A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux

J Neurosci Methods. 2010 May 30;189(1):14-22. doi: 10.1016/j.jneumeth.2010.03.001. Epub 2010 Mar 16.

Abstract

Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9+/-6.4 fmol cm(-2)s(-1)). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50-150 fmol cm(-2)s(-1) range. Uptake was inhibited using threo-beta-benzyloxyaspartate, and average surface flux in replicate cells (1.1+/-7.4 fmol cm(-2)s(-1)) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Aspartic Acid / pharmacology
  • Biological Transport, Active / physiology
  • Biosensing Techniques / instrumentation*
  • Biosensing Techniques / methods
  • Brain Chemistry / physiology*
  • Cells, Cultured
  • Electric Stimulation
  • Electrophysiology / instrumentation*
  • Electrophysiology / methods
  • Glutamic Acid / analysis
  • Glutamic Acid / metabolism*
  • Mice
  • Neurochemistry / instrumentation*
  • Neurochemistry / methods
  • Neurons / metabolism*
  • Oxidoreductases / chemistry
  • Reaction Time / physiology
  • Synaptic Transmission / physiology
  • Time Factors

Substances

  • benzyloxyaspartate
  • Aspartic Acid
  • Glutamic Acid
  • Oxidoreductases