Marta Rodríguez-Arias

Neurobiological mechanisms of the reinstatement of drug-conditioned place preference

Drug addiction is a chronic disorder characterized by a high rate of relapse following detoxification. There are two main versions of the reinstatement model that are employed to study relapse to drug abuse; one based on the operant self-administration procedure, and the other on the classical conditioned place preference procedure. In the last seven years, the use of the latter version has become more widespread, and the results obtained complement those obtained in self-administration studies. It has been observed that the conditioned place preference induced by opioids, psychostimulants, nicotine, ethanol and other drugs of abuse can be extinguished and reinstated by drug priming or exposure to stressful events. Herein, the neuroanatomical and neurochemical basis of drug priming- and stress-induced reinstatement of morphine and cocaine, together with the molecular correlates of reinstatement behavior, are reviewed. Differences between the conditioned place preference and self-administration studies are also discussed. Evidence suggests that data of reinstatement with the CPP are to be viewed with caution until more extensive analysis of operant procedures has been performed, and that further research will undoubtedly improve our understanding of the neurobiological mechanisms of relapse to drug seeking.

Effects of risperidone and SCH 23390 on isolation-induced aggression in male mice

In this study, the antiaggressive effects of risperidone and SCH 23390 have been explored. Using the paradigm of isolation-induced aggression, 150 albino male mice of the OF1 strain were allocated to control and experimental groups which received three doses of risperidone (0.01, 0.05 and 0.1 mg/kg) or two doses of SCH 23390 (0.05 and 0.1 mg/kg). Only the highest doses of risperidone decreased threat and attack behaviours but all doses significantly impaired motor behaviour. SCH 23390 decreased attack with the two doses used and also produced significant increases in immobility. Although both antipsychotics are antiaggressive, this action seems to be more specific in the case of risperidone. Finally, both drugs failed to affect animals with short attack latency, being antiaggressive only in subjects with long attack latency, which suggests that these two types of animals are different in their dopamine and serotonin neurotransmission.

Changes in histone acetylation in the prefrontal cortex of ethanol-exposed adolescent rats are associated with ethanol-induced place conditioning.

Alcohol drinking during adolescence can induce long-lasting effects on the motivation to consume alcohol. Abnormal plasticity in reward-related processes might contribute to the vulnerability of adolescents to drug addiction. We have shown that binge-like ethanol treatment in adolescent rats induces alterations in the dopaminergic system and causes histone modifications in brain reward regions. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced alterations in gene expression and behavior, we addressed the hypothesis that ethanol is capable of inducing transcriptional changes by histone modifications in specific gene promoters in adolescent brain reward regions, and whether these events are associated with acquisition of place conditioning. After treating juvenile and adult rats with intermittent ethanol administration, we found that ethanol treatment upregulates histone acetyl transferase (HAT) activity in adolescent prefrontal cortex and increases histone (H3 or H4) acetylation and H3(K4) dimethylation in the promoter region of cFos, Cdk5 and FosB. Inhibition of histone deacetylase by sodium butyrate before ethanol injection enhances both up-regulation of HAT activity and histone acetylation of cFos, Cdk5 and FosB. Furthermore, co-administration of sodium butyrate with ethanol prolongs the extinction of conditioned place aversion and increased the reinstatement effects of ethanol in ethanol-treated adolescents, but not in ethanol-treated adult rats. These results indicate that ethanol exposure during adolescence induces chromatin remodeling, changes histone acetylation and methylation, and modify the effects of ethanol on place conditioning. They also suggest that epigenetic mechanisms might open up avenues to new treatments for binge drinking-induced drug addiction during adolescence.

Rewarding Effects and Reinstatement of MDMA-Induced CPP in Adolescent Mice

Although the rewarding effects of 3,4-methylenedioxy-metamphetamine (MDMA) have been demonstrated in self-administration and conditioned place preference (CPP) procedures, its addictive potential (ie, the vulnerability to relapse, measured by its ability to induce reinstatement of an extinguished response), remains poorly understood. In this study, the effects of MDMA (5, 10, and 20 mg/kg) on the acquisition, extinction and reinstatement of CPP were evaluated in mice, using two different protocols during acquisition of CPP. In the first experiment, animals were trained using a two-session/day schedule (MDMA and saline for 4 consecutive days), whereas in the second experiment, they were trained using an alternating day schedule (MDMA and saline each 48 h). After extinction, the ability of drug priming to reinstate CPP was evaluated. In Experiment 1, MDMA did not significantly increase the time spent in the drug-paired compartment during the post-conditioning (Post-C) test, although the preference was evident a week afterwards, lasting between 2 and 21 weeks. No reinstatement was observed after MDMA priming. In Experiment 2, all doses produced CPP in Post-C, which lasted between 1 and 4 weeks. MDMA induces reinstatement at doses up to 4 times lower than those used in conditioning. The analyses of brain monoamines revealed that the daily schedule of treatment induces a non-dose-dependent decrease in dopamine and serotonin (5-HT) in the striatum, whereas the alternating schedule produces a dose-dependent decrease of 5-HT in the cortex. These results demonstrate that MDMA produces long-lasting rewarding effects and reinstatement after extinction, suggesting the susceptibility of this drug to induce addiction.

