Groundbreaking work within the NIH BRAIN Initiative has revealed many new types of neurons and their
genetic signatures. The dividends from this research will include sophisticated tools allowing selective genetic
access to these cell-types, such as for imaging, optogenetic or tracing studies. To complement these powerful
genetic tools, it will be equally important to have new imaging techniques that can reveal how multiple neuron-
types work together in the live brain to support information-processing and construct different brain states.
To address this challenge, Stanford University and The John B. Pierce Laboratory at Yale University
will create optical techniques for imaging the concurrent voltage dynamics of up to 4 separate neuron-types in
behaving animals. First, we will combine machine learning methods and an automated, high-throughput protein
screening platform to engineer 4 different categories of genetically encoded fluorescent optical indicators of
neuronal transmembrane voltage. We will then innovate several types of optical instruments tailored to work in
conjunction with the new voltage indicators. These instruments will enable unprecedented studies of voltage
rhythms and spiking dynamics in 2–4 genetically identified neuron-types in superficial and deep brain areas of
awake behaving animals. One instrument will allow us to track the concurrent, population voltage oscillations of
2 neuron-types in freely behaving rodents. Another instrument, an optical mesoscope, will enable imaging
studies of voltage waves and oscillations across the entire neocortical surface of behaving mice. A third device
will be a high-speed miniature microscope for tracking neural dynamics at single cell-, single spike-resolution in
freely behaving mice. Lastly, we will develop the capability to image with millisecond-scale precision the
simultaneous spiking dynamics of 4 targeted neuron-types in either cortical or deep brain areas. Five external
beta-tester labs will evaluate all these innovations in live mice and flies and provide critical user-feedback.
If our work succeeds, it will be a ‘game-changer’ for studies of brain dynamics, yielding vital knowledge
about how different neuron-types synergize their dynamics to shape animal behavior and the brain’s global
states in health and disease. To facilitate this outcome, we plan a 5-fold strategy for resource sharing: (i) All
voltage-indicator constructs, viral vectors, transgenic flies, software and screening data will be deposited at
public repositories for open distribution; (ii) All instrument designs will be published in extensive detail to
facilitate replication; (iii) Our novel imaging devices will be integrated into an existing NIH-supported, publicly
accessible facility for brain-imaging in rodents; (iv) In project years 2–4, we will conduct 4 training workshops
for 40 visiting scientists per year (120 in total) to learn the new technologies firsthand. These visitors will also
provide extensive user-feedback; (v) We will license our imaging instruments for commercial distribution.
Overall, we expect our project will lead to major conceptual advances in brain science and multiple new
technologies that will reshape the practice of mammalian brain imaging.