Enhanced sucrose and cocaine self-administration and cue-induced drug seeking after loss of VGLUT2 in midbrain dopamine neurons in mice

Abstract

The mesostriatal dopamine (DA) system contributes to several aspects of responses to rewarding substances and is implicated in conditions such as drug addiction and eating disorders. A subset of DA neurons has been shown to express the type 2 Vesicular glutamate transporter (Vglut2) and may therefore corelease glutamate. In the present study, we analyzed mice with a conditional deletion of Vglut2 in DA neurons (Vglut2(f/f;DAT-Cre)) to address the functional significance of the glutamate-DA cophenotype for responses to cocaine and food reinforcement. Biochemical parameters of striatal DA function were also examined by using DA receptor autoradiography, immediate-early gene quantitative in situ hybridization after cocaine challenge, and DA-selective in vivo chronoamperometry. Mice in which Vglut2 expression had been abrogated in DA neurons displayed enhanced operant self-administration of both high-sucrose food and intravenous cocaine. Furthermore, cocaine seeking maintained by drug-paired cues was increased by 76%, showing that reward-dependent plasticity is perturbed in these mice. In addition, several lines of evidence suggest that adaptive changes occurred in both the ventral and dorsal striatum in the absence of VGLUT2: DA receptor binding was increased, and basal mRNA levels of the DA-induced early genes Nur77 and c-fos were elevated as after cocaine induction. Furthermore, in vivo challenge of the DA system by potassium-evoked depolarization revealed less DA release in both striatal areas. This study demonstrates that absence of VGLUT2 in DA neurons leads to perturbations of reward consumption as well as reward-associated memory, features of particular relevance for addictive-like behavior.

Figure 1.

Self-administration of sucrose is elevated in cKO mice. A, The threshold for sucrose preference was lower in cKO than in Ctrl mice (3% sucrose for cKO and 10% sucrose for Ctrl mice). Significant preference in the cKO group is denoted by ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.0001. Significant preference in the Ctrl mice is denoted by ^¤¤¤p < 0.0001. Ctrl, n = 9; cKO, n = 10. B, Food-restricted cKO mice self-administer more sucrose reinforcers than do controls (controls, n = 6; cKO, n = 5). Max, Maximum. C, Hunger-motivated responding, i.e., self-administration of low-sucrose (grain-based) food pellets on an FR5 schedule during food restriction. D, Refeeding after food restriction does not differ between genotypes. E, Palatability-motivated feeding, i.e., %kcal from high-sucrose pellets during ad libitum (nonrestricted) conditions. In C–E, n = 9 controls and n = 8 cKO were used. Group data represent mean ± SEM. *p < 0.05 versus controls.

Figure 2.

Elevated operant responding for low-dose cocaine and drug-paired cues during extinction in cKO mice. A, Nosepoke response of food-trained mice implanted with indwelling intravenous catheters and allowed to nosepoke for cocaine infusions (controls, n = 7; cKO, n = 7). B, Total dose self-administered at the different cocaine concentrations in A. C, Cocaine-seeking, i.e., responding for light and sound cues previously associated with cocaine in the absence of the drug, of mice from A that had been responding for 0.75 mg/kg per infusion (inf) and subsequently subjected to 21 d of forced cocaine abstinence (controls, n = 6; cKO, n = 5). Group data represent mean ± SEM. *p < 0.05; **p < 0.01 versus Ctrl.

Figure 3.

Dopamine receptor binding is altered in cKO mice. A, D₁R binding sites in cKO (n = 6) and control (n = 9) mice were measured by auroradiography using [³H]SCH-23390. B, D₂R binding sites were measured by autoradiography using [³H]raclopride binding on sections adjacent to those used in A. DA receptor binding site levels were evaluated in the AcSh and AcC and in striatal subterritories (StDM, StDL, StVM, StVL) in Ctrl and cKO mice. Group data represent mean ± SEM. *p < 0.05 and **p < 0.01 versus Ctrl.

Figure 4.

High expression of Nur77 under baseline conditions in cKO mice. A–D, Quantitative in situ hybridization of Nur77 mRNA levels in Ctrl and cKO mice treated with saline (Ctrl, n = 6; cKO, n = 7) or cocaine (Ctrl, n = 5; cKO, n = 4). A, Levels of Nur77 mRNA in AcSh and AcC (bregma, 1.70 mm). B, Nur77 mRNA levels in medial (StM) and lateral (StL) rostral striatum (bregma, 1.70 mm). C, Nur77 transcript levels in the caudodorsal striatum (bregma, 0.48 mm). D, Nur77 levels in the caudoventral striatum (bregma, 0.48). E, F, Representative autoradiograms of Nur77 mRNA in situ hybridization signals in the rostral striatal area (E; bregma, 1.70 mm) and in the caudal striatal area (F; bregma, 0.48 mm) with schematic illustrations to the right showing location of the analyzed regions. Group data represent mean ± SEM expressed as percentage of Ctrl (saline). *p < 0.05, **p < 0.01, and ***p < 0.001 versus Ctrl saline group; ^#p < 0.05 versus Ctrl cocaine group. cc, Corpus callosum.

Figure 5.

Decreased DA release in ventral and dorsal striatum of cKO mice. A, Graphical illustration of placement of DA-selective electrode for in vivo amperometric measurements in DStr and AcbC (bregma, 1.10 mm). B, Representative DA trace illustrating the calculation of redox ratio for each peak. C, G, DA amplitude, defined as the peak DA concentration (micromoles) from baseline in AcbC and DStr in response to four consecutive ejections of 120 mm KCl. D, H, t_rise, defined as time (seconds) between ejection and peak maximum (F, J), in AcbC and DStr for the four releases of DA. E, I, t_80, defined as the time (seconds) from peak amplitude until the signal reached 20% of the peak amplitude (F, J), in AcbC and DStr for the four KCl-evoked releases of DA. F, J, Schematic illustrations of the first post-KCl ejection recordings of DA, based on graph values, for AcbC (F) and DStr (J). Insets show analysis of peak area, defined as area under curve. Four consecutive KCl ejections (numbered 1–4 on x-axes) were used, spaced by 5 min for DStr and 10 min for AcbC. AcbC data are from n = 7 Ctrl and n = 7 cKO mice. DStr data are from n = 11 controls and n = 12 cKO mice. Group data represent mean ± SEM. Significant main genotype effects observed by ANOVA analysis are illustrated by ^##p < 0.01 and ^###p < 0.001; differences by post hoc test are shown by *p < 0.05, **p < 0.01, and ***p < 0.001.