Reporting Checklist for Nature Neuroscience

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1 Corresponding Author: Manuscript Number: Manuscript Type: Atsushi Miyawaki NNT51493A Technical Report Reporting Checklist Nature Neuroscience # Main Figures: 7 # Supplementary Figures: 12 # Supplementary Tables: 3 # Supplementary Videos: 2 This checklist is used to ensure good reporting stards to improve the reproducibility of published results. For more inmation, please read Reporting Life Sciences Research. Please note that in the event of publication, it is matory that authors include all relevant methodological statistical inmation in the manuscript. Statistics reporting, by figure example example Please specify the following inmation each panel reporting quantitative data, where each item is reported (section, e.g. Results, paragraph number). Each figure should ideally contain an exact sample size (n) each experimental group/condition, where n is an exact number not a range, a clear definition of how n is defined ( example x cells from x slices from x animals from x litters, collected over x days), a description of the statistical test used, the results of the tests, any descriptive statistics clearly defined error bars if applicable. For any experiments using custom statistics, please indicate the test used stats obtained each experiment. Each figure 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 figures, please use the paragraph number instead of the figure number. Note: Mean stard deviation are not appropriate on small samples, plotting independent data points is usually more inmative. When technical replicates are reported, error significance measures reflect the experimental variability not the variability of the biological process; it is misleading not to state this clearly. FIGURE NUMBER 1a results, TEST USED WHICH TEST? oneway ANOVA unpaired t test SECTION Fig. Results EXACT VALUE 9, 9, 10, 15 n DEFINED? mice from at least 3 litters/group 15 slices from 10 mice SECTION Methods para 8 Results DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? error bars are mean / SEM error bars are mean / SEM SECTION Fig. Results P VALUE EXACT VALUE SECTION Fig. Results DEGREES OF FREEDOM F/t/z/R/ETC VALUE VALUE F(3, 36) = 2.97 t(28) = SECTION Fig. Results 1

2 FIGURE NUMBER 2b 2b TEST USED WHICH TEST? Oneway ANOVA with Bonferroni's multiple s test Oneway ANOVA with Bonferroni's multiple s test SECTION Fig,2 Fig,2 EXACT VALUE 9, 6, 4, 4 9, 6, 4, 4 n DEFINED? transmittance ScaleStreated hemispheres(9) CUBICtreated(6), 3DISCOtreated(4), or SeeDBtreated(4) hemispheres. normalized YFP fluorescence ScaleStreated hemispheres(9) CUBICtreated(6), 3DISCOtreated(4), or SeeDBtreated(4) hemispheres. SECTION Fig. 2 Fig. 2 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? mean / SD mean / SD SECTION Fig. 2 Fig. 2 P VALUE EXACT VALUE [Oneway ANOVA] E15 [Bonferroni test] E6 ScaleS CUBIC E11 ScaleS 3DISCO E10 ScaleS SeeDB [Oneway ANOVA] E11 [Bonferroni test] E10 ScaleS CUBIC E11 ScaleS 3DISCO E7 ScaleS SeeDB SECTION DEGREES OF FREEDOM F/t/z/R/ETC VALUE VALUE [Oneway ANOVA] F(3, 20)= [Bonferroni test] t = ScaleS CUBIC. t = ScaleS 3DISCO. t = ScaleS SeeDB. [Oneway ANOVA] F(3, 20)= [Bonferroni test] t = ScaleS CUBIC. t = ScaleS 3DISCO. t = ScaleS SeeDB. SECTION 2

3 2d 2d 3y S 10d S Tab3 S Tab3 Welch's unpaired ttest, twotailed Welch's unpaired ttest, twotailed Mann Whitney's Utest Welch's unpaired ttest, twotailed Welch's unpaired ttest, twotailed Welch's unpaired ttest, twotailed Fig,2 Fig,2 Fig.10 6, 7 6, 7 6, 8 10, 10 3, 6 3, 9 transmittance ScaleStreated brain slices(6) PACTtreated brain slices(7). normalized YFP fluorescence ScaleStreated brain slices(6) PACTtreated brain slices(7). presynaptic fabric postsynaptic fabric ScaleS PBS( ) treated brain hemispheres. Six regions from 3 mice (ScaleS). Eight regions from 3 mice (PBS( )). synaptic density inside outside amyloid plaques. Ten areas from a 24monthsold (APPknockin) mouse brain were analyses. Density of diffuse amyloid plaques in AD patient brains. diffuse plaque density cored plaque () samples (3) cored plaque ( ) samples (6). microglial association cored plaques (3) diffuse plaques (9). Nine AD patient samples were used. Diffuse plaques were found in all 9 samples.; cored plaques were found in 3 of 9. Fig. 2 Fig. 10 Fig. 10 mean / SD mean / SD mean / SEM mean / SEM mean / SD mean / SD Fig. 10 Fig.10 Table 3 Table P = E4 For presynaptic fabric: For postsynaptic fabric: Fig.10 t = df = t = df = For presynaptic fabric: U=11 Z= For postsynaptic fabric: U=9.5 Z= t = 4.807, df = 18 t = , df = t = df = Fig.10 3

4 Representative figures 1. Are any representative images shown (including Western blots immunohistochemistry/staining) in the paper? If so, what figure(s)? (Main Figs) <Fig.1a,b> At least 3 different samples were examined each condition. <Fig. 1d,e> See Fig. 2 Fig. 3. <Fig. 2a, c> Representative images (#1) are shown. All the images other than #1 are shown in Supplementary Fig. 3. <Fig. 3a x> See Table 2. <Fig. 4b h> The same results were obtained from another AppNLF/NLF mouse (20 months old). <Fig. 5a,b> Observation from different angles revealed that plaques were mostly located in the cerebral cortex. <Fig. 5d> A similar movie was created using another 20monthold AppNLF/ NLF mouse. <Fig.5e h> See Fig. 10. <Fig. 5in> More than 3 single plaques were successfully tracked from hemispheres of 2 different animals. <Fig.6b h> Similar images were obtained from 3 other AppNLF/NLF mice (10, 12, 14 month old) (See Fig. 11). <6j,k> See. <6n q> See. <Fig.7a l> Similar results were obtained using 11 6 mice ScaleSQ(0) ScaleSQ(5) experiments, respectively. (Supplementary Figs) <Supplementary Fig. 5> The experiment was permed in triplicate each using different mouse brains. <Supplementary Fig. 6a f> Similar TEM images were obtained from 3 other mice of the same age. <Supplementary Fig. 6g i> Similar TEM images were obtained from 3 other mice of the same age. <Supplementary Fig. 9b> Similar results were obtained with another cube from #1625. <Supplementary Fig. 10a c> Three representative images of 10. 4

5 2. For each representative image, is there a clear statement of how many times this experiment was successfully repeated a discussion of any limitations in repeatability? If so, where is this reported (section, paragraph #)? Statistics general methods 1. Is there a justification of the sample size? If so, how was it justified? Even if no sample size calculation was permed, authors should report why the sample size is adequate to measure their effect size. 2. Are statistical tests justified as appropriate every figure? a. If there is a section summarizing the statistical methods in the methods, is the statistical test each experiment clearly defined? b. Do the data meet the assumptions of the specific statistical test you chose (e.g. normality a parametric test)? Where is this described (section, paragraph #)? c. Is there any estimate of variance within each group of data? Is the variance similar groups that are being statistically compared? Where is this described (section, paragraph #)? In the s of the abovementioned figures, supplementary figures, supplementary tables. The sample size was determined by the availability of Tg mice (YFP H ChR2YFP) or APP knockin mice. No statistical methods were used to predetermine sample sizes, but our sample sizes (n > 3 mice per group imaging analysis) are similar to those generally used in this field. When N = 2 or 3, individual data are shown (e.g., Fig. 8). Yes, they are justified in "Statistical analysis" in the Online Methods, indicated in the s of Fig. 2, Fig. 3, Supplementary Fig. 10, Supplementary Table. 3. Also, a correlation coefficient is shown in Supplementary Fig.7b. Yes, there is a section "Statistical analysis" in the Online Methods. Yes, the statistical test each experiment is defined in the. The distribution of data in each experiment was checked normality /or equal variation. In the section "Statistical analysis" in the Online Methods. Yes, the variance of each data group is shown with the stard deviation (S.D.) or stard error of mean (S.E.M.). Yes, equal variation was checked. In the s of Fig. 2, Fig. 3, Supplementary Fig. 10, Supplementary Table. 3. d. Are tests specified as one or twosided? Unpaired ttest was used twogroup in Fig. 2 Supplementary Fig. 10 Supplementary Table. 3. Both tests are specified as twosided. e. Are there adjustments multiple s? Yes, Bonferroni adjustments were used. 3. Are criteria excluding data points reported? Was this criterion established prior to data collection? Where is this described (section, paragraph #)? 4. Define the method of romization used to assign subjects (or samples) to the experimental groups to collect process data. If no romization was used, state so. No. There are no data excluded. No romization of mice or samples was used prior to analysis. Where does this appear (section, paragraph #)? 5

6 5. Is a statement of the extent to which investigator knew the group allocation during the experiment in assessing outcome included? If no blinding was done, state so. 6. For experiments in live vertebrates, is a statement of compliance with ethical guidelines/regulations included? 7. Is the species of the animals used reported? 8. Is the strain of the animals (including background strains of KO/ transgenic animals used) reported? 9. Is the sex of the animals/subjects used reported? 10. Is the age of the animals/subjects reported? 11. For animals housed in a vivarium, is the light/dark cycle reported? 12. For animals housed in a vivarium, is the housing group (i.e. number of animals per cage) reported? 13. For behavioral experiments, is the time of day reported (e.g. light or dark cycle)? 14. Is the previous history of the animals/subjects (e.g. prior drug administration, surgery, behavioral testing) reported? When evaluating different methods comparatively, the analyzers were blinded to all conditions during data analysis. In "Statistical analysis" of the Online Methods. The experimental procedures housing conditions animals were approved by the Institute s Animal Experiments Committee all animals were cared treated humanely in accordance with the Institutional Guidelines Experiments using animals. This is stated in the Online Methods section: Mouse lines. In the Online Methods section Figure Legends. In the Online Methods section: Mouse lines, Figure Legends. A general statement is made in the Online Methods section: Mouse lines. For mouse brain study, the gender inmation is shown in Fig. 2 Supplementary Fig. 3. For human postmortem study, the gender inmation is shown in Fig. 6 Supplementary. Yes, the age is always specified in all display items their s. Very often in the main text too. Mice were housed under 12 hrs light/12 hrs dark cycle, the statement is made in the Online Methods section: Mouse lines. Five mice per cage. In the Online Methods section: Mouse lines. 6

7 a. If multiple behavioral tests were conducted in the same group of animals, is this reported? 15. If any animals/subjects were excluded from analysis, is this reported? Reagents a. How were the criteria exclusion defined? Where is this described (section, paragraph #)? b. Specify reasons any discrepancy the number of animals at the beginning end of the study. Where is this described (section, paragraph #)? 1. Have antibodies been validated use in the system under study (assay species)? a. Is antibody catalog number given? Where does this appear (section, paragraph #)? b. Where were the validation data reported (citation, supplementary inmation, Antibodypedia)? Where does this appear (section, paragraph #)? 2. If cell lines were used to reflect the properties of a particular tissue or disease state, is their source identified? Male mice (wildtype, C57BL/6J) were used examining clearing capability ultrastructure preservation of methods. Otherwise, no animal exclusion was made. In the online Methods: Mouse lines. The antibodies used 3Dimmunohistochemistry (3DIHC) were validated by 2DIHC that used cryosections prepared from C57BL/6J or APP(NLF/NLF) mouse brain tissues AD patient brain tissues. Their catalog number are given in the Online Methods section (see Immunohistochemistry). The mouse monoclonal antiamyloid beta antibody (clone 6E10) was validated by Vidal et al. [FASEB J 26, (2012)]. The mouse monoclonal antineun antibody (clone A60) was validated by Zhu et al. [J. Neurosci. 23, (2003)]. The rabbit polyclonal antiiba1 antibody was validated by Yoshikawa et al. [Plos ONE 5 e13932 (2010)]. These papers are cited in the Online Methods section. The validation data of the rabbit polyclonal antineun antibody was given by the Millipore (URL:http: product/antineun%28rabbit%29antibody,mm_nfabn78? iscountryemd=yes). Rabbit polyclonal antivglut1 serum, rabbit polyclonal antiglur1 antibody, mouse monoclonal antipsa NCAM antibody (clone 22B) were validated in our previous paper [Hama et al. Nat. Neurosci. 14, (2011)]. Other antibodies, rabbit polyclonal antimap2 antibody (Millipore, Chemicon), mouse monoclonal antigfap antibody [clone GA5 (Sigma)], mouse monoclonal antisynaptophysin antibody [clone SVP38 (Synaptic Systems)], rabbit polyclonal antisv2a serum (Synaptic Systems), mouse monoclonal antiglur2 antibody [clone 6C4 (Millipore Chemicon)] were validated by their suppliers via product specification data. 7

8 a. Were they recently authenticated? Where is this inmation reported (section, paragraph #)? Data deposition Data deposition in a public repository is matory : a. Protein, DNA RNA sequences b. Macromolecular structures c. Crystallographic data small molecules d. Microarray data Deposition is strongly recommended many other datasets which structured public repositories exist; more details on our data policy are available here. We encourage the provision of other source data in supplementary inmation or in unstructured repositories such as Figshare Dryad. We encourage publication of Data Descriptors (see Scientific Data) to maximize data reuse. 1. Are accession codes deposit dates provided? Computer code/software Any custom algorithm/software that is central to the methods must be supplied by the authors in a usable readable m readers at the time of publication. However, referees may ask this inmation at any time during the review process. 1. Identify all custom software or scripts that were required to conduct the study where in the procedures each was used. 2. If computer code was used to generate results that are central to the paper's conclusions, include a statement in the Methods section under "Code availability" to indicate whether how the code can be accessed. Include version inmation as necessary any restrictions on availability. Human subjects 1. Which IRB approved the protocol? Where is this stated (section, paragraph #)? Open CV Threshold is stated in the Online Methods section: Image Segmentation, it was used in the experiments Fig. 5, Fig. 6, Supplementary Fig. 11. Distance Calculation from Volocity is stated in the Methods section: Measurement of distances, it was used in the experiments Fig. 6 Supplementary Fig D Reslicer is stated in the Online Methods section: Image reslicing in the zaxis, it was used in the experiments Fig. 6 Supplementary Fig. 11. Jinarashi is cited in the Online Methods section: Image segmentation, it was used 3D volume rendering in the experiments Fig. 5a, b, d, Fig. 6b h, 6i l, 6n r, Supplementary Fig. 11b d. Computer code was not used. The software programs "Image segmentation", "Measurement of 3D distances", "Image reslicing in the zaxis" are available upon request (stated in the Online Methods). The hling ethics human specimens were approved by the Institute s Research Ethics Committee all the human tissues were cared treated in accordance with the Institutional Guidelines Experiments with Human Specimens (RIKEN). This is stated in the Online Methods section (see "Human sample preparation"). 8

9 2. Is demographic inmation on all subjects provided? 3. Is the number of human subjects, their age sex clearly defined? 4. Are the inclusion exclusion criteria (if any) clearly specified? 5. How well were the groups matched? Where is this inmation described (section, paragraph #)? 6. Is a statement included confirming that inmed consent was obtained from all subjects? 7. For publication of patient photos, is a statement included confirming that consent to publish was obtained? fmri studies No. We examined postmortem brain tissues of Alzheimer's disease patients. The inmation of age sex is provided in Fig. 6, Supplementary. For papers reporting functional imaging (fmri) results please ensure that these minimal reporting guidelines are met that all this inmation is clearly provided in the methods: 1. Were any subjects scanned but then rejected the analysis after the data was collected? a. If yes, is the number rejected reasons rejection described? 2. Is the number of blocks, trials or experimental units per session / or subjects specified? 3. Is the length of each trial interval 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. 9

10 5. Is the task design clearly described? 6. How was behavioral permance measured? 7. Is an ANOVA 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, TE/TR/ flip angle clearly stated? 10. Are the software specific parameters (model/functions, smoothing kernel size if applicable, etc.) used data processing preprocessing clearly stated? 11. Is the coordinate space the anatomical/functional imaging data clearly defined as subject/native space or stardized stereotaxic space, e.g., original Talairach, MNI305, ICBM152, etc? Where (section, paragraph #)? 12. If there was data normalization/stardization to a specific space template, are the type of transmation (linear vs. nonlinear) used image types being transmed clearly described? Where (section, paragraph #)? 13. How were anatomical locations determined, e.g., via an automated labeling algorithm (AAL), stardized coordinate database (Talairach daemon), probabilistic atlases, etc.? 14. Were any additional regressors (behavioral covariates, motion etc) used? 15. Is the contrast construction clearly defined? 16. Is a mixed/rom effects or fixed inference used? a. If fixed effects inference used, is this justified? 17. Were repeated measures used (multiple measurements per subject)? 10

11 a. If so, are the method to account within subject correlation the assumptions made about variance clearly stated? 18. If the threshold used inference visualization in figures varies, is this clearly stated? 19. Are statistical inferences corrected multiple s? a. If not, is this labeled as uncorrected? 20. Are the results based on an ROI (region of interest) analysis? a. If so, is the rationale clearly described? b. How were the ROI s defined (functional vs anatomical localization)? 21. Is there correction multiple s within each voxel? 22. For clusterwise significance, is the clusterdefining threshold the corrected significance level defined? Additional comments Additional Comments 11