Striatal regulation of ΔFosB, FosB, and cFos during cocaine self-administration and withdrawal.

Chronic drug exposure induces alterations in gene expression profiles that are thought to underlie the development of drug addiction. The present study examined regulation of the Fos-family of transcription factors, specifically cFos, FosB, and ΔFosB, in striatal subregions during and after chronic intravenous cocaine administration in self-administering and yoked rats. We found that cFos, FosB, and ΔFosB exhibit regionally and temporally distinct expression patterns, with greater accumulation of ΔFosB protein in the nucleus accumbens (NAc) shell and core after chronic cocaine administration, whereas ΔFosB increases in the caudate-putamen (CPu) remained similar with either acute or chronic administration. In contrast, tolerance developed to cocaine-induced mRNA for ΔFosB in all three striatal subregions with chronic administration. Tolerance also developed to FosB expression, most notably in the NAc shell and CPu. Interestingly, tolerance to cocaine-induced cFos induction was dependent on volitional control of cocaine intake in ventral but not dorsal striatal regions, whereas regulation of FosB and ΔFosB was similar in cocaine self-administering and yoked animals. Thus, ΔFosB-mediated neuroadaptations in the CPu may occur earlier than previously thought with the initiation of intravenous cocaine use and, together with greater accumulation of ΔFosB in the NAc, could contribute to addiction-related increases in cocaine-seeking behavior.

Environmental enrichment produces a behavioral phenotype mediated by low cyclic adenosine monophosphate response element binding (CREB) activity in the nucleus accumbens.

BACKGROUND: Previous research has shown that rats reared in an enriched condition (EC) are more sensitive to the acute effects of amphetamine than rats reared in an isolated condition (IC); yet, EC rats self-administer less amphetamine than IC rats. The present study used cocaine to further explore this environmental enrichment behavioral phenotype, as well as the underlying molecular mechanisms involved. METHODS: Enriched condition and IC rats were studied in a broad battery of behavioral tests, including cocaine conditioned place preference (CPP) and self-administration and several measures of anxiety- and depression-related behavior. The involvement of the transcription factor, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), in mediating EC versus IC differences was investigated. RESULTS: Enriched condition rats exhibited less cocaine self-administration, despite showing enhanced cocaine CPP. Enriched condition rats also displayed less depression-like behavior but higher levels of anxiety-like behavior. This behavioral phenotype is consistent with low CREB activity in the nucleus accumbens, a key brain reward region. Indeed, EC rats have less phospho-CREB (the transcriptionally active form of the protein) in the nucleus accumbens than IC rats, and a selective knockdown of CREB in this brain region of normally reared rats, by use of a novel viral vector expressing a short hairpin RNA (shRNA) directed against CREB, reproduced the EC behavioral phenotype. CONCLUSIONS: These studies identify a potential molecular mechanism for how rearing environment-a nonpharmacological, nonsurgical manipulation-can modify a wide range of complex emotional behaviors.

Induction of activating transcription factors (ATFs) ATF2, ATF3, and ATF4 in the nucleus accumbens and their regulation of emotional behavior.

Previous research has shown that cAMP response element (CRE) binding protein (CREB) in the nucleus accumbens gates behavioral responses to emotional stimuli. For example, overexpression of CREB decreases anxiety, sucrose preference, and sensitivity to drugs of abuse and increases depression-like behavior, whereas blocking CREB via overexpression of inducible cAMP early repressor (ICER) or other dominant-negative inhibitors of CRE-mediated transcription has the opposite effects. However, CREB and ICER are but two members of a larger family of leucine zipper-containing transcription factors composed of multiple products of the creb, crem (cAMP response element modulator), and atf (activating transcription factor) genes. We demonstrate here that ATF2, ATF3, and ATF4 are each robustly induced in the nucleus accumbens and dorsal striatum by restraint stress or by amphetamine administration. In contrast, little induction is seen for ATF1 or CREM. Using viral-mediated gene transfer, we show that ATF2 overexpression in nucleus accumbens produces increases in emotional reactivity and antidepressant-like responses, a behavioral phenotype similar to that caused by dominant-negative antagonists of CREB. In contrast, ATF3 or ATF4 overexpression in nucleus accumbens decreases emotional reactivity and increases depression-like behavior, consistent with the behavioral phenotype induced by CREB. Because amphetamine and stress induce ATF2, ATF3, and ATF4 in nucleus accumbens, and overexpression of these transcription factors in this brain region in turn alters behavioral responsiveness to amphetamine and stress, our findings support novel roles for these ATF family members in regulating emotional behavior.

