U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Abstract

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.

Estrogen suppresses epileptiform activity by enhancing Kv4.2-mediated transient outward potassium currents in primary hippocampal neurons

“…These results indicate that the effect of 17-β-estradiol on the epileptiform bursting discharges is U-shape like, and 17-β-estradiol, in a certain low dose range, has a suppressing effect, similar to the effect that we previously reported (13).…”

“…1). Similar to the previous study (13), all the neurons (7/7, 100%) were showing epileptiform bursting activities while the neurons were cultured in phenol red-free medium with no added 17-β-estradiol (0 ng/ml). However, when the neurons were cultured in low dose 17-β-estradiol (0.1 ng/ml)-added medium, the percentage of neurons showing abnormal epileptiform bursting discharges (7/24, 29%) was significantly reduced (P<0.001) when compared to the vehicle control.…”

“…By contrast, estrogen receptor activation has also been reported to decrease neuronal excitability by indirectly changing the local neurotransmitter release (29), particularly by changing the interaction with GABAergic neurons (30,31). Our previous study also indicated that on the same hippocampal neuron, a weak estrogen receptor agonist, phenol red, could have a U-shape-like activation-inhibition-activation effect on the epileptiform bursting activities: Low and high concentrations of phenol red all induced the epileptiform bursting activities in the cultured hippocampal neurons, while the middle concentration (~28 µM) of phenol red suppressed this activity (13). Similar to the previous reports, a large proportion of the neurons cultured in the estrogen-free medium had epileptiform bursting activities in the present study, and the low dose 17-β-estradiol (~0.1 ng/ml) had a suppressive effect, whereas the high dose 17-β-estradiol (1 ng/ml) had a promoting effect on the neuronal excitability change, forming a U-shape-like dose-dependent action on the neuronal excitation change.…”

“…In a previous study, we reported that phenol red, a weak estrogen receptor agonist, at the concentration of a supplement ingredient in the commercially available culture medium, has a suppressive action on epileptiform bursting activities in cultured neurons by activation of the estrogen receptors (13). Thus, in the present study, all the experiments were performed in phenol red-free medium cultured hippocampal neurons.…”

“…In one of our previous studies, we reported that phenol red, a weak estrogen receptor agonist, as well as 17-β-estradiol, by activation of estrogen receptors, exhibited a dose-dependent U-shape-like effect on the spontaneous epileptiform bursting activities in cultured hippocampal neurons (13); however, the underlying mechanism remains unknown. Hoffman et al (14) also reported that modulation of voltage-gated potassium channels by estrogen reduced seizure and induced the spread and degree of neuronal loss in the kainate acid (KA) epilepsy model, which was modulated by progesterone (15).…”