Reporting Checklist for Nature Neuroscience - Semantic Scholar

Andreas Luthi

# Main Figures:

6

Manuscript Number:

NN-A51870A

# Supplementary Figures:

10

Manuscript Type:

Article

# Supplementary Tables:

0

# Supplementary Videos:

0

nature neuroscience | reporting checklist

Corresponding Author:

Reporting Checklist for Nature Neuroscience This checklist is used to ensure good reporting standards and 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 statistical information in the manuscript.



Statistics reporting, by figure Please specify the following information for each panel reporting quantitative data, and where each item is reported (section, e.g. Results, & paragraph number).

z

Each figure legend 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 statistical test used, the results of the tests, any descriptive statistics and clearly defined error bars if applicable.

z

z

For any experiments using custom statistics, please indicate the test used and stats obtained for each experiment.

z

Each figure legend 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.

z

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 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.

Fig. legend

9, 9, 10, 15

results, para 6

unpaired ttest

Results para 6

15

mice from at least 3 Methods litters/group para 8

slices from 10 mice

Results para 6

error bars are mean +/- SEM error bars are mean +/- SEM

EXACT VALUE

Fig. legend

p = 0.044

Results para 6

p = 0.0006

VALUE

SECTION & PARAGRAPH #

one-way ANOVA

REPORTED?



example

1a

DEFINED?


FIGURE NUMBER

EXACT VALUE

Fig. legend

F(3, 36) = 2.97

Fig. legend

Results para 6

t(28) = 2.808

Results para 6

November 2014

WHICH TEST?

(AVERAGE, VARIANCE)

DEGREES OF FREEDOM & F/t/z/R/ETC VALUE

P VALUE


DESCRIPTIVE STATS

n

example

TEST USED

1

-

+

-

1b

11 for CS Fig. only Paired t-test Legend 12 for CS-US

Yes

Fig. Legend

1d

Unpaired ttest

11 for CS only 12 for CS-US

Yes

Fig. legend

Fig. legend

EXACT VALUE

error bars are mean +/- SEM

***p < 0.001 for CS-US (CS+ Fig. vs CS-) legend p = 0.404 for CS only (CS+ vs CS-)

Fig. legend


Fig. legend

Fig. legend

p = 0.865

Panel 1e, CS-/ CS+ ratio vs time in open arms (p < 0.001)

+

-

1e, f

Linear regression

Fig. legend

12

Yes

Fig. legend

Each dot represent an animal

Fig. legend

Panel 1f, CSratio vs time in open arms for CS only group (p= 0.260)

Panel 1e, CS-/CS+ ratio vs time in open arms (R = 0.6)

Fig. legend

-

+

+

+

+

+

+

+

-

Figure legend

1g

One-Way ANOVA

7 ChR, 9 control, 6 ARCH for Fig. EPM legend baseline tacklengt h

2l

Two-Way ANOVA

Fig. legend

7, 6

Yes

Fig. legend


Fig. legend

p = 0.005

Fig. legend

2l

Paired t-test

Fig. legend

7, 6

Yes

Fig. legend


Fig. legend

p = 0.011

Fig. legend

2m

Paired t-test

Fig. legend

7, 9, 6

Yes

Fig. legend


Fig. legend

p = 0.009

Fig. legend

2m

Two-Way ANOVA

Fig. legend

7, 9, 6

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend

2p

Paired t-test

Fig. legend

7, 8, 7

Yes

Fig. legend


Fig. legend

p = 0.008

Fig. legend

2p

Two-Way ANOVA

Fig. legend

7, 8, 7

Yes

Fig. legend


Fig. legend

P < 0.01

Fig. legend

3b

Paired t-test

Fig. legend

6

Yes

Fig. legend


Fig. legend

p = 0.005

Fig. legend

3c

One-Way ANOVA followed by Post-hoc Tukey Test

Fig. legend

4

Yes

Fig. legend


Fig. legend

p = 0.013

Fig. legend

Yes

Fig. legend


Fig. legend

p = 0.766

Fig. legend

F(1,11) = 11.83

Fig. legend

F(2,19) = 26.72

Fig. legend

F(2,19) = 58.587

Fig. legend November 2014

+

Panel 1f, CSratio vs time in open arms for CS only group (R = 0.298) Panel 1f, CSratio vs time in open arms for CS only group (R= 0.287)

Panel 1f, CSratio vs time in open arms for CS only group (p= 0.335)

+

VALUE


REPORTED?