Proteasome-dependent and -independent mechanisms for FosB destabilization: identification of FosB degron domains and implications for DeltaFosB stability.

The transcription factor DeltaFosB (Delta FosB) accumulates in a region-specific manner in the brain during chronic exposure to stress, drugs of abuse or other chronic stimuli. Once induced, DeltaFosB persists in the brain for at least several weeks following cessation of the chronic stimulus. The biochemical basis of the persistent expression of DeltaFosB has remained unknown. Here, we show that the FosB C-terminus, absent in DeltaFosB as a result of alternative splicing, contains two degron domains. Pulse-chase experiments of C-terminal truncation mutants of full-length FosB indicate that removal of its most C-terminal degron increases its half-life approximately fourfold, and prevents its proteasome-mediated degradation and ubiquitylation, properties similar to DeltaFosB. In addition, removal of a second degron domain, which generates DeltaFosB, further stabilizes FosB approximately twofold, but in a proteasome-independent manner. These data indicate that alternative splicing specifically removes two destabilizing elements from FosB in order to generate a longer-lived transcription factor, DeltaFosB, in response to chronic perturbations to the brain.

Regulation of fosB and DeltafosB mRNA expression: in vivo and in vitro studies.

The transcription factor DeltaFosB, a truncated splice isoform of FosB, accumulates in brain after several types of chronic stimulation. This accumulation is thought to be mediated by the unique stability of DeltaFosB compared to all other Fos family proteins. The goal of the present study was to determine if the relative expression of the two fosB isoforms is also regulated at the mRNA level, thereby further contributing to the selective accumulation of DeltaFosB after chronic stimulation. First, unlike the protein, the half-life of DeltafosB mRNA is only slightly longer than that of full-length fosB mRNA both in cultured cells in vitro and in the brain in vivo. Additionally, similar to c-fos, both fosB isoforms are induced abundantly in striatum after acute administration of amphetamine or stress, and partially desensitize after chronic exposures. Surprisingly, the relative ratio of DeltafosB to fosB mRNA increases most significantly after acute, not chronic, stimulation. Finally, overexpression of polypyrimidine tract binding protein (PTB1), which regulates RNA splicing, in cultured cells decreases the relative expression of DeltafosB compared to fosB mRNA. Together, these findings suggest that splicing of fosB pre-mRNA is regulated by the quantity of unspliced transcript available to the splicing machinery. These data provide fundamental information concerning the generation of DeltafosB mRNA, and indicate that the selective accumulation of DeltaFosB protein with chronic stimulation does not involve its preferential generation by RNA splicing.

Induction of inducible cAMP early repressor expression in nucleus accumbens by stress or amphetamine increases behavioral responses to emotional stimuli.

Previous research has shown that cAMP response element (CRE)-mediated transcription is activated in the nucleus accumbens, a major brain reward region, by a variety of environmental stimuli and contributes to neuroadaptations to these stimuli. CRE-binding protein (CREB) is the most studied activator of CRE transcription and has been implicated in this brain region as a gating mechanism for behavioral responses to emotional stimuli. Little attention, however, has been given to naturally occurring inhibitors of CRE-mediated transcription, such as the inducible cAMP early repressor (ICER), an inhibitory product of the CRE modulator gene. In the present study, we investigated the extent to which ICER is induced in the nucleus accumbens by two types of environmental stimuli, stress and amphetamine, and characterized how induction of ICER in this region affects complex behavior. We show that stress and amphetamine each induces ICER expression and that overexpression of ICER in the nucleus accumbens, using viral-mediated gene transfer, increases behavioral responses to both rewarding and aversive emotional stimuli. For example, ICER overexpression increases sensitivity to amphetamine-stimulated locomotor activity as well as to natural rewards such as sucrose and social grooming. However, ICER overexpression also increases measures of anxiety in the elevated plus maze and neophobia to novel tastes. Finally, ICER produces an antidepressant-like effect in the forced swim test, further indication of an enhanced active response to stress. These results suggest that ICER is an important mechanism for modulating CRE-mediated transcription in the nucleus accumbens.