Korean J Physiol Pharmacol.  2022 Jan;26(1):59-65. 10.4196/kjpp.2022.26.1.59.

Decrease of glycogen synthase kinase 3β phosphorylation in the rat nucleus accumbens shell is necessary for amphetamineinduced conditioned locomotor activity

• Shin JK
• Kim WY
• Rim H
• Kim JH

• KMID: 2524531
• DOI: http://doi.org/10.4196/kjpp.2022.26.1.59

Abstract

Phosphorylation levels of glycogen synthase kinase 3β (GSK3β) negatively correlated with psychomotor stimulant-induced locomotor activity. Locomotor sensitization induced by psychomotor stimulants was previously shown to selectively accompany the decrease of GSK3β phosphorylation in the nucleus accumbens (NAcc) core, suggesting that intact GSK3β activity in this region is necessary for psychomotor stimulants to produce locomotor sensitization. Similarly, GSK3β in the NAcc was also implicated in mediating the conditioned effects formed by the associations of psychomotor stimulants. However, it remains undetermined whether GSK3β plays a differential role in the two sub-regions (core and shell) of the NAcc in the expression of drug-conditioned behaviors. In the present study, we found that GSK3β phosphorylation was significantly lower in the NAcc shell obtained from rats expressing amphetamine (AMPH)-induced conditioned locomotor activity. Further, we demonstrated that these effects were normalized by treatment with lithium chloride, a GSK3β inhibitor. These results suggest that the behavior produced by AMPH itself and a conditioned behavior formed by associations with AMPH are differentially mediated by the two sub-regions of the NAcc.

Keyword

Amphetamine; Classical conditioning; GSK3beta; Lithium chloride; Nucleus accumbens

Figure

• 

  Fig. 1 Experiment outline. (A) An experimental timeline indicates a conditioning development schedule and the point at which challenge and brain extraction were performed. (B) A table summarizes three groups showing where they received AMPH and saline. AMPH, amphetamine.

• 

  Fig. 2 Paired group shows increased conditioned locomotor activity. All rats were tested following an IP saline injection. Data are shown as group mean + SEM total locomotor activity counts observed during the 1 h test. Symbols indicate significant differences revealed by post-hoc Bonferroni comparison following one-way ANOVA. ***p < 0.001, significantly more counts in the Paired rats relative to those in the Control and Unpaired groups. Time-course data is shown in the inset as group mean + SEM locomotor activity counts at 20 min intervals. Numbers for each group are 15. IP, intraperitoneal; CTL, control.

• 

  Fig. 3 AMPH conditioning lowers GSK3β phosphorylation levels in the NAcc shell. (A) The core (circle) and the shell (rectangular), two sub-regions of the NAcc, are shown. Punches were prepared bilaterally and pooled for each individual animal’s protein isolation. Line drawing is from Paxinos and Watson [39] and depicts the caudal surface of a coronal section (1.0 mm thick) extending 1.70–2.70 mm from bregma. (B) Representative Western blots are shown. Values for the band intensities were first normalized to β-actin and then the average values for the ratio of phosphorylated to total proteins in each group were expressed as group mean + SEM. relative to Control group. Symbols indicate significant differences as revealed by post-hoc Bonferroni comparisons following one-way ANOVA. **p < 0.01, significantly different compared to Control group. Numbers for each group are 15. AMPH, amphetamine; GSK3β, glycogen synthase kinase 3β; NAcc, nucleus accumbens.

• 

  Fig. 4 Lithium chloride inhibits the expression of AMPH-induced conditioned locomotor activity. All rats were tested following an IP saline or lithium chloride (50 mg/kg) injection. Data are shown as group mean + SEM total locomotor activity counts observed during the 1 h test. Symbols indicate significant differences revealed by post-hoc Bonferroni comparison following two-way ANOVA. ***p < 0.001, significantly more counts in the Paired rats relative to those in the Control and Unpaired groups. †††p < 0.001, significantly different from no lithium treated the Paired group. Time-course data is shown in the inset as group mean + SEM locomotor activity counts at 20 min intervals. Numbers for each group are 8. AMPH, amphetamine; IP, intraperitoneal; CTL, control; LiCl, lithium chloride.

