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. 2013 Dec 1;34(2):277–288. doi: 10.1007/s10571-013-0011-z

Changes in the Levels of p-ERK, p-CREB, and c-fos in Rat Mesocorticolimbic Dopaminergic System After Morphine-Induced Conditioned Place Preference: The Role of Acute and Subchronic Stress

Abbas Haghparast 1,2,✉, Zahra Fatahi 1, Shabnam Zeighamy Alamdary 1, Zahra Reisi 3, Fariba Khodagholi 1
PMCID: PMC11488949  PMID: 24292370

Abstract

ERK pathway plays a critical role in the cellular adaptive responses to environmental changes. Stressful conditions can induce the activation of activate ERK, and its downstream targets, CREB and c-fos, in neural cells. Exposure to opioids has the same effect. In this study, we investigated the effects of morphine-induced conditioned place preference (CPP) on p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the mesocorticolimbic dopaminergic system including the nucleus accumbens (NAc), amygdala (AMY), striatum (Str), and prefrontal cortex (PFC).Our aim was to determine if acute and subchronic stress would affect these alterations. Male Wistar rats were divided into two saline- and morphine-treated groups. Each group contained of control, acute stress, and subchronic stress subgroups. The CPP procedure was performed for all of the rats. We dissected out the NAc, AMY, Str, and PFC regions and measured the mentioned ratios and c-fos level by Western blot analysis. The results revealed that in saline-treated animals, all factors enhanced significantly after performing acute and subchronic stress while there was an exception in p-ERK/ERK ratio in the Str and PFC; the changes were not significant during acute stress. Conditioning score decreased after applying the subchronic but not acute stress. In morphine-treated animals, all factors were increased after application of acute and subchronic stress, and conditioning scores also decreased after stress. Our findings suggest that in saline- or morphine-treated animals, acute and subchronic stress increases p-ERK, p-CREB, and c-fos levels in the mesocorticolimbic system. It has been shown that morphine induces the enhancement of the mentioned factors; on the other hand, our result demonstrates that stress can amplify these changes.

Electronic supplementary material

The online version of this article (doi:10.1007/s10571-013-0011-z) contains supplementary material, which is available to authorized users.

Keywords: Mesocorticolimbic dopaminergic system, ERK, CREB, c-fos, Conditioned place preference, Forced swim stress

Introduction

Mesocorticolimbic (MCL) dopaminergic system consists of the dopamine producing neurons which are located in the ventral tegmental area (VTA). These neurons send projections to the nucleus accumbens (NAc), amygdala (AMY), striatum (Str), and prefrontal cortex (PFC) (Guo et al. 2009; Wang et al. 2004). This system serves as a neural pathway for mediating reinforcement processes (Guo et al. 2009) and behavioral effects of reward (Kest et al. 2012). Previous studies have revealed that exposure to all known drugs of abuse directly or indirectly activates dopamine neurotransmission in the NAc and repeated drug exposure results in enduring alterations of MCL regions, particularly the VTA and NAc (Young et al. 2011). Furthermore, this system has a critical role in the development of morphine dependence (Guo et al. 2009). In this manner, a lesion of the VTA or NAc blocks the reactivation of the conditioned place preference (CPP) induced by morphine priming (Daza-Losada et al. 2008).

Clinical literature shows that exposure to stress in adolescence is a predisposing factor that increases the risk for drug abuse in the upcoming years (McCormick 2010). Early life stressors, both adverse and nurturing, can modify physiological development, notably the limbic–hypothalamic–pituitary–adrenal and associated systems which then results in vulnerability to drug-seeking, drug-taking behavior, addiction, or dependence (Gordon 2002). Nevertheless, it was found that chronic stress in adult rats (without exposure to earlier stress) paired with morphine suppresses the acquisition of preferences for a distinctive environment (Papp et al. 1992). Stress can stimulate the activation of kappa opioid receptors in the MCL, and thereby causes conditioned place aversion. Kappa receptors inhibit dopamine release in the MCL and also have an important role in cocaine or morphine-induced aversions (Davis et al. 2009; Kim et al. 2004; Land et al. 2009).

