Therapeutic potential of CB1R activation by Qingyangshen glycoside M1 for seizure relief.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchum otophyllum C.K.Schneid.PI.Wilson, commonly referred as ''Qingyangshen'' (QYS), is a traditional folk medicine from Yunnan, renowned for its efficacy in neurological and psychiatric disorders. Glycosides isolated from QYS have shown promise in alleviating epilepsy, however, mechanisms of action and specific molecular targets remain to be elucidated. AIM OF THE STUDY: The study aimed to evaluate the anticonvulsant effects of Qingyangshen glycosides M1 (M1), a C21 steroidal glycoside from QYS, on pentylenetetrazol (PTZ)-induced convulsions in zebrafish (Danio rerio), and its neuroprotective effect on Glutamate (Glu)-induced damage to PC12 cells, and importantly to identify its potential molecular targets. MATERIALS AND METHODS: To evaluate anticonvulsant activity of M1, 7 days-post-fertilization (7-dpf) animals were pretreated (by immersion) and then exposed to PTZ (10 mM) solution. Furthermore, Glu-induced PC12 cell damage was employed to investigate the neuroprotective and anti-apoptotic capacity. Cells were pretreated with various concentrations of M1 (0-10 µM) for 12 h and then co-treated with Glu (15 mM) for an additional 24 h. The cell viability, apoptosis rate and apoptosis-related proteins (p-PI3K, PI3K, Akt, p-Akt, CREB, p-CREB, BDNF, Bax and Bcl-2) were measured using CCK-8, annexin V/PI and Western blot assays. To model the expected interaction between M1 and candidate cannabinoid receptor type 1 (CB1R), ERK phosphorylation, molecular docking, and drug affinity responsive target stability (DARTS) techniques were employed. Finally, CB1R antagonist Rimonabant (Rim) was validated by co-administration in both zebrafish and cells to confirm the requirement of CB1R for M1 efficacy. RESULTS: At a concentration of 400 µM, M1 dramatically reversed PTZ-induced convulsive-like behaviors in zebrafish, as evidenced by a significant reduction in locomotor activity. In the context of Glu-induced cytotoxicity, M1 (10 µM) demonstrated a notable increase in cell viability and suppressed apoptosis through modulation of the Bax/Bcl-2 ratio and activation of the PI3K/Akt/CREB/BDNF signaling axis. These effects were facilitated through CB1R activation. In contrast, Rim dampened the beneficial activities of M1 as a cannabinoid agonist. CONCLUSIONS: These results demonstrated that M1 as a potential CB1R activator, exhibiting anticonvulsive effects in a PTZ-induced zebrafish model and neuroprotective properties via the PI3K/Akt/CREB/BDNF signaling axis in a Glu-induced PC12 cell injury model. Notably, the observed seizure relief attenuated by CB1R chemical antagonism.

Epilepsy: Mitochondrial connections to the 'Sacred' disease.

Over 65 million people suffer from recurrent, unprovoked seizures. The lack of validated biomarkers specific for myriad forms of epilepsy makes diagnosis challenging. Diagnosis and monitoring of childhood epilepsy add to the need for non-invasive biomarkers, especially when evaluating antiseizure medications. Although underlying mechanisms of epileptogenesis are not fully understood, evidence for mitochondrial involvement is substantial. Seizures affect 35%-60% of patients diagnosed with mitochondrial diseases. Mitochondrial dysfunction is pathophysiological in various epilepsies, including those of non-mitochondrial origin. Decreased ATP production caused by malfunctioning brain cell mitochondria leads to altered neuronal bioenergetics, metabolism and neurological complications, including seizures. Iron-dependent lipid peroxidation initiates ferroptosis, a cell death pathway that aligns with altered mitochondrial bioenergetics, metabolism and morphology found in neurodegenerative diseases (NDDs). Studies in mouse genetic models with seizure phenotypes where the function of an essential selenoprotein (GPX4) is targeted suggest roles for ferroptosis in epilepsy. GPX4 is pivotal in NDDs, where selenium protects interneurons from ferroptosis. Selenium is an essential central nervous system micronutrient and trace element. Low serum concentrations of selenium and other trace elements and minerals, including iron, are noted in diagnosing childhood epilepsy. Selenium supplements alleviate intractable seizures in children with reduced GPX activity. Copper and cuproptosis, like iron and ferroptosis, link to mitochondria and NDDs. Connecting these mechanistic pathways to selenoproteins provides new insights into treating seizures, pointing to using medicines including prodrugs of lipoic acid to treat epilepsy and to potential alternative therapeutic approaches including transcranial magnetic stimulation (transcranial), photobiomodulation and vagus nerve stimulation.

