Cocaine-Induced Synaptic Redistribution of NMDARs in Striatal Neurons Alters NMDAR-Dependent Signal Transduction.

The consequence of repeated cocaine exposure and prolonged abstinence on glutamate receptor expression in the nucleus accumbens has been extensively studied. However, the early effects of cocaine on NMDAR signaling remain unknown. NMDAR signaling depends on the subunit composition, subcellular localization, and the interaction with proteins at the postsynaptic density (PSD), where NMDARs and other proteins form supercomplexes that are responsible for the signaling pathways activated by NMDAR-induced Ca2+ influx. Here, we investigated the effect of cocaine on NMDAR subunit composition and subcellular localization after both intraperitoneal non-contingent cocaine and response-contingent intravenous cocaine self-administration in mice. We found that repeated cocaine exposure, regardless of the route or contingency of drug administration, decreases NMDAR interactions with the PSD and synaptic lipid rafts in the accumbens shell and dorsal striatum. We provide evidence that cocaine triggers an early redistribution of NMDARs from synaptic to extrasynaptic sites, and that this adaptation has implications in the activation of downstream signaling pathways. Thus, consistent with a loss of NMDAR function, cocaine-induced ERK phosphorylation is attenuated. Because early NMDAR activity contributes to the initiation of lasting addiction-relevant neuroadaptations, these data may hold clues into cellular mechanisms responsible for the development of cocaine addiction.

Knockdown of the histone di-methyltransferase G9a in nucleus accumbens shell decreases cocaine self-administration, stress-induced reinstatement, and anxiety.

Comorbid neuropsychiatric disorders such as addiction and anxiety could involve common underlying mechanisms. One potential mechanism involves epigenetic regulation of histone 3 dimethylation at lysine 9 residues (H3K9me2) by the histone dimethyltransferase G9a. Here we provide evidence that local AAV-RNAi-mediated knockdown of G9a expression in nucleus accumbens shell (NAcSh) of male rats reduces both addictive-related and anxiety-related behaviors. Specifically, G9a knockdown reduces sensitivity to low dose cocaine reinforcement when cocaine is freely available (fixed ratio schedule). Similarly, G9a knockdown reduces motivation for cocaine under higher effort demands (progressive ratio schedule). Following several weeks of forced abstinence, G9a knockdown attenuates extinction responding and reinstatement triggered by either cocaine-priming injections or footshock stress. This decrease in addictive behavior is associated with a long-term reduction in anxiety-like behavior as measured by the elevated plus maze (EPM). G9a knockdown also reduces basal anxiety-like behavior in EPM and marble burying tests in drug-naïve rats. These results complement our previous work showing that increased G9a expression in NAcSh enhances addictive-related and anxiety-related behaviors, indicating that G9a bi-directionally controls these responses. These results also suggest that regulation of G9a-influenced gene expression could be a common epigenetic mechanism for co-morbid anxiety and psychostimulant addiction.

Overexpression of the Histone Dimethyltransferase G9a in Nucleus Accumbens Shell Increases Cocaine Self-Administration, Stress-Induced Reinstatement, and Anxiety.