It is well known that exposure to the drug of abuse and stressful situations can affect intracellular signaling pathways and cause changes in extracellular signal-regulated kinase (ERK) activation which, in turn, triggers an essential pathway within cells that generates adaptive response to environmental alterations. ERK has many cellular targets and influences a wide range of cellular functions, including synaptic plasticity, learning, memory formation, arousal, apoptosis, and so on. In this manner, ERK inhibition prevents the formation of lasting memories of an event or association, including spatial memory, fear memory, and object recognition memory (Imbe et al. 2004; Shiflett and Balleine 2011). Cyclic AMP responsive element-binding protein (CREB) is a transcription factor and a downstream target of ERK pathway (Qi et al. 2008). CREB has been shown to be essential for different physiological responses like development of behavioral sensitization, increased drug-taking, and emotional behaviors by binding to different target genes (Leao et al. 2012; Wan et al. 2009). CREB also plays an important role as a molecular basis in cannabinoid, opioid, and alcohol dependencies (Haghparast et al. 2011). On the other hand, it was propounded that c-fos is an immediate early gene (lEG) and another ERK downstream target. The IEGs are considered to reflect neuronal activities through controlling gene transcription by binding of their protein forms to regulatory sites on the DNA (Nathaniel et al. 2012). Therefore, in the present study, we aimed to investigate the effects of morphine-induced CPP on the alterations of p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the MCL dopaminergic system and/or determine if acute and subchronic stress could affect these alterations or no.

Materials and Methods

Animals

Male Wistar rats (W: 230–280 g; Pasture Institute, Tehran, Iran) were housed in standard cages under controlled temperature (22 ± 2 °C) and light conditions (lights on at 06:00, lights off at 18:00). Food and water were supplied with no limitation, except during the experiments. All investigations and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the local ethical committee, Shahid Beheshti University of Medical Sciences.

Materials

In the current study, the following materials were used: morphine sulfate was bought from Temed Co., Iran. Antibodies directed against p-ERK 1/2, ERK 1/2, p-CREB, CREB, c-fos, β-actin, and secondary antibody IgG were purchased from Cell Signaling Technology, USA. Enhancedchemiluminescence (ECL) kit was obtained from Amersham Bioscience, USA. Morphine sulfate was dissolved in 0.9 % sterile saline at a concentration of 5 mg/ml and injected subcutaneously (s.c.).

Behavioral Test

Conditioning Apparatus and Paradigm

A three-compartment CPP apparatus was used in this study. The apparatus was made of Plexiglas and two compartments were identical in size (30 × 30 × 40 cm3), but differed in shading and texture. Compartment A was white with black horizontal stripes 2 cm wide on the walls and also had a net-like floor. Compartment B was black with vertical white stripes, 2 cm wide and also had a smooth floor. The third compartment, C, was a red tunnel (30 × 15 × 40 cm3). It protruded from the rear of the two large compartments and connected the entrances of them. In this apparatus, rats showed no consistent preference for either large compartments (A and B), which supports our un-biased CPP paradigm. CPP protocol was performed in 5 continuous days consisting three distinct phases: pre-conditioning, conditioning and post-conditioning.

Pre-conditioning Phase

On the first day, each rat was placed separately into the apparatus for 10 min with free access to all compartments (A, B, and C), and the amount of time spent in each compartment was measured to assess unconditioned preference. In the particular experimental setup that we used in our study, the animals did not show a significant preference for either of the compartments (A and B) during preconditioning phase.

Conditioning Phase

This phase started 1 day after pre-conditioning phase. It consisted of six 30-min sessions (three saline and three morphine pairing) in a 3-day procedure. These sessions were performed twice daily, with a 6-h interval. On each day, two conditioning session was performed for each group one with morphine and the other with saline. During 30-min session intervals for morphine/saline, the rats were confined to one of the compartments by closing the removable wall of the apparatus. This phase consisted of a 3-day schedule of conditioning sessions. Three trials in which rats ordered morphine (5 mg/kg), while confined to one large compartment for 30 min, and the other three trials in which they ordered saline (1 ml/kg) while confined to the other large compartment by closing the removable wall. Access to the compartments was blocked on these days (acquisition period).

