Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients

Abstract

Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

“…We note that this is a magnitude of approximately 3-10 times larger concentration than previously stated for this neurotransmitter that has been so difficult to quantify. 10,11 This is in line with recent estimations using postsynaptic receptor saturation measurement and computer modeling of quantal release. 33 This result could be related to that our newly developed ultrafast glutamate sensor allows direct measurement of glutamate at single synaptic vesicle level and perhaps also that we might have used a synaptic vesicle protocol that better retain the vesicle glutamate content before analysis.…”

“…16 At the last step of the glutamate sensor fabrication, an ultra-thin layer of enzyme was immobilized onto the AuNP-coated carbon electrode using the protocol developed from our lab that control the enzyme layer thickness to a monolayer. 11 To completely dissolve the enzymes used for coating the electrode, 500 L of 10 mM sodium bicarbonate (pH 8.2) was added into the L-Glutamate Oxidase lyophilized powder bottle (5U) and allowed the enzyme to dissolve by gently rotate the bottle. GluOx was allowed to self-adsorb onto the sensor surface by immersing the AuNP-coated electrode tip into 100 l freshly prepared enzyme solution for approximate 2-3 hours at room temperature.…”

“…With a 45° beveled electrode angle, the sensor tip was placed at the NAc region of coronal planes that contain glutamatergic terminals from various brain regions with the assistance of an upright Nikon E600FN (Nikon Inc., Japan) fluorescence microscope mounted with 4× lens, and by placement of the sensor in the brain slice using methods as previously published. 11 The amperometric recordings were carried out by applying a constant potential of -0.5 V to the sensor surface versus a Ag/AgCl disc reference electrode, using the same MultiClamp 700B. Except for two measurements that were recorded using a digitizer (model Digidata 1440A, Molecule Devices, Sunnyvale, CA, USA) at 10 kHz and filtered at 4 kHz using the internal low pass 4-pole Bessel filter, all the other measurements were sampled at 20 kHz and filtered at 10 kHz.…”

“…[3][4][5][6][7] However, there is still an analytical challenge for direct, reliable quantitative measurements of glutamate stored in the ultra-small volume of a synaptic vesicle with an average diameter of 40 nm. 8,9 Methods such as radioreceptor assay (3640 glutamate molecules/vesicle) 10 and immunoisolation of synaptic vesicles from synaptosomes (60 mM) 11 have been used to try to solve the debate about how many glutamate molecules are stored inside synaptic vesicles.…”

Drug testing complementary metal‐oxide‐semiconductor chip reveals drug modulation of transmitter release for potential therapeutic applications

“…The CMOS chips used in our study are not designed for measurement of glutamate. However, with the recent development of fast responding glutamate sensors (Wang et al ), the CMOS chip technology may in the future also be applicable to directly perform testing of drugs with respect to modulation of glutamate release.…”