Ceftriaxone and selenium mitigate seizures and neuronal injury in pentylenetetrazole-kindled rats: Oxidative stress and inflammatory pathway.

Epilepsy is one of the most serious worldwide neurological disorders that lead to the cognitive-psychosocial insults in recurrent seizures. About one third of the patients are drug-resistant, so innovative drugs are needed to manage seizures to improve the quality of life. Ceftriaxone is a cephalosporin antibiotic that increases the expression of glutamate transporters-1 and improves the neurobehavioral effects caused by increased glutamate level in the CNS. Selenium is well known antioxidant. The present study aimed to investigate ceftriaxone and selenium therapeutic effects against epilepsy in rats. Epilepsy was induced by PTZ given at a dose (50 mg/kg I.P) on alternative days for 13 days. Eighty rats were randomly divided into 8 groups: Group1-2; normal and vehicle control, Group 3; PTZ group, Group 4-8; kindled rats received selenium, ceftriaxone100, ceftriaxone200, selenium + ceftriaxone100 and selenium + ceftriaxone200 mg/kg/day respectively for a week. At the end of the study, behavioral tests were performed. Oxidative stress, inflammatory markers, neurotransmitters and GLT-1 were measured in brain tissue homogenate. Brain histopathological investigation was also done. PTZ-kindled rats exhibited increased Racine score, besides behavioral tests and histopathological changes, significant elevation in oxidative stress and inflammatory markers, with decrease in serotonin, dopamine, GABA levels and GLT-1 expressions. Selenium and Ceftriaxone alone or combined treatment decreased Racine score with remarkable improvement in behavioral and histopathological changes. The antioxidant enzymes, neurotransmitters and GLT-1 expressions were increased, along with reduced TNF-α, IL-1 levels. Current study showed that selenium + ceftriaxone100 group represents a possible approach to improve epilepsy particularly through inhibiting oxidative stress and inflammation.

GluN2D subunit-containing NMDA receptors regulate reticular thalamic neuron function and seizure susceptibility.

Thalamic regulation of cortical function is important for several behavioral aspects including attention and sensorimotor control. This region has also been studied for its involvement in seizure activity. Among the NMDA receptor subunits GluN2C and GluN2D are particularly enriched in several thalamic nuclei including nucleus reticularis of the thalamus (nRT). We have previously found that GluN2C deletion does not have a strong influence on the basal excitability and burst firing characteristics of reticular thalamus neurons. Here we find that GluN2D ablation leads to reduced depolarization-induced spike frequency and reduced hyperpolarization-induced rebound burst firing in nRT neurons. Furthermore, reduced inhibitory neurotransmission was observed in the ventrobasal thalamus (VB). A model with preferential downregulation of GluN2D from parvalbumin (PV)-positive neurons was generated. Conditional deletion of GluN2D from PV neurons led to a decrease in excitability and burst firing. In addition, reduced excitability and burst firing was observed in the VB neurons together with reduced inhibitory neurotransmission. Finally, young mice with GluN2D downregulation in PV neurons showed significant resistance to pentylenetetrazol-induced seizure and differences in sensitivity to isoflurane anesthesia but were normal in other behaviors. Conditional deletion of GluN2D from PV neurons also affected expression of other GluN2 subunits and GABA receptor in the nRT. Together, these results identify a unique role of GluN2D-containing receptors in the regulation of thalamic circuitry and seizure susceptibility which is relevant to mutations in GRIN2D gene found to be associated with pediatric epilepsy.

