Electroshock Seizure Threshold Research Articles

Transient anticonvulsant effects of time-restricted feeding in the 6-Hz mouse model

IntroductionIntermittent fasting enhances neural bioenergetics, is neuroprotective, and elicits antioxidant effects in various animal models. There are conflicting findings on seizure protection, where intermittent fasting regimens often cause severe weight loss resembling starvation which is unsustainable long-term. Therefore, we tested whether a less intensive intermittent fasting regimen such as time-restricted feeding (TRF) may confer seizure protection. MethodsMale CD1 mice were assigned to either ad libitum-fed control, continuous 8 h TRF, or 8 h TRF with weekend ad libitum food access (2:5 TRF) for one month. Body weight, food intake, and blood glucose levels were measured. Seizure thresholds were determined at various time points using 6-Hz and maximal electroshock seizure threshold (MEST) tests. Protein levels and mRNA expression of genes, enzyme activity related to glucose metabolism, as well as mitochondrial dynamics were assessed in the cortex and hippocampus. Markers of antioxidant defence were evaluated in the plasma, cortex, and liver. ResultsBody weight gain was similar in the ad libitum-fed and TRF mouse groups. In both TRF regimens, blood glucose levels did not change between the fed and fasted state and were higher during fasting than in the ad libitum-fed groups. Mice in the TRF group had increased seizure thresholds in the 6-Hz test on day 15 and on day 19 in a second cohort of 2:5 TRF mice, but similar seizure thresholds at other time points compared to ad libitum-fed mice. Continuous TRF did not alter MEST seizure thresholds on day 28. Mice in the TRF group showed increased maximal activity of pyruvate dehydrogenase in the cortex, which was accompanied by increased protein levels of mitochondrial pyruvate carrier 1 in the cortex and hippocampus. There were no other major changes in protein or mRNA levels associated with energy metabolism and mitochondrial dynamics in the brain, nor markers of antioxidant defence in the brain, liver, or plasma. ConclusionsBoth continuous and 2:5 TRF regimens transiently increased seizure thresholds in the 6-Hz model at around 2 weeks, which coincided with stability of blood glucose levels during the fed and fasted periods. Our findings suggest that the lack of prolonged anticonvulsant effects in the acute electrical seizure models employed may be attributed to only modest metabolic and antioxidant adaptations found in the brain and liver. Our findings underscore the potential therapeutic value of TRF in managing seizure-related conditions.

Changes in neuronal excitability in the rat hippocampus in a prolonged febrile seizures model

Febrile seizures (FSs) are a prevalent neurological disorder among children aged 3 months to 5 years, with the highest incidence observed in the second year of life [1]. Considering that neuronal and glial cell development and synaptic contact formation are ongoing during this period [2, 3], FSs can potentially influence these processes. However, the present data on the effect of FSs on brain development remain inconsistent. This study aims to examine the impact of extended seizures on the attributes of hippocampal pyramidal neurons in rats of varying ages. Wistar rats were used in this study. FSs were induced on postnatal day (P)10 through placing pups onto the bottom of a glass chamber for 30 minutes and exposing them to a controlled stream of heated air, causing their body temperature to rise to 39 °C and trigger the occurrence of FSs. Only animals that underwent FSs that persisted for at least 15 minutes were included in the study. Littermates were employed as controls and were placed away from the nest for the same duration but kept at room temperature. At postnatal days 12, 21–23, and 51–55, the rats were decapitated, and their brains were extracted. Horizontal brain slices (400 micrometers) were sliced. The study examined the biophysical properties of CA1 pyramidal neurons using the whole-cell patch-clamp method. 1.5-second current pulses were injected, and subthreshold membrane properties such as resting membrane potential, input resistance, membrane time constant, and intrinsic firing properties, were assessed. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum of the hippocampus to assess the efficacy of synaptic neurotransmission at CA3-CA1 pyramidal neuron synapses. The amplitude and fiber volley (FV) amplitude of each fEPSP were measured by applying high amplitude currents to each slice. A sigmoidal Gompertz function was used to evaluate the efficacy of neurotransmission. Paired-pulse stimulation was used to examine potential alterations in short-term synaptic plasticity. Paired pulses were administered at intervals spanning from 10 to 500 ms, and the paired-pulse ratio (PPR) was assessed as the proportion of the amplitude of the second to the first fEPSP for each interval. Maximum electroshock seizure threshold (MEST) was determined two months after FS to assess the animals’ susceptibility to seizures. The lowest current at which tonic hind limb extension was exhibited was determined for every animal. No significant changes in subthreshold firing properties were observed in P12 or P21 rats following FSs. However, we did observe several changes in intrinsic firing properties. For example, the maximum firing frequency decreased by 23% in P12 rats exposed to FSs, relative to age-matched control rats. Additionally, we noticed that firing frequency adaptation was significantly less pronounced in P12 rats that underwent FSs, compared to control rats of the same age. Seizure-induced alterations in firing characteristics were absent in P21 rats. No significant variations in fEPSP or FV amplitudes were found among the different groups of P12 and P55 rats at different current intensities. However, a significant increase in FV amplitudes and decrease in neuronal input-output (I/O) relationships between fEPSP and FV amplitudes were observed in P21 rats following FSs. At P12, short-term synaptic plasticity was disrupted, evidenced by a significant increase in PPR in rats two days post-FS. However, at P21 and P55, experimental and control groups were not significantly different. MEST test displayed a significant rise in the hind limb extension threshold in rats two months following FS as compared to control animals. Overall, these findings suggest alterations in neuronal excitability after prolonged FS. Two days after FS, neuronal excitability transiently decreased while PPR increased, indicating a decrease in the probability of presynaptic glutamate release in hippocampal neurons. The efficiency of synaptic neurotransmission in CA3-CA1 was reduced in three-week-old animals. Additionally, the MEST test demonstrated that rats, two months following FS, exhibited a higher hind limb extension threshold in comparison to control animals.

