Neuromuscular Transmission in a Biological Context.

Neuromuscular transmission is the process by which motor neurons activate muscle contraction and thus plays an essential role in generating the purposeful body movements that aid survival. While many features of this process are common throughout the Animal Kingdom, such as the release of transmitter in multimolecular "quanta," and the response to it by opening ligand-gated postsynaptic ion channels, there is also much diversity between and within species. Much of this diversity is associated with specialization for either slow, sustained movements such as maintain posture or fast but brief movements used during escape or prey capture. In invertebrates, with hydrostatic and exoskeletons, most motor neurons evoke graded depolarizations of the muscle which cause graded muscle contractions. By contrast, vertebrate motor neurons trigger action potentials in the muscle fibers which give rise to all-or-none contractions. The properties of neuromuscular transmission, in particular the intensity and persistence of transmitter release, reflect these differences. Neuromuscular transmission varies both between and within individual animals, which often have distinct tonic and phasic subsystems. Adaptive plasticity of neuromuscular transmission, on a range of time scales, occurs in many species. This article describes the main steps in neuromuscular transmission and how they vary in a number of "model" species, including C. elegans , Drosophila , zebrafish, mice, and humans. © 2024 American Physiological Society. Compr Physiol 14:5641-5702, 2024.

Wirelessly steerable bioelectronic neuromuscular robots adapting neurocardiac junctions.

Biological motions of native muscle tissues rely on the nervous system to interface movement with the surrounding environment. The neural innervation of muscles, crucial for regulating movement, is the fundamental infrastructure for swiftly responding to changes in body tissue requirements. This study introduces a bioelectronic neuromuscular robot integrated with the motor nervous system through electrical synapses to evoke cardiac muscle activities and steer robotic motion. Serving as an artificial brain and wirelessly regulating selective neural activation to initiate robot fin motion, a wireless frequency multiplexing bioelectronic device is used to control the robot. Frequency multiplexing bioelectronics enables the control of the robot locomotion speed and direction by modulating the flapping of the robot fins through the wireless motor innervation of cardiac muscles. The robots demonstrated an average locomotion speed of ~0.52 ± 0.22 millimeters per second, fin-flapping frequency up to 2.0 hertz, and turning locomotion path curvature of ~0.11 ± 0.04 radians per millimeter. These systems will contribute to the expansion of biohybrid machines into the brain-to-motor frontier for developing autonomous biohybrid systems capable of advanced adaptive motor control and learning.

Incorporation of decellularized-ECM in graphene-based scaffolds enhances axonal outgrowth and branching in neuro-muscular co-cultures.

Peripheral nerve and large-scale muscle injuries result in significant disability, necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials, graphene is favored for its high conductivity, but its low bioactivity requires enhancement through biomimetic components. In this study, we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds, aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia, observing significant improvements in axon outgrowth. Next, we tested the scaffolds with C2C12 mouse and human primary myoblasts, finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally, using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts, we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration, offering robust support for the growth and development of diverse neuromuscular tissues.

Long-chain n-3 polyunsaturated fatty acid supplementation and neuromuscular function in older adults.

PURPOSE OF REVIEW: This review aims to explore the latest research investigating the effects of marine-derived long-chain n -3 polyunsaturated fatty acid (LC n -3 PUFA) supplementation on neuromuscular function in older adults. RECENT FINDINGS: Ageing results in a decline in skeletal muscle strength and mass. There is growing evidence that LC n -3 PUFA supplementation increases muscle strength and mass in healthy older adults, yet the mechanisms underlying these effects remain elusive. Recent studies investigating LC n -3 PUFA supplementation have demonstrated effects on neuromuscular function such as increases in the compound muscle action potential (M-wave) amplitude and surface electromyography alongside increases in muscular strength. Therefore, evidence suggests that LC n -3 PUFA may elicit a beneficial effect at the neuromuscular junction and possess neuroprotective properties in older adults. SUMMARY: LC n -3 PUFA supplementation may increase or maintain neuromuscular function throughout the ageing process. Further research is warranted to investigate the long-term effects LC n -3 PUFA supplementation on neuromuscular outcomes such as single motor unit properties and cortical/supraspinal networks, utilizing state-of-the-art techniques in neuromuscular physiology.

Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6 months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post-injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s-1, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5 days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Transplantation of embryonic spinal motor neurons into peripheral nerves enables functional reconstruction of a denervated diaphragm.