Post-conditioning Phase

On the fifth day (test day), the wall was removed, and each animal was allowed to freely access to all parts of the apparatus. The mean time spent for each rat in both large compartments was recorded by Ethovision software (Version 3.1), a video tracking system for automation of behavioral experiments (Noldus Information Technology, The Netherlands). The conditioning score was calculated as the time spent in the drug-paired compartment minus the time spent in saline-paired compartment.

Forced Swim Stress

The forced swim stress (FSS) was performed in a Plexiglas tank (50 cm height × 30 cm diameter) filled with 35-cm depth of water (24–27 °C). Each rat was forced to swim individually for 6 min once a day. Acute stress conducted in a single session, while the animals in the subchronic group swam for three consecutive days 6 min per day.

Western Blot Analysis

For Western blot analysis, rats were sacrificed after CPP paradigm performed. The brains were dissected out and put into a glass plate on the ice, and then the NAc, AMY, Str, and PFC were collected according to Paxinos and Watson atlas (Paxinos and Watson 2007). Tissues were sonicated in 1 % sodium dodecyl sulfate buffer in Tris–EDTA, pH 7.4, containing 1× protease inhibitor cocktail, 5 mM NaF, and 1× phosphatase inhibitor cocktail. Samples were boiled for 5 min and centrifuged at 16,100×g for 10 min. Then, the total proteins were electrophoresed in 12 % SDS–PAGE gels, transferred to polyvinylidene fluoride membranes and probed with specific antibodies. Immunoreactive polypeptides were detected by chemiluminescence using enhanced ECL reagents and subsequent autoradiography. Quantification of the results was performed by densitometric scan of films. Data analysis was done by ImageJ, measuring integrated density of bands after background subtraction. Protein concentrations were determined according to Bradford’s method (1976).

Experimental Design

Animals were divided into two main supergroups, saline- and morphine-treated animals. Each supergroup consists of control, acute stress and subchronic stress groups. In all groups (n = 6–7/group for behavioral studies and 3–4/group for molecular studies), animals passed CPP protocol (Electronic supplementary Fig. 1A). In saline-treated animals, control group received saline (1 ml/kg) during the conditioning phase without any stress, acute stress group received saline during the conditioning phase and also FSS on the post-conditioning phase (test day) 10 min before the CPP test, and subchronic stress group received FSS during 3-day schedule conditioning phase, 10 min before saline injection once a day. In morphine-treated animals, control group was treated with morphine (5 mg/kg) during the conditioning phase, acute stress group received morphine in the conditioning phase, also FSS on the post-conditioning phase 10 min before the test (Electronic supplementary Fig. 1B), and in the subchronic stress group, animals exposed to FSS during the conditioning phase 10 min prior to the administration of morphine (Electronic supplementary Fig. 1C). Conditioning score was calculated for each rat on the test day.

For measuring the alterations of p-ERK, p-CREB and c-fos levels, on the fifth day, the animals were sacrificed and the brains were removed. Then the NAc, AMY, Str, and PFC were immediately dissected out and delivered into the liquid nitrogen. Tissues were prepared for Western blot analysis. In each area, p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level were evaluated.

Statistics

All behavioral and molecular data are shown as mean ± SEM (standard error of mean) and in order to compare the conditioning scores in control and experimental groups, two- and one-way analysis of variance (ANOVA) followed by post-hoc Newman–Keuls and Bonferroni tests were used, respectively. In molecular section, Western blot analysis, the optical densitometric data were analyzed by two- or one-way ANOVA followed by post-hoc Bonferroni test or Newman–Keuls multiple comparison test is needed, respectively. The level of statistical significance was set at P < 0.05 and calculations were performed by using GraphPad Prism (Version 5.0) software.

Results

In behavioral experiments, for determining the effects of acute and subchronic stress on the change in conditioning scores, the animals passed CPP stages according to the CPP protocol and afterward, conditioning score was calculated. Electronic supplementary Fig. 2 shows that there are significant differences in conditioning scores between with/without stress groups in saline- and morphine-treated animals [stress effect: F(2,35) = 25.77, P = 0.0004; morphine treatment: F(1,35) = 1.02, P = 0.4413; stress × morphine treatment: F(2,35) = 26.4, P = 0.0003]. These data also clarified that in saline-treated animals, conditioning scores did not have any alteration in respective saline-control group after application of acute stress but it decreased after performing subchronic stress [F(5,40) = 8.271, P < 0.0001]. On the other hand, in morphine-treated animals, conditioning scores decreased after application of both acute and subchronic stress (Electronic supplementary Fig. 2).

Alterations of p-ERK/ERK Ratio, p-CREB/CREB Ratio, and c-fos Level in the Nucleus Accumbens After Application of Acute and Subchronic Stress in the CPP Paradigm

As shown in Fig. 1, two-way ANOVA followed by Bonferroni test for p-ERK/ERK ratio [stress effect: F(2,12) = 54.59, P < 0.0001; morphine treatment: F(1,12) = 23.96, P = 0.0004; stress × morphine treatment: F(2,12) = 13.37, P = 0.0009; Fig. 1a], p-CREB/CREB ratio [stress effect: F(2,12) = 97.97, P < 0.0001; morphine treatment: F(1,12) = 59.45, P < 0.0001; stress × morphine treatment: F(2,12) = 17.79, P = 0.0003; Fig. 1b] and for c-fos level [stress effect: F(2,12) = 775, P < 0.0001; morphine treatment: F(1,12) = 250.2, P < 0.0001; stress × morphine treatment: F(2,12) = 14.31, P = 0.0007; Fig. 1c] revealed that application of acute and subchronic stress significantly increased the p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the saline- or morphine-treated animals, also the increasing of these proteins in the morphine-control group was rather than those in the saline-control group. One-way ANOVA followed by Newman–Keuls test showed that increasing of p-ERK/ERK ratio [F(5,17) = 31.98, P < 0.0001] in the morphine-treated animals was more considerable than that of the saline-treated animals after application of acute (P < 0.01) and subchronic stress (P < 0.05) as shown in Fig. 1a. The increasing of p-CREB/CREB ratio after application of acute and subchronic stress (P < 0.01) was also significant between the saline- and morphine-treated animals [F(5,17) = 58.2, P < 0.0001; Fig. 1b]. Furthermore, one-way ANOVA [F(5,17) = 365.5, P < 0.0001] showed that increase of c-fos level in morphine-treated animals was more remarkable than that of saline-treated animals after application of acute and subchronic stress (P < 0.001; Fig. 1c).

Fig. 1.

Fig. 1

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Effects of acute and subchronic stress on the change in a p-ERK/ERK ratio, b p-CREB/CREB ratio, c c-fos level in the nucleus accumbens in saline- and morphine-treated animals in conditioned place preference paradigm. Upper panels are the representative immunoblots of proteins in this area. Bottom graphs show the mean p-ERK/ERK ratio, the mean p-CREB/CREB ratio and the mean c-fos level calculated from densitometric quantification of the corresponding bands. In first two bars the animals received saline or morphine in conditioned place preference protocol, respectively. Each point shows the mean ± SEM for 3–4 rats. *P < 0.05, **P < 0.01, ***P < 0.001 different from the saline-control group. † P < 0.05, †† P < 0.01, ††† P < 0.001 different from the morphine-control group

Alterations of p-ERK/ERK Ratio, p-CREB/CREB Ratio, and c-fos Level in the Amygdala After Application of Acute and Subchronic Stress in the CPP Paradigm

