A Glass Capillary Microelectrode Based on Capillarity and Its Application to the Detection of l-Glutamate Release from Mouse Brain Slices

Abstract

The detection of local and dynamic concentration changes of various bioactive substances in the mammalian central nervous system (CNS) is important for understanding the physiological function of excitatory synaptic transmission and metabolism. Many methods have been developed for the in vivo and in vitro detection of bioactive substances in biological fluids, including in vivo voltammetry, 1-3 patch-clamp biosensors, 4-7 microdialysis (MD) combined with various analytical methods 8 and ionselective microelectrode methods. 9 These methods have been widely used for detecting neurotransmitters, second messengers and other bioactive substances that play a key role in neuronal signal transmission.L-Glutamate is an excitatory neurotransmitter, and its release is thought to play a crucial role in brain development, synaptic plasticity, memory formation and neurotoxicity. 10 25 which is much larger than the electrodes for direct measurements, and the time resolution is on the order of minutes. In addition, the recovery of MD sampling is usually low because released L-glutamate is diluted by a perfusion solution. Niwa et al. used a glass capillary, instead of an MD probe, for sampling L-glutamate in a suction mode, followed by flow-through detection at a GluOx/Os-gel-HRP modified electrode. 26 On-line approaches using liquid chromatography, flow injection analysis and capillary electrophoresis in combination with MD probes have also been reported. 8 Improvements of the spatial dissolution of L-glutamate measurements by miniaturizing the size of sampling probe and/or that of enzyme-based electrodes are important, because the neuronal activity that releases and metabolizes L-glutamate is a very fast event, 10 and hence the concentration of Lglutamate detected at a sensing probe depends on the location and distance of the probe from the neurons. For the purpose of miniaturizing the size of the sensing probes, pulled glass capillaries 27,28 have a unique feature: the tip diameters range from submicro-to several µm, and the glass surface has an affinity to lipids and biocompatibility.In the present paper, we describe a new glass capillary microelectrode (tip diameter, ∼12 µm) based on capillarity for measuring L-glutamate. The inner solution provides a reservoir for enzymes, i.e., L-glutamate oxidase and L-ascorbate oxidase, and a built-in three-electrode system Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Japan A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, ∼12.5 µm) with a very small volume (∼2 µl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, re...

A Glass Capillary-Based Microsensor for L-Glutamate in in Vitro Uses

“…When an aqueous solution contained 5% dextrane, the sampling rate was deteriorated significantly due to an increase in viscosity. In another set of experiments, we quantified the capillary action by measuring the weight of a capillary dipped in an aqueous solution (Nakajima et al, 2003). With a tip diameter of 2.5 m or less, a rise of the solution by capillarity is not observed.…”

In Vitro Measurements of Extracellular l-Glutamate Level in Region CA3 of Mouse Hippocampal Slices under Chemical Stimulation

“…15,16 Amperometric measurements were performed at 0 V vs. Ag-AgCl with a computer-controlled electroanalytical system Cypress Model CS-1200 (Cypress Systems, KA). The sensor was implanted in region CA3 to a depth of ~10 μm with a manipulator.…”

An Enzyme-entrapped Agarose Gel for Visualization of Ischemia-induced L-Glutamate Fluxes in Hippocampal Slices in a Flow System

“…The monitoring of extracellular L-glutamate has been performed by using a variety of invasive and non-invasive methods. [1][2][3] Although the methods are invasive, in situ microdialysis [4][5][6][7] and enzyme based microsensors [8][9][10][11] are useful for monitoring L-glutamate at a single point with temporal resolution of seconds. On the other hand, physically non-invasive techniques for visualizing the distribution of L-glutamate release with cellular resolution have also been exploited by using glutamate binding and related proteins coupled to a fluorescence molecule [12][13][14][15][16] and various glutamate-sensitive techniques.…”