Long-lasting potentiation of GABAergic synapses in dopamine neurons after a single in vivo ethanol exposure

Abstract

The mesolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) is involved in many drug-related behaviors, including ethanol self-administration. In particular, VTA activity regulating ethanol consummatory behavior appears to be modulated through GABA(A) receptors. Previous exposure to ethanol enhances ethanol self-administration, but the mechanisms underlying this phenomenon are not well understood. In this study, we examined changes occurring at GABA synapses onto VTA DA neurons after a single in vivo exposure to ethanol. We observed that evoked GABA(A) IPSCs in DA neurons of ethanol-treated animals exhibited paired-pulse depression (PPD) compared with saline-treated animals, which exhibited paired-pulse facilitation (PPF). Furthermore, PPD was still present 1 week after the single exposure to ethanol. An increase in frequency of spontaneous miniature GABA(A) IPSCs (mIPSCs) was also observed in the ethanol-treated animals. Additionally, the GABA(B) receptor antagonist (3-aminopropyl)(diethoxymethyl) phosphinic acid shifted PPD to PPF, indicating that presynaptic GABA(B) receptor activation, likely attributable to GABA spillover, might play a role in mediating PPD in the ethanol-treated mice. The activation of adenylyl cyclase by forskolin increased the amplitude of GABA(A) IPSCs and the frequency of mIPSCs in the saline- but not in the ethanol-treated animals. Conversely, the protein kinase A (PKA) inhibitor N-[z-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide significantly decreased both the frequency of spontaneous mIPSCs and the amplitude of GABA(A) IPSCs in the ethanol-treated mice but not in the saline controls. The present results indicate that potentiation of GABAergic synapses, via a PKA-dependent mechanism, occurs in the VTA after a single in vivo exposure to ethanol, and such potentiation might be a key synaptic modification underlying increased ethanol intake.

Fig. 1.

Increased probability of GABA release 24 hr after a single in vivo exposure to ethanol. GABA_AIPSCs from ethanol-treated mice show PPD compared with saline controls, which show PPF. a, b, Examples of recordings from saline-treated (a) and ethanol-treated (b) animals. c, No correlation was found between the amplitude of IPSC1 and the IPSC2/IPSC1 ratio in both saline-treated mice (n = 16) and ethanol-treated mice (n = 17). d, The IPSC2/IPSC1 ratio is independent of the stimulus strength. Results are the average from four cells in each group of animals. e, PPD in ethanol-treated mice is a long-lasting phenomenon. The bar graph shows the average IPSC2/IPSC1 ratio (mean ± SEM) of saline- and ethanol-treated mice after 1 d (n= 16 and 17 for saline and ethanol, respectively; *p < 0.05), 1 week (n = 9 per group; *p < 0.05), and 2 weeks (n = 8 per group; p > 0.05) after ethanol pre-exposure. f, Ethanol pre-exposure does not change either number or function of postsynaptic GABA_Areceptors. Bath application of GABA (100 μm, 3 min) in the presence of GABA_B receptor antagonist CGP 35348 (100 μm) elicited a similar current (30 sec bins) in both groups of animals (n = 4; p > 0.05) when neurons were voltage-clamped at −70 mV.

Fig. 2.

Ethanol pre-exposure increased the frequency, but not amplitude, of spontaneous mIPSCs. a, Samples of mIPSCs from saline-treated mice (top traces) and ethanol-treated mice (bottom traces). b,Bar graph showing the average (mean ± SEM) frequency for saline-treated animals (n = 7) and ethanol-treated animals (n = 9; *p < 0.05). c, Bar graph (10 pA bins) showing an amplitude histogram of mIPSCs for ethanol-treated (n = 9) versus saline-treated (n = 7) mice. d, Bar graph showing the average (mean ± SEM) amplitude for saline-treated animals (n = 7) and ethanol-treated animals (n = 9).

Fig. 3.

One week after the pre-exposure, ethanol-treated mice show increased ethanol intake and preference compared with saline controls. a, Voluntary 24 hr ethanol consumption (grams per kilogram) in C57BL/6J mice pretreated with ethanol (n = 8; *p < 0.05) and saline (n = 8). b, Ethanol preference, calculated as 100 × milliliters of ethanol per total milliliters consumed. C57BL/6J mice pretreated with ethanol demonstrated a significant increase in ethanol preference (n = 8; *p < 0.05) when compared with saline controls (n = 8). c, Blood ethanol clearance after acute administration of ethanol (4 gm/kg, i.p.) did not differ between ethanol- and saline-treated mice. Data (mean ± SEM) represent four animals per each group at every time point.

Fig. 4.

Effect of the GABA_B receptor antagonist CGP35348 (100 μm) on IPSC2/IPSC1 ratio and spontaneous mIPSCs. a, CGP35348 (100 μm, 5 min) shifts the PPD to PPF in ethanol-treated mice (n = 10; *p < 0.05), without affecting the PPF in saline-treated mice (n = 5).b, The IPSC2/IPSC1 ratio is plotted as a function of time in cells recorded from saline- and ethanol-treated mice and normalized against the mean of the first 10 min for each cell.c, CGP35348 does not change the frequency of mIPSCs in either group (n = 7 per each group).d, No changes in amplitude of spontaneous mIPSCs were found in either group (n = 7 per each group).

Fig. 5.

Effect of the GABA_B receptor agonist baclofen on evoked IPSCs. a, Concentration–response curve for baclofen measuring the amplitude of IPSC1 from saline- and ethanol-treated animals (n = 5 per each group at all doses tested). b, Baclofen (1 μm, 10 min) decreases the amplitude of IPSC1 in both saline- and ethanol-treated mice to the same extent (n = 5 per each group). c, Baclofen (10 μm, 10 min) shifts the PPD to PPF in ethanol-treated mice (n = 5; *p < 0.05) without affecting the PPF in saline-treated mice (n = 5). d, IPSC2/IPSC1 ratio plotted as a function of time in cells recorded from saline- and ethanol-treated mice (n = 5 per group; *p < 0.05) and normalized against the mean of the first 10 min for each cell.

Fig. 6.

Effect of forskolin on evoked and spontaneous IPSCs. a, Forskolin (10 μm, 10 min) increases the amplitude of evoked IPSC1 in saline-treated mice (n = 5; *p < 0.05) but not ethanol-treated mice (n = 5). b, Forskolin (10 μm, 10 min) shifts the PPF to PPD in saline-treated mice (n = 5; *p< 0.05) without affecting the PPD in ethanol-treated mice (n = 5). The IPSC2/IPSC1 ratio is plotted as function of time in cells recorded from saline- and ethanol-treated mice and normalized against the mean of the first 10 min for each cell.c, Forskolin (10 μm, 10 min) induces a significant increase in the frequency of mIPSCs in saline-treated mice (n = 7; *p < 0.05) but not in ethanol-treated mice (n = 9). d, No changes in amplitude were found in either group.

Fig. 7.

Effect of H89 on evoked and spontaneous IPSCs.a, H89 (10 μm, 20 min) decreases the amplitude of evoked IPSCs in ethanol-treated mice (n = 5; *p < 0.05), but not saline-treated mice (n = 5). b, H89 (10 μm, 20 min) shifts the PPD to PPF in ethanol-treated mice (n = 5; *p < 0.05) without affecting the PPF in saline-treated mice. c, H89 (10 μm, 20 min) induces a significant decrease in the frequency of mIPSCs in ethanol-treated mice (n = 6; *p < 0.05) but not saline-treated mice (n = 5). d, No changes in amplitude were found in either group.