Ca2+ influx through muscle-type nicotinic AChRs contributes to contractions and development of slow muscle cells in early developmental stages

Ca2+ influx through muscle-type nicotinic AChRs contributes to contractions and development of slow muscle cells in early developmental stages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ca 2+ influx through muscle-type nicotinic AChRs contributes to contractions and development of slow muscle cells in early developmental stages Buntaro Zempo, Fumihito Ono, Koichi Nakajo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6525282/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca 2+ permeability compared to that of AChRs of fast muscles. To analyze the significance of the Ca 2+ influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca 2+ -impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1-3 days post fertilization (dpf) compared to those of zebrafish expressing Ca 2+ -permeable AChRs in its slow muscles. Ca 2+ imaging suggested that Ca 2+ influx via AChRs is crucial for the Ca 2+ response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca 2+ response. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca 2+ -permeable AChRs. These short Ca 2+ responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca 2+ influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles. Biological sciences/Physiology/Neurophysiology Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Muscle contraction mechanisms have long been studied and are well understood 1 – 3 . Neuromuscular junctions (NMJs) play crucial roles in the process of muscle contraction. At the NMJs, motor neurons release acetylcholine (ACh) and stimulate nicotinic acetylcholine receptors (AChRs). AChRs are ligand-gated cation-permeable ion channels. Binding of ACh induces cation influx through AChRs, depolarizes the membrane potential, activates the voltage-gated Na + channels (Nav) in the sarcolemma, and generates an action potential. Depolarization of the sarcolemma opens the L-type Ca 2+ channels that are in physical contact with ryanodine receptors (RyRs). As the L-type Ca 2+ channels open, RyRs also open and release Ca 2+ from the sarcoplasmic reticulum (SR), which induces muscle contractions. In this process, AChRs are essential molecular components that play a key role in the initial step, receiving signals from motor neurons. Muscle-type AChRs are pentamers composed of two α1s, β1, δ, and ε (or γ in early development) subunits 4 . AChRs in NMJs tend to be highly permeable to Na + , whereas they show relatively low permeability for Ca 2+ 5,6 . However, recent studies have revealed a different type of AChR composed of only α1, β1, and δ subunits in zebrafish 7 – 9 . It was also found in those studies that slow muscles and fast muscles specifically express αβδ-type and αβδγ/ε-type AChRs, respectively. This finding begs the next question: why do slow muscles express AChRs that are different from those of fast muscles? To address this question, we focused on the difference between channel properties of slow and fast muscle-type AChRs. Although αβδ-type AChRs have been reported only in zebrafish, the larval tunicate Ciona has been shown to possess AChRs composed of only three types of subunits: α1, B2/4, and BGDE3 10 . The slow muscle-type AChRs of zebrafish show characteristics similar to those of the AChRs of Ciona : both AChRs exhibit inward rectification 7 , 10 . This property is unique and not found in muscle-type AChRs described to date, including fast muscle-type AChRs of zebrafish. On the other hand, neuronal AChRs show inward rectification and high Ca 2+ permeability 11 , 12 , which are bestowed by negatively charged amino acid residues of the “intermediate ring” (part of the ion permeation pathway of AChRs). Nishino et al. showed that muscle-type AChRs of Ciona display inward rectification and high Ca 2+ permeability. Moreover, the intermediate rings of AChRs of Ciona are composed of glutamate (E), a negatively charged amino acid 10 . The authors further showed that Ciona AChRs lose the rectification and Ca 2+ permeability by mutating E of the intermediate ring to glutamine (Q) (uncharged amino acid). In zebrafish, fast muscle-type AChRs are composed of α1, β1, δ, ε, or γ subunits, among which ε and γ subunits possess Q at the intermediate ring. In contrast, intermediate rings in slow muscle-type AChRs are composed of only E, because γ and ε subunits are absent. Based on these facts, we hypothesized that slow muscle-type AChRs in zebrafish are highly permeable to Ca 2+ and that the Ca 2+ influx through AChRs may contribute to the functions of slow muscles. The results of the present study suggested that the slow muscle-type AChRs of zebrafish have actually high Ca 2+ permeability. We further analyzed the physiological significance of the Ca 2+ permeability. Materials and methods Maintenance of fish lines Zebrafish were kept in water at 28°C with 14 h of light (8:00–22:00) and 10 h of darkness (22:00–8:00). Animal experiments using zebrafish were approved by the Animal Experiment Committee of Jichi Medical University (Approval No. 23013-01, 23013-02). Electrophysiology cDNAs for the muscle ACh subunits α1, β1, δ, and ε were previously cloned from zebrafish 7 . The cDNA of each subunit was subcloned into the pTNT vector (Promega, WI, USA) for in vitro transcription. The purified plasmid was linearized at the BamHI site and in vitro transcribed with T7 RNA polymerase (mMESSAGE mMACHINE T7 Transcription kit; Thermo Fisher Scientific, MA, USA). Oocytes were isolated and defolliculated by treatment with 2 mg/ml collagenase (Sigma-Aldrich, C0130, MO, USA) for 4–6 hours in MBSH solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3 , 10 mM HEPES, 0.3 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , pH 7.6). For expression of the α1β1δε receptor, the cRNA solution of each subunit was mixed to final concentrations of 0.2, 0.1, 0.1, and 0.1 ng/nl, and 50 nl of the solution was injected per oocyte. For expression of αβδ, cRNAs were mixed at 0.2, 0.1, and 0.2 ng/nl. Injected oocytes were incubated at 17ºC for 2–3 days in MBSH solution supplemented with 0.1% penicillin-streptomycin (Sigma-Aldrich). Electrophysiology was performed with a two-electrode voltage clamp using OC-725C (Warner Instruments, CT, USA). Generation of voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface (Molecular Devices, CA, USA) and Clampex 10.7 software (Molecular Devices). All experiments were performed at room temperature. A glass electrode with a resistance of 0.2–0.5 MΩ was prepared from a borosilicate glass capillary (GC150TF-10, MA, United States) using a micropipette puller (P-1000, Sutter Instrument, CA, USA). The glass electrode was filled with 3 M KCl. Low Ca 2+ solution or high Ca 2+ solution was used as the bath solution. Low Ca 2+ solution consisted of (in mM) 5 HEPES, 10 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , and 86 NMDG, pH 7.5, and high Ca 2+ solution consisted of 5 HEPES, 10 NaCl, 2 KCl, 10 CaCl 2 , and 1 MgCl 2 , 69.6 NMDG, pH 7.5. An ionic current was induced by puff applying 20 µM acetylcholine diluted in bath solution to the oocytes. The I-V relationship was examined by applying a ramp pulse. Generation of a transgenic line We designed a gene construct that expresses a mutated δ subunit and enhanced green fluorescent protein (EGFP) under the regulation of a slow muscle-specific promoter, psmyhc1 13 . The coding sequence of the mutated δ subunit, P2A, and EGFP were cloned into the Tol2 plasmid 14 . The plasmid was injected along with transposase mRNA into one-cell stage embryos. The established transgenic (Tg) line was crossed with the sofa potato (sop) mutant zebrafish line, and an smyhc1 : mutated δ-P2A-EGFP; sop-/- line ( pure slow Ca 2+ -imperm ) was generated. Immunohistochemistry After generation of the Tg line, pure slow Ca 2+ -imperm , we confirmed the specificity of EGFP expression in slow muscle cells by labeling slow muscle fibers and EGFP. First, 3 days post fertilization larvae of the Tg line were deeply anesthetized with 0.03% MS-222 (Sigma-Aldrich). Then the trunk regions of the fish were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The fixed samples were frontally cryosectioned at 20 µm with a cryostat (CM 3050S; Leica Microsystems, Wetzlar, Germany) and mounted onto a micro slide glass (CREST; Matsunami, Osaka, Japan). Sections were incubated overnight with a mixture of anti-EGFP antibody raised in rabbits (Thermo Fisher Scientific, MA, USA) that was diluted 1:500 with phosphate-buffered saline containing 0.3% Tween 20 (PBST) and F59 antibody raised in mice (DSHB, Iowa, USA) that was diluted 1:500 with PBST for 2 hours and then rinsed twice with PBST and incubated for 2 hours with a mixture of CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech, Illinois, USA) diluted 1:500 with PBST, anti-mouse IgG H&L (Alexa Fluor 555; Abcam, Cambridge, UK) diluted 1:500 with PBST, and DAPI (Nacalai, Kyoto, Japan) diluted 1:2000 with PBST. The sections were then washed with PBST and coverslipped with CC/Mount (Diagnostic BioSystems, Pleasanton, CA). Fluorescence was observed with a confocal microscope (Dragonfly; OXFORD INSTRUMENTS, Oxford, UK). For the labeling of slow muscle fibers in whole-mount samples, 2 dpf or 5 dpf larval zebrafish were fixed in 4% PFA at 4℃ for 2 hours and then washed several times in PBST. The samples were then incubated in PBS containing 1 mg/ml collagenase (Sigma-Aldrich) for 60–90 min at room temperature and permeabilized with acetone for 10 min at -20℃. After washing with PBST, the samples were incubated with F59 antibody diluted 1:20 with PBST overnight at room temperature. Embryos were rinsed in PBST and incubated with anti-mouse IgG H&L (Alexa Fluor 555; Abcam) diluted 1:500 in PBST for 2 hours at room temperature. Fluorescence was observed and pictures were taken with a camera (Digital Sight 10; Nikon, Tokyo, Japan) on an MVX10 microscope (OLYMPUS) and a confocal microscope (Dragonfly; OXFORD). The data were analyzed with ImageJ. Locomotor analysis High-speed image capturing of larval zebrafish was performed with a Photron camera (INFINICAM; Photron, Tokyo, Japan) at 1000 frames/s. Captured images were saved as JPEG files and processed with ImageJ and software for motion analysis (Mova-tr/2 and Wriggle Tracker; Library, Tokyo, Japan). For each of the larvae (2–5 dpf), the head was touched gently with an eyelash 15 to induce escape behavior. For 1 dpf larvae, the head and yolk were embedded in an agarose gel, and spontaneous activities were recorded. The tail bend angle, swimming speed, tail beat speed, and maximum tail bend angle were calculated using Mova-tr/2 and Wriggle Tracker. Kinetics of tail bend angles were drawn by plotting degrees of tail bend angles against time. The frame at 0 ms was set immediately preceding the detection of the first motion. Ca 2+ imaging We designed a gene construct that expresses a red fluorescent Ca 2+ sensor protein, jRGECO1a 16 (obtained from Addgene #61563), under the regulation of a skeletal muscle-specific promoter, pactc1b 17 . The promoter activity of pactc1b is stronger than that of a slow muscle-specific promoter ( psmyhc1 ). Thus, to make observation easier, we chose the pactc1b promoter instead of the slow muscle-specific promoter. The coding sequence of jRGECO1a and the sequence of pactc1b were cloned into the Tol2 plasmid. We injected the constructed plasmid into one cell–stage embryos from AChR γ subunit −/− ε subunit +/− pairs and pure slow Ca 2+ -imperm (sop +/− ) pairs. The genotype of the injected embryos was determined by sequence analyses after Ca 2+ imaging. jRGECO1a-expressing fish at 2 or 5 dpf were embedded in the lateral position in 2% low-melting agarose in a 35 mm dish. The heads were touched gently with glass pipettes to induce swimming. Ca 2+ responses were recorded with a Zyla 4.2 sCMOS camera (OXFORD) on an MVX10 microscope (OLYMPUS, Tokyo, Japan) at 200 Hz for 3 sec. The data were analyzed with ImageJ. ΔF/F was calculated for each frame using the formula (F-F 0 )/F 0 , where F represents the fluorescence in that frame and F 0 is the resting intensity before the Ca 2+ rise. The experiments were performed for slow muscle cells of the pure slow Ca 2+ -imperm and those of the γ/ε subunits double KO line ( pure slow Ca 2+ -perm ). For in vitro Ca 2+ imaging, fish at 2 or 5 dpf were deeply anesthetized with 0.03% MS-222 (Sigma-Aldrich). After dissecting the trunk region into several pieces with surgical knives, tissue samples were incubated in 10 mg/ml collagenase solution in a bath solution (112 mM NaCl, 2 mM CaCl 2 , 3 mM Glu, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, pH 7.4) at room temperature for 30 minutes. MgCl 2 was used to replace CaCl 2 in Ca 2+ -free solution. After washing twice with the bath solution, the samples were incubated in Rhod-4 solution (Abcam, Cat: ab112157) following the manufacturer’s instructions at room temperature for 30 min. Then the samples were washed with the bath solution and placed in a recording chamber filled with the bath solution. Ca 2+ responses were induced by puff application of 30 µM ACh using a glass pipette. Ca 2+ responses were recorded with a Zyla 4.2 sCMOS camera (OXFORD) on an MVX10 microscope (OLYMPUS) at 200 Hz for 14 sec. The data were analyzed with ImageJ. ΔF/F was calculated for each frame using the formula (F-F 0 )/F 0 , where F represents the fluorescence in that frame and F 0 is the resting intensity before the Ca 2+ rise. The experiments were performed for slow muscle cells of the pure slow Ca 2+ -imperm and those of the pure slow Ca 2+ -perm . Statistical analysis Unpaired Student’s t -test (two-tailed) was performed for statistical analysis. Averages and SEM are displayed in bar graphs. Results AChRs of slow muscles show high Ca 2+ permeability. First, we compared the sequence alignment of subunits that compose zebrafish muscle-type AChRs (Fig. 1 A). The γ and ε subunits possess a glutamine (Q) residue in the intermediate ring. Fast muscle-type AChRs comprising α1, β1, δ, ε, or γ subunits (Fig. 1 B) therefore contain one Q at the intermediate ring. On the other hand, slow muscle-type AChRs lack γ/ε-subunits (Fig. 1 B), and their intermediate rings are therefore composed only of E, as in the case of AChRs of Ciona , which show high Ca 2+ permeability 10 . Based on these observations, we analyzed the Ca 2+ permeability of fast muscle-type and slow muscle-type AChRs of zebrafish by electrophysiology. We recorded ACh-induced ionic currents mediated by AChRs expressed in Xenopus laevis oocytes by a two-electrode voltage clamp and measured reversal potential with different Ca 2+ concentrations (1.8 and 10 mM Ca 2+ ). The current-voltage (I-V) relationships of the AChRs revealed that slow muscle-type AChRs showed inward rectification, whereas fast muscle-type AChRs showed no clear rectification (Fig. 1 C). While the reversal potential of fast muscle-type AChRs was unaffected by external Ca 2+ level (-34.1 ± 24 mV in 1.8 mΜ Ca 2+ , -32.7 ± 2.0 mV in 10 mM Ca 2+ ; P = 0.67), the reversal potential significantly changed in slow muscle-type AChRs (Fig. 1 D) (-39.3 ± 0.48 mV in 1.8 mΜ Ca 2+ , -25.2 ± 2.5 mV in 10 mM Ca 2+ ; P = 0.003). To confirm the importance of amino acid residues in the intermediate ring for Ca 2+ permeability, we mutated E of the intermediate ring in the δ subunit to Q (Fig. 1 E). We recorded ACh-induced ionic currents mediated by AChRs that are composed of α1, β1, and mutated δ subunits. Mutated slow muscle-type AChRs showed