Repeated exposure to cocaine induces lasting epigenetic changes in neurons that promote the development and persistence of addiction. One epigenetic alteration involves reductions in levels of the histone dimethyltransferase G9a in nucleus accumbens (NAc) after chronic cocaine administration. This reduction in G9a may enhance cocaine reward because overexpressing G9a in the NAc decreases cocaine-conditioned place preference. Therefore, we hypothesized that HSV-mediated G9a overexpression in the NAc shell (NAcSh) would attenuate cocaine self-administration (SA) and cocaine-seeking behavior. Instead, we found that G9a overexpression, and the resulting increase in histone 3 lysine 9 dimethylation (H3K9me2), increases sensitivity to cocaine reinforcement and enhances motivation for cocaine in self-administering male rats. Moreover, when G9a overexpression is limited to the initial 15 d of cocaine SA training, it produces an enduring postexpression enhancement in cocaine SA and prolonged (over 5 weeks) increases in reinstatement of cocaine seeking induced by foot-shock stress, but in the absence of continued global elevations in H3K9me2. The increase in stress-induced reinstatement is paralleled by heightened anxiety measures, suggesting that countering the cocaine-induced decreases in endogenous G9a with ectopic G9a overexpression leads to lasting anxiogenic effects. Finally, we found an enduring reduction in phosphorylated cAMP-response element binding protein levels in the NAcSh that could account for the increased anxiety. These data demonstrate a novel role for G9a in promoting comorbid cocaine addiction and anxiety and suggest that increased epigenetic repression of transcription through H3K9 during cocaine use can have long-lasting and unexpected negative consequences on behavior.SIGNIFICANCE STATEMENT Cocaine addiction is a neuropsychiatric disorder that is detrimental to society and currently has no effective treatments. The difficulty in treating drug addiction is compounded by the high comorbidity with other psychiatric illnesses, including anxiety disorders. Here, we demonstrate that G9a, an epigenetic repressor of gene expression, acting in the nucleus accumbens, a brain reward region, is capable of increasing both addiction- and anxiety-like behaviors in rats. These findings are intriguing because repeated cocaine exposure decreases G9a in this region and thereby enhances expression of certain addiction-promoting genes. However, our results suggest that countering this cocaine-induced decrease in G9a activity actually exacerbates addiction and sensitivity to relapse under stressful situations.

Inactivation of NMDA Receptors in the Ventral Tegmental Area during Cocaine Self-Administration Prevents GluA1 Upregulation but with Paradoxical Increases in Cocaine-Seeking Behavior.

Cocaine self-administration increases expression of GluA1 subunits in ventral tegmental area (VTA) dopamine neurons, which subsequently enhance the motivation for cocaine. This increase in GluA1 may be dependent on concomitant NMDA receptor (NMDAR) activation during self-administration, similar to cocaine-induced long-term potentiation in the VTA. In this study, we used viral-mediated expression of a dominant-negative GluN1 subunit (HSV-dnGluN1) in VTA neurons to study the effect of transient NMDAR inactivation on the GluA1 increases induced by chronic cocaine self-administration in male rats. We found that dnGluN1 expression in the VTA limited to the 3 weeks of cocaine self-administration prevents the subsequent increase in tissue GluA1 levels when compared with control infusions of HSV-LacZ. Surprisingly, dnGluN1 expression led to an enhancement in the motivation to self-administer cocaine as measured using a progressive ratio reinforcement schedule and to enhanced cocaine seeking measured in extinction/reinstatement tests following an extended 3 week withdrawal period. Despite blocking tissue GluA1 increases in cocaine self-administering animals, the HSV-dnGluN1 treatment resulted in increased membrane levels of GluA1 and GluN2B, along with markedly higher locomotor responses to intra-VTA infusions of AMPA, suggesting a paradoxical increase in VTA AMPA receptor responsiveness. Together, these data suggest that NMDARs mediate cocaine-induced increases in VTA GluA1 expression, but such transient NMDAR inactivation also leads to compensatory scaling of synaptic AMPA receptors that enhance the motivational for cocaine.SIGNIFICANCE STATEMENT Dopamine neurons in the ventral tegmental area (VTA) are critical substrates of drug rewards. Animal models indicate that chronic cocaine use enhances excitatory glutamatergic input to these neurons, making them more susceptible to environmental stimuli that trigger drug craving and relapse. We previously found that self-administration of cocaine increases AMPA glutamate receptors in the VTA, and this effect enhances motivation for cocaine. Here we report that the mechanism for this upregulation involves NMDA receptor activity during cocaine use. While interference with NMDA receptor function blocks AMPA receptor upregulation, it also produces a paradoxical enhancement in membrane AMPA receptor subunits, AMPA responsiveness, and the motivation for cocaine. Thus, pharmacotherapy targeting NMDA receptors may inadvertently produce substantial adverse consequences for cocaine addiction.

HDAC5 and Its Target Gene, Npas4, Function in the Nucleus Accumbens to Regulate Cocaine-Conditioned Behaviors.

