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A cholinergic hub in the nucleus accumbens gates opioid-reward learning

Nature作者：S. Aryana Yousefzadeh2026年8月5日正文已收录本站

Data availability

Source data are available at https://github.com/tadrosslab/CIN_NaloxoneDART. Source data are provided with this paper.

Code availability

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Acknowledgements

We thank members of the Mouse Behavioral and Neuroendocrine Analysis Core Facility at Duke, including S. P. Steffens, A. Hellman and C. Means for help with collecting behavioural data; members of the Rodent Genetics and Breeding Core, including K. Cleveland, C. Jimenez and L. Russell for breeding and colony maintenance; A. Min for assistance with the elevated plus maze assay; A. West, S. Lisberger and Z. Farahbakhsh for insightful feedback on the manuscript. LLMs were used to aid in editing for clarity.

Funding

This work was funded by the National Institute on Drug Abuse (NIDA) R61-DA051530, R33-DA051530 and BRAIN initiative RF1-MH117055 and R01-MH132592 (to M.R.T.).

Author information

Author notes

  1. These authors contributed equally: Yunju Oh, Pyeonghwa Jeong

Authors and Affiliations

  1. Department of Neurosurgery, Duke University, Durham, NC, USA

    S. Aryana Yousefzadeh, Haidun Yan, Brenda C. Shields & Michael R. Tadross

  2. Department of Psychology and Neuroscience, Duke University, Durham, NC, USA

    S. Aryana Yousefzadeh

  3. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Haidun Yan, Shaun S. X. Lim, James M. Roach, Brenda C. Shields & Michael R. Tadross

  4. Department of Chemistry, Duke University, Durham, NC, USA

    Seung-Hwa Kwak, Yunju Oh, Pyeonghwa Jeong & Jiyong Hong

  5. Department of Psychiatry and Behavioral Sciences, Duke University, Durham, NC, USA

    Vladimir Pogorelov, Ramona M. Rodriguiz & William C. Wetsel

  6. Department of Neurobiology, Duke University, Durham, NC, USA

    J. Russell Ravenel, William C. Wetsel & Michael R. Tadross

  7. Mouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University, Durham, NC, USA

    Ramona M. Rodriguiz & William C. Wetsel

  8. Department of Cell Biology, Duke University, Durham, NC, USA

    William C. Wetsel

  9. Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA

    Jiyong Hong

Authors

  1. S. Aryana Yousefzadeh
  2. Haidun Yan
  3. Seung-Hwa Kwak
  4. Yunju Oh
  5. Pyeonghwa Jeong
  6. Vladimir Pogorelov
  7. J. Russell Ravenel
  8. Shaun S. X. Lim
  9. James M. Roach
  10. Brenda C. Shields
  11. Ramona M. Rodriguiz
  12. William C. Wetsel
  13. Jiyong Hong
  14. Michael R. Tadross

