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Involvement of extracellular signal-regulated protein kinase in acute cocaine-induced c-fos in nucleus accumbens
Neuroscience Letters 438 (2008) 155–158
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Involvement of extracellular signal-regulated protein kinase in acute cocaine-induced c-fos in nucleus accumbens Xiaowei Guan a,b , Jue Hu a , Shengnan Li a,∗ a b
Department of Pharmacology, Nanjing Medical University, 140 HanZhong Road, Nanjing 210029, China Department of Human Anatomy, Nanjing Medical University, Nanjing 210029, China
a r t i c l e
i n f o
Article history: Received 25 January 2008 Received in revised form 23 March 2008 Accepted 7 April 2008 Keywords: C-fos Cocaine Nucleus accumbens Extracellular signal-regulated protein kinase
a b s t r a c t It is well known that acute cocaine administration increases c-Fos expression that is involved in cocaineinduced persistent changes in the central nervous system. In the present study, we investigated a possible involvement of extracellular signal-regulated protein kinase (ERK) in induction of c-fos expression in response to acute cocaine treatment in nucleus accumbens (NAc). We found that inhibition of ERK activation significantly attenuated cocaine-induced c-fos expression at both protein and mRNA levels in the NAc. Furthermore, using an immunofluorescent staining approach, we found that inhibition of ERK activation completely abolished cocaine-induced increase in number of c-Fos-positive cells in the core region of NAc, whereas, in shell region of NAc, inhibition of ERK activation partially attenuated cocaine-induced c-Fos expression. Our findings suggest that ERK might participate in cocaine-induced c-fos expression in the NAc, particularly in the core region of NAc. © 2008 Elsevier Ireland Ltd. All rights reserved.
The nucleus accumbens (NAc) that is composed of the shell and the core parts, plays an important role in the reinforcing effects of drugs [3]. Currently, drug addition is defined solely on the basis of behavioral abnormalities, such as compulsive drug-seeking and taking. The stability of these behavioral abnormalities has been reported to be mediated, at least in part, through changes of gene expression [12,20,21]. c-fos, one of the immediate early genes (IEGs), can be induced by drugs such as amphetamine or cocaine in several brain areas, including NAc [12]. Although c-fos is generally thought as a marker for an increase in neuronal activity, recent studies demonstrated that c-fos might contribute to the cocaineinduced persistent changes in biochemistry and structure of the brain [1,12,22]. Several signaling pathways have been identified to be involved in cocaine-induced c-fos expression [1,11,17]. Recently, the role of extracellular signal-regulated protein kinase (ERK) has attracted much attention. Previous studies showed that acute cocaine administration increased ERK activation in several brain areas, including NAc [8]. Blockade of ERK pathway prevented the cocaine-induced long-lasting behaviors, such as conditioned place preference (CPP) and hyperlocomotor activity [8,19]. These results suggest that ERK might be a critical mediator for cocaine-induced behavioral abnormalities.
