Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: J Neurochem. 2008 Aug 20;107(2):570–577. doi: 10.1111/j.1471-4159.2008.05632.x

Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

Perrine SA 1,*, Miller JS 1, Unterwald EM 1
PMCID: PMC2581649  NIHMSID: NIHMS70515  PMID: 18717814

Abstract

Protein kinase B (Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase-3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of 3 daily injections of 15 mg/kg cocaine or 1 ml/kg saline spaced one hour apart for 1, 3 or 14 days. Amygdala, nucleus accumbens, caudate putamen and hippocampus tissues were dissected 30 minutes following the last injection and analyzed for phosphorylated and total Akt and GSK3(α & β) protein levels using Western blot analysis. Phosphorylation of Akt on the threonine-308 residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 were significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.

Keywords: cocaine, Akt, GSK3, rat, amygdala, nucleus accumbens

Introduction

Protein kinase B (also known as Akt) is a serine/threonine kinase that is widely expressed in brain and has classically been associated with its ability to regulate cellular growth and apoptosis (Song et al. 2005). Akt activity has been shown to play a key role in dopamine mediated behaviors, an effect independent of its role in cell survival and dependent on decreased phosphorylation, thus deactivation, of striatal Akt (Beaulieu et al. 2007a). Akt is the downstream mediator of two key signaling pathways in the cell, the inositol tri-phosphate pathway and the cAMP/protein kinase A pathway (Song et al. 2005). Akt activity is regulated by phosphorylation at serine-473 and threonine-308 (T308). Studies have shown that full activation of Akt in the role of cell survival pathways requires the phosphorylation of both residues (Song et al. 2005), while the role of Akt in dopamine-mediated behaviors is dependent on phosphorylation only at the T308 residue (Beaulieu et al. 2007a). Akt is upstream to many signaling molecules, notably glycogen synthase kinase-3 (GSK3). Akt negatively regulates the activity of GSK3 via increased phosphorylation of the inhibitory serine-9 residue in the beta form (Manning & Cantley 2007).

Originally isolated from skeletal muscle (Rylatt et al. 1980), GSK3 was first characterized based on its effects on glycogen synthase (Embi et al. 1980); however, it has been shown to be widely expressed in brain (Woodgett 1990, Leroy & Brion 1999) and its ability to regulate the transcription factors cAMP response element-binding protein (CREB) and β-catenin (Frame et al. 2001) have led researchers to study its role in neuronal function. GSK3 plays a key role in the Wnt signaling pathway, and it is unique in that its phosphorylation at serine-21/9 (serine-21 in GSK3α and serine-9 in GSK3β) leads to decreased kinase activity (Frame & Cohen 2001). GSK3 plays a critical role in diabetes, stroke, Alzheimer’s disease (Frame & Cohen 2001) and schizophrenia (Alimohamad et al. 2005), and the mechanism responsible for the mood stabilizing effect of lithium is dependent on inhibition of GSK3β activity (Gould et al. 2004).

Previous studies have documented that Akt and/or GSK3 activity is altered by dopamine receptor agonists (Brami-Cherrier et al. 2002), amphetamine (Beaulieu et al. 2004, McGinty et al. 2008, Svenningsson et al. 2003, Wei et al. 2007), methamphetamine (Chen et al. 2007), cocaine (Gil et al. 2003, Novikova et al. 2005, Wei et al. 2007), or by genetic deletion of the dopamine transporter (Beaulieu et al. 2006, Beaulieu et al. 2004). Typically, following dopamine receptor activation, a time-dependent decrease in phosphorylated Akt in striatum is observed which leads to increased GSK3 activity (by decreasing phosphorylated GSK3) and subsequent increase in gene transcription (Beaulieu et al. 2004, Chen et al. 2007). Although the clinical relevance of GSK3 has been noted, the role of Akt in neuropsychiatric disorders particularly psychostimulant abuse has largely gone unstudied; however, a positive association between a single nucleotide polymorphism and Akt1 haplotype in methamphetamine abusers has been identified (Ikeda et al. 2006). With these observations in mind, we measured the effects of acute (1 day), repeated (3 days) or chronic (14 days) binge-pattern cocaine administration on levels of phosphorylated and total Akt and GSK3 proteins in key dopaminergic-projection areas, including amygdala, nucleus accumbens, caudate putamen and hippocampus, in order to determine the temporal and spatial patterns of Akt and GSK3 regulation.

