Distinct patterns of DeltaFosB induction in brain by drugs of abuse.

The transcription factor DeltaFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of DeltaFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Delta(9)-tetrahydrocannabinol (Delta(9)-THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Delta(9)-THC, robustly induces DeltaFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of DeltaFosB induction in dorsal striatum. In addition, all four drugs induced DeltaFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced DeltaFosB to a small extent in amygdala. Furthermore, all drugs induced DeltaFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of DeltaFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of DeltaFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces DeltaFosB in a region-specific manner in brain.

Antisense-induced reduction in nucleus accumbens cyclic AMP response element binding protein attenuates cocaine reinforcement.

Repeated cocaine exposure up-regulates cyclic AMP signaling and increases the transcriptional activity of cyclic AMP response element binding protein (CREB) in the nucleus accumbens. To study the possibility that nucleus accumbens CREB activity regulates self-administration behavior, we tested the effects of a single, bilateral infusion of CREB antisense oligonucleotide into nucleus accumbens core and shell sub-regions on cocaine self-administration in rats. Nucleus accumbens core infusions of CREB antisense reduced CREB and the CREB-regulated immediate early gene brain-derived neurotrophic factor by 31 and 27%, respectively, but failed to alter levels of the homologous CREB family proteins cyclic AMP response element modulator and activating transcription factor 1, and had no effect on CREB levels in adjacent nucleus accumbens shell tissue. Similar infusions of CREB antisense in either core or shell produced a transient downward shift in cocaine self-administration dose-response curves on a fixed ratio 5 (five responses/injection) reinforcement schedule, indicating a reduction in cocaine reinforcement that fully recovered 3 days after treatment. CREB antisense also increased the threshold dose of cocaine required for reinstating cocaine self-administration, indicating that nucleus accumbens CREB levels regulate the incentive properties of cocaine. When access to cocaine was less restricted on a fixed ratio 1 schedule, infusion of CREB antisense in the core, but not shell, caused a transient (1-2 days) reduction in stabilized cocaine self-administration, but had no effect on responding maintained by sucrose pellets, indicating that basal CREB levels in the nucleus accumbens core regulate drug intake. None of these effects were produced by nucleus accumbens infusions of complementary sense oligonucleotide. These results suggest a necessary role for nucleus accumbens CREB activity in cocaine reinforcement, and, by converse analogy, up-regulation in CREB activity after chronic cocaine use could contribute to addiction-related increases in cocaine self-administration.

Effects of dopamine D2 receptor (DRD2) and transporter (SLC6A3) polymorphisms on smoking cue-induced cigarette craving among African-American smokers.

Cue-induced craving for addictive substances has long been known to contribute to the problem of persistent addiction in humans. Research in animals over the past decade has solidly established the central role of dopamine in cue-induced craving for addictive substances, including nicotine. Analogous studies in humans, however, are lacking, especially among African-American smokers, who have lower quit rates than Caucasian smokers. Based on the animal literature, the study's objective was to test the hypothesis that smokers carrying specific variants in dopamine-related genes previously associated with risk for addictive behaviors would exhibit heightened levels of cigarette craving following laboratory exposure to cues. To this end, cigarette craving was induced in healthy African-American smokers (n=88) through laboratory exposure to smoking cues. Smokers carrying either the DRD2 (D2 dopamine receptor gene) TaqI A1 RFLP or the SLC6A3 (dopamine transporter gene) 9-repeat VNTR polymorphisms had stronger cue-induced cravings than noncarriers (Ps <0.05 and 0.01, respectively). Consistent with the separate biological pathways involved (receptor, transporter), carriers of both polymorphisms had markedly higher craving responses compared to those with neither (P<0.0006), reflecting additive effects. Findings provide support for the role of dopamine in cue-induced craving in humans, and suggest a possible genetic risk factor for persistent smoking behavior in African-American smokers.

Stress-induced cigarette craving: effects of the DRD2 TaqI RFLP and SLC6A3 VNTR polymorphisms.