a linear I-V relationship (Fig. 1 F), and its reversal potential was not affected by the external Ca 2+ concentrations (Fig. 1 G) (-40.8 ± 3.0 mV in 1.8 mΜ Ca 2+ , -38.8 ± 2.9 mV in 10 mM Ca 2+ ; P = 0.65). Generation of a transgenic zebrafish line that expresses AChRs with low Ca 2+ permeability in its slow muscle. To clarify the physiological roles of Ca 2+ influx through slow muscle-type AChRs, we generated a transgenic (Tg) zebrafish line that expresses slow muscle-type AChRs with low Ca 2+ permeability in its slow muscle cells. The mutated δ subunit (Fig. 1 E), P2A, and EGFP sequences were fused to the promoter region of the slow myosin heavy chain 1 ( smyhc1 ) gene (Fig. 2 A). In the Tg line, EGFP signals were observed in trunk regions (Fig. 2 B). We first examined the specificity of EGFP signals by double labeling. EGFP and slow muscle fibers were visualized by anti-EGFP antibody and anti-skeletal muscle myosin antibody (F59), respectively (Fig. 2 C). F59 antibody is widely used for detecting slow muscle cells in zebrafish. The obtained image confirmed the specificity of EGFP expression in slow muscle fibers, suggesting that slow muscle cells specifically express mutated δ subunits. Moreover, to remove the effects of endogenous δ subunits and fast muscle-type AChRs, we crossed the Tg line and the sofa potato ( sop ) mutant line. sop harbors a point mutation in its δ subunit gene 18 , and previous studies showed that the sop mutant completely lacks both slow muscle-type and fast muscle-type AChRs 19 . By crossing the newly established Tg and sop , we generated a zebrafish line that expresses mutated slow muscle-type AChRs with low Ca 2+ permeability in its slow muscles, while fast muscle cells lack AChRs (Fig. 2 D). In this article, we call this line harboring an E-Q mutated δ subunit in the sop background “ pure slow Ca 2+ -imperm” . To clarify the physiological importance of Ca 2+ permeability of slow muscle-type AChRs, we compared locomotor activities of pure slow Ca 2+ -imperm with those of another mutant, γ/ε subunits double KO (γεDKO) 8 . The latter line expresses AChRs only in slow muscles. Of note, the γεDKO line expresses intact slow muscle-type AChRs that allow permeation of Ca 2+ . In this article, we call γεDKO “ pure slow Ca 2+ -perm” . Comparison of pure slow Ca 2+ -perm and pure slow Ca 2+ -imperm was therefore expected to reveal the physiological significance of Ca 2+ influx through slow muscle-type AChRs (Fig. 2 D). Ca 2+ influx through AChRs plays an important role in the locomotor activities of slow muscles at early developmental stages. Using a high-speed camera, we analyzed locomotor activities of the pure slow Ca 2+ -perm (γεDKO) line and the pure slow Ca 2+ -imperm line at 1, 2, 3, and 5 days post fertilization (dpf). At the 1 dpf stage, spontaneous tail movement (coiling) was recorded (Fig. 3 A), and we analyzed tail bend angles, measured as the angle of the trunk deviation from the longitudinal axis of the head. Kinematics for representative traces showed that tail bend angles of pure slow Ca 2+ -imperm were smaller than those of pure slow Ca 2+ -perm (Fig. 3 A). Maximum tail bend angles in pure slow Ca 2+ -imperm were significantly smaller than those in pure slow Ca 2+ -perm (142.5 ± 15.0° in Ca 2+ -perm , 16.8 ± 10.8° in Ca 2+ -imperm ; P < 0.001). At the 2 dpf stage, we induced an escape response by tactile stimuli and recorded the locomotor activity with a high-speed camera, analyzing tail bend angles (Fig. 3 B). The pure slow Ca 2+ -perm fish showed weak but significant swimming behavior despite the lack of fast muscle activity (Fig. 3 B). On the other hand, as shown in video 1, the pure slow Ca 2+ -imperm line wagged its tail only slightly. Kinematics for representative traces suggested that the amplitude of tail bend angle is much smaller in pure slow Ca 2+ -imperm than in pure slow Ca 2+ -perm (see also video 1 and video 2). Calculated average tail bend angles of pure slow Ca 2+ -imperm were significantly smaller than those of Ca 2+ -perm (53.1 ± 3.9° in Ca 2+ -perm , 2.0 ± 0.1° in Ca 2+ -imperm ; P < 0.001). We also analyzed escape behavior at the 5 dpf stage (Fig. 3 C). At this stage, unexpectedly, tail beat kinematics of pure slow Ca 2+ -imperm larvae appeared to be almost identical to those of pure slow Ca 2+ -perm , displaying no significant difference in average tail bend angles (43.6 ± 1.2° in Ca 2+ -perm , 43.1 ± 1.1° in Ca 2+ -imperm ; P = 0.73, see also video 3 and video 4). We also calculated swimming speed, tail beat speed, and maximum tail beat angle in 2–5 dpf larvae (Fig. 3 D-F). As a result, tail beat speed and amplitude were decreased in 1–3 dpf pure slow Ca 2+ -imperm , resulting in slower swimming. However, at the 5 dpf stage, there was no difference in locomotor functions between pure slow Ca 2+ -perm and Ca 2+ -imperm . These results suggest that the Ca 2+ permeability of AChRs is important for the contraction process of slow muscle cells at early developmental stages. Ca 2+ imaging suggests that Ca 2+ influx through AChRs contributes to a sustained Ca 2+ response of slow muscle cells in early developmental stages. To analyze the physiological roles of Ca 2+ influx through AChRs, we examined the Ca 2+ response to tactile stimulation in vivo with a fluorescent Ca 2+ sensor protein (jRGECO1a) (Fig. 4 A). We injected a construct containing a slow muscle-specific promotor sequence and jRGECO1a into one cell–stage embryos from pure slow Ca 2+ -perm pairs or pure slow Ca 2+ -imperm pairs and carried out Ca 2+ imaging. At the 2 dpf stage, fluorescence traces showed that the Ca 2+ response of slow muscle cells in pure slow Ca 2+ -imperm decreased faster than that of slow muscle cells of pure slow Ca 2+ -perm (Fig. 4 B). We calculated the peak amplitude of the Ca 2+ response (ΔF/F 0 ) and the decay time to 50% of the peak amplitude (Fig. 4 C). There was no significant difference in peak amplitude between Ca 2+ -perm and imperm (0.318 ± 0.068 in Ca 2+ -perm , 0.47 ± 0.072 in Ca 2+ -imperm ; P = 0.15). However, decay time was shorter in pure slow Ca 2+ -imperm (0.50 ± 0.044 sec in Ca 2+ -perm , 0.36 ± 0.036 sec in Ca 2+ -imperm ; P < 0.05, P = 0.019). We also analyzed 5 dpf larvae (Fig. 4 D-E). In the 5dpf stage, peak amplitude of Ca 2+ -imperm was not significantly different from that of Ca 2+ -perm (Fig. 4 E; 0.30 ± 0.13 in Ca 2+ -perm , 0.40 ± 0.046 in Ca 2+ -imperm ; P = 0.13). In addition, there was no difference in decay time between Ca 2+ -perm and imperm (Fig. 4 E; 0.21 ± 0.028 sec in Ca 2+ -perm , 0.29 ± 0.040 sec in Ca 2+ -imperm ; P = 0.15). To confirm the importance of Ca 2+ influx through AChRs for the activities of slow muscles, the Ca 2+ response of slow muscle cells was assessed under a Ca 2+ -free condition. We analyzed Ca 2+ response of dissociated slow muscle cells of pure slow Ca 2+ -perm and Ca 2+ -imperm fish to ACh in Ca 2+ -free bath solution and Ca 2+ -containing (normal) bath solution with a fluorescent Ca 2+ indicator (Rhod-4) (Fig. 5 A). Unlike the in vivo Ca 2+ imaging, in this experiment, we evaluated the kinetics of Ca 2+ response by calculating fluorescent decay rate (ΔF/F 0 of 3 sec after the peak divided by the peak ΔF/F 0 ) because ΔF/F 0 did not reach 50% of the peak amplitude, which is arguably due to the absence of acetylcholine esterase in the bath solution. As a result, the peak amplitude of the Ca 2+ response of cells from pure slow Ca 2+ -perm in Ca 2+ -free bath solution was not significantly different from that in normal bath solution at the 2 dpf stage (Fig. 5 B; 0.52 ± 0.11 in normal bath solution, 0.37 ± 0.05 in Ca 2+ -free bath solution; P = 0.22). On the other hand, fluorescence decay rate significantly decreased in the Ca 2+ -free condition (Fig. 5 B; 87.7 ± 2.4% in pure slow Ca 2+ -perm in normal bath solution, 50.7 ± 12.3% in Ca 2+ -free bath solution; P < 0.01, P = 0.009). In slow muscle cells of pure slow Ca 2+ -imperm , both peak amplitude (Fig. 5 C; 0.78 ± 0.18 in normal bath solution, 0.48 ± 0.08 in Ca 2+ -free bath solution; P = 0.13) and decay rate (Fig. 5 C; 72.4 ± 3.3% in normal bath solution, 59.4 ± 7.2% in Ca 2+ -free bath solution; P = 0.54) did not change with the external Ca 2+ . In addition, Ca 2+ response of slow muscle cells in pure slow Ca 2+ -perm was unaffected by external Ca 2+ level at the 5 dpf stage (Fig. 5 D; peak amplitude = 0.54 ± 0.08 in normal bath solution, 0.74 ± 0.09 in Ca 2+ -free bath solution; P = 0.11, decay rate = 63.3 ± 5.7% in normal bath solution, 67.7 ± 6.5% in Ca 2+ -free bath solution; P = 0.62). Therefore, slow muscle cells require Ca 2+ influx through AChRs for a sustained Ca 2+ response at the early developmental stage (2 dpf). The sustained Ca 2+ response may be important for locomotion. However, at 5 dpf, Ca 2+ response of slow muscle cells in both pure slow Ca 2+ -perm and Ca 2+ -imperm shortened. Thus, slow muscle cells need a sustained Ca 2+ response in relatively early developmental stages around 2 dpf, and the Ca 2+ influx through AChRs plays a crucial role in prolonging the Ca 2+ response. The Ca 2+ influx through AChRs becomes less important by the 5 dpf stage. Ca 2+ influx through AChRs is important for the development of slow muscle cells. In addition to the Ca 2+ response, inhibition of Ca 2+ influx through AChRs affects the morphology of the slow muscle cell. We labeled slow muscle cells of the pure slow Ca 2+ -perm and Ca 2+ -imperm lines by immunohistochemistry (Fig. 6 ). We then observed these slow muscle cells in a lateral view and measured their width and length. In 2 dpf larvae, slow muscle cells of pure slow Ca 2+ -imperm were significantly thinner than those of pure slow Ca 2+ -perm (5.62 ± 0.27 µm in Ca 2+ -perm , 2.52 ± 0.11 µm in Ca 2+ -imperm ; P < 0.001) (Fig. 6 B). However, there was no significant difference in their length (78.4 ± 1.5 µm in Ca 2+ -perm , 81.4 ± 1.9 µm in Ca 2+ -imperm ; P = 0.22) (Fig. 6 B). Although the width of pure slow Ca 2+ -imperm was still smaller than that of pure slow Ca 2+ -perm at 5 dpf (7.84 ± 0.44 µm in Ca 2+ -perm , 5.61 ± 0.28 µm in Ca 2+ -imperm ; P < 0.001) (Fig. 6 C and D), it had grown more than twice as thick compared to 2 dpf larvae. The length of slow muscle cells of pure slow Ca 2+ -imperm was larger than that of slow muscle cells of pure slow Ca 2+ -perm (98.5 ± 1.5 µm in Ca 2+ -perm , 107.8 ± 2.8 µm in Ca 2+ -imperm ; P < 0.01) (Fig. 6 D) at 5 dpf. Discussion Ca 2+ influx through AChRs contributes to the growth of slow muscles at early developmental stages. The results of the present study suggest that slow muscle-type AChRs of zebrafish show much higher Ca 2+ permeability than that of fast muscle-type AChRs (Fig. 1 ). We generated a Tg line that expresses only mutated slow muscle-type AChRs with low Ca 2+ permeability and compared its locomotor activities with those of the γεDKO line, which lacks fast muscle-type AChRs and expresses only Ca 2+ -permeable slow muscle-type AChRs (Figs. 2 , 3 ) 8 . These zebrafish lines allowed us to assess the significance of Ca 2+ influx through AChRs in slow muscle cells with the influence of fast muscles eliminated. The results of locomotion analyses suggest that Ca 2+ influx from AChRs plays an important role in the contraction of slow muscle cells in the early developmental stages (Fig. 3 ). Ca 2+ imaging also showed that slow muscle cells require Ca 2+ influx through AChRs for sufficient Ca 2+ response to ACh at the 2 dpf stage (Fig. 4 , 5 ). In addition, morphological analysis revealed that the slow muscle cells became thinner when the Ca 2+ permeability of their AChRs was decreased (Fig. 6 ). Inhibition of Ca 2+ influx through AChRs delays the growth of slow muscle cells, which presumably compromises motor functions at the 2 dpf stage. The muscle force is proportional to diameter or cross-sectional area of muscles 20 . Unexpectedly, the slow muscle fibers of Pure slow Ca 2+ -imperm form thick bundles by 5 dpf (Fig. 6 C, D), which may underlie the improved locomotion. The morphological alterations in slow muscle cells of pure slow Ca 2+ -imperm are consistent with results of previous studies suggesting that intracellular Ca 2+ signaling plays an important role in the development of slow muscle cells 21 – 23 . Pharmacological inhibition of AChRs or RyRs in early developmental stages induced abnormal organization in slow muscle fibers 23 . The authors suggested that the slow muscle fibers were not aligned and they could not form thick bundles. Cheung et al . (2011) also reported that RyR antagonist treatment during early developmental stages disrupted banding of slow muscle fibers 22 . Therefore, the elevation of intracellular Ca 2+ levels during embryonic stages is important for the development of slow muscle cells, and the source of the Ca 2+ is the SR. The results of the present study suggested that not only Ca 2+ stores in the SR but also the influx of extracellular Ca 2+ through AChRs contributes to the thickening of slow muscle cells. The Ca 2+ influx through AChRs may be required to stimulate Ca 2+ release from RyRs to promote the growth of slow muscle cells. Additionally, Brennan et al . (2005) reported that the slow muscle fibers of mutant zebrafish lacking AChRs tend to be longer than those of wild-type zebrafish because of abnormal organization of myofibrils 23 . Interestingly, in the present study, slow muscle cells of pure slow Ca 2+ -imperm also became longer at the 5 dpf (Fig. 6 ). Taken together, Ca 2+ influx through AChRs may play a critical role in the proper development of slow muscle cells during early developmental stages. Ca 2+ influx through AChRs contributes to slow muscle contraction. In the present study, locomotor activities of pure slow Ca 2+ -imperm were markedly decreased at the 2 dpf. One possible reason we proposed above for the loss of motor function is that disruptions in the development of slow muscle cells result in thinner slow muscle fibers, leading in turn to motor dysfunction. In addition to the morphological effect, we need to consider the possibility that Ca 2+ influx through AChRs is involved in the physiological process of muscle contraction. In zebrafish, the contraction process of slow muscle cells is different from that of fast muscle cells. Slow muscle cells of zebrafish do not fire action potentials due to the lack of voltage-gated Na + channels (Nav) 24 . Thus, cation influx through AChRs needs to activate L-type Ca 2+ channels directly (AChRs-L-type Ca 2+ channel pathway) to induce Ca 2+ release from the SR. In this process, Ca 2+ influx through AChRs not only stimulates L-type Ca 2+ channels but may also directly activate RyRs through the Ca 2+ -induced Ca 2+ release (CICR) mechanism, as suggested by the results of a study on the muscle cells of Ciona 10 . Ca 2+ influx through AChRs may support Ca 2+ release during muscle contraction by these mechanisms. The results of Ca 2+ imaging in the present study showed that the inhibition of Ca 2+ influx through AChRs results in a shortened Ca 2+ response. Consequently, RyRs in the SR are possibly unable to release sufficient Ca 2+ for the contractions of slow muscles without the Ca 2+ influx through AChRs. This could be another reason for the reduced locomotor activity in pure slow Ca 2+ -imperm at the 2 dpf stage. Ca 2+ influx through AChRs becomes less important for slow muscle cells at later stages. At the 5 dpf stage, slow muscle cells in both pure slow Ca 2+ -perm and Ca 2+ -imperm tended to display shorter Ca 2+ response than that at the 2 dpf stage, and there was no significant difference in decay time