In the present study, we examined the effects of acute and chronic intravenous cocaine exposure or chronic self-administration on regulation of ΔFosB, FosB, and cFos levels in the NAc shell, NAc core, and CPu striatal subregions. Previous studies have consistently found that ΔFosB is increased only after repeated exposure, and not after acute cocaine administration using passive IP cocaine injections (Hope et al. 1994
, Nye et al. 1995
; Chen et al. 1995
). Similarly, we found that chronic IV cocaine exposure increased ΔFosB in all striatal subregions examined, regardless of whether it was administered in a volitional or passive fashion. However, a major difference from previous studies is that acute cocaine administration increased ΔFosB protein levels in both NAc core and CPu, and approached significance in the NAc shell (p
< 0.1). One possible explanation for this difference may be the dose and/or duration of cocaine exposure, as rats in the AY group received multiple IV cocaine infusions over the single 4 h session resulting in total cocaine intake that ranged from 25.5 to 57.5 mg/kg across individual animals, which far exceeds doses of 10-20 mg/kg typically used with a single bolus IP injection (Hope et al. 1994
; Lee et al. 2006
). In addition, cocaine was administered via a more direct IV route of administration which produces higher peak brain levels of cocaine and dopamine that persist throughout the session, whereas these effects typically wane within an hour after IP injection (Bradberry, 2002
). Thus, the ability of ΔFosB to accumulate after a single acute exposure to cocaine is likely dependent on both the strength and duration of the cocaine stimulus used in the present study. In any event, the finding that ΔFosB can accumulate after a single exposure to cocaine indicates that ΔFosB could exert its effects more rapidly than previously thought, possibly resulting from an initial self-administration binge.
Interestingly, the amount of ΔFosB accumulation differed between dorsal and ventral striatal regions over the course of chronic cocaine administration. In the NAc core, the amount of ΔFosB found immediately after the final day of chronic administration (0 h WD) was more than double the amount that was found after acute administration, and smaller ΔFosB increases in the NAc shell reached significance only after chronic administration, regardless of whether cocaine was self-administered or received by passive yoked infusion. Increases with chronic cocaine administration probably reflect the accumulation of highly stable ΔFosB protein since they persisted for at least 24 hours after the last exposure. In contrast, large increases in the amount of ΔFosB in the CPu failed to differ with acute or chronic exposure, potentially reflecting a ceiling produced by acute exposure in this brain region. However, even in the CPu, the accumulation of ΔFosB protein likely contributed to persistently increased ΔFosB levels after chronic exposure, since substantial tolerance developed to cocaine-induced mRNA for ΔFosB in all 3 brain regions with chronic administration.
Acute administration of IV cocaine also increased full length FosB protein levels, with greater increases in the CPu and NAc shell than NAc core. However, mRNA for FosB was induced by almost 10 fold in the NAc shell, and less than 5 fold in CPu and NAc core. Substantial tolerance developed to cocaine's ability to induce both mRNA and protein for FosB with chronic administration, although a lower induction of FosB protein remained and could potentially compete with ΔFosB for AP-1 binding partners. The relative ratio of FosB/ΔFosB mRNA also was reduced by acute cocaine administration due to relatively greater induction of ΔFosB, consistent with previous reports using amphetamine (Alibhai et al. 2007
). In contrast to previous findings with repeated amphetamine treatments, the reduction in the relative ratio of FosB/ΔFosB mRNA by acute cocaine remained after chronic administration, reflecting the relatively higher residual induction of ΔFosB than FosB.
The fact that ΔFosB levels increase after even acute cocaine using patterns and duration of administration more typical of human intravenous drug use has important implications for the addiction process. Thus, ΔFosB could contribute to AP-1 binding activity with initial cocaine use if adequate doses were self-administered. However, ΔFosB would compete with both FosB and cFos for AP-1 binding activity, leading to downstream gene expression and neuroplasticity that is distinct from chronic administration when ΔFosB is elevated with substantially reduced cFos and FosB. Hence, ΔFosB may have greater effects after chronic cocaine administration due to both greater accumulation in the ventral striatum and decreased competition for AP-1 binding partners in both dorsal and ventral striatum. Given that striatal-specific over-expression of ΔFosB increases the motivation for cocaine (Colby et al. 2003
), such rapid accumulation of ΔFosB with initial cocaine exposure could perpetuate cocaine use in very early stages of the addiction process. Moreover, such prominent and widespread ΔFosB expression throughout the striatum with acute exposure would alter AP-1 binding activity in a manner that could facilitate the formation of compulsive habits through early engagement of dorsal striatal circuits (Belin and Everitt, 2008
Considering the stability of the ΔFosB isoforms, ΔFosB levels remained markedly elevated 24 hours after the last cocaine administration session, consistent with previous studies using chronic intravenous cocaine administration (Pich et al. 1997
; Perotti et al. 2008
). Other studies using passive experimenter administration of IP cocaine injections found that ΔFosB accumulation can persist for 1-2 weeks of withdrawal (Hope et al. 1994
; Brenhouse and Stellar, 2006
; Lee et al. 2006
), although we found no evidence for these changes 3 weeks after cessation of cocaine administration. Together, these studies suggest that ΔFosB accumulation may persist for relatively short withdrawal periods (< 3 weeks), and directly contribute to ongoing cocaine use, but may not directly contribute to a greater propensity for relapse in prolonged withdrawal. However, ΔFosB immunoreactivity has been detected in D1 receptor-containing striatal neurons after 30 days withdrawal from repeated cocaine in mice (Lee et al. 2006
