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Published in final edited form as: Behav Pharmacol. 2009 Oct;20(7):576–583. doi: 10.1097/FBP.0b013e32832ec57e

Extracellular signal-regulated kinase activation in the amygdala mediates elevated plus maze behavior during opioid withdrawal

Rebecca S Hofford 1, Stephen R Hodgson 1, Kris W Roberts 2, Camron D Bryant 3, Christopher J Evans 2, Shoshana Eitan 1,*
PMCID: PMC4494789  NIHMSID: NIHMS202793  PMID: 19738463

Abstract

This study examined whether activation of extracellular signal-regulated kinase (ERK) contributes to the increased open-arm time observed in the elevated plus maze (EPM) during opioid withdrawal. We applied SL327, a selective ERK kinase (MEK) inhibitor, to specific limbic areas and examined the effect on EPM behaviors of controls and during naloxone-precipitated morphine withdrawal. We next confirmed that ERK activation increased in limbic areas of mice undergoing naloxone-precipitated morphine withdrawal. Direct injection of SL327 into the amygdala blocked the withdrawal-induced increase in open-arm time; however, injecting SL327 into the septum had no effect. Consistent with these results, both 0.2 and 2 mg/kg naloxone increased ERK activation in the central amygdala of morphine-dependent mice. In drug-naïve mice, 2 mg/kg naloxone, but not 0.2 mg/kg, increased ERK activation in the central amygdala. During withdrawal, increased ERK activation was also observed in the lateral septum. In the LC, a significant increase was only observed in morphine-dependent mice receiving 2 mg/kg, but not 0.2 mg/kg, naloxone. It is concluded that ERK activation in limbic areas is likely involved in both the aversive properties of naloxone and in the affective/emotional symptoms of opioid withdrawal, including mediating EPM behaviors.

Keywords: Morphine, Naloxone, Dependence, Mitogen-activated protein kinase (MAPK), Opioid signaling, mouse

INTRODUCTION

Theories of addiction stipulate that the maintenance of drug abuse, despite the severe consequences, is in part driven by the desire to avoid the precipitation of withdrawal or to escape the withdrawal state (Schulteis and Koob, 1996; Koob et al., 1997). Like humans, opioid withdrawal in rodents also elicits numerous somatic and affective signs. We recently demonstrated that during both naloxone-precipitated and spontaneous morphine withdrawal, mice exhibit an increase in the time spent in the open arms of the elevated plus maze (EPM) (Hodgson et al., 2008, Buckman et al., 2008). We hypothesized that this increase in open-arm time might be explained by the different emotionality, motivation and defensive patterns triggered by withdrawal. Thus, the EPM behaviors of mice undergoing withdrawal may represent a change in their defensive strategies due to an increased motivation to escape. An increase in the motivation to escape might also induce an increase in exploration and/or risk-taking behaviors similar to the behaviors observed in addicts (Hodgson et al., 2008).

Multiple studies point to the importance of the central amygdala, the extended amygdala and the lateral septum in regulating the affective responses during morphine withdrawal (Aston-Jones et al, 1999; Gracy et al, 2001; Frenois et al, 2002; Watanabe et al., 2002, 2003; Veinante et al., 2003; Hamlin et al, 2004; Jin et al., 2005; Nakagawa et al., 2005). Moreover, an increase in ERK activation in the central amygdala was recently demonstrated to be involved in cue-induced drug-seeking during opioid withdrawal (Li et al., 2008). An increase in phospho-ERK (the activated form of ERK) during naloxone-precipitated withdrawal was also observed in the locus coeruleus (LC), solitary tract, hypothalamus (Schulz and Hollt, 1998), cortex, and striatum (Asensio et al., 2006), as well as in the spinal cord (Cao et al., 2005, 2006) and the heart (Almela et al., 2007). In the spinal cord, ERK activation contributes to the precipitation of somatic signs (Cao et al., 2005, 2006). In the heart, ERK was demonstrated to contribute to adaptive processes induced by opioid withdrawal, such as c-Fos expression (Almela et al., 2007).