Protective effect of Nardostachys jatamansi extract against lithium-pilocarpine-induced spontaneous recurrent seizures and associated cardiac irregularities in a rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D.Don) DC. is a perennial herbaceous medicinal plant widely used for the ethnomedical treatment of various ailments. The underground parts of the plants are used in traditional medicine to manage epilepsy and other cardiovascular conditions. AIM OF THE STUDY: The present study was undertaken to investigate the efficacy of a characterized hydroalcoholic extract (NJET) of Nardostachys jatamansi in the lithium-pilocarpine rat model of spontaneous recurrent seizures (SRS) and associated cardiac irregularities. MATERIALS AND METHODS: NJET was prepared by percolation using 80% ethanol. The dried NEJT was subjected to UHPLC-qTOF-MS/MS for chemical characterization. Molecular docking studies were performed using the characterized compounds to understand mTOR interactions. The animals showing SRS following lithium-pilocarpine administration were treated with NJET for 6 weeks. Afterward, seizure severity, cardiac parameters, serum biochemistry, and histopathological parameters were studied. The cardiac tissue was processed for specific protein and gene expression studies. RESULTS: The UHPLC-qTOF-MS/MS characterized 13 compounds in NJET. The identified compounds subjected to molecular docking showed promising binding affinities toward mTOR. There was a dose-dependent decrease in the severity of SRS following the extract administration. A reduction in mean arterial pressure and serum biochemical markers (lactate dehydrogenase and creatine kinase) was also observed following NJET treatment in epileptic animals. Histopathological investigations revealed reduced degenerative changes and decreased fibrosis following the extract treatment. The cardiac mRNA level of Mtor, Rps6, Hif1a, and Tgfb3 was reduced in the extract-treated groups. Further, a similar reduction in the protein expression of p-mTOR and HIF-1α was also observed following NJET treatment in the cardiac tissue. CONCLUSIONS: The results concluded that NJET treatment reduces lithium-pilocarpine-induced recurrent seizures and associated cardiac irregularities via downregulation of the mTOR signalling pathway.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

Neuroprotective effect of Geijigadaehwang-tang against trimethyltin-induced hippocampal neurodegeneration: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Patients with dementia are diagnosed with deficiency patterns and interior patterns in traditional Chinese medicine due to decreased physical strength, mental atrophy including cognitive function, and decreased motor function in the gastrointestinal tract. Since "greater yin symptom" in Shanghanlun has been interpreted as interior, deficiency, and cold pattern in traditional Chinese medicine, it is necessary to determine whether Geijigadaehwang-tang (GDT) has therapeutic effects on neurodegenerative diseases and the underlying mechanism if it has such effects. AIMS OF THE STUDY: Trimethyltin (TMT), a neurotoxic organotin compound, has been used to induce several neurodegenerative diseases, including epilepsy and Alzheimer's disease. This study aimed to evaluate the therapeutic efficacy of GDT for TMT-induced hippocampal neurodegeneration and seizures and to determine the mechanisms involved at the molecular level. MATERIALS AND METHODS: The main components of GDT were analyzed using ultra-performance liquid chromatography. TMT was used to induce neurotoxicity in microglial BV-2 cells and C57BL6 mice. GDT was administered at various doses to determine its neuroprotective and seizure inhibition effects. The inhibitory effects of GDT on TMT-induced apoptosis, inflammatory pathways, and oxidative stress pathways were determined in the mouse hippocampal tissues. RESULTS: GDT contained emodin, chrysophanol, albiflorin, paeoniflorin, 6-gingerol, and liquiritin apioside. In microglial BV-2 cells treated with TMT, GDT showed dose-dependent neuroprotective effects. Oral administration of GDT five times for 2.5 days before and after TMT injection inhibited seizures at doses of 180 and 540 mg/kg and inhibited neuronal death in the hippocampus. In hippocampal tissues extracted from mice, GDT inhibited the protein expression of ionized calcium binding adaptor molecule 1, glial fibrillary acidic protein, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3, and phosphorylated nuclear factor (NF)-κB/total-NFκB ratio. Additionally, GDT inhibited the messenger RNA levels of tumor necrosis factor-α, inducible nitric oxide synthase, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, interleukin-1ß, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1. CONCLUSION: This study's results imply that GDT might have neuroprotective potential in neurodegenerative diseases through neuronal death inhibition and anti-inflammatory and antioxidant mechanisms.

