Reporting Checklist for Nature Neuroscience

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1 Corresponding Author: Manuscript Number: Manuscript Type: Ofer Yizhar, PhD NNA54616C Article Reporting Checklist for Nature Neuroscience # Main s: 5 # lementary s: 10 # lementary Tables: 0 # lementary Videos: 0 This checklist is used to ensure good reporting standard to improve the reproducibility of published results. For more information, please read Reporting Life Sciences Research. Please note that in the event of publication, it is mandatory that authors include all relevant methodological and cal information in the manuscript. Statisti reporting, by example example Please specify the following information for each panel reporting quantitative data, and where each item is reported (section, e.g., & number). Each should ideally contain an exact sample size (n) for each experimental group/condition, where n is an exact number and not a range, a clear definition of how n is defined (for example x cells from x slices from x animals from x litters, collected over x days), a description of the cal used, the results of the s, any descriptive and clearly defined error bars if applicable. For any experiments using custom, please indicate the used and stats obtained for each experiment. Each should include a statement of how many times the experiment shown was replicated in the lab; the details of sample collection should be sufficiently clear so that the replicability of the experiment is obvious to the reader. For experiments reported in the text but not in the s, please use the number instead of the number. Note: Mean and standard deviation are not appropriate on small samples, and plotting t data points is usually more informative. When technical replicates are reported, error and significance measures reflect the experimental variability and not the variability of the biological process; it is misleading not to state this clearly. FIGURE NUMBER 1a results, para 6 TEST USED WHICH TEST? oneway unpaired t para 6 EXACT VALUE 9, 9, 10, 15 n DEFINED? mice from at least 3 litters/group 15 slices from 10 mice ds para 8 para 6 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? error bars are mean / error bars are mean / para 6 P VALUE EXACT VALUE p = p = para 6 DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE F(3, 36) = 2.97 t(28) = para 6 1

2 FIGURE NUMBER 1c 1d 1d 1e,f 1g 1i 1j TEST USED WHICH TEST? twotailed unpaired Students t Wilcoxon for Wilcoxon for Twoways Tukey posthoc Twoways Tukey posthoc paired twotailed Students t paired twotailed Students t n 1, pa n 1, pa n 1, pa ; c 3 ; c 3 s, Result, II, 5 Result II, 5 EXACT VALUE 79,11 79,10 79,10 11,10,10 appears in the 7 7 n DEFINED? averaged lightresponses at 100% light intensity variance of 5 repeats at 100% light intensity average of 5 repeats at 100% light intensity normalized average of 5 pulse to Tpre normalized average of 5 pulse to Tpre average of 60 repeats before and after normalized to first 20 sweeps mean of 50second bins 1 Fig ; result sec tion II and in 1 g; result sec tion II and in 1 1 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? bars indicate / EPSP(mean,RS) 3.85 mv EPSP(mean,in) 3.48 mv ms (median RS) ms(median, FS) 3.85 ms (median RS) 3.28 ms(median, FS) mean indicate / mean indicate / mean of 50 second bins / mean of 50 second bins / 2 Result s n 2,para gr 3 Result s n 2,para grpra ph 3 metho ds statist ical metho ds statist ical P VALUE EXACT VALUE P=0.26 P = 0.52 P = P < P < P(oEPSC1) = P(oEPSC2) = P = n 1, pa n 1, pa n 1, pr 2 n 1, pr 3 n 1, pr 3 Result n 1, ap h 5 Result n 1, ah 5 DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE t(55) = W=3505 W=3680 Main over amplitude F(4,54)=8.419 Main for experiment F(2,104) = t(6) = (Pk1) t(6) = (Pk2) t(6) = n1 n 1. ap n 1a p n 1,a ph 3 n 1,a ph 3 Result n 1, ap h 5 ds Statisti 2

