Comparison of Cortical Epileptic Afterdischarges in Immature Genetic Absence Epilepsy WAG/Rij Rats with Those in Two Other Strains (ACI and Wistar)

Summary:  The aim of this study was to examine the development of cortical epileptic afterdischarges (ADs) in genetic absence epilepsy WAG/Rij rats, and to compare them with two strains with minimal incidence of spike-and-wave (SW) episodes (ACI and Wistar). Epileptic ADs were elicited by stimulation of sensorimotor cortex in 12-, 18-, and 25-day-old rats of the three strains. The threshold current intensities were established for movements accompanying stimulation, for ADs of the SW type and accompanying clonic seizures and for transition into limbic type of ADs (characterized by behavioral automatisms). Individual groups were formed by 7–12 rats. There were no differences among the three strains in the thresholds for elicitation of stimulation-bound movements. In contrast, WAG/Rij and ACI rats exhibited easier elicitation of SW ADs than Wistar rats at the age of 18 and 25 days. There was no difference among the three strains in transition into the limbic type of ADs in 18- and 25-day-old rats. Lower thresholds for SW ADs in 18- and 25-day-old WAG/Rij and ACI rats in comparison with Wistar rats are in agreement with our data from adult animals as well as with development of pharmacologically induced models of absence seizures. The failure to find a specific difference between WAG/Rij rats and the other two strains might indicate a difference in generation of SW episodes and SW cortical AD.     

The Effect of Generalized Absence Seizures on the Progression of Kindling in the Rat

Summary:  The involvement of the thalamus in limbic epileptogenesis has recently drawn attention to the connectivity between the nuclei of the thalamus and limbic structures. Thalamo-limbic circuits are thought to regulate limbic seizure activity whereas thalamocortical circuits are involved in the expression and generation of spike-and-wave discharges (SWDs) in the absence epilepsy models. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) and WAG/Rij (Wistar Albino Glaxo from Rijswijk) are well-defined genetic animal models of absence epilepsy. We aimed to examine the duration of behavioral changes in the kindling process and the relation of SWD activity to the kindling progress in the GAERS and WAG/Rij animals. Electrodes were stereotaxically implanted into the basolateral amygdala and the cortex of rats for stimulation and recording. The animals were stimulated at the threshold for producing afterdischarges. EEG was recorded to analyze SWDs and afterdischarge durations. The seizure severity was evaluated using Racine's 5-stage scale. None of the GAERS animals reached stage 3, 4, or 5 after application of 30 stimulations. The WAG/Rij animals showed different rate of kindling, therefore they were further categorized into the kindling-resistant, slow-kindled, and rapid-kindled groups. The kindling-resistant animals demonstrated a significantly longer duration of SWDs on the first day of the experiment before kindling stimulation and shorter duration of afterdischarge than did the kindled WAG/Rij animals. Behavioral durations at stage 2 were longer in kindled Wistar and WAG/Rij animals compared to kindling-resistant WAG/Rij and GAERS. These results suggest that mechanisms involved in the generation of SWDs act as a counterbalance to the excitability induced by kindling.     

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.     

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.     

Audiogenic kindling in Wistar and WAG/Rij rats: kindling-prone and kindling-resistant subpopulations

Purpose: Audiogenic kindling (AK) is a model of naturally occurring epileptogenesis triggered by repeated sound stimulation of rats genetically prone to audiogenic seizures. It is accepted that limbic seizure networks underlie progressive changes in behavioral seizure pattern during AK. The present study investigated AK progression in rats susceptible and unsusceptible to absence seizures. Methods: Progression of AK as indicated by an appearance and intensification of limbic clonus was examined in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats with genetic absence epilepsy and in Wistar rats. Results: Subpopulations of kindling‐prone and kindling‐resistant rats were found in both Wistar and WAG/Rij strains. Despite identical seizure responses to the first sound stimulation, AK progression dramatically differed between the two subpopulations. AK‐prone rats exhibited rapid kindling development up to maximal stage‐5 severity. In AK‐resistant rats, limbic clonus did not appear after 30 stimulations or if it appeared, it did not progress beyond stage 2. The proportions of AK‐prone and AK‐resistant animals within Wistar and WAG/Rij strains were similar. Comparison of Wistar and WAG/Rij rats within the kindling‐prone and kindling‐resistant groups did not reveal a significant strain effect on AK progression. However, within the WAG/Rij strain, a significantly higher incidence of absence seizures was found in AK‐resistant rats compared to AK‐prone rats. Conclusions: The present study demonstrates that sensitivity to sound‐induced epileptogenesis differs dramatically within Wistar and WAG/Rij strains, whereas genetic susceptibility to absence seizures does not change AK progression significantly. It is supposed that an increased incidence of nonconvulsive seizures and resistance to kindling result from a common seizure modulating mechanism.     