Contributions

S.A.Y.—conceptualization: evolution of research goals; methodology: piloting and optimization of Tango assays, microdialysis and behavioural paradigms; validation: validation of DART compounds, intracerebroventricular DART delivery and virus conditions; investigation: data collection for Tango assays, behavioural paradigms and microdialysis; data curation: management of the datasets; analysis: formal analysis of the data from the Tango assay, microdialysis and behavioural paradigms; writing: original draft, review and editing; visualization: preparation and presentation of figures; project administration: coordination of experiments among laboratories and laboratory members. H.Y.—investigation: data collection for the patch clamp experiments; analysis: formal analysis of the patch clamp data; writing: review and editing. J.R.R.—investigation: data collection for the open field assays and histology; analysis: formal analysis of the open field data; writing: review and editing. S.S.X.L.—methodology: design and molecular cloning of viral constructs; investigation: data collection for the elevated plus maze assay. J.M.R.—methodology: development of open field arenas and behavioural-tracking pipeline; analysis: formal analysis of behavioural-tracking data. B.C.S.—methodology: original DART platform development, preparation of DART aliquots for cell and behavioural assays, molecular cloning of viral constructs and piloting of Tango assay; writing: review and editing; project administration: coordination of experiments among laboratories. S.-H.K.—methodology: synthetic approaches to naloxoneDART; investigation: synthesis and analytical characterization of naloxoneDART; validation: verification of the overall replication and reproducibility of the synthesis of naloxoneDART; writing: review and editing. Y.O.—methodology: synthetic approaches to naloxoneDART; investigation: synthesis and analytical characterization of naloxoneDART; validation: verification of the overall replication and reproducibility of the synthesis of naloxoneDART; writing: review and editing. P.J.—methodology: synthetic approaches to naloxoneDART; investigation: synthesis and analytical characterization of naloxoneDART; validation: verification of the overall replication and reproducibility of the synthesis of naloxoneDART; writing: review and editing. V.P.—methodology: piloting of the microdialysis experiments; investigation: data collection for microdialysis experiments. R.M.R.—investigation: data collection for CPP and analgesia paradigms; project administration: coordination of behavioural experiments. W.C.W.—conceptualization: evolution of research goals; supervision: oversight and leadership responsibility for the microdialysis and behavioural experiments; writing: review and editing. J.H.—conceptualization: evolution of research goals; methodology: synthetic approaches to naloxoneDART; supervision: oversight and leadership responsibility for the synthesis of naloxoneDART; writing: review and editing. M.R.T.—senior author and lead contact; conceptualization: original conception and evolution of goals. supervision: oversight and leadership of overall project; methodology: reagent design, experimental design and troubleshooting throughout the project; software: MATLAB code; analysis: formal analysis of microdialysis, behaviour, histology, fitting and bootstrap statistics; visualization: preparation and presentation of figures; writing: review and editing; project administration: coordination of experiments among laboratories and laboratory members; funding acquisition: NIH R61-DA051530, R33-DA051530, RF1-MH117055 and R01-MH132592.

Corresponding author

Correspondence to Michael R. Tadross.

Ethics declarations

Competing interests

M.R.T. and B.C.S. are named as inventors on patents describing DART.2. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Julia Lemos 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 The efficacy of naloxone.xDART.2 variants against different opioid receptors.

Dose-response curves measuring Tango μOR, δOR, and κOR activity (y-axes) versus naloxone.xDART.2 concentration. Data were normalized to min/max of naloxone data from each plate. Each symbol represents mean ± SEM from two plate replicates; Lines are Langmuir binding curve fits, yielding AC50 for each ligand. Table shows the structure of the drug module for each variant as well as its AC50 against the opioid receptors.

Source data

Extended Data Fig. 2 Viral targeting strategy.

(a) Traditional strategy: cDIO (Cre-dependent HTP in ChAT::Cre mice). Example histology showing surface Alexa647DART capture in the nucleus accumbens. Representative image from 1 of 6 sections per mouse across 2 mice with similar results. The exemplar represents the best cDIO expression that we were able to achieve after comprehensive tests of six serotypes (AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10), each tested across three titers. (b) Optimized strategy: fDIO (Flp-dependent HTP + Cre-dependent FlpO in ChAT::Cre mice). Example histology under matched conditions (same ligand infusion, imaging parameters, and display contrast as panel a). Representative image from 1 of 6 sections per mouse across 2 mice with similar results. (c) fDIO yields approximately 6-fold higher expression than cDIO. Error bars are mean ± SEM Alexa647DART intensity (each symbol is one hemisphere; n = 12 cDIO hemispheres from 2 mice; n = 12 fDIO hemispheres from 2 mice). (d-e) Motivating hypothesis for the optimized viral strategy. Single-stranded AAV genomes contain reverse-complementary loxP sites that thermodynamically favor annealing, which we hypothesized could render them susceptible to reaction with Cre, even in single-stranded DNA, leading to trapped intermediates (panel d). We reasoned that this may be a particular problem for ChAT::Cre mice because the ChAT gene is expressed at high levels, resulting in high levels of pre-existing Cre. The fDIO approach was designed to avoid this trap by providing delayed FlpO expression, giving fDIO-HTP genomes enough time to become double-stranded before encountering FlpO (panel e).

Source data

Extended Data Fig. 3 Tethered naloxoneDART has no detectable off-target effects.

