The neuromuscular junction (NMJ) is a chemical synapse that is present between nerve cells and muscle cells in the peripheral nervous system (PNS), mainly deep within skeletal muscle tissue. Structurally, in addition to motor neurons (pre-synapse) and muscle cells (post-synapse), terminal Schwann cells in contact with them at the synaptic cleft also serve as major components of the NMJ. The presynaptic nerve terminals contain numerous synaptic vesicles filled with the neurotransmitter acetylcholine (ACh). The postsynaptic muscle membrane is densely packed with ACh receptors (AChRs) and has a highly folded structure due to specialized proteins such as agrin, MuSK, and rapsyn, which play important roles in NMJ formation and maintenance [1-3]. When an action potential reaches the nerve terminal, ACh is released into the synaptic cleft, which contains a special basal lamina that anchors acetylcholinesterase (AChE) and binds to AChRs on the muscle fiber. This interaction ultimately leads to muscle contraction. AChE quickly hydrolyzes ACh, thus terminating synaptic transmission and facilitating the recycling of neurotransmitter signals through the NMJ [4]. Peripheral neuropathies, including congenital myasthenic syndrome (CMS) and myasthenia gravis, are well-known diseases associated with the NMJ that result in abnormalities in neurotransmitter release or signal transmission [5]. Damage to the PNS is particularly severe, as it leads not only to neurological deficits but also to broader issues affecting bodily movement.

Studies using zebrafish as a model for NMJ research are increasingly reported in biomedical literature [6,7]. The zebrafish model is particularly appealing for NMJ disease research because it allows for relatively easy observation of NMJ formation and function in vivo during embryonic and larval stages [8,9]. Above all, the high degree of similarity between human and zebrafish NMJ-related disease genes significantly strengthens its relevance [10-13]. These advantages make zebrafish an effective in vivo platform for high-throughput drug screening and for investigating disease mechanisms. By integrating advanced imaging technologies and genetic manipulation tools, research using the zebrafish model has accelerated our understanding of NMJ disease mechanisms and the development of targeted treatments. The aim of this review is to present practical methods for studying the functions of genes in NMJ diseases and assessing the efficacy of therapeutic candidates in zebrafish, drawing on data from recent publications. It also aims to identify and discuss necessary improvements to address the limitations of the zebrafish model.

The zebrafish PNS shows structural and functional homology with humans, particularly in the organization and development of motor neurons, sensory neurons, and NMJs [14]. Both humans and zebrafish share a conserved basic structure of the NMJ, which consists of presynaptic motor neuron terminals, a synaptic cleft, and highly specialized postsynaptic muscle fibers. In addition to structural similarities, zebrafish are suitable for neuromuscular disease modeling due to their high similarity in NMJ component proteins and NMJ-related disease genes [6,15,16].

Despite these similarities, human NMJs generally exhibit mono-neuronal innervation during the early developmental stages, whereas zebrafish NMJs initially exhibit multi-neuronal innervation and undergo synaptic pruning during development [9,17]. Furthermore, the regenerative capacity of the nervous system, including the repair and remodeling of damaged NMJs, is generally more robust in zebrafish than in humans. This robustness provides a significant advantage for studies on neuronal regeneration and synaptic plasticity [18]. Therefore, zebrafish, with their similarities in human NMJ structure and function and their specific advantages as an experimental animal model, are attracting increasing attention in research on the pathogenesis of NMJ-related diseases and the development of treatments.

The experimental advantage of zebrafish in NMJ studies lies in the ease of in vivo visualization of NMJ development and function. This is achievable through both standard and advanced fluorescence microscopy techniques at embryonic and larval stages [9,19]. Additionally, real-time imaging of synaptic remodeling, axon guidance, and neurotransmission can be conducted in living organisms—experiments that are challenging to perform in mammalian models [20]. Another experimental advantage of zebrafish is that many offspring can be obtained through in vitro fertilization and embryo development is rapid. This allows large-scale genetic and chemical screening, enabling rapid and simple processing and screening of a variety of candidate therapeutics for NMJ-related diseases that currently lack targeted therapies.

Several examples of research on NMJ diseases using zebrafish models have been reported [12,21,22]. For instance, the use of zebrafish is increasing in research on the pathogenesis of Charcot-Marie-Tooth (CMT) disease [11-13,23,24]. Details related to NMJ disease modeling are discussed in detail in the next section. Previous studies have suggested that zebrafish are not only useful for studying disease mechanisms due to the anatomical similarity of the NMJ to humans, but also for evaluating drug efficacy, as drug responses in zebrafish are similar to those in humans [23,25]. Collectively, the benefits of embryonic optical transparency, rapid development, and species-specific regenerative capabilities, combined with genetic and anatomical similarities to humans, have established zebrafish as a preferred model for studying NMJ diseases. In this review, we aim to emphasize the utility of zebrafish in understanding the pathology of NMJ diseases at the molecular level, in verifying the physiological phenomena that underpin these mechanisms, and in their application as a disease model.

