Fluoxetine accelerates epileptogenesis and magnifies disease severity in a rat model of acquired epilepsy.

OBJECTIVE: Many people with epilepsy experience comorbid anxiety and depression, and antidepressants remain a primary treatment for this. Emerging evidence suggests that these agents may modulate epileptogenesis to influence disease severity. Here, we assessed how treatment with the selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine impacts epileptogenic, behavioral, and pathological sequelae following status epilepticus. METHODS: Male Wistar rats received kainic acid to induce status epilepticus (SE) or vehicle (sham). Animals then received either fluoxetine (10 mg/kg/day) or vehicle for 8 weeks via subcutaneous osmotic pump. Video-electroencephalography was recorded continuously until behavioral testing at day 56, including assessments of anxiety- and depression-like behavior and spatial cognition. Postmortem immunocytochemistry studies examined mossy fiber sprouting. RESULTS: Fluoxetine treatment significantly accelerated epileptogenesis following SE, reducing the average period to the first spontaneous seizure (from 32 days [vehicle] to 6 days [fluoxetine], p < .01). Also, fluoxetine exposure magnified the severity of the resultant epilepsy, increasing seizure frequency compared to vehicle (p < .01). Exposure to fluoxetine was associated with improved anxiety- and depression-like behaviors but significantly worsened cognition. Mossy fiber sprouting was more pronounced in fluoxetine-treated rats compared to vehicle (p < .0001). SIGNIFICANCE: Our studies demonstrate that, using a model exhibiting spontaneous seizures, epileptogenesis is accelerated and magnified by fluoxetine, an effect that may be related to more severe pathological neuroplasticity. The differential influence of fluoxetine on behavior indicates that different circuitry and mechanisms are responsible for these comorbidities. These findings suggest that caution should be exercised when prescribing SSRI antidepressants to people at risk of developing epilepsy.

Metyrapone abolishes spike-wave discharge seizures in genetic absence epilepsy rats from Strasbourg by reducing stress hormones.

OBJECTIVE: Stress is one of the most commonly reported triggers for seizures in patients with epilepsy, although the mechanisms that mediate this effect are not established. The clinical evidence supporting this is derived from patients' subjective experience of stress, and how this influences their own seizures. Animal models can be used to explore this phenomenon in controlled environments, free from subjective bias. Here, we used genetic absence epilepsy rats from Strasbourg (GAERS), a genetic rat model of absence epilepsy, to explore the influence of stress and stress hormones on spontaneous seizures. METHODS: Adult male GAERS (n = 38) and nonepileptic control (NEC) rats (n = 4) were used. First, rats were subjected to 30-min restraint stress to assess hypothalamic-pituitary-adrenal axis function. Next, we assessed the effects of 30-min noise stress, and cage tilt stress, on spike-wave discharge seizures in GAERS. We then performed pharmacological experiments to assess the direct effects of stress hormones on seizures, including corticosterone, metyrapone, and deoxycorticosterone. RESULTS: GAERS exhibited elevated baseline corticosterone levels, compared to NEC rats. Noise stress and cage tilt stress significantly enhanced seizure incidence (p < .05), but only during stress periods. Exogenous corticosterone administration also significantly increased seizure occurrence (p < .05). Metyrapone, an inhibitor of corticosterone synthesis, completely abolished seizures in GAERS, and seizures remained suppressed for >2 h. However, deoxycorticosterone, the precursor of corticosterone, increased seizures. SIGNIFICANCE: These results suggest that GAERS exhibit elevations in stress hormones, and this may contribute to seizures. Inhibiting corticosterone synthesis with metyrapone prevents seizures in GAERS, and shows potential for repurposing this drug as a future antiseizure medication.

Cognitive deficits in a rat model of temporal lobe epilepsy using touchscreen-based translational tools.

