Russian Journal of PhysiologyRussian Journal of PhysiologyРоссийский физиологический журнал им. И.М. Сеченова0869-81392658-655XThe Russian Academy of Sciences69670310.7868/S2658655X25090096EXPERIMENTAL ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИResearch ArticleRegulation of intracellular calcium during high-frequency rhythmic stimulation of the motor nerve ending of a frogРегуляция внутриклеточного кальция при высокочастотной ритмической стимуляции двигательного нервного окончания лягушкиSamigullinD. V.СамигуллинД. В.samid75@mail.ruFatikhovN. F.ФатиховН. Ф.KhazievE. F.ХазиевЭ. Ф.BukharaevaE. A.БухараеваЭ. А.Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific CenterКазанский институт биохимии и биофизики Казанского научного центра РАНFederal State Budgetary Educational Institution of Higher Education “Kazan National Research Technical University named after A.N. Tupolev – KAI”Федеральное государственное бюджетное образовательное учреждение высшего образования «Казанский национальный исследовательский технический университет им. А.Н. Туполева – КАИ»150920251119VOL 111, NO9 (2025)ТОМ 111, №9 (2025)1558157521112025Copyright ©; 2025, Russian Academy of SciencesCopyright ©; 2025, Российская академия наук2025Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0869-8139/article/view/696703

Under physiological conditions, chemical synapses, including neuromuscular junctions, operate rhythmically at different frequencies depending on the functional type of muscle and the state of synaptic contact. Calcium ions (Ca2+) entering the axoplasm through voltage-gated Ca2+ channels during each action potential activate exocytosis of synaptic vesicles and play a key role in modulating the secretory process. The endoplasmic reticulum (ER), which can release Ca2+ ions via Ca2+-dependent release, may contribute significantly to intracellular Ca2+ dynamics. Optical recording techniques using Ca2+-sensitive fluorescent dyes are used to monitor changes in intracellular Ca2+. However, such an evaluation must pay special attention to the dye's binding characteristics with Ca²⁺ ions, specifically its affinity, because the degree of dye saturation affects the parameters of the Ca²⁺ response being investigated. In this study, the low-affinity dye Magnesium Green was used to analyze changes in the intracellular Ca2+ ions concentration in the neuromuscular synapse m. cutaneus pectoris of the frog during rhythmic stimulation, which allows correct assessment of Ca2+ signals. With increasing frequency of motor nerve stimulation, the smooth increase at 20 Hz of Ca2+ response was replaced by a biphasic increase at 50 Hz and a sharp increase at 70 Hz. This indicates the inclusion of additional Ca2+ sources, which may be the ER. Blocking ryanodine and inositol receptors abolished the increase in Ca2+ response at higher frequencies of nerve stimulation. Blocking Ca2+ ATPases ER (SERCA) resulted in a dramatic increase in the Ca2+ response and eliminated its multiphasic character. It is shown that the change in Ca2+ transient reflects the accumulation of intracellular Ca2+ in the axoplasm and depends on the activity of SERCA, ryanodine and inositol receptors. The use of a low affinity fluorescent Ca2+ dye makes it possible to track the contribution of these systems to the formation of the intracellular concentration of the main ions that determine the process of neurosecretion.

В физиологических условиях химические синапсы, включая нервно-мышечные соединения, работают ритмически с разной частотой в зависимости от функционального типа мышц и состояния синаптического аппарата. Ионы кальция (Ca2+), входящие в аксоплазму через потенциал-зависимые Ca2+-каналы во время каждого потенциала действия, активируют экзоцитоз синаптических везикул и играют ключевую роль в модуляции секреторного процесса. Существенный вклад в динамику внутриклеточного Ca2+ может вносить эндоплазматический ретикулум (ЭР), способный высвобождать ионы Ca2+ посредством Ca2+-зависимого освобождения. Для отслеживания изменений внутриклеточного Ca2+ применяются методы оптической регистрации с использованием Ca2+-чувствительных флуоресцентных красителей. Особое внимание при такой оценке необходимо уделять характеристикам связывания красителя с ионами Ca2+, а именно их аффинности, поскольку степень насыщения красителя влияет на параметры Ca2+-ответа. В данном исследовании для анализа изменений внутриклеточного содержания ионов Ca2+ в нервно-мышечном синапсе кожно-грудинной мышцы лягушки (m. cutaneus pectoris) при ритмической стимуляции был использован низкоаффинный краситель Magnesium Green, который позволяет корректно оценивать Ca2+-сигналы. При увеличении частоты стимуляции двигательного нерва плавное нарастание Ca2+-ответа при частоте стимуляции 20 Гц сменялось двухфазным увеличением при 50 Гц и резким ростом при 70 Гц. Это свидетельствует о включении дополнительных источников Ca2+, каковым может быть ЭР. Блокирование рианодиновых и инозитольных рецепторов устраняло возрастание Ca2+-ответа при повышенных частотах стимуляции нерва. Блокирование кальциевых АТФаз ЭР (SERCA) приводило к резкому увеличению Ca2+-ответа и устраняло его многофазность. Установлено, что изменение Ca2+-транзиента отражает накопление внутриклеточного Ca2+ в аксоплазме и зависит от активности SERCA, рианодиновых и инозитольных рецепторов. Применение никоаффинного флуоресцентного Ca2+-красителя дает возможность отслеживать включение этих систем в формирование внутриклеточного содержания основных ионов, определяющих процесс нейросекреции.