Animal models have long implicated dopamine in stress-induced craving for a variety of addictive substances. However, translational studies of dopamine, stress and craving in humans are lacking. Based on the animal literature, this study's objective was to test the hypothesis that cigarette smokers carrying specific variants in dopamine-related genes would have heightened levels of cigarette craving following exposure to a laboratory stressor. Cigarette craving induced by controlled exposure to a laboratory stressor was assessed in healthy adult smokers (n=108) recruited by advertisement. Significantly stronger stress-induced cigarette craving was found for individuals carrying either the DRD2 (D2 dopamine receptor gene) A1, or the SLC6A3 (dopamine transporter gene) nine-repeat allelic variants. Stress-induced craving was markedly higher for those carrying both alleles, compared to those with neither, consistent with the separate biological pathways involved (receptor, transporter). Findings provide strong support for the possibility that dopamine involvement in stress-induced craving well established in animal models also applies to humans, and suggest a potential genetic risk factor for persistent smoking behavior.

Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in transgenic mice decreases sensitivity to cocaine.

Administration of cocaine induces the Fos family of transcription factors in the striatum, including the nucleus accumbens (NAc), a brain region important for the rewarding effects of addictive drugs. Several Fos proteins are induced acutely by cocaine, with stable isoforms of DeltaFosB predominating after chronic drug administration. However, it has been difficult to study the functional consequences of these Fos responses in vivo. Fos proteins heterodimerize with members of the Jun family to form active AP-1 transcription factor complexes. In the present study, we took advantage of this property and generated transgenic mice, using the tetracycline gene regulation system, that support the inducible, brain region-specific expression of a dominant negative mutant form of c-Jun (Deltac-Jun), which can antagonize the actions of Fos proteins. Expression of Deltac-Jun in the striatum and certain other brain regions of adult mice decreases their development of cocaine-induced conditioned place preference, suggesting reduced sensitivity to the rewarding effects of cocaine. In contrast, Deltac-Jun expression had no effect on cocaine-induced locomotor activity or sensitization. However, expression of Deltac-Jun in adult mice blocked the ability of chronic cocaine administration to induce three known targets for AP-1 in the NAc: the AMPA glutamate receptor subunit GluR2, the cyclin-dependent protein kinase Cdk5, and the transcription factor nuclear factor-kappaB (NFkappaB), without affecting several other proteins examined for comparison. Taken together, these results provide further support for an important role of AP-1-mediated transcription in some of the behavioral and molecular mechanisms underlying cocaine addiction.

DeltaFosB: a sustained molecular switch for addiction.

The longevity of some of the behavioral abnormalities that characterize drug addiction has suggested that regulation of neural gene expression may be involved in the process by which drugs of abuse cause a state of addiction. Increasing evidence suggests that the transcription factor DeltaFosB represents one mechanism by which drugs of abuse produce relatively stable changes in the brain that contribute to the addiction phenotype. DeltaFosB, a member of the Fos family of transcription factors, accumulates within a subset of neurons of the nucleus accumbens and dorsal striatum (brain regions important for addiction) after repeated administration of many kinds of drugs of abuse. Similar accumulation of DeltaFosB occurs after compulsive running, which suggests that DeltaFosB may accumulate in response to many types of compulsive behaviors. Importantly, DeltaFosB persists in neurons for relatively long periods of time because of its extraordinary stability. Therefore, DeltaFosB represents a molecular mechanism that could initiate and then sustain changes in gene expression that persist long after drug exposure ceases. Studies in inducible transgenic mice that overexpress either DeltaFosB or a dominant negative inhibitor of the protein provide direct evidence that DeltaFosB causes increased sensitivity to the behavioral effects of drugs of abuse and, possibly, increased drug seeking behavior. This work supports the view that DeltaFosB functions as a type of sustained "molecular switch" that gradually converts acute drug responses into relatively stable adaptations that contribute to the long-term neural and behavioral plasticity that underlies addiction.

A neurobiological basis for substance abuse comorbidity in schizophrenia.

It is commonly held that substance use comorbidity in schizophrenia represents self-medication, an attempt by patients to alleviate adverse positive and negative symptoms, cognitive impairment, or medication side effects. However, recent advances suggest that increased vulnerability to addictive behavior may reflect the impact of the neuropathology of schizophrenia on the neural circuitry mediating drug reward and reinforcement. We hypothesize that abnormalities in the hippocampal formation and frontal cortex facilitate the positive reinforcing effects of drug reward and reduce inhibitory control over drug-seeking behavior. In this model, disturbances in drug reward are mediated, in part, by dysregulated neural integration of dopamine and glutamate signaling in the nucleus accumbens resulting form frontal cortical and hippocampal dysfunction. Altered integration of these signals would produce neural and motivational changes similar to long-term substance abuse but without the necessity of prior drug exposure. Thus, schizophrenic patients may have a predilection for addictive behavior as a primary disease symptom in parallel to, and in many, cases independent from, their other symptoms.

