We measured spontaneous inhibitory postsynaptic currents (sIPSCs) and separately spontaneous excitatory postsynaptic currents (sEPSCs, described below). To examine Proteasome inhibitor GABAergic activity, we blocked glutamate receptors with DNQX (20 μM) and AP5 (50 μM). Nicotine pretreatment did not significantly alter the mean basal sIPSC frequency between the groups (saline pretreatment control, 2.7 ±

0.3 Hz; nicotine pretreatment, 3.4 ± 0.6 Hz; n = 7, 8; p > 0.05) or the mean basal sIPSC amplitudes (saline pretreatment control, 27.9 ± 4.2 pA; nicotine pretreatment, 27.9 ± 2.8 pA; n = 7, 8; p > 0.05). In control DA neurons from saline-pretreated rats, bath-applied ethanol (50 mM) induced a marginal increase in the sIPSC frequency (black data, Figures 4A and 4C; n = 7) (Theile et al., 2008). By contrast, in DA neurons from nicotine-pretreated rats, ethanol caused a much greater potentiation of the sIPSC frequency above the control response (red data, Figures 4B and 4C; n = 8;

p < 0.01) with no change in sIPSC amplitude. We repeated this experiment using a lower bath ethanol concentration (25 mM) and find more determined that the effect of nicotine and ethanol on sIPSC frequency was still present. Nicotine pretreatment increased the sIPSC frequency induced by 25 mM ethanol by approximately 24% (n = 6/group, p < 0.05) compared to the saline pretreatment response, whereas in 50 mM ethanol the percent increase between Cell press the nicotine and saline pretreatment was approximately 56% (Figure 4C; n = 7, 8; p < 0.01). An increase in the frequency, but not the amplitude, of the sIPSCs after nicotine pretreatment suggested a presynaptic change in GABA transmission. To investigate whether a presynaptic mechanism rather than a postsynaptic mechanism was at work, we measured the paired-pulse ratio of evoked IPSCs under different pretreatment conditions after ethanol application. Differences in the amplitudes

between two consecutively evoked IPSCs (i.e., the paired-pulse ratio) suggest a transient change in the probability of GABA release. Baseline paired-pulse ratios were not different between the saline pretreatment and nicotine pretreatment groups (p > 0.05). Application of ethanol decreased the paired-pulse ratio in both the saline pretreatment control (n = 14) and the nicotine pretreatment group (n = 21). The magnitude of this paired-pulse depression, however, was significantly greater (p < 0.05) after the nicotine pretreatment (78.6% ± 3.4%) compared to the saline pretreatment (90.0% ± 3.8%) (Figure 4D), which is consistent with a presynaptic change in GABA transmission. To confirm that changes in ethanol-induced GABA transmission contribute to changes in DA neuron responses, we blocked GABAA receptors prior to the bath application of ethanol.