Figure 1.

Ca²⁺ dynamics and glutamate signaling are present during neural tube formation. A, Developmental stages of X. laevis neural tube formation. hpf: hours post fertilization, hpf at 23°C. Adapted from the study by Nieuwkoop and Faber (1994). B, Representative trace of in vivo spontaneous changes in the Ca²⁺ sensor GCaMP6s fluorescence in a neural plate cell from a neurulating stage 14 embryo. C, Frequency of Ca²⁺ transients (per 40 min period, percentage of total number of transients in 2 h period, stages 12.5–14); values are the mean ± SEM, n = 12 embryos, *p < 0.05, ANOVA followed by Dunnett's test, compared with first 40 min period. St., Stage. D, Distribution of Ca²⁺ transients (per 2 h period) in the dorsal ectoderm (percentage of the total number of transients per 2 h period, stages 12.5–14); values are the mean ± SEM, n = 15 embryos. ****p < 0.0001, ANOVA followed by Dunnett's test compared with the most lateral region. E, RT-PCR from the dorsal half of stage 13 embryos. F, Western blot assay from whole-embryo lysates at neural plate (stage 18) and tailbud (stage 28) stages (top), and from neural plate stage (stage 18) embryos injected at the two-cell stage with C-Mo or GluN1-Mo2 (bottom). Similar results were obtained in three independent experiments. G, Traces from stage 15-dissociated neural plate cells loaded with the Ca²⁺-sensitive dye Fluo4-AM and imaged during the addition of 10 μm NMDA or 5 mm glutamate. Lines under the traces indicate the time of incubation with NMDA or glutamate. Samples were time-lapse imaged at a rate of 0.2 Hz for 5 min. Regions of interest were selected to measure the fluorescence intensity over time for individual neural plate cells with NIS Elements software (Nikon). Fluorescence (F) is normalized to the baseline for each cell (F₀) as a percentage. H, Examples of GCaMP6s-expressing embryos imaged in the absence (vehicle) or presence of the indicated drugs for 2 h starting at stage 12.5. Circles indicate the occurrence of Ca²⁺ transients. Ten micromolar NMDA was preincubated for 30 min before the addition of 1 mm VPA (NMDA+VPA). I, Frequency of Ca²⁺ transients (per 2 h period, stages 12.5–14); values are the mean ± SEM, numbers between parentheses are the number of embryos per treatment. *p < 0.05, one-way ANOVA, followed by Dunnett's test compared with vehicle-treated samples.