Alterations in Rat Hippocampal Glutamatergic System Properties after Prolonged Febrile Seizures

Febrile seizures during early childhood may result in central nervous system developmental disorders. However, the specific mechanisms behind the impact of febrile seizures on the developing brain are not well understood. To address this gap in knowledge, we employed a hyperthermic model of febrile seizures in 10-day-old rats and tracked their development over two months. Our objective was to determine the degree to which the properties of the hippocampal glutamatergic system are modified. We analyzed whether pyramidal glutamatergic neurons in the hippocampus die after febrile seizures. Our findings indicate that there is a reduction in the number of neurons in various regions of the hippocampus in the first two days after seizures. The CA1 field showed the greatest susceptibility, and the reduction in the number of neurons in post-FS rats in this area appeared to be long-lasting. Electrophysiological studies indicate that febrile seizures cause a reduction in glutamatergic transmission, leading to decreased local field potential amplitude. This impairment could be attributable to diminished glutamate release probability as evidenced by decreases in the frequency of miniature excitatory postsynaptic currents and increases in the paired-pulse ratio of synaptic responses. We also found higher threshold current causing hind limb extension in the maximal electroshock seizure threshold test of rats 2 months after febrile seizures compared to the control animals. Our research suggests that febrile seizures can impair glutamatergic transmission, which may protect against future seizures.

Alterations in the Properties of the Rat Hippocampus Glutamatergic System in the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy

Status epilepticus (SE) triggers many not yet fully understood pathological changes in the nervous system that can lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in the lithium-pilocarpine model of temporal lobe epilepsy in rats. The studies were performed 1 day (acute phase), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. According to RT-qPCR data, expression of the genes coding for the AMPA receptor subunits GluA1 and GluA2 was downregulated in the latent phase, which may lead to the increased proportion of calcium-permeable AMPA receptors that play an essential role in the pathogenesis of many CNS diseases. The efficiency of excitatory synaptic neurotransmission in acute brain slices was decreased in all phases of the model, as determined by recording field responses in the CA1 region of the hippocampus in response to the stimulation of Schaffer collaterals by electric current of different strengths. However, the frequency of spontaneous excitatory postsynaptic potentials increased in the chronic phase, indicating an increased background activity of the glutamatergic system in epilepsy. This was also evidenced by a decrease in the threshold current causing hindlimb extension in the maximal electroshock seizure threshold test in rats with temporal lobe epilepsy compared to the control animals. The results suggest a series of functional changes in the properties of glutamatergic system associated with the epilepsy development and can be used to develop the antiepileptogenic therapy.

Dietary Zinc Differentially Regulates the Effects of the GPR39 Receptor Agonist, TC-G 1008, in the Maximal Electroshock Seizure Test and Pentylenetetrazole-Kindling Model of Epilepsy.

