Dandiset 001692

Reproducing Bouabid, Vu et al. (2026) from dandiset 001692 33

Notebook reproduce_bouabid_2026.ipynb

• ✕Not reproducedExtended Data Figure 1i-k; Results, first section

  Mice expressing non-functional sensors (a mutant ACh sensor and tdTomato, n = 3) show only sparse, weak positive red-green correlations with no anatomical organization; no hotspot is found.

  In AD4 to AD6 (ACh4.3mut and tdTomato) the hotspot procedure finds a hotspot with mean smoothed r +0.33 (551 voxels, centroid AP 0.68, ML 1.29, DV -4.14 mm), so by the paper's test the claim of no organization fails. The procedure also finds a hotspot in 73 of 100 shuffles of the control values across sites, but none of those reaches 551 voxels (31 have a mean of at least +0.33), so by the paper's own test the control correlations are organized in space. The correlations are mostly positive (68% of 124 sites, median 0.20), as stated, but not weak; the median absolute dominant r is 0.238 against 0.240 in the sensor mice, and 65% of control sites exceed 0.2 against 54%. One control site has no defined correlation (a constant red trace) and is dropped; 19 site-sessions have constant red traces. Keeping the reward periods gives median r 0.22. Two deviations could add correlated red-green signal and so bear on this outcome; the controls use the high-pass series without artifact removal (none is in their files), and only their 15 reward sessions, where the paper used all ITI data.

  How the test differed from the paper’s method

  • The 15 uncued reward sessions of the control mice are used, with 6 s after rewards and first licks left out; the paper used the ITI data of all their sessions, which the files' event tables would allow but which would take most of the time limit to stream.
  • The control files hold no artifact-removed series; the high-pass series is used.
• ✕Not reproducedResults, fourth section; Figure 4a

  After Pavlovian conditioning, the conditioned light cue evokes an ACh peak, then a DA peak, then an ACh dip at a majority of sites (49 of 75).

  The sequence is found at 58 of 75 sites under both readings (code percentiles 55 and 52), above the tolerance of 44.1 to 53.9. The qualitative statement holds; a majority of sites shows the sequence overall and in each mouse (12 of 17, 19 of 24, 27 of 34). The criterion counted only a shortfall as partial; a count above the range is not reproduced by its wording.

  How the test differed from the paper’s method

  • The paper does not define how the sequence is identified; significant ACh peak, DA peak and ACh dip with ordered extremes in the mean trace was chosen here.
  • Learning sessions are identified from the events (every light cue rewarded); the last 3 per mouse are used, following the code's intent (see findings on the code).
  • Tone trials cannot be analysed (no tone events); the paper's figure uses light trials.
• ✕Not reproducedResults, fifth section; Figure 5e-g

  Initiation-related DA dips are concentrated in the aDLS and DA peaks in dorsomedial and posterior striatum; ACh dips are localized to the aDLS.

  Dominant DA dips are more anterior than DA rises (median AP 0.36 against -0.48 mm, p < 0.0001) but more medial (ML 1.88 against 2.30 mm) and ventral; ACh-dip sites (148 of 174) are not more anterior or lateral than the 26 others (p = 0.33 and 0.25). All 28 hotspot sites have a dominant DA dip and an ACh dip. The paper describes maps; with ACh dips at 85% of sites a contrast between sites with and without dips may not capture where dips are strongest.

  How the test differed from the paper’s method

  • The paper shows the topography as smoothed maps; site-level coordinate tests were chosen here.
• ◐Partially reproducedResults, first section; Figure 1f

  The dominant correlations form a significant hotspot, identified by local Moran's I, in the anterior dorsolateral striatum (aDLS), where the strongest correlations are negative.