Anticonvulsive and Neuroprotective Effects of Eupafolin in Rats Are Associated with the Inhibition of Glutamate Overexcitation and Upregulation of the Wnt/ß-Catenin Signaling Pathway.

Several plant compounds have been found to possess neuroactive properties. The aim of this study was to investigate the anticonvulsant effect of eupafolin, a major active component extracted from Salvia plebeia, a herb used in traditional medicine for its anti-inflammatory properties. To this end, we assessed the anticonvulsant effects of eupafolin in rats intraperitoneally (i.p.) injected with kainic acid (KA) to elucidate this mechanism. Treatment with eupafolin (i.p.) for 30 min before KA administration significantly reduced behavioral and electrographic seizures induced by KA, similar to carbamazepine (i.p.), a widely used antiepileptic drug. Eupafolin treatment also significantly decreased KA seizure-induced neuronal cell death and glutamate elevation in the hippocampus. In addition, eupafolin notably reversed KA seizure-induced alterations in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR2, glutamate decarboxylase 67 (GAD67, GABAergic enzyme), and Wnt signaling-related proteins, including porcupine, Wnt1, phosphorylated-glycogen synthase kinase-3ß, ß-catenin, and Bcl-2 in the hippocampus. Furthermore, the increased level of Dickkopf-related protein 1 (Dkk-1, a Wnt signaling antagonist) and the decreased level of Disheveled1 (Dvl-1, a Wnt signaling activator) in the hippocampus of KA-treated rats were reversed by eupafolin. This study provides evidence of the anticonvulsant and neuroprotective properties of eupafolin and of the involvement of regulation of glutamate overexcitation and Wnt signaling in the mechanisms of these properties. These findings support the benefits of eupafolin in treating epilepsy.

Contribution of non-selective membrane channels and receptors in epilepsy.

Overcoming refractory epilepsy's resistance to the combination of antiepileptic drugs (AED), mitigating side effects, and preventing sudden unexpected death in epilepsy are critical goals for therapy of this disorder. Current therapeutic strategies are based primarily on neurocentric mechanisms, overlooking the participation of astrocytes and microglia in the pathophysiology of epilepsy. This review is focused on a set of non-selective membrane channels (permeable to ions and small molecules), including channels and ionotropic receptors of neurons, astrocytes, and microglia, such as: the hemichannels formed by Cx43 and Panx1; the purinergic P2X7 receptors; the transient receptor potential vanilloid (TRPV1 and TRPV4) channels; calcium homeostasis modulators (CALHMs); transient receptor potential canonical (TRPC) channels; transient receptor potential melastatin (TRPM) channels; voltage-dependent anion channels (VDACs) and volume-regulated anion channels (VRACs), which all have in common being activated by epileptic activity and the capacity to exacerbate seizure intensity. Specifically, we highlight evidence for the activation of these channels/receptors during epilepsy including neuroinflammation and oxidative stress, discuss signaling pathways and feedback mechanisms, and propose the functions of each of them in acute and chronic epilepsy. Studying the role of these non-selective membrane channels in epilepsy and identifying appropriate blockers for one or more of them could provide complementary therapies to better alleviate the disease.

6-Gingerol, a Major Constituent of Zingiber officinale Rhizoma, Exerts Anticonvulsant Activity in the Pentylenetetrazole-Induced Seizure Model in Larval Zebrafish.

