Effect of ΔFosB overexpression on opioid and cannabinoid receptor-mediated signaling in the nucleus accumbens (2011)

Neuropharmacology. 2011 Dec;61(8):1470-6. doi: 10.1016/j.neuropharm.2011.08.046.

Sim-Selley LJ, Cassidy MP, Sparta A, Zachariou V, Nestler EJ, Selley DE.

Source

Department of Pharmacology and Toxicology and Institute for Drug and Alcohol Studies, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.

Abstract

The stable transcription factor ΔFosB is induced in the nucleus accumbens (NAc) by chronic exposure to several drugs of abuse, and transgenic expression of ΔFosB in the striatum enhances the rewarding properties of morphine and cocaine. However, the mechanistic basis for these observations is incompletely understood. We used a bitransgenic mouse model with inducible expression of ΔFosB in dopamine D(1) receptor/dynorphin-containing striatal neurons to determine the effect of ΔFosB expression on opioid and cannabinoid receptor signaling in the NAc. Results showed that mu opioid-mediated G-protein activity and inhibition of adenylyl cyclase were enhanced in the NAc of mice that expressed ΔFosB. Similarly, kappa opioid inhibition of adenylyl cyclase was enhanced in the ΔFosB expressing mice. In contrast, cannabinoid receptor-mediated signaling did not differ between mice overexpressing ΔFosB and control mice. These findings suggest that opioid and cannabinoid receptor signaling are differentially modulated by expression of ΔFosB, and indicate that ΔFosB expression might produce some of its effects via enhanced mu and kappa opioid receptor signaling in the NAc.

Keywords: G-protein, adenylyl cyclase, striatum
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1. Introduction

Opioid receptors and cannabinoid CB1 receptors (CB1R) are the neurobiological targets for two widely used drug classes that include morphine, heroin and prescription opioids, and marijuana (Δ9-tetrahydrocannabinol (THC)), respectively. The acute effects of opioids and cannabinoids are mediated by G-protein-coupled receptors that activate primarily Gi/o proteins and produce downstream effector responses such as inhibition of adenylyl cyclase (Childers, 1991, Childers, et al., 1992, Howlett, et al., 2002). The motor, memory impairing and psychoactive effects of Δ9-THC are produced by CB1R (Huestis, et al., 2001, Zimmer, et al., 1999), which are widely distributed in the brain, with high levels in the basal ganglia, hippocampus and cerebellum (Herkenham, et al., 1991). The analgesic and rewarding effects of most clinically relevant and abused opioid drugs are mediated mainly by mu opioid receptors (MOR) (Matthes, et al., 1996), which are enriched in the limbic system and brainstem (Mansour, et al., 1994). The mesolimbic system, composed of dopaminergic projections from the ventral tegmental area (VTA) to nucleus accumbens (NAc), plays an important role in the rewarding effects of opioids and cannabinoids (Bozarth and Wise, 1984, Vaccarino, et al., 1985, Zangen, et al., 2006), as well as other drugs of abuse (Koob and Volkow, 2010). Moreover, endogenous opioid and cannabinoid systems are involved in the rewarding effects of multiple classes of psychoactive drugs (Maldonado, et al., 2006, Trigo, et al., 2010). Thus, it is important to elucidate mechanisms by which opioid and CB1R signaling are regulated in the NAc.

A central question in the drug abuse field has been to identify proteins that mediate the transition from acute to long-term effects of psychoactive drugs. The AP-1 transcription factor ΔFosB is particularly interesting because it is a stable truncated splice variant product of the fosb gene that accumulates upon repeated exposure to drugs of abuse or natural rewards (McClung, et al., 2004, Nestler, 2008, Nestler, et al., 1999). We have found that ΔFosB is induced in the brain following repeated exposure to morphine, Δ9-THC, cocaine or ethanol, with each drug producing a unique regional pattern of ΔFosB expression (Perrotti, et al., 2008). A consistent finding across drugs was that ΔFosB was highly induced in the striatum, where all four drugs induced ΔFosB in the NAc core and all except Δ9-THC significantly induced expression in the NAc shell and caudate-putamen.

Pharmacological studies showed that co-administration of the dopamine D1 receptor (D1R) antagonist SCH 23390 blocked ΔFosB induction in the NAc and caudate-putamen following intermittent cocaine or morphine administration, suggesting the potential importance of D1R-expressing neurons (Muller and Unterwald, 2005, Nye, et al., 1995). The effect of ΔFosB induction on drug-mediated behaviors has been investigated using bitransgenic mice that express ΔFosB in specific neuronal populations of the NAc and dorsal striatum (Chen, et al., 1998). Mice that express ΔFosB in dynorphin/D1R positive neurons in the NAc and dorsal striatum (line 11A) show altered responses to drugs of abuse, notably enhanced sensitivity to the rewarding effects of cocaine or morphine (Colby, et al., 2003, Kelz, et al., 1999, Zachariou, et al., 2006). These alterations occurred in the absence of changes in the levels of MOR or various G-protein subunits. However, dynorphin mRNA levels were reduced in the NAc of ΔFosB expressing mice (Zachariou, et al., 2006), suggesting that one target of ΔFosB is a gene encoding an endogenous opioid peptide. ΔFosB induction might also produce behavioral changes by regulating receptor signaling in the NAc, but this possibility has not been investigated. Therefore, the present studies used the bitransgenic mouse model to determine whether overexpression of ΔFosB in dynorphin/D1R containing striatal neurons alters MOR-mediated G-protein activity and MOR- and KOR-mediated adenylyl cyclase inhibition in the NAc. The effect of ΔFosB on CB1R-mediated G-protein activity was also assessed because Δ9-THC administration induces ΔFosB in the NAc (Perrotti, et al., 2008) and the endocannabinoid system is known to regulate brain reward circuits (Gardner, 2005, Maldonado, et al., 2006), but the effect of ΔFosB on the endocannabinoid system has not been investigated.

