Selection of Laboratory Mice for the Cognitive Task Successful Solution and for the Inability to Solve It.

Using the selected mouse strain EX as the founding population (selection for extrapolation ability) three selection generations of mice were obtained, which were selected for successful solution of object permanence test (plus-sub-strain) and for lack of such solution (minus-sub-strain). The successful solution required not only the ability to operate the object permanence rule (by J. Piajet), but the performance of complicated action (executive function) which was significantly higher in plus-substrain, and this is the unique example of successful selection for cognitive trait.

Effect of Ethosuximide on Audiogenic Epilepsy in Krushinsky-Molodkina Rats.

The anticonvulsant effect of ethosuximide (T-type calcium channel blocker) was evaluated in Krushinsky-Molodkina rats predisposed to audiogenic epilepsy. Ethosuximide given with drinking water (300 mg/kg/day) over 45 days slightly reduced proneness to audiogenic epilepsy and increased locomotor activity of the animals at the periphery of the open field. Neonatal administration of ethosuximide (3-4 mg per animal, from 2 to 10 days of life) insignificantly modulated the parameters of audiogenic epilepsy in these animals at the age of 1.5 months and reduced manifestation of audiogenic myoclonic convulsion that developed after long daily sound presentation started at the age of 3 months. The findings attested to a weak anticonvulsant effect of ethosuximide on tonic convulsions with its predominant effect on convulsions with forebrain focus location.

Study of Uridine Effect on the Development of Audiogenic Tonic Seizures in Krushinsky-Molodkina Strain Rats.

The latency of tonic seizure in response to loud sound (in rats of the Krushinsky-Molodkina strain with audiogenic epilepsy) had been slightly (although statistically significantly) longer after chronic uridine injections (100 mg/kg, i.p., three times a day during 9 or 12 days). The recovery time from the tonic seizure was shorter after 12 days of injections in comparison to the 9-day injection period. At the same time, the intensity of tonic seizures provoked by loud sound did not change after chronic uridine injections. The lack of uridine anticonvulsive effect demonstrated in the audiogenic epilepsy model contradicts the anticonvulsant effects of uridine in experiments with other seizure models, in which the epileptic foci were localized in the forebrain structures.

Audiogenic Epilepsy and Structural Features of Superior Colliculus in KM Rats.

Using immunoblotting, we showed that in rats of audiogenic epilepsy (AE) prone strain (Krushinsky- Molodkina, KM) the superior colliculus tissue (SC) contains significantly less quantity of glial neurotrophic factor (GDNF), beta-tubulin and actin in comparison to the same brain region in "0" rats, nonprone to AE. This fact led to the suggestion that the histological structure of the SC in KM rats could differ significantly from that of the "0" strain. Using neuromorphologу technique, we demonstrated that the total number of SC cells, as well as the number of neurons were significantly less in KM rats than in the "0" strain rats. Particularly strong differences were found in the deep layers of SC, the area of terminals from IC. Further studies of the midbrain structures, will help to identify the novel aspects of neural networks, involved in the genesis of AE in rats of KM strain.

Influence of "Enriched Environment" on Behavior and Neurogenesis in Mice Selected by Cognitive Trait.

Mice selected for high score in the extrapolation test (EX line) and kept under conditions of "enriched environment" for 3 months demonstrated changes in locomotor and exploratory activity and enhanced reaction to novelty. The relative brain weight was higher and neurogenesis in the hippocampal fascia dentate was more intensive in this group. In non-selected mice, the changes were similar, but insignificant in many cases.

Intermale aggression in mice, selected for the cognitive trait.

The data are presented on intermale aggression in mice which were selected for high scores of cognitive trait (the ability for extrapolation of movement direction) in comparison to the data of control mice performance. The changes in aggression level in the course of selection are presumably connected with anxiety level which also changed during selection generations.

Remote effects of short-term neonatal hyperthermia in Krushinsky-Molodkina rats prone to audiogenic seizures strain.

Using the audiogenic seizure prone Krushinsky-Molodkina rat strain, it was demonstrated that short-term (5 min) exposure of 14-day-old pups to an elevated temperature (42°C) resulted in a significant decrease in audiogenic seizure severity at the age of 1 month. Presumably, this effect is determined by the activation of the heat-shock protein system (stress proteins).

The Krushinsky-Molodkina rat strain: The study of audiogenic epilepsy for 65years.

