Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.

Frailty is a feature of neuromuscular ageing. Here we provide insight into the relative contribution of pre- and postsynaptic dysfunction to neuromuscular ageing using the nematode Caenorhabditis elegans. Assays of C. elegans motility highlight a precipitous decline during ageing. We describe a novel deployment of pharmacological assays of C. elegans neuromuscular function to resolve pre- and postsynaptic dysfunction that underpin this decreased motility during ageing. The cholinergic agonist levamisole and the cholinesterase inhibitor aldicarb elicited whole worm contraction and allowed a direct comparison of neuromuscular integrity, from 1 to 16 days old: measurements could be made from aged worms that were otherwise almost completely immobile. The rapidity and magnitude of the drug-induced contraction provides a measure of neuromuscular signalling whilst the difference between levamisole and aldicarb highlights presynaptic effects. Presynaptic neuromuscular transmission increased between 1 and 5 days old in wild-type but not in the insulin/IGF1 receptor mutant daf-2 (e1370). Intriguingly, there was no evidence of a role for insulin-dependent effects in older worms. Notably in 16-day-old worms, which were virtually devoid of spontaneous movement, the maximal contraction produced by both drugs was unchanged. Taken together the data support a maturation of presynaptic function and/or upstream elements during early ageing that is lost after genetic reduction of insulin signalling. Furthermore, this experimental approach has demonstrated a counterintuitive phenomenon: in aged worms neuromuscular strength is maintained despite the absence of motility.

The “connectome,” a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond.