Quantitative Analysis of Single Glutamatergic Vesicles in the Brain

Book chapter, 2024

Amperometry is an electrochemical method that can provide quantitative as well as kinetic information on neurotransmitter release from single exocytosis events and quantify the neurotransmitter content in single secretory vesicles. However, electrochemical recording of glutamate, the primary excitatory neurotransmitter in the brain, remains challenging due to the non-electroactive nature of glutamate. Hence, glutamate cannot be detected using conventional electrochemical methods but is feasible by creating an electrochemical glutamate biosensor. This can be achieved by immobilizing enzymes that catalyze glutamate at an electrode surface. The catalytic reaction of glutamate produces hydrogen peroxide, which can serve as an electrochemically detectable reporter molecule for glutamate detection. Although glutamate biosensors for a long time have suffered from a low temporal resolution (sub-seconds to seconds) and thus are too slow for detection of single sub-millisecond exocytosis events, we here present the technological development of a novel ultrafast (20 kHz) enzyme-based gold nanoparticle-coated amperometric microsensor designed for detection of glutamate release from synaptic vesicles. Our goal in this chapter is to describe the ultrafast glutamate biosensor design and explain how it can be manually fabricated for detecting and dissecting glutamate released from single synaptic vesicles. Additionally, we introduce a novel biosensor calibration method for quantifying the number of glutamate molecules released from isolated synaptic vesicles and single exocytosis events.