Comparison of brain structure and volume in tlr2?/? and control adult zebrafish. (A) Representative sagittal and coronal slices of DTI of control and tlr2?/?adult zebrafish obtained at 17.6 T. (B) 2D and 3D representation of the zebrafish brain, divided into its main regions, providing a comprehensive view of its anatomical organisation. (C) Comparison of the cortical thickness of the TL, the CCem and TeO shows a significant increase in tlr2?/? zebrafish compared with controls. Furthermore, significant increases in the volume of the CB and RC were observed. In all other brain regions, as for the total brain volume, a similar trend is observed, albeit nonsignificantly (p > 0.05). Acquisition details: TR 2000 ms, TE 9 ms, 4 averages, isotropic resolution 35 ?m, effective b-value range of 100, 1000 or 2500 s/mm2, with 4, 12 and 24 directions, respectively. Statistical analysis was performed using the unpaired t-test, assuming Gaussian distribution, with p less than 0.05 considered to imply significant differences between the control and the tlr2?/? group. CB, cerebellum; CCem, molecular layer of the cerebellar corpus; DC, diencephalon; DTI, diffusion tensor imaging; MC, mesencephalon; OB, olfactory bulb; Pa, pallium; RC, rhombencephalon; TeO, optic tectum; TL, longitudinal torus.

T2 relaxation time and ADC measurement in various brain regions of control and tlr2 mutant (tlr2?/?) zebrafish. (A) Anatomical DWI (b-value 1000 s/mm2), sagittal slice, showing ROIs used for T2 and ADC estimation. (B) Estimated T2 relaxation times in selected ROIs of control and tlr2?/? zebrafish. (C) Estimated ADC in selected ROIs of control and tlr2?/? zebrafish. Acquisition details for MSME: TR 3000, ? 6.0 ms, 4 averages, resolution 47 × 47 ?m and a slice thickness of 500 ?m; and for DWI: TR 1000 ms, TE 13 ms, 4 averages, resolution 47 × 47 ?m, slice thickness 200 ?m and effective b-value range 50, 500, 1000, 1500, 2000, 2500 or 3000 s/mm2. Statistical analysis was performed using the unpaired t-test, assuming Gaussian distribution, with p less than 0.05 considered to imply significant differences between the control and the tlr2?/? group. *p < 0.05, **p < 0.01, ****p < 0.001. ADC, apparent diffusion coefficient; CCeg, granular layer of the cerebellar corpus; CCem, molecular layer of the cerebellar corpus; DIL, diffuse nucleus of the inferior lobe; DM, medial zone of the dorsal telencephalon; DWI, diffusion-weighted imaging; MLF, medial longitudinal fascicle; MSME, multislice multiecho; ROI, region of interest; TL, longitudinal torus.

Phasor analysis of the experimental ADC curves. (A) Phasor plot of the CCem of tlr2?/? and control, relative to the phasor plot semicircle (red). On the semicircle, 0 mm2/s is located at [0,0], with steps of 1·10?4 mm2/s indicated as red dots on the semicircle. (B) Monocomponent analysis shows that the ADC in tlr2?/? is significantly reduced, while (C?E) Multicomponent analysis of the ADC decay curve in the CCem shows a significant increase in the area of the slow diffusion component in tlr2?/?, associated with restricted diffusion. Acquisition details for the DWI used for phasor plot analysis: TR 1000 ms, TE 13 ms, 4 averages, resolution 47 × 47 ?m, slice thickness 200 ?m and effective b-value range 50, 500, 1000, 1500, 2000, 2500 or 3000 s/mm2. Statistical analysis was performed using the unpaired t-test, assuming Gaussian distribution, with p less than 0.05 considered to imply significant differences between the control and the tlr2?/? group. * p < 0.05. ADC, apparent diffusion coefficient; CCem, molecular layer of the cerebellar corpus; DWI, diffusion-weighted imaging.

Comparison of DTI results in the main brain regions of tlr2?/? and control adult zebrafish. Representative central slice of DTI measurements of control and tlr2?/? adult zebrafish. (A) From these slices, the diffusion tensor metrics were estimated: D?, D?, MD and FA, as well as (B) the diffusion kurtosis metrics: K?, K?, MK and KFA. (C) DTI and DKI results of tlr2?/? adult zebrafish compared with controls reveal a noteworthy trend towards decreased D?, D? and MD, reduced FA, as well as increased K?, K? and MK. Additionally, a significant decrease in KFA is observed in the tlr2?/? group. Acquisition details: TR 2000 ms, TE 12.4 ms, 32 averages, resolution 25 × 25 × 200 ?m, effective b-value range 4, 1000, 3500 or 6000 s/mm2, with 8, 12, 24 or 36 directions, respectively. Statistical analysis was performed using the unpaired t-test, assuming Gaussian distribution, with p less than 0.05 considered to imply significant differences between the control and the tlr2?/? group. *p < 0.05, ***p < 0.005. CB, cerebellum; D?, axial diffusivity; D?, radial diffusivity; DC, diencephalon; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; FA, fractional anisotropy; K?, axial kurtosis; K?, radial kurtosis; KFA, kurtosis fractional anisotropy; MC, mesencephalon; MD, mean diffusivity; MK, mean kurtosis; OB, olfactory bulb; Pa, pallium; RC, rhombencephalon.

Comparison of DTI results of white matter structures of tlr2?/? and control adult zebrafish. (A) Sagittal and coronal slices of the DEC stTDI msmt-CSD map, used for the identification of white matter structures in the zebrafish brain. Acquisition details: TR 2000 ms, TE 9 ms, 4 averages, isotropic resolution 35 ?m, effective b-value range 100, 1000 or 2500 s/mm2, with 4, 12 and 24 directions, respectively. (B) Diffusion metrics estimated by DTI results show reduced D?, D? and MD, reduced FA, increased K?, K? and MK and KFA in white matter structures in the tlr2?/? zebrafish brain. Acquisition details: TR 2000 ms, TE 12.4 ms, 32 averages, resolution 25 × 25 × 200 ?m, effective b-value range 4, 1000, 3500 or 6000 s/mm2, with 8, 12, 24 or 36 directions, respectively. Statistical analysis was performed using the unpaired t-test, assuming Gaussian distribution, with p less than 0.05 considered to imply significant differences between the control and the tlr2?/? group. *p < 0.05, ***p < 0.005, ****p < 0.001. Cans, ansulate commissure; Cgus, commissure of the secondary gustatory nuclei; Cpost, posterior commissure; CSD, constraint spherical deconvolution; Cven, ventral rhombencephalic commissure; D?, axial diffusivity; D?, radial diffusivity; DEC, directional encoded colour; DTI, diffusion tensor imaging; FA, fractional anisotropy; K?, axial kurtosis; K?, radial kurtosis; KFA, kurtosis fractional anisotropy; LLF, lateral longitudinal fascicle; MD, mean diffusivity; MK, mean kurtosis; MLF, medial longitudinal fascicle; msmt, multishell multitissue; OT, optic tract; stTDI, short-track track-density imaging.