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 saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. 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.

Neuromodulatory silencing of nerve terminals

“…To do so, we utilized iGluSnFR3 v857 (iGluSnFR), a genetically-encoded, fluorescent biosensor of extracellular glutamate concentrations with an excellent signal-to-noise ratio and temporal resolution enabling quantification of glutamate release from one AP at individual nerve terminals 27 . Co-expression of mRuby-synapsin allowed measurements to be localized to neurotransmitter release sites (Figure 6B); however, because iGluSnFR is anchored by glycophosphatidylinositol (GPI) to the cell surface, the indicator does not necessarily discriminate between glutamate released by the transfected cell or neighboring terminals from non-transfected neurons 27 . Accordingly, while fluorescent signals at mRuby-synapsin puncta may receive contributions from additional terminals, silent terminals are definitively assigned as inactive.…”

“…The following published DNA constructs were used: VGLUT1-pHluorin (vGpH) 25 , synaptophysin-GCaMP6f (physin-GCaMP) 49 , and GPI iGluSnFR3 v857(iGluSnFR) 27 , which was a gift from Kasper Podgorski. mRuby-synapsin (Addgene plasmid #187896) was generated by removing GFP from GFP-synapsin 50 using restriction sites AgeI and BGIII, and substituting it in frame with mRuby obtained from pKanCMV-mRuby3-18aa-actin, which was a gift from Michael Lin (Addgene plasmid #74255).…”

“…In experiments with physin-GCaMP, the chamber perfusate was exchanged for Tyrode’s solution with Ca 2+ 4 mM, pH 6.9, and ionomycin 500 μM to saturate the fluorophore and allow conversion of fluorescence to absolute [Ca 2+ ] i 49 . In experiments with iGluSnFR, glutamate 100 mM was applied to saturate the sensor and normalize to maximum fluorescence 27 . Recordings of physin-GCaMP6f and vGpH included ~200 nerve terminals on average while recordings of iGluSnFR included ~50 nerve terminals due to the limited EMCCD dimensions at this frame rate.…”

“…For vGpH, fluorescence was normalized to the total internal pool of vesicles following perfusion of NH4Cl 18 . For iGluSnFR, fluorescence was normalized to the maximum following perfusion of a saturating concentration of glutamate 27 . For physin-GCaMP, fluorescence was converted to absolute [Ca 2+ ]i using 49 :…”

NMDAR-dependent presynaptic homeostasis in adult hippocampus: Synapse growth and cross-modal inhibitory plasticity

“…When this analysis was repeated, determining the average QC per cell, inclusive of all synaptic ROIs within a field of view, we arrived at the same conclusion (Figure S7D). We subsequently deployed the most recent version of GluSnFR3 (Aggarwal et al, 2022), which has greater photo-stability, enabling two additional analyses. We assessed the ratio of successes to failures across a field of 6G).…”