Search

Abstract—Experiments were carried out on rats of the Krushinsky–Molodkina (KM) strain without sound stimulation (KM-baseline group) and after the development of audiogenic kindling (AuK) in them (KM-AuK group). The control group was rats of the "0" strain, in which convulsions in response to sound were completely absent. AuK was generated using 20 repeated sound stimulation (120 dB). Neurochemical analysis was performed by HPLC/ED in the frontal cortex, hippocampus, hypothalamus, striatum, nucleus accumbens, and brainstem. It was shown that in KM rats AuK leads to the appearance of myoclonic seizures and a decrease in the intensity of generalized convulsive seizures, which is accompanied by a change in the functional activity of the noradrenergic and serotonergic systems of the brain. KM rats in the baseline (without the action of sound and the development of seizures) show a low content of norepinephrine in the hippocampus and hypothalamus, and during the development of AuK, noradrenaline deficiency is observed in the frontal cortex. After the formation of AuK, the serotonin metabolism, which is more intense than in the "0" rats, slows in the hippocampus, nucleus accumbens, and, especially, in the brainstem, and the serotonin deficiency in the striatum disappears. The features of noradrenaline metabolism in KM rats before and after AuK emphasize the important role of the cortex in the development of myoclonic seizures, as well as the possible involvement of the hippocampus and hypothalamus in tonic–clonic seizures. The high functional activity of the serotonergic system, which was revealed in KM rats in a number of brain structures, weakens in the baseline during the development of AuK. The results we obtained demonstrate and confirm the significant role of a brain monoamine imbalance in the genesis of epileptiform seizures in KM rats with genetically determined audiogenic epilepsy and in the development of AuK in them. [ABSTRACT FROM AUTHOR]

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"., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

The novel antiepileptic drug levetiracetam (keppra, 80 mg/kg, i.p.) produces a pronounced anticonvulsant effect on all parameters of the audiogenic locomotion response in Wistar rats, leading to the prevention of acoustic seizures approximately in 50% animals. Keppra injection also led to a decrease in the intensity of single audiogenic convulsive episode, a twofold prolongation of the latency of motor reaction, and a change in the pattern of seizure reaction toward increasing number of rats with "one-wave" response. In contrast, a lack of sound resistant animals and the change from one- to two-wave audiogenic response were observed in Krushinsky-Molodkina strain rats.

Proportion of animals which developed pinch-induced catalepsy and the duration of this state were analyzed in rats of several genotypes which differed in audiogenic epilepsy proneness and compared with "audiogenic" catalepsy after a sound-induced seizure fit. The following genotypes were studied: Wistar, KM (Krushinsky-Molodkina) strain and substrains "4" and "0" (selected from KM and Wistar hybrid population for high "4" and low "0" audiogenic epilepsy proneness). Adult KM and substrain "4" rats developed the most intense pinch induced catalepsy, whereas Wistar and 2-month-old KM showed practically no catalepsy. After a single sound exposure pinch-induced catalepsy developed in all animals which demonstrated an audiogenic seizure fit--in KM, substrain "4", part of Wistar rats and several animals of substrain "0", latency of the fit onset in all rats being shorter than initially. After sound exposure pinch-induced catalepsy was revealed even in those substrain "0" rats, which demonstrated no audiogenic fits. It is suggested that despite the phenomenological similarity between cataleptic states of different origin (pinch-induced, "audiogenic") their neurophysiologic substrates overlap only partially. The findings are considered as presenting genetic model for further analysis of catalepsy.

The duration and severity of cataleptic freezing after audiogenic seizures were investigated in rats of several genotypes with different predisposition to audiogenic epilepsy. These genotypes were the Krushinsky-Molodkina strain (KM--the audiogenic seizure prone strain), the Wistar and two new substrains selected from hybrids KM x Wistar for high ("4") and low ("0") audiogenic seizure susceptibility. The drug injections, which modulated the audiogenic fit severity, induced the changes in catalepsy too. It was found, that in intact animals cataleptic freezing developed after audiogenic seizures only and was more intense after severe seizures, the correlation coefficient being statistically significant. Levetiracetam (anticonvulsant drug) injections resulted in significant decrease of audiogenic fit severity and cataleptic state in Wistar and KM rats. Caffeine injections resulted in audiogenic seizures accompanied by cataleptic freezing in rats of the substrain "0", selected for audiogenic non-pronness.

The new antiepileptic drug levetiracetam (LVT) at doses of 40 and 80 mg/kg exhibits a pronounced anticonvulsant effect, influencing all the parameters of audiogenic epileptiform seizures (AES) in Krushinsky-Molodkina (KM) rats. The latent period of the motor reaction was increased 3-15 times compared to control, and the intensity of single convulsive episodes was significantly decreased. Changes were also detected in the profile of the first stage of motor activity, and the "two-wave" reaction was observed in 50-80% of animals. The mechanism of LVT anticonvulsant action in KM rats is probably associated with an increase in the inhibitory and a decrease in the excitatory processes in the CNS.

