (Miller et al. 2009). These results are consistent with our previous findings, in which either systemic LiCI administration or infusion of the GSK-3β inhibitor SB216763 could attenuate the initiation and expression of cocaine-induced behavioral sensitization, suggesting a critical role for GSK-3β in drug sensitization (Xu et al. 2009). In our previous experiments, we found that LiCl and cocaine co-administration inhibited the initiation of cocaine sensitization compared with the saline plus cocaine group on day 14. During a drug-free period, a challenge injection was administered to test the expression of cocaine sensitization after LiCl elimination, demonstrating the inhibition of expression in the LiCl plus cocaine group compared with controls. These results may demonstrate plasticity during the initiation period (Xu et al. 2009). The disparate findings on pGSK-3 level after acute psychostimulant injection might be attributable to the different experimental methods or different animal models (Wei et al. 2007; Perrine et al. 2008).

Our present study found that the NAc core but not shell is critical for the initiation and expression of methamphetamine sensitization, and this was in line with our previous study (Xu et al. 2009), this also supported the note that NAc core and shell are heterogeneous structures with distinct afferent and efferent connections and immunohistochemical properties (Voorn et al. 1986; Jongen-Relo et al. 1994). Indeed, some previous studies have also investigated the differential roles of the NAc core and shell in the effects of drugs of abuse and motivated behavior (Pontieri et al. 1995; Di Chiara et al. 2004). Behavioral sensitization to morphine (Cadoni and Di Chiara 1999), amphetamine (Robinson et al. 1988; Paulson and Robinson 1995), and nicotine (Cadoni and Di Chiara 2000; Cadoni et al. 2000) is associated with an increase in dopamine responsiveness in the NAc core. Conversely, dopamine responsiveness in the NAc shell was not affected or was even acutely reduced (Di Chiara 2002; Di Chiara et al. 2004; Ikemoto and Wise 2004;  ; Bossert et al. 2007). All these results indicated that NAc core is a component of the neural circuitry involved in the storage of reward-related information derived from conditioned reinforcers and that NAc shell dopamine is essential for the stimulant effect of psychostimulants (Ito et al. 2004).

In conclusion, the present study demonstrated that GSK-3β activity in the NAc core is critical for the development and expression of methamphetamine-induced sensitization. However, the molecular mechanisms involved in the control of GSK-3β in methamphetamine-induced behavioral sensitization remain unclear. Determination of the precise downstream targets of GSK-3β and its molecular mechanism will provide a better understanding of the biological mechanisms of behavioral sensitization and addiction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by the National Basic Research Program of China (No. 2009CB522000), the Natural Science Foundation of China (No. 81071079), and the Natural Science Foundation of Beijing Municipality (No. 7091003 and 7092058). The authors declare that they do not have any conflicts of interest (financial or otherwise) related to the data presented in this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References