Live confocal imaging of Purkinje cells

MiniTol2 plasmid DNA encoding the aR3H or iR4H mutant subunits or exogenous wild-type Kv3.3a and mEGFP, or mEGFP alone under the control of the aldoca promotor was injected into TLN embryos. Purkinje cell development and viability were assessed by repeated confocal imaging in live zebrafish between 3 and 8 dpf. Isolated Purkinje cells expressing mEGFP or traceable cells in small groups of 2–3 expressing cells were selected for analysis starting at 3 dpf. Zebrafish were anesthetized with pharmaceutical grade 0.02% MS-222 (Tricaine-S) (Syndel, Ferndale WA) and embedded dorsal side up in 1% low melt ultra-pure agarose (ThermoFisher Scientific, Waltham MA). Images were acquired as 1 μm optical sections using an Olympus Fluoview FV300 laser scanning confocal microscope (Olympus, Tokyo, Japan). The same cells or small groups of cells were imaged on consecutive days. Zebrafish were carefully removed from the agarose between time points and re-embedded for subsequent imaging.

Single expressing Purkinje cells were traced from confocal image stacks for three-dimensional digital reconstruction using Imaris 8.0 (Bitplane, Concord MA). Total process length and the number of branches for each cell were obtained from the traced images. Spine number was counted manually using ImageJ (NIH, Bethesda MD). Spines in each cell were counted three times and the values, which differed by less than 10%, were averaged. Statistical analyses were performed using SPSS software (IBM, Armonk NY). Values were log₁₀-transformed because individual data points were right-skewed rather than normally distributed. Comparisons were made using a Linear Mixed Model with Bonferroni post-hoc tests. Statistical significance was assessed as p<0.05. Figures were prepared using Adobe Illustrator (Adobe, San Jose CA).