As demonstrated in Fig. 2, two-way ANOVA followed by Bonferroni test for p-ERK/ERK ratio [stress effect: F(2,12) = 127.1, P < 0.0001; morphine treatment: F(1,12) = 97.94, P < 0.0001; stress × morphine treatment: F(2,12) = 36.32, P < 0.0001; Fig. 2a], p-CREB/CREB ratio [stress effect: F(2,12) = 176.2, P < 0.0001; morphine treatment: F(1,12) = 103.5, P < 0.0001; stress × morphine treatment: F(2,12) = 20.2, P = 0.0002; Fig. 2b], and for c-fos level [stress effect: F(2,12) = 85.52, P < 0.0001; morphine treatment: F(1,12) = 11.58, P = 0.0052; stress × morphine treatment: F(2,12) = 7.436, P = 0.0079; Fig. 2c] indicated that in the saline- or morphine-treated animals, aforementioned proteins significantly increased after applying acute and subchronic stress models as well as enhancement of proteins in the morphine-control group was more than the saline-control group. One-way ANOVA followed by Newman–Keuls test [F(5,17) = 84.96, P < 0.0001] showed that in the morphine-treated animals, increasing of p-ERK/ERK ratio was more remarkable than the saline-treated animals after acute (P < 0.001) and subchronic stress (P < 0.01) application as shown in Fig. 2a. The ratio of p-CREB/CREB after applying acute (P < 0.001) and subchronic stress (P < 0.01) showed a more noteworthy increase in the morphine-treated animals than that in the saline-treated animals [F(5,17) = 99.27, P < 0.0001; Fig. 2b]. In addition, c-fos level [F(5,17) = 39.5, P < 0.0001; Fig. 2c] increased in the morphine-treated animals rather than the saline-treated animals after application of acute (P < 0.01) and subchronic stress (P < 0.001).

Fig. 2.

Fig. 2

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Effects of acute and subchronic stress on the alternation of a p-ERK/ERK ratio, b p-CREB/CREB ratio, c c-fos level in the amygdala in saline- and morphine-treated animals in conditioned place preference paradigm. Upper panels are the representative immunoblots of proteins in this area. Bottom graphs show the mean p-ERK/ERK ratio, the mean p-CREB/CREB ratio and the mean c-fos level calculated from densitometric quantification of the corresponding bands. In first two bars the animals received saline or morphine in conditioned place preference protocol, respectively. Each point shows the mean ± SEM for 3–4 rats. **P < 0.01, ***P < 0.001 different from the saline-control group. † P < 0.05, †† P < 0.01, ††† P < 0.001 different from the morphine-control group

Alterations of p-ERK/ERK Ratio, p-CREB/CREB Ratio, and c-fos Level in the Striatum After Application of Acute and Subchronic Stress in the CPP Paradigm

Two-way ANOVA followed by Bonferroni test for p-ERK/ERK ratio [stress effect: F(2,12) = 195.9, P < 0.0001; morphine treatment: F(1,12) = 94.83, P < 0.0001; stress × morphine treatment: F(2,12) = 49.16, P < 0.0001; Fig. 3a], p-CREB/CREB ratio [stress effect: F(2,12) = 190.6, P < 0.0001; morphine treatment: F(1,12) = 69.57, P < 0.0001; stress × morphine treatment: F(2,12) = 27.01, P < 0.0001; Fig. 3b], and for c-fos level [stress effect: F(2,12) = 592.1, P < 0.0001; morphine treatment: F(1,12) = 466.7, P < 0.0001; stress × morphine treatment: F(2,12) = 175.7, P < 0.0001; Fig. 3c] revealed that in the saline-treated animals p-CREB/CREB ratio and c-fos level increased significantly after application of acute and subchronic stress as compared with the saline-control group but increasing of p-ERK/ERK ratio after application of the acute stress model was not significant despite its significant increase after application of subchronic stress model. In the morphine-treated animals, mentioned factors enhanced significantly after application of acute and subchronic stress these factors also showed a more considerable increasing in the morphine-control group compared to the saline-control group. One-way ANOVA followed by Newman–Keuls test revealed that increase of p-ERK/ERK ratio [F(5,17) = 117, P < 0.0001] after performing the acute and subchronic stress models (P < 0.001) indicated a significant difference between the saline- and morphine-treated animals (Fig. 3a). Enhancement of p-CREB/CREB ratio after application of acute (P < 0.01) and subchronic stress (P < 0.001) was more significant in morphine-treated animals [F(5,17) = 101.3, P < 0.0001; Fig. 3b]. Also, the level of c-fos [F(5,17) = 760.2, P < 0.0001] increased in the morphine-treated animals more considerable than that of the saline-treated rats after application of acute and subchronic stress (P < 0.001; Fig. 3c).