The G-protein coupled receptor 39 (GPR39) is gaining increasing attention as a target for future drugs, yet there are gaps in the understanding of its pharmacology. Zinc is an endogenous agonist or an allosteric modulator, while TC-G 1008 is a synthetic, small molecule agonist. Zinc is also a positive allosteric modulator for the activity of TC-G 1008 at GPR39. Activation of GPR39 by TC-G 1008 facilitated the development of epileptogenesis in the pentylenetetrazole (PTZ)-induced kindling model of epilepsy. Congruently, TC-G 1008 decreased the seizure threshold in the maximal electroshock seizure threshold (MEST) test. Here, we investigated the effects of TC-G 1008 under the condition of zinc deficiency. Mice were fed a zinc-adequate diet (ZnA, 50 mg Zn/kg) or a zinc-deficient diet (ZnD, 3 mg Zn/kg) for 4 weeks. Following 4 weeks of dietary zinc restriction, TC-G 1008 was administered as a single dose and the MEST test was performed. Additional groups of mice began the PTZ-kindling model during which TC-G 1008 was administered repeatedly and the diet was continued. TC-G 1008 administered acutely decreased the seizure threshold in the MEST test in mice fed the ZnD diet but not in mice fed the ZnA diet. TC-G 1008 administered chronically increased the maximal seizure severity and the percentage of fully kindled mice in those fed the ZnA diet, but not in mice fed the ZnD diet. Our data showed that the amount of zinc in a diet is a factor contributing to the effects of TC-G 1008 in vivo.

Caffeine impairs anticonvulsant effects of levetiracetam in the maximal electroshock seizure threshold test in mice.

Caffeine is the most widely used psychoactive substance in the world. Animal studies indicate that acute caffeine exposure at high doses may induce seizures anddiminish the anticonvulsant activity of antiepileptic drugs (AEDs) at much lower doses. The aim of the current study was to assess the effect of caffeine on the anticonvulsant action of levetiracetam (LEV) and vigabatrin (VGB). The anticonvulsant activity of LEV and VGB was examined in the maximal electroshock seizure threshold test in mice (MEST test). All drugs were administered intraperitoneally by single injections, and caffeine was applied at doses capable of interfering with AEDs. Effects of caffeine exposure on AEDs were also investigated in tests of memory and motor performance. Caffeine reduced the protective effect of LEV against electroconvulsions. Total brain concentration of LEV was unaffected by caffeine as well as inversely; LEVhad no significant impact on the brain caffeine concentration, suggesting a pharmacodynamic nature of theinteraction between LEV and caffeine in the MEST test.VGB at applied doses did not affect the convulsive threshold. Administration of VGB, but not LEV, alone orincombination with caffeine, impaired memory retention. In the chimney test, the combined treatment with AEDs and caffeine did not cause motor coordination impairment. It is suggested that caffeine may negatively affect the anticonvulsant action of LEV in patients with epilepsy.

Glyceryl triacetate feeding in mice increases plasma acetate levels but has no anticonvulsant effects in acute electrical seizure models

IntroductionAcetate has been shown to have neuroprotective and anti-inflammatory effects. It is oxidized by astrocytes and can thus provide auxiliary energy to the brain in addition to glucose. Therefore, we hypothesized that it may protect against seizures, which is investigated here by feeding glyceryl triacetate (GTA), to provide high amounts of acetate without raising sodium or acid levels. MethodCD1 male mice were fed controlled diets with or without GTA for up to three weeks. Body weights, blood glucose levels, plasma short-chain fatty acid levels, and other hematological parameters were monitored. Seizure thresholds were determined in 6 Hz and maximal electroshock seizure threshold (MEST) tests. Antioxidant capacities were evaluated in the cerebral cortex and plasma using a ferric reducing antioxidant power (FRAP) assay and Trolox equivalent antioxidant capacity assay. ResultsBody weight gain was similar with both diets with and without GTA in two experiments. Glyceryl triacetate-fed groups showed 2–3- and 1.6-fold increased acetate and propionate levels in plasma, respectively. Glucose levels were unaltered in blood collected from the tail tip but increased in trunk blood. No differences were found in the activity of cerebral cortex acetyl-CoA synthetase. In the 6 Hz threshold test, seizure thresholds were lower by 3 mA and 2.4 mA after 8 and 14 days, respectively, in the GTA compared to the control diet-fed group, but showed no difference on day 16, showing that GTA has small, but inconsistent proconvulsant effects in this model. In MEST tests, a slightly increased seizure threshold (1 mA) was found on day 19 in the GTA-fed group, but not in another experiment on day 21. There were no differences in antioxidant capacity in plasma or cortex between the two groups. ConclusionGlyceryl triacetate feeding showed no antioxidant effects nor beneficial changes in acute electrical seizure threshold mouse models, despite its ability to increase plasma acetate levels.

