Reporting Checklist for Nature Neuroscience · z Each figure legend should ideally contain an exact sample size (n) for each experimental group/condition, where n is an exact number

1

nature neuroscience | reporting checklistApril 2015

Corresponding Author: Berislav V. Zlokovic

Manuscript Number: NN-BC53203F

Manuscript Type: Article

# Main Figures: 6

# Supplementary Figures: 8

# Supplementary Tables: 1

# Supplementary Videos: 2

Reporting Checklist for Nature NeuroscienceThis 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).

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.

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

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.

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.

TEST USED n DESCRIPTIVE STATS (AVERAGE, VARIANCE)

P VALUEDEGREES OF FREEDOM &

F/t/z/R/ETC VALUE

FIG

URE

N

UM

BER

WHICH TEST?

SECT

ION

&

PARA

GRA

PH #

EXACT VALUE DEFINED?

SECT

ION

&

PARA

GRA

PH #

REPORTED?

SECT

ION

&

PARA

GRA

PH #

EXACT VALUE

SECT

ION

&

PARA

GRA

PH #

VALUE

SECT

ION

&

PARA

GRA

PH #

exam

ple

1a one-way ANOVA

Fig. legend

9, 9, 10, 15

mice from at least 3 litters/group

Methods para 8

error bars are mean +/- SEM

Fig. legend p = 0.044 Fig.

legend F(3, 36) = 2.97 Fig. legend

exam

ple

results, para 6

unpaired t-test

Results para 6 15 slices from 10 mice Results

para 6error bars are mean +/- SEM

Results para 6 p = 0.0006 Results

para 6 t(28) = 2.808 Results para 6

Nature Neuroscience: doi:10.1038/nn.4489

2


TEST USED n DESCRIPTIVE STATS (AVERAGE, VARIANCE)

P VALUEDEGREES OF FREEDOM &

F/t/z/R/ETC VALUE

FIG

URE

N

UM

BER

WHICH TEST?SE

CTIO

N &

PA

RAG

RAPH

#EXACT VALUE DEFINED?

SECT

ION

&

PARA

GRA

PH #

REPORTED?

SECT

ION

&

PARA

GRA

PH #

EXACT VALUE

SECT

ION

&

PARA

GRA

PH #

VALUE

SECT

ION

&

PARA

GRA

PH #

+- 1a t-test Fig.

Legend 10, 910 Pdgfrb+/+ and 9 Pdgfrb+/- mice per

group

Fig. Legend

error bars are 95% CI

Fig & main text

p=0.02 Figure F(8,9)=1.379 t(17)=2.524

Excel sheet,

Fig. Legend

+- 1c N/A N/A 37,33

37 capillaries from 12 Pdgfrb+/+ mice, and 33 capillaries from 9 Pdgfrb+/- mice per group

Fig. Legend

error bars are 95% CI Fig. N/A N/A N/A N/A

+- 1d t-test Fig.

Legend 12,9

Capillaries from 12 Pdgfrb+/+ mice and 9 Pdgfrb+/-mice per group,

averaged by mouse

Fig. Legend


Fig & main text

p=0.00016 Figure F(8,11)=5.527 t(10)=6.479

Excel sheet,

Fig. Legend

+- 1e N/A N/A 18,20

18 arterioles from 8 Pdgfrb+/+ mice, and 20 arterioles from 11 Pdgfrb+/-

mice per group

Fig. Legend


+- 1f t-test Fig.

Legend 8,11

arterioles from 8 Pdgfrb+/+ mice

and 11 Pdgfrb+/-mice per group,

averaged by mouse

Fig. Legend


Fig & main text

p=0.59894 Figure F(10,7)=1.5554 t(17)=0.5360

Excel sheet,

Fig. Legend

+- 1h N/A N/A 39,38

39 capillaries from 9 Pdgfrb+/+ mice, and 38 capillaries 12 Pdgfrb+/- mice

per group

Fig. Legend


+- 1i t-test Fig.

Legend 9,12

capillaries from 9 Pdgfrb+/+ mice


averaged by mouse

Fig. Legend


Fig & main text

p=0.02991 Figure F(11,8)=3.6517 t(19)=2.3472

Excel sheet,

Fig. Legend

+- 1k t-test Fig.