• 

  Fig. 5 Lithium chloride recovers a reduction in GSK3β phosphorylation levels in the NAcc shell. Representative Western blots are shown. Values for the band intensities were first normalized to β-actin and then the average values for the ratio of phosphorylated to total proteins in each group were expressed as group mean + SEM relative to saline challenged Control group. Symbols indicate significant differences as revealed by post-hoc Bonferroni comparisons following two-way ANOVA. *p < 0.05, significantly different compared to Control group. ††p < 0.01, significantly different from no lithium chloride treated the Paired group. Lithium chloride treatment also recovered the lowered GSK3β phosphorylation levels in the Paired group, but it did not reach statistical significance, in the NAcc core. Numbers for each group are 8. GSK3β, glycogen synthase kinase 3β; NAcc, nucleus accumbens; CTL, control; LiCl, lithium chloride.

Reference

1. Robbins TW, Cador M, Taylor JR, Everitt BJ. 1989; Limbic-striatal interactions in reward-related processes. Neurosci Biobehav Rev. 13:155–162. DOI: 10.1016/S0149-7634(89)80025-9. PMID: 2682402.

2. Kalivas PW, Stewart J. 1991; Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res Brain Res Rev. 16:223–244. DOI: 10.1016/0165-0173(91)90007-U. PMID: 1665095.

3. Hyman SE. 1996; Addiction to cocaine and amphetamine. Neuron. 16:901–904. DOI: 10.1016/S0896-6273(00)80111-7.

4. Goto Y, Grace AA. 2008; Limbic and cortical information processing in the nucleus accumbens. Trends Neurosci. 31:552–558. DOI: 10.1016/j.tins.2008.08.002. PMID: 18786735. PMCID: PMC2884964.

5. Beaulieu JM, Gainetdinov RR, Caron MG. 2007; The Akt-GSK-3 signaling cascade in the actions of dopamine. Trends Pharmacol Sci. 28:166–172. DOI: 10.1016/j.tips.2007.02.006. PMID: 17349698.

6. Beaulieu JM, Gainetdinov RR, Caron MG. 2009; Akt/GSK3 signaling in the action of psychotropic drugs. Annu Rev Pharmacol Toxicol. 49:327–347. DOI: 10.1146/annurev.pharmtox.011008.145634. PMID: 18928402.

7. Leroy K, Brion JP. 1999; Developmental expression and localization of glycogen synthase kinase-3beta in rat brain. J Chem Neuroanat. 16:279–293. DOI: 10.1016/S0891-0618(99)00012-5. PMID: 10450875.

8. Dajani R, Fraser E, Roe SM, Young N, Good V, Dale TC, Pearl LH. 2001; Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell. 105:721–732. DOI: 10.1016/S0092-8674(01)00374-9. PMID: 11440715.

9. Frame S, Cohen P, Biondi RM. 2001; A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell. 7:1321–1327. DOI: 10.1016/S1097-2765(01)00253-2. PMID: 11430833.

10. Miller JS, Tallarida RJ, Unterwald EM. 2009; Cocaine-induced hyperactivity and sensitization are dependent on GSK3. Neuropharmacology. 56:1116–1123. DOI: 10.1016/j.neuropharm.2009.03.006. PMID: 19328817. PMCID: PMC2721824.

11. Xu CM, Wang J, Wu P, Zhu WL, Li QQ, Xue YX, Zhai HF, Shi J, Lu L. 2009; Glycogen synthase kinase 3beta in the nucleus accumbens core mediates cocaine-induced behavioral sensitization. J Neurochem. 111:1357–1368. DOI: 10.1111/j.1471-4159.2009.06414.x. PMID: 19799712.

12. Xu CM, Wang J, Wu P, Xue YX, Zhu WL, Li QQ, Zhai HF, Shi J, Lu L. 2011; Glycogen synthase kinase 3β in the nucleus accumbens core is critical for methamphetamine-induced behavioral sensitization. J Neurochem. 118:126–139. DOI: 10.1111/j.1471-4159.2011.07281.x. PMID: 21517846.

