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Title:Neuronal filopodia borne along tips and shafts of dendrites comprise two distinct populations as evidenced by differences in structure and dynamics
Author(s):Jain, Anika
Director of Research:Gillette, Martha U
Doctoral Committee Chair(s):Gillette, Martha U
Doctoral Committee Member(s):Bashir, Rashid; Ceman, Stephanie; Brieher, William
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Filopodia
Imaging
Abstract:Ever since their discovery in 1880 by Ramon y Cajal, dendritic spines have evoked considerable interest in the field of cellular and molecular neuroscience. Subsequent studies into their morphogenesis, and into synaptogenesis, brought into the spotlight their putative precursors – the dendritic filopodia. This set off several lines of investigation into filopodial structure and function, notable among which is the work by Portera-Cailliau et al. who showed in 2003 that growth cone filopodia differ from shaft filopodia in terms of densities and lengths, and in their response to blocking of synaptic transmission, and of ionotropic glutamate receptors. However, they observed these differences only up to postnatal day 5. In 2010, Korobova and Svitkina reported the existence of a different actin organization in shaft filopodia at 10 days in vitro (DIV). This work fills the gap between those two studies, investigating differences between tip and shaft filopodia at 4, 7, 10 and 14 DIV, and examining structure and dynamics, as well as responses to developmental cues, specifically, Semaphorin3A (Sema3A). Using confocal microscopy to visualize filopodial membrane and actin we found that shaft filopodia are shorter than tip filopodia, and show a less dense presentation along the dendrite. We then employed the quantitative phase imaging technology of Spatial Light Interference Microscopy (SLIM) for analysis of mass change dynamics of individual filopodia. We found that tip and shaft filopodia show similar dynamics early on, but further on in development by 7 DIV shaft filopodia slow down considerably while tip filopodia continue to show fast increases and decreases in mass. Further analysis of growth rates showed that both types filopodia exhibit exponential growth during their extension, implying that the bigger the filopodium the faster it grows. Next we sought to examine the functional ramifications of these differences in tip and shaft filopodia. We investigated the differential responses of the two populations to Sema3A. We found that a 24 h exposure to Sema3A at 0-1 DIV leads to accelerated maturation of shaft filopodia as evidenced by (1) an increase in dendritic branching, (2) an acceleration of maturation into spines, and (3) into synapses. An analysis of the underlying dynamics showed that Sema3A treatment results in (1) tip filopodial movement becoming more deterministic, (2) an increase in average growth and shrinkage rates in shaft filopodia, and, (3) an increase in speed of the fastest growth and shrinkage in tip and shaft filopodia at 4 and 7 DIV. Together these findings show that Sema3A is a unique cue that acts on both tip filopodia and shaft filopodia, but with different outcomes – the former to increase dendrite lengths, and the latter to increase branching, spinogenesis and synaptogenesis. Bath application of Sema3A also elicits an axonal response, which might itself affect the cells as a whole, and could confound the filopodial read out. To avoid this, we supplemented bath application studies with investigations using microfluidic devices that enable focal, dendrite specific application of Sema3A, and, also, better replicate the in vivo layered structure of the hippocampus. Our results held true even with this sub-cellular administration of Sema3A. Taken together our findings provide further evidence for differences in the two dendritic filopodial populations – those borne on the tips, and those along the shafts, and help deconstruct the role of Sema3A in dendritic development. A greater comprehension of this diversity in the filopodial population, and its role in shaping the development of neuronal networks will not only further our understanding of the nervous system, but will also help unravel the mechanistic bases of developmental disorders and diseases.
Issue Date:2017-04-19
Type:Thesis
URI:http://hdl.handle.net/2142/97587
Rights Information:Copyright 2017 Anika Jain
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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