Zingiber officinale is one of the most frequently used medicinal herbs in Asia. Using rodent seizure models, it was previously shown that Zingiber officinale hydroethanolic extract exerts antiseizure activity, but the active constituents responsible for this effect have not been determined. In this paper, we demonstrated that Zingiber officinale methanolic extract exerts anticonvulsant activity in the pentylenetetrazole (PTZ)-induced hyperlocomotion assay in larval zebrafish. Next, we isolated 6-gingerol (6-GIN)-a major constituent of Zingiber officinale rhizoma. We observed that 6-GIN exerted potent dose-dependent anticonvulsant activity in the PTZ-induced hyperlocomotion seizure assay in zebrafish, which was confirmed electroencephalographically. To obtain further insight into the molecular mechanisms of 6-GIN antiseizure activity, we assessed the concentration of two neurotransmitters in zebrafish, i.e., inhibitory γ-aminobutyric acid (GABA) and excitatory glutamic acid (GLU), and their ratio after exposure to acute PTZ dose. Here, 6-GIN decreased GLU level and reduced the GLU/GABA ratio in PTZ-treated fish compared with only PTZ-bathed fish. This activity was associated with the decrease in grin2b, but not gabra1a, grin1a, gria1a, gria2a, and gria3b expression in PTZ-treated fish. Molecular docking to the human NR2B-containing N-methyl-D-aspartate (NMDA) receptor suggests that 6-GIN might act as an inhibitor and interact with the amino terminal domain, the glutamate-binding site, as well as within the ion channel of the NR2B-containing NMDA receptor. In summary, our study reveals, for the first time, the anticonvulsant activity of 6-GIN. We suggest that this effect might at least be partially mediated by restoring the balance between GABA and GLU in the epileptic brain; however, more studies are needed to prove our hypothesis.

Impact of Dysfunctional Feed-Forward Inhibition on Glutamate Decarboxylase Isoforms and γ-Aminobutyric Acid Transporters.

Absence seizures are associated with generalised synchronous 2.5-4 Hz spike-wave discharges causing brief and sudden alteration of awareness during childhood, which is known as childhood absence epilepsy (CAE). CAE is also associated with impaired learning, psychosocial challenges, and physical danger. Absence seizures arise from disturbances within the cortico-thalamocortical (CTC) network, including dysfunctional feed-forward inhibition (FFI); however, the precise mechanisms remain unclear. In epileptic stargazers, a genetic mouse model of CAE with chronic seizures, levels of γ-aminobutyric acid (GABA), and expression of GABA receptors are altered within the CTC network, implicating altered GABAergic transmission in absence seizures. However, the expression of GABA synthesising enzymes (GAD65 and GAD67) and GABA transporters (GAT-1 and 3) have not yet been characterised within absence seizure models. We found a specific upregulation of GAD65 in the somatosensory cortex but not the thalamus of epileptic stargazer mice. No differences were detected in GAD67 and GAT-3 levels in the thalamus or somatosensory cortex. Then, we assessed if GAD65 upregulation also occurred in Gi-DREADD mice exhibiting acute absence seizures, but we found no change in the expression profiles of GAD65/67 or GAT-3. Thus, the upregulation of GAD65 in stargazers may be a compensatory mechanism in response to long-term dysfunctional FFI and chronic absence seizures.

BACE inhibitor treatment of mice induces hyperactivity in a Seizure-related gene 6 family dependent manner without altering learning and memory.

