Distinct dynamics and intrinsic properties in ventral tegmental area populations mediate reward association and motivation

Abstract

Ventral tegmental area (VTA) dopamine neurons regulate reward-related associative learning and reward-driven motivated behaviors, but how these processes are coordinated by distinct VTA neuronal subpopulations remains unresolved. Here, we compare the contribution of two primarily dopaminergic and largely non-overlapping VTA subpopulations, all VTA dopamine neurons and VTA GABAergic neurons of the mouse midbrain, to these processes. We find that the dopamine subpopulation that projects to the nucleus accumbens (NAc) core preferentially encodes reward-predictive cues and prediction errors. In contrast, the subpopulation that projects to the NAc shell preferentially encodes goal-directed actions and relative reward anticipation. VTA GABA neuron activity strongly contrasts VTA dopamine population activity and preferentially encodes reward outcome and retrieval. Electrophysiology, targeted optogenetics, and whole-brain input mapping reveal multiple convergent sources that contribute to the heterogeneity among VTA dopamine subpopulations that likely underlies their distinct encoding of reward-related associations and motivation that defines their functions in these contexts.

Keywords: CP: Neuroscience; GABA; dopamine; learning; motivation; reward; subpopulations.

Figure 1.. Fiber photometry during cued reinstatement

(A) Schematic of a fiber photometry session. (B) Schematic depicting training and performance of mice (n = 57 mice; solid lines indicate mean across mice, and gray lines indicate individual replicates). (C) Example recording trace during acquisition showing GCaMP fluorescence (top) aligned to event timestamps (bottom). (D) Schematic of viral injection and optic fiber implant and example histology image from the VTA showing GCaMP6 (green) in the DAT_VTA group. Scale bar: 500 μm. (E) Z scored GCaMP fluorescence from DAT_VTA population recordings aligned to task events (n = 9 mice, 63 sessions). Data are from all trials during the first, third, and last acquisition and extinction training sessions and from all trials during the reinstatement session. (F) Average Z scored GCaMP fluorescence from DAT_VTA population recordings during LP, CS, reward, port entry, and omission periods (n = 9 mice, 63 sessions, bars and error bars indicate mean ± SEM across mice; see Table S1 for statistical values). (G) Same as in (D) but for Cck_VTA population recordings. Scale bar: 500 μm. (H) Same as in (E) but for Cck_VTA population recordings (n = 16 mice, 112 sessions). (I) Same as in (F) but for Cck_VTA population recordings (n = 16 mice, 112 sessions). (J) Same as in (D) but for Crhr1_VTA population recordings. Scale bar: 500 μm. (K) Same as in (E) but for Crhr1_VTA population recordings (n = 13 mice, 91 sessions). (L) Same as in (F) but for Crhr1_VTA population recordings (n = 13 mice, 91 sessions). (M) Same as in (D) but for Vgat_VTA population recordings. Scale bar: 500 μm. (N) Same as in (E) but for Vgat_VTA population recordings (n = 8 mice, 56 sessions). (O) Same as in (F) but for Vgat_VTA population recordings (n = 8 mice, 56 sessions). (P) Average latency to peak of Z scored GCaMP fluorescence following reward delivery during acquisition for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. (Q) Average Z scored GCaMP fluorescence during CS presentation period following reward delivery during acquisition for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. (R) Average Z scored GCaMP fluorescence during port entry period during extinction phase of cued reinstatement for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. (S) Average Z scored GCaMP fluorescence during CS presentation periods during reinstatement presession for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. (T) Average Z scored GCaMP fluorescence during CS presentation periods during reinstatement session for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13), and Vgat_VTA (n = 8 mice) groups. (U) Z scored GCaMP fluorescence aligned to reward omission period during reinstatement phase in DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. Dotted rectangle indicates mean Z score analysis epoch. (V) Average latency to minimum GCaMP fluorescence during reward omission period during reinstatement for DAT_VTA (n = 9 mice), Cck_VTA (n = 16 mice), Crhr1_VTA (n = 13 mice), and Vgat_VTA (n = 8 mice) groups. Bars and error bars indicate mean ± SEM across mice (see Table S1 for statistical values).

