Motor neuron (MN) diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) lead to progressive degeneration of MNs, presenting first with muscle weakness, followed by locomotor deficits and frequently death due to respiratory failure. While progress has been made in identifying genes associated with MN degeneration, the molecular and cellular processes underlying disease onset and progression remain unclear, and no effective therapies are available. Our project aims to identify the underlying causes of MN degeneration, using stem cell-derived MNs harboring genetic mutations associated with SMA as a model system. We first developed an innovative assay platform for stimulating and recording the activity of stem cell derived MNs using optical methods rather than more traditional electrical activity measurements. This modification improves the speed with which MN function can evaluated, allowing us to measure activity differences at a population level rather than individual cells. We used this platform to characterize defects in how SMA MNs communicate with muscle cells. Two major defects were seen: some SMA MNs fail to respond to stimulation, while others show activity independent of stimulation, leading to inappropriate spontaneous muscular contractions reminiscent of fasciculations seen in MN disease patients. We have further discovered that these MN activity differences are most likely related to changes in the inherent electrical properties of the SMA MNs, resulting in hyperexcitability in most cases. We have also observed differences in the size and molecular features of the SMA MNs, and are actively investigating how these changes affect MN excitability and degeneration. Our ongoing studies seek to discover the root causes of these differences in MN morphology and activity, and means for correcting. Through this approach, we hope to identify therapeutic agents that could improve MN function and combat MN disease progression.