Joanna Miszkiel

The effects of cannabinoid CB1, CB2 and vanilloid TRPV1 receptor antagonists on cocaine addictive behavior in rats

There is evidence that indicates that tonic activation of cannabinoid CB1 receptors plays a role in extinction/reinstatement of cocaine seeking-behavior but is not involved in the maintenance of cocaine self-administration. To further explore the importance of other endocannabinoid-related receptors in an animal model of cocaine addiction, the present paper examines cannabinoid CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and the transient receptor potential vanilloid type-1 (TRPV1) receptor antagonist N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) on intravenous (i.v.) cocaine self-administration and extinction/reinstatement of cocaine-seeking behavior in rats. For comparison and reference purposes, the effect of the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) was also examined. Moreover, for comparison effects of those drugs on operant lever responding for artificial (cocaine) vs. natural (food) reward, food self-administration was also evaluated. Our findings show that AM251 (1-3mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.3-1mg/kg) did not affect cocaine self-administration. However, AM251 (0.1-1mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.1-1mg/kg) decreased cocaine-induced reinstatement of cocaine-seeking behavior, and AM251 (0.3-1mg/kg) decreased cue-induced reinstatement. Moreover, AM251 (3mg/kg), SR144528 (0.1-1mg/kg) and SB366791 (0.1-1mg/kg) slightly decreased food self-administration behavior, but only AM251 (3mg/kg) reduced food reward. In conclusion, our results indicate for the first time, that tonic activation of CB2 or TRPV1 receptors is involved in cocaine-induced reinstatement of cocaine-seeking behavior, but their activity is not necessary for the rewarding effect of this psychostimulant. In contrast to CB1 receptors, neither CB2 nor TRPV1 receptors play a role in cue-induced reinstatement of cocaine-seeking behavior.

N-acetylcysteine possesses antidepressant-like activity through reduction of oxidative stress: behavioral and biochemical analyses in rats.

The growing body of evidence implicates the significance of oxidative stress in the pathophysiology of depression. The aim of this paper was to examine N-acetylcysteine (NAC) - a putative precursor of the most important tissue antioxidant glutathione - in an animal model of depression and in ex vivo assays to detect oxidative stress parameters. Imipramine (IMI), a classical and clinically-approved antidepressant drug was also under investigation. Male Wistar rats which underwent either bulbectomy (BULB; removal of the olfactory bulbs) or sham surgery (SHAM; olfactory bulbs were left undestroyed) were treated acutely or repeatedly with NAC (50-100mg/kg, ip) or IMI (10mg/kg, ip). Following 10-daily injections with NAC or IMI or their solvents, or 9-daily injections with a corresponding solvent plus acute NAC or acute IMI forced swimming test on day 10, and locomotor activity were performed; immediately after behavioral tests animals were decapitated. Biochemical tests (the total antioxidant capacity - TAC and the superoxide dismutase activity - SOD) were performed on homogenates in several brain structures. In behavioral studies, chronic (but not acute) administration of NAC resulted in a dose-dependent reduction in the immobility time seen only in BULB rats while chronic IMI produced a significant decrease in this parameter in both SHAM and BULB animals. On the other hand, chronic administration of NAC and IMI resulted in a significant increase in cellular antioxidant mechanisms (SOD activity) that reversed the effects of BULB in the frontal cortex, hippocampus and striatum. Our study further supports the antidepressant-like activity of NAC and links its effect as well as IMI actions with the enhancement of brain SOD activity.

Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats

BACKGROUND Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. METHODS To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. RESULTS The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. CONCLUSION Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.

Prolonged Induction of miR-212/132 and REST Expression in Rat Striatum Following Cocaine Self-Administration.

Chronic exposure to cocaine in vivo induces long-term synaptic plasticity associated with the brain’s circuitry that underlies development of repetitive and automatic behaviors called habits. In fact, prolonged drug consumption results in aberrant expression of protein-coding genes and small regulatory RNAs, including miRNAs that are involved in synaptic plasticity and neuroadaptations. However, the mechanisms mediating cocaine use disorder are still not fully understood. The present study is designed to examine the expression of miR-124, miR-132, miR-134, and miR-212, as well as the levels of the Ago2, Pum2, and REST mRNAs and proteins implicated in their regulation. We applied rat cocaine self-administration (SA) and extinction training procedures with a yoked triad to assess the changes in the levels of four miRNAs and three protein-coding genes and corresponding proteins in the dorsal striatum. We demonstrated that elevated expression of mature miR-212 and miR-132 is long-lasting and persists in the drug-free period (till 10-day abstinence). Moreover, mRNA and protein of REST, a regulator of neuronal transcription, was raised selectively in cocaine self-administering rats and Ago2 transcript decreased after cocaine treatment. Unexpectedly, the expression level of Ago2 and Pum2 proteins changed only in the active cocaine-receiving animals. These results point out the important aspects of long-lasting alterations in microRNAs, genes, and protein expressions involved in the control of synaptic plasticity associated with reward and motivation learning related to cocaine addiction.

