Ventral tegmental area GABAergic inhibition of cholinergic interneurons in the ventral nucleus accumbens shell promotes reward reinforcement

Abstract

The long-range GABAergic input from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is relatively understudied, and therefore its role in reward processing has remained unknown. In the present study, we show, in both male and female mice, that long-range GABAergic projections from the VTA to the ventral NAc shell, but not to the dorsal NAc shell or NAc core, are engaged in reward and reinforcement behavior. We show that this GABAergic projection exclusively synapses on to cholinergic interneurons (CINs) in the ventral NAc shell, thereby serving a specialized function in modulating reinforced reward behavior through the inhibition of ventral NAc shell CINs. These findings highlight the diversity in the structural and functional topography of VTA GABAergic projections, and their neuromodulatory interactions across the dorsoventral gradient of the NAc shell. They also further our understanding of neuronal circuits that are directly implicated in neuropsychiatric conditions such as depression and addiction.

Figure 1.. VTA GABAergic terminals in the vNAcSh are preferentially engaged during reward reinforcement.

(a) Coronal brain schematic of viral injection of GCaMP6s into VTA and fiber expression in the NAc of VGAT-Cre+ mice (right panel – top), and representative spread of expression in the dNAcSh or vNAcSh (right panel – bottom). Representative 20X coronal image (left panel) showing expression of GCaMP6s (green), DAPI (blue) and fiber placement in dNAcSh (top; scalebar – 200 μm) and vNAcSh (bottom; scalebar – 200 μm). (b) Schematic of fiber photometry setup connected to mouse performing pavlovian task. (c) Pavlovian training schedule with photometry recordings, as indicated. (d) Latency to eat following reward delivery (0–6s) for Pavlovian training. Top – vNAcSh (n = 7 mice; P1 vs. P5 paired t test p=0.0163, t=3.308, df=6). Bottom – dNAcSh (n = 5 mice; P1 vs. P5 paired t test p=0.0268, t=2.709, df=4). Data represented as mean +/− SEM. (e) Representative recorded GCaMP activity in the vNAcSh (top) or dNAcSh (bottom) during pavlovian training (highlights represent cue light during reward period). (f) Peak fluorescence during cue period (0–6s) for Pavlovian training. Top – vNAcSh (n = 7 mice; P1 vs. P5 paired t test p=0.8437, t=0.19175, df=135). Bottom – dNAcSh (n = 5 mice; P1 vs. P5 paired t test p<0.0001, t=4.067, df=97). Data represented as mean +/− SEM. (g) Peak fluorescence during reward period (6–30s) for Pavlovian training. Top – vNAcSh (n = 7 mice; P1 vs. P5 paired t test p=0.0031, t=3.016, df=135). Bottom – dNAcSh (n = 5 mice; P1 vs. P5 paired t test p=0.2438, t=1.175, df=97). Data represented as mean +/− SEM. (h) Top – Peak fluorescence during cue period (0–6s) comparing vNAcsh vs. dNAcSh for Pavlovian training (unpaired t test, p=0.9638, t=0.0454, df=680). Bottom – Peak fluorescence during reward period (6–30s) comparing vNAcsh vs. dNAcSh for Pavlovian training (unpaired t test, p<0.0001, t=5.106, df=682). Data represented as mean +/− SEM. (i) Mean vNAcSh recorded calcium activity averaged across all trials from day 1 of Pavlovian conditioning (n = 7 mice). (j) Mean vNAcSh recorded calcium activity averaged across all trials from day 5 of Pavlovian conditioning (n = 7 mice). (k) Heatmap raster plot raster plot of vNAcSh recorded calcium activity for every trial of Pavlovian conditioning on day 1 (left) and day 5 (right). Trials are displayed in ascending order by average activity following pellet delivery (n = 7 mice). (l) Mean dNAcSh recorded calcium activity averaged across all trials from day 1 of Pavlovian conditioning (n = 5 mice). (m) Mean dNAcSh recorded calcium activity averaged across all trials from day 5 of Pavlovian conditioning (n = 5 mice). (n) Heatmap raster plot of dNAcSh recorded calcium activity for every trial of Pavlovian conditioning on day 1 (left) and day 5 (right). Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice).

Figure 2.. Bi-directional photo-manipulation of VTA GABAergic projections to the vNAcSh impacts reward reinforcement.

