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Endocannabinoids facilitate reward engagement through retrograde gain control

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Data availability

All data associated with this project have been uploaded to Figshare (https://figshare.com/authors/David_Marcus/23255861).

Code availability

All code associated with this project has been uploaded to Figshare (https://figshare.com/authors/David_Marcus/23255861).

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Acknowledgements

We thank the Molecular Genetics Resource Core for the Center in Neurobiology of Addiction, Pain, and Emotion and its director, S. Schattauer, for generating the GRABeCB2.0 and CRISPR viruses used in this study; A. Suko for laboratory management and organization; T. Hobbs, C. Pizzano and V. Lau for colony management; and the entire Bruchas laboratory as well as other members of the NAPE Center at the University of Washington for resources and critical feedback.

Funding

This work was supported by the National Institute on Drug Abuse: F32 DA054709 (to D.J.M.), K99/R00 DA059617 (to D.J.M.), R37 DA033396 (to M.R.B.) and R21s DA056816 and DA057186 (to M.R.B. and N.S.); the National Institute of Mental Health R01 MH112355 (to M.R.B.); and the National Center for Complementary and Integrative Health RO1 AT011524 (to B.B.L.). Further support was provided by the UW Addictions, Drug, and Alcohol Institute research grant (to D.J.M.) and the Scan Design Foundation Innovative Pain Research Grant (to D.J.M.).

Author information

Authors and Affiliations

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

    David J. Marcus, Anthony E. English, Gunn Chun, Emmaline F. Seth, Rachel Oommen, Sabrina Hwang, Bailey A. Wells, Sean C. Piantadosi, Azra Suko, Sayaka J. Kenmochi, Anupritaa A. Parasnis & Michael R. Bruchas

  2. Center of Excellence in the Neurobiology of Addiction, Pain, and Emotion (NAPE), University of Washington, Seattle, WA, USA

    David J. Marcus, Anthony E. English, Gunn Chun, Emmaline F. Seth, Rachel Oommen, Sabrina Hwang, Bailey A. Wells, Sean C. Piantadosi, Azra Suko, Sayaka J. Kenmochi, Anupritaa A. Parasnis, Ethan Ancell, Larry S. Zweifel, Benjamin B. Land, Nephi Stella & Michael R. Bruchas

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

    Anthony E. English, Larry S. Zweifel, Benjamin B. Land, Nephi Stella & Michael R. Bruchas

  4. BioSyft Inc., Seattle, WA, USA

    Anthony E. English & Gunn Chun

  5. Department of Statistics, University of Washington, Seattle, WA, USA

    Ethan Ancell

  6. School of Life Sciences, Peking University, Beijing, China

    Yulong Li

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

    Michael R. Bruchas

Authors

  1. David J. Marcus
  2. Anthony E. English
  3. Gunn Chun
  4. Emmaline F. Seth
  5. Rachel Oommen
  6. Sabrina Hwang
  7. Bailey A. Wells
  8. Sean C. Piantadosi
  9. Azra Suko
  10. Sayaka J. Kenmochi
  11. Anupritaa A. Parasnis
  12. Ethan Ancell
  13. Yulong Li
  14. Larry S. Zweifel
  15. Benjamin B. Land
  16. Nephi Stella
  17. Michael R. Bruchas

Contributions

D.J.M. and M.R.B. conceptualized the study. D.J.M., M.R.B., B.B.L., L.S.Z., Y.L., N.S., A.E.E. and G.C. formed the methodology. D.J.M., G.C., E.F.S., R.O., S.H., B.A.W., S.J.K., A.A.P. and A.S. conducted the investigation. D.J.M., G.C., S.C.P., A.E.E. and E.A. performed the data analysis. D.J.M. and M.R.B. performed the visualization. D.J.M., B.B.L. and M.R.B. acquired funding. D.J.M. and M.R.B. wrote the original draft of the manuscript. D.J.M., M.R.B., B.B.L. and N.S. reviewed and edited the manuscript.

Corresponding author

Correspondence to Michael R. Bruchas.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Anna Beyeler, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 2-AG is released in the NAc following rewarding stimuli.