∗ Corresponding author. Tel.: +86 25 86863364; fax: +86 25 86863050. E-mail address: [email protected] (S. Li). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.04.035
In the present study, using an acute cocaine treatment models, we determined whether ERK was involved in acute cocaineinduced c-fos expression in the NAc. Male C57BL/6J mice weighing 23–26 g were maintained in standard housing on a 12 h/12 h light/dark cycle. All experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice received intraperitoneal (i.p.) injection of SL 327 [Sigma, dissolved in 20% dimethylsulfoxide (DMSO)] or vehicle (20% DMSO). One hour later, cocaine hydrochloride (20 mg/kg, dissolved in saline, Sigma) or saline was injected intraperitoneally. For immunofluorescent and Western blot, NAc was collected at 2 h post-cocaine or post-saline. For RT-PCR, NAc was collected at 1.5 h post-cocaine or post-saline. The immunofluorescent procedure was based on the protocol described previously [18]. In brief, the sections (at the thickness of 20 m) were pre-incubated with 0.1% BSA and 5% goat serum in PBS for 1 h and then with primary rabbit anti-c-Fos (1:200, CST) dissolved in 4% Triton X-100 and 0.1% BSA in PBS at 4 ◦ C overnight. The sections were incubated with the secondary goat anti-rabbit antibody labeled with Cy3 (1:100, Sigma) at 37 ◦ C for 2 h. Immunofluorescent control was performed by replacing the primary antibodies with PBS buffer. All immunofluorescent positive cells were quantified by software (VNT QuantLab-ST). Nuclear protein was extracted from NAc in accordance with the guidelines of Nuclear Extraction Kit (KangChen Bio-tech). Protein was separated by 10% SDS-PAGE and electrophoretically transferred
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Fig. 1. Effect of inhibition of ERK activation on acute cocaine-induced c-Fos protein expression in the NAc. Top: example of Western blot results. Bottom: statistical analysis. Data are presented as mean ± S.E.M. (n = 12 mice/group). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group.
Fig. 2. Effect of inhibition of ERK activation on acute cocaine-induced c-fos mRNA level in the NAc. Top: example of PCR products. Bottom: statistical analysis. Data are presented as mean ± S.E.M. (n = 12 mice/group). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group.
onto polyvinylidene difluoride membranes. The blots were blocked with 5% non-fat dry milk and 0.1% Tween 20 in 10 mM Tris–HCl (pH 7.5) at room temperature for 2 h, and then incubated with rabbit anti-c-Fos (1:200, CST) or mouse anti--actin (1:1000, Santa Cruz) at 4 ◦ C overnight. The blots were subsequently incubated with HRP-conjugated secondary antibody (1:4000, Santa Cruz) at room temperature for 1.5 h. The bound antibodies were visualized by the ECL system (Amershan Pharmacia Biotech) and exposed to X-OMAT film (Kodak). -Actin was served as an internal control. The densities of immunoblotting bands were quantified by software (Leica Qwin Standard V 2.8). Western blot were repeated at least three times and NAc samples were obtained from different mice. RNA from NAc was extracted using a RNeasy Mini kit (QIAGEN). Total RNA was denatured at 70 ◦ C for 5 min and then reverse transcribed at 42 ◦ C for 1 h. The synthesized cDNA products (4 l) were subjected to PCR in a 20-l reaction mixture containing 2 mM MgCL2 , and 0.5 U Tag DNA polymerase (Promega). Amplification was performed on thermocycler (BioRad, Hemel Hempstead, Herts) with 35 cycles as follows: 15 s at 96 ◦ C, 30 s at 60 ◦ C, and 1 min at 72 ◦ C. Amplified products were run on a 2% agarose gel containing 0.06% g/ml ethidium bromide and visualized under Molecular Image FX (Bio-Rad, Herculer). The sequences of the c-fos primers are as follows: 5 -GGGACAGCCTTTCCTACTACCAT-3 (sense primer) and 5 CAACGCAGACTTCTCATCTTCAA-3 (anti-sense primer). The level of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) mRNA was used as an internal control. The intensity