Perturbations in risk/reward decision making and frontal cortical catecholamine regulation induced by mild traumatic brain injury.

Mild traumatic brain injury (mTBI) disrupts cognitive processes that influence risk taking behavior. Little is known regarding the effects of repetitive mild injury (rmTBI) or whether these outcomes are sex specific. Risk/reward decision making is mediated by the prefrontal cortex (PFC), which is densely innervated by catecholaminergic fibers. Aberrant PFC catecholamine activity has been documented following TBI and may underlie TBI-induced risky behavior. The present study characterized the effects of rmTBI on risk/reward decision making behavior and catecholamine transmitter regulatory proteins within the PFC. Rats were exposed to sham, single (smTBI), or three closed-head controlled cortical impact (CH-CCI) injuries and assessed for injury-induced effects on risk/reward decision making using a probabilistic discounting task (PDT). In the first week post-final surgery, mTBI increased risky choice preference. By the fourth week, males exhibited increased latencies to make risky choices following rmTBI, demonstrating a delayed effect on processing speed. When levels of tyrosine hydroxylase (TH) and the norepinephrine reuptake transporter (NET) were measured within subregions of the PFC, females exhibited dramatic increases of TH levels within the orbitofrontal cortex (OFC) following smTBI. However, both males and females demonstrated reduced levels of OFC NET following rmTBI. These results indicate the OFC is susceptible to catecholamine instability after rmTBI and suggests that not all areas of the PFC contribute equally to TBI-induced imbalances. Overall, the CH-CCI model of rmTBI has revealed time-dependent and sex-specific changes in risk/reward decision making and catecholamine regulation following repetitive mild head injuries.

Cocaine Exposure Increases Excitatory Synaptic Transmission and Intrinsic Excitability in the Basolateral Amygdala in Male and Female Rats and across the Estrous Cycle.

INTRODUCTION: Sex and ovarian hormones influence cocaine seeking and relapse vulnerability, but less is known regarding the cellular and synaptic mechanisms contributing to these behavioral sex differences. One factor thought to influence cue-induced seeking behavior following withdrawal is cocaine-induced changes in the spontaneous activity of pyramidal neurons in the basolateral amygdala (BLA). However, the mechanisms underlying these changes, including potential sex or estrous cycle effects, are unknown. METHODS: Ex vivo whole-cell patch clamp electrophysiology was conducted to investigate the effects of cocaine exposure, sex, and estrous cycle fluctuations on two properties that can influence spontaneous activity of BLA pyramidal neurons: (1) frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and (2) intrinsic excitability. Recordings of BLA pyramidal neurons were conducted in adult male and female rats and across the estrous cycle following 2-4 weeks of withdrawal from extended-access cocaine self-administration (6 h/day for 10 days) or drug-naïve conditions. RESULTS: In both sexes, cocaine exposure increased the frequency, but not amplitude, of sEPSCs and neuronal intrinsic excitability. Across the estrous cycle, sEPSC frequency and intrinsic excitability were significantly elevated only in cocaine-exposed females in the estrus stage of the cycle, a stage when cocaine-seeking behavior is known to be enhanced. CONCLUSIONS: Here, we identify potential mechanisms underlying cocaine-induced alterations in the spontaneous activity of BLA pyramidal neurons in both sexes along with changes in these properties across the estrous cycle.

mGlu5 inhibition in the basolateral amygdala prevents estrous cycle-dependent changes in cue-induced cocaine seeking.