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2. Materials and Methods

2.1. Reagents

[35S]GTPγS (1250 Ci/mmol), [α-32P]ATP (800 Ci/mmol) and [3H]cAMP (26.4 Ci/mmol) were purchased from PerkinElmer (Shelton, CT). ATP, GTP, GDP, cAMP, bovine serum albumin, creatine phosphokinase, papaverine, imidazole and WIN-55212-2, were purchased from Sigma Aldrich (St. Louis, MO). GTPγS was purchased from Roche Diagnostic Corporation (Chicago, IL). DAMGO was provided by the Drug Supply Program of the National Institute on Drug Abuse (Rockville, MD). Econo-1 scintillation fluid was obtained from Fisher Scientific (Norcross, GA). Ecolite scintillation fluid was obtained from ICN (Costa Mesa, CA). All other chemicals were obtained from Sigma Aldrich or Fisher Scientific.

2.2. Mice

Male bitransgenic mice derived from NSE-tTA (line A) × TetOp-ΔFosB (line 11) were generated as described in Kelz et al. (Kelz, et al., 1999). Bitransgenic mice were conceived and raised on doxycycline (100 µg in drinking water) to suppress transgene expression. At 8 weeks of age, doxycycline was omitted from the water for half of the mice to allow transgene expression, whereas remaining mice were maintained on doxycycline to suppress the transgene. Brains were collected 8 weeks later, the time at which transcriptional effects of ΔFosB are maximal (McClung and Nestler, 2003). A second transgenic mouse line was used in which Δc-Jun, a dominant negative antagonist of c-Jun, is expressed in D1R/dynorphin and D2R/enkephalin cells of the striatum, hippocampus and parietal cortex (Peakman, et al., 2003). C-Jun and related Jun family proteins dimerize with Fos family proteins and bind to the AP-1 site of target genes to regulate transcription. However, truncation of the N-terminus of c-Jun (Δc-Jun) renders the complex transcriptionally inactive and able to obstruct the DNA binding of active AP-1 complexes. Male bitransgenic mice derived from NSE-tTA (line A) × TetOp-FLAG-Δc-Jun (line E) were generated as described in Peakman et al. (Peakman, et al., 2003). Bitransgenic mice were conceived and raised on doxycycline (100 µg in drinking water) to suppress transgene expression. Pups were weaned at 3 weeks, genotyped, and separated into groups, with half maintained on doxycycline-containing water and half on regular drinking water to induce FLAG-Δc-Jun expression. Brains were collected 6 weeks later, the time at which maximal levels of FLAG-Δc-Jun have been measured (Peakman, et al., 2003). All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.3. Membrane Preparation

Brains were stored at −80°C until the day of the assay. Prior to assay, each brain was thawed, and the NAc was dissected on ice. Each sample was homogenized in 50 mM Tris-HCl, 3 mM MgCl2, 1 mM EGTA, pH 7.4 (membrane buffer) with 20 strokes from a glass homogenizer at 4°C. The homogenate was centrifuged at 48,000 × g at 4°C for 10 min, resuspended in membrane buffer, centrifuged again at 48,000 × g at 4°C for 10 min and resuspended in 50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.4 (assay buffer). Protein levels were determined by the method of Bradford (Bradford, 1976) using bovine serum albumin (BSA) as the standard.

2.4. Agonist-Stimulated [35S]GTPγS Binding

Membranes were pre-incubated for 10 minutes at 30°C with adenosine deaminase (3 mU/ml) in assay buffer. Membranes (5–10 µg protein) were then incubated for 2 hr at 30°C in assay buffer containing 0.1% (w/v) BSA, 0.1 nM [35S]GTPγS, 30 µM GDP and adenosine deaminase (3 mU/ml) with and without appropriate concentrations of DAMGO or WIN55,212-2. Nonspecific binding was measured with 20 µM GTPγS. The incubation was terminated by filtration through GF/B glass fiber filters, followed by 3 washes with 3 ml ice-cold 50 mM Tris-HCl, pH 7.4. Bound radioactivity was determined by liquid scintillation spectrophotometry after overnight extraction of the filters in Econo-1 scintillation fluid.

2.5. Adenylyl Cyclase Assay

Membranes (5–25 µg protein) were preincubated with adenosine deaminase as described above, then incubated for 15 min at 30°C in the presence or absence of 1µM forskolin, with or without DAMGO, U50,488H or WIN55,212-2, in assay buffer containing 50 µM ATP, [α-32P]ATP (1.5 µCi), 0.2 mM DTT, 0.1% (w/v) BSA, 50 µM cyclic AMP, 50 µM GTP, 0.2 mM papaverine, 5 mM phosphocreatine, 20 units/ml creatine phosphokinase and adenosine deaminase (3 mU/ml) in a final volume of 100 µl. Under these conditions, total [α-32P]cAMP recovered was generally less than 1% of the total amount of added [α-32P]ATP in each sample. The reaction was terminated by boiling for 3 min and [32P]Cyclic AMP was isolated by the dual column (Dowex and alumina) method of Salomon (Salomon, 1979). [3H] cAMP (10,000 dpm) was added to each tube prior to column chromatography as an internal standard. Radioactivity was determined by liquid scintillation spectrophotometry (45% efficiency for 3H) after 4.5 ml of the eluate were dissolved in 14.5 ml of Ecolite scintillation fluid.

