Neuroscience. 2014 Oct 10. pii: S0306-4522(14)00856-2. doi: 10.1016/j.neuroscience.2014.10.002.
Previous work has implicated the transcription factor, ΔFosB, acting in the nucleus accumbens, in mediating the pro-rewarding effects of drugs of abuse such as cocaine as well as in mediating resilience to chronic social stress. However, the transgenic and viral gene transfer models used to establish these ΔFosB phenotypes express, in addition to ΔFosB, an alternative translation product of ΔFosB mRNA, termed Δ2ΔFosB, which lacks the N-terminal 78 aa present in ΔFosB. To study the possible contribution of Δ2ΔFosB to these drug and stress phenotypes, we prepared a viral vector that overexpresses a point mutant form of ΔFosB mRNA which cannot undergo alternative translation as well as a vector that overexpresses Δ2ΔFosB alone. Our results show that the mutant form of ΔFosB, when overexpressed in the nucleus accumbens, reproduces the enhancement of reward and of resilience seen with our earlier models, with no effects seen for Δ2ΔFosB. Overexpression of full length FosB, the other major product of the FosB gene, also has no effect. These findings confirm the unique role of ΔFosB in nucleus accumbens in controlling responses to drugs of abuse and stress.
ΔFosB is encoded by the FosB gene and shares homology with other Fos family transcription factors, which include c-Fos, FosB, Fra1, and Fra2. All Fos family proteins are induced rapidly and transiently in specific brain regions after acute administration of many drugs of abuse [see Nestler, 2008]. These responses are seen most prominently in nucleus accumbens (NAc) and dorsal striatum, which are important mediators of the rewarding and locomotor actions of the drugs. All of these Fos family proteins, however, are highly unstable and return to basal levels within hours of drug administration. In contrast, ΔFosB, due to its unusual stability in vitro and in vivo (Ulery et al., 2006; Carle et al., 2006; Ulery-Reynolds et al., 2009), accumulates uniquely within the same brain regions after repeated drug exposure (Hope et al., 1994; Hiroi et al., 1997; Perrotti et al., 2008). More recent studies have demonstrated that chronic exposure to certain forms of stress also induces the accumulation of ΔFosB in the NAc, and that such induction occurs preferentially in animals that are relatively resistant to the deleterious effects of the stress (i.e., resilient animals) (Perrotti et al., 2004; Vialou et al., 2010a, 2010b).
We have demonstrated that overexpression of ΔFosB in the NAc, either in inducible bitransgenic mice or by local viral-mediated gene transfer, increases an animal’s sensitivity to the rewarding and locomotor-activating effects of cocaine and other drugs of abuse (Kelz et al., 1999; Colby et al., 2003; Zachariou et al., 2006; Grueter et al., 2013; Robison et al., 2013). Such induction also boosts consumption of and motivation for natural rewards (Werme et al., 2002; Teegarden et al., 2007; Wallace et al., 2008; Hedges et al., 2009; Been et al., 2013; Pitchers et al., 2009; 2013), increases brain stimulation reward in intra-cranial self-stimulation paradigms (Muschamps et al., 2012), and renders animals more resilient to several forms of chronic stress (Vialou et al., 2010a, 2010b). Likewise, mice that constitutively lack expression of full length FosB, but show increased expression ΔFosB, display reduced sensitivity to stress (Ohnishi et al., 2011). Together, these findings support the view that ΔFosB, acting in the NAc, boosts an animal’s state of reward, mood, and motivation.