Respiratory muscle paralysis due to trauma or neurodegenerative diseases can have devastating consequences. Only a few studies have investigated the reconstruction of motor function in denervated diaphragms caused by such conditions. Here, we studied the efficacy of transplanting E14 embryonic spinal motor neurons (SMNs) into peripheral nerve grafts for functionally reconstructing a denervated diaphragm in a rat model. The diaphragms of 8-week-old male Fischer 344 rats were first denervated by transecting the phrenic nerves. Subsequently, peripheral nerve grafts taken from the lower limb were used for neurotization of the denervated diaphragms. One week later, fetal E14 SMNs were transplanted into the peripheral nerve grafts. After 3 months, we observed functional contraction of the diaphragm following neuromuscular electrical stimulation (NMES) of the peripheral nerve graft. Additionally, we confirmed that SMN transplantation into the peripheral nerve graft had an inhibitory effect on diaphragm muscle atrophy. The SMNs transplanted into the peripheral nerve grafts formed a structure similar to the spinal cord, and the neuromuscular junction of the denervated diaphragm was reinnervated. These findings suggest the establishment of an ectopic motor neuron pool in the peripheral nerve graft. Free peripheral intra-nerve SMN transplantation in combination with NMES, which can be applied for diaphragmatic pacing, offers novel insights into the development of neuroregenerative therapies for treating life-threatening and intractable respiratory muscle paralysis caused by severe nerve damage and degenerative diseases.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

Mechanisms of stationary voltage fluctuation in the neuromuscular junction endplate and corresponding denoising paradigms.

The neuromuscular junction (NMJ) has an elaborate anatomy to ensure agile and accurate signal transmission. Based on our formerly obtained expressions of the thermal and conductance induced voltage fluctuations, in this paper, the mechanisms underlying the conductance-induced voltage fluctuation are characterized from two aspects: the scaling laws with respect to either of the two system-size factors, the number of receptors or the membrane area; and the "seesaw effect" with respect to the intensive parameter, the concentration of acetylcholine. According to these mechanisms, several aspects of the NMJ anatomy are explained from a denoising perspective. Finally, the power spectra of the two types of voltage fluctuations are characterized by their specific scaling laws, based on which we explain why the endplate noise has the low-frequency property that is described by the term "seashell sound".

Beadex, the Drosophila LIM only protein, is required for the growth of the larval neuromuscular junction.

The appropriate growth of the neurons, accurate organization of their synapses, and successful neurotransmission are indispensable for sensorimotor activities. These processes are highly dynamic and tightly regulated. Extensive genetic, molecular, physiological, and behavioral studies have identified many molecular candidates and investigated their roles in various neuromuscular processes. In this article, we show that Beadex (Bx), the Drosophila LIM only (LMO) protein, is required for motor activities and neuromuscular growth of Drosophila. The larvae bearing Bx7, a null allele of Bx, and the RNAi-mediated neuronal-specific knockdown of Bx show drastically reduced crawling behavior, a diminished synaptic span of the neuromuscular junctions (NMJs) and an increased spontaneous neuronal firing with altered motor patterns in the central pattern generators (CPGs). Microarray studies identified multiple targets of Beadex that are involved in different cellular and molecular pathways, including those associated with the cytoskeleton and mitochondria that could be responsible for the observed neuromuscular defects. With genetic interaction studies, we further show that Highwire (Hiw), a negative regulator of synaptic growth at the NMJs, negatively regulates Bx, as the latter's deficiency was able to rescue the phenotype of the Hiw null mutant, HiwDN. Thus, our data indicate that Beadex functions downstream of Hiw to regulate the larval synaptic growth and physiology.NEW & NOTEWORTHY A novel role for Beadex (Bx) regulates the larval neuromuscular junction (NMJ) structure and function in a tissue-specific manner. Bx is expressed in a subset of Toll-6-expressing neurons and is involved in regulating synaptic span and physiology, possibly through its negative interaction with Highwire (Hiw). The findings of this study provide insights into the molecular mechanisms underlying NMJ development and function and warrant further investigation to understand the role of Bx in these processes fully.

[Effects of different exercise modes on neuromuscular junction and metabolism of skeletal muscle-related proteins in aging rats].

