Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses

Abstract

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ~10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 M and 600 M, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons. Significance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells. INTRODUCTIONThe efficacy of synaptic transmission is not constant, but changes dynamically during high-frequency activity. In terms of information processing, different forms of short-term plasticity act as specific frequency filters: Facilitating synapses are most effective during high frequency bursts, while depressing synapses preferentially transmit isolated spikes preceded by silent periods (Markram et al., 1998). Mechanistically, a number of pre-and postsynaptic parameters change dynamically during high frequency activity, e.g. the number of readily releasable vesicles, presynaptic Ca 2+ dynamics, and the properties of po...

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR

“…S5b). (Recently, another iGluSnFR mutant at this site, S72T, was published 12 ; in our hands, SF-iGluSnFR.S72T shows lower ΔF/F than S72A. )…”

Genetic Dissection of Neural Circuits: A Decade of Progress

“…GluSNFR reports changes in extracellular glutamate concentration by changing fluorescence intensity with glutamate binding (Marvin et al, 2013). Sensitive new versions of GluSNFR can detect changes in cleft glutamate corresponding to the release of single vesicles (Helassa et al, 2017; Marvin et al, 2017). These sensors are very useful to image synaptic transmission, especially at large synapses, such as the retinal bipolar synapse (Franke et al, 2017).…”

Targeted sensors for glutamatergic neurotransmission

“…In contrast, optical report of neurotransmission via membrane dyes (Griesinger et al, 2005), calcium dyes (Oertner et al, 2002;Enoki et al, 2009), voltage-sensing dyes (Popovic et al, 2015) or neurotransmitterbinding reporters offers direct spatial resolution of synaptic events, usually with a time resolution about 2 orders of magnitude slower. Quantitation and analysis of the relevant optical signals remains challenging (Helassa et al, 2018;James et al, 2019;Soares et al, 2019).…”

“…The advent of bright, single-wavelength genetically-encoded reporters that directly bind neurotransmitters, such as iGluSnFR, has expanded the scope of optical report of synaptic transmission by speeding up report to the limit of optical microscopy (Marvin et al, 2013). This class of sensor has also been adapted to mature faster, and offer affinity and colour variants (Marvin et al, 2018;Helassa et al, 2018). For the purpose of subcellular targeting, the compact sensor architecture of iGluSnFR is easy to transplant into other host proteins.…”

“…Even when signals from the latter are localised to putative dendritic spines, the unknown proximity of the sensor to the release site introduces unwanted variability (Soares et al, 2019), because the glutamate signal diverges rapidly in space and time due to diffusion (Raghavachari and Lisman, 2004). Where spatial confinement of the reporter was achieved by exploiting targeting (Kim et al, 2020) or anatomy (Helassa et al, 2018;James et al, 2019;Duerst et al, 2020), the quantitative improvement of the fluorescent report is striking.…”