  The hotspot procedure finds a significant hotspot of 1.01 mm3 with mean smoothed r -0.368 (hull mean -0.185). Its centroid (AP 0.63, ML 2.02, DV -3.14 mm) is anterior and dorsal of the median site (0.10, 2.04, -3.21 mm) but 0.02 mm medial of it, which fails the lateral part of the criterion fixed beforehand. With the 99th percentile of the null the hotspot has 518 voxels and its centroid is at ML 2.09 mm, lateral of the median. 28 sites from all five mice are inside. The location test against the median site is a coarse stand-in for the paper's maps, in which the hotspot lies in aDLS; the shortfall is small. Finding a hotspot is not evidence on its own, as the procedure finds one in 67 of 100 shuffles of the values across sites; none of these is as large (at most 482 voxels) or has a mean as low.

  How the test differed from the paper’s method

  • The hotspot procedure is simplified from the paper's code (linear in place of natural-neighbor interpolation, 100 um voxels in place of 50 um, the convex hull of the sites in place of the Allen striatum mask, 200 in place of 10000 shuffles).
  • The paper does not define the aDLS location in numbers; the criterion compares the centroid with the median site coordinates.
• ◐Partially reproducedResults, second section; Figure 2d

  Magnitudes of paired transients are negatively correlated at many sites for opposite-sign pairs (121 of 174 for ACh troughs with DA peaks, 81 of 174 for ACh peaks with DA troughs) and positively for same-sign pairs (97 of 174 peak-peak, 78 of 174 trough-trough), with no site significant in the other direction.

  Significant sites of the stated sign are 114, 69, 97 and 78 against 121, 81, 97 and 78; the second is 3.9 below its tolerance. The counts move by several sites with the random draws (the first run gave 115, 70, 90 and 76). No site is significant in the opposite direction, as stated. All sites have at least 100 pairs. The code's computation (Pearson r over all pairs, p < 0.05) finds 142 to 149 sites per type, so the paper's counts fit the Methods' bootstrap.

  How the test differed from the paper’s method

  • 1000 in place of 10000 repetitions of the 100-pair bootstrap.
• ◐Partially reproducedFigure 2d; Results, second section

  Magnitude correlations of opposite-sign pairs, but not same-sign pairs, are stronger inside the aDLS hotspot than outside (rank-sum p < 0.05 for opposite-sign pairs, p > 0.05 for same-sign pairs).

  ACh troughs with DA peaks are more negative inside (median -0.33 against -0.23, p = 0.0001); ACh peaks with DA troughs do not differ (-0.19 against -0.16, p = 0.11). Same-sign pairs do not differ (p = 0.11 and 0.22), as stated.

• ◐Partially reproducedResults, third section; Figure 3b

  After unpredicted rewards, trial-by-trial DA peak magnitude correlates with ACh; dominant correlations are negative at 114 of 174 sites with DA leading (mean latency 177.9 +/- 96.7 ms) and positive at 58 of 174 with ACh leading (-41.2 +/- 218.9 ms).

  122 sites are dominant negative (mean latency 172.1 ms, 120 with DA leading) and 52 dominant positive (mean latency -59.8 ms, 37 with ACh leading). Three values are within tolerance; the positive count is 0.2 below its range (52.2 to 63.8). Negative sites outnumber positive ones in every mouse.

  How the test differed from the paper’s method

  • The latency tolerances are the standard errors implied by the paper's spreads.
  • One UG27 reward session (240313r) is skipped for lacking the artifact-removed green series.
• ◐Partially reproducedResults, fourth section; Figure 4c

  For conditioned light cues, ACh correlates with the trial-by-trial DA peak negatively at lagging times at 39 of 75 sites (ACh lagging, 202.28 +/- 128.4 ms) and positively at leading times at 26 of 75 (ACh leading, -79.1 +/- 224.0 ms).

  All 75 sites have a significant cue DA peak, so the two readings agree. 41 sites are negative (mean latency 206.0 ms, 37 with ACh lagging) and 34 positive (-83.3 ms, 22 with ACh leading). The positive count is above its range (23.4 to 28.6); the paper's 39 plus 26 make 65, which fits neither reading here. The code's percentiles also give 75 sites, and the null draws from 68868 to 74196 candidate times per mouse, so neither explains the difference. The difference remains unexplained.

  How the test differed from the paper’s method

  • The denominator is ambiguous (75 sites or those with a significant DA peak); both readings are shown and agree here.
• ◐Partially reproducedResults, fifth section; Figure 5d

  At locomotion initiations, ACh rises and then falls at 131 of 174 sites; DA dips at 119 of 174 sites, rises at 73, and both rises and dips at 32.

