Aversion But Not Aggression: Emotional Traits of KM Rats in Sociability Tests

Animal models of human diseases are commonly used in experimental research. Autism Spectrum Disorder (ASD) is characterized by reduced social motivation and often co-occurs with epilepsy, representing a distinct clinical subgroup. This study investigated social deficits in Krushinsky-Molodkina (KM) rats, which present with latent epilepsy and an autistic-like phenotype, by measuring ultrasonic communications during social interaction. The three-chamber test for social preference/novelty, accompanied by registration of ultrasonic vocalizations (USVs), was conducted in 12 KM and 12 control Wistar rats. For analysis, each individual vocalization trajectory was mapped and the results were derived from aggregating the individual data. To assess potential anhedonia, sucrose preference was tested in a separate group (10 KM and 20 Wistar rats) by measuring the consumption of 1% sucrose and water in individual rats over a 24-h period. All animals used were seizure-naive males, aged 4-6 months. A longer duration of aversive USV calls was registered during the sociability tests in KM rats (p < 0.05, compared with controls). The majority (p < 0.05) of aversive USVs occurred when KM rats distanced themselves from the social stimuli, and the duration of these calls showed a positive correlation with freezing behavior (Spearman coefficient Rs = 0.68, p < 0.05). Reduced sucrose preference was not observed in KM rats; instead, an increase in daytime sucrose consumption was noted. KM rats exhibit negative emotional states in sociability tests, as evidenced by enhanced aversive vocalizations and distancing behavior. The social aversion observed in KM rats is not associated with anhedonia.

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

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 3months. At the age of 1-2months, 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.

The aim of present work was to evaluate functional state of the hypothalamic–pituitary–adrenal axis (HPA) in both naive control Wistar rats and Krushinsky-Molodkina (KM) rats, genetically prone to audiogenic epilepsy, and analyzed the changes of activity of HPA in KM rats during and after expression of audiogenic seizure (AGS). Obtained data demonstrated that the activity of corticotropin-releasing hormone (CRH) neurons in KM rats was reduced, due to the increased inhibitory effect of GABAergic neurons. In the anterior pituitary, we observed accumulation of proopiomelanocortin and adrenocorticotropic hormone (ACTH). However, basal levels of ACTH and corticosterone were equal in the blood in both naive KM rats and Wistar rats. Thus, the data indicate the disturbances in the interaction between the hypothalamus and the pituitary gland in KM rats, which can be one of the regulatory mechanisms for maintaining hormone concentrations in the blood. Analysis of AGS effects on HPA demonstrated increasing secretion of CRH and vasopressin into the portal system of KM rats and ACTH in the blood at clonus-tonus stages, which expresses in 6-10 seconds after sound stimulation. Therein corticosterone level was increased in KM rats during 24 hours. In Wistar rats, the sound stimulation did not change the levels of ACTH and corticosterone in the blood. Thus, we demonstrated specific activation of HPA in KM rats by AGS as a result of epileptiform activity overspreading in the brain that revealed participation of HPA is the expression of seizure activity.

Accumulating evidence indicates that inherited astrocyte dysfunction can be a primary trigger for epilepsy development; however, the available data are rather limited. In addition, astrocytes are considered as a perspective target for the design of novel and improvement of the existing antiepileptic therapy. Piracetam and related nootropic drugs are widely used in the therapy of various epileptic disorders, but detailed mechanisms of racetams action and, in particular, their effects on glial functions are poorly understood. In this study, we explored the functional state of astrocytes in the dentate gyrus (DG) of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures and compared the action of piracetam on the DG astrocytes in KM and normal Wistar rats. Wistar and naïve KM rats which received injections of saline (control) or piracetam (100 mg/kg) for 21 days were recruited in our studies. Comparative analysis of control Wistar and KM rats revealed genetically determined abnormalities in DG astrocytes of KM rats including an increased expression of NFIA but a decreased GFAP, ALDH1L1, EAATs, and glutamine synthetase (GS). Piracetam treatment normalized the expression of all studied markers, except NFIA, in KM rats, while in Wistar rats, it potentiated only GS and NFIA. The results suggested that the nootropic and antiepileptic effects of piracetam may be, at least partially, mediated by the modulation of astroglia functions. In addition, analysis of NFIA and GS colocalization revealed the novel pattern of astrocyte heterogeneity in the DG which was significantly altered in epileptic rats but corrected by piracetam.

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

The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H2O2 generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.

