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).