Methods

Male Sprague Dawley rats (275–375 g) were purchased from Charles River Labs (Wilmington, MA), group-housed on a 12 h light/dark cycle (7 AM – 7 PM) and given water and food ad libitum. Rats were acclimated to their environment for 5 days during which time they were weighed and handled daily. All procedures were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and approved by Temple University School of Medicine Institutional Animal Care and Use Committee. Cocaine HCl was provided by NIDA Drug Supply Program. All other reagents and supplies were purchased from Sigma-Aldrich (St Louis, MI) or Bio-Rad (Hercules, CA).

Rats were injected intraperitoneally (ip) with cocaine (15 mg/kg) or saline (1 ml/kg) in a binge-pattern (3 injections at 1 hour intervals beginning at 9 AM) for 1, 3 or 14 days. Binge-pattern administration is a commonly used animal model of cocaine abuse and was developed to better approximate the temporal pattern and relationship to circadian rhythm that is seen in human abuse of cocaine (Unterwald et al. 1992, Unterwald et al. 2001). Rats were killed 30 minutes after the last injection and brains were dissected rapidly on ice. The amygdala, nucleus accumbens, caudate putamen and hippocampus were dissected according to the rat brain atlas of Paxinos and Watson (Paxinos & Watson 2006). Samples were immediately prepared by homogenizing brain regions from individual animals in boiling 1% (g/L) sodium dodecyl sulfate (SDS) using a sonicator. Homogenized samples were boiled for 5 minutes and then stored at −80°C until use.

Protein concentrations were measured using the Folin-Lowery assay (Lowry et al. 1951). Proteins were separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to nitrocellulose for Western blot analysis of phosphorylated and total protein levels. Equal amounts (20 μg) of protein were loaded in 10% (g/L) Tris-HCl BioRad Ready-gels. Proteins were visualized by Ponceau S Red (general protein dye) to ensure transfer and equal protein loading. Nitrocellulose blots were incubated in blocking solution (tris-buffered saline with 5% (g/L) non-fat dry milk), then in primary antibody overnight at 4°C (except tubulin antibody which was incubated for 1 h at room temperature). Blots were washed with Tween-20 Tris-buffered saline (TTBS) then incubated with anti-rabbit (1:2,000 for pAkt(T308), pGSK3α-β and total Akt) or anti-mouse (1:5,000 for total GSK3α-β and tubulin) secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Blots were washed with TTBS. Nitrocellulose-bound proteins were visualized using the Pierce Supersignal enhanced chemiluminescent substrate and Fujifilm image gauge hardware and quantified using Science Lab 99 L-Process v1.96 software. To determine phosphorylated protein levels as a ratio of total protein levels, blots were first probed for phosphorylated protein (pAkt(T308) or pGSK3α-β), then stripped using Restore Western Blot Stripping Buffer (Pierce), washed with TTBS and re-probed for total protein levels (Akt or GSK3α-β). Primary antibodies were diluted in TBS (with 5% (g/L) non fat dry milk) and those used include phosphorylated Akt T308 [pAkt(T308); Cell Signaling 1:500], Akt [total Akt; Cell Signaling 1:1,000], phosphorylated GSK3α-β [pGSK3α-β Cell Signaling 1:2,000], GSK3α-β [total GSK3α-β Santa Cruz 1:5,000] and tubulin [Sigma 1:40,000]. The antibody used to detect GSK3 (phosphorylated and total) recognizes both a (S21, 51 kDa) and β (S9, 48 kDa) isoforms. Immunoreactivity to tubulin was used to verify equal protein loading and transfer. All data are expressed as phosphorylated protein/total protein and graphed as a percent of control. The extent of enzyme phosphorylation is used as a proxy indicator of enzymatic activity. A correlation between phosphorylation of Akt(T308) and Akt enzymatic activity has been established (Ma et al. 2008).

Data were analyzed by independent two-tailed t-tests comparing group means between cocaine- and saline-injected controls following 1, 3 or 14 days of treatment. Statistical significance was considered at p<0.05 with a 95% confidence interval. Data were analyzed using Graphpad Prism 4. Samples sizes ranged from 6–10 and were generally 8/group.

Results

Levels of phosphorylated and total protein were determined for Akt(T308) and its downstream mediator GSK3α-β in the amygdala, nucleus accumbens, caudate putamen, and hippocampus from rats injected with cocaine for 1, 3 or 14 days. In the amygdala, acute binge-pattern cocaine administration selectively increased pAkt(T308), while chronic cocaine administration decreased phosphorylation of both Akt(T308) and GSK3α-β. Representative immunoblots of tissue extracts from the amygdala of saline- and cocaine-injected rats at each time point (1, 3 or 14 days) are shown in Figure 1.