Given the role of the ERK pathway in withdrawal-induced behaviors, this study examines the involvement of ERK in mediating EPM behavior during withdrawal. Moreover, given the importance of the limbic system in the manifestation of different withdrawal affective signs, we specifically examined the effects of localized ERK inhibition in the amygdala and septum during naloxone-precipitated withdrawal.

METHODS

Subjects and drugs

All procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee. Male C57BL/6 mice (9-10 weeks old; Harlan Lab, Houston, TX) were housed 4-5 per cage with food and water ad lib. They were housed in a temperature-controlled vivarium with a 12 h/12 h light/dark cycle (lights on 07.00h). Morphine sulfate, naloxone hydrochloride, and SL327 were purchased from Sigma (St. Louis, MO). Placebo and 25 mg morphine pellets were supplied by the National Institute on Drug Abuse (NIDA). Separate mice were used for the immunohistochemical and behavioral analyses.

Stereotaxic manipulation

Cannulae (23 gauge) were inserted bilaterally into the amygdala [position relative to bregma: anteroposterior (AP) -1 mm, lateral (Lat) ±3.2 mm] and unilaterally into the septum [position relative to bregma: AP +1.5 mm, Lat 0 mm] using Kopf's 1900 Stereotaxic Alignment instrument (Tujunga, CA). Mice were given at least one week to recover prior to starting the experiments. To keep the cannulae open and flowing, mice received intra-cannula saline injections during this week. All intra-cannula injections were performed using 30 gauge inserts 4.2mm deep for the amygdala and 3mm deep for the septum. At the end of the experiment, the placement of the cannulae was confirmed via dye injections by an observer blind to the treatments, and any mice with incorrect cannula placement were excluded. We have about 90% success rate in the correct placement of bilateral cannulas.

Elevated plus maze (EPM)

All procedures, except the SL327 and DMSO injections, were performed in identical manner to our earlier EPM study as described in Hodgson et al. (2008). Mice were injected twice daily (9 a.m. and 5 p.m.) for 3 consecutive days with saline or increasing doses of morphine (10-40 mg/kg, s.c.) for a total of 6 injections. Specifically, on day 1 the mice were injected with saline or 10 mg/kg morphine. On day 2, mice were injected with saline or 20 mg/kg morphine. On day 3, they were injected with saline or 40 mg/kg morphine. A final dose of 20 mg/kg (s.c.) was administered on day 4. One hour later, the mice were injected with SL327 or DMSO directly into the amygdala or septum. Intra-cannula injections of SL327 and DMSO were 0.2 μl per side for the amygdala and 0.4 μl for the septum, and were administered over a 10-minute period. Each insert was withdrawn 5 minutes after completing the injection. One hour later (i.e. 2 hours post-morphine), the mice were injected s.c. with saline or 0.2 mg/kg naloxone. A volume of 10 ml/kg was used for the saline, morphine, and naloxone injections. Five minutes following the saline or naloxone injections, mice were examined for EPM behaviors. EPM tests were performed between 11.00m and 13.00h. Mice were habituated to the room for at least 30 minutes prior to testing. The plus-maze apparatus consisted of four arms elevated 63.8 cm above the floor, with each arm (87 mm wide, 155 mm long) positioned at 90° relative to the adjacent arms. Two arms were enclosed on two sides by 16.3 cm high opaque walls, and the other two arms were open. Mice were placed in the center of the maze facing toward a closed arm and recorded for 10 minutes by an overhead camera. The apparatus was thoroughly cleaned between mice. Behaviors were scored by an observer blind to the treatments. For each mouse, the following behaviors were monitored: 1) length of time (in seconds) spent in the open arms, starting when all four legs had crossed the entrance line to one of the open arms; 2) total activity, defined as the total number of entries or exits into or out of any arm.

