The remarkable functional range of the human brain emerges from interactions of myriad neuronal filopodia that generate specific patterns of connectivity. This neuronal wiring during brain development is shaped by sub-cellular spatial heterogeneity, such as dendritic `hot-spots' and filopodia. Even adjacent filopodia of developing dendrites encounter distinct local micro-environments that alter their fate. Consequently, many disease origins, including altered cognition, can be attributed to aberrant behavior(s) of filopodia during brain wiring. A fundamental, unsolved question is how the local environment modulates developing dendrites and how these interactions control dendritic fate, in normal differentiation and in disease. We hypothesize that local redox state provides crucial context, shaping neurite responses to signals. In particular, there is an unmet need for probing local regulators of filopodia during the sculpting of the dendritic arbor. This proposal addresses this need by integrating our expertise in cell signaling, the neurobiology of redox dynamics, and high-resolution imaging in living cells with our expertise in designing and fabricating nanoliter micro-environments for low density neuronal cultures. We will use microfluidic device (?FD) environments and high-resolution imaging of fluorescent intracellular redox reporters to probe changes in localization, activity, and function of redox signaling in developing hippocampal dendrites. We will use this system to map and influence redox dynamics 1) in isolated, dendritic segments during their development, and 2) in their response to semaphorin 3A, which has a dual nature as an axon repulsion cue and a promoter of dendrite growth. We will gain new insights on how local redox dynamics in early developmental stages and in response to Sema3A stimulation contribute to filopodia/dendritic maturation. These novel studies will address the need for high resolution localization, regulation, and function of redox dynamics in developing dendrites, a topic that has received little attention. This approach will provide fresh insights on this putative regulator, new tools for studying regulation of redox-dynamics during dendrogenesis, and contribute to developing effective strategies for restoring defects in affective disorders, Alzheimer's, schizophrenia, Fragile X syndrome, autism, and chronic stress.
Highly organized neural circuits generate the range of functions controlled by our nervous system. The goal of this proposal is to elucidate the way in which the fine process of individual neurons develop, identifying the signals by which they interconnect and integrate to form proper networks. Novel methods and protocols developed under this innovation award will enable us to understand heterogeneous, subcellular processes that mediate dendrite formation and maintenance, accelerating the development of new solutions that diminish the dysfunction associated with dendritic defects, including affective dysfunctions (e.g., schizophrenia and chronic stress), cognitive disorders (e.g., Alzheimer's disease), and developmental disorders (e.g., Fragile X Mental Retardation and autism).