Figure 1. Representative immunoblots of amygdala tissue from saline (sal) and cocaine (coc) treated rats at each time point (1, 3 or 14 days of cocaine or saline administration).

Figure 1

Open in a new tab

Bands represent phosphorylated Akt(T308), total Akt, phosphorylated GSK3α-β, total GSK3α-β and tubulin (from top to bottom). The molecular weights of the proteins are stated on the left (kilodaltons).

Levels of pAkt (pAkt/total Akt) were significantly increased after 1 day (acute; t12=2.82, p=0.015), not changed after 3 days (repeated; p>0.05), and decreased after 14 days (chronic; t12=2.77, p=0.017) of repeated cocaine administration in the amygdala (Figure 2A). Phosphorylation of GSK3α-β in the amygdala followed an unusual pattern compared to its upstream mediator pAkt(T308). The increase in pAkt after 1 day of binge-pattern cocaine administration (Figure 2A) was not accompanied by a significant increase in pGSK3α(pGSK3α/total GSK3α) or pGSK3β (pGSK3β/total GSK3β) in the amygdala (Figure 2B; p>0.05). However, the decrease in pAkt after 14 days of cocaine administration (Figure 2A) was paralleled by a significant decrease in pGSK3α(pGSK3α/total GSK3α) and pGSK3β(pGSK3β/total GSK3β) in the amygdala (Figure 2B; t13=3.04, p=0.009 and t13=2.70, p=0.018, respectively).

Figure 2. Acute binge-pattern cocaine administration selectively increases phosphorylated Akt, while chronic treatment decreases phosphorylation of both Akt and GSK3 in the amygdala of rats.

Figure 2

Open in a new tab

A. Levels of pAkt(T308) to total Akt (pAkt/total Akt) and B. pGSK3α to total GSK3α (pGSK3α/total GSK3α) and pGSK3β to total GSK3β (pGSK3β/total GSK3β) were measured by PAGE and Western blot analysis from the amygdala of rats treated with binge-pattern cocaine administration for 1, 3 or 14 days. Total-Akt/tubulin, total-GSK3α/tubulin and total- GSK3β/tubulin levels were not changed in any group (data not shown). Bars represent the mean ± SEM (N=6–10/group) and are expressed as a percent of the saline-matched control for each group. *p<0.05, **p<0.01 compared to saline-matched control.

Acute (1 day) binge-pattern cocaine administration significantly decreased pAkt (pAkt/total Akt) in the nucleus accumbens of rats (t16=2.81, p=0.013), as shown in Figure 3A. No significant differences were found after 3 or 14 days of cocaine administration suggesting that tolerance develops to the regulation of Akt(T308) phosphorylation by cocaine (p>0.05; Figure 3A). The levels of pGSK3α(pGSK3α/total GSK3α) and pGSK3β (pGSK3β/total GSK3β) in the nucleus accumbens were not significantly affected by cocaine administration at any day of treatment (p>0.05; Figure 3B).

Figure 3. Acute binge-pattern cocaine administration decreases phosphorylated Akt in the nucleus accumbens of rats.

Figure 3

Open in a new tab

A. Levels of pAkt(T308) to total Akt (pAkt/total Akt) and B. pGSK3α to total GSK3α (pGSK3α/total GSK3α) and pGSK3β to total GSK3β (pGSK3β/total GSK3β) were measured by PAGE and Western blot analysis from the nucleus accumbens of rats injected with cocaine in a binge-pattern for 1, 3 or 14 days. Total-Akt/tubulin, total-GSK3α/tubulin and total- GSK3β/tubulin levels were not changed in any group (data not shown). Bars represent the mean ± SEM (N=6–9/group) and are expressed as a percent of the saline-matched control for each group. *p<0.05 compared to saline-injected controls.

In the caudate putamen, the levels of pAkt(T308)/total Akt (Figure 4A) and pGSK3α-β/total GSK3α-β (Figure 4B) were not altered by 1, 3 or 14 days of binge-pattern cocaine (p>0.05). Likewise, in the hippocampus, pAkt(T308)/total Akt (Figure 5A) and pGSK3α-β/total GSK3α-β (Figure 5B) levels following cocaine were not statistically different from saline-injected controls (p>0.05). Total Akt, total GSK3α-β, or tubulin in cocaine-injected animals were not significantly different from those in controls injected with saline for an equal number of days in any brain region tested (p>0.05; data not shown).