2.6. Data analysis

Unless otherwise indicated, data are reported as mean values ± S.E. of 4–8 separate experiments, each of which was performed in triplicate. Net-stimulated [35S]GTPγS binding is calculated as agonist-stimulated binding minus basal binding. Net forskolin-stimulated adenylyl cyclase activity is defined as forskolin-stimulated activity – basal activity (pmol/mg/min). Percent inhibition of forskolin-stimulated adenylyl cyclase activity is defined as (net forskolin-stimulated activity in the absence of agonist – net forskolin-stimulated activity in the presence of agonist/net forskolin-stimulated activity in the absence of agonist) × 100. All curve-fitting and statistical analyses were performed using Prism 4.0c (GraphPad Software, Inc., San Diego, CA). Concentration-effect curves were analyzed by iterative non-linear regression to obtain EC50 and Emax values. Statistical significance of the concentration-effect data was determined by two-way analysis of variance (ANOVA), using agonist dose and gene induction (on or off) as the main factors. Statistical significance of curve-fit values (Emax or EC50) was determined by the non-paired two-tailed Student’s t-test, using Welch’s correction or square root transformation of the data where necessary to correct for unequal variances (detected by F-test) in EC50 values.

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3. Results

3.1. Effect of ΔFosB expression on opioid and cannabinoid receptor-mediated G-protein activation

To determine whether MOR- or CB1R-mediated G-protein activation was altered by inducible transgenic expression of ΔFosB in the NAc, agonist-stimulated [35S]GTPγS binding was examined in isolated membranes prepared from this region of bitransgenic mice conditionally expressing (ΔFosB on) or not expressing (ΔFosB off) the ΔFosB transgene. The MOR-selective enkephalin analog DAMGO was used to activate MOR and the cannabinoid aminoalkylindole WIN55,212-2 was used to activate CB1R. These ligands were previously shown to be full agonists at MOR and CB1R, respectively (Breivogel, et al., 1998, Selley, et al., 1997). It was not feasible to examine KOR-mediated G-protein activity because the signal is too low in rodent brain (Childers, et al., 1998). Results showed concentration-dependent stimulation of G-protein activity by both DAMGO and WIN55,122-2 in NAc from ΔFosB off and ΔFosB on mice (Figure 1). For DAMGO-stimulated activity (Figure 1A), two-way ANOVA of the concentration-effect data revealed significant main effects of ΔFosB status (p<0.0001, F=22.12, df=1) and DAMGO concentration (p<0.0001, F=29.65, df=5) with no significant interaction (p=0.857, F=0.387, df=5). Nonlinear regression analysis of the concentration-effect curves revealed a significantly greater DAMGO Emax value in ΔFosB on mice (Emax = 73 ± 5.2% stimulation) relative to ΔFosB off mice (Emax = 56 ± 4.1% stimulation; p<0.05 different from ΔFosB on mice by Student’s t-test). DAMGO EC50 values were not different between ΔFosB on and ΔFosB off mice (302 ± 72 nM versus 212 ± 56 nM, respectively, p = 0.346).

Figure 1 

Effect of ΔFosB expression on agonist-stimulated [35S]GTPγS binding in the NAc. Membranes from ΔFosB-expressing (ΔFosB on) or control (ΔFosB off) mice were assayed as described in Methods using varying concentrations …

In contrast to results obtained with the MOR agonist DAMGO, no ΔFosB status-dependent differences in G-protein activation were observed with the cannabinoid agonist WIN55,212-2 (Figure 1B). Two-way ANOVA of the WIN55,212-2 concentration-effect data revealed a significant main effect of WIN55,212-2 concentration (p < 0.0001, F = 112.4, df = 7), but not of ΔFosB status (p = 0.172, F = 1.90, df=1) and there was no interaction (p = 0.930, F = 0.346, df = 7). Similarly, there was no effect of ΔFosB status on WIN55,212-2 Emax values (103 ± 6% versus 108 ± 8% stimulation in ΔFosB on and off mice, respectively, p = 0.813 by Student’s t-test) or EC50 values (103 ± 20 nM versus 170 ± 23 nM in ΔFosB on and off mice, respectively, p = 0.123).

