Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

Abstract

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.

Optical analysis of the action range of glutamate in the neuropil

“…Our finding of small but regular synaptic cross-talk violates modelling studies which predict that cross-talk at AMPA-Rs is negligible and at NMDA-Rs at least two-fold smaller than what we observe experimentally [11,[15][16][17][18]. To some degree this difference could be explained by more recent estimates of certain biological parameters such as a higher glutamate content of synaptic vesicles (7000-8000 molecules) [56,57], a wider synaptic cleft (>=24 nm) [72][73][74] and a deeper understanding of glutamate transporter reaction schemes [75]. A further shortcoming of existing modelling studies may be the approaches used to approximate diffusible signaling on the nanoscale in the brain.…”

“…The number n of glutamate molecules released by our uncaging pulse can then be estimated by: n = 0.2 fl * 5 mM * ε * ExVF * NAv ~ 36000, with ExVF being the extracellular volume fraction (0.2) and ε the estimated fraction of glutamate uncaged (0.3, see discussion). Assuming recent estimates for the number of glutamate molecules per synaptic vesicle, ~7000-8000 [56,57], this calculation shows that our uncaging releases approximately the same number of glutamate molecules contained in 5 synaptic vesicles. We combined those estimates and calculated the approximate glutamate concentration profile around the uncaging position and superimposed it on an electron micrograph of a cortical synapse for a rough spatial comparison (Fig.…”

The Role of Amino Acids in Neurotransmission and Fluorescent Tools for Their Detection

“…Glutamate concentrations in the synapse can range from less than 20 nM to 5 mM and a recent study found that glutamate concentration in isolated synaptic vesicles was approximately 700 mM [ 13 , 14 , 15 ]. Glutamate binds to three ionotropic receptors (i.e., N -methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate receptors), which are all channels that allow the passage of Na + , K + and sometimes Ca 2+ .…”

Electrochemical Quantification of Neurotransmitters in Single Live Cell Vesicles Shows Exocytosis is Predominantly Partial

“…If these experiments are representative, it is very likely that many transmitters are released by partial release. Preliminary evidence with a glutamate biosensor suggests partial release might occur for this transmitter as well [48] . The number of glutamate molecules in an isolated vesicle of rodent brain was about 8 000, while only 5 200 molecules were released during exocytosis, resulting in 65 % fraction of glutamate release; however, control experiments blocking glutamate and without glutamate dehydrogenase are needed to give confidence that glutamate is indeed being measured in these experiments, leaving this still as an area to explore and verify.…”