(a-b) Assay to test whether tethered naloxoneDART alters CIN excitability or spike waveform. Whole-cell current-clamp CIN recordings were obtained while the bath contained a traditional opioid receptor blocker (10 µM naloxone; throughout). Because opioid receptors were blocked throughout, this assay tests for non-opioid, off-target effects of tethered naloxoneDART on cellular excitability or spike shape (e.g., direct modulation of sodium or potassium channels). (a) Left: normalized CIN pacemaker firing rate (FR) before and after naloxoneDART tethering. Gray shading defines pre and post intervals; mean ± SEM over 5 cells. Right: each connected pair of symbols represents one cell’s FR pre- and post-naloxoneDART; error bars are mean ± SEM. FR did not differ pre vs post naloxoneDART (t4 = 1.67, P = 0.17, paired two-sided t-test). (b) Parametrization of spike shape. Table shows each parameter mean ± SEM over 5 cells, showing broad agreement between pre- and post-naloxoneDART conditions. (c) Assay to test whether tethered naloxoneDART directly modulates AMPARs on CINs. Whole-cell voltage-clamp CIN recordings were obtained while the bath contained GABAA, NMDA, and opioid receptor blockers (10 µM gabazine, 10 µM CPP, and 10 µM naloxone; throughout). Because opioid receptors were blocked throughout, this assay tests for off-target effects of tethered naloxoneDART on the AMPAR itself. Left: evoked AMPAR-mediated EPSC amplitude, normalized to baseline, mean ± SEM over 7 cells for controlDART and 8 cells for naloxoneDART. Gray shading indicates DART application. Right: representative EPSC waveforms before (dark) and after (light) DART application. Baseline-normalized EPSC amplitudes did not differ between naloxoneDART and controlDART (t13 = 0.68, P = 0.50, unpaired two-sided t-test). (d) Assay to test whether tethered naloxoneDART directly modulates GABAARs on CINs. Whole-cell voltage-clamp recordings were obtained in the presence of AMPA, NMDA, and opioid receptor blockers (10 µM DNQX, 10 µM CPP, and 10 µM naloxone; throughout). Because opioid receptors were blocked throughout, this assay tests for off-target effects of naloxoneDART on the GABAAR itself. IPSC amplitude normalized to baseline, mean ± SEM over 8 cells for controlDART and 9 cells for naloxoneDART (format as in c). Baseline-normalized IPSC amplitudes did not differ between naloxoneDART and controlDART (t15 = 0.44, P = 0.66, unpaired two-sided t-test). (e-f) Experiment to assay presynaptic opioid receptors on GABAergic afferents to CINs (related to Fig. 3a-b). The bath contained AMPA and NMDA blockers (10 µM DNQX, 10 µM CPP), and CINs were recorded in whole-cell voltage-clamp with intracellular Cs+ to block postsynaptic GIRK responses. Presynaptic opioid receptors were activated with DAMGO and then blocked by the addition of traditional naloxone. (e) Paired-pulse ratio, PPR (P2/P1), normalized to baseline, mean ± SEM over 7 cells. DAMGO applied at time zero; gray shading indicates naloxone application. (f) Summary of PPR in CINs following consecutive application of DAMGO and naloxone. One-way repeated-measures ANOVA revealed a significant effect of treatment (F1.58,9.45 = 89.68, P = 6.1 × 10−8). Tukey test showed that DAMGO significantly increased the PPR (P = 7.7 × 10−6), while naloxone significantly reduced the PPR (P = 0.0001).

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Extended Data Fig. 4 Specificity and penetrance of viral targeting.

(a) Example histology: cytosolic ChAT, nuclear NLSTomato, and surface Alexa647DART. Representative image from 1 of 3 sections per mouse across 28 mice with similar results. Dashed lines depict areas defined as NAc core, mShell, and mSeptum used for quantification. (b) Left: percentage of naloxoneDART recipients that are not CINs in the NAc mShell (X0) plotted as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1). Middle: percentage of CINs receiving naloxoneDART in the NAc core (X2) plotted as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1). Right: percentage of CINs receiving naloxoneDART in the medial septum (X3) plotted as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1). Each symbol represents one mouse. Curves are obtained by collapsing each scatterplot onto one-dimensional histograms and fitting those with lognormal functions along each axis.

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Extended Data Fig. 5 Dominant contribution of mShell to CPP.

