Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

Abstract

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR’s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

A deep learning framework for automated and generalized synaptic event analysis

“…For example, an existing miniML model can be easily retrained to eliminate potential false positives, e.g., due to sudden changes in noise characteristics. miniML's approach to detect events is not restricted to electrophysiological data, but can also be applied to time-series data derived from live imaging experiments using, e.g., reporters of glutamate (A. Aggarwal et al, 2023), Ca 2+ (Tran and Stricker, 2021), or membrane voltage (Li et al, 2020). The development of novel optical sensors with improved sensitivity and temporal resolution (Evans et al, 2023; necessitates efficient and robust event detection methods.…”

“…The development of highly sensitive fluorescent probes enables the recording of synaptic events using fluorescence imaging techniques (Abdelfattah et al, 2023;A. Aggarwal et al, 2023;Ralowicz et al, 2024).…”

“…In addition, a published dataset (A. Aggarwal et al, 2023) was used to probe the application of miniML to imaging data [https://doi.org/10.25378/janelia.21985406].…”

The adhesion GPCR ADGRV1 controls glutamate homeostasis in hippocampal astrocytes supporting neuron development: first insights into to pathophysiology ofADGRV1-associated epilepsy

“…Podgorski (Aggarwal et al, 2023). Images were acquired by Nikon eclipse Ti2 microscope (Nikon Instruments Inc) equipped with CSU-W1-T2 automatic Spinning Disk Confocal Scanning unit with 63x water objective (Yokogawa Electric Corporation).…”

“…To follow the uptake of glutamate from the media into the primary astrocytes, we used the genetically encoded glutamate reporter GFAP.iGluSnFR3.v857.GPI (Aggarwal et al, 2023). This glutamate reporter contains a glycosylphosphatidylinositol anchor that allows it to bind to glycosylphosphatidylinositols in biological membranes and thereby to monitor changes in glutamate concentration (Kinoshita and Fujita, 2016).…”