The effects of the immature rat model of febrile seizures on the occurrence of later generalized tonic-clonic and absence epilepsy

This study was designed to investigate whether the intensity of experimental febrile seizures reduces the threshold to generalized tonic-clonic epilepsy and its effects on the development of generalized absence epilepsy in adulthood. For the evaluation of absence epilepsy, WAG/Rij rats and for the tonic-clonic seizures, PTZ injected Wistar rats were used. Our results showed that while the frequency of the experimental febrile seizures facilitates PTZ-induced generalized tonic-clonic seizures, it does not influence the properties of absence epileptic seizures in adulthood.     

Dynamic fMRI and EEG recordings during spike-wave seizures and generalized tonic-clonic seizures in WAG/Rij rats.

Generalized epileptic seizures produce widespread physiological changes in the brain. Recent studies suggest that "generalized" seizures may not involve the whole brain homogeneously. For example, electrophysiological recordings in WAG/Rij rats, an established model of human absence seizures, have shown that spike-and-wave discharges are most intense in the perioral somatosensory cortex and thalamus, but spare the occipital cortex. Is this heterogeneous increased neuronal activity matched by changes in local cerebral blood flow sufficient to meet or exceed cerebral oxygen consumption? To investigate this, we performed blood oxygen level-dependent functional magnetic resonance imaging (fMRI) measurements at 7T with simultaneous electroencephalogram recordings. During spontaneous spike-wave seizures in WAG/Rij rats under fentanylhaloperidol anesthesia, we found increased fMRI signals in focal regions including the perioral somatosensory cortex, known to be intensely involved during seizures, whereas the occipital cortex was spared. For comparison, we also studied bicuculline-induced generalized tonic-clonic seizures under the same conditions, and found fMRI increases to be larger and more widespread than during spike-and-wave seizures. These findings suggest that even in regions with intense neuronal activity during epileptic seizures, oxygen delivery exceeds metabolic needs, enabling fMRI to be used for investigation of dynamic cortical and subcortical network involvement in this disorder.     

Altered distribution and function of A2A adenosine receptors in the brain of WAG/Rij rats with genetic absence epilepsy, before and after appearance of the disease

The involvement of excitatory adenosine A_2A receptors (A_2ARs), which probably contribute to the pathophysiology of convulsive seizures, has never been investigated in absence epilepsy. Here, we examined the distribution and function of A_2ARs in the brain of Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a model of human absence epilepsy in which disease onset occurs 2–3 months after birth. In the cerebral areas that are mostly involved in the generation of absence seizures (somatosensory cortex, reticular and ventrobasal thalamic nuclei), A_2AR density was lower in presymptomatic WAG/Rij rats than in control rats, as evaluated by immunohistochemistry and western blotting. Accordingly, in cortical/thalamic slices prepared from the brain of these rats, A_2AR stimulation with the agonist 2-[4-(-2-carboxyethyl)-phenylamino]-5'- N -ethylcarboxamido-adenosine failed to modulate either cAMP formation, mitogen-activated protein kinase system, or K⁺-evoked glutamate release. In contrast, A_2AR expression, signalling and function were significantly enhanced in brain slices from epileptic WAG/Rij rats as compared with matched control animals. Additionally, the in vivo injection of the A_2AR agonist CGS21680, or the antagonist 5-amino-7-(2-phenylethyl)-2-(2-fuyl)-pyrazolo-(4,3-c)1,2,4-triazolo(1,5-c)-pyrimidine, in the examined brain areas of epileptic rats, increased and decreased, respectively, the number/duration of recorded spontaneous spike–wave discharges in a dose-dependent manner during a 1–5 h post-treatment period. Our results support the hypothesis that alteration of excitatory A_2AR is involved in the pathogenesis of absence seizures and might represent a new interesting target for the therapeutic management of this disease.     

Vigabatrin in low doses selectively suppresses the clonic component of audiogenically kindled seizures in rats

PURPOSE: The effect of systemic administration of the gamma-aminobutyric acid (GABA)-transaminase inhibitor vigabatrin (VGB) on different components of convulsions was tested in the model of audiogenically kindled seizures, which consist of brainstem (running, tonus) and forebrain (clonus) elements. METHODS: Audiogenically susceptible rats of Krushinsky-Molodkina (KM), Wistar, and WAG/Rij strains received repeated sound stimulation (60 dB, 10-80 kHz) until kindled audiogenic seizures were reliably elicited. Kindled audiogenic seizures consisted of running, tonic, and generalized clonic phases in KM rats (severe audiogenic seizures) and of running and Racine stage 5 facial/forelimb clonus in Wistar and WAG/Rij rats (moderate seizures). Vehicle, 100, or 200 mg/kg of VGB was intraperitoneally injected 2, 4 and 24 h before the induction of kindled audiogenic seizures. RESULTS: At both doses, VGB did not change the seizure latency and the duration of running and tonic convulsions, but suppressed clonic ones in all rat strains. In KM rats, the mean duration of posttonic clonus was significantly reduced at 24 h after 100 mg/kg and from 4 h after 200 mg/kg. In Wistar and WAG/Rij rats, the mean duration of facial/forelimb clonus was reduced from 4 and 2 h after 100- and 200-mg/kg administration, respectively; 24 h after the high-dose injection, clonus was completely blocked in all rats of both strains. No difference in efficacy of VGB between Wistar and WAG/Rij rats was observed. CONCLUSIONS: VGB more effectively suppresses clonic convulsions than running and tonic ones in audiogenically kindled rats. It is supposed that this selective anticonvulsive effect of VGB results from different sensitivities of forebrain and brainstem epileptic networks to the presumed GABA enhancement.     

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.     

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.     

Unilateral cortical spreading depression is an early marker of audiogenic kindling in awake rats

Spreading depression (SD), a self-propagating wave of reversible cellular depolarization, is thought to play an important role in brain pathophysiology. SD and seizures are closely related events but little is known about involvement of SD in chronic epileptogenesis. Here we show that cortical SD is the first and highly reproducible manifestation of audiogenic kindling induced by repeated sound stimulation of WAG/Rij rats with genetic audiogenic and absence epilepsy. Repetition of sound-induced running seizures in freely moving rats led to an appearance and gradual intensification of post-running facial and forelimb clonic convulsions coupled with afterdischarge in the fronto-parietal cortex. Before the development of these traditional manifestations of audiogenic kindling, an unilateral cortical SD wave began to be triggered by audiogenic seizures. Once cortical SD appeared, it became a permanent component of subsequent seizures. SD was always recorded in the hemisphere ipsilateral to the running direction. Only at the late stages of audiogenic kindling SD developed bilaterally. To estimate the contribution of SD in postictal effects of audiogenic seizures, we compared cortical activity after seizures induced SD or not. It was found that only seizures with cortical SD were followed by postictal suppression of spontaneous spike-wave discharges displayed by WAG/Rij rats. The results show that (1) cortical SD is readily triggered by brief sensory-induced seizures in awake animals; (2) SD may be responsible for postictal changes in cortical activity; (3) unilateral initiation of SD suggests asymmetrical recruitment of the cortex into seizure network during audiogenic kindling.     

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.     

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.     

Mixed forms of epilepsy in a subpopulation of WAG/Rij rats

Mixed forms of epilepsy in patients are often refractory. Therefore, animal models of comorbid convulsive and nonconvulsive seizure are needed for experimental research. Susceptibility to audiogenic convulsions was studied in a large group of young and adult WAG/Rij rats with inherited absence epilepsy. In 30% of adult rats, sound stimulation provoked audiogenic seizures of moderate intensity. The seizures had two excitation periods separated by a remarkably stable "arrest" of paroxysmal movements. Up to 20% of young WAG/Rij rats were also susceptible to audiogenic seizures, with a longer latency, lower intensity, and more simple seizure patterns. No difference in manifestations of spike-wave discharges was observed between the WAG/Rij rats with and without audiogenic seizures. This subpopulation of WAG/Rij rats genetically predisposed to absence and audiogenic seizures is proposed as an animal model suitable for investigation of basal mechanisms and pharmacological profiles of this mixed form of epilepsy.     

Cardiovascular effects of intracerebroventricular bicuculline in rats with absence seizures