of the products was semi-quantified by software (Leica Qwin Standard V 2.8). The experiments were repeated at least three times in different mice brains. All results were analyzed by one-way ANOVA. Individual mean comparisons were made by using the Newman–Keuls when required. Significant levels were set at p < 0.05. To investigate the role of ERK activation in c-Fos protein expression after acute cocaine treatment, a selective ERK inhibitor, SL 327 (40 mg/kg), was administered i.p. 1 h before cocaine injection. Western blot was performed to quantify the levels of c-Fos protein in the NAc. As shown in Fig. 1, the level of c-Fos protein was significantly increased at 120 min after acute cocaine injection (0.881 ± 0.022) compared to the salinetreated group (0.372 ± 0.018) (**p < 0.01 versus the saline-treated
group). We found that pre-treatment with SL 327 significantly attenuated cocaine-induced increase in c-Fos protein expression (0.395 ± 0.018) (## p < 0.01 versus the cocaine-treated group). SL 327 pretreatment alone had no influence on basal level of c-Fos protein expression (0.364 ± 0.020). In order to investigate the role of ERK activation in transcription of c-fos during cocaine exposure, we detected c-fos mRNA levels in NAc using RT-PCR. As shown in Fig. 2, the level of c-fos mRNA significantly increased after acute cocaine injection (0.368 ± 0.020) compared to the saline-treated group (0.104 ± 0.013) (**p < 0.01 versus the saline-treated group). This increase was significantly reversed by SL 327 pretreatment (0.238 ± 0.010) (## p < 0.01 versus the cocaine-treated group) (Fig. 2), although this reverse was not completed (**p < 0.01 versus the saline-treated group) (Fig. 2). To further observe whether inhibition of ERK activation affected the distribution of c-Fos in the NAc after acute cocaine treatment, we performed c-Fos immunofluorescent staining. As shown in Fig. 3, c-Fos-positive cells were increased markedly in both the core and the shell regions of the NAc in response to acute cocaine treatment. SL 327 pretreatment completely abolished cocaine-induced increase in number of Fos-positive cells in the core region (Fig. 3). However, cocaine-induced increase in number of Fos-positive cells was partially attenuated by SL327 in the shell region (Fig. 3). Our results demonstrated that ERK might be involved in acute cocaine-induced c-fos expression at both the transcription and protein levels. In addition, these ERK-dependent c-fos changes induced by acute cocaine exposure occur predominantly in the core region of the NAc. Although c-fos is considered as an indicator of neuronal activity in the central nervous system, evidences have been shown that induction of c-fos is one of the key steps to form drug-induced synaptic plasticity and neuroadaptations [4,14,22]. Several signaling pathways have been identified to participate in cocaine-induced c-fos expression, such as PKA and PKC [11,17]. ERK, which is crucial for cell responses to changes of the environment [16], could be activated after acute cocaine treatment in the brain [8,19]. Radwanska and his college showed that ERK was involved in c-Fos expression induced by chronic (but not acute) cocaine exposure in the mouse amygdala, another brain structure known to be related to an addicted state [15]. In the present study, we found that inhibition of ERK activation by SL 327 could significantly attenuated
X. Guan et al. / Neuroscience Letters 438 (2008) 155–158
157
Fig. 3. Effect of inhibition of ERK on the distribution of c-Fos-positive neurons in the NAc. White arrows mark c-Fos immunopositive cells on the boundary of NAc core. White squares mark c-Fos immunopositive cells on the boundary of NAc shell. Scale bar: 20 m. Data are presented as mean ± S.E.M. (n = 4 mice/group, 3 sections/mouse). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group; # p < 0.05 versus the cocaine-treated group. NAc, nucleus accumbens; aca, anterior part of commissure anterior.