However, a major caveat of these studies is that another product of the FosB gene, termed Δ2ΔFosB, is also expressed in all of these genetic mutant mice and viral vector systems, leaving open the possible contribution of Δ2ΔFosB to the behavioral phenotypes observed. Δ2ΔFosB is translated from an alternative start codon located within the ΔFosB mRNA transcript (Chen et al., 1997). This alternative translation leads to the formation of Δ2ΔFosB, which lacks the 78 N-terminal aa of ΔFosB. In this study, we examined the role of Δ2ΔFosB in drug abuse and stress models by overexpressing it, or ΔFosB or FosB, with AAV (adeno-associated virus) vectors; we used a mutant form of ΔFosB mRNA that cannot undergo this alternative translation mechanism. Our results confirm that the pro-reward and pro-resilient actions seen in earlier studies are indeed mediated via ΔFosB and not by the two other protenproducts of the FosB gene, full-length FosB or Δ2ΔFosB.
Prior to experimentation, 9- to 11-week-old C57BL/6J male mice (The Jackson Laboratory, Bar Harbor, ME, USA) were group housed at five per cage in a colony room set at constant temperature (23°C) on a 12 hr light/dark cycle (lights on at 7 AM) with ad libitum access to food and water. Some experiments utilized bitransgenic mice in which overexpression of ΔFosB is under the control of the tetracycline gene regulation system, as described (Kelz et al., 1999). Mice were used on doxycycline (to maintain gene expression off) or off doxycycline which enables ΔFosB expression. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Mount Sinai.
We used AAV2 serotype for packaging AAV vectors expressing full-length FosB, ΔFosB, or Δ2ΔFosB under the human immediate early cytomegalovirus (CMV) promoter with Venus fluorescent protein encoded after an intervening IRES2 (internal ribosome re-entry site 2). The AAV-ΔFosB construct expressed a mutant form of ΔFosB mRNA, where the codon representing Met79 was mutated to Leu to obliterate the alternative translation start site that generates Δ2ΔFosB.
Viral-mediated gene transfer
Mice were positioned in small animal stereotaxic instruments under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia, and their cranial surfaces were exposed. Thirty-three gauge syringe needles were bilaterally lowered into the NAc to infuse 0.5 μl of AAV vector at a 10° angle (anterior/posterior + 1.6; medial/lateral + 1.5; dorsal/ventral − 4.4 mm). Infusions occurred at a rate of 0.1 μl/min. Animals receiving AAV injections were allowed to recover for at least 24 hr after surgery. For confirmation of expression, mice were anesthetized and perfused intracardially with 4% paraformaldehyde/PBS (phosphate-buffered saline). Brains were cryoprotected with 30% sucrose, and then frozen and stored at −80°C until use. Coronal sections (40 μm) were cut on a cryostat and processed for scanning by confocal microscopy.
Mice were studied with several standard behavioral assays according to published protocols as follows:
Chronic (10 days) social defeat stress was performed exactly as described (Berton et al., 2006; Vialou et al., 2010a). Briefly, one experimental mouse and one CD1 aggressor were put together for 5 min in the CD1 mouse’s home cage. They were then separated by a plastic divider, which was perforated to allow for sensory contact for the reminder of the day. Every morning for 10 days, the experimental mouse was moved into a different aggressor mouse’s cage. Non-defeated control mice underwent similar exposures, but with other C57BL/6J mice. Tests for social interaction were performed as previously described (Berton et al., 2006; Vialou et al., 2010a). Briefly, the test mouse was placed within a novel arena that included a small cage on one side. Movement (e.g., distance traveled, spent time in the vicinity of this small cage) was monitored initially for 150 sec when the small cage was empty, followed by an additional 150 sec with a CD1 mouse in that cage. Movement information was obtained using EthoVision 5.0 software (Noldus).
We used a standard, unbiased conditioned place preference (CPP) procedure (Kelz et al., 1999; Robison et al., 2013). Briefly, animals were pretested for 20 min in a photo-beam monitored three-chambered box with free access to environmentally distinct side-chambers. The mice were then divided into control and experimental groups with equivalent pretest scores. After experimental manipulation, mice underwent four 30 min training sessions (alternating cocaine and saline pairing). On the test day, mice had 20 min of unrestricted access to all chambers, and a CPP score was calculated by subtracting time spent in the cocaine-paired chamber minus time spent in the saline-paired chamber. Cocaine-induced locomotor activity was measured via photobeam breaks in the CPP box for 30 min following each test injection.