). Such cell-specific sampling may be more sensitive to residual ΔFosB accumulation than whole tissue analysis used in the present study, or perhaps ΔFosB changes merely persist longer in mice than in rats. It also is possible that ΔFosB induces a cascade of transcriptional events leading to long-lasting morphological changes such as dendritic spine formation in D1-containing striatal neurons (Lee et al. 2006
; Maze et al. 2010
). In this regard, several ΔFosB targets including Cdk5 and NFκB are increased after chronic cocaine, and these factors may modify nucleus accumbens circuitry through changes in neuronal structural and/or function (Ang et al. 2001
; Benavides and Bibb, 2004
; Nestler, 2008
). Thus, it is possible that sustained ΔFosB accumulation during withdrawal is not necessary for its long-lasting impact on future drug-taking or -seeking behavior, but instead could represent a “molecular switch” that triggers multiple cellular processes that facilitate the transition to more addicted biological states (Nestler et al. 2001
The present study found that cocaine-mediated ΔFosB accumulation is not influenced by the volitional control of cocaine intake in self-administering animals consistent with previous studies using immunohistochemical procedures and multiple drugs of abuse (Perotti et al. 2008
; Pich et al. 1997
). This indicates that cocaine-induced increases in ΔFosB and FosB are likely related to the pharmacological response to cocaine or other downstream events of monoaminergic receptor signaling. In contrast to ΔFosB, we found that the development of tolerance to cocaine-induced cFos was substantially influenced by volitional control over cocaine intake in the NAc, but not in the CPu. Thus, tolerance to cocaine-induced cFos in the NAc failed to occur in animals receiving cocaine passively by chronic yoked infusion when compared to acute yoked infusion. These findings differ markedly from numerous reports of tolerance to psychostimulant-induced cFos in the NAc when drugs are given by passive IP injection (Hope et al. 1994
; Nye et al. 1995
; Chen et al. 1995
; Alibhai et al. 2007
). Given that tolerance to cFos in cocaine self-administering animals parallels several studies with repeated IP injections, the lack of tolerance with chronic intravenous yoked administration may be related to the stress associated with multiple and unpredictable yoked cocaine injections (Goeders 1997
). The loss of tolerance in ventral rather than dorsal striatum would be consistent with a selective effect on limbic circuits involved in motivational and emotional responses. In addition, while tolerance to induction of cFos did occur in animals self-administering cocaine, there remained a substantial ~50% increase in cFos protein in the NAc shell immediately after their final self-administration session, and a trend (p
< 0.1) for cFos increases also occurred in the core. Reasons for this discrepancy likely reflect differences between IP injection and multiple IV infusions over a 4 h period as discussed above. The residual induction of cFos in the NAc after chronic cocaine self-administration is a novel finding that forces the reconsideration of its role in the addiction process, whereby AP-1 complexes containing cFos, ΔFosB and FosB would all coexist to some extent after chronic exposure.
Given recent evidence that cFos is directly down-regulated by ΔFosB accumulation in dorsal striatum (Renthal et al. 2008
), it is interesting that cocaine-induced cFos in the CPu was paralleled by increases in ΔFosB with acute cocaine exposure. One possibility is that the accumulation of ΔFosB with acute administration occurs too late in the 4 h session to affect cFos induction, while its presence 24 h after cocaine in chronically treated animals impedes the induction of cFos with subsequent cocaine exposure. This idea is consistent with a trend (p = 0.067) for a moderate positive correlation between cFos and ΔFosB levels in the CPu with acute cocaine administration (0 h WD). This notion also is consistent with the strong positive correlation between cFos induction and cocaine intake in the CPu of acute yoked animals. These findings suggest that, similar to ΔFosB, the cFos response may reflect the dose of cocaine that was received. However, in the NAc, the greater accumulation of ΔFosB with chronic yoked cocaine administration cannot account for the lack of tolerance in the cFos response in these animals. Moreover, although tolerance to cFos induction was evident in self-administering animals, the strong positive correlation between residual cFos and ΔFosB levels in the NAc shell after 24 h withdrawal does not support a negative interaction between cFos and ΔFosB in the ventral striatum. Another difference from the CPu data is that cFos in the NAc core was negatively rather than positively correlated with cocaine intake immediately after acute cocaine administration, which could reflect a within-session tachyphylaxis that occurs with higher dose exposure in the ventral striatum.
Overall, the findings from the present study indicate that cFos, FosB, and ΔFosB undergo distinct regional patterns of expression after acute and chronic intravenous cocaine administration. These expression patterns are uniquely dependent on both the duration and amount of drug exposure, and tolerance to cocaine-induced cFos is highly dependent on volitional cocaine self-administration. The results also show that ΔFosB can accumulate with both acute and chronic cocaine administration by intravenous injection, supporting the idea that ΔFosB accumulation may be important in early processes that promote increased cocaine-seeking behavior and contribute to the development of cocaine addiction. Ultimately, it will be important to understand how ΔFosB can indirectly influence persistent drug craving in withdrawal via relatively short-term influences on gene expression during cocaine use and early withdrawal periods. Efforts to identify the various downstream targets and their effects on neuronal morphology and/or function will ultimately clarify the role of ΔFosB and other Fos-related antigens in the expression of addictive behavior.