Chemogenetic activation of ventral tegmental area GABA neurons, but not mesoaccumbal GABA terminals, disrupts responding to reward-predictive cues

Abstract

Cues predicting rewards can gain motivational properties and initiate reward-seeking behaviors. Dopamine projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) are critical in regulating cue-motivated responding. Although, approximately one third of mesoaccumbal projection neurons are GABAergic, it is unclear how this population influences motivational processes and cue processing. This is largely due to our inability to pharmacologically probe circuit level contributions of VTA-GABA, which arises from diverse sources, including multiple GABA afferents, interneurons, and projection neurons. Here we used a combinatorial viral vector approach to restrict activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to GABA neurons in the VTA of wild-type rats trained to respond during a distinct audiovisual cue for sucrose. We measured different aspects of motivation for the cue or primary reinforcer, while chemogenetically activating either the VTA-GABA neurons or their projections to the NAc. Activation of VTA-GABA neurons decreased cue-induced responding and accuracy, while increasing latencies to respond to the cue and obtain the reward. Perseverative and spontaneous responses decreased, yet the rats persisted in entering the reward cup when the cue and reward were absent. However, activation of the VTA-GABA terminals in the accumbens had no effect on any of these behaviors. Together, we demonstrate that VTA-GABA neuron activity preferentially attenuates the ability of cues to trigger reward-seeking, while some aspects of the motivation for the reward itself are preserved. Additionally, the dense VTA-GABA projections to the NAc do not influence the motivational salience of the cue.

Fig. 1

Combinatorial adeno-associated virus (AAV) targeting of GABA neurons in the VTA. a Viral constructs used in this study, white and black arrow blocks indicate the lox sites constituting the DIO configuration. b Double immunofluorescence staining for tyrosine hydroxylase (TH; red) and channelrhodopsin-2-enhanced yellow fluorescent protein (ChR2-EYFP; green) in the VTA, scale bar indicates 20 µm, and (c) proportion of dopamine (DA) and non-DA neurons expressing ChR2 (406 neurons counted, n = 4). d ChR2-EYFP terminals in the NAc, scale bar indicates 20 µm. e Illustrates optical post synaptic current (oPSC) induced by photostimulation of ChR2 in the VTA and recorded from nearby putative DA neuron voltage clamped at −90 mV, −58 mV, and 0 mV (n = 4, mean of 10 trials at 0.05 Hz). The GABA_A antagonist picrotoxin (100 µM) decreases the amplitude of oPSCs. f oPSCs reverse polarity at E_cl (i.e., −58 mV). g Representative double fluorescent image of GAD1 targeted hM3D (top), TH (middle) and merged (bottom) of the left and right VTA, scale bar indicates 100 µm. h Double immunofluorescence staining for tyrosine hydroxylase (TH; cyan) and hM3D-mCherry (magenta) in the VTA, scale bar indicates 20 µm. i Illustrates representative recording traces from spontaneously firing DA neurons in a GFP sham rat (top) and a GAD1 directed hM3D rat (bottom), respectively. At 1 μM, CNO had no effects on the spontaneous firing of a DA neuron recorded from a GFP sham rat. In contrast, CNO at 0.1 μM completely inhibited the spontaneous firing of a DA neuron recorded from a GAD1 directed hM3D rat. j Diagram of events for a successful response during the IC task denoting the rat snout (gray cone) responding to the IC (A), by nosepoking (B), followed by a reward cup entry (C)

Fig. 2

Activation of VTA-GABA neurons regulate incentive cue (IC) task performance. All data are shown as mean ± SEM. a Response ratio and accuracy significantly decreased, and latency to nosepoke and enter reward cup significantly increased after systemic CNO in rats expressing hM3D compared to saline and rats expressing GFP. b Intra-NAc injections of CNO had no effect on IC performance across the behavioral metrics, in either hM3D or GFP expressing rats. Asterisks represent significant differences determined by a Holms-Sidak post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

VTA-GABA neuron mediated decreases in incentive cue (IC) task performance are not mediated by inappropriate nosepoking. All data are presented as mean ± SEM. a Systemic CNO significantly decreased the response ratio and accuracy throughout the 1-h session when compared to saline control while the latency to nosepoke after the presentation of an IC and enter the reward cup significantly increased. b The total number of rewarded and unrewarded nosepokes during a session after systemic and intra-NAc CNO administration. The number of rewarded nosepokes significantly decreased after systemic but not intra-NAc CNO pretreatment compared to saline and GFP controls. For clarity, we report all significant differences in (a) as #p < 0.0001. Asterisks represent significant differences between saline and CNO determined by a Holms-Sidak post hoc test in all other panels (***p < 0.001)

Fig. 4

VTA-GABA neuron activation disrupts instrumental performance while retaining some reward-seeking behaviors. All data are presented as mean ± SEM. a Systemic but not intra-NAc CNO pretreatment significantly reduced reward cup entries during a session compared to saline control (top left), and systemic CNO pretreated rats achieved significantly less rewards than controls (top middle) so that reward cup entries per reward significantly increased after systemic CNO compared to saline (top right). There were no differences between intra-NAc pretreatments (bottom). b Systemic but not intra-NAc increased the mean first rewarded IC at the start of the session, determined by a planned two-tailed Wilcoxon matched-pairs signed-rank test. Note, in some sessions all rats responded to the first IC, thus some bars have no error bars. Far right panel indicate the location of the tip of the cannula in rats used for intra-NAc infusion experiments. c Systemic CNO pretreatment disrupted the appropriate behavioral response pattern to an IC. The number of initial rewarded action sequences after the presentation of the IC (left). Systemic CNO significantly decreased the number of IC (A₊)→nosepoke (B)→reward cup (C) sequences. Conversely, CNO had no effect on the IC→reward cup→nosepoke→reward cup response. The number of unrewarded responses sequences after the IC (right) show that CNO significantly increased the number of reward cup entries during an IC presentation (i.e., bypassing a nosepoke: IC (A₊)→reward cup (C)), the number of reward cup entries when the IC was not present (end of IC (A₀)→reward cup (C)) or when there was no detectable behavioral response to the IC (A₀). Significant difference between treatment groups determined by a Holms-Sidak post hoc test. Asterisks represent significant differences in the statistical tests indicated (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

VTA-GABA neuron activation does not impair sucrose consumption or locomotor behavior, and clozapine (CZP) has no effect on the incentive cue (IC) task. Data presented as mean ± SEM. a Total volume of sucrose (ml) during the first 2 min of a free-drinking session is equivalent to that obtained during an entire IC session; however, CNO decreased only the total volume consumed only during the IC task (t₁₆ = 10.97, p < 0.0001) and not free-drinking. b Distance traveled (normalized as a percent of saline control) during a 20-min open-field locomotor test did not statistically differ between saline and CNO pretreatment. c Rats injected with a GFP sham virus into the VTA did not show a behavioral effect after systemic pretreatment with 0.3 mg/kg i.p. CZP across IC task metrics. Asterisks represent significant differences (****p < 0.0001)