DANDI:000971

Reproducing Seiler et al. (2022) from dandiset 000971 34

Notebook reproduce_seiler_2022.ipynb

• ✕Not reproducedResults, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 2; Figure 1D

  Mice trained on RI60 accept more shocks in the shock probes than mice trained on RR20 (main effect of schedule, F1,43 = 6.37, p < 0.05; schedule by time interaction, F1,43 = 4.54, p < 0.05), and RI60 mice accept more shocks on the late probe than on the early one (p < 0.01).

  We observed a significant main effect of schedule (RI60 vs RR20) on the number of shocks mice were willing to receive on the shock probes ... after extended RI60 training, mice increased the number of shocks they were willing to receive

  Results, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 2; Figure 1D

  What the paper reports

  • main effect of schedule: two-way ANOVA, F1,43 = 6.37, p < 0.05 (n = 36 RI60 and 9 RR20 mice)
  • interaction of schedule and training time: two-way ANOVA, F1,43 = 4.54, p < 0.05
  • RI60 late against early probe: Bonferroni, p < 0.01, Bonferroni-corrected

  In the 32 RI60 and 5 RR20 mice with both probes, RI60 mice accept more shocks (early 11.38, late 16.31, against 4.80 and 6.60), but the schedule effect has p = 0.071 (F1,35 = 3.46) and the interaction p = 0.59. The RI60 rise has paired p = 0.032, 0.063 after Bonferroni correction, where the claim needs p < 0.01. Summing a probe day's files gives the same picture. The dandiset holds 6 RR20 mice (5 with both probes) against 9 in Figure 1D, so the test has little power on the RR20 side.

  How the test differed from the paper’s method

  • 5 RR20 mice with both probes where the paper has 9, and 32 RI60 mice where it has 36 (4 lack a probe in the dandiset).
  • The test is a two-way mixed ANOVA on complete cases, in Python; the paper used Prism.
• ✕Not reproducedResults, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 2; Figure 3F

  The DMS rewarded-unrewarded peak score differs by phenotype (F2,33 = 8.16, p < 0.01) and changes across training (p < 0.001).

  The rewarded-unrewarded peak score changed across training ... and was significantly different by phenotype

  Results, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 2; Figure 3F

  What the paper reports

  • phenotype: mixed-effects analysis, F2, 33 = 8.16, p < 0.01
  • training: mixed-effects analysis, F1.61,29.8 = 13.83, p < 0.001

  Mean DMS score PR 2.35, DPR 1.66, PS 1.05 z, the paper's order, but the phenotype effect has likelihood ratio p = 0.100 and neither stage alone is significant (p = 0.124 early, 0.270 late). The score rises from early to late (paired p = 0.046, 17 mice; model stage p = 0.088). Without the dropout step the phenotype p is 0.389. 51 scores from 34 mice. The mice with a score differ from Figure 3E (late PR 4, DPR 7, PS 10 against 5, 6, 13), so the comparison with F2,33 is not like for like.

  How the test differed from the paper’s method

  • Early and late RI60 only; the paper's test also includes RI30, which is not in the dandiset.
  • Channel dropouts are left out (samples below half the channel median, with 1 s around them), a step the authors' code does not have; it was added after seeing that dropouts made some session z-scores near zero, and after the per-session scores had been seen. The authors' processing without it is shown and gives p = 0.389.
  • One DLS recording with dropouts over 10% of samples is excluded, and one session without rewarded nosepokes (mixed clocks) has no score.
  • A linear mixed model with a random intercept per mouse replaces Prism's mixed-effects analysis.
• ✕Not reproducedResults, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4G

  The DLS rewarded-unrewarded peak score does not correlate with shocks received (r = -0.01, ns).