Figure 4. Binge-pattern cocaine administration does not affect Akt or GSK3α-β phosphorylation in the caudate putamen of rats.

Figure 4

Open in a new tab

Levels of (A.) pAkt(T308) to total Akt (pAkt/total Akt) or (B.) pGSK3α-β to total GSK3α-β (pGSK3α-β/total GSK3α-β) were measured by PAGE and Western blot analysis from the caudate putamen of rats injected with binge-pattern cocaine for 1, 3 or 14 days. Total- Akt/tubulin was not changed in any group (data not shown). Bars represent the mean ± SEM (N=8–10/group) and are expressed as a percent of the saline-matched control for each group.

Figure 5. Binge-pattern cocaine administration does not affect Akt or GSK3α-β phosphorylation in the hippocampus of rats.

Figure 5

Open in a new tab

Levels of (A.) pAkt(T308) to total Akt (pAkt/total Akt) or (B.) pGSK3α-β to total GSK3α-β (pGSK3α-β/total GSK3α-β) were measured by PAGE and Western blot analysis from the hippocampus of rats injected with binge-pattern cocaine for 1, 3 or 14 days. Total-Akt/tubulin was not changed in any group (data not shown). Bars represent the mean ± SEM (N=8–10/group) and are expressed as a percent of the saline-matched control for each group.

Discussion

Cocaine inhibits the function of dopamine transporters (DAT) and thus the uptake of synaptic dopamine, resulting in increased extracellular dopamine levels and increased activation of both dopamine D1 and D2 receptors. The ascending dopamine system consists of three pathways: 1) the nigrostriatal pathway extending from the substantia nigra to the caudate putamen, 2) the mesolimbic pathway projecting from ventral tegmental area to nucleus accumbens and amygdala, and 3) the mesocortical pathway originating in the ventral tegmental area and innervating cerebral cortex and hippocampus. The data presented here show that the effect of cocaine on Akt activity (as assessed by pAkt levels) is spatially restricted to the nucleus accumbens and amygdala, and does not occur in the caudate putamen or hippocampus, indicating the selective involvement of the mesolimbic dopamine system. To our knowledge, these are the first data to show a spatial pattern of Akt regulation following cocaine exposure. In addition, our data also show the temporal pattern of regulation of Akt and GSK3 activity between acute and chronic cocaine administration.

Several studies document the regulation of Akt activity by dopaminergic receptor signaling. Dopamine D1 or D2 receptor agonists rapidly increase pAkt(T308) in primary striatal neuronal cultures, an effect mediated by extracellular signal-regulated kinase (ERK) (Brami-Cherrier et al. 2002). While an increase in pAkt(T308) in rat striatum occurs 15 min after an amphetamine injection, a decrease is found two hours later (McGinty et al. 2008, Shi & McGinty 2007). The decrease in pAkt(T308) is dependent on the calcium/calmodulin-dependent protein kinase-II pathway following acute amphetamine administration (Wei et al. 2007) and requires an increase in ERK phosphorylation via dopamine D3 receptor following methamphetamine treatment (Chen et al. 2007). A decrease in Akt(T308) activity is also observed in mice lacking DAT, which have high levels of synaptic dopamine (Beaulieu et al. 2006, Beaulieu et al. 2004). Our results agree with these findings by demonstrating that pAkt(T308) levels are reduced in the nucleus accumbens (ventral striatum) following acute binge-pattern cocaine administration.

The downstream consequences of Akt activation remain to be fully characterized. However, a clear association between Akt and GSK3 has been demonstrated (Beaulieu et al. 2004). GSK3 has been shown to be the target of the mood stabilizer lithium (Gould et al. 2004) and dopamine D2 receptor-dependent antipsychotic drugs (Alimohamad et al. 2005). GSK3 activity is negatively regulated by Akt activity; therefore as pAkt levels decrease so do those of pGSK3 resulting in increased GSK3 enzymatic activity. It has been demonstrated that increased striatal GSK3α-β activity (decreased pGSK3) accompanies decreased pAkt levels in DAT-knockout mice and in animals exposed to amphetamine (Beaulieu et al. 2004). GSK3 can regulate dopaminergic signaling and behaviors. GSK3-knockout animals have reduced dopamine-dependent locomotor behavior (Beaulieu et al. 2004), whereas GSK3β-overexpressing transgenic mice display increased general locomotor activity, reduced immobility in the forced swim test (i.e. antidepressant-like effect) and most notably an upregulation of Akt expression suggesting a compensatory mechanism (Prickaerts et al. 2006). These studies suggest a dopamine-dependent mechanism of regulation of GSK3 activity, but it may be that GSK3 is one of several key downstream effectors of DARPP-32 in a common signaling pathway activated by dopaminergic agonists (e.g. cocaine or amphetamine), serotonergic drugs (e.g. LSD; see discussion below) and glutamatergic antagonists (e.g. PCP) (Svenningsson et al. 2003).

