EFFECTS OF DC FIELDS ON VERTEBRATE NEURONS

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DC electric fields are thought to stimulate axonal regeneration and growing axons, and so have been suggested as a method for inducing axonal regeneration following nerve injuries. If true, the application of such fields could be important in treating nerve injuries. Although several studies have reported the use of DC fields as a treatment for spinal and peripheral nerve injuries in animals, there are a variety of concerns with each of these studies that have left he general scientific community skeptical of the findings. In contrast, the general phenomena of axonal orientation, growth, and migration towards an electric field in cultaure are well established. While it has been possible to unequivocally demonstrate galvanic responses in vitro, comparable effects have been difficult to produce reliably in vivo. DC fields have low information content and can only provide cells with a directional or stimulatory cue. Either of these cues would be mediated by biasing one side of the cell in terms of ion channels, membrane receptors, membrane potential, etc. In the uniform environment of a culture dish, an applied field may be the only asymmetric influence, and thus may have a significant effect on cellular behavior. In in vivo condiitons, however, there a variety of competing signals for cell behavior such as gradients of diffusible factors, cell to cell interactions, and gradients of substrate adhesiveness. In this situation, the slight directional bias supplied by an electric field may be inconsequential. The long term objectives of the proposed study are to determine the mechanism of the effect of applied fields on cells in vitro and to determine if such a mechanism is functional in the in vivo situation. Specific aims are to; 1. compare the galvanic response of chick neurons grown on simple and complex substrates. 2. determine the effect of developmental stage on the response. 3. examine the effects of cell type and substrate source on the response. 4. to develop the means for systematically and reliable performing such experiments. To test this hypothesis, the galvanic response of neurons will be examined in complex culture conditions such as slice culture. These conditions retain the control, accessibility, and manipulability of the in vitro condition but provide a complex environment ot simulate the natural in vivo situation more closely. The basic method is to acquire time lapse videos of individual cells responding to applied DC fields. The cells will either be fluorescently labeled cells seeded on top of a complex substrate, or cells within a tissue slice that have been dye filled by microinjections. Using fluorescence videomicroscopy, images of individual cells will be made at high magnification. Stepper motors ont he microscope stage will move between many preselected cells and acquire images at regulate time intervals. These images will be recombined into a video sequence detailing the behavior of each cell that will then be statistically analyzed.