Neuron
Volume 3, Issue 6, December 1989, Pages 715-720
Journal home page for Neuron

Article
Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate

https://doi.org/10.1016/0896-6273(89)90240-7Get rights and content

Abstract

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.

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      Notably, glutamate transport into these vesicles is considered a critical step in diverting glutamate from its metabolic role to its neurotransmitter function. It has been estimated that glutamate can reach intravesicular concentrations of 60 mM in vivo (Burger et al., 1989) while the cytosolic glutamate concentration is approximately 5–10 mM (Danbolt, 2001; Featherstone and Shippy, 2008). The mechanism for glutamate storage into synaptic vesicles involves an energy-dependent system coupling glutamate accumulation with ATP hydrolysis (Naito and Ueda, 1985; Ozkan and Ueda, 1998).

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