Dandiset 001084

Reproducing Vu et al. (2024) from dandiset 001084 21

Notebook reproduce_vu_2024.ipynb

• ◐Partially reproducedFigure 1D; Results, second section; code Fig01/D

  Dopamine release to unpredicted water reward is reliable and of comparable amplitude over at least 12 weeks after implantation (median over 44 fibers, normalized to week 5).

  In the 17 sessions with rewards and 25 rewards or fewer (2021-08-19 to 2021-09-13), the median over the 44 fibers of the weekly peak reward response, relative to week 5, is 0.903, 0.932 and 1.196 in weeks 6 to 8, within the band of 0.7 to 1.3. These sessions end at week 8; from 2021-09-10 the sessions have 44 to 70 rewards, of which the paper keeps only those of one task phase, which the files do not record, so weeks 9 to 12 cannot be tested. With all rewards of all sessions the median falls to 0.767 in week 8, 0.609 in week 10 and 0.392 in week 13, as expected if predicted rewards are included; this variant does not test the claim.

  How the test differed from the paper’s method

  • Weeks are counted from the first session, labelled week 5, because the files give no implantation date; the paper's code puts the first session on day 35, which gives the same weeks.
  • Only weeks 5 to 8 can be tested.
• ◐Partially reproducedFigure 4E; Figure S4A; Results, fourth section

  Light-evoked dopamine increases follow an amplitude gradient from the anterior striatum into the posterior tail, where increases are less prevalent and smaller.

  The light value is larger anteriorly (Spearman rho(AP, value) = 0.168, p = 0.0047) and smaller in the tail (median 0.0055 against 0.0117 DF/F, p = 0.0013), but increases are as prevalent there (83% of 35 tail fibers against 86% elsewhere, p = 0.38). The result is the same with the tail at -0.8 or -1.3 mm and with the code's lags (rho = 0.162). Per mouse the gradient is positive in DL18, DL20 and DL27 (rho 0.45 to 0.69) and negative in DL23 (-0.43), DL29 and DL30. With AP and the light value ranked within each mouse and a permutation test within mice, so that differences between mice cannot drive it, the correlation is r = 0.152 (p = 0.013); (a) holds this way too.

  How the test differed from the paper’s method

  • The tail boundary (AP at most -1.0 mm) was fixed here; the paper gives none.
• ◐Partially reproducedFigure 4F; Results, fourth section

  Tone-evoked decreases are slightly more prevalent in the anterior lateral and ventral striatum, with many sites showing both increases to the light and decreases to the tone.

  Of 245 anterior fibers, the 103 with a significant tone decrease are more ventral than the others (median DV 3.66 against 3.30 mm, p = 0.0031) but not more lateral (median ML 2.07 against 1.97 mm, p = 0.43); the same at the other tail boundaries. They are also more anterior (p = 1e-06). 79 anterior fibers (32%) have both a significant light increase and a significant tone decrease, so "many sites" holds by the 20% reading.

• ◐Partially reproducedResults, fourth section (Figure S4A, C)

  The modality-specific release patterns are consistent within and across mice.

  The tone correlation with AP is negative in all 7 mice (rho -0.44 to -0.70, each p < 0.03). The light correlation is positive in 4 of 7 mice (significant in DL18, DL20 and DL27) and negative in DL23 (rho -0.43, p = 0.026), DL29 and DL30, below the 5 required.

  How the test differed from the paper’s method

  • The supplementary legend of Figure S4 was not available; the test per mouse was chosen here.
• ◐Partially reproducedResults, fourth section (Figure S4); Methods; code SuppFig04/EFGH/sal_stim_bubbles_loco.m

  Locomotor changes to the stimuli are variable and bi-directional, while the dopamine release patterns to light and tone are consistent, so the patterns are stimulus and not movement driven.

  Locomotion after the stimuli varies between mice and presentations (acceleration and deceleration splits from 0 and 21 in DL29 light to 21 and 23 in DL18 tone; mean velocity change of either sign for both stimuli). The fiber maps of the two splits correlate (light r = 0.873 over 235 fibers, tone r = 0.784 over 229). The tone pattern holds after acceleration (tail median 0.0073, anterior -0.0031 DF/F) but not after deceleration, where the anterior median is positive (0.0042; tail 0.0098). The test uses raw DF/F, as the paper's code for Figure S4E-H (sal_stim_bubbles_loco.m) does, with no baseline and without the GLM's locomotion regressors. With each split's mean over the 1 s before onset subtracted (a variant, which does not decide the outcome), the maps correlate at r = 0.755 (light) and 0.823 (tone) and the tone pattern holds in both splits (anterior median -0.0043 after acceleration and -0.0024 after deceleration, tail 0.0069 and 0.0101), so the failure under the code's measure follows from the level before onset in the deceleration trials.

