Abstract
Large-scale data analysis of synaptic morphology is becoming increasingly important to the field of neurobiological research (e.g. ‘connectomics’). In particular, a detailed knowledge of neuromuscular junction (NMJ) morphology has proven to be important for understanding the form and function of synapses in both health and disease. The recent introduction of a standardized approach to the morphometric analysis of the NMJ—‘NMJ-morph’—has provided the first common software platform with which to analyse and integrate NMJ data from different research laboratories. Here, we describe the design and development of a novel macro—‘automated NMJ-morph’ or ‘aNMJ-morph’—to update and streamline the original NMJ-morph methodology. ImageJ macro language was used to encode the complete NMJ-morph workflow into seven navigation windows that generate robust data for 19 individual pre-/post-synaptic variables. The aNMJ-morph scripting was first validated against reference data generated by the parent workflow to confirm data reproducibility. aNMJ-morph was then compared with the parent workflow in large-scale data analysis of original NMJ images (240 NMJs) by multiple independent investigators. aNMJ-morph conferred a fourfold increase in data acquisition rate compared with the parent workflow, with average analysis times reduced to approximately 1 min per NMJ. Strong concordance was demonstrated between the two approaches for all 19 morphological variables, confirming the robust nature of aNMJ-morph. aNMJ-morph is a freely available and easy-to-use macro for the rapid and robust analysis of NMJ morphology and offers significant improvements in data acquisition and learning curve compared to the original NMJ-morph workflow.
Highlights
Synaptic connectivity is central to the structure and functioning of the mammalian nervous system
The sensitivity of neuromuscular junction (NMJ)-morph analysis has identified subtle changes in NMJ morphology found in Charcot–Marie–Tooth disease [8] and helped characterize NMJ degeneration in CHCHD10-encoded mitochondrial myopathy associated with motor neuron disease [9]
To support the continued uptake of NMJ-morph in the field of synaptic biology and related disciplines, we developed a macro update of the original workflow—‘aNMJ-morph’
Summary
Synaptic connectivity is central to the structure and functioning of the mammalian nervous system. Given the complexity of the central nervous system, the study of ‘model synapses’ has been critical to the progress of synaptic biology, with the neuromuscular junction (NMJ)—the synapse between lower motor neuron and skeletal muscle fibre—representing the paradigm example. Finding effective treatments for these conditions depends on a greater understanding of the normal and pathological architecture of mammalian synapses, including NMJs. Until recently, even basic quantification of the gross cellular anatomy of the NMJ has been hampered by the lack of a standardized approach to morphometric analysis. NMJ-morph revealed in detail the unique ‘nummular’ morphology of the human NMJ and further demonstrated its structural stability over the lifespan, in direct contrast to age-related fragmentation of rodent NMJs. The sensitivity of NMJ-morph analysis has identified subtle changes in NMJ morphology found in Charcot–Marie–Tooth disease [8] and helped characterize NMJ degeneration in CHCHD10-encoded mitochondrial myopathy associated with motor neuron disease [9]. We present a macro update to the original NMJ-morph workflow—‘automated NMJ-morph’ or ‘aNMJ-morph’—to streamline and expedite data acquisition
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