(a) Schematic for fiber photometry recordings of GRABeCB2.0 in the NAc during Pavlovian reward conditioning. (b) Fiber placements for GRABeCB2.0 cohort displayed in Fig. 1. (c) Photometry trace of cue-aligned GRABeCB2.0 signal in SR141716A (10 mg/kg) treated animals (N = 11). (d-f) Photometry trace of lick-aligned GRABeCB2.0 signal in vehicle, SR141716A (10 mg/kg), and DO34 (20 mg/kg) treated animals (N = 11). (g) Comparison of cue-aligned GRABeCB2.0 signal (0-26 s post cue) showing attenuation of GRABeCB2.0 signal (0-20 s post lick) in SR141716A (p = 0.0131) and DO34 (p = 0.0079) treated animals compared to vehicle (N = 11). (h) Comparison of lick-aligned (0-20 s) GRABeCB2.0 signal in SR141716A (p = 0.0343) and DO34 (p = 0.0469) treated animals compared to vehicle (N = 11). (i-k) Photometry traces of cue-aligned GRABeCB2.0 signal in vehicle, JZL184 (5 mg/kg), and URB597 3 mg/kg) treated animals (N = 8). (l) Comparison of cue-aligned (0-26 s) GRABeCB2.0 signal in JZL184 (p = 0.0914) and URB597 (p = 0.6983) treated animals compared to vehicle (N = 8) (m) Schematic for Pavlovian reward conditioning with different cue modalities (n,o) Photometry traces of light and tone cue aligned GRABeCB2.0 signal (N = 7). (p) Comparison of light and tone aligned GRABeCB2.0 signal (N = 7). (q) Comparison of number of licks in light and tone Pavlovian reward conditioning sessions (N = 7). (r) Schematic for reward omission assay. (s,t) Photometry trace and quantification of cue-aligned (0-26 s) GRABeCB2.0 signal during rewarded trials in the reward omission assay (N = 11). (u,v) Photometry trace and quantification of cue-aligned (0-26 s) GRABeCB2.0 signal during non-rewarded trials in the reward omission assay (N = 11). (w) Schematic for Pavlovian fear conditioning/extinction. (x,y) Photometry trace of GRABeCB2.0 signal during Pavlovian fear conditioning and extinction with representative heatmaps (N = 11). (z,aa) Comparison of GRABeCB2.0 signal during foot shock (0-5 s, p = 0.0049) and cued recall (0-20 s, p = 0.8987) (N = 11). (bb) Summary schematic of elicited eCB release in the NAc. Error bars represent ± SEM (p,q,t,v,z,aa), or min/max values (g,h,l); box plots represent interquartile range with line at median; N represents number of mice. p values reported from one-way ANOVA (g,h,l) and two-tailed paired t test (p,q,t,v,z,aa). *p < 0.05, **p < 0.01.

Extended Data Fig. 2 Histological analysis of CB1R expressing inputs to the NAc and histological/electrophysiological characterization of excitatory aPVTNTS projections.

(a-c) (Top) TdTomato positive NAc projecting neurons in the medial prefrontal cortex (mPFC) Basolateral Amygdala (BLA) and ventral Hippocampus (vHip) and (bottom) RNAscope image and quantification of Cre and CB1R transcript levels in the mPFC BLA and vHip (N = 3,3,3). (d-f) Optically evoked input-output curve from D1R+ and D1R- neurons in vehicle (D1R + n = 19, D1- n = 20, 7.36 · 10−5), SR141617A (D1R+ n = 14 D1R- n = 12, p = 0.0050), and JZL184 (D1+ n = 17, D1R- n = 15, p = 0.9698) treated slices. (g,h) 1μM JZL184 treatment significantly reduces the maximum oEPSC amplitude (p = 0.0193) and increases the Paired Pulse Ratio (p = 0.0355) in D1(+) neurons compared to vehicle treated slices (Veh: n = 19, SR141716A: n = 14, JZL184: n = 16). (i,j) 1μM SR141716A significantly increases the maximum oEPSC amplitude (p = 0.0016) and reduces the Paired Pulse Ratio (p = 0.0092) in D1(−) neurons (Veh: n = 20, SR141716A: n = 11, JZL184: n = 15). (k-n) Electrophysiological comparison of sag current amplitude (p = 0.3871) afterhyperpolzarization voltage (p = 0.3404), holding current (p = 0.6077), and membrane resistance (p = 0.6166) between D1(+) (n = 14) and D1(−) (n = 13) neurons. (o) Schematic for electrophysiological characterization of aPVT output to NAc, BNST, CeA, and ZI. (p-s) Optically evoked input/output curves for aPVT input to the NAc (n = 21), ZI (n = 8) BNST (n = 4), and CeA (n = 4). (t) Comparison of maximal aPVT evoked oEPSC in the NAc, ZI, BNST, and CeA. (u-w) Schematics, representative images, and quantification of collateralization of aPVT projections to the NAc and the ZI, BNST, and CeA, respectively (n = 3,3,3). (x-z) Representative images and quantification of collateralization of pPVT projections to the NAc and the ZI, BNST, and CeA, respectively. Error bars represent ± SEM (d-f,p-s), or min/max values (g-n,t); box plots represent interquartile range with line at median; n represent number of neurons. p values reported from two-tailed unpaired t-test (k-n), one-way ANOVA (g-j), and two-way ANOVA with Holm-Sidak post-hoc correction (d-f). *p < 0.05, **p < 0.01, ****p < 0.0001.

Extended Data Fig. 3 Inhibition of aPVTNTS-NAc terminals is not dependent on reward receipt and is not associated with locomotor activity.

(a) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during ad libitum sucrose consumption and Pavlovian reward conditioning. (b) Fiber placements for aPVTNTS-NAc GCaMP cohort displayed in Fig. 2. (c) Photometry trace of GCaMP6s signal aligned to first and last licks in a licking bout during ad libitum consumption (N = 10). (d,e) Transition diagram showing probabilities of transitions between approach, engage, and null/exit behaviors on day 1 and day 5 of conditioning during the post-sipper time window, with size of circles corresponding to number of events. (f,g) Photometry traces of GCaMP6s signal aligned to first lick and last lick on day 1 of reward conditioning. (h,i) Photometry traces of GCaMP6s signal aligned to first lick and last lick on day 5 of reward conditioning. (j,k) Quantification of first lick (0-20 s, p = 0.8736, N = 10) and last lick (0-20 s, p = 0.0004, N = 10) aligned GCaMP6s signals on day 1 and day 5 of reward conditioning. (l) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during reward omission. (m,n) Photometry traces of GCaMP6s signal during rewarded and non-rewarded trials during the reward omission test, demonstrating aPVTNTS-NAc terminal inhibition in the absence of reward receipt. (o) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during reward conditioning. (p) Photometry trace of GCaMP6s signal on day 5 of reward conditioning during ignored cue/reward trials (no reward engagement), demonstrating a lack of inhibition when the cue/reward is ignored. (q) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during Fixed Ratio 1 (FR1) training and Progressive Ratio (PR) training. (r,s) Photometry traces of GCaMP6s signal aligned to cue on day 1 and day 3 of FR1 conditioning, demonstrating inhibition during active operant responding for reward. (t,u) Photometry traces of GCaMP6s signal aligned to cue and nose poke during PR conditioning. (v) Schematic for fiber photometry recordings of GCaMP6s from aPVTNTS-NAc terminals during the open field assay. (w,x) Photometry traces of GCaMP6s signal aligned to walk and rear events during the open field assay. All error bars represent ± SEM; n represent number of neurons. p values reported from two-tailed paired t-test (j,k). ***p < 0.001.

Extended Data Fig. 4 aPVTNTS-NAc terminal activity is negatively correlated with engagement in defensive freezing behaviors.

(a) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during Pavlovian fear conditioning and extinction. (b) Photometry trace of GCaMP6s signal during Pavlovian fear conditioning. (c) Quantification of GCaMP6s signal during foot shock (0-5 s, N = 11, p = 0.0002). (d) Photometry trace of GCaMP6s signal aligned to freeze end on extinction day 1. (e) Quantification of GCaMP6s signal aligned to freeze end (0-20 s, N = 11, p = 0.0024). (f) Correlation between freezing time and tone-aligned photometry Z-score (0-20 s) across 5 days of extinction (N = 11, R2 = 0.13, p = 0.0068). (g) Schematic for optogenetic manipulation of aPVT-NAc terminals using DIO-ChR2 and DIO-PPO. (h) 20hz optical stimulation ChR2 (N = 9) expressing aPVTNTS-NAc terminals during the 20 second cue presentation decreases freezing time compared to eYFP (N = 9) controls (p = 0.0053). (i) 10hz optical stimulation of PPO (N = 14) expression aPVTNTS-NAc terminals does not alter freezing behavior compared to eYFP (N = 11) controls (p = 0.1470). Error bars represent ± SEM (c,e), or min/max values (h,i); box plots represent interquartile range with line at median; N represent number of mice. p values reported from paired two-tailed t-test (c,e), unpaired two-tailed t-test (h,i) and simple linear regression (f). **p < 0.01, ***p < 0.001.

