Cyfip1 haploinsufficient rats show white matter changes, myelin thinning, abnormal oligodendrocytes and behavioural inflexibility

The biological basis of the increased risk for psychiatric disorders seen in 15q11.2 copy number deletion is unknown. Previous work has shown disturbances in white matter tracts in human carriers of the deletion. Here, in a novel rat model, we recapitulated low dosage of the candidate risk gene CYFIP1 present within the 15q11.2 interval. Using diffusion tensor imaging, we first showed extensive white matter changes in Cyfip1 mutant rats, which were most pronounced in the corpus callosum and external capsule. Transmission electron microscopy showed that these changes were associated with thinning of the myelin sheath in the corpus callosum. Myelin thinning was independent of changes in axon number or diameter but was associated with effects on mature oligodendrocytes, including aberrant intracellular distribution of myelin basic protein. Finally, we demonstrated effects on cognitive phenotypes sensitive to both disruptions in myelin and callosal circuitry.


Statistics
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Software and code
Policy information about availability of computer code Data collection MRI scanner (Bruker, Karlsruhe, Germany) Data analysis ExploreDTI 4.8.3 and SPM (version 12, UCL, London, UK) were used for preprocessing of the DTI data, and FSL was used for whole-brain statistical analyses. All other analyses and plotting was performed in RStudio statistical software version 1.1.463 (R Foundation for Statistical Computing, Vienna, Austria). ImageJ software (version 1.51) was used for cell quantification.
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Data
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Life sciences study design
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Sample size
A cohort of 24 rats (12 per genotype) were used for diffusion tensor imaging, a number used typically in rodent imaging studies, and with sufficient power to detect genotype effects in this study. For electron microscopy 9 rats were used (5 WT and 4 Cyfip1 hets); in this detailed ultrastructural analyses around 13 000 axons were analysed in total as a representative sample, which is significantly in excess of published reports where similar analyses have been done. For immunofluorescence 14 rats were used (7 per genotype). Here, at least 5 random regions were quantified. For the in vitro oligodendrocyte culture experiments 3 biological repeats were used (which is the recommended standard procedure) and at least 5 images were collected from each well (8 wells per replicate), resulting in a total representative sample of ~14 000 cells. For the reversal learning work we used 7 WT and 9 hets on the basis of published reports using touch-screen methods in rats and our own experience with the paradigm. There was less previous information on the mismatch task so we increased the n accordingly, WT 21/hets 15.

Replication
In all the experiments each animal was a biological repeat. The oligodendrocyte culture experiments also utilised a triple technical repeat design. In both the electron microscope and cell culture experiments the representative samples were obtained from very large numbers of determinations (for the EM over 13,000 separate determinations, for the culture work 14,000 in total including 1688 cells analysed for the MBP area data). We did not perform additional repeats of the DTI study. For the behaviour we did not repeat the study for the individual paradigms but we did however, as noted in the manuscript, obtain converging evidence from both assays of effects on psychological processes underlying behavioural flexibility.
Randomization All allocations were random.

Blinding
The investigator was blind to the genotype in all analyses done.
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Animals and other organisms
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Laboratory animals
All the rats used in this study were Long Evans males. The age of the rats slightly differed in each experiment: the rats used in DTI were 5 months old and were euthanized 1 month after the scanning for immunofluorescence. The rats used for electron microscopy were 6 months old, and the rats used for behavioural experiments were between 6 to 9 months old.
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Sequence & imaging parameters
The MRI protocol included DTI acquisition with a diffusion-weighted (DW) spin-echo echo-planar-imaging (EPI) pulse sequence. T2 weighted images were also acquired for anatomical reference. For DTI acquisition two EPI segments were applied with gradient strength up to 600 mT/m and duration of 4.5 ms and separation of 10.5 ms, with 60 noncollinear gradient directions with a single b-value shell at 1000 s/mm^2 and one image with a b-value of 0 s/mm^2. TR = 4000 ms and a TE = 22 ms. Geometrical parameters were: 34 slices, each 0.32 mm thick (brain volume) and with in-plane resolution of 0.32x0.32 mm^2 (matrix size 80x96; FOV 25.6x30.73 mm^2). For the T2 weighted images acquisition a multi-slice multi-echo pulse sequence was used with the following parameters: TR = 7200ms, TE = 15ms and effective TE of 45ms, rare factor was 8. Image resolution was set to 0.22 mm^3 with matrix size of 128x160x50 to cover the entire brain.
Area of acquisition whole brain Diffusion MRI Used Not used Parameters 60 noncollinear gradient directions with a single b-value shell at 1000 s/mm^2 and one image with a b-value of 0 s/mm^2.

Preprocessing
Preprocessing software ExploreDTI 4.8.371 and SPM (version 12, UCL, London, UK) were used in the preprocessing of the rat DTI data. Brain was extracted following the protocol described in the paper and using mainly SPM and ExploreDTI masking option.

Normalization template
We used a population-specific template.
Noise and artifact removal Eddy-current induced distortion and motion correction were performed, and data was also corrected for field inhomogeneities using ExploreDTI 4.8.371 (the protocol is described in the manuscript). The data was visually inspected and data quality was performed using ExploreDTI 4.