(a) Top: CPP as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage of naloxoneDART recipients that are not CINs in the NAc mShell (X0). Each symbol is one mouse with color denoting the CPP score (blue = high CPP score, pink = low CPP score). Colored surface is the best-fit joint model: CPP = f(β1·X1 + β0·X0) where f() is the sigmoidal function (Methods). Gray shading denotes the 95% confidence interval over bootstraps. Bottom: joint distribution of normalized regression weights βx1 and βx0. The shaded triangular region (βx1 > βx0) indicates iterations where X1 is the dominant predictor of behavior; P is the proportion in this region. (b) CPP as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage of CINs receiving naloxoneDART in the NAc core (X2; Format as in a). (c) CPP as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage of CINs receiving naloxoneDART in the medial septum (X3; Format as in a). (d) Left: CPP score of individual mice (symbols), kernel density estimate (shaded violin), bootstrap of the mean (outlined violin), and mean (dashed line). Right: absolute difference in mean CPP score (|naloxoneDART − controlDART|; dashed gray line) differs significantly from its null distribution (black; P = 0.032, two-sided permutation test). (e) Left: CPP score as a function of the percentage of CINs receiving naloxoneDART in the NAc mShell (X1). Each symbol represents one mouse. Fit parameters: FitC (controlDART, 0% CINs), fitN (naloxoneDART, 100% CINs), and %mid (X1 at sigmoidal midpoint). Black curve (sigmoid fit) and sigmoid shading (bootstrap 95% confidence interval). Right: floating y-axis shows that |fitN − fitC| (dashed gray line) differs significantly from its null distribution (black; H∅: fitN = fitC; P = 0.0017, two-sided permutation test). (f) Left: CPP score as a function of the percentage of CINs receiving naloxoneDART in the NAc core (X2; format as in e). Right: |fitN − fitC| differs significantly from its null distribution (P = 0.028, two-sided permutation test). (g) Left: CPP score as a function of the percentage of CINs receiving naloxoneDART in the medial septum (X3; format as in e). Right: |fitN − fitC| differs significantly from its null distribution (P = 0.034, two-sided permutation test). (h) Left: ∆post of individual mice (format as in d). Right: absolute difference in mean ∆post (|naloxoneDART − controlDART|; dashed gray line) differs significantly from its null distribution (P = 0.035, two-sided permutation test). (i) Left: ∆post as a function of the percentage of CINs receiving naloxoneDART in the NAc mShell (X1; format as in e). Right: |fitN − fitC| differs significantly from its null distribution (P = 0.0011, two-sided permutation test). (j) Left: ∆post as a function of the percentage of CINs receiving naloxoneDART in the NAc core (X2; format as in e). Right: |fitN − fitC| differs significantly from its null distribution (P = 0.017, two-sided permutation test). (k) Left: ∆post as a function of the percentage of CINs receiving naloxoneDART in the medial septum (X3; format as in e). Right: |fitN − fitC| differs significantly from its null distribution (P = 0.033, two-sided permutation test).

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Extended Data Fig. 6 Dominant contribution of mSeptum to between-session habituation.

(a) Left: between-session habituation (BSH; speed in second divided by first saline session). Individual mice (symbols), kernel density estimate (shaded violin), bootstrap of the mean (outlined violin), and mean (dashed line). Right: absolute difference in mean BSH (|naloxoneDART − controlDART|; dashed gray line) differs significantly from its null distribution (black; P = 0.011, two-sided permutation test). (b) Left: habituation as a function of the percentage of CINs receiving naloxoneDART in the NAc mShell (X1). Each symbol represents one mouse. Fit parameters: fitC (controlDART, 0% CINs), fitN (naloxoneDART, 100% CINs), and %mid (X1 at sigmoidal midpoint). Black curve (sigmoid fit) and sigmoid shading (bootstrap 95% confidence interval). Right: floating y-axis shows that |fitN − fitC|(dashed gray line) does not significantly differ from its null distribution (black; H∅: fitN = fitC; P = 0.062, two-sided permutation test). (c) Left: BSH as a function of the percentage of CINs receiving naloxoneDART in the NAc core (X2; format as in b). Right: |fitN − fitC| does not significantly differ from its null distribution (P = 0.148, two-sided permutation test). (d) Left: BSH as a function of the percentage of CINs receiving naloxoneDART in the medial septum (X3; format as in b). Right: floating y-axis shows that |fitN − fitC| (dashed gray line) significantly differs from its null distribution (P = 0.030, two-sided permutation test). (e) Top: BSH as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage naloxoneDART recipients that are not CINs in the NAc mShell (X0). Each symbol is one mouse with color denoting the BSH (blue = high BSH, pink = low BSH). Colored surface is the best-fit joint model: BSH = f(β1·X1 + β0·X0) where f() is the sigmoidal function (Methods). Gray shading denotes the 95% confidence interval over bootstraps. Bottom: joint distribution of normalized regression weights βx1 and βx0. The shaded triangular region (βx1 > βx0) indicates iterations where X1 is the dominant predictor of behavior; P is the proportion in this region. (f) BSH as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage of CINs receiving naloxoneDART in the NAc core (X2; Format as in e). (g) BSH as a function of percentage of CINs receiving naloxoneDART in the NAc mShell (X1) and percentage of CINs receiving naloxoneDART in the medial septum (X3; Format as in e). (h-k) Within-session habituation (WSH; speed in last 15 min divided by lastfirst 15 min, average over saline sessions). Format as in panels a-d, showing no significant naloxoneDART effects (two-sided permutation test).

