Developing enhanced methods for reporting and manipulating brain activity is a major focus of the BRAIN Initiative. A major aspect of these efforts is aimed at developing genetically encoded probes for large-scale sensing and/or manipulation of neural activity in vivo. Major advances have been made in developing probes with enhanced intrinsic properties as to efficacy of reporting and/or manipulating neural activity. However, the utility of these probes in vivo has been limited by an inability to direct their localization to specific subcellular sites in brain neurons. We propose to develop a pipeline of genetically encoded localization modules or GELMs to direct probes for large-scale sensing and/or manipulation of neural activity to specific subcellular sites in brain neurons in vivo. This will yield enhanced signal to noise ratio at any specific site, and allows researchers and clinicians to more effectively report and/or modulate neuronal activity, an important step towards developing new ways to treat, cure, and even prevent brain disorders. An interdisciplinary consortium comprising neurobiologists, binder developers, and neural activity probe developers assembled here proposes development of a very ambitious pipeline for development of a robust and diverse set of genetically encoded localization modules, or GELMs, that when fused to activity reporters and modulators, or ARMs, will lead to the localization and concentration of the fusion proteins at specific subcellular sites in neurons. The ability to effectively and reliably target ARMs, and other genetically encoded probes, to specific subcellular sites in brain neurons in vivo will transform the methodology for large-scale sensing and/or manipulation of neural. The novel high-throughput pipeline for GELM development is based on a convergence of powerful new methods. The first is advances in developing high affinity, specific binders for neuronal target proteins in a format that can be used as intrabodies within neurons. These will be developed into Intrabody-based GELMs or I-GELMs. The pipeline also takes advantage of emerging data on targeting motifs present in otherwise highly related proteins that exhibit highly specific yet distinct subcellular localizations in brain neurons, and that affords an opportunity to develop Targeting motif-based GELMs or T-GELMs based on these motifs. We take advantage of high throughput systems for evaluating the expression, localization and function of GELM-ARM fusions in brain neurons. Lastly, we will make GELMs and GELM-ARM fusions widely available through open source plasmid repositories.

Developing enhanced methods for reporting and manipulating brain activity is a major focus of the BRAIN Initiative. A major aspect of these efforts is aimed at developing genetically encoded probes for large-scale sensing and/or manipulation of neural activity in vivo. Major advances have been made in developing probes with enhanced intrinsic properties as to efficacy of reporting and/or manipulating neuronal activity. However, the utility of these probes in vivo has been limited by an inability to direct their localization to specific subcellular sites in brain neurons. We propose to develop a pipeline of genetically encoded localization modules or GELMs to direct probes for large-scale sensing and/or manipulation of neural activity to specific subcellular sites in brain neurons in vivo, and make them freely available. This will yield enhanced signal to noise ratio at any specific site, and will allow researchers and clinicians to more effectively report and/or modulate neuronal activity, an important step towards developing new ways to treat, cure, and even prevent brain disorders.