Audiogenic kindling activates glutamatergic system in the hippocampus of rats with genetic predisposition to audiogenic seizures.

Temporal lobe epilepsy (TLE) development is associated with dysregulation of glutamatergic transmission in the hippocampus; however, detailed molecular mechanisms of pathological changes are still poorly understood. In the present study, we performed the complex analysis of glutamatergic system in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). Daily AGS stimulations (audiogenic kindling) were used to reproduce the dynamics of TLE development. Naïve KM rats were used as a control. After 14 AGS, at the stage of developing TLE, KM rats demonstrated significant upregulation of extracellular signal-regulated kinases (ERK) 1 and 2, cAMP response element-binding protein (CREB), and c-Fos in the hippocampus indicating activation of the hippocampal cells. These changes were accompanied with an increase in glutaminase and vesicular glutamate transporter (VGLUT) 2 suggesting the activation of glutamate production and loading into the synaptic vesicles. After 21 AGS, when TLE was fully-established, alterations were similar but more pronounced, with higher activation of glutaminase, increase in glutamate production, upregulation of VGLUT1 and 2, and Fos-related antigen 1 (Fra-1) along with c-Fos. Analysis of glutamate receptors showed variable changes. Thus, after 14 AGS, simultaneous increase in metabotropic glutamate receptor mGluR1 and decrease in ionotropic N-methyl-D-aspartate (NMDA) receptors could reflect compensatory anti-epileptic mechanism, while further kindling progression induced upregulation of ionotropic receptors, probably, contributing to the hippocampal epileptization. However, we revealed practically no alterations in the expression of synaptic proteins. Altogether, obtained results suggested that overactivation of glutamate production in the hippocampus strongly contributed to TLE development in KM rats.

Postnatal development of the hippocampal GABAergic system in rats genetically prone to audiogenic seizures.

Epileptogenesis can be associated with altered genetic control of the GABAergic system. Here we analyzed Krushinsky-Molodkina (KM) rats genetically prone to audiogenic epilepsy. KM rats express fully formed audiogenic seizures (AGSs) not early, then they reach 3 months. At the age of 1-2 months, KM rats either do not express AGS or demonstrate an incomplete pattern of seizure. Such long-term development of AGS susceptibility makes KM rats an especially convenient model to investigate the mechanisms and dynamics of the development of inherited epilepsy. The analysis of the GABAergic system of the hippocampus of KM rats was done during postnatal development at the 15th, 60th, and 120th postnatal days. Wistar rats of corresponding ages were used as a control. In the hippocampus of KM pups, we observed a decrease in the expression of glutamic acid decarboxylase 67 (GAD67) and parvalbumin (PV), which points to a decrease in the activity of GABAergic neurons. Analysis of the 2-month-old KM rats showed an increase in GAD67 and PV expression while synapsin I and vesicular GABA transporter (VGAT) were decreased. In adult KM rats, the expression of GAD67, PV, and synapsin I was upregulated. Altogether, the obtained data indicate significant alterations in GABAergic transmission in the hippocampus of audiogenic KM rats during the first postnatal months.

In search of stress: analysis of stress-related markers in mice after hindlimb unloading and social isolation.

OBJECTIVES: Hindlimb unloading (HU), widely used to simulate microgravity effects, is known to induce a stress response. However, as single-housed animals are usually used in such experiments, social isolation (SI) stress can affect experimental results. In the present study, we aimed to delineate stressful effects of 3-day HU and SI in mice. METHODS: Three animal groups, HU, SI, and group-housed (GH) control mice, were recruited. A comprehensive analysis of stress-related markers was performed using ELISA, western blotting, and immunohistochemistry. RESULTS: Our results showed that blood corticosterone and activity of glucocorticoid receptors and cAMP response element-binding protein (CREB) in the hippocampus of SI and HU animals did not differ from GH control. However, SI mice demonstrated upregulation of the hippocampal corticotropin-releasing hormone (CRH), inducible NO synthase (iNOS), vesicular glutamate transporter 1 (VGLUT1), and glutamate decarboxylases 65/67 (GAD65/67) along with activation of Fos-related antigen 1 (Fra-1) in the amygdala confirming the expression of stress. In HU mice, the same increase in GAD65/67 and Fra-1 indicated the contribution of SI. The special HU effect was expressed only in neurogenesis attenuation. DISCUSSION: Thus, our data indicated that 3-day HU could not be characterized as physiological stress, but SI stress contributed to the negative effects of HU.

