Fig. 6. Fentanyl vapor self-administration reduces GABA_B receptor currents in VTA dopamine neurons through pre- and postsynaptic mechanisms.

(A) Representative traces of GABA_B IPSCs evoked by one, three, five, seven, or nine electrical stimuli (60 Hz) in slices from mice with a history of short access to vehicle versus fentanyl self-administration. Stimulus artifacts were blanked for clarity. (B) The amplitude of GABA_B IPSCs was reduced in neurons from fentanyl versus vehicle mice, shown as the peak amplitude for one, three, five, seven, or nine stimuli (left; two-way RM ANOVA; F_1,29 = 5.44; P = 0.027) and full time course for IPSCs evoked by five stimuli (right) (vehicle area under the curve = 6454, CI: 6117 to 6791; fentanyl area under the curve = 4709, CI: 4476 to 4942). (C) The plot of GABA_B IPSC amplitudes that were normalized to the intensity of electrical stimulation shows a greater difference between vehicle and fentanyl GABA_B IPSCs (stimulus × treatment interaction F_4,116 = 9.12; P < 0.0001). (D) Representative traces of the arithmetically summed GABA_B IPSCs (1 stimulus × 9) versus measured GABA_B IPSC that were evoked by nine stimuli (9 stims) in slices from vehicle mice (left) (measured < calculated), suggesting paired-pulse depression, and fentanyl mice, (right) (measured > calculated), suggesting paired-pulse facilitation. (E) Ratio of the measured GABA_B-IPSC amplitude over the calculated amplitude for each number of stimuli. Linear regression shows a significantly negative slope for vehicle (a = −0.02; F_1,63 = 12.3; P = 0.0008) and a significantly positive slope for fentanyl (a = 0.021; F_1,55 = 7.282; P = 0.009). (F) Representative traces of whole-cell voltage-clamp recordings (baselined and peak-aligned) from vehicle versus fentanyl mice, demonstrating the concentration-dependent outward current produced by the application of (R)-baclofen. (G) The maximal outward current that was produced by (R)-baclofen was reduced in neurons from fentanyl mice compared with vehicle mice (left). The data were analyzed by nonlinear regression (sigmoidal fit; vehicle, E_max = 446.9 pA; fentanyl, E_max = 279.6 pA; F_1,51 = 7.35; P = 0.009). No change in the EC₅₀ was observed (vehicle, EC₅₀ = 1.87 μM; fentanyl, EC₅₀ = 1.77 μM). When the amplitude of the outward current was normalized to membrane capacitance (current density, pA/pF), the response to (R)-baclofen was still lower in fentanyl neurons (right). Solid lines represent the sigmoidal fit of the data. n, number of cells. The data are expressed as means ± SEM. *P < 0.05.