BACE inhibitors, which decrease BACE1 (ß-secretase 1) cleavage of the amyloid precursor protein, are a potential treatment for Alzheimer's disease. Clinical trials using BACE inhibitors have reported a lack of positive effect on patient symptoms and, in some cases, have led to increased adverse events, cognitive worsening and hippocampal atrophy. A potential drawback of this strategy is the effect of BACE inhibition on other BACE1 substrates such as Seizure-related gene 6 (Sez6) family proteins which are known to have a role in neuronal function. Mice were treated with an in-diet BACE inhibitor for 4-8 weeks to achieve a clinically-relevant level of amyloid-ß40 reduction in the brain. Mice underwent behavioural testing and postmortem analysis of dendritic spine number and morphology with Golgi-Cox staining. Sez6 family triple knockout mice were tested alongside wild-type mice to identify whether any effects of the treatment were due to altered cleavage of Sez6 family proteins. Wild-type mice treated with BACE inhibitor displayed hyperactivity on the elevated open field, as indicated by greater distance travelled, but this effect was not observed in treated Sez6 triple knockout mice. BACE inhibitor treatment did not lead to significant changes in spatial or fear learning, reference memory, cognitive flexibility or anxiety in mice as assessed by the Morris water maze, context fear conditioning, or light-dark box tests. Chronic BACE inhibitor treatment reduced the density of mushroom-type spines in the somatosensory cortex, regardless of genotype, but did not affect steady-state dendritic spine density or morphology in the CA1 region of the hippocampus. Chronic BACE inhibition for 1-2 months in mice led to increased locomotor output but did not alter memory or cognitive flexibility. While the mechanism underlying the treatment-induced hyperactivity is unknown, the absence of this response in Sez6 triple knockout mice indicates that blocking ectodomain shedding of Sez6 family proteins is a contributing factor. In contrast, the decrease in mature spine density in cortical neurons was not attributable to lack of shed Sez6 family protein ectodomains. Therefore, other BACE1 substrates are implicated in this effect and, potentially, in the cognitive decline in longer-term chronically treated patients.

Neuroendocrine changes in the hypothalamic-neurohypophysial system in the Wistar audiogenic rat (WAR) strain submitted to audiogenic kindling.

The Wistar audiogenic rat (WAR) strain is used as an animal model of epilepsy, which when submitted to acute acoustic stimulus presents tonic-clonic seizures, mainly dependent on brainstem (mesencephalic) structures. However, when WARs are exposed to chronic acoustic stimuli (audiogenic kindling-AK), they usually present tonic-clonic seizures, followed by limbic seizures, after recruitment of forebrain structures such as the cortex, hippocampus and amygdala. Although some studies have reported that hypothalamic-hypophysis function is also altered in WAR through modulating vasopressin (AVP) and oxytocin (OXT) secretion, the role of these neuropeptides in epilepsy still is controversial. We analyzed the impact of AK and consequent activation of mesencephalic neurocircuits and the recruitment of forebrain limbic (LiR) sites on the hypothalamic-neurohypophysial system and expression of Avpr1a and Oxtr in these structures. At the end of the AK protocol, nine out of 18 WARs presented LiR. Increases in both plasma vasopressin and oxytocin levels were observed in WAR when compared to Wistar rats. These results were correlated with an increase in the expressions of heteronuclear (hn) and messenger (m) RNA for Oxt in the paraventricular nucleus (PVN) in WARs submitted to AK that presented LiR. In the paraventricular nucleus, the hnAvp and mAvp expressions increased in WARs with and without LiR, respectively. There were no significant differences in Avp and Oxt expression in supraoptic nuclei (SON). Also, there was a reduction in the Avpr1a expression in the central nucleus of the amygdala and frontal lobe in the WAR strain. In the inferior colliculus, Avpr1a expression was lower in WARs after AK, especially those without LiR. Our results indicate that both AK and LiR in WARs lead to changes in the hypothalamic-neurohypophysial system and its receptors, providing a new molecular basis to better understaind epilepsy.

The Transcription Factor Shox2 Shapes Neuron Firing Properties and Suppresses Seizures by Regulation of Key Ion Channels in Thalamocortical Neurons.