Effect of Ellagic Acid on Seizure Threshold in Two Acute Seizure Tests in Mice.

Ellagic acid (EA) is a natural dietary polyphenol that has many beneficial properties, including anti-inflammatory, antioxidant, antiviral, antibacterial, and neuroprotective effects. Studies have revealed that EA may modulate seizure activity in chemically induced animal models of seizures. Therefore, the aim of the present study was to investigate the effect of EA on the seizure threshold in two acute seizure tests in male mice, i.e., in the intravenous (i.v.) pentylenetetrazole (PTZ) seizure test and in the maximal electroshock seizure threshold (MEST) test. The obtained results showed that EA (100 mg/kg) significantly elevated the threshold for both the first myoclonic twitch and generalized clonic seizure in the i.v. PTZ seizure test. At the highest dose tested (200 mg/kg), EA increased the threshold for tonic hindlimb extension in the MEST test. EA did not produce any significant changes in motor coordination (assessed in the chimney test) or muscular strength (investigated in the grip-strength test). The plasma and total brain concentration-time profiles of EA after intraperitoneal and oral administration were also determined. Although further studies are necessary to confirm the anticonvulsant activity of EA, our findings suggest that it may modulate seizure susceptibility in animal models.

Effects of the NKCC1 inhibitors bumetanide, azosemide, and torasemide alone or in combination with phenobarbital on seizure threshold in epileptic and nonepileptic mice

The sodium-potassium-chloride (Na–K–Cl) cotransporter NKCC1 is found in the plasma membrane of a wide variety of cell types, including neurons, glia and endothelial cells in the brain. Increased expression of neuronal NKCC1 has been implicated in several brain disorders, including neonatal seizures and epilepsy. The loop diuretic and NKCC inhibitor bumetanide has been evaluated as an antiseizure agent alone or together with approved antiseizure drugs such as phenobarbital (PB) in pre-clinical and clinical studies with varying results. The equivocal efficacy of bumetanide may be a result of its poor brain penetration. We recently reported that the loop diuretic azosemide is more potent to inhibit NKCC1 than bumetanide. In contrast to bumetanide, azosemide is not acidic, which should favor its brain penetration. Thus, azosemide may be a promising alternative to bumetanide for treatment of brain disorders such as epilepsy. In the present study, we determined the effect of azosemide and bumetanide on seizure threshold in adult epileptic mice. A structurally related non-acidic loop diuretic, torasemide, which also blocks NKCC1, was included in the experiments. The drug effects were assessed by determing the maximal electroshock seizure threshold (MEST) in epileptic vs. nonepileptic mice. Epilepsy was induced by pilocarpine, which was shown to produce long-lasting increases in NKCC1 in the hippocampus, whereas MEST did not alter NKCC1 mRNA in this region. None of the three loop diuretics increased MEST or the effect of PB on MEST in nonepileptic mice. In epileptic mice, all three diuretics significantly increased PB's seizure threshold increasing efficacy, but the effect was variable upon repeated MEST determinations and not correlated with the drugs' diuretic potency. These data may indicate that inhibition of NKCC1 by loop diuretics is not an effective means of increasing seizure threshold in adult epilepsy.