Short-term hindlimb unloading negatively affects dopaminergic transmission in the nigrostriatal system of mice.

The nigrostriatal system composed of the dorsal striatum and the substantia nigra (SN) is highly involved in the control of motor behavior. Various extremal and pathological conditions as well as social isolation (SI) may cause an impairment of locomotor function; however, corresponding alterations in the nigrostriatal dopaminergic pathway are far from full understanding. Here, we analyzed the effect of 3-day hindlimb unloading (HU) and SI on the key players of dopamine transmission in the nigrostriatal system of CD1 mice. Three groups of mice were analyzed: group-housed (GH), SI, and HU animals. Our data showed a significant decrease in the expression and phosphorylation of tyrosine hydroxylase (TH) in the SN and dorsal striatum of HU mice that suggested attenuation of dopamine synthesis in response to HU. In the dorsal striatum of HU mice, the downregulation of TH expression was also observed indicating the effect of unloading; however, TH phosphorylation at Ser40 was mainly affected by SI pointing on an impact of isolation too. Expression of dopamine receptors D1 in the dorsal striatum of HU mice was increased suggesting a compensatory response, but the activity of downstream signaling pathways involving protein kinase A and cAMP response element-binding protein was inhibited. At the same time, SI alone did not affect expression of DA receptors and activity of downstream signaling in the dorsal striatum. Obtained data let us to conclude that HU was the main factor which impaired dopamine transmission in the nigrostriatal system but SI made some contribution to its negative effects.

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.

Audiogenic kindling stimulates aberrant neurogenesis, synaptopodin expression, and mossy fiber sprouting in the hippocampus of rats genetically prone to audiogenic seizures.

Temporal lobe epilepsy is associated with considerable structural changes in the hippocampus. Pharmacological and electrical models of temporal lobe epilepsy in animals strongly suggest that hippocampal reorganization is based on seizure-stimulated aberrant neurogenesis but the data are often controversial and hard to interpret. The aim of the present study was to estimate neurogenesis and synaptic remodeling in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). In our experiments we exposed KM rats to audiogenic kindling of different durations (4, 14, and 21 AGS) to model different stages of epilepsy development. Naïve KM rats were used as a control. Our results showed that even 4 AGS stimulated proliferation in the subgranular layer of the dentate gyrus (DG) accompanied with increase in number of doublecortin (DCX)-positive immature granular cells. Elevated number of proliferating cells was also observed in the hilus indicating the enhancement of abnormal migration of neural progenitors. In contrast to the DG, all DCX-positive cells in the hilus expressed VGLUT1/2 and their number was increased indicating that seizure activity accelerates glutamatergic differentiation of ectopic hilar cells. 14-day kindling further stimulated proliferation, abnormal migration, and glutamatergic differentiation of new neurons both in the DG granular and subgranular layers and in the hilus. However, after 21 AGS increased proliferation was observed only in the DG, while the numbers of immature neurons expressed VGLUT1/2 were still enhanced in both hippocampal areas. Audiogenic kindling also stimulated sprouting of mossy fibers and enhanced expression of synaptopodin in the hippocampus indicating generation of new synaptic contacts between granular cells, mossy cells, and CA3 pyramid neurons. Thus, our data suggest that epilepsy progression is associated with exacerbation of aberrant neurogenesis and reorganization of hippocampal neural circuits that contribute to the enhancement and spreading of epileptiform activity.

Plantar Stimulations during 3-Day Hindlimb Unloading Prevent Loss of Neural Progenitors and Maintain ERK1/2 Activity in the Rat Hippocampus.