Phospho-ERK staining

Mice were injected twice daily (09.00 and 17.00h) for 3 consecutive days with saline or increasing doses of morphine (10-40 mg/kg, s.c.) as described for the EPM study. A final dose was administered on day 4, where mice were injected with saline or 20 mg/kg morphine. Two hours later, the mice were injected s.c. with saline, 0.2 mg/kg naloxone or 2 mg/kg naloxone. Seven minutes after the naloxone injection, they were injected with pentobarbital (100 mg/kg, i.p.) and after another five minutes mice were perfused with 50 ml ice-cold PBS followed by 200 ml of 4% paraformaldehyde. Brains were removed, post-fixed overnight at 4°C, cryo-protected in 30% sucrose at 4°C for 2 days, and frozen embedded in a Tissue-Tek OCT compound (Sakura Finetek Inc., Torrance, CA). Brains were then cryo-sectioned at 40 μm and stored in PBS containing 0.1% thimerosal at 4°C. We performed simultaneous staining on control and treated brain sections using the free-floating method. Briefly, sections were incubated overnight with antibodies against phospho-p42/44 ERK (Cell Signaling Technology, Danvers, MA) diluted 1:300 at 4°C. Sections were then washed and incubated with biotin-conjugated anti-rabbit antibodies, followed by ABC enhancement (Vector Lab Inc., Burlingame, CA). Staining was revealed using a 3,3'-diaminobenzidine (DAB) kit (Vector Lab Inc., Burlingame, CA).

Following staining, digital pictures were taken using an Olympus BX51 microscope equipped with a digital camera (Microfire A/R, Optronics) and stored using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Particle analysis was performed in regions of interest using NIH Image v1.62 as previously described (Eitan et al., 2003). For each mouse, three brain areas were analyzed - the central amygdala, lateral septum and the LC. Areas of interest within each of these sections were determined based on the mouse atlas (Paxinos and Franklin, 2001). For the central amygdala, we used coronal sections falling between −0.82 mm to −1.94 mm antero-posterior relative to bregma. In these sections, we analyzed areas identified in the atlas as central amygdaloid nucleus, capsular part (CeC), lateral division (CeL), medial division (CeM), and medial posteroventral part (CeMPV). For the lateral septum, we used coronal sections falling between 1.18 mm to 0.14 mm antero-posterior relative to bregma. In these sections, we analyzed areas identified in the atlas as lateral septal nucleus, dorsal part (LSD), intermediate part (LSI) and ventral part (LSV). Finally, the sections between −5.34 mm and −5.68 mm antero-posterior to the bregma were analyzed for the area identified in the atlas as LC. Examples of the areas of interest for each location are marked in Fig 3.

Fig. 3. Naloxone administration increases ERK activation in the brain.

Fig. 3

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Mice were injected with increasing doses of morphine for 3 days (10-40 mg/kg, s.c.). On day 4, withdrawal was precipitated using 2 mg/kg naloxone. A naloxone-induced increase in ERK activation was observed in the central amygdala (A), lateral septum (B), and the LC (C). (A), (B) and (C) are representative DAB stained sections for each brain area. (Control) drug-naive mice receiving saline; (Withdrawal) morphine-dependent mice receiving 2 mg/kg naloxone. The scale bar is 300 μm. The regions of interests for the semi-quantification analysis are outlined on the representative DAB stained sections for withdrawal.

In a similar experiment, mice were implanted s.c. with placebo or 25 mg morphine pellets under light isoflurane anesthesia. Three days later, pellets were removed under light anesthesia. An hour later mice were injected with saline or 2 mg/kg naloxone. Brains were then processed for immunohistochemistry and analyzed as described above.

Data analysis

EPM data were analyzed using two-way ANOVA followed by Bonferroni's post-hoc comparisons, with condition (control/withdrawal) and treatment (DMSO/SL327) as variables. To analyze the phospho-ERK staining, each brain area of each mouse in the experimental group was normalized to the corresponding staining in the saline control group. The change in staining compared with saline controls was calculated using the following formula: [(experimental value/saline control average) × 100] – 100. Statistical analysis was conducted using two-way ANOVA with pretreatment (placebo/morphine) and treatment (saline/naloxone) as variables, followed by Bonferroni's post-hoc comparisons.