DEGREES OF FREEDOM & F/t/z/R/ETC VALUE

P VALUE


DEFINED?


EXACT VALUE

(AVERAGE, VARIANCE)



FIGURE NUMBER

+

WHICH TEST?

DESCRIPTIVE STATS

n


TEST USED

2

-

+

-

+

-

+

-

+

+

-

+

-

+

-

+

-

+

+

+

+

Fig. legend

4

Yes

Fig. legend


Fig. legend

p = 0.029

Fig. legend

3e

Paired t-test Fig. (Wilcoxon Signed Rank legend Test)

5

Yes

Fig. legend


Fig. legend

p = 0.01

Fig. legend

4a

One-Way ANOVA followed by Dunn’s pairwise multiple comparison test

CS+ in CS-US p < 0.001

Fig. legend

11 CS only, 12 CS-US

Yes

4c

Unpaired ttest(MannWhitney Rank Sum Test)

Fig. legend

26 (CSUS), 14 (CS only)

4c


Fig. legend

4d

Linear regression

Fig. legend

5b

One-Way ANOVA followed by Dunn’s pairwise multiple comparison test

Fig. 5, 4, 5, 5, legend 6, 6

Yes

Fig. legend


Fig. legend

p = 0.006

Fig. legend

5c

Unpaired ttest (MannWhitney Rank Sum Test)

Fig. legend

9, 13

Yes

Fig. legend


Fig. legend

p = 0.008

Fig. legend

5d

Two tailed unpaired ttest (Equal variance Test)

Fig. legend

8

Yes

Fig. legend


Fig. legend

p = 0.043

Fig. legend

6c


Fig. legend

7, 8

Yes

Fig. legend


Fig. legend

p = 0.006

Fig. legend

6d

Unpaired tFig. test(Shapiro legend -Wilk)

7

Yes

Fig. legend


Fig. legend

p = 0.007

Fig. legend

6e


7

Yes

Fig. legend


Fig. legend

p = 0.005

Fig. legend

6f


5

Yes

Fig. legend


Fig. legend

p = 0.007

Fig. legend

Fig. legend


Fig. legend

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend

28 (CSUS), 14 (CS only)

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend

8 values

Yes. Each dot 2-4 cells


CS only p = 0.412

Fig. legend

p = 0.041

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-

3d

Unpaired ttest (MannWhitney Rank Sum Test)


+

3

-

+

-

+

-

S1d

Fig. legend

5

Yes

Fig. legend


Fig. legend

p < 0.05

Fig. legend

q = 3.8

S1g

One-Way ANOVA on Ranks followed by Tukey’s pairwise multiple comparison test

Fig. legend

6

Yes

Fig. legend


Fig. legend

p < 0.05

Fig. legend

q = 3.9

Yes

Fig. legend


Fig. legend

P = 0.766

Fig. legend

S2a

One-Way ANOVA

7 ChR, 9 control, 6 ARCH for Fig. EPM legend baseline tacklengt h


+


p = 0.656 for on/off in ChR

+

-

+

-

S2b

S2c

Paired t-test 5 ChR, 7 between Fig. control, 5 light on and legend ARCH off for each group

Yes

Paired t-test between Fig. light on and legend off

Yes

7

Fig. legend


Fig. legend

p = 0.445 for on/off in control

Fig. legend

p = 0.464 for on/off in ARCH Fig. legend


Fig. legend

p = 0.421

Fig. legend

p < 0.001 between on/ off in ChR group

+

-

S2d

Paired t-test between Fig. light on and legend off for each group

7 each group

Yes

Fig. legend


Fig. legend

p = 0.895 between on/ off in Control group

Fig. legend

p =0.049 between on/ off in Arch group p < 0.001 between on/ off in ChR group

+

-

S2e

Paired t-test between Fig. light on and legend off for each group

7 each group

Yes

Fig. legend


Fig. legend

p = 0.081 between on/ off in Control group

Fig. legend

+

+

-

S2f

Unpaired ttest

Fig. legend

7 each group

Yes

Fig. legend


Fig. legend

p= 0.014

Fig. legend

S2g

Unpaired ttest

Fig. legend

7 each groups

Yes

Fig. legend


Fig. legend

p = 0.011

Fig. legend

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p = 0.001 between on/ off in ARCH group