In vitro and in vivo characterization of Lu AA41178: A novel, brain penetrant, pan-selective Kv7 potassium channel opener with efficacy in preclinical models of epileptic seizures and psychiatric disorders

Activation of the voltage-gated Kv7 channels holds therapeutic promise in several neurological and psychiatric disorders, including epilepsy, schizophrenia, and depression. Here, we present a pharmacological characterization of Lu AA41178, a novel, pan-selective Kv7.2–7.5 opener, using both in vitro assays and a broad range of in vivo assays with relevance to epilepsy, schizophrenia, and depression.Electrophysiological characterization in Xenopus oocytes expressing human Kv7.2-Kv7.5 confirmed Lu AA41178 as a pan-selective opener of Kv7 channels by significantly left-shifting the activation threshold. Additionally, Lu AA41178 was tested in vitro for off-target effects, demonstrating a clean Kv7-selective profile, with no impact on common cardiac ion channels, and no potentiating activity on GABAA channels.Lu AA41178 was evaluated across preclinical in vivo assays with relevance to neurological and psychiatric disorders. In the maximum electroshock seizure threshold test and PTZ seizure threshold test, Lu AA41178 significantly increased the seizure thresholds in mice, demonstrating anticonvulsant efficacy. Lu AA41178 demonstrated antipsychotic-like activity by reducing amphetamine-induced hyperlocomotion in mice as well as lowering conditioned avoidance responses in rats. In the mouse forced swim test, a model with antidepressant predictivity, Lu AA41178 significantly reduced immobility. Additionally, behavioral effects typically observed with Kv7 openers was also characterized. In vivo assays were accompanied by plasma and brain exposures, revealing minimum effective plasma levels <1000 ng/ml.Lu AA41178, a potent opener of neuronal Kv7 channels demonstrate efficacy in assays of epilepsy, schizophrenia and depression and might serve as a valuable tool for exploring the role of Kv7 channels in both neurological and psychiatric disorders.

Anticonvulsant Activity of Pterostilbene in Zebrafish and Mouse Acute Seizure Tests

Pterostilbene (PTE), a natural dimethylated analog of resveratrol, possesses numerous health-beneficial properties. The ability of PTE to cross the blood–brain barrier raised the possibility that this compound may modulate central nervous system functions, including seizure activity. The aim of our study was to investigate the activity of PTE in the larval zebrafish pentylenetetrazole (PTZ) seizure assay and three acute seizure tests in mice, i.e., in the maximal electroshock seizure threshold (MEST), 6 Hz-induced psychomotor seizure threshold and intravenous (iv) PTZ tests. Additionally, potential antidepressant activity of PTE was estimated in the forced swim test in mice. The chimney test was used to determine the influence of PTE on motor coordination in mice, while its influence on neuromuscular strength was assessed in the grip strength test in mice. Locomotor activity was determined to verify the results from the forced swim test. PTE revealed an evident anticonvulsant effect both in zebrafish larvae (10 µM; 2 h-incubation) and mice (at doses of 100 and 200 mg/kg, intraperitoneally) but it did not exhibit antidepressant potential in the forced swim test. Furthermore, it did not cause any statistically significant changes in motor coordination, neuromuscular strength and locomotor activity in mice. In conclusion, our present findings demonstrate for the first time the anticonvulsant potential of PTE. The aforementioned results suggest that it might be employed in epilepsy treatment, however, further precise studies are required to verify its activity in other experimental seizure and epilepsy models and its precise mechanism of action should be determined.

Effect of Tadalafil on Seizure Threshold and Activity of Antiepileptic Drugs in Three Acute Seizure Tests in Mice

Tadalafil, a selective phosphodiesterase type 5 inhibitor, is a long-acting oral agent for the treatment of erectile dysfunction of multiple etiologies. Although generalized tonic-clonic seizures were reported in a healthy man after taking tadalafil, the influence of tadalafil on seizure susceptibility has not been studied so far. Therefore, the aim of the present study was to investigate the effect of tadalafil on seizure threshold as well as on the activity of some first- and second-generation antiepileptic drugs in three acute seizure tests in mice. The obtained results showed that tadalafil, at the highest dose tested (20 mg/kg), significantly decreased the threshold for the first myoclonic twitch in the intravenous pentylenetetrazole (i.v. PTZ) seizure test. It did not affect the threshold for generalized clonic seizure and forelimb tonus in the i.v. PTZ, for tonic hindlimb extension in the maximal electroshock seizure threshold test, and for psychomotor seizure in the 6-Hz-induced seizure threshold test. Tadalafil did not alter the anticonvulsant activity of any of the studied antiepileptic drugs in electrically induced seizure tests. Interestingly, tadalafil potentiated the anticonvulsant activity of clonazepam and decreased the anticonvulsant activity of oxcarbazepine in the i.v. PTZ test. These interactions were pharmacodynamic in nature, as tadalafil did not alter clonazepam and oxcarbazepine concentrations both in serum and brain tissue. Furthermore, neither tadalafil alone nor its combinations with the studied antiepileptic drugs produced any significant impairment of motor coordination (assessed in the chimney test), muscular strength (investigated in the grip-strength test), and long-term memory (assessed in the passive avoidance task). In conclusion, tadalafil may increase the risk of myoclonic seizure and decrease the anticonvulsant efficacy of oxcarbazepine. Further studies are warranted to evaluate the safety of tadalafil usage in patients with epilepsy.