Role of CB2 cannabinoid receptors in the rewarding, reinforcing, and physical effects of nicotine.

This study was aimed to evaluate the involvement of CB2 cannabinoid receptors (CB2r) in the rewarding, reinforcing and motivational effects of nicotine. Conditioned place preference (CPP) and intravenous self-administration experiments were carried out in knockout mice lacking CB2r (CB2KO) and wild-type (WT) littermates treated with the CB2r antagonist AM630 (1 and 3 mg/kg). Gene expression analyses of tyrosine hydroxylase (TH) and α3- and α4-nicotinic acetylcholine receptor subunits (nAChRs) in the ventral tegmental area (VTA) and immunohistochemical studies to elucidate whether CB2r colocalized with α3- and α4-nAChRs in the nucleus accumbens and VTA were performed. Mecamylamine-precipitated withdrawal syndrome after chronic nicotine exposure was evaluated in CB2KO mice and WT mice treated with AM630 (1 and 3 mg/kg). CB2KO mice did not show nicotine-induced place conditioning and self-administered significantly less nicotine. In addition, AM630 was able to block (3 mg/kg) nicotine-induced CPP and reduce (1 and 3 mg/kg) nicotine self-administration. Under baseline conditions, TH, α3-nAChR, and α4-nAChR mRNA levels in the VTA of CB2KO mice were significantly lower compared with WT mice. Confocal microscopy images revealed that CB2r colocalized with α3- and α4-nAChRs. Somatic signs of nicotine withdrawal (rearings, groomings, scratches, teeth chattering, and body tremors) increased significantly in WT but were absent in CB2KO mice. Interestingly, the administration of AM630 blocked the nicotine withdrawal syndrome and failed to alter basal behavior in saline-treated WT mice. These results suggest that CB2r play a relevant role in the rewarding, reinforcing, and motivational effects of nicotine. Pharmacological manipulation of this receptor deserves further consideration as a potential new valuable target for the treatment of nicotine dependence.

Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence

Studies in humans and experimental animals have demonstrated the vulnerability of the adolescent brain to actions of ethanol and the long-term consequences of binge drinking, including the behavioral and cognitive deficits that result from alcohol neurotoxicity, and increased risk to alcohol abuse and dependence. Although the mechanisms that participate in these effects are largely unknown, we have shown that ethanol by activating innate immune receptors, toll-like receptor 4 (TLR4), induces neuroinflammation, impairs myelin proteins and causes cognitive dysfunctions in adolescent mice. Since neuroimmune signaling is also involved in alcohol abuse, the aim of this study was to assess whether ethanol treatment in adolescence promotes the long-term synaptic and molecular events associated with alcohol abuse and addiction. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3g/kg) for 2 weeks, we showed that binge-like ethanol treatment in adolescent mice promotes short- and long-term alterations in synaptic plasticity and epigenetic changes in the promoter region of bdnf and fosb, which increased their expression in the mPFC of young adult animals. These molecular events were associated with long-term rewarding and anxiogenic-related behavioral effects, along with increased alcohol preference. Our results further showed the participation of neuroimmune system activation and the TLR4 signaling response since deficient mice in TLR4 (TLR4-KO) are protected against molecular and behavioral alterations of ethanol in the adolescent brain. Our results highlight a new role of the neuroimmune function and open up new avenues to develop pharmacological treatments that can normalize the immune signaling responsible for long-term effects in adolescence, including alcohol abuse and related disorders.

Effects of DA D1 and D2 antagonists on the sensitisation to the motor effects of morphine in mice

Acute morphine administration produces hyperactivity in mice and repeated treatment induces an enhancement of this effect. In this experiment, we study the sensitisation to the hyperactivity induced by intermittent morphine administration (40 mg/kg) and the effects of dopamine (DA) antagonists on this phenomenon. Animals received three injections, separated by 48 h, and after each injection, their activity was registered between 30 and 60 min. In Experiment 1, animals were divided into two groups, which received saline and morphine (S-S-M) or only morphine (M-M-M). In Experiment 2, animals were divided into 12 groups. Half, which was designed to study the effects of DA antagonists on the acquisition of morphine sensitisation, received morphine plus 0.125, 0.25, or 0.5 mg/kg SCH 23390 or raclopride in the two first administrations and only morphine in the third (M+SCH-M+SCH-M; M+R-M+R-M). The other groups, designed to study the effects of DA antagonists on the expression of morphine sensitisation, received morphine in the two first administrations and morphine plus DA antagonists in the last injection (M-M-M+SCH; M-M-M+R). Intermittent morphine administration produces greater hyperactivity than acute morphine. DA D1 antagonists reduce acquisition and block expression of sensitisation, while DA D2 antagonists only affect expression with the intermediate and high dose. These results support the implication of DA in the behavioural sensitisation of morphine in mice.