Figure 2.. Photostimulation and photoinhibition of Crhr1_VTA and Cck_VTA neurons during cued reinstatement

(A) Schematic of viral injection and optic fiber implant and example histology image from the VTA showing staining for ChR2-YFP (green) in VTA subpopulations. (B) Schematic of the training phases of the optogenetic cued reinstatement task. (C) Schematic of an optogenetic cued reinstatement session. (D) Mean number of lever presses across mice during acquisition and extinction sessions in control, Cck_VTA, and Crhr1_VTA groups (n = 11–21 mice, error bars represent SEM). (E) Mean number of cumulative lever presses on day 14 (Stim + CS) with optogenetic activation or in control mice without opsin expression (n = 11–21 mice, error bars indicate SEM). (F) Mean number of cumulative trials completed on day 14 (Stim + CS) with optogenetic activation or in control mice without opsin expression (n = 11–21 mice, error bars indicate SEM). (G) Schematic of viral injection and optic fiber implant and example histology image from the VTA showing staining for JAWS-GFP (green) in VTA subpopulations. (H) Schematic of the training phases of the opto-genetic cued reinstatement task. (I) Schematic of an optogenetic cued reinstatement session. (J) Mean number of lever presses on day 12 (extinction [Ext] D12), and day 13 (Light + CS) with optogenetic inhibition or in control mice without opsin expression (n = 12–14 mice, error bars indicate SEM). (K) Mean number of cumulative lever presses on day 13 (Light + CS) with optogenetic inhibition or in control mice without opsin expression (n = 12–14 mice, error bars indicate SEM). (L) Mean number of cumulative trials completed on day 13 (Light + CS) with optogenetic inhibition or in control mice without opsin expression (n = 12–14 mice, error bars indicate SEM; see Table S1 for statistical values).

Figure 3.. Time-locked activation of VTA subpopulations during random reward omission

(A) Schematic of the random reward omission task; probability of reward was 50%. (B) Example behavioral session showing lever press, reward, and omission times. (C) Trial initiation latency following the intertrial interval period (n = 25 mice, bars and error bars indicate mean ± SEM across mice). (D) Z scored GCaMP fluorescence and heatmaps aligned to reward (color) or reward omission (gray) task events from the DAT_VTA group (n = 7 mice). (E) Average Z scored GCaMP fluorescence during reward, omission, and port entry periods for the DAT_VTA (n = 7 mice) group. (F) Same as in (D) but for the Cck_VTA (n = 12 mice) group. (G) Same as in (E) but for the Cck_VTA (n = 12 mice) group. (H) Same as in (D) but for the Crhr1_VTA (n = 13 mice) group. (I) Same as in (E) but for the Crhr1_VTA (n = 13 mice) group. (J) Same as in (D) but for the Vgat_VTA (n = 8 mice) group. (K) Same as in (E) but for the Vgat_VTA (n = 8 mice) group. (L) Z scored GCaMP fluorescence (left) and average Z scored GCaMP fluorescence (right) during reward omission periods shaded according to previous trial outcome type for the DAT_VTA group (n = 7 mice). (M) Same as in (L) but for the Cck_VTA group (n = 12 mice). (N) Same as in (L) but for the Crhr1_VTA group (n = 13 mice). (O) Same as in (L) but for the Vgat_VTA group (n = 8 mice). (P) Schematic of outcome history regression model approach. The current previous five trial outcomes were used with a multiple linear regression to predict the GCaMP signal during the current trial outcome. (Q) Average regression coefficients across mice for the outcome history linear regression for DAT_VTA (n = 7 mice), Cck_VTA neurons (left) (n = 12 mice), and Crhr1_VTA neurons (right) (n = 13 mice; see Table S1 for statistical values). (R) Same as in (Q) but for the Cck_VTA group (n = 12 mice). (S) Same as in (Q) but for the Crhr1_VTA group (n = 13 mice).

