Cystine/glutamate exchange regulates metabotropic glutamate receptor presynaptic inhibition of excitatory transmission and vulnerability to cocaine seeking

Abstract

Withdrawal from chronic cocaine reduces extracellular glutamate levels in the nucleus accumbens by decreasing cystine/glutamate exchange (xc-). Activating xc- with N-acetylcysteine restores extracellular glutamate and prevents cocaine-induced drug seeking. It was hypothesized that the activation of xc- prevents drug seeking by increasing glutamatergic tone on presynaptic group II metabotropic glutamate receptors (mGluR2/3) and thereby inhibiting excitatory transmission. In the first experiment, the capacity of glutamate derived from xc- to regulate excitatory transmission via mGluR2/3 was determined. Physiological levels of cystine (100-300 nm) were restored to acute tissue slices from the nucleus accumbens or prefrontal cortex. Cystine increased glutamate efflux and decreased miniature EPSC (mEPSC) and spontaneous EPSC (sEPSC) frequency as well as evoked EPSC amplitude. These effects of cystine were presynaptic, because there was no change in mEPSC or sEPSC amplitude, and an increase in the evoked EPSC paired-pulse facilitation ratio. The cystine-induced reduction in EPSCs was reversed by blocking either xc- or mGluR2/3. In the second experiment, blocking mGluR2/3 prevented the ability of N-acetylcystine to inhibit the reinstatement of drug seeking in rats trained to self-administer cocaine. These data demonstrate that nonsynaptic glutamate derived from xc- modulates synaptic glutamate release and thereby regulates cocaine-induced drug seeking.

Figure 1.

Cystine regulates glutamate levels and excitatory transmission. A, Cystine increased extracellular glutamate in NA slices, as revealed by microdialysis (n = 5 per dose). The 1/10 μm dose is pooled data for perfusion of 1 μm (n = 2) and 10 μm (n = 3). Data were normalized to the average of the three baseline samples and are shown as mean ± SEM percentage change. Basal levels of glutamate (picomoles per sample) are as follows: 0 = 0.176 ± 0.023; 0.1 = 0.134 ± 0.016; 0.3 = 0.125 ± 0.019; 1/10 = 0.136 ± 0.040. B, Cystine (0.3 μm) reduced mEPSC (n = 11; 0.9 ± 0.2 Hz) and sEPSC (n = 9 baseline; 0.8 ± 0.2 Hz) frequency in medium spiny cells (V_m = -80 mV) in the NA. Data are presented as a percentage change from baseline ± SEM. C, Cystine (0.1 μm) increased extracellular glutamate in prefrontal cortex slices (n = 6). Data were normalized to percentage change from baseline; the basal level was 0.084 ± 0.015. D, Cystine (0.1 μm) reduced mEPSC (n = 12, 2.3 ± 0.6 Hz) and sEPSC (n = 8; 4.9 ± 0.8 Hz) frequency in PFC slices. ^*p < 0.05, comparing cystine treatment to the average baseline value using a one-way ANOVA with repeated measures and least significant difference post hoc comparison.

Figure 2.

Cystine inhibition of excitatory transmission is concentration dependent. A, Evoked EPSCs were normalized to the average of baseline (2-10 min) and are shown as mean percentage change. Legend and n at each dose are shown in B. Cells in the 0.3 μm group were exposed to a wash period (22-30 min). Representative traces are from a single cell during baseline, cystine, and wash. Calibration: 60 pA, 10 ms. B, Data presented in A are presented as mean ± SEM percentage change from baseline (samples 8-10) by cystine (samples 18-20). Symbols correspond to A, and n at each dose is shown below the symbol. C, Cells exposed to 0.3 μm cystine (Cys) exhibit enhanced paired-pulse facilitation. The data are expressed as a percentage change in the ratio of the second response to the first. D, Normalized sEPSC during baseline and cystine. See E for legend and n at each concentration of cystine. A representative 1 s current trace from a single cell after 10 min of baseline, cystine (0.3 μm), and wash is shown. Calibration: 20 pA, 200 ms. E, Data in D presented as mean ± SEM percentage change from baseline (samples 8-10) by cystine (samples 18-20). Symbols corresponding to D and n at each dose are shown below the symbol. F, The distribution of time intervals between successive sEPSC during baseline (10 min) and cystine (20 min). G, Distribution of sEPSC amplitude during baseline (10 min) and cystine (20 min). The asterisk denotes a significant difference from baseline (p ≤ 0.05).

Figure 3.

xc- and mGluR2/3 regulate excitatory transmission in the NA. A, C, E, G, Data were normalized to the average of baseline and are shown as mean ± SEM percentage change. Data in B, D, F, and H were collapsed during 8-10, 18-20, and 28-30 min of each experiment and compared with baseline (n is shown in the first bar). A, B, Cystine (0.3 μm) reduced sEPSC frequency and evoked EPSC amplitude after 20 min of baseline. Evoked EPSC traces from a single cell (calibration: 300 pA, 10 ms) and 1 s current traces from the same cell during aforementioned treatments are shown. C, D, The xc- antagonist CPG (1 μm) prevented cystine (0.3 μm) from reducing sEPSC frequency and EPSC amplitude. Evoked EPSC traces from a single cell (calibration: 200 pA, 10 ms) and 1 s current traces from the same cell during aforementioned treatments are shown. E, F, Cystine (0.3 μm) reduces sEPSC frequency and EPSC amplitude in the presence of the group I metabotropic glutamate receptor antagonist AIDA (1 mm). Evoked EPSC traces from a single cell (calibration: 100 pA, 10 ms) and 1 s current traces from the same cell during aforementioned treatments are shown. G, H, The mGluR2/3 antagonist LY341495 (LY) (0.3 μm) prevented cystine (0.3 μm) from reducing sEPSC frequency and EPSC amplitude. Evoked EPSC traces from a single cell (calibration: 100 pA, 10 ms) and 1 s current traces from the same cell during aforementioned treatments are shown. ^*p < 0.05, comparing cystine treatment with the average baseline value using a one-way ANOVA with repeated measures and least significant difference post hoc comparison.

Figure 4.

Blocking mGluR2/3 prevents the capacity of N-acetylcysteine to inhibit cocaine-induced reinstatement. Each rat was given injections of either N-acetylcysteine (60 mg/kg, s.c.) or saline 4 h before a cocaine injection (10 mg/kg, i.p.). In addition, rats were given injections once every hour after the injection of N-acetylcysteine or saline with either saline or the mGluR2/3 antagonist LY341495 (1 mg/kg, i.p.) before the cocaine priming injection. The data are shown as the mean ± SEM total number of active lever presses during the 120 min after a cocaine-priming injection (n > 5 for each treatment). ^*p < 0.05, compared with control (first bar on the left) using a one-way ANOVA followed by a Dunnett's test.