c-Fos protein expression induced by acute cocaine exposure in the NAc. This suggests that ERK might be a critical mediator for acute cocaine-induced c-Fos protein expression in the NAc. NAc, one of main targeting regions for drug of abuse in the central nervous system, has been considered as one of brain structures related to drug-induced behavioral alterations, such as sensitization and tolerance [3]. In the present study, we found that acute cocaine-induced c-Fos expressions in both the core and the shell of the NAc, but inhibition of ERK activation affected cocaine-induced c-Fos protein mainly in the core region of NAc. The core and the shell regions might mediate distinct processes associated with cocaine-induced behavioral abnormalities [5]. For example, selective lesion of the core region could impair drugcue-controlled cocaine-seeking behavior [6]. In contrast, selective damage of the shell region did not affect the acquisition of cocaine-seeking behavior, but it increased the rate of responding after cocaine treatment [2,6,13]. It has been shown that chronic cocaine-induced CPP activated ERK and Fos in the core region, but not in the shell region. In addition, intra-core infusions of U0126, an inhibitor of ERK kinase MEK, prevented both the activation of ERK and Fos, and impaired cocaine-induced CPP [13]. Taken together, these results indicate that the core ERK signaling pathway may be necessary for cocaine-related behavioral alterations. In the current study, we found that ERK was involved in acute cocaine-induced c-fos mRNA levels in the NAc. However, inhibition of ERK activation only partially blocked cocaine-induced c-fos mRNA expression in the NAc. This suggests that other ERKindependent pathway might be involved in cocaine-elevated c-fos mRNA level. Given that the enhanced c-Fos protein expression by cocaine administration in the shell was not strongly attenuated by SL 327 (Fig. 2), it may explain the partial effect of SL 327 on mRNA level in the NAc. The mechanism by which ERK regulate c-fos mRNA is poorly understood. Previous studies reported that gene expression after acute cocaine treatment mostly depended on cAMP response element binding protein (CREB) [7,10]. CREB could regulate c-fos transcription through cAMP response element
(CRE) [10]. It was reported that ERK played a key role in cocaineincreased CREB activation [9]. Thus, it is very likely that CREB might be involved in regulation of ERK in cocaine-induced c-fos mRNA expression.
References [1] D.I. Cho, W. Quan, M.H. Oak, H.J. Choi, K.Y. Lee, K.M. Kim, Functional interaction between dopamine receptor subtypes for the regulation of c-fos expression, Biochem. Biophys. Res. Commun. 357 (2007) 1113–1118. [2] P. Di Ciano, B.J. Everitt, Dissociable effects of antagonism of NMDA and AMPK/KA receptors in the nucleus accumbens core and shell on cocaine-seeking behavior, Neuropsychopharmacology 25 (2001) 341–360. [3] B.J. Everitt, T.W. Robbins, Neural system of reinforcement for drug addiction: from actions to habits to compulsion, Nat. Neurosci. 8 (2005) 1481–1489. [4] A.M. Graybiel, R. Moratalla, H.A. Robertson, Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limibic subdivisions of the striatum, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 6912–6916. [5] R. Ito, J.W. Dalley, S.R. Howes, T.W. Robbins, B.J. Everitt, Dissociation inconditioned dopamine release in the nuleus accumbens core and shell in response to cocaine cues and during cocaine-seeking behavior in rats, J. Neurosci. 20 (2000) 7489–7495. [6] R. Ito, T.W. Robbins, B.J. Everitt, Differential control over cocaine-seeking behavior by nucleus accumbens core and shell, Nat. Neurosci. 7 (2004) 389–397. [7] C. Konradi, R.L. Cole, S. Heckers, S.E. Hyman, Amphetamine regulates gene expression in rat striatum via transcription factor CREB, J. Neurosci. 14 (1994) 5623–5634. [8] L. Lu, E. Koya, H. Zhai, B.T. Hope, Y. Shaham, Role of ERK in cocaine addiction, Trends Neurosci. 29 (2006) 695–703. [9] B.J. Mattson, J.M. Bossert, D.E. Simmons, N. Nozaki, D. Nagarkar, J.D. Kreuter, B.T. Hope, Cocaine-induced CREB phosphorylation in nucleus accumbens of cocaine-sensitized rats is enabled by enhanced activation of extracellular signal-related kinase, but not protein kinase A, J. Neurochem. 95 (2005) 1481–1494. [10] C.A. McClung, E.J. Nestler, Regulation of gene expression and cocaine reward by CREB and DeltaFOSB, Nat. Neurosci. 6 (2003) 1208–1215. [11] J.P. McGillis, C.N. Miller, D.B. Schneider, S. Fernandez, M. Knopt, Calcitonin generelated peptide induces AP-1 activity by a PKA and c-fos-dependent mechanism in pre-Bcells, J. Neuroimmunol. 123 (2002) 83–90. [12] C.A. Miller, J.F. Marshall, Altered Fos expression in neural pathways underlying cue-elicited drug seeking in the rat, Eur. J. Neurosci. 21 (2005) 1385–1393. [13] C.A. Miller, J.F. Marshall, Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory, Neuron 47 (2005) 873–884.
