Rob Binnekade

Drug‐induced reinstatement of heroin‐ and cocaine‐seeking behaviour following long‐term extinction is associated with expression of behavioural sensitization

The present study was designed to evaluate the relationship between reinstatement of drug‐seeking behaviour following long‐term extinction of intravenous (i.v.) drug self‐administration (an animal model for craving) and long‐term behavioural sensitization. Rats were allowed to self‐administer heroin (50 μg/kg per inj., 14 daily sessions), cocaine (500 μg/kg per inj., 10 daily sessions) or saline. Following a 3‐week extinction period, reinstatement tests were performed to evaluate priming effects of amphetamine, cocaine and heroin on non‐reinforced drug‐seeking behaviour. In addition, the occurrence of long‐term behavioural sensitization in rats with a history of heroin or cocaine self‐administration was determined. Heroin‐seeking behaviour was reinstated by heroin (0.25 mg/kg), amphetamine (1.0 mg/kg) and cocaine (10 mg/kg). In addition, animals with a history of heroin self‐administration displayed locomotor sensitization to both heroin and amphetamine. Cocaine‐seeking behaviour was reinstated by cocaine and amphetamine, but not by heroin. Interestingly, locomotor sensitization to amphetamine, but not heroin, was observed in animals with a history of cocaine self‐administration. In other words, the induction of drug‐seeking behaviour following a prolonged drug‐free period was found to be associated with the expression of long‐term behavioural sensitization. These data provide experimental evidence for a role of behavioural sensitization in the incentive motivation underlying drug‐seeking behaviour. If drug hyperresponsiveness would indeed be a crucial factor in drug‐induced craving in human addicts, pharmacological readjustment of the neuroadaptations underlying drug sensitization may prevent relapse to drug use long after detoxification.

Dopaminergic mechanisms mediating the incentive to seek cocaine and heroin following long-term withdrawal of IV drug self-administration

Abstract  Rationale: The neurobiological mechanisms underlying the persistence of drug craving in detoxified addicts are still poorly understood. Objective: The present study was designed to evaluate dopaminergic mechanisms in drug-seeking behaviour following long-term (>3 weeks) extinction of IV drug self-administration in rats. Methods: To that end, we studied the effects of direct and indirect dopamine (DA) agonists on reinstatement of previously extinguished responding for heroin (50 μg/kg per injection; 14–15 daily 3-h sessions) and cocaine (500 μg/kg per injection; 10–11 daily 2-h sessions). Results: In animals with a cocaine history, priming with cocaine, the selective DA reuptake inhibitor GBR-12909 and the DA D2 receptor agonist quinpirole resulted in robust and selective reinstatement of non-reinforced nose poking behaviour in the previously drug-paired hole. In contrast, the D1 agonist SKF-82958 failed to reinstate responding and the non-selective DA agonist apomorphine even suppressed responding in these animals. In heroin-trained rats, heroin and GBR-12909 strongly reinstated responding, whereas all direct DA agonists were ineffective. Again, the two highest doses of apomorphine decreased responding in these animals. In a parallel study, the ability of DA ligands to express behavioural sensitization in animals pretreated with amphetamine or morphine was evaluated. Interestingly, all agonists that reinstated responding in the present study caused expression of locomotor sensitization and vice versa. Conclusions: The differences between direct and indirect agonists indicate a clear, but complex, involvement of DA in drug-seeking behaviour long after detoxification. Moreover, the results show an important role of D2 receptor activation in the persistence of cocaine- but not heroin-seeking behaviour. Finally, the results from both studies suggest a relationship between drug-induced reinstatement and drug hyperresponsiveness in long-term abstinent rats.

Relapse to Cocaine- and Heroin-Seeking Behavior Mediated by Dopamine D2 Receptors Is Time-Dependent and Associated with Behavioral Sensitization ☆

The sensitizing properties of drugs of abuse have been proposed to play an important role in the persistence of drug seeking behavior. We decided to evaluate the temporal relationship of dopamine D2 receptor-mediated drug seeking behavior and behavioral sensitization in animals with a history of cocaine and heroin self-administration. During early phases of withdrawal (<1 week), activation of dopamine D2 receptors with quinpirole resulted in robust, dose-dependent, reinstatement of (non-reinforced) responding in both cocaine- and heroin-trained rats. Cocaine and heroin seeking induced by quinpirole was associated with a dramatic enhancement of the psychomotor stimulant effects of the D2 agonist, indicating that sensitization to D2-mediated events had developed. During the late phase of withdrawal (>3 weeks), reinstatement of cocaine seeking by quinpirole was still apparent, but less robust. In heroin-trained rats, increases of responding were no longer observed. Interestingly, behavioral sensitization to quinpirole was still observed in cocaine-trained rats, but was absent in heroin-trained rats. Thus, it appears that dopamine D2 receptors have a time-dependent role in relapse to cocaine and heroin seeking which is strongly associated with a behaviorally sensitized state.

Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking

Associative learning processes have an important role in the initiation and persistence of heroin-seeking. Here we show in a rat self-administration model that reexposure to cues previously associated with heroin results in downregulation of AMPA receptor subunit GluR2 and concomitant upregulation of clathrin-coat assembly protein AP2m1 in synaptic membranes of the medial prefrontal cortex (mPFC). Reduced AMPA receptor expression in synaptic membranes was associated with a decreased AMPA/NMDA current ratio and increased rectification index in mPFC pyramidal neurons. Systemic or ventral (but not dorsal) mPFC injections of a peptide inhibiting GluR2 endocytosis attenuated both the rectification index and cue-induced relapse to heroin-seeking, without affecting sucrose-seeking. We conclude that GluR2 receptor endocytosis and the resulting synaptic depression in ventral mPFC are crucial for cue-induced relapse to heroin-seeking. As reexposure to conditioned stimuli is a major cause for heroin relapse, inhibition of GluR2 endocytosis may provide a new target for the treatment of heroin addiction.

Short Stressor Induced Long-Lasting Increases of Vasopressin Stores in Hypothalamic Corticotropin-Releasing Hormone (CRH) Neurons in Adult Rats

Recently, we demonstrated that single administration of interleukin‐1β(IL‐1) to adult rats induces a long‐lasting (weeks) increase of vasopressin (AVP) stores in terminals of CRH neurons in the external zone of the median eminence (ZEME). This is accompanied by hypersecretion of AVP into the pituitary portal circulation and long‐lasting hyperresponsiveness of the hypothalamo‐pituitary‐adrenal (HPA) axis to stressors. Here, we determine whether this form of plasticity of hypothalamic CRH neurons is specific for IL‐1 or represents a general response to a stressor. Single exposure of rats to lipopolysaccharide (LPS), IL‐1, brain surgery or electric footshocks increases the AVP stores in the ZEME 7 and 11 days later. Exposure to insulin or ether does not affect the AVP stores. The stressors have little or no effect on the CRH stores in the ZEME. The amplitude of the increase in AVP as measured 7–11 days after stimulation correlates with the overall ACTH response to the stressor (area under curve, r=0.89, P<0.0001), with the peak ACTH levels (r=0.52, P<0.05), but not with the duration of the ACTH response nor with any parameter of the corticosterone response. Administration of ACTH or corticosterone at doses that mimic stress‐induced plasma levels does not increase AVP stores 7 days later. We conclude that long‐lasting increases of AVP stores in CRH terminals in the ZEME can be induced by various stressors and postulate that the amplitude of such increases depends on the degree of activation of the CRH neurons by the stressor. (NWO grant: 900–543–101.)

Enhanced motivation to self-administer cocaine is predicted by self-grooming behaviour and relates to dopamine release in the rat medial prefrontal cortex and amygdala

Rats, like humans, show strong individual differences in their response to anxiogenic and stressful stimuli. In the present study we evaluated whether differences in stress‐induced self‐grooming behaviour may predict an individuals vulnerability to engage in drug self‐administration behaviour. From a population of Wistar rats, the lower and upper quartile with respect to time spent self‐grooming on an elevated plus maze (EPM) were selected and trained to intravenously self‐administer cocaine under fixed and progressive ratio schedules of reinforcement. High grooming (HG) rats reached considerably higher breakpoints than low grooming (LG) rats but showed no differences in acquisition rate and dose–response relationships. Further, EPM exposure elicited higher anxiety levels and enhanced plasma corticosterone secretion in HG rats. In addition, HG rats did not display enhanced novelty‐seeking and still spent more time self‐grooming during an EPM re‐test following the cocaine self‐administration procedure, indicating that stress‐induced self‐grooming is a stable behavioural trait marker. Neurochemically, electrically evoked [3H]dopamine release in vitro was profoundly lower in brain slices from the substantia nigra, medial prefrontal cortex and amygdala of naive HG rats as compared to LG rats, whereas no differences were found in the nucleus accumbens shell and core, the ventral tegmental area and caudate putamen. In conclusion, stress‐induced self‐grooming specifically predicts enhanced motivation to self‐administer cocaine rather than sensitivity to its reinforcing effects. Responsiveness of dopaminergic nerve terminals in the medial prefrontal cortex and amygdala may represent pre‐existing underlying factors.

Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

Single administration of interleukin-1 increased corticotropin releasing hormone and corticotropin releasing hormone-receptor mRNA in the hypothalamic paraventricular nucleus which paralleled long-lasting (weeks) sensitization to emotional stressors

