The goal of the proposed research is to optimize the ability to control with great spatial and temporal precision the levels of cAMP in specific subsets of vertebrate neurons by using a light-activated adenylyl cyclase, PAC. By placing this cyclase under the control of cell specific promoters it should be possible to control cAMP levels non-invasively by light in dissociated neurons, slice cultures and in intact developing embryos in vivo. Using FRET based measurements via a cAMP reporter, the cAMP transients produced by brief flashes of 455nm light in explant cultures of embryonic chick and mouse motoneurons electroporated with PAC will be characterized in somas and growth cones and compared to those produced by endogenous spontaneous electrical bursts in the same cells. Such transients will also be characterized in more intact cord-limb bud slice cultures. The effect of cAMP transients on the ability of motoneurons to respond to inhibitory and attractive guidance cues will be tested in dissociated neurons. Their effect on motoneuron dorso-ventral pathfinding will be tested in cord-limb bud chick and mouse slice cultures and in intact developing chick embryos in ovo. PAC will be placed under the motoneuron-specific promoter Hb9 to determine if it can selectively drive cAMP transients in motoneurons. Since intracellular cAMP has been shown to modulate how neurons interpret attractive and inhibitory growth/guidance cues, different patterns and frequencies of light activation of PAC will be used to determine if these can influence the response of cultured rodent DRG neurons to inhibitory signals. Based on a positive outcome, light activation of PAC in adult rodent DRG cells in vivo will be carried out to determine if this can enhance the regeneration of either the peripheral or central processes of transected DRGs. Given that cAMP modulates a vast array of important processes in the nervous system, the information gained from this study should facilitate the application of light-activated adenylate cyclase to control cAMP levels non-invasively in a variety of circumstances in intact organisms with widespread basic and translational applications, including enhancing the regeneration of axons in the CNS.
By providing a tool to non-invasively control cAMP levels in specific neurons in intact organisms this research will provide new strategies for enhancing regeneration and restoration of function following brain or spinal cord injury.
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