Drug associated cues are a common relapse trigger for individuals recovering from cocaine use disorder. Sex and ovarian hormones influence patterns of cocaine use and relapse vulnerability, with studies indicating that females show increased cue-induced craving and relapse vulnerability compared to males. In a rodent model of cocaine craving and relapse vulnerability, cue-induced cocaine seeking behavior following weeks of withdrawal from extended-access cocaine self-administration is higher in females in the estrus stage of the reproductive (estrous) cycle (Estrus Females) compared to both Males and females in all other stages (Non-Estrus Females). However, the neuronal substrates and cellular mechanisms underlying these sex differences is not fully understood. One region that contributes to both sex differences in behavioral responding and cue-induced cocaine seeking is the basolateral amygdala (BLA), while one receptor known to play a critical role in mediating cocaine seeking behavior is metabotropic glutamate receptor 5 (mGlu5). Here we assessed the effects of BLA mGlu5 inhibition following prolonged withdrawal from cocaine self-administration on observed estrous cycle-dependent changes in cue-induced cocaine seeking behavior. We found that BLA microinjections of the mGlu5 antagonist MTEP selectively reduced the enhanced cue-induced cocaine seeking normally observed in Estrus Females while having no effect on cocaine seeking in Males and Non-Estrus Females. These findings identify a unique interaction between cocaine-exposure, estrous cycle fluctuations and BLA mGlu5-dependent transmission on cue-induced cocaine seeking behavior.

Age- and Sex-Dependent Changes in Locus Coeruleus Physiology and Anxiety-Like Behavior Following Acute Stressor Exposure.

Adolescence is a critical period of development with increased sensitivity toward psychological stressors. Many psychiatric conditions emerge during adolescence and animal studies have shown that that acute stress has long-term effects on hypothalamic-pituitary-adrenal axis function and behavior. We recently demonstrated that acute stress produces long-term electrophysiological changes in locus coeruleus and long-lasting anxiety-like behavior in adolescent male rats. Based on prior reports of increased stress sensitivity during adolescence and increased sensitivity of female locus coeruleus toward corticotropin releasing factor, we hypothesized that the same acute stressor would cause different behavioral and physiological responses in adolescent female and adult male and female rats one week after stressor exposure. In this study, we assessed age and sex differences in how an acute psychological stressor affects corticosterone release, anxiety-like behavior, and locus coeruleus physiology at short- and long-term intervals. All groups of animals except adult female responded to stress with elevated corticosterone levels at the acute time point. One week after stressor exposure, adolescent females showed decreased firing of locus coeruleus neurons upon current injection and increased exploratory behavior compared to controls. The results were in direct contrast to changes observed in adolescent males, which showed increased anxiety-like behavior and increased spontaneous and induced firing in locus coeruleus neurons a week after stressor exposure. Adult males and females were both behaviorally and electrophysiologically resilient to the long-term effects of acute stress. Therefore, there may be a normal developmental trajectory for locus coeruleus neurons which promotes stress resilience in adults, but stressor exposure during adolescence perturbs their function. Furthermore, while locus coeruleus neurons are more sensitive to stressor exposure during adolescence, the effect varies between adolescent males and females. These findings suggest that endocrine, behavioral, and physiological responses to stress vary among animals of different age and sex, and therefore these variables should be taken into account when selecting models and designing experiments to investigate the effects of stress. These differences in animals may also allude to age and sex differences in the prevalence of various psychiatric illnesses within the human population.

GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

Effects of Sex and Estrous Cycle on the Time Course of Incubation of Cue-Induced Craving following Extended-Access Cocaine Self-Administration.

Cocaine addiction is a devastating public health epidemic that continues to grow. Studies focused on identifying biological factors influencing cocaine craving and relapse vulnerability are necessary to promote abstinence in recovering drug users. Sex and ovarian hormones are known to influence cocaine addiction liability and relapse vulnerability in both humans and rodents. Previous studies have investigated sex differences in the time-dependent intensification or "incubation" of cue-induced cocaine craving that occurs during withdrawal from extended-access cocaine self-administration and have identified changes across the rat reproductive cycle (estrous cycle). Female rats in the estrus stage of the cycle (Estrus Females), the phase during which ovulation occurs, show an increase in the magnitude of incubated cue-induced cocaine craving compared with females in all other phases of the estrous cycle (Non-Estrus Females). Here we extend these findings by assessing incubated craving across the estrous cycle during earlier withdrawal periods (withdrawal day 1 and 15) and later withdrawal periods (withdrawal day 48). We found that this increase in the magnitude of incubated craving during estrus (Estrus Females) is present on withdrawal day 15, but not on withdrawal day 1, and further increases by withdrawal day 48. No difference in the magnitude of incubated craving was observed between Males and Non-Estrus Females. Our data indicate that the effects of hormonal fluctuations on cue-induced cocaine craving intensify during the first month and a half of withdrawal, showing an interaction among abstinence length, estrous cycle fluctuations, and cocaine craving.

mGlu5 function in the nucleus accumbens core during the incubation of methamphetamine craving.

Many studies have demonstrated that negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGlu5) reduce cocaine and methamphetamine seeking in extinction-reinstatement animal models of addiction. Less is known about effects of mGlu5 NAMs in abstinence models, particularly for methamphetamine. We used the incubation of drug craving model, in which cue-induced craving progressively intensifies after withdrawal from drug self-administration, to conduct the first studies of the following aspects of mGlu5 function in the rat nucleus accumbens (NAc) core during abstinence from methamphetamine self-administration: 1) functionality of the major form of synaptic depression in NAc medium spiny neurons, which is induced postsynaptically via mGlu5 and expressed presynaptically via cannabinoid type 1 receptors (CB1Rs), 2) mGlu5 surface expression and physical associations between mGlu5, Homer proteins, and diacylglycerol lipase-α, and 3) the effect of systemic and intra-NAc core administration of the mGlu5 NAM 3-((2-methyl-4-)ethynyl)pyridine (MTEP) on expression of incubated methamphetamine craving. We found that mGlu5/CB1R-dependent synaptic depression was lost during the rising phase of methamphetamine incubation but then recovered, in contrast to its persistent impairment during the plateau phase of incubation of cocaine craving. Furthermore, whereas the cocaine-induced impairment was accompanied by reduced mGlu5 levels and mGlu5-Homer associations, this was not the case for methamphetamine. Systemic MTEP reduced incubated methamphetamine seeking, but also reduced inactive hole nose-pokes and locomotion, while intra-NAc core MTEP had no significant effects. These findings provide the first insight into the role of mGlu5 in the incubation of methamphetamine craving and reveal differences from incubation of cocaine craving.

Cocaine and chronic stress exposure produce an additive increase in neuronal activity in the basolateral amygdala.

Cocaine addiction is a chronic, relapsing disorder. Stress and cues related to cocaine are two common relapse triggers. We have recently shown that exposure to repeated restraint stress during early withdrawal accelerates the time-dependent intensification or "incubation" of cue-induced cocaine craving that occurs during the first month of withdrawal, although craving ultimately plateaus at the same level observed in controls. These data indicate that chronic stress exposure during early withdrawal may result in increased vulnerability to cue-induced relapse during this period. Previous studies have shown that chronic stress exposure in drug-naïve rats increases neuronal activity in the basolateral amygdala (BLA), a region critical for behavioral responses to stress. Given that glutamatergic projections from the BLA to the nucleus accumbens are critical for the incubation of cue-induced cocaine craving, we hypothesized that cocaine withdrawal and chronic stress exposure produce separate increases that additively increase BLA neuronal activity. To assess this, we conducted in vivo extracellular single-unit recordings from the BLA of anesthetized adult male rats following cocaine or saline self-administration (6 h/day for 10 days) and repeated restraint stress or control conditions on withdrawal days (WD) 6-14. Recordings were conducted from WD15 to WD20. Interestingly, cocaine exposure alone increased the spontaneous firing rate in the BLA to levels observed following chronic stress exposure in drug-naïve rats. Chronic stress exposure during cocaine withdrawal further increased firing rate. These studies may identify a potential mechanism by which both cocaine and chronic stress exposure drive cue-induced relapse vulnerability during abstinence.

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.

mGlu1 tonically regulates levels of calcium-permeable AMPA receptors in cultured nucleus accumbens neurons through retinoic acid signaling and protein translation.

In several brain regions, ongoing metabotropic glutamate receptor 1 (mGlu1) transmission has been shown to tonically suppress synaptic levels of Ca2+ -permeable AMPA receptors (CP-AMPARs) while pharmacological activation of mGlu1 removes CP-AMPARs from these synapses. Consistent with this, we previously showed in nucleus accumbens (NAc) medium spiny neurons (MSNs) that reduced mGlu1 tone enables and mGlu1 positive allosteric modulation reverses the elevation of CP-AMPAR levels in the NAc that underlies enhanced cocaine craving in the "incubation of craving" rat model of addiction. To better understand mGlu1/CP-AMPAR interactions, we used a NAc/prefrontal cortex co-culture system in which NAc MSNs express high CP-AMPAR levels, providing an in vitro model for NAc MSNs after the incubation of cocaine craving. The non-specific group I orthosteric agonist dihydroxyphenylglycine (10 min) decreased cell surface GluA1 but not GluA2, indicating CP-AMPAR internalization. This was prevented by mGlu1 (LY367385) or mGlu5 (MTEP) blockade. However, a selective role for mGlu1 emerged in studies of long-term antagonist treatment. Thus, LY367385 (24 hr) increased surface GluA1 without affecting GluA2, whereas MTEP (24 hr) had no effect. In hippocampal neurons, scaling up of CP-AMPARs can occur through a mechanism requiring retinoic acid (RA) signaling and new GluA1 synthesis. Consistent with this, the LY367385-induced increase in surface GluA1 was blocked by anisomycin (translation inhibitor) or 4-(diethylamino)-benzaldehyde (RA synthesis inhibitor). Thus, mGlu1 transmission tonically suppresses cell surface CP-AMPAR levels, and decreasing mGlu1 tone increases surface CP-AMPARs via RA signaling and protein translation. These results identify a novel mechanism for homeostatic plasticity in NAc MSNs.

Repeated restraint stress exposure during early withdrawal accelerates incubation of cue-induced cocaine craving.

A major challenge for treating cocaine addiction is the propensity for abstinent users to relapse. Two important triggers for relapse are cues associated with prior drug use and stressful life events. To study their interaction in promoting relapse during abstinence, we used the incubation model of craving and relapse in which cue-induced drug seeking progressively intensifies ('incubates') during withdrawal from extended-access cocaine self-administration. We tested rats for cue-induced cocaine seeking on withdrawal day (WD) 1. Rats were then subjected to repeated restraint stress or control conditions (seven sessions held between WD6 and WD14). All rats were tested again for cue-induced cocaine seeking on WD15, 1 day after the last stress or control session. Although controls showed a time-dependent increase in cue-induced cocaine seeking (incubation), rats exposed to repeated stress in early withdrawal exhibited a more robust increase in seeking behavior between WD1 and WD15. In separate stressed and control rats, equivalent cocaine seeking was observed on WD48. These results indicate that repeated stress in early withdrawal accelerates incubation of cocaine craving, although craving plateaus at the same level were observed in controls. However, 1 month after the WD48 test, rats subjected to repeated stress in early withdrawal showed enhanced cue-induced cocaine seeking following acute (24 hours) food deprivation stress. Together, these data indicate that chronic stress exposure enhances the initial rate of incubation of craving during early withdrawal, resulting in increased vulnerability to cue-induced relapse during this period, and may lead to a persistent increase in vulnerability to the relapse-promoting effects of stress.

Trafficking of calcium-permeable and calcium-impermeable AMPA receptors in nucleus accumbens medium spiny neurons co-cultured with prefrontal cortex neurons.

AMPA receptor (AMPAR) transmission onto medium spiny neurons (MSNs) of the adult rat nucleus accumbens (NAc) is normally dominated by GluA2-containing, Ca2+-impermeable AMPAR (CI-AMPARs). However, GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs) accumulate after prolonged withdrawal from extended-access cocaine self-administration and thereafter their activation is required for the intensified (incubated) cue-induced cocaine craving that characterizes prolonged withdrawal from such regimens. These findings suggest the existence of mechanisms in NAc MSNs that differentially regulate CI-AMPARs and CP-AMPARs. Here, we compared trafficking of GluA1A2 CI-AMPARs and homomeric GluA1 CP-AMPARs using immunocytochemical assays in cultured NAc MSNs plated with prefrontal cortical neurons to restore excitatory inputs. We began by evaluating constitutive internalization of surface receptors and found that this occurs more rapidly for CP-AMPARs. Next, we studied receptor insertion into the membrane; combined with past results, the present findings suggest that activation of protein kinase A accelerates insertion of both CP-AMPARs and CI-AMPARs. We also studied constitutive cycling (net loss of receptors from the membrane under conditions where internalization and recycling are both occurring). Interestingly, although CP-AMPARs exhibit faster constitutive internalization, they cycle at similar rates as CI-AMPARs, suggesting faster reinsertion of CP-AMPARs. In studies of synaptic scaling, long-term (24 h) activity blockade increased surface expression and cycling rates of CI-AMPARs but not CP-AMPARs, whereas long-term increases in activity produced more pronounced scaling down of CI-AMPARs than CP-AMPARs but did not alter receptor cycling. These findings can be used to evaluate and generate hypotheses regarding AMPAR plasticity in the rat NAc following cocaine exposure.

AMPA Receptor Plasticity in Accumbens Core Contributes to Incubation of Methamphetamine Craving.

BACKGROUND: The incubation of cue-induced drug craving in rodents provides a model of persistent vulnerability to craving and relapse in human addicts. After prolonged withdrawal, incubated cocaine craving depends on strengthening of nucleus accumbens (NAc) core synapses through incorporation of Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (CP-AMPARs). Through metabotropic glutamate receptor 1 (mGluR1)-mediated synaptic depression, mGluR1 positive allosteric modulators remove CP-AMPARs from these synapses and thereby reduce cocaine craving. This study aimed to determine if similar plasticity accompanies incubation of methamphetamine craving. METHODS: Rats self-administered saline or methamphetamine under extended-access conditions. Cue-induced seeking tests demonstrated incubation of methamphetamine craving. After withdrawal periods ranging from 1 to >40 days, rats underwent one of the following procedures: 1) whole-cell patch clamp recordings to characterize AMPAR transmission, 2) intra-NAc core injection of the CP-AMPAR antagonist 1-naphthyl acetyl spermine followed by a seeking test, or 3) systemic administration of a mGluR1 positive allosteric modulator followed by a seeking test. RESULTS: Incubation of methamphetamine craving was associated with CP-AMPAR accumulation in NAc core, and both effects were maximal after ~1 week of withdrawal. Expression of incubated craving was decreased by intra-NAc core 1-naphthyl acetyl spermine injection or systemic mGluR1 positive allosteric modulator administration. CONCLUSIONS: These results are the first to demonstrate a role for the NAc in the incubation of methamphetamine craving and describe adaptations in synaptic transmission associated with this model. They establish that incubation of craving and associated CP-AMPAR plasticity occur much more rapidly during withdrawal from methamphetamine compared with cocaine. However, a common mGluR1-based therapeutic strategy may be helpful for recovering cocaine and methamphetamine addicts.

Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration.

It is well established that cocaine-induced changes in glutamate receptor expression in the nucleus accumbens (NAc) play a significant role in animal models of cocaine addiction. Far less is known about cocaine-induced changes in GABA transmission, despite its importance in regulating NAc output via local interneurons and medium spiny neuron (MSN) axon collaterals (GABA 'microcircuit'). Here we investigated whether GABAA receptor surface or total expression is altered following an extended-access cocaine self-administration regimen that produces a time-dependent intensification (incubation) of cue-induced cocaine craving in association with strengthening of AMPA receptor (AMPAR) transmission onto MSN. Rats self-administered cocaine or saline (control condition) 6h/day for 10 days. NAc tissue was obtained and surface proteins biotinylated on three withdrawal days (WD) chosen to span incubation of craving and associated AMPAR plasticity: WD2, WD25 and WD48. Immunoblotting was used to measure total and surface expression of three GABAA receptor subunits (α1, α2, and α4) that are strongly expressed in the NAc. We found a transient increase in surface, but not total, expression of the α2 subunit on WD2 from cocaine self-administration, an effect that was no longer observed by WD25. The expression of α1 and α4 subunits was not altered at these withdrawal times. On WD48, when AMPAR transmission is significantly potentiated, we did not find any alteration in GABAA receptor surface or total expression. Our findings suggest that the strengthening of AMPAR-mediated glutamate transmission in the NAc is not accompanied by compensatory strengthening of GABAergic transmission through insertion of additional GABAA receptors.

Response of the Ubiquitin-Proteasome System to Memory Retrieval After Extended-Access Cocaine or Saline Self-Administration.

The ubiquitin-proteasome system (UPS) has been implicated in the retrieval-induced destabilization of cocaine- and fear-related memories in Pavlovian paradigms. However, nothing is known about its role in memory retrieval after self-administration of cocaine, an operant paradigm, or how the length of withdrawal from cocaine may influence retrieval mechanisms. Here, we examined UPS activity after an extended-access cocaine self-administration regimen that leads to withdrawal-dependent incubation of cue-induced cocaine craving. Controls self-administered saline. In initial experiments, memory retrieval was elicited via a cue-induced seeking/retrieval test on withdrawal day (WD) 50-60, when craving has incubated. We found that retrieval of cocaine- and saline-associated memories produced similar increases in polyubiquitinated proteins in the nucleus accumbens (NAc), compared with rats that did not undergo a seeking/retrieval test. Measures of proteasome catalytic activity confirmed similar activation of the UPS after retrieval of saline and cocaine memories. However, in a subsequent experiment in which testing was conducted on WD1, proteasome activity in the NAc was greater after retrieval of cocaine memory than saline memory. Analysis of other brain regions confirmed that effects of cocaine memory retrieval on proteasome activity, relative to saline memory retrieval, depend on withdrawal time. These results, combined with prior studies, suggest that the relationship between UPS activity and memory retrieval depends on training paradigm, brain region, and time elapsed between training and retrieval. The observation that mechanisms underlying cocaine memory retrieval change depending on the age of the memory has implications for development of memory destabilization therapies for cue-induced relapse in cocaine addicts.

BDNF contributes to both rapid and homeostatic alterations in AMPA receptor surface expression in nucleus accumbens medium spiny neurons.

Brain-derived neurotrophic factor (BDNF) plays a critical role in plasticity at glutamate synapses and in the effects of repeated cocaine exposure. We recently showed that intracranial injection of BDNF into the rat nucleus accumbens (NAc), a key region for cocaine addiction, rapidly increases α-amino-3-hyroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) surface expression. To further characterize BDNF's role in both rapid AMPAR trafficking and slower, homeostatic changes in AMPAR surface expression, we investigated the effects of acute (30 min) and long-term (24 h) treatment with BDNF on AMPAR distribution in NAc medium spiny neurons from postnatal rats co-cultured with mouse prefrontal cortex neurons to restore excitatory inputs. Immunocytochemical studies showed that acute BDNF treatment increased cell surface GluA1 and GluA2 levels, as well as their co-localization, on NAc neurons. This effect of BDNF, confirmed using a protein crosslinking assay, was dependent on ERK but not AKT signaling. In contrast, long-term BDNF treatment decreased AMPAR surface expression on NAc neurons. Based on this latter result, we tested the hypothesis that BDNF plays a role in AMPAR 'scaling down' in response to a prolonged increase in neuronal activity produced by bicuculline (24 h). Supporting this hypothesis, decreasing BDNF signaling with the extracellular BDNF scavenger TrkB-Fc prevented the scaling down of GluA1 and GluA2 surface levels in NAc neurons normally produced by bicuculline. In conclusion, BDNF exerts bidirectional effects on NAc AMPAR surface expression, depending on duration of exposure. Furthermore, BDNF's involvement in synaptic scaling in the NAc differs from its previously described role in the visual cortex.

Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving.

Cue-induced cocaine craving is a major cause of relapse in abstinent addicts. In rats, cue-induced craving progressively intensifies (incubates) during withdrawal from extended-access cocaine self-administration. After ~1 month of withdrawal, incubated craving is mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs) that accumulate in the nucleus accumbens (NAc). We found that decreased mGluR1 surface expression in the NAc preceded and enabled CP-AMPAR accumulation. Thus, restoring mGluR1 transmission by administering repeated injections of an mGluR1 positive allosteric modulator (PAM) prevented CP-AMPAR accumulation and incubation, whereas blocking mGluR1 transmission at even earlier withdrawal times accelerated CP-AMPAR accumulation. In studies conducted after prolonged withdrawal, when CP-AMPAR levels and cue-induced craving are high, we found that systemic administration of an mGluR1 PAM attenuated the expression of incubated craving by reducing CP-AMPAR transmission in the NAc to control levels. These results suggest a strategy in which recovering addicts could use a systemically active compound to protect against cue-induced relapse.

Adaptations in AMPA receptor transmission in the nucleus accumbens contributing to incubation of cocaine craving.

Cue-induced cocaine craving in rodents intensifies or "incubates" during the first months of withdrawal from long access cocaine self-administration. This incubation phenomenon is relevant to human users who achieve abstinence but exhibit persistent vulnerability to cue-induced relapse. It is well established that incubation of cocaine craving involves complex neuronal circuits. Here we will focus on neuroadaptations in the nucleus accumbens (NAc), a region of convergence for pathways that control cocaine seeking. A key adaptation is a delayed (~3-4 weeks) accumulation of Ca(2+)-permeable AMPAR receptors (CP-AMPARs) in synapses on medium spiny neurons (MSN) of the NAc. These CP-AMPARs mediate the expression of incubation after prolonged withdrawal, although different mechanisms must be responsible during the first weeks of withdrawal, prior to CP-AMPAR accumulation. The cascade of events leading to CP-AMPAR accumulation is still unclear. However, several candidate mechanisms have been identified. First, mGluR1 has been shown to negatively regulate CP-AMPAR levels in NAc synapses, and it is possible that a withdrawal-dependent decrease in this effect may help explain CP-AMPAR accumulation during incubation. Second, an increase in phosphorylation of GluA1 subunits (at the protein kinase A site) within extrasynaptic homomeric GluA1 receptors (CP-AMPARs) may promote their synaptic insertion and oppose their removal. Finally, elevation of brain-derived neurotrophic factor (BDNF) levels in the NAc may contribute to maintenance of incubation after months of withdrawal, although incubation-related increases in BDNF accumulation do not account for CP-AMPAR accumulation. Receptors and pathways that negatively regulate incubation, such as mGluR1, are promising targets for the development of therapeutic strategies to help recovering addicts maintain abstinence. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure.

Hypocretin peptides are critical for the effects of cocaine on excitatory synaptic strength in the ventral tegmental area (VTA). However, little is known about their role in cocaine-induced synaptic plasticity in the nucleus accumbens (NAc). First, we tested whether hypocretin-1 by itself could acutely modulate glutamate receptor surface expression in the NAc, given that hypocretin-1 in the VTA reproduces cocaine's effects on glutamate transmission. We found no effect of hypocretin-1 infusion on AMPA or NMDA receptor surface expression in the NAc, measured by biotinylation, either 30 min or 3h after the infusion. Second, we were interested in whether changes in hypocretin receptor-2 (Hcrtr-2) expression contribute to cocaine-induced plasticity in the NAc. As a first step towards addressing this question, Hcrtr-2 surface expression was compared in the NAc after withdrawal from extended-access self-administration of saline (control) versus cocaine. We found that surface Hcrtr-2 levels remain unchanged following 14, 25 or 48 days of withdrawal from cocaine, a time period in which high conductance GluA2-lacking AMPA receptors progressively emerge in the NAc. Overall, our results fail to support a role for hypocretins in acute modulation of glutamate receptor levels in the NAc or a role for altered Hcrtr-2 expression in withdrawal-dependent synaptic adaptations in the NAc following cocaine self-administration.