Khaled Moussawi

N -Acetylcysteine reverses cocaine-induced metaplasticity

Cocaine addiction is characterized by an impaired ability to develop adaptive behaviors that can compete with cocaine seeking, implying a deficit in the ability to induce plasticity in cortico-accumbens circuitry crucial for regulating motivated behavior. We found that rats withdrawn from cocaine self-administration had a marked in vivo deficit in the ability to develop long-term potentiation (LTP) and long-term depression (LTD) in the nucleus accumbens core subregion after stimulation of the prefrontal cortex. N-acetylcysteine (NAC) treatment prevents relapse in animal models and craving in humans by activating cystine-glutamate exchange and thereby stimulating extrasynaptic metabotropic glutamate receptors (mGluR). NAC treatment of rats restored the ability to induce LTP and LTD by indirectly stimulating mGluR2/3 and mGluR5, respectively. Our findings show that cocaine self-administration induces metaplasticity that inhibits further induction of synaptic plasticity, and this impairment can be reversed by NAC, a drug that also prevents relapse.

Altered Dendritic Spine Plasticity in Cocaine-Withdrawn Rats

Chronic cocaine treatment is associated with changes in dendritic spines in the nucleus accumbens, but it is unknown whether this neuroplasticity alters the effect of a subsequent cocaine injection on spine morphology and protein content. Three weeks after daily cocaine or saline administration, neurons in the accumbens were filled with the lipophilic dye, DiI. Although daily cocaine pretreatment did not alter spine density compared with daily saline, there was a shift from smaller to larger diameter spines. During the first 2 h after an acute cocaine challenge, a bidirectional change in spine head diameter and increase in spine density was measured in daily cocaine-pretreated animals. In contrast, no change in spine diameter or density was elicited by a cocaine challenge in daily saline animals during the first 2 h after injection. However, spine density was elevated at 6 h after a cocaine challenge in daily saline-pretreated animals. The time-dependent profile of proteins in the postsynaptic density subfraction elicited by a cocaine challenge in daily cocaine-pretreated subjects indicated that the changes in spine diameter and density were associated with a deteriorating actin cytoskeleton and a reduction in glutamate signaling-related proteins. Correspondingly, the amplitude of field potentials in accumbens evoked by stimulating prefrontal cortex was reduced for up to 6 h after acute cocaine in daily cocaine-withdrawn animals. These data indicate that daily cocaine pretreatment dysregulates dendritic spine plasticity elicited by a subsequent cocaine injection.

Extinction Training after Cocaine Self-Administration Induces Glutamatergic Plasticity to Inhibit Cocaine Seeking

Learning to inhibit drug seeking can be an important strategy for inhibiting relapse, and this can be modeled by extinguishing drug seeking in response to a drug-paired context. Rats were either extinguished or withdrawn without extinction training (abstinence) from cocaine self-administration, and measurements of postsynaptic density proteins in the core and shell subcompartments of the nucleus accumbens were compared with yoked-saline controls. Only extinguished rats had elevations of PSD-95, Homer1b/c, and Narp in the postsynaptic density of the core, whereas no proteins measured were altered in the postsynaptic density of the shell in either extinguished or abstinent rats. Using a biotinylation strategy, it was found that surface expression of mGluR5 was reduced only in the core of extinguished animals. Although both extinguished and abstinent animals showed a reduction in long-term potentiation elicited in the core by stimulating prefrontal cortex, blunted long-term depression was observed only in extinguished rats. These data indicate that the elevation in Homer1b/c in the core may have sequestered mGluR5 away from the membrane surface and that the loss of surface mGluR5 inhibits long-term depression. Accordingly, when Homer1c was overexpressed in the core of cocaine-naive rats with an adenoassociated virus, long-term depression was inhibited. This mechanism may contribute to the inhibition of cocaine seeking by extinction training because overexpression of Homer1c in the core also inhibited cue-induced reinstatement of cocaine seeking. These data identify a cellular mechanism that may contribute to extinction-induced inhibition of cocaine seeking.

Reversing cocaine-induced synaptic potentiation provides enduring protection from relapse

Cocaine addiction remains without an effective pharmacotherapy and is characterized by an inability of addicts to inhibit relapse to drug use. Vulnerability to relapse arises from an enduring impairment in cognitive control of motivated behavior, manifested in part by dysregulated synaptic potentiation and extracellular glutamate homeostasis in the projection from the prefrontal cortex to the nucleus accumbens. Here we show in rats trained to self-administer cocaine that the enduring cocaine-induced changes in synaptic potentiation and glutamate homeostasis are mechanistically linked through group II metabotropic glutamate receptor signaling. The enduring cocaine-induced changes in measures of cortico-accumbens synaptic and glial transmission were restored to predrug parameters for at least 2 wk after discontinuing chronic treatment with the cystine prodrug, N-acetylcysteine. N-acetylcysteine produced these changes by inducing an enduring restoration of nonsynaptic glutamatergic tone onto metabotropic glutamate receptors. The long-lasting pharmacological restoration of cocaine-induced glutamatergic adaptations by chronic N-acetylcysteine also caused enduring inhibition of cocaine-seeking in an animal model of relapse. These data mechanistically link nonsynaptic glutamate to cocaine-induced adaptations in excitatory transmission and demonstrate a mechanism to chronically restore prefrontal to accumbens transmission and thereby inhibit relapse in an animal model.

