Synapses are formed, broken and reformed dynamically both during development, normal function and in response to activity. Although this general principle is well-established, the way in which this is manifested in specific subtypes of neurons across a complex network, and how altered patterns of synaptic input will determine network function, have not been quantitatively investigated. Here we propose to develop molecular genetic tools for defining synaptic organization and connectivity in the mouse brain using fluorogen activating proteins (FAPs), a robust and modular system that enables multiplexed fluorescence identification of synapses and cell-specific connectivity. Our preliminary data indicate that we can target FAPs to synapses for quantitative analysis, as well as import 3D fluorescence image data for automated synapse detection using the image processing platform Imaris. Here we will create and validate pre- and postsynaptic targeting of fluorescent and FAP proteins respectively, acheiveing trans-synaptic FRET signal with high signal-to-background sensitized emission, allowing selective detection of synaptic connections formed between two genetically selected cell populations. These constructs, and the associated imaging and analysis approach, establish a pipeline for high- throughput data acquisition and analysis for assignment of cell-type specific contacts. As a test- bed for this technology, we will employ it to determine the synaptic input map for an important subset of cortical interneurons, somatostatin-expressing GABAergic cells, in the mouse neocortex.
The cerebral cortex is critical for sensation, higher-ordering reasoning, and memory. Composed of more than a dozen different cell types across 6 layers, it is highly plastic, rapidly changing in response to new inputs. Without knowledge of the fine-scale wiring principles of this structure, the computations that it carries out will remain obscure, and we will have little hope of understanding how genetic lesions are linked to cognitive malfunction, or how specific experiences are encoded into long-lasting memories. The scope of this problem is beyond what can be achieved by human observers, and requires automated synapse detection and contact assignment. We will develop molecular genetic tools that enable fluorescence identification of cell-type specific connections for high-throughput imaging and analysis. !