(a) Top – Coronal brain cartoon of viral injection of ChR2 into VTA and fiber expression in the NAc of VGAT-Cre+ mice. Bottom – Representative 20X coronal image (left panel) showing expression of ChR2 (green), Nissl (blue) and fiber placement in vNAcSh (left; scalebar – 100 μm), dNAcSh (middle; scalebar – 100 μm) and NAc core (right; scalebar – 100 μm). (b) Photostimulation of vGAT terminal in vNAcSh at 40Hz; 20ms pulse width shows significant preference. Two-way repeated measures ANOVA (Control vs. ChR2; F(1,8)=15.83, p=0.0040; 0 vs 40 Hz; F(1,8)=47.48, p=0.0001; Interaction; F(1,8)=22.96, p=0.0014); Bonferroni post-hoc analysis. Control vs ChR2, (****p<0.0001, t=6.069, DF=16); ChR2 group 0 vs 40Hz,(****p<0.0001, t=9.236, DF=8). Data represented as mean +/− SEM, n = 6–7. (c) Photostimulation of vGAT terminal in dNAcSh at 40Hz; 20ms pulse width. Two-way repeated measures ANOVA, (Control vs. ChR2; F(1,15)=2.840, p=0.1126; 0 vs 40 Hz; F(1,15)=0.09334, p=0.7642; Interaction; F(1,15)=0.7677, p=0.3947); Bonferroni post-hoc analysis. Control vs ChR2, (p>0.9999, t=0.4447, DF=15); ChR2 group 0 vs 40Hz, (p=0.9061, t=0.7704, DF=15). Data represented as mean +/− SEM, n = 8–10). (d) Photostimulation of vGAT terminal in NAcc at 40Hz; 20ms pulse width. Two-way repeated measures ANOVA (Control vs. ChR2; F(1,30)=0.3, p=0.5773; 0 vs 40 Hz; F(1,30)=0.1, p=0.6830; Interaction; F(1,30)=3.9, p=0.0550); Bonferroni post-hoc analysis. Control vs ChR2, (p=0.1606, t=1.810, DF=30); ChR2 group 0 vs 40Hz, (p=0.6380, t=1.013, DF=30). Data represented as mean +/− SEM, n = 8–9). (e) Schematic outlining experimental procedure of operant testing. (f) Data showing number of nose pokes during fixed-ratio 1 testing versus fixed-ratio 3 testing. Control vs ChR2, (**p=0.0296, t=2.694, DF=18); ChR2 FR1 vs FR3, *p=0.031, t=2.974, DF=9). Data represented as mean +/− SEM, n = 4–7. (g) Significant differences in active nose pokes in response to 5 Hz photostimulation reward versus 40 Hz photostimulation reward and a 40Hz photostimulation reward versus 5 Hz photostimulation reward again. Both ChR2 5Hz vs 40Hz, (****p<0.0001, DF=4). Data represented as mean +/− SEM, n = 5–7. (h) Data showing number of nose pokes during progressive ratio testing with no significant differences between control and experimental groups. Unpaired Student’s T-test, two-tailed (p=0.6048, t=0.7902, DF=12). Data represented as mean +/− SEM, n = 6–8). (i) Top – Coronal brain cartoon of viral injection of JAWS into VTA and fiber expression in the NAc of VGAT-Cre+ mice. Bottom – Representative 40X coronal image (left panel) showing expression of JAWS (green), DAPI (blue) and fiber placement in vNAcSh (scalebar – 100 μm). (j) Cartoon outlining experimental procedure of uncued reward delivery testing. (k) Total pellets consumed in matched, counterbalanced sessions comparing laser on vs. off (pulse - 2s on, 2s ramp down, 1s off). Left – JAWS group (n = 7 mice; Off vs. On paired t test p=0.0582, t=2.335, df=6). Right – YFP group (n = 7 mice; Off vs. On paired t test p=0.8461, t=0.2027, df=6). Data represented as mean +/− SEM. (l) Pellet retrieval latencies in matched, counterbalanced sessions comparing laser on vs. off (pulse - 2s on, 2s ramp down, 1s off). Left – JAWS group (n = 7 mice; Off vs. On paired t test p=0.0308, t=2.810, df=6). Right – YFP group (n = 7 mice; Off vs. On paired t test p=0.6443, t=0.4859, df=6). Data represented as mean +/− SEM.

Figure 3.. VTA GABAergic terminals preferentially project to, and inhibit cholinergic interneurons in the vNAcSh.

