To a remarkable extent, progress in our understanding of neural structure and function has depended on a series of improvements in our ability to image individual neurons. In our previous studies, we generated many lines of transgenic mice in which a small number of individual neurons are labeled with green fluorescent proteins (GFP) in a Golgi-like fashion, allowing novel analyses of processes such as axonal guidance, dendritic growth, stability and modification, synapse formation, maturation and plasticity. Of particular importance is that the combination of a genetically coded chromophore and Golgi-like labeling of isolated individual neurons makes these mice an excellent tool for long-term imaging of dendritic dynamics, synaptic development and plasticity in living mice. In this application, we propose to explore a new technology in transgenic mice that would allow the inactivation of loxP-flanked genes at any given time in single neurons and simultaneous labeling of the same neurons with GFP for imaging. Like mosaic analysis in Drosophila, these mice will permit phenotypic analysis of single mutant neurons in an otherwise wildtype environment. Importantly, by inactivating genes in individual neurons and labeling the same neurons with GFP in a Golgi-like fashion, we will be able to image the dynamics of neuronal connectivity, synaptic structure and plasticity in mutant neurons, thus allowing us to investigate the molecular mechanisms underlying these (and many other) fundamental neurobiological phenomena.
Our first aim i s to generate transgenic mice that express a tamoxifen-inducible Cre recombinase and GFP in the same subset of neurons.
The second aim of the proposal is to characterize these transgenic mice by crossing them to a Cre reporter line of mice to ensure the functionality and inducibility of the Cre recombinase in individual neurons. If successful, these mice will provide the neuroscience community a novel tool to study the molecular and cellular mechanisms regulating the formation, maturation, modification and plasticity of neural circuits. ? ?