13. Enman NM, Unterwald EM. 2012; Inhibition of GSK3 attenuates amphetamine-induced hyperactivity and sensitization in the mouse. Behav Brain Res. 231:217–225. DOI: 10.1016/j.bbr.2012.03.027. PMID: 22649795. PMCID: PMC3566781.

14. Kim WY, Jang JK, Lee JW, Jang H, Kim JH. 2013; Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine-induced hyper-locomotor activity. J Neurochem. 125:642–648. DOI: 10.1111/jnc.12222. PMID: 23439225.

15. Jongen-Rêlo AL, Voorn P, Groenewegen HJ. 1994; Immunohistochemical characterization of the shell and core territories of the nucleus accumbens in the rat. Eur J Neurosci. 6:1255–1264. DOI: 10.1111/j.1460-9568.1994.tb00315.x. PMID: 7526940.

16. Zahm DS. 2000; An integrative neuroanatomical perspective on some subcortical substrates of adaptive responding with emphasis on the nucleus accumbens. Neurosci Biobehav Rev. 24:85–105. DOI: 10.1016/S0149-7634(99)00065-2. PMID: 10654664.

17. Meredith GE, Baldo BA, Andrezjewski ME, Kelley AE. 2008; The structural basis for mapping behavior onto the ventral striatum and its subdivisions. Brain Struct Funct. 213:17–27. DOI: 10.1007/s00429-008-0175-3. PMID: 18256852. PMCID: PMC2556127.

18. Stewart J, Vezina P. Kalivas PW, Barnes CD, editors. 1988. Conditioning and behavioral sensitization. Sensitization in the nervous system. Telford Press;Caldwell: p. 207–224.

19. O'Brien CP, Childress AR, McLellan AT, Ehrman R. 1992; Classical conditioning in drug-dependent humans. Ann N Y Acad Sci. 654:400–415. DOI: 10.1111/j.1749-6632.1992.tb25984.x. PMID: 1632593.

20. Domjan M. 2005; Pavlovian conditioning: a functional perspective. Annu Rev Psychol. 56:179–206. DOI: 10.1146/annurev.psych.55.090902.141409. PMID: 15709933.

21. Tzschentke TM. 2007; Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict Biol. 12:227–462. DOI: 10.1111/j.1369-1600.2007.00070.x. PMID: 17678505.

22. Miller JS, Barr JL, Harper LJ, Poole RL, Gould TJ, Unterwald EM. 2014; The GSK3 signaling pathway is activated by cocaine and is critical for cocaine conditioned reward in mice. PLoS One. 9:e88026. DOI: 10.1371/journal.pone.0088026. PMID: 24505362. PMCID: PMC3913742.

23. Shi X, Miller JS, Harper LJ, Poole RL, Gould TJ, Unterwald EM. 2014; Reactivation of cocaine reward memory engages the Akt/GSK3/mTOR signaling pathway and can be disrupted by GSK3 inhibition. Psychopharmacology (Berl). 231:3109–3118. DOI: 10.1007/s00213-014-3491-8. PMID: 24595501. PMCID: PMC4110417.

24. Wickens RH, Quartarone SE, Beninger RJ. 2017; Inhibition of glycogen synthase kinase-3 by SB 216763 affects acquisition at lower doses than expression of amphetamine-conditioned place preference in rats. Behav Pharmacol. 28:262–271. DOI: 10.1097/FBP.0000000000000283. PMID: 27984209.

25. Shi X, Barr JL, von Weltin E, Wolsh C, Unterwald EM. 2019; Differential roles of accumbal GSK3β in cocaine versus morphine-induced place preference, U50,488H-induced place aversion, and object memory. J Pharmacol Exp Ther. 371:339–347. DOI: 10.1124/jpet.119.259283. PMID: 31420527. PMCID: PMC6800444.

26. Franklin TR, Druhan JP. 2000; Involvement of the nucleus accumbens and medial prefrontal cortex in the expression of conditioned hyperactivity to a cocaine-associated environment in rats. Neuropsychopharmacology. 23:633–644. DOI: 10.1016/S0893-133X(00)00162-7. PMID: 11063919.

