Phenytoin attenuates the hyper-exciting neurotransmission in cultured embryonic cortical neurons

• We study the effects of phenytoin on neurotransmission among cultured neurons.
• Phenytoin suppresses action potential firing and neurotransmission among neurons.
• Phenytoin decreases the amplitude and frequency of hyper-exciting EPSCs.
• Phenytoin preferentially inhibits abnormal high-frequency neurotransmission.

Phenytoin is an effective anti-epileptic drug that inhibits Na+ channel activities; however, how phenytoin modulates synaptic transmission to soothe epileptic symptoms is not clear. To characterize the effects of phenytoin regulation on neurotransmission, we studied the electrophysical properties of cultured embryonic cortical neurons. Phenytoin inhibited the inward Na+ current in a dose-dependent manner with an IC50 of 16.8 μM, and at 100 μM, the inhibitory effect of phenytoin on the Na+ current was proportional to the frequency applied. In cultured neurons, phenytoin significantly decreased the action potential firing rate and the peak potential. To study the effect of phenytoin in neurotransmission, we measured the Ca2+ responses from stimulated target neurons and their neighboring neurons. Phenytoin significantly suppressed the Ca2+ responses evoked by strong stimulations in the target and neighboring neurons, and exerted a decreased inhibitory effect under moderate stimulation. Picrotoxin, a GABAA receptor antagonist, enhanced the recorded spontaneous excitatory postsynaptic current activities. After picrotoxin-induced enhancement, phenytoin had a more pronounced effect on the suppression of the spontaneous hyper-exciting excitatory postsynaptic current (>100 pA), but it only mildly inhibited the general excitatory postsynaptic current. Our results demonstrate that phenytoin suppresses the efficacy of neurotransmission especially for the high-frequency stimulation by reducing the Na+ channel activity and can potentially alleviate epileptiform activity.