The more recent history and main experimental data for the Krushinsky-Molodkina (KM) audiogenic rat strain are presented. The strain selection started in late 1940. Now this strain is inbred, and two new strains are maintained in a laboratory in parallel. These strains originated from KM×Wistar hybrids and were bred (starting from 2000) for no-seizure and intense audiogenic seizure phenotypes, respectively. The experimental evidences of audiogenic seizure physiology were accumulated in parallel with (and usually ahead of) data on other audiogenic-prone strains. The peculiar feature of the KM strain is its vulnerability to brain hemorrhages. Thus, the KM strain is used not only as a genetic model of seizure states, but also as a model of blood flow disturbances in the brain. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

The ability to solve elementary logic tasks in mice with the knockout of sodium-calcium exchanger gene 2 (NCX2).

Mice with a knockout of the sodium-calcium exchanger 2 (NCX2) gene were statistically significantly more successful than wild-type controls in the solution of two cognitive tasks, the test for the capacity to extrapolate the direction of the stimulus movement and the "puzzle-box" test for the capacity to find a hidden route to safe environment, which were based on food and aversive motivations, respectively. In both tests, the success of task solution was based on the animal's ability to use the object's "permanence" rule (according to J. Piaget). The data confirm that the knockout of this gene, which is accompanied by modulation of the temporal pattern of calcium membrane flux, also induces changes in mouse CNS plasticity.

Effect of chronic fluoxetine treatment on audiogenic epilepsy, symptoms of anxiety and depression in rats of four lines.

Anxiety (Anx) and depression (Dp) levels were evaluated in rats of 4 lines: 2 of them (KM and "4") exhibited audiogenic seizures (AS), and 2 (Wistar and "0") had no AS. In KM rats (with AS), Anx and Dp levels were higher than in Wistars (without AS), while in "4" and "0" rats with the related genetic background but contrasting in AS severity, Anx and Dp indices were not different. Fluoxetine treatment exerted antidepressant effect in all rat lines irrespective of its effect on AS. Thus, phenotypic expression of AS is not directly associated with the mechanisms of Anx and Dp development.

Audiogenic epilepsy and GABAergic system of the colliculus inferior in Krushinsky-Molodkina rats.

GABAergic brain system is an important link in the pathological circuits of rodent audiogenic epilepsy (AE). The number of GABAergic neurons in the inferior colliculi of KM strain rats (AE prone) was not different from that of the control non-prone strain. At the same time, the glutamate decarboxylase mRNA expression (the key enzyme of GABA synthesis) was 5 times higher than in control. The data evidence that the GABAergic system does play an important role in AE proneness.

Maternal methyl-enriched diet in rat reduced the audiogenic seizure proneness in progeny.

Audiogenic epilepsy proneness was analyzed in the progeny of rats from two strains (audiogenic seizure prone-strain "4"-and audiogenic seizure non-prone, strain "0"). Females were fed by a diet which contained substances enriched with methyl-groups during 1week before mating (MED), during pregnancy period and 1week after the delivery. This MED treatment resulted in a decrease of audiogenic seizure fit intensity, which was more evident in rats of strain "0". Control rats of strain "4" displayed intense seizures (tonic seizure, 3.85 arbitrary units). Med "4" rats seizures were less intense (3.23, tonic seizure of lower intensity), control "0" strain rats demonstrated the seizure with mean 3.09 arbitrary units, "0" MED rats only 2.03 arbitrary unit intensity (only clonic seizures, significantly, p<0.05, different from controls). Methyl-enriched diet resulted in the significant changes in methylation status of several genes (Cpne6, Gtf2i, Sctr,1 Sfmbt, Phe2). These genes among others were chosen for analysis as their expression was analyzed in other methylation study. These genes were hypermethylated after "epileptic tolerance". Due to this procedure, the intensity of status epilepticus, produced by kainate in mice, decreased (Miller-Delaney et al., 2012). The modulation of audiogenic seizure intensity as the result of methyl-enriched diet during prenatal and early postnatal ontogeny was demonstrated for the first time.

[The behavior of mice selected for cognitive trait in hyponeophagia test].

