Reporting Checklist for Nature Neuroscience

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1 Corresponding Author: Manuscript Number: Manuscript Type: Benjamin R. Rost NNT51693 Technical Report Reporting Checklt for Nature Neuroscience # Main Figures: 6 # Supplementary Figures: 9 # Supplementary Tables: 0 # Supplementary Videos: 0 Th checklt used to ensure good reporting standards and to improve the reproducibility of publhed results. For more information, please read Reporting Life Sciences Research. Please note that in the event of publication, it mandatory that authors include all relevant methodological and stattical information in the manuscript. Stattics reporting, by figure example example Please specify the following information for each panel reporting quantitative data, and where each item reported (section, e.g. Results, & paragraph number). Each figure should ideally contain an exact sample size (n) for each experimental group/condition, where n an exact number and not a range, a clear definition of how n defined (for example x cells from x slices from x animals from x litters, collected over x days), a description of the stattical used, the results of the s, any descriptive stattics and clearly defined error bars if applicable. For any experiments using custom stattics, please indicate the used and stats obtained for 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 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 and standard deviation are not appropriate on small samples, and plotting independent data points 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 mleading not to state th clearly. FIGURE NUMBER 1a results, para 6 TEST USED WHICH TEST? oneway ANOVA unpaired t Fig. Results para 6 EXACT VALUE 9, 9, 10, 15 n DEFINED? mice from at least 3 litters/group 15 slices from 10 mice ods para 8 Results para 6 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? Fig. Results para 6 P VALUE EXACT VALUE p = p = Fig. Results para 6 DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE F(3, 36) = 2.97 t(28) = Fig. Results para 6 1

2 FIGURE NUMBER 1c 1d 2c 3a 3b 3c 3c 3d 3d 3e TEST USED WHICH TEST? unpaired twotailed t No stattical performed. Aim of th experiment to demonstrat e the feasability of the method. Mann, twotailed Mann, twotailed Mann, twotailed t Mann, twotailed t Mann, twotailed Pearson correlation, twotailed EXACT VALUE Arch3: n=13; n=8 syphy: n=16; n=12 15 control: n=19; n=24 control: n=19; n=24 control: n=16; n=17 control: n=16; n=17 control: n=16; n=17 control: n=16; n=17 n=17 n DEFINED? n cells from N =2 n cells from N=3 n cells from N=2 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? P VALUE EXACT VALUE P= by Gaussian Approximatio n DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE U=0 P=0.7 t(26)=0.378 P< U=55 P=0.1 U=159 P=0.5; P=0.1 t(15)=0.701; t(16)=1.809 P=0.2 U=102 P=0.13; P< na na P = 0.04 t(15)=1.596; t(16)=5.719 P=0.02 U=72 Pearson r= 0.5 R^2 =

3 4b 5a 5b 5c 5d 5g 5h 5j 6 c 6 d t; Wilcoxon signed rank Wilcoxon signedrank t; Wilcoxon signedrank t t t t RRP: Pearson correlation, twotailed Pvr: Spearman correlation Oneway ANOVA with Tukey's post No stattical performed n=15 n=26 n=21 n=10 n=26 n=15 n=15 n=15 n=10 for all groups lysophoenix: n=18; CD63 phluorin: n=15 n cells from N=6 n cells from N=6 n cells from N=2 n cells from N=6 n cells from N=2 n cells from N=2 (5j) n.a. P<0.0001; P<0.001 t(14)=5.847 W=120 P< W=351 P<0.0001; P= t(20)=9.811 W=217 P=0.003 t(9)=4.155 P=0.004 t(25)=3.222 P= t(14)=5.116 P=0.02 t(14)=2.665 RRP: P< Pvr: P=0.003 (Gaussian Approx.) P<0.001 for LAMP2 vs. other markers. P>0.05 for all other comparons RRP: Pearson r=0.87; R^2=0.75 Pvr: Spearman r=0.71 only R^2 stated in the F(4,45)=