(a) Coronal brain schematic of viral injection of ChR2 into VTA and fiber expression in the NAc of VGAT-Cre+ mice. (b) Top – Saggital brain cartoon depicting rostral-caudal position. Bottom - 40X confocal image of rostral NAc (left; +1.8 A/P relative to bregma, scalebar – 100 μm) and caudal NAc (right; +0.9 A/P relative to bregma, scalebar – 100 μm) showing expression of ChR2 (green), CHAT (red) and DAPI (blue); zoomed images showing expression of ChR2 fibers and CHAT soma in the dNAcSh (top) and vNAcSh (bottom). (c) GABAergic fiber density (maximum pixel intensity/mm²) in the vNAcSh or dNAcSh (n = 2 mice, 6 slices; vNAcSh vs. dNAcSh paired t test p=0.0009, t=7.041, df=5). Data represented as mean +/− SEM. (d) CHAT soma density (normalized to DAPI) in the vNAcSh or dNAcSh (n = 2 mice, 8 slices; vNAcSh vs. dNAcSh paired t test p=0.0264, t=2.329, df=7). Data represented as mean +/− SEM. (e) Schematic depicting top – percentage of VGAT fiber density and bottom – percentage of CHAT soma (relative to 100% total in the NAcSh) in the vNAcSh vs. dNAcSh. (f) Top - Representative image of biocytin (blue), ChAT-ir (red), and ChR2-YFP (green) in the vNAcSh (scalebar – 100 μm). White inset: Spontaneous activity measured in cell-attached configuration. (g) Example traces of evoked whole-cell voltage-clamp recordings of CIN activity (left) and MSN activity (right) in response to VTA-vNAcSh vgat+ stimulation. (h) Graph showing amplitude +/− SEM of oIPSCs recorded in CINs and MSNs (n=12–18, Mean ± SEM: CIN: 394± 100 pA; MSN: 8 ± 2 pA, unpaired t-test, t = 3.132, p = 0.0040). Data represented as mean +/− SEM. (i) Graph showing change in amplitude +/− SEM of oIPSCs recorded in CINs and MSNs with treatment of gabazine (n=12–18, Two-way repeated measure ANOVA: cell type × time interaction: F_1,18 = 6.212, p = 0.0227; post-hoc Sidak’s comparison of baseline vs gabazine: CIN: p = 0.0018; MSN: p = 0.9996). Data represented as mean +/− SEM. (j) Example traces of evoked whole-cell voltage-clamp recordings of CIN activity in response to ChR2 stimulation in the vNAcSh or dNAcSh and plots of responders vs. non-responders in the vNAcSh (7 responders, no non-responders) and dNAcSh (7 responders, 5 non-responders). (k) Graph showing amplitude +/− SEM of oIPSCs recorded in CINs in the vNAcSh vs. dNAcSh (unpaired t test, p<0.0001, t=5.343, df=18). Data represented as mean +/− SEM.

Figure 4.. Photo-manipulation of vNAcSh cholinergic interneuron activity bi-directionally modulates preference and reward reinforcement.

(a) Top – Coronal brain schematic of viral injection of ChR2 into vNAcSh of CHAT-Cre+ mice. Bottom – Representative 20X coronal image (left panel) showing expression of ChR2 (green), Nissl (blue) and fiber placement in vNAcSh (left; scalebar – 100 μm). (b) Representative heatmap raster plot showing behavioral response to ventral photoactivation. (c) Significant aversion to vNAc photostimulation of ChAT neurons at 20Hz. Two-way repeated measures ANOVA (Control vs. ChR2; F(1,34)=4.729, p=0.0367; 0 vs 40 Hz; F(1,34)=1.718, p=0.1987; Interaction; F(1,34)=10.41, p=0.028); Bonferoni post hoc test. ChR2 0 vs 20Hz, (*p<0.0004, t=4.162, DF=34). 20Hz ChR2 vs Control, (**p=0.0058, t=3.209, DF=34). Data represented as mean +/− SEM, n = 8–11. (d) Top – Coronal brain cartoon of viral injection of eNpHR into vNAcSh of CHAT-Cre+ mice. Bottom – Representative 20X coronal image (left panel) showing expression of eNpHR (green), Nissl (blue) and fiber placement in vNAcSh (left; scalebar – 100 μm). (e) Representative heatmap raster plot showing behavioral response to constant ventral photoinhibition. (f) Real-time behavior following constant photo-inhibition of ChAT neurons in ventral NAcSh. (Control vs. eNPHR; F(1,19)=5.182, p=0.0296; 0 vs constant; F(1,19)=10.66, p=0.0041; Interaction; F(1,19)=22.65, p=0.0001); Bonferoni post hoc test. Control vs eNPHR, **p=0.0081; Control vs eNPHR, ***p=0.0002; (data represented as mean +/− SEM, n = 8–12). (g) Cartoon outlining experimental procedure of uncued reward delivery testing. (h) Total pellets consumed in matched, counterbalanced sessions comparing laser on vs. off (20Hz, 5ms pulse width). Left – ChR2 group (n = 9 mice; Off vs. On paired t test p=0.0014, t=4.797, df=8). Right – YFP group (n = 6 mice; Off vs. On paired t test p=0.4896, t=0.7454, df=5). Data represented as mean +/− SEM. (l) Pellet retrieval latencies in matched, counterbalanced sessions comparing laser on vs. off (pulse – 20 Hz). Left – ChR2 group (n = 9 mice; Off vs. On paired t test p=0.0275, t=2.690, df=8). Right – YFP group (n = 6 mice; Off vs. On paired t test p=0.9728, t=0.03587, df=5). Data represented as mean +/− SEM.

