In examining the relationship between the molecular and behavioral changes, it is possible to speculate on the roles of molecular adaptations in the development and/or expression of increased drug-seeking and reinforcing efficacy. This study and previous reports using the DT4 model have established that there is an increase in drug-seeking and reinforcing efficacy measures beginning at 7 days of abstinence and still evident at 100 days of abstinence. Clearly, some form of incubation is occurring in the first week of abstinence and these changes then persist for months, in a manner similar to the previous work of Shaham, Hope and colleagues (Shaham and Hope, 2005
; Lu et al., 2004
The gene expression results demonstrate that molecular changes persist even following 100 days of forced abstinence. Rats generated for the gene expression and chromatin experiments did not undergo the extinction session to avoid extinction responding-induced gene expression changes. It is noteworthy that the genes displayed different temporal expression profiles. Decreases in all of the immediate early genes examined were evident at 1 day of forced abstinence either in the mPFC or NAc. The increased extinction responding observed at 30 and 100 days of forced abstinence and our previous examinations of progressive ratio breakpoints (Morgan et al., 2002
; Morgan et al., 2005
) indicate that at least 7-10 days of forced abstinence are required for the incubation of behavioral changes. The fact that gene expression changes are occurring before the behavioral changes may indicate that these genes contribute to the development of the behavioral phenotype. As such, they may play a role in development of the incubation phenotype through the secondary effects they cause (McClung and Nestler, 2003
). Further studies will be needed to address whether their induction returns with relapse of cocaine self-administration following forced abstinence. The continued repression of these genes even after 100 days of forced abstinence suggests that they may also play a role in the expression of the incubation phenotype.
Both of the neuropeptides NPY and CART are significantly increased in the mPFC at 1 day of forced abstinence, but return to normal levels with 10 or 100 days of abstinence. These changes would seem to be in response to cocaine self-administration and do not continue with removal of the cocaine stimulus. Further studies will be needed to address whether their induction returns with relapse of cocaine self-administration following extended periods of forced abstinence.
The individual genes found to be altered in their expression have a number of points of convergence with the existing cocaine literature. First, the neuropeptide CART was initially reported as an uncharacterized cocaine-responsive transcript by Douglass and colleagues (Douglass et al., 1995
). Subsequent work has determined that CART may play a critical role in the rewarding and reinforcing properties of natural rewards such as food and also psychostimulants (for review see (Jaworski and Jones, 2006
)). There has been some controversy as to the responsiveness of CART to psychostimulant administration with reports demonstrating increases in NAc mRNA levels (Brenz Verca et al., 2001
; Fagergren and Hurd, 1999
) and a possible dependency on binge administration (Hunter et al., 2005
), while other reports finding no differences (Marie-Claire et al., 2003
). We observed no changes in accumbal CART mRNA levels, but instead observed a novel and dramatic increase in mPFC mRNA levels (over 6 times higher than controls). While much of the existing literature on cocaine-responsive CART mRNA expression has been in the NAc, equivalent levels of expression are found in prefrontal cortices (Hurd and Fagergren, 2000
). As previously noted (Jaworski and Jones, 2006
), precise definitions of CART's role in rewarding properties of cocaine remain to be made, but CART knockout mice respond less for cocaine and have lower cocaine intake than wild-type mice. If ablation of the CART gene reduces cocaine intake, increases such as those seen in this study may have the opposite effect.
Neuropeptide Y (NPY) is a 36 amino acid peptide that is widely distributed throughout the nervous system. While NPY has been extensively studied in alcohol abuse (Thiele and Badia-Elder, 2003
), reports relating to NPY and cocaine abuse are less abundant. Most relevant is a report of decreased frontal cortex NPY mRNA for up to six weeks of forced abstinence following 1 week of non-contingent cocaine administration (Wahlestedt et al., 1991
). This is the opposite of our findings and the most likely basis of this difference is the dissimilar mode and dose of cocaine administered. The significant increase in acetylated histone H3 at the NPY promoter at 1 day of abstinence is in agreement with the gene expression data as actetylated histone H3 (K9-14) is associated with increased transcription (Roh et al., 2005
). The return to levels of control rats of acetylated histone H3 at 10 days mirrors the return to levels of the mRNA abundance in the drug-naïve rats.
