Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue

Abstract

Brain function relies in large part on Ca 2+ -dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca 2+ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca 2+ -sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spikeevoked presynaptic Ca 2+ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca 2+ dynamics and transmitter release in an intact brain at the level of individual synapses.

Multiplex imaging of quantal glutamate release and presynaptic Ca2+ at multiple synapses in situ

“…We next asked whether the kinetic features of the iGluSnFR sensors were suited for simultaneous imaging of multiple axonal boutons. This consideration was important because the laser beam has to dwell for 1-2 ms at each bouton to achieve signal-to-noise ratio comparable to that obtained under tornado-scanning of individual boutons 8 . Thus, reliable imaging of fluorescent signals concurrently at 4-5 locations requires the signals to last (preferably near its peak) for at least 10-15 ms. Fortuitously, this requirement is normally met by high-affinity Ca 2+ indicators including Cal-590 14 .…”

“…In parallel, advances in the imaging techniques suited to monitor membrane retrieval at presynaptic terminals have enabled detection of synaptic vesicle exocytosis in cultured neurons 5 . Recently developed optical glutamate sensors 6,7 have drastically expanded the sensitivity and the dynamic range of glutamate discharge detection in organised brain tissue 8 . However, such methods on their own cannot relate neurotransmitter release to presynaptic Ca 2+ dynamics, which is the key to understanding presynaptic release machinery.…”

“…Registered emission intensity can be strongly affected by focal drift, photobleaching, or any experimental concomitants (such as cell swelling or temperature fluctuations) impacting on light scattering. To overcome such difficulties in Ca 2+ imaging, we have recently developed a technique to employ Ca 2+ -sensitive fluorescence lifetime imaging (FLIM) of the Ca 2+ indicator OGB-1 8,12,13 . The FLIM readout provides nanomolar-range Ca 2+ sensitivity and is insensitive to light scattering, dye concentration, focus drift or photobleaching.…”

“…The FLIM readout provides nanomolar-range Ca 2+ sensitivity and is insensitive to light scattering, dye concentration, focus drift or photobleaching. However, OGB-1 emission is chromatically inseparable from that of existing glutamate sensors, thus prohibiting simultaneous imaging of glutamate release 8 . To deal with this challenge, we have systematically explored time-resolved fluorescence properties of Ca 2+ indicators and discovered that the fluorescence lifetime of red-shifted Cal-590, an indicator that has been successfully used in deep-brain imaging 14 , is also sensitive to low nanomolar Ca 2+ .…”

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

“…The electronic excited state lifetime of some fluorescent reporters can also provide an expression level-independent measure of activation. These techniques have been applied for voltage [109, 110] and calcium[111, 112], but not yet in a high-throughput format. Ultimately, quantitative measurements come from careful experimental design, including e.g.…”