Abstract <p>Under physiological conditions, chemical synapses, includingneuromuscular junctions, operate rhythmically at different frequenciesdepending on the functional type of muscle and the state of the synapticcontact. During each action potential, calcium ions (Ca²⁺)enter the axoplasm through voltage-gated Ca²⁺ channels,activating exocytosis of synaptic vesicles and playing a key rolein modulating the secretory process. The endoplasmic reticulum (ER),which can release Ca²⁺ ions through itsCa²⁺-gated release channels, may significantlycontribute to intracellular Ca²⁺ dynamics.Optical recording techniques using Ca²⁺-sensitivefluorescent dyes are used to monitor changes in intracellular Ca²⁺. Specialattention during such assessment must be paid to the dye’s bindingcharacteristics with Ca²⁺ ions, specificallyits affinity, because the degree of dye saturation affects the parametersof the Ca²⁺ response. In this study,Magnesium Green, a low-affinity dye, was used to analyze changesin the intracellular concentration of Ca²⁺ ionsin the neuromuscular synapse of the frog's m. cutaneus pectoris duringrhythmic stimulation because it allows correct assessment of Ca²⁺ signals.With increasing frequency of motor nerve stimulation, the smoothincrease in the Ca²⁺ response at 20 Hzwas replaced by a biphasic increase at 50 Hz and a sharp increaseat 70 Hz. This indicates the involvement of additional Ca²⁺ sources,such as the ER. Blocking ryanodine and inositol receptors abolishedthe increase in the Ca²⁺ response athigher stimulation frequencies. Blocking endoplasmic reticulum Ca²⁺ ATPases(SERCA) resulted in a dramatic increase in the Ca²⁺ response,eliminating its multiphasic character. It was established that thechange in the Ca²⁺ transient reflectsthe accumulation of intracellular Ca²⁺ inthe axoplasm and depends on the activity of SERCA, ryanodine, andinositol receptors. Using a low-affinity fluorescent Ca²⁺ dyemakes it possible to track these systems’ contribution to formingthe intracellular concentration of key ions that determine neurosecretion.</p>