Spike–wave discharges are necessary for the expression of behavioral depression-like symptoms

Purpose:   The WAG/Rij strain of rats, a well-established model for absence epilepsy, has comorbidity for depression. These rats exhibit depression-like behavioral symptoms such as increased immobility in the forced swimming test and decreased sucrose intake and preference (anhedonia). These depression-like behavioral symptoms are evident in WAG/Rij rats, both at 3–4 and 5–6 months of age, with a tendency to aggravate in parallel with an increase in seizure duration. Here we investigated whether the behavioral symptoms of depression could be prevented by the suppression of absence seizures.
Methods:   Ethosuximide (ETX; 300 mg/kg/day, in the drinking water) was chronically applied to WAG/Rij rats from postnatal day 21 until 5 months. Behavioral tests were done before the cessation of the treatment. Electroencephalography (EEG) recordings were made before and after cessation of treatment to measure seizure severity at serial time-points.
Results:   ETX-treated WAG/Rij rats exhibited no symptoms of depression-like behavior in contrast to untreated WAG/Rij rats of the same age. Moreover, treated WAG/Rij rats did not differ from control age-matched Wistar rats. ETX treatment led to almost complete suppression of spike-wave discharges (SWDs) in 5–6 month old WAG/Rij rats. Discontinuation of chronic treatment was accompanied by a gradual emergence of SWDs; however, a persistent reduction in seizure activity was still present 47 days after discontinuation of the chronic treatment.
Discussion:   The results suggest that seizure activity is necessary for the expression of depression-like behavioral symptoms and confirm that epileptogenesis can be prevented by early and chronic treatment.     

NMDA-NR1 and AMPA-GluR4 receptor subunit immunoreactivities in the absence epileptic WAG/Rij rat

From an age of 2-3 months onwards, the WAG/Rij rat, a genetic model for absence epilepsy, develops spike-wave discharges (SWD). SWD start in the peri-oral somatosensory cortex (POsc), whereas the rostral reticular thalamic nucleus (rRTN) contributes to synchronizing the thalamo-cortical oscillations. We hypothesize that N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors in the POsc and rRTN are involved in, respectively, the initiation and synchronization of SWD activity. As a first step to test this hypothesis, 3 months old non-epileptic and 6 months old absence epileptic WAG/Rij rats were compared with age-matched non-epileptic ACI control rats. The presence of NMDA and AMPA receptors was assessed by quantifying immunostaining for the NMDA-NR1 subunit and the AMPA-GluR4 subunit, respectively. In the POsc, WAG/Rij rats of both ages showed less NMDA-NR1 (-14.7%) and AMPA-GluR4 (-8.7%) subunit staining than ACI rats. From 3 to 6 months, AMPA-GluR4 subunit staining more strongly increased in the rRTN of WAG/Rij rats than of ACI rats. Further studies should support our assumption that in the POsc of the WAG/Rij rat, SWD start as a result of reduced NMDA- and AMPA-mediated glutamatergic stimulation, and that AMPA-GluR4 containing neurons in the rRTN of this rat strain contribute to synchronization of thalamic and cortical neurons.     

The WAG/Rij strain: a genetic animal model of absence epilepsy with comorbidity of depression [corrected