RESULTS

ERK activation in the amygdala, but not the septum, mediates the increase in open-arm time in the EPM during morphine withdrawal

We first examined the effect of administering SL327, an ERK kinase (MEK) inhibitor, into the amygdala on EPM behaviors in saline-injected control mice and the morphine-dependent mice undergoing withdrawal (Fig. 1). Two-way ANOVA revealed significant main effects of both withdrawal (F1,26=23.59; p<0.005) and SL327 (F1,26=13.02; p<0.025) on open-arm time, but no significant interaction. Bonferroni's post-hoc comparisons revealed a significant increase in open-arm time during naloxone-precipitated morphine withdrawal (p<0.01). Injecting SL327 bilaterally (4 μg per side) into the amygdala of mice undergoing withdrawal completely blocked the increase in open-arm time compared to mice injected with vehicle (p<0.05, Fig. 1A). The effect of SL327 was not simply due to an effect overall motor activity. Two-way ANOVA revealed a significant main effect of withdrawal (F1,26=34.48; p<0.001) on total activity, but no significant effect of SL327 or interaction. Bonferroni's post-hoc comparisons revealed a significant decrease in total activity during naloxone-precipitated morphine withdrawal in both vehicle- and SL327-treated mice (p<0.05) compared to controls. However, there was no significant difference in the total activity between mice undergoing withdrawal that were injected with vehicle and those injected with SL327 (Fig. 1B). For the drug naïve mice, injecting SL327 into the amygdala had no effect on time spent in the open arms or total activity (Fig. 1A, B). In mice undergoing morphine withdrawal, SL327 (8 μg) injected into the septum did not change the time spent in the open arms or total activity (Fig. 2A, B).

Fig. 1. Blockade of ERK in the amygdala attenuates the withdrawal-induced increase in open-arm time.

Fig. 1

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Mice were injected with increasing doses of morphine for 3 days (10-40 mg/kg, s.c.). On day 4, mice were injected SL327 or DMSO directly into the amygdala. Withdrawal was precipitated using 0.2 mg/kg naloxone. (A) shows open-arm time and (B) shows total activity. White bars represent vehicle treatment; Black bars represent SL327 treatment. Drug-naive mice receiving vehicle or SL327, n=6; Morphine-dependent mice undergoing withdrawal receiving vehicle, n=10; Morphine-dependent mice undergoing withdrawal receiving SL327, n=8. Results are presented as mean ± SEM. (*) indicates a significant difference from control (p<0.05). (§) indicates a significant difference from morphine-dependent mice undergoing withdrawal receiving vehicle (p<0.05).

Fig. 2. Blockade of ERK in the septum has no effect on EPM behaviors during withdrawal.

Fig. 2

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Mice were injected with increasing doses of morphine for 3 days (10-40 mg/kg, s.c.). On day 4, mice were injected SL327 or DMSO directly into the septum. Withdrawal was precipitated using 0.2 mg/kg naloxone. No significant effect of SL327 was found. (A) shows open-arm time and (B) shows total activity. Results are presented as mean + SEM, n=9-10.

Naloxone differentially induces an increase in ERK activation in drug naïve and morphine-dependent mice

In rats, increased ERK activation was previously observed in the central, but not basolateral, amygdala during morphine withdrawal (Li et al., 2008). Since mice exhibit species-dependent differences in EPM behaviors during withdrawal (Hodgson et al., 2008), we next confirmed that indeed in mice there is a comparable increase in ERK activation in the amygdala during morphine withdrawal. In this experiment we examined ERK activation in drug-naïve and morphine-dependent mice receiving low (0.2 mg/kg) and high (2 mg/kg) naloxone doses. As expected, an increase in ERK activation was observed in the central amygdala. ERK activation was not observed in the basolateral amygdala or any other surrounding areas (data not shown). We also observed increased ERK activation in the lateral septum and LC. An increase in ERK activation was also observed in the bed nucleus of stria terminalis (BNST) in morphine-dependent mice receiving naloxone, but was not quantified (data not shown).

In the central amygdala (Fig. 3A, 4A), two-way ANOVA (n=10-14) revealed a significant main effect of naloxone treatment (F2,59=57.68; p<0.001) and a significant interaction between naloxone treatment and morphine pretreatment (F2,59=14.29; p<0.001). Bonferroni's post-hoc comparisons revealed that 2 mg/kg naloxone, but not 0.2 mg/kg, significantly increased ERK activation in drug-naïve mice (p<0.001). In morphine-dependent mice, a significant decrease was observed in mice receiving saline compared to drug-naïve saline controls (p<0.01). For mice undergoing withdrawal, both 0.2 and 2 mg/kg naloxone significantly increased ERK activation in morphine-dependent mice as compared to their drug-naïve counterparts (p<0.001 and p<0.05, respectively). Moreover, 2 mg/kg naloxone significantly increased ERK activation as compared to 0.2 mg/kg naloxone (p<0.05).