Legend 7,7capillaries from 7 mice per group,

averaged by mouse

Fig. Legend


Fig & main text

p=0.02380 Figure F(6,6)=1.037 t(12)=2.586

Excel sheet,

Fig. Legend

+- 2c t-test Fig.

Legend 7,7 7 mice per group. Fig. Legend


Fig & main text

p=0.018 Figure F(6,6)=3.072 t(12)=2.346

Excel sheet,

Fig. Legend

+- 2d t-test Fig.

Legend 7,7 7 mice per group. Fig. Legend


Fig & main text

p=0.038 Figure F(6,6)=5.354 t(12)=1.949

Excel sheet,

Fig. Legend

+- 2g t-test Fig.

Legend 5,65 Pdgfrb+/+ mice, and 6 Pdgfrb+/- mice per group

Fig. Legend


Fig & main text

p=0.95 Figure F(5,4)=2.161 t(9)=0.069

Excel sheet,

Fig. Legend

+- 2h t-test Fig.


Fig. Legend


Fig & main text

p=0.80 Figure F(5,4)=10.831 t(5)=0.122

Excel sheet,

Fig. Legend


3


+- 3a t-test Fig.

Legend 7,10

arterioles from 7 Pdgfrb+/+ mice


averaged by mouse

Fig. Legend


Fig & main text

p=0.47 Figure F(9,6)=2.3324 t(15)=0.7351

Excel sheet,

Fig. Legend

+- 3c N/A N/A 5,4

5 Pdgfrb+/+ mice, and 4 Pdgfrb+/-

per group

Fig. Legend


+- 3d t-test Fig.

Legend 5,45 Pdgfrb+/+ mice, and 4 Pdgfrb+/-

per group

Fig. Legend


Fig & main text

p=0.33 Figure F(4,3)=1.306 t(7)=1.046

Excel sheet,

Fig. Legend

+- 3e t-test Fig.

Legend 5,45 Pdgfrb+/+ mice, and 4 Pdgfrb+/-

per group

Fig. Legend


Fig & main text

p=0.69 Figure F(4,3)=3.183 t(7)=0.414

Excel sheet,

Fig. Legend

+- 3f

Mann-Whitney U

test

Fig. Legend 3,3 n=3 mice per group Fig.

Legend

error bars are 95% CI,

bootstrapped

Fig & main text

p>0.99 Figure U=4 Excel sheet

+- 3g

Mann-Whitney U

test


Legend


bootstrapped

Fig & main text

p>0.99 Figure U=4 Excel sheet

+- 3h

Mann-Whitney U

test


Legend


bootstrapped

Fig & main text

p=0.70 Figure U=3 Excel sheet

+- 4c

Mann-Whitney U

test


Legend


bootstrapped

Fig & main text

p=0.03 at 100um

p=0.05 at 200um

Figure U=0 U=0

Excel sheet

+- 4f t-test Fig.

Legend 4,54 Pdgfrb+/+ mice, and 5 Pdgfrb+/+ mice per group

Fig. Legend


Fig & main text

p=0.003 Figure F(3,4)=1.247 t(7)=4.394

Excel sheet,

Fig. Legend

+- 5b t-test Fig.


Fig. Legend


Fig & main text

p=0.036 Figure F(6,5)=11.612 t(6)=1.813

Excel sheet,

Fig. Legend

+- 5c t-test Fig.

Legend 6,6 n=6 mice per group Fig. Legend


Fig & main text

p=0.043 Figure F(5,5)=1.660 t(10)=1.902

Excel sheet,

Fig. Legend

+- 5d t-test Fig.


Fig. Legend


Fig & main text

p=0.308 Figure F(5,4)=1.784 t(9)=1.080

Excel sheet,

Fig. Legend

+- 5e t-test Fig.


Fig. Legend


Fig & main text

p=0.022 Figure F(4,3)=1.426 t(7)=2.912

Excel sheet,

Fig. Legend

+- 6b t-test Fig.

Legend 5,5 n= 5 mice per group

Fig. Legend


Fig & main text

p=0.72 Figure F(4,4)=1.154 t(8)=0.368

Excel sheet,

Fig. Legend

+- 6c t-test Fig.

Legend 5,5 n= 5 mice per group

Fig. Legend


Fig & main text

p=0.73 Figure F(4,4)=1.193 t(8)=0.351

Excel sheet,

Fig. Legend

+- 6d t-test Fig.

Legend 4,4 n = 4 mice per group

Fig. Legend


Fig & main text

p=0.29 Figure F(3,3)=4.300 t(6)=1.169

Excel sheet,

Fig. Legend

+- 6e

Mann-Whitney U

test

Fig. Legend 3,3 n = 3 mice per

groupFig.

Legend


bootstrapped

Fig & main text

p>0.99 Figure U=3.5 Excel sheet


4


+- 6f t-test Fig.


Fig. Legend


Fig & main text

p=0.76 Figure F(6,5)=4.621 t(11)=0.313

Excel sheet,

Fig. Legend

+- 6i t-test Fig.


Fig. Legend


Fig & main text

p=0.049 Figure F(3,4)=8.190 t(7)=1.908

Excel sheet,

Fig. Legend

+- 6j t-test Fig.


Fig. Legend


Fig & main text

p=0.038 Figure F(3,4)=1.573 t(7)=2.087

Excel sheet,

Fig. Legend

+- 6l t-test Fig.

Legend 5,5,5,5

Cortex: 5 mice per group,

Hippocampus: 5 mice per group

Fig. Legend


Fig & main text

p=0.026 p=0.037 Figure

Cortex: F(4,4)=1.251 t(8)=2.276

Hippocampus: F(4,4)=1.284 t(8)=2.054

Excel sheet,

Fig. Legend

+- 6m t-test Fig.

Legend 6,6,6,6

Cortex: 6 mice per group,

Hippocampus: 6 mice per group

Fig. Legend


Fig & main text

p=0.016 p=0.014 Figure

Cortex: F(5,5)=1.021 t(10)=2.480

Hippocampus: F(5,5)=1.452 t(10)=2.557

Excel sheet,

Fig. Legend

+- 6n t-test Fig.

Legend 4,4 n = 4 mice per group

Fig. Legend


Fig & main text

p=0.013 Figure F(3,3)=1.354 t(6)=3.515

Excel sheet,

Fig. Legend

+- 6o

Mann-Whitney U

test

Fig. Legend 5,5 n = 5 mice per

groupFig.

Legend


bootstrapped

Fig & main text

p=0.05 Figure U=3.5 Excel sheet

+- 6p t-test Fig.


Fig. Legend


Fig & main text

p=0.019 Figure F(6,5)=1.943 t(11)=2.760

Excel sheet,

Fig. Legend

+-

Results

paragraph 1

t-testResults paragraph 1

12,8

Pdgfrb+/+ capillaries from 12

mice, and arterioles from 8

mice

Page 3, para 1;

Fig 1

Data appears as parts of Fig. 1d,f

with error bars as 95% CI

main text p=0.04552 page 3,

line 90F(7,11)=5.033

t(9)=1.892Excel sheet

+-

Results

paragraph 1

t-testResults paragraph 1

9,11

Pdgfrb+/- capillaries from 9

mice, and arterioles from 11

mice

Page 3, para 1;

Fig 1

Data appears as parts of Fig. 1d,f

with error bars as 95% CI

main text p=0.01886 page 3,

line 92F(10,8)=1.416 t(18)=2.243

Excel sheet

+-

Sup Fig 2b

t-test Fig. Legend 5,6

5 Pdgfrb+/+ mice, and 6 Pdgfrb+/- mice per group

Fig. Legend


Fig & main text

p=6.13E-7 FIgure F(5,4)=7..325 t(9)=11.361

Excel sheet,

Fig. Legend

+-

Sup Fig 3a

t-test Fig. Legend

9,11 10,10

Arterioles: 9 Pdgfrb+/+ mice, and 11

Pdgfrb+/- mice per group; Capillaries: 10 Pdgfrb+/+ mice, and 10 Pdgfrb+/- mice per group