Few studies have investigated the effects of cocaine on Akt and GSK3 signaling. Mouse fetuses from cocaine-injected dams show changes in cerebral wall Akt genes (Novikova et al. 2005), and young rabbits (PND 20) exposed in utero to cocaine have increased basal levels of pGSK3β in striatum and prefrontal cortex, but not hippocampus (Gil et al. 2003). One study found that cocaine does not affect pGSK3α but blocks the amphetamine-induced decrease in pGSK3α in striatal synaptosomes (Wei et al. 2007). Our data agree in that pGSK3α (or β) levels are unaffected by acute cocaine and, in addition directly demonstrate that Akt enzymatic activity is decreased in the accumbens following acute cocaine administration.

The results of the present study also demonstrate that the effects of cocaine are dependent on the length of drug exposure. In the nucleus accumbens, Akt activity is decreased after 1 day, but not after 3 or 14 days of repeated cocaine administration. These data are in agreement with the finding that acute amphetamine decreases pAkt(T308) (Beaulieu et al. 2004, McGinty et al.2008). However, our data show that pGSK3α-β activity is not statistically affected by 1 day of cocaine treatment, which is consistent with the published report showing a lack of effect of cocaine on pGSK3α (Wei et al. 2007), but contrary to those studies reporting an amphetamine-induced decrease in pGSK3α-β (Beaulieu et al. 2004, Chen et al. 2007). It should be noted that the lack of effect of cocaine on striatal pGSK3 in contrast to that seen with amphetamine may be a reflection of the fact that cocaine is less effective at elevating extracellular dopamine than amphetamine (Ramsey et al. 2008), which not only blocks DAT but reverses its function leading to higher levels of synaptic dopamine independent of dopaminergic cell firing (Jones et al.1998). The pharmacological effects of the two agents also differ in terms of their dependence on glutamatergic neurotransmission (Ramsey et al. 2008).

This discussion has thus far focused on the dopaminergic effects of cocaine because of the well-characterized effects of the drug on this neurotransmitter and because of the demonstrated effects of dopamine signaling on Akt or GSK3 activity (Beaulieu et al. 2007b, Brami-Cherrier et al. 2002). While it remains likely that the effects in the striatum are dopamine-dependent, the effects in other brain regions may not be dependent on dopamine receptor signaling. In fact, serotonin-deficient mice have elevated levels of pGSK3 in the frontal cortex. Further, genetic or pharmacological inhibition of GSK3 activity reduces the behavioral abnormalities induced by 5-HT deficiency in these transgenic mice (Beaulieu et al. 2008). These data suggest that lack of 5-HT signaling increases GSK3-dependent behaviors, including anxiety- and depression-like behaviors. Furthermore, the effects of 5-HT deficiency were localized to the frontal cortex and not observed in striatum and hippocampus (amygdala was not studied) (Beaulieu et al. 2008).

The data presented herein demonstrate that cocaine alters Akt and GSK activity selectively in the nucleus accumbens and the interconnected amygdala, but not caudate putamen albeit statistically non-significant decreases in pAkt(T308) and pGSK3 levels were observed in this region. This suggests that regulation of Akt by cocaine selectively occurs in the projection fields (i.e. nucleus accumbens and amygdala) of the mesolimbic dopaminergic pathway. This is important because dopamine release onto medium spiny neurons in the accumbens and amygdala is a key regulator of initial drug perception, reinforcement, and reward, orientation and memory of emotional response to drug salience (Berke & Hyman 2000, Kalivas & Volkow 2005). On the other hand, the projection fields of the nigrostriatal and mesocortical dopamine pathways govern compulsive habit and contextual learning behaviors (Berke & Hyman 2000) and regulation of motivational salience and receives, integrates and communicates these stimuli to subcortical areas (Kalivas & Volkow 2005).

