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VOL. 73 (2), 441-452, 2007 GLUTAMATE DETERMINATIONS USING AMPLEX RED...

to start the experiment, Locke’s solution was changed by a new one
containing 50 µM PDC in order to block the glutamate reuptake
systems. Then, granule neurons were stimulated for 1 min with
BzATP (100 nM-200 µM) in Locke’s solution without Mg2+. All
stimulations were performed in the presence of 3 U/ml of ADA
in order to avoid the presence of adenosine in the medium. Finally,
the medium was aspired and the glutamate content was measured
using the Amplex Red Glutamic Acid Assay Kit (Molecular Probes)
according to the protocol provided by the manufacturer. The
resorfurin fluorescence was detected with a BMG Labtechnologies
96 plate reader using excitation filter at 544 nM and emission filter
at 590 nM.

Analyses of membrane pore formation

To analyze wheter BzATP induced pore formation, cultured
granule cells (14 days old) were exposed for 5 min with BzATP
(without Mg2+) in presence of 2 µM YO-PRO-1, the cell-impermeant
dye, that becomes fluorescent when interacting with DNA. Changes
in fluorescence were continuosly monitored using a fluorescence
microscopy setting excitation wavelength at 490 nm and emission at
510 nm. At the end of each experiment positive control was carried
out making permeable granule cell with 10% triton X-100.

Statistical analysis

Data are presented as mean ± S.E.M. of al least 3 independent
experiments. Comparisons between experimental samples and
controls were carried out using Student’s t-test . Dose-response curve
showing the effect of BzATP on glutamate release were fitted using
GraphPad Prism version 3.00 for Windows, GraphPad Software, San
Diego California.

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