OBJECTIVE: Cognitive deficits are commonly observed in people with epilepsy, but the biologic causation of these is challenging to identify. Animal models of epilepsy can be used to explore pathophysiologic mechanisms leading to cognitive problems, as well as to test novel therapeutics. We utilized a well-validated animal model of epilepsy to explore cognitive deficits using novel translational assessment tools/automated rodent touchscreen assays. METHODS: To induce epilepsy, adult Wistar rats were subjected to kainic acid-induced status epilepticus or sham control (n = 12/group). Two months following induction, animals underwent the Pairwise Discrimination and Reversal learning touchscreen tasks, novel object recognition, and the Y maze test of spatial memory. RESULTS: In the Pairwise Discrimination paradigm, only 40% of epilepsy animals acquired the discrimination learning criterion, compared to 100% of sham animals (P = 0.003). Epilepsy and sham animals that successfully acquired the discrimination progressed onto the reversal phase, which measures cognitive flexibility. Of interest, there were no differences in the rate of reversal learning; however, on the first reversal session, epilepsy rats committed more perseverative errors than shams (mean ± SEM: 6.3 ± 0.9 vs 1.8 ± 0.5, P < 0.0001). Additional behavioral analysis revealed that epilepsy rats were significantly impaired in novel object recognition and short-term spatial learning and memory. SIGNIFICANCE: Using translationally relevant behavioral tools in combination with traditional assays to measure cognition in animal models, here we identify impairments in learning and memory, and enhanced perseverative behaviors in rats with epilepsy. These tools can be used in future research to explore biologic mechanisms and treatments for cognitive deficits associated with epilepsy.

Disease-Modifying Effects of Neural Regeneration Peptide 2945 in the GAERS Model of Absence Epilepsy.

Epilepsy is a common neurological condition characterised by spontaneous recurrent seizures. Current anti-epileptic drugs are only effective and tolerated in ~70% of patients, leaving a substantial proportion of patients untreated. As such, there is a pressing need to develop new therapies. We assessed the anti-seizure activity of Neural Regeneration Peptide 2945 (NRP 2945) in the GAERS model of absence epilepsy. Drug effects on seizures were assessed using two study designs. Male adult GAERS were implanted with EEG electrodes to measure seizure frequency. The first study compared the effects of acute sc injection of vehicle, NRP 10 µg/kg, NRP 20 µg/kg, and controlled against the active comparator Valproaic acid (200 mg/kg). In the second study, animals received one of four treatments for 4 weeks: vehicle, NRP 60 µg/kg/day, NRP 120 µg/kg/day (delivered by continuous infusion) or NRP 20 µg/kg sc injected every second day (e.s.d). In the acute study, we found significant (p < 0.01) anti-seizure effects in animals treated with NRP2945 (20 µg/kg) and VPA, with NRP2945 slightly more efficacious, despite the 70,000 times lower molar dosage. In the chronic study, animals receiving 120 µg/kg/day and NRP 20 µg/kg e.s.d had significantly fewer seizures (p < 0.001), compared with vehicle. These effects were sustained for at least 10 days after drug treatment had ceased, indicative of disease-modifying activity. We demonstrate sustained anti-seizure effects of NRP2945, a potent small molecule peptide which enters the brain and is devoid of adverse effects. Early stage first-in-man trials have been initiated for subcutaneously delivered NRP2945 which is a promising step to providing therapeutic benefits for refractory epilepsy patients.

Sodium selenate retards epileptogenesis in acquired epilepsy models reversing changes in protein phosphatase 2A and hyperphosphorylated tau.

There are no treatments in clinical practice known to mitigate the neurobiological processes that convert a healthy brain into an epileptic one, a phenomenon known as epileptogenesis. Downregulation of protein phosphatase 2A, a protein that causes the hyperphosphorylation of tau, is implicated in neurodegenerative diseases commonly associated with epilepsy, such as Alzheimer's disease and traumatic brain injury. Here we used the protein phosphatase 2A activator sodium selenate to investigate the role of protein phosphatase 2A in three different rat models of epileptogenesis: amygdala kindling, post-kainic acid status epilepticus, and post-traumatic epilepsy. Protein phosphatase 2A activity was decreased, and tau phosphorylation increased, in epileptogenic brain regions in all three models. Continuous sodium selenate treatment mitigated epileptogenesis and prevented the biochemical abnormalities, effects which persisted after drug withdrawal. Our studies indicate that limbic epileptogenesis is associated with downregulation of protein phosphatase 2A and the hyperphosphorylation of tau, and that targeting this mechanism with sodium selenate is a potential anti-epileptogenic therapy.

Environmental enrichment imparts disease-modifying and transgenerational effects on genetically-determined epilepsy and anxiety.

