Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

Abstract

Ventral tegmental area (VTA) dopamine (DA) neurons perform diverse functions in motivation and cognition, but their precise roles in addiction-related behaviors are still debated. Here, we targeted VTA DA neurons for bidirectional chemogenetic modulation during specific tests of cocaine reinforcement, demand, and relapse-related behaviors in male rats, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of G_q signaling, but not G_s signaling, in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory G_i/o signaling in DA neurons blunted the reinforcing and priming effects of cocaine, reduced stress-potentiated reinstatement, and altered behavioral strategies for cocaine seeking and taking. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein-signaling cascades, and DA neuron efferent targets, highlighting their multifaceted roles in addiction.SIGNIFICANCE STATEMENT G-protein-coupled receptors are crucial modulators of ventral tegmental area (VTA) dopamine neuron activity, but how this metabotropic signaling impacts the complex roles of dopamine in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs (designer receptors exclusively activated by designer drugs) during a variety of cocaine-seeking behaviors, revealing nuanced, pathway-specific roles in cocaine reward, effortful seeking, and relapse-like behaviors. G_q and G_s stimulation activated dopamine neurons, but only G_q stimulation robustly enhanced cocaine seeking. G_i/o inhibitory signaling reduced some, but not all, types of cocaine seeking. Results show that VTA dopamine neurons modulate numerous distinct aspects of cocaine addiction- and relapse-related behaviors, and point to potential new approaches for intervening in these processes to treat addiction.

Keywords: DREADDs; addiction; conditioned cues; motivation; neural circuits; reinstatement.

Figure 1.

Behavioral testing timeline: following surgery and recovery, G_q-, G_s-, G_i-DREADD rats and WT controls were trained to self-administer cocaine plus a tone/light cue over 10 daily 2 h sessions, then extinguished over more than seven additional sessions. In a repeated-measures design, we examined the effects of counterbalanced injections of CNO (0, 1, 10 mg/kg) on cocaine seeking in a CNO-induced reinstatement test (no cues/cocaine), during reinstatement elicited by cocaine cues, cocaine priming injection, or yohimbine (YOH) plus cocaine cues, on a test of cocaine behavioral economic (BE) demand, and on locomotor activity in a habituated environment.

Figure 2.

DREADD expression and function in VTA DA Neurons. a, Midbrain TH staining (green) and mCherry-tagged DREADD expression (red) in VTA is shown, with yellow indicating colabeling. Inset shows schematic of AAV DIO-DREADD vectors used. b, Percentage of midbrain TH+ cells located within borders of SN (white bars), or VTA (black bars) that coexpress mCherry-tagged DREADDs. Individual rat data shown with red diamond (G_q), yellow triangle (G_s), or blue square (G_i) symbols. c, Percentage of TH+ VTA neurons expressing Fos after Veh or CNO (10 mg/kg) in G_q-, G_s-, or G_i-coupled DREADD rats (individual rat data represented with symbols on top of bars). d, Firing rates are depicted for baseline and post-CNO periods, and individual cells are depicted with lines. e, CNO increases the mean (SEM) firing rate of VTA DREADD+ neurons in vitro, relative to baseline firing. f, In anesthetized rats with G_q DREADDs, more type 1 cells were encountered per electrophysiological recording track after 10 mg/kg CNO injection (colored bars, symbols) than before CNO injection (white bars, symbols). No significant effects on population activity were observed after CNO injection in the G_s, G_i, or mCherry groups. g, Interspike interval before CNO injection (black lines, error bars indicate the SEM) and after 10 mg/kg, i.p., CNO injection (red, G_q; yellow, G_s; blue, G_i; black, mCherry). *p < 0.05.

Figure 3.

Reinstatement of cocaine seeking is modulated by chemogenetic DA neuron manipulations. a, In G_q-DREADD rats, CNO (1 mg/kg, gray bars; 10 mg/kg, black bars) increased cocaine seeking (relative to vehicle, white bars) in the absence (CNO-induced reinstatement) or the presence of cues, and after a cocaine prime (no cues) or yohimbine (YOH + cues) test (10 mg/kg). b, CNO failed to increase reinstatement in G_s-DREADD rats. c, In G_i-DREADD rats, CNO reduced the priming effects of cocaine and the potentiation of cued responding by YOH (10 mg/kg). d, No effects of CNO were observed in WT rats with no DREADD expression. Light gray bars indicate inactive lever presses for each test. *p < 0.05 e, f, Effects of CNO (0, 1, 10 mg/kg) on horizontal locomotion (e) and rearing behavior is shown for DREADD-expressing and WT animals (f). G_q stimulation increased and G_i stimulation decreased locomotor activity in a familiar environment. Individual rat data are shown in Figure 3-1, and locomotion time course is shown in Figure 3-2.