4 S2 a S2 b S2 c S2 d S2 e S2 f S3 b S4 d S5 f & g S6 a S6 b Twotailed Mann Test Twotailed Mann Test Twotailed Mann Test Twotailed Mann Test Twotailed Mann Test No stattical performed No stattical performed No stattical performed No stattical performed Twotailed Mann repeated measures twoway ANOVA with Sidak's multiple comparons post n=40; n=46 n=40; n=46 n=40; n=46 n=36; phoenix n=43 n=31; phoenix n=40 untreate d, phluorin: n=6; Baftreated, phluorn: n=8; Baftreated, n=15 n=5 both groups: n=8 n=42; Arch3: n=10 untreate d: n=20; Baftreated: n=21 n cells from N=2 n cells from 5 slices from 2 animals n cells from N=3 n cells, from N=7 (phoenix); N=2 (Arch3) n cells, from N=3 P=0.6 U=681 P=0.9 U=726 P=0.8 U=715 P=0.5 U=589 P=0.06 U=583 P< U=0 pre light untreated vs. treated: multiplicity adjusted P value = 0.02 postlight untreated vs. treated: multiplicity adjusted P value = Interaction: F (1, 39) = 11.7 Illumination: F (1, 39) = 14.9 Baftreatment: (1, 39) = 2.71 Subjects (matching): F (39, 39) = 8.96 not stated 4

5 S6 c S7 b S7 d S8 twoway ANOVA with Sidak's multiple comparons post for comparons repeated measures twoway ANOVA with Sidak's multiple comparons post Wilcoxon signedrank t No stattical performed untreate d: n=16; Baftreated: n=19 n=29 n=29 n=9 Representative figures n cells from N=3 n cells from N=5 n cells from N=5 n cells from N=2 1. Are any representative images shown (including Western blots and immunohtochemtry/staining) in the paper? If so, what figure(s)? amplitudes: pre light untreated vs. treated: multiplicity adjusted P value = postlight untreated vs. treated: multiplicity adjusted P value = 0.88 freq: pre light untreated vs. treated: multiplicity adjusted P value < postlight untreated vs. treated: multiplicity adjusted P value = Interaction: F (1, 63) = Illumination: F (1, 63) = Baftreatment: F (1, 63) = freq: Interaction F (1, 33 = Illumination: F (1, 33) = Baftreatment: F (1, 33) = Subjects (matching): F (33, 33) = not stated P < W = 435 P < t(28)=6 Yes. Representative immunocytochemtry images are shown in Fig. 1b, Fig. 6b, and Supplementary Fig. 9. Typical live cell imaging experiments are dplayed in Fig. 2a, Fig. 6d, Supplementary Fig. 1b and c, Supplementary Fig. 3a, Supplementary Fig. 4b, and Supplementary Fig. 5d and e. 5

6 2. For each representative image, there a clear statement of how many times th experiment was successfully repeated and a dcussion of any limitations in repeatability? If so, where th reported (section, paragraph #)? Stattics and general methods 1. Is there a justification of the sample size? If so, how was it justified? Even if no sample size calculation was performed, authors should report why the sample size adequate to measure their effect size. 2. Are stattical s justified as appropriate for every figure? a. If there a section summarizing the stattical methods in the meth the stattical for each experiment clearly defined? b. Do the data meet the assumptions of the specific stattical you chose (e.g. normality for a parametric )? Where th described (section, paragraph #)? c. Is there any estimate of variance within each group of data? Is the variance similar between groups that are being stattically compared? Where th described (section, paragraph #)? The immunocytochemtry images are representatives of two to three independent experiments, as stated in section Immunocytochemtry. Live cell imaging experiments of phluorin signals in Fig. 2a, Supplementary Fig. 1c, Supplementary Fig. 3a and Supplementary Fig. 5d and e were quantified, images are shown to illustrate the effects. The number of ed cells are stated in the figure s. Live cell fluorescence of infected cells, as shown in Supplementary Fig. 1b and Supplementary Fig. 4b, were routinely checked for electrophysiological experiments, but not further ed. Images are representative for these experiments. Sample sizes were not predetermined by stattical meth but are similar to those reported in previous publications. Th stated in the Online methods. Appropriate stattical s were applied for each set of data. Details of the s are provided in the ods section under the title " ". Stattical methods are summarized under. For each experiment, the stattical specified in the figure. dtribution was ed for each set of data using the D'Agostino and Pearson omnibus normality. Stated in. We have not systematically ed for equal variances, but report SEM providing a reliable measure of variance within and between groups. d. Are s specified as one or twosided? Yes. We only used twosided s. e. Are there adjustments for multiple comparons? We performed Sidak's multiple comparons and report multiplicityadjusted Pvalues for the twoway ANOVAs. 3. Are criteria for excluding data points reported? Was th criterion establhed prior to data collection? Where th described (section, paragraph #)? During the of miniature EPSCs (Fig. 4, Fig. 5, Supplementary Fig. 2, Supplementary Fig. 6), cells were excluded posthoc when falsepositive events occured at a frequency of > 2 Hz. Described in. 6

7 4. Define the method of randomization used to assign subjects (or samples) to the experimental groups and to collect and process data. If no randomization was used, state so. Where does th appear (section, paragraph #)? 5. 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. 6. For experiments in live vertebrates, 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, the light/dark cycle reported? 12. For animals housed in a vivarium, the housing group (i.e. number of animals per cage) reported? 13. For behavioral experiments, the time of day reported (e.g. light or dark cycle)? 14. Is the previous htory of the animals/subjects (e.g. prior drug admintration, surgery, behavioral ing) reported? Viral particles encoding controls and phoenix constructs were randomly applied on different wells of the same 6 or 12 well plate. Recordings from multiple groups were performed on the same day, in a randomized order. No blinding was done throughout the study. Analys of s was performed by an automatic miniature event detection algorithm implemented in Axograph X, which does not require any manual selection of events. Therefore, we concluded the blinding was not necessary in th study. We did not perform experiments in live animals, but obtained primary cell from newborn mice or rats. Animals were handled in accordance with the regulations of local authorities and the animal welfare committee of the Charité Universitätsmedizin Berlin, Germany. Stated in section "Autaptic of primary hippocampal neurons". Yes. Stated in section "Cell Culture": Mice were used for autaptic, while rats were used for organotypic slice. Yes. We used C57/BL6N mice for autaptic and Wtar rats for organotypic slice. Stated in sections "Autaptic of primary hippocampal neurons" and "Hippocampal organotypic slice " Genders were equally used for the preparation of primary. Stated in section "Cell Culture". Yes. Stated in Cell culture, sections "Autaptic of primary hippocampal neurons" and "Hippocampal organotypic slice " 7

8 a. If multiple behavioral s were conducted in the same group of animals, th reported? 15. If any animals/subjects were excluded from, th reported? Reagents a. How were the criteria for exclusion defined? Where th described (section, paragraph #)? b. Specify reasons for any dcrepancy between the number of animals at the beginning and end of the study. Where th described (section, paragraph #)? 1. Have antibodies been validated for use in the system under study (assay and species)? a. Is antibody catalog number given? Where does th appear (section, paragraph #)? We only use commercially available antibodies that have been extensively used in our and other labs before. Optimal dilutions were determined in pilot experiments. Antibody catalog numbers are stated in section Immunocytochemtry. 8