INTRODUCTION: The absence epilepsies are presumed to be caused by genetic factors, but the influence of environmental exposures on epilepsy development and severity, and whether this influence is transmitted to subsequent generations, is not well known. We assessed the effects of environmental enrichment on epilepsy and anxiety outcomes in multiple generations of GAERS - a genetic rat model of absence epilepsy that manifests comorbid elevated anxiety-like behaviour. METHODS: GAERS were exposed to environmental enrichment or standard housing beginning either prior to, or after epilepsy onset, and underwent EEG recordings and anxiety testing. Then, we exposed male GAERS to early enrichment or standard housing and generated F1 progeny, which also underwent EEG recordings. Hippocampal CRH mRNA expression and DNA methylation were assessed using RT-PCR and pyrosequencing, respectively. RESULTS: Early environmental enrichment delayed the onset of epilepsy in GAERS, and resulted in fewer seizures in adulthood, compared with standard housed GAERS. Enrichment also reduced the frequency of seizures when initiated in adulthood. Anxiety levels were reduced by enrichment, and these anti-epileptogenic and anxiolytic effects were heritable into the next generation. We also found reduced expression of CRH mRNA in GAERS exposed to enrichment, but this was not due to changes in DNA methylation. CONCLUSIONS: Environmental enrichment produces disease-modifying effects on genetically determined absence epilepsy and anxiety, and these beneficial effects are transferable to the subsequent generation. Reduced CRH expression was associated with these phenotypic improvements. Environmental stimulation holds promise as a naturalistic therapy for genetically determined epilepsy which may benefit subsequent generations.

Seizure expression, behavior, and brain morphology differences in colonies of Genetic Absence Epilepsy Rats from Strasbourg.

OBJECTIVE: Originally derived from a Wistar rat strain, a proportion of which displayed spontaneous absence-type seizures, Genetic Absence Epilepsy Rats from Strasbourg (GAERS) represent the most widely utilized animal model of genetic generalized epilepsy. Here we compare the seizure, behavioral, and brain morphometric characteristics of four main GAERS colonies that are being actively studied internationally: two from Melbourne (MELB and STRAS-MELB), one from Grenoble (GREN), and one from Istanbul (ISTAN). METHODS: Electroencephalography (EEG) recordings, behavioral examinations, and structural magnetic resonance imaging (MRI) studies were conducted on GAERS and Non-Epileptic Control (NEC) rats to assess and compare the following: (1) characteristics of spike-and-wave discharges, (2) anxiety-like and depressive-like behaviors, and (3) MRI brain morphology of regions of interest. RESULTS: Seizure characteristics varied between the colonies, with MELB GAERS exhibiting the least severe epilepsy phenotype with respect to seizure frequency, and GREN GAERS exhibiting four times more seizures than MELB. MELB and STRAS-MELB colonies both displayed consistent anxiety and depressive-like behaviors relative to NEC. MELB and GREN GAERS showed similar changes in brain morphology, including increased whole brain volume and increased somatosensory cortical width. A previously identified mutation in the Cacna1h gene controlling the CaV 3.2 T-type calcium channel (R1584P) was present in all four GAERS colonies, but absent in all NEC rats. SIGNIFICANCE: This study demonstrates differences in epilepsy severity between GAERS colonies that were derived from the same original colony in Strasbourg. This multi-institute study highlights the potential impact of environmental conditions and/or genetic drift on the severity of epileptic and behavioral phenotypes in rodent models of epilepsy.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Ethosuximide reduces epileptogenesis and behavioral comorbidity in the GAERS model of genetic generalized epilepsy.