Актуальность. Селектированная на проявление судорог в ответ на сильный звук (аудиогенную эпилепсию, АЭ) линия крыс Крушинского-Молодкиной (КМ) - модель судорожных состояний человека, c быстрым развитием судорожного припадка в ответ на включение звука (100-120 дБ). Однако у крыс с АЭ изменений уровня кортикостерона (КС) в крови в связи с судорогами не определяли, хотя анализ связи АЭ и стресс-реакции - важная практическая задача. Методы. Уровень КС в образцах сыворотки крови определяли с помощью набора у крыс линии КМ, а также у крыс линии «0», селектированных из гибридной популяции КМ х Вистар на отсутствие АЭ, и у крыс популяции Вистар. Результаты. У всех крыс КМ был зарегистрирован аудиогенный припадок, тогда как у крыс линии «0» и Вистар судорог не было. Через 30 мин после действия звука (и судорог) у крыс КМ повышеается уровень КС, тогда как сразу после судорог (через 1-3 мин) данная реакция не обнаруживается. У крыс линии «0» обнаружен достоверно более высокий фоновый уровень КС, по сравнению с КМ и Вистар. Уровень КС у них, как и у крыс Вистар, после действия звука не изменялся. Заключение. Подъем уровня КС в крови после действия звука наблюдали только у крыс КМ через 30 мин после припадка АЭ. Крысы линии «0», у которых нет судорог в ответ на звук, обнаружили более высокий, чем у КМ и Вистар уровень КС в фоне. Aims. The Krushinsky-Molodkina (KM) rat strain, which was selected for a seizure response to a sound (audiogenic epilepsy, AE), is known as a model of human seizure states. Although the association of blood CS with stress reaction is an important practical issue, blood levels of corticosterone (CS) have never been analyzed in rats with AE in relation with seizures. Methods. Serum concentration of CS was measured using IBL International Corticosterone ELISA kits in KM rats, rats of the «0» strain, which was selected (based on F2 KM х Wistar hybrids) for the lack of seizures in response to a sound, and Wistar rats. Results. All KM rats developed AE seizures in response to a sound while no seizures were observed in rats of the «0» strain and Wistar rats. The increase in blood CS was observed only in KM rats at 30 min after AE seizure. At background, the level of CS was significantly higher in rats of the «0» strain, which did not develop seizures in response to a sound, than in KM and Wistar rats. CS levels remained unchanged in both «0» and Wistar rats after the sound exposure. Conclusions. The increase in blood CS occurred in KM rats at 30 min after the seizure episode. At background, the CS level was higher in rats of the «0» strain, which did not respond with seizures to a sound, than in Wistar and KM rats. Therefore, the blood level of CS depends in a complicated way on both AE and the selection history of the «0» rat strain.

Abstract—Functional activities of dopaminergic, GABAergic and glutamatergic neurons were studied in the nigrostriatal system of Krushinsky–Molodkina (KM) rats genetically prone to audiogenic seizures as compared with control Wistar rats. In KM rats, a reduced activity of GABA-and dopaminergic neurons was detected in the substania nigra, while glutamatergic transmission remained intact. In the striatum of both KM and Wistar rats, no significant differences were found in levels of the GABA-synthesizing enzymes, GAD65/67 and phospho-tyrosine hydroxylase, however, an increased level of D1 dopamine receptor and decreased of D2 dopamine receptor imply possible upregulation of glutamatergic neurotransmission. Indeed, an increased expression of vesicular glutamate transporter VGlut2 and the NMDA receptor NR2B subunit was revealed in the striatum of KM rats. While the striatum glutamatergic fibers were demonstrated to express phosphorylated ERK1/2 kinases, both the striatum and substantia nigra in KM rats exhibit an increased activity of ERK1/2. This correlates with intense exocytosis, as evidenced by a decreased level of SNAP25 protein. Activation of the glutamatergic system may result, among other reasons, from the disruption of the inhibitory effect of the substantia nigra dopamine- and GABAergic systems innervating the striatum. We suggest that the increased activity of the striatum glutamatergic neurons in KM rats lacking adequate dopamine- and GABAergic inhibitory feedback may, at least in part, account for heightened convulsive readiness in KM rats.

The study of the role of neurotransmitter systems in the pathogenesis of epilepsy is one of the priorities of epileptology. New data on the functions of free neurotransmitter-like amino acid in the central nervous system are of the greatest importance and determine the prospects for the development of novel effective anticonvulsants. It is widely believed in clinical medicine that epilepsy has distinct gender characteristics. The aim of this study was to investigate the gender peculiarities in the content of neurotransmitter amino acids in the brain of Krushinsky-Molodkina (KM) rats, which were used as model organisms for the study of genetically induced audiogenic epilepsy. The content of Asp, Glu, GABA, Gly, and Tau of the medulla oblongata, hippocampus and cerebral cortex were determined using high-performance liquid chromatography (HPLC) in intact KM rats, KM rats exposed to a series of epileptiform seizures, and Wistar rats (control group). Both the Wistar and KM rats had gender distinctions in the distribution of free amino acids among the investigated brain parts. The audiogenic epilepsy was characterized by smoothing gender differences as well as differences between the concentrations of free amino acids in the cortex and medulla oblongata, specific for Wistar rats. The changes observed in male rats after the set of seizures included the increase in GABA concentration and a decrease in the Gly level in all investigated brain parts, as well as the decrease of the Tau content in the cortex and hippocampus. At the same time, the Glu content in cortex increased, while the Asp level decreased. After 6 days of audiogenic stimulations the female KM rats demonstrated the increase in the Glu level in all investigated brain parts, the increase in Gly and Asp levels in hippocampus, and no changes in the GABA content. Thus, after the set of epileptiform seizures the KM rats achieved a new steady state of the studied amino acids pool, which differed in males and females. In this case, gender differences significantly changed after the 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.

The present study analysed the effects of audiogenic kindling on the functional state of the vasopressinergic system of Krushinsky-Molodkina (KM) rats. KM rats represent a genetic model of audiogenic reflex epilepsy. Multiple audiogenic seizures in KM rats lead to the involvement of the limbic structures and neocortex in the epileptic network. The phenomenon of epileptic activity that overspreads from the brain stem to the forebrain is called audiogenic kindling and represents a model of limbic epilepsy. In the present study, audiogenic kindling was induced by 25 repetitive audiogenic seizures (AGS) with 1 AGS per day. A proportion of KM rats did not express AGS to sound stimuli, and these rats were characterised as the AGS-resistant group. The data demonstrated that audiogenic kindling did not change activity of extracellular signal-regulated kinase 1/2 or cAMP response element-binding protein, although it led to an increase in vasopressin (VP) expression in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN). Additionally, we observed a decrease in GABAergic innervation of the hypothalamic neuroendocrine neurones after audiogenic kindling, whereas glutamatergic innervation of the SON and PVN was not altered. By contrast, analysis of AGS-resistant KM rats did not reveal any changes in the activity of the VP-ergic system, confirming that the activation of VP expression was caused by repetitive AGS expression, rather than by repetitive acoustic stress. Thus, we suggest that overspread of epileptiform activity in the brain is the main factor that affects VP expression in the hypothalamic magnocellular neurones.

Inherited and seizure-induced abnormalities in the temporal cortex astroglia of Krushinsky-Molodkina audiogenic rats: A pilot study.

In clinical practice, epilepsy is often comorbid with the autism spectrum disorders (ASDs). This warrants a search of animal models to uncover putative overlapping neuronal mechanisms. The Krushinsky-Molodkina (KM) rat strain is one of the oldest inbred animal models for human convulsive epilepsies. We analyzed the behavioral response of adult seizure-naive KM males in three-chambered tests for social preference. We found that a presence of social stimuli (encaged unfamiliar Wistar rats of the same age and sex) evoked a reduced or reversed exploratory response in freely moving KM individuals. The epilepsy-prone rats demonstrated remarkably shortened bouts of social contacts and displayed less locomotion around the stranger rat-containing boxes, together with a pronounced freezing response. The decrease in social preference was not due to a general decrease in activity, since relative measures of activity, the index of sociability, were decreased, too. The susceptibility to audiogenic seizures was verified in the KM cohort but not seen in the control Wistar group. We propose the KM rat strain as a new animal model for comorbid ASD and epilepsy.

Epilepsy or epileptic syndromes affect more than 70 million people, often comorbid with autism spectrum disorders (ASD). Seizures are concerned as a factor for social regression in ASD. A stepwise experimental approach to this problem requires an animal model to provoke seizures and monitor subsequent behavior. We used rats of the Krushinsky-Molodkina (KM) strain as a validated inbred genetic model for human temporal lobe epilepsy, with recently described social deficiency and hypolocomotion. Generalized tonic-clonic seizures in KM rats are sound-triggered, thus being controlled events in drug-naïve animals. We studied whether seizure experience would aggravate contact deficits in these animals. Locomotor and contact parameters were registered in "the elevated plus maze", "socially enriched open field", and "social novelty/social preference tests" before and after sound-provoked seizures. The triple seizure provocations minimally affected the contact behavior. The lack of social drive in KM rats was not accompanied by a submissive phenotype, as tested in "the tube dominance test", but featured with a poor contact repertoire. Here, we confirmed our previous findings on social deficits in KM rats. The contact deficiency was dissociated from hypolocomotion and anxiety and did not correlate with seizure experience. It was established that experience of rare, generalized tonic-clonic convulsions did not lead to an impending regress in contact motivation, as seen in an animal model of genetic epilepsy and comorbid social deficiency. One of the oldest animal models for epilepsy has a translational potential to study mechanisms of social behavioral deficits in future neurophysiological and pharmacological research.