Single exposure to the proinflammatory cytokine interleukin-1 induces sensitization of the adrenocorticotropin hormone and corticosterone responses to stressors weeks later (hypothalamus-pituitary-adrenal sensitization). Hypothalamus-pituitary-adrenal responses are controlled by corticotropin-releasing hormone and arginine-vasopressin secreted from parvocellular corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus and may involve autoexcitatory feedback mechanisms. Therefore, we studied the temporal relationship between resting levels of corticotropin-releasing hormone, corticotropin-releasing hormone-R1 and arginine-vasopressin receptor (V1a, V1b) mRNAs in the paraventricular nucleus and the development of hypothalamus-pituitary-adrenal sensitization to an emotional stressor (novelty). The adrenocorticotropin hormone precursor molecule proopiomelanocortin hnRNA in the pituitary gland served as an index for acute activation. Single administration of interleukin-1 induced sensitization of the hypothalamus-pituitary-adrenal to novelty from 3 to 22 days later, but not after 42 days. Single administration of interleukin-1 induced biphasic increases in corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNAs in the paraventricular nucleus: an early peak within 24 h, followed by a delayed (>7 days) increase that peaked after 22 days. Hypothalamic V1a and V1b mRNA levels were unaffected. In contrast, in the pituitary gland, there was an early decrease in corticotropin-releasing hormone-R1 mRNA (from 10.5 to 3 h after interleukin-1) and V1b receptor mRNA (3 to 6 h), which returned to control levels from 24 h onwards. Thus, interleukin-1-induced long-lasting hypothalamus-pituitary-adrenal sensitizations associated with prolonged activation of corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNA expression in the paraventricular nucleus, but not with changes in the expression of proopiomelanocortin hnRNA or V1b receptor or corticotropin-releasing hormone R1 mRNAs in the pituitary gland. We propose that transient exposure to immune events can induce long-lasting hypothalamus-pituitary-adrenal sensitization, which at least in part involves long-term hypothalamic adaptations that enhance central corticotropin-releasing hormone signaling.

Stressor- or drug-induced sensitization of the corticosterone response is not critically involved in the long-term expression of behavioural sensitization to amphetamine

Repeated exposure to drugs of abuse induces long-lasting behavioural sensitization, which is thought to play a role in the persistence of drug-seeking behaviour. Recently, we showed that repeated exposure of rats to cocaine resulted in a long-lasting (weeks) sensitization of the hypothalamus-pituitary-adrenal axis, i.e. hypersecretion of adrenocorticotropic hormone and of the glucocorticoid corticosterone. Moreover, we found that the administration of a glucocorticoid receptor antagonist abolished the expression of psychostimulant-induced behavioural sensitization. In the present study we tested whether stressor- or drug-induced long-term hypersecretion of corticosterone is associated with the long-term expression of behavioural sensitization to psychostimulant drugs. To that end, groups of male Wistar rats were exposed once to interleukin-1beta or to footshocks, treatments that are known to induce long-term sensitization of the hypothalamus-pituitary-adrenal axis, or were treated with amphetamine or morphine, according to protocols known to induce long-lasting behavioural (locomotor) sensitization. Three weeks later, the groups and their controls were challenged with amphetamine or vehicle. Previous exposure to interleukin-1beta or footshocks enhanced adrenocorticotropic hormone and corticosterone responses, but did not affect the long-term locomotor sensitization to amphetamine. Prior amphetamine treatment enhanced the locomotor response and the adrenocorticotropic hormone and corticosterone responses to amphetamine. Prior morphine treatment resulted in long-term locomotor sensitization, whereas the adrenocorticotropic hormone and corticosterone responses to amphetamine were decreased. From these findings and the absence of within-group correlation between corticosterone and locomotor responses in interleukin-1beta and morphine-pretreated rats, we conclude that there is no correlation between sensitization of the corticosterone response and behavioural sensitization to amphetamine. Apparently, sensitization of the corticosterone response is not a prerequisite for the long-term expression of behavioural sensitization, which suggests that drug-induced long-term behavioural sensitization may involve corticosteroid receptor-dependent (central) mechanisms that occur independent of hypothalamus-pituitary-adrenal axis responsiveness.

Enhanced cortical and accumbal molecular reactivity associated with conditioned heroin, but not sucrose-seeking behaviour.

Re‐exposure to drug‐related cues elicits drug‐seeking behaviour and relapse in both humans and laboratory animals even after months of abstinence. Identifying neural and molecular substrates underlying conditioned heroin‐seeking behaviour will be helpful in understanding mechanisms behind opiate relapse. In humans and animals, brain areas activated by natural reward‐related stimuli (e.g. food, sex) do not show a complete overlap with those activated by stimuli associated with drugs of abuse, suggesting the involvement of different circuitry. To that end, we investigated neural reactivity by measuring immediate early gene (IEG) expression patterns in mesocorticolimbic system target areas following cue‐induced reinstatement of heroin seeking and compared those IEG expression patterns to what was measured during natural reward (sucrose)‐seeking behaviour. Animals were trained to administer heroin associated with a compound audio‐visual cue. Re‐exposure to the cue after 3 weeks of withdrawal reinstated heroin‐seeking behaviour, which resulted in IEG expression of ania‐3, MKP‐1, c‐fos and Nr4a3 in the medial prefrontal cortex (mPFC), and of ania‐3 in the orbital frontal cortex (OFC) and nucleus accumbens core (NAC). The expression patterns for heroin‐seeking behaviours did not generalize to sucrose‐seeking behaviours, indicating that the two behaviours involve different connectivity pathways of neuronal signalling.