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[14] E.J. Nestler, Molecular basis of long-term plasticity underlying addiction, Nat. Rev. Neurosci. 2 (2001) 119–128. [15] K. Radwanska, J. Caboche, L. Kaczmarek, Extracellular signal-regulated kinases (ERKs) modulate cocaine-induced gene expression in the mouse amygdala, Eur. J. Neurosci. 22 (2005) 939–948. [16] H.J. Schaeffer, M.J. Weber, Mitogen-activated protein kinases: a specific messages from ubiquitous messengers, Mol. Cell Biol. 19 (1999) 2435–2444. [17] H.W. Suh, S.S. Choi, J.K. Lee, H.K. Lee, E.J. Han, J. Lee, Regulation of c-fos and c-jun gene expression by lipopolysaccharide and cytokines in primary cultured astrocytes: effect of PKA and PKC pathways, Arch. Pharm. Res. 27 (2004) 396–401. [18] J. Tao, Y. Zhang, T.W. Soong, S. Li, Expression of urocortin2 and its inhibitory effects on intracellular Ca2+ via L-type voltage-gated calcium channels in rat pheochromocytoma (PC12) cells, Neuropsychopharmacology 31 (2006) 2600–2609.
[19] E. Valjent, A.G. Corbille, J. Bertran-Gonzalez, D. Herve, J.A. Girault, Inhibition of ERK pathway or protein synthesis during reexposure to drugs of abuse erases previously learned place preference, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 2932–2937. [20] V. Zachariou, C.A. Bolanos, D.E. Selley, D. Theobald, M.P. Cassidy, M.B. Kelz, T. Shaw-Lutchman, O. Berton, L.J. Sim-Selley, R.J. Dileone, A. Kumar, E.J. Nestler, An essential role for DeltaFosB in the nucleus accumbens in morphine action, Nat. Neurosci. 9 (2006) 205–211. [21] L. Zhang, D. Lou, H. Jiao, D. Zhang, X. Wang, Y. Xia, J. Zhang, M. Xu, Cocaine-induced intracellular signaling and gene expression are oppositely regulated by the dopamine D1 and D3 receptors, J. Neurosci. 24 (2006) 3344– 3354. [22] J. Zhang, L. Zhang, H. Jiao, Q. Zhang, D. Zhong, D. Lou, J.L. Katz, M. Xu, c-Fos facilitates the acquisition and extinction of cocaine-induced persistent changes, J. Neurosci. 26 (2006) 13287–13296.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Involvement of extracellular signal-regulated protein kinase in acute cocaine-induced c-fos in nucleus accumbens Xiaowei Guan a,b , Jue Hu a , Shengnan Li a,∗ a b
Department of Pharmacology, Nanjing Medical University, 140 HanZhong Road, Nanjing 210029, China Department of Human Anatomy, Nanjing Medical University, Nanjing 210029, China
a r t i c l e
i n f o
Article history: Received 25 January 2008 Received in revised form 23 March 2008 Accepted 7 April 2008 Keywords: C-fos Cocaine Nucleus accumbens Extracellular signal-regulated protein kinase
a b s t r a c t It is well known that acute cocaine administration increases c-Fos expression that is involved in cocaineinduced persistent changes in the central nervous system. In the present study, we investigated a possible involvement of extracellular signal-regulated protein kinase (ERK) in induction of c-fos expression in response to acute cocaine treatment in nucleus accumbens (NAc). We found that inhibition of ERK activation significantly attenuated cocaine-induced c-fos expression at both protein and mRNA levels in the NAc. Furthermore, using an immunofluorescent staining approach, we found that inhibition of ERK activation completely abolished cocaine-induced increase in number of c-Fos-positive cells in the core region of NAc, whereas, in shell region of NAc, inhibition of ERK activation partially attenuated cocaine-induced c-Fos expression. Our findings suggest that ERK might participate in cocaine-induced c-fos expression in the NAc, particularly in the core region of NAc. © 2008 Elsevier Ireland Ltd. All rights reserved.
