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

Nature Neuroscience volume 24, pages 1414–1428 (2021)Cite this article

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An Author Correction to this article was published on 09 September 2021

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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.

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Fig. 1: VTA GABAergic terminals in the vNAcSh are preferentially engaged during reward reinforcement.
Fig. 2: Bidirectional photomanipulation of VTA GABAergic projections to the vNAcSh impacts reward reinforcement.
Fig. 3: VTA GABAergic terminals preferentially project to, and inhibit, CINs in the vNAcSh.
Fig. 4: Photomanipulation of vNAcSh CIN activity bidirectionally modulates preference and reward reinforcement.
Fig. 5: CIN activity and ACh release in the vNAcSh are inhibited during reward reinforcement.
Fig. 6: ACh release in the vNAcSh is inhibited by VTA-vNAcShVGAT activity.

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Data availability

The raw data that support the findings of the present study are available from the corresponding authors upon request.

Code availability

Custom MATLAB code used for data analysis is available at https://github.com/BruchasLab.

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Acknowledgements

We thank all at M.R.B.’s lab and R.A.-H.’s lab, and J. McCall for their helpful insight and discussion throughout the preparation of the manuscript, and in particular C. Stander, L. Lawson, D. Blumenthal and A. Suko for their technical support. We thank our funding sources: NIH/NIDA K99/R00 DA038725 (to R.A.-H.), NIH/NIDA F31 DA051124 (to C.E.P), Addictions, Drug and Alcohol Institute (ADAI) Fellowship (to D.J.M), SCAN Design Fellowship (to S.E.S), Mary Gates Fellowship (to A.E.J), NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation, grant no. 28243 (to R.A.-H.), NIH/NIMH K99/R00 MH109627 (to J.C.L) and NIH/NIDA R37 DA033396, P30 DA048736 (to M.R.B).

Author information

Author notes

  1. These authors contributed equally: Ream Al-Hasani, Raajaram Gowrishankar, Gavin P. Schmitz.

Authors and Affiliations

  1. Center for Clinical Pharmacology, University of Health Sciences and Pharmacy and Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Ream Al-Hasani & Gavin P. Schmitz

  2. Department of Pharmaceutical and Administrative Sciences, University of Health Science and Pharmacy, St. Louis, MO, USA

    Ream Al-Hasani

  3. Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, USA

    Ream Al-Hasani, Gavin P. Schmitz, Sophie J. Renaud & Michael R. Bruchas

  4. Center for the Neurobiology of Addiction, Pain and Emotion, University of Washington, Seattle, WA, USA

    Raajaram Gowrishankar, Christian E. Pedersen, David J. Marcus, Sofia E. Shirley, Taylor E. Hobbs, Abigail J. Elerding & Michael R. Bruchas

  5. Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA

    Raajaram Gowrishankar, Christian E. Pedersen & Michael R. Bruchas

  6. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Christian E. Pedersen & Michael R. Bruchas

  7. Chinese Institute for Brain Research, Beijing, China

    David J. Marcus & Miao Jing

  8. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing, China

    Miao Jing & Yulong Li

  9. PKU-IDG/McGovern Institute for Brain Research, Beijing, China

    Miao Jing & Yulong Li

  10. Peking-Tsinghua Center for Life Sciences, Beijing, China

    Miao Jing & Yulong Li

  11. Laboratory on Neurobiology of Compulsive Behaviors, Intramural Research Program, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Veronica A. Alvarez & Julia C. Lemos

  12. Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA

    Julia C. Lemos

  13. Department of Pharmacology, University of Washington, Seattle, WA, USA

    Michael R. Bruchas

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  1. Ream Al-Hasani
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Contributions