Influence of propafenone on the anticonvulsant activity of various novel antiepileptic drugs in the mouse maximal electroshock model.

The main mechanism of action of propafenone (antiarrhythmic drug) involves the inhibition of the fast inward sodium current during phase 0 of the action potential. Sodium channel-blocking activity is also characteristic for some antiepileptic drugs. Therefore, it could be assumed that propafenone may also affect seizures. In the present study, we evaluated the effect of propafenone on the protective effect of oxcarbazepine, lamotrigine, topiramate and pregabalin against the maximal electroshock-induced seizures in mice. Anticonvulsant activity of propafenone was assessed with the maximal electroshock seizure threshold (MEST) test. Influence of propafenone on the anticonvulsant activity of antiepileptic drugs was estimated in the mouse maximal electroshock model (MES). Drug-related adverse effects were determined in the chimney test (motor coordination) and passive-avoidance task (long-term memory). Brain concentrations of antiepileptics were assessed by fluorescence polarization immunoassay. Propafenone at doses 60-90mg/kg significantly increased the threshold of seizures, in turn at doses 5-50mg/kg did not affect this parameter. Administration of propafenone at the subthreshold dose of 50mg/kg increased antielectroshock activity of oxcarbazepine, topiramate and pregabalin, but not that of lamotrigine. As regards adverse effects, propafenone alone and in combination with antiepileptic drugs did not significantly impair motor coordination or long-term memory in mice. Propafenone (50mg/kg) significantly increased the brain level of pregabalin. Brain concentrations of topiramate and oxcarbazepine were not affected. Our findings show that propafenone has own anticonvulsant action and enhances efficacy of oxcarbazepine, topiramate and pregabalin, but not that of lamotrigine, at least in experimental condition.

HBK-14 and HBK-15, triple 5-HT1A, 5-HT7 and 5-HT3 antagonists with potent antidepressant- and anxiolytic-like properties, increase seizure threshold in various seizure tests in mice

Most antidepressants lower seizure threshold, which might be due to the modulation of serotonergic neurotransmission. Here, we investigated the effects of two 5-HT1A, 5-HT7 and 5-HT3 antagonists, i.e., 1-(2-(2-(2,6-dimethylphenoxy)ethoxy)ethyl)-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-{2-[2-(2-chloro-6-methylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15), with antidepressant- and anxiolytic-like properties, on seizure thresholds in three acute seizure tests, i.e., the intravenous pentylenetetrazole, maximal electroshock seizure threshold (MEST), and 6-Hz corneal stimulation test in mice. We also evaluated their affinity for voltage-gated sodium channels. Our results indicate that HBK-14 increased seizure thresholds in three seizure tests in mice, while HBK-15 was active in the MEST and 6-Hz tests. None of the compounds affected neuromuscular strength or motor coordination at active doses. We showed that both compounds had high affinity for voltage-dependent sodium channels, which combined with the influence on 5-HT1A, 5-HT7 and 5-HT3 receptors, might underlie their anticonvulsant effects. Since most antidepressants lower the seizure threshold, the fact that both compounds with potent antidepressant-like activity, increased or had no influence on seizure threshold is worth investigating.

Evaluation of the Anticonvulsant Effect of Brilliant Blue G, a Selective P2X7 Receptor Antagonist, in\xa0the iv PTZ-, Maximal Electroshock-, and 6\xa0Hz-Induced Seizure Tests in Mice