3 2b Right,c 2b Left,c 2d Top 2d Bott om 2e Left 2e Right twotailed paired t for dependent samples twotailed paired t for dependent samples Twoways oneway Pearsons correlation and correlation significance Twoways (27<5, 30>=5), 38(20<5, 18>=5) Light responsive units before and after HFS schedule Light responsive units before and after control time period Light responsive units stimulated in different intensities at different time points. Light responsive units spontaneous FR in different time points correlation between basal FR and change in response after ohfs Evoked response change by splitting both Control and ohfs groups according to the basal FR of the unit Smaller and higher than 5Hz raw data scatter with xaxis representing before values and y axis representing after values descriptive represented in 3c Error bars indicate / raw data scatter with xaxis representing before values and y axis representing after values descriptive represented in 3c Error bars indicate / Circles represent bars indicate / Line represent average and shaded regions indicate / raw data scatter with xaxis representing Basal FR and y axis representing change in evoked response bars indicate / Fig 2 Fig 2 Fig 2 Fig 2 P = 8.9*10^(8) P = main of time P <1.0*10^(6) (post ho all P<0.005) main of intensity P <1.0*10^(6) (all P<1.0*10^(5) ) time * intensity P <1.0*10^(6) (post ho all P<0.001) P = Fig 2 P =5.7*10^(4) Fig 2 Experiment*u nit type p= Posthoc (HFS group P =0.0079) t(56) = t(37) = main of time F(4,23380)=230 main of intensity F(3,23380)= F(12,23380)= p< F(4,220)=0.05 r=0.46 F(1,88)=7.965, ap, ap, ap h 3, ap h 3, ap h 4, ap h 4 3

4 3cd 3ef 4a 4b 4d Twoways Twoways Twoways Twoways Threeways 7,9 47,50 20,18 20,18 13,13 Evoked responses to light stimulation at T1 and T0 Responses to CS of CS responsive units during the Extinction fear acquisition in extinction training in control/hfs mice fear acquisition in c: raw data scatter with xaxis representing before values and y axis representing after values 3d: Circles represent average and Error bars indicate / Circles represent bars indicate / Diamonds represent average and Error bars indicate / bars indicate / Diamonds represent average and Error bars indicate / Fig 3 Fig Interaction : p= Posthoc ohfs: p= control: p=0.962 Trial main P <1.0*10^(6) Group main post ho all p< Trial X Group P =0.009 posthoc both P<0.001 Trial main P <1.0*10^(6) Trial main P <1.0*10^(6) Group main P =0.05 Trial X Group P =0.08 posthoc P= Trial main P <1.0*10^(6) n3 n3 n4 n4 n4 F(1,908)=9.79 Trial main F(3,1392)= Group main Trial X Group : F(3,1392)=2.462 F(5,180)=17.62 Trial main F(1,36)= Group main F(1,36)=4.08 Trial X Group F(1,36)= F(5,300)= n3, ap n3 ap h 3 n4, ap h 1 n4, ap h 1 n4, ap 4

5 4e 5b 5c 5e 5f 1c Threeways Twoways Twoways Twoways Twoways twotailed paired Students t l. 13,13 8,10 8,10 9,10 9,10 10,10 (control) 11,11 (short HFS) 19,19 (long HFS) extinction training in control/hfs mice the Extinction retention in the Extinction retention in the Extinction retention in the Extinction retention in spontanous EPSPs during calibration pre and post ohfs l. 1 s, ds bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / mean error bar indicate / l. 1 Trial main P <1.0*10^(6) Experiment X Trial X Group P =0.02 pre extinction postho: early extinction: p=0.41 late extinction P=0.047 pre acquisition postho: early extinction: p=0.01 late extinction P= p= p= p= p= P = 0.74 (control) P = 0.85 (short HFS) P = 0.42 (long HFS) n4 n6 n6 n6 n6 l. 1 Trial main F(1,60)= Experiment X Trial X Group F(1,60)= F(1,16)= F(1,16)= F(1,17)= F(1,17)= t(9) = t(10) = t(18) = n4, ap n6 n6 n6 n6, 1d twotailed paired Students t l. 7,7 Amplitude of 1st Peak, 2nd Peak and Paired Pulse Ratio pre and post ohfs l. 1 s, ds mean error bar indicate / l. 1 P = (control) P = (short) P = 0.43 (PPR) l. 1 t(6)= t(6)= t(6)=0.0678, 5

6 1e Fig 2b 4a 4b 4c Left 4c Right 5d 5g 6c twotailed t for mean of Vm trace repeated measure oneway for binomial distribution Pearsons correlation and correlation significance twotailed t for repeated measure oneway Pearsons correlation and correlation significance Twoway l. l. ap n2 n2 n2 ap ap 6 7, , ,6 in each region Failure Rate of evoking oepsp in the 1st pulse and 2nd pulse pre and post ohfs acute slice replicate Light responsive units before, during and after HFS schedule Histograms depicting change in evoked FR for each responsive unit, at each of the measured time points, from T0 (top) to T90 (bottom). Scatter plot depicting the correlation between the dorsoventral location of the recording electrode and the ohfsinduced change in evoked response. Mean change in lightevoked spiking in units within the PL and IL. firing rate Before/ during/after train ohfsinduced attenuation in evoked response (% of baseline) Quantification of fluorescence normalized to baseline l. 1 s, ds l. 1, ds mean error bar indicate / mean and individual traces Circles represent bars indicate / distribution of units between 1 rand 1 Scatter plot depicting the correlation bars indicate / bars indicate / Circles represent bars indicate / bars indicate / l. 1 l P = (1st Peak) P = (2nd Peak) p=0.084 T0: p= ; T30: p= ; T60: p=0.003; T90: p=0.124; P= p= p=0.35 p= experiment X Region p=0.017 l t(6) = t(6) = F(2,54)=2.531 r= T(52)= F(2,94)= r=0.335 experiment X Region F(8,112)=2.47, , ap h 5, ap h 5 6 7a Left Twoway n3 5,5 fear acquisition in 7 Line represent average and shaded regions indicate / 7 p=0.027 n3 F(19,152)=1.796 n3, ap 6

7 7a Right 8 Cent er 8 Right 9a right 9b cent er 9b right 9c left 9c right 9d left 9d right Twoway Twoway Twoway twotailed t for Twoways twotailed t for twotailed t for twotailed t for Twoways twotailed t for n3 n4 n4 n5 n5 ap ap 5,5 10,8 10,8 7,8 9,10 9,10 10,10 10,10 9,10 9,10 fear acquisition in the Extinction training in control/hfs mice the Extinction retention in fear context extinction in fear context acquisition in fear context acquisition in Velocity in open field in control/hfs mice Percent of time spent in center of open field in Percent of time spent in elevated plus maze different zones in control/ HFS mice Velocity in elevated plus maze in control/hfs mice bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / bars indicate / p=0.28 p= p= p= Trial main P <1.0*10^(6) P = p=0.949 p= Main group p=0.85 Main zone P <7.0*10^(5) Post hoc (all p<0.001) Interaction p=0.158 p=0.132 n3 n4 n F(1,8)=1.32 F(1,16)= F(1,16)= T(13)= F(5,85)=20.13 t(17) = t(18)= t(18)=0.718 Main group F(1,17)=0.038 Main zone F(2,34)=12.92 Interaction F(2,34)=1.95 t(17)= n3, ap,,

8 Representative s 1. Are any representative images shown (including Western blot immunohistochemistry/staining) in the paper? If so, what (s)? 2. For each representative image, is there a clear statement of how many times this experiment was successfully repeated and a discussion of any limitations in repeatability? If so, where is this reported (section, #)? Statisti and general methods 1. Is there a justification of the sample size? If so, how was it justified? Where (section, #)? Even if no sample size calculation was performed, authors should report why the sample size is adequate to measure their size. 2. Are cal s justified as appropriate for every? Where (section, #)? a. If there is a section summarizing the cal methods in the metho is the cal for each experiment clearly defined? b. Do the data meet the assumptions of the specific cal you chose (e.g. normality for a parametric )? Where is this described (section, #)? c. Is there any estimate of variance within each group of data? Is the variance similar between groups that are being cally compared? Where is this described (section, #)? Yes; 3b, 3e. 2. 3a. 5c supp. Fig 6a, 6b 3b, 3e: n 3. 2a. 3a: n 3. 5c: n 2 supp. Fig 6a, 6b: supp. Fig 6 No cal s were run to predetermine sample size, stated in methods Yes, (methods ) Yes, (methods ) Yes, we used parametric if we could assume population normality or or had an appropriate n to assume sample distribution normality, otherwise we used nonparametric (for the cal s used see methods ) Yes, and if not df is corrected accordingly. d. Are s specified as one or twosided? Yes, two sided. methods e. Are there adjustments for multiple comparisons? Yes, post hoc Tukey HSD s were used. 8

9 3. To promote transparency, Nature Neuroscience has stopped allowing bar grs to report in the papers it publishes. If you have bar grs in your paper, please make sure to switch them to dotplots (with central and dispersion displayed) or to boxandwhisker plots to show data distributions. 4. Are criteria for excluding data points reported? Was this criterion established prior to data collection? Where is this described (section, #)? 5. Define the method of randomization used to assign subjects (or samples) to the experimental group to collect and process data. If no randomization was used, state so. Where does this appear (section, #)? 6. Is a statement of the extent to which investigator knew the group allocation during the experiment and in assessing outcome included? If no blinding was done, state so. Where (section, #)? 7. For experiments in live vertebrates, is a statement of compliance with ethical guidelines/regulations included? Where (section, #)? 8. Is the species of the animals used reported? Where (section, #)? 9. Is the strain of the animals (including background strains of KO/ transgenic animals used) reported? Where (section, #)? 10. Is the sex of the animals/subjects used reported? Where (section, #)? 11. Is the age of the animals/subjects reported? Where (section, #)? 12. For animals housed in a vivarium, is the light/dark cycle reported? Where (section, #)? OK Yes, methods No randomization was used, but automated s was used whenever possible. Stated in the Online ds section. Blinding was not performed, but automated s was used whenever possible. Stated in the Online ds section. Yes, methods section 2 Behavioral paradigms Yes, methods 1 Yes, methods 1 Yes, methods 1 Yes, methods 1 Yes, methods For animals housed in a vivarium, is the housing group (i.e. number of animals per cage) reported? Where (section, #)? Yes, methods 2 9

10 14. For behavioral experiments, is the time of day reported (e.g. light or dark cycle)? Where (section, #)? 15. Is the previous history of the animals/subjects (e.g. prior drug administration, surgery, behavioral ing) reported? Where (section, #)? a. If multiple behavioral s were conducted in the same group of animals, is this reported? Where (section, #)? 16. If any animals/subjects were excluded from s, is this reported? Where (section, #)? Reagents a. How were the criteria for exclusion defined? Where is this described (section, #)? b. Specify reasons for any discrepancy between the number of animals at the beginning and end of the study. Where is this described (section, #)? 1. Have antibodies been validated for use in the system under study (assay and species)? a. Is antibody catalog number given? Where does this appear (section, #)? b. Where were the validation data reported (citation, supplementary information, Antibodypedia)? Where does this appear (section, #)? 2. Cell line identity a. Are any cell lines used in this paper listed in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample? Where (section, #)? Yes, methods 2 Yes, methods 2 yes, methods 3 Context after cued conditioning extinction No animals were excluded from s. No experiments were conducted with cell lines. HEK 293 cells were used for virus production only. b. If yes, include in the ds section a scientific justification of their useindicate here in which section and the justification can be found. 10