The data presented herein from the amygdala indicate that Akt activity is increased after 1 day, not changed after 3 days and decreased after 14 days of binge-pattern cocaine administration. While the levels of pGSK3α-β showed a trend towards an elevation after 1 and 3 days, only after 14 days of cocaine administration were there significant changes in GSK3 phosphorylation and at this time point levels of pGSK3α-β were significantly reduced. These data suggest that GSK3 may not be the sole substrate of Akt in the amygdala following acute cocaine, but rather GSK3 may be important in long-term plasticity induced by repeated cocaine exposure. The amygdala is a limbic structure classically recognized for its role in the neuronal underpinnings of emotion and mood-related behaviors in humans (Trimble 1988, Kalia 2005). Although additional studies are required, it is tempting to speculate that the temporal and opposite effects of acute versus chronic cocaine on Akt and GSK3 activity in the amygdala reflect the emotional states observed in humans. We have previously shown that acute withdrawal from 14-day binge-pattern cocaine administration induces anxiety- and depression-like behaviors in rats (Perrine et al. 2008). The significant decrease in pAkt and pGSK3α-β levels and resulting increase in GSK3 activity after 14 days of cocaine exposure is consistent with the finding that GSK3 inhibitors have mood stabilizing and antidepressant-like effects (Gould et al. 2004). It may be that the initial euphoric and emotional perception of cocaine is dependent on dopamine-induced increase in Akt activity in the amygdala, while the anxiety and mood-related effects resulting from long-term abuse of cocaine are influenced by serotonin-mediated decreases in Akt activity and subsequent high GSK3 activity in the amygdala. Chronic cocaine administration does not decrease serotonin levels in basolateral amygdala of rat (Baumann et al. 1993). However, cocaine exposure upregulates serotonin transporter levels in amygdala in humans (Mash et al. 2000) and downregulates 5-HT1A receptors in the central medial amygdala of rats (Cunningham et al. 1992). These latter actions of chronic cocaine on components of the 5-HT system could result in decreases in post-synaptic receptor-mediated events and potentially reduce GSK3 activity and increase aberrant behaviors.

In summary, the results presented here show that cocaine induces a spatially restricted pattern of phosphorylation of Akt and GSK3, wherein the nucleus accumbens and amygdala but not caudate putamen and hippocampus are affected. Regulation of these signaling molecules is also dependent on the length of cocaine exposure. More research is needed to understand the behavioral implications of these findings, but the results provide evidence that the Akt-GSK3 pathway is impacted by cocaine exposure. Future studies will investigate the relationship between cocaine-mediated behavioral sensitization, anxiety- and depression-like behaviors and the involvement of Akt-GSK pathway in these effects.

Acknowledgments

The authors would like to thank Ms. Mary McCafferty, Mr. Imran Sheikh, and Dr. Joseph A. Schroeder for their assistance. This work was supported in part by NIDA/NIH grants DA009580, DA018326, and T32 DA07237 (EMU).

