Figure 1.
Phenol red suppressed epileptiform bursting in cultured hippocampal pyramidal neurons.
A. (a) Bright field microscope picture showing an example of the pyramidal shaped neuron chosen for electrophysiological recordings; (b)–(e) pictures of immunohistochemical staining of cultured hippocampal neurons with (b, in bright field NeuN (c) and GAD67 (d). Pyramidal shaped neuron (white arrow) was GAD67 negative (d) and the oval shaped neuron (yellow arrow) was GAD67 positive (e). B. Current clamp recording traces from pyramidal neurons cultured in the media that was either phenol red free (a) or contained 21.5 micromoles of phenol red (b). In phenol red free medium, pyramidal cells generated repeated bursting of action potentials fired on top of the sustained membrane potential depolarization (a). In neurons cultured with phenol red, only single isolated action potentials riding on top of the EPSPs were recorded. C. Group analysis showed that the percentage of the bursting neurons in phenol red free culture medium (n = 19) was significantly higher than in phenol red added (21.5 µM) culture medium (n = 24, P<0.001). ***P<0.001.
Figure 2.
U-shape inhibition of the bursting activities in cultured hippocampal pyramidal neurons by phenol red.
A. Current clamp traces showing hippocampal pyramidal neurons with different firing patterns in culture media with different phenol red concentrations (0, 10, 21.5, 30, 100 µM). Low and high concentrations of phenol red induced the most prominent and longer lasting depolarizations and bursting activity. B. Group data showing the U-shape suppressive effect of phenol red on the percentage of the bursting neurons in different concentrations of the phenol red (0–200 µM). C. Group data analysis showing the most significant decrease of the burst frequency by phenol red at 30 µM pooled either in all tested neurons (P<0.001) (a) or in only the bursting neurons (P<0.01) (b). **P<0.01, ***P<0.001; #P<0.05.
Figure 3.
Estrogen receptor antagonist ICI 182,780 inhibits the suppressive effect of phenol red on the burst formation in cultured hippocampal pyramidal neurons.
A. Electrical traces showing different spontaneous firing pattern in neurons cultured in medium containing either phenol red (30 µM) (a) or phenol red (30 µM) plus ICI 182,780 (100 nM) (b); B. Group data showing ICI 182,780 significantly (P<0.001) prevented the decrease of the percentage of the bursting neurons by phenol red at 30 µM; C. Group data analysis showing a significant blockade of the burst frequency by ICI 182,780 on the phenol red inhibition pooled either in all tested neurons (a) or in those bursted neurons (b). **, ##P<0.01; ***, ###P<0.001.
Figure 4.
17-β-estradiol dose related inhibition of the epileptiform bursting activities in cultured hippocampal pyramidal neurons.
A. Different firing patterns of neurons cultured in medium with either alcohol control or 0.1 and 1 ng/ml 17-β-estradiol. B. Group data showing the dose related suppressive effect of the 17-β-estradiol on the percentage of the neurons having the epileptiform bursting activities. C. Group data analysis showing a significant decrease of the burst frequency by 17-β-estradiol at 0.1 ng/ml pooled either in all tested neurons (P<0.01) (a) or a significant increase of the burst frequency in the estradiol at 1 ng/ml in the bursting neurons (P<0.05) (b). **P<0.01, ***P<0.001; #P<0.05, ###P<0.001.
Figure 5.
Effect of phenol red concentration at 21.5 µM and 30 µM in the burst activities in the cultured hippocampal pyramidal neurons.
A–B. Bar histograms showing stronger suppressive effect of phenol red at 30 µM on the epileptiform bursting activities than at the commercially available medium with the added concentration of phenol red (21.5 µM). C. Curve of the two phase effect of phenol red on the epileptiform bursting activities with the inhibitory phase at the low concentration (IC50 = 14 µM) and the enhancing phase at the high concentration (EC50 = 45 µM); with the most effective concentration for the burst suppression seen at 28 µM. * P<0.05.