  DA dips at 129 sites and rises at 77 are within 10% (the paper 119 and 73); both at 46 is not (32). ACh rise then fall at 107 sites under reading (a) and a rise at 147 under reading (b), neither within 117.9 to 144.1. Code percentiles give 114 dips, 61 rises and 26 both. The code's bout of 2.2 s gives 132, 74 and 44. UG27 contributes 501 initiations against 92 to 149 for the others; its wheel velocity exceeds the threshold 49% of the time.

  How the test differed from the paper’s method

  • A zero-phase Butterworth filter replaces MATLAB's lowpass.
  • 1000 bootstrap samples in place of 10000.
  • The Methods' bout of 2.27 s is used; the code's 2.2 s is a variant.
• ◐Partially reproducedResults, seventh section; Figure 7e

  DA stimulation evokes significant ACh suppression at a subset of sites (116 of 180, 64%).

  147 of 180 sites (82%) show a significant ACh dip against 116 (64%); 128 (71%) with the code's percentiles. The 33 sites without a dip are mostly in ADS19 (17 of 27) and ADS16 (9 of 37).

  How the test differed from the paper’s method

  • 1000 bootstrap samples in place of 10000.
• ◐Partially reproducedExtended Data Figure 9a-d; Results, seventh section

  The stimulation cohort replicates the aDLS anti-correlation hotspot in spontaneous release outside stimulation periods; its dominant correlations are more negative inside the original hotspot, and its own hotspot overlaps the original.

  The 9 cohort sites inside the original hotspot are more negative (median -0.42 against -0.30, p = 0.049, exact p = 0.048; OR exclusion p = 0.041). The cohort's own hotspot is negative (mean -0.44, 1536 voxels, centroid AP 0.71, ML 2.00, DV -3.45 mm) and shares 564 voxels with the original against a null median of 65, but overlap p = 0.085 with 200 random volumes. 167 of 180 cohort sites are dominant negative. The AND exclusion leaves out 6.5% of samples and the OR exclusion 32.7%. Of the samples within 3 s after a stimulation, AND keeps 42% and OR none. With OR the cohort hotspot is similar (mean -0.41, centroid AP 0.72, ML 2.08, DV -3.43 mm), with overlap p = 0.085.

  How the test differed from the paper’s method

  • The code's AND of the three exclusions is followed; OR is a variant.
• ✓ReproducedResults, first section; Figure 1e

  Across 174 sites in 5 mice, the dominant DA-ACh cross-correlation (the largest magnitude Pearson r within 1 s lags) during spontaneous behavior is negative at 132 sites, 127 of them with DA leading, and positive at 42 sites, 31 of them with ACh leading.

  Of 174 sites, 133 have a dominant negative correlation (128 with DA leading) and 41 a dominant positive one (32 with ACh leading); 6 have their extreme at lag 0. All four counts are within 10% of the paper's. Every mouse has more negative than positive sites. Correlation over concatenated sessions gives 133, 128, 41 and 30.

  How the test differed from the paper’s method

  • One UG27 reward session (240313r) lacks the artifact-removed green series and is skipped, as the code skips empty series; it is not a spontaneous session, so this claim is unaffected.
• ✓ReproducedFigure 1h

  Sites inside the aDLS hotspot have more negative dominant correlations than sites outside (Wilcoxon rank-sum p = 1.67e-16).

  The 28 sites inside have median dominant r -0.41 against -0.14 for the 146 outside, rank-sum p = 1.9e-13 (the paper 1.67e-16). The test is partly circular, as in the paper.

  How the test differed from the paper’s method

  • The hotspot is the simplified one of fig1f-adls-hotspot.
• ✓ReproducedResults, first section; Figure 1f

  Dominant positive correlations lie in the posterior striatum.

  Sites with a positive dominant r have median AP -0.40 mm (41 sites) against 0.24 mm for negative sites (133), one-sided rank-sum p = 4.3e-07. The direction holds in all five mice and is significant in UG27, UG29 and UG31.