Fig. Legend


Fig & main text

p=0.79 p=0.31 Figure

F(10,8)=1.334 t(18)=0.271 F(9,9)=2.275 t(18)=1.041

Excel sheet,

Fig. Legend

+-

Sup Fig 3b

t-test Fig. Legend

6,10 9,12

Arterioles: 6 Pdgfrb+/+ mice, and 10

Pdgfrb+/- mice per group; Capillaries: 9 Pdgfrb+/+ mice, and 12 Pdgfrb+/- mice per group

Fig. Legend


Fig & main text

p=0.82 p=0.74 Figure

F(9,5)=2.542 t(14)=0.233

F(8,11)=3.525 t(19)=0.334

Excel sheet,

Fig. Legend

+-

Sup Fig 3d

t-test Fig. Legend 7,6


Fig. Legend


Fig & main text

p=0.28 Figure F(6,5)=1.340 t(11)=1.138

Excel sheet,

Fig. Legend


5


+-

Sup Fig 4b

t-test Fig. Legend 5,5 5 mice per group Fig.

Legenderror bars are

95% CI

Fig & main text

p=0.82 Figure F(4,4)=1.098 t(8)=0.238

Excel sheet,

Fig. Legend

+-

Sup Fig 4c



95% CI

Fig & main text

p=0.38 Figure F(4,4)=1.299 t(8)=0.931

Excel sheet,

Fig. Legend

+-

Sup Fig 4d



95% CI

Fig & main text

p=0.80 Figure F(4,4)=1.159 t(8)=0.260

Excel sheet,

Fig. Legend

+-

Sup Fig 5b

Mann-Whitney U

test

Fig. Legend 3,3 3 mice per group FIg.

Legend


bootstrapped

Fig & main text


+-

Sup Fig 5c

Mann-Whitney U

test

Fig. Legend 5,3

5 Control mice, and 3 Meox2+/- mice per group

FIg. Legend


bootstrapped

Fig & main text

p=1 Figure U=7 Excel sheet

+-

Sup Fig 5d

Mann-Whitney U

test

Figure Legend 7,3


Fig. Legend


bootstrapped

Fig & main text


+-

Sup Fig 5e

Mann-Whitney U

test

Figure Legend 6,3


Fig. Legend


bootstrapped

Fig & main text

p=0.55 FIgure U=6 Excel sheet

+-

Sup Fig 6a

t-test Figure Legend 5,5 5 mice per group Fig.


95% CI

Fig & main text

p=0.63 Figure F(4,4)=2.602 t(8)=0.51

Excel sheet,

Fig. Legend

+-

Sup FIg 6b

t-test Figure Legend 6,4


Fig. Legend


Fig & main text

p=0.94 Figure F(3,5)=1.216 t(8)=0.81

Excel sheet,

Fig. Legend

+-

Sup Fig 7c

Mann-Whitney U

test

Figure Legend 5,5 5 mice group Fig.

Legend


bootstrapped

Fig & main text


+-

Sup Fig 7d



95% CI

Fig & main text

p=0.92 Figure F(4,4)=1.038 t(8)=0.105

Excel sheet,

Fig. Legend

+-

Sup Fig 8a

Mann-Whitney U

test

Figure Legend 4,4 4 mice per group Fig.

Legend


bootstrapped

Fig & main text


+-

Sup Fig 8c



95% CI

Fig & main text

p=0.001 Figure F(3,3)=2.436 t(6)=4.894

Excel sheet,

Fig. Legend

+-

Sup Fig 4f t-test Figure

Legend 4,4 4 mice per group Fig. Legend


Main text

<6um, p=0.06 >6um, p=0.92 total, p=0.07

Figure

<6um: F(3,3)=2.964 t(6)=1.772

>6um: F(3,3)=2.243

t(6)=0.10 total:

F(3,3)=2.926 t(6)=1.681

Excel sheet,

Fig. Legend

Representative figures

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

If so, what figure(s)?

Figure 1b, g, j; Figure 2a,b,e, f; Figure 3b, c, Figure 4a, d, e; Figure 5a; Figure 6a,g, h, k., Sup. Fig. 1, Sup Fig. 2a, c, Sup. Fig. 3c, Sup Fig. 4 a, e, Sup Fig. 5a, Sup Fig. 7a, b, Sup Fig. 8b


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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, paragraph #)?

