LATERAL CEREBELLAR NUCLEUS (LCN) CONTRIBUTION TO MYOCLONUS PATHOGENESIS IN PTZ-KINDLED RATS
Description
In recent years, the cerebellum and its nuclei have become an essential target for understanding and suppressing the mechanisms of seizures. Тus, Kros et al. (2015) reported that cerebellar nuclei are potent modulators of thalamocortical network pathological oscillations observed during generalized spike-wave discharges (SWDs) generation. Accordingly, cerebellar nuclei neurons can be identified as therapeutic targets, especially in controlling absence seizures (Kros et al. 2015). Another study showed that single-pulse optogenetic stimulation of cerebellar nuclei disrupted neuronal thalamic oscillations during SWD generation (Rooda et al., 2021). Cerebellar crosstalk with forebrain epileptic activity has been well-documented since the sixteenth of the previous century, with a wide range of data showing both suppressive and facilitative characteristics (Akyuz et al., 2021; Zangiabadi et al., 2019; Beckinghausen et al., 2023). Hence, the next steps in clarifying factors contributing to data controversies and the role of cerebellar structures in seizure control are necessary. This investigation aims to observe the EEG and behavioral peculiarities of PTZ-kindled manifestations with an emphasis on myoclonus seizure, at early and fully developed stages under LCN electrical stimulation (ES) conditions. The male Wistar rats were kindled with pentylenetetrazol (PTZ) (35.0 mg/kg, i.p.) to myoclonus (9-11 PTZ injections) and generalized tonic-clonic seizures (21 PTZ injections). Unilateral ES (100 Hz) was delivered daily for five days after the last kindled PTZ administration, with PTZ seizure testing after ES. Seizures were videotaped, and the severity score was determined in a blinded manner. ES of LCN performed at the early stage of kindling facilitated the appearance of myoclonus and increased seizure severity by 30.2% points compared to the control group (H=6.567; p = 0.037) with the spikes frequency generation during the poststimulation period (F(5,42)=15.41, p < 0.001). In fully developed kindling, ES prevented generalized seizure and reduced seizure severity by 27.5% (H=11.058; p = 0.004), while myoclonuses were present with spikes generation in brain structures. The data obtained showed that repeated ES of LCN at the early stage promoted myoclonic seizures, while in fully PTZ-kindled rats, it suppressed generalized seizure fits, which were substituted with myoclonus. Hence, LCN activity might be facilitative for seizure myoclonus precipitation while suppressive for spreading generalized activity in wider neuronal networks.
Files
Steps to reproduce
Experiments were performed on 31 male Wistar rats with an initial body weight of 180-220 g. All experiments followed the National Institutes of Health guidelines for the care and use of laboratory animals, the European Council Directive on 24 November 1986 for Care and Use of Laboratory Animals (86/609/EEC) and Odesa National Medical University Bioethics Committee (UBC) approval No. 3 dated 14/03/2018. Epilepsy model Kindled seizures were induced with PTZ (Sigma Aldrich, St. Louis, MO, USA) that was given intraperitoneally (i.p.) in a daily dose of 35.0 mg/kg for 21 days. The seizure responses were observed over a 30-minute cutoff period and were classified as initially described by Racine: zero: no response; one: vibrissae twitching, mouth, ear, and facial twitching; two: convulsive waves through the body; three: whole body myoclonic jerks, rearing; four: tonic-clonic convulsions with rearings and falling; five: repeated seizures as at stage four, loss of postural control. Seizure severity score was determined in a blinded manner. Criteria for inclusion of rats into observation The early kindled stage was modeled in rats demonstrating minor absence manifestations, including myoclonus of muscle groups of extremities and body (one-two scored the severity of seizures). Rats with whole body myoclonus or rearings (three-scored severity) until the 11th PTZ administration composed the group treated with the PTZ for induction fully kindled seizures. Groups of animals Seven rats composed the early kindling control group, and seven formed the control group of fully kindled animals (Design). Experimental groups subjected to ES trials included eight rats with early kindling and nine with fully kindled seizures. Thus, control and experimental groups included animals with an equally proportioned number of rats concerning their seizure severity score. Surgery In animals, anesthesia was performed with i.p. ketamine administration (100 mg/kg, i.p.). The nichrome monopolar electrodes were implanted in the ventral hippocampus and frontal cortex of both hemispheres, according to the rat brain atlas. The custom-made nichrome electrodes had a diameter of 0.15 mm, and an interelectrode distance of 0.25 mm. They were implanted at the left LCN (AP=-10.8; ML=3.5; DV=6.0). The reference electrode was located in the nasal bones. Electrical stimulation After setting the ES intensity, which does not induce behavioral reactions, stimulations were delivered one per day during next five days with a 100 Hz impulse frequency, 0.25 ms monophasic pulse duration, 50-100 mcA intensity, and 4.5-5.0 s. Sham stimulations were conducted in control group. Statistics Values were compared using one-way ANOVA and Newman-Keuls test for the latency of seizures; Kruskal-Wallis followed with a post hoc Dunn`s was used for seizure severity; “z” criteria for comparing two proportions. The Shapiro-Wilk test for normality was used for the latency. P values <0.05 were considered significant.