Project Summary The long-term goal of our research project is to define mechanisms that govern the development and maintenance of motor neurons, as well as the formation and maturation of the synaptic connection between the motor neuron and the skeletal muscle - the neuromuscular junction (NMJ). The mammalian NMJ is a classic model of cholinergic synapses; it contains all elements of cholinergic synapses, including the neurotransmitter acetylcholine (ACh) and the ACh receptor (acetylcholine receptor, AChR), as well as the enzymes responsible for biosynthesis and degradation of ACh, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively. We use the NMJ as a model for understanding synapses as it can provide information pertinent to both peripheral and central synapses. Its relatively simple structure and easy accessibility make it more amenable to investigation than those of the central nervous system (CNS). Furthermore, the NMJ has a significant advantage over the CNS because genes can be deleted separately in the pre verses post-synaptic compartment, thereby facilitating the study of compartment- specific functions. This project addresses a fundamental question in the interaction between the nerve and the muscle – how does muscle signal to the nerve to promote motor neuron survival, and to regulate synapse formation and maturation? Using targeted gene deletions in mice, we found that muscle activity, mediated through muscle dihydropyridine receptors (DHPRs) and a newly identified protein, STAC3 (SH3 and cysteine-rich domain- containing protein 3), plays a key role in regulating motor neurons and the NMJ. Like DHPR, STAC3 is localized at the T-tubules and is required for excitation contraction coupling in skeletal muscles. Our preliminary findings open a new avenue of investigation for identifying feedback mechanisms from the muscle to the nerve, and will provide important insights supporting future development of therapeutic strategies to prevent motor neuron loss and treat neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).