calcium ionsneuromuscular junctionryanodine receptorsinositol receptorscalcium ATPasesионы кальциянервно-мышечное соединениерианодиновые рецепторыинозитольные рецепторыкальциевые АТФазыKatz B, Miledi R (1965) The Effect of Calcium on Acetylcholine Release from Motor Nerve Terminals. Proc R Soc B Biol Sci 161: 496–503. https://doi.org/10.1098/rspb.1965.0017Schweizer FE, Betz H, Augustine GJ (1995) From vesicle docking to endocytosis: Intermediate reactions of exocytosis. Neuron 14: 689–696. https://doi.org/10.1016/0896-6273(95)90213-9Narita K, Akita T, Osanai M, Shirasaki T, Kijima H, Kuba K (1998) A Ca2+-induced Ca2+ Release Mechanism Involved in Asynchronous Exocytosis at Frog Motor Nerve Terminals. J Gen Physiol 112: 593–609. https://doi.org/10.1085/jgp.112.5.593Suzuki S ichi, Osanai M, Murase M, Suzuki N, Ito K, Shirasaki T, Narita K, Ohnuma K, Kuba K, Kijima H (2000) Ca2+ dynamics at the frog motor nerve terminal. Pflugers Arch Eur J Physiol 440: 351–365. https://doi.org/10.1007/S004240000278Narita K, Suzuki N, Himi N, Murayama T, Nakagawa T, Okabe N, Nakamura-Maruyama E, Hayashi N, Sakamoto I, Miyamoto O, Kuba K (2019) Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals. Eur J Neurosci 50: 1700–1711. https://doi.org/10.1111/ejn.14353Galante M, Marty A (2003) Presynaptic Ryanodine-Sensitive Calcium Stores Contribute to Evoked Neurotransmitter Release at the Basket Cell-Purkinje Cell Synapse. J Neurosci 23: 11229–11234. https://doi.org/10.1523/JNEUROSCI.23-35-11229.2003Emptage NJ, Reid CA, Fine A (2001) Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release. Neuron 29: 197–208. https://doi.org/10.1016/S0896-6273(01)00190-8Conti R, Tan YP, Llano I (2004) Action Potential-Evoked and Ryanodine-Sensitive Spontaneous Ca2+ Transients at the Presynaptic Terminal of a Developing CNS Inhibitory Synapse. J Neurosci 24: 6946–6957. https://doi.org/10.1523/JNEUROSCI.1397-04.2004Smith AB, Cunnane TC (1996) Ryanodine-sensitive calcium stores involved in neurotransmitter release from sympathetic nerve terminals of the guinea-pig. J Physiol 497: 657–664. https://doi.org/10.1113/JPHYSIOL.1996.SP021797Verkhratsky A (2005) Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol Rev 85: 201–279. https://doi.org/10.1152/physrev.00004.2004McGraw CF, Somlyo AV, Blaustein MP (1980) Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis. J Cell Biol 85: 228–241. https://doi.org/10.1083/JCB.85.2.228Hartter DE, Burton PR, Laveri LA (1987) Distribution and calcium-sequestering ability of smooth endoplasmic reticulum in olfactory axon terminals of frog brain. Neuroscience 23: 371–386. https://doi.org/10.1016/0306-4522(87)90297-1Bouchard R, Pattarini R, Geiger JD (2003) Presence and functional significance of presynaptic ryanodine receptors. Prog Neurobiol 69: 391–418. https://doi.org/10.1016/S0301-0082(03)00053-4Castejón OJ, Apkarian RP (1992) Conventional and high resolution scanning electron microscopy of outer and inner surface features of cerebellar nerve cells. J Submicrosc Cytol Pathol 24: 549–562.Blaustein MP, McGraw CF, Somlyo AV, Schweitzer ES (1980) How is the cytoplasmic calcium concentration controlled in nerve terminals? J Physiol (Paris) 76: 459–470.Westrum LE, Gray EG (1986) New observations on the substructure of the active zone of brain synapses and motor endplates. Proc R Soc London Ser B Biol Sci 229: 29–38. https://doi.org/10.1098/rspb.1986.0072Balezina OP, Bogacheva PO, Orlova TY (2007) Effect of L-type calcium channel blockers on activity of newly formed synapses in mice. Bull Exp Biol Med 143: 171–174. https://doi.org/10.1007/s10517-007-0041-yБалезина ОП, Букин АН, Лаптева ВИ (2001) Влияние дантролена и рианодина на вызванную активность нервно-мышечных синапсов у мышей. 