Dynamics of neurodegeneration in the hippocampus of Krushinsky-Molodkina rats correlates with the progression of limbic seizures.

Audiogenic seizures (AGS) (audiogenic kindling) in genetically selected audiogenic rodents are a reliable model of temporal lobe epilepsy (TLE). Temporal lobe epilepsy is accompanied with neurodegeneration in the hippocampus, but how the cells die is not fully understood. We analyzed the dynamics and mechanisms of cell loss in the hippocampus of audiogenic Krushinsky-Molodkina (KM) rats during the development of TLE. Audiogenic kindling of different durations was carried out to reproduce TLE progression in KM rats. Behavioral analysis showed the development of post-tonic clonus, the main indicator of TLE, by the 14th AGS. The severity and duration of post-tonic clonus positively correlated with the increase in the number of AGS. Temporal lobe epilepsy development was accompanied with two peaks of cell loss. The first peak was detected after 7 AGS in the dentate gyrus (DG) granular layer and associated with activation of p53- and mitochondria-dependent apoptosis. After a 7-day rest period, activation of autophagy and restoration of cell number were revealed. The second peak occurred after 14 AGS, affected both granular and hilar mossy cells and persisted further after 21 AGS, but no compensation was observed. Thus, activation of autophagy probably plays a neuroprotective role and supports survival of hippocampal cells at the beginning of epileptogenesis, but exacerbation of limbic seizures during TLE development causes irreversible neurodegeneration.

Impaired postnatal development of the hippocampus of Krushinsky-Molodkina rats genetically prone to audiogenic seizures.

The hippocampus plays an important role in epilepsy progression even if it is not involved in seizure generalization. We hypothesized that abnormal development of the hippocampus may underlie epileptogenesis. Here we analyzed postnatal development of the hippocampus of Krushinsky-Molodkina (KM) rats, which are the animal model of reflex audiogenic epilepsy. KM rats are genetically prone to audiogenic seizures that are expressed in age-dependent manner. The study was performed on seizure-naïve KM rats at several days of postnatal development (P15, P30, P60, P120). Wistar rats of the corresponding ages were used as a control. We showed that at early stages (P15, P30), the hippocampus of KM rats was characterized by significantly smaller cell population, but the number of proliferated cells was increased in comparison with control Wistar rats. Only at P60 proliferation and the total number of the hippocampal cells reached a level equal to Wistar rats. These data suggest delayed postnatal development of the hippocampus of KM rats. Analysis of apoptosis demonstrated significantly increased number of TUNEL-positive cells in the dentate gyrus (DG) of KM rats at P30 that was accompanied with expression of p53, Bcl-2 and cleaved caspases 3 and 9. Additionally, at all analyzed stages in the hilus of KM rats, the number of new-born glutamatergic cells was significantly increased that suggests formation of hilar ectopic granular cells. Our data suggest that in the case of hereditary epilepsy aberrant neurogenesis may be genetically determined.

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.

Apoptosis and proliferation in the inferior colliculus during postnatal development and epileptogenesis in audiogenic Krushinsky-Molodkina rats.

It is known that audiogenic seizure (AGS) expression is based on the activation of the midbrain structures such as the inferior colliculus (IC). It was demonstrated that excessive sound exposure during the postnatal developments of the IC in rats led to AGS susceptibility in adulthood, which correlated with underdevelopment of the IC. In adult rodents, noise overstimulation induced apoptosis in the IC. The purpose of this study was to investigate postnatal development of the IC in rats genetically prone to AGS and to check if audiogenic kindling would activate apoptosis and/or proliferation in the IC. In our study, we used inbred audiogenic Krushinsky-Molodkina (KM) rats, which are characterized by age-dependent seizure expression. Analysis of postnatal development showed the increased number of proliferating cells in the IC central nucleus of KM rats on the 14th postnatal day (P14) in comparison with those of Wistar rats. Moreover, we also observed increased apoptosis level and decreased general cell population in the IC central nucleus. These data pointed towards a delayed development of the IC in KM rats. Analysis of the IC central nucleus of KM rat after audiogenic kindling for a week, with one AGS per day, demonstrated dramatically increased cell death, which was accompanied with a reduction of general cell population. Audiogenic kindling also decreased proliferation in the IC central nucleus. However, a week after the last AGS, the number of proliferating cells was increased, which supposes a certain compensatory mechanism to prevent cell loss.

Effects of ERK1/2 kinases inactivation on the nigrostriatal system of Krushinsky-Molodkina rats genetically prone to audiogenic seizures.

Recently, we demonstrated that inhibition of ERK1/2 activity by SL-327 treatment blocks seizure behavior in Krushinsky-Molodkina (KM) rats, which was mediated by altering of GABA and glutamate release mechanism in the hippocampus. Basal ganglia representing various subcortical cell groups play a significant role in the regulation of motor activity, including epileptiform seizures. OBJECTIVES:  To verify if nigrostriatal system could be also affected by SL-327 treatment we analyzed the expression of tyrosine hydroxylase, D1 and D2 dopamine receptors, NR2B subunit of NMDA receptor as well as vesicular glutamate transporter VGLUT2 and glutamic acid decarboxylases GAD65/67 in the striatum and substantia nigra of KM rats. METHODS: Animals were injected i.p. with SL-327 (50 mg/kg) 60 min before audio stimulation. After audiogenic stimulation the brains of control and SL 327 treated rats were removed for further immunohistochemical and biochemical analysis. RESULTS:  Obtained results demonstrated a decrease activity in synapsin I, and accumulation of VGLUT2 in the striatum after blockade of audiogenic seizure (AGS) by SL 327 that could lead to inhibition of glutamate release. While in the striatum GAD65/67 level was diminished, in the substantia nigra GAD65/67 was increased showing enhanced inhibitory output to the compact part of the substantia nigra. Analysis of dopaminergic system showed a significant reduction of tyrosine hydroxylase activity and expression in the substantia nigra, and decreased D1 and D2 receptor expression in the striatum. In summary, we propose that changes in the nigrostriatal system could be mediated by inhibitory effect of SL 327 on AGS expression.

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.

Effects of selective Bcl-2 inhibitor HA14-1 treatments on functional activity of magnocellular vasopressinergic neurons of rat hypothalamus.

In this study we examined whether in vivo treatments with Bcl-2 inhibitor HA14-1 can affect the function of vasopressinergic system of rat. HA14-1 is a novel organic compound that has micromolar affinity for Bcl-2 and Bcl-xL and acts as a mimetic of BH3-only proteins by antagonizing the anti-apoptotic Bcl-2 proteins and triggering Bax-dependent apoptosis. We found that intrahypothalamic injections of HA14-1 did not induce apoptosis of vasopressin (VP) cells of supraoptic nucleus, but led to activation of VP synthesis and release, resulting in decreased diuresis. Our data has also demonstrated that injections of HA14-1 increased phospho-MEK1/2, phospho-CREB and phospho-Elk-1 levels in magnocellular neurons. Thus we propose that injections of HA14-1 into the hypothalamus do not lead to neuronal death, but change the functional activity of VP neurons of hypothalamus centres.