Elsevier

Brain Research Protocols

Volume 14, Issue 2, February 2005, Pages 61-66
Brain Research Protocols

Protocols
Measurement of GABA and glutamate in vivo levels with high sensitivity and frequency

https://doi.org/10.1016/j.brainresprot.2004.03.005Get rights and content

Abstract

In the present protocol, we demonstrate a high-performance liquid chromatography (HPLC) system that enables detection of very low amounts of γ-aminobutyric acid (GABA) (0.03 pmol) and glutamate (0.8 pmol). The HPLC system consists of two pumps, an electrochemical detector, a high-pressure six-way switching valve, a guard column, a microbore column, and a column oven. A microdialysis probe was implanted in the right parietal cortex in rats. Dialysates were collected every 5 min and were split into two equal aliquots for separate analysis of GABA and glutamate. After derivatization with o-phthalaldehyde (OPA), samples were isocratically separated and purified by the guard column. To make the peak of GABA or glutamate appear in an opportune place in a chromatogram, a six-way switching valve was used to control the eluate containing GABA or glutamate to be led to the microbore column and electrochemical detector. By the use of this system, decrease in extracellular concentration of GABA, which precedes the appearance of electrical discharge initiated by hyperbaric oxygen (HBO2) exposure, was detected by microdialysis at the time resolution of 5 min.

Introduction

γ-Aminobutyric acid (GABA) and glutamate are thought to be the most important inhibitory and excitatory amino acid neurotransmitters in the central nervous system (CNS) [3], [10], so the measurement of amino acids concentration becomes popular. Quantification of amino acid neurotransmitters in brain microdialysis samples has conventionally been performed by using a high-performance liquid chromatography (HPLC) system with electrochemical detection. However, because of the lower concentration of GABA, it makes it difficult to be measured. Thus, the dialysis sampling time must be prolonged (general 20 min) [3], [7] and resulted in a poor time resolution. It may be possible to measure a very low amount of GABA (0.03 pmol) if the sensitivity is raised with a microbore, but it will bring out many peaks and will need washout for several hours, thus becoming impractical [5], [6], [7]. In the present study, we utilized an HPLC system to analyze GABA and glutamate concentrations in 5-μl dialysate samples collected at intervals of 5 min by a microdialysis technique from the right parietal cortex in rats. A general guard column was used as a primary column to preseparate the dialysate and to prevent degradation of the main microbore column (analytical column) from contamination. GABA in the mobile phase was subsequently isolated in an analytical column, and a high-pressure six-way switching valve was combined with two pumps in the HPLC system to control the eluate containing GABA or glutamate to be led to the microbore column and electrochemical detector, making it possible to let the peak of GABA or glutamate appear in an opportune place in a chromatogram. By using the present HPLC system, we not only assayed very low amounts of GABA (0.03 pmol) and glutamate (0.8 pmol) but also grasped the reduction of extracellular GABA concentration prior to onset of a seizure induced by hyperbaric oxygen (HBO2).

Section snippets

Type of research

  • In vivo investigations of amino acid neurotransmitters in experimental preparations of CNS or other systems of animals (rats, gerbils, mice, etc.).

  • Monitoring extracellular levels of amino acid neurotransmitters in the CNS in models of acute conditions such as stroke, HBO2 seizure, and trauma.

  • Studies on neurochemical correlations of neurotoxicity and amino acid neurotransmitters.

Time required

  • Implantation of microdialysis probe: 30 min.

  • Exposure to HBO2: 1 h/animal.

  • Collection of dialysate samples: 45 min/animal.

  • HPLC analysis of GABA: 40 min/sample.

  • HPLC analysis of glutamate: 30 min/sample.

Animals

Twenty-eight male Wistar rats (Charles River Japan, Yokohama) weighing 310±58 g were used in this study. The animals were fed ad libitum before the experiments. All experiments were performed in accordance with the National Institutes of Health Animal Care Guidelines and were approved by the Animal Research Control Committee of Okayama University Medical School.

Microdialysis equipment

Microdialysis probes (A-I-4-02; membrane length=2 mm, molecular mass cutoff=50,000, and O.D.=0.22 mm; Eicom, Kyoto, Japan) and an

Animals were anesthetized with a mixture of 1% halothane and 30% oxygen–70% nitrogen

Following oral tracheal intubation and placement on a mechanical ventilator, catheters were inserted into the right femoral artery and right femoral vein for the purposes of blood sampling, blood pressure monitoring, and continuous infusion of analgesics during HBO2 exposure. After each rat had been placed in a stereotaxic apparatus, a burr hole was made 3 mm posterior to and 3 mm left of the bregma. A laser Doppler flow meter probe was placed on the surface of the dura through the hole for

Results

Fig. 3A shows a chromatogram of a 0.025-μM GABA standard, and Fig. 3B shows a chromatogram of a 5-min brain dialysate sample obtained from the cortex of an anesthetized rat.

Fig. 4A shows a chromatogram of a 0.625-μM glutamate standard, and Fig. 4B shows a 5 min-brain dialysate sample obtained from the cortex of an anesthetized rat.

Linearity of the detector response to standards of known concentrations was determined. As seen in Table 1, a linear response was obtained for both five standards in

Discussion

Although microbore columns provide high precision and sensitivity, if the biological specimen in dialysate samples are pumped directly into the microbore column, many hid peaks, unstable baselines were puzzled, it took times to washout, and, moreover, the column deteriorated early [2], [6]. To prevent the analytical column from deterioration by the foreign substances, we used a guard column upstream of the analytical column. The guard column washed all non-GABA and glutamate compounds and

Surgery and microdialysis

  • The animal is anesthetized and fixed on a stereotaxic frame.

  • A microdialysis probe is implanted into the right parietal cortex of a rat. The probe is perfused with Ringer's solution at a flow rate of 2 μl/min, and dialysate samples are collected every 5 min.

HPLC with electrochemical detection

  • Preparation of OPA derivatization.

  • Separate analysis of GABA and glutamate.

Essential literature references

References [1], [7], [9], [10]

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