R.A.-H., G.P.S., R.G. and M.R.B. conceptualized and designed the experiments. R.A.-H., R.G., G.P.S., T.E.H., S.E.S., A.J.E. and J.C.L. performed the surgeries. J.C.L. and D.J.M. performed the electrophysiology. R.G., S.E.S. and C.E.P. performed the fiber photometry. R.G. and C.E.P. recorded the ITI. R.A.-H., R.G., A.J.E., T.E.H., G.P.S. and J.C.L. carried out the immunohistochemistry. R.G. did the uncued reward delivery. R.A.-H., R.G. and G.P.S. performed the operant self-stimulation and real-time place testing. R.G., C.E.P. and S.E.S. carried out the operant behavior paradigm (photometry) and the pavlovian behavior paradigm (photometry). Y.L. and M.J. provided the sensor resources. R.A.-H., R.G., G.P.S., C.E.P., J.C.L., V.A.A. and M.R.B. wrote the original draft, and reviewed and edited it. R.A.-H. and M.R.B. acquired funds. R.A.-H. and M.R.B. administered the project.

Corresponding authors

Correspondence to Ream Al-Hasani or Michael R. Bruchas.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks William Howe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 VTA GABAergic terminals in the vNAcSh are preferentially engaged during reward reinforcement.

(a) Coronal brain cartoon of fiber placements in the dNAcSh (light blue; n = 5) or vNAcSh (dark blue; n = 7) of VGAT-Cre+ mice. (b) Inter-trial interval (ITI) head entries for Pavlovian training, vNAcSh (n = 7 mice; P1 vs. P5 paired t test p = 0.0008, t = 6.274, df=6). (c) Mean vNAcSh recorded calcium activity during pellet retrieval averaged across all trials from day 1-5 of Pavlovian conditioning (n = 7 mice). Data represented as mean +/− SEM. (d) Mean vNAcSh recorded calcium activity during unrewarded, ITI head entries averaged across all trials from day 1-5 of Pavlovian conditioning (n = 7 mice). Data represented as mean +/− SEM. (e) Mean vNAcSh recorded calcium activity averaged across all trials from day 3 of Pavlovian conditioning (n = 7 mice). Data represented as mean +/− SEM. (f) Mean dNAcSh recorded calcium activity averaged across all trials from day 3 of Pavlovian conditioning (n = 5 mice). Data represented as mean +/− SEM. (g) Mean vNAcSh recorded calcium activity averaged across all trials from day 7 of Pavlovian conditioning extinction (n = 7 mice). Data represented as mean +/− SEM. (h) Mean dNAcSh recorded calcium activity averaged across all trials from day 7 of Pavlovian conditioning extinction (n = 5 mice). Data represented as mean +/− SEM. (i) Operant training schedule with photometry recordings, as indicated. (j) Top – vNAcSh active nosepokes for Operant training days (n = 7 mice; O1 vs. O3 paired t test p > 0.0001, t = 14.24, df=6). Bottom – dNAcSh active nosepokes for Operant training days (n = 5 mice; O1 vs. O3 paired t test p = 0.0035, t = 6.180, df=4). Data represented as mean +/− SEM. (k) Peak fluorescence during cue period (0-6 s) for Operant training days. Top – vNAcSh (n = 7 mice; O1 vs. O3 paired t test p = 0.5002, t = 0.6759, df=142). Bottom – dNAcSh (n = 5 mice; O1 vs. O3 paired t test p = 0.0047, t = 2.871, df=146). Data represented as mean +/− SEM. (l) Peak fluorescence during reward period (6-30 s) for Operant training days. Top – vNAcSh (n = 7 mice; O1 vs. O3 paired t test p = 0.9724, t = 03463, df=141). Bottom – dNAcSh (n = 5 mice; O1 vs. O3 vs. P5 paired t test p = 0.0003, t = 3.702, df=146). Data represented as mean +/− SEM. (m) Top – Peak fluorescence during cue period (0-6 s) comparing vNAcsh (n = 7 mice) vs. dNAcSh (n = 5 mice) for Operant training (unpaired t test, p = 0.6729, t = 0.4222, df=1038). Bottom - Peak fluorescence during reward period (6-30 s) comparing vNAcsh vs. dNAcSh for Operant training (unpaired t test, p < 0.0001, t = 9.513, df=1038). Data represented as mean +/− SEM. (n) Mean vNAcSh recorded calcium activity averaged across all trials from day 1-5 of Operant conditioning (n = 7 mice). Data represented as mean +/− SEM. (o) Heatmap raster plot of vNAcSh recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 7 mice). (p) Mean dNAcSh recorded calcium activity averaged across all trials from day 1-5 of Operant conditioning (n = 5 mice). Data represented as mean +/− SEM. (q) Heatmap raster plot of vNAcSh recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice).