4

-

+

-

S2h

Unpaired ttest

Fig. legend

7 each groups

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend

S3b


Fig. legend

4

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend


+

p = 0.259 for SR(1 uM) +

-

S3d

Sample ttest vs control 0

Fig. legend

6, 15, 12

Yes

Fig. legend


Fig. legend

p = 0.130 for SR(50 uM)

Fig. legend

p < 0.001 for PTX (100uM) +

-

+

-

S3e

Paired t-test

Fig. legend

S3i


Fig. legend

6 for SR (1 uM), 15 for SR (50 uM)

4, 5

Yes

Fig. legend


Fig. legend

Yes

Fig. legend


Fig. legend

p < 0.001 for each SR concentration p = 0.188 for SR p = 0.031 for PTX

Fig. legend

Fig. legend

p = 0.206 for baseline vs SR +

-

+

-

+

-

+

-

+

+

+

-

-

Paired t-test

S3l


Fig. legend

4 for SR, 5 for PTX

Fig. legend

8 for SR, 10 for PTX

Yes

Fig. legend

Yes

Yes

Fig. legend

Fig. legend

S3m

Paired t-test

Fig. legend

8 for SR, 10 for PTX

S4a

Repeated measures One-Way ANOVA followed by Dunnett's multiple comparison test

Fig. legend

6

Yes

Fig. legend

S4b

Paired t-test

Fig. legend

7

Yes

Fig. legend

S4c

Paired t-test

Fig. legend

7

Yes

Fig. legend

S4d


Fig. legend

Liner regression

Fig. legend

S4e

7

Yes

Yes

Each dot is from a Fig. recorded cell legend


Fig. legend

p = 0.031 for baseline vs PTX p = 0.214 for SR p = 0.008 for PTX

p = 0.428 for baseline vs SR Each dot is from a Fig. p = 0.008 for recorded cell legend baseline vs PTX


Fig. legend

Fig. legend

p = 0.005 for SR vs PTX

Fig. legend


p = 0.01

Fig. legend


p = 0.904

Fig. legend

Fig. legend


Fig. legend

Each dot is from a recorded cell

Fig. legend

Fig. legend

Fig. legend

p = 0.016 for TTX p < 0.001 for PTX p = 0.028

Fig. legend

Fig. legend

R = 0.848

Fig. legend

November 2014

+

S3j

5

p = 0.004 L-655,708 (5 uM) p = 0.006 L-655,708 (50 uM) p = 0.009 PWZ (100 nM)

L-655,70 8 (6-9)

+

-

S6b

Sample ttest versus control 0

p = 0.019 PWZ (1 uM)

PWZ (4-5) Fig. legend

THDOC (8) Zolpidem (8)

Yes

Fig. legend


p = 0.945 Fig. THDOC 10 nM legend

Fig. legend


p = 0.024 L655,708 (50 nM)

p = 0.988 THDOC (100 nM)

PTX (14)

p = 0.379 Zolpidem (20 nM) p = 0.855 Zolpidem (100 nM) p = 0.938 Zolpidem (300 nM) p < 0.001 PTX (100 uM)

+

-

S6b

One-Way ANOVA followed by Tukey's multiple comparison test

Fig. legend

L-655,70 8 (6-9) Yes PWZ (4-5)

Fig. legend


p = 0.012 L655,708 (50 uM) vs THDOC Fig. (100 nM) legend p = 0.024 PTX (100 uM) vs all conditions

Fig. legend

p = 0.593 L-655,708 (50 nM)

+

-

S6c


Fig. legend

L-655,70 8 (6-9) Yes PWZ (4)

Fig. legend


Fig. legend

p = 0.325 L-655,708 (5 uM)

Fig. legend

p = 0.351 L-655,708 (50 uM) p = 0.902 PWZ (1 uM)