  We also looked at whether these signals correlated with shock or omission probe performance on an individual basis and found no correlations

  Results, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4G

  Pooled DLS score against shocks, r = 0.326, p = 0.024, n = 48, where the paper finds r = -0.01. Late pairs r = 0.374, p = 0.10; early pairs r = 0.103, p = 0.60. Without the dropout step r = 0.113, p = 0.45 (late 0.033, early 0.182), so the outcome depends on the artifact handling, a step chosen here and in neither the Methods nor the authors' code. The DLS labels are consistent with the data (the DLS response has the larger 2 to 10 s area, as the paper describes for DLS).

  How the test differed from the paper’s method

  • The pairing of scores and probes follows reading (a) of fig3g-dms-score-shock-correlation; the paper does not state it.
• ✕Not reproducedResults, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5F

  Mice receiving DMS dopamine terminal stimulation on rewarded nosepokes (ChR2) escalate their nosepoking faster than EYFP and ChR2 Scrambled controls during FR1 and RI30.

  ChR2 mice escalated their nosepoking much faster than the control groups during FR1 and RI30

  Results, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5F

  In the pooled fit no ChR2 slope differs from a control (FR1 interaction p = 0.63 and 0.98, RI30 p = 0.82 and 0.49), and the pooled FR1 ChR2 slope (0.011) is not above the controls (0.051 and 0.005). Per-mouse slopes, a variant, put ChR2 above EYFP (p = 0.015) and Scrambled (p = 0.021) in FR1 but not in RI30. ChR2 mice nosepoke more in RI30 (7.75 against 4.01 and 4.21 per minute). The pooled fit mixes mice with 1 to 13 FR1 sessions, which the per-mouse slopes avoid. A mixed model with a random intercept and slope per mouse puts the FR1 ChR2 slope above ChR2 Scrambled (p = 0.034) and EYFP (p = 0.0075) but not in RI30 (p = 0.68 and 0.70); under that model the rule would give partially_reproduced. That FR1 fit converged only after a retry with lbfgs, warns that the MLE may be on the boundary and has a random intercept variance near zero, and a model with a random intercept only puts ChR2 above neither control (p = 0.46 and 0.33). The criterion fixed the pooled fit before the analysis, so the outcome stays.

  How the test differed from the paper’s method

  • The paper fits a segmental linear regression in Prism; the pooled least squares fit with a group by session interaction was chosen before the analysis.
  • 33 DMS excitatory mice in the dandiset where the paper has 39.
• ✕Not reproducedResults, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5G

  On the first shock probe ChR2 mice accept more shocks than EYFP mice (interaction of time and manipulation, F2,35 = 3.65, p < 0.05; Tukey p < 0.05); the difference fades by the second probe.

  On the first shock probe, ChR2 mice were significantly more resistant to punishment than EYFP mice ... This difference faded by the second probe

  Results, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5G

  What the paper reports

  • interaction of time and manipulation: two-way ANOVA, F2,35 = 3.65, p < 0.05
  • ChR2 against EYFP on the first probe: Tukey's multiple comparison, p < 0.05, Tukey-corrected

  First probe ChR2 28.69, EYFP 21.86, Scrambled 20.67 shocks, the paper's order; Tukey ChR2 vs EYFP p = 0.51; group by probe interaction F2,29 = 1.95, p = 0.16 (paper F2,35 = 3.65).

  How the test differed from the paper’s method

  • 32 mice with both probes (13 ChR2, 12 Scrambled, 7 EYFP) where the paper's degrees of freedom imply 38.
• ✕Not reproducedResults, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7F

  Mice receiving DLS dopamine terminal stimulation (ChR2, EYFP, ChR2 Scrambled) escalate their nosepoking at the same rates.

  All groups of mice (ChR2, EYFP, and ChR2 Scrambled) behaved similarly, escalating their nosepoking at the same rates

  Results, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7F

  In FR1 the Scrambled slope (0.087) is below the EYFP slope (0.224), interaction p = 0.013 (F test of all group terms p = 0.034). ChR2 differs from neither control (p = 0.12 and 0.98); in RI30 no pair differs (p = 0.09 to 0.92). The difference is between the two control groups, not a ChR2 effect. A mixed model with a random intercept and slope per mouse finds no difference in slope (p = 0.43 to 0.96, FR1 EYFP vs Scrambled p = 0.76), but its FR1 fit has a singular random effects covariance, so those p values are weak evidence. A model with a random intercept only fits without warnings and finds no FR1 difference either (EYFP vs Scrambled p = 0.23). The FR1 difference rests on the pooled fit the criterion fixed.

