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. 2014 Dec 19;8:147. doi: 10.3389/fncir.2014.00147

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Copyright © 2014 Hsieh, Ulrich, Issa, Wan and Papazian.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Rapid emergence and maturation of excitability in zebrafish Purkinje cells. (A) Dorsal view of la118Tg:Tg(aldoca:gap43-Venus) transgenic zebrafish at 5 dpf shows that membrane-bound Venus is specifically and exclusively expressed in cerebellar Purkinje cells (Tanabe et al., 2010). Anterior is to the left. A z-projection of 168 1 μm confocal images has been superimposed on the corresponding bright field image. Images were acquired with an Olympus Fluoview FV300 laser scanning confocal microscope. Labels: E, eye; OT, optic tectum; *, otic vesicle; arrows, cerebellovestibular axon tracts. Scale bar, 100 μm. (B) The recording configuration is shown. A transgenic zebrafish at 5 dpf has been fixed in the recording chamber. A loose-patch electrode (arrow) was inserted from the anterior side and advanced to contact the cell body of a Purkinje cell. In some experiments, a theta pipette (arrowhead) was placed in the inferior olive to stimulate climbing fibers. (C–F) Shown are representative loose-patch recordings acquired at (C) 4 dpf, (D) 5 dpf, (E) 6 dpf, and (F) 7 dpf. In (D), red bar marks portion of trace that is shown on an expanded time scale below the main trace. In (D), (E), and (F), complex spike-like events are indicated by red dots. (G) The average simple spike firing frequency has been plotted vs. age (n = 7–31 cells from 6–24 animals). The frequency at 4 dpf, 5.4 Hz, was significantly different from the frequency on all subsequent days, which averaged ~9 Hz (*, ANOVA: 4–9 dpf, F_(5,113) = 2.41, p = 0.04, followed by Tukey’s post hoc test: 4 and 5 dpf, p < 0.05). Values obtained between 5 and 9 dpf did not differ significantly (NS, ANOVA: 4–9 dpf, F_(5,113) = 2.41, p = 0.04, followed by Holm-Bonferroni post hoc test: 5–9 dpf, p ≥ 0.05). (H) The regularity of spontaneous tonic firing was quantified by determining the coefficient of variation of adjacent intervals (CV2), which has been plotted against age(n = 7–31 cells from 6–24 animals). CV2 declined significantly from 4 dpf (CV2 = 0.76) to 6 dpf (CV2 = 0.48); CV2 did not differ significantly from 6–9 dpf (ANOVA: 4–9 dpf, F_(5,113) = 5.83, p = 7.96 × 10⁻⁵, followed by Holm-Bonferroni post hoc test: 4–6 dpf, p < 0.05 [*] and 6–9 dpf, p ≥ 0.05 [NS]).