Extended Data Fig. 5 Neither activation nor inhibition of aPVTNTS-NAc terminals alters locomotion, drives real-time place preference (RTPP), or drive intracranial self-stimulation (ICSS).

(a) Schematic for optogenetic manipulation of aPVT-NAc terminals using DIO-ChR2 and DIO-PPO. (b) 20hz photo-stimulation of ChR2 expressing aPVTNTS-NAc terminals during cue presentation reduces engagement in consummatory behaviors (ChR2: N = 9 p = 0.0427, eYFP: N = 9), p = 0.8190). (c,d) Neither 20hz photo-stimulation (2 mins off, 2 mins on, 2 mins off) of ChR2 expressing aPVTNTS-NAc terminals (N = 10, p = 0.3655) nor eYFP expressing aPVTNTS-NAc terminals (N = 8, p = 0.8947) alters locomotor behavior. (e) 20hz photo-activation of aPVTNTS-NAc terminals does not support RTPP (ChR2: N = 10 p = 0.8141, eYFP: N = 9 p = 0.7191). (f) 20hz photo-activation (2 second burst per nose poke) of aPVTNTS-NAc terminals does not support ICSS (ChR2: N = 10 p = 0.0968, eYFP: N = 8, p = 0.1670). (g) 10hz photo-inhibition of aPVTNTS-NAc terminals does not support RTPP (PPO: N = 7 p = 0.7492, eYFP: N = 6 p = 0.3216). (h) 10hz photo-inhibition of aPVTNTS-NAc terminals does not support ICSS (PPO: N = 13 p = 0.2565, eYFP: N = 11 p = 0.4527). (i) Fiber placements for optogenetics cohort displayed in Fig. 2. Error bars represent ± SEM (b,e-h), or min/max values (c,d); box plots represent interquartile range with line at median; N represent number of mice. p values reported from paired two-tailed t-test (b,e-h), and one-way ANOVA (c,d). *p < 0.05.

Extended Data Fig. 6 Effect of CB1R antagonism on inhibition of aPVTNTS-NAc terminals and characterization of AAV1-FLEX-sgCNR1 deletion of CB1R from aPVT NTS neurons.

(a) Schematic for fiber photometry recordings from GCaMP6s expressing aPVTNTS-NAc terminals during Pavlovian reward conditioning. (b,c) Photometry trace with representative lick raster and heatmap of cue-aligned GCaMP6s signal in control mice or mice treated with 10 mg/kg SR141716A (N = 10). (d,e) Effect SR141716A on cue-aligned photometry Z-score (p = 0.0064) and total number of licks (p = 0.0270) throughout the session (N = 10). (f,g,h) Diagram showing % of neurons expressing NTS, CB1R, or NTS and CB1R in the aPVT from NTS-Cre mice injected with sgROSA (N = 6) or sgCNR1 (N = 6). (i) Quantification of % of total neurons with CB1R transcript (p = 0.0064). (j) % of NTS neurons CB1R transcript (p = 0.0333). (k) % of NTS neurons expressing CB1R (p = 0.0055). (l)% of total neurons expressing NTS (p = 0.6507). (m) % of total neurons that are NTS positive and CB1R negative (p = 0.0354). (n) % of total neurons that are CB1R positive and NTS negative (p = 0.1512). Error bars represent ± SEM (d,e), or min/max values (i-n); box plots represent interquartile range with line at median; N represent number of mice. p values reported from paired (d,e) and unpaired (i-n) two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Extended Data Fig. 7 Effect of AAV1-FLEX-sgCNR1 deletion of CB1R from aPVT NTS neurons and BLA Vglut1 neurons on reward consumption and Pavlovian reward conditioning.

