Incubation Of Cocaine Research Articles

AMPA receptor and metabotropic glutamate receptor 1 adaptations in the nucleus accumbens core during incubation of methamphetamine craving.

Cue-induced drug craving progressively intensifies after withdrawal from self-administration of cocaine, methamphetamine, and other drugs of abuse, a phenomenon termed incubation of craving. For cocaine and methamphetamine, expression of incubated craving ultimately depends on strengthening of nucleus accumbens (NAc) synapses through an accumulation of high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) that is detectable with electrophysiological approaches. This study sought to further characterize glutamate receptor adaptations in NAc core during methamphetamine incubation. Previous biochemical studies revealed that the CP-AMPARs accumulating after cocaine incubation are mainly homomeric GluA1 receptors and that their accumulation is reflected by increased cell surface GluA1. Here, for methamphetamine, we observed no significant change in surface or total GluA1 (GluA2 and GluA3 were also unchanged). Nonetheless, GluA1 translation was elevated after incubation of methamphetamine craving, as recently found for cocaine. Additionally, for cocaine, we previously observed a withdrawal-dependent decrease in mGlu1 surface expression that precedes and enables CP-AMPAR accumulation and incubation of craving, reflecting weakening of mGlu1-dependent mechanisms that normally limit synaptic CP-AMPAR levels in the NAc core. Here, we observed no change in surface or total mGlu1 protein or its coupling to Homer scaffolding proteins after methamphetamine withdrawal, nor did elevation of mGlu1 tone through repeated injections of an mGlu1-positive allosteric modulator delay incubation of craving. These findings suggest a common role for increased GluA1 translation, but not decreased mGlu1 function, in the incubation of methamphetamine and cocaine craving. We speculate that increased GluA1 translation near synapses may drive formation and synaptic insertion of homomeric GluA1 receptors in the absence of detectable changes in GluA1 protein levels.

Extinction of cocaine-related cues reduces incubation of cocaine craving in adolescent and adult rats

Extinction of cocaine-related cues reduces incubation of cocaine craving in adolescent and adult rats

The central amygdala nucleus is critical for incubation of methamphetamine craving.

Cue-induced methamphetamine seeking progressively increases after withdrawal but mechanisms underlying this 'incubation of methamphetamine craving' are unknown. Here we studied the role of central amygdala (CeA), ventral medial prefrontal cortex (vmPFC), and orbitofrontal cortex (OFC), brain regions implicated in incubation of cocaine and heroin craving, in incubation of methamphetamine craving. We also assessed the role of basolateral amygdala (BLA) and dorsal medial prefrontal cortex (dmPFC). We trained rats to self-administer methamphetamine (10 days; 9 h/day, 0.1 mg/kg/infusion) and tested them for cue-induced methamphetamine seeking under extinction conditions during early (2 days) or late (4-5 weeks) withdrawal. We first confirmed that 'incubation of methamphetamine craving' occurs under our experimental conditions. Next, we assessed the effect of reversible inactivation of CeA or BLA by GABAA+GABAB receptor agonists (muscimol+baclofen, 0.03+0.3 nmol) on cue-induced methamphetamine seeking during early and late withdrawal. We also assessed the effect of muscimol+baclofen reversible inactivation of vmPFC, dmPFC, and OFC on 'incubated' cue-induced methamphetamine seeking during late withdrawal. Lever presses in the cue-induced methamphetamine extinction tests were higher during late withdrawal than during early withdrawal (incubation of methamphetamine craving). Muscimol+baclofen injections into CeA but not BLA decreased cue-induced methamphetamine seeking during late but not early withdrawal. Muscimol+baclofen injections into dmPFC, vmPFC, or OFC during late withdrawal had no effect on incubated cue-induced methamphetamine seeking. Together with previous studies, results indicate that the CeA has a critical role in incubation of both drug and non-drug reward craving and demonstrate an unexpected dissociation in mechanisms of incubation of methamphetamine vs cocaine craving.

Covalent Thiol Adducts Arising from Reactive Intermediates of Cocaine Biotransformation

Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic pathway that may be of particular significance to the clinical and forensic toxicology of cocaine and provides analytical tools and methods that can be applied to the determination of these conjugates in humans, opening a new area of research on cocaine biotransformation and toxicology.

