Delayed audiogenic seizure development in a genetic rat model is associated with overactivation of ERK1/2 and disturbances in glutamatergic signaling.

Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizure are characterized by age-dependent expression of audiogenic seizures (AGS). It is known that the critical period of enhanced seizure susceptibility in rodents occurs at 2nd-3rd weeks of postnatal development. However, KM rats do not express AGS at this time-point, but start to demonstrate a stable AGS only after the age of 3 months. We hypothesized that this delay in AGS susceptibility in KM rats is genetically determined and may depend on some alterations in the development of the hippocampal glutamatergic system during the early postnatal period. We analyzed the expression and activity of seizure-related proteins, such as vesicular glutamate transporter 2 (VGLUT2), extracellular signal-regulated kinases 1 and 2 (ERK1/2), synapsin I, and NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor (NR2B) in the hippocampus of KM rats during postnatal development. A significantly higher activity of ERK1/2 in KM rats was observed at 14th, 30th, and 60th days of postnatal development (P14, P30, P60) in comparison with control Wistar rats of the corresponding ages, while in adult (P120) KM rats it was at the same level with Wistar rats. Despite the increased activity of ERK1/2 at P14 and P30, the phosphorylation of synapsin I at Ser62/67 was significantly lower in the hippocampus of KM rats than in Wistar rats of the same ages; however, at P60 and P120, the phosphorylation of synapsin I was enhanced. Our data also revealed the increase of VGLUT2 and NR2B expression at P14, which dramatically decreased at the later stages. Our data indicate that a genetically determined increase in ERK1/2 kinase activity during postnatal ontogenesis in KM rats may be associated with the disturbances in synthesis and activity of the proteins, which are responsible for glutamatergic transmission in the KM rat hippocampus during the seizure susceptibility development.

The expression and distribution of seizure-related and synaptic proteins in the insular cortex of rats genetically prone to audiogenic seizures.

It is known that perirhinal/insular cortices participate in the transmission of sensory stimuli to the motor cortex, thus coordinating motor activity during seizures. In the present study we analysed seizure-related proteins, such as GABA, glutamate, ERK1/2 and the synaptic proteins in the insular cortex of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). We compared seizure-naïve and seizure-experienced KM rats with control Wistar rats in order to distinguish whether seizure-related protein changes are associated with seizure event or representing an inhered pathological abnormality that determines predisposition to AGS. Our data demonstrated an increased level of vesicular glutamate transporter VGLUT2 in naïve and seizure-experienced KM rats, while glutamic acid decarboxylases GAD65 and GAD67 levels were unchanged. Evaluation of the synaptic proteins showed a decrease in SNAP-25 and upregulation of synapsin I phosphorylation in both groups of KM rats in comparison to Wistar rats. However, when phosphorylation level of ERK1/2 in naïve KM rats was significantly increased, several episodes of AGS diminished ERK1/2 activity. Obtained data indicate that changes in ERK1/2 phosphorylation status and glutamate release controlling synaptic proteins in the insular cortex of KM rats could contribute to the AGS susceptibility.

Inhibition of ERK1/2 signaling prevents epileptiform behavior in rats prone to audiogenic seizures.

It has recently been proposed that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. Here we report that inhibition of ERK1/2 by SL 327 prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. Accumulation of VGLUT2 was associated with differential changing of synaptic proteins VAMP2, SNAP-25 and synapsin I. The obtained data indicate that the inhibition of ERK1/2 alters neurotransmitter release by changing the exocytosis machinery, thus preventing seizures.