Calcium transient recording
Nerve motor endings were loaded with high-affinity calcium-sensitive dye
Oregon Green 488 BAPTA-1 Hexapotassium Salt 1 mM (Molecular Probes,
Eugene, OR, USA) through the nerve stump, as described previously
(Samigullin, Khaziev, Zhilyakov, Sudakov, et al., 2017). The
fluorescence signal was recorded using an imaging setup based on an
Olympus BX-51 microscope with a x40 water-immersion objective (Tokyo,
Japan). Calcium transient registration performed via high-sensitivity
Red Shirt Imaging NeuroCCD-smq camera (RedShirtImaging, Decatur, GA,
USA), 500 fps (exposure time 2 ms) at 80x80 pixels, what was sufficient
for calcium transient registration with good temporal resolution. As the
source of light Polychrome V (Till Photonics, Munich, Germany) was used,
with set light wavelength 488 nm. The following filter set was used to
isolate the fluorescent signal: 505DCXT dichroic mirror, E520LP emission
(Chroma, Bellows Falls, VT, USA). We used Turbo-SM software
(RedShirtImaging, Decatur, GA, USA) for data recording. In each
experiment, 8 fluorescence responses were recorded then averaged. This
was an optimal amount to obtain the data of sufficient quality and to
reduce excitotoxicity and photobleaching of fluorophore.
To analyze recorded images ImageJ software (NIH, Bethesda, MD, USA) was
used. We picked regions of interest in motor nerve ending image and
background manually. Subsequent data processing was performed in Excel
(Microsoft, Redmond, WA, USA). Background values were averaged and
subtracted from signal ones. Data were represented as a ratio: (ΔF/F0 −
1) × 100 %, where ΔF is the fluorescence intensity during stimulation,
F0 is the fluorescence intensity at rest (Samigullin, Khaziev,
Zhilyakov, Bukharaeva, et al., 2017).