The present study aimed to explore the effects of different exercise modes on neuromuscular junction (NMJ) and metabolism of skeletal muscle-related proteins in aging rats. Ten from 38 male Sprague-Dawley (SD) rats (3-month-old) were randomly selected into young (Y) group, while the rest were raised to 21 months old and randomly divided into elderly control (O), endurance exercise (EN) and resistance exercise (R) groups. After 8 weeks of corresponding exercises training, the gastrocnemius muscles of rats were collected, and the expression of S100B in Schwann cells was detected by immunofluorescence staining. Western blot was used to detect the protein expression levels of agglutinate protein (Agrin), low-density lipoprotein receptor-related protein 4 (Lrp4), muscle- specific kinase protein (MuSK), downstream tyrosine kinase 7 (Dok7), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target rapamycin (p-mTOR), and phosphorylated forkhead box O1 (p-FoxO1) in rat gastrocnemius muscles. The results showed that, endurance and resistance exercises increased the wet weight ratio of gastrocnemius muscle in the aging rats. The protein expression of S100B in the R group was significantly higher than those in the O and EN groups. Proteins related to NMJ function, including Agrin, Lrp4, MuSK, and Dok7 were significantly decreased in the O group compared with those in the Y group. Resistance exercise up-regulated these four proteins in the aging rats, whereas endurance exercise could not reverse the protein expression levels of Lrp4, MuSK and Dok7. Regarding skeletal muscle-related proteins, the O group showed down-regulated p-Akt, and p-mTOR protein expression levels and up-regulated p-FoxO1 protein expression level, compared to the Y group. Resistance and endurance exercises reversed the changes in p-mTOR and p-FoxO1 protein expression in the aging rats. These findings demonstrate that both exercise modes can enhance NMJ function, increase protein synthesis and reduce the catabolism of skeletal muscle-related proteins in aging rats, with resistance exercise showing a more pronounced effect.

Extreme Tolerance of Extraocular Muscles to Diseases and Aging: Why and How?

The extraocular muscles (EOMs) possess unique characteristics that set them apart from other skeletal muscles. These muscles, responsible for eye movements, exhibit remarkable resistance to various muscular dystrophies and aging, presenting a significant contrast to the vulnerability of skeletal muscles to these conditions. In this review, we delve into the cellular and molecular underpinnings of the distinct properties of EOMs. We explore their structural complexity, highlighting differences in fiber types, innervation patterns, and developmental origins. Notably, EOM fibers express a diverse array of myosin heavy-chain isoforms, retaining embryonic forms into adulthood. Moreover, their motor innervation is characterized by a high ratio of nerve fibers to muscle fibers and the presence of unique neuromuscular junctions. These features contribute to the specialized functions of EOMs, including rapid and precise eye movements. Understanding the mechanisms behind the resilience of EOMs to disease and aging may offer insights into potential therapeutic strategies for treating muscular dystrophies and myopathies affecting other skeletal muscles.

PTPN11/Corkscrew Activates Local Presynaptic Mapk Signaling to Regulate Synapsin, Synaptic Vesicle Pools, and Neurotransmission Strength, with a Dual Requirement in Neurons and Glia.

Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models.

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

Ca2+ imaging of synaptic compartments using subcellularly targeted GCaMP8f in Drosophila.

GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail "quantal" imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4.

The Mechanism of α2 adrenoreceptor-dependent Modulation of Neurotransmitter Release at the Neuromuscular Junctions.

α2-Adrenoreceptors (ARs) are main Gi-protein coupled autoreceptors in sympathetic nerve terminals and targets for dexmedetomidine (DEX), a widely used sedative. We hypothesize that α2-ARs are also potent regulators of neuromuscular transmission via G protein-gated inwardly rectifying potassium (GIRK) channels. Using extracellular microelectrode recording of postsynaptic potentials, we found DEX-induced inhibition of spontaneous and evoked neurotransmitter release as well as desynchronization of evoked exocytotic events in the mouse diaphragm neuromuscular junction. These effects were suppressed by SKF-86,466, a selective α2-AR antagonist. An activator of GIRK channels ML297 had the same effects on neurotransmitter release as DEX. By contrast, inhibition of GIRK channels with tertiapin-Q prevented the action of DEX on evoked neurotransmitter release, but not on spontaneous exocytosis. The synaptic vesicle exocytosis is strongly dependent on Ca2+ influx through voltage-gated Ca2+ channels (VGCCs), which can be negatively regulated via α2-AR - GIRK channel axis. Indeed, inhibition of P/Q-, L-, N- or R-type VGCCs prevented the inhibitory action of DEX on evoked neurotransmitter release; antagonists of P/Q- and N-type channels also suppressed the DEX-mediated desynchronization of evoked exocytotic events. Furthermore, inhibition of P/Q-, L- or N-type VGCCs precluded the frequency decrease of spontaneous exocytosis upon DEX application. Thus, α2-ARs acting via GIRK channels and VGCCs (mainly, P/Q- and N-types) exert inhibitory effect on the neuromuscular communication by attenuating and desynchronizing evoked exocytosis. In addition, α2-ARs can suppress spontaneous exocytosis through GIRK channel-independent, but VGCC-dependent pathway.