Based on the shape of the curves and the fact that our previous studies have shown biphasic WIN55,212-2 concentration-effect curves in brain (Breivogel, et al., 1999, Breivogel, et al., 1998), the WIN55,212-2 curves were also analyzed using a two-site model. Analysis of the averaged data showed a slight improvement in goodness of fit using the two-site model (R2 = 0.933 and 0.914, sum of squares = 3644 and 5463 in ΔFosB on and off mice, respectively) compared to the single-site model (R2 = 0.891 and 0.879, sum of squares = 6561 and 6628 in ΔFosB on and off mice, respectively). However, no significant differences were found between ΔFosB on and off mice in either the Emax or EC50 values of the high or low potency sites (Supplementary Table 1), although there was a trend towards a lower EC50 value at the high potency site in mice with ΔFosB on (EC50high = 28.0 ± 10.6 nM) compared to those with ΔFosB off (EC50high = 71.5 ± 20.2 nM; p = 0.094). Moreover, there was no effect of ΔFosB status on basal [35S]GTPγS binding in NAc membranes (253 ± 14 versus 226 ± 14 fmol/mg in ΔFosB on and off mice, respectively, p = 0.188). These data indicate that inducible transgenic expression of ΔFosB in the NAc of mice increased MOR-mediated G-protein activation without significantly affecting CB1R-mediated or basal G-protein activity.

3.2. Effect of ΔFosB on opioid and cannabinoid receptor-mediated inhibition of adenylyl cyclase

To evaluate the effect of inducible transgenic expression of ΔFosB on modulation of downstream effector activity by MOR and CB1R, inhibition of 1 µM forskolin-stimulated adenylyl cyclase activity was examined in NAc membranes. In addition to MOR- and CB1R-mediated inhibition of adenylyl cyclase activity, effects of KOR activity were also examined using the KOR-selective full agonist U50,488 (Zhu, et al., 1997), because previous results showed that dynorphin mRNA was a target of ΔFosB in the bitransgenic model (Zachariou, et al., 2006). Results showed that DAMGO, U50,488 and WIN55,212-2 each produced concentration-dependent inhibition of adenylyl cyclase activity in both ΔFosB off and ΔFosB on mice (Figure 2). Two-way ANOVA of DAMGO concentration-effect data (Figure 2A) revealed significant main effects of ΔFosB status (p = 0.0012, F = 11.34, df = 1) and DAMGO concentration (p < 0.0001, F = 29.61, df = 6), but no significant interaction (p = 0.441, F = 0.986, df = 6). Nonlinear regression analysis of DAMGO concentration-effect curves revealed a significantly lower DAMGO EC50 value in ΔFosB on mice (101 ± 11 nM) compared to ΔFosB off mice (510 ± 182 nM, p < 0.05 by student’s t-test). However, there was no significant difference in DAMGO Emax values (20.9 ± 1.26% versus 19.8 ± 1.27% inhibition in ΔFosB on and off mice, respectively, p = 0.534).

Figure 2 

Effect of ΔFosB expression on inhibition of adenylyl cyclase activity in the NAc. Membranes from ΔFosB-expressing (ΔFosB on) or control (ΔFosB off) mice were assayed as described in Methods in the presence of 1 µM …

KOR-mediated adenylyl cyclase inhibition also differed as a function of inducible transgenic expression of ΔFosB (Figure 2B). Two-way ANOVA of U50,488 concentration-effect data showed significant main effects of ΔFosB status (p = 0.0006, F = 14.53, df = 1) and U50,488 concentration (p < 0.0001, F = 26.48, df = 3), with no significant interaction (p = 0.833, F = 0.289, df = 3). Nonlinear regression analysis of concentration-effect curves revealed a greater U50,488 Emax value in ΔFosB on mice (18.3 ± 1.14% inhibition) compared to ΔFosB off mice (12.5 ± 2.03% inhibition; p < 0.05 different from ΔFosB on by Student’s t-test), with no significant difference in U50,488 EC50 values (310 ± 172 nM versus 225 ± 48 nM in ΔFosB on and off mice, respectively, p = 0.324).

In contrast to effects observed with MOR and KOR, there was no significant effect of inducible transgenic ΔFosB expression on inhibition of adenylyl cyclase by the cannabinoid agonist WIN55212-2 (Figure 2C). Two-way ANOVA of WIN55,212-2 concentration-effect data showed a significant effect of drug concentration (p < 0.0001, F = 23.6, df = 2), but not of ΔFosB status (p = 0.735, F = 0.118, df = 1) nor was there a significant interaction (p = 0.714, F = 0.343, df = 2). Furthermore, there was no effect of ΔFosB status on basal or forskolin-stimulated adenylyl cyclase activity in the absence of any agonist. Basal adenylyl cyclase activity was 491 ± 35 pmol/mg/min in ΔFosB on mice compared to 546 ± 44 in ΔFosB off mice (p = 0.346 by Student’s t-test). Likewise, adenylyl cyclase activity in the presence of 1 µM forskolin was 2244 ± 163 pmol/mg/min in ΔFosB on mice versus 2372 ± 138 pmol/mg/min in ΔFosB off mice (p = 0.555).

