Cajal revolutionized the study of the brain through the use of the Golgi stain to label cells sparsely and stochastically in a fashion that revealed a neuron's morphology in its entirety. Although genetic tools for sparse and stochastic labeling and manipulation of single neurons in Drosophila have been used extensively over the past 15 years, they have only recently become available for mammalian systems, but the latter tools are limited to only a few systems for which cell-type specific reagents (e.g. enhancers) are available or otherwise involve cumbersome manipulations. Thus, there is an important need in the field to develop robust reagents for analysis of neurons at the level of single cells. Indeed, analysis of neurons at the single identified cellular level provides critical information on the control of neuronal morphology, connectivity, physiology and plasticity. This application is in response to BRAIN Initiative RFA-MH-14-216. We provide proof-of-concept preliminary data for a general method to label and manipulate single neurons in the mouse central nervous system. Using this method (called MORF), we created a novel dopamine D1 receptor BAC transgenic mouse that can sparsely and stochastically label a subset of D1-expressing striatal direct pathway medium spiny neurons as well as hippocampal pyramidal neurons. The labeled neurons reveal detailed morphology including dendritic arbors and synapses. We propose to further validate and expand this technique to be of general use for both single-neuron genetic labeling as well as genetic manipulation for multiple neuronal cell types in the mammalian brain. In addition, we propose to modify MORF to facilitate epitope tagging of synaptic proteins from their endogenous loci to image synapses of single identified cell types. We will develop and streamline imaging and computational tools to acquire and register brain-wide single neuron morphological information in a standard brain atlas for rapid dissemination of data to the research community. In summary, our proposed plan will develop a novel genetically-directed single neuron labeling tool that is conceptually different and drastically simpler than those currently available, along with streamlined imaging and mapping methods to facilitate the use of rich single neuron information provided by the models. The novel tools and mouse resources developed here should be immediately useful and impactful for neuroscience and brain disease-related fields.
In-depth understanding on how neural circuits regulate normal brain function and how dysfunction of these circuits lead to neurological or neuropsychiatric disorders requires novel tools to visualize and functionally manipulate its most fundamental units, the single neurons, in the intact brain. The current tools, however, have been restricted to only a few systems for which cell-type specific reagents (e.g. enhancers) are available or otherwise involve cumbersome manipulations. This application will provide validation and further development of a novel genetically-directed single neuron labeling tool that is conceptually different and drastically simpler than those currently available, along with streamlined imaging and brain mapping methods with proof-of-concept utility for normal and diseased brains so that the mouse resources developed from the application should be immediately useful and impactful for neuroscience and brain disease-related fields.