Yes, the number of samples and the number of experiments/replicates were stated in each figure legend. The number of animals varied from 3-12 depending on analysis. There was no limitation in repeatability.

Statistics and general methods

1. Is there a justification of the sample size?

If so, how was it justified?

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.

Yes, it is stated in the supplementary method, section of "Statistical analysis", 1st paragraph: "Sample sizes were calculated using nQUERY assuming a two-sided alpha-level of 0.05, 80% power, and homogenous variances for the two samples to be compared, with the means and common standard deviation for different parameters predicted from published data and our previous studies."

2. Are statistical tests justified as appropriate for every figure?


Yes. It is summarized in the supplementary method, section of "All data are expressed as mean ± 95% confidence interval, unless otherwise noted. Grubb’s outlier test was used to determine significant outliers, which were excluded from further data analysis (GraphPad). Lilliefors test was used to test normality of the data (XLSTAT). For parametric analysis, the F test was used to determine the equality of variances between the groups compared; statistical significance across two groups was tested by Student’s t-test (Microsoft Excel 2010). Mann–Whitney U test was used for non-parametric analysis (Prism, GraphPad). Bootstrapping (1,000 sampling size) was performed on data that could not be statistically distinguished from a non-normal distribution to calculate 95% confidence intervals (XLSTAT)60,61. Effect size (e.g. Cohen’s d) was calculated for all comparisons made (http://www.socscistatistics.com/effectsize/). The accepted level of statistical significance was p≤0.05." For every figure, the statistical tests were listed in the corresponding legend.

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

Yes. It is stated in the supplementary method, section of "Statistical analysis", 2nd paragraph:"All data are expressed as mean ± 95% confidence interval, unless otherwise noted. Grubb’s outlier test was used to determine significant outliers, which were excluded from further data analysis (GraphPad). Lilliefors test was used to test normality of the data (XLSTAT). For parametric analysis, the F test was used to determine the equality of variances between the groups compared; statistical significance across two groups was tested by Student’s t-test (Microsoft Excel 2010). Mann–Whitney U test was used for non-parametric analysis (Prism, GraphPad). Bootstrapping (1,000 sampling size) was performed on data that could not be statistically distinguished from a non-normal distribution to calculate 95% confidence intervals (XLSTAT)60,61. Effect size (e.g. Cohen’s d) was calculated for all comparisons made (http://www.socscistatistics.com/effectsize/). The accepted level of statistical significance was p≤0.05." Statistical test for each experiment can be found in the raw data submitted in Excel.


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

Where is this described (section, paragraph #)?

Yes. It is stated in the supplementary method, section of "Statistical analysis", 2nd paragraph: "All data are expressed as mean ± 95% confidence interval, unless otherwise noted. Grubb’s outlier test was used to determine significant outliers, which were excluded from further data analysis (GraphPad). Lilliefors test was used to test normality of the data (XLSTAT). For parametric analysis, the F test was used to determine the equality of variances between the groups compared; statistical significance across two groups was tested by Student’s t-test (Microsoft Excel 2010). Mann–Whitney U test was used for non-parametric analysis (Prism, GraphPad). Bootstrapping (1,000 sampling size) was performed on data that could not be statistically distinguished from a non-normal distribution to calculate 95% confidence intervals (XLSTAT)60,61. Effect size (e.g. Cohen’s d) was calculated for all comparisons made (http://www.socscistatistics.com/effectsize/). The accepted level of statistical significance was p≤0.05." F-test for each experiment can be found in the raw data submitted in Excel.

c. Is there any estimate of variance within each group of data?

Is the variance similar between groups that are being statistically compared?


Yes. It is stated in the supplementary method, section of "Statistical analysis", 2nd paragraph:" F test was used to determine the equality of variances between the groups compared."

d. Are tests specified as one- or two-sided? Two-tailed were used, except where noted as single tailed.

e. Are there adjustments for multiple comparisons? No. We only compared between two groups of mice with different Pdgfrb genotypes. No multiple comparison is needed

3. Are criteria for excluding data points reported?

Was this criterion established prior to data collection?