Elevated plus maze tests were performed using black Plexiglass fitted with white bottom surfaces to provide contrast (Monteggia et al., 2007). Mice were placed in the center of the plus maze and allowed to freely explore the maze for 5 min under red-light conditions. The position of each mouse over time in the open and closed arms was monitored with videotracking equipment (Ethovision) and a ceiling-mounted camera.
General, ambulatory locomotor activity during the night phase was assessed in home cages with a photocell grid device (Med Associates Inc., St. Albans, VT, USA) that counted the number of ambulatory photo beam breaks during a 12 hr period (Roybal et al., 2007).
NAc samples were subjected to Western blotting as described (Vialou et al., 2010a, 2010b). Frozen NAc dissections were homogenized in 100 μl of buffer containing phosphatase inhibitor cocktails I and II (Sigma, St. Louis, MO, USA) and protease inhibitors (Roche, Basel, Switzerland) using an ultrasonic processor (Cole Parmer, Vemon Hills, IL, USA). Protein concentrations were determined using a DC protein assay (Bio-Rad, Hercules, CA, USA), and 10–30 μg of protein were loaded onto 12.5% or 4%–15% gradient Tris-HCl plyacrylamide gels for electrophoresis fractionation (Bio-Rad). After transferring proteins to nitrocellulose filters, the filters were incubated with an anti-FosB antibody that recognizes all FosB gene products, then with secondary antibody, and finally quantified using the Odyssey system (Li-Cor) according to manufacturer protocols.
ANOVAs and student’s t-tests were used, corrected for multiple comparisons, with significance set at p<0.05.
As shown in Figure 1A, the FosB gene encodes mRNAs for full-length FosB and for ΔFosB. ΔFosB mRNA is generated from an alternative splicing event within Exon 4 of the FosB primary transcript; this results in the generation of a premature stop codon and to the truncated ΔFosB protein, which lacks the C-terminal 101 aa present in FosB. FosB and ΔFosB mRNA share the same ATG start codon, located toward the 3′ end of Exon 1. It has been known since the original cloning of FosB products that the two mRNAs also share alternative translation start sites within Exon 2, termed the Δ1, Δ2, and Δ3 ATGs. Previous work showed that a minor protein product is generated from ΔFosB mRNA, but not FosB mRNA, via the Δ2 ATG; this protein is termed Δ2ΔFosB and lacks the 78 aa N-terminal region of ΔFosB (Chen et al., 1997). In contrast, the Δ1 and Δ3 ATGs appear to be silent, since there is no evidence for their use in translation of the FosB or ΔFosB transcripts.
Figure 1B illustrates the induction of FosB gene products in NAc after a course of repeated cocaine administration, with animals examined 2 hr after the last cocaine dose. At this time point, both ΔFosB and FosB proteins show significant induction by cocaine, with no consistent induction of Δ2ΔFosB. Note that the induction of both ΔFosB and FosB is different from the pattern seen at 24 hr or more after the last drug dose, when only ΔFosB is induced due to the unique stability of the ΔFosB protein (Hope et al., 1994; Chen et al., 1997; Perrotti et al., 2008). However, in contrast to the lack of induction of Δ2ΔFosB by repeated cocaine administration, the bitransgenic mouse system that we have used to overexpress ΔFosB and to thereby study its behavioral consequences (Kelz et al., 1999; Colby et al., 2003; Zachariou et al., 2006) leads to a significant, albeit lower levels of, overexpression of Δ2ΔFosB in addition to ΔFosB (Figure 1C). A similar level of induction of Δ2ΔFosB is seen with our viral vectors that overexpress wildtype ΔFosB (e.g., see Figure 2). These observations raise the possibility that some of the purported actions of ΔFosB reported previously might be mediated in part via Δ2ΔFosB.