Figure 4.

Time course of cue-induced reinstatement. a–d, Time course of active lever (solid lines) and inactive lever (dashed lines) are shown for G_q (a), G_s (b), G_i DREADD (c), and WT control (d) rats after vehicle or CNO administration (1, 10 mg/kg). Data are shown in 30 min bins throughout the 120 min session. Lines represent the mean, and error bars represent the SEM. *p < 0.05, interaction of dose × time.

Figure 5.

DA neuron modulation affects low-effort cocaine intake and demand elasticity. a, Effects of CNO (0, 1, or 10 mg/kg) in G_q, G_s, and G_i DREADD rats, or WT rats on free cocaine intake (Q₀) are depicted. b, Effects of CNO on demand elasticity (α) are depicted. When VTA DA neurons were stimulated, rats preferred less cocaine but worked harder to obtain this lower dose. When DA neurons were inhibited, subjects preferred more cocaine but were less willing to work hard to obtain it. c, No effects of CNO were found on control (inactive) lever responding during economic demand tests. *p < 0.05. Individual rat data are shown in Figure 5-1.

Figure 6.

DREADDs do not affect behavior in the absence of CNO. No significant effects of DREADD expression are observed in the absence of CNO. a–d, Rats with DREADDs (G_q, red; G_s, yellow; G_i, blue) or no DREADDs (WT controls, black) did not differ in the total number of milligrams of cocaine self-administered throughout training (a), the number of daily cocaine infusions across the 10 d training period (b), active lever pressing across the first 7 d of extinction training (c), or active lever pressing on reinstatement (d). Bars and lines represent the mean, and error bars represent the SEM.

Figure 7.

CNO does not have persistent effects on behavior. a–d, No evidence for long-lasting effects of CNO in G_q-DREADD (a), G_s-DREADD (b), G_i-DREADD (c), or WT control (d) rats were found, as demonstrated with active lever presses on the extinction retraining trial held 24 h after the injection of vehicle (white bars), 1 mg/kg CNO (light red, yellow, blue, black bars), or 10 mg/kg CNO (dark color bars). e, f, Similarly, no persistent effects of prior-day CNO were observed on demand elasticity (α; e) or free cocaine intake (Q₀; f). Bars represent the percentage of preinjection baseline on day following administration of vehicle, or 1 or 10 mg/kg CNO.

Figure 8.

Chemogenetic pathway-specific stimulation of VTA DA terminals. a, In acute coronal NAc slices from rats with G_q DREADDs in VTA DA neurons, DA release was evoked with local electrical stimulation (single pulse, 20 or 40 Hz) before or after bath application of CNO (1 μm), measured using cyclic voltammetry. Bars depict the percentage of baseline (pre-CNO) DA release after CNO administration in G_q-, G_s-, or G_i- DREADD rats, or WT rats. CNO facilitated DA release in G_q-DREADD rats at all stimulation parameters and decreased DA release in G_i-DREADD rats. No effects of CNO were seen in G_s-DREADD or WT rats. b, c, G_q DREADDs are robustly transported to targets including NAc (b) and mPFC (c). d, axon terminals of VTA TH(+) neurons, labeled with mCherry (red) colocalized with tyrosine hydroxylase (TH; green) to a greater extent in NAc compared with mPFC. e–l, Effects of CNO microinjection into NAc of WT rats without DREADDs (e, i), NAc of G_q DREADD rats (f, j), mPFC of G_q rats (g, k), or BLA of G_q rats (h, l) on reinstatement with (right bars) or without cues (left bars; e–h), or on 2 h chow intake in a separate test with the same rats (i–l). *p < 0.05, **p < 0.01. m–p, Microinjection sites are shown for WT (m), NAc G_q (n), mPFC G_q (o), and BLA G_q (p) animals. Individual rat data are shown in Figure 8-1.