Individuals suffering from substance-use disorders develop strong associations between the drug's rewarding effects and environmental cues, creating powerful, enduring triggers for relapse. We found that dephosphorylated, nuclear histone deacetylase 5 (HDAC5) in the nucleus accumbens (NAc) reduced cocaine reward-context associations and relapse-like behaviors in a cocaine self-administration model. We also discovered that HDAC5 associates with an activity-sensitive enhancer of the Npas4 gene and negatively regulates NPAS4 expression. Exposure to cocaine and the test chamber induced rapid and transient NPAS4 expression in a small subpopulation of FOS-positive neurons in the NAc. Conditional deletion of Npas4 in the NAc significantly reduced cocaine conditioned place preference and delayed learning of the drug-reinforced action during cocaine self-administration, without affecting cue-induced reinstatement of drug seeking. These data suggest that HDAC5 and NPAS4 in the NAc are critically involved in reward-relevant learning and memory processes and that nuclear HDAC5 limits reinstatement of drug seeking independent of NPAS4.

BDNF-TrkB controls cocaine-induced dendritic spines in rodent nucleus accumbens dissociated from increases in addictive behaviors.

Chronic cocaine use is associated with prominent morphological changes in nucleus accumbens shell (NACsh) neurons, including increases in dendritic spine density along with enhanced motivation for cocaine, but a functional relationship between these morphological and behavioral phenomena has not been shown. Here we show that brain-derived neurotrophic factor (BDNF) signaling through tyrosine kinase B (TrkB) receptors in NACsh neurons is necessary for cocaine-induced dendritic spine formation by using either localized TrkB knockout or viral-mediated expression of a dominant negative, kinase-dead TrkB mutant. Interestingly, augmenting wild-type TrkB expression after chronic cocaine self-administration reverses the sustained increase in dendritic spine density, an effect mediated by TrkB signaling pathways that converge on extracellular regulated kinase. Loss of TrkB function after cocaine self-administration, however, leaves spine density intact but markedly enhances the motivation for cocaine, an effect mediated by specific loss of TrkB signaling through phospholipase Cgamma1 (PLCγ1). Conversely, overexpression of PLCγ1 both reduces the motivation for cocaine and reverses dendritic spine density, suggesting a potential target for the treatment of addiction in chronic users. Together, these findings indicate that BDNF-TrkB signaling both mediates and reverses cocaine-induced increases in dendritic spine density in NACsh neurons, and these morphological changes are entirely dissociable from changes in addictive behavior.

Incubation of cue-induced reinstatement of cocaine, but not sucrose, seeking in C57BL/6J mice.

Prior studies have shown that drug-seeking behaviors increase, rather than dissipate, over weeks to months after withdrawal from drug self-administration. This phenomenon - termed incubation - suggests that drug-craving responses elicited by conditioned environmental or discrete cues may intensify over pronged abstinence. While most of this work is conducted in rats with intravenous drug self-administration models, there is less evidence for incubation in mice that have greater utility for molecular genetic analysis and perturbation. We tested whether incubation of cocaine-seeking behavior is evident in C57BL/6J mice following 3weeks (5days/week) of cocaine self-administration in 2h self-administration sessions. We compared cocaine-seeking (drug-paired lever) responses 1, 7, or 28days after withdrawal from cocaine self-administration, and over similar times following sucrose pellet self-administration. We found that the initial re-exposure to the self-administration test chambers elicited increased reward-seeking behavior in both sucrose and cocaine self-administering mice, with maximal responses found at 7days compared to 1 or 28days after self-administration with either reinforcer. However, following extinction training, reinstatement of cocaine seeking reinforced by response-contingent presentation of reward-associated cues (tone/light) was significantly higher after 28days compared to 1 or 7days following cocaine self-administration. In contrast, cue-induced reinstatement of sucrose-paired lever pressing did not increase over this time frame, demonstrating a drug-specific incubation effect not seen with a natural reward. Thus, C57BL/6J mice display incubation of cue-induced reinstatement of cocaine seeking similar to findings with rats, but only show a transient incubation of context-induced cocaine seeking.

It's only a matter of time: longevity of cocaine-induced changes in dendritic spine density in the nucleus accumbens.