A great number of clinical observations show a relationship between epilepsy and depression. Idiopathic generalized epilepsy, including absence epilepsy, has a genetic basis. The review provides evidence that WAG/Rij rats can be regarded as a valid genetic animal model of absence epilepsy with comorbidity of depression. WAG/Rij rats, originally developed as an animal model of human absence epilepsy, share many EEG and behavioral characteristics resembling absence epilepsy in humans, including the similarity of action of various antiepileptic drugs. Behavioral studies indicate that WAG/Rij rats exhibit depression-like symptoms: decreased investigative activity in the open field test, increased immobility in the forced swimming test, and decreased sucrose consumption and preference (anhedonia). In addition, WAG/Rij rats adopt passive strategies in stressful situations, express some cognitive disturbances (reduced long-term memory), helplessness, and submissiveness, inability to make choice and overcome obstacles, which are typical for depressed patients. Elevated anxiety is not a characteristic (specific) feature of WAG/Rij rats; it is a characteristic for only a sub-strain of WAG/Rij rats susceptible to audiogenic seizures. Interestingly, WAG/Rij rats display a hyper-response to amphetamine similar to anhedonic depressed patients. WAG/Rij rats are sensitive only to chronic, but not acute, antidepressant treatments, suggesting that WAG/Rij rats fulfill a criterion of predictive validity for a putative animal model of depression. However, more and different antidepressant drugs still await evaluation. Depression-like behavioral symptoms in WAG/Rij rats are evident at baseline conditions, not exclusively after stress. Experiments with foot-shock stress do not point towards higher stress sensitivity at both behavioral and hormonal levels. However, freezing behavior (coping deficits) and blunted response of 5HT in the frontal cortex to uncontrollable sound stress, increased c-fos expression in the terminal regions of the meso-cortico-limbic brain systems and greater DA response of the mesolimbic system to forced swim stress suggest that WAG/Rij rats are vulnerable to some, but not to all types of stressors. We propose that genetic absence epileptic WAG/Rij rats have behavioral depression-like symptoms, are vulnerable to stress and might represent a model of chronic low-grade depression (dysthymia). Both 5HT and DAergic abnormalities detected in the brain of WAG/Rij rats are involved in modulation of vulnerability to stress and provocation of behavioral depression-like symptoms. The same neurotransmitter systems modulate SWDs as well. Recent studies suggest that the occurrence and repetition of absence seizures are a precipitant of depression-like behavior. Whether the neurochemical changes are primary to depression-like behavioral alterations remains to be determined. In conclusion, the WAG/Rij rats can be considered as a genetic animal model for absence epilepsy with comorbidity of dysthymia. This model can be used to investigate etiology, pathogenic mechanisms and treatment of a psychiatric comorbidity, such as depression in absence epilepsy, to reveal putative genes contributing to comorbid depressive disorder, and to screen novel psychotropic drugs with a selective and/or complex (dual) action on both pathologies.     

Midfrequency cortico-thalamic oscillations and the sleep cycle: genetic, time of day and age effects

WAG/Rij rats have various types of mid frequency cortico-thalamic oscillations, such as anterior and posterior sleep spindles and two types of spike-wave discharges (SWD). The generalized SWD (type I) preferentially occur at transitions from wake to sleep, type II can be found at the occipital cortex during quite wakefulness. In the present experiment sleep spindles, SWD and sleep cycle characteristics of 6-month-old WAG/Rij rats were studied and compared with those of younger WAG/Rij rats with much less SWD and age-matched control (ACI) rats. EEG recordings were made during the beginning (morning) and end (afternoon) of the light period in these four groups of rats. Quantitative characteristics of SWD, sleep spindles and the sleep cycle were determined. There were strain-related and age-dependent effects in the various cortico-thalamic oscillations, older WAG/Rij had more SWDs than younger WAG/Rij rats (both types I and II) and there were more type I SWDs at the end of the light period compared to the beginning. Large strain, age and time of day effects on the sleep cycle were found. The duration of non-REM sleep and the sleep cycle was shorter in WAG/Rij rats but only at the end of the light period and only in older WAG/Rij rats. It can be concluded that the various phasic events and the length of the sleep cycle are under genetic control, and that the sleep cycle length is also controlled by time of day, age and genetic factors. Non-REM sleep and the sleep cycle are disrupted by absence seizures but only in fragile periods when drowsiness and light slow wave sleep dominate.     