Figure 5.. Cholinergic interneuron activity and acetylcholine release in the vNAcSh are inhibited during reward reinforcement.

(a) Left – Coronal brain cartoon (top panel) of viral injection of either GRAB-Ach4.3 (top) or GCaMP6s (bottom) into the vNAcSh of WT mice. Right – representative coronal image showing expression of GCaMP6s (green), DAPI (blue) and fiber placement (top; scalebar – 200 μm), or GRAB-Ach3.0 (green), DAPI (blue) and fiber placement (bottom; scalebar – 200 μm) in the vNAcSh. (b) Representative recorded activity in the vNAcSh for GRAB-Ach3.0 (top) or GcAMP6s (bottom) (highlights represent cue light during reward period). (c) Latency to eat following reward delivery (0–6s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p=0.0302, t=2.416, df=5). Bottom – vNAcSh CHAT GcAMP6s (n = 5 mice; P1 vs. P5 paired t test p=0.0038, t=6.201, df=4). (d) Peak fluorescence during cue period (0–6s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p=0.0122, t=2.546, df=114). Bottom – vNAcSh CHAT GcAMP6s (n = 5 mice; P1 vs. P5 paired t test p<0.0001, t=4.355, df=95). Data represented as mean +/− SEM. (e) Peak fluorescence during reward period (6–30s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p<0.0001, t=5.424, df=114). Bottom – vNAcSh CHAT GcAMP6s (n = 5 mice; P1 vs. P5 paired t test p=0.4182, t=0.8140, df=95). Data represented as mean +/− SEM. (f) Minimum fluorescence during reward period (6–30s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p=0.0077, t=2.711, df=114). Bottom – vNAcSh CHAT GcAMP6s (n = 5 mice; P1 vs. P5 paired t test p=0.0001, t=4.038, df=95). Data represented as mean +/− SEM. (g) Mean fluorescence during reward period (6–30s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p=0.0355, t=2.128, df=114). Bottom – vNAcSh CHAT GcAMP6s (n = 5 mice; P1 vs. P5 paired t test p=0.8999, t=0.1263, df=95). Data represented as mean +/− SEM. (h) Mean fluorescence comparison during reward period (6–30s) for Pavlovian training. Top – vNAcSh GRAB-Ach3.0 vs. vNAcSh VGAT terminal GcAMP6s (unpaired t test p<0.0001, t=8.513, df=529). Bottom – vNAcSh CHAT GcAMP6s vs. vNAcSh VGAT terminal GcAMP6s (unpaired t test p<0.0001, t=6.007, df=451). Data represented as mean +/− SEM. (i) Minimum fluorescence comparison for baseline (−10–0s) and reward period (6–30s) during all pavlovian training days. Top – GRAB-Ach3.0 (paired t test, p<0.0001, t=9.185, df=385). Bottom - vNAcSh VGAT terminal GcAMP6s (paired t test, p<0.1886, t=1.318, df=298). Data represented as mean +/− SEM. (j) Minimum fluorescence comparison for baseline (−10–0s) and reward period (6–30s) during all pavlovian training days. Top – vNAcSh CHAT GcAMP6s (paired t test, p<0.0001, t=7.85, df=230). Bottom - vNAcSh VGAT terminal GcAMP6s (paired t test, p<0.1886, t=1.318, df=298). Data represented as mean +/− SEM. (k) Mean vNAcSh GRAB-Ach3.0 recorded calcium activity averaged across all trials from day 1 of Pavlovian conditioning (n = 6 mice). (l) Mean vNAcSh GRAB-Ach3.0 recorded calcium activity averaged across all trials from day 5 of Pavlovian conditioning (n = 6 mice). (m) Heatmap raster plot raster plot of vNAcSh GRAB-Ach3.0 recorded calcium activity for every trial of Pavlovian conditioning on day 1 (left) and day 5 (right). Trials are displayed in ascending order by average activity following pellet delivery (n = 6 mice). (n) Mean vNAcSh CHAT GcAMP6s recorded calcium activity averaged across all trials from day 1 of Pavlovian conditioning (n = 5 mice). (o) Mean vNAcSh CHAT GcAMP6s recorded calcium activity averaged across all trials from day 5 of Pavlovian conditioning (n = 5 mice). (p) Heatmap raster plot of vNAcSh CHAT GcAMP6s recorded calcium activity for every trial of Pavlovian conditioning on day 1 (left) and day 5 (right). Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice).

