Targeted sensors for glutamatergic neurotransmission

Abstract

Optical report of neurotransmitter release allows visualization of excitatory synaptic transmission. Sensitive genetically-encoded glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, retained function and reported spontaneous glutamate release in hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic neurons on micro island cultures, evoked release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

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

“…This enables more quantitative measurements of glutamate release over space and time that were previously confounded by saturation effects. Similar advantages were recently demonstrated for another novel variant in culture (13). Secondly, we demonstrated reliable detection of glutamate release associated with electrophysiologically-identified APs in vivo (Figure 5a-g).…”

“…The spatial specificity of glutamate transmission relies on nanoscopic localization of receptors to post-synaptic densities apposed to presynaptic active zones. However, electrophysiological and imaging studies have suggested that iGluSnFR may be excluded from the post-synaptic density, impacting the indicator's ability to report synaptic transmission when expressed post-synaptically (13). We therefore compared a variety of surface display sequences for their performance at reporting synaptic signals, using the amplitude, SNR, and detection rate of optical minis as readouts.…”

“…The intensity-based Glutamate-Sensing Fluorescent Reporter (iGluSnFR) and its variants have been widely adopted for neural glutamate measurements. iGluSnFR has been used to image synaptic release, including spontaneous and evoked quantal release (12)(13)(14)(15)(16), as well as extra-synaptic signaling and glutamate clearance (17,18). However, the ability of commonly-used iGluSnFR variants to distinguish synaptic from extra-synaptic signals has not been established.…”

“…However, the ability of commonly-used iGluSnFR variants to distinguish synaptic from extra-synaptic signals has not been established. Experiments have measured or bounded the spatial extent of iGluSnFR and SF-iGluSnFR signals under multiple conditions (10,13,(18)(19)(20)(21), but these bounds have been larger than the mean inter-synaptic distance. For example, SF-Venus-iGluSnFR.A184S exhibited signal correlations spanning 3.6 micrometers in visual cortex in vivo (18), corresponding to a volume containing roughly 30 glutamatergic synapses (1,2).…”

“…Existing variants have low values of K M , meaning that the indicator ON rate is saturated during high-concentration glutamate transients and responses are therefore reduced at release sites ("kinetic saturation"; Supplemental Note 1). Experiments also suggested that the existing approach for displaying iGluSnFR on cell surfaces using the PDGFR transmembrane domain results in poor nanoscopic localization at post-synapses; iGluSnFR signals have generally been larger at axonal boutons than on dendrites (20) and fusion to a post-synaptically-localized protein dramatically increased signals of a low-affinity variant (13).…”