To distinguish the differential roles of ΔFosB versus Δ2ΔFosB, we generated an AAV vector that overexpresses Δ2ΔFosB alone, as well as a new vector that overexpresses a mutant form of ΔFosB mRNA (mΔFosB mRNA) which cannot be subject to alternative translation to generate Δ2ΔFosB. Both vectors also express Venus as a marker of expression. We compared the effects of these two vectors to others, which express FosB plus Venus or Venus alone as a control. The ability of these new AAV vectors to selectively overexpress their encoded transgenes is depicted in Figure 2.
Next, to test the effect of each FosB gene product, acting in the NAc. on complex behavior, we injected each of these AAVs into this brain region bilaterally of separate groups of mice and, 3 weeks later when transgene expression is maximal (Figure 3A), performed a battery of tests. We first evaluated the ability of the FosB gene products to influence the pro-resilience phenotype reported previously for ΔFosB in the social defeat paradigm (Vialou et al., 2010a, 2010b), As shown in Figure 3A, control mice expressing Venus alone displayed the expected decrement in social interaction behavior, a well-established behavioral marker of susceptibility (Berton et al., 2006; Vialou et al., 2010a). Overexpression of mΔFosB completely reversed this phenotype, in contrast to Δ2ΔFosB and FosB which had no effect.
To test the relative contribution of each FosB gene product to the rewarding effects of cocaine, we overexpressed Δ2ΔFosB itself, mΔFosB, or FosB bilaterally in the NAc and studied the animals in the conditioned place preference paradigm. As shown in Figure 3B, bilateral overexpression of mΔFosB in the NAc increases the place conditioning effects of a threshold dose of cocaine, which did not produce a significant place preference in Venus-expressing control animals. In contrast, overexpression of Δ2ΔFosB or FosB had no effect on cocaine place conditioning. Since we used a threshold dose of cocaine, which did not produce a significant place preference in control animals, we cannot exclude the possibility that FosB or Δ2ΔFosB might reduce the rewarding effects of cocaine.
Finally, to evaluate baseline behaviors, we examined locomotor activity in the animals’ home cage as well as anxiety-like behavior in the elevated plus maze. FosB, mΔFosB, nor Δ2ΔFosB overexpression in the NAc had an effect on locomotor activity, although FosB and Δ2ΔFosB—but not mΔFosB—produced a small but significant decrease in anxiety-like behavior in the elevated plus maze (Figure 3D,E). These data suggest that FosB gene expression does not appreciably alter behavior under normal conditions.
Results of the present study confirm that the phenotype reported previously for ΔFosB is indeed mediated via ΔFosB and not by Δ2ΔFosB, an alternatively translated product of ΔFosB mRNA which lacks ΔFosB’s N-terminus. While our previously used tools to overexpress ΔFosB also result in the generation of low levels of Δ2ΔFosB, we show here that overexpression in NAc of a mutated form of ΔFosB mRNA, which cannot generate Δ2ΔFosB due to mutation of the alternative start codon involved, recapitulates the increase both in cocaine reward and in resilience to social defeat stress reported previously for ΔFosB (Kelz et al., 1999; Vialou et al., 2010a). Moreover, overexpression of Δ2ΔFosB itself has no effect on either cocaine or stress responses. We also show, for the first time, that overexpression of full length FosB in NAc likewise has no effect on behavioral responses to cocaine or stress.
While these results do not rule out the possibility that Δ2ΔFosB, as a minor protein product of the FosB gene, might exert functional effects in other brain regions or in peripheral tissues, our findings nonetheless confirm the unique contribution of ΔFosB, acting in the NAc reward circuit, in promoting cocaine reward and stress resilience.
- ΔFosB mRNA gives rise to ΔFosB and to the minor alternatively translated Δ2ΔFosB.
- Overexpression of ΔFosB alone confirms its pro-reward and pro-resilience phenotype.
- In contrast, Δ2ΔFosB has no effect on cocaine reward or stress vulnerability.
