Abhi Aggarwal discussed gene-encoded optical sensors iGluSnFR and iGABASnFR.
Mon, Aug 29th at 3:00PM in Plenary Hall
Abstract:
Genetically encoded fluorescent indicators for calcium, neurotransmitters, and neuromodulators (GECIs, GENIs, SnFRs) that modulate their fluorescent intensity in response to changes in ligand concentrations are powerful optogenetic tools for the understanding of neuronal signaling. The most widely used set of neurotransmitter-based indicators are the intensity-based Glutamate-Sensing Fluorescent Reporter (iGluSnFR) and its variants, that are engineered by fusing the glutamate binding protein GltI from Escherichia coli to a green fluorescent protein (GFP) or its derivatives. Variants of iGluSnFR have been used to image synaptic glutamate release, including spontaneous and evoked quantal release, as well as extra-synaptic signaling and glutamate clearance. Following the precent of the second generation iGluSnFR, SF-iGluSnFR-A184V, we have used an optimized multi-assay screen in bacteria, soluble protein, and cultured neurons, to develop an improved third generation, termed iGluSnFR3, with improved kinetics and signal-to-noise ratio. Furthermore, we have developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses.
Glutamate and GABA are the most abundant excitatory and inhibitory neurotransmitters, respectively, in the vertebrate brain. Current techniques for monitoring GABA are suboptimal for experiments requiring high signal to noise ratios, and imaging of high frequency transients in neural circuits. Moreover, the current intensity-based GABA-Sensing Fluorescent Reporter (iGABASnFR) is also engineered using green fluorescent protein scaffold, and therefore cannot provide completely crosstalk-free coupling when imaging glutamate using iGluSnFR3. To enable simultaneous imaging of both chief excitatory and inhibitory neurotransmitters, glutamate and GABA respectively, and to enhance the photophysical properties of the existing iGABASnFR, we undertook the development of a new iGABASnFR based on a cyan fluorescent protein, mTurquoise2. Based on the multi-assay screen that was used to develop iGluSnFR3, and surface display constructs that improved iGluSnFR’s localization to post-synapses, we show an improved cyan iGABASnFR that surpasses the performance of its predecessor and enables multiplex imaging with iGluSnFR3.
I will discuss our work on the development of iGluSnFR3, as well as recent, unpublished advances in our development of cyan-iGABASnFR, and share preliminary results validating them in vitro.
Title: Optical sensors for detecting neurotransmitters and neuromodulators
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