The responsivity of EGR1, also known as NGFI-A, krox24, and zif268, to cocaine administration has been known for over a decade (Helton et al., 1993
; Moratalla et al., 1992
). More recently though, decreases in EGR1 mRNA expression have been documented during forced abstinence from high dose cocaine self-administration binges (Mutschler et al., 2000
). The potential importance of EGR1 in cocaine-related behaviors has been demonstrated in antisense injection and mutant mouse experiments (Lee et al., 2005
; Lee et al., 2006
; Valjent et al., 2006
The finding of decreases in histone H3 acetylation at 1 and 10 days of abstinence suggests a decrease in active transcription of EGR1. The previous report of increased acetylated histone H3 at specific promoters following cocaine self-administration was restricted to 1 day after the last self-administration session (Kumar et al., 2005
). Our findings extend the timeline of histone modifications as persisting for up to 10 days. Examination of longer abstinence periods and histone acetylation at promoters of genes with persistent expression changes is required in future studies. The work by Meany and colleagues has demonstrated that EGR1 is a target of epigenetic modification and that this manifests itself in persistently altered behavior. The combination of the known behavioral relevance of EGR1 and known epigenetic modifications suggests that further specific study of EGR1 may be fruitful.
While fos induction is generally associated with cocaine administration, there have been reported decreases in mPFC fos with forced abstinence (Todtenkopf et al., 2002
). The suppression of fos mRNA levels was similar in both the mPFC and NAc and occurred by 1 day of abstinence with no return towards control levels at 10 or 100 days of abstinence. Unlike the previous examination of histone H3 acetylation at the Fos promoter with chronic cocaine self-administration which found increases (Kumar et al., 2005
), no changes were observed in this study. This may be due to the difference in the cocaine self-administration procedure: DT4 in this study and FR1 in the previous report.
Nr4a1, known previously as NGFI-B/Nur77, is a nuclear orphan receptor expressed in many dopaminergic terminal regions (Zetterstrom et al., 1996
). Previously, both we and others have described induction of mPFC and/or NAc mRNA levels of Nr4a1 with non-contingent and self-administered cocaine (Werme et al., 2000
; Freeman et al., 2002b
; Freeman et al., 2002a
). As with the other immediate early genes, we found long lasting decreases in mRNA expression in opposition to the previous reports of acute induction with cocaine administration.
Arc induction was first reported by Fosnaugh et al (Fosnaugh et al., 1995
). Previously, we found increases in Arc protein in the mPFC after chronic non-contingent (Freeman et al., 2002a
), but not self-administered, cocaine (Freeman et al., 2002b
). Recently, a temporal analysis of Arc protein levels was examined after non-contingent cocaine administration (Fumagalli et al., 2006
). Increases in mPFC levels were found to occur during the initial hours of forced abstinence, but the levels returned to baseline within 1 day for a single administration and 2 weeks following chronic administration (Fumagalli et al., 2006
). Most relevant to this report, induction of Arc mRNA within the mPFC and NAc were greater when cocaine was infused rapidly in a manner that engendered locomotor sensitization (Samaha et al., 2004
No changes in glutamate receptor transcripts, previously described as cocaine-responsive, were found in the study. In fact, all of the transcripts varied less than 15% from control values in either brain region or at any time point. This finding may reflect different administration procedures. For example, the previous report of Gria2 induction in the mPFC used non-contingent administration (Ghasemzadeh et al., 1999
) and previous reports clearly indicate that contingency affects the gene expression response (Mutschler et al., 2000
). The most similar previous report of expression changes with long-term abstinence following cocaine self-administration (Lu et al., 2003
) examined protein expression levels. While we have previously found close correlations between cocaine-responsive mRNA and protein expression (Freeman et al., 2001b
), we have also described changes in protein expression that are not always accompanied by mRNA expression changes, depending on the brain region (Bennett et al., 1999
). Future protein expression studies of these glutamatergic genes and the other genes examined will be needed.
In total, these data point to several novel findings. Behaviorally, the DT4 paradigm can engender increases in drug seeking that persist for up to 100 days of drug abstinence. At the molecular level, there is persistent depression of several immediate early genes in both the NAc and mPFC for up to 100 days. This repression of gene expression suggests that long term forced abstinence may present opposing gene expression changes to acute cocaine-induced changes. Last, for at least some of these genes, expression may be in part controlled through histone modifications.