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Extended Data Fig. 7 Other behavioral measurements versus histology.

(a-d) Hyperlocomotion (mean speed [cm/sec] during first morphine session). Black curve (sigmoid fit) and sigmoid shading (bootstrap 95% confidence interval). Format as in Extended Data Fig. 6a-d, showing no significant naloxoneDART effects (two-sided permutation test). (e-h) Sensitization (mean speed [cm/sec] during the challenge morphine session). Format as in panels a-d, showing no significant naloxoneDART effects. (i-l) Mean hot plate latency (sec). Format as in panels a-d, showing no significant naloxoneDART effects. (m-p) Mean tail flick latency (sec). Format as in panels a-d, showing no significant naloxoneDART effects.

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Extended Data Fig. 8 Absence of anxiety-like behavior in naloxoneDART mice.

Left: percentage of time spent in the open arms (time in open arm/total time). Each symbol is one mouse (male: square, female: circle). The box plot spans the 25th to 75th percentiles with the line as median. Whiskers extend to 1.5 times the interquartile range. Separate error bars are mean ± SEM. Two-way repeated-measures ANOVA revealed no main effect of DART (F1,10 = 0.063, P = 0.81) or DART × systemic drug interaction (F1,10 = 0.47, P = 0.51). Middle: percentage of time spent in the closed arms (time in closed arm/total time). Two-way repeated-measures ANOVA revealed no main effect of DART (F1,10 = 0.41, P = 0.54) or DART × systemic drug interaction (F1,10 = 0.21, P = 0.65). Right: percentage of time spent in the center (time in center zone/total time). Two-way repeated-measures ANOVA revealed no main effect of DART (F1,10 = 1.70, P = 0.22) or DART × systemic drug interaction (F1,10 = 1.97, P = 0.19). Post hoc tests were corrected for multiple comparisons using the Bonferroni method.

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Extended Data Fig. 9 Effects of CIN-specific naloxoneDART on morphine-induced dopamine metabolites.

(a) Time course of normalized change in dialysate 3,4-dihydroxyphenylacetic acid (DOPAC) (format as in Fig. 5b). (b) Δ[DOPAC]norm averaged 1-2 hr post injection (format as in Fig. 5c). Morphine increased Δ[DOPAC]norm in controlDART (left: P = 0.0002; morphine − saline = +0.22 [+0.16, +0.29]) and naloxoneDART mice (right: P = 0.0005; morphine − saline = +0.15 [+0.09, +0.21]). Post-morphine Δ[DOPAC]norm did not differ in controlDART vs naloxoneDART mice (center: P = 0.46; controlDART − naloxoneDART = +0.03 [−0.04, +0.09]). (c) Time course of normalized change in homovanillic acid (HVA) (format as in Fig. 5b). (d) Δ[HVA]norm averaged 1-2 hr post injection (format as in Fig. 5c). Morphine increased Δ[HVA]norm in controlDART (left: P = 0.00004; morphine − saline = +0.18 [+0.13, +0.23]) and naloxoneDART mice (right: P = 0.001; morphine − saline = +0.13 [+0.08, +0.19]). Post-morphine Δ[HVA]norm did not differ in controlDART vs naloxoneDART mice (center: P = 0.99; controlDART − naloxoneDART = 0.00 [−0.05, +0.05]).

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Yousefzadeh, S.A., Yan, H., Kwak, SH. et al. A cholinergic hub in the nucleus accumbens gates opioid-reward learning. Nature (2026). https://doi.org/10.1038/s41586-026-10887-9

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