Thalamocortical neurons (TCNs) play a critical role in the maintenance of thalamocortical oscillations, dysregulation of which can result in certain types of seizures. Precise control over firing rates of TCNs is foundational to these oscillations, yet the transcriptional mechanisms that constrain these firing rates remain elusive. We hypothesized that Shox2 is a transcriptional regulator of ion channels important for TCN function and that loss of Shox2 alters firing frequency and activity, ultimately perturbing thalamocortical oscillations into an epilepsy-prone state. In this study, we used RNA sequencing and quantitative PCR of control and Shox2 knockout mice to determine Shox2-affected genes and revealed a network of ion channel genes important for neuronal firing properties. Protein regulation was confirmed by Western blotting, and electrophysiological recordings showed that Shox2 KO impacted the firing properties of a subpopulation of TCNs. Computational modeling showed that disruption of these conductances in a manner similar to Shox2's effects modulated frequency of oscillations and could convert sleep spindles to near spike and wave activity, which are a hallmark for absence epilepsy. Finally, Shox2 KO mice were more susceptible to pilocarpine-induced seizures. Overall, these results reveal Shox2 as a transcription factor important for TCN function in adult mouse thalamus.

The modulator action of thiamine against pentylenetetrazole-induced seizures, apoptosis, nitric oxide, and oxidative stress in rats and SH-SY5Y neuronal cell line.

Accumulating evidences indicate that thiamine plays a vital role in the nervous system. However, questions exist as to how it causes epilepsy, neuronal damage, and antiepileptic mechanisms. The study looked at how the thiamine supplement impacted pentylenetetrazole (PTZ)-induced seizures in rats and pentylenetetrazole-induced neurotoxicity in the SH-SY5Y cell line. We used twenty-four male rats and they were randomly divided into 4 groups as control, saline (1 mL/kg/day serum physiologic) + PTZ, thiamine (50 mg/kg/day) + PTZ, and thiamine (50 mg/kg/day) for 10 days. PTZ (45 mg/kg) was given to activate the seizure on day 10. Memory efficiency was measured by using passive avoidance. The brain levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) were analyzed by using ELISA kits. SH-SY5Y cells were treated with/without thiamine for 1 h, followed by PTZ (30 µm) at a medium level to trigger neurotoxicity. Cell viability, total antioxidant status, total oxidant status, and apoptosis were assayed in the SH-SY5Y cells. Thiamine delayed the initiation of epileptic seizures and increased memory damage. In addition, 8-OHdG, caspase-3, NO, and cGMP levels were significantly reduced in the brain and prevented pentylenetetrazole-induced neurotoxicity, apoptosis, enhanced antioxidant, and reduced oxidant in SH-SY5Y cells. Thiamine dramatically altered seizures, memory loss, oxidative stress, and apoptosis. Thiamine has a preventative effect on PTZ-induced seizures in rats and PTZ-induced neurotoxicity in SH-SY5Y neuroblastoma cells. It could prevent oxidative stress and signaling of NO/cGMP. Thiamine supplement could be used as an additional therapeutic agent in epilepsy.

The ω-3 endocannabinoid docosahexaenoyl ethanolamide reduces seizure susceptibility in mice by activating cannabinoid type 1 receptors.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most recognized omega-3 unsaturated fatty acids showing neuroprotective activity in animal and clinical studies. Docosahexaenoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) are non-oxygenated endogenous metabolites of DHA and EPA, which might be in charge of the anti-seizure activity of the parent molecules. We examined the effect of these metabolites on the threshold of clonic seizures induced by pentylenetetrazole (PTZ). DHEA and EPEA possess similar chemical structure to the endogenous cannabinoids. Therefore, involvement of cannabinoid (CB) receptors in the anti-seizure effect of these metabolites was also investigated. DHA, DHEA, EPEA, AM251 (CB1 receptor antagonist), and AM630 (CB2 receptor antagonist) were administered to mice by intracerebroventricular (i.c.v.) route. Threshold of clonic seizures was determined 10 and/or 15 min thereafter by intravenous infusion of PTZ. The effect of DHA and DHEA on seizure threshold was then determined in mice, which were pretreated with AM251 and/or AM630. DHA (300µM), and DHEA (100 and 300 µM) significantly increased seizure threshold, 15 (p < 0.05) and 10 min (p < 0.01) after administration, respectively. DHEA was more potent than its parent lipid, DHA in decreasing seizure susceptibility. EPEA (300 and 1000 µM) did not change seizure threshold. AM251 fully prevented the increasing effect of DHA and DHEA on seizure threshold (p < 0.05). AM630 did not inhibit the effect of DHA and DHEA on seizure threshold. This is the first report indicating that DHEA but not EPEA, possesses anti-seizure action via activating CB1 receptors. DHEA is more potent than its parent ω-3 fatty acid DHA in diminishing seizure susceptibility.