Extinction training regulates tyrosine hydroxylase during withdrawal from cocaine self-administration.

Chronic exposure to drugs of abuse is known to modulate tyrosine hydroxylase (TH) levels in the mesolimbic dopamine system. In this study, 12 d of cocaine self-administration in rats (4 hr/d) reduced TH immunoreactivity by 29% in the nucleus accumbens (NAc) shell, but not core, after a 1 week withdrawal period. In contrast, TH immunoreactivity in the NAc was completely restored in animals that experienced extinction training (4 hr/d) during the same withdrawal period. Extinction training also increased TH levels in the ventral tegmental area (VTA) by 45%, whereas TH was not altered in the VTA by cocaine withdrawal alone. Thus, extinction-induced normalization of NAc TH levels could involve increased TH synthesis, stability, and/or transport from the VTA to the NAc. A similar extinction training regimen failed to alter TH levels in the NAc or VTA of rats trained to self-administer sucrose pellets, indicating that TH regulation in cocaine-trained animals is not a generalized effect of extinction learning per se. Rather, these data suggest that neuroadaptative responses during cocaine withdrawal ultimately are determined by a complex interaction between chronic drug exposure and drug-seeking experience. The ability of extinction training to restore NAc TH levels is hypothesized to accelerate recovery from dopamine depletion and anhedonia during cocaine withdrawal.

Effects of the novel D1 dopamine receptor agonist ABT-431 on cocaine self-administration and reinstatement.

Selective D1 dopamine agonists represent a potential pharmacotherapy for the treatment of cocaine addiction. Here we report that systemic injections of the novel D1 agonist ABT-431 lack the ability to induce cocaine-seeking behavior, and completely attenuate the ability of cocaine to induce this behavior in rats tested in a reinstatement paradigm. Similar doses suppress the initiation of cocaine self-administration, and produce an extinction-like response pattern in animals that subsequently initiate self-administration, without altering responding maintained by food pellets. There was no tolerance to this effect over 4 days of testing. The results suggest that ABT-431 attenuates the motivation to seek cocaine, and masks the reinforcing effects of cocaine during self-administration. The profile of ABT-431 is similar to the behavioral effects of other structurally distinct D1 agonists, and is consistent with the desired profile of a "methadone-like" compounds for cocaine addiction.

Opiates inhibit neurogenesis in the adult rat hippocampus.

Recent work implicates regulation of neurogenesis as a form of plasticity in the adult rat hippocampus. Given the known effects of opiates such as morphine and heroin on hippocampal function, we examined opiate regulation of neurogenesis in this brain region. Chronic administration of morphine decreased neurogenesis by 42% in the adult rat hippocampal granule cell layer. A similar effect was seen in rats after chronic self-administration of heroin. Opiate regulation of neurogenesis was not mediated by changes in circulating levels of glucocorticoids, because similar effects were seen in rats that received adrenalectomy and corticosterone replacement. These findings suggest that opiate regulation of neurogenesis in the adult rat hippocampus may be one mechanism by which drug exposure influences hippocampal function.

Factors that determine a propensity for cocaine-seeking behavior during abstinence in rats.

Individual differences in locomotor responses to novelty and psychostimulants, and sensitization following repeated drug exposure, predict increased sensitivity to the reinforcing effects of psychostimulants and are thought to underlie vulnerability to drug addiction. This study tested whether these factors determine another core feature of drug addiction, the propensity for drug-seeking behavior during abstinence in rats with prior cocaine-self-administration experience. Low and high response groups for each of these factors were determined in outbred rats by the median locomotor response to novelty and amphetamine prior to cocaine self-administration (pre-test), and to amphetamine during abstinence (post-test). Cocaine-seeking behavior during abstinence was measured by the level of drug-paired lever responding during extinction, and also during reinstatement induced by cocaine-associated cues, an amphetamine priming injection, and footshock stress. Animals with low and high locomotor responses to novelty and the amphetamine pre-test showed similar levels of cocaine-seeking behavior during extinction and reinstatement testing. Locomotor responses to amphetamine following cocaine self-administration (post-test) also failed to determine amphetamine's ability to reinstate cocaine-seeking behavior. Conversely, high levels of amphetamine-induced reinstatement were associated specifically with escalating cocaine intake during prior self-administration. These animals also developed locomotor sensitization to amphetamine following cocaine self-administration (post-test vs. pre-test), but the capacity to develop locomotor sensitization was not sufficient to determine a propensity for cocaine-seeking behavior. The findings suggest that the relationship between locomotor responses to novelty, amphetamine and behavioral sensitization a,nd the propensity for cocaine-seeking behavior during abstinence is complex, while the level of drug intake during prior self-administration is a primary determinant of this behavior.

