Reversing cocaine-induced synaptic potentiation provides enduring protection from relapse

Enduring Relapse Protection by Chronic N-Acetylcysteine.

We adapted the extinction–reinstatement model of relapse to cocaine-seeking (27). Rats were trained to lever press for i.v. cocaine for 12 d, followed by 12 d of extinction training, during which lever pressing no longer delivered cocaine (Fig. 1A). Two hours before each extinction session, animals received the cystine prodrug N-acetylcysteine (100 mg/kg, i.p.) or vehicle. The rats then underwent 2–3 additional weeks of cocaine withdrawal in the absence of extinction training or N-acetylcysteine treatments to evaluate long-lasting effects of N-acetylcysteine on glutamate homeostasis, synaptic physiology, and conditioned cue- or cocaine-induced reinstatement of lever pressing (cocaine-seeking behavior). Control animals received an i.v. saline infusion that was yoked to cocaine infusions in rats self-administering cocaine. This protocol of cocaine self-administration, N-acetylcysteine treatment, and withdrawal was used throughout the study.

In the first experiment, reinstatement trials were conducted to estimate the effect of N-acetylcysteine on reinstated cocaine-seeking 2 h after N-acetylcysteine or vehicle on days 25 and 29 and again on days 43 and 47 after leaving the rats in their home cages for 2–3 wk without drug treatments (Fig. 1A). Animals trained to self-administer cocaine were divided into two groups such that levels of active lever pressing across the last 3 d of cocaine self-administration were equivalent in each group (Fig. 1b). The N-acetylcysteine-treated animals showed an initial reduction in lever pressing during extinction training, although by 2 wk the levels of extinction pressing were equivalent between the vehicle and N-acetylcysteine groups (Fig. 1B). Reduced extinction lever pressing unlikely resulted from nonspecific motor inhibition since acute N-acetylcysteine does not inhibit locomotor activity or operant responding (25).

Cocaine-seeking was elicited by presenting cues (tone and light) previously paired with cocaine delivery during self-administration or by injecting cocaine (10 mg/kg, i.p) just before the cue-reinstatement session. Reinstated lever pressing induced by either modality was inhibited in the N-acetylcysteine pretreated group (days 25 and 29; Fig. 1C) compared with the vehicle-treated group. More importantly, 2–3 wk after discontinuing N-acetylcysteine or vehicle treatments, both cue-induced and cocaine- and cue-induced reinstatement remained inhibited in the N-acetylcysteine group (Fig. 1D).

Enduring Restoration of Glutamate Homeostasis in the NAcore.

The reduction in basal extracellular concentrations of glutamate in the NAcore by chronic cocaine use is a marker of disrupted glutamate homeostasis, arising from an enduring reduction in cystine–glutamate exchange (20, 21, 25). Basal extracellular glutamate concentrations assayed in the NAcore by in vivo microdialysis arise largely from cystine–glutamate exchange rather than from synaptic activity (19). In the next experiment, we measured basal glutamate levels after the daily treatment regimen of N-acetylcysteine to determine whether the reduction in cocaine-seeking was associated with restoration of nonsynaptic glutamate in the extracellular space (Fig. 2A). Animals trained to self-administer cocaine and treated with saline vehicle during extinction training showed reduced basal levels of glutamate compared with yoked-saline controls using no-net-flux microdialysis (Fig. 2B). However, basal extracellular glutamate levels in cocaine-trained animals were restored to control levels when measured 2–3 wk after the last daily injection of N-acetylcysteine. In contrast, the N-acetylcysteine regimen did not alter glutamate in yoked-saline rats. Fig. 2C shows the location of the active zone of the microdialysis probes in the NAcore.

Enduring Restoration of Synaptic Strength and Role of mGluR2/3.

The long-lasting normalization of extracellular glutamate by N-acetylcysteine (Fig. 2B) may increase glutamatergic tone onto perisynaptic mGluR2/3 (28) that negatively regulate synaptic glutamate release (23, 29–31). To examine this possibility, stimulating electrodes were placed into the prefrontal cortex and recording electrodes into the NAcore to elicit field potentials as an estimate of the overall strength of prefrontal to NAcore synapses (Fig. 3B; ref. 7). This protocol permits selective activation of prefrontal afferents to the NAcore in an in vivo environment where glutamate homeostasis is not disrupted (32). We previously found a long-term potentiation (LTP)-like shift to the left of the input–output curve 3 wk after the last cocaine self-administration session (7). Similarly, 4–5 wk after the last cocaine exposure, the input–output curve for rats trained to self-administer cocaine was potentiated compared with yoked-saline controls (Fig. 3A). This LTP-like shift was abolished 2–3 wk after completing daily N-acetylcysteine. In contrast, N-acetylcysteine did not alter the input–output curve in yoked-saline animals.

The importance of mGluR2/3 in N-acetylcysteine-induced restoration of synaptic strength was examined by infusing the mGluR2/3 antagonist LY341495 through a microdialysis probe attached to the recording electrode (Fig. 3B). To validate this methodology, reverse-dialysis of the AMPA receptor antagonist, CNQX, or the mGluR2/3 agonist, LY379268, to drug-naive animals reduced field potential amplitude (Fig. S1). Infusing receptor-selective concentrations (33) of LY341495 into the NAcore potentiated evoked field potentials in yoked-saline rats (Fig. 3C). The increase in field amplitude was absent in cocaine-trained rats treated with vehicle during extinction training, indicating decreased tone on mGluR2/3 commensurate with reduced basal extracellular concentrations of glutamate (Fig. 1B). However, similar to controls, LY341495 potentiated prefrontal-to-NAcore synaptic transmission in rats receiving N-acetylcysteine during extinction training. Therefore, the capacity of LY341495 to block inhibitory mGluR2/3 tone and potentiate field potentials was restored by N-acetylcysteine administration during extinction training, corroborating the enduring restoration of extracellular glutamate levels (Fig. 2B).

Enduring Relapse Protection by Restoring Glutamatergic Tone to mGluR2/3.

Up to this point the data show an enduring normalization of glutamate homeostasis by N-acetylcysteine, and thereby a mGluR2/3-dependent depotentiation of prefrontal-to-NAcore excitatory synapses. Given the postulated role by heightened accumbens glutamatergic transmission in cocaine relapse (4), we asked whether the long-lasting protection from reinstated cocaine-seeking provided by the chronic N-acetylcysteine treatment regimen was also mGluR2/3-dependent. In this experiment, animals treated with N-acetylcysteine during extinction were microinjected with the mGluR2/3 antagonist LY341495 into the NAcore (Fig. 4A). Blockade of mGluR2/3 prevented N-acetylcysteine-induced inhibition of reinstated cocaine-seeking in the presence of N-acetylcysteine (days 25 and 29) and 2–3 wk after the last N-acetylcysteine injection (days 43 and 47) (Fig. 4B). In contrast, LY341495 did not alter reinstated responding in control animals (Fig. 4C). These data demonstrate that N-acetylcysteine induced a long-lasting restoration of glutamatergic tone onto mGluR2/3, thereby normalizing synaptic strength and causing an enduring reduction in reinstated cocaine-seeking.

Enduring Restoration of Both Presynaptic and Postsynaptic Glutamate Transmission.

The experiments so far demonstrate that N-acetylcysteine produced an enduring increase in nonsynaptic glutamate (Fig. 2B) that impinges on mGluR2/3 (Fig. 3D) to normalize synaptic strength (Fig. 3A). Since the measure of synaptic strength was via field potentials in vivo, it is unclear if N-acetylcysteine's effects were primarily presynaptic, postsynaptic, or both. To further examine the role of glutamate homeostasis in presynaptic regulation of excitatory transmission in the NAcore, we measured mEPSC in MSNs using whole-cell patch recordings in tissue slices obtained from rats treated as in Fig. 2A. Presynaptic release probability was estimated by measuring the frequency of mEPSC after eliminating action potential-evoked glutamate release with tetrodotoxin (TTX) (29, 34, 35). The frequency of mEPSC was elevated in NAcore MSNs recorded in cocaine-trained subjects compared with yoked-saline controls, and this increase was abolished when measured 2–3 wk after the last N-acetylcysteine injection (Fig. 5 A and B). In contrast to mEPSC frequency, there was no effect by either cocaine or N-acetylcysteine on mEPSC amplitude (Fig. 5C). Also, spontaneous EPSC (sEPSC) recorded in the absence of TTX showed an increased frequency in cocaine rats treated with vehicle, but not in cocaine rats that received N-acetylcysteine treatment, and no treatment group differed in sEPSC amplitude (Fig. S2). These results indicate that by restoring extracellular glutamate (Fig. 2B) and glutamatergic tone on presynaptic release-regulating mGluR2/3s (Fig. 3 C and D), chronic N-acetylcysteine normalizes presynaptic glutamate release probability.

To estimate the postsynaptic effects of cocaine and N-acetylcysteine on glutamatergic synaptic transmission, we recorded the ratio of peak AMPA to NMDA receptor-mediated currents. Recordings were restricted to the dorsomedial NAcore, and stimulation electrodes were placed dorsomedial to the recording electrode to preferentially stimulate prefrontal afferents (36). Akin to a previous report with chronic cocaine followed by withdrawal (8), cocaine-trained rats showed an increase in AMPA:NMDA relative to yoked-saline controls (Fig. 5D). The AMPA:NMDA ratio elevation was reversed in animals treated with N-acetylcysteine during extinction training, supporting an enduring normalization of postsynaptic strength by N-acetylcysteine. Considering the observed changes in AMPA:NMDA ratio, the lack of effect on mEPSC or sEPSC amplitude was surprising. This discrepancy between mEPSC amplitude and AMPA:NMDA ratio is not uncommon and is thought to result from mEPSC arriving from diverse excitatory synapses onto a neuron, whereas the evoked EPSC is largely thought to result from prefrontal afferents (35, 37).

Animal Housing and Surgery.

All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the Institutional Animal Care and Use Committee at the Medical University of South Carolina approved all procedures. Male Sprague-Dawley rats were single housed, and food intake was maintained at ∼25 g/d. Rats were anesthetized with ketamine/xylazine and implanted with i.v. catheters. For in vivo intracranial experiments, rats were stereotaxically implanted with bilateral guide cannulae aimed at the NAcore according to Paxinos and Watson (47).

Self-Administration, Extinction, and Reinstatement Procedures.

Daily self-administration consisted of 12 d at >10 infusions/2-h session (lever press = 0.2 mg in 0.05 mL cocaine infusion over 2 s and a 5-s presentation of light- and tone-conditioned cues). Paired yoked saline controls were used. Extinction sessions began on the day after the final self-administration session and continued for 2 wk, after which rats were left in their home cages for 2–3 wk. Following the extinction period, animals underwent cue and cocaine- and cue-primed (10 mg/kg) reinstatement trials (Fig. 4A). For the microinjection reinstatement study, a counterbalanced cross-over design was used, and LY341495 (2 μg/0.5 μL per side) or artificial cerebrospinal fluid (aCSF) were infused over 2 min.

In Vivo Microdialysis and Glutamate Quantification.

Probes were constructed as described (48). The night before the experiment, a probe was inserted through an indwelling guide cannula, and the following morning 20-min samples were collected. Glutamate was measured using HPLC with electrochemical detection (48).

In Vivo Field Potentials.

Rats were anesthetized with urethane and mounted in a stereotaxic apparatus. Concentric bipolar stimulating electrodes were placed in the ventral prelimbic prefrontal cortex, glass recording electrodes were aimed at the dorsomedial region of the accumbens, and field potential amplitude was recorded (7). In some field recordings, microdialysis probes were attached to recording electrodes (0.5- to 1.0-mm separation) and lowered into the NAcore to deliver an mGluR2/3 agonist or antagonist (2 μL/min flow rate).

Slice Preparation and In Vitro Whole Cell Recording.

Rats were administered ketamine/xylazine and transcardially perfused with aCSF. Coronal brain slices were collected and incubated in recording aCSF at 35°. Recordings targeted the dorsomedial NAcore where in vivo recordings were made and prefrontal inputs are most dense (36, 49). MSNs were visualized using video microscopy. Picrotoxin was used to block inhibitory synaptic transmission. EPSC were recorded under −80 mV voltage clamp whole-cell configuration using glass microelectrodes and were evoked using bipolar stimulation at ∼100–200 μm dorsomedial of the recorded cell. AMPA currents were isolated by applying the NMDA receptor antagonist d-AP5 at +40 mV, and NMDA currents were obtained by subtracting the AMPA current from the total current (8). mEPSC were recorded in presence of bath applied TTX, with detection criteria set at >7 pA (37). Cumulative probability plots were generated using 400–600 consecutive events.

Histology and Statistics.

For the microinjection and microdialysis studies, fixed brains were sectioned (100 μm thick) and stained with a Nissl stain. For field potential studies, pontamine sky blue was used to mark the recording site at the end of each recording session (7). Parametric statistical analysis were used for behavioral and electrophysiology data as indicated, and for greater than two groups, a Bonferroni multiple comparison post hoc comparison was used. For the no-net-flux analysis, linear regression with a Bonferroni correction for multiple comparisons was used. F values are reported in the figure legends when significant (P < 0.05).