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

Medina-Ceja L, Pardo-Peña K, Morales-Villagrán A, Ortega-Ibarra J, López-Pérez S - BMC Neurosci (2015)

Bottom Line: 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.These 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurophysiology and Neurochemistry, Department of Cellular and Molecular Biology, CUCBA, University of Guadalajara, Jalisco, Mexico. lauramedcej@gmail.com.

ABSTRACT

Background: Glutamate 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.

Methods: A 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.

Results: The 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.

Conclusions: These 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.

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Related in: MedlinePlus

The device developed for sampling and reading of the glutamate concentration in dialysates. Top of A and B show the schematic device with all of its components: 1, the cover; 2, the stage movable in two axes; 3, the Z axis with the needle for sampling; 4, polycarbonate plate; 5, the control box used to program the device in terms of the number of rows and the frequency of the sampling process, as well as for reading; and 6, space for a wet sponge to create the humidity chamber when the cover slides over it. Actual images of the schematic device are shown at the bottom. C, Schematic and actual images (top and bottom, respectively) of fluorescence samples obtained at the end of an experiment.
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Fig1: The device developed for sampling and reading of the glutamate concentration in dialysates. Top of A and B show the schematic device with all of its components: 1, the cover; 2, the stage movable in two axes; 3, the Z axis with the needle for sampling; 4, polycarbonate plate; 5, the control box used to program the device in terms of the number of rows and the frequency of the sampling process, as well as for reading; and 6, space for a wet sponge to create the humidity chamber when the cover slides over it. Actual images of the schematic device are shown at the bottom. C, Schematic and actual images (top and bottom, respectively) of fluorescence samples obtained at the end of an experiment.

Mentions: The designed device constructed for these studies fulfilled the sampling and fluorescence reading requirements according to the desired protocol. The device could be satisfactorily programmed without errors, neither during deposition nor during the reading of the samples. Additionally, 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. The incubation time (60 min) for each sample prior to reading was maintained constant, and the fiber optic arrangement enabled the light to be emitted directly over the samples and to detect the light emitted following excitation. The fiber optics was mounted through a micromanipulator to assess their exact position in each experiment. Different intensities of laser power were evaluated to obtain the best response, and 5 mW was found to be the optimal power to excite the samples and obtain the strongest fluorescence response. For the CCD-based fluorescence detector, it was possible to use the software to synchronize the time of reading (every five seconds) with the movement of the platform such that the reading could be performed automatically. Accordingly, the time required for reading depended on the total number of deposited samples. This process offers the advantage of measuring an experimental series in a single session, and using the SpectraSuite software (Ocean Optics), the analysis could be performed simultaneously during the reading. Thus, a total of 900 samples could be evaluated in approximately 75 min. A scheme of the device is presented in Figure 1.Figure 1


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

Medina-Ceja L, Pardo-Peña K, Morales-Villagrán A, Ortega-Ibarra J, López-Pérez S - BMC Neurosci (2015)

The device developed for sampling and reading of the glutamate concentration in dialysates. Top of A and B show the schematic device with all of its components: 1, the cover; 2, the stage movable in two axes; 3, the Z axis with the needle for sampling; 4, polycarbonate plate; 5, the control box used to program the device in terms of the number of rows and the frequency of the sampling process, as well as for reading; and 6, space for a wet sponge to create the humidity chamber when the cover slides over it. Actual images of the schematic device are shown at the bottom. C, Schematic and actual images (top and bottom, respectively) of fluorescence samples obtained at the end of an experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363345&req=5

Fig1: The device developed for sampling and reading of the glutamate concentration in dialysates. Top of A and B show the schematic device with all of its components: 1, the cover; 2, the stage movable in two axes; 3, the Z axis with the needle for sampling; 4, polycarbonate plate; 5, the control box used to program the device in terms of the number of rows and the frequency of the sampling process, as well as for reading; and 6, space for a wet sponge to create the humidity chamber when the cover slides over it. Actual images of the schematic device are shown at the bottom. C, Schematic and actual images (top and bottom, respectively) of fluorescence samples obtained at the end of an experiment.
Mentions: The designed device constructed for these studies fulfilled the sampling and fluorescence reading requirements according to the desired protocol. The device could be satisfactorily programmed without errors, neither during deposition nor during the reading of the samples. Additionally, 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. The incubation time (60 min) for each sample prior to reading was maintained constant, and the fiber optic arrangement enabled the light to be emitted directly over the samples and to detect the light emitted following excitation. The fiber optics was mounted through a micromanipulator to assess their exact position in each experiment. Different intensities of laser power were evaluated to obtain the best response, and 5 mW was found to be the optimal power to excite the samples and obtain the strongest fluorescence response. For the CCD-based fluorescence detector, it was possible to use the software to synchronize the time of reading (every five seconds) with the movement of the platform such that the reading could be performed automatically. Accordingly, the time required for reading depended on the total number of deposited samples. This process offers the advantage of measuring an experimental series in a single session, and using the SpectraSuite software (Ocean Optics), the analysis could be performed simultaneously during the reading. Thus, a total of 900 samples could be evaluated in approximately 75 min. A scheme of the device is presented in Figure 1.Figure 1

Bottom Line: 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.These 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurophysiology and Neurochemistry, Department of Cellular and Molecular Biology, CUCBA, University of Guadalajara, Jalisco, Mexico. lauramedcej@gmail.com.

ABSTRACT

Background: Glutamate 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.

Methods: A 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.

Results: The 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.

Conclusions: These 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.

Show MeSH
Related in: MedlinePlus