Periodic Reporting for period 3 - MULTIVIsion (Multiphoton Voltage Imaging)
Berichtszeitraum: 2022-12-01 bis 2024-05-31
Information in the brain is encoded in changes in the voltage across the membrane of brain cells. Voltage imaging with genetically encoded voltage indicators (GEVIs) is a revolutionary method that allows faithful recording of the fast electrical dynamics of many genetically targeted cells in parallel. This provides an unprecedented means to record how patterns of change in this membrane voltage, called action potentials, manifest in subcellular compartments, cells, and networks across the brain, which is the only way to arrive at a fundamental understanding of brain functions like learning and memory, and of neurogenerative diseases. This is not only a means to unravel the secrets of the brain, but also a way to acquire information about the function of brain cells that could help us understand and develop treatments for diseases related to malfunctioning of brain cells.
For this promise to be fulfilled, we need voltage imaging in 3-dimensioal tissue, so we can look at the functionality of netowrks of neurons in their native environments.
The overall objective of this project are to develop a GEVI and an excitation-and imaging protocol that would allow voltage imaging deep in tissue, so that we can start looking at the detailed functionality of nerve cells in their native environments. we will then do a proof-of-principle experiment in which a model animal learns a movement task: we will try to record the change in functionality in the relevant neuronal networks.
- We developed an optical setup that helps us evolve GEVIs by imaging a large number of cells, which each express a mutated version of a GEVI. We can then select the cells that show a good response to our stimulus, and sequence the plasmid in it, to find the mutations that provided better functionality. A version of this setup was also used to identify aggressive cancer cells (L. You et al; Nature Biomedical Engineering, 6, 667–675 (2022) )
- We developed a setup for nonlinear screening and in vivo imaging of GEVIs, including all the hardware and software needed for the evolution and nonlinear optogenetic experiments (X. Meng et al; Journal of Optics, 24, 054004 (2022)
- We created a setup for pump-probe experiments on GEVIS in cellular environments, to understand their response to nonlinear excitation under different voltages (project in development)
- Covid delayed the progress on some aspects of the project. To catch up, we created a separate in vivo imaging setup, which aims for more superficial voltage imaging in vivo, which can already start working on the proof-of-principle learning task while the other parts of the project develop further. (project in development)
Apart from the optical work:
- We set up a number of cell lines that are helpful to us in our GEVI evolution, characterization of GEVI dynamics and chanracterization of neural dynamics (used in a.o. Q. Li et al; Optics Express, 29, 21, 34097-34108 (2021) )
- We set up a theoretical framework for the analysis and manipulation of GEVI photocycles (project in development)
- We evaluated different GEVI families for dynamics under nonlinear excitations, and screened them for evolved brightness and functionality. (project in development)
The results of the screening are promising and we expect until the end of the project to deliver an optimized GEVI for multiphoton imaging that goes beyond the state of the art in sensitivity, brightness and kinetics. Our in situ investigation of nonlinear dynamics of GEVIs is also beyond the state of the art. These experiments turned out harder than expected in terms of the needed alignment precision (M.-P. Chien et al; Science Advances, 7, 19, eabe3216 (2021))., but we expect to be able to deliver interesting evaluations of GEVI dynamics and a framework for how they can be used in optimized imaging protocols. Finally, we took a head start on the in vivo experiments and expect that we will be able to do multiple interesting proof-of-principle experiments on GEVI-imaged in vivo activity before the end of the project.