From organisms using the most basic of nervous systems to the intricate circuits found within the human brain, a fundamental requirement of neuronal function is that it be able to modulate its activity based upon previous experience. In many neurobiological systems, specific patterns of electrical activity will alter the responsiveness of a cell. Long-term changes in cellular excitability due to synaptic stimulation require the synthesis of new protein. This can occur through several mechanisms, including the activation of transcription factors leading to changes in gene expression. Mammalian neurons utilize a variety of second messenger systems sensitive to synaptic stimulation in order to trigger activity-dependent gene expression. Moreover, these signaling proteins regulate many different transcription factors. Using rodent hippocampal neurons, we have been particularly interested in the regulation of cAMP response element binding protein (CREB)- and nuclear factor of activated T-cells (NF-AT)-dependent transcription following brief episodes of heightened synaptic activity, as this is an established model for learning and memory. Under these conditions, both transcription factors are regulated by calcium. Yet remarkably, CREB- and NF-AT-dependent transcription is activated only following the opening of L-type voltage-gated calcium channels. Increases in intracellular calcium via other avenues are ineffective in inducing changes in gene expression. The experiments outlined within this proposal will determine the mechanism by which L-type channels are privileged in signaling to CREB and NF-AT as well as examine the physiological significance for having L-type, and not other calcium entry pathways, responsible for CREB- and NF-AT-dependent transcription. It is our central hypothesis that clustering of L-type channels results in localized calcium signaling within the regions of a neuron containing the signaling machinery responsible for triggering activity-dependent gene expression. Moreover, we believe CREB and NF-AT are differentially regulated by synaptic activity because the second messenger systems responsible for their activation exhibit differences in calcium-sensitivity. Further, we will look to determine the downstream genes being regulated by CREB- and NF-AT-dependent transcription. We will use DNA transfection and manipulation, field stimulation, pharmacology and DNA microarray techniques to achieve our goals. Our results will have implications in clinical disorders arising from dysfunction in activity-dependent gene expression such as Rubenstein-Taybi syndrome, epilepsy and drug addiction, and may help in our understanding of illnesses that result in memory deficits including Alzheimer's disease.
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