Many reports show that repeated cocaine administration increases dendritic spine density in medium spiny neurons of the nucleus accumbens, but there is less agreement regarding the persistence of these changes. In this review we examine these discrepancies by systematically categorizing papers that measured cocaine-induced changes in accumbal spine density. We compare published reports based on withdrawal time, short versus long duration of cocaine administration, environmental pairing with cocaine, and core/shell subregion specificity. Together, these studies suggest that cocaine exposure induces rapid and dose-dependent increases in spine density in accumbens neurons that may play a role in the maintenance of cocaine use and vulnerability to early relapse, but are not a factor in behavioral changes associated with longer abstinence.

Optogenetic stimulation of accumbens shell or shell projections to lateral hypothalamus produce differential effects on the motivation for cocaine.

Previous studies suggest that pharmacological or molecular activation of the nucleus accumbens shell (AcbSh) facilitates extinction of cocaine-seeking behavior. However, overexpression of CREB, which increases excitability of AcbSh neurons, enhances cocaine-seeking behavior while producing depression-like behavior in tests of mood. These discrepancies may reflect activity in differential AcbSh outputs, including those to the lateral hypothalamus (LH), a target region known to influence addictive behavior and mood. Presently, it is unknown whether there is a causal link between altered activity in the AcbSh-LH pathway and changes in the motivation for cocaine. In this study, we used an optogenetics approach to either globally stimulate AcbSh neurons or to selectively stimulate AcbSh terminal projections in the LH, in rats self-administering cocaine. We found that stimulation of the AcbSh-LH pathway enhanced the motivation to self-administer cocaine in progressive ratio testing, and led to long-lasting facilitation of cocaine-seeking behavior during extinction tests conducted after withdrawal from cocaine self-administration. In contrast, global AcbSh stimulation reduced extinction responding. We compared these opposing motivational effects with effects on mood using the forced swim test, where both global AcbSh neuron and selective AcbSh-LH terminal stimulation facilitated depression-like behavioral despair. Together, these findings suggest that the AcbSh neurons convey complex, pathway-specific modulation of addiction and depression-like behavior, and that these motivation and mood phenomenon are dissociable.

Direct regulation of diurnal Drd3 expression and cocaine reward by NPAS2.

BACKGROUND: Circadian gene disruptions are associated with the development of psychiatric disorders, including addiction. However, the mechanisms by which circadian genes regulate reward remain poorly understood. METHODS: We used mice with a mutation in Npas2 and adeno-associated virus-short hairpin RNA mediated knockdown of Npas2 and Clock in the nucleus accumbens (NAc). We performed conditioned place preference assays. We utilized cell sorting quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation followed by deep sequencing. RESULTS: Npas2 mutants exhibit decreased sensitivity to cocaine reward, which is recapitulated with a knockdown of neuronal PAS domain protein 2 (NPAS2) specifically in the NAc, demonstrating the importance of NPAS2 in this region. Interestingly, reducing circadian locomotor output cycles kaput (CLOCK) (a homologue of NPAS2) in the NAc had no effect, suggesting an important distinction in NPAS2 and CLOCK function. Furthermore, we found that NPAS2 expression is restricted to Drd1 expressing neurons while CLOCK is ubiquitous. Moreover, NPAS2 and CLOCK have distinct temporal patterns of DNA binding, and we identified novel and unique binding sites for each protein. We identified the Drd3 dopamine receptor as a direct transcriptional target of NPAS2 and found that NPAS2 knockdown in the NAc disrupts its diurnal rhythm in expression. Chronic cocaine treatment likewise disrupts the normal rhythm in Npas2 and Drd3 expression in the NAc, which may underlie behavioral plasticity in response to cocaine. CONCLUSIONS: Together, these findings identify an important role for the circadian protein, NPAS2, in the NAc in the regulation of dopamine receptor expression and drug reward.

ΔFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli.

The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.

Adenosine A1 and dopamine d1 receptor regulation of AMPA receptor phosphorylation and cocaine-seeking behavior.

AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptor) stimulation in the nucleus accumbens (NAc) is critical in cocaine seeking. Here, we investigate the functional interaction between D1 dopamine receptors (D1DR) and AMPARs in the NAc, and explore how A1 adenosine receptor (A1AR) stimulation may reduce dopamine-induced facilitation of AMPARs and cocaine seeking. All animals were trained to self-administer cocaine and were tested for reinstatement of cocaine seeking following extinction procedures. The role of AMPARs in both AMPA- and D1DR-induced cocaine seeking was assessed using viral-mediated gene transfer to bi-directionally modulate AMPAR activity in the NAc core. The ability of pharmacological AMPAR blockade to modulate D1DR-induced cocaine seeking also was tested. Immunoblotting was used to determine whether stimulating D1DR altered synaptic AMPA GluA1 phosphorylation (pGluA1). Finally, the ability of an A1AR agonist to modulate D1DR-induced cocaine seeking and synaptic GluA1 receptor subunit phosphorylation was explored. Decreasing AMPAR function inhibited both AMPA- and D1DR-induced cocaine seeking. D1DR stimulation increased AMPA pGluA1(S845). Administration of the A1AR agonist alone decreased synaptic GluA1 expression, whereas coadministration of the A1AR agonist inhibited both cocaine- and D1DR-induced cocaine seeking and reversed D1DR-induced AMPA pGluA1(S845). These findings suggest that D1DR stimulation facilitates AMPAR function to initiate cocaine seeking in D1DR-containing direct pathway NAc neurons. A1AR stimulation inhibits both the facilitation of AMPAR function and subsequent cocaine seeking, suggesting that reducing AMPA glutamate neurotransmission in direct pathway neurons may restore inhibitory control and reduce cocaine relapse.

Cocaine self-administration behaviors in ClockΔ19 mice.

RATIONALE: A key role has been identified for the circadian locomotor output cycles kaput (Clock) gene in the regulation of drug reward. Mice bearing a dominant negative mutation in the Clock gene (ClockΔ19 mice) exhibit increased cocaine-induced conditioned place preference, reduced anxiety- and depression-like behavior, increased sensitivity to intracranial self-stimulation, and increased dopaminergic cell activity in the ventral tegmental area. OBJECTIVES: We sought to determine if this hyperhedonic phenotype extends to cocaine self-administration and measures of motivation. METHODS: Two separate serial testing procedures were carried out (n = 7-10/genotype/schedule). Testing began with acquisition of sucrose pellet self-administration, implantation of intravenous catheter, acquisition of cocaine self-administration, and dose-response testing (fixed ratio or progressive ratio). To evaluate diurnal variations in acquisition behavior, these sessions occurred at Zeitgeber 2 (ZT2) or ZT14. RESULTS: WT and ClockΔ19 mice exhibited similar learning and readily acquired food self-administration at both ZT2 and ZT14. However, only ClockΔ19 mice acquired cocaine self-administration at ZT2. A greater percentage of ClockΔ19 mice reached acquisition criteria at ZT2 and ZT14. ClockΔ19 mice self-administered more cocaine than WT mice. Using fixed ratio and progressive ratio schedules of reinforcement dose-response paradigms, we found that cocaine is a more efficacious reinforcer in ClockΔ19 mice than in WT mice. CONCLUSION: Our results demonstrate that the Clock gene plays an important role in cocaine reinforcement and that decreased CLOCK function increases vulnerability for cocaine use.

Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.

Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.

Striatal signal transduction and drug addiction.

Drug addiction is a severe neuropsychiatric disorder characterized by loss of control over motivated behavior. The need for effective treatments mandates a greater understanding of the causes and identification of new therapeutic targets for drug development. Drugs of abuse subjugate normal reward-related behavior to uncontrollable drug-seeking and -taking. Contributions of brain reward circuitry are being mapped with increasing precision. The role of synaptic plasticity in addiction and underlying molecular mechanisms contributing to the formation of the addicted state are being delineated. Thus we may now consider the role of striatal signal transduction in addiction from a more integrative neurobiological perspective. Drugs of abuse alter dopaminergic and glutamatergic neurotransmission in medium spiny neurons of the striatum. Dopamine receptors important for reward serve as principle targets of drugs abuse, which interact with glutamate receptor signaling critical for reward learning. Complex networks of intracellular signal transduction mechanisms underlying these receptors are strongly stimulated by addictive drugs. Through these mechanisms, repeated drug exposure alters functional and structural neuroplasticity, resulting in transition to the addicted biological state and behavioral outcomes that typify addiction. Ca(2+) and cAMP represent key second messengers that initiate signaling cascades, which regulate synaptic strength and neuronal excitability. Protein phosphorylation and dephosphorylation are fundamental mechanisms underlying synaptic plasticity that are dysregulated by drugs of abuse. Increased understanding of the regulatory mechanisms by which protein kinases and phosphatases exert their effects during normal reward learning and the addiction process may lead to novel targets and pharmacotherapeutics with increased efficacy in promoting abstinence and decreased side effects, such as interference with natural reward, for drug addiction.