Electroencephalographic characterization of spike-wave discharges in cortex and thalamus in WAG/Rij rats

PURPOSE: The waveform of spontaneous spike-wave discharges (SWD) in the electroencephalogram (EEG) was delineated in the WAG/Rij rat model of absence epilepsy according to the definitions of clinical electroencephalography. We defined four elements in SWD based on the schema of Weir (1965): Spike 1 and 2, Positive Transient (PT), and Wave. The EEG patterns of generalized type I and local type II SWD in cortical and thalamic areas were analyzed. METHODS: EEGs were recorded in freely moving rats epidurally from different cortical regions and with deep electrodes from the specific and reticular thalamic nuclei. Grand average SWD waveforms were computed to assess spatiotemporal patterns of seizures. RESULTS: SWD I in the frontal cortex comprised of a large Spike 2 + Wave, and in the thalamus PT + Wave. Small transient spikes were associated with SWD I in the anterior-middle part of the cortex. SWD II were found in the occipital cortex as a sequence of (occasional) Spike 1 + PT + Wave. CONCLUSIONS: The EEG structure of SWD in WAG/Rij rats was comparable with that of epileptic patients, suggesting face validity of the WAG/Rij model. Fast transients spikes are an integrative part of SWD I. Time-amplitude linkage between cortical and thalamic counterparts of SWD I suggests a complex spatiotemporal organization of SWD I. The thalamus sustained SWD I, but not SWD II.     

Brain protein expression changes in WAG/Rij rats,a genetic rat model of absence epilepsy after peripheral lipopolysaccharide treatment

Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8–10 Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1 mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12 h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep–wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep–wake stages and decreases body temperature.     

Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy

Due to the involvement of cortical neurons in spike-wave discharge (SWD) initiation, and the contribution of voltage-gated sodium channels (VGSCs) to neuronal firing, we examined alterations in the expression of VGSC mRNA and protein in cortical neurons in the WAG/Rij absence epileptic rat. WAG/Rij rats were compared to age-matched Wistar control rats at 2, 4, and 6 months. Continuous EEG data was recorded, and percent time in SWD was determined. Tissue from different cortical locations from WAG/Rij and Wistar rats was analyzed for VGSC mRNA (by quantitative PCR) and protein (by immunocytochemistry). SWDs increased with age in WAG/Rij rats. mRNA levels for sodium channels Nav1.1 and Nav1.6, but not Nav1.2, were found to be up-regulated selectively within the facial somatosensory cortex (at AP +0.0, ML +6.0 mm). Protein levels for Nav1.1 and Nav1.6 were up-regulated in layer II–IV cortical neurons in this region of cortex. No significant changes were seen in adjacent regions or other brain areas, including the pre-frontal and occipital cortex. In the WAG/Rij model of absence epilepsy, we identified a specific region of cortex, in layer II–IV neurons on the lateral convexity of the cortex in the facial somatosensory area, where mRNA and protein expression of sodium channel genes Nav1.1 and Nav1.6 are up-regulated. This region of cortex approximately matches the electrophysiologically determined region of seizure onset. Changes in the expression of Nav1.1 and Nav1.6 parallel age-dependent increases in seizure frequency and duration.     

Cortical and limbic excitability in rats with absence epilepsy

The classical cortico-reticular theory on absence epilepsy suggests that a hyperexcitable cortex is a precondition for the occurrence of absence seizures. In the present experiment seizure thresholds and characteristics of cortical and limbic epileptic afterdischarges (AD) were determined in a comparative cortical stimulation study in young and old adult genetically epileptic WAG/Rij, congenic ACI and Wistar rats. Fifteen-second series of 8Hz stimulation of the sensory-motor cortex were applied in 80- and 180-day-old rats with implanted electrodes. Strain differences were found for the threshold for movements directly induced by stimulation, low frequency spike-and-wave AD, maximal clonic intensity of seizures accompanying direct stimulation, and frequency characteristics of low frequency AD. None of these results agreed with a higher cortical excitability exclusively in WAG/Rij rats. However, WAG/Rij rats had the longest duration of the low frequency AD, and the lowest threshold for the transition to the limbic type of AD. The decrease of this threshold correlated with the increase of the incidence and total duration of spontaneous SWDs in WAG/Rij rats. It is concluded that the elevated excitability of the limbic system or pathways mediating the spread of the epileptic activity into this system can be attributed to the development of genetic epileptic phenotype in WAG/Rij rats.     

