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. 2018 Dec 15;17(1):A40-A49.
eCollection 2018 Fall.

Teaching Dose-response Relationships Through Aminoglycoside Block of Mechanotransduction Channels in Lateral Line Hair Cells of Larval Zebrafish

Hannah Payette Peterson  1 , Eileen L Troconis  1 , Alexander J Ordoobadi  1 , Stacey Thibodeau-Beganny  1 , Josef G Trapani  1
Affiliations

Teaching Dose-response Relationships Through Aminoglycoside Block of Mechanotransduction Channels in Lateral Line Hair Cells of Larval Zebrafish

Hannah Payette Peterson et al. J Undergrad Neurosci Educ. .

Abstract

Here we introduce a novel set of laboratory exercises for teaching about hair cell structure and function and dose-response relationships via fluorescence microscopy. Through fluorescent labeling of lateral line hair cells, students assay aminoglycoside block of mechanoelectrical transduction (MET) channels in larval zebrafish. Students acquire and quantify images of hair cells fluorescently labeled with FM 1-43, which enters the hair cell through MET channels. Blocking FM 1-43 uptake with different concentrations of dihydrostreptomycin (DHS) results in dose-dependent reduction in hair-cell fluorescence. This method allows students to generate dose-response curves for the percent fluorescence reduction at different concentrations of DHS, which are then visualized to examine the blocking behavior of DHS using the Hill equation. Finally, students present their findings in lab reports structured as scientific papers. Together these laboratory exercises give students the opportunity to learn about hair cell mechanotransduction, pharmacological block of ion channels, and dose-dependent relationships including the Hill equation, while also exposing students to the zebrafish model organism, fluorescent labeling and microscopy, acquisition and analysis of images, and the presentation of experimental findings. These simple yet comprehensive techniques are appropriate for an undergraduate biology or neuroscience classroom laboratory.

Keywords: DHS; FM 1-43; MET channel; dihydrostreptomycin; dose-response curves; drug competition; fluorescence microscopy; hair cells; lateral line; mechanoelectrical transduction channel; zebrafish.

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Figures

Figure 1
Figure 1
MET channels in lateral line hair cells of zebrafish. (A) Two adjacent stereocilia connected by a tip link (dashed line) at the apical end of a hair cell. Deflection of stereocilia opens MET channel (orange) located at their tips, which results in the influx of K+ and other cations. (B) Cation influx through MET channels causes depolarization of the membrane potential resulting in activation of voltage-gated calcium channels, fusion of synaptic vesicles (purple circles), and release of glutamate neurotransmitter onto afferent neurons (brown lines) at the basal end of the hair cell. (C) A collection of hair cells forms a neuromast, which together (D) makeup the lateral line system of zebrafish (purple asterisk = neuromast).
Figure 2
Figure 2
FM 1-43 entry and DHS block of hair cells. (A) FM 1-43 (green rectangle) enters hair cells via open MET channels (orange) and fluorescently labels the hair cell membrane. (B) DHS (blue circle) permeates slowly through MET channels, blocking ion flow and preventing FM 1-43 entry into hair cells.
Figure 3
Figure 3
Experimental method for labeling larvae with FM 1-43. (A) Pre-labeling dishes for control and experimental larvae. DHS (blue drop) is added at the test concentration to all experimental dishes. (B) For control larva, the labeling dish contains FM 1-43 (green drop) and the experimental dish contains both FM 1-43 and DHS. (C) Rinse dishes for experimental larvae contain DHS. Multiple rinses (2 or 3x) with several different dishes is recommended for both control and experimental larvae. (D, E) The larva is moved to an anesthesia dish and then an agarose conical for immobilization of larvae prior to mounting on a microscope slide for fluorescence microscopy.
Figure 4
Figure 4
Figure of fluorescent neuromasts taken from a student lab report. (A) FM1-43-labeled neuromast from a control larva at 40× magnification. The circular region of interest (ROI) was drawn in FIJI. (B) Fluorescent image of a neuromast from an experimental larva (0.3 mM DHS). (C) Image showing fluorescently labeled neuromasts surrounding the head of a zebrafish larva at 10× magnification (overlay of DIC light and GFP fluorescent images). Figure by Alex Ordoobadi, Amherst College class of 2015.
Figure 5
Figure 5
Two different plots created by students using data collected in the laboratory. Both graphs plot the percent reduction in fluorescence versus the concentration of DHS (note X-axis on a log scale). (A) Shown are two hypothetical Hill equations with slopes of n = 1 (red) and n = 2 (green) and the student’s data connected by a line (blue). Plot by Kyra Shapiro, Amherst College class of 2015. (B) A plot where the student did not normalize the percent-reduction to 100% block. The Hill equation with slope of 1 did not fit to the data as well, but still allowed the student to visualize the IC50 value, a concentration that would produce a half-maximal decrease in fluorescence. Plot by Alex Ordoobadi, Amherst College class of 2015.
Figure 6
Figure 6
The results of the evaluation filled out by students from the fall 2016 laboratory. There were 16 out of 20 student responses to the survey. Students rated the effectiveness of the teaching lab from 1 to 5 (one was not at all effective and 5 was very effective) in achieving each of the learning objectives, as outlined in the Methods section.
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