Mice of two strains with different levels of male aggression (RSB and RLB) were subjected to daily injections of 5-HT1A receptor agonist buspirone (25 microg) on the 2nd - 6th postnatal days. This neonatal treatment augmented the aggressive behavior (tested in the dyadic contests with non-aggressive A/Sn males) in aggressive RSB mice and reduced aggression in less aggressive RLB. Correlations with different signs were found between the 5-HT and 5-HIAA levels in the neocortex, hippocampus, and hypothalamus and behavioral indices of aggression in RSB and RLB males. The remote effects of neonatal buspirone in these two mice strains presumably depend on genotype-related features of ontogeny of the 5-HT system.

Physiological, biochemical and genetic studies of audiogenic epilepsy in Krushinsky-Molodkina (KM) rat strain are analyzed. In brief, the history of KM strain selection and its current state are reviewed. Physiological mechanisms of the typical audiogenic seizure development and the audiogenic myoclonus (audiogenic kindling phenomena) are considered. The EEG correlates of these phenomena are presented. Biochemical features of the strain (neurotransmitter and neuronal membrane biochemistry) are described.

The expression of audiogenic seizure fits has been studied in F1 hybrids between audiogenic seizure-prone Krushinsky-Molodkina rat strain and Wistar rats not prone to audiogenic seizures, as well as in two backcross generations. Only 10% of F1 hybrids exhibit audiogenic seizure fits, whereas the frequency of this character in two generations of their backcrosses with Krushinsky-Molodkina rats is about 50%. A digenic model with incomplete penetrance has been put forward to explain the control of audiogenic seizure fits. This model fits the data obtained: the theoretically expected distributions of the character in offsprings of different crosses do not differ significantly from those observed in experiments. The model explains why the distribution of the character is the same in the first and second backcross offsprings.

Rats Krushinsky-Molodkina inbred strain (KM) genetically prone to audiogenic seizures were injected with pineal hormone melatonin (50 mg/kg, s.c.) within the period 7th to the 14th posnatal days (PND). The remote effects of this injection adult KM rats consisted in a decrease in the latency and increase in severity of myoclonic seizures produced by audiogenic kindling (20 sound stimuli, 100 dB and 12-15 kHz). As compared to the control, in the cortex and hippocampus of rats of melatonin group, we also found a significant reduction of both total and functional activity of Ca2+/calmodulin-dependent protein kinase II (CAMK II) after audiogenic kindling. On the contrary, melatonin administration within the 1st to 7th PND and the 14th to the 21st PND resulted in a decrease in seizure activity. In the first case, both the total (cortical) and functional (hippocampal) CAMK II activities in melatonin-injected rats were increased as compared to control, whereas in the second case, only a slight increase in Ca2+-independent CAMK II activity in the hippocampus of melatonin-injected rats was observed. Probably, the melatonin administration in the period of early postnatal development changes the features of expression and/or regulation of CAMK II activity, and this could be one of the mechanisms of audiogenic seizure modulation in KM rats.

The reaction to a single sound stimulation (a bell) applied daily and the time of the first appearance of myoclonus were recorded in Krushinsky-Molodkina (KM) rats of different ages (30, 60, and 150-200 days). The audiogenic seizures in adult animals were of short latency and always resulted in tonic convulsions, which were stable in their patten. In contrast, in young animals (in particular, in 30-day-old rats), the seizures were less pronounced and unstable. The daily sound stimulation produced a gradual development of myoclonic seizures in young and adult rats. In old rats, the stable myoclonus appeared on the 17th day of stimulation, and in 30-day-old animals the myoclonus was recorded on the 26th day. In 60-day-old rats the myoclonus appeared for the first time on the 20th day of stimulation. It is suggested that these age differences can be explained by the btain immaturity (in particular, of neurotransmitter system) in young animals.

It was shown that the content of serotonin (5-OT) and 5-oxyindoleacetic acid (5-OIAA) in the temporal cortex, hippocampus and medulla oblongata and the content of dopamine and 3,4-dihydroxyphenilacetic acid in the striatum are increased initially, while the level of norepinephrine in the striatum is reduced in Krushinskiĭ-Molodkina rats genetically predisposed to audiogenic epilepsy in comparison with Wistar rats. Carbamazepine levelled out the changes in the 5-OT and 5-OIAA levels at the peak of the audiogenic attack in the temporal cortex and hippocampus, causing at the same time a sum increase in the content of all monoamines and their metabolites in the medulla oblongata and striatum.