Figure 4.. Differential encoding of task-relevant behavioral variables during random reward omission

(A) Schematic of the linear encoding model. Task event timestamps were convolved with a set of cubic splines to generate a predictor set of ten behavior event types. The GCaMP signal was predicted based on task events. (B) Example observed (gray) and predicted (color) GCaMP traces from Cck_VTA (top), Crhr1_VTA (middle), and Vgat_VTA (bottom) groups. (C) Relative contribution of each task event type to the explained variance of the GCaMP signal during the action cue period averaged across mice for the Cck_VTA group (n = 12 mice) (see Table S1 for statistical values). (D) Same as in (C) but for the Crhr1_VTA group (n = 11 mice). (E) Same as in (C) but for the Vgat_VTA group (n = 8 mice). (F) Relative contribution of each task event type to the explained variance of the GCaMP signal during the trial outcome period averaged across mice for the Cck_VTA group (n = 12 mice). (G) Same as in (F) but for the Crhr1_VTA group (n = 11 mice). (H) Same as in (F) but for the Vgat_VTA group (n = 8 mice). (I) Relative contribution of trial lever press during action cue period average across mice for Cck_VTA (n = 12 mice), Crhr1_VTA (n = 11 mice), and Vgat_VTA (n = 8 mice) groups (see Table S1 for statistical values). (J) Relative contribution of the cue during action cue period average across mice for Cck_VTA (n = 12 mice), Crhr1_VTA (n = 11 mice), and Vgat_VTA (n = 8 mice) groups (see Table S1 for statistical values). (K) Relative contribution of reward during trial outcome period average across mice for Cck_VTA (n = 12 mice), Crhr1_VTA (n = 11 mice), and Vgat_VTA (n = 8 mice) groups (see Table S1 for statistical values). (L) Relative contribution of reward entry during trial outcome period average across mice for Cck_VTA (n = 12 mice), Crhr1_VTA (n = 11 mice), and Vgat_VTA (n = 8 mice) groups (see Table S1 for statistical values).

Figure 5.. Differential encoding of motivated responses

Schematic of the progressive ratio (PR) task. (A) Cumulative lever presses (blue) and rewards (red) during PR sessions. Lines indicate individual mice (n = 42 mice). (B) Lever press (blue) and reward (red) event times shown for all sessions (n = 42 mice). (C) Z scored GCaMP fluorescence from photometry recordings aligned to lever press bout onset, port entry bout onset, and reward retrieval for Cck_VTA group (n = 17 mice). (D) Average Z scored GCaMP fluorescence during lever press bout onset, port entry bout onset, and reward retrieval periods for the Cck_VTA group (n = 17 mice). Bars and error bars indicate mean ± SEM across mice. (E) Same as in (D) but for the Crhr1_VTA group (n = 17 mice). (F) Same as in (E) but for the Crhr1_VTA group (n = 17 mice). (G) Same as in (D) but for the Vgat_VTA group (n = 8 mice). (H) Same as in (E) but for the Vgat_VTA group (n = 8 mice). (I) Z scored GCaMP fluorescence from recordings aligned to lever press bout onset and separated by bouts occurring prior to (lighter shade) and after (darker shade) 50% of all completed reinforcement ratios for the Cck_VTA group (n = 17 mice). (J) Same as in (J) but for the Crhr1_VTA group (n = 17 mice). (K) Same as in (J) but for the Vgat_VTA group (n = 8 mice). (L) Correlations across bouts between percentage of breakpoint and mean Z scored GCaMP signal during bout onset for the Cck_VTA group (n = 165 bouts). Correlation coefficient (r) and p values are shown on the top right of the plot. (M) Same as in (M) but for the Crhr1_VTA group (n = 172 bouts). (N) Same as in (M) but for the Vgat_VTA group (n = 172 bouts). (O) Pearson’s correlation coefficient per mouse between percentage of breakpoint and mean Z scored GCaMP signal for Cck_VTA (n = 17 mice), Crhr1_VTA (n = 17 mice), and Vgat_VTA (n = 8 mice) groups (see Table S1 for statistical values).