  How the test differed from the paper’s method

  • The paper gives no test; the fit of fig5f-dms-chr2-escalation was used, judged on all three pairs.
• ◐Partially reproducedResults, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 3; Figure 1E

  RI60-trained mice take longer to complete the omission probe than RR20-trained mice (unpaired t-test, p < 0.05; n = 20 and 7).

  RI60-trained mice took longer to complete the omission probe than RR20-trained mice

  Results, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 3; Figure 1E

  What the paper reports

  • unpaired t-test, p < 0.05 (n = 20 RI60 and 7 RR20 mice)

  RI60 mice take 33.97 min (n = 19) and RR20 mice 17.72 min (n = 4); unpaired t-test p = 0.070, Welch p = 0.0005. The direction holds without significance by the paper's test. The RR20 variance is 251 times smaller than the RI60 one, so the Student and Welch tests disagree.

  How the test differed from the paper’s method

  • 4 RR20 omission probes in the dandiset against 7 in the paper, and 19 RI60 against 20.
  • Completion time is the time of the 50th reward, or 60 min when fewer were earned; the Methods give the 50 reward and 60 min limits but not how incomplete sessions were scored.
• ◐Partially reproducedFigure 2A; STAR Methods, Behavioral Analysis, paragraph 3

  RI60 mice are split by their shock probe counts into 9 punishment resistant (PR), 9 delayed punishment resistant (DPR) and 18 punishment sensitive (PS) mice, by the rule in the Methods (DPR when the count rises by more than 85%; otherwise PR when the early count exceeds 13).

  Mice in the top quartile of changers (who increased the number of shocks received by greater than 85%) were classified as delayed punishment resistant (DPR; n=9) ... with mice receiving more than 13 shocks on the first probe classified as punishment resistant (PR; n=9) and those earning fewer as punishment sensitive

  Figure 2A; STAR Methods, Behavioral Analysis, paragraph 3

  The stored labels give 9 PR, 9 DPR and 18 PS, which match by construction; only the rule check is informative. The Methods rule agrees with the stored label for 31 of 32 mice with both probes; 048.392 (13 early, 18 late) is stored as PR but the rule's "more than 13" makes it PS. With 13 or more, 32 of 32 agree. The 85% cut is near the top quartile of change (87.7%), but the median early count is 9.0, so 13 is not the median of these mice. Summing a day's files gives 30 of 32.

  How the test differed from the paper’s method

  • Four RI60 mice have one probe in the dandiset and cannot be checked against the rule.
• ◐Partially reproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 4; Figure 2K

  After a reward PS mice wait longer before nosepoking again (38 +- 15 s) than PR (21 +- 5 s) and DPR (22 +- 4 s) mice (one-way ANOVA p < 0.01; Tukey p < 0.01 for each).

  PS mice waited an average of 38 ± 15 seconds, significantly longer than the other groups

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 4; Figure 2K

  What the paper reports

  • phenotype: one-way ANOVA, F2,33 = 8.57, p < 0.01
  • PS against PR: Tukey's multiple comparison, p < 0.01, Tukey-corrected
  • PS against DPR: Tukey's multiple comparison, p < 0.01, Tukey-corrected

  PS mice wait longest (53.94 s, SD 44.67, maximum 171 s), PR 21.05 s, DPR 21.86 s. PR and DPR are within tolerance, the PS mean is above it (23 to 53 s); with each mouse's median session PS is 32.2 s. The two highest PS mice (139.298 at 171.0 s, 348.393 at 168.7 s) each have one session with a mean wait over 1000 s. ANOVA p = 0.017; Tukey PS > PR p = 0.045, PS > DPR p = 0.051.

  How the test differed from the paper’s method

  • The rewarded port entry (first port entry after a reward) and the wait (to the next active nosepoke) are defined here from the Results; the code for them is not published.
• ◐Partially reproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 4; Figure 2L

  PS mice make fewer extra nosepokes between a rewarded nosepoke and the rewarded port entry than PR mice (PS 1.28 +- 0.85, PR 3.23 +- 1.65, DPR 2.1 +- 2.63; Tukey PS vs PR p < 0.05).

  PS mice made significantly fewer extra nosepokes than PR mice

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 4; Figure 2L

  What the paper reports

  • phenotype: one-way ANOVA, F2,33 = 4.24, p < 0.05
  • PS against PR: Tukey's multiple comparison, p < 0.05, Tukey-corrected

  PS 1.61, PR 3.33, DPR 2.28 extra nosepokes per reward, all within the stated SDs; PS < PR but Tukey p = 0.117 (ANOVA p = 0.139).

  How the test differed from the paper’s method

  • Extra nosepokes are defined here from the Results (active nosepokes after a reward and before the rewarded port entry).
• ◐Partially reproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 5; Figure 2N

  Averaged over RI60 training, PS mice earn fewer rewards per session (39.91 +- 7.39) than DPR mice (46.56 +- 4.49; PR 45.33 +- 4.33); one-way ANOVA p < 0.05, Tukey PS vs DPR p < 0.05.

  when the number of rewards per session was averaged over all days, PS mice received significantly fewer rewards

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 5; Figure 2N

  What the paper reports

  • phenotype: one-way ANOVA, F2,33 = 4.45, p < 0.05
  • PS against DPR: Tukey's multiple comparison, p < 0.05, Tukey-corrected

  Rewards per RI60 session, PS 39.12, DPR 44.98, PR 44.66, all within tolerance; ANOVA F = 4.02, p = 0.028; Tukey PS vs DPR p = 0.059, so the pairwise test fails. The result sits at the threshold. With the false start of 140.306 kept as a session of 0 rewards Tukey p is 0.048 (the first version of this attempt), and with one file per mouse-day (PS 39.98, DPR 46.16, PR 44.66) it is 0.044. The rule used (no active nosepoke and no reward) was set after round 1 and is the only one of the three that gives p above 0.05; other kept short files of 1 to 4 rewards fit the same false-start reason. The claim is borderline.

  How the test differed from the paper’s method

  • Training files with no active nosepoke and no reward are left out (one PS file of 170 s); other files count as sessions, also when a mouse has two or three on one day.
• ◐Partially reproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 6; Figure 2O

  PR mice escalate their nosepoking as soon as they enter RI30, while DPR mice escalate later, during RI60.

  PR mice escalate their nosepoking as soon as they enter RI30 (Fig. 2O). DPR mice escalate their nosepoking later in RI60

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 6; Figure 2O

  RI30 rate PR 4.85 per minute, DPR 3.03 (Mann-Whitney p = 0.114), PS 2.22 (p = 0.0063), so part (i) fails on DPR. Median RI60 slope DPR 0.809, PR 0.494 nosepokes/min per day, so part (ii) holds (p = 0.289).

  How the test differed from the paper’s method

  • The paper shows this with a segmental linear regression in Prism; the measures here (RI30 rate and RI60 slope per mouse) were chosen before the analysis.
• ◐Partially reproducedResults, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 3; Figure 3G

  The DMS rewarded-unrewarded peak score correlates with the shocks a mouse receives in the shock probes (r = 0.53, p < 0.0001).

  the shocks received were significantly correlated with DMS rewarded-unrewarded peak score on a mouse-by-mouse basis

  Results, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 3; Figure 3G

  What the paper reports

  • r = 0.53, p < 10⁻⁴

  Reading (a), pooled early and late pairs, r = 0.170, p = 0.24, n = 49 (given as measured); (b) late pairs r = 0.014, p = 0.95; (c) early pairs r = 0.375, p = 0.045, n = 29. Only (c) is positive and significant, and it is below the tolerance around 0.53. The support is weak. Corrected for the three readings (c) has p = 0.13, and without the dropout step (c) gives r = 0.362, p = 0.053 and the pooled r is 0.100. The step leaves out more than 1% of samples in one DMS session of 51, but changes some scores a lot where it leaves out less (88.239 from 0.09 to 1.92, 048.392 from 0.02 to 1.81, 346.394 from 0.00 to 1.10).

  How the test differed from the paper’s method

  • The paper does not say which scores and probes form the pairs; three readings were listed before the analysis, none of which matches.
  • RI30 scores, which may be among the paper's points, are not in the dandiset.
• ◐Partially reproducedResults, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure S5F (legend not available)

  Under DMS ChR2 stimulation 100% of male mice and about 71% of female mice are PR.

  Notably, under ChR2 stimulation, 100% of male mice were PR mice (Fig. S5F). A large majority of female mice (~71%) were also PR

  Results, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure S5F (legend not available)

  7 of 7 male ChR2 mice are PR; 5 of 6 female ChR2 mice (83.3%), outside 71 +- 7.1%.

  How the test differed from the paper’s method

  • One female ChR2 mouse (126.286) has a single session in the dandiset and no probes.
• ◐Partially reproducedResults, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6G

  DMS dopamine terminal inhibition on rewarded nosepokes delays punishment resistance; NpHR mice accept fewer shocks than EYFP mice on the second probe (Tukey p < 0.01).

  NpHR inhibition of DMS dopamine terminals during RI60 training delayed the development of punishment-resistant reward-seeking, with a significant effect on the second shock probe

  Results, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6G

  What the paper reports

  • Tukey's multiple comparisons, p < 0.01, Tukey-corrected

  Second probe NpHR 9.33 against EYFP 23.47 shocks (34 mice with both probes), Tukey p = 0.069. With all 35 mice that have a second probe p = 0.043. Mixed ANOVA group p = 0.056.

  How the test differed from the paper’s method

  • 34 DMS inhibitory mice with both probes; the paper's degrees of freedom (F2,36) imply 39.
• ✓ReproducedResults, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 5; Figure 1F

  Among RI60 mice, shocks received on the late probe and omission completion time are not significantly correlated (r = 0.42, ns).

  We found that these two measures were not significantly correlated

  Results, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 5; Figure 1F

  Late probe shocks and omission time, r = 0.335, p = 0.175, n = 18 (paper r = 0.42, ns). With the early probe r = 0.592, p = 0.0076.

• ✓ReproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 1; Figure 2B

  There is an interaction of phenotype and probe on shocks received (p < 0.0001), and only DPR mice receive significantly more shocks on the late probe than the early one.

  There was a significant interaction of phenotype and training time ... only DPR mice showed a significant increase in shocks received across the probes

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 1; Figure 2B

  What the paper reports

  • interaction of phenotype and training time: F3, 40 = 24.18, p < 10⁻⁴
  • DPR increase across the probes: p < 10⁻⁴

  Phenotype by probe interaction F2,29 = 26.37, p = 3e-07. DPR mice rise from 11.56 to 32.11 shocks (Bonferroni p = 0.00041); PR (23.50 to 20.50) and PS (7.00 to 6.47) do not change.

  How the test differed from the paper’s method

  • 32 mice with both probes against 36; F2,29 where the paper gives F3,40.
• ✓ReproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 3; Figure 2C

  During RI60 training PR and DPR mice nosepoke at higher rates than PS mice (main effect of phenotype, p < 0.0001).

  PR and DPR mice had higher rates of nosepoking than PS mice

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 3; Figure 2C

  What the paper reports

  • mixed-effects analysis, main effect of phenotype, F2,33 = 18.59, p < 10⁻⁴

  Active nosepokes per minute over RI60 training, PR 14.20, DPR 12.79, PS 5.27; ANOVA p = 4.9e-06; Tukey PR > PS p below 0.0001, DPR > PS p = 0.0003; mixed model over 492 sessions p = 2.3e-06. With the empty files kept p = 4.3e-06, with one file per mouse-day p = 7.3e-06. Sex and phenotype are confounded (17 of 18 PS mice female, 8 of 9 PR male).

  How the test differed from the paper’s method

  • The main test is a one-way ANOVA on per-mouse means; the paper used a mixed-effects analysis over days, shown as a variant.
  • Training files with no active nosepoke and no reward are left out (one PS file of 170 s here, which holds inactive nosepokes and port entries).
• ✓ReproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 3; Figure 2D

  PR mice take longer than PS mice to complete the omission probe (PR 51.50 +- 17, PS 27.92 +- 13.65; one-way ANOVA F2,18 = 4.36, p < 0.05; Tukey PR vs PS p < 0.05).

  PR mice took significantly longer than PS mice to complete the omission probe

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 3; Figure 2D

  What the paper reports

  • phenotype: one-way ANOVA, F2,18 = 4.36, p < 0.05 (n = 4 PR, 5 DPR and 11 PS mice)
  • PR against PS: Tukey's multiple comparison, p < 0.05, Tukey-corrected

  PR 51.65 min (n = 4), PS 28.06 min (n = 10), DPR 31.67 (n = 5); ANOVA F2,16 = 3.85, p = 0.043; Tukey PR vs PS p = 0.037. Three of four PR mice reach 60 min.

  How the test differed from the paper’s method

  • The Results print the values in seconds; they are read as minutes (the Methods' median split is at 29 minutes).
  • 10 PS mice where the paper has 11.
• ✓ReproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 5; Figure 2M

  PS mice have longer inter-reward intervals (79.77 +- 26.54 s) than PR (71.2 +- 32.3 s) and DPR (69.58 +- 35.18 s) mice (Kolmogorov-Smirnov, p < 0.0001 for each).

  PS mice pay for their efficiency with longer inter-reward intervals (Fig. 2M). The average inter-reward interval for PS mice is 79.77 ± 26.54s compared to 71.2 ± 32.3s for PR and 69.58 ± 35.18s for DPR

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 5; Figure 2M

  What the paper reports

  • PR against PS: K-S Test, p < 10⁻⁴
  • DPR against PS: K-S Test, p < 10⁻⁴

  Pooled RI60 intervals average PS 81.75 s, PR 71.96 s, DPR 69.57 s; KS PS vs PR p = 5e-30, PS vs DPR p = 3.3e-33. Per-mouse means give ANOVA p = 0.033.

  How the test differed from the paper’s method

  • The Methods define the interval from rewarded nosepoke to rewarded nosepoke and cite Figure 2L for it; reward times are used, which coincide with rewarded nosepokes in these files.
• ✓ReproducedResults, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 4; Figure S1I (legend not available)

  RI60-trained mice earn more rewards per training session than RR20-trained mice (unpaired t-test, p < 0.0001).

  RI60-trained mice ... earned significantly more rewards per training session than RR20-trained mice

  Results, A random interval schedule of reinforcement promotes punishment-resistant reward-seeking, paragraph 4; Figure S1I (legend not available)

  What the paper reports

  • unpaired t-test, p < 10⁻⁴

  RI60 41.97 against RR20 22.63 rewards per session, t-test p = 5.4e-07, not below the 0.0001 stated. With the 21 empty files kept as sessions of 0 rewards (the 20 RR20 files that hold port entries only and the RI60 false start), RR20 15.38, RI60 41.89 and p = 1.4e-11.

  How the test differed from the paper’s method

  • 6 RR20 mice where the paper has 9 (or 7 in the Methods).
  • 20 RR20 files of four mice hold port entries only and are left out, with one RI60 false start.
• ✓ReproducedResults, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 2; Figure S2B (legend not available)

  Male mice accept more shocks than female mice in the shock probes (main effect of sex, F1,35 = 26.19, p < 0.0001).

  Male mice tolerated more shocks than females

  Results, Three punishment-related phenotypes emerge with extended RI60 training, paragraph 2; Figure S2B (legend not available)

  What the paper reports

  • two-way ANOVA, main effect of sex, F1,35 = 26.19, p < 10⁻⁴

  Males early 16.58 and late 23.25, females 8.25 and 12.15; sex effect F1,30 = 9.81, p = 0.0039. The paper's p < 0.0001 does not hold. Sex is confounded with phenotype (PR 8 males of 9, PS 17 females of 18).

  How the test differed from the paper’s method

  • The supplementary legend was not available; the test is a two-way mixed ANOVA of sex and probe.
• ✓ReproducedResults, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 3; Figure 3H

  The DMS rewarded-unrewarded peak score does not correlate with omission completion time (r = 0.1, ns).

  Performance on the omission probe was not correlated with this score

  Results, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 3; Figure 3H

  Late DMS score against omission time, r = 0.014, p = 0.96, n = 12 (95% interval -0.56 to 0.58); pooled r = -0.050, p = 0.80. With 12 mice the test has little power.

  How the test differed from the paper’s method

  • 12 mice with a late score and an omission probe.
• ✓ReproducedResults, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4F

  The DLS rewarded-unrewarded peak score has no significant effect of phenotype.

  There was a significant effect of training stage ... but no significant effect of group

  Results, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4F

  Phenotype likelihood ratio p = 0.48 (per stage p = 0.61 and 0.47); stage p = 0.034, as the paper also finds a training effect in DLS.

  How the test differed from the paper’s method

  • No RI30 stage; dropout step as for fig3f-dms-score-phenotype (without it p = 0.68).
• ✓ReproducedResults, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4H

  The DLS rewarded-unrewarded peak score does not correlate with omission completion time (r = -0.21, ns).

  We also looked at whether these signals correlated with shock or omission probe performance on an individual basis and found no correlations

  Results, Dopamine signals in the DLS do not predict punishment-resistant reward-seeking, paragraph 1; Figure 4H

  Late DLS score against omission time, r = 0.311, p = 0.32, n = 12 (paper r = -0.21, ns); 95% interval -0.32 to 0.75, so the test has little power.

• ✓ReproducedResults, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5H

  ChR2 mice are very likely to be classified PR, while EYFP and ChR2 Scrambled mice are distributed across phenotypes as expected.

  ChR2 mice were extremely likely to be categorized as PR, whereas EYFP and ChR2 Scrambled mice were distributed as expected across groups

  Results, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5H

  PR in 12 of 13 ChR2 (92%), 5 of 7 EYFP (71%), 6 of 12 Scrambled (50%); Fisher ChR2 against both controls p = 0.0497. The control PR fractions are higher than in the fiber photometry cohort (19%).

  How the test differed from the paper’s method

  • Phenotypes are computed with the Methods rule, as the dandiset stores none for optogenetic mice.
• ✓ReproducedResults, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5I

  DMS dopamine terminal stimulation does not change omission probe performance.

  We also looked to see whether DMS dopamine terminal stimulation influenced performance on the omission probe. It did not

  Results, Optogenetic excitation of DMS dopamine terminals at the time of a rewarded nosepoke accelerates the development of punishment-resistant reward-seeking, paragraph 3; Figure 5I

  ChR2 25.79, Scrambled 35.53, EYFP 37.29 min; ANOVA p = 0.17, Kruskal-Wallis p = 0.14.

• ✓ReproducedResults, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6H

  The NpHR group has more PS mice, and the NpHR Scrambled group more PR mice, than the EYFP group.

  the NpHR group had an increased incidence of PS mice, while the NpHR Scrambled group had an increased incidence of PR mice

  Results, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6H

  PS in 8 of 9 NpHR (89%) against 5 of 15 EYFP (33%), Fisher p = 0.013; PR in 6 of 10 NpHR Scrambled (60%) against 6 of 15 EYFP (40%), p = 0.43. The NpHR effect is in both sexes here (3 of 4 females and 5 of 5 males PS).