Zebrafish have been widely used to genetically investigate the pathogenesis of various diseases, including those affecting the NMJ. Researchers have induced mutations in genes associated with NMJ diseases in zebrafish to better understand the functions linked to each disease mechanism and to explore their potential as targets for therapeutic intervention [26]. For instance, a zebrafish model has been developed that expresses mutations in genes associated with CMT disease, such as ganglioside-induced differentiation-associated protein 1 (GDAP1) and mitofusin 2 (MFN2) [11-13]. These genetically engineered zebrafish display marked motor deficits and NMJ abnormalities, a phenotype very similar to that of CMT patients [11-13]. Additionally, zebrafish models of CMS have been created by targeting genes involved in NMJ formation and function, such as docking protein 7 (DOK7) and AGRIN [27,28]. These zebrafish models effectively mimic the CMS phenotype, including reduced motility, altered NMJ morphology, and impaired synaptic transmission [27,28].

In addition to genetic models, zebrafish can also be used to elucidate the pathological mechanisms of NMJ diseases through chemical treatment and injury induction. Neurotoxins such as botulinum toxin, organophosphorus compounds, and caffeine have been effectively employed to induce transient NMJ dysfunction in zebrafish. This approach allows researchers to investigate the mechanisms of synapse destruction and subsequent recovery in vivo [29-32]. These toxin-based zebrafish models are valuable for studying the plasticity of the neuromuscular system and for screening potential therapeutics that could accelerate NMJ regeneration.

Zebrafish models of acquired myasthenia gravis have been developed through exposure to AChE inhibitors or by manipulating AChR expression using a chemical induction system [33]. These models have facilitated studies on antibody-mediated NMJ control mechanisms, akin to those conducted using mouse models [34,35]. Additionally, zebrafish models created by inducing physical injuries, such as spinal cord transections, are employed in research focused on NMJ remodeling and regeneration post-injury [36]. The unique nerve regeneration capabilities of zebrafish are particularly advantageous for exploring the molecular and cellular mechanisms that support NMJ repair and functional recovery.

A variety of sophisticated methods have been developed and further optimized to investigate NMJ dysfunction in zebrafish models. The combined use of these techniques enables a comprehensive analysis of NMJ structure, function, and dynamics: (1) Immunohistochemistry of NMJ: High-resolution imaging of NMJ structures is achieved through immunostaining techniques, such as fluorescently conjugated α-bungarotoxin for AChR visualization and antibodies against synaptic vesicle proteins for presynaptic staining [15,23,37]. These methods facilitate a detailed examination of NMJ structures, including the assessment of AChR cluster size, density, and distribution, as well as presynaptic terminal morphology. (2) Behavioral analysis: Quantitative analysis of motor function and coordination is conducted using a variety of complex behavioral tests, including touch-evoked escape response, spontaneous swimming analysis, and fine motor control assessment [23]. These analyses provide a valuable readout of NMJ function and can reveal subtle motor deficits associated with NMJ dysfunction. (3) Electrophysiological testing: NMJ function is assessed at the cellular level using advanced electrophysiological techniques, including patch clamp recordings and extracellular field potential measurements [38]. These methods allow accurate quantification of synaptic transmission parameters, such as miniature endplate potential frequency and amplitude, quantum content, and muscle fiber excitability [38]. Electrophysiological analysis is particularly useful in identifying subtle functional abnormalities that may not be apparent from morphological or behavioral studies. (4) In vivo live imaging: Transgenic zebrafish larvae expressing motor neuron- or muscle cell-specific fluorescent proteins can be used to observe axonal outgrowth during neural development or regeneration and muscle patterning in real time [39,40]. Advanced microscopy techniques, such as two-photon microscopy and light-sheet microscopy, enable long-term, high-resolution imaging of NMJ development, maintenance, and remodeling in living animals [39]. This approach could be particularly powerful for studying the progression of NMJ disorders and assessing the efficacy of therapeutic interventions in real time. (5) Molecular and biochemical analyses: RNA sequencing for transcriptome analysis, proteomics to identify alterations in protein expression and post-translational modifications, and clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 genome editing to generate precise genetic modifications are molecular and biochemical approaches for studying NMJ in zebrafish [41,42]. These approaches may provide important clues about the molecular pathways and processes resulting in NMJ disorders, thereby guiding the development of targeted treatment strategies.

By integrating various methods applicable to zebrafish models, it is possible to comprehensively investigate the multifaceted characteristics of NMJ disorders, ranging from genetic and molecular alterations to functional and behavioral abnormalities of the NMJ (Fig. 1).