Reduced GABAB receptor subunit expression and paired-pulse depression in a genetic model of absence seizures

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABA(B) receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABA(B(1)) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABA(B(2)) mRNA is unchanged. Next, we investigated the cellular distribution of GABA(B(1)) and GABA(B(2)) subunits by confocal microscopy and discovered that GABA(B(1)) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABA(B) receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABA(B) receptor dysfunction. In conclusion, our data identify changes in GABA(B) receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocortical hyperexcitability and thus to SW generation in absence epilepsy.     

Auditory evoked potentials from auditory cortex, medial geniculate nucleus, and inferior colliculus during sleep-wake states and spike-wave discharges in the WAG/Rij rat

OBJECTIVE: Click auditory evoked potentials (AEP) were simultaneously recorded from the auditory cortex (ACx), the medial geniculate nucleus (MGN), and the inferior colliculus (IC) in the freely moving WAG/Rij rat, to investigate state-dependent changes of the AEP in different anatomical locations along the auditory pathway. METHODS: AEPs obtained during active (AW) and passive wakefulness (PW), slow wave sleep (SWS), rapid-eye-movement sleep (REM) and generalized spike-wave discharges (SWD; a specific trait of the WAG/Rij rat, a genetic model for absence epilepsy), were compared. RESULTS: The early components in ACx, MGN and IC were stable throughout the sleep-wake cycle and SWD, apart from a slight increase in the IC during SWD. At all three locations a prominent enlargement of a later component (i.e., N32 in IC, N33 in MGN, and N44 in ACx) was found during SWS and SWD. CONCLUSIONS: The early AEP components are not modulated by the normal sleep-wake states, and are not impaired during SWD. A strong state-dependent modulation of a later AEP component occurs at all three anatomical locations investigated. This suggests that apart from the thalamic burst firing mode, additional mechanisms must exist for the enlargement of the AEP during EEG-synchronized states at the prethalamic and cortical level.     

Effect of intracranial administration of ethosuximide in rats with spontaneous or pentylenetetrazol-induced spike-wave discharges

Purpose: Generalized absence seizures are characterized by bilateral spike‐wave discharges (SWDs), particularly in the frontoparietal cortical region. In WAG/Rij and GAERS rats with absence epilepsy, recent evidence indicates that SWDs arise first from the lateral somatosensory cortex (LSC), that is, the cortical focus theory. To further understand the cortical role in SWD generation, two epileptic rat models were assessed. Methods: Two models, Long‐Evans rats with spontaneous SWDs and Wistar rats with low‐dose pentylenetetrazol‐induced SWDs (20 mg/kg, i.p.), were administered intracortical or intrathalamic ethosuximide (ESM) or saline. Electroencephalographic recordings were analyzed before and after intracranial microinfusion to evaluate onset, frequency, and duration of SWDs. Key Findings: In both epileptic rat models, ESM in the LSC significantly reduced SWD number, shortened SWD duration, and delayed SWD onset compared to saline. By contrast, ESM in the medial somatosensory cortex had little effect compared to saline. Intrathalamic infusion of ESM only delayed SWD onset. Significance: These findings suggest that the LSC may be essential for the occurrence of SWDs. Our data support the cortical focus theory for the generation of absence seizures.     

Mechanisms of the development and integration of nerve processes: Age-related dynamics of the development of absence epilepsy,changes in the concentration of monoamines and their metabolites in the brain structures of WAG/Rij and Wistar rats,and the dynamics of the disruption of learning and memory

We studied peak-wave activity in WAG/Rij rats at ages of 2 and 6 months. We found age-dependent enhancement of peak-wave discharges. At 2 months, the discharges were rare and weak, whereas 6-month-old rats had robust peak-wave discharges. We measured the concentrations of monoamines and their metabolites in 2- and 6-month-old WAG/Rij and Wistar rats in five brain structures: the prefrontal cortex, nucleus accumbens, hypothalamus, striatum, and hippocampus. In 2-month-old WAG/Rij rats, we found a decrease in the concentration of dopamine (DA) metabolites (HVA) in the prefrontal cortex and striatum compared to Wistar rats. In 6-month-old WAG/Rij rats, we found a considerable decrease in the activity of DA system compared to Wistar rats in all five studied brain structures. Concentrations of both DA and its metabolites decreased. We found impairments of learning and memory of WAG/Rij rats compared to Wistar rats. Impairment of learning and memory were more pronounced in 6-month-old WAG/Rij rats compared to 2-month-old WAG/Rij rats. We believe that the DA system not only responds to emotionally positive states but is also a reward and reinforcement system. An increase in the concentration of DA and its metabolites by madopar prevents disturbances of learning and memory. Our general conclusion is that enhancement of absence epilepsy induces a strong deficit of activity of the mesocorticolimbic and nigrostriatal DA system, which induces depression-like behavior and disturbance of learning and memory in WAG/Rij rats. These are the mechanisms of the development of impairments and the integration of these impairments, as well as the aggravation and widening of pathological states.     

Auditory evoked potentials from auditory cortex, medial geniculate nucleus, and inferior colliculus during sleep–wake states and spike-wave discharges in the WAG/Rij rat

Objective: Click auditory evoked potentials (AEP) were simultaneously recorded from the auditory cortex (ACx), the medial geniculate nucleus (MGN), and the inferior colliculus (IC) in the freely moving WAG/Rij rat, to investigate state-dependent changes of the AEP in different anatomical locations along the auditory pathway. Methods: AEPs obtained during active (AW) and passive wakefulness (PW), slow wave sleep (SWS), rapid-eye-movement sleep (REM) and generalized spike-wave discharges (SWD; a specific trait of the WAG/Rij rat, a genetic model for absence epilepsy), were compared. Results: The early components in ACx, MGN and IC were stable throughout the sleep–wake cycle and SWD, apart from a slight increase in the IC during SWD. At all three locations a prominent enlargement of a later component (i.e., N32 in IC, N33 in MGN, and N44 in ACx) was found during SWS and SWD. Conclusions: The early AEP components are not modulated by the normal sleep–wake states, and are not impaired during SWD. A strong state-dependent modulation of a later AEP component occurs at all three anatomical locations investigated. This suggests that apart from the thalamic burst firing mode, additional mechanisms must exist for the enlargement of the AEP during EEG-synchronized states at the prethalamic and cortical level.     

WAG/Rij rats show a reduced expression of CB1 receptors in thalamic nuclei and respond to the CB1 receptor agonist,R(+)WIN55,212‐2, with a reduced incidence of spike‐wave discharges

Purpose: Genetically epileptic WAG/Rij rats develop spontaneous absence‐like seizures after 3 months of age. We used WAG/Rij rats to examine whether absence seizures are associated with changes in the expression of type‐1 cannabinoid (CB₁) receptors. Methods: Receptor expression was examined by in situ hybridization and western blot analysis in various brain regions of “presymptomatic” 2‐month old and “symptomatic” 8‐month‐old WAG/Rij rats relative to age‐matched nonepileptic control rats. Furthermore, we examined whether pharmacologic activation of CB₁ receptor affects absence seizures. We recorded spontaneous spike‐wave discharges (SWDs) in 8‐month old WAG/Rij rats systemically injected with the potent CB₁ receptor agonist, R(+)WIN55,212‐2 (3–12 mg/kg, s.c.), given alone or combined with the CB₁ receptor antagonist/inverse agonist, AM251 (12 mg/kg, s.c.). Results: Data showed a reduction of CB₁ receptor mRNA and protein levels in the reticular thalamic nucleus, and a reduction in CB₁ receptor protein levels in ventral basal thalamic nuclei of 8‐month‐old WAG/Rij rats, as compared with age‐matched ACI control rats. In vivo, R(+)WIN55,212‐2 caused a dose‐dependent reduction in the frequency of SWDs in the first 3 h after the injection. This was followed by a late increase in the mean SWD duration, which suggests a biphasic modulation of SWDs by CB₁ receptor agonists. Both effects were reversed or attenuated when R(+)WIN55,212‐2 was combined with AM251. Discussion: These data indicate that the development of absence seizures is associated with plastic modifications of CB₁ receptors within the thalamic‐cortical‐thalamic network, and raise the interesting possibility that CB₁ receptors are targeted by novel antiabsence drugs.     