87: 1511–1517. [Balezina OP, Bukin AN, Lapteva VI (2001) Effects of dantrolene and ryanodine on the evoked activity of neuromuscular synapses in mice. Russ J Physiol 87: 1511–1517. (In Russ)].Zucker RS, Regehr WG (2002) Short-Term Synaptic Plasticity. Annu Rev Physiol 64: 355–405. https://doi.org/10.1146/annurev.physiol.64.092501.114547Narita K, Akita T, Hachisuka J, Huang SM, Ochi K, Kuba K (2000) Functional coupling of Ca2+ channels to ryanodine receptors at presynaptic terminals: Amplification of exocytosis and plasticity. J Gen Physiol 115: 519–532. https://doi.org/10.1085/JGP.115.4.519Verkhratsky A (2004) Endoplasmic reticulum calcium signaling in nerve cells. Biol Res 37: 693–699. https://doi.org/10.4067/s0716-97602004000400027Khuzakhmetova VF, Samigullin DV, Bukharaeva EA (2014) The role of presynaptic ryanodine receptors in regulation of the kinetics of the acetylcholine quantal release in the mouse neuromuscular junction. Biochem Suppl Ser A Membr Cell Biol 8: 144–152. https://doi.org/10.1134/S199074781305005XTazerart S, Vinay L, Brocard F (2008) The persistent sodium current generates pacemaker activities in the central pattern generator for locomotion and regulates the locomotor rhythm. J Neurosci 28: 8577–8589. https://doi.org/10.1523/JNEUROSCI.1437-08.2008,Bullen A, Saggau P (1999) Optical Recording from Individual Neurons in Culture. In: Windhorst U, Johansson H (eds) Modern Techniques in Neuroscience Research. Springer, Berlin, pp 89–126.Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450. https://doi.org/10.1016/S0021-9258(19)83641-4Tsien RY (1989) Fluorescent Indicators of Ion Concentrations. Methods Cell Biol 30: 127–156. https://doi.org/10.1016/S0091-679X(08)60978-4Adams SR (2010) How Calcium Indicators Work. Cold Spring Harb Protoc 2010(3): pdb.top70. https://doi.org/10.1101/pdb.top70Tank DW, Regehr WG, Delaney KR (1995) A quantitative analysis of presynaptic calcium dynamics that contribute to short-term enhancement. J Neurosci 15: 7940–7952. https://doi.org/10.1523/JNEUROSCI.15-12-07940.1995Regehr WG, Atluri PP (1995) Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J 68: 2156–2170. https://doi.org/10.1016/S0006-3495(95)80398-XSamigullin D, Fatikhov N, Khaziev E, Skorinkin A, Nikolsky E, Bukharaeva E (2015) Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes. Front Synaptic Neurosci 6: 29. https://doi.org/10.3389/FNSYN.2014.00029Samigullin DV, Bukharaeva EA (2025) Monitoring presynaptic calcium dynamics with membrane-impermeant fluorescent indicators in motor nerve endings. Biophys Rev. https://doi.org/10.1007/s12551-025-01336-4Vyshedskiy A, Allana T, Lin J-W (2000) Analysis of Presynaptic Ca2+ Influx and Transmitter Release Kinetics during Facilitation at the Inhibitor of the Crayfish Neuromuscular Junction. J Neurosci 20: 6326–6332. https://doi.org/10.1523/JNEUROSCI.20-17-06326.2000Vyshedskiy A, Lin JW (2000) Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution. J Neurophysiol 83: 552–562. https://doi.org/10.1152/jn.2000.83.1.552Borst JGG, Sakmann B (1998) Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem. J Physiol 506: 143–157. https://doi.org/10.1111/j.1469-7793.1998.143bx.xPeng Y, Zucker RS (1993) Release of LHRH is linearly related to the time integral of presynaptic Ca+ elevation above a threshold level in bullfrog