Extended Data Fig. 2 Photo-activation of VTA GABAergic projections to the vNAcSh promotes positive reinforcement.

(a) Cartoon of ventral NAcSh fiber optic placement. (b) Representative examples of real-time mouse heatmap raster plots following no stimulation, 2 Hz, 10 Hz, 20 Hz, 40 Hz, and 60 Hz ventral NAcSh photostimulation. (c) Hit map showing fiber optic placements of each individual mouse included in the ventral NAcSh ChR2 group. (d) Frequency response curve showing photostimulation of vGAT terminal in vNAcSh at no stimulation, 2 Hz, 5 Hz, 10 Hz, 20 Hz, 40 Hz, and 60 Hz; 20 ms pulse width. Photostimulation shows significant preference at 40 and 60 Hz. Unpaired Student’s t Test, two-tailed, Control vs ChR2, *p < 0.05; Unpaired Student’s t Test, two-tailed, ChR2 group 0 vs 40 Hz,**p < 0.01 (data represented as mean +/− SEM, n = 6 mice/Control,7 mice/ChR2). (e) Cartoon of dorsal NAcSh fiber optic placement. (f) Representative examples of real-time mouse heatmap raster plots following no stimulation, 2 Hz, 10 Hz, 20 Hz, 40 Hz, and 60 Hz dorsal NAcSh photostimulation. (g) Hit map showing fiber optic placements of each individual mouse included in the dorsal NAcSh ChR2 group. (h) Data showing number of nose pokes during fixed-ratio 1 testing versus fixed-ratio 3 testing (data represented as mean +/− SEM, n = 6 mice/FR-3, 9 mice/FR-1). (i) Data showing number of nose pokes during progressive ratio testing with no significant differences between control and experimental groups ((data represented as mean +/− SEM, n = 6 mice/Control, 9 micee/Experimental). (j) Frequency response curve showing photostimulation of vGAT terminal in dNAcSh at no stimulation, 2 Hz, 5 Hz, 10 Hz, 20 Hz, 40 Hz, and 60 Hz; 20 ms pulse width. Photostimulation shows no significant preference. Control vs ChR2 (data represented as mean + - SEM, n = 6 mice/Control, n = 10 mice/ChR2). (k) Cartoon of NAcc fiber optic placement. (l) Hit map showing fiber optic placements of each individual mouse included in the NAcC ChR2 group. (m) Data showing number of nose pokes during fixed-ratio 1 testing versus fixed-ratio 3 testing (data represented as mean + - SEM, n = 7 mice/FR-3, 11 mice/FR-1). (n) Data showing number of nose pokes during progressive ratio testing with no significant differences between control and experimental groups ((data represented as mean + - SEM, n = 7 mice/Control, 11 mice/Experimental). (o) Frequency response curve showing photostimulation of vGAT terminal in NAcC at no stimulation, 2 Hz, 5 Hz, 10 Hz, 20 Hz, 40 Hz, and 60 Hz; 20 ms pulse width. Photostimulation shows no significant preference. Control vs ChR2 (data represented as mean + - SEM, n = 6 mice/Control, 8 mice/ChR2). (p) Total head entries performed in matched, counterbalanced sessions comparing laser on vs. off (pulse − 2 s on, 2 s ramp down, 1 s off) in the JAWS group (n = 7 mice; Off vs. On paired t test p = 0.0269, t = 2.913, df=6). Right – YFP group (n = 7 mice; Off vs. On paired t test p = 0.2739, t = 1.204, df=6). Data represented as mean +/− SEM. (q) Total head entries performed in matched, counterbalanced sessions comparing laser on vs. off (pulse − 2 s on, 2 s ramp down, 1 s off) in the YFP group (n = 7 mice; Off vs. On paired t test p = 0.2739, t = 1.204, df=6). Data represented as mean +/− SEM.