Upholding WAG/Rij rats as a model of absence epileptogenesis: Hidden mechanisms and a new theory on seizure development

The WAG/Rij rat model has recently gathered attention as a suitable animal model of absence epileptogenesis. This latter term has a broad definition encompassing any possible cause that determines the development of spontaneous seizures; however, most of, if not all, preclinical knowledge on epileptogenesis is confined to the study of post-brain insult models such as traumatic brain injury or post-status epilepticus models. WAG/Rij rats, but also synapsin 2 knockout, Kv7 current–deficient mice represent the first examples of genetic models where an efficacious antiepileptogenic treatment (ethosuximide) was started before seizure onset. In this review, we have critically reconsidered all articles published regarding WAG/Rij rats, from the perspective that the period before SWD onset is considered as the latent period. In our new theory on seizure development, it is proposed that genes might be considered as the initial ‘insult’ responsible for all plastic changes underpinning the development of spontaneous seizures. According to this idea, in WAG/Rij rats, genetic predisposition would lead to the development of abnormal bilateral cortical epileptic foci, which would then non-genetically stimulate the rest of the brain to rearrange networks in order to phenotypically develop seizures similarly to what happens during electrical kindling.     

IL-6 Receptor Blockade by Tocilizumab Has Anti-absence and Anti-epileptogenic Effects in the WAG/Rij Rat Model of Absence Epilepsy

Increased expression of interleukin-6 (IL-6) both in cerebrospinal fluid (CSF) and plasma is closely associated with convulsive epilepsy and symptom severity of depression. By comparison, at present, little is known about the role of this cytokine in childhood (non-convulsive) absence epilepsy. The aim of this work was to investigate the potential effects of acute and chronic treatment with tocilizumab (TCZ, 10 and 30 mg/kg/day), on absence seizures, their development, and related psychiatric comorbidity in WAG/Rij rats. It is known that lipopolysaccharide (LPS)-induced changes in inflammatory processes increase absence epileptic activity. In order to study the central effects of TCZ, we investigated whether administration of this anti-IL-6R antibody could modulate the lipopolysaccharide (LPS) or IL-6-evoked changes in absence epileptic activity in WAG/Rij rats. Our results demonstrate that TCZ, at both doses, significantly reduced the development of absence seizures in adult WAG/Rij rats at 6 months of age (1 month after treatment suspension) compared with untreated controls, thus showing disease-modifying effects. Decreased absence seizure development at 6 months of age was also accompanied by reduced comorbid depressive-like behavior, whereas no effects were observed on anxiety-related behavior. Acute treatment with TCZ, at 30 mg/kg, had anti-absence properties lasting ~25 h. The co-administration TCZ with i.c.v. LPS or IL-6 showed that TCZ inhibited the worsening of absence seizures induced by both proinflammatory agents in the WAG/Rij rats, supporting a central anti-inflammatory-like protective action. These results suggest the possible role of IL-6 and consequent neuroinflammation in the epileptogenic process underlying the development and maintenance of absence seizures in WAG/Rij rats. Accordingly, IL-6 signaling could be a promising pharmacological target in absence epilepsy and depressive-like comorbidity.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00893-8) contains supplementary material, which is available to authorized users.Key Words: Absence epilepsy, epileptogenesis, neuroinflammation, tocilizumab, depressive-like behavior, anxiety.     