9 b. Where were the validation data reported (citation, supplementary information, Antibodypedia)? Where does th appear (section, paragraph #)? 2. If cell lines were used to reflect the properties of a particular tsue or dease state, their source identified? a. Were they recently authenticated? Where th information reported (section, paragraph #)? References for antibodies are not stated in the manuscript for space limitations. Examples for references are given below: The polyclonal antivglut1 antibody (# , Synaptic Systems) has been K.O. verified by the company ( products/vglut1/facts php) and has been used before, e.g. Wahl, Katiyar, Schmitz, 2013, The Journal of Neuroscience ( The polyclonal antimap2 antibody (AB5543, Chemicon, now EMD Millipore) has been used by our and other labs before, e.g. Chao, Zhogbi, Rosenmund, 2007, Neuron ( science/article/pii/s ), and Xu et al., 2013, Journal of Neuroscience ( content/33/13/5867.long). The polyclonal antigfp antibody (#A11122, Life Technologies) has been used before to detect phluorin molecules in immunocytochemtry assays, e.g. Matsuda et al., 2009, The Journal of Neuroscience ( content/29/45/14185.full). The monoclonal antilamp2 /CD107b antibody (clone H4B4 by Developmental Studies Hybridoma Bank, provided by Hs Diagnostics, Cat. No 10672C100) has been publhed before, e.g. Lorents et al., 2012, The Journal of Biological Chemtry ( The monoclonal antirab 5 antibody (clone 621.3; , Synaptic Systems) has been publhed before, e.g. Das et al. 2013, Neuron ( S ). The monoclonal antipdi antibody (RL90; MA 3019, Affinity BioReagents, now Thermo Scientific) has been publhed before, e.g. Markovic et al. 2004, Blood ( content/103/5/1586.long?ssochecked=true#sec2). The monoclonal antigm 130 antibody (Clone 35/GM130; #610823, BD Biosciences) has been publhed before, e.g. Kovacs et al., 2006, Journal of Cell Science ( content/119/13/2715.full). The monoclonal antihsp60 antibody (Clone 24/HSP60; #611563, BD Biosiences) has been publhed before, e.g. Ulett et. al., 2005, Journal of Immunology ( content/175/4/2555.full.html). 9

10 deposition deposition in a public repository mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Microarray data Deposition strongly recommended for many other datasets for which structured public repositories ext; more details on our data policy are available here. We encourage the provion 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. 1. Are accession codes for deposit dates provided? Computer code/software Constructs will be made available to the scientific community via Addgene. Genbank accession numbers are provided in the ods. Genbank accession number KT lyso Genbank accession number KT lysophluorin: Genbank accession number KT Any custom algorithm/software that 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 th 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. 2. If computer code was used to generate results that are central to the paper's conclusions, include a statement in the ods 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 th stated (section, paragraph #)? 2. Is demographic information on all subjects provided? 3. Is the number of human subjects, their age and sex clearly defined? 4. Are the inclusion and exclusion criteria (if any) clearly specified? 10

11 5. How well were the groups matched? Where th information described (section, paragraph #)? 6. Is a statement included confirming that informed consent was obtained from all subjects? 7. For publication of patient photos, a statement included confirming that consent to publh was obtained? fmri studies For papers reporting functional imaging (fmri) results please ensure that these minimal reporting guidelines are met and that all th information clearly provided in the methods: 1. Were any subjects scanned but then rejected for the after the data was collected? a. If yes, the number rejected and reasons for rejection described? 2. Is the number of blocks, trials or experimental units per session and/ or subjects specified? 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? 6. How was behavioral performance measured? 7. Is an ANOVA or factorial design being used? 8. For data acquition, a whole brain scan used? If not, state area of acquition. a. How was th region determined? 11

12 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, paragraph #)? 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, paragraph #)? 13. How were anatomical locations determined, e.g., via an automated labeling algorithm (AAL), standardized coordinate database (Talairach daemon), probabiltic atlases, etc.? 14. Were any additional regressors (behavioral covariates, motion etc) used? 15. Is the contrast construction clearly defined? 16. Is a mixed/random effects or fixed inference used? a. If fixed effects inference used, th 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 vualization in figures varies, th clearly stated? 19. Are stattical inferences corrected for multiple comparons? a. If not, th labeled as uncorrected? 12

13 20. Are the results based on an ROI (region of interest)? a. If so, the rationale clearly described? b. How were the ROI s defined (functional vs anatomical localization)? 21. Is there correction for multiple comparons within each voxel? 22. For clusterwe significance, the clusterdefining threshold and the corrected significance level defined? Additional comments Additional Comments 13