- Full-length FosB, encoded by FosB mRNA, also does not affect reward or resilience.
This work was supported by grants from the National Institute of Mental Health and National Institute on Drug Abuse, and by the Ishibashi foundation and the Japan Society for the Promotion of Science (JSPS KAKENHI numbers: 24591735).
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- Been LE, Hedges VL, Vialou V, Nestler EJ, Meisel RL. Delta JunD expression in the nucleus accumbens prevents sexual reward in female Syrian hamsters. Genes Brain Behav. 2013;12:666–672. [PMC free article] [PubMed]
- Berton O, McClung CA, DiLeone RJ, Krishnan V, Russo S, Graham D, Tsankova NM, Bolanos CA, Rios M, Monteggia LM, Self DW, Nestler EJ. Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science. 2006;311:864–868. [PubMed]
- Carle TL, Ohnishi YN, Ohnishi YH, Alibhai IN, Wilkinson MB, Kumar A, Nestler EJ. Absence of conserved C-terminal degron domain contributes to ΔFosB’s unique stability. Eur J Neurosci. 2007;25:3009–3019. [PubMed]
- Chen JS, Kelz MB, Hope BT, Nakabeppu Y, Nestler EJ. Chronic Fos-related antigens: stable variants of deltaFosB induced in brain by chronic treatments. J Neurosci. 1997;17:4933–4941. [PubMed]
- Colby CR, Whisler K, Steffen C, Nestler EJ, Self DW. ΔFosB enhances incentive for cocaine. J Neurosci. 2003;23:2488–2493. [PubMed]
- Grueter BA, Robison AJ, Neve RL, Nestler EJ, Malenka RC. ΔFosB differentially modulates nucleus accumbens direct and indirect pathway function. Proc Natl Acad Sci USA. 2013;110:1923–1927. [PMC free article] [PubMed]
- Hedges VL, Chakravarty S, Nestler EJ, Meisel RL. ΔFosB overexpression in the nucleus accumbens enhances sexual reward in female Syrian hamsters. Genes Brain Behav. 2009;8:442–449. [PMC free article] [PubMed]
- Hiroi N, Brown J, Haile C, Ye H, Greenberg ME, Nestler EJ. FosB mutant mice: Loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to cocaine’s psychomotor and rewarding effects. Proc Natl Acad Sci USA. 1997;94:10397–10402. [PMC free article] [PubMed]
- Hope BT, Nye HE, Kelz MB, Self DW, Iadarola MJ, Nakabeppu Y, Duman RS, Nestler EJ. Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments. Neuron. 1994;13:1235–1244. [PubMed]
- Kelz MB, Chen J, Carlezon WA, Jr, Whisler K, Gilden L, Beckmann AM, Steffen C, Zhang Y-J, Marotti L, Self DW, Tkatch R, Baranauskas G, Surmeier DJ, Neve RL, Duman RS, Picciotto MR, Nestler EJ. Expression of the transcription factor ΔFosB in the brain controls sensitivity to cocaine. Nature. 1999;401:272–276. [PubMed]
- Monteggia LM, Luikart B, Barrot M, Theobald D, Malkovska I, Nef S, Parada LF, Nestler EJ. BDNF conditional knockouts show gender differences in depression related behaviors. Biol Psychiatry. 2007;61:187–197. [PubMed]
- Muschamp JW, Nemeth CL, Robison AJ, Nestler EJ, Carlezon WA., Jr ΔFosB enhances the rewarding effects of cocaine while reducing the pro-depressive effects of the kappa-opioid agonist U50488. Biol Psychiatry. 2012;71:44–50. [PMC free article] [PubMed]
- Nestler EJ. Transcriptional mechanisms of addiction: role of deltaFosB. Philos Trans R Soc London B Biol Sci. 2008;363:3245–3255. [PMC free article] [PubMed]
