Supplementary Information – Ellegood et al. 2014

Supplementary Methods

Animal Models

Experimenters were not blinded to genotype, as the MRI scanning, registration, and volume measurements are completely automated and unbiased. The data presented here was compliant with all ethical regulations concerning animal experimentation and was approved by the animal care committee at the Toronto Centre for Phenogenomics and all the other institutions that provided animals.

Perfusion Protocol

Mice were anesthetized with a ketamine/xylazine mix (10mg/kg) then intracardially perfused with 30mL of 0.1 M PBS containing 10 U/mL heparin (Sigma) and 2mM ProHance (a Gadolinium contrast agent) followed by 30 mL of 4% paraformaldehyde (PFA) containing 2mM ProHance. After perfusion, mice were decapitated and the skin, lower jaw, ears and cartilaginous nose tip were removed as previously described1, 2. The brain within the skull was incubated in 4% PFA containing 2mM ProHance overnight at 4 degrees Celsius then transferred to 0.1M PBS containing 2mM ProHance and .02% sodium azide for at least 7 days prior to MRI scanning.

MRI Sequence Details

Images were acquired on a 7 Tesla MRI scanner (Agilent, Palo Alto, CA) with either an insert gradient with an inner bore diameter of 6 cm or an outer gradient with a 30 cm inner bore diameter as previously described3-5. As this project was done over 5+ years the anatomical MRI scan that we use has been modified over time in an effort to increase throughput from 3 mouse brains per scanning session to 16. Both sequences gave similar SNR and tissue contrast with the main difference being the resolution. The original 3 brain sequence resolution was 32 µm isotropic, and the newer 16 brain sequence resolution, using the outer gradient coil, was 56 µm isotropic. The majority of autism mouse models were investigated with the current 16 brain sequence therefore the 32 µm images were resampled to 56 µm prior to registration.

Original Sequence – 3 Brains per overnight session

Three custom-built solenoid coils were used to image the brains in parallel6. The sequence used was a T2 weighted Fast Spin Echo (FSE): parameters for this sequence were optimized for high contrast between the gray and white matter, which was required for the registration performed later. Parameters were as follows: TR – 325 ms, TEs of 10 ms per echo for 6 echoes, with the centre of k-space acquired on the 4th echo, 4 averages, field-of-view (FOV) of 14 x 14 x 25 mm3 and a matrix size of 432 x 432 x 780 yielding an image with 32 µm isotropic voxels. Total imaging time for this MRI sequence was ~ 12 hrs2, 7.

Current Sequence – 16 Brains per overnight session

An in-house custom built 16-coil solenoid array was used to acquire anatomical images in parallel, allowing acquisition of the MRI images for 16 samples in one overnight session2, 8, 9. Parameters used in this anatomical MRI were optimized for high efficiency and gray/white matter contrast. The sequence was a T2-weighted 3D FSE, with TR=2000 ms, echo train length = 6, TEeff=42 ms, FOV of 25 mm × 28 mm × 14 mm, and a matrix size of 450 × 504 × 250. This yields an isotropic (3D) resolution of 56 µm. In the first phase encode dimension, consecutive k-space lines were assigned to alternating echoes to move discontinuity related ghosting artifacts to the edges of the FOV10. This sequence involves oversampling in the phase encoding direction by a factor of 2 to avoid the interference of these artifacts. This FOV direction was subsequently cropped to 14 mm after reconstruction. Total imaging time was ~12 hours.

Registration Details

To visualize and compare the mouse brains, the images from the anatomical MRI scans were linearly and nonlinearly registered together for each of the individual models using a combination of the mni_autoreg tools11 and ANTS12, 13. All scans were then resampled with the appropriate transform and averaged to create a population atlas representing the average anatomy of the given model. The result of the registration was to have the scans for each model deformed into exact alignment with each other in an unbiased fashion. For the volume measurements, this allowed for the analysis of the deformations needed to take each individual mouse’s anatomy into this final atlas space, the goal being to model how the deformation fields relate to genotype4, 14. The Jacobian determinants of the deformation fields are then calculated as measures of volume at each voxel. Significant volume changes and intensity differences were then calculated by warping a pre-existing classified MRI atlas onto the population atlas, which allowed for either the volume of 62 segmented structures to be assessed in all brains. The volumes of each of the 62 regions were then calculated as percentages of total brain volume for each of the 26 different models.

