Visual experience modulates the development circuits in the brain that process visual information. One way that such neuronal activity can modify neuronal development is by changing the structure of the component parts, the individual neurons themselves. Considerable evidence supports the hypothesis that neuronal growth can be increased or decreased by changes in the pattern of electrical activity experienced by the neuron. The biochemical mechanisms by which such changes in neuronal growth occur are likely to be mediated through enzymes which themselves are controlled by neuronal activity. One such class of enzymes is the calcium-sensitive protein kinases, including. This protein kinase has been implicated in neuronal development, synapse formation and synaptic plasticity in the visual system. Unfortunately the pharmacological agents used in earlier studies could not clearly distinguish CaMKII and another calcium-sensitive protein kinase, protein kinases C. To address the problem of poor specificity of pharmacological agents, we now propose to use Vaccinia virus to introduce into neurons of the frog visual system a gene for a form of CaMKII that is no longer regulated by calcium and calmodulin. With this method, we can obtain expression of the constitutively active kinase only in the postsynaptic neurons, but not in the presynaptic retinal cells. Using confocal microscopy, we will then take time-lapse images of dye-labeled retinal axons or optic tectal neurons to observe their pattern of growth over a period of up to 4 days This latter method offers the unique opportunity to observe neurons grow in their normal complex environment. Previous experiments in fixed tissue have provided static images of neuronal development, from which active processes were inferred. Thus far, our observations of neuronal growth in situ have shown that in fact neuronal structure is much more dynamic than previously recognized and that retinal axonal structure can be modified by activity at earlier developmental stages than previously thought. By combining these 2 techniques, viral transfection of neurons in the optic tectum and confocal imaging of developing neurons in situ, we will be able to test the hypothesis that postsynaptic CaMKII activity can modify the development of neuronal structure and synaptic circuits in the visual system.
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