Measuring clusters of spontaneous spike-wave discharges in absence epileptic rats

Spike-wave discharges (SWDs) characterizing absence epilepsy appear in closely packed aggregated sequences, which gave rise to the name "pyknolepsy" for this disease. In WAG/Rij rats, genetically prone to absence epilepsy, spontaneous SWDs seem to occur in clusters as well. Here, we aimed to quantify the seizures' clusters. SWDs sequences were extracted from long-term (complete estrous cycle) EEG recordings of adult female WAG/Rij rats. Spectral characteristics and half-decay time of autocorrelation functions (AC-tau) were calculated for time series of i(SWD) (proportion of time occupied by spike-wave activity), measured for subsequent periods. The clusters were characterized by means of AC-tau calculated for time series of i(SWD). The absence seizures were indeed clustered in a minute range. The clustering had a non-periodical character, since no significant and consistent periodicity was found in the minute range. AC-tau correlated positively with propensity of SWDs: i.e. the aggravation of absence epilepsy led to longer sequences of paroxysms and thus to a less random distribution. AC-tau was not sensitive to various phases of the estrous cycle, but was larger in the dark than in the light periods. We suggest that AC-tau can be used to quantify aggregation of epileptic events in the search for physiological basis of its temporal clustering.     

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I_h). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I_h properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I_h was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I_h found in WAG/Rij rats compensate each other in a way that leaves I_h availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I_h on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I_h in older animals.     

Endogenous rhythm of absence epilepsy: relationship with general motor activity and sleep-wake states

The rhythms of spontaneously occurring seizures (spike-wave discharges, SWD) and motor activity, as well as the relationship between SWD and sleep-wake states were investigated in the WAG/Rij rat model of absence epilepsy. In order to establish whether SWD are controlled by external (Zeitgebers) or by endogenous factors such as circadian influences or the state of vigilance, the study was performed in entrained and constant dim light conditions. EEG and motor activity were recorded in the 12:12 light-dark cycle and in constant dim light conditions. Circadian rhythmicity was found both for motor activity and the occurrence of SWD in conditions of entrainment. In constant dim light conditions also circadian rhythms emerged, however, the change in circadian parameters was opposite for the rhythm of SWD and motor activity. SWD were preceded mostly by passive wakefulness and by slow-wave sleep in both experimental conditions. It can be concluded that the rhythm of SWD seems to be generated and controlled by an endogenous mechanism distinct from that which controls the rhythm of motor activity. The relationship between SWD and sleep-wake states preceding their occurrences appeared to be unchanged, suggesting that the mechanism of generation of SWD is independent of the circadian timing system.     

Intracerebroventricularly administered lipopolysaccharide enhances spike-wave discharges in freely moving WAG/Rij rats

Peripheral lipopolysaccharide (LPS) injection enhances spike-wave discharges (SWDs) in the genetic rat model of absence epilepsy (Wistar Albino Glaxo/Rijswijk rats: WAG/Rij rats) parallel with the peripheral proinflammatory cytokine responses. The effect of centrally administered LPS on the absence-like epileptic activity is not known, however despite the important differences in inflammatory mechanisms. To examine the effect of centrally administered LPS on the pathological synchronization we intracerebroventricularly (i.c.v.) injected LPS into WAG/Rij rats and measured the number and duration of SWDs. I.c.v. injected LPS increased the number and duration of SWDs for 3 h, thereafter, a decrease in epileptic activity was observed. To further investigate the nature of this effect, a non-steroid anti-inflammatory drug (indomethacin; IND) or a competitive N-methyl-d-aspartate (NMDA) receptor antagonist (2-amino-5-phosphonopentanoic acid; AP5) was injected intraperitoneally (i.p.), preceding the i.c.v. LPS treatment. IND abolished the i.c.v. LPS induced changes in SWDs, while AP5 extended it for 5 h. As control treatments, both IND and AP5 application by themselves decreased the number of SWDs for 2 and 3 h, respectively. Our results show that centrally injected LPS, likewise the peripheral injection, can increase the number and duration of SWDs in the WAG/Rij rat, and the effect invoke inflammatory cytokines as well as excitatory neurotransmitters.