Lighting up the brain: Optogenetic tools to record, trace, and manipulate brain circuits at cellular resolution

PROJECT SUMMARY
Brain circuits are dynamic networks of neurons that process information in the form of electrical and chemical
signals to form memories and shape behaviors. To investigate how brain circuits instantiate fundamental
computations underlying behaviors, we need to map their wiring diagrams coupled with functional analysis at
cellular resolution. However, the electrical (voltage) and chemical (e.g. neuropeptides) signals are not directly
visible, and current circuit tracing tools are insufficient for meaningful functional analysis. Using protein
engineering this proposal aims to develop a toolbox of genetically-encoded fluorescent reporters and tracers
specifically tailored to study neural circuits. At the electrical level, voltage sensors can image the precise timing
of action potentials and subthreshold voltage not detectable by other means. However, even the latest voltage
sensors do not perform well with high-resolution microscopes that use 2-photon illumination for imaging deep
in the brain. To overcome these limitations, we are taking a two-pronged approach by evolving amino acids at
the mechanistic heart of voltage sensor proteins and by using spectroscopy to aid our protein engineering efforts.
We believe directed evolution will improve voltage sensitivity and 2-photon functionality >10 fold, enabling us
to image currently invisible signals, like synaptic potentials, deep inside the brain. At the chemical level,
neuropeptides are highly expressed in almost all cortical neurons, but their role and impact in animals can only
be inferred because current detection methods, like microdialysis, are invasive and lack spatiotemporal
resolution. We are using phage display to evolve nanobodies capable of recognizing neuropeptides and coupling
their conformational changes to fluorescence changes from reporter molecules. These sensors will provide
visualization of neuropeptide release at cellular resolution throughout an animal’s brain during behavior
paradigms that mimic human health and disease states. At the cellular connectivity level, current tools for circuit-
mapping, like rabies virus, exhibit substantial neurotoxicity, prohibiting meaningful functional analyses. We are
engineering proteins with a natural propensity to assemble into structures capable of delivering a genetic
payload to specific cells to produce more effective and less toxic tools to map and manipulate brain circuits.
Effective and robust tools to map the brain will bridge functional and structural analysis and finally allow long-
term studies of neural networks based on their connectivity. Overall, the optogenetic tools developed in this
proposal will translate the chemical and electrical signals between neural circuits into fluorescence that can be
easily measured. Consequently, they can be used to unravel the functional basis and causes of neuronal disorders
at a level of detail that has not been accessible to date and empower us to develop novel treatments.

Grant Number: 4DP2MH129956-02
NIH Institute/Center: NIH
Principal Investigator: Ahmed Abdelfattah