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Purpose: We have previously described a novel type of multiterminal en plaque motor endplates in the human extraocular muscles (EOMs). This study aimed to investigate whether multiterminal en plaque motor endplates are conserved in EOMs among vertebrates.

Methods: The motor endplates were identified with α-bungarotoxin (α-BTx) and antibodies against synaptic proteins and neurofilament in the EOMs of zebrafish, rabbits and mice. Transcriptomic data were re-analyzed to identify acetylcholine receptor (AChR) subunits in EOMs and trunk muscles of wild-type zebrafish at five and 20 months of age.

Results: In addition to the two typical types of single en plaque and multiple en grappe motor endplates, the third type of multiterminal en plaque motor endplates were observed in the EOMs of zebrafish, rabbits, and mice. The EOMs of zebrafish showed a significantly higher proportion of myofibers containing multiterminal en plaque motor endplates compared to EOMs of rabbits and mice. RNA sequencing data revealed significantly higher AChR subunits in the zebrafish EOMs compared to trunk muscles.

Conclusions: Multiterminal en plaque motor endplates are not exclusive to human EOMs but are also present in the EOMs of other vertebrate species, suggesting a conserved feature of the EOMs.