Investigating postsynaptic effects of a Drosophila neuropeptide on muscle contraction.

The Drosophila neuropeptide, DPKQDFMRFamide, was previously shown to enhance excitatory junctional potentials (EJPs) and muscle contraction by both presynaptic and postsynaptic actions. Since the peptide acts on both sides of the synaptic cleft, it has been difficult to examine postsynaptic modulatory mechanisms, particularly when contractions are elicited by nerve stimulation. Here, postsynaptic actions are examined in 3rd instar larvae by applying peptide and the excitatory neurotransmitter, l-glutamate, in the bathing solution to elicit contractions after silencing motor output by removing the central nervous system (CNS). DPKQDFMRFamide enhanced glutamate-evoked contractions at low concentrations (EC50 1.3 nM), consistent with its role as a neurohormone, and the combined effect of both substances was supra-additive. Glutamate-evoked contractions were also enhanced when transmitter release was blocked in temperature-sensitive (Shibire) mutants, confirming the peptide's postsynaptic action. The peptide increased membrane depolarization in muscle when co-applied with glutamate, and its effects were blocked by nifedipine, an L-type channel blocker, indicating effects at the plasma membrane involving calcium influx. DPKQDFMRFamide also enhanced contractions induced by caffeine in the absence of extracellular calcium, suggesting increased calcium release from the sarcoplasmic reticulum (SR) or effects downstream of calcium release from the SR. The peptide's effects do not appear to involve calcium/calmodulin-dependent protein kinase II (CaMKII), previously shown to mediate presynaptic effects. The approach used here might be useful for examining postsynaptic effects of neurohormones and cotransmitters in other systems.NEW & NOTEWORTHY Distinguishing presynaptic and postsynaptic effects of neurohormones is a long-standing challenge in many model organisms. Here, postsynaptic actions of DPKQDFMRFamide are demonstrated by assessing its ability to potentiate contractions elicited by direct application of the neurotransmitter, glutamate, when axons are silent and when transmitter release is blocked. The peptide acts at multiple sites to increase contraction, increasing glutamate-induced depolarization at the cell membrane, acting on L-type channels, and acting downstream of calcium release from the sarcoplasmic reticulum.

Immunohistochemical Analysis of the Drosophila Larval Neuromuscular Junction.

Synapses are specialized junctions between cells that mediate neurotransmission to modify brain activity and body function. Studies on synapse structure and function play an important role in understanding how neurons communicate and the consequences of their dysfunction in neurological disorders. The Drosophila larval neuromuscular junction is an excellent model for dissecting the cellular and molecular mechanisms of the synapse, with its large size, accessibility, and well-characterized genetics. This protocol describes the steps required for morphological and immunohistochemical analysis of the Drosophila larval neuromuscular junction including its dissection and multiplex labeling of synaptic proteins. This technique can be used to assess the impact of genetic manipulations on synaptic development, integrity, and plasticity, thus providing a valuable tool for probing complex neurological processes in a whole animal system.

NMJ Analyser: a novel method to quantify neuromuscular junction morphology in zebrafish.

MOTIVATION: Neuromuscular junction (NMJ) structural integrity is crucial for transducing motor neuron signals that initiate skeletal muscle contraction. Zebrafish has emerged as a simple and efficient model to study NMJ structural morphology and function in the context of developmental neurobiology and neuromuscular diseases. However, methods to quantify NMJ morphology from voluminous data of NMJ confocal images accurately, rapidly, and reproducibly are lacking. RESULTS: We developed an ImageJ macro called "NMJ Analyser" to automatically and unbiasedly analyse NMJ morphology in zebrafish. From the Z-stack of a zebrafish hemisomite, both presynaptic and postsynaptic fluorescently labeled termini at NMJs are extracted from background signal, with larger clusters of termini being segmented into individual termini using an unbiased algorithm. The program then determines whether each presynaptic terminus is co-localized with a postsynaptic terminus and vice versa, or whether it is orphaned, and tabulates the number of orphan and co-localized pre- and postsynaptic termini. The usefulness of this ImageJ macro plugin will be helpful to quantify NMJ parameters in zebrafish, particularly during development and in disease models of neuromuscular diseases. It can enable high-throughput NMJ phenotypic screens in the drug discovery process for neuromuscular diseases. It could also be further applied to the investigation of NMJ of other developmental systems. AVAILABILITY AND IMPLEMENTATION: NMJ Analyser is available for download at https://github.com/PattenLab/NMJ-Analyser.git.