Study / Model / Control Strain / # of Mice per Group / Age / Refs
15q11-13 / 15q11-13 (patdp/+) / C57BL/6J / 10 / 10-11 wks / 15
16p11 / 16p11.2 (df/+) / B6/129F2 / 9-10 / 32-35 wks / 16
16p11.2 (df/dp)
16p11.2(dp/+)
AndR / AndR 12Q h/mAR / C57BL/6J / 9-10 / P60 / 17
AndR 48Q h/mAR
BALB/c / BALB/cJ / C57BL/6J / 10 / P90 / 18
BTBR / BTBR T+tf/J / C57BL/6J / 12 / P77 / 19, 20
FVB/(AntJ)
CNTNAP2 / CNTNAP2(-/-) / C57BL/6J / 9 / P112 / 21
En2 / En2 (-/-) / C57BL/6J / 9 / P60 / 22
FMR1 / FMR1 (-/y) / C57BL/6J / 8 / P30 / 23
FMR1 (-/y) / FVB / 10 / P60
GTF2i / GTF2i (-/+) / CD-1 / 10 / 13-15 wks / 24
GTF2i (dp/dp) / 10
ITGβ3 / ITGβ3 (-/-) / B6/129F2 / 11 / P60 / 3
Mecp2 / Mecp2308(-/y) / C57BL/6J / 10 / P60 / 25, 26
NLGN3 / NLGN3 R451C KI / B6/129F2 / 8 / P108 / 7
NRXN1α / NRXN1α (-/+) / B6/SV129 / 9-12 / 11-13 wks / 27
NRXN1α (-/-)
SLC6A4 / SLC6A4 Ala56 KI / C57BL/6J / 10 / P60 / 28, 29
129 / 10 / P60
SLC6A4 KO / C57BL/6J / 10 / P60 / 30
SHANK3 / SHANK3 e4-9 (+/-) / B6/SV129 / 10 / 22-23 wks / 31
SHANK3 e4-9 (-/-)
XO / XO / B6CBACaF1/J-Aw-J/A / 11 / P80 / 32

Supplementary Table 1 – Additional details about each of the 26 models used in this study.

Randomized Data Sets –100 Trials
% Threshold / Connections between Models
With Overlapping Controls / Without Overlapping Controls
> 0.20 / 28 / 12
> 0.25 / 16 / 0
> 0.30 / 16 / 0
> 0.50 / 16 / 0

Supplementary Table 2 - Two random simulated data sets were created inorder to confirm the connections presented in Figure 5. This table shows that in a randomized data set without overlapping controls no connections would exist above 25%.

Study / Model / Regional Differences (out of 62) / Total Brain Volume Difference
Relative
(% total brain volume)
15q11-13 / 15q11-13 (patdp/+) / 15 / -
16p11 / 16p11.2 (df/+) / 11 / -
16p11.2 (df/dp) / 0 / -
16p11.2(dp/+) / 2 / -
AndR / AndR 12Q h/mAR / 4 / -
AndR 48Q h/mAR / 0 / -
BALB/c / BALB/cJ / 50 / -
BTBR / BTBR T+tf/J (vs. B6) / 47 / Yes
BTBR T+tf/J (vs. FVB) / 48 / Yes
CNTNAP2 / CNTNAP2(-/-) / 0 / -
En2 / En2 (-/-) / 43 / -
FMR1 / FMR1 (-/y) (B6) / 1 / -
FMR1 (-/y) (FVB) / 24 / -
GTF2i / GTF2i (-/+) / 0 / -
GTF2i (dp/dp) / 6 / -
ITGβ3 / ITGβ3 (-/-) / 39 / Yes
Mecp2 / Mecp2308(-/y) / 30 / -
NLGN3 / NLGN3 R451C KI / 20 / Yes
NRXN1α / NRXN1α (-/+) / 1 / -
NRXN1α (-/-) / 1 / Yes
SLC6A4 / SLC6A4 Ala56 KI (B6) / 0 / -
SLC6A4 Ala56 KI (129) / 0 / -
SLC6A4 KO / 0 / -
SHANK3 / SHANK3 e4-9 (+/-) / 0 / -
SHANK3 e4-9 (-/-) / 0 / Yes
XO / XO / 6 / -

Supplementary Table 3 – Summary of the significant differences found in the 26 models. Anything indicated here has a false discovery rate of less than 10%.

Group 1
En2 (-/-)
FMR1 (-/Y) (B6)
FMR1 (-/Y) (FVB)
NRXN1a (-/+)
NRXN1a (-/-)
SHANK3 (-/+)
SHANK3 (-/-) / Larger
Cerebellar peduncle: superior
Corpus callosum
Fimbria
Fornix
Frontal Lobe
Lateral Ventricle
Parieto-Temporal Lobe
Ventral Tegmental Decussation / Smaller
Cerebellar Cortex
Cerebellar peduncle: middle
Inferior Colliculus
Medulla
Nucleus Accumbens
Pontine Nucleus
Group 2
15q11-13
AndR 12Q
AndR 48Q
BTBR (B6)
BTBR (FVB)
GTF2i (dp/dp)
ITGB3 (-/-)
NL3 R451C KI
SERT Ala56 KI (129) / Larger
Amygdala
Anterior Commisure: Anterior
Cerebral Aqueduct
Entorhinal Cortex
Fourth Ventricle
Olfactory bulbs
Olfactory tubercle
Pons
Rhinocele
Superior Colliculus / Smaller
Anterior Commisure: Posterior
Cerebral peduncle
Corpus callosum
Dentate gyrus
Internal capsule
Medial Longitudinal Fasciculus
Parieto-Temporal Lobe
Pre-para subiculum
Stratum granulosum
Stria Medullaris
Stria Terminallis
Group 3
16p11.2 (df/+)
16p11.2 (df/dp)
16p11.2 (dp/+)
BALBC (B6)
CNTNAP2 (-/-)
GTF2i (+/-)
Mecp2 (-/y)
SERT Ala56 KI (B6)
SERT KO (-/-)
XO / Larger
Arbor vita of the cerebellum
Cerebellar Cortex
Cuneate nucleus
Inferior Olivary Complex
Medial Longitudinal Fasciculus
Medulla / Smaller
Anterior Commisure: Anterior
Anterior Commisure: Posterior
Frontal Lobe
Fimbria
Lateral septum
Lateral Ventricle
Parieto-Temporal Lobe
Rhinocele

Supplementary Table 4 – List of all the regions which had positive or negative median effect sizes greater than 0.5 or less than -0.5.

Supplementary Table 5 – Summary of the published behavioural findings for the models examined in this study.

Model / Behavioural Findings
Social Behaviour / Communication / Repetitive Behaviours / Cognitive / Motor
Group 1
En2 (-/-) 22 / Deficit / No Diff. / No Diff. / Deficit / Deficit
FMR1 (-/y) (B6) 33-37 / Mixed / Deficit / Mixed / Mixed / No Diff.
FMR1 (-/y) (FVB)36 / Deficit / - / - / Mixed / -
NRXN1α (-/+)27 / No Diff. / - / Increased / No Diff. / Enhanced
NRXN1α (-/-)27
SHANK3 (-/+)38, 39 / Deficit / Mixed / Increased / Deficit / Impaired
SHANK3 (-/-)38, 39
Group 2
15q11-1315 / Deficit / Increased / - / Deficit / -
AndR (12Q) / - / - / - / - / -
AndR (48Q)
BTBR (B6) 20, 40, 41 / Deficit / Decreased / Increased / Deficit / Imparied
BTBR (FVB) 20, 40, 41
GTF2i (dp/dp)42 / Enhanced / - / - / No Diff. / Impaired
ITGβ3 (-/-)43 / Deficit (Novelty) / - / Increased / - / -
NL3 R451C 44-46 / Mixed / Decreased / - / Enhanced / Mixed
SLC6A4 Ala56 (129) 28, 29 / Mixed / Decreased / - / No Diff. / No Diff.
Group 3
16p11.2 (df/+) 16 / No Diff / - / Decreased / - / -
16p11.2 (df/dp) 16 / - / - / - / - / -
16p11.2 (dp/+) 16
BALBC (B6) 18, 47, 48 / Deficit / Increased / Increased / Deficit / Impaired
CNTNAP2 21 / Deficit / - / - / Deficit / Enhanced
GTF2i (+/-) / - / - / - / - / -
Mecp2 308 (-/y) 25, 26, 49 / Deficit / - / No Diff. / Mixed / Mixed
SLC6A4 Ala56 (B6) 29 / Mixed / Increased / - / No Diff. / No Diff.
SCL6A4 KO (-/-)34, 50, 51 / Deficit / - / No Diff. / No Diff. / No Diff.
XO / - / - / - / - / -

Social Behaviour was defined using the 3 Chamber Apparatus when possible

Communication was defined using ultrasonic vocalizations

Repetitive Behaviour was defined by assessing the grooming behaviour

Cognitive Behaviour was defined by performance on in the water maze (reversal learning when possible)

Motor performance was defined by the rotarod test

‘-‘ indicates the particular behaviour has not been tested

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