Fig. 4. The differential effect of low and high naloxone doses on ERK activation in drug-naïve and morphine-dependent mice.

Fig. 4

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Mice were injected with increasing doses of morphine for 3 days (10-40 mg/kg, s.c.). On day 4, withdrawal was precipitated using 0.2 or 2 mg/kg naloxone. (A) central amygdala, (B) lateral septum, and (C) the LC. White bars represent saline treatment; gray bars represent 0.2 mg/kg naloxone treatment; black bars represent 2 mg/kg naloxone treatment. Results are presented as mean ± SEM. (*) indicates a significant difference compared to drug-naïve mice receiving saline (p<0.05); (#) indicates a significant difference compared to drug-naïve mice receiving 0.2 mg/kg naloxone (p<0.05); (§) indicates a significant difference compared to drug-naïve mice receiving 2 mg/kg naloxone (p<0.05); (£) indicates a significant difference compared to morphine-dependent mice receiving saline (p<0.05).

In the lateral septum (Fig. 3B, 4B), two-way ANOVA (n=10-14) revealed significant main effects of naloxone treatment (F2,59=14.4; p<0.001) and morphine pretreatment (F1,59=7.92; p=0.01), and a significant interaction between naloxone treatment and morphine pretreatment (F2,59=13.95; p<0.001). Bonferroni's post-hoc comparisons revealed a significant decrease in ERK activation in morphine-dependent mice receiving saline compared to drug-naïve saline controls (p<0.05). Both 0.2 and 2 mg/kg naloxone significantly increased ERK in morphine-dependent mice as compared to their drug-naïve counterparts (p<0.01). Naloxone did not significantly modulate ERK activation in the lateral septum for drug-naïve mice.

In the LC (Fig. 3C, 4C), two-way ANOVA (n=10-14) revealed a significant main effect of naloxone treatment (F2,59=6.193; p<0.005) and a significant interaction between naloxone treatment and morphine pretreatment (F2,59=8.672; p<0.001). Bonferroni's post-hoc comparisons revealed a significant decrease in ERK activation in morphine-dependent mice receiving saline compared to drug-naïve saline controls (p<0.05) In contrast, 2 mg/kg naloxone significantly increased ERK activation in the LC for morphine-dependent mice as compared to their drug-naïve counterparts (p<0.01). However, 0.2 mg/kg naloxone did not significantly change ERK activation levels in morphine-dependent mice as compared to drug-naïve mice.

Similar results were also observed when morphine dependency was established using 25 mg morphine pellets and withdrawal was precipitated with 2 mg/kg naloxone. Namely, increased ERK activation was observed in the central amygdala but not in the basolateral amygdala or other surrounding brain regions (data not shown). Similarly, increased ERK activation was also observed in the lateral septum, BNST and LC (data not shown).

DISCUSSION

This study demonstrates the involvement of the ERK pathway in the increased open-arm time observed during naloxone-precipitated morphine withdrawal. Notably, inhibition of ERK in the amygdala blocked the increase in open-arm time, yet inhibiting ERK in the septum had no effect. Additionally, increases in phospho-ERK staining (the active form of ERK) were observed in the central amygdala of morphine-dependent mice receiving both 0.2 and 2 mg/kg naloxone. In morphine-dependent mice, the dose required to increase ERK activation in the central amygdala was lower than the dose necessary to precipitate significant jumping behavior (Hodgson et al., 2008) - a behavioral measure commonly taken to determine the severity of somatic signs (Iorio et al., 1975). This low dose, however, is sufficient to precipitate both the physiological signs of stress from withdrawal, as indicated by significant increases in plasma corticosterone levels, as well as the increase in EPM open-arm time (Hodgson et al., 2008). Note that although total levels of ERK protein were not measured in our experiments, increases in total protein levels are highly unlikely during the 13 minute period.

