Spatially defined sub-cellular heterogeneity determines neuron function. Thus, it is not surprising that disease origins can be traced back to the aberrant behavior(s) of dendritic filopodia that wire the brain. Interactions of the myriad filopodia extended by dendrites of individual neurons in their spatial contexts generate the remarkable range of functionalities of the human brain. Even adjacent filopodia encounter distinct local micro-environments and develop individual functionalities. Only by overcoming ensemble averaging of populations and measuring molecular signatures with single- filopodium resolution can we understand the interplay of the diverse intrinsic and extrinsic regulators, and explain the spectrum of neurological functions encompassing healthy and disease states. In particular, there is an unmet need for ways of probing of local regulators of filopodia during the emergence and sculpting of the dendritic arbor. This innovation proposal addresses this need by integrating our expertise in designing and fabricating nanoliter microfluidic environments for ultra-low density neuronal cultures with our expertise in the cell biology of neurons. We propose to use microfluidic device (?FD) environments and high resolution image analysis to probe changes in localization, activity, and function of specific microRNAs (miRNAs) in developing hippocampal dendrites. miRNAs are short, non-coding RNAs that act as regulators of local protein synthesis, especially during dendrogenesis and local wiring of the nervous system. Our objective is to control the structure and function of individual dendrites within micro-channels of fabricated ?FDs to isolate individual dendrites. We will use this system to map and influence miR125b functioning in filopodia during their development and in response to glutamatergic stimulation. This novel set of studies will address the need for understanding with high resolution the localization, activation, and function of miR125b during wiring of the hippocampus. This approach will provide new insights on this putative regulator, new tools for studying properties of miRNA control of dendrogenesis in single neurons, and contribute to effective strategies for restoring defects in models of affective dysfunctions, chronic stress, Alzheimer's disease, and autism.
The proposed research is relevant to public health because our new techniques and protocols will enable us to elucidate the function a critical regulator of dendrite development during brain wiring, microRNA (miRNA) 125b, with unprecedented sub-cellular resolution. The methods developed can be adapted to study the perturbations and defects in dendrites in affective dysfunctions (e.g., schizophrenia and chronic stress), aging disorders (e.g., Alzheimer's disease), and developmental disorders (e.g., Fragile X Mental Retardation and autism). Outcomes will enhance understanding of local, subcellular processes that mediate dendrite formation and maintenance, and accelerate development of new diagnostic, interventional, and therapeutic opportunities.
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