3.3. Effect of ΔcJun on opioid and cannabinoid receptor-mediated inhibition of adenylyl cyclase

Because inducible transgenic expression of ΔFosB enhanced inhibitory signal transduction from MOR and KOR to adenylyl cyclase in the NAc, it was of interest to determine whether a dominant negative inhibitor of ΔFosB-mediated transcription would modulate opioid receptor signaling in an opposite manner. To address this question, inhibition of forskolin-stimulated adenylyl cyclase activity by DAMGO and U50,488 was examined in membranes prepared from the NAc of bitransgenic mice conditionally expressing ΔcJun. The results showed no significant effect of ΔcJun expression on inhibition of adenylyl cyclase activity by MOR or KOR (Figure 3). Two-way ANOVA of DAMGO concentration-effect curves showed a significant main effect of DAMGO concentration (p < 0.0001, F = 20.26, df = 6), but not of ΔcJun status (p = 0.840, F= 0.041, df = 1) and there was no significant interaction (p = 0.982, F= 0.176, df = 6). Similarly, there was no significant difference in Emax or EC50 values between mice with ΔcJun on (Emax = 23.6 ± 2.6%; EC50 = 304 ± 43 nM) or ΔcJun off (Emax = 26.1 ± 2.5%, p = 0.508; EC50 = 611 ± 176 nM, p = 0.129). Similar results were seen with U50,488, such that two-way ANOVA of the concentration-effect curves showed a significant effect of concentration (p < 0.0001, F = 11.94, df = 6), but not of ΔcJun status (p = 0.127, F= 2.391, df = 1) and there was no significant interaction (p = 0.978, F= 0.190, df = 6). Likewise, there were no significant differences in Emax or EC50 values between mice with ΔcJun on (Emax = 14.8 ± 2.9%; EC50 = 211 ± 81 nM) or off (Emax = 16.7 ± 1.8%, p = 0.597; EC50 = 360 ± 151 nM, p = 0.411).

Figure 3 

Effect of ΔcJun expression on inhibition of adenylyl cyclase activity in the NAc. Membranes from ΔcJun-expressing (ΔcJun on) or control (ΔcJun off) mice were incubated in the presence of DAMGO (A), U50,488H (B) or WIN55,212-2 …

ΔcJun expression also did not significantly affect inhibition of adenylyl cyclase in the NAc by the cannabinoid agonist. Two-way ANOVA of the WIN55,212-2 concentrationeffect curves showed a significant main effect of WIN55,212-2 concentration (p < 0.0001, F = 15.53, df = 6), but not of genotype (p = 0.066, F = 3.472, df = 1) and there was no significant interaction (p = 0.973, F = 0.208, df = 6). Likewise, there were no significant differences in WIN55,212-2 Emax values (13.0 ± 2.3% and 13.6 ± 0.9% inhibition in ΔcJun on versus off mice, respectively, p = 0.821) and or EC50 values (208 ± 120 nM and 417 ± 130 nM in ΔcJun on versus off mice, respectively, p = 0.270). Thus, although there was a slight trend toward decreased potency of WIN55,212-2 in mice expressing ΔcJun, the transgene did not significantly alter cannabinoid inhibition of adenylyl cyclase. Moreover, there was no effect of ΔcJun status on basal or forskolin-stimulated adenylyl cyclase activity. Basal adenylyl cyclase activity was 1095 ± 71 pmol/mg/min and 1007 ± 77 pmol/mg/min (p = 0.403) in mice with ΔcJun on or off, respectively. Adenylyl cyclase activity stimulated by 1 µM forskolin was 4185 ± 293 pmol/mg/min versus 4032 ± 273 pmol/mg/min (p = 0.706) in mice with ΔcJun on or off, respectively.

3.4. Discussion

The results of this study revealed enhanced MOR-mediated G-protein activation and inhibition of adenylyl cyclase in the NAc of mice with inducible transgenic expression of ΔFosB in dynorphin/D1R containing neurons. KOR-mediated inhibition of adenylyl cyclase activity was also enhanced in the NAc of ΔFosB expressing mice, suggesting that ΔFosB regulates the endogenous opioid system in the NAc. The DAMGO Emax value was greater for MOR-stimulated [35S]GTPγS binding, and its EC50 value was lower for adenylyl cyclase inhibition, in ΔFosB over-expressing mice compared to control mice. These findings suggest the possibility of receptor reserve for effector modulation but not G-protein activation under the assay conditions examined. The finding that maximal inhibition of adenylyl cyclase by the KOR agonist was affected by ΔFosB expression suggests low receptor reserve for the KOR-mediated response, consistent with the low levels of KOR binding sites in mouse brain (Unterwald, et al., 1991). In contrast, CB1R-mediated G-protein activity and inhibition of adenylyl cyclase were unaffected by ΔFosB expression, suggesting that the opioid and cannabinoid systems differ in their response to ΔFosB in these NAc neurons.

The effect of ΔFosB on opioid receptor-mediated signaling is consistent with our previous report that ΔFosB expression in the striatum altered acute and chronic effects of morphine (Zachariou, et al., 2006). One finding of that study was that mice with transgenic expression of ΔFosB in dynorphin/D1R striatal neurons were more sensitive to morphine in place conditioning than controls. Furthermore, this effect was mimicked by virally mediated expression of ΔFosB by site-specific injection into the NAc. These observations are consistent with the current results that show enhanced MOR signaling in the NAc.

We previously identified the gene encoding dynorphin as a target of ΔFosB, and proposed that reduced dynorphin would be consistent with enhanced rewarding properties of morphine in ΔFosB bitransgenic mice (Zachariou, et al., 2006). The present results show that KOR-mediated inhibition of adenylyl cyclase in the NAc is enhanced in ΔFosB expressing mice, which might reflect a compensatory increase in KOR sensitivity following reduced dynorphin. Previous studies have shown that KOR was upregulated in certain brain regions of prodynorphin knockout mice, including NAc (Clarke, et al., 2003).

In contrast to ΔFosB, inducible transgenic expression of ΔcJun, the dominant negative truncated mutant of the ΔFosB binding partner cJun, did not alter adenylyl cyclase inhibition by MOR or KOR agonists. These results suggest that basal levels of ΔFosB expression, which are relatively low, do not play a significant role in maintaining opioid receptor signaling at this level of signal transduction in the NAc. The fact that the conditioned rewarding effect of morphine was decreased by ΔcJun expression in our previous study (Zachariou, et al., 2006) suggests either that morphine induction of ΔFosB during the conditioning procedure is important in regulating behavioral responses to the drug or that transcriptional effects of ΔFosB other than those affecting proximal signaling by opioid receptors might influence opioid reward. In any event, results of the present study show clearly that, when ΔFosB expression is elevated above basal levels in striatal dynorphin/D1R-expressing neurons, there is a robust increase in the coupling of MOR and KOR to inhibition of adenylyl cyclase in the NAc.

The mechanisms by which MOR- and KOR-mediated signaling are enhanced by ΔFosB overexpression are unclear, but we have previously shown that MOR levels, assessed by [3H]naloxone binding, are not different in the NAc of ΔFosB on versus off mice (Zachariou, et al., 2006). The same study found that Gαi1 and 2 protein levels were not affected in this region by ΔFosB expression. However, previous gene expression array analyses showed that Gαo mRNA was upregulated in NAc of ΔFosB on mice (McClung and Nestler, 2003). It will be of interest in future studies to comprehensively examine the effect of transgenic ΔFosB expression on G-protein subunit expression at the protein level as well as on the expression of many G-protein modulatory proteins.

It is interesting that ΔFosB expression did not enhance CB1R-mediated signaling in the NAc. It is possible that alterations in CB1R signaling occur in a discrete population of neurons that is obscured in the whole NAc preparation. For example, administration of Δ9– THC significantly induced ΔFosB in the core, but not shell, of the NAc (Perrotti, et al., 2008). Indeed, it has been shown that challenge with Δ9-THC following repeated administration of Δ9-THC increased dopamine release in the NAc core, but decreased release in the shell (Cadoni, et al., 2008). It is also important to note that the 11A line of bitransgenic mice express ΔFosB only in dynorphin/D1R positive medium spiny neurons of the striatum, but CB1R are expressed in both dynorphin/D1R and enkephalin/D2R positive striatal neurons (Hohmann and Herkenham, 2000), as well as on terminals of cortical afferents (Robbe, et al., 2001). Expression of the dominant negative regulator of ΔFosB-mediated transcription, ΔcJun, also had no significant effect on cannabinoid receptor signaling, although ΔcJun is inducibly expressed in both D1 and D2-containing populations of medium spiny neurons in these mice (Peakman, et al., 2003). It is possible, however, that basal ΔFosB expression is sufficiently low that ΔcJun would not affect receptor signaling, as suggested by results with MOR and KOR. It is also possible that CB1R signaling is modestly enhanced by basal ΔFosB expression, such that further increasing ΔFosB expression or blocking its actions with ΔcJun had only slight effects that did not reach the level of statistical significance. Indirect support for this interpretation can be seen by comparing WIN55,212-2 EC50 values between mice expressing ΔcJun versus ΔFosB. The ratio of the WIN55,212-2 EC50 value for adenylyl cyclase inhibition in mice with induced expression of ΔcJun to its EC50 value for G-protein activation in mice with induced expression of ΔFosB was 4.0, whereas the same ratio in mice without induction of either transgene was 1.2.

Alternatively, cannabinoids might induce ΔFosB expression without any direct effect on CB1R signaling. In this scenario, cannabinoids could modulate responsiveness to the psychoactive effects of other drugs via ΔFosB-mediated transcriptional regulation. In fact, administration of Δ9-THC produces cross-sensitization to opioids and amphetamine (Cadoni, et al., 2001, Lamarque, et al., 2001), consistent with this hypothesis. Moreover, repeated administration of the cannabinoid agonist CP55,940 was reported to increase MOR-mediated G-protein activation in the NAc, similarly to mice inducibly expressing ΔFosB in the present study (Vigano, et al., 2005). The effect of ΔFosB expression on Δ9-THC-mediated behaviors has not been evaluated, but the present results do not preclude an interaction. The results of this and our previous study (Zachariou, et al., 2006) show ΔFosB-induced alterations in MOR and KOR/dynorphin in the striatum. The rewarding effects of Δ9-THC, as measured by place preference, are abolished in MOR null mice, whereas deletion of KOR attenuated Δ9-THC place aversion and revealed Δ9-THC place preference (Ghozland, et al., 2002). Similarly, conditioned place aversion to Δ9-THC is absent in pro-dynorphin knockout compared to wild-type mice (Zimmer, et al., 2001). These data suggest that Δ9-THC might be more rewarding after ΔFosB induction and consequent induction of MOR signaling with reductions in dynorphin expression.

In summary, the results of this study showed that expression of ΔFosB in D1R/dynorphin positive striatal neurons enhanced MOR- and KOR-mediated signaling at the level of G-protein mediated inhibition of adenylyl cyclase activity in the NAc. This finding is consistent with studies that have demonstrated a role for the endogenous opioid system in reward (Trigo, et al., 2010), and provide a potential mechanism for ΔFosB-mediated effects on reward. In contrast, CB1R-mediated signaling in the NAc was not significantly affected by striatal ΔFosB expression under the conditions examined, although further studies are warranted to determine the effect of ΔFosB induction on the endocannabinoid system.

