Computational protocol: Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

Similar protocols

Protocol publication

[…] Zebrafish processed via immunohistochemistry were laterally mounted on a slide in PBST, lightly cover slipped, and sealed. Single focal plane images (zn5 labeled fish) and image stacks (zn5, znp-1, anti-chrn2b and F59 labeled fish) were obtained with an Orca ER CCD camera mounted to a Zeiss inverted microscope (Axiovert 200M or Axio Observer) equipped with epifluorescence and a Zeiss ApoTome (Carl Zeiss). Images were acquired with a 20× dry (0.6 N.A, 0.8 N.A), a 25× water (0.8 N.A.), or a 40× oil (1.3 N.A.) objective. Multiple single focal plane images were acquired for each fish capturing motoneuron axon morphologies from both sides within each body segment. Image stacks were acquired to reveal high-resolution motoneuron axon morphology using volume-rendering software (Imaris 5.7.2, Bitplane Inc., Saint Paul, MN).Evaluation of motoneuron axon morphology following different nicotine exposure paradigms was performed on single focal plane images taken from multiple hemisegments for control and nicotine-exposed fish. For every fish, we focused our analysis between the 6th and 20th body segment. Each body segment consists of two hemisegments, one on each side of the body. The number of hemisegments imaged and analyzed per fish varied depending on the quality of the fish’s lateral mount on the slide.Each hemisegment typically has one motoneuron nerve bundle that exits spinal cord and its axons take three distinct trajectories to the periphery; dorsal, ventral and mediolateral (see ). zn5 labeling was used to label SMN axons and if a hemisegment possessed SMN axon errors in any of the three axonal trajectories, that hemisegment was then considered abnormal. The number of normal hemisegments over the total number of hemisegments analyzed per fish was then expressed as a percentage. Each nicotine exposure paradigm was replicated multiple times (5 experiments for 22–72 hpf, ; 3 experiments for 12–30 hpf; ).To quantify the effects of nicotine exposure on individual axonal trajectories (dorsal, ventral and mediolateral), a subset of the fish analyzed for (22–72 hpf exposure window) and the fish analyzed for (12–30 hpf exposure window) were used. Hence, the quantification shown in and the subsequent motoneuron phenotyping of the dorsal projecting SMN axons was performed from the same experimental dataset for each nicotine exposure paradigm. For , each hemisegment was evaluated for errors in any of the three main trajectories (dorsal, ventral and mediolateral). For example, the number of hemisegments that possessed abnormal dorsal projecting axons was divided by the total number of hemisegments analyzed and was then expressed as a percentage. This quantification revealed the relative effects of nicotine on different axonal projections.Evaluation of muscle fiber morphology was performed using high-resolution image stacks following F59 labeling (). Slow muscle measurements (fiber widths) were acquired using Axiovision 4.7/4.8 (Carl Zeiss) software. For each fish, multiple slow muscle fibers were measured but only a single measurement was taken for a given fiber (29.7 ± 5.8 fibers/fish; mean ± standard deviation). All of the measurements were taken from 2–3 consecutive hemisegments in the mid body over the yolk sac extension, corresponding to body segments 10–15. Each measurement was arbitrarily taken from the fiber and we avoided taking measurements from fibers close to the segmental boundary or from muscle fibers adjacent to the horizontal myoseptum.For the characterization of the anti-chrn2b antibody, image stacks were acquired to reveal the anti-chrn2b labeling in different transgenic lines or following dual labeling with either zn5 or znp-1 antibodies (). Multiple segments from several fish were imaged to capture motoneuron morphologies at different developmental stages.Representative high-resolution photomicrographs shown in the figures were acquired using volume-rendering software (Imaris 5.7.2, Bitplane Inc., Saint Paul, MN). Images were cropped using Photoshop 7.0 (Adobe, San Jose, CA) and CorelDraw Graphics Suite 12 (Ottawa, Ontario, CA) was used to organize the figures. All of the images are presented with rostral to the left and dorsal to the top. Cartoons were created using CorelDraw by tracing axonal trajectories from the photomicrographs. [...] All data were tested for normality prior to statistical analysis. For normally distributed data, comparison of the means was performed using one-way analysis of variance (ANOVA), followed by a Holm-Sidak post hoc test. For data that failed the normality test, we performed a Kruskal-Wallis analysis of variance on ranks, followed by Dunn's post hoc test. All multiple comparisons using post hoc tests were performed for each nicotine group against the control. All statistical analysis was performed using SigmaPlot 11 (Systat Software Inc., San Jose, USA). For each test, we parenthetically report the degrees of freedom with its corresponding F or H values according to convention. All values are reported as means ± standard error (SE). Significance was assigned at p <0.05 and is reported in the figure legends where appropriate. […]

Pipeline specifications

Software tools Imaris, CorelDraw, SigmaPlot
Application Miscellaneous
Organisms Danio rerio, Solanum tuberosum
Diseases Intestinal Pseudo-Obstruction, Muscular Diseases
Chemicals Nicotine