In Vivo Electrochemical Measurements of Exogenously Applied Dopamine in Drosophila melanogaster

Carbon-fiber microelectrodes coupled with electrochemical detection have been used extensively for the analysis of biogenic amines. In order to determine the functional role of these amines, in vivo studies have primarily used rats and mice as model organisms. Here, we report on the development of these microanalytical techniques for in vivo electrochemical detection of dopamine in the adult Drosophila melanogaster central nervous system (CNS). A triple-barrel micropipet injector was used to exogenously apply three different concentrations of dopamine, and a cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial brain area where dopamine neurons are densely populated. Background-subtracted fast-scan cyclic voltammetry was used to measure dopamine concentration in the fly CNS. Distinct differences are shown for the clearance of exogenously applied dopamine in the brains of wild type flies versus fumin (fmn) mutants lacking a functional dopamine transporter. The current response due to oxidation of dopamine increased significantly from baseline for wild type flies following cocaine incubation. Interestingly, the current remained unchanged for mutant flies under the same conditions. These data confirm the accepted theory that cocaine blocks dopamine transporter function and validates the use of in vivo electrochemical methods to monitor dopamine uptake in Drosophila. Furthermore, after incubation with tetrodotoxin (TTX), a sodium channel blocker, there was a significant increase in peak oxidation current in the wild type flies; however, the current did not significantly change in the fmn mutant. These data suggest that factors that affect neuronal activity via ion channels such as TTX also influence the function of the dopamine transporter in Drosophila.

SKF 525-A INDUCES COCAINE N-DEMETHYLASE, ETHOXYRESORUFIN O-DEETHYLASE, AND PENTOXYRESORUFIN O-DEALKYLASE ACTIVITIES BY INDUCTION OF CYTOCHROME P-450 2B IN FEMALE B6C3F1 MICE

Studies demonstrated that cocaine-induced immunosuppression is mediated by metabolites of cocaine. Although SKF 525-A inhibited cocaine N-demethylation in liver S9 fractions isolated from female B6C3F1 mice, our study showed that pretreatment of mice with SKF 525-A potentiated cocaine-induced suppression of the antibody response to sheep red blood cells. An increase in formaldehyde generation was subsequently shown following incubation of cocaine with the S9 fractions prepared from SKF 525-A-treated mice, indicating the possibility of cytochrome P-450 (CYP) induction. Therefore, the inductive effects of SKF 525-A on CYP enzyme activities and proteins were investigated in female B6C3F1 mice to elucidate the potentiation of cocaine-induced immunosuppression by SKF 525-A. When SKF 525-A was administered at 10, 20, or 40 mg/kg/d intraperitoneally for 7 consecutive days, both ethoxyresorufin O-deethylase and pentoxyresorufin O-dealkylase activities were induced dose-dependently. Furthermore, the induction of enzymatic activity was time dependent. Meanwhile, when the type of isozyme induced by SKF 525-A was analyzed by Western immunoblotting with monospecific anti-CYP 1A and anti-CYP 2B antibodies, only the CYP 2B appeared to be induced. From in vitro inhibition studies with monoclonal antibodies, it was confirmed that the induced activity of ethoxyresorufin O-deethylase by SKF 525-A was due to increased levels of CYP 2B proteins. Our present results provide an explanation for the potentiation of cocaine-induced immunosuppression by repeated exposure to SKF 525-A. Our results also indicate that ethoxyresorufin O-deethylase, a selective substrate for CYP 1A, may also be catalyzed by CYP 2B.

A comprehensive study of the stability of cocaine and its metabolites.

The stability of cocaine (COC) in blood bank blood, postmortem human whole blood, and buffers was evaluated with consideration for the presence of the degradation products, benzoylecgonine (BE) and ecgonine methyl ester (EME). At COC concentrations commonly seen, the rate of COC hydrolysis was independent of concentration. COC was stable in blood for at least 150 days if the blood was adjusted to pH 5 and preserved with 2% NaF or organophosphates and maintained at 4 degrees C or lower. Without preservation, most COC hydrolyzed to EME. The addition of a pseudocholinesterase (PChE) inhibitor without a reduction of pH caused COC to hydrolyze to BE. COC also hydrolyzed to BE in phosphate buffer. The rate of COC hydrolysis in all studies increased with increasing pH and temperature. COC was more stable in unpreserved postmortem blood than blood bank blood due to the lower pH of the former. The incubation of COC in enzyme solutions provided further evidence of the generally accepted hypothesis that COC is hydrolyzed to EME by PChE and to BE by chemical hydrolysis. In unpreserved blood, BE was more stable than EME at room temperature. There was little loss of BE or EME at refrigerated temperature over a period of 35 days and no evidence that EME or BE could be hydrolyzed enzymatically.