Increase in the extracellular glutamate level during seizures and electrical stimulation determined using a high temporal resolution technique

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.

Central GPR109A Activation Mediates Glutamate-Dependent Pressor Response in Conscious Rats

“…We followed the protocols described in the online Supplemental Material RVLM GPR109A Mediates Glutamate-Dependent Pressor Response and in our previous studies (Ibrahim and Abdel-Rahman, 2012;Penumarti and Abdel-Rahman, 2014 (Medina-Ceja et al, 2015) as provided in the Supplemental Material. NO was measured using 4-amino-5-methylamino-29,79-difluorofluorescein diacetate (DAF-FM Diacetate; Molecular Probes) according to the manufacturer's instructions and reported studies (Cortese-Krott et al, 2012) as described under Methods in the online Supplemental Material.…”

An electrochemiluminescent method for glutamate measurement in small microdialysate samples in asphyxiated young rats

“…Also, a multiple bio‐sensing chip to quantify choline, glucose, lactate, lysine, urate including Glu was constructed using a similar principle proving that H 2 O 2 measurement can be set also in a static format . Also, the well‐known Amplex Red method has been used in different works and formats, such as fluorescence measurement in capillary tubing and fluorescence measurement in samples deposited on polycarbonate plates in Cartesian coordinates …”

“…[6] Also, the well-known Amplex Red method has been used in different works and formats, such as fluorescence measurement in capillary tubing [7] and fluorescence measurement in samples deposited on polycarbonate plates in Cartesian coordinates. [8] In this work, we propose an additional optical method to measure Glu through the detection of H 2 O 2 based on the electrochemiluminescence (ECL) reaction. In ECL, light is generated by several steps, the first is the oxidation of the luminol in an alkaline buffer which forms an anion, then this intermediate molecule again undergoes an electrooxidation reaction with its co-reactant H 2 O 2 and the subsequent formation of 3-aminophthalate, leading to the excited state and finally emits a photon of a wavelength at 425 nm ( Figure 1).…”

A microdialysis and enzymatic reactor sensing procedure for the simultaneous registration of online glutamate measurements at high temporal resolution during epileptiform activity

“…; Medina‐Ceja et al . ). In these examples, different formats of fluorescence readings were used, and the time resolution was substantially greater than that obtained using classical microdialysis coupled with HPLC.…”

“…Galvan et al (2003) used Glu dehydrogenase to measure nicotinamide adenine dinucleotide (NADH) as a fluorescent product of the reaction of this enzyme with Glu, although the detection limit and temporal resolution obtained were low. Glu concentrations have also been measured based on fluorescence using the well-known Amplex Red method, which measures hydrogen peroxide (H 2 O 2 ), which is a reaction product (Slaney et al 2011;Morales-Villagr an et al 2012;Medina-Ceja et al 2015). In these examples, different formats of fluorescence readings were used, and the time resolution was substantially greater than that obtained using classical microdialysis coupled with HPLC.…”