Computational protocol: TRPM8 and Nav1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

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Protocol publication

[…] Between 12 and 24 hr after plating, sensory neurons, grown on coverslips, were loaded with Fluo-4 AM calcium indicator (1 μM, Invitrogen, CA, USA) in Hanks balanced salt solution (HBSS) without phenol red (Sigma-Aldrich, MO, USA) for 7 min, rinsed with fresh HBSS and incubated for a further 15 min at 37°C to allow complete de-esterification. All imaging experiments were carried out at 24-26°C. Calcium-free experiments were performed in calcium-free HBSS (Sigma-Aldrich, MO, USA) supplemented with 300 μM EGTA (glycol-bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid). Growth cones from small neurons (<15 μm in diameter) were selected for analysis. Freshly prepared TTR proteins were added to culture dishes to a final concentration of 0.5 mg/ml for all experiments and remained in contact with growth cones for the duration of the imaging period. Calcium experiments on aged (36 hr) protein were only performed using the V30M variant. Fluorescence images were acquired at 1-5 Hz using a confocal microscope (Zeiss LSM 510, Jena, Germany) using a 20× (1.0 NA) water immersion objective. Fluorescence images and pixel intensities were analysed using ImageJ software [] and custom software (Matlab, MathWorks, MA, USA). ΔF for each image frame was calculated by subtracting integrated pixel intensity of a region of interest encompassing the entire growth cone from the average integrated pixel intensity or F0, of the first 20-30 frames of the imaging session for each cell. Maximal ΔF/F0 was calculated for each growth cone over the entire 7 min imaging period. Prism 4 (GraphPad Software, CA, USA) and Adobe Illustrator CS3 (Adobe Systems, CA, USA) were used for graphical and statistical analysis of data. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Alzheimer Disease, Peripheral Nervous System Diseases, Polyneuropathies, Drug-Related Side Effects and Adverse Reactions
Chemicals Calcium, Sodium