Yes. 1. Grubb’s outlier test was used to determine significant outliers which were excluded from further data analysis. Stated in Supplementary methods "Statistical Analysis" section, 2nd paragraph, and established after collection. 2. Vessels outside the IOS (intrinsic optical imaging) response area were excluded from analysis. Stated in Supplementary methods "Diameter and Velocity Measurements" section, 2nd paragraph, and established before collection. 3. For vessels within the IOS response area, "... any vessel that showed a focal drift occurring during the experiment, or if there were excessive breathing or movement artifacts were excluded. Only vessels with a diameter change larger than 1% after stimulus start were considered as responding vessels and taken for further analysis." Stated in Supplementary methods "Diameter and Velocity Measurements" section, 2nd paragraph, and established after collection.

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 this appear (section, paragraph #)?

It is stated in the supplementary methods section "Statistical Analysis", 1st paragraph:"All animals were randomized for their genotype information."


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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.


It is stated in the supplementary methods section "Statistical Analysis", 1st paragraph: "Immunostaining experiments were blinded; the operators responsible for the experimental procedures and data analysis were blinded and unaware of group allocation throughout the experiments. Other experiments were analyzed, but not performed, in a blinded fashion."

6. For experiments in live vertebrates, is a statement of compliance with ethical guidelines/regulations included?


It is stated in the supplementary method, section of "Animals", 1st paragraph:"All procedures were approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines and the Massachusetts General Hospital Subcommittee on Research Animal Care."

7. Is the species of the animals used reported?


It is stated in the supplementary method, section of "Animals", 1st paragraph that mice were used in this study.

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


It is stated in the supplementary method, section of "Animals", 1st paragraph: "Pdgfrb+/- mice and their littermate controls on mixed 129S1/SvlmJ background were generated as we previously described9. ....Pdgfrb+/- mice with pericytes expressing dsRed (Pdgfrb+/-;dsRed) were generated by crossing Pdgfrb+/- mice with NG2-dsRed mice on C57Bl6 background11. Meox2+/- mice and their controls on C57Bl6 background were generated as we previously described28."

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


Yes, It is stated in the supplementary method, section "Animals", 1st paragraph:"Both male and female animals were used."

10. Is the age of the animals/subjects reported?


Yes, supplementary method, section "Animals", 1st paragraph: "All experiments were performed on 1-2 mo old or 6-8 mo old Pdgfrβ+/- mice and littermate Pdgfrβ+/+ controls unless otherwise specified. Pdgfrβ+/- mice with pericytes expressing dsRed (Pdgfrβ+/-;dsRed) were generated by crossing Pdgfrβ+/- mice with NG2-dsRed mice11. Meox2+/- were generated as we previously described26 and used at 1-2 mo of age. " Also, supplementary method, section "Tissue Oxygen partial pressure (pO2) measurements" 1st paragraph: "the 5-6 mo old Pdgfrβ+/- or Pdgfrβ+/+ mice..."

11. For animals housed in a vivarium, is the light/dark cycle reported?


Yes, supplementary method, section "Animals", 1st paragraph: "Mice were housed in plastic cages on a 12 h light cycle with ad libitum access to water and a standard laboratory diet."

12. For animals housed in a vivarium, is the housing group (i.e. number of animals per cage) reported?


No. However, "All procedures were approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines.", which means the maximum mice per cage is 5.

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


No. However, in the supplementary methods section "Behavior testing" we reference a previous publication that describes all testing parameters used in detail (ref. 42).


9


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


No. However, all animals used in this study were used exclusively for the experiments presented in the manuscript.

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


No. The animals were analyzed with all 3 behavior tests, including nesting, borrowing, and NOR. None of these tests were performed in consecutive days.

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


No. Exclusions were made based on vessel responses and outlier tests, not on a per animal basis. See question 3 above or 15a below.

a. How were the criteria for exclusion defined?