  How the test differed from the paper’s method

  • The supplementary legend was not available; the correlation threshold and the tone-pattern test were chosen here.
• ◐Partially reproducedResults, fourth section (Figure S5E-H); code SuppFig05/EFGH

  With isosbestic (415 nm) excitation, large fluorescence changes and spatial patterns to light and tone are not observed, ruling out a hemodynamic origin of the release patterns.

  In DL31 and DL32 (96 fibers), 2 fibers respond significantly to light and 1 to tone at 415 nm, against 45 and 18 at 470 nm, and the AP correlations of the tone values at 470 nm (-0.49, -0.43) are absent at 415 nm (-0.19, 0.16). The median absolute value at 415 nm is 0.49 of that at 470 nm for light but 0.68 for tone, above the criterion's half; for tone most 470 nm fibers are not significant either, and both medians (0.0027 and 0.0018 DF/F) are near the noise level. With random onsets the median absolute value is 0.0025 at 470 nm and 0.0019 at 415 nm, so the tone median at 470 nm is only 0.0002 above this floor and the ratio cannot fall to half; for light the 470 nm median is 0.0017 above the floor and the 415 nm one 0.0001. With 13 spline functions (the Methods' number for the main GLM) in place of the code's 9 at 18 Hz, the ratios are 0.55 (light) and 0.70 (tone), with 36 and 15 significant fibers at 470 nm against 1 and 0 at 415 nm.

  How the test differed from the paper’s method

  • The spline basis has 9 functions at 18 Hz, as the paper's code (run_sal_stim_glm_470_415.m, knots every 3 frames) has; the Methods give 13 for the GLM at 30 Hz. The 13-function basis is shown as a variant.
  • Events are placed by behaviour sample index for both channels, because the stored regular frame rate drifts from the frame timestamps by up to half a second; the clock check for these two sessions is weak (decoding 0.90 and 0.80 with true onsets against 0.40 to 0.65 with shifted ones, 20 presentations each).
  • The 470 and 405 nm sessions of Figure S5A-D are not in the dandiset.
• ◐Partially reproducedFigure 5A-B; Results, fifth section

  Latencies to half-maximum are shorter for dopamine increases to light than to tone, and shorter for increases than decreases to tone (light increase 221.6 ms, SD 42.5; tone increase 305.4 ms, SD 100.0; tone decrease 428.3 ms, SD 197.1).

  Counted from the onset frame as the Methods define latency, the means are 187.5 ms (240 light increases), 271.7 ms (94 tone increases) and 393.2 ms (108 tone decreases); the order holds and the tone decrease is within tolerance, the other two are below it. Counted as the paper's code counts (its 1-based index divided by the frame rate, one frame later), they are 220.8, 305.0 and 426.5 ms, within 2 ms of the paper's values, with SDs of 38.1, 98.7 and 196.5 ms against 42.5, 100.0 and 197.1. One tone increase has no half-maximum crossing before its peak.

  How the test differed from the paper’s method

  • Latency follows the Methods (from the onset frame); the code's count, one frame later, is shown as a variant and matches the paper.
  • Tone onsets are shifted by 11 frames, the speaker delay in the paper's code, which the Methods do not mention.
• ◐Partially reproducedResults, fifth section (Figure S6C-D)

  The spatial latency gradients are also present in individual mice.

  The tone gradient is positive in 6 of 7 mice (significant in DL20 and DL23); the light gradient is negative in 4 of 7 (significant in DL18 and DL27), zero in DL21 and positive in DL23 and DL29.

  How the test differed from the paper’s method

  • The supplementary legend was not available; the count of mice was chosen here.
• ◐Partially reproducedMethods, "DA release to optogenetic stimulation"; Figure 6G legend

  In the mouse with ChR2 in dopamine axons and rDAm3.0 in the striatum, 24 recording locations lie within or on the border of the striatum; 18 of the 24 stimulated locations evoke a significant response at another location, and 22 of the 24 recording locations respond to at least one stimulation.

  In Opto2, 22 fibers are flagged included and 20 of them respond to at least one stimulation, both within tolerance; 14 of the 19 stimulated included fibers evoke a significant response at another included fiber, below 18; as a fraction (0.74) this is close to the paper's 18 of 24 (0.75), but the denominators differ because fewer included fibers were stimulated in the files. Counting only responses to another fiber's stimulation, so that a stimulated fiber's own photoswitching does not count, still gives 20 responding locations. Adding the two fibers labelled Caudoputamen but not flagged (16 and 34) gives the paper's 24 locations and 22 responding ones, and 16 of 21 stimulated fibers with a response elsewhere; the files have 29 single-fiber sessions, of which 21 stimulate a striatal fiber, so 24 stimulated locations cannot be reached. With the code's further exclusions of fibers 8, 9 and 21 the counts are 19, 14 of 19 and 18. Session 2023-03-20f, with the laser artefact on 13 fibers, is left out.

  How the test differed from the paper’s method

  • Stimulation epochs and the stimulated fiber are found from the DF/F alone (artefact above 0.4), without the code's check against the trigger line, which the files do not hold.
  • The bootstrap counts responses of either sign as significant, as the code does.
• ◐Partially reproducedFigure 6G (D/V distance); Results, seventh section

  Evoked release drops off less with distance dorsal to the stimulated fiber than ventral to it, consistent with dorsally extending dopamine axon branches.

  The decay rate is -0.971 per mm for 156 dorsal pairs and -1.403 for 109 ventral pairs, in the claimed order, but the 95% bootstrap interval of the difference (-0.094 to 0.969) includes 0.

  How the test differed from the paper’s method

  • The paper shows binned means; the comparison of fitted decay rates was chosen here.
• ✓ReproducedFigure 1C; Results, second section

  In a D1-cre mouse expressing GCaMP7f in D1 spiny projection neurons, fluorescence under 470 nm excitation shows Ca2+ transients across fibers that are absent under 405 nm (isosbestic) excitation.

  Mouse 842, session 2022-11-23, 61 included fibers. The SD of DF/F under 470 nm exceeds that under 405 nm in 61 of 61 fibers (median ratio 3.01, range 1.56 to 19.41). Frames above 5 times the median 405 nm SD make up a median 6.7% of each 470 nm trace (range 0.13% to 35.9%) and a median 0.0% of each 405 nm trace (at most 1.2%; 11 of 61 have none). The session of 2022-11-18 gives a median ratio of 2.84.

  How the test differed from the paper’s method

  • The paper shows example traces; the SD ratio and its thresholds were chosen here before the analysis.
  • Figure 1C describes a 93-fiber array; the mouse its code reads (842) has 82 fibers per channel, 61 included.
• ✓ReproducedResults, second section; Figure S2D; code SuppFig02/DEF/surface_brain_dist.m

  Pairwise correlations of D1 SPN Ca2+ signals are generally low but variable, and drop off significantly with the distance between fiber tips.

  Over 1830 pairs of the 61 included fibers (470 nm, 2022-11-23), the median pairwise correlation is 0.163 (IQR -0.025 to 0.361, range -0.678 to 0.904), and correlations fall with tip distance (r = -0.190, p = 2.8e-16; mean 0.293 in the closest quarter of pairs against 0.133 in the farthest). The session of 2022-11-18 gives r = -0.238. The p-value treats pairs as independent, as the paper's code does.

  How the test differed from the paper’s method

  • The code's set of fibers (goodROIs.mat) is not published; the included fibers are used.
  • The threshold of 0.3 for "generally low" was chosen here.
• ✓ReproducedResults, second section; Figure S2E-F; code SuppFig02/DEF/surface_brain_dist.m

  After accounting for distance in the brain, signal correlations do not depend on the distance between fibers on the bundle surface, so nearby fibers in the bundle do not cross-talk.

  In the code's linear model of pairwise correlation on Euclidean, AP, ML and DV tip distances and bundle-surface distance (ROI centroids), the surface coefficient is -3.4e-05 per pixel (95% CI -2.7e-04 to 2.0e-04, p = 0.77), while Euclidean tip distance has -0.278 (p < 1e-5). The other session gives p = 0.41. Pairs adjacent on the bundle correlate at 0.222 against 0.156 for others, which the model attributes to their tips also being closer (tip and surface distances correlate at r = 0.437).

  How the test differed from the paper’s method

  • Surface distance is in pixels of the bundle image; the code converts it to mm, which does not change the p-value.
• ✓ReproducedFigure 4E-F; the paper's intermediate file glm_spline_results.mat (Zenodo, Fig04)uses data from outside the dandiset

  The dandiset's DF/F traces, fibers and stimulus times are those behind the paper's salient-stimulus analysis, so that its per-fiber responses can be linked to the paper's.

  Fibers matched by order within each mouse. The raw triggered averages from the dandiset equal the published cue1_roi_mu for light in all 7 mice and cue2_roi_mu for tone in the 6 mice other than DL18 (largest difference 0.0). The GLM spline estimates correlate with the published ones at r = 0.9957 for light and 0.9922 for tone over all fibers, and significance agrees for 93.6% of fiber and stimulus pairs (lowest DL18 tone 0.684, where 44 tones are used in place of 14; DL29 light 0.822). DL18's 14 published tone trials match 14 distinct tones of the file (r at least 0.952, best competitor at most 0.693), whose raw average equals the published one. Refitted with these 14 tones, DL18's tone spline estimates correlate with the published ones at r = 0.9977 and significance agrees for 0.947 of its fibers; with them, agreement over all pairs is 94.5% and the pooled tone r is 0.9974. Per mouse, r is below 0.99 for DL29 light (0.979) and DL27 and DL29 tone (0.989, 0.986).

  How the test differed from the paper’s method

  • The GLM is a re-implementation in Python; filtering and lag selection differ in detail (see the salient-stimulus claims), which explains the significance disagreements.
• ✓ReproducedFigure 4B, 4E; Results, fourth section

  The light evokes transient increases in dopamine release across many striatal locations.

  240 of 280 fibers (86%) have a significant increase to light and 38 a significant decrease; by the signed largest value 237 are increases and 11 decreases. Each mouse has increases in most fibers (34 of 51 in DL21 to 50 of 53 in DL20). The chance rate of a significant increase per fiber, from 200 null refits per mouse, is 0.024 (about 7 of 280 fibers), and 0.043 for an increase or a decrease.

  How the test differed from the paper’s method

  • The paper gives p < 1e-6 for its test (3 consecutive frames outside the 99% band), which is 0.01 cubed and assumes independent frames. With the same test, three given consecutive frames are all outside the band at a rate of 0.0037 in the null refits, and the per-fiber chance rate is 0.024. The protocol blocks a claim when the chance rate of a selection is far from the paper's, as a sign that the procedures differ; here the procedure is shown to be the paper's (raw averages identical, significance agreeing with the published calls for 93.6 to 94.5% of pairs, fig4-link-published-results), so the gap is in how the paper states the rate, and the outcome is kept. 240 increases against about 7 expected by chance.
  • The low-pass filter of overall velocity is a zero-phase Butterworth filter in place of MATLAB's lowpass; locomotion lags come from a cross-correlation that shifts negative lags, where the paper's code does not (its version correlates at r = 0.997 with the light values used).
  • DL18's tone regressors include all 44 tones (the files do not give tone frequency).
• ✓ReproducedFigure 4D, 4F; Figure S4C; Results, fourth section

  The tone evokes the strongest dopamine increases at sites in the posterior tail, while anterior to the tail it evokes primarily decreases.

  Tail fibers have larger tone values than anterior fibers (median 0.0092 against -0.0026 DF/F, p = 5.5e-13), all 10 of the largest values are in the tail, and 32 of 35 tail fibers have a significant increase (none a decrease). Anterior to the tail 103 significant responses are decreases and 60 increases (binomial p = 0.00047). The amplitude comparison and the anterior excess of decreases hold at every tail boundary, with the code's lags, and with DL18 restricted to its 14 tones of 12 kHz identified from the paper's results (108 against 60). At the -1.3 mm boundary only 5 of the 10 largest values are in the tail, so part (a) of the criterion, which needs 6, fails at that variant boundary. The anterior decrease dominates in DL21 (36 against 3) and DL23 (17 against 6) but not in DL18, DL20, DL27 or DL30.