Fig. 3.

Fig. 3

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Effects of acute and subchronic stress on the alternation of a p-ERK/ERK ratio, b p-CREB/CREB ratio, c c-fos level in the striatum (Str) in saline- and morphine-treated animals in conditioned place preference paradigm. Upper panels are the representative immunoblots of proteins in this area. Bottom graphs show the mean p-ERK/ERK ratio, the mean p-CREB/CREB ratio and the mean c-fos level calculated from densitometric quantification of the corresponding bands. In first two bars the animals received saline or morphine in conditioned place preference protocol, respectively. Each point shows the mean ± SEM for 3–4 rats. *P < 0.05, **P < 0.01, ***P < 0.001 different from the saline-control group. † P < 0.05, †† P < 0.01, ††† P < 0.001 different from the morphine-control group

Alterations of p-ERK/ERK Ratio, p-CREB/CREB Ratio, and c-fos Level in the Prefrontal Cortex After Application of Acute and Subchronic Stress in the CPP Paradigm

As shown in Fig. 4, two-way ANOVA followed by Bonferroni test for for p-ERK/ERK ratio [stress effect: F(2,12) = 34.84, P < 0.0001; morphine treatment: F(1,12) = 15.55, P = 002; stress × morphine treatment: F(2,12) = 2.361, P = 0.1365; Fig. 4a], p-CREB/CREB ratio [stress effect: F(2,12) = 109.6, P < 0.0001; morphine treatment: F(1,12) = 42.14, P < 0.0001; stress × morphine treatment: F(2,12) = 8.756, P = 0.0045; Fig. 4b], and for c-fos level [stress effect: F(2,12) = 228.6, P < 0.0001; morphine treatment: F(1,12) = 130.3, P < 0.0001; stress × morphine treatment: F(2,12) = 24.45, P < 0.0001; Fig. 4c] shows that in the saline- or morphine-treated animals all proteins increased significantly after application of acute and subchronic stress compared to the saline- or morphine-control groups except for the p-ERK/ERK ratio in the saline-treated animals which was not significant after application of acute stress. We also observed a significant increase in the levels of these factors in the morphine-control group in comparison to the saline-control group. One-way ANOVA followed by Newman–Keuls test revealed that enhancement of p-ERK/ERK ratio [F(5,17) = 17.99, P < 0.0001; Fig. 4a] after exerting acute (P < 0.05) and subchronic stress (P < 0.01) models, p-CREB/CREB ratio [F(5,17) = 55.79, P < 0.0001; Fig. 4b] after application of acute (P < 0.01) and subchronic stress (P < 0.001), and c-fos level [F(5,17) = 127.3, P < 0.0001; Fig. 4c] after exertion of acute and subchronic stress (P < 0.01) in the morphine-treated animals had a more noticeable increase than the saline-treated animals.

Fig. 4.

Fig. 4

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Effects of acute and subchronic stress on the change in a p-ERK/ERK ratio, b p-CREB/CREB ratio, c c-fos level in the prefrontal cortex (PFC) in saline- and morphine-treated animals in conditioned place preference paradigm. Upper panels are the representative immunoblots of proteins in this area. Bottom graphs show the mean p-ERK/ERK ratio, the mean p-CREB/CREB ratio and the mean c-fos level calculated from densitometric quantification of the corresponding bands. In first two bars the animals received saline or morphine in conditioned place preference protocol, respectively. Each point shows the mean ± SEM for 3–4 rats. *P < 0.05, **P < 0.01, ***P < 0.001 different from the saline-control group. † P < 0.05, †† P < 0.01 different from the morphine-control group