  How the test differed from the paper’s method

  • The paper shows this on maps; the test of AP coordinates was chosen here.
• ✓ReproducedExtended Data Figure 2d; Results, first section

  The hotspot is consistent across mice; in each mouse, sites inside the group hotspot have more negative dominant correlations than sites outside.

  All five mice have lower median r inside the group hotspot; p < 0.05 in UG27, UG28, UG30 and UG31, and p = 0.084 (exact p = 0.087) in UG29, which has 2 sites inside.

  How the test differed from the paper’s method

  • The per-mouse hotspots of Extended Data Figure 2b-c are not computed.
• ✓ReproducedFigure 2a; Results, second section

  For coincident transients of opposite polarity (ACh trough with DA peak, ACh peak with DA trough) the DA transient leads; for same-polarity pairs (peak with peak, trough with trough) the ACh transient leads.

  Median directionality indices are -0.27 (ACh trough with DA peak), -0.30 (ACh peak with DA trough), +0.33 (peak with peak) and +0.12 (trough with trough) over 174 sites, each with Wilcoxon p below 1e-24, and each mouse has the same four signs.

  How the test differed from the paper’s method

  • Pairs are matched by the Methods' rule (each first transient takes its earliest partner, each partner used once), a simplification of the code's two-way matching.
  • The code keeps only sites flagged as having signal in both channels (`sig`); these flags are not in the files and all 174 included sites are used, as the paper reports 174.
• ✓ReproducedFigure 2b-c; Results, second section

  The frequency of paired transients is highest in aDLS, in a region overlapping the correlation hotspot, for all four pair types (volume overlap test, p < 0.01 for each).

  The occurrence hotspot of each pair type overlaps the correlation hotspot by 780 to 826 voxels against null medians of 88 to 111 (overlap p 0.005, 0.015, 0.005 and below 0.005; the paper 0.005, 0.005, 0.002 and below 0.001). Occurrence is higher inside than outside for each type (p < 0.001). With the chance occurrence subtracted (DA and ACh transients from different sessions of the same site, as in the Methods), every site keeps an excess for every type, the excess is higher inside (p < 0.001), and its hotspots overlap the correlation hotspot with p 0.030, 0.005, 0.015 and below 0.005.

  How the test differed from the paper’s method

  • 200 random volumes in the overlap test, so p cannot fall below 0.005.
  • The chance occurrence is the mean of 10 shuffles of sessions, in place of 10000.
• ✓ReproducedExtended Data Figure 3d-e

  DA and ACh transient rates are higher at sites inside the global correlation hotspot than outside.

  All four transient types are more frequent inside the hotspot (for example DA peaks 0.226 against 0.176 per second of usable data), p of 0.0013 or less. Rates are now per second of data that is not NaN and not within 6 s after a reward; the first run divided by the whole session and gave the same result.

• ✓ReproducedFigure 3d

  Reward-evoked DA-ACh correlations are significantly more negative inside the global hotspot than outside.

  Median r -0.52 inside against -0.31 outside, p = 7.3e-09; same sign in all five mice, significant in three.

• ✓ReproducedFigure 3c, 3e

  Reward anti-correlations form an aDLS hotspot that significantly overlaps the hotspot of spontaneous correlations (volume overlap test, p = 0.008).

  The reward hotspot has mean -0.53 (1022 voxels, centroid AP 0.54, ML 2.07, DV -3.26 mm) and shares 721 voxels with the spontaneous hotspot against a null median of 116, overlap p = 0.01 (the paper 0.008).

  How the test differed from the paper’s method

  • 200 random volumes in the overlap test.
• ✓ReproducedResults, fourth section; Figure 4g-h

  After cue-specific extinction (light cue rewarded on 20% of trials), cue-evoked DA dips correlate negatively with lagging ACh (DA dip then ACh late peak) at 41 of 74 sites, mean lag 117.9 +/- 176.2 ms.