Cocaine-induced glutamate receptor trafficking is abrogated by extinction training in the rat hippocampus

BACKGROUND It has been demonstrated that long-term exposure to cocaine leads to plastic changes in the brain that contribute to the manifestation of addictive behaviors. While attention has mostly focused on the meso-cortico-limbic pathway, the hippocampus seems to play a role in the craving induced by cues in drug addicts, in particular in cue- and drug-induced reinstatement of cocaine seeking. Since glutamate appears to be critical for context-induced drug seeking behaviors, the major aim of our work was to investigate the expression of hippocampal AMPA and NMDA glutamate receptors following repeated cocaine exposure and during extinction training. METHODS We thus employed the yoked control operant paradigm and exposed the animals to contingent or non-contingent cocaine exposure for 2 weeks and sacrificed the animals after the last self-administration (SA) session and following 1 or 10 days of extinction. Protein levels of glutamate receptors were analyzed by Western blotting. RESULTS We found increased levels of the main subunits of both NMDA and AMPA receptors in the post-synaptic density (PSD) fraction, but not in the whole homogenate, of the hippocampus of animals repeatedly exposed to cocaine indicating increased trafficking toward the membrane of these receptors. Also, we found that extinction abolished such effect, suggesting that the trafficking was tightly linked to the presence of the psychostimulant. CONCLUSIONS These data reveal a novel, previously unappreciated role of glutamate receptors in the action of cocaine and cocaine-extinction behavior in rat hippocampus.

The Alterations in Mitochondrial DNA Copy Number and Nuclear-Encoded Mitochondrial Genes in Rat Brain Structures after Cocaine Self-Administration

The repeated intake of cocaine evokes oxidative stress that is present even during drug withdrawal. Recent studies demonstrate that cocaine-induced oxidative and/or endoplasmic reticulum stress can affect mitochondrial function and dynamics as well as the expression of mitochondrial and nuclear genes. These alterations in mitochondrial function may determine synaptic and behavioral plasticity. Mitochondria and mitochondrial DNA (mtDNA) seem to play an important role in the initiation of drug addiction. We used a microarray approach to investigate the expression patterns of nuclear-encoded genes relevant for mitochondrial functions and quantitative real-time PCR assays to determine the numbers of copies of mtDNA and of mRNAs corresponding to two mitochondrial proteins in the prefrontal cortex and hippocampus of rats during early cocaine abstinence. We found a significant elevation in the copy number of mtDNA and concomitant increased expression of mitochondrial genes. Moreover, microarray analysis revealed changes in the transcription of nuclear genes engaged in mtDNA replication, nucleoid formation, the oxidative phosphorylation pathway, and mitochondrial fission and fusion. Finally, we observed the upregulation of endoplasmic reticulum stress-induced genes. Cocaine self-administration influences the expression of both nuclear and mitochondrial genes as well as mtDNA replication. To determine whether these alterations serve as compensatory mechanisms to help maintain normal level of ATP production, further studies are necessary.

Effects of the histamine H3 receptor antagonist ABT-239 on cognition and nicotine-induced memory enhancement in mice

BACKGROUND The strong correlation between central histaminergic and cholinergic pathways on cognitive processes has been reported extensively. However, the role of histamine H(3) receptor mechanisms interacting with nicotinic mechanisms has not previously been extensively investigated. METHODS The current study was conducted to determine the interactions of nicotinic and histamine H(3) receptor systems with regard to learning and memory function using a modified elevated plus-maze test in mice. In this test, the latency for mice to move from the open arm to the enclosed arm (i.e., transfer latency) was used as an index of memory. We tested whether ABT-239 (4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl), an H(3) receptor antagonist/inverse agonist, had influence on two different stages of memory, i.e., memory acquisition and consolidation (administered prior to or immediately after the first trial, respectively) and whether ABT-239 influenced nicotine-induced memory enhancement. RESULTS Our results revealed that the acute administration of nicotine (0.035 and 0.175 mg/kg), but not of ABT-239 (0.1-3 mg/kg) reduced transfer latency in the acquisition and consolidation phases. In combination studies, concomitant administration of either ABT-239 (1 and 3 mg/kg) and nicotine (0.035 mg/kg), or ABT-239 (0.1 mg/kg) and nicotine (0.0175 mg/kg) further increased nicotine-induced improvement in both memory acquisition and consolidation. CONCLUSION The present data confirm an important role for H(3) receptors in regulating nicotine-induced mnemonic effects since inhibition of H(3) receptors augmented nicotine-induced memory enhancement in mice.