Epilepsy is one of the most common neurological disorders which is diagnosed in around 65 million people worldwide. Clinically available antiepileptic drugs fail to control epileptic activity in about 30% of patients and they are merely symptomatic treatments and cannot cure or prevent epilepsy. There remains a need for searching new therapeutic strategies for epileptic disorders. The P2X7 receptor has been recently investigated as a new target in epilepsy treatment. Preclinical studies revealed that P2X7 receptor antagonists have anticonvulsant properties in some models of epilepsy. We aimed to investigate whether P2X7 receptor antagonist—brilliant blue G (BBG)—is able to change seizure threshold in three acute seizure models in mice, i.e., in the intravenous pentylenetetrazole seizure threshold, maximal electroshock seizure threshold and 6 Hz psychomotor seizure threshold tests. BBG was administered acutely (50–200 mg/kg, 30 min before the tests) and sub-chronically (25–100 mg/kg, once daily for seven consecutive days). Moreover, the chimney and grip strength tests were used to estimate the influence of BBG on the motor coordination and muscular strength in mice, respectively. Our results revealed only a week anticonvulsant potential of the studied P2X7 receptor antagonist because it showed anticonvulsant action only in the 6 Hz seizure test, both after acute and sub-chronic administration. BBG did not significantly influence seizure thresholds in the remaining tests. Motor coordination and muscular strength were not affected by the studied P2X7 receptor antagonist. In summary, BBG does not possess any remarkable anticonvulsant potential in acute seizure models in mice.

Design, synthesis and anticonvulsant-analgesic activity of new N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols.

New derivatives of N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols have been synthesized and evaluated for their anticonvulsant activity in maximal electroshock (MES), maximal electroshock seizure threshold (MEST), and pentylenetetrazol (PTZ) tests. Their neurotoxicity was evaluated via rotarod and chimney tests. The compounds exhibiting the most beneficial activity and protection indices were evaluated for analgesic activity using the formalin test for neurogenic pain. They were also evaluated for their influence on cytotoxic activity using in vitro cellular models (HepG2 and CRL-2534 cell lines). Experiments performed using MTT and neutral red cytotoxicity assays showed that all evaluated compounds were safe for normal, glial cells (astrocytes) and did not induce hepatotoxic effects. Based on the results from the in vitro studies, the safety of the evaluated compounds was inferred. The most promising compound in this research was 1-{2-[2-(2,3-dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol hydrochloride. Additionally, in silico metabolism prediction for the compound has been performed.

Proconvulsant effects of the ketogenic diet in electroshock-induced seizures in mice

Among non-pharmacological treatments, the ketogenic diet (KD) has the strongest demonstrated evidence of clinical success in drug resistant epilepsy. In an attempt to model the anticonvulsant effects of the KD pre-clinically, the present study assessed the effects of the KD against electroshock-induced convulsions in mice. After confirming that exposure to the KD for 2 weeks resulted in stable ketosis and hypoglycemia, mice were exposed to electroshocks of various intensities to establish general seizure susceptibility. When compared to mice fed the standard rodent chow diet (SRCD), we found that mice fed the KD were more sensitive to electroconvulsions as reflected by a significant decrease in seizure threshold (3.86 mA in mice on the KD vs 7.29 mA in mice on the SRCD; P < 0.05) in the maximal electroshock seizure threshold (MEST) test. To examine if this increased seizure sensitivity to electroconvulsions produced by the KD would affect anticonvulsant effects of antiepileptic drugs (AEDs), anticonvulsant potencies of carbamazepine (CBZ), phenobarbital (PB), phenytoin (PHT), and valproate (VPA) against maximal electroshock (MES)-induced convulsions were compared in mice fed the KD and SRCD. We found that potencies of all AEDs studied were decreased in mice fed the KD in comparison to those on the SRCD, with decreases in the anticonvulsant potencies ranging from 1.4 fold (PB) to 1.7 fold (PHT). Finally, the lack of differences in brain exposures of the AEDs studied in mice fed the KD and SRCD ruled out a pharmacokinetic nature of the observed findings. Taken together, exposure to the KD in the present study had an overall pro-convulsant effect. Since electroconvulsions require large metabolic reserves to support their rapid spread throughout the brain and consequent generalized tonic-clonic convulsions, this effect may be explained by a high energy state produced by the KD in regards to increased energy storage and utilization.

The bumetanide prodrug BUM5, but not bumetanide, potentiates the antiseizure effect of phenobarbital in adult epileptic mice.