between those two zebrafish lines. A possible reason for the shortened Ca 2+ response at 5 dpf could be the development of the Ca 2+ reuptake system such as SR Ca 2+ -ATPase (SERCA) and mitochondria. The expression of SERCA changes throughout the developmental process in mammals 25 , 26 , and the expression of SERCA possibly increases between 2 and 5 dpf in zebrafish. Mitochondria also play important roles in reuptake of Ca 2+ in muscle cells. In mice, mitochondria gradually shift their position within the muscle cell during development, positioning themselves close to the SR to effectively reuptake Ca 2+ from the SR 27 . Thus, it is possible that similar positional changes occurred in zebrafish between 2 and 5 dpf. These developments of Ca 2+ reuptake systems may have shortened the Ca 2+ response in slow muscle cells at 5 dpf. The results of locomotor analysis suggested that the Ca 2+ permeability of AChRs becomes less important for the contraction of slow muscles at the 5 dpf stage (Fig. 3 ). Although the slow muscle cells of pure slow Ca 2+ -imperm were still thinner than those of pure slow Ca 2+ -perm , they become thicker at 5 dpf than at 2 dpf (Fig. 6 ). Thus, the growth of slow muscle cells may have improved locomotion. The Ca 2+ influx through AChRs may still be important for the development of slow muscle cells at 5 dpf. However, the effect of reduction in the Ca 2+ influx on locomotor activities may be more apparent at 2 dpf, when the muscle cells are thinner. Ca 2+ signaling pathway in slow muscle cells in zebrafish development In addition to development, Ca 2+ entry through AChRs is possibly more important for the contraction process of slow muscle cells at 2 dpf than at 5 dpf. We hypothesize that the mechanism for controlling muscle contraction is not mature at 2 dpf and the immature slow muscle cells therefore require Ca 2+ influx through AChRs for their functions. Based on the results of previous studies and the results of the present study, we propose that the following series of events occurs in activated immature slow muscle cells. AChRs are first stimulated by ACh from motor neurons. Na + and Ca 2+ influx through the AChRs depolarize membrane potential. L-type Ca 2+ channels are then activated, which in turn triggers opening of RyRs in the SR to release Ca 2+ . Note that electrophysiological analysis has shown that the L-type Ca 2+ channel of zebrafish does not allow permeation of Ca 2+ 28 . In this process, the Ca 2+ influx through AChRs may contribute to both the development of slow muscle cells and membrane depolarization. Ca 2+ entry through AChRs may also directly stimulate RyRs and thereby induce Ca 2+ release from the SR probably in the manner of CICR 29 , 30 . The released Ca 2+ facilitates the development of slow muscle cells while inducing muscle contraction. In general, CICR is important for the contraction of cardiac muscles 31 , but it is not essential for functions of skeletal muscles. However, in amphibians, previous studies suggested that CICR contributes to the contraction of skeletal muscles. In amphibians, RyRβs (corresponding to RyR3s of mammals) are localized outside the junctions between T-tubules and the SR, and the RyRβs are activated by CICR 32 , 33 . Besides, CICR is observed in embryonic or early postnatal myofibers of mammals. These immature myofibers express RyR3 and exhibit spontaneous Ca 2+ sparks, which represent Ca 2+ release from the SR, independent from functions of the L-type Ca 2+ channel 34 . The Ca 2+ spark is considered to be initiated by CICR because its frequency is increased by elevation of the cytosolic Ca 2+ level 35 . During development, Ca 2+ sparks are rarely observed in mammalian muscle cells 36 , 37 because most RyRs interact with L-type Ca 2+ channels and spontaneous Ca 2+ release from RyRs ceases to occur. In zebrafish, T-tubules are almost fully developed by 2 dpf 38 , but the number of RyR1 contacts with the L-type Ca 2+ channel is possibly fewer than that in matured animals at this stage. Actually, the expression of RyR1 at 1–3 dpf stages is less than that in 6 dpf larvae 39 . Therefore, the AChRs-L-type Ca 2+ channel pathway may not be fully developed at 1–3 dpf stages. Thus, the CICR mechanism might be involved in contractions of muscle cells with the immature AChRs-L-type Ca 2+ channel pathway. At 5 dpf, the L-type Ca 2+ channel-RyR complex increases, and the AChRs-L-type Ca 2+ channel pathway becomes mature (Fig. 7 ). Declarations Acknowledgement We are grateful to Ms. Seiko Ohkuma at JMU for her excellent care of zebrafish used in this study. This study was supported by JSPS KAKENHI Grant Numbers 21K15138 and 23K05856 (to B.Z.). References Herzog, W., Schappacher-Tilp, G.: Molecular mechanisms of muscle contraction: A historical perspective. J. Biomech. 155 , 111659 (2023) Sandow, A.: Excitation-contraction coupling in muscular response. Yale J. Biol. Med. 25 , 176–201 (1952) Egashira, Y., Zempo, B., Sakata, S., Ono, F.: Recent advances in neuromuscular junction research prompted by the zebrafish model. Curr. Opin. Physiol. 4 , 70–75 (2018) Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C., Sakmann, B.: Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 321 , 406–411 (1986) Adams, D.J., Dwyer, T.M., Hille, B.: The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol. 75 , 493–510 (1980) Huang, L.Y., Catterall, W.A., Ehrenstein, G.: Selectivity of cations and nonelectrolytes for acetylcholine-activated channels in cultured muscle cells. J. Gen. Physiol. 71 , 397–410 (1978) Mongeon, R., Walogorsky, M., Urban, J., Mandel, G., Ono, F., Brehm, P.: An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses. J. Gen. Physiol. 138 , 353–366 (2011) Zempo, B., Yamamoto, Y., Williams, T., Ono, F.: Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle. Sci. Adv. 6 , eaax8382 (2020) Park, J.-Y., Mott, M., Williams, T., Ikeda, H., Wen, H., Linhoff, M., Ono, F.: A single mutation in the acetylcholine receptor δ-subunit causes distinct effects in two types of neuromuscular synapses. J. Neurosci. 34 , 10211–10218 (2014) Nishino, A., Baba, S.A., Okamura, Y.: A mechanism for graded motor control encoded in the channel properties of the muscle ACh receptor. Proc. Natl. Acad. Sci. U. S. A. 108 , 2599–2604. (2011) Haghighi, A.P., Cooper, E.: A molecular link between inward rectification and calcium permeability of neuronal nicotinic acetylcholine alpha3beta4 and alpha4beta2 receptors. J. Neurosci. 20 , 529–541 (2000) Bertrand, D., Galzi, J.L., Devillers-Thiéry, A., Bertrand, S., Changeux, J.P.: Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor. Proc. Natl. Acad. Sci. U. S. A. 90 , 6971–6975. (1993) Elworthy, S., Hargrave, M., Knight, R., Mebus, K., Ingham, P.W.: Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity. Development. 135 , 2115–2126 (2008) Urasaki, A., Asakawa, K., Kawakami, K.: Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proc. Natl. Acad. Sci. U. S. A. 105 , 19827–19832. (2008) Nakano, Y., Fujita, M., Ogino, K., Saint-Amant, L., Kinoshita, T., Oda, Y., Hirata, H.: Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation. Development. 137 , 1689–1698 (2010) Dana, H., Mohar, B., Sun, Y., Narayan, S., Gordus, A., Hasseman, J.P., Tsegaye, G., Holt, G.T., Hu, A., Walpita, D., et al.: Sensitive red protein calcium indicators for imaging neural activity. Elife 5 . (2016). https://doi.org/10.7554/eLife.12727 Housley, M.P., Reischauer, S., Dieu, M., Raes, M., Stainier, D.Y.R., Vanhollebeke, B.: Translational profiling through biotinylation of tagged ribosomes in zebrafish. Development. 141 , 3988–3993 (2014) Ono, F., Mandel, G., Brehm, P.: Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish. J. Neurosci. 24 , 5475–5481 (2004) Ono, F., Higashijima, S., Shcherbatko, A., Fetcho, J.R., Brehm, P.: Paralytic zebrafish lacking acetylcholine receptors fail to localize rapsyn clusters to the synapse. J. Neurosci. 21 , 5439–5448 (2001) Krivickas, L.S., Dorer, D.J., Ochala, J., Frontera, W.R.: Relationship between force and size in human single muscle fibres: Muscle fibre size and force. Exp. Physiol. 96 , 539–547 (2011) Kelu, J.J., Chan, H.L.H., Webb, S.E., Cheng, A.H.H., Ruas, M., Parrington, J., Galione, A., Miller, A.L.: Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca(2+) signaling during myogenesis in intact zebrafish. Int. J. Dev. Biol. 59 , 313–325 (2015) Cheung, C.Y., Webb, S.E., Love, D.R., Miller, A.L.: Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos. Int. J. Dev. Biol. 55 , 153–174 (2011) Brennan, C., Mangoli, M., Dyer, C.E.F., Ashworth, R.: Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo. J. Cell. Sci. 118 , 5181–5190 (2005) Buckingham, S.D., Ali, D.W.: Sodium and potassium currents of larval zebrafish muscle fibres. J. Exp. Biol. 207 , 841–852 (2004) Anger, M., Samuel, J.L., Marotte, F., Wuytack, F., Rappaport, L., Lompré, A.M.: In situ mRNA distribution of sarco(endo)plasmic reticulum Ca(2+)-ATPase isoforms during ontogeny in the rat. J. Mol. Cell. Cardiol. 26 , 539–550 (1994) Periasamy, M., Kalyanasundaram, A.: SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve. 35 , 430–442 (2007) Boncompagni, S., Rossi, A.E., Micaroni, M., Beznoussenko, G.V., Polishchuk, R.S., Dirksen, R.T., Protasi, F.: Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures. Mol. Biol. Cell. 20 , 1058–1067 (2009) Schredelseker, J., Shrivastav, M., Dayal, A., Grabner, M.: Non-Ca2+-conducting Ca2 + channels in fish skeletal muscle excitation-contraction coupling. Proc. Natl. Acad. Sci. U. S. A. 107 , 5658–5663. (2010) Endo, M.: Calcium-induced calcium release in skeletal muscle. Physiol. Rev. 89 , 1153–1176 (2009) Ríos, E.: Calcium-induced release of calcium in muscle: 50 years of work and the emerging consensus. J. Gen. Physiol. 150 , 521–537 (2018) DelPrincipe, F., Egger, M., Niggli, E.: Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation. Nat. Cell. Biol. 1 , 323–329 (1999) Felder, E., Franzini-Armstrong, C.: Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position. Proc. Natl. Acad. Sci. U. S. A. 99 , 1695–1700. (2002) Shirokova, N., García, J., Pizarro, G., Ríos, E.: Ca2 + release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle. J. Gen. Physiol. 107 , 1–18 (1996) Conklin, M.W., Barone, V., Sorrentino, V., Coronado, R.: Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle. Biophys. J. 77 , 1394–1403 (1999) Zhou, J., Launikonis, B.S., Ríos, E., Brum, G.: Regulation of Ca2 + sparks by Ca2 + and Mg2 + in mammalian and amphibian muscle. An RyR isoform-specific role in excitation-contraction coupling? J. Gen. Physiol. 124 , 409–428 (2004) Brown, L.D., Rodney, G.G., Hernández-Ochoa, E., Ward, C.W., Schneider, M.F.: Ca2 + sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers. Am. J. Physiol. Cell. Physiol. 292 , C1156–C1166 (2007) Zhou, J., Yi, J., Royer, L., Launikonis, B.S., González, A., García, J., Ríos, E.: A probable role of dihydropyridine receptors in repression of Ca2 + sparks demonstrated in cultured mammalian muscle. Am. J. Physiol. Cell. Physiol. 290 , C539–C553 (2006) Hall, T.E., Martel, N., Ariotti, N., Xiong, Z., Lo, H.P., Ferguson, C., Rae, J., Lim, Y.-W., Parton, R.G.: In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation. Nat. Commun. 11 , 3711 (2020) Wu, H.H., Brennan, C., Ashworth, R.: Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development. BMC Res. Notes. 4 , 541 (2011) Additional Declarations There is NO Competing Interest. Supplementary Files supplementalvideo1.avi Video 1 Startle response in the pure slow Ca 2+ -perm line at 2 dpf in response to tactile stimuli (40 frames/sec). supplementalvideo2.avi Video 2 Startle response in the pure slow Ca 2+ -imperm line at 2 dpf in response to tactile stimuli (40 frames/sec). supplementalvideo3.avi Video 3 Startle response in the pure slow Ca 2+ -perm line at 5 dpf in response to tactile stimuli (40 frames/sec). supplementalvideo4.avi Video 4 Startle response in the pure slow Ca 2+ -imperm line at 5 dpf in response to tactile stimuli (40 frames/sec). Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6525282","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455583831,"identity":"a82736fd-d862-417f-9af2-bb22b56a028e","order_by":0,"name":"Buntaro Zempo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACAwhlw8zGwMNwAMRkbCBOSxrpWg4DMQ+RDjPnP3x0c0HFeXY+9rMHDzDU2DEwzyZgjWXDsbTbM87cZmbjyUs4wHAsmYFxzgECDjvYY3abtw2oRYLH4AAD2wEGxhkJBLQc5v8G1HIOquUfMVqO8bABtRyAaGFsI0KLZQ+bGdAvyUC/5BgcSOxL5iHoF2CIPbtdUGGXLN9+xvjDh292coaEQgwEmIE4GcwCOonHcAZhHWAtdnCevAQRWkbBKBgFo2BEAQCCmUAbQkmydwAAAABJRU5ErkJggg==","orcid":"","institution":"Jichi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Buntaro","middleName":"","lastName":"Zempo","suffix":""},{"id":455583832,"identity":"098e5e72-219a-4a80-8c3b-1d4692b6fa47","order_by":1,"name":"Fumihito Ono","email":"","orcid":"https://orcid.org/0000-0001-7532-5262","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Fumihito","middleName":"","lastName":"Ono","suffix":""},{"id":455583833,"identity":"7e09e37c-30d0-478f-91e6-a958ad030357","order_by":2,"name":"Koichi Nakajo","email":"","orcid":"https://orcid.org/0000-0003-0766-7281","institution":"Jichi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Koichi","middleName":"","lastName":"Nakajo","suffix":""}],"badges":[],"createdAt":"2025-04-25 05:00:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6525282/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6525282/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82885492,"identity":"99ade7aa-8a0b-4f03-bcbf-061046d309eb","added_by":"auto","created_at":"2025-05-16 11:47:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":212224,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) Sequence alignment around the intermediate ring. Sequences of α1, β1, δ, γ, and ε are shown. Amino acids that compose the intermediate ring are boxed.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e) Schematic illustrations of subunit compositions of muscle-type nicotinic acetylcholine receptors (AChRs) of zebrafish. Slow muscle cells express γ/ε-less AChRs.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eC\u003c/em\u003e) Current-voltage (I-V) relationship for acetylcholine (ACh)-induced currents through fast muscle-type or slow muscle-type AChRs with external Ca\u003csup\u003e2+\u003c/sup\u003e concentration of 10 mM (black lines) or 1.8 mM (gray lines). Slow muscle-type AChRs-mediated current showed inward rectification.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eD\u003c/em\u003e) Changes in reversal potential in response to external Ca\u003csup\u003e2+\u003c/sup\u003e level. In slow muscle-type AChRs, reversal potential was significantly shifted with changes in the external Ca\u003csup\u003e2+\u003c/sup\u003e level. On the other hand, reversal potential did not change in fast muscle-type AChRs.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eE\u003c/em\u003e) Sequence alignment around the intermediate ring of the mutated d subunit (asterisk). An amino acid that composes an intermediate ring is boxed. Glu (E) of the intermediate ring is mutated to Gln (Q). A schematic illustration of the subunit composition of mutated slow muscle-type AChRs is shown on the right.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eF\u003c/em\u003e) Current-voltage (I-V) relationship for ACh-induced currents through mutated slow muscle-type AChRs in 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e solution (black line) or 1.8 mM Ca\u003csup\u003e2+\u003c/sup\u003e solution (gray line).