Computational protocol: Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

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Protocol publication

[…] The potentiometric fluorescent indicator dye di-8-amino-naphthylethenylpyridinium (Di-8-ANEPPS) was used to measure muscle fiber APs. The lipophilic dye accumulates in the sarcolemma and the T-tubular system. The loading protocol was adapted from . Fibers were subjected to 10 µM Di-8-ANEPPS in culture medium for 30 min at 37°C. APs were measured immediately after washing with the experimental Ringer’s solution. For fluorescence measurements, we used an excitation filter (BP 470/20), a dichroic beam splitter (FT 493), and an emission filter (BP 505–530; all from Carl Zeiss). The signal from the PM was analogue filtered at 50 kHz and sampled at 100 kHz. A short screening protocol with four sequential pulses of different voltage (+6, −7, +8, and −9 V) was used to check cells in the dish for the presence of APs. The AP threshold was then determined with pairs of rectangular pulses of equal amplitude but opposite polarity spaced 50 ms apart. The amplitude was gradually increased from 1 to 10 V, with a 1-V increment per sweep. The AP trigger voltage was then individually set to a supra-maximal value (1 V above threshold). Pulses were applied at 1 Hz. For analysis, 20 consecutive all-or-none APs were averaged offline and smoothed by cubic spline interpolation. Averaged and smoothed signals were baseline corrected and normalized to the peak of the signal. AP recordings were used for kinetic analysis. A comparison of absolute amplitudes could not be performed because the basal background fluorescence showed considerable fiber-to-fiber variation (probably mainly resulting from dye in the Matrigel coating used for plating the cells). […]

Pipeline specifications

Software tools MUSCLE, EMBOSS
Application Nucleotide sequence alignment
Organisms Mus musculus
Diseases Huntington Disease
Chemicals Ryanodine