  How the test differed from the paper’s method

  • DL18 uses all 44 tones in the main analysis.
• ✓ReproducedFigure 5C, E, G; Results, fifth section; code Fig05/general_functions

  Dopamine release latencies to light follow an anterior-posterior gradient, with faster increases in the anterior striatum, consistent with wave-like dynamics.

  Over 240 fibers with a light latency (median 18 presentations each), relative latency is shorter anteriorly (Spearman rho(AP, latency) = -0.285, p = 7e-06); it also increases laterally (rho = 0.263, p = 3.8e-05) and not with depth (p = 0.93). Ranked within each mouse and tested by permuting within mice, which removes differences between mice and the dependence of relative latency on the number of fibers in a mouse, the gradient remains (r = -0.223, p = 0.0014).

  How the test differed from the paper’s method

  • Relative latencies do not depend on the code's one-frame offset, which cancels in the difference.
• ✓ReproducedFigure 5D, F, H; Figure S6A-B; Results, fifth section

  Dopamine release latencies to tone follow a different gradient, faster in the posterior striatum than in the anterior striatum.

  Over 196 fibers with a tone latency (earlier of increase and decrease), relative latency is shorter posteriorly (rho(AP, latency) = 0.254, p = 0.00033; tail median 308 ms against 368 ms anterior). Increases alone (rho = -0.07, p = 0.5) and decreases alone (rho = -0.21, p = 0.028) do not show it, so the gradient comes from the tail's fast increases and the slower anterior decreases, as the paper says of Figure S6A-B. Ranked within each mouse and tested by permuting within mice, the gradient remains (r = 0.214, p = 0.003).

• ✓ReproducedFigure 6E-H; Results, seventh section

  Stimulating dopamine axons through one fiber evokes release at neighbouring non-stimulated sites that drops off sharply with distance in all three dimensions.

  Over 266 pairs of 14 stimulated and 20 recording fibers (378 pairs, 18 by 21, in the paper), the fit is a = 0.0147 DF/F and b = -1.096 per mm (95% bootstrap interval over stimulation sites -1.325 to -0.890), and the response falls with absolute distance along ML, AP and DV (Spearman rho -0.30, -0.27, -0.39, all p < 1e-5). The binned mean falls from 0.0079 DF/F at 0.54 mm to 0.0013 to 0.0017 beyond 2 mm.

  How the test differed from the paper’s method

  • Fewer stimulation and recording sites than the paper, from the inclusion of fig6-inclusion-counts.
• ✓ReproducedResults, seventh section; Figure 6G controls

  In control mice expressing tdTomato in place of rDAm3.0, single-fiber stimulation evokes no significant fluorescence increase at non-stimulated sites (0 of 48 sites, 26 stimulations in 2 mice), so the release is not a hemodynamic effect.

  No included non-stimulated fiber has a significant increase after stimulation in 304 fiber and session pairs of Opto4 (13 sessions) or 203 of Opto9 (12 sessions); median responses are -0.0002 and 0.0003 DF/F, and one Opto9 pair has a significant decrease. The same test finds increases in 142 of 619 pairs (20 distinct fibers) in Opto2. The files hold 25 tdTomato stimulations against the paper's 26.

  How the test differed from the paper’s method

  • In these mice the laser artefact spreads to many fibers, and the stimulated fiber is taken as the one with the largest artefact, which can be wrong; the true stimulated fiber is then among the fibers tested, which makes the test stricter.
• ✓ReproducedResults, seventh section; Figure 6E, Figure S7A

  In mice expressing rDAm3.0 without opsin, light through one fiber raises fluorescence at the stimulated site by photoswitching, but at no other site (0 of 22 non-stimulated sites for 3 stimulations).

  In each of UG21's 3 sessions the stimulated fiber (12, 18 and 24, the numbers of the paper's code folders) has a significant increase after the laser (0.0118 to 0.0144 DF/F), and none of the 21 other included fibers responds significantly in either direction.

  How the test differed from the paper’s method

  • The paper included these sites only if they responded to reward; UG21 has no reward events in the files, so that step is not applied.
  • The files give UG21's indicator as tdTomato; it is treated as the paper's rDAm3.0 mouse without opsin from its sessions, fiber counts and stimulated fibers (see findings).