Gamma-aminobutyric acid (GABA) plays an important role in both central cardiovascular homeostasis and pathogenesis of epileptic seizures. Previous studies have indicated a critical role of the amygdala in the spread of seizures from brainstem to forebrain and in the regulation of autonomic responses such as blood pressure and heart rate. The purpose of the present study was to examine blood pressure and heart rate effects of bicuculline, a GABA(A) antagonist, and the effect of lesions of the central or the basolateral nuclei of the amygdala on bicuculline-induced cardiovascular responses in conscious WAG/Rij rats with absence epilepsy. Intracerebroventricular administration of 0.3 and 0.5 nmol of bicuculline produces an increase in blood pressure and a slight bradycardia in non-epileptic Wistar rats. The blood pressure response to intracerebroventricular bicuculline is significantly potentiated in epileptic WAG/Rij rats. Bilateral lesions of the basolateral nucleus of the amygdala of WAG/Rij rats completely prevent the pressor response to 0.5 nmol of bicuculline, whereas unilateral lesion of the basolateral nucleus does not affect blood pressure changes in epileptic WAG/Rij rats. Additionally, the pressor effect of 0.5 nmol of bicuculline is not attenuated by bilateral electrolytic ablation of the central nucleus of the amygdala in WAG/Rij rats. Heart rate response to bicuculline is not significantly changed in the lesioned groups. These findings indicate (a) altered GABAergic function in blood pressure regulation; and (b) a critical role of the basolateral nucleus in GABA(A)-mediated blood pressure control in epileptic WAG/Rij rats.     

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.     

Changes in electroencephalographic characteristics and blood-brain barrier permeability in WAG/Rij rats with cortical dysplasia

PurposeThis study investigated the effects of cortical dysplasia (CD) on electrophysiology and blood-brain barrier (BBB) permeability in WAG/Rij rats with genetic absence epilepsy.MethodsPregnant WAG/Rij rats were exposed to 145 cGy of gamma-irradiation on embryonic day 17 to induce CD. An electroencephalogram was recorded from cortices subdurally in the offspring of the pregnant animals. Horseradish peroxidase (HRP) was used as determinant of BBB permeability.ResultsA massive tissue loss in the cerebral cortex was seen in WAG/Rij rats with CD (p < 0.05). There was a significant decrease in the number and duration of spike-and-wave discharges (SWDs) and an increase in the frequency of SWDs in the WAG/Rij rats with CD when compared with the properties of SWDs in intact WAG/Rij rats (p < 0.01). Ultrastructurally, the accumulation of HRP reaction products in the cerebral cortex and thalamus of WAG/Rij rats was significantly higher than that of control values (p < 0.01). The accumulation of HRP reaction products in the cerebral cortex and thalamus regions of WAG/Rij rats with CD increased and was higher than that of the control and WAG/Rij animals (p < 0.01).ConclusionIn our study, we showed that number and duration of SWDs decreased and SWD frequency increased in WAG/Rij rats with CD, suggesting a shift in seizure pattern. The association of these alterations with significant loss of cortical thickness and increased BBB permeability to HRP tracer may represent a causal relation of the EEG abnormalities with cerebral structural changes in these animals.     

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.     

Thalamocortical oscillations in a genetic model of absence seizures

We used field potential recordings in an in vitro thalamocortical slice preparation to compare the rhythmic oscillations generated by reciprocally connected networks of the thalamus and cerebral cortex obtained from epileptic (> 160 days old) WAG/Rij and age-matched, nonepileptic control (NEC) rats. To increase neuronal excitability, and thus to elicit spontaneous field potential activity in vitro, we applied medium containing: (i) zero [Mg2+]; (ii) high [K+] (8.25 mm); or (iii) low concentrations of the K+ channel blocker 4-aminopyridine (4AP, 0.5-1 micro m). Of these procedures, only the last was effective in triggering oscillatory activity that depended on the type of tissue. Thus, during 4AP application: (i) sequences of fast (intraburst frequency 9.5-16.1 Hz) and slower (5-8.9 Hz) field potential oscillations (FPOs) were recorded in WAG/Rij slices (n = 23), but (ii) only fast FPOs were seen in NEC slices (n = 7). Slower FPOs in WAG/Rij slices reflected a larger degree of thalamocortical synchronization than fast FPOs, and disappeared after surgical separation of cortex and thalamus (n = 5); under these conditions fast FPOs continued to occur in thalamus only. In addition, fast and slower FPOs disappeared in all areas of the WAG/Rij slice during thalamic application of the excitatory amino acid receptor antagonist kynurenic acid (n = 3), while fast FPOs continued to occur in thalamus when kynurenic acid was applied to the cortex (n = 4). Bath application of the N-methyl-D-aspartic acid (NMDA) receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP) abolished slower FPOs in WAG/Rij cortex and thalamus (n = 6) without infuencing fast FPOs recorded in WAG/Rij (n = 6) or NEC slices (n = 4). Moreover, cortical application of CPP (n = 6) abated slower FPOs although they persisted following CPP application to the thalamus (n = 7). Our data demonstrate that highly synchronized, slower FPOs can occur during 4AP application in WAG/Rij but not in NEC slices. This activity, which may represent an in vitro hallmark of thalamocortical epileptogenicity, requires the function of reciprocally connected thalamic and cortical networks and depends on cortical NMDA receptor-mediated mechanisms.