A novel BRET system to optimize and image in vivo recording of neural activity in freely behaving animals

About This Grant

Title: A novel BRET system to optimize and image in vivo recording of neural activity in freely behaving animals Project Summary/Abstract: To define the brain mechanisms that underlie complex behaviors and mental disorders, neurobiologists need tools to monitor cell-type specific activity from freely behaving animals and in response to stimuli. The currently available optical methods for measuring neural activity are based on fluorescence methods that have inherent limitations due to the need for excitation of the sensor, which leads to unintended and undesirable consequences that can damage neurons and distort neural activity. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. A novel luminescence-based technology for monitoring neural activity as a new tool for functional neuroscience will be developed in this project. Luminescence intrinsically circumvents the complications associated with fluorescence because it is an enzymatic reaction that is independent of any need for excitatory illumination. The luminescence sensor responds to the calcium ion fluxes generated by neuronal activity, is genetically encodable, and can be targeted to specific cell types and to specific cellular loci that are involved in neural activity. In particular, our novel ratiometric sensor based on Bioluminescence Resonance Energy Transfer (BRET) is modulated by neural activity so that the spectrum of luminescent emission is altered when neurons are activated. This new luminescence methodology avoids drawbacks of electrophysiology and fluorescence excitation (esp. off-target optogenetic stimulation, photobleaching, & tissue autofluorescence), and therefore optimally partners with optogenetic methods for neural manipulation. The BRET sensor will be developed into an optimal monitor of neural activity in vivo for imaging of deep brain regions in fiber-tethered and in unrestrained, freely behaving mice in combination with optogenetics. Benchmarking of the BRET sensor with existing fluorescence sensors of neural activity will be accomplished in freely behaving mice, and the novel sensor will be applied to concurrent use with multiple optogenetic actuators to excite or inhibit neurons in a single experiment. Finally, the sensor technology will be reciprocally coupled with the design, testing, and dissemination of new open-source miniature microscopes that are optimized for BRET-based imaging of single neurons and neural modulation in freely behaving animals. This project is responsive to the FOA in its invention/characterization of new probes for sensing and modulating neuronal activity in vivo, as well as the development of novel instrumentation optimized for imaging and recording BRET-based sensors of neural activity in conjunction with optogenetic actuators to manipulate neural activity. Ultimately these new sensor technologies and tools will advance our understanding of the complex circuits in the brain that underlie behavior and neural function which are altered in mental disorders.