The nucleus accumbens (NAc) that is composed of the shell and the core parts, plays an important role in the reinforcing effects of drugs [3]. Currently, drug addition is defined solely on the basis of behavioral abnormalities, such as compulsive drug-seeking and taking. The stability of these behavioral abnormalities has been reported to be mediated, at least in part, through changes of gene expression [12,20,21]. c-fos, one of the immediate early genes (IEGs), can be induced by drugs such as amphetamine or cocaine in several brain areas, including NAc [12]. Although c-fos is generally thought as a marker for an increase in neuronal activity, recent studies demonstrated that c-fos might contribute to the cocaineinduced persistent changes in biochemistry and structure of the brain [1,12,22]. Several signaling pathways have been identified to be involved in cocaine-induced c-fos expression [1,11,17]. Recently, the role of extracellular signal-regulated protein kinase (ERK) has attracted much attention. Previous studies showed that acute cocaine administration increased ERK activation in several brain areas, including NAc [8]. Blockade of ERK pathway prevented the cocaine-induced long-lasting behaviors, such as conditioned place preference (CPP) and hyperlocomotor activity [8,19]. These results suggest that ERK might be a critical mediator for cocaine-induced behavioral abnormalities.
∗ Corresponding author. Tel.: +86 25 86863364; fax: +86 25 86863050. E-mail address: [email protected] (S. Li). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.04.035
In the present study, using an acute cocaine treatment models, we determined whether ERK was involved in acute cocaineinduced c-fos expression in the NAc. Male C57BL/6J mice weighing 23–26 g were maintained in standard housing on a 12 h/12 h light/dark cycle. All experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice received intraperitoneal (i.p.) injection of SL 327 [Sigma, dissolved in 20% dimethylsulfoxide (DMSO)] or vehicle (20% DMSO). One hour later, cocaine hydrochloride (20 mg/kg, dissolved in saline, Sigma) or saline was injected intraperitoneally. For immunofluorescent and Western blot, NAc was collected at 2 h post-cocaine or post-saline. For RT-PCR, NAc was collected at 1.5 h post-cocaine or post-saline. The immunofluorescent procedure was based on the protocol described previously [18]. In brief, the sections (at the thickness of 20 m) were pre-incubated with 0.1% BSA and 5% goat serum in PBS for 1 h and then with primary rabbit anti-c-Fos (1:200, CST) dissolved in 4% Triton X-100 and 0.1% BSA in PBS at 4 ◦ C overnight. The sections were incubated with the secondary goat anti-rabbit antibody labeled with Cy3 (1:100, Sigma) at 37 ◦ C for 2 h. Immunofluorescent control was performed by replacing the primary antibodies with PBS buffer. All immunofluorescent positive cells were quantified by software (VNT QuantLab-ST). Nuclear protein was extracted from NAc in accordance with the guidelines of Nuclear Extraction Kit (KangChen Bio-tech). Protein was separated by 10% SDS-PAGE and electrophoretically transferred
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Fig. 1. Effect of inhibition of ERK activation on acute cocaine-induced c-Fos protein expression in the NAc. Top: example of Western blot results. Bottom: statistical analysis. Data are presented as mean ± S.E.M. (n = 12 mice/group). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group.
Fig. 2. Effect of inhibition of ERK activation on acute cocaine-induced c-fos mRNA level in the NAc. Top: example of PCR products. Bottom: statistical analysis. Data are presented as mean ± S.E.M. (n = 12 mice/group). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group.
onto polyvinylidene difluoride membranes. The blots were blocked with 5% non-fat dry milk and 0.1% Tween 20 in 10 mM Tris–HCl (pH 7.5) at room temperature for 2 h, and then incubated with rabbit anti-c-Fos (1:200, CST) or mouse anti--actin (1:1000, Santa Cruz) at 4 ◦ C overnight. The blots were subsequently incubated with HRP-conjugated secondary antibody (1:4000, Santa Cruz) at room temperature for 1.5 h. The bound antibodies were visualized by the ECL system (Amershan Pharmacia Biotech) and exposed to X-OMAT film (Kodak). -Actin was served as an internal control. The densities of immunoblotting bands were quantified by software (Leica Qwin Standard V 2.8). Western blot were repeated at least three times and NAc samples were obtained from different mice. RNA from NAc was extracted using a RNeasy Mini kit (QIAGEN). Total RNA was denatured at 70 ◦ C for 5 min and then reverse transcribed at 42 ◦ C for 1 h. The synthesized cDNA products (4 l) were subjected to PCR in a 20-l reaction mixture containing 2 mM MgCL2 , and 0.5 U Tag DNA polymerase (Promega). Amplification was performed on thermocycler (BioRad, Hemel Hempstead, Herts) with 35 cycles as follows: 15 s at 96 ◦ C, 30 s at 60 ◦ C, and 1 min at 72 ◦ C. Amplified products were run on a 2% agarose gel containing 0.06% g/ml ethidium bromide and visualized under Molecular Image FX (Bio-Rad, Herculer). The sequences of the c-fos primers are as follows: 5 -GGGACAGCCTTTCCTACTACCAT-3 (sense primer) and 5 CAACGCAGACTTCTCATCTTCAA-3 (anti-sense primer). The level of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) mRNA was used as an internal control. The intensity of the products was semi-quantified by software (Leica Qwin Standard V 2.8). The experiments were repeated at least three times in different mice brains. All results were analyzed by one-way ANOVA. Individual mean comparisons were made by using the Newman–Keuls when required. Significant levels were set at p < 0.05. To investigate the role of ERK activation in c-Fos protein expression after acute cocaine treatment, a selective ERK inhibitor, SL 327 (40 mg/kg), was administered i.p. 1 h before cocaine injection. Western blot was performed to quantify the levels of c-Fos protein in the NAc. As shown in Fig. 1, the level of c-Fos protein was significantly increased at 120 min after acute cocaine injection (0.881 ± 0.022) compared to the salinetreated group (0.372 ± 0.018) (**p < 0.01 versus the saline-treated
group). We found that pre-treatment with SL 327 significantly attenuated cocaine-induced increase in c-Fos protein expression (0.395 ± 0.018) (## p < 0.01 versus the cocaine-treated group). SL 327 pretreatment alone had no influence on basal level of c-Fos protein expression (0.364 ± 0.020). In order to investigate the role of ERK activation in transcription of c-fos during cocaine exposure, we detected c-fos mRNA levels in NAc using RT-PCR. As shown in Fig. 2, the level of c-fos mRNA significantly increased after acute cocaine injection (0.368 ± 0.020) compared to the saline-treated group (0.104 ± 0.013) (**p < 0.01 versus the saline-treated group). This increase was significantly reversed by SL 327 pretreatment (0.238 ± 0.010) (## p < 0.01 versus the cocaine-treated group) (Fig. 2), although this reverse was not completed (**p < 0.01 versus the saline-treated group) (Fig. 2). To further observe whether inhibition of ERK activation affected the distribution of c-Fos in the NAc after acute cocaine treatment, we performed c-Fos immunofluorescent staining. As shown in Fig. 3, c-Fos-positive cells were increased markedly in both the core and the shell regions of the NAc in response to acute cocaine treatment. SL 327 pretreatment completely abolished cocaine-induced increase in number of Fos-positive cells in the core region (Fig. 3). However, cocaine-induced increase in number of Fos-positive cells was partially attenuated by SL327 in the shell region (Fig. 3). Our results demonstrated