  How the test differed from the paper’s method

  • Phenotypes computed with the Methods rule.
• ✓ReproducedResults, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6I

  DMS dopamine terminal inhibition does not change omission completion time.

  No significant differences in omission time were observed

  Results, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 3; Figure 6I

  EYFP 28.74, NpHR 21.47, NpHR Scrambled 27.82 min; ANOVA p = 0.21.

• ✓ReproducedResults, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7G

  DLS dopamine terminal stimulation does not change the number of shocks received.

  All groups of mice (ChR2, EYFP, and ChR2 Scrambled) behaved similarly ... and receiving similar numbers of shocks

  Results, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7G

  Group p = 0.055, group by probe p = 0.068, 31 mice with both probes. EYFP mice rise most (19.11 to 32.89).

  How the test differed from the paper’s method

  • 4 DLS mice lack a late probe in the dandiset.
• ✓ReproducedResults, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7H

  DLS ChR2, EYFP and ChR2 Scrambled mice have similar distributions of PR, DPR and PS.

  All groups had similar distributions of PR, DPR, and PS mice

  Results, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7H

  Chi-square p = 0.12 on a 3 x 3 table with smallest expected count 1.5; ChR2 mice include no DPR.

  How the test differed from the paper’s method

  • The paper gives no test; a chi-square test was chosen before the analysis.
• ✓ReproducedResults, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7I

  DLS dopamine terminal stimulation does not change omission completion time.

  No significant differences were observed in omission completion time

  Results, Optogenetic excitation of dopamine terminals in DLS at the time of a rewarded nosepoke does not influence instrumental learning or behavioral flexibility, paragraph 2; Figure 7I

  ChR2 28.44, Scrambled 33.51, EYFP 36.08 min; ANOVA p = 0.53.

• ✓ReproducedResults, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 1; Figure S3A-B (legend not available)data in Zenodo doi:10.5281/zenodo.11266654 (Densitometry.xlsx)

  Heterozygous DAT-IRES-cre mice do not have reduced DAT protein in DMS or DLS compared with wild-type mice.

  heterozygous mice in our colony do not have a significant reduction in DAT protein levels in DMS or DLS

  Results, Dopamine axon signals in the DMS predict punishment-resistant reward-seeking, paragraph 1; Figure S3A-B (legend not available)

  DAT/actin of het samples is 131.3% of the WT mean in DMS (t-test p = 0.41) and 74.7% in DLS (p = 0.20). The table holds females only, 3 WT and 4 het samples per region, named as lanes of one WT mouse (410) and one het mouse (484); the male samples of Figure S3A are not in it.

  How the test differed from the paper’s method

  • Females only; the paper's test is in a legend that was not available, so an unpaired t-test was used.
• ⊘BlockedResults, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 2; Figure 6F· Data missing from the datasetverifier disagreed at first

  DMS dopamine terminal inhibition impairs initial learning; 25% of NpHR and 22% of NpHR Scrambled mice in group 1 failed FR1, and 29% of NpHR mice in group 2 failed RI30, and were dropped.

  25% of NpHR mice and 22% of NpHR Scrambled mice were dropped from the study after >14 days (mean+2SD) of unsuccessful FR1 training ... In this case, 29% of NpHR mice were dropped from the study because they could not learn RI30

  Results, Optogenetic inhibition of dopamine terminals in DMS delays the development of punishment-resistant reward-seeking, paragraph 2; Figure 6F

  The dandiset holds 13 NpHR, 13 NpHR Scrambled and 16 EYFP mice, where Figure 6F counts 16, 15 and 16. The shortfall (3, 2, 0) equals the mice the paper drops (about 4 NpHR, 2 NpHR Scrambled) less the one NpHR mouse deposited and flagged as not learning (270.476, no RI60). So the dropped mice are, but for that one, not in the dandiset. The 'Did Not Learn' flag also marks an EYFP mouse that reached RI60 (244.465), so it is not the paper's dropout criterion, and no file says which group (1 or 2) a mouse is in. Blocked under the criterion, as the dandiset does not say which mice were dropped.