11 c. For each cell line, include in the ds section a statement that specifies: the source of the cell lines have the cell lines been authenticated? If so, by which method? have the cell lines been ed for mycoplasma contamination? Where (section, #)? availability Provide a availability statement in the ds section under " availability", which should include, where applicable: Accession codes for deposited data Other unique identifiers (such as DOI hyperlinks for any other datasets) At a minimum, a statement confirming that all relevant data are available from the authors Formal citations of datasets that are assigned DOIs A statement regarding data available in the manuscript as source data A statement regarding data available with restrictions See our data availability and data citations policy page for more information. deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallogric data for small molecules d. Microarray data Deposition is strongly recommended for many other datasets for which structured public repositories exist; more details on our data policy are available here. We encourage the provision of other source data in supplementary information or in unstructured repositories such as Figshare and Dryad. We encourage publication of Descriptors (see Scientific ) to maximize data reuse. Where is the Availability statement provided (section, #)? Computer code/software Any custom algorithm/software that is central to the methods must be supplied by the authors in a usable and readable form for readers at the time of publication. However, referees may ask for this information at any time during the review process. 1. Identify all custom software or scripts that were required to conduct the study and where in the procedures each was used. 11

12 2. If computer code was used to generate results that are central to the paper's conclusions, include a statement in the ds section under "Code availability" to indicate whether and how the code can be accessed. Include version information as necessary and any restrictions on availability. Human subjects 1. Which IRB approved the protocol? Where is this stated (section, #)? 2. Is demogric information on all subjects provided? Where (section, #)? 3. Is the number of human subjects, their age and sex clearly defined? Where (section, #)? 4. Are the inclusion and exclusion criteria (if any) clearly specified? Where (section, #)? 5. How well were the groups matched? Where is this information described (section, #)? 6. Is a statement included confirming that informed consent was obtained from all subjects? Where (section, #)? 7. For publication of patient photos, is a statement included confirming that consent to publish was obtained? Where (section, #)? fmri studies For papers reporting functional imaging (fmri) results please ensure that these minimal reporting guidelines are met and that all this information is clearly provided in the methods: 1. Were any subjects scanned but then rejected for the s after the data was collected? a. If yes, is the number rejected and reasons for rejection described? Where (section, #)? 12

13 2. Is the number of blocks, trials or experimental units per session and/ or subjects specified? Where (section, #)? 3. Is the length of each trial and interval between trials specified? 4. Is a blocked, eventrelated, or mixed design being used? If applicable, please specify the block length or how the eventrelated or mixed design was optimized. 5. Is the task design clearly described? Where (section, #)? 6. How was behavioral performance measured? 7. Is an or factorial design being used? 8. For data acquisition, is a whole brain scan used? If not, state area of acquisition. a. How was this region determined? 9. Is the field strength (in Tesla) of the MRI system stated? a. Is the pulse sequence type (gradient/spin echo, EPI/spiral) stated? b. Are the fieldofview, matrix size, slice thickness, and TE/TR/ flip angle clearly stated? 10. Are the software and specific parameters (model/functions, smoothing kernel size if applicable, etc.) used for data processing and preprocessing clearly stated? 11. Is the coordinate space for the anatomical/functional imaging data clearly defined as subject/native space or standardized stereotaxic space, e.g., original Talairach, MNI305, ICBM152, etc? Where (section, #)? 12. If there was data normalization/standardization to a specific space template, are the type of transformation (linear vs. nonlinear) used and image types being transformed clearly described? Where (section, #)? 13. How were anatomical locations determined, e.g., via an automated labeling algorithm (AAL), standardized coordinate database (Talairach daemon), probabilistic atlases, etc.? 13

14 14. Were any additional regressors (behavioral covariates, motion etc) used? 15. Is the contrast construction clearly defined? 16. Is a mixed/random s or fixed inference used? a. If fixed s inference used, is this justified? 17. Were repeated measures used (multiple measurements per subject)? a. If so, are the method to account for within subject correlation and the assumptions made about variance clearly stated? 18. If the threshold used for inference and visualization in s varies, is this clearly stated? 19. Are cal inferences corrected for multiple comparisons? a. If not, is this labeled as uncorrected? 20. Are the results based on an ROI (region of interest) s? a. If so, is the rationale clearly described? b. How were the ROI s defined (functional vs anatomical localization)? 21. Is there correction for multiple comparisons within each voxel? 22. For clusterwise significance, is the clusterdefining threshold and the corrected significance level defined? Additional comments Additional Comments 14