An enzyme-entrapped agarose gel for visualization of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system.

An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 µmol L(-1) for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler's Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated.

Simultaneous monitoring of excitatory postsynaptic potentials and extracellular L-glutamate in mouse hippocampal slices.

Simultaneous monitoring of amperometric currents at a glass capillary sensor based on recombinant GluOx and field excitatory postsynaptic potentials (fEPSPs) were performed in region CA1 of mouse hippocampal slices. A transient increase in the glutamate current relative to the basal one at control stimulation (0.052Hz) was evoked by stimulation at 2 Hz for 2 min. The magnitude of the glutamate current was dependent on the intensity (current) of a 2 Hz stimulus and reflected the slope of the fEPSP. The in situ calibration of the L-glutamate sensor revealed that the extracellular concentration of L-glutamate released by 2 Hz stimulation before tetanus is in the range from 0.8 to 2.2 µM and it is enhanced after tetanic stimulation. The L-glutamate level at a test stimulus (0.052 Hz) was estimated to be 32 nM. The recombinant GluOx-based sensor exhibited weak responses to glutamine above 300 µM and L-aspartic acid above 200 µM. The potential use of a glass capillary sensor in combination with fEPSP measurements for electrophysiological study is discussed.

Visualizing L-glutamate fluxes in acute hippocampal slices with glutamate oxidase-immobilized coverslips.

We used a glutamate oxidase (GluOx)-immobilized glass coverslip for reducing diffusional blur and improving the temporal resolution of visualizing L-glutamate fluxes in acute brain slices. The immobilization of GluOx on an avidin modified glass coverslips was achieved by optimized the amine coupling method. The GluOx coverslip was applied to the imaging of L-glutamate fluxes in acute hippocampal slices under hypoxia and KCl stimulation. A slice from mouse brain was loaded with horseradish peroxidase (HRP) and substrate DA-64, and placed on the GluOx coverslip for stimulation. The regional distribution of hypoxia-induced L-glutamate fluxes was analyzed. The maximum flux at 3 min after the onset of hypoxia increased in the order CA1>CA3>DG. The time-courses of the L-glutamate fluxes at CA1 and DG were biphasic, while that at CA3 decreased monotonously. The KCl-stimulated release of L-glutamate in the presence of the DL-TBOA uptake inhibitor was imaged. While no noticeable change was observed in the absence of DL-TBOA, L-glutamate fluxes in the presence of the inhibitor increased in the order CA1>CA3>DG, reflecting the effect of uptake processes. The present approach suppressed diffusional blur of the glutamate signal and improved the temporal resolution as compared with the BSA-HRP membrane method described earlier.

Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition.

Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.