Ian R. Wickersham - US grants

This project consists of engineering a system for producing selective expression of light-inducible molecules in targeted neuron population in non-genetically modified animals of any species. The result will be a set of reagents that will be made freely available to the scientific community through nonprofit repositories and service centers. This new set of tools will enable the study of neural circuitry with greater resolution, power, and throughput than is currently possible, allowing major advances to be made in understanding the organization of the complex neural systems underlying perception, cognition, and behavior. This increased understanding could also result in improved artificial intelligence and machine learning. Finally, the future direct application of the technology in human patients holds promise for potentially treating conditions such as Parkinson's disease and epilepsy, by allowing the selective activation or inactivation of distinct components of the compromised neural circuitry that is associated with these disorders.

Over the last decade, sophisticated genetic tools have been developed that allow control and monitoring of neuron electrical activity using light alone. "Optogenetics", as this area of technology has become known, is only useful if optogenetic molecules can be specifically expressed in functionally meaningful groups of neurons instead of broadly in all the diverse neuron types that are present in any brain region. This requirement has confined their use almost entirely to genetically modified (transgenic) mice and rats. The approach of using transgenic animals has three major disadvantages. First, the production and maintenance of transgenic rodents is very expensive. Second, even within transgenic rodents, it allows the optogenetic study and manipulation of only one or two cell types at a time, preventing powerful combinatorial experiments in which different neuron types are independently controlled within the same tissue. These combinatorial experiments will be critical for deciphering the complex interactions between cell types. Third, it restricts the experiments to rodents, preventing studies in other important taxa including primates, in which optogenetic experimentation during complex cognitive tasks would almost certainly provide major insights into the neural circuitry underlying cognition. This project aims to create engineered binding proteins that recognize selected endogenous proteins that will then act as scaffolds for assembly of transcription factors that will activate gene expression in specific neurons.

? DESCRIPTION (provided by applicant): Genetic tools have dramatically increased the power and resolution of neuroscientific experiments, allowing monitoring and perturbation of specific neuronal populations within the brain, often in the context of complex cognitive and behavioral paradigms. However, the usefulness of these tools is limited by the available means of delivering them in circuit-specific ways, a major drawback in view of the critical importance of specific connectivity between individual neurons and between neuronal classes. The primary available means of achieving transgene expression based on neurons' synaptic connections is virus-based transsynaptic tracing, which allows identification, activity imaging, optogenetic control, and perturbation of gene expression in networks of synaptically connected neurons in vivo. The required viruses, however, are toxic within a few days, precluding longer-term experiments that are needed to address many central questions in neuroscience. We will solve this problem by engineering viral transsynaptic tracing systems with either greatly reduced or entirely eliminated toxicity, so that the role of neuronal networks of known connectivity in cognition and behavior. The result will be a set of tools that will allow optical imaging, physiological recording, and manipulation of the activity and gene expression of neuronal networks of known synaptic connectivity in the context of behavioral and other experimental paradigms lasting weeks, months, or years, in any mammalian model species. This will greatly enhance our understanding of the neural bases of normal cognition as well as neurological and mental disorders.

? DESCRIPTION (provided by applicant): Monosynaptic tracing using rabies virus has become a standard component of the systems neuroscience toolkit, allowing identification and manipulation of neurons directly presynaptic to any targeted neuronal population in the brain. However, while this retrograde monosynaptic tracing system is now well established, an anterograde counterpart, which would allow identification and manipulation of neurons directly postsynaptic to a target cell group, has never been constructed. Here we propose to meet this important outstanding need, with a multipronged and tiered strategy designed to ensure the delivery of at least one anterograde monosynaptic tracing system to the field, and possibly of several such systems, of differing levels of ease of use, power, and sophistication. Success of any of our aims will provide neuroscience with a transformative new tool for understanding and manipulating neural circuits throughout the brain, in many species including rodents and primates. Achievement of all of our aims will provide a versatile and powerful array of techniques to systems neuroscientists, allowing a broad set of new experimental designs that are likely to quickly yield major insights into the organization of neuronal circuits and the biological bases of normal cognition and behavior and the dysfunctions underlying neurological and mental disorders.

PROJECT SUMMARY We will use high throughput techniques to produce a set of viral vectors that will allow selective expression of transgenes in specific populations of neurons in the brain. Along with many other applications, this will allow optogenetic control, recording, and genomic modification of targeted neuronal populations without the need for production of transgenic or knock-out lines. This will provide neuroscience with a versatile and powerful set of tools that will make possible a broad set of new experimental designs that are likely to yield major insights into the organization of neuronal circuits and the biological bases of normal cognition and behavior and the dysfunctions underlying neurological and mental disorders. Additionally, because the new tools are also designed to work in humans, they are likely to result in important new therapies for mental and neurological diseases.

PROJECT SUMMARY Every part of the brain is composed of dense tangles of heavily-interconnected neurons of many different types, each playing completely different roles in the circuitry. Rabies viral vectors have become indispensable tools for revealing the organization of this otherwise generally indecipherable jumble, because they allow the identification of synaptically-connected networks of neurons within the tissue. They also allow monitoring and manipulation of the activity of the virally-labeled neurons by optogenetics and other techniques, so that the contributions to mental processes of identified components of neural circuitry can be revealed. Despite their usefulness, however, rabies viral vectors have drawbacks, chief among which is their toxicity to the labeled neurons, which prevents their use in longer-term physiological and behavioral experiments that would provide major insights into the biological bases of cognition. In this project, we will develop a new generation of rabies viral vectors and monosynaptic tracing systems based on them that will allow completely nontoxic fluorescent labeling, optical monitoring, and optogenetic manipulation of synaptically-connected neuronal networks.