Extended Data Fig. 3 VTA GABAergic terminals project to and inhibit cholinergic interneurons in the NAc.

(a) Top – Saggital brain cartoon depicting rostral-caudal position. Bottom − 20X confocal image of rostral VTA (left; −2.9 A/P relative to bregma, scalebar – 100 𝜇m), middle VTA (middle; −3.4 A/P relative to bregma, scalebar – 100 𝜇m) and caudal VTA (right; −3.8 A/P relative to bregma, scalebar – 100 𝜇m) showing expression of ChR2 (green) and DAPI (blue). (b) Table comparing electrophysiological parameters for CINs vs. MSNs – Rm: unpaired t-test: t = 3.000, p = 0.0056, n = 12 (MSNs)−18(CINs), Cm: unpaired t-test: t = 0.3852, p = 0.7030, n = 12-18, sIPSC frequency: unpaired t-test: t = 3.617, p = 0.0040, n = 8 (CINs), 5(MSNs), sIPSC amplitude: unpaired t-test: t = 0.5700, p = 0.5802, n = 8,5, Holding current: 2-way RM ANOVA, time x cell type interaction: F1,18 = 5.031, p = 0.0377; main effect of cell type: F1,18 = 28.29, p < 0.0001, post-hoc Sidak t-test – baseline vs. gabazine: CINs: p = 0.02, MSNs: p = 0.8194). Data represented as mean +/− SEM. (c) oIPSC amplitude comparing 5 Hz vs. 40 Hz stimulation (5 Hz: 269 ± 64 pA; 40 Hz: 280 ± 62 pA; paired t-test, t = 0.7367, p = 0.4755, n = 13 cells/4 mice). Data represented as mean +/− SEM. (d) oIPSC area (nA*s) over 250 ms period comparing 5 Hz vs. 40 Hz stimulation (5 Hz: 8 ± 3 nA*s; 40 Hz: 18 ± 4 nA*s; paired t-test, t = 3.804, p = 0.0025, n = 13 cells/4 mice). Data represented as mean +/− SEM. (e) Image showing relative position of patch pipette for recordings in the vNAcSh. (f) Image showing relative position of patch pipette for recordings in the dNAcSh.

Extended Data Fig. 4 Photo-activation of vNAcSh cholinergic interneuron activity does not impact reinforcement.

(a) Cartoon of ventral NAcSh virus and fiber optic placement. (b) Placement map showing fiber optic placements of each individual mouse included in the vNAcSh ChR2 group. (c) Data showing number of nose pokes during FR-1 self stimulation with no significant differences between control and experimental groups (data represented as mean +/− SEM, n = 7 each, unpaired t test, p = 0.4165, t = 0.8416, df=12). (d) Cartoon of bilateral ventral NAcSh virus and fiber optic placement. (e) Representative examples of real-time mouse heatmap raster plots following no stimulation, 2 s phasic stimulation, and constant ventral NAcSh photostimulation. (f) Cartoon of bilateral ventral NAcSh virus and fiber optic placement. (g) No aversion real-time behavior following phasic photo-inhibition of ChAT neurons in ventral NAcSh (data represented as mean +/− SEM, n = 8 mice/Control, 12 mice/Halo). (h) Total head entries performed in matched, counterbalanced sessions comparing laser on vs. off (pulse − 2 s on, 2 s ramp down, 1 s off). Left – ChR2 group (n = 9 mice; Off vs. On paired t test p = 0.1328, t = 1.673, df=8). Right – YFP group (n = 6 mice; Off vs. On paired t test p = 0.1629, t = 1.635, df=5). Data represented as mean +/− SEM.

