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

Abstract

The ventral pallidum (VP) is a central node in the reward system that is strongly implicated in reward and addiction. Although the majority of VP neurons are GABAergic and encode reward, recent studies revealed a novel glutamatergic neuronal population in the VP [VP neurons expressing the vesicular glutamate transporter 2 (VP_VGluT2)], whose activation generates aversion. Withdrawal from drugs has been shown to induce drastic synaptic changes in neuronal populations associated with reward, such as the ventral tegmental area (VTA) or nucleus accumbens neurons, but less is known about cocaine-induced synaptic changes in neurons classically linked with aversion. Here, we demonstrate that VP_VGluT2 neurons contact different targets with different intensities, and that cocaine conditioned place preference (CPP) training followed by abstinence selectively potentiates their synapses on targets that encode aversion. Using whole-cell patch-clamp recordings combined with optogenetics in male and female transgenic mice, we show that VP_VGluT2 neurons preferentially contact aversion-related neurons, such as lateral habenula neurons and VTA GABAergic neurons, with minor input to reward-related neurons, such as VTA dopamine and VP GABA neurons. Moreover, after cocaine CPP and abstinence, the VP_VGluT2 input to the aversion-related structures is potentiated, whereas the input to the reward-related structures is depressed. Thus, cocaine CPP followed by abstinence may allow VP_VGluT2 neurons to recruit aversion-related targets more readily and therefore be part of the mechanism underlying the aversive symptoms seen after withdrawal.SIGNIFICANCE STATEMENT The biggest problem in drug addiction is the high propensity to relapse. One central driver for relapse events is the negative aversive symptoms experienced by addicts during withdrawal. In this work, we propose a possible mechanism for the intensification of aversive feelings after withdrawal that involves the glutamatergic neurons of the ventral pallidum. We show not only that these neurons are most strongly connected to aversive targets, such as the lateral habenula, but also that, after abstinence, their synapses on aversive targets are strengthened, whereas the synapses on other rewarding targets are weakened. These data illustrate how after abstinence from cocaine, aversive pathways change in a manner that may contribute to relapse.

Keywords: Lateral habenula; VGluT2 neurons; Ventral pallidum; cocaine; optogenetics; slice electrophysiology.

Figure 1.

Projection patterns of VP_VGluT2 neurons. A, Schematic representation of the recording setup. An AAV expressing ChR2 in a Cre-dependent manner (AAV2-DIO-ChR2-eYFP) was injected into the VP of crossed VGluT2-IRES-Cre × Ai9 mice. Thus, VP_VGluT2 neurons exclusively expressed ChR2 and tdTomato. Recordings were performed in each of the depicted targets while transmitter release was evoked optogenetically. B, Photomicrographs of injection site in the VP (top), VP_VGluT2 axons in the MDT/LHb (middle), and VTA (bottom). Note the stronger fluorescence in LHb compared with adjacent MDT. ac, Anterior commissure; LPO, lateral preoptic area; MHb, medial habenula; ml, medial lemniscus; PN, paranigral nucleus; SI, substantia innominate; sm, stria medularis of the thalamus. C, Photomicrographs showing tdTomato expression in VP_VGluT2 neurons (top), ChR2-eYFP expression (middle), and the merge (bottom). Only two cells of 141 that expressed ChR2-eYFP did not coexpress tdTomato, indicating ChR2 expression was restricted to VP_VGluT2 neurons (right). D, Proportion of recorded neurons that showed VP_VGluT2 input (of all recorded neurons in that region) in each region/cell type. Gray, no response; color, showed response. VP_VGluT2 neurons inset, Representative evoked postsynaptic glutamatergic currents (Glu; white) and presumed ChR2-mediated currents [in the presence of 10 μm CNQX and 50 μm picrotoxin (green)] recorded from VP_VGluT2 neurons. ChR2-mediated currents were negligible in amplitude.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

Changes in VP_VGluT2 synapses after cocaine CPP and abstinence. A, System level. Drawings are arranged such that synapses on aversive targets are on top (VP_VGluT2, LHb, VTA_GABA) and synapses on rewarding (VP_GABA, VTA_DA) or neutral (MDT) targets are at the bottom. Left, Saline mice. VP_VGluT2 neurons make the strongest synapses on each other and on LHb neurons. Right, After cocaine CPP and abstinence. The synapses of VP_VGluT2 neurons on LHb and VTA_GABA are strengthened, whereas synapses on all rewarding targets are weakened. The synapse on MDT does not seem to change. B, Hypothesized synaptic mechanisms. Top, Saline. Bottom, After cocaine CPP and abstinence. Width of arrows at their bases and ends reflects the strength of the synapse in control and after abstinence, respectively. Saline mice, VP_VGluT2 synapses on each other and on LHb neurons show the highest number of AMPA receptors (based on highest AMPA/NMDA ratios) compared with the inputs to the VTA, MDT, and VP_GABA neurons. Presynaptic parameters (represented in the drawing by the number of vesicles in the terminal) are similar between synapses. After abstinence, synapses on aversive targets (except VP_VGluT2 neurons) are potentiated, either presynaptically (VTA_GABA) or both presynaptically and postsynaptically (LHb). Synapses on other VP_VGluT2 neurons seem to weaken. All synapses on reward targets are depressed through a postsynaptic mechanism (fewer postsynaptic AMPA receptors).