The Effect of Thrombin in the Serum-Adapted Ionic Environment on the Induction of Epileptiform Firing Activity of Hippocampal Cultured Neurons

Abstract

The mechanisms of epileptiform neuronal activity develop- ment under blood-brain barrier (BBB) dysfunction remains relevant in modern psychoneurology. In the present work we mimic some effects of BBB disruption in the culture of hip- pocampal neurons to examined the effect of serum-adapted ionic environment on the impulse activity of hippocampal neurons and the role of serum protein thrombin in induction of epileptiform neuronal activity. Using the whole-cell patch- clamp method under current-clamp mode we analyzed the spontaneous action potentials (AP) in the single hippocampal neurons. The changing of ionic extracellular neuronal environ- ment to such serum-adapted contributed to the development of epileptiform tonic activity of cultured hippocampal neurons and led to increase the average APs frequency by 65.1 ± 17.9% (n = 5) in neurons with spontaneous firing activity (FA) and to occurrence of tonic electrical activity (1.65 ± 0.4 s-1) in neurons without firing activity. Glutamate NMDA receptors significantly contribute to epileptiform tonic activity formation in neurons with FA, while their role in tonic activity providing in neurons without FA was insignificant. Thrombin (5 U/ml) in the serum-adapted ionic solution significantly enhanced of epileptiform activity in neurons with and without spontaneous FA: APs frequency increased in these neuronal groups by 117.3 ± 25.6% (n = 3) and by 61.8 ± 11.5% (n = 3), respective- ly, compared with that in the serum-adapted ionic solution only. Blockade of thrombin protease activated receptor 1 (PAR-1) by application of SCH 79797 (10 μm) canceled the thrombin’s effect in neurons without spontaneous FA, and significantly reduced such in neurons with FA. Therefore, the change of ionic extracellular neuronal environment to serum-adapted stimulates the occurrence of epileptiform activity in hippo- campal neurons, that is apparently associated with NMDA- receptors activation in neurons with FA. The proepileptiform action of thrombin was mostly mediated by PAR-1 activation. Thrombin-dependent regulation of the hippocampal single neurons firing activity involves the mechanisms different from the modulation of glutamate NMDA receptors in these cells.

The role of thrombin in early-onset seizures

“…In our study application of 5 U/ml thrombin together with blood plasma ionic saline induced SLA in hippocampal CA1 pyramidal layer with the frequency of 3.39 ± 0.35 Hz and amplitude 0.66 ± 0.09 mV ( n = 18, Figure 1A2 ). These data are in agreement with our previous report when in similar conditions the epileptiform activity in cultured hippocampal neurons was significantly enhanced (Shypshyna et al, 2021 ). Extracellular recordings from the slices of SE-treated rats demonstrated high-frequency oscillations with the frequency of 3.31 ± 0.35 Hz and amplitude 0.51 ± 0.03 mV ( n = 14, Figure 1A3 ).…”

“…In our recent study, the application of serum-adapted solution increases the average action potentials frequency in neurons with spontaneous firing activity as well as tonic electrical activity in neurons. Increasing neuronal activity by blood plasma ACSF led to the development of epileptiform tonic activity in cultured hippocampal neurons (Shypshyna et al, 2021 ). Thrombin was shown to facilitate the effects of proconvulsants in the hippocampal slices from adult rats (Maggio et al, 2008 ).…”