Reinforcement-related regulation of AMPA glutamate receptor subunits in the ventral tegmental area enhances motivation for cocaine.

Chronic cocaine use produces numerous biological changes in brain, but relatively few are functionally associated with cocaine reinforcement. Here we show that daily intravenous cocaine self-administration, but not passive cocaine administration, induces dynamic upregulation of the AMPA glutamate receptor subunits GluR1 and GluR2 in the ventral tegmental area (VTA) of rats. Increases in GluR1 protein and GluR1(S845) phosphorylation are associated with increased GluR1 mRNA in self-administering animals, whereas increased GluR2 protein levels occurred despite substantial decreases in GluR2 mRNA. We investigated the functional significance of GluR1 upregulation in the VTA on cocaine self-administration using localized viral-mediated gene transfer. Overexpression of GluR1(WT) in rat VTA primarily infected dopamine neurons (75%) and increased AMPA receptor-mediated membrane rectification in these neurons with AMPA application. Similar GluR1(WT) overexpression potentiated locomotor responses to intra-VTA AMPA, but not NMDA, infusions. In cocaine self-administering animals, overexpression of GluR1(WT) in the VTA markedly increased the motivation for cocaine injections on a progressive ratio schedule of cocaine reinforcement. In contrast, overexpression of protein kinase A-resistant GluR1(S845A) in the VTA reduced peak rates of cocaine self-administration on a fixed ratio reinforcement schedule. Neither viral vector altered sucrose self-administration, and overexpression of GluR1(WT) or GluR1(S845A) in the adjacent substantia nigra had no effect on cocaine self-administration. Together, these results suggest that dynamic regulation of AMPA receptors in the VTA during cocaine self-administration contributes to cocaine addiction by acting to facilitate subsequent cocaine use.

Emergence of context-associated GluR(1) and ERK phosphorylation in the nucleus accumbens core during withdrawal from cocaine self-administration.

Reexposure to cocaine-associated environments promotes relapse to cocaine seeking and represents a persistent impediment to successful abstinence. Neurobiological adaptations are thought to underlie the preservation of drug-seeking behavior during protracted withdrawal periods, possibly including changes associated specifically with cocaine-paired contexts. We measured GluR(1) (S845) and extracellular signal-regulated kinase (ERK) phosphorylation in rat striatal subregions in an animal model of cocaine relapse. Animals with cocaine self-administration experience and their yoked partners were exposed to extinction conditions for one hour in the drug-paired environmental context after one day or three weeks withdrawal to measure protein phosphorylation induced by the cocaine-paired context in the absence of cocaine reinforcement. GluR(1) (S845) (an index of protein kinase A (PKA) activity) and ERK phosphorylation increased in the nucleus accumbens core of self-administering but not yoked animals after three weeks (but not one day) withdrawal, indicating a time-dependent emergence of context-associated protein phosphorylation in this accumbens subregion. In comparison, animals trained to self-administer sucrose displayed a similar increase in ERK, but not GluR(1) (S845) , phosphorylation following reexposure to a sucrose-paired environment three weeks later, indicating that GluR(1) (S845) phosphorylation did not result solely from lever press behavior per se. In contrast, basal (home cage) GluR(1) (S845) phosphorylation was elevated in the nucleus accumbens shell and caudate-putamen after one day or three weeks cocaine withdrawal regardless of context exposure. These results suggest that time-dependent emergence of context-associated GluR(1) (S845) phosphorylation in the nucleus accumbens core may contribute to the persistence of cocaine-seeking behavior, whereas ERK phosphorylation may be a consequence of this behavior.