Figure 1

The effect of TD on AMPA-stimulated [Ca²⁺] _i in cultured cortical neurons. A: Cortical neurons were maintained in vitro for 7 days (DIV7), and TD was induced by the treatment of amprolium (1 mmol/L) for 1 or 4 days (TD1 or TD4). The resting [Ca²⁺]_i was measured as described under the Materials and Methods. B: Cortical neurons of DIV7 were treated with amprolium (0 or 1 mmol/L) for 4 days and perfused with AMPA (30 μM) for 5 sec. The calcium image (Fluo-3 fluorescence) was recorded as described under the Material and Methods. There were 240 cells for each treatment group. Scale bar = 20 μm. The colored scale bar indicates the fluorescence intensity of Fluo-3. C: Cortical neurons of DIV7 were treated with amprolium (0 or 1 mmol/L) for 1 or 4 days and then perfused with AMPA (30 μM) for 5 seconds. Two minutes after the first AMPA perfusion, neurons were subjected to a second AMPA perfusion with/without a general AMPAR antagonist GYKI (30 μM). Intracellular free levels [Ca²⁺]_i were determined by single cell Fluo-3 fluorescence imaging. The data show a representative response to AMPA by a single cell. D: The intensity of Fluo-3 fluorescence was quantified as described under the Materials and Methods. The results were calculated based on 75 cells. E: Cortical neurons of DIV7 were treated with amprolium (1 mmol/L) for 4 days and perfused with AMPA (30 μM) for 5 sec, followed by a second perfusion with AMPA plus tetrodotoxin (TTX; 0.5 μM)/bicuculline (BIC; 10 μM). A third AMPA perfusion was performed in the presence of AMPAR antagonist GKKI (30 μM). The perfusions were 2 min apart. The results were expressed as the mean ± SEM. *p < 0.001. The experiments were replicated three times.