  Under reading (a), 38 of 61 sites with a significant DA dip are negative with mean lag 118.4 ms (the paper 41 of 74, 117.9 ms). Over all 75 sites, 47 negative with mean lag 114.7 ms. Only 28 of the 38 have ACh lagging, and AD2's negative sites have a mean lag of -79.4 ms. The code's polarity (DA peak) gives 44 sites with mean lag 137.6 ms.

  How the test differed from the paper’s method

  • The DA trough is used, as the legend and Methods state; the code correlates the DA peak in this step, shown as a variant.
  • The paper's denominator (74) and the legend's 48 sites in 5 mice do not match each other or the 75 sites in 3 mice of the files; two readings are shown.
• ✓ReproducedFigure 4d-e

  Conditioned-cue DA-ACh correlations are more negative at sites inside the global aDLS hotspot than outside.

  The 9 AD1 to AD3 sites inside the global hotspot have median r -0.31 against 0.02 for the 66 outside, p = 0.024 (exact p = 0.022). The cue hotspot's own overlap with the global hotspot is not significant (p = 0.175), unlike the paper's Figure 4e.

  How the test differed from the paper’s method

  • Sites of the Pavlovian cohort are placed in the global hotspot by their coordinates on the shared grid.
• ✓ReproducedFigure 4i-j

  After extinction, DA dip to ACh correlations are more negative at sites inside the global aDLS hotspot than outside.

  Among dip sites, median r -0.35 inside (8 sites) against -0.21 outside (53), p = 0.035 (exact p = 0.034); all sites p = 0.018. The extinction hotspot's overlap with the global one is not significant (p = 0.17).

• ✓ReproducedResults, fifth section

  Initiation-related signals occur in sequence; DA dips first (median latency 111.11 ms), then ACh peaks (222.22 ms) and DA peaks (227.78 ms), then ACh dips (611.11 ms).

  Medians are 111.1 ms (DA dips), 277.8 ms (ACh peaks), 277.8 ms (DA peaks) and 666.7 ms (ACh dips), in the stated order; the ACh peak and dip medians are exactly one sample later than the paper's, at the edge of the tolerance. The per-mouse order is irregular (DA peak medians from -388.9 to 527.8 ms).

  How the test differed from the paper’s method

  • The Methods do not say how latency is measured; the time of the extreme of the triggered average in -0.5 to 1 s was chosen here.
• ✓ReproducedResults, sixth section; Figure 6a

  At sites with initiation-related DA dips (119 sites), trial-by-trial DA dip magnitudes correlate negatively with ACh with DA leading (mean latency 193.80 +/- 177.68 ms).

  Of 129 sites with a DA dip (the paper 119), 98 are dominant negative (88 with ACh lagging), mean latency 188.8 ms under reading (a); 98.2 ms under reading (b). Most dip sites are negative in every mouse. The trough search now ignores NaNs; before the fix a trough within 2 samples of a NaN was missed. 2197 of 30341 site-trials have a NaN in the range searched, so at most that many were affected. Before the fix the result was 99 sites and 182.4 ms.

  How the test differed from the paper’s method

  • The latency tolerance is the standard error implied by the paper's spread.
• ✓ReproducedFigure 6b-d

  Initiation DA dip to ACh correlations are concentrated in the aDLS and more negative inside the global hotspot than outside.

  Median r -0.35 inside (28 sites) against -0.21 outside (101), p = 1.7e-05 (6.1e-08 before the fix of the trough search).

• ✓ReproducedResults, sixth section; Figure 6e-h; Methods, generalized linear model

  At locomotion invigorations, GLM-estimated DA peak magnitudes correlate negatively with ACh at lagging times, and these correlations are concentrated in the aDLS, more negative inside the global hotspot (131 sites with invigoration DA peaks).

  The GLM finds invigoration DA peaks at 160 sites (the paper 131); 105 are dominant negative (97 with ACh lagging, mean lag 179.4 ms); median r -0.34 inside (28) against -0.21 outside (132), p = 4.5e-05.

  How the test differed from the paper’s method

  • Ordinary least squares in numpy in place of MATLAB fitglm (normal family, identical model).
• ✓ReproducedResults, seventh section; Extended Data Figure 10b

  Optogenetic stimulation of midbrain DA neurons evokes significant DA release at 179 of 180 sites.