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

(a) Coronal brain cartoon of fiber placements in the vNAcSh for VGAT-Cre+ mice injected with GRAB Ach3.0 (violet; n = 6). (b) Inter-trial interval (ITI) head entries for Pavlovian training, vNAcSh GRAB Ach3.0 (n = 6 mice; P1 vs. P5 paired t test p = 0.0053, t = 4.713, df=5). (c) Mean vNAcSh GRAB Ach3.0 recorded calcium activity during pellet retrieval averaged across all trials from day 1-5 of Pavlovian conditioning (n = 6 mice). Data represented as mean +/− SEM. (d) Mean vNAcSh GRAB Ach3.0 recorded calcium activity during unrewarded, ITI head entries averaged across all trials from day 1-5 of Pavlovian conditioning (n = 6 mice). Data represented as mean +/− SEM. (e) Coronal brain cartoon of fiber placements in the vNAcSh for CHAT-Cre+ mice injected with GCaMP6s (majenta; n = 5). (f) Inter-trial interval (ITI) head entries for Pavlovian training, vNAcSh GCaMP6s (n = 5 mice; P1 vs. P5 paired t test p = 0.0034, t = 6.20, df=4). (g) Mean vNAcSh GCaMP6s recorded calcium activity during pellet retrieval averaged across all trials from day 1-5 of Pavlovian conditioning (n = 5 mice). Data represented as mean +/− SEM. (h) Mean vNAcSh GCaMP6s recorded calcium activity during unrewarded, ITI head entries averaged across all trials from day 1-5 of Pavlovian conditioning (n = 5 mice). Data represented as mean +/− SEM. (i) Mean vNAcSh GRAB Ach3.0 recorded calcium activity averaged across all trials from day 3 of Pavlovian conditioning (n = 6 mice). Data represented as mean +/− SEM. (j) Mean vNAcSh GCaMP6s recorded calcium activity averaged across all trials from day 3 of Pavlovian conditioning (n = 5 mice). (k) Mean vNAcSh GRAB Ach3.0 recorded calcium activity averaged across all trials from day 7 of Pavlovian conditioning extinction (n = 6 mice). (l) Mean vNAcSh GCaMP6s recorded calcium activity averaged across all trials from day 7 of Pavlovian conditioning extinction (n = 5 mice). (m) Top – vNAcSh GRAB Ach3.0 active nosepokes for Operant training days (n = 6 mice; O1 vs. O3 paired t test p = 0.0287, t = 2.457, df=5). Bottom – vNAcSh GCaMP6s active nosepokes for Operant training days (n = 5 mice; O1 vs. O3 paired t test p = 0.0129, t = 4.274, df=4). Data represented as mean +/− SEM. (n) Peak fluorescence during cue period (0-6 s) for Operant training days. Top – vNAcSh GRAB Ach3.0 (n = 6 mice; O1 vs. O3 paired t test p = 0.0364, t = 1.810, df=263). Bottom – vNAcSh GCaMP6s (n = 5 mice; O1 vs. O3 paired t test p = 0.0197, t = 2.070, df=299). Data represented as mean +/− SEM. (o) Peak fluorescence during reward period (6-30 s) for Operant training days. Top – vNAcSh GRAB Ach3.0 (n = 6 mice; O1 vs. O3 paired t test p = 0.1010, t = 1.658, df=263). Bottom – vNAcSh GCaMP6s (n = 5 mice; O1 vs. O3 paired t test p = 0.1423, t = 1.471, df=299). Data represented as mean +/− SEM. (p) Minimum fluorescence during reward period (6-30 s) for Operant training days. Top – vNAcSh GRAB Ach3.0 (n = 6 mice; O1 vs. O3 paired t test p = 0.0005, t = 3.608, df=263). Bottom – vNAcSh GCaMP6s (n = 5 mice; O1 vs. O3 paired t test p = 0.5695, t = 0.5702, df=299). Data represented as mean +/− SEM. (q) Mean vNAcSh GRAB Ach3.0 recorded calcium activity averaged across all trials from day 1-5 of Operant conditioning (n = 6 mice). Data represented as mean +/− SEM. (r) Heatmap raster plot of vNAcSh GRAB Ach3.0 recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 7 mice). (s) Mean vNAcSh GCaMP6s recorded calcium activity averaged across all trials from day 1-5 of Operant conditioning (n = 5 mice). Data represented as mean +/− SEM. (t) Heatmap raster plot of vNAcSh GCaMP6s recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice).