- Ohnishi YN, Ohnishi YH, Hokama M, Nomaru H, Yamazaki K, Tominaga Y, Sakumi K, Nestler EJ, Nakabeppu Y. FosB Is essential for the enhancement of stress tolerance and antagonizes locomotor sensitization by FosB. Biol Psychiatry. 2011;70:487–495. [PMC free article] [PubMed]
- Perrotti LI, Hadeishi Y, Ulery P, Barrot M, Monteggia L, Duman RS, Nestler EJ. Induction of ΔFosB in reward-related brain regions after chronic stress. J Neurosci. 2004;24:10594–10602. [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 ΔFosB induction in brain by drugs of abuse. Synapse. 2008;62:358–369. [PMC free article] [PubMed]
- Pitchers KK, Frohmader KS, Vialou V, Mouzon E, Nestler EJ, Lehman MN, Coolen LM. ΔFosB in the nucleus accumbens is critical for reinforcing effects of sexual reward. Genes Brain Behav. 2010;9:831–840. [PMC free article] [PubMed]
- Pitchers KK, Vialou V, Nestler EJ, Lehman MN, Coolen LM. Sexual experience increases amphetamine reward and nucleus accumbens spinogenesis via dopamine D1 receptor activity and induction of deltaFosB. J Neurosci. 2013;33:3434–3442. [PMC free article] [PubMed]
- Roybal K, Theobold D, DiNieri JA, Graham A, Russo S, Krishnan V, Chakravarty S, Peevey J, Oehrlein N, Birnbaum S, Vitaterna MH, Orsulak P, Takahashi JS, Nestler EJ, Carlezon WA, Jr, McClung CA. Mania-like behavior induced by disruption of CLOCK. Proc Natl Acad Sci USA. 2007;104:6406–6411. [PMC free article] [PubMed]
- Teegarden SL, Bale TL. Decreases in dietary preference produce increased emotionality and risk for dietary relapse. Biol Psychiatry. 2007;61:1021–1029. [PubMed]
- Ulery PG, Rudenko G, Nestler EJ. Regulation of ΔFosB stability by phosphorylation. J Neurosci. 2006;26:5131–5142. [PubMed]
- Ulery-Reynolds PG, Castillo MA, Vialou V, Russo SJ, Nestler EJ. Phosphorylation of ΔFosB mediates its stability in vivo. Neuroscience. 2009;158:369–372. [PMC free article] [PubMed]
- Vialou V, Robison AJ, LaPlant QC, Covington HE, III, Dietz DM, Ohnishi YN, Mouzon E, Rush AJ, III, Watts EL, Wallace DL, Iñiguez SD, Ohnishi YH, Steiner MA, Warren B, Krishnan V, Neve RL, Ghose S, Berton O, Tamminga CA, Nestler EJ. ΔFosB in brain reward circuits mediates resilience to stress and antidepressant responses. Nature Neurosci. 2010a;13:745–752. [PMC free article] [PubMed]
- Vialou V, Maze I, Renthal W, LaPlant QC, Watts EL, Mouzon E, Ghose S, Tamminga CA, Nestler EJ. Serum response factor promotes resilience to chronic social stress through the induction of ΔFosB. J Neurosci. 2010b;30:14585–14592. [PMC free article] [PubMed]
- Wallace DL, Vialou V, Rios L, Carle-Florence TL, Chakravarty S, Kumar A, Graham D, Green TA, Iniguez SD, Perrotti LI, Barrot M, DiLeone RJ, Nestler EJ, Bolaños CA. The influence of ΔFosB in the nucleus accumbens on natural reward-related behavior. J Neurosci. 2008;28:10272–10277. [PMC free article] [PubMed]
- Werme M, Messer C, Olson L, Gilden L, Thorén P, Nestler EJ, Brené S. ΔFosB regulates wheel running. J Neurosci. 2002;22:8133–8138. [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. ΔFosB: An essential role for ΔFosB in the nucleus accumbens in morphine action. Nature Neurosci. 2006;9:205–211. [PubMed]