Research Highlights

  • MOR signaling is enhanced in the nucleus accumbens of mice that express ΔFosB
  • KOR inhibition of adenylyl cyclase is also enhanced in mice expressing ΔFosB
  • Expression of ΔFosB does not alter CB1R signaling in the nucleus accumbens
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Supplementary Material

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Acknowledgements

The authors thank Hengjun He, Jordan Cox and Aaron Tomarchio for technical assistance with the [35S]GTPγS binding assays. This study was supported by USPHS Grants DA014277 (LJS), DA10770 (DES) and P01 DA08227 (EJN).

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Footnotes

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References

  • Bozarth MA, Wise RA. Anatomically distinct opiate receptor fields mediate reward and physical dependence. Science. 1984;224:516–517. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. [PubMed]
  • Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Vogt LJ, Sim-Selley LJ. Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptor-activated G-proteins in brain. J. Neurochem. 1999;73:2447–2459. [PubMed]
  • Breivogel CS, Selley DE, Childers SR. Cannabinoid receptor agonist efficacy for stimulating [35S]GTPγS binding to rat cerebellar membranes correlates with agonist-induced decreases in GDP affinity. J. Biol. Chem. 1998;273:16865–16873. [PubMed]
  • Cadoni C, Pisanu A, Solinas M, Acquas E, Di Chiara G. Behavioural sensitization after repeated exposure to Delta 9-tetrahydrocannabinol and cross-sensitization with morphine. Psychopharmacology (Berl) 2001;158:259–266. [PubMed]
  • Cadoni C, Valentini V, Di Chiara G. Behavioral sensitization to delta 9-tetrahydrocannabinol and cross-sensitization with morphine: differential changes in accumbal shell and core dopamine transmission. J. Neurochem. 2008;106:1586–1593. [PubMed]
  • Chen J, Kelz MB, Zeng G, Sakai N, Steffen C, Shockett PE, Picciotto MR, Duman RS, Nestler EJ. Transgenic animals with inducible, targeted gene expression in brain. Mol. Pharmacol. 1998;54:495–503. [PubMed]
  • Childers SR. Opioid receptor-coupled second messengers. Life Sci. 1991;48:1991–2003. [PubMed]
  • Childers SR, Fleming L, Konkoy C, Marckel D, Pacheco M, Sexton T, Ward S. Opioid and cannabinoid receptor inhibition of adenylyl cyclase in brain. Ann. N. Y. Acad. Sci. 1992;654:33–51. [PubMed]
  • Childers SR, Xiao R, Vogt LJ, Sim-Selley LJ. Kappa opioid receptor stimulation of [35S]GTPγS binding in guinea pig brain: Lack of evidence for kappa2-selective activation of G-proteins. Biochem. Pharmacol. 1998;56:113–120. [PubMed]
  • Clarke S, Zimmer A, Zimmer AM, Hill RG, Kitchen I. Region selective up-regulation of micro-, delta- and kappa-opioid receptors but not opioid receptor-like 1 receptors in the brains of enkephalin and dynorphin knockout mice. Neuroscience. 2003;122:479–489. [PubMed]
  • Colby CR, Whisler K, Steffen C, Nestler EJ, Self DW. Striatal cell type-specific overexpression of DeltaFosB enhances incentive for cocaine. J. Neurosci. 2003;23:2488–2493. [PubMed]
  • Gardner EL. Endocannabinoid signaling system and brain reward: emphasis on dopamine. Pharmacol. Biochem. Behav. 2005;81:263–284. [PubMed]
  • Ghozland S, Matthes HW, Simonin F, Filliol D, Kieffer BL, Maldonado R. Motivational effects of cannabinoids are mediated by mu-opioid and kappa-opioid receptors. J. Neurosci. 2002;22:1146–1154. [PubMed]
  • Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J. Neurosci. 1991;11:563–583. [PubMed]
  • Hohmann AG, Herkenham M. Localization of cannabinoid CB(1) receptor mRNA in neuronal subpopulations of rat striatum: a double-label in situ hybridization study. Synapse. 2000;37:71–80. [PubMed]
  • Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacological Review. 2002;54:161–202.
  • Huestis MA, Gorelick DA, Heishman SJ, Preston KL, Nelson RA, Moolchan ET, Frank RA. Blockade of effects of smoked marijuana by the CB1-selective cannabinoid receptor antagonist SR141716. Arch. Gen. Psychiatry. 2001;58:322–328. [PubMed]
  • Kelz MB, Chen J, Carlezon WA, Jr, Whisler K, Gilden L, Beckmann AM, Steffen C, Zhang YJ, Marotti L, Self DW, Tkatch T, Baranauskas G, Surmeier DJ, Neve RL, Duman RS, Picciotto MR, Nestler EJ. Expression of the transcription factor deltaFosB in the brain controls sensitivity to cocaine. Nature. 1999;401:272–276. [PubMed]
  • Koob GF, Volkow ND. Neurocircuitry of addiction. Neuropsychopharmacology. 2010;35:217–238. [PMC free article] [PubMed]
  • Lamarque S, Taghzouti K, Simon H. Chronic treatment with Delta(9)-tetrahydrocannabinol enhances the locomotor response to amphetamine and heroin. Implications for vulnerability to drug addiction. Neuropharmacology. 2001;41:118–129. [PubMed]
