A hallmark of memory is that events that are fleeting or might occur only once can be remembered for a lifetime. How this is accomplished in the brain is thus of critical importance for understanding brain function and its dysfunction in disease. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify how nerve cells respond to stimuli and how nerve cells are connected. Traditional studies to examine how neural activity drives gene activation rely on the time-averaged behavior of thousands or millions of cells. These suffer from several limitations. For example, the stimulus is poorly controlled, and averaging across cells obscures how individual cells respond to stimuli offers only a static snapshot of a dynamic process. This proposal will develop a novel platform for single cell analysis of neuronal activity-dependent transcription by combining light-gated, temporally precise activation of neuronal firing with real time, quantitative imaging of intracellular calcium and transcription. Neurons will be engineered to express a light-sensitive ion channel, Channelrhodopsin 2, to drive neural activity with millisecond precision. Intracellular calcium will be measured with fluorescent imaging to study the extent of cell activation. Finally, the brain specific gene, Fos, will be tagged to produce a fluorescent signal at the moment it is transcribed into messenger RNA, which provides a blueprint for protein synthesis. This experimental platform will allow interrogation of the quantitative relationship between neuronal activity and transcriptional regulation in different activity-dependent genes. The cell lines developed as part of this project will be made freely available to other investigators following their publication. The training of graduate and undergraduate students will be an integral part of the work.
A hallmark of memory is that events that are fleeting or might occur only once can be remembered for a lifetime. How this is accomplished in the brain is thus of critical importance for understanding brain function and its dysfunction in disease. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify how nerve cells respond to stimuli and how nerve cells are connected. Traditional studies to examine how neural activity drives gene activation rely on the time-averaged behavior of thousands or millions of cells. These suffer from several limitations: the stimulus is poorly controlled, averaging across cells obscures how individual cells respond to stimuli and only offers a static snapshot of a dynamic process. Supported by this award, we have developed a novel platform for analysis of the synthesis of new genes in single living brain cells (neurons). We have genetically engineered cells to express a brain specific gene, Fos, tagged such that new messenger RNA synthesized in response to an electrical stimulus can be observed in real time and have shown that we can visualize them. This study will now allow us to examine how neurons synthesize new genes in response to environmental stimuli in order to alter neural circuits. The cell lines developed as part of this project will be made freely available to other investigators following their publication. One graduate student has been trained in advanced molecular biology and imaging techniques.