Plasma profile of pro-inflammatory cytokines and chemokines in cocaine users under outpatient treatment: influence of cocaine symptom severity and psychiatric co-morbidity.

The treatment for cocaine use constitutes a clinical challenge because of the lack of appropriate therapies and the high rate of relapse. Recent evidence indicates that the immune system might be involved in the pathogenesis of cocaine addiction and its co‐morbid psychiatric disorders. This work examined the plasma pro‐inflammatory cytokine and chemokine profile in abstinent cocaine users (n = 82) who sought outpatient cocaine treatment and age/sex/body mass‐matched controls (n = 65). Participants were assessed with the diagnostic interview Psychiatric Research Interview for Substance and Mental Diseases according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM‐IV‐TR). Tumor necrosis factor‐alpha, chemokine (C‐C motif) ligand 2/monocyte chemotactic protein‐1 and chemokine (C‐X‐C motif) ligand 12 (CXCL12)/stromal cell‐derived factor‐1 (SDF‐1) were decreased in cocaine users, although all cytokines were identified as predictors of a lifetime pathological use of cocaine. Interleukin‐1 beta (IL‐1β), chemokine (C‐X3‐C motif) ligand 1 (CX3CL1)/fractalkine and CXCL12/SDF‐1 positively correlated with the cocaine symptom severity when using the DSM‐IV‐TR criteria for cocaine abuse/dependence. These cytokines allowed the categorization of the outpatients into subgroups according to severity, identifying a subgroup of severe cocaine users (9–11 criteria) with increased prevalence of co‐morbid psychiatric disorders [mood (54%), anxiety (32%), psychotic (30%) and personality (60%) disorders]. IL‐1β was observed to be increased in users with such psychiatric disorders relative to those users with no diagnosis. In addition to these clinical data, studies in mice demonstrated that plasma IL‐1β, CX3CL1 and CXCL12 were also affected after acute and chronic cocaine administration, providing a preclinical model for further research. In conclusion, cocaine exposure modifies the circulating levels of pro‐inflammatory mediators. Plasma cytokine/chemokine monitoring could improve the stratification of cocaine consumers seeking treatment and thus facilitate the application of appropriate interventions, including management of heightened risk of psychiatric co‐morbidity. Further research is necessary to elucidate the role of the immune system in the etiology of cocaine addiction.

Effect of memantine and CNQX in the acquisition, expression and reinstatement of cocaine-induced conditioned place preference.

The present study evaluates the effect of memantine, a non-competitive N-methyl-d-aspartate (NMDA) glutamate receptor antagonist and CNQX, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist on the rewarding effects of cocaine in mice, using the conditioned place preference (CPP) paradigm. Cocaine-induced CPP was studied pairing this drug with different memantine or CNQX doses during either the acquisition or the expression phase of the procedure. Once CPP was established, and the preference extinguished, reinstatement was induced by a priming dose of cocaine. Both antagonists, which in themselves do not present motivational actions on the preference shown by the animals, abolished the acquisition and expression of the cocaine-induced CPP. Neither of the antagonists precipitated reinstatement of the preference induced by cocaine but memantine blocked the cocaine-primed reinstatement. Our results suggest that cocaine-induced CPP and reinstatement is largely dependent on glutamate neurotransmission, and confer a putative role for memantine among the tools useful for cocaine management and treatment.

Memantine presents different effects from MK-801 in motivational and physical signs of morphine withdrawal

Adaptive changes in neural systems due to chronic opiate exposure are related to the neural plasticity phenomenon, NMDA receptors being implicated in these processes, e.g. tolerance, dependence or withdrawal. In this work, we investigated the effect of two non-competitive NMDA antagonists, memantine and MK-801, in motivational (Conditioned Place Aversion paradigm, CPA) and physical aspects of morphine withdrawal. After the induction of morphine dependence, animals in which the CPA was studied, received memantine (5 and 10 mg/kg) or MK-801 (0.3-0.006 mg/kg) either during the acquisition (conditioning) or expression (test) phase of this procedure. Both drugs were capable of inhibiting conditioned aversion when administered in any phase. In a second experiment, the effects of these drugs were evaluated in the intensity of the physical signs of withdrawal, only memantine administration being efficient. In addition to these studies, the intensity of morphine dependence was investigated under the blockade of NMDA receptors, i.e. morphine was co-administered with memantine or MK-801. These animals did not develop CPA and present less intensity in the physical signs of morphine withdrawal. Our results support the idea that NMDA receptors are involved in the behavioural changes and therefore in the neural adaptations produced by repeated morphine administration.