Discussion

The gathered data in this study revealed that in the saline- or morphine-treated animals, application of acute and subchronic stress reduced conditioning scores (except for acute stress in saline-treated animals) and enhanced p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the mesocorticolimbic dopaminergic system. In this study, we used forced swimming to induce stress because it has been shown that forced swimming results in adrenocorticotrophic hormone and corticosterone release. Both hormones increased significantly up to 40 min after stress (Abel 1994; Rittenhouse et al. 2002; Shishkina et al. 2010). We observed that in saline-treated animals, p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level enhanced significantly after application of acute and subchronic stress in all investigated regions. However, the increase of p-ERK/ERK ratio in the Str and PFC after application of acute stress was not significant. It has been shown that ERK is one of the important regulators of neuronal functions (Shiflett and Balleine 2011). A large number of extra cellular stimuli such as exposure to stress or drug of abuse activates ERK and its downstream targets including CREB and c-fos. ERK pathway plays a critical role in the induction of processes such as proliferation, differentiation, development, learning, apoptosis, and morphology determination. This pathway influences translation and new protein synthesis (Shaul and Seger 2007; Xu et al. 2012). c-Fos protein binds to the DNA and regulates transcription of various target genes. The basal level of fos expression in neurons is low, but it transiently increases via second messenger systems following stimulation (Nathaniel et al. 2012). Fos contributes to the initiation and coordination of molecular cascades and cellular events that are required for neuroplasticity, and therefore has an established role in learning and memory (Johnson et al. 2010). In this study, application of acute and subchronic stress caused a significant increase in the p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the mentioned areas except for the p-ERK/ERK ratio in the Str and PFC after acute stress, but since in these regions ERK downstream targets (p-CREB and c-fos) increased significantly, it is conceivable that p-ERK/ERK ratio increased significantly after performing acute stress, but this enhancement was transient and returned to the basal level when we dissected out the tissues. Although our findings revealed that acute and subchronic stress enhanced the p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the MCL, there is contradictory evidence concerning the effect of stress on alteration of these factors. Imbe et al. (2004) showed that chronic restraint stress induced a significant increase and decrease in p-ERK in rostral ventromedial medulla (RVM) and in locus coeruleus (LC), respectively. Furthermore, acute restraint stress produced a significant increase in fos-immunoreactive in the RVM. Beside, Gronli et al. (2006) reported that the application of unpredictable mild stress for 5 weeks inhibits phosphorylation of CREB in the dentate gyrus, whereas it does not affect the hippocampus (HIP) considerably. Another study cited that chronic forced swim stress for 14 consecutive days decreases the ratio of p-ERK to total ERK in the HIP and PFC (Qi et al. 2006). Additionally, it was demonstrated that exposure to footshock stress enhances CREB activity in the NAc shell (Muschamp et al. 2011) and acute tail shock stress induces hyperactivation of ERK in the HIP (Yang et al. 2004). Therefore, it seems that the effect of stress on the increase or decrease of p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the neural cells depends on three parameters (i) stress protocol; (ii) duration of stress application; and (iii) the experimented regions of the brain. In addition, behavioral results in these animals explained that, conditioning score decreased after application of subchronic, but not acute stress. It was reported that stress results in conditioned place aversion (Land et al. 2009) and chronic exposure to mild unpredictable stress reduces or abolishes the acquisition of place preference conditioning (Papp et al. 1992). In this manner, we can conclude that repeated (but not single) exposure to swim stress could induce place aversion.

