Recombinant Immunolabels for Nanoprecise Brain Mapping Across Scales Understanding brain function and dysfunction requires an understanding of the circuitry of the brain from molecules to cells to circuits. While no single technique can achieve this, a strategic combination of techniques applied across scales can provide information that when integrated can lead to a more complete picture. These techniques can range from analyses of single molecules, including their localization in nanodomains, to subcellular compartments such as synapses and synaptic networks to entire brains. One common theme of these techniques is that they require affinity probes that specifically label subcellular structures, cell types, and circuits within the brain. We propose to enhance an existing resource of renewable affinity probes in the form of an extensive collection of highly-validated monoclonal antibodies. We will enhance our ongoing dissemination of low cost, high quality antibodies to neuroscience researchers by converting these to recombinant form. This will also ensure permanence of this valuable collection of renewable reagents for future researchers. We will use established methods to miniaturize these conventional antibodies into a nanoscale form. These miniaturized antibodies will allow for labeling of brain targets with single nanometer precision, which will provide more spatially precise labeling and overcome the limits to imaging resolution that conventional Abs represent. Moreover, the miniaturized Abs will have much better sample penetration, allowing for more efficient labeling of larger samples of the type used for defining intact circuits. We have formed a consortium of experts who developed and/or are experts in a powerful set of advanced techniques that together allow for brain mapping across scales. This consortium will validate nanoscale antibodies in their respective techniques, a key component of rigor and reproducibility of any antibody-based research. We will also generate and validate novel nanoscale antibodies against targets of great interest to brain investigators for which no suitable reagents exist. Together, these efforts will further enhance the dissemination of this valuable resource, and fundamentally accelerate the pace of brain circuit mapping across scales.

Understanding brain function and dysfunction requires an understanding of the circuitry of the brain from molecules to cells to circuits. This proposal is aimed at enhancing dissemination of a valuable resource of renewable affinity labels, in the form of monoclonal antibodies, for brain mapping across scales. We will enhance dissemination of this resource by using cutting-edge high-throughput sequencing to transform the frozen archive of hybridoma cells that produce these affinity labels into a permanent archive of DNA sequence, engineer miniaturized versions of the affinity probes to enhance imaging resolution and sample penetration, and validate them in a set of powerful cutting-edge techniques developed to map brain circuits across scales.