Zebrafish have been extensively utilized as an in vivo system for high-throughput drug screening to identify therapeutic candidates, as well as for physiological studies exploring various pathologies of hereditary motor neuron diseases caused by NMJ abnormalities [25,43-45]. Indeed, numerous studies have identified potential therapeutic candidates for NMJ diseases by testing a range of compounds that are effective in restoring NMJ formation or function in the zebrafish model. In this review, we summarize (1) the methods used to target causative genes for each NMJ disorder and the approaches taken to develop zebrafish disease models, and (2) the types of therapeutic candidates and in vivo delivery methods that have been employed in each zebrafish model (Table 1) [11-13,23,24,46-56].

Currently, drug screening in zebrafish models primarily involves administering drugs directly into culture dishes or wells, leveraging the aquatic nature of zebrafish. This approach is effective for high-content screening as the drug passively diffuses into the organism. However, this method faces challenges in delivering drugs to specific target tissues, such as the NMJ of the PNS. These limitations can obscure the interpretation of phenotype restoration, making it difficult to determine whether the therapeutic effects of a drug are due to its direct action on NMJs or are the result of broader systemic effects. To address the shortcomings of these drug treatment methods in zebrafish NMJ disease models, several advanced drug delivery techniques may be employed. One promising approach involves the use of targeted drug delivery systems, such as nanoparticles or liposomes, that are engineered to specifically bind to NMJ-related proteins such as AChR [57-59]. These vehicles can be designed to encapsulate drugs and release them precisely at the NMJ, thereby minimizing off-target effects. Another strategy involves the direct administration of drugs into the PNS region of zebrafish using sophisticated microinjection techniques [60]. This method offers the advantage of being able to monitor the drug delivery process through real-time imaging, which helps predict the specific area affected by the drug. The implementation of an automated robotic injection system could further increase drug sensitivity and optimize the concentration for drug administration.

A final drug delivery method worth considering is photoactivated drug design, in which the drug remains inactive until it is activated by light of a specific wavelength. This approach allows for precise spatial and temporal control of drug activation and facilitates highly localized validation in the NMJ region. Integrating these advanced drug delivery methods could revolutionize drug screening and efficacy validation experiments using the zebrafish NMJ model.

In this review, we discuss the practical advantages and utility of zebrafish as a model for NMJ disease. Recent advances in gene editing technologies, ranging from morpholino oligomers to the CRISPR/Cas9 system, have enabled the modification of disease genes in zebrafish. The creation of these gene-targeted zebrafish models has enhanced our understanding of the pathology of gene-mediated NMJ diseases in vivo and has facilitated the development of targeted gene therapies. NMJ diseases, which often involve motility disorders due to abnormalities in nerve transmission, require in vivo models for accurate reproduction and verification of the pathology, as in vitro studies alone are insufficient. Zebrafish models allow for relatively easy induction of NMJ pathology compared to mammals, and the assessment of NMJ structure and function can be conducted quickly and simply.

Zebrafish has become a crucial animal model for high-throughput drug screening, favored for its small size, rapid development, and the simplicity of observing phenotypic changes. High-content screening utilizing this model has facilitated the identification of numerous promising compounds with potential applications in human medicine [23,25,43-45]. The similarity between the structure and function of the zebrafish NMJ and that of humans allows for the selection of candidate substances to treat NMJ diseases through in vivo experiments, providing an advantage over traditional in vitro methods alone.

The integration of advanced genetics and imaging techniques in zebrafish research has enabled the visualization of NMJ dynamics in real time. Transgenic lines facilitating neuron-targeted imaging in the PNS have allowed for real-time observation of NMJ disorders and immediate assessment of drug screening effectiveness [39]. These advancements in imaging and genetic modification have improved the analysis of NMJ formation and function, leveraging the practical strengths of the zebrafish model. The zebrafish fin represents a promising new frontier for future NMJ studies in the PNS. The extensive innervation and remarkable regenerative capabilities of the fin's dorsal structures offer a unique model for exploring NMJ formation, degeneration, and regeneration [21,61]. Therefore, expanding NMJ research from the zebrafish body to the fins could provide fresh insights into the mechanisms underlying NMJ diseases and foster the development of more innovative treatment strategies.

In conclusion, zebrafish are a widely used animal model that facilitates disease modeling through genetic manipulation, analysis of replicated disease phenotypes, and efficient large-scale drug screening. It is particularly valuable in studying the pathogenesis and developing treatments for peripheral neuropathy, including NMJ diseases. However, more sophisticated methods for analyzing NMJ structure and function, particularly physiological analysis at the molecular level, would make the zebrafish an even more effective model for understanding the complexities of NMJ disorders and advancing treatments for peripheral neuropathy.