Optogenetic inhibition of cocaine seeking in rats

Inhibitory optogenetics was used to examine the roles of the prelimbic cortex (PL), the nucleus accumbens core (NAcore) and the PL projections to the NAcore in the reinstatement of cocaine seeking. Rats were microinjected into the PL or NAcore with an adeno‐associated virus containing halorhodopsin or archaerhodopsin. After 12 days of cocaine self‐administration, followed by extinction training, animals underwent reinstatement testing along with the presence/absence of optically induced inhibition via laser light. Bilateral optical inhibition of the PL, NAcore or the PL fibers in the NAcore inhibited the reinstatement of cocaine seeking.

Group II metabotropic glutamate receptors (mGlu2/3) in drug addiction

Drug addiction is characterized by maladaptive decision-making and dysfunctional brain circuitry regulating motivated behaviors, resulting in loss of the behavioral flexibility needed to abstain from drug seeking. Hence, addicts face high risk of relapse even after prolonged periods of abstinence from drug use. This is thought to result from long-lasting drug-induced neuroadaptations of glutamate and dopaminergic transmission in the mesocorticolimbic and cortico-striatal circuits where group II metabotropic glutamate receptors (mGlu(2/3) receptors) are densely expressed. mGlu(2/3) receptors presynaptically control glutamate as well as dopamine release throughout the mesocorticolimbic structures involved in reward processing and drug seeking, and their function is reduced after prolonged exposure to drugs of abuse. In pre-clinical models, mGlu(2/3) receptors have been shown to regulate both reward processing and drug seeking, in part through the capacity to control release of dopamine and glutamate respectively. Specifically, mGlu(2/3) receptor agonists administered systemically or locally into certain brain structures reduce the rewarding value of commonly abused drugs and inhibit the reinstatement of drug seeking. Given the ability of mGlu(2/3) receptor agonists to compensate for and possibly reverse drug-induced neuroadaptations in mesocorticolimbic circuitry, this class of receptors emerges as a new therapeutic target for reducing relapse in drug addiction.

Heroin relapse requires long-term potentiation-like plasticity mediated by NMDA2b-containing receptors

Persistent relapse to addictive drugs constitutes the most challenging problem in addiction therapy, and is linked to impaired prefrontal cortex regulation of motivated behaviors involving the nucleus accumbens. Using a rat model of heroin addiction, we show that relapse requires long-term potentiation (LTP)-like increases in synaptic strength in the prefrontal cortex projection to the nucleus accumbens. The increased synaptic strength was paralleled by dendritic spine enlargement in accumbens spiny neurons and required up-regulated surface expression of NMDA2b-containing receptors (NR2B). Accordingly, blocking NR2B before reinstating heroin-seeking prevented the induction of LTP-like changes in spine remodeling and synaptic strength, and inhibited heroin relapse. These data show that LTP-like neuroplasticity in prefrontal-accumbens synapses is initiated by NR2B stimulation and strongly contributes to heroin relapse. Moreover, the data reveal NR2B-containing NMDA receptors as a previously unexplored therapeutic target for treating heroin addiction.

The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine

BACKGROUND Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). METHODS Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. RESULTS NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. CONCLUSIONS The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efficacy of NAC can be improved by simultaneously inhibiting mGluR5.

Extracellular Glutamate: Functional Compartments Operate in Different Concentration Ranges

Extracellular glutamate of glial origin modulates glial and neuronal glutamate release and synaptic plasticity. Estimates of the tonic basal concentration of extracellular glutamate range over three orders of magnitude (0.02–20 μM) depending on the technology employed to make the measurement. Based upon binding constants for glutamate receptors and transporters, this range of concentrations translates into distinct physiological and pathophysiological roles for extracellular glutamate. Here we speculate that the difference in glutamate measurements can be explained if there is patterned membrane surface expression of glutamate release and transporter sites creating extracellular subcompartments that vary in glutamate concentration and are preferentially sampled by different technologies.

Chronic N-Acetylcysteine during Abstinence or Extinction after Cocaine Self-Administration Produces Enduring Reductions in Drug Seeking

The cysteine prodrug N-acetylcysteine (NAC) has been shown to reduce reinstatement of cocaine seeking by normalization of glutamatergic tone. However, enduring inhibition of cocaine seeking produced by NAC has not been explored under different withdrawal conditions. Thus, the present study determined whether chronic NAC administered during daily extinction training or daily abstinence after withdrawal from cocaine self-administration would reduce cocaine seeking. Rats self-administered intravenous cocaine during daily 2-h sessions for 12 days, followed by daily extinction or abstinence sessions. During this period, rats received daily injections of saline or NAC (60 or 100 mg/kg). Subsequently, rats were tested for cocaine seeking via conditioned cue, cue + cocaine-primed, and context-induced relapse. Chronic NAC administration blunted cocaine seeking under multiple experimental protocols. Specifically, NAC attenuated responding during cue and cue + cocaine-primed reinstatement tests after extinction and context, cue, and cue + cocaine relapse tests after abstinence. Protection from relapse by NAC persisted well after treatment was discontinued, particularly when the high dose was combined with extinction trials. The finding that NAC reduced cocaine seeking after drug treatment was discontinued has important implications for the development of effective antirelapse medications. These results support recent preclinical and clinical findings that NAC may serve as an effective treatment for inhibiting relapse in cocaine addicts.