Effects of the histamine (H)3 receptor antagonist ABT-239 on acute and repeated nicotine locomotor responses in rats

The addictive potential of nicotine is linked to psychomotor and cognition-enhancing effects. Histamine (H)(3) receptor antagonism has similarly received attention for a role in cognition, however, the role of H(3) receptors are far less studied for affects on nicotine-induced locomotor responses. In the present study we tested whether the H(3) receptor antagonist 4-(2-{2-[(2R)-2 methylpyrrolidinyl] ethyl}-benzofuran-5-yl) benzonitrile (ABT-239) influenced the psychomotor responses to acute and repeated nicotine, including sensitization and conditioned locomotion. ABT-239 (0.3-3 mg/kg) did not alter basal, nicotine-evoked (0.4 mg/kg) locomotor responses, the expression of sensitization, or cue-conditioned locomotion. However, in combination studies rats pretreated with a separate dose of ABT-239 (1 mg/kg) prior to nicotine (0.4 mg/kg) for 5 days and then challenged with nicotine (0.4 mg/kg) after a 5-day withdrawal period, showed significantly higher locomotor hyperactivity in comparison with the effect observed in nicotine-pretreated and challenged rats. Our findings implicate a limited role for H(3) receptors in locomotor responses to nicotine.

Neuroadaptive changes in metabotropic glutamate mGlu2/3R expression during different phases of cocaine addiction in rats.

BACKGROUND In the cocaine addiction the development from transient into persistent neuroplastic changes strongly involves the glutamatergic system. In this respect, among glutamatergic receptors special attention is paid to the group II of metabotropic glutamatergic receptors (mGlu2/3R) which are involved in the transition from drug use to drug addiction including the relapse mechanisms. METHODS The present study employed radioligand binding and Western blot assays to study mGlu2/3R density, affinity and protein expression in selected rat brain areas after cocaine self-administration, extinction training and cocaine-induced reinstatement. Rats were randomly assigned in triads to one of three conditions: contingent cocaine intravenous self-administration, non-contingent injections of cocaine (yoked cocaine), or saline yoked to the intake of the self-administering subject. RESULTS Cocaine self-administration and yoked cocaine delivery resulted in a significant increase in the mGlu2/3R density in the prefrontal cortex and the dorsal striatum, while 10-day extinction training provoked a reduction in the prefrontal cortex and the nucleus accumbens. Cocaine abstinence also enhanced an increase in the [3H]ligand binding to mGlu2/3R in the prefrontal cortex. During reinstatement the cocaine challenge dose (10mg/kg, ip) led to important elevation in the mGlu2/3R density in the prefrontal cortex. CONCLUSIONS Our study demonstrated the role of mGlu2/3R localized in the prefrontal cortex-striatum pathways to cocaine repeated exposure.

Increased 5-hydroxymethylation levels in the hippocampus of rat extinguished from cocaine self-administration

Drug craving and relapse risk during abstinence from cocaine are thought to be caused by persistent changes in transcription and chromatin regulation. Although several brain regions are involved in these processes, the hippocampus seems to play an important role in context‐evoked craving and drug‐seeking behavior. Only a few studies have examined epigenetic alterations during a period of cocaine abstinence. To investigate the effects of cocaine abstinence on DNA methylation and gene expression, rats that self‐administered the drug underwent cocaine abstinence in two time points with extinction training. During the cocaine extinction, we observed elevated global 5‐hydroxymethylcytosine(5‐hmC) levels with a concurrent increase in Tet3 transcript levels. Moreover, we did not find significant alterations in the levels of Tet3 mRNA and 5‐hmC in rats subjected to cocaine abstinence without extinction training. Additionally, our findings demonstrated that the expression of Tet3 target genes was activated. Besides, altered DNA methylation was detected at promoter regions of miRNAs, such as miR‐30d and miR‐let7i. Further in silico analysis provided evidence that these two molecules targeted the 3′ UTR region of the Tet3 gene and thus may contribute to its post‐transcriptional regulation. This study has presented novel findings in the hippocampus of rats that underwent extinction training following cocaine self‐administration. The alterations in the Tet3 gene expression and the level of 5‐hmC may play an important role in extinction learning and the reduction of subsequent cocaine seeking.