Figure 2. Acute nicotine exposure alters Cl− homeostasis in the VTA. A, GABAergic input onto VTA GABA neurons was measured using gramicidin perforated-patch whole-cell recordings at different holding potentials to measure nicotine-induced alterations in anion homeostasis. GABAA IPSCs were evoked by electrical stimulation in the presence of ionotropic glutamate and GABAB receptor antagonists. B, Representative eIPSC recordings from saline (black) and nicotine (red)-treated animals at the given holding potentials (Vh). The eIPSCs reverse direction at EGABA. For display, the traces were filtered, and stimulus artifacts were removed. C, VTA GABA neurons from nicotine-treated animals (red) showed a significantly more positive EGABA compared with saline-treated animals (black); **p < 0.01, significantly different by t test, n = 13, n = 14 cells/group, n = 7 rats/group. D, Activity-dependent synaptic depression in VTA GABA neurons was measured during whole-cell patch clamp recordings under repetitive GABAAR stimulation. Traces on top: representative GABA neurons from saline (black) and nicotine-treated rats (red) demonstrated a similar depression in eIPSC amplitude when stimulated at 20 Hz and clamped at −90 mV. Bottom graph, At −90 mV, VTA GABA neurons from control and stressed animals showed no significant difference in rate of eIPSC amplitude depression (p > 0.05, n = 8 cells, n = 4 rats for the nicotine group and n = 16 cells, n = 6 rats for the saline group). Amplitude values were averaged for five eIPSCs and shown as a percent of the first five eIPSCs mean amplitude. E, Cl− accumulation was measured as in D, but VTA GABA neurons were clamped at 0 mV. Top traces, Upon stimulation, VTA GABA neuron from control animal (black) demonstrated a minor depression of eIPSC amplitude compared with the significantly greater depression seen in a GABA neuron from a stressed animal (red). Bottom graph, At 0 mV, GABA neurons from nicotine treated animals (red) demonstrated a significantly greater rate of eIPSC amplitude depression than GABA neurons from saline-treated animals (black); **p < 0.01, significantly different by ANOVA with repeated measures, n = 8 cells, n = 4 rats for the nicotine group and n = 16 cells, n = 6 rats for the saline group. F, Western blot analysis was conducted for KCC2 protein expression with GAPDH as a loading control. A representative Western blotting shows reduced expression of KCC2 in nicotine-treated animals. G, Densiometric analysis showed a significant reduction in KCC2 protein in nicotine-treated animals (red bars) compared with saline-treated controls (horizontal dashed line); *p < 0.05, **p < 0.01, significantly different by paired t test, n = 6 animals/group.