We investigated the effect of chronic hypoinsulinemia on the level of synaptic activity and short-term plasticity in cultured hippocampal neurons. Hypoinsulinemia was induced by culturing mature (16-20 days in vitro) rat’s hippocampal neurons without insulin for 1, 2, and 4 days. The control insulin concentration was 100 nM. Spontaneous and evoked glutamatergic excitatory postsynaptic currents (sEPSC and eEPSC, respectively) in these neurons were analyzed using the whole-cell patch-clamp method and the method of local electrical stimulation of individual axon. Hypoinsulinemia during the 1st, 2nd and 4th days led to significantly reduction of the mean sEPSC’s frequency to 49.9 ± 15.8% (n = 6), 8.5 ± 7.7% (n = 6) and 16.6 ± 5.2% (n = 8) respectively, relative to control. Also, there was a decrease of the average sEPSC’s amplitudes to 52.6 ± 5.5% (n = 6), 36.6 ± 5.8% (n = 6) and 43.9 ± 8.4% (n = 8), respectively, relative to control. Quantal analysis of the sEPSC’s amplitudes showed a decrease of multivesicular glutamate release at the synapses under such conditions. Hypoinsulinemia caused a shift in the direction of short-term plasticity in glutamatergic hippocampal synapses from potentiation to depression. The paired-pulse ratio decreased from 1.83 ± 0.25 in the control to 0.59 ± 0.07, 0.77 ± 0.07, and 0.80 ± 0.06 after the 1st, 2nd, and 4th days under cultivation without insulin. Accordingly, the ratio of the coefficients of variation of eEPSC’s amplitudes (CV2/ CV1) increased from 0.82 ± 0.07 to 1.30 ± 0.28, 1.52 ± 0.27, and 1.61 ± 0.24. The presented results indicate a significant reduction of synaptic activity and decrease in the probability of multivesicular release of glutamate at the synapses of cultured hippocampal neurons under hypoinsulinemia.

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.

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