Figure 6.. Acetylcholine release in the vNAcSh is inhibited by VTA-vNAcSh^vGAT activity.

(a) Mean vNAcSh GRAB-Ach3.0 recorded calcium activity preceding a nosepoke averaged across all trials from day 1 (left) and day 3 (right) of Operant conditioning (n = 6 mice). (b) Heatmap raster plot of vNAcSh GRAB-Ach3.0 recorded calcium activity preceding a nosepoke for every trial of Operant conditioning on day 1 (left) and day 3 (right). Trials are displayed in ascending order by average activity preceding a nosepoke (n = 6 mice). (c) Mean vNAcSh CHAT GcAMP6s recorded calcium activity preceding a nosepoke averaged across all trials from day 1 (left) or day 3 (right) of Operant conditioning (n = 6 mice). (d) Heatmap raster plot of vNAcSh CHAT GcAMP6s recorded calcium activity preceding a nosepoke for every trial of Operant conditioning on day 1 (left) and day 3 (right). Trials are displayed in ascending order by average activity preceding a nosepoke (n = 5 mice). (e) Mean fluorescence during nosepoke ramp (−10–1s) for Operant training days. Top – vNAcSh GRAB Ach3.0 Pavlovian days (n = 6 mice; O1 vs. O3 paired t test p=0.0012, t=3.35, df=85). Bottom – vNAcSh CHAT GcAMP6s Operant days (n = 5 mice; O1 vs. O3 paired t test p=0.0048, t=2.879, df=109). Data represented as mean +/− SEM. (f) Coronal brain cartoon (top panel) of viral injection of Chrimson into the VTA and GRAB-Ach4.3 into the vNAcSh of VGAT-Cre+ mice and representative 40x coronal image (bottom panel) showing expression of GRAB-Ach4.3 (green), Chrimson (red) and DAPI (blue) in the vNAcSh. (g) Cartoon schematic of fiber photometry setup connected to mouse performing operant task where active nosepoke results in 40 Hz red light stimulation. (h) Total nosepokes performed (active vs. inactive) in counterbalanced sessions resulting in Chrimson stimulation and no stimulation (Two Way ANOVA, *Active vs. Inactive in Stim – p=0.0001, t=4.982, df=32, #Active Stim vs. Active No Stim – p=0.0022, t=3.979, df=32). Data represented as mean +/− SEM. (i) Top – Mean vNAcSh GRAB-Ach3.0 fluorescence comparison across active (resulting in chrimson stimulation) vs. inactive nosepoke trials (unpaired t test, p=0.0002, t=3.844, df=119). Bottom – Mean vNAcSh GRAB-Ach3.0 fluorescence comparison of active nosepoke trials across stimulation (resulting in chrimson stimulation) vs. no stimulation days (unpaired t test, p=0.0436, t=2.309, df=126). Data represented as mean +/− SEM. (j) Mean vNAcSh recorded GRAB-Ach3.0 activity during the stimulation session averaged across all active (resulting in chrimson stimulation), nosepoke trials (n = 6 mice). (k) Mean vNAcSh recorded GRAB-Ach3.0 activity during the stimulation session averaged across all inactive (resulting in chrimson stimulation), nosepoke trials (n = 6 mice). (l) Heatmap raster plot of vNAcSh recorded GRAB-Ach3.0 activity for every active nosepoke trial (resulting in chrimson stimulation). Trials are displayed in ascending order by mean activity during active nosepokes (n = 6 mice). (m) Mean vNAcSh recorded GRAB-Ach3.0 activity during the no stimulation session averaged across all active (resulting in no chrimson stimulation) nosepoke trials (n = 6 mice). (n) Mean vNAcSh recorded GRAB-Ach3.0 activity during the no stimulation session averaged across all inactive (resulting in no chrimson stimulation) nosepoke trials (n = 6 mice). (o) Heatmap raster plot raster plot of vNAcSh recorded GRAB-Ach3.0 activity for every active nosepoke trial (resulting in no chrimson stimulation). Trials are displayed in ascending order by mean activity during active nosepokes (n = 6 mice).