Hungry mice from strain which is under selection for high scores of capacity for extrapolation the direction of stimulus movement (line EX) the hyponeophagia level (reaction to new food in novel environment) was significantly lower than in mice of control unselected population (CoEX). This signify their lower anxiety as well as higher neophilia (which in turn is connected to cognitive abilities). These mice displayed more:approaches to food, they ate food for longer time intervals and their consumed more food. At the same time these mice revealed higher anxiety indices in the EPM test controls. The data made it possible to suggest that anxiety states which develop in different situations are non-uniform, and this view finds the confirmation in the literature.

[Laboratory rat selection for the trait "the absence of audiogenic seizure proneness"].

The hybrids between Krushinsky-Molodkina (KM) inbred strain, selected for high predisposition to audiogenic epilepsy (AE), and Wistar rats non-prone to audiogenic seizure were the initial population for selection. Rats were selected for the trait "the absence of audiogenic seizure proneness". The creation of such strain in which the significant proportion of animals develop no AE in response to sound and share partly the genetic background of the KM strain is very important for the correct use of RV strain as the laboratory model of seizure states. As alleles which determine the AE proneness are recessive the selection for the "opposite" trait proceeds necessarily slow.

Binding of specific ligand by D2- and NMDA-receptors of striatum cells in two rat strains predisposed and resistant to audiogenic seizures.

We studied parameters of specific receptor binding of D2-dopamine receptor ligand [(3)H]-sulpiride and NMDA-receptor ligand [(3)H]-MK-801 on the membranes of striatum cells in Krushinsky-Molodkina rats (predisposed to audiogenic seizures) and strain "0" selected for the absence of audiogenic seizures. No interstrain differences were observed in affinity (K(d)) of both D2- and NMDA-receptors to ligands. At the same time, significant interstrain differences in receptor density (B(max)) were found for both D2-receptors and NMDA-receptors. The reduced number of dopamine and glutamate receptors in the striatum can be associated with neurological peculiarities of Krushinsky-Molodkina rat strain (audiogenic seizures and postictal catalepsy).

[Neonatal injections of pharmacological agents and their remote genotype-dependent effects in mice and rats].

Experimental data were reviewed which demonstrated that the neonatal injection effects of certain biologically active drugs (ACTH(4-10) fragment and its analogue Semax, piracetam, caffeine, levetiracetam, busperone, etc.) could be detected in adult animals as changes in physiological and behavioral reactions and in several morphological traits as well. Audiogenic seizures proneness, anxiety-fear and exploration behavior as well as pain sensitivity were analyzed. The remote effects discovered were either similar in direction to those applied to an adult organism, or opposite to it. Pharmacological treatments of such type presumably interfere the CNS development during early postnatal ontogeny and change the normal pattern ofbrain development. These modulatory influences could be due to changes in neurotransmitter system development and are presumably capable to induce CNS morphological deviations (numbers of neurons, adult neurogenesis).

Anxiety and predisposition to audiogenic epilepsy in rats of different genotypes.

We studied plus-maze behavior of inbred Krushinskii-Molodkina, Wistar, and black-hooded rats (originating from the Long-Evans outbred strain) differing by predisposition to audiogenic seizures. The severity of audiogenic seizures partially correlated with anxiety and negatively correlated with the total level of locomotor activity in the elevated plus-maze. The anxiety parameters in Krushinskii-Molodkina rats were evaluated after injection of anticonvulsant levetiracetam and anxiolytic afobazol. Levetiracetam and afobazol somewhat stimulated locomotor activity.

[Selection of mice for high level of extrapolation capacity with cobcommitant low anxiety level].

The behavior scores were assessed in mice selected simultaneously for high percentage of correct extrapolation task solutions and for low anxiety during test performance. Extrapolation test requires that the hungry animal searches for the food bait which disappeared from the view moving in the direction of food bait movement. In the 4th selection generation no significant changes occurred in the percentage of correct task solutions neither in comparison to control unselected population, nor against 50% chance level. Although the proportion of mice in selected strain which performed with 80-100% of correct solutions increased and in F4 was higher in comparison to controls (approaching significance). The proportion of "0" solution (when mouse made no choice) and of "refusals" of performance (anxiety indices in this test) were lower in selected line and the proportion of refusals in F4 was significantly lower than in controls. Elevated plus maze, closed plus maze and inescapable slippery funnel tests demonstrated significantly lower anxiety in mice of selected strain. These data demonstrate much more complex genetic basis for the capacity for extrapolation (lack of response to selection) in comparison with that of anxiety traits in mice (changes in the response to selection).