[R01] Development of genetically encoded sensors and high-speed FLIM for in vivo quantitative multiplexed voltage imaging
Ente: National Institute of Mental Health
Scadenza: 2031-08-31
Importo max: $889,894
Paese: US
Descrizione
PROJECT SUMMARY
Neural cell types play distinct roles in brain computation, and cell-type-specific activity patterns are crucial for
learning. Cell-type-specific recordings of rapid electrical signals are critical for fully understanding neural
computation and the vulnerabilities of different cell types in disease contexts. However, current technologies for
measuring membrane potential have limitations: while whole-cell recording is the gold standard, it is not
high-throughput. Our team previously developed the genetically encoded voltage sensor, Voltron, which offers
high sensitivity and fast speed. Despite these advancements, the existing membrane voltage sensor toolkit
imposes severe constraints on the number of different cell types that can be imaged in vivo and does not allow
for quantification of absolute membrane voltage, rather reports only relative membrane voltage changes, and
has restricted depth penetration. This project aims to address these challenges by developing and validating a
palette of novel chemigenetic sensors for in vivo quantitative multiplexed FLIM voltage imaging. Close
collaboration among the Abdelfattah, Yasuda, and Inagaki labs will leverage their complementary expertise in
protein engineering, instrument design, and in vivo neurophysiology to achieve this goal. Our preliminary data
have yielded several voltage sensor variants with distinct fluorescence lifetimes at resting membrane potential
and robust spike detection capabilities. By combining these sensors with high-speed two-photon fluorescence
lifetime imaging microscopy (hs-2pFLIM), we will measure absolute membrane potentials (on the millivolt
scale) and distinguish up to six sensor types expressed by distinct cell types simultaneously, a feat that was
previously not possible. These innovations will enable multiplexing and absolute voltage quantification in vivo,
which is essential for analyzing subthreshold voltage activity and synaptic input. Specifically, we aim to: (1)
develop and characterize novel genetically encoded voltage sensor variants for fluorescence lifetime
multiplexed imaging, (2) design and optimize a hs-2pFLIM system for absolute voltage quantification, and (3)
deploy this system in the frontal cortex of mice in vivo to investigate multi-cell-type neural dynamics during
learning. This quantitative, multiplexed approach will offer unprecedented insights into membrane potential
dynamics and synaptic interactions across cell types that drive learning. Furthermore, we will validate our novel
voltage imaging tool by comparing the imaging results with cell type-specific extracellular electrophysiology
and whole-cell recordings. Together, our novel FLIM voltage sensor toolkit combined with hs-2pFLIM imaging
will provide neuroscientists with unprecedented means of investigating animal models of human learning with
optical visualization of brain circuits deep in the brain The successful outcome of this project will deliver a
powerful and accessible im
Istituzione: BROWN UNIVERSITY
PI: Ahmed Abdelfattah, Hidehiko Inagaki, Ryohei Yasuda
Progetto: 1R01MH141441-01A1
Settori: National Institute of Mental Health
Vai al bando originale
Registrati gratis su Bandolo per trovare bandi compatibili con la tua azienda.