Adult neurogenesis is a flexible process that depends on the environment and correlates with cognitive functions. Cognitive functions are impaired by various factors including space flight conditions and reduced physical activity. Physically active life significantly improves both cognition and the hippocampal neurogenesis. Here, we analyzed how 3-day simulated microgravity caused by hindlimb unloading (HU) or dynamic foot stimulation (DFS) during HU can affect the hippocampal neurogenesis. Adult Wistar rats were recruited in the experiments. The results demonstrated a decrease in the number of doublecortine (DCX) positive neural progenitors, but proliferation in the subgranular zone of the dentate gyrus was not changed after 3-day HU. Analysis of the effects of DFS showed restoration of neural progenitor population in the subgranular zone of the dentate gyrus. Additionally, we analyzed activity of the cRaf/ERK1/2 pathway, which is one of the major players in the regulation of neuronal differentiation. The results demonstrated inhibition of cRaf/ERK1/2 signaling in the hippocampus of HU rats. In DFS rats, no changes in the activity of cRaf/ERK1/2 were observed. Thus, we demonstrated that the process of neurogenesis fading during HU begins with inhibition of the formation of immature neurons and associated ERK1/2 signaling activity, while DFS prevents the development of mentioned alterations.

Dynamic Foot Stimulations During Short-Term Hindlimb Unloading Prevent Dysregulation of the Neurotransmission in the Hippocampus of Rats.

Spaceflight and simulated microgravity both affect learning and memory, which are mostly controlled by the hippocampus. However, data about molecular alterations in the hippocampus in real or simulated microgravity conditions are limited. Adult Wistar rats were recruited in the experiments. Here we analyzed whether short-term simulated microgravity caused by 3-day hindlimb unloading (HU) will affect the glutamatergic and GABAergic systems of the hippocampus and how dynamic foot stimulation (DFS) to the plantar surface applied during HU can contribute in the regulation of hippocampus functioning. The results demonstrated a decreased expression of vesicular glutamate transporters 1 and 2 (VGLUT1/2) in the hippocampus after 3 days of HU, while glutamate decarboxylase 67 (GAD67) expression was not affected. HU also significantly induced Akt signaling and transcriptional factor CREB that are supposed to activate the neuroprotective mechanisms. On the other hand, DFS led to normalization of VGLUT1/2 expression and activity of Akt and CREB. Analysis of exocytosis proteins revealed the inhibition of SNAP-25, VAMP-2, and syntaxin 1 expression in DFS group proposing attenuation of excitatory neurotransmission. Thus, we revealed that short-term HU causes dysregulation of glutamatergic system of the hippocampus, but, at the same time, stimulates neuroprotective Akt-dependent mechanism. In addition, most importantly, we demonstrated positive effect of DFS on the hippocampus functioning that probably depends on the regulation of neurotransmitter exocytosis.

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.

Glutamatergic Fate of Neural Progenitor Cells of Rats with Inherited Audiogenic Epilepsy.

Epilepsy is associated with aberrant neurogenesis in the hippocampus and may underlie the development of hereditary epilepsy. In the present study, we analyzed the differentiation fate of neural progenitor cells (NPC), which were isolated from the hippocampus of embryos of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic epilepsy. NPCs from embryos of Wistar rats were used as the control. We found principal differences between Wistar and KM NPC in unstimulated controls: Wistar NPC culture contained both gamma-aminobutyric acid (GABA) and glutamatergic neurons; KM NPC culture was mainly represented by glutamatergic cells. The stimulation of glutamatergic differentiation of Wistar NPC resulted in a significant increase in glutamatergic cell number that was accompanied by the activation of protein kinase A. The stimulation of KM NPC led to a decrease in immature glutamatergic cell number and was associated with the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B/ glycogen synthase kinase 3 beta (Akt/GSK3ß), which indicates the activation of glutamatergic cell maturation. These results suggest genetically programmed abnormalities in KM rats that determine the glutamatergic fate of NPC and contribute to the development of audiogenic epilepsy.