A major challenge for the neurosciences in the twenty-first century is to understand the mechanisms by which signal transduction networks within neurons lead to higher order functions such as learning and memory. These processes originate from the interplay of multiple different pathways rather than a single linear cascade of events. The mechanisms by which the networks operate are not likely to be intuitive since they employ sophisticated circuitry components such as positive and negative feedback loops, timing devices, thresholding mechanisms, and competition between network members. The goal of this proposal is to develop a completely new paradigm for the study of these intriguing intraneuronal networks. The technology will draw upon principles from the fields of chemistry, physics and biology to enable the simultaneous measurement of the activities of multiple, key signal transduction enzymes in a single neuron. This innovative approach will combine newly developed optical methods for neuronal sampling, a fluorescence-based assay of enzyme activity, and highly efficient and sensitive analytical chemistry techniques. Once fully developed, this technology will quantitate the activation of a multitude of enzymes in a single neuron while permitting measurement of other neuronal properties such as the intracellular concentration of free Ca2+ or membrane potential. This information can be used to map the temporal interrelationships of neuronal signaling networks. As a result many fundamental questions regarding neuronal development, synaptic plasticity, brain injury, and aging can be answered.
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