Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

The problem of measuring fast calcium waves in excitable cells is one of the most important and challenging aspects of studying signal transmission in the central and peripheral nervous systems. Calcium ions play an important role in triggering neurotransmitter release, synaptic plasticity, and modulation of the activity of various intracellular proteins^1,2,3,4,5. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases⁶. To measure changes in the calcium levels, fluorescent calcium-sensitive dyes are commonly used, and changes in their fluorescence level are analyzed^7,8,9.

Loading of calcium dyes into cells can be achieved in different ways. Predominantly, cell-permeant dyes are used^10,11. However, in such a case, it is not only difficult to control the concentration of a dye inside the cell, but it is also hard to select target cells for loading. This method is not applicable for studying peripheral nerve endings since the dye enters postsynaptic cells. Instead, cell impermeant dyes are more suitable for such preparations. In this case, the dyes are delivered to the cells by microinjection or through a patch pipette^12,13,14. There is also a method of loading through a nerve stump. The latter method is most suitable for neuromuscular junction preparations^{15,16,17,18,19,20}. It allows performing staining for only cells of interest. Although this method does not provide an accurate evaluation of the concentration of the dye in the target cell, the concentration can be estimated approximately by comparing the level of fluorescence of the cells at rest in solutions with a known concentration of calcium²¹. In this study, a modification of this method applied to synapses of mammals is presented.

Calcium entry during the depolarizing phase of the action potential is a fast process, especially in the neuromuscular junction; therefore, for its registration, appropriate equipment is required¹. A recent study using a voltage-sensitive fluorescent dye demonstrated that the duration of the action potential in the peripheral synapse of a mouse is approximately 300 µs²². Calcium transient, evaluated using calcium-sensitive dyes in the peripheral synapses of the frog, has a longer duration: the rise time is about 2-6 ms and the decay time is about 30-90 ms, depending on the calcium dye used^23,24. To measure fast processes with the help of fluorescent dyes, CCD or CMOS cameras are generally used, with fast and sensitive CCD matrices. However, these cameras have the disadvantage of low resolution, limited by the size of the sensitive elements of the matrix^25,26,27,28. The fastest cameras with sufficient sensitivity to record both action potentials and calcium transients in response to low frequency stimulation of cells have a scanning frequency of 2,000 Hz, and a matrix with a dimension of 80 x 80²⁹. To obtain signals with a higher spatial resolution, confocal microscopy is used, especially if it is necessary to assess some volumetric changes in the signal^30,31,32. But it should be kept in mind that confocal microscopy has a high scanning speed in line scan mode, but there are still significant limitations on the speed of recordings of fast processes when building a spatial image³³. There are confocal microscopes based on rotating Nipkow disks (slit-scanning microscopy) and Multipoint-Array Scanners, which have a higher scanning speed. At the same time, they are inferior to the classical confocal microscopes in confocal image filtering (pinholes crosstalk for microscopes with a Nipkow disk)^32,34,35. Confocal imaging with resonance scanning can also provide a high spatio-temporal resolution required for high temporal measurements³⁶. However, take into account that the registration of weak fluorescent responses at a high scanning speed when using resonance scanners requires highly sensitive detectors such as hybrid detectors³⁶.

This article presents a method for increasing the temporal resolution of signals recorded with the Laser Scanning Confocal Microscopy (LSCM) while maintaining the spatial resolution³⁷. The current method is a further development of the methods described earlier and transferred to the LSCM platform^38,39,40. This approach does not require changes in the microscope hardware and is based on the application of an algorithm for recording periodically evoked fluorescent signals with a time shift relative to the moment of stimulation.