Previously, correlations were made between the motivational components of naloxone-precipitated morphine withdrawal and the activation of c-Fos in the central amygdala, extended amygdala, and lateral septum (Gracy et al., 2001; Veinante et al., 2003; Hamlin et al., 2004; Jin et al., 2004). The lateral septum is implicated in the modulation of anxiety via the mu opioid receptor (Le Merrer et al., 2006). The central and extended amygdala regulate withdrawal-induced conditioned place aversion (Aston-Jones et al., 1999; Gracy et al., 2001; Jin et al., 2005) and are involved in altered hedonic processing during withdrawal (Harris and Aston-Jones, 2007). Additionally, activation of ERK and CREB in the central amygdala is involved in mediating drug-seeking and craving during withdrawal (Li et al., 2008). However, these brain regions were demonstrated to have little involvement in the precipitation of somatic signs of withdrawal (Nakagawa et al., 2005; Maldonado et al., 1992).

Consistent with these prior findings, this study implicates ERK activation in the central amygdala in mediating EPM behaviors in mice during withdrawal. It is important to note that in this study, SL327 was injected one hour prior to the precipitation of withdrawal and the EPM test. Although only a very small quantity was administered, we can not exclude the possibility of some diffusion of SL327 to surroundings areas. Thus, it is possible that the effect on EPM behaviors is due to inhibition of ERK in nearby brain regions. However, consistent with other studies, there were no conditions (i.e. repeated morphine injections, morphine pellets, high and low naloxone doses) in which we observed an increase in phospho-ERK in the areas surrounding the central amygdala. The closest areas in which we observed increased ERK activation were the lateral septum and BNST. Yet, injection of SL327 into the septum had no effect. Thus, it is very likely that the activation of ERK in the central amygdala mediates the effect on EPM behaviors. Nevertheless, we cannot exclude the possibility that changes occurring below the detection threshold in nearby areas are involved in mediating the EPM behaviors during withdrawal.

Naloxone is aversive in both drug-naïve (Skoubis et al., 2005) and morphine-dependent mice (Maldonado et al., 2004; Broseta et al., 2005; Shoblock and Maidment, 2006). However, in drug-naïve mice, a high naloxone dose is required in order to establish significant levels of aversion. In contrast, for morphine-dependent mice, withdrawal precipitation even by lower naloxone doses is sufficient to produce a significant emotional reaction. Just as high doses are required to induce aversion in opioid-naive mice, drug-naïve mice also require 2 mg/kg naloxone, not just 0.2 mg/kg, to increase ERK activation in the central amygdala. Thus, we hypothesize that the activation of ERK in limbic areas is most likely also involved in the aversive properties of naloxone. The central amygdala is known to have high tonic levels of enkephalin and other endogenous opioid peptides (Mansour et al., 1993; Chieng et al., 2006; Marchant et al., 2007; Poulin et al., 2008). Because drug-naive mice lacking preproenkephalin do not exhibit increased ERK activation in the central amygdala (Eitan et al, 2002) this supports the hypothesis that naloxone increases ERK activation via inhibition of tonic signaling of endogenous opioid peptides. Similar to ERK, an increase of c-Fos in the central amygdala was observed in drug-naïve rats following naloxone administration (Gestreau et al., 2000).

As mentioned above, increased ERK activation was observed in the central amygdala of drug-naïve mice administered 2 mg/kg naloxone, but not 0.2 mg/kg naloxone. However, drug-naïve mice did not exhibit a significant increase in open-arm time even when administered 10 mg/kg naloxone (Hodgson et al., 2008). Therefore, it is very unlikely that the differences in EPM behaviors between drug-naïve and morphine-dependent mice receiving naloxone can be explained solely by different levels of ERK activation. This leads to the conclusion that the increase in ERK activation is necessary to drive the increased EPM open-arm time during withdrawal. However, the lack of naloxone-induced increases in EPM open-arm time in drug-naïve mice suggests that other signaling cascades are required in addition to ERK activation.