The obtained result in the morphine-treated animals revealed that acute and subchronic stress increases the p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level significantly in all cited areas compared to the saline- or morphine-control groups. Notably, these proteins significantly enhanced in the morphine-control group in comparison to the saline-control group. This finding points to the role of morphine-CPP on the increase of mentioned proteins in the MCL system. Consistently, it was shown that p-CREB increased in the HIP by morphine and nicotine-induced CPP (Moron et al. 2010; Pascual et al. 2009). Also, a significant increase in the ERK activity was observed in the VTA following morphine-induced CPP (Lin et al. 2010). Additionally, it was explained that acute nicotine administration increases c-fos mRNA expression in several brain regions, including the NAc, AMY, LC, paraventricular nucleus of the hypothalamus, and lateral septum in adolescent and adult rats (Shram et al. 2007). Another study revealed that chronic morphine administration could cause an up-regulation in the levels of CREB mRNA in the HIP and PFC (Wan et al. 2009). Shiflett and Balleine (2011) clarified that pairing a context with drug exposure resulted in a CPP, and ERK inhibition prior to drug-context pairing prevented the formation of this preference, and Walters et al. (2005) also reported that a single administration of an opioid receptor antagonist, naloxone, blocks both CREB phosphorylation and nicotine reward circuit in a place preference paradigm. Nonetheless, there is some evidence to confirm that drugs of abuse have a decreasing effect on the level of these factors. For example, Guitart et al. (1992) demonstrated that acute administration of morphine into the LC results in the decrease of CREB phosphorylation in this region and Yang and Pu (2009) showed that chronic morphine treatment twice daily for four consecutive days with increasing doses on each day leads to the decrease of p-CREB in the HIP. Also, another study revealed that acute administration of morphine intravenously produces a decrease in the level of c-fos in the spinal cord (Catheline et al. 1999). So, it can be supposed that the effect of drug of abuse on the alterations of p-ERK, p-CREB, and c-fos relates to different parameters including duration of drug receiving, route of administration and dosage of the drug. In these animals induction of both acute and subchronic stress decreased conditioning scores. Provided evidence showed that stress leads to release of dynorphin, a neuropeptide that binds to kappa opioid receptors and activates ERK/CREB pathway (Bruchas et al. 2010). ERK signaling in the nervous system has a critical role in memory formation and adaptive responses to environmental changes (Shiflett and Balleine 2011). Therefore, we can suppose that ERK/CREB pathway resulted in memory formation and consequently aversion to the compartment that the animal placed in after receiving stress. However, further studies are needed to prove this result.

On the other hand, the effects of acute and subchronic stress on the enhancement of p-ERK, p-CREB, and c-fos levels in morphine-treated animals were more noticeable than saline-treated animals. It is well known that stressful experiences causes the release of dopamine in the NAc and leads to profound and long-lasting alterations of MCL system function (Fanous et al. 2010; Trainor 2011). Also, Ikemoto and Goeders (1998) demonstrated that stress stimulates hypothalamic–pituitary–adrenal axis activity which in turn results in the MCL dopamine release. Hence, with regard to the effect of stress on the increase of dopamine release in the MCL and as for significant enhancement of p-ERK, p-CREB, and c-fos levels after application of acute and subchronic stress in the morphine-treated animals in comparison the saline-treated animals, we suggest that stress amplifies the effects of morphine on the increase of cited factors in the MCL system. In addition, although our results showed that stress suppressed the acquisition of morphine-CPP but as mentioned above stress results in the release of dopamine in the NAc, causes long-lasting changes in MCL system function and increases risk of drug abuse. Base on this evidence, stress may cause the aversion to the environment which the animal received stress there but it generally increases risk for drug abuse and drug-taking behavior. Our results accompany with future supplementary studies maybe helpful to find a new clinical approach for treatment of addiction. In conclusion, these findings points that in the saline- or morphine-treated animals application of acute and subchronic forced swim stress decreases conditioning scores (excluding acute stress in the saline-treated animals) and increases the p-ERK/ERK ratio, p-CREB/CREB ratio, and c-fos level in the MCL system. We also suggest that concurrent exposure to stress and morphine can amplify the increase of these factors compared with those when stress or morphine is applied alone.

Electronic supplementary material

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Supplementary material 2 (DOCX 71 kb) (71.1KB, docx)

Acknowledgments

This work was supported by the Grant (No. 91003540) from Iran National Science Foundation, Tehran, Iran.

Conflict of interest

There are no conflicts of interest.

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Supplementary material 1 (DOCX 64 kb) (64.3KB, docx)
Supplementary material 2 (DOCX 71 kb) (71.1KB, docx)

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