that ERK might be involved in acute cocaine-induced c-fos expression at both the transcription and protein levels. In addition, these ERK-dependent c-fos changes induced by acute cocaine exposure occur predominantly in the core region of the NAc. Although c-fos is considered as an indicator of neuronal activity in the central nervous system, evidences have been shown that induction of c-fos is one of the key steps to form drug-induced synaptic plasticity and neuroadaptations [4,14,22]. Several signaling pathways have been identified to participate in cocaine-induced c-fos expression, such as PKA and PKC [11,17]. ERK, which is crucial for cell responses to changes of the environment [16], could be activated after acute cocaine treatment in the brain [8,19]. Radwanska and his college showed that ERK was involved in c-Fos expression induced by chronic (but not acute) cocaine exposure in the mouse amygdala, another brain structure known to be related to an addicted state [15]. In the present study, we found that inhibition of ERK activation by SL 327 could significantly attenuated
X. Guan et al. / Neuroscience Letters 438 (2008) 155–158
157
Fig. 3. Effect of inhibition of ERK on the distribution of c-Fos-positive neurons in the NAc. White arrows mark c-Fos immunopositive cells on the boundary of NAc core. White squares mark c-Fos immunopositive cells on the boundary of NAc shell. Scale bar: 20 m. Data are presented as mean ± S.E.M. (n = 4 mice/group, 3 sections/mouse). **p < 0.01 versus the saline-treated group; ## p < 0.01 versus the cocaine-treated group; # p < 0.05 versus the cocaine-treated group. NAc, nucleus accumbens; aca, anterior part of commissure anterior.
c-Fos protein expression induced by acute cocaine exposure in the NAc. This suggests that ERK might be a critical mediator for acute cocaine-induced c-Fos protein expression in the NAc. NAc, one of main targeting regions for drug of abuse in the central nervous system, has been considered as one of brain structures related to drug-induced behavioral alterations, such as sensitization and tolerance [3]. In the present study, we found that acute cocaine-induced c-Fos expressions in both the core and the shell of the NAc, but inhibition of ERK activation affected cocaine-induced c-Fos protein mainly in the core region of NAc. The core and the shell regions might mediate distinct processes associated with cocaine-induced behavioral abnormalities [5]. For example, selective lesion of the core region could impair drugcue-controlled cocaine-seeking behavior [6]. In contrast, selective damage of the shell region did not affect the acquisition of cocaine-seeking behavior, but it increased the rate of responding after cocaine treatment [2,6,13]. It has been shown that chronic cocaine-induced CPP activated ERK and Fos in the core region, but not in the shell region. In addition, intra-core infusions of U0126, an inhibitor of ERK kinase MEK, prevented both the activation of ERK and Fos, and impaired cocaine-induced CPP [13]. Taken together, these results indicate that the core ERK signaling pathway may be necessary for cocaine-related behavioral alterations. In the current study, we found that ERK was involved in acute cocaine-induced c-fos mRNA levels in the NAc. However, inhibition of ERK activation only partially blocked cocaine-induced c-fos mRNA expression in the NAc. This suggests that other ERKindependent pathway might be involved in cocaine-elevated c-fos mRNA level. Given that the enhanced c-Fos protein expression by cocaine administration in the shell was not strongly attenuated by SL 327 (Fig. 2), it may explain the partial effect of SL 327 on mRNA level in the NAc. The mechanism by which ERK regulate c-fos mRNA is poorly understood. Previous studies reported that gene expression after acute cocaine treatment mostly depended on cAMP response element binding protein (CREB) [7,10]. CREB could regulate c-fos transcription through cAMP response element
(CRE) [10]. It was reported that ERK played a key role in cocaineincreased CREB activation [9]. Thus, it is very likely that CREB might be involved in regulation of ERK in cocaine-induced c-fos mRNA expression.
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