Figure 6.. Properties and connectivity of Crhr1_VTA and Cck_VTA neurons

(A) Schematic of the viral injection strategy and ex vivo electrophysiology recordings. (B) Representative evoked excitability traces (90 pA current injection). (C) Current-voltage plot for Crhr1_VTA and Cck_VTA neurons (n = 13–15 cells). Bars and error bars indicate mean ± SEM across cells. (D) Mean spike latency following current injection for Crhr1_VTA and Cck_VTA neurons (n = 13–15). Bars and error bars indicate mean ± SEM across cells. (E) Representative spontaneous inhibitory postsynaptic current (sIPSC) traces from Crhr1_VTA or Cck_VTA neurons. (F) Mean sIPSC frequency for Crhr1_VTA and Cck_VTA neurons (n = 13–15 cells). (G) Mean sIPSC amplitude for Crhr1_VTA and Cck_VTA neurons (n = 13–15 cells). (H) Representative traces of rebound spiking from Crhr1_VTA or Cck_VTA neurons following injection of a −120 pA hyperpolarizing current. (I) Mean time to first spike following hyperpolarization for Crhr1_VTA and Cck_VTA neurons (n = 5–6 cell). (J) Mean ramp slope prior to first spike following hyperpolarization for Crhr1_VTA and Cck_VTA neurons (n = 5–6 cells; see Table S1 for statistical values). (K) Schematic of viral injection strategy for Flp-dependent Chrimson and Cre-dependent GCaMP into the VTA with optical fiber implanted above VTA. Scale bar: 500 μm. (L) Z scored GCaMP fluorescence aligned to Vgat_VTA stimulation in Cck_VTA and Crhr1_VTA groups. (M) Average Z scored GCaMP fluorescence during stimulation and post-stimulation periods from (L). Bars and error bars indicate mean ± SEM across mice. (N) Schematic of viral injection strategy for expression of Flp-dependent Chrimson into the LH and Cre-dependent GCaMP into the VTA. Stimulation and recording fibers were implanted above the LH and VTA, respectively. Scale bar: 500 μm. (O) Z scored GCaMP fluorescence aligned to LH GABA stimulation in Cck_VTA and Crhr1_VTA groups. (P) Average Z scored GCaMP fluorescence during stimulation and post-stimulation periods from (O) for Cck_VTA (n = 5 mice, 10 sessions) and Crhr1_VTA (n = 3 mice, 6 sessions) groups (bottom). Bars and error bars indicate mean ± SEM across mice. (Q) Schematic of viral injection strategy for expression of Flp-dependent Chrimson into the NAc mshell and Cre-dependent GCaMP into the VTA. Stimulation and recording fibers were implanted above the NAc mshell and VTA, respectively. Scale bar: 500 μm. (R) Z scored GCaMP fluorescence aligned to NAc shell stimulation in Cck_VTA and Crhr1_VTA groups. (S) Average Z scored GCaMP fluorescence during stimulation and post-stimulation periods from (R) for Cck_VTA (n = 3 mice, 6 sessions) and Crhr1_VTA (n = 3 mice, 6 sessions) groups (bottom). Bars and error bars indicate mean ± SEM across mice (see Table S1 for statistical values).

Figure 7.. Whole-brain mapping of inputs to Crhr1_VTA and Cck_VTA neurons

(A) Schematic of viral injection strategy, whole-brain clearing, and light sheet fluorescence microscopy (LSFM). Cre-dependent helper virus (AAV-syn-DIO-TC66T-2A-EGFP-2A-oG) was injected, and 2 weeks later, rabies virus (EnvA-SADDG-RV-dsRed) was injected into the VTA. Nine days later, intact brains were cleared and imaged. (B) Location of input cells to Cck_VTA and Crhr1_VTA neurons in example mice. (C) Voxelized heatmap of input cell density in coronal sections across the whole brain. Mean density of dsRed-positive cells per mm³ across mice (left). Voxelized results of group one-way ANOVA pairwise comparison (right) (n = 3 mice). Scale bar: 2 mm. (D) Mean number of input cells normalized to total number of input cells for all input regions (n = 3 mice/group). (E) Heatmap of group pairwise comparison one-way ANOVA results for clustered input regions (see Table S1 for statistical values).