The BRAIN initiative objective is to unlock the mystery of the brain. An aspect of cracking this mystery is to map out the intricate neuronal signaling pathways happening between neurotransmitter for various activities. Building onto pre-existing methods and technologies, creating a map of the human brain continued to expand. A technology built on to provide key insight into these major neural networks is the use of genetically encoded fluorescent sensors. The explosive movement towards advancing the knowledge of the brain led to a major acceleration with the technology associated with achieving this goal.
A common method to track different chemical changes had always been fluorescence imaging. Within the few years, these techniques were being used to record neural dynamics in specific networks. Genetically encoded fluorescent sensors started to be introduced in in vivo, ex vivo, and behaving animals to mimic neurotransmitter to expand optical imaging. As discussed previously, genetically encoded Ca2+ indicators were one of the fluorescence imaging tools that were starting to be considered useful in the study of these neuronal networks.
Genetically encoded Ca2+ indicators, also known as GECIs, are able to track neuronal activity and synaptic transmission because of the changing Ca2+ concentration within the cell. A new advancement for this method was done by the Isacoff group. These researchers designed photoactivatable GCaMP, GECIs, GCaMP6s, and GCaMP6f to allow the ability to selectively activate GECIs in individual neurons from a large cell population. A new red fluorescent protein sensor, R-CaMP2, in combination with a green Ca2+ indicator created dual-color images of brain activities. R-CaMP2 assists mapping in the deeper parts of the brain because of the reduction of tissue that scatters at longer excitation wavelengths which in turn enables the easier detection and quantification of action potential signals and fast kinetics in vivo. GECIs started to become highly integrated with other technologies like intensity-based glutamate-sensing fluorescent reporters and optogenetic activation with channelrhodopsin for more multi-color neural activity imaging. GECI is a positive tool to study live mammals over longer periods of times because of its stability. Therefore; leading to long-term imaging of neural processes.
Another tool with advancements were genetically encoded voltage indicators, GEVIs. This type of indicator is able to provide measurements of neural activity in cells in milliseconds about membrane potential changes. The recent fusion of Acetabularia acetabulum rhodopsin (Ace) and mNeonGreen fluorescent protein was a new type of GEVI that allowed voltage sensitive fluorescence resonance energy transfer (FRET). This coupling solved the limitation of lacking sufficient signaling speeds and dynamic ranges to measure action potentials in vivo. The new GEVI allowed studies to be done on live mice and flies that yielded high-precision imaging of spikes in action potentials because of its improved brightness in its fluorescence and faster kinetics. GEVIs have the ability to monitor activity of larger number of neurons, voltage-sensing domains (VSDs) because the sensors are able to exhibit this faster kinetics and are used in living subjects to record cortical population responses. These VSD sensors were inserted with permutated GFP constructed from GCaMP3 that generated adequate brightness, fast kinetics, and large dynamic range. Therefore; allowing the detection of the quick pathways of action potentials.
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