Non-standard abbreviations

Akt

protein kinase B

GSK3

glycogen synthase kinase 3

References

  1. Alimohamad H, Rajakumar N, Seah YH, Rushlow W. Antipsychotics alter the protein expression levels of beta-catenin and GSK-3 in the rat medial prefrontal cortex and striatum. Biol Psychiatry. 2005;57:533–542. doi: 10.1016/j.biopsych.2004.11.036. [DOI] [PubMed] [Google Scholar]
  2. Baumann MH, Raley TJ, Partilla JS, Rothman RB. Biosynthesis of dopamine and serotonin in the rat brain after repeated cocaine injections: a microdissection mapping study. Synapse. 1993;14:40–50. doi: 10.1002/syn.890140107. [DOI] [PubMed] [Google Scholar]
  3. Beaulieu JM, Gainetdinov RR, Caron MG. The Akt-GSK-3 signaling cascade in the actions of dopamine. Trends Pharmacol Sci. 2007a;28:166–172. doi: 10.1016/j.tips.2007.02.006. [DOI] [PubMed] [Google Scholar]
  4. Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG. Paradoxical striatal cellular signaling responses to psychostimulants in hyperactive mice. J Biol Chem. 2006;281:32072–32080. doi: 10.1074/jbc.M606062200. [DOI] [PubMed] [Google Scholar]
  5. Beaulieu JM, Sotnikova TD, Yao WD, Kockeritz L, Woodgett JR, Gainetdinov RR, Caron MG. Lithium antagonizes dopamine-dependent behaviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade. Proc Natl Acad Sci U S A. 2004;101:5099–5104. doi: 10.1073/pnas.0307921101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beaulieu JM, Tirotta E, Sotnikova TD, Masri B, Salahpour A, Gainetdinov RR, Borrelli E, Caron MG. Regulation of Akt signaling by D2 and D3 dopamine receptors in vivo. J Neurosci. 2007b;27:881–885. doi: 10.1523/JNEUROSCI.5074-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beaulieu JM, Zhang X, Rodriguiz RM, Sotnikova TD, Cools MJ, Wetsel WC, Gainetdinov RR, Caron MG. Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. Proc Natl Acad Sci U S A. 2008;105:1333–1338. doi: 10.1073/pnas.0711496105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berke JD, Hyman SE. Addiction, dopamine, and the molecular mechanisms of memory. Neuron. 2000;25:515–532. doi: 10.1016/s0896-6273(00)81056-9. [DOI] [PubMed] [Google Scholar]
  9. Brami-Cherrier K, Valjent E, Garcia M, Pages C, Hipskind RA, Caboche J. Dopamine induces a PI3-kinase-independent activation of Akt in striatal neurons: a new route to cAMP response element-binding protein phosphorylation. J Neurosci. 2002;22:8911–8921. doi: 10.1523/JNEUROSCI.22-20-08911.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen PC, Lao CL, Chen JC. Dual alteration of limbic dopamine D1 receptor-mediated signalling and the Akt/GSK3 pathway in dopamine D3 receptor mutants during the development of methamphetamine sensitization. J Neurochem. 2007;100:225–241. doi: 10.1111/j.1471-4159.2006.04203.x. [DOI] [PubMed] [Google Scholar]
  11. Cunningham KA, Paris JM, Goeders NE. Chronic cocaine enhances serotonin autoregulation and serotonin uptake binding. Synapse. 1992;11:112–123. doi: 10.1002/syn.890110204. [DOI] [PubMed] [Google Scholar]
  12. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem. 1980;107:519–527. [PubMed] [Google Scholar]
  13. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J. 2001;359:1–16. doi: 10.1042/0264-6021:3590001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frame S, Cohen P, Biondi RM. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell. 2001;7:1321–1327. doi: 10.1016/s1097-2765(01)00253-2. [DOI] [PubMed] [Google Scholar]
  15. Gil M, Zhen X, Friedman E. Prenatal cocaine exposure alters glycogen synthase kinase-3beta (GSK3beta) pathway in select rabbit brain areas. Neurosci Lett. 2003;349:143–146. doi: 10.1016/s0304-3940(03)00852-8. [DOI] [PubMed] [Google Scholar]
  16. Gould TD, Chen G, Manji HK. In vivo evidence in the brain for lithium inhibition of glycogen synthase kinase-3. Neuropsychopharmacology. 2004;29:32–38. doi: 10.1038/sj.npp.1300283. [DOI] [PubMed] [Google Scholar]
  17. Ikeda M, Iwata N, Suzuki T, et al. Positive association of AKT1 haplotype to Japanese methamphetamine use disorder. Int J Neuropsychopharmacol. 2006;9:77–81. doi: 10.1017/S1461145705005481. [DOI] [PubMed] [Google Scholar]
  18. Jones SR, Gainetdinov RR, Wightman RM, Caron MG. Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. J Neurosci. 1998;18:1979–1986. doi: 10.1523/JNEUROSCI.18-06-01979.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kalia M. Neurobiological basis of depression: an update. Metabolism. 2005;54:24–27. doi: 10.1016/j.metabol.2005.01.009. [DOI] [PubMed] [Google Scholar]