\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eG\u003c/em\u003e) Changes in reversal potential in response to external Ca\u003csup\u003e2+\u003c/sup\u003e level. In mutated slow muscle-type AChRs, reversal potential was not significantly shifted by changes in the external Ca\u003csup\u003e2+\u003c/sup\u003e level.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/872aa5593b2fb8f8a77327a3.png"},{"id":82885494,"identity":"0471f9c4-2827-4f76-b06a-57894a8d90e7","added_by":"auto","created_at":"2025-05-16 11:47:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":588437,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) DNA construct used for making a transgenic line that expresses mutated δ subunit and EGFP under control of a slow muscle-specific promotor sequence.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e) 1 dpf Tg[\u003cem\u003esmyhc1\u003c/em\u003e:mutated δ-P2A-EGFP] embryo. The right panel shows a bright field image. Scale bars: 200 mm.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eC\u003c/em\u003e) Photographs showing double immunohistochemistry for EGFP (green) and\u0026nbsp;slow muscle fibers (magenta, F59 antibody) in a frontal section of the trunk region of a 3 dpf larva, which indicate that EGFP and mutated d subunits are specifically expressed in slow muscle cells. Scale bars: 50 mm.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eD\u003c/em\u003e) Transgenic lines that were used in the present study. The \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm \u003c/em\u003eline expresses mutated (Ca\u003csup\u003e2+\u003c/sup\u003e-impermeable) AChRs in its slow muscles. To remove the effects of endogenous δ subunits and fast muscle-type AChRs, we crossed the Tg line and the \u003cem\u003esofa potato\u003c/em\u003e (\u003cem\u003esop\u003c/em\u003e) mutant, which carries a mutation in the δ subunit gene leading to failure of assembly of AChRs in muscle cells. The \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm \u003c/em\u003eline expresses Ca\u003csup\u003e2+\u003c/sup\u003e-permeable slow muscle-type AChRs in its slow muscle cells. Note that both lines lack AChRs in fast muscles.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/2a49fc71d32b437c377e2f0c.png"},{"id":82885495,"identity":"75656975-1006-4a32-8c4f-0f2b22f056e8","added_by":"auto","created_at":"2025-05-16 11:47:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1440630,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) Spontaneous locomotor activities of 1 dpf γεDKO (\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e) and Tg (\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e) lines. Images of representative larvae on the left show superimposed frames of the spontaneous activity. The head and yolk of the embryos were partially restrained in an agarose gel, leaving the trunk and tail free to move. Kinematics for representative traces of larvae are shown in the middle panels. Tail bend angles are shown in degrees, with 0 indicating a straight body and positive and negative values indicating tail bends in opposite directions. In the right panel, maximum turn angles were calculated for each group of fish (n=9 fish).\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e) Touch-evoked startle responses of 2 dpf or 5 dpf \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e larvae. Startle response was elicited by touching the head with an eyelash. Images of representative larvae on the left show superimposed frames of the startle response. Kinematics for representative traces of larvae are shown in the middle panels. In the right panel, average turn angles were calculated for each group of fish (n=40: 5 turns from 8 fish). In 1-2 dpf larvae, the tail bend angles of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were significantly smaller than those in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e. However, at 5 dpf, tail bend angles of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e improved to the same level as that of \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eD\u003c/em\u003e-\u003cem\u003eF\u003c/em\u003e) Swimming speed, tail beat speed, and maximum turn angles were calculated for each group of fish (2-5 dpf). In the 2-3 dpf \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e line, the swimming speed, tail beat speed, and maximum tail bend angle were notably reduced. However, at 5 dpf, these values improved to be comparable to those of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/50b36bf7cf633eb708b96b08.png"},{"id":82885502,"identity":"9be174b0-8b17-4e77-bf82-1cce36e7a47b","added_by":"auto","created_at":"2025-05-16 11:47:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3300281,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) Ca\u003csup\u003e2+\u003c/sup\u003e response elicited by tactile stimulation in a single slow muscle cell was analyzed with the Ca\u003csup\u003e2+\u003c/sup\u003e sensor protein jRGECO1a. The transgenic construct expresses jRGECO1a under the control of a slow muscle-specific promotor sequence. The construct was injected into an embryo at the one-cell stage. Ca\u003csup\u003e2+ \u003c/sup\u003eresponse to tactile stimulation was recorded using an sCMOS camera at 200 frames/sec.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e) The pseudo color panels represent changes in fluorescence intensity of slow muscle cells of 2 dpf \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e. Arrowheads indicate one of the target slow muscle cells expressing jRGECO1a. The other cells include fast muscle cells, which are silent in those zebrafish lines. Consecutive panels show the peak fluorescence intensity at and before and after the peak. Representative traces showing the increase of ΔF/F\u003csub\u003e0\u003c/sub\u003e in slow muscle cells (cells indicated by arrowheads in the left panels) from \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (black trace) and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e (gray trace).\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eC\u003c/em\u003e) The peak amplitude of ΔF/F\u003csub\u003e0 \u003c/sub\u003eand decay time to 50% of the peak amplitude were calculated. The decay time was significantly shorter in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e than in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e. There was no significant difference in the peak amplitude.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eD\u003c/em\u003e) The pseudo color panels represent changes in fluorescence intensity of slow muscle cells of 5 dpf \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e. Arrowheads indicate slow muscle cells expressing jRGECO1a. Consecutive panels show the peak fluorescence intensity at rest, at the peak amplitude and at 50% of the peak. Representative traces show the increase of ΔF/F\u003csub\u003e0\u003c/sub\u003e in slow muscle cells (cells indicated by arrowheads in the left panels) from \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (black trace) and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e (gray trace).\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eE\u003c/em\u003e) The peak amplitude of ΔF/F\u003csub\u003e0 \u003c/sub\u003eand decay time to 50% of the peak amplitude were calculated. There was no significant difference between the two lines in either peak amplitude or decay time.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/ddd11cd5d698e0677d78719c.png"},{"id":82885516,"identity":"ce37dcb7-e191-4d65-8464-4d4e1afdc15d","added_by":"auto","created_at":"2025-05-16 11:47:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":730760,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) Ca\u003csup\u003e2+\u003c/sup\u003e response elicited by acetylcholine (ACh) in a dissociated single slow muscle cell was analyzed with the fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e indicator Rhod-4. Ca\u003csup\u003e2+ \u003c/sup\u003esignals were recorded using the sCMOS camera at 200 frames/sec.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e) Ca\u003csup\u003e2+\u003c/sup\u003e imaging for slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e performed in normal or Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution. The experiments were performed in 2 dpf larvae. Representative traces showing the increase of ΔF/F\u003csub\u003e0\u003c/sub\u003e in slow muscle cells in normal Ringer’s solution (left panel) and Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution (right panel). The peak amplitude of ΔF/F\u003csub\u003e0 \u003c/sub\u003eand fluorescence decay rate\u003csub\u003e \u003c/sub\u003ewere calculated. At the 2 dpf stage, the fluorescence decay rate was significantly lower in muscle cells in the Ca\u003csup\u003e2+\u003c/sup\u003e-free solution than in muscle cells in the normal solution. There was no significant difference in the peak amplitude.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eC\u003c/em\u003e) Ca\u003csup\u003e2+\u003c/sup\u003e imaging for slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e performed in normal or Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution. The experiments were performed in 2 dpf larvae. Representative traces showing the increase of ΔF/F\u003csub\u003e0\u003c/sub\u003e in slow muscle cells in normal Ringer’s solution (left panel) and Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution (right panel). The peak amplitude of ΔF/F\u003csub\u003e0 \u003c/sub\u003eand fluorescence decay rate\u003csub\u003e \u003c/sub\u003ewere calculated. There was no significant difference in either peak amplitude or decay time.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eD\u003c/em\u003e) Ca\u003csup\u003e2+\u003c/sup\u003e imaging for slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e performed in normal or Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution. The experiments were performed in 5 dpf larvae. Representative traces showing the increase of ΔF/F\u003csub\u003e0\u003c/sub\u003e in slow muscle cells in normal Ringer’s solution (left panel) and Ca\u003csup\u003e2+\u003c/sup\u003e-free Ringer's solution (right panel). The peak amplitude of ΔF/F\u003csub\u003e0 \u003c/sub\u003eand fluorescence decay rate\u003csub\u003e \u003c/sub\u003ewere\u003cbr\u003e\ncalculated. There was no significant difference in either peak amplitude or decay time.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/e5c7c33d7d202f819e826a24.png"},{"id":82885509,"identity":"0356b233-3a86-4d33-8cd0-76c65ca6d4b3","added_by":"auto","created_at":"2025-05-16 11:47:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":943500,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eA\u003c/em\u003e) Photographs showing trunk regions of a \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e larva (2 dpf) and a \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e larva (2 dpf) at low magnification (upper panels) and high magnification (lower panels). Slow muscle cells were labeled with slow muscle-specific F59 antibody.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eB\u003c/em\u003e) The width and length of single slow muscle cells were measured. In \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, slow muscle cells were significantly thinner than those in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e. There was no significant difference in the length of slow muscle cells between \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm \u003c/em\u003eand\u003cem\u003e Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003eC\u003c/em\u003e) Photographs showing trunk regions of a \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e larva (5 dpf) and a \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e larva (5 dpf) at low magnification (upper panels) and high magnification (lower panels). Slow muscle cells were labeled with slow muscle-specific F59 antibody.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(\u003cem\u003eD\u003c/em\u003e) The width and length of single slow muscle cells were measured. In \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, slow muscle cells were significantly thinner than those in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e. In \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, slow muscle cells were significantly longer than those in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/484d9b3364e3e5c157b9403a.png"},{"id":82886967,"identity":"4c45e6ac-67c6-4ef7-8e0d-0776eb36b693","added_by":"auto","created_at":"2025-05-16 11:55:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":607329,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustrations show the functions of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs suggested by the results of the present study.\u003c/p\u003e\n\u003cp\u003eAt 2 dpf, Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs stimulate development of slow muscle cells. Thus, slow muscle cells became thinner in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e. Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs also contribute to Ca\u003csup\u003e2+\u003c/sup\u003e release during the contraction process by depolarizing the membrane potential or CICR system. The AChR-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway (yellow arrow) is still immature at the 2 dpf stage, which makes the support of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs important for activating the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel-RyR complexes efficiently. Reduction of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs results in insufficient Ca\u003csup\u003e2+\u003c/sup\u003e release, which was observed in slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAt 5 dpf, the slow muscle cells are thicker than those at 2 dpf even in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e. This morphological development allows \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e larvae to exhibit locomotion comparable to that of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e. Additionally, the AChR-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway is well developed by this stage, decreasing the contribution of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs to Ca\u003csup\u003e2+\u003c/sup\u003e release. At the 5 dpf stage, slow muscle cells can induce sufficient Ca\u003csup\u003e2+\u003c/sup\u003e release primarily through the AChR-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway, without relying on Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs. Besides, the Ca\u003csup\u003e2+\u003c/sup\u003e reuptake system is also developed by 5 dpf, and the maturation of this reuptake system may lead to shorter Ca\u003csup\u003e2+\u003c/sup\u003e responses.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/9a29c3606da8a7f7fa55e6da.png"},{"id":83837974,"identity":"a1a0b4c6-3fd0-45b9-9902-f614b0dd90e0","added_by":"auto","created_at":"2025-06-03 13:29:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10357743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/88da8147-b0aa-4303-ae52-8b3a9d092b53.pdf"},{"id":82888596,"identity":"19ff4edf-eac0-42b2-8d1a-1fba7e56416f","added_by":"auto","created_at":"2025-05-16 12:03:03","extension":"avi","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23182000,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 1\u003c/p\u003e\n\u003cp\u003eStartle response in the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e line at 2 dpf in response to tactile stimuli (40 frames/sec).\u003c/p\u003e","description":"","filename":"supplementalvideo1.avi","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/f58488978b7951afab3c46e7.avi"},{"id":82885507,"identity":"53d475d0-bcb7-41cc-8b6a-fb709239b1c6","added_by":"auto","created_at":"2025-05-16 11:47:03","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27828476,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 2\u003c/p\u003e\n\u003cp\u003eStartle response in the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e line at 2 dpf in response to tactile stimuli (40 frames/sec).\u003c/p\u003e","description":"","filename":"supplementalvideo2.avi","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/a5f992f1cd9bad12a7d4395e.avi"},{"id":82885517,"identity":"d73958f6-9b57-40e6-a197-1f90bd1734ca","added_by":"auto","created_at":"2025-05-16 11:47:03","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":29613834,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 3\u003c/p\u003e\n\u003cp\u003eStartle response in the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e line at 5 dpf in response to tactile stimuli (40 frames/sec).\u003c/p\u003e","description":"","filename":"supplementalvideo3.avi","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/52300862f29e0d8c254df7a1.avi"},{"id":82886969,"identity":"95c6f909-546d-40ad-99f7-a2907564edb9","added_by":"auto","created_at":"2025-05-16 11:55:03","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14401630,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 4\u003c/p\u003e\n\u003cp\u003eStartle response in the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e line at 5 dpf in response to tactile stimuli (40 frames/sec).\u003c/p\u003e","description":"","filename":"supplementalvideo4.avi","url":"https://assets-eu.researchsquare.com/files/rs-6525282/v1/1afd8b3ac4fbd782fdfa0c00.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e influx through muscle-type nicotinic AChRs contributes to contractions and development of slow muscle cells in early developmental stages\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMuscle contraction mechanisms have long been studied and are well understood \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Neuromuscular junctions (NMJs) play crucial roles in the process of muscle contraction. At the NMJs, motor neurons release acetylcholine (ACh) and stimulate nicotinic acetylcholine receptors (AChRs). AChRs are ligand-gated cation-permeable ion channels. Binding of ACh induces cation influx through AChRs, depolarizes the membrane potential, activates the voltage-gated Na\u003csup\u003e+\u003c/sup\u003e channels (Nav) in the sarcolemma, and generates an action potential. Depolarization of the sarcolemma opens the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels that are in physical contact with ryanodine receptors (RyRs). As the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels open, RyRs also open and release Ca\u003csup\u003e2+\u003c/sup\u003e from the sarcoplasmic reticulum (SR), which induces muscle contractions. In this process, AChRs are essential molecular components that play a key role in the initial step, receiving signals from motor neurons.\u003c/p\u003e \u003cp\u003eMuscle-type AChRs are pentamers composed of two α1s, β1, δ, and ε (or γ in early development) subunits \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. AChRs in NMJs tend to be highly permeable to Na\u003csup\u003e+\u003c/sup\u003e, whereas they show relatively low permeability for Ca\u003csup\u003e2+ 5,6\u003c/sup\u003e. However, recent studies have revealed a different type of AChR composed of only α1, β1, and δ subunits in zebrafish \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It was also found in those studies that slow muscles and fast muscles specifically express αβδ-type and αβδγ/ε-type AChRs, respectively. This finding begs the next question: why do slow muscles express AChRs that are different from those of fast muscles?\u003c/p\u003e \u003cp\u003eTo address this question, we focused on the difference between channel properties of slow and fast muscle-type AChRs. Although αβδ-type AChRs have been reported only in zebrafish, the larval tunicate \u003cem\u003eCiona\u003c/em\u003e has been shown to possess AChRs composed of only three types of subunits: α1, B2/4, and BGDE3 \u003csup\u003e10\u003c/sup\u003e. The slow muscle-type AChRs of zebrafish show characteristics similar to those of the AChRs of \u003cem\u003eCiona\u003c/em\u003e: both AChRs exhibit inward rectification \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This property is unique and not found in muscle-type AChRs described to date, including fast muscle-type AChRs of zebrafish.\u003c/p\u003e \u003cp\u003eOn the other hand, neuronal AChRs show inward rectification and high Ca\u003csup\u003e2+\u003c/sup\u003e permeability \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which are bestowed by negatively charged amino acid residues of the \u0026ldquo;intermediate ring\u0026rdquo; (part of the ion permeation pathway of AChRs). Nishino \u003cem\u003eet al.\u003c/em\u003e showed that muscle-type AChRs of \u003cem\u003eCiona\u003c/em\u003e display inward rectification and high Ca\u003csup\u003e2+\u003c/sup\u003e permeability. Moreover, the intermediate rings of AChRs of \u003cem\u003eCiona\u003c/em\u003e are composed of glutamate (E), a negatively charged amino acid \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The authors further showed that \u003cem\u003eCiona\u003c/em\u003e AChRs lose the rectification and Ca\u003csup\u003e2+\u003c/sup\u003e permeability by mutating E of the intermediate ring to glutamine (Q) (uncharged amino acid).\u003c/p\u003e \u003cp\u003eIn zebrafish, fast muscle-type AChRs are composed of α1, β1, δ, ε, or γ subunits, among which ε and γ subunits possess Q at the intermediate ring. In contrast, intermediate rings in slow muscle-type AChRs are composed of only E, because γ and ε subunits are absent.\u003c/p\u003e \u003cp\u003eBased on these facts, we hypothesized that slow muscle-type AChRs in zebrafish are highly permeable to Ca\u003csup\u003e2+\u003c/sup\u003e and that the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may contribute to the functions of slow muscles. The results of the present study suggested that the slow muscle-type AChRs of zebrafish have actually high Ca\u003csup\u003e2+\u003c/sup\u003e permeability. We further analyzed the physiological significance of the Ca\u003csup\u003e2+\u003c/sup\u003e permeability.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of fish lines\u003c/h2\u003e \u003cp\u003eZebrafish were kept in water at 28\u0026deg;C with 14 h of light (8:00\u0026ndash;22:00) and 10 h of darkness (22:00\u0026ndash;8:00). Animal experiments using zebrafish were approved by the Animal Experiment Committee of Jichi Medical University (Approval No. 23013-01, 23013-02).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElectrophysiology\u003c/h3\u003e\n\u003cp\u003ecDNAs for the muscle ACh subunits α1, β1, δ, and ε were previously cloned from zebrafish \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The cDNA of each subunit was subcloned into the pTNT vector (Promega, WI, USA) for \u003cem\u003ein vitro\u003c/em\u003e transcription. The purified plasmid was linearized at the BamHI site and \u003cem\u003ein vitro\u003c/em\u003e transcribed with T7 RNA polymerase (mMESSAGE mMACHINE T7 Transcription kit; Thermo Fisher Scientific, MA, USA). Oocytes were isolated and defolliculated by treatment with 2 mg/ml collagenase (Sigma-Aldrich, C0130, MO, USA) for 4\u0026ndash;6 hours in MBSH solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 10 mM HEPES, 0.3 mM Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, 0.41 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.82 mM MgSO\u003csub\u003e4\u003c/sub\u003e, pH 7.6). For expression of the α1β1δε receptor, the cRNA solution of each subunit was mixed to final concentrations of 0.2, 0.1, 0.1, and 0.1 ng/nl, and 50 nl of the solution was injected per oocyte. For expression of αβδ, cRNAs were mixed at 0.2, 0.1, and 0.2 ng/nl. Injected oocytes were incubated at 17\u0026ordm;C for 2\u0026ndash;3 days in MBSH solution supplemented with 0.1% penicillin-streptomycin (Sigma-Aldrich). Electrophysiology was performed with a two-electrode voltage clamp using OC-725C (Warner Instruments, CT, USA). Generation of voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface (Molecular Devices, CA, USA) and Clampex 10.7 software (Molecular Devices). All experiments were performed at room temperature. A glass electrode with a resistance of 0.2\u0026ndash;0.5 MΩ was prepared from a borosilicate glass capillary (GC150TF-10, MA, United States) using a micropipette puller (P-1000, Sutter Instrument, CA, USA). The glass electrode was filled with 3 M KCl. Low Ca\u003csup\u003e2+\u003c/sup\u003e solution or high Ca\u003csup\u003e2+\u003c/sup\u003e solution was used as the bath solution. Low Ca\u003csup\u003e2+\u003c/sup\u003e solution consisted of (in mM) 5 HEPES, 10 NaCl, 2 KCl, 1.8 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, and 86 NMDG, pH 7.5, and high Ca\u003csup\u003e2+\u003c/sup\u003e solution consisted of 5 HEPES, 10 NaCl, 2 KCl, 10 CaCl\u003csub\u003e2\u003c/sub\u003e, and 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 69.6 NMDG, pH 7.5. An ionic current was induced by puff applying 20 \u0026micro;M acetylcholine diluted in bath solution to the oocytes. The I-V relationship was examined by applying a ramp pulse.\u003c/p\u003e\n\u003ch3\u003eGeneration of a transgenic line\u003c/h3\u003e\n\u003cp\u003eWe designed a gene construct that expresses a mutated δ subunit and enhanced green fluorescent protein (EGFP) under the regulation of a slow muscle-specific promoter, \u003cem\u003epsmyhc1\u003c/em\u003e \u003csup\u003e13\u003c/sup\u003e. The coding sequence of the mutated δ subunit, P2A, and EGFP were cloned into the Tol2 plasmid \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The plasmid was injected along with transposase mRNA into one-cell stage embryos. The established transgenic (Tg) line was crossed with the \u003cem\u003esofa potato\u003c/em\u003e (sop) mutant zebrafish line, and an \u003cem\u003esmyhc1\u003c/em\u003e: mutated δ-P2A-EGFP; sop-/- line (\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e) was generated.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eAfter generation of the Tg line, \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, we confirmed the specificity of EGFP expression in slow muscle cells by labeling slow muscle fibers and EGFP. First, 3 days post fertilization larvae of the Tg line were deeply anesthetized with 0.03% MS-222 (Sigma-Aldrich). Then the trunk regions of the fish were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The fixed samples were frontally cryosectioned at 20 \u0026micro;m with a cryostat (CM 3050S; Leica Microsystems, Wetzlar, Germany) and mounted onto a micro slide glass (CREST; Matsunami, Osaka, Japan). Sections were incubated overnight with a mixture of anti-EGFP antibody raised in rabbits (Thermo Fisher Scientific, MA, USA) that was diluted 1:500 with phosphate-buffered saline containing 0.3% Tween 20 (PBST) and F59 antibody raised in mice (DSHB, Iowa, USA) that was diluted 1:500 with PBST for 2 hours and then rinsed twice with PBST and incubated for 2 hours with a mixture of CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech, Illinois, USA) diluted 1:500 with PBST, anti-mouse IgG H\u0026amp;L (Alexa Fluor 555; Abcam, Cambridge, UK) diluted 1:500 with PBST, and DAPI (Nacalai, Kyoto, Japan) diluted 1:2000 with PBST. The sections were then washed with PBST and coverslipped with CC/Mount (Diagnostic BioSystems, Pleasanton, CA). Fluorescence was observed with a confocal microscope (Dragonfly; OXFORD INSTRUMENTS, Oxford, UK).\u003c/p\u003e \u003cp\u003eFor the labeling of slow muscle fibers in whole-mount samples, 2 dpf or 5 dpf larval zebrafish were fixed in 4% PFA at 4℃ for 2 hours and then washed several times in PBST. The samples were then incubated in PBS containing 1 mg/ml collagenase (Sigma-Aldrich) for 60\u0026ndash;90 min at room temperature and permeabilized with acetone for 10 min at -20℃. After washing with PBST, the samples were incubated with F59 antibody diluted 1:20 with PBST overnight at room temperature. Embryos were rinsed in PBST and incubated with anti-mouse IgG H\u0026amp;L (Alexa Fluor 555; Abcam) diluted 1:500 in PBST for 2 hours at room temperature.\u003c/p\u003e \u003cp\u003eFluorescence was observed and pictures were taken with a camera (Digital Sight 10; Nikon, Tokyo, Japan) on an MVX10 microscope (OLYMPUS) and a confocal microscope (Dragonfly; OXFORD). The data were analyzed with ImageJ.\u003c/p\u003e\n\u003ch3\u003eLocomotor analysis\u003c/h3\u003e\n\u003cp\u003eHigh-speed image capturing of larval zebrafish was performed with a Photron camera (INFINICAM; Photron, Tokyo, Japan) at 1000 frames/s. Captured images were saved as JPEG files and processed with ImageJ and software for motion analysis (Mova-tr/2 and Wriggle Tracker; Library, Tokyo, Japan). For each of the larvae (2\u0026ndash;5 dpf), the head was touched gently with an eyelash \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e to induce escape behavior. For 1 dpf larvae, the head and yolk were embedded in an agarose gel, and spontaneous activities were recorded. The tail bend angle, swimming speed, tail beat speed, and maximum tail bend angle were calculated using Mova-tr/2 and Wriggle Tracker. Kinetics of tail bend angles were drawn by plotting degrees of tail bend angles against time. The frame at 0 ms was set immediately preceding the detection of the first motion.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCa\u003csup\u003e2+\u003c/sup\u003e imaging\u003c/h2\u003e \u003cp\u003eWe designed a gene construct that expresses a red fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e sensor protein, jRGECO1a \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (obtained from Addgene #61563), under the regulation of a skeletal muscle-specific promoter, \u003cem\u003epactc1b\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The promoter activity of \u003cem\u003epactc1b\u003c/em\u003e is stronger than that of a slow muscle-specific promoter (\u003cem\u003epsmyhc1\u003c/em\u003e). Thus, to make observation easier, we chose the \u003cem\u003epactc1b\u003c/em\u003e promoter instead of the slow muscle-specific promoter. The coding sequence of jRGECO1a and the sequence of \u003cem\u003epactc1b\u003c/em\u003e were cloned into the Tol2 plasmid. We injected the constructed plasmid into one cell\u0026ndash;stage embryos from AChR γ subunit\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e ε subunit\u003csup\u003e+/\u0026minus;\u003c/sup\u003e pairs and \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm (sop\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e pairs. The genotype of the injected embryos was determined by sequence analyses after Ca\u003csup\u003e2+\u003c/sup\u003e imaging.\u003c/p\u003e \u003cp\u003ejRGECO1a-expressing fish at 2 or 5 dpf were embedded in the lateral position in 2% low-melting agarose in a 35 mm dish. The heads were touched gently with glass pipettes to induce swimming. Ca\u003csup\u003e2+\u003c/sup\u003e responses were recorded with a Zyla 4.2 sCMOS camera (OXFORD) on an MVX10 microscope (OLYMPUS, Tokyo, Japan) at 200 Hz for 3 sec. The data were analyzed with ImageJ. ΔF/F was calculated for each frame using the formula (F-F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e, where F represents the fluorescence in that frame and F\u003csub\u003e0\u003c/sub\u003e is the resting intensity before the Ca\u003csup\u003e2+\u003c/sup\u003e rise. The experiments were performed for slow muscle cells of the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e and those of the γ/ε subunits double KO line (\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e imaging, fish at 2 or 5 dpf were deeply anesthetized with 0.03% MS-222 (Sigma-Aldrich). After dissecting the trunk region into several pieces with surgical knives, tissue samples were incubated in 10 mg/ml collagenase solution in a bath solution (112 mM NaCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 3 mM Glu, 2 mM KCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM HEPES, pH 7.4) at room temperature for 30 minutes. MgCl\u003csub\u003e2\u003c/sub\u003e was used to replace CaCl\u003csub\u003e2\u003c/sub\u003e in Ca\u003csup\u003e2+\u003c/sup\u003e-free solution. After washing twice with the bath solution, the samples were incubated in Rhod-4 solution (Abcam, Cat: ab112157) following the manufacturer\u0026rsquo;s instructions at room temperature for 30 min. Then the samples were washed with the bath solution and placed in a recording chamber filled with the bath solution. Ca\u003csup\u003e2+\u003c/sup\u003e responses were induced by puff application of 30 \u0026micro;M ACh using a glass pipette. Ca\u003csup\u003e2+\u003c/sup\u003e responses were recorded with a Zyla 4.2 sCMOS camera (OXFORD) on an MVX10 microscope (OLYMPUS) at 200 Hz for 14 sec. The data were analyzed with ImageJ. ΔF/F was calculated for each frame using the formula (F-F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e, where F represents the fluorescence in that frame and F\u003csub\u003e0\u003c/sub\u003e is the resting intensity before the Ca\u003csup\u003e2+\u003c/sup\u003e rise. The experiments were performed for slow muscle cells of the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e and those of the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eUnpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (two-tailed) was performed for statistical analysis. Averages and SEM are displayed in bar graphs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAChRs of slow muscles show high Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003epermeability.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFirst, we compared the sequence alignment of subunits that compose zebrafish muscle-type AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The γ and ε subunits possess a glutamine (Q) residue in the intermediate ring. Fast muscle-type AChRs comprising α1, β1, δ, ε, or γ subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) therefore contain one Q at the intermediate ring. On the other hand, slow muscle-type AChRs lack γ/ε-subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and their intermediate rings are therefore composed only of E, as in the case of AChRs of \u003cem\u003eCiona\u003c/em\u003e, which show high Ca\u003csup\u003e2+\u003c/sup\u003e permeability \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on these observations, we analyzed the Ca\u003csup\u003e2+\u003c/sup\u003e permeability of fast muscle-type and slow muscle-type AChRs of zebrafish by electrophysiology. We recorded ACh-induced ionic currents mediated by AChRs expressed in \u003cem\u003eXenopus laevis\u003c/em\u003e oocytes by a two-electrode voltage clamp and measured reversal potential with different Ca\u003csup\u003e2+\u003c/sup\u003e concentrations (1.8 and 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e). The current-voltage (I-V) relationships of the AChRs revealed that slow muscle-type AChRs showed inward rectification, whereas fast muscle-type AChRs showed no clear rectification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). While the reversal potential of fast muscle-type AChRs was unaffected by external Ca\u003csup\u003e2+\u003c/sup\u003e level (-34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;24 mV in 1.8 mΜ Ca\u003csup\u003e2+\u003c/sup\u003e, -32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 mV in 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.67), the reversal potential significantly changed in slow muscle-type AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) (-39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 mV in 1.8 mΜ Ca\u003csup\u003e2+\u003c/sup\u003e, -25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 mV in 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). To confirm the importance of amino acid residues in the intermediate ring for Ca\u003csup\u003e2+\u003c/sup\u003e permeability, we mutated E of the intermediate ring in the δ subunit to Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). We recorded ACh-induced ionic currents mediated by AChRs that are composed of α1, β1, and mutated δ subunits. Mutated slow muscle-type AChRs showed a linear I-V relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), and its reversal potential was not affected by the external Ca\u003csup\u003e2+\u003c/sup\u003e concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) (-40.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 mV in 1.8 mΜ Ca\u003csup\u003e2+\u003c/sup\u003e, -38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 mV in 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.65).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of a transgenic zebrafish line that expresses AChRs with low Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003epermeability in its slow muscle.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo clarify the physiological roles of Ca\u003csup\u003e2+\u003c/sup\u003e influx through slow muscle-type AChRs, we generated a transgenic (Tg) zebrafish line that expresses slow muscle-type AChRs with low Ca\u003csup\u003e2+\u003c/sup\u003e permeability in its slow muscle cells. The mutated δ subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), P2A, and EGFP sequences were fused to the promoter region of the slow myosin heavy chain 1 (\u003cem\u003esmyhc1\u003c/em\u003e) gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the Tg line, EGFP signals were observed in trunk regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe first examined the specificity of EGFP signals by double labeling. EGFP and slow muscle fibers were visualized by anti-EGFP antibody and anti-skeletal muscle myosin antibody (F59), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). F59 antibody is widely used for detecting slow muscle cells in zebrafish. The obtained image confirmed the specificity of EGFP expression in slow muscle fibers, suggesting that slow muscle cells specifically express mutated δ subunits.\u003c/p\u003e \u003cp\u003eMoreover, to remove the effects of endogenous δ subunits and fast muscle-type AChRs, we crossed the Tg line and the \u003cem\u003esofa potato\u003c/em\u003e (\u003cem\u003esop\u003c/em\u003e) mutant line. \u003cem\u003esop\u003c/em\u003e harbors a point mutation in its δ subunit gene \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and previous studies showed that the \u003cem\u003esop\u003c/em\u003e mutant completely lacks both slow muscle-type and fast muscle-type AChRs \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. By crossing the newly established Tg and \u003cem\u003esop\u003c/em\u003e, we generated a zebrafish line that expresses mutated slow muscle-type AChRs with low Ca\u003csup\u003e2+\u003c/sup\u003e permeability in its slow muscles, while fast muscle cells lack AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In this article, we call this line harboring an E-Q mutated δ subunit in the \u003cem\u003esop\u003c/em\u003e background \u0026ldquo;\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u0026rdquo;\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo clarify the physiological importance of Ca\u003csup\u003e2+\u003c/sup\u003e permeability of slow muscle-type AChRs, we compared locomotor activities of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e with those of another mutant, γ/ε subunits double KO (γεDKO) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The latter line expresses AChRs only in slow muscles. Of note, the γεDKO line expresses intact slow muscle-type AChRs that allow permeation of Ca\u003csup\u003e2+\u003c/sup\u003e. In this article, we call γεDKO \u0026ldquo;\u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u0026rdquo;\u003c/em\u003e. Comparison of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e was therefore expected to reveal the physiological significance of Ca\u003csup\u003e2+\u003c/sup\u003e influx through slow muscle-type AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux through AChRs plays an important role in the locomotor activities of slow muscles at early developmental stages.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing a high-speed camera, we analyzed locomotor activities of the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (γεDKO) line and the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e line at 1, 2, 3, and 5 days post fertilization (dpf). At the 1 dpf stage, spontaneous tail movement (coiling) was recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and we analyzed tail bend angles, measured as the angle of the trunk deviation from the longitudinal axis of the head. Kinematics for representative traces showed that tail bend angles of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were smaller than those of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Maximum tail bend angles in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were significantly smaller than those in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (142.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15.0\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eAt the 2 dpf stage, we induced an escape response by tactile stimuli and recorded the locomotor activity with a high-speed camera, analyzing tail bend angles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e fish showed weak but significant swimming behavior despite the lack of fast muscle activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On the other hand, as shown in video 1, the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e line wagged its tail only slightly. Kinematics for representative traces suggested that the amplitude of tail bend angle is much smaller in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e than in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (see also video 1 and video 2). Calculated average tail bend angles of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were significantly smaller than those of \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (53.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e , 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eWe also analyzed escape behavior at the 5 dpf stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At this stage, unexpectedly, tail beat kinematics of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e larvae appeared to be almost identical to those of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, displaying no significant difference in average tail bend angles (43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 43.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u0026deg; in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.73, see also video 3 and video 4).\u003c/p\u003e \u003cp\u003eWe also calculated swimming speed, tail beat speed, and maximum tail beat angle in 2\u0026ndash;5 dpf larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). As a result, tail beat speed and amplitude were decreased in 1\u0026ndash;3 dpf \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, resulting in slower swimming. However, at the 5 dpf stage, there was no difference in locomotor functions between \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e. These results suggest that the Ca\u003csup\u003e2+\u003c/sup\u003e permeability of AChRs is important for the contraction process of slow muscle cells at early developmental stages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eimaging suggests that Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003einflux through AChRs contributes to a sustained Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eresponse of slow muscle cells in early developmental stages.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo analyze the physiological roles of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs, we examined the Ca\u003csup\u003e2+\u003c/sup\u003e response to tactile stimulation \u003cem\u003ein vivo\u003c/em\u003e with a fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e sensor protein (jRGECO1a) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We injected a construct containing a slow muscle-specific promotor sequence and jRGECO1a into one cell\u0026ndash;stage embryos from \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e pairs or \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e pairs and carried out Ca\u003csup\u003e2+\u003c/sup\u003e imaging. At the 2 dpf stage, fluorescence traces showed that the Ca\u003csup\u003e2+\u003c/sup\u003e response of slow muscle cells in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e decreased faster than that of slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We calculated the peak amplitude of the Ca\u003csup\u003e2+\u003c/sup\u003e response (ΔF/F\u003csub\u003e0\u003c/sub\u003e) and the decay time to 50% of the peak amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). There was no significant difference in peak amplitude between \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eimperm\u003c/em\u003e (0.318\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068 in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072 in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; P\u0026thinsp;=\u0026thinsp;0.15). However, decay time was shorter in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e (0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044 sec in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036 sec in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;=\u0026thinsp;0.019). We also analyzed 5 dpf larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). In the 5dpf stage, peak amplitude of \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e was not significantly different from that of \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.046 in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; P\u0026thinsp;=\u0026thinsp;0.13). In addition, there was no difference in decay time between \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eimperm\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028 sec in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040 sec in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; P\u0026thinsp;=\u0026thinsp;0.15).