FIGURE SUMMARY
Title

Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish

Authors
Fame, R.M., Chang, J.T., Hong, A., Aponte-Santiago, N.A., Sive, H.
Source
Full text @ Fluids Barriers CNS

Zebrafish brain ventricle anatomy at early and late larval stages. Using 2 nL rhomencephalic ventricular injections of fluorescein-labeled dextran and imaging by Selective Plane Illumination Microscopy (SPIM, or Lightsheet Microscopy), ventricular system anatomy, volume, and connectivity was visualized in three dimensions. a Dorsal, b lateral view, 30 hpf zebrafish larvae with neuroepithelium labeled in red (mApple-caax) and the ventricular system labeled in green (fluorescein dextran injection). c H&E stain of 4 μm thick parasagittal section of 30 hpf zebrafish. Dorsal, Lateral view, 30 hpf larval zebrafish ventricular system. inset Magnification of boxed region in to show spinal canal (S) branching from the ventral aspect of the D/M ventricle (arrow). H&E stain of 4 μm thick section of 30 hpf zebrafish. cʹʹ magnification of boxed region in to show spinal canal (S) branching from D/M ventricle. d Dorsal, e Lateral view, 54 hpf larval zebrafish ventricular system. e inset Magnification of boxed region in d to show SC branching from the ventral aspect of the diencephalon (arrow). f H&E stain of 4 μm thick section of 54 hpf zebrafish. magnification of boxed region in f to show spinal canal (S) branching from diencephalon. g Dorsal, h Lateral view of the 5 dpf larval zebrafish ventricular system. h insets Magnification of boxed regions in h to show SC branching from the ventral aspect of the diencephalon (arrow) and rhombencephalon (arrow). i H&E stain of 4 μm thick section of 5 dpf zebrafish. magnification of boxed region in i to show spinal canal (S) branching from rhombencephalon. T telencephalic ventricle, D/M diencephalic/mesencephalic ventricle, D diencephalic ventricle, Te tectal ventricle, R rhombencephalic ventricle, S spinal canal, hpf hours post-fertilization, dpf days post-fertilization. Scale bar: , e, h insets, cʹʹ, , : 50 μm; all others: 100 μm

Bulk movement of CSF at early and late larval stages. a, d, g, j Schematics of brain ventricular regions analyzed. b At 27–30 hpf, the average vmax is significantly higher in the anterior to posterior (red) direction through the telencephalic-to-diencephalic/mesencephalic aqueduct than in the posterior to anterior direction (blue). c Overlaid thresholded volumes generated by Imaris (Bitplane) software of a representative 27–30 hpf T → D/M dataset. e At 27–30 hpf, the average vmax is significantly higher in the anterior to posterior (red) direction through the diencephalic/mesencephalic-to-rhombencephalic aqueduct than in the posterior to anterior direction (blue). f Overlaid thresholded volumes generated by Imaris (Bitplane) software of a representative 27–30 hpf D/M → R dataset. h At 51–54 hpf, through the telencephalic-to-diencephalic aqueduct, directionality is reversed such that the vmax is significantly higher in the posterior to anterior (blue) direction through the telencephalic-to-diencephalic aqueduct than in the anterior to posterior direction (red). i Overlaid thresholded volumes generated by Imaris (Bitplane) software of a representative 51–54 hpf T → D dataset. k At 51–54 hpf, the average vmax is significantly higher in the anterior to posterior (red) direction through the diencephalic-to-rhombencephalic aqueduct than in the posterior to anterior direction (blue). l Overlaid thresholded volumes generated by Imaris (Bitplane) software of a representative 51–54 hpf D → R dataset. Red circles indicate photoconversion region. Lines represent average vmax and error bars denote SEM. p value calculated using unpaired Student’s t test, **p < 0.001; ***p < 0.0005; ****p < 0.0001. T telencephalic ventricle, D/M diencephalic/mesencephalic ventricle, Te tectal ventricle, R rhombencephalic ventricle, hpf hours post-fertilization. Scale bar: 50 μm

Brain cilia are non-motile at early larval stage; some are motile at late larval stage. a, c Arl13b: GFP is expressed in cilia throughout the developing fish including the neuroepithelium and spinal canal. b, d Quantification of ciliary beat frequency (Hz) for motile cilia in each region at early (27 hpf) and late (51 hpf) larval stages, if any. e, f Visualization of ciliary movement for cilia in each region at 27 and 51 hpf, if any. Each of the three panels is an overlay of two sequential image acquisition frames (images were taken in the same region at distinct times at 57.22 fps, so each overlay represents a time difference of ~0.017 s). See Additional file 2: Movie 1 and Additional file 3: Movie 2 for full dataset. Colors were selected such that unmoved pixels appear black (Frame 1: green + magenta; Frame 2: red + cyan). Horizontal lines represent average vmax and error bars denote SEM. T telencephalic ventricle, D/M diencephalic/mesencephalic ventricle, R rhombencephalic ventricle, SC spinal canal ventricle analysis region, hpf hours post-fertilization, N.A. quantification of cilia frequency is not applicable because no cilia are motile. Scale bars: a, c: 100 μm; e, f: 5 μm

Ventricle injections do not disrupt gross ventricular morphology.

Larvae were imaged under brightfield (first and third columns) and fluorescence (second and fourth columns, merged with brightfield) microscopy both before and after ventricular injection. Injections comprised 2nL of 2000kDa dextran FITC at 27 hpf (top) and 56 hpf (bottom). No change in ventricular morphology is observed in either dorsal (first two columns) or lateral (last two columns) views. hpf, hours post-fertilization. Scale bar: 200μm. N ≥10 observed at each stage, representative images shown.

BDM treatment does not disrupt gross ventricular morphology over 2 hours.

Larvae treated with 40mM BDM show no change in gross ventricular shape compared to untreated larvae. Brightfield and florescence microscopy (merged images shown) is shown after ventricular injection of 2nL of 2000kDa dextran FITC. BDM, 2,3 butanedione monoxime ; hpf, hours post-fertilization; t= time after treatment (minutes). Scale bar: 200μm. N ≥10 observed for each treatment, representative images shown.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Fluids Barriers CNS