Arline J. & Curtis F. Garvin Professor
Member of NAS
Chair of Department of Neurosciences
Case Western Reserve University
School of Medicine, Room E653
We are interested in understanding the cellular and molecular mechanisms responsible for the formation of spinal motor circuits in vertebrates; the phenomena studied include mechanisms of motor axon pathfinding, the formation of functionally effective synapses, the assembly of motor- and interneurons into locomotor circuits, and the role of spontaneous rhythmic electrical activity in these processes. We believe that the mechanisms used during development to assemble the nervous system will be relevant to strategies for restoring neural circuits that have been damaged by injury or disease. Using embryonic mouse and avian spinal cords as models, the laboratory applies a wide range of techniques including electrophysiology and Ca2+ imaging of isolated spinal cord preparations or cord slices, molecular approaches to alter the expression of molecules in-vivo or in motoneuron-myotube cell cultures, specific tract tracing, dynamic imaging of fluorescently labeled cells, and biochemistry.
By combining electrophysiology and imaging of synaptic vesicle cycling at mouse neuromuscular junctions we recently discovered that different isoforms of NCAM play distinct and essential roles in both synaptic maturation and in maintaining effective transmission when adult junctions are activated repetitively. A specific domain on NCAM 180 is needed to activate an activity dependent signaling pathway, involving MLCK and myosin II, which is required to rapidly replenish synaptic vesicles during high frequency stimulation. We are now using bioinformatic, molecular, and physiological approaches to further decipher this pathway as well as a frequency dependent switch in the mode of synaptic vesicle cycling. The role of NCAM isoforms (and other molecules such as CD24) in the selective targeting of transmitter release machinery to synapses is being studied in motoneuron-myotube cultures in which one or more isoforms is selectively expressed pre- or post-synaptically. We believe that these observations are relevant to other parts of the nervous system as NCAM null mice also exhibit impaired secretion of adrenalin from chromaffin cells, and defects in hippocampal LTP, learning, and memory.
A second area of interest is to understand the role that rhythmic spontaneous activity, which is widespread throughout the developing nervous system, plays in the development of spinal motor circuits. By elucidating the transmitters and circuit that drives this activity we were able to alter it during precise periods of chick embryo development via in-ovo drug application. We made the surprising discovery that modest alterations in the frequency of this activity, as axons were growing to their targets, caused either dorsal-ventral or motoneuron pool-specific axon pathfinding errors depending on the sign of the alteration. We are now using channels that can be activated by light to drive activity in intact developing embryos with different stimulus patterns to elucidate downstream signaling pathways responsible for these pathfinding errors. By altering activity even earlier and by using 2-photon Ca2+ imaging to asses the alterations, we will test whether the generation and phenotypic differentiation of specific subtypes of motor- and intern-neurons and their assembly into circuits is sensitive to activity. Our observations show that any maternally taken drugs, which affect such early activity, have the potential to cause defects in the formation of spinal cord circuits.