1. Grubb’s outlier test was used to determine significant outliers which were excluded from further data analysis. Stated in Supplementary methods "Statistical Analysis" section, 2nd paragraph, and established after collection. 2. Vessels outside the IOS (intrinsic optical imaging) response area were excluded from analysis. Stated in Supplementary methods "Diameter and Velocity Measurements" section, 2nd paragraph, and established before collection. 3. For vessels within the IOS response area, "... any vessel that showed a focal drift occurring during the experiment, or if there were excessive breathing or movement artifacts were excluded. Only vessels with a diameter change larger than 1% after stimulus start were considered as responding vessels and taken for further analysis." Stated in Supplementary methods "Diameter and Velocity Measurements" section, 2nd paragraph, and established after collection.

b. Specify reasons for any discrepancy between the number of animals at the beginning and end of the study.


N/A

Reagents

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

Yes, we only chose antibodies validated by previous publications, either from our own group (e.g. ref: Bell et al Neuron 2010, Winkler, et al, Mol. Neurodegener, 2010, Bell et al, Nature 2012), or from other labs (Armulik et al Nature 2010, etc.).

a. Is antibody catalog number given?


Yes, the publications reporting antibody details and catalog numbers are referenced in the Supplemental Methods section.

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


we only chose antibodies validated by previous publications, either from our own group (e.g. ref: Bell et al Neuron 2010, Winkler, et al, Mol. Neurodegener, 2010, Bell et al, Nature 2012), or from other labs (Armulik et al Nature 2010, etc.).


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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?


N/A

b. If yes, include in the Methods section a scientific justification of their use--indicate here in which section and paragraph the justification can be found.

N/A

c. For each cell line, include in the Methods 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 tested for mycoplasma

contamination?


N/A

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?


N/A

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.

Suplemental Methods section "Diameter and Velocity Measurements," 4th and 5th paragraphs. Custom procedures in Igor Pro 6 were used to automate the analysis of vessel diameter and velocity measurements described in the paragraphs.


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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.

We have included the phrase "Igor Pro code can be made available upon request" in Suplemental Methods section "Diameter and Velocity Measurements," 5th paragraph.

Human subjects

1. Which IRB approved the protocol?

Where is this stated (section, paragraph #)?

N/A

2. Is demographic information on all subjects provided?


N/A

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


N/A

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


N/A

5. How well were the groups matched?

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

N/A

6. Is a statement included confirming that informed consent was obtained from all subjects?


N/A

7. For publication of patient photos, is a statement included confirming that consent to publish was obtained?


N/A

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 analysis after the data was collected?

N/A

a. If yes, is the number rejected and reasons for rejection described?


N/A


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2. Is the number of blocks, trials or experimental units per session and/or subjects specified?


N/A

3. Is the length of each trial and interval between trials specified? N/A

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.

N/A

5. Is the task design clearly described?


N/A

6. How was behavioral performance measured? N/A

7. Is an ANOVA or factorial design being used? N/A

8. For data acquisition, is a whole brain scan used?

If not, state area of acquisition.

N/A

a. How was this region determined? N/A

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

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

N/A

b. Are the field-of-view, matrix size, slice thickness, and TE/TR/flip angle clearly stated?

N/A

10. Are the software and specific parameters (model/functions, smoothing kernel size if applicable, etc.) used for data processing and pre-processing clearly stated?

N/A

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 #)?

N/A

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 #)?

N/A

13. How were anatomical locations determined, e.g., via an automated labeling algorithm (AAL), standardized coordinate database (Talairach daemon), probabilistic atlases, etc.?

N/A


13


14. Were any additional regressors (behavioral covariates, motion etc) used?

N/A

15. Is the contrast construction clearly defined? N/A

16. Is a mixed/random effects or fixed inference used? N/A

a. If fixed effects inference used, is this justified? N/A

17. Were repeated measures used (multiple measurements per subject)? N/A

a. If so, are the method to account for within subject correlation and the assumptions made about variance clearly stated?

N/A

18. If the threshold used for inference and visualization in figures varies, is this clearly stated?

N/A

19. Are statistical inferences corrected for multiple comparisons? N/A

a. If not, is this labeled as uncorrected? N/A

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

a. If so, is the rationale clearly described? N/A

b. How were the ROI’s defined (functional vs anatomical localization)?

N/A

21. Is there correction for multiple comparisons within each voxel? N/A

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

N/A

Additional comments

Additional Comments N/A


Documents

Reporting Checklist for Nature Neuroscience · z Each figure legend should ideally contain an exact sample size (n) for each experimental group/condition, where n is an exact number