  • Maldonado R, Valverde O, Berrendero F. Involvement of the endocannabinoid system in drug addiction. Trends Neurosci. 2006;29:225–232. [PubMed]
  • Mansour A, Fox CA, Thompson RC, Akil H, Watson SJ. mu-Opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding. Brain Res. 1994;643:245–265. [PubMed]
  • Matthes HWD, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, LeMeur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid receptor gene. Nature. 1996;383:819–823. [PubMed]
  • McClung CA, Nestler EJ. Regulation of gene expression and cocaine reward by CREB and DeltaFosB. Nat. Neurosci. 2003;6:1208–1215. [PubMed]
  • McClung CA, Ulery PG, Perrotti LI, Zachariou V, Berton O, Nestler EJ. DeltaFosB: a molecular switch for long-term adaptation in the brain. Brain Res. Mol. Brain Res. 2004;132:146–154. [PubMed]
  • Muller DL, Unterwald EM. D1 dopamine receptors modulate deltaFosB induction in rat striatum after intermittent morphine administration. J. Pharmacol. Exp. Ther. 2005;314:148–154. [PubMed]
  • Nestler EJ. Review. Transcriptional mechanisms of addiction: role of DeltaFosB. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008;363:3245–3255. [PMC free article] [PubMed]
  • Nestler EJ, Kelz MB, Chen J. DeltaFosB: a molecular mediator of long-term neural and behavioral plasticity. Brain Res. 1999;835:10–17. [PubMed]
  • Nye HE, Hope BT, Kelz MB, Iadarola M, Nestler EJ. Pharmacological studies of the regulation of chronic FOS-related antigen induction by cocaine in the striatum and nucleus accumbens. J. Pharmacol. Exp. Ther. 1995;275:1671–1680. [PubMed]
  • Peakman MC, Colby C, Perrotti LI, Tekumalla P, Carle T, Ulery P, Chao J, Duman C, Steffen C, Monteggia L, Allen MR, Stock JL, Duman RS, McNeish JD, Barrot M, Self DW, Nestler EJ, Schaeffer E. Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in transgenic mice decreases sensitivity to cocaine. Brain Res. 2003;970:73–86. [PubMed]
  • Perrotti LI, Weaver RR, Robison B, Renthal W, Maze I, Yazdani S, Elmore RG, Knapp DJ, Selley DE, Martin BR, Sim-Selley L, Bachtell RK, Self DW, Nestler EJ. Distinct patterns of DeltaFosB induction in brain by drugs of abuse. Synapse. 2008;62:358–369. [PMC free article] [PubMed]
  • Robbe D, Alonso G, Duchamp F, Bockaert J, Manzoni OJ. Localization and mechanisms of action of cannabinoid receptors at the glutamatergic synapses of the mouse nucleus accumbens. J. Neurosci. 2001;21:109–116. [PubMed]
  • Salomon Y. Adenylate cyclase assay. Adv. Cyclic Nucleotide Res. 1979;10:35–55. [PubMed]
  • Selley DE, Sim LJ, Xiao R, Liu Q, Childers SR. Mu opioid receptor-stimulated [35S]GTPγS binding in rat thalamus and cultured cell lines: Signal transduction mechanisms underlying agonist efficacy. Mol. Pharmacol. 1997;51:87–96. [PubMed]
  • Trigo JM, Martin-Garcia E, Berrendero F, Robledo P, Maldonado R. The endogenous opioid system: a common substrate in drug addiction. Drug Alcohol Depend. 2010;108:183–194. [PubMed]
  • Unterwald EM, Knapp C, Zukin RS. Neuroanatomical localization of κ1 and κ2 opioid receptors in rat and guinea pig brain. Brain Res. 1991;562:57–65. [PubMed]
  • Vaccarino FJ, Bloom FE, Koob GF. Blockade of nucleus accumbens opiate receptors attenuates intravenous heroin reward in the rat. Psychopharmacology (Berl) 1985;86:37–42. [PubMed]
  • Vigano D, Rubino T, Vaccani A, Bianchessi S, Marmorato P, Castiglioni C, Parolaro D. Molecular mechanisms involved in the asymmetric interaction between cannabinoid and opioid systems. Psychopharmacology (Berl) 2005;182:527–536. [PubMed]
  • Zachariou V, Bolanos CA, Selley DE, Theobald D, Cassidy MP, Kelz MB, Shaw-Lutchman T, Berton O, Sim-Selley LJ, Dileone RJ, Kumar A, Nestler EJ. An essential role for DeltaFosB in the nucleus accumbens in morphine action. Nat. Neurosci. 2006;9:205–211. [PubMed]
  • Zangen A, Solinas M, Ikemoto S, Goldberg SR, Wise RA. Two brain sites for cannabinoid reward. J. Neurosci. 2006;26:4901–4907. [PubMed]
  • Zhu J, Luo LY, Li JG, Chen C, Liu-Chen LY. Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPγS binding to membranes: determination of potencies and efficacies of ligands. J. Pharmacol. Exp. Ther. 1997;282:676–684. [PubMed]
  • Zimmer A, Valjent E, Konig M, Zimmer AM, Robledo P, Hahn H, Valverde O, Maldonado R. Absence of delta -9-tetrahydrocannabinol dysphoric effects in dynorphin-deficient mice. J. Neurosci. 2001;21:9499–9505. [PubMed]
  • Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc. Natl. Acad. Sci. U. S. A. 1999;96:5780–5785. [PMC free article] [PubMed]