27. Kim WY, Vezina P, Kim JH. 2008; Blockade of group II, but not group I, mGluRs in the rat nucleus accumbens inhibits the expression of conditioned hyperactivity in an amphetamine-associated environment. Behav Brain Res. 191:62–66. DOI: 10.1016/j.bbr.2008.03.010. PMID: 18433894.

28. Yoon HS, Kim WY, Kim JH. 2010; Microinjection of CART peptide 55-102 into the nucleus accumbens core inhibits the expression of conditioned hyperactivity in a cocaine-associated environment. Behav Brain Res. 207:169–173. DOI: 10.1016/j.bbr.2009.10.003. PMID: 19818812.

29. Bossert JM, Poles GC, Wihbey KA, Koya E, Shaham Y. 2007; Differential effects of blockade of dopamine D1-family receptors in nucleus accumbens core or shell on reinstatement of heroin seeking induced by contextual and discrete cues. J Neurosci. 27:12655–12663. DOI: 10.1523/JNEUROSCI.3926-07.2007. PMID: 18003845. PMCID: PMC2117350.

30. Fuchs RA, Ramirez DR, Bell GH. 2008; Nucleus accumbens shell and core involvement in drug context-induced reinstatement of cocaine seeking in rats. Psychopharmacology (Berl). 200:545–556. DOI: 10.1007/s00213-008-1234-4. PMID: 18597075. PMCID: PMC2613506.

31. Chaudhri N, Sahuque LL, Schairer WW, Janak PH. 2010; Separable roles of the nucleus accumbens core and shell in context- and cue-induced alcohol-seeking. Neuropsychopharmacology. 35:783–791. DOI: 10.1038/npp.2009.187. PMID: 19924113. PMCID: PMC2813976.

32. Xie X, Lasseter HC, Ramirez DR, Ponds KL, Wells AM, Fuchs RA. 2012; Subregion-specific role of glutamate receptors in the nucleus accumbens on drug context-induced reinstatement of cocaine-seeking behavior in rats. Addict Biol. 17:287–299. DOI: 10.1111/j.1369-1600.2011.00325.x. PMID: 21521425. PMCID: PMC4384648.

33. Cruz FC, Babin KR, Leao RM, Goldart EM, Bossert JM, Shaham Y, Hope BT. 2014; Role of nucleus accumbens shell neuronal ensembles in context-induced reinstatement of cocaine-seeking. J Neurosci. 34:7437–7446. DOI: 10.1523/JNEUROSCI.0238-14.2014. PMID: 24872549. PMCID: PMC4035511.

34. Singer BF, Forneris J, Vezina P. 2014; Inhibiting cyclin-dependent kinase 5 in the nucleus accumbens enhances the expression of amphetamine-induced locomotor conditioning. Behav Brain Res. 275:96–100. DOI: 10.1016/j.bbr.2014.08.055. PMID: 25196634. PMCID: PMC4253310.

35. Singer BF, Bubula N, Li D, Przybycien-Szymanska MM, Bindokas VP, Vezina P. 2016; Drug-paired contextual stimuli increase dendritic spine dynamics in select nucleus accumbens neurons. Neuropsychopharmacology. 41:2178–2187. DOI: 10.1038/npp.2016.39. PMID: 26979294. PMCID: PMC4908651.

36. Beaulieu JM, Caron MG. 2008; Looking at lithium: molecular moods and complex behaviour. Mol Interv. 8:230–241. DOI: 10.1124/mi.8.5.8. PMID: 19015387.

37. Beaulieu JM, Del'guidice T, Sotnikova TD, Lemasson M, Gainetdinov RR. 2011; Beyond cAMP: the regulation of Akt and GSK3 by dopamine receptors. Front Mol Neurosci. 4:38. DOI: 10.3389/fnmol.2011.00038. PMID: 22065948. PMCID: PMC3206544.

38. Kim WY, Cai WT, Jang JK, Kim JH. 2020; Ezrin-radixin-moesin proteins are regulated by Akt-GSK3β signaling in the rat nucleus accumbens core. Korean J Physiol Pharmacol. 24:121–126. DOI: 10.4196/kjpp.2020.24.1.121. PMID: 31908581. PMCID: PMC6940492.

39. Paxinos G, Watson C. 2004. The rat brain in stereotaxic coordinates. Elsevier Academic Press;Boston: