Aging is accompanied with attenuation of the inhibitory γ-aminobutyric acid (GABA) neurotransmission, while age-related neurological disorders including neurodegenerative diseases and epilepsy can exacerbate this deficit. However, the interrelation between age-dependent dysfunction of GABAergic transmission and inherited epileptic predisposition remains unclear. In this study, we analyzed the markers of GABAergic system in the hippocampus of aging (18-month-old) Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). To delineate the inherited background, we compared naïve KM rats with no AGS experience with Wistar rats of the corresponding age. The results showed that GABA-positive cell numbers and glutamate decarboxylase 67 (GAD67) expression in the hippocampus of aging KM rats were increased indicating the higher activity of GABAergic interneurons and GABA production. Increased colocalization of Fos-related antigen 1 and GAD67, along with elevated parvalbumin, additionally confirmed this suggestion. At the same time, unchanged expression of vesicular GABA transporter, synaptic vesicle glycoprotein 2A, and synaptosomal-associated protein, 25 kD, together with unaltered GABA content in fibers and terminals, suggested that the activity of GABA exocytosis corresponded to the age-related norm. Analysis of proteins, regulating postsynaptic GABA action, revealed normal expression of cation-chloride transporters, however, GABA-A receptors were significantly decreased. Thus, we suppose that the increased activity of the inhibitory GABAergic interneurons of aging KM rats may restrict hyperexcitability of the hippocampus, in particular, during single AGS. However, GABA receptor deficit may, in opposite, contribute to overexcitation of the hippocampus during repetitive seizures and promote excitotoxic damage of the hippocampal cells at the advanced age.

Susceptibility to audiogenic epilepsy (AE) seizures and phenotypic manifestations of epileptic activity in rats of the Krushinsky-Molodkina (KM) strain were shown to develop in parallel with the appearance of seizure EEG patterns from 2 to 7 months of age. The seizure latency decreased with age, while the intensity of convulsive seizures increased. Two types of epileptiform discharges (EDs) were identified in background EEGs (no exposure to sound) of KM rats. One type had a form of high-amplitude generalized packs of waves with the animal shuddering. The other type had a form of generalized non-seizure absence-like spike-and-wave discharges with the animal freezing. The total and mean durations of single absence-like discharges increased with age. Parallel age-related increases in the severity of AE seizures and the number of generalized absence-like discharges in the forebrain EEG suggests the development of the epileptic system in KM rats.

Epilepsy is a multifaceted neurological disorder characterized by recurrent seizures and associated with molecular and immune alterations in key brain regions. The GASH/Sal (Genetic Audiogenic Seizure Hamster, Salamanca), a genetic model for audiogenic epilepsy, provides a powerful tool to study seizure mechanisms and resistance in predisposed individuals. This study investigates the proteomic and immune responses triggered by audiogenic kindling in the inferior colliculus, comparing non-responder animals exhibiting reduced seizure severity following repeated stimulation versus GASH/Sal naïve hamsters. To assess auditory pathway functionality, Auditory Brainstem Responses (ABRs) were recorded, revealing reduced neuronal activity in the auditory nerve of non-responders, while central auditory processing remained unaffected. Cytokine profiling demonstrated increased levels of proinflammatory markers, including IL-1 alpha (Interleukin-1 alpha), IL-10 (Interleukin-10), and TGF-beta (Transforming Growth Factor beta), alongside decreased IGF-1 (Insulin-like Growth Factor 1) levels, highlighting systemic inflammation and its interplay with neuroprotection. Building on these findings, a proteomic analysis identified 159 differentially expressed proteins (DEPs). Additionally, bioinformatic approaches, including Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-expression Network Analysis (WGCNA), revealed disrupted pathways related to metabolic and inflammatory epileptic processes and a module potentially linked to a rise in the threshold of seizures, respectively. Differentially expressed genes, identified through bioinformatic and statistical analyses, were validated by RT-qPCR. This confirmed the upregulation of six genes (Gpc1-Glypican-1; Sdc3-Syndecan-3; Vgf-Nerve Growth Factor Inducible; Cpne5-Copine 5; Agap2-Arf-GAP with GTPase domain, ANK repeat, and PH domain-containing protein 2; and Dpp8-Dipeptidyl Peptidase 8) and the downregulation of two (Ralb-RAS-like proto-oncogene B-and S100b-S100 calcium-binding protein B), aligning with reduced seizure severity. This study may uncover key proteomic and immune mechanisms underlying seizure susceptibility, providing possible novel therapeutic targets for refractory epilepsy.

Advancing age strongly correlates with an increased risk of epilepsy development. On the other hand, epilepsy may exacerbate the negative effects of aging making it pathological. In turn, the possible link between aging and epileptogenesis is dysregulation of glutamatergic transmission. In the present study, we analyzed the functional state of the glutamatergic system in the hippocampus of aging (18-month-old) Krushinsky-Molodkina (KM) audiogenic rats to disclose alterations associated with aging on the background of inherited predisposition to audiogenic seizures (AGS). Naïve KM rats with no AGS experience were recruited in the experiments. Wistar rats of the corresponding age were used as a control. First of all, aging KM rats demonstrated a significant decrease in cell population and synaptopodin expression in the hippocampus indicating enhanced loss of cells and synapses. Meanwhile, elevated phosphorylation of ERK1/2 and CREB and increased glutamate in the neuronal perikarya were revealed indicating increased activity of the rest hippocampal cells and increased glutamate production. However, glutamate in the fibers and synapses was mainly unchanged, and the proteins regulating glutamate exocytosis showed variable changes which could compensate each other and maintain glutamate release at the unchanged level. In addition, we revealed downregulation of NMDA-receptor subunit GluN2B and upregulation of AMPA-receptor GluA2 subunit, which could also prevent overexcitation and support cell survival in the hippocampus of aging KM rats. Nevertheless, abnormally high glutamate production, observed in aging KM rats, may provide the basis for hyperexcitability of the hippocampus and increased seizure susceptibility in old age.

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.