  20. Kalivas PW, Volkow ND. The neural basis of addiction: a pathology of motivation and choice. Am J Psychiatry. 2005;162:1403–1413. doi: 10.1176/appi.ajp.162.8.1403. [DOI] [PubMed] [Google Scholar]
  21. Leroy K, Brion JP. Developmental expression and localization of glycogen synthase kinase-3beta in rat brain. J Chem Neuroanat. 1999;16:279–293. doi: 10.1016/s0891-0618(99)00012-5. [DOI] [PubMed] [Google Scholar]
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  23. Ma K, Cheung SM, Marshall AJ, Duronio V. PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity. Cell Signal. 2008;20:684–694. doi: 10.1016/j.cellsig.2007.12.004. [DOI] [PubMed] [Google Scholar]
  24. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129:1261–1274. doi: 10.1016/j.cell.2007.06.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mash DC, Staley JK, Izenwasser S, Basile M, Ruttenber AJ. Serotonin transporters upregulate with chronic cocaine use. J Chem Neuroanat. 2000;20:271–280. doi: 10.1016/s0891-0618(00)00102-2. [DOI] [PubMed] [Google Scholar]
  26. McGinty JF, Shi XD, Schwendt M, Saylor A, Toda S. Regulation of psychostimulant-induced signaling and gene expression in the striatum. J Neurochem. 2008;104:1440–1449. doi: 10.1111/j.1471-4159.2008.05240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Novikova SI, He F, Bai J, Badan I, Lidow IA, Lidow MS. Cocaine-induced changes in the expression of apoptosis-related genes in the fetal mouse cerebral wall. Neurotoxicol Teratol. 2005;27:3–14. doi: 10.1016/j.ntt.2004.08.004. [DOI] [PubMed] [Google Scholar]
  28. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Academic Press; London: 2006. [Google Scholar]
  29. Perrine SA, Sheikh IS, Nwaneshiudu CA, Schroeder JA, Unterwald EM. Withdrawal from chronic administration of cocaine decreases delta opioid receptor signaling and increases anxiety- and depression-like behaviors in the rat. Neuropharmacology. 2008;54:355–364. doi: 10.1016/j.neuropharm.2007.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Prickaerts J, Moechars D, Cryns K, Lenaerts I, van Craenendonck H, Goris I, Daneels G, Bouwknecht JA, Steckler T. Transgenic mice overexpressing glycogen synthase kinase 3beta: a putative model of hyperactivity and mania. J Neurosci. 2006;26:9022–9029. doi: 10.1523/JNEUROSCI.5216-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramsey AJ, Laakso A, Cyr M, Sotnikova TD, Salahpour A, Medvedev IO, Dykstra LA, Gainetdinov RR, Caron MG. Genetic NMDA Receptor Deficiency Disrupts Acute and Chronic Effects of Cocaine but not Amphetamine. Neuropsychopharmacology. 2008 doi: 10.1038/sj.npp.1301663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rylatt DB, Aitken A, Bilham T, Condon GD, Embi N, Cohen P. Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur J Biochem. 1980;107:529–537. [PubMed] [Google Scholar]
  33. Shi X, McGinty JF. Repeated amphetamine treatment increases phosphorylation of extracellular signal-regulated kinase, protein kinase B, and cyclase response element-binding protein in the rat striatum. J Neurochem. 2007;103:706–713. doi: 10.1111/j.1471-4159.2007.04760.x. [DOI] [PubMed] [Google Scholar]
  34. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 2005;9:59–71. doi: 10.1111/j.1582-4934.2005.tb00337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Svenningsson P, Tzavara ET, Carruthers R, et al. Diverse psychotomimetics act through a common signaling pathway. Science. 2003;302:1412–1415. doi: 10.1126/science.1089681. [DOI] [PubMed] [Google Scholar]
  36. Trimble MR. The neurology of anxiety. Postgrad Med J. 1988;64(Suppl 2):22–26. [PubMed] [Google Scholar]
  37. Unterwald EM, Horne-King J, Kreek MJ. Chronic cocaine alters brain mu opioid receptors. Brain Res. 1992;584:314–318. doi: 10.1016/0006-8993(92)90912-s. [DOI] [PubMed] [Google Scholar]
  38. Unterwald EM, Kreek MJ, Cuntapay M. The frequency of cocaine administration impacts cocaine-induced receptor alterations. Brain Res. 2001;900:103–109. doi: 10.1016/s0006-8993(01)02269-7. [DOI] [PubMed] [Google Scholar]
  39. Wei Y, Williams JM, Dipace C, Sung U, Javitch JA, Galli A, Saunders C. Dopamine transporter activity mediates amphetamine-induced inhibition of Akt through a Ca2+/calmodulin-dependent kinase II-dependent mechanism. Mol Pharmacol. 2007;71:835–842. doi: 10.1124/mol.106.026351. [DOI] [PubMed] [Google Scholar]
  40. Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A. Embo J. 1990;9:2431–2438. doi: 10.1002/j.1460-2075.1990.tb07419.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES