Movement restraint reduces cell proliferation in the larval forebrain.

By 6 dpf, movement restraint reduces the proportion of PCNA+ cells in the forebrain (A-C; control n = 5, restraint n = 6). This reduction in PCNA +cells is maintained when movement restraint is continued until 9 dpf (D-F; control n = 6, restraint n = 7). Movement restraint until 9 dpf also reduces tbr2+ cells in the pallium (G-I; n = 9) without affecting the number of GFAP+ radial glia stem cells in the pallium (J-L; n = 7; scale bar for micrographs in B-L = 30 µm). Following a 24 hr pulse with EdU starting on 5 dpf, fewer EdU+ cells in the subpallium (M) and pallium (N; n = 4) co-label for the neuronal fate marker Elavl3 in controls (O–Q) compared to movement restrained larvae (R-T; scale bar = 20 µm). White dotted lines mark the boundaries of Elavl3+ expression to highlight the increased overlap between EdU+ cell cohorts and Elavl3+ in restrained larvae. *p<0.05. Data are represented as mean ± SEM.

Example traces of brain regions sampled through coronal sections in the larval zebrafish brain.

Micrographs (20 µm thickness) with example boundaries traced for the olfactory bulb, pallium, subpallium, and optic tectum (white dotted line) along with their approximate rostrocaudal position on a schematic of a dorsal view of the larval zebrafish head. Scale bars = 20 µm.

Rearing larvae against a displacing current increases pallial cell proliferation.

From 3 dpf, larvae were reared in groups in a plastic canal with water flow producing (A) a weak current that did not displace larvae or (B) a strong current that would displace larvae on a daily schedule (C). By 9 dpf, larvae reared against a strong current exhibited significantly more PCNA +cells in the pallium (D; control n = 10, current n = 7), but not subpallium (E; control n = 11, current n = 10) compared to controls. Larvae reared against a strong current exhibited significantly less Elavl3/EdU co-labeling compared to controls (F; n = 6) and did not differ in body length from controls (G; control n = 17, current n = 14). *p<0.05. n.s. = not significant. Data are represented as mean ± SEM.

Tail movement, not visual stimulation, associated with locomotion maintains pallial cell proliferation.

Larvae were fully embedded in agarose at 3 dpf and reared able to perceive either (A) visual stimulation associated with movement (a moving gradient; purple arrows) and tail movement (tail cut free from agarose; green arrows), (B) tail movement only, or (C; scale bar = 20 µm) visual stimulation only. By 6 dpf, larvae capable of tail movement exhibited more PCNA+ cells in the pallium (D) compared to larvae perceiving only visual stimulation associated with movement. Isolating visual or physical cues of movement had no significant affect on PCNA+ cell counts in the subpallium (E; control n = 6, physical only n = 6, visual only n = 5) by 6 dpf. *p<0.05. n.s. = not significant. Data are represented as mean ± SEM.

Ablation of the lateral line does not affect pallial cell proliferation.

On 3 dpf, 30 min exposure to CuSO₄ destroyed kinocilia (as visualized by acetylated tubulin) associated with hair cell cupulae (white arrows) along the larval zebrafish lateral line (A). Prior ablation of lateral line hair cells on 3 dpf did not affect the number of PCNA+ cells in the pallium reared in unrestrained conditions (B; n = 7). Scale bar = 40 µm. n.s., not significant. Data are represented as mean ± SEM.

Impairing trunk DRG formation attenuates movement-dependent pallial neurogenesis.

(Ai) Dorsal root ganglia (white arrow) and Rohon-Beard neurons (white asterisk) were visualized in Tg(isl2b:mgfp) larvae (scale bar = 40 µm). Treatment with AG1478 from 8 to 30 hpf prevented development of DRG along the trunk in larvae by 3 dpf without affecting RB neuron populations dorsal to the spinal cord (Aii-iii). Earlier treatment with AG1478 did not affect swimming compared to DMSO-treated controls on 8 dpf (B; n = 6). By 9 dpf, restrained larvae in both DMSO (F-G; control n = 6, restraint n = 7) and AG1478 (H-I; control n = 8, restraint n = 6) treatments exhibited fewer pallial PCNA+ cells compared to controls, however, AG1478-treated controls exhibited fewer pallial PCNA+ cells compared to DMSO-treated controls, despite similar swimming behaviour. *p<0.05. Data are represented as mean ± SEM.