November 2014

6


p = 0.208 L-655,708 (50 nM)

+

-

S6d


Fig. legend

L-655,70 8 (6-9) Yes PWZ (4)

Fig. legend


Fig. legend

p = 0.195 L-655,708 (5 uM)

Fig. legend

p = 0.157 L-655,708 (50 uM) p = 0.144 PWZ (1 uM)

+

-

+

+

+

-

+

-

+

+

+

-

+

-


Fig. legend

5-6

S7d

Unpaired ttest

Fig. legend

8 for vehicle 6 for PWZ group

S8b

Upaired ttest

Fig. legend

Linear regression

Fig. legend

S6e

S8c

S8e

Repeated measures One-Way ANOVA

S8f

One-way ANOVA followed by Bonferroni Post-hoc test

S8g

Linear regression

S8h


S8i


Fig. legend

Yes

Yes

8 CS only

7

5 each group


Fig. legend

Fig. legend


Fig. legend

p < 0.001

Fig. legend

Fig. legend

p = 0.008

Fig. legend

Fig. legend

p = 188

Fig. legend

Fig. legend

12 CS-US Yes

Yes

p = 0.025 for PWZ (100 nM)

Fig. legend

Fig. legend

Fig. legend

Each dot is from 2-4 cells from a single animal


Fig. legend

p = 0.046 for PWZ (1 uM)

p < 0.001 PTX versus THDOC (10 and 100 nM) for CS only

Fig. legend

R = 0.145

Fig. legend

Fig. legend

p = 0.001 PTX versus THDOC (10 and 100 nM) for CS-US

Fig. legend

11 (CS only) 12 (CSUS) 10 (US only)

Fig. legend

11

Fig. legend

6 neurons for US only (the rest is in fig. 3)

Fig. legend

6 neurons for US only (the rest is in fig. 3)

Yes

Fig. legend


Fig. legend

p < 0.001 CS only versus CS-US or US only

Fig. legend

Yes

Fig. legend


Fig. legend

p < 0.001

Fig. legend

Yes

Fig. legend


Fig. legend


Fig. legend

Yes

Fig. legend


Fig. legend


Fig. legend

November 2014

7

Representative figures

1. Are any representative images shown (including Western blots and immunohistochemistry/staining) in the paper?

Yes, there are representative traces in Figs. 1, 2, 3, 4,5 and Supplementary Figs. 1, 2, 3, 4, 5, 6, 8, 9.

If so, what figure(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?

The statistical sample size is indicated in each figure legend.

If so, where is this reported (section, paragraph #)?






Statistics and general methods

1. Is there a justification of the sample size? If so, how was it justified?

The sample size is justified by checking on the overall standard deviation and variability of the groups. Highly variable samples were hard to define and compared with another group.

Where (section, paragraph #)? Even if no sample size calculation was performed, authors should report why the sample size is adequate to measure their effect size.

2. Are statistical tests justified as appropriate for every figure? Where (section, paragraph #)?

No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications.

Standard statistical tests were used to compare either a change from baseline or between sample groups. No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications. Statistics

a. If there is a section summarizing the statistical methods in the methods, is the statistical test for each experiment clearly defined?

Different statistical methods, such as ANOVAs or paired t-tests, are not explained because we assume they are standard statistics for analyze the variability among groups. Analyses and averages are indicated in the method session.

b. Do the data meet the assumptions of the specific statistical test you chose (e.g. normality for a parametric test)?

Normal Data distribution and equal variance was formally tested using Sigma Plot 13.0 or GraphPad Prism 6 for all statistical tests.

Where is this described (section, paragraph #)?

Figure legends

c. Is there any estimate of variance within each group of data? Figure legends Is the variance similar between groups that are being statistically compared?

d. Are tests specified as one- or two-sided?

Yes

e. Are there adjustments for multiple comparisons?

Yes

November 2014

Where is this described (section, paragraph #)?

8

Was this criterion established prior to data collection? Where is this described (section, paragraph #)?

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.

Methods - Optogenetic, electrophysiology paragraphs The criteria for data exclusion were considered for technical problems such as stability of the recordings, loss of a recorded neuron, missed injection site, etc. They are indicated in the material and method session.

Methods - behavior. Usually, all the experimental groups were interleaved in order to have a similar time and experimental condition.

Where does this appear (section, paragraph #)? Data were collected and processed randomly apart from the data collected from grouped animals.

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. Where (section, paragraph #)?


3. Are criteria for excluding data points reported?

Methods - Electrophysiology, Virus injection and optogenetic All the recording data were collected on the basis of their technical validity and the stability of the recordings and injection site. There was no further group allocation. Data collection and analysis were not performed blind to the conditions of the experiments. Methods - Statistics

6. For experiments in live vertebrates, is a statement of compliance with Methods - "Animals". ethical guidelines/regulations included? Where (section, paragraph #)?

7. Is the species of the animals used reported? Where (section, paragraph #)?

8. Is the strain of the animals (including background strains of KO/ transgenic animals used) reported?

Methods - Animals Results - first paragraph

Results - first paragraph

Where (section, paragraph #)?

9. Is the sex of the animals/subjects used reported?

Methods - "Animals".


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 Methods - "Animals". animals per cage) reported?

November 2014



9


14. Is the previous history of the animals/subjects (e.g. prior drug administration, surgery, behavioral testing) reported?

Methods - "Animals". All the experiments were performed between 8 am and 5 pm during the light cycle. Methods - Behavior


a. If multiple behavioral tests were conducted in the same group of animals, is this reported?

Methods - "Behavior" and "Electrophysiology".


15. If any animals/subjects were excluded from analysis, is this reported?


13. For behavioral experiments, is the time of day reported (e.g. light or dark cycle)?

Methods - "Behavior" and "Electrophysiology".


a. How were the criteria for exclusion defined? Where is this described (section, paragraph #)?

Methods - Virus injections and optogenetic experiments The animals were excluded from the data if bilateral viral injection and expression was not successful.

b. Specify reasons for any discrepancy between the number of The number of animals used was between 6 and 15 in each data set. There is no major discrepancy from the beginning to the end of animals at the beginning and end of the study. the project. Where is this described (section, paragraph #)?



Reagents

1. Have antibodies been validated for use in the system under study (assay and species)?

a. Is antibody catalog number given?

Yes, they have been validated for other research groups or on knock out animals.

Methods - "Immunohistochemistry".

Where does this appear (section, paragraph #)?

b. Where were the validation data reported (citation, supplementary information, Antibodypedia)?

Methods - "Immunohistochemistry". Supplementary figure 5 for the alpha5 antibody.

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?

No cell lines were used.

a. Were they recently authenticated? Where is this information reported (section, paragraph #)?

-

November 2014


10

Data deposition

Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic 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 Data Descriptors (see Scientific Data) to maximize data reuse. 1. Are accession codes for 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 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.

Custom softwares are commercially available and their name can be found in the method session.

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 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?

No human studies were conducted.

Where is this stated (section, paragraph #)? 2. Is demographic information on all subjects provided?

-


3. Is the number of human subjects, their age and sex clearly defined?

-



-

November 2014

4. Are the inclusion and exclusion criteria (if any) clearly specified?

11

-

Where is this information described (section, paragraph #)?

6. Is a statement included confirming that informed 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


5. How well were the groups matched?

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 analysis after the data was collected?

a. If yes, is 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, event-related, or mixed design being used? If applicable, please specify the block length or how the event-related or mixed design was optimized.

-

5. Is the task design clearly described?

-


-

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?

November 2014

6. How was behavioral performance measured?

-

12

a. Is the pulse sequence type (gradient/spin echo, EPI/spiral) stated?

-

b. Are the field-of-view, 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 pre-processing 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 #)?


9. Is the field strength (in Tesla) of the MRI system stated?

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), probabilistic 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, 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 figures varies, is this clearly stated?

a. If not, is this labeled as uncorrected?

-

November 2014

19. Are statistical inferences corrected for multiple comparisons?

13

-

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 cluster-wise significance, is the cluster-defining threshold and the corrected significance level defined?

-




20. Are the results based on an ROI (region of interest) analysis?

Additional comments Additional Comments

-

November 2014

14