PURPOSE: Ethosuximide (ESX) is a drug of choice for the symptomatic treatment of absence seizures. Chronic treatment with ESX has been reported to have disease-modifying antiepileptogenic activity in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (1) possesses antiepileptogenic effects in the genetic absence epilepsy rats from Strasbourg (GAERS) model of GGE, (2) is associated with a mitigation of behavioral comorbidities, and (3) influences gene expression in the somatosensory cortex region where seizures are thought to originate. METHODS: GAERS and nonepileptic control (NEC) rats were chronically treated with ESX (in drinking water) or control (tap water) from 3 to 22 weeks of age. Subsequently, all animals received tap water only for another 12 weeks to assess enduring effects of treatment. Seizure frequency and anxiety-like behaviors were serially assessed throughout the experimental paradigm. Treatment effects on the expression of key components of the epigenetic molecular machinery, the DNA methyltransferase enzymes, were assessed using quantitative polymerase chain reaction (qPCR). KEY FINDINGS: ESX treatment significantly reduced seizures in GAERS during the treatment phase, and this effect was maintained during the 12-week posttreatment phase (p < 0.05). Furthermore, the anxiety-like behaviors present in GAERS were reduced by ESX treatment (p < 0.05). Molecular analysis revealed that ESX treatment was associated with increased expression of DNA methyltransferase enzyme messenger RNA (mRNA) in cortex. SIGNIFICANCE: Chronic ESX treatment has disease-modifying effects in the GAERS model of GGE, with antiepileptogenic effects against absence seizures and mitigation of behavioral comorbidities. The cellular mechanism for these effects may involve epigenetic modifications.

Early life adversity accelerates epileptogenesis and enhances depression-like behaviors in rats.

OBJECTIVE: Early life stressors are well-established risk factors for psychiatric disorders, and evidence also suggests that these promote vulnerability to epilepsy. Given the high prevalence of psychiatric disorders in epilepsy, early life stress may represent a common driver for these comorbidities. We used animal modelling to investigate the effects of early life stress on epileptogenesis and depressive behaviors, also exploring HPA axis programming as a potential associative mechanism. METHODS: From post-natal day 2-9, Wistar rat dams (n = 3) and their offspring were exposed to the Limited Bedding and Nesting (LBN) model of early life adversity. Control dams (n = 3) were undisturbed. Maternal care was video-recorded, and behavior scored. As adults, rats (n = 7/group) underwent kainic acid-induced status epilepticus (SE), to trigger epilepsy development. Spontaneous seizures, depression-like behavior and HPA axis function were quantified. RESULTS: LBN significantly altered aspects of maternal care, including markedly reducing the consistency of care (p < 0.05), compared to control conditions. Following SE, LBN rats exhibited significantly accelerated epileptogenesis (p = 0.01) and greater disease severity (p = 0.001), compared to control rats. Anhedonia and behavioral despair were observed in epileptic rats exposed to LBN. LBN rats showed significantly dampened HPA axis responsivity, but epileptic rats showed greater corticosterone responses to CRH administration (all p < 0.05). SIGNIFICANCE: Early life adversity promotes a vulnerability to experimental epileptogenesis. These two 'hits' (early life stress and epilepsy) interact to create a depressive-like phenotype, but effects on HPA axis are complex and contrasting. This has implications for the mechanisms underpinning the increased prevalence of psychiatric disorders observed in people with epilepsy.

High sucrose diet does not impact spatial cognition in rats using advanced touchscreen technology.

INTRODUCTION: Western diets, including those consisting of saturated fats, simple sugars and processed foods, is rising at an unprecedented rate. These lead to obesity and metabolic diseases, and possibly cognitive deficits. Exploring this, recent studies demonstrate marked impairment in spatial learning in rodents exposed to high-sugar diets. We utilised advanced touchscreen technology to assess several spatial and non-spatial components of cognition in rats chronically exposed to a high sucrose diet. METHODS: Male Wistar rats received 70 ml of 10% sucrose solution each day, or control tap water, persisting for the experiment duration (total n = 32). After 5 weeks of diet, rats performed Pairwise Discrimination, Location Discrimination, or Progressive Ratio tasks on automated touchscreens, and performance compared between groups. RESULTS: Sucrose rats consumed all the sugar solution provided to them, and had significantly increased caloric intake, compared to controls (p < 0.0001). However, in all tests, we found no significant difference in cognitive performance between Sucrose and Control treated rats. This included the number of trials for acquisition, and reversal, in Pairwise Discrimination, and number of trials required to complete Location Discrimination (p > 0.05 for all outcomes). No differences were observed in perseverative behaviour, motivation levels, or processing speed. CONCLUSION: Our study found no evidence to suggest that chronic consumption of sucrose impairs cognition, including both spatial and non-spatial learning tasks. These findings suggest that not all aspects of spatial cognition are negatively impacted by high sugar diet in rodents, and that particular use of touchscreen technology may probe different aspects of cognition than traditional tasks.