Expression of the transcription factor deltaFosB in the brain controls sensitivity to cocaine.

Acute exposure to cocaine transiently induces several Fos family transcription factors in the nucleus accumbens, a region of the brain that is important for addiction. In contrast, chronic exposure to cocaine does not induce these proteins, but instead causes the persistent expression of highly stable isoforms of deltaFosB. deltaFosB is also induced in the nucleus accumbens by repeated exposure to other drugs of abuse, including amphetamine, morphine, nicotine and phencyclidine. The sustained accumulation of deltaFosB in the nucleus accumbens indicates that this transcription factor may mediate some of the persistent neural and behavioural plasticity that accompanies chronic drug exposure. Using transgenic mice in which deltaFosB can be induced in adults in the subset of nucleus accumbens neurons in which cocaine induces the protein, we show that deltaFosB expression increases the responsiveness of an animal to the rewarding and locomotor-activating effects of cocaine. These effects of deltaFosB appear to be mediated partly by induction of the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole) glutamate receptor subunit GluR2 in the nucleus accumbens. These results support a model in which deltaFosB, by altering gene expression, enhances sensitivity to cocaine and may thereby contribute to cocaine addiction.

Neural substrates of drug craving and relapse in drug addiction.

Drug addiction is characterized by motivational disturbances such as compulsive drug taking and episodes of intense drug craving. Recent advances using animal models of relapse have shown that drug-seeking behaviour can be triggered by drug-associated cues, by stress and by 'priming' injections of the drugs themselves, events also known to trigger drug craving in human drug addicts. Current evidence suggests that these stimuli all induce relapse, at least in part, by their common ability to activate the mesolimbic dopamine system. Drug-associated cues and stress can activate this system via neural circuits from the prefrontal cortex and amygdala and through activation of the hypothalamic-pituitary-adrenal axis. Our studies suggest that dopamine triggers relapse to drug-seeking behaviour by stimulating D2-dopamine receptors which inhibit the cyclic AMP second messenger pathway in the neurones of the nucleus accumbens. In contrast, compounds which activate D1 receptors prevent relapse to drug-seeking behaviour, possibly through satiation of reward pathways. Chronic neuroadaptations in dopamine receptor signalling pathways in the nucleus accumbens caused by repeated drug use are hypothesized to produce tolerance to the rewarding effects of D1-receptor stimulation, leading to increased drug intake during drug self-administration. Conversely, these same neuroadaptations are hypothesized to enhance drug craving by potentiating D2 receptor-mediated signals during abstinence. These findings identify D1 and D2-dopamine receptor mechanisms as potential targets for developing anticraving compounds to treat drug addiction.

Relapse to drug-seeking: neural and molecular mechanisms.

A central determinant of addictive disorders in people is increased risk of relapse to drug use even after prolonged periods of abstinence. Recent advances in animal models of relapse indicate that drug-seeking behavior can be triggered by priming injections of the drugs themselves, by drug-associated environmental stimuli, and by footshock stress. The neural mechanisms underlying this relapse can be viewed in general terms as drug-like or proponent processes. Considerable evidence points to the mesolimbic dopamine system, and more specifically to activation of D2-like dopamine receptors in the nucleus accumbens, as a crucial neural substrate utilized by various stimuli that induce relapse. Drug-associated stimuli and stress may activate this system via neural circuits from the prefrontal cortex and amygdala as well as via the hypothalamo-pituitary-adrenal axis. There is also evidence for dopamine-independent mechanisms in relapse as well. A major effort of current research is to identify the long-lasting neuroadaptations within these various brain regions that contribute to relapse in addicted people. One potential neuroadaptation is up-regulation of the cAMP pathway in the nucleus accumbens, which occurs after chronic drug exposure, and represents a drug-opposite or opponent process. Modulation of this system has been related directly to relapse to drug-seeking behavior. Given the long-lasting nature of increased risk of relapse, it is likely that the relevant neuroadaptations are mediated via drug-induced changes in gene expression. A detailed understanding of the neural and molecular basis of relapse will facilitate efforts to develop truly effective treatments and preventive measures.