This study also demonstrated increased ERK activation in the LC during opioid withdrawal. This is consistent with a previous study that observed an increase in phospho-ERK staining in the LC (Schulz and Hollt, 1998). In contrast to the limbic areas, increased ERK activation in the LC was observed only in morphine-dependent mice receiving the higher naloxone dose – a dose needed to precipitate significant jumping behavior (Hodgson et al., 2008). The LC was suggested to be essential for the development of morphine dependence and the precipitation of withdrawal signs, especially the physical symptoms (Aghajanian, 1978; Crawley et al., 1979; Maldonado et. al, 1992). Specifically, inhibition of adrenergic tone, mediated by GIRK2 and GIRK3, was suggested to be required for the development of dependence (Cruz et al., 2008). Likewise, various receptors and signaling molecules in the LC were demonstrated to be involved in the precipitation of withdrawal's somatic signs. Examples include alpha-2 adrenoceptors (Aghajanian, 1982; Engberg et al., 1982), GABA-A receptor (Mirzaii-Dizgah et al., 2008), glutamate transporters (Ozawa et al., 2004; Nakagawa and Satoh, 2004), and the cAMP pathway, including adenylyl cyclases 1 and 8 and cAMP response element-binding protein (Valverde et al., 2004; Oh et al., 2007; Zachariou et al., 2008; Han et al., 2006; Lane-Ladd et al., 1997). However, the role of the LC in the precipitation of somatic withdrawal signs remains somewhat debatable, given that lesioning of noradrenergic terminals arising from the LC does not inhibit the precipitation of physical symptoms of opioid withdrawal (Chieng and Christie, 1995).

In this study, we also observed a decreased ERK activation in the central amygdala, lateral septum and LC of morphine-dependent mice receiving saline (i.e. in which withdrawal was not precipitated with naloxone). We previously observed decreased ERK activation in the central amygdala 30 minutes following acute or repeated morphine administration (Eitan et al., 2003). This was the only brain region in which we did not observe the development of tolerance for ERK modulation following repeated morphine administration, although antinociceptive tolerance was observed (Eitan et al., 2003). Thus, the decreased ERK activation in the central amygdala is most likely a persistent morphine effect that is already observed at 30 minutes post-morphine and lasts for at least 2 hours. This is unlikely due to spontaneous withdrawal, given that the half-life of morphine (Kalvass et al., 2007) requires a longer duration before withdrawal spontaneously precipitates.

For the LC, we previously observed that acute morphine administration produces an increase in ERK activation (Eitan et al., 2003). However, following repeated morphine administration, tolerance for ERK activation developed; namely, we did not observe an increase or a decrease in ERK activation 30 minutes post-morphine in mice receiving repeated morphine administration, as compared to controls (Eitan et al., 2003). In the present study, morphine-dependent mice showed a decrease in ERK activation at 2 hours post-morphine. This decreased ERK activation might represent a delayed adaptive response to a morphine challenge in morphine-dependent mice. Such opposite responses are known to emerge upon cessation of prolonged administration of opioids. These opponent processes in many cases counteract the acute effects of opioids (e.g. hyperalgesia vs. antinociception) and can contribute to the development of tolerance and physical dependence (Bryant et al., 2005). Thus, the decrease in ERK activation may represent one of several cellular opponent processes. Interestingly, this cellular event parallels the opioid-induced hyperalgesia (behavioral rebound) observed at the same time in morphine-tolerant mice (Eitan et al., 2003).

Although opiates are commonly used for pain management, the neuroplastic mechanisms underlying their adaptive processes remain somewhat elusive. The purpose of this study was to examine the involvement of the ERK pathway in EPM behaviors of mice undergoing opioid withdrawal. We focused on the ERK pathway because it plays an important role in synaptic plasticity, and has been implicated in the chronic adaptations to opioids. We observed an increase in ERK activation at the onset of morphine withdrawal. This increase was observed in brain areas previously implicated in mediating the emotional response to withdrawal. Finally, we found that ERK signaling is necessary for mediating the withdrawal-induced increase in EPM open-arm time. Future studies will examine the receptor mechanisms involved, as well as other brain pathways and signaling cascades that are important in driving behavioral adaptations during drug withdrawal.

Acknowledgements

These studies were supported by NIDA (DA022402 and DA05010) and by the Shirley and Stefan Hatos Neuroscience Research Foundation. We also would like to thank Mr. Menachum M Slodowitz for his editorial assistance.

Footnotes

Disclosure/Conflict of Interest: We have no financial interests to disclose.

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