The loop diuretic bumetanide has been reported to potentiate the antiseizure activity of phenobarbital in rodent models of neonatal seizures, most likely as a result of inhibition of the chloride importer Na-K-Cl cotransporter isoform 1 (NKCC1) in the brain. In view of the intractability of neonatal seizures, the preclinical findings prompted a clinical trial in neonates on bumetanide as an add-on to phenobarbital, which, however, had to be terminated because of ototoxicity and lack of efficacy. We have recently shown that bumetanide penetrates only poorly into the brain, so that we developed lipophilic prodrugs such as BUM5, the N,N-dimethylaminoethylester of bumetanide, which penetrate more easily into the brain and are converted to bumetanide. In the present study, we used a new strategy to test whether BUM5 is more potent than bumetanide in potentiating the antiseizure effect of phenobarbital. Adult mice were made epileptic by pilocarpine, and the antiseizure effects of bumetanide, BUM5, and phenobarbital alone or in combination were determined by the maximal electroshock seizure threshold test. In nonepileptic mice, only phenobarbital exerted seizure threshold-increasing activity, and this was not potentiated by the NKCC1 inhibitors. In contrast, a marked potentiation of phenobarbital by BUM5, but not bumetanide, was determined in epileptic mice. Thus, bumetanide is not capable of potentiating phenobarbital's antiseizure effect in an adult mouse model, which, however, can be overcome by using the prodrug BUM5. These data substantiate that BUM5 is a promising tool compound for target validation and proof-of-concept studies on the role of NKCC1 in brain diseases.

SB 334867, a selective orexin receptor type 1 antagonist, elevates seizure threshold in mice

AimOrexins A and B are hypothalamic neuropeptides involved in a number of physiological and behavioral processes. They work via OX1 and OX2 receptors. Recent studies revealed that orexins may be implicated in seizure activity. Therefore, the present study was undertaken to evaluate the influence of SB 334867 (a selective OX1 receptor antagonist) and EMPA (a selective OX2 receptor antagonist) on the seizure thresholds in mice. We also aimed to determine the changes of orexin A level following different types of seizures. Main methodsThe intravenous pentylenetetrazole (i.v. PTZ) seizure test, the maximal electroshock seizure threshold (MEST) test and the 6Hz seizure test were used in the present study. Brain orexin A level was determined via enzyme-linked immunoassay (ELISA). Key findingsSB 334867 did not affect the seizure threshold for myoclonic twitches and tonic seizures in the i.v. PTZ seizure test. This compound, however, significantly raised the threshold for the PTZ-induced clonic seizures, for tonic hindlimb extension in the MEST test as well as for psychomotor seizures induced by 6Hz stimulation. In comparison, EMPA did not alter the seizure thresholds in the i.v. PTZ test. Both EMPA and SB 334867 did not affect motor coordination and muscular strength. ELISA showed the increase of total brain orexin A level following different types of seizures. SignificanceOur results provide further evidence for the role of orexins in seizure activity and suggest that pharmacological blockade of the OX1 receptors may represent a novel therapeutic approach in the treatment of seizure disorders.

A Highly Selective Inhibitor of Glycine Transporter-1 Elevates the Threshold for Maximal Electroshock-Induced Tonic Seizure in Mice.

Many anti-epileptic drugs (AEDs) that mainly target ion channels or post-synaptic receptors are in clinical use, but a proportion of patients are resistant to these traditional AEDs and experience repeated severe break-out seizures. Given its involvement in the etiology of epilepsy, the neurotransmitter glycine may serve as a novel target for epilepsy treatment. Increasing evidence suggests that inhibitors of glycine transporter 1 (GlyT1) exhibit anti-seizure properties in mouse models and show potential as anti-convulsions drugs. In the present study, we investigated the effect of a highly selective GlyT1 inhibitor (named M22) on glycine transport kinetics using a radioactive substrate uptake assay and investigated the anti-seizure effects of M22 on the maximal electroshock seizure threshold (MEST) test and the timed intravenous (i.v.) pentylenetetrazole (PTZ) intravenous test. Our results demonstrate that M22 was capable of elevating the seizure threshold in the MEST test but did not alter the seizure threshold in the PTZ i.v. test. Strychnine, an inhibitor of glycine receptor activity, reversed the threshold elevation at a subconvulsive dosage (0.1 mg/kg subcutaneously) in the MEST test and did not affect M22 plasma levels in mice, suggesting that the anti-seizure effect in this model may be mediated by increased glycine receptor activity. Moreover, M22 administration did not influence motor function and coordination in mice. In combination with the previously reported excellent pharmacokinetic features of M22, our present results suggested that M22 has the potential to serve as a new anti-convulsive drug or as a lead compound for the development of AEDs.