Involvement of cAMP-dependent protein kinase in the nucleus accumbens in cocaine self-administration and relapse of cocaine-seeking behavior.

cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc) has been implicated in cocaine addiction because (1) cocaine reinforcement is mediated by dopamine receptors that modulate cAMP formation, and (2) repeated exposure to cocaine upregulates the cAMP system in NAc neurons. This study tested PKA involvement in cocaine self-administration and relapse of cocaine-seeking behavior by infusing cAMP analogs that activate or inhibit PKA into the NAc of rats. Bilateral intra-NAc infusions of the PKA inhibitor Rp-cAMPS reduced baseline cocaine self-administration, shifted the dose-response curve for cocaine self-administration to the left, and induced relapse of cocaine-seeking behavior after extinction from cocaine self-administration, consistent with an enhancement of cocaine effects in each paradigm. In contrast, pretreatment with intra-NAc infusions of a PKA activator, Sp-cAMPS or dibutyryl cAMP, increased baseline cocaine self-administration during the second hour of testing and shifted the dose-response curve to the right, consistent with an antagonist-like action. After extinction from cocaine self-administration, similar infusions of Sp-cAMPS induced generalized responding at both drug-paired and inactive levers. As an index of PKA activity in vivo, NAc infusions of Rp-cAMPS reduced basal levels of dopamine-regulated phosphoprotein-32 phosphorylation and blocked amphetamine-induced increases in cAMP response element-binding protein (CREB) phosphorylation. Conversely, NAc infusions of Sp-cAMPS increased phosphorylation of CREB. Together, these results suggest that sustained upregulation of the cAMP system in the NAc after repeated cocaine exposure could underlie tolerance to cocaine reinforcement, whereas acute inhibition of this system may contribute to drug craving and relapse in addicted subjects.

Opposite modulation of opiate withdrawal behaviors on microinfusion of a protein kinase A inhibitor versus activator into the locus coeruleus or periaqueductal gray.

Chronic opiate administration upregulates the cAMP pathway in the locus coeruleus (LC). This adaptation is thought to increase the electrical excitability of LC neurons and contribute to the dramatic increase in LC firing induced by opioid receptor antagonists in opiate-dependent animals. The goal of the present study was to evaluate directly a role of the cAMP pathway in opiate withdrawal behaviors by studying, in vivo, whether withdrawal is influenced by intra-LC infusion of compounds known to activate or inhibit protein kinase A (PKA). Infusions into amygdala or periaqueductal gray (PAG) were studied for comparison. In one series of experiments the effect of intra-LC, intra-amygdala, or intra-PAG infusions of the PKA inhibitor Rp-cAMPS on naloxone-precipitated withdrawal from morphine was examined. Intra-LC infusions of Rp-cAMPS significantly attenuated several prominent behavioral signs of morphine withdrawal. Intra-PAG infusions of Rp-cAMPS also significantly attenuated opiate withdrawal behaviors, although different behaviors were affected. In contrast, intra-amygdala infusions of Rp-cAMPS were without significant effect. In a second series of experiments the effect of intra-LC or intra-PAG infusions of the PKA activator Sp-cAMPS on behavior in nondependent drug-naive animals was determined. Sp-cAMPS infusions into either brain region induced a quasi-withdrawal syndrome, but the observed behaviors differed between the two groups. Analysis of the phosphorylation state of tyrosine hydroxylase, a well characterized substrate for PKA, confirmed the ability of Rp-cAMPS and Sp-cAMPS to inhibit and activate, respectively, PKA activity in vivo. Together, these data provide direct evidence for involvement of the cAMP-PKA system in the LC, as well as in the PAG, in opiate withdrawal and withdrawal-related behaviors.

Opposite modulation of cocaine-seeking behavior by D1- and D2-like dopamine receptor agonists.

Activation of the mesolimbic dopamine system is known to trigger relapse in animal models of cocaine-seeking behavior. We found that this "priming" effect was selectively induced by D2-like, and not by D1-like, dopamine receptor agonists in rats. Moreover, D1-like receptor agonists prevented cocaine-seeking behavior induced by cocaine itself, whereas D2-like receptor agonists enhanced this behavior. These results demonstrate an important dissociation between D1- and D2-like receptor processes in cocaine-seeking behavior and support further evaluation of D1-like receptor agonists as a possible pharmacotherapy for cocaine addiction.