(a) Schematic for CB1R deletion from the aPVT and photometry recordings ofGCaMP6s aPVTNTS-NAc terminals during adlib sucrose consumption and Pavlovian reward conditioning. (b) Fiber placements for aPVT CB1R deletion/aPVTNTS-NAc cohort in Fig. 3. (c,d) Photometry traces of GCaMP6s signal aligned first lick during adlib sucrose consumption from sgROSA (N = 7) and sgCNR1 (N = 7) injected mice. (e) Comparison of first lick-aligned (0-20 s) GCaMP6s signal between sgROSA (N = 7) and sgCNR1 (N = 7) injected mice (p = 0.1741) during adlib sucrose consumption. (f-h) Comparison of total lick number (p = 0.3705), bout length (p = 0.0393, and bout number (p = 0.2828), between sgROSA (N = 7) and sgCNR1 (N = 7) injected mice during adlib sucrose consumption. (i) Comparison GCaMP6s signal (0-20 s) during rewarded trials in vehicle treated sgROSA (N = 6) and sgCNR1 (N = 7) injected mice during Pavlovian reward conditioning (p = 0.0373). (j) Comparison of # of licks showing a main effect of DO34 treatment on reducing sucrose consumption (Main effects: Treatment p = 0.0025, genotype p = 0.0146, interaction p = 0.5607). (k,l) Photometry traces of GCaMP6s signal aligned to footshock during Pavlovian fear conditioning. (m,n) Photometry traces of GCaMP6s signal aligned to tone presentation of day 1 of fear extinction. (o) Schematic for CB1R deletion from the BLA and photometry recordings of GCaMP6s BLAVGlut-NAc terminals during adlib sucrose consumption and Pavlovian reward conditioning. (p) Fiber placements for BLA CB1R deletion/BLAVGlut-NAc photometry. (q,r) Photometry traces of GCaMP6s signal during adlib sucrose consumption from sgROSA (n = 7) and sgCNR1 (n = 7) mice. (s) # of licks during adlib sucrose consumption (sgROSA N = 7, sgCNR1 N = 7). (t) Comparison of lick-aligned GCaMP6s signal during adlib sucrose consumption. (u-x) Photometry traces of GCaMP6s signal from vehicle and DO34 treated sgROSA (N = 7) and sgCNR1 (N = 7) mice. (y) Comparison of engagement time demonstrating a main effect of both treatment and genotype (Main effects: Treatment p = 0.0025, genotype p = 0.0146, interaction p = 0.5607, (sgROSA N = 7, sgCNR1 N = 7). (z) Comparison of GCaMP6s signal (0-26 s post-cue) demonstrating a main effect of both treatment and genotype (Main effects: Treatment p = 0.0395, genotype p = 0.0066, interaction p = 0.6757, (sgROSA N = 7, sgCNR1 N = 7). (aa) Comparison of total lick number demonstrating a trend toward a main effect of treatment (Main effects: Treatment p = 0.0109, genotype p = 0.4333, interaction p = 0.4333, (sgROSA N = 7, sgCNR1 N = 7). All error bars represent min/max values; box plots represent interquartile range with line at median; N represent number of mice. p values reported from unpaired two-tailed t-test (e-i, s,t) or Two-way ANOVA (j, y-aa). *p < 0.05, **p < 0.01, ***p < 0.001.

Extended Data Fig. 8 AAV1-FLEX-sgCNR1 deletion of CB1R from aPVT NTS neurons does not affect fear conditioning or extinction and foot shock induced eCB release and binding to aPVT terminals in the NAc.

