Uncovering the mechanism(s) responsible for a novel neurite co-orientation process.

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The primary goal of this project is to uncover the mechanism(s) by which neural explants exhibit co-oriented neurite growth in tissue culture. The primary observation is that explants of chick embryonic neural tissue, either sympathetic or sensory ganglia, show asymmetric outgrowth with neighbouring explants exhibiting preferential growth in the same direction. The possibility of a role for a global field effect, such as gravity, has been excluded. Alternative mechanisms, such as diffusible or substrate gradients, cannot be excluded but are difficult to reconcile with the available data. An intriguing alternative is that the explants establish electric fields that mediate communication with nearby explants. Although the generation of electrical fields within neuronal tissues, and the responsiveness of such tissues to external fields have both been well established, generation and response to such fields in order to coordinate biological processes within the same tissue would be entirely novel. There are two research aims. The first is to determine whether this phenomenon occurs in analogous explants from other species (specifically mammalian and insect). The second is to examine the potential role of electrical activity in mediating the co-oriented outgrowth. The applicant will engage in a multi-disciplinary research program designed to draw on the expertise of four different scientists who will provide training and oversight in the following areas: primary neural cultures, morphometric analysis, multi-electrode array recordings, activity-imaging and single cell patch-clamp recordings. The three initial 8-week training rotations will be: 1) primary neural tissue culture methods under the supervision of Keith Crutcher and Curtis Dobson, 2) the use of multi-electrode array electrophysiology under the supervision of Tim Brown and, 3) the use of single cell electrophysiological and activity-imaging methods supervised by Richard Baines. After completing the rotations, the initial experiments will involve establishing cultures of embryonic chick sympathetic ganglia to replicate the initial findings if not already accomplished during the rotation. The next step will be to determine whether the phenomenon observed with chick explants also occurs with mouse sensory ganglia and/or Drosophila central neuron cultures. These experiments will involve: 1) dissecting the tissue, 2) establishing primary cultures and 3) staining and analyzing the resulting outgrowth. If co-oriented outgrowth occurs with mouse and/or Drosophila explants (thereby demonstrating ubiquity), this opens up a possible future line of investigation to take advantage of the numerous genetic tools available using these two additional models to probe underlying mechanisms. If mouse/Drosophila tissue does not show the same results as the chick tissue, the focus will remain on using the chick tissue to probe the possibility that electric fields are mediating the growth effect. These experiments will be supervised by Tim Brown who currently uses multi-electrode arrays to study the activity of mouse brain slices. Training during the initial rotation will use the existing methods to ensure the applicant is familiar with the approach. Only then will the method be adapted to the use of the chick (and/or mouse/fly) explants with the first question being whether or not there is evidence of electrical activity and, if there is, whether there is any indication of co-ordination or synchronicity. If no such evidence is found, alternative mechanisms will be explored, such as diffusible chemical gradients. If electrical activity is implicated, additional studies using single cell recording techniques and/or global activity imaging (using Ca2+-reporters) under the direction of Richard Baines will be carried out with the goal of uncovering possible cellular mechanisms.