Effect of valproate exposure on the differentiation of serotonergic and other brainstem neuronal subtypes. The timing of drug treatment is indicated in this and all subsequent schematics by a coloured bar. Arrowheads mark the developmental stage of interest (hpf). Dashed lines with arrows indicate the developmental time point at which embryos were harvested. (A) Immunostaining for brainstem neuronal subtypes in 48 hpf zebrafish embryos exposed to VPA from 50% epiboly to 27 hpf [condition (i)] shows the failure of 5HT neuronal differentiation (n=22/22, P<0.0001), marked by 5HT (yellow arrowheads in control panel), and lack of Mauthner neurons (n=22/22), marked by the 3A10 anti-neurofilament monoclonal antibody (yellow arrowheads in control panel). Isl1+ motor neurons (yellow arrowheads in control and VPA panels) (n=10/10) and GABA-ergic neurons (n=10/10) persist. Scale bar: 50 µm. (B) Failure of serotonergic differentiation in embryos treated with VPA from 24-48 hpf [condition (ii)], as indicated by lack of 5HT (n=32/32, P<0.0001) and Tph2 expression. Pet1 (arrowhead) is severely reduced (n=3/15) or absent (n=12/15) in VPA-treated embryos. (C) Persistence of Mauthner, motor and GABA-ergic neurons in VPA treatment condition (ii). (D) At 72 hpf, embryos treated with VPA from 24-48 hpf [condition (iii)] show a limited recovery of 5HT expression (n=30/30). Treatment with VPA from 48-72 hpf [condition (iv)] does not affect serotonergic differentiation (n=30/30). hpf, hours post-fertilisation. Scale bar: 50 µm.
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