  179 of 180 sites show a significant DA peak after ITI stimulation (178 with the code's percentiles); 115 ITI trains in total (20 per mouse, 15 in ADS6).

• ✓ReproducedFigure 7d, 7f-g

  Stimulation-evoked ACh dips are larger inside the cohort's correlation hotspot than outside, and their own hotspot overlaps it.

  ACh minima are deeper inside the cohort hotspot (median -0.0179 against -0.0149 dF/F, 17 against 163 sites, p = 0.0005). The AND exclusion keeps 42% of the samples within 3 s after a stimulation, so its cohort hotspot is built partly from stimulation responses; with the hotspot of the OR exclusion, which leaves them out, the result holds (19 against 161 sites, median -0.0188 against -0.0149, p = 0.0003). Against the original hotspot p = 0.094; the ACh-minimum hotspot's overlap with the cohort hotspot has p = 0.065, so the claim's second part is not supported at p < 0.05; the criterion treats it as a variant. Inside sites are lower in all six mice, significantly in ADS19.

• ✓ReproducedFigure 8bdata in Zenodo doi:10.5281/zenodo.18202873, file Bouabid_Vu (2026)_Source_Data_Fig8.xlsx

  The D2 agonist quinpirole suppresses evoked ACh release in DLS and NAcC, more strongly in DLS; two-way ANOVA region effect F(1,40) = 105.9, region by stimulus interaction F(4,40) = 3.014, stimulus effect F(4,40) = 17.80.

  Region F 105.87, interaction F 3.014 and stimulus F 17.80 match the paper; DLS mean 25.1 against NAcC 68.1. Type III sums of squares are used, as Prism computes them; Type II gives the same values here.

• ✓ReproducedFigure 8everifier disagreed at firstdata in Zenodo doi:10.5281/zenodo.18202873, file Bouabid_Vu (2026)_Source_Data_Fig8.xlsx

  Cocaine reduces evoked ACh release more in DLS than in NAcC; two-way ANOVA stimulus by region interaction F(4,55) = 10.75, stimulus effect F(4,55) = 36.37, region effect F(1,55) = 199.9.

  With Type III sums of squares, interaction F 10.75, stimulus F 36.37 and region F 199.86 match the paper (10.75, 36.37 and 199.9); DLS 32.8 against NAcC 74.1. The design is unbalanced (8 DLS and 5 NAcC slices). The first run used Type II sums of squares, which give stimulus F 47.68, and scored the claim partially reproduced.

• ✓ReproducedFigure 8f-gdata in Zenodo doi:10.5281/zenodo.18202873, file Bouabid_Vu (2026)_Source_Data_Fig8.xlsx

  The D2 antagonist L-741,626 attenuates the cocaine-induced suppression in both regions; D2R block effect F(1,55) = 231.9 in DLS and F(1,40) = 32.57 in NAcC.

  Block F 231.87 (DLS) and 32.57 (NAcC) match; the antagonist raises ACh in both (32.8 to 78.6, 74.1 to 107.9). With Type III sums of squares the DLS stimulus F is 34.46, as in the legend (Type II 45.04).

• ⊘BlockedFigure 8c· Methods underspecifieddata in Zenodo doi:10.5281/zenodo.18202873, file Bouabid_Vu (2026)_Source_Data_Fig8.xlsx

  Cocaine elevates evoked DA release to a similar degree in DLS and NAcC (two-way ANOVA, cocaine by region interaction F(8,36) = 0.20, p = 0.98).

  The sheet Figure_8c_TP holds a voltammogram (voltage and current). Figure_8c_LP and _RP hold mean DA time courses (24 time points from 0.625 to 3.5 s) for three mice per region and two conditions. The legend's ANOVA has 8 degrees of freedom for cocaine and 36 residual, which fits neither a two-level cocaine factor nor the 24 time points, so the test cannot be repeated. Descriptively, the cocaine peak is 1.48 times the control peak on average in both regions (per mouse 1.31 to 1.77 in DLS, 1.10 to 2.09 in NAcC).