SignificanceExcitatory 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.

This Protocol describes the design, in vitro characterisation and imaging applications of iGluSnFR-based genetically-encoded glutamate indicators (GEGIs) in tissue culture of rat hippocampus TWEET A new protocol for the design, characterisation and high-speed imaging applications of genetically-encoded glutamate indicators (GEGIs). COVER TEASER High-speed imaging of glutamate release Up to three primary research articles where the protocol has been used and/or developed. 1. Helassa, N. et al. Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.

Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s−1, compared with 109 s−1 for f-RGECO1 and f-RGECO2 (37 °C). Thus, the CaM–peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM–peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.

Information processing in the brain is controlled by quantal release of glutamate, a tightly regulated process. Even in a single axon, presynaptic boutons differ in the number of docked vesicles, but it is not known if the vesicular release probability (pves) is homogenous or variable between individual boutons. We optically measured evoked transmitter release at individual Schaffer collateral synapses using the genetically encoded glutamate sensor iGluSnFR, localizing the fusion site on the bouton with high spatiotemporal precision. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters n, pves, and q. Schaffer collateral boutons typically released only a single vesicle under low pves conditions and switched to multivesicular release in high calcium saline. We found that pves was highly variable between individual boutons and had a dominant impact on presynaptic output.

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