Projection-Specific Potentiation of Ventral Pallidal Glutamatergic Outputs after Abstinence from Cocaine

VP_VGluT2 neurons make the strongest synapses on VP_VGluT2 and LHb neurons based on postsynaptic, but not presynaptic, parameters. A, The A/N ratio of VP_VGluT2 synapses was the highest in VP_VGluT2 neurons and in LHb neurons [one-way ANOVA main effect of target, F_(5,25) = 9.63, p < 0.0001; p < 0.05 using Tukey's post hoc multiple-comparisons test, compared with VP_VGluT2 (*) or LHb (#)]. Insets, Representative AMPA (red) and NMDA (blue) currents for each region. NMDA currents normalized between regions to ease comparison. B, The decay time constant (τ) of the NMDA current was the slowest in VP_VGluT2 neurons (one-way ANOVA main effect of target, F_(5,25) = 9.40, p < 0.0001; *p < 0.05 using Tukey's post hoc multiple-comparisons test, comparing to VP_VGluT2 neurons). Inset, Representative NMDA currents from each target (normalized to peak). C, The CV of the EPSCs recorded at −70 mV did not differ between targets (one-way ANOVA, p = 0.86). D, The PPR recorded at −70 mV did not differ between targets (one-way ANOVA, p = 0.13), although MDT shows a trend toward lower PPR values. Numbers in bars represent the number of cells, and the number of mice is in parentheses. n.s. = not significant.

Differences in NMDA current kinetics between VP_VGluT2 and LHb neurons stem from different membrane properties and not different NMDA receptor subunit composition. A, B, Both the decay (A) and rise time (B) of the NMDA currents were slower in VP_VGluT2 neurons compared with LHb. C, Representative NMDA traces. D, E, The selective GluN2B NMDA receptor subunit antagonist Ro-256981 (1 μm) decreased the NMDA decay time constant in the synapse that VP_VGluT2 neurons make on both VP_VGluT2 neurons (average decrease of 47.7 ± 17.4 ms, which represents ∼17.8% decrease) and LHb neurons (average decrease of 6.0 ± 4 ms, which represents 14.6% decrease). F, G, The decay (F) of the AMPA currents was slower in VP_VGluT2 neurons compared with LHb neurons; the rise time was not different between the two cell populations (G). H, Representative AMPA traces. I, J, The membrane input resistance (I) did not differ between VP_VGluT2 and LHb neurons, but the capacitance (J) was significantly higher in LHb neurons. D, E, Paired or unpaired two-tailed Student's t tests were used. n.s. = not significant.

LHb, VP_VGluT2, and VTA_GABA neurons are the most sensitive to VP_VGluT2 input. Recordings were performed as in Figure 1, but VP_VGluT2 neurons were infected with the inhibitory opsin ArchT. Recordings of sEPSCs were performed in the presence of picrotoxin (50 μm) before (4 s) and during (4 s) the activation of ArchT using a 560 nm LED. A–C, Effect in the VP. A, Inhibiting the VP_VGluT2 input significantly decreased the sEPSC frequency but not amplitude in VP_VGluT2 neurons. B, No effect was seen in VP_GABA neurons. C, Representative traces (note the step in outward current in the VP_VGluT2 neuron when ArchT is activated, indicating that the recorded cell was infected with ArchT). D–F, Effect in the VTA. Inhibiting the VP_VGluT2 input did not alter sEPSC frequency in either VTA cell type but significantly decreased the amplitude in VTA_GABA neurons. F, Representative traces. G, Inhibiting VP_VGluT2 input did not affect the frequency or amplitude of sEPSCs in MDT neurons. H, Inhibiting the VP_VGluT2 terminals in the LHb decreased both the frequency and amplitude of sEPSCs in the LHb. I, Representative traces for MDT and LHb recordings. All statistical tests are paired Student's t tests. n.s. = not significant.

Cocaine CPP and abstinence strengthen VP_VGluT2 input to the LHb and VTA_GABA neurons but weaken input to all other targets. A, Left, Timeline of the CPP protocol (from left to right). Mice were habituated to the arena on the first day and then received eight alternating intraperitoneal injections of either cocaine (15 mg/kg) or saline, one injection per day. Control (cocaine-naive) mice received only saline injections. Cocaine was paired with one of the sides, and saline was given on the other side. After conditioning, mice went through 14 d of abstinence from cocaine and then were either tested for preference or used for electrophysiological recordings. Right, Preference for the cocaine-paired side in the cocaine group (C) was significantly higher than zero (one-sample t test, CPP score 0.39 ± 0.26, p = 0.0009) and different from the saline (S) group (p = 0.0019). B–G, Postsynaptic effects. A/N ratios in each target of VP_VGluT2 neurons in saline (full bars) and cocaine-abstinent (open bars) mice. B–F, Cocaine CPP and abstinence decreased the A/N ratio in VP_VGluT2 (B), VP_GABA (C), and VTA_DA (E); generated a nonsignificant decrease in MDT neurons (p = 0.07; F); and did not affect the A/N ratio in VTA_GABA neurons (D). G, In contrast, cocaine CPP and abstinence significantly increased the A/N ratio in the LHb by more than twofold, from 1.55 ± 0.3 to 3.84 ± 1.5. Insets, Representative AMPA (red) and NMDA (blue) currents for each region. NMDA currents normalized between regions to ease comparison. H–L, Presynaptic effects. Cocaine CPP and abstinence decreased the coefficient of variation of evoked EPSCs (CV) and the PPR in both VTA_GABA (H–J) and LHb (K–L) neurons. J, Representative traces for the presynaptic effects of abstinence from cocaine on VP_VGluT2 input to VTA_GABA neurons. All statistical tests are unpaired Student's t tests. The asterisk indicates p < 0.05 when comparing to zero using a one-sample t-test.