The effect of Lamotrigine and carbamazepine on the development and character of the epileptiform seizures after strong sonic stimulation were studied on Krushinskiĭ-Molodkina line of rats. Both drugs manifested a dose-dependent increase in the latency of motor reaction Lamotrigine administration in the dose range of 5-40 mg/kg, i.p., resulted in a moderate decrease in the seizure intensity preventing predominantly the tonic component of the seizure and failed to modify the character of convulsive reaction. Carbamazepine administered in doses of 7.5-30 mg/kg effectively prevented the epileptiform seizures. The "two-wave" motor reaction was observed in 15% of carbamazepine-treated rats.

The anticonvulsant action of diphenin, sodium valproate, and pentobarbital was studied in Krushinskiĭ-Molodkina (KM) rats subjected to audiogenic seizures. The anticonvulsant effects of the drugs were different and dose-dependent. Valproate in a dose of 120 mg/kg completely eliminated the tonic phase of seizures. Given in a dose of 300 mg/kg it in 50% of cases prevented from the development of the initial seizure stage of motor excitation. The peculiarity of the pentobarbital action consisted in its ability to change the character of motor excitation during sound stimulation from the one-wave reaction for the two-wave.

Genetical homogeneity of Krushinskiĭ-Molodkina (KM) stock was studied by the method of reciprocal transplantation of skin. The rats were selected inside genealogic card from 5 different substrains of the 20th generation of brother-sister crossing (F 20). The tail-to-tail transplantation was made by the scheme of double reciprocal ring in 9 females from the KM stock. Observations within 314 days showed a good condition of the transplants. The obtained data suggest that Krushinskiĭ-Molodkina rats are an inbred strain.

Male Wistar rats were trained to perform dissociated conditioned reactions with food reinforcement in norm and under the action of pentobarbital (15 mg/kg). Retrieval of the learned tasks was investigated after isolated injections of synthetic antioxidant phenozan-K (40 mg/kg) and in combination with pentobarbital (7.5 mg/kg). In contrast to the results obtained with KM rats, in Wistar rats the influence of the antioxidant on pentobarbital effects was not detected. Comparative study of brain phospholipid content in rats revealed significant distinctions between the strains after pharmacological influences. It was supposed that the effects of phenozan-K depended on the initial brain phospholipid content. This may be one of the reasons of phenozan-K-induced modifications of retrieval in KM but not Wistar rats.

Brain tribulin activity in rats with an inherited predisposition to audiogenic epilepsy was studied after seizures of different intensity were induced by an electric bell. Weak seizures (from 0 to 2 arbitrary units) did not produce any changes in endogenous inhibitory activity towards either monoamine oxidase (MAO) A or B. Moderate seizures were characterized by increases in both MAO A and MAO B inhibitory activity (up to 1.9-fold). Complete tonic epileptiform seizures with total areflexia (4 arbitrary units) induced further augmentation (up to 2.5-fold) of MAO A but not of MAO B inhibitory activity. This dissociation between the two inhibitory activities points to the existence of a separate MAO A-inhibiting component of brain tribulin which is different from isatin.

Catalytic properties of mitochondrial monoamine oxidases (MAO) were studied in brain, heart, liver and kidney of rats of Krushinskii-Molodkina strain with inherited audiogenic epilepsia. In brain of these rats a. 2.5-fold decrease of Vmax of serotonin (MAO-A substrate) deamination has been found. During audiogenic epileptiformic attack 2.5-5-fold decrease of Vmax of serotonin deamination in brain and liver and also decrease of benzylamine (MAO-B substrate) oxidation in brain were found. Simultaneously oxidation of glucosamine and gamma-aminobutyric acid was markedly increased (up to 5-fold and more) in brain. The data obtained showed that qualitative transformation occurred in catalytic properties of MAO which may be an important pathogenic factor in the development of epileptiformic attack.

Dependence was studied of pentobarbital effects at dissociated learning in rats of KM line after intraperitoneal injection of antioxidant phenozan-K. It was shown that preliminary (before 1.5 hour) injection of 40 mg/kg of phenozan-K to animals changed the composition (index of oxidation S) of cells lipids of rats brain membranes and simultaneously increased the sensitivity to pentobarbital. Injection of half a dose of pentobarbital, which caused dissociated state, against the background of antioxidant action had the same effect as the full dose (15 mg/kg).

On rats of KM (Krushinskiĭ-Molodkina) line the influence was studied of antioxidant (phenozan-K) on the ability to solve extrapolation task and behaviour in the open field test. It is shown that intraperitoneal injection of the antioxidant in a dose of 30 mg/kg significantly increases the number of individuals solving the extrapolation task at its first presentations and also activates the orienting-investigatory behaviour in the open field test. Proceeding from the supposition about the necessity of sufficiently high (optimum) level of excitability of the central nervous system for realization of mental act, a conclusion is made about the possibility of its correction by means of antioxidants.