Abstract
BackgroundGlutamate has been measured using different methods to determine its role under normal and pathological conditions. Although microdialysis coupled with HPLC is the preferred method to study glutamate, this technique exhibits poor temporal resolution and is time consuming. The concentration of glutamate in dialysis samples can be measured via glutamate oxidase using the Amplex Red method.MethodsA new device has been designed and constructed to rapidly deposit dialysis samples onto a polycarbonate plate at Cartesian coordinates (every five seconds). The samples were added to an enzymatic reaction that generates hydrogen peroxide from glutamate, which was quantified using fluorescence detection. Fluorescence emission was induced by laser excitation, stimulating each spot automatically, in addition to controlling the humidity, temperature and incubation time of the enzymatic reaction.ResultsThe measurement of standard glutamate concentrations was linear and could be performed in dialysis samples. This approach was used to determine the effect of the convulsant drugs bicuculline and 4-aminopyridine on the extracellular glutamate concentration. Seizure activity was associated with a considerable increase in glutamate that correlated with altered EEG patterns for both drugs.ConclusionsThese results indicate that this method is able to read samples with high temporal resolution, and it is easy to use compared with classical methods such as high-performance liquid chromatography, with the advantage that a large number of samples can be measured in a single experimental series. This method provides an alternative approach to determine the concentrations of neurotransmitters or other compounds that generate hydrogen peroxide as a reaction product.
Highlights
Glutamate has been measured using different methods to determine its role under normal and pathological conditions
The samples were allowed to react, and the fluorescence was subsequently measured using a chargecoupled device (CCD) detector following laser excitation of each sample. We evaluated this method in seizure models induced by the GABAA receptor antagonist bicuculline and by administration of the potassium channel blocker 4aminopyridine (4-AP), as well as through electrical stimulation
The humidified chamber that was coupled to the sampling stage was crucial in preventing sample evaporation, allowing the reaction to proceed despite incubation at room temperature
Summary
Glutamate has been measured using different methods to determine its role under normal and pathological conditions. Microdialysis is the preferred method to measure extracellular Glu concentrations, whereby samples are Medina-Ceja et al BMC Neuroscience (2015) 16:11 periodically collected (with a resolution of minutes) and quantified by high-performance liquid chromatography (HPLC) This method is reliable, and it is considered to be the method of reference, it is frequently assumed to exhibit poor temporal resolution because a minimum volume is required to perform this analysis and a flow rate of 2.0 μl/min is generally used. By coupling the microdialysis technique to capillary electrophoresis (CE) with laser-induced fluorescence detection (LIFD), it has been possible to measure extracellular Glu concentrations in dialysate samples at one and ten second resolution, respectively [9,10] This procedure offers the advantage of improving the temporal resolution with respect to the classic HPLC methods, handling such small sample volumes may be an important issue to consider due to evaporation. Another important advantage of HPLC and CE is that a group of compounds sharing similar physicochemical properties, such as monoamines or amino acids, can be separated, identified and quantified in the same sample
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