sympathetic ganglia. Neuron 10: 465–473. https://doi.org/10.1016/0896-6273(93)90334-NWu LG, Betz WJ (1996) Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction. Neuron 17: 769–779. https://doi.org/10.1016/s0896-6273(00)80208-1Samigullin DV, Khaziev EF, Zhilyakov NV, Bukharaeva EA, Nikolsky EE (2017) Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction. J Vis Exp (125): 55122. https://doi.org/10.3791/55122Fill M, Copello JA (2002) Ryanodine receptor calcium release channels. Physiol Rev 82: 893–922. https://doi.org/10.1152/physrev.00013.2002De Smet P, Parys JB, Callewaert G, Weidema AF, Hill E, De Smedt H, Erneux C, Sorrentino V, Missiaen L (1999) Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca2+ pumps. Cell Calcium 26: 9–13. https://doi.org/10.1054/CECA.1999.0047Castonguay A, Robitaille R (2002) Xestospongin C is a potent inhibitor of SERCA at a vertebrate synapse. Cell Calcium 32: 39–47. https://doi.org/10.1016/s0143-4160(02)00093-3Kubota M, Narita K, Murayama T, Suzuki S, Soga S, Usukura J, Ogawa Y, Kuba K (2005) Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals. Cell Calcium 38: 557–567. https://doi.org/10.1016/j.ceca.2005.07.008Suzuki S, Osanai M, Mitsumoto N, Akita T, Narita K, Kijima H, Kuba K (2002) Ca2+ -Dependent Ca2+ Clearance Via Mitochondrial Uptake and Plasmalemmal Extrusion in Frog Motor Nerve Terminals. J Neurophysiol 87: 1816–1823. https://doi.org/10.1152/jn.00456.2001Soga-Sakakibara S, Kubota M, Suzuki S, Akita T, Narita K, Kuba K (2010) Calcium dependence of the priming, activation and inactivation of ryanodine receptors in frog motor nerve terminals. Eur J Neurosci 32: 948–962. https://doi.org/10.1111/j.1460-9568.2010.07381.xSinha SR, Wu L-G, Saggau P (1997) Presynaptic Calcium Dynamics and Transmitter Release Evoked by Single Action Potentials at Mammalian Central Synapses. Biophys J 72: 637–651. https://doi.org/10.1016/S0006-3495(97)78702-2Sala F, Hernández-Cruz A (1990) Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties. Biophys J 57: 313–324. https://doi.org/10.1016/S0006-3495(90)82533-9Lin J-W, Fu Q, Allana T (2005) Probing the endogenous Ca2+ buffers at the presynaptic terminals of the crayfish neuromuscular junction. J Neurophysiol 94: 377–386. https://doi.org/10.1152/jn.00617.2004Neher E (1998) Usefulness and limitations of linear approximations to the understanding of Ca++ signals. Cell Calcium 24: 345–357. https://doi.org/10.1016/S0143-4160(98)90058-6Narita K, Akita T, Osanai M, Shirasaki T, Kijima H, Kuba K (1998) A Ca2+-induced Ca2+ Release Mechanism Involved in Asynchronous Exocytosis at Frog Motor Nerve Terminals. J Gen Physiol 112: 593–609. https://doi.org/10.1085/JGP.112.5.593Castonguay A, Robitaille R (2001) Differential regulation of transmitter release by presynaptic and glial Ca2+ internal stores at the neuromuscular synapse. J Neurosci 21: 1911–1922. https://doi.org/10.1523/jneurosci.21-06-01911.2001Bennett MR, Farnell L, Gibson WG, Dickens P (2007) Mechanisms of calcium sequestration during facilitation at active zones of an amphibian neuromuscular junction. J Theor Biol 247: 230–241. https://doi.org/10.1016/j.jtbi.2007.03.022John LM, Lechleiter JD, Camacho P (1998) Differential Modulation of SERCA2 Isoforms by Calreticulin. J Cell Biol 142: 963. https://doi.org/10.1083/JCB.142.4.963He XP, Yang F, Xie ZP, Lu B (2000) Intracellular Ca2+ and Ca2+/Calmodulin-Dependent Kinase II Mediate Acute Potentiation of Neurotransmitter Release by Neurotrophin-3. J Cell Biol 149: 783. https://doi.org/10.1083/JCB.149.4.783