The dyad model was developed to explain the extremely rapid kinetics of synaptic vesicle exocytosis. In contrast to most hypotheses which invoke interactions among synaptotagmins, SNAREs and other regulatory molecules, the dyad model features a quartet of synaptotagmins arrayed at the synaptic vesicle-plasma membrane interface. Ca(2+)-triggered movements of these synaptotagmins initiate a sequence of events culminating in the fusion of the vesicular and plasma membranes. The relative simplicity of this model and its amenability to empirical testing provide a useful template for future investigations of the molecular events underlying the exocytotic cascade.

Investigations over the last two decades have made major inroads in clarifying the cellular and molecular events that underlie the fast, synchronous release of neurotransmitter at nerve endings. Thus, appreciable progress has been made in establishing the structural features and biophysical properties of the calcium (Ca2+) channels that mediate the entry into nerve endings of the Ca2+ ions that trigger neurotransmitter release. It is now clear that presynaptic Ca2+ channels are regulated at many levels and the interplay of these regulatory mechanisms is just beginning to be understood. At the same time, many lines of research have converged on the conclusion that members of the synaptotagmin family serve as the primary Ca2+ sensors for the action potential-dependent release of neurotransmitter. This identification of synaptotagmins as the proteins which bind Ca2+ and initiate the exocytotic fusion of synaptic vesicles with the plasma membrane has spurred widespread efforts to reveal molecular details of synaptotagmin's action. Currently, most models propose that synaptotagmin interfaces directly or indirectly with SNARE (soluble, N-ethylmaleimide sensitive factor attachment receptors) proteins to trigger membrane fusion. However, in spite of intensive efforts, the field has not achieved consensus on the mechanism by which synaptotagmins act. Concurrently, the precise sequence of steps underlying SNARE-dependent membrane fusion remains controversial. This review considers the pros and cons of the different models of SNARE-mediated membrane fusion and concludes by discussing a novel proposal in which synaptotagmins might directly elicit membrane fusion without the intervention of SNARE proteins in this final fusion step.

Spinal motor neurons (MNs) control diverse motor tasks including respiration, posture and locomotion that are disrupted by neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Methods directing MN differentiation from stem cells have been developed to enable disease modelling in vitro. However, most protocols produce only a limited subset of endogenous MN subtypes. Here we demonstrate that limb-innervating lateral motor column (LMC) MNs can be efficiently generated from mouse and human embryonic stem cells through manipulation of the transcription factor Foxp1. Foxp1-programmed MNs exhibit features of medial and lateral LMC MNs including expression of specific motor pool markers and axon guidance receptors. Importantly, they preferentially project axons towards limb muscle explants in vitro and distal limb muscles in vivo upon transplantation-hallmarks of bona fide LMC MNs. These results present an effective approach for generating specific MN populations from stem cells for studying MN development and disease.

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.