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Laser System for Optogenetic Stimulation for the subcellular investigation of Neural Networks

Subject Area Biomedical Systems Technology
Term from 2018 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 402988941
 
Final Report Year 2023

Final Report Abstract

Cell cultures from human induced pluripotent stem cells promise opportunities for bottom-up experiments on developing human tissues. One notable potential application is the general or even patient-specific disease modeling of neurological diseases. Optogenetics, a combination of methods for the cell-specific optical control of genetically light-sensitized excitable cells, has emerged as a tool for the probing of neural activity and network functional connectivity, which are regarded as the basis of high-level cognitive modalities like learning and memory. Systems engineering challenges for optogenetics-based experiments on human neuronal tissue are manifold: Strong, cell-type-specific genetic manipulations suited for stem-cell-derived human neurons in vitro have to be established to reproducibly create organoids suited for targeted optical manipulations. Furthermore, the optical system has to allow for multi-cell stimulation to trigger groups of neurons. High temporal resolution is required to act on the time basis of neuron action potentials. To address these issues, we implemented an optical setup using two lasers (blue and yellow) for the individual actuation of cell membrane ion channels and pumps. Using two ferroelectric spatial light modulators, alternating light patterns based on optically reconstructed computergenerated holograms can be projected to tissue samples at a rate of up to 1.7 kHz to generate fast network stimuli, e.g. to test time-dependent signal processing. In addition, we implemented iterative procedures based on Zernike polynomials to correct for system-induced aberrations. We furthermore developed an optimized long-term cell culture protocol based on a coculture of induced human neurons (iNGN) and primary rat astrocytes, which facilitated investigations covering the maturation and neuronal circuit development periods of human iPSC-derived neurons. We performed electrophysiological studies to access multiple functional features of developing iNGN networks providing over time. Our results showed that iNGN networks reflect several maturation patterns similar to primary cortical and hippocampal networks. Our holographic stimulation platform revealed functional subunits within neuronal networks, which were otherwise masked by full field stimulation. We further demonstrated that holographic stimulation enabled correlating electrode activity to neurons located far from recording electrodes, and thus increased the spatial resolution of the connectivity maps which is defined by the MEA electrode pitch. Our platform, consisting of cell culture and optical systems, is therefore a methodological entry point to capture high-resolution long-term functional data of individual neurons organized in sub-circuits within entire random networks. Our data provide an extended view on precise functional differences during network maturation. Having fully functional human neurons and single-cellular activation control are important steps to transfer this model system as a complementary platform to animal research within the neuroscience community.

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