\u003c/p\u003e \u003cp\u003eTo confirm the importance of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs for the activities of slow muscles, the Ca\u003csup\u003e2+\u003c/sup\u003e response of slow muscle cells was assessed under a Ca\u003csup\u003e2+\u003c/sup\u003e-free condition. We analyzed Ca\u003csup\u003e2+\u003c/sup\u003e response of dissociated slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e fish to ACh in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution and Ca\u003csup\u003e2+\u003c/sup\u003e-containing (normal) bath solution with a fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e indicator (Rhod-4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Unlike the \u003cem\u003ein vivo\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e imaging, in this experiment, we evaluated the kinetics of Ca\u003csup\u003e2+\u003c/sup\u003e response by calculating fluorescent decay rate (ΔF/F\u003csub\u003e0\u003c/sub\u003e of 3 sec after the peak divided by the peak ΔF/F\u003csub\u003e0\u003c/sub\u003e) because ΔF/F\u003csub\u003e0\u003c/sub\u003e did not reach 50% of the peak amplitude, which is arguably due to the absence of acetylcholine esterase in the bath solution. As a result, the peak amplitude of the Ca\u003csup\u003e2+\u003c/sup\u003e response of cells from \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution was not significantly different from that in normal bath solution at the 2 dpf stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 in normal bath solution, 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;=\u0026thinsp;0.22). On the other hand, fluorescence decay rate significantly decreased in the Ca\u003csup\u003e2+\u003c/sup\u003e-free condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; 87.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e in normal bath solution, 50.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3% in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;=\u0026thinsp;0.009). In slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e, both peak amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 in normal bath solution, 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;=\u0026thinsp;0.13) and decay rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; 72.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3% in normal bath solution, 59.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2% in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;=\u0026thinsp;0.54) did not change with the external Ca\u003csup\u003e2+\u003c/sup\u003e. In addition, Ca\u003csup\u003e2+\u003c/sup\u003e response of slow muscle cells in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e was unaffected by external Ca\u003csup\u003e2+\u003c/sup\u003e level at the 5 dpf stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; peak amplitude\u0026thinsp;=\u0026thinsp;0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 in normal bath solution, 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;=\u0026thinsp;0.11, decay rate\u0026thinsp;=\u0026thinsp;63.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7% in normal bath solution, 67.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5% in Ca\u003csup\u003e2+\u003c/sup\u003e-free bath solution; P\u0026thinsp;=\u0026thinsp;0.62). Therefore, slow muscle cells require Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs for a sustained Ca\u003csup\u003e2+\u003c/sup\u003e response at the early developmental stage (2 dpf). The sustained Ca\u003csup\u003e2+\u003c/sup\u003e response may be important for locomotion. However, at 5 dpf, Ca\u003csup\u003e2+\u003c/sup\u003e response of slow muscle cells in both \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e shortened. Thus, slow muscle cells need a sustained Ca\u003csup\u003e2+\u003c/sup\u003e response in relatively early developmental stages around 2 dpf, and the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs plays a crucial role in prolonging the Ca\u003csup\u003e2+\u003c/sup\u003e response. The Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs becomes less important by the 5 dpf stage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux through AChRs is important for the development of slow muscle cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn addition to the Ca\u003csup\u003e2+\u003c/sup\u003e response, inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs affects the morphology of the slow muscle cell. We labeled slow muscle cells of the \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e lines by immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We then observed these slow muscle cells in a lateral view and measured their width and length. In 2 dpf larvae, slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were significantly thinner than those of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, there was no significant difference in their length (78.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 81.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.22) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Although the width of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e was still smaller than that of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e at 5 dpf (7.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D), it had grown more than twice as thick compared to 2 dpf larvae. The length of slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e was larger than that of slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e (98.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, 107.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 \u0026micro;m in \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) at 5 dpf.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux through AChRs contributes to the growth of slow muscles at early developmental stages.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe results of the present study suggest that slow muscle-type AChRs of zebrafish show much higher Ca\u003csup\u003e2+\u003c/sup\u003e permeability than that of fast muscle-type AChRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We generated a Tg line that expresses only mutated slow muscle-type AChRs with low Ca\u003csup\u003e2+\u003c/sup\u003e permeability and compared its locomotor activities with those of the γεDKO line, which lacks fast muscle-type AChRs and expresses only Ca\u003csup\u003e2+\u003c/sup\u003e-permeable slow muscle-type AChRs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These zebrafish lines allowed us to assess the significance of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs in slow muscle cells with the influence of fast muscles eliminated. The results of locomotion analyses suggest that Ca\u003csup\u003e2+\u003c/sup\u003e influx from AChRs plays an important role in the contraction of slow muscle cells in the early developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Ca\u003csup\u003e2+\u003c/sup\u003e imaging also showed that slow muscle cells require Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs for sufficient Ca\u003csup\u003e2+\u003c/sup\u003e response to ACh at the 2 dpf stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, morphological analysis revealed that the slow muscle cells became thinner when the Ca\u003csup\u003e2+\u003c/sup\u003e permeability of their AChRs was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs delays the growth of slow muscle cells, which presumably compromises motor functions at the 2 dpf stage. The muscle force is proportional to diameter or cross-sectional area of muscles \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Unexpectedly, the slow muscle fibers of \u003cem\u003ePure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e form thick bundles by 5 dpf (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D), which may underlie the improved locomotion.\u003c/p\u003e \u003cp\u003eThe morphological alterations in slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e are consistent with results of previous studies suggesting that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e signaling plays an important role in the development of slow muscle cells \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Pharmacological inhibition of AChRs or RyRs in early developmental stages induced abnormal organization in slow muscle fibers \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The authors suggested that the slow muscle fibers were not aligned and they could not form thick bundles. Cheung \u003cem\u003eet al\u003c/em\u003e. (2011) also reported that RyR antagonist treatment during early developmental stages disrupted banding of slow muscle fibers \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, the elevation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels during embryonic stages is important for the development of slow muscle cells, and the source of the Ca\u003csup\u003e2+\u003c/sup\u003e is the SR. The results of the present study suggested that not only Ca\u003csup\u003e2+\u003c/sup\u003e stores in the SR but also the influx of extracellular Ca\u003csup\u003e2+\u003c/sup\u003e through AChRs contributes to the thickening of slow muscle cells. The Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may be required to stimulate Ca\u003csup\u003e2+\u003c/sup\u003e release from RyRs to promote the growth of slow muscle cells.\u003c/p\u003e \u003cp\u003eAdditionally, Brennan \u003cem\u003eet al\u003c/em\u003e. (2005) reported that the slow muscle fibers of mutant zebrafish lacking AChRs tend to be longer than those of wild-type zebrafish because of abnormal organization of myofibrils \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Interestingly, in the present study, slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e also became longer at the 5 dpf (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Taken together, Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may play a critical role in the proper development of slow muscle cells during early developmental stages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux through AChRs contributes to slow muscle contraction.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, locomotor activities of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were markedly decreased at the 2 dpf. One possible reason we proposed above for the loss of motor function is that disruptions in the development of slow muscle cells result in thinner slow muscle fibers, leading in turn to motor dysfunction. In addition to the morphological effect, we need to consider the possibility that Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs is involved in the physiological process of muscle contraction. In zebrafish, the contraction process of slow muscle cells is different from that of fast muscle cells. Slow muscle cells of zebrafish do not fire action potentials due to the lack of voltage-gated Na\u003csup\u003e+\u003c/sup\u003e channels (Nav) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thus, cation influx through AChRs needs to activate L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels directly (AChRs-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway) to induce Ca\u003csup\u003e2+\u003c/sup\u003e release from the SR. In this process, Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs not only stimulates L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels but may also directly activate RyRs through the Ca\u003csup\u003e2+\u003c/sup\u003e-induced Ca\u003csup\u003e2+\u003c/sup\u003e release (CICR) mechanism, as suggested by the results of a study on the muscle cells of \u003cem\u003eCiona\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may support Ca\u003csup\u003e2+\u003c/sup\u003e release during muscle contraction by these mechanisms. The results of Ca\u003csup\u003e2+\u003c/sup\u003e imaging in the present study showed that the inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs results in a shortened Ca\u003csup\u003e2+\u003c/sup\u003e response. Consequently, RyRs in the SR are possibly unable to release sufficient Ca\u003csup\u003e2+\u003c/sup\u003e for the contractions of slow muscles without the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs. This could be another reason for the reduced locomotor activity in \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e at the 2 dpf stage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux through AChRs becomes less important for slow muscle cells at later stages.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt the 5 dpf stage, slow muscle cells in both \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e and \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e tended to display shorter Ca\u003csup\u003e2+\u003c/sup\u003e response than that at the 2 dpf stage, and there was no significant difference in decay time between those two zebrafish lines. A possible reason for the shortened Ca\u003csup\u003e2+\u003c/sup\u003e response at 5 dpf could be the development of the Ca\u003csup\u003e2+\u003c/sup\u003e reuptake system such as SR Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase (SERCA) and mitochondria. The expression of SERCA changes throughout the developmental process in mammals \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and the expression of SERCA possibly increases between 2 and 5 dpf in zebrafish. Mitochondria also play important roles in reuptake of Ca\u003csup\u003e2+\u003c/sup\u003e in muscle cells. In mice, mitochondria gradually shift their position within the muscle cell during development, positioning themselves close to the SR to effectively reuptake Ca\u003csup\u003e2+\u003c/sup\u003e from the SR \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, it is possible that similar positional changes occurred in zebrafish between 2 and 5 dpf. These developments of Ca\u003csup\u003e2+\u003c/sup\u003e reuptake systems may have shortened the Ca\u003csup\u003e2+\u003c/sup\u003e response in slow muscle cells at 5 dpf.\u003c/p\u003e \u003cp\u003eThe results of locomotor analysis suggested that the Ca\u003csup\u003e2+\u003c/sup\u003e permeability of AChRs becomes less important for the contraction of slow muscles at the 5 dpf stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the slow muscle cells of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-imperm\u003c/em\u003e were still thinner than those of \u003cem\u003epure slow Ca\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-perm\u003c/em\u003e, they become thicker at 5 dpf than at 2 dpf (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, the growth of slow muscle cells may have improved locomotion. The Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may still be important for the development of slow muscle cells at 5 dpf. However, the effect of reduction in the Ca\u003csup\u003e2+\u003c/sup\u003e influx on locomotor activities may be more apparent at 2 dpf, when the muscle cells are thinner.