(a) Schematic for CB1R deletion from the BLA and photometry recordings of GCaMP6s BLAVGlut -NAc terminals during Pavlovian fear conditioning and extinction. (b,c) Photometry traces of GCaMP6s signal aligned to foot shock from sgROSA (N = 7) and sgCNR1 (N = 7) injected mice during Pavlovian fear conditioning. (d,e) Comparison of freezing behavior (p = 0.1880) and GCaMP6s signal (0-5 s, p = 0.4953) between sgROSA (N = 7) and sgCNR1 (N = 7) injected animals during fear conditioning. (f,g) Photometry traces of GCaMP6s signal aligned to cue presentation from sgROSA (N = 7) and sgCNR1 (N = 7) injected mice during fear extinction. (h,i) Comparison of freezing behavior (p = 0.9355) and GCaMP6s signal (0-20 s, p = 0.5312) from sgROSA (N = 7) and sgCNR1 (N = 7) injected mice. (j) Schematic for photometry recordings of GRABeCB2.0 or GRABeCBMUT during fear conditioning and fear extinction. (k) Photometry traces of GRABeCB2.0 (n = 8) or GRABeCBMUT (n = 4) signal during fear conditioning. (l) Photometry trace of GRABeCB2.0 (n = 8) or GRABeCBMUT (n = 4) signal during fear extinction. (m) Fiber placements for GRABeCB mice used in j–m and Fig. 4j–n. All error bars represent min/max values; box plots represent interquartile range with line at median; N represent number of mice. p values reported from unpaired two-tailed t-test (d,e,h,i).

Extended Data Fig. 9 Characterization of transsynaptic labeling, specificity of closed-loop optogenetic stimulation, and cluster classification.

(a) Schematic for validation of transsynaptic labeling approach. Transsynaptic AAV1-DIO-FLP was injected into the aPVT, AAV5-DIO-tdTomato was injected into the dorsolateral striatum (DLS), and a cocktail of AAV5-DIO-tdTomato and AAV5-fDIO-eYFP was injected in the NAc of Penk-Cre mice. The DLS, which expresses Penk but does not receive aPVT input, showed expression of AAV5-DIO-tdTomato but not AAV5-DIO-eYFP. The NAc, which expresses Penk and does receives aPVT input, showed expression of both AAV5-DIO-tdTomato and AAV5-DIO-eYFP. (b) Fiber placements for transsynaptic optogenetics cohort in Fig. 4 and representative image. (c-f) Closed-loop optogenetic stimulation during sipper port engagement does not affect % time disengaging, walking, rearing, or grooming. (g) Schematic for closed-loop optogenetic activation of aPVT-NAcPENK neurons during walk events. (h,i) Effect of walk elicited closed-loop activation of aPVT-NAcPENK neurons on % engagement time in ChRimson (N = 7) and eYFP (N = 5) expressing animals. (j,k) Effect of walk elicited closed-loop activation of aPVT-NAcPENK neurons on % walking time in ChRimson (N = 7) and eYFP (N = 5) expressing animals. (l) Lens placements for transsynaptic 1-photon imaging cohort in Fig. 5. (m) Dendrogram plot of hierarchical clustering of 206 tracked aPVT-NAcPenk neurons based on Principal Component Analysis (PCA) of the activity of each neuron in the 60 second window following cue-onset. (n) Percent of variance explained by the top 20 PCs. (o) Support Vector Machine decoding of cluster identity, trained on PavD5 cluster activity.

Extended Data Fig. 10 Validation of generalized linear model and Hilbert and Rayleigh analysis of cluster entrainment to aPVTNTS-NAc GCaMP6s and aPVT-NAc GRABeCB2.0 signal.

(a-e) Cue-aligned traces of % time engaging with sipper port, disengaging from sipper port, walking, rearing, and grooming on PavD1 and PavD5 (N = 10) from GRIN lens implanted animals. (f-g) Heatmap of % of neurons within each cluster encoding each behavioral state on PavD5, with a β coefficient threshold of +/− 0.5. (h,i) Heatmap of % of neurons within each cluster encoding each behavioral state on PavD1, with a β coefficient threshold of +/− 0.5. (j,k) Effect of individually dropping each predictor on model accuracy for PavD5 and PavD1. (l-p) Contribution of effect sizes for each neuron in each cluster to the generalized linear model (T values). (q-u) Overlay of Pavlovian reward conditioning day 5 aPVT-NAcPenk cluster GCaMP6s traces with aPVTNTS-NAc terminal GCaMP6s traces and Rayleigh plots from cluster 1, 2, 3, 4, and 5. (v-z) Overlay of Pavlovian reward conditioning day 5 aPVT-NAcPenk cluster GCaMP6s traces with aPVT-NAc terminal GRABeCB2.0 traces and Rayleigh plots from cluster 1, 2, 3, 4, and 5.

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Marcus, D.J., English, A.E., Chun, G. et al. Endocannabinoids facilitate reward engagement through retrograde gain control. Nature (2026). https://doi.org/10.1038/s41586-026-10967-w

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