Extended Data Fig. 6 Acetylcholine release in the dNAcSh is unchanged during reward reinforcement.

(a) Coronal brain cartoon of fiber placements in the dNAcSh for WT mice injected with GRAB Ach3.0 (plum; n = 5). (b) Top – dNAcSh GRAB Ach3.0 latency to eat for Pavlovian training days (n = 5 mice; P1 vs. P5 paired t test p = 0.0284, t = 2.654, df=4). Bottom – dNAcSh GRAB Ach3.0 active nosepokes for Operant training days (n = 5 mice; O1 vs. O3 paired t test p = 0.0057, t = 5.395, df=4). Data represented as mean +/− SEM. (c) Peak fluorescence during cue period (0-6 s) for Pavlovian or Operant training days. Top – dNAcSh GRAB Ach3.0 Pavlovian days (n = 5 mice; P1 vs. P5 paired t test p = 0.6406, t = 0.4683, df=95). Bottom – dNAcSh GRAB Ach3.0 Operant days (n = 5 mice; O1 vs. O3 paired t test p = 0.7091, t = 0.3753, df=46). Data represented as mean +/− SEM. (d) Peak fluorescence during reward period (6-30 s) for Pavlovian or Operant training days. Top – dNAcSh GRAB Ach3.0 Pavlovian days (n = 5 mice; P1 vs. P5 paired t test p = 0.3495, t = 0.9401, df=95). Bottom – dNAcSh GRAB Ach3.0 Operant days (n = 5 mice; O1 vs. O3 paired t test p = 0.3006, t = 1.047, df=46). Data represented as mean +/− SEM. (e) Minimum fluorescence during reward period (6-30 s) for Pavlovian or Operant training days. Top – dNAcSh GRAB Ach3.0 Pavlovian days (n = 5 mice; P1 vs. P5 paired t test p = 0.0016, t = 3.241, df=95). Bottom – dNAcSh GRAB Ach3.0 Operant days (n = 5 mice; O1 vs. O3 paired t test p = 0.7585, t = 0.3093, df=46). Data represented as mean +/− SEM. (f) Top – Minimum fluorescence during reward period (6-30 s) comparing vNAcsh (n = 6 mice) vs. dNAcSh (n = 5 mice) GRAB Ach3.0 for Pavlovian training (unpaired t test, p < 0.0001, t = 9.760, df=422). Bottom – Minimum fluorescence during reward period (6-30 s) comparing vNAcsh vs. dNAcSh GRAB Ach3.0 for Operant training (unpaired t test, p < 0.0001, t = 5.470, df=278). Data represented as mean +/− SEM. (g) Mean dNAcSh GRAB Ach3.0 recorded calcium activity averaged across all trials from day 1-5 of Pavlovian conditioning (n = 5 mice). Data represented as mean +/− SEM. (h) Heatmap raster plot of dNAcSh GRAB Ach3.0 recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice). (i) Mean dNAcSh GRAB Ach3.0 recorded calcium activity averaged across all trials from day 1-5 of Operant conditioning (n = 5 mice). Data represented as mean +/− SEM. (j) Heatmap raster plot of dNAcSh GRAB Ach3.0 recorded calcium activity for every trial of Operant conditioning across all days. Trials are displayed in ascending order by average activity following pellet delivery (n = 5 mice).

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Al-Hasani, R., Gowrishankar, R., Schmitz, G.P. et al. Ventral tegmental area GABAergic inhibition of cholinergic interneurons in the ventral nucleus accumbens shell promotes reward reinforcement. Nat Neurosci 24, 1414–1428 (2021). https://doi.org/10.1038/s41593-021-00898-2

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