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCa\u003csup\u003e2+\u003c/sup\u003e signaling pathway in slow muscle cells in zebrafish development\u003c/h2\u003e \u003cp\u003eIn addition to development, Ca\u003csup\u003e2+\u003c/sup\u003e entry through AChRs is possibly more important for the contraction process of slow muscle cells at 2 dpf than at 5 dpf. We hypothesize that the mechanism for controlling muscle contraction is not mature at 2 dpf and the immature slow muscle cells therefore require Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs for their functions. Based on the results of previous studies and the results of the present study, we propose that the following series of events occurs in activated immature slow muscle cells. AChRs are first stimulated by ACh from motor neurons. Na\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e influx through the AChRs depolarize membrane potential. L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels are then activated, which in turn triggers opening of RyRs in the SR to release Ca\u003csup\u003e2+\u003c/sup\u003e. Note that electrophysiological analysis has shown that the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel of zebrafish does not allow permeation of Ca\u003csup\u003e2+ 28\u003c/sup\u003e. In this process, the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs may contribute to both the development of slow muscle cells and membrane depolarization. Ca\u003csup\u003e2+\u003c/sup\u003e entry through AChRs may also directly stimulate RyRs and thereby induce Ca\u003csup\u003e2+\u003c/sup\u003e release from the SR probably in the manner of CICR \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The released Ca\u003csup\u003e2+\u003c/sup\u003e facilitates the development of slow muscle cells while inducing muscle contraction. In general, CICR is important for the contraction of cardiac muscles \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, but it is not essential for functions of skeletal muscles. However, in amphibians, previous studies suggested that CICR contributes to the contraction of skeletal muscles. In amphibians, RyRβs (corresponding to RyR3s of mammals) are localized outside the junctions between T-tubules and the SR, and the RyRβs are activated by CICR \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Besides, CICR is observed in embryonic or early postnatal myofibers of mammals. These immature myofibers express RyR3 and exhibit spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e sparks, which represent Ca\u003csup\u003e2+\u003c/sup\u003e release from the SR, independent from functions of the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The Ca\u003csup\u003e2+\u003c/sup\u003e spark is considered to be initiated by CICR because its frequency is increased by elevation of the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e level \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. During development, Ca\u003csup\u003e2+\u003c/sup\u003e sparks are rarely observed in mammalian muscle cells \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e because most RyRs interact with L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels and spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e release from RyRs ceases to occur. In zebrafish, T-tubules are almost fully developed by 2 dpf \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, but the number of RyR1 contacts with the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel is possibly fewer than that in matured animals at this stage. Actually, the expression of RyR1 at 1\u0026ndash;3 dpf stages is less than that in 6 dpf larvae \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Therefore, the AChRs-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway may not be fully developed at 1\u0026ndash;3 dpf stages. Thus, the CICR mechanism might be involved in contractions of muscle cells with the immature AChRs-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway. At 5 dpf, the L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel-RyR complex increases, and the AChRs-L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel pathway becomes mature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eWe are grateful to Ms. Seiko Ohkuma at JMU for her excellent care of zebrafish used in this study. This study was supported by JSPS KAKENHI Grant Numbers 21K15138 and 23K05856 (to B.Z.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHerzog, W., Schappacher-Tilp, G.: Molecular mechanisms of muscle contraction: A historical perspective. J. Biomech. \u003cb\u003e155\u003c/b\u003e, 111659 (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandow, A.: Excitation-contraction coupling in muscular response. Yale J. Biol. Med. \u003cb\u003e25\u003c/b\u003e, 176\u0026ndash;201 (1952)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEgashira, Y., Zempo, B., Sakata, S., Ono, F.: Recent advances in neuromuscular junction research prompted by the zebrafish model. Curr. Opin. Physiol. \u003cb\u003e4\u003c/b\u003e, 70\u0026ndash;75 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C., Sakmann, B.: Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. \u003cb\u003e321\u003c/b\u003e, 406\u0026ndash;411 (1986)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdams, D.J., Dwyer, T.M., Hille, B.: The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol. \u003cb\u003e75\u003c/b\u003e, 493\u0026ndash;510 (1980)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, L.Y., Catterall, W.A., Ehrenstein, G.: Selectivity of cations and nonelectrolytes for acetylcholine-activated channels in cultured muscle cells. J. Gen. Physiol. \u003cb\u003e71\u003c/b\u003e, 397\u0026ndash;410 (1978)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMongeon, R., Walogorsky, M., Urban, J., Mandel, G., Ono, F., Brehm, P.: An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses. J. Gen. Physiol. \u003cb\u003e138\u003c/b\u003e, 353\u0026ndash;366 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZempo, B., Yamamoto, Y., Williams, T., Ono, F.: Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle. Sci. Adv. \u003cb\u003e6\u003c/b\u003e, eaax8382 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, J.-Y., Mott, M., Williams, T., Ikeda, H., Wen, H., Linhoff, M., Ono, F.: A single mutation in the acetylcholine receptor δ-subunit causes distinct effects in two types of neuromuscular synapses. J. Neurosci. \u003cb\u003e34\u003c/b\u003e, 10211\u0026ndash;10218 (2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishino, A., Baba, S.A., Okamura, Y.: A mechanism for graded motor control encoded in the channel properties of the muscle ACh receptor. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e108\u003c/em\u003e, 2599\u0026ndash;2604. (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaghighi, A.P., Cooper, E.: A molecular link between inward rectification and calcium permeability of neuronal nicotinic acetylcholine alpha3beta4 and alpha4beta2 receptors. J. Neurosci. \u003cb\u003e20\u003c/b\u003e, 529\u0026ndash;541 (2000)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertrand, D., Galzi, J.L., Devillers-Thi\u0026eacute;ry, A., Bertrand, S., Changeux, J.P.: Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e90\u003c/em\u003e, 6971\u0026ndash;6975. (1993)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElworthy, S., Hargrave, M., Knight, R., Mebus, K., Ingham, P.W.: Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity. Development. \u003cb\u003e135\u003c/b\u003e, 2115\u0026ndash;2126 (2008)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrasaki, A., Asakawa, K., Kawakami, K.: Efficient transposition of the \u003cem\u003eTol2\u003c/em\u003e transposable element from a single-copy donor in zebrafish. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e105\u003c/em\u003e, 19827\u0026ndash;19832. (2008)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakano, Y., Fujita, M., Ogino, K., Saint-Amant, L., Kinoshita, T., Oda, Y., Hirata, H.: Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation. Development. \u003cb\u003e137\u003c/b\u003e, 1689\u0026ndash;1698 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDana, H., Mohar, B., Sun, Y., Narayan, S., Gordus, A., Hasseman, J.P., Tsegaye, G., Holt, G.T., Hu, A., Walpita, D., et al.: Sensitive red protein calcium indicators for imaging neural activity. Elife \u003cem\u003e5\u003c/em\u003e. (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.12727\u003c/span\u003e\u003cspan address=\"10.7554/eLife.12727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHousley, M.P., Reischauer, S., Dieu, M., Raes, M., Stainier, D.Y.R., Vanhollebeke, B.: Translational profiling through biotinylation of tagged ribosomes in zebrafish. Development. \u003cb\u003e141\u003c/b\u003e, 3988\u0026ndash;3993 (2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOno, F., Mandel, G., Brehm, P.: Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish. J. Neurosci. \u003cb\u003e24\u003c/b\u003e, 5475\u0026ndash;5481 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOno, F., Higashijima, S., Shcherbatko, A., Fetcho, J.R., Brehm, P.: Paralytic zebrafish lacking acetylcholine receptors fail to localize rapsyn clusters to the synapse. J. Neurosci. \u003cb\u003e21\u003c/b\u003e, 5439\u0026ndash;5448 (2001)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrivickas, L.S., Dorer, D.J., Ochala, J., Frontera, W.R.: Relationship between force and size in human single muscle fibres: Muscle fibre size and force. Exp. Physiol. \u003cb\u003e96\u003c/b\u003e, 539\u0026ndash;547 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelu, J.J., Chan, H.L.H., Webb, S.E., Cheng, A.H.H., Ruas, M., Parrington, J., Galione, A., Miller, A.L.: Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca(2+) signaling during myogenesis in intact zebrafish. Int. J. Dev. Biol. \u003cb\u003e59\u003c/b\u003e, 313\u0026ndash;325 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung, C.Y., Webb, S.E., Love, D.R., Miller, A.L.: Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos. Int. J. Dev. Biol. \u003cb\u003e55\u003c/b\u003e, 153\u0026ndash;174 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrennan, C., Mangoli, M., Dyer, C.E.F., Ashworth, R.: Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo. J. Cell. Sci. \u003cb\u003e118\u003c/b\u003e, 5181\u0026ndash;5190 (2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuckingham, S.D., Ali, D.W.: Sodium and potassium currents of larval zebrafish muscle fibres. J. Exp. Biol. \u003cb\u003e207\u003c/b\u003e, 841\u0026ndash;852 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnger, M., Samuel, J.L., Marotte, F., Wuytack, F., Rappaport, L., Lompr\u0026eacute;, A.M.: In situ mRNA distribution of sarco(endo)plasmic reticulum Ca(2+)-ATPase isoforms during ontogeny in the rat. J. Mol. Cell. Cardiol. \u003cb\u003e26\u003c/b\u003e, 539\u0026ndash;550 (1994)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeriasamy, M., Kalyanasundaram, A.: SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve. \u003cb\u003e35\u003c/b\u003e, 430\u0026ndash;442 (2007)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoncompagni, S., Rossi, A.E., Micaroni, M., Beznoussenko, G.V., Polishchuk, R.S., Dirksen, R.T., Protasi, F.: Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures. Mol. Biol. Cell. \u003cb\u003e20\u003c/b\u003e, 1058\u0026ndash;1067 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchredelseker, J., Shrivastav, M., Dayal, A., Grabner, M.: Non-Ca2+-conducting Ca2\u0026thinsp;+\u0026thinsp;channels in fish skeletal muscle excitation-contraction coupling. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e107\u003c/em\u003e, 5658\u0026ndash;5663. (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEndo, M.: Calcium-induced calcium release in skeletal muscle. Physiol. Rev. \u003cb\u003e89\u003c/b\u003e, 1153\u0026ndash;1176 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026iacute;os, E.: Calcium-induced release of calcium in muscle: 50 years of work and the emerging consensus. J. Gen. Physiol. \u003cb\u003e150\u003c/b\u003e, 521\u0026ndash;537 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelPrincipe, F., Egger, M., Niggli, E.: Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation. Nat. Cell. Biol. \u003cb\u003e1\u003c/b\u003e, 323\u0026ndash;329 (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelder, E., Franzini-Armstrong, C.: Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e99\u003c/em\u003e, 1695\u0026ndash;1700. (2002)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShirokova, N., Garc\u0026iacute;a, J., Pizarro, G., R\u0026iacute;os, E.: Ca2\u0026thinsp;+\u0026thinsp;release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle. J. Gen. Physiol. \u003cb\u003e107\u003c/b\u003e, 1\u0026ndash;18 (1996)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConklin, M.W., Barone, V., Sorrentino, V., Coronado, R.: Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle. Biophys. J. \u003cb\u003e77\u003c/b\u003e, 1394\u0026ndash;1403 (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, J., Launikonis, B.S., R\u0026iacute;os, E., Brum, G.: Regulation of Ca2\u0026thinsp;+\u0026thinsp;sparks by Ca2\u0026thinsp;+\u0026thinsp;and Mg2\u0026thinsp;+\u0026thinsp;in mammalian and amphibian muscle. An RyR isoform-specific role in excitation-contraction coupling? J. Gen. Physiol. \u003cb\u003e124\u003c/b\u003e, 409\u0026ndash;428 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, L.D., Rodney, G.G., Hern\u0026aacute;ndez-Ochoa, E., Ward, C.W., Schneider, M.F.: Ca2\u0026thinsp;+\u0026thinsp;sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers. Am. J. Physiol. Cell. Physiol. \u003cb\u003e292\u003c/b\u003e, C1156\u0026ndash;C1166 (2007)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, J., Yi, J., Royer, L., Launikonis, B.S., Gonz\u0026aacute;lez, A., Garc\u0026iacute;a, J., R\u0026iacute;os, E.: A probable role of dihydropyridine receptors in repression of Ca2\u0026thinsp;+\u0026thinsp;sparks demonstrated in cultured mammalian muscle. Am. J. Physiol. Cell. Physiol. \u003cb\u003e290\u003c/b\u003e, C539\u0026ndash;C553 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall, T.E., Martel, N., Ariotti, N., Xiong, Z., Lo, H.P., Ferguson, C., Rae, J., Lim, Y.-W., Parton, R.G.: In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation. Nat. Commun. \u003cb\u003e11\u003c/b\u003e, 3711 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, H.H., Brennan, C., Ashworth, R.: Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development. BMC Res. Notes. \u003cb\u003e4\u003c/b\u003e, 541 (2011)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6525282/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6525282/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca\u003csup\u003e2+\u003c/sup\u003e permeability compared to that of AChRs of fast muscles. To analyze the significance of the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca\u003csup\u003e2+\u003c/sup\u003e-impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1-3 days post fertilization (dpf) compared to those of zebrafish expressing Ca\u003csup\u003e2+\u003c/sup\u003e-permeable AChRs in its slow muscles. Ca\u003csup\u003e2+\u003c/sup\u003e imaging suggested that Ca\u003csup\u003e2+\u003c/sup\u003e influx via AChRs is crucial for the Ca\u003csup\u003e2+\u003c/sup\u003e response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca\u003csup\u003e2+ \u003c/sup\u003eresponse. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca\u003csup\u003e2+\u003c/sup\u003e-permeable AChRs. These short Ca\u003csup\u003e2+\u003c/sup\u003e responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca\u003csup\u003e2+\u003c/sup\u003e influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles.\u003c/p\u003e","manuscriptTitle":"Ca2+ influx through muscle-type nicotinic AChRs contributes to contractions and development of slow muscle cells in early developmental stages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 11:46:57","doi":"10.21203/rs.3.rs-6525282/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecff97f2-61f5-4875-8c57-31dc10ffb6f3","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48568774,"name":"Biological sciences/Physiology/Neurophysiology"},{"id":48568775,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-06-03T13:21:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 11:46:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6525282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6525282","identity":"rs-6525282","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}