Cannabidiol-enriched oil in children and adults with treatment-resistant epilepsy-does tolerance exist?

AIM: To evaluate the long-term effectiveness of cannabidiol (CBD)-enriched oil for the treatment of refractory epilepsy and to assess the development of tolerance to its anti-seizure effect. METHODS: A prospective study of 92 consecutive patients (age 1-37 years, mean-11.8 years) with treatment resistant epilepsy who were treated with cannabis oil extract (CBD/tetrahydrocannabinol [THC] ratio of 20:1). Mean monthly seizure frequency was reported by the patients/their parents during monthly clinic visits. Tolerance was defined as either the need to increase the dose by ≥30% due to reduced treatment efficacy or as an increase of ≥30% in mean monthly seizure frequency in patients treated for at least 3 months with no change in other anti-seizure medications. RESULTS: Mean follow-up time was 19.8 ± 12.5 months (range 3-45). Mean CBD dose was 11.3 (4-38) mg/kg/day. Twenty-nine (31%) patients discontinued treatment due to lack of effect or adverse reactions, which were reported in 51% (47/87) of the patients. Overall responder rate (>50% seizures reduction) was 54%, whereas 8 patients (9%) became seizure-free. Eighty-four patients were included in the tolerance analysis. Tolerance was observed in 21 (25%) patients after a mean duration of 7.3 ± 5.4 months of CBD-enriched oil treatment. There was a negative correlation between epilepsy duration and tolerance development (p = 0.038). CONCLUSIONS: We report for the first time the plausible appearance of tolerance to cannabidiol-enriched oil. This may limit treatment efficacy in the long-term clinical management of refractory epilepsy in both pediatric and adult population. Further studies are needed to investigate potential mechanisms.

Preconditioning by ultra-low dose of tramadol reduces the severity of tramadol-induced seizure: Contribution of glutamate receptors.

Tramadol, a weak agonist of mu-opioid receptors, causes seizure via several mechanisms. Preconditioning has been purposed to reduce the epileptic seizures in animal models of epilepsy. The preconditioning effect of tramadol on seizure is not studied yet. This study was designed to evaluate the preconditioning effect of ultra-low dose of tramadol on the seizures induced by tramadol at high dose. Furthermore, regarding the critical role of glutamate signaling in the pathogenesis of epilepsy, the effect of preconditioning on some glutamate signaling elements was also examined. Male Wistar rats received tramadol (2 mg/kg, i.p) or normal saline (1 mL/kg, i.p) in preconditioning and control groups, respectively. After 4 days, the challenging tramadol dose (150 mg/kg) was injected to all rats. Epileptic behaviors were recorded during 50 min. The expression of Norbin (as a regulator of metabotropic glutamate receptor 5), Calponin3 (as a regulator of excitatory synaptic markers), NR1 (NMDA receptor subunit 1) and GluR1 (AMPA receptor subunit 1) was measured in hippocampus, prefrontal cortex (PFC) and amygdala. Preconditioning decreased the number and duration of tremors and tonic-clonic seizures. Norbin, Calponin3, NR1 and GluR1 expression were decreased in hippocampus, and preconditioning had no effect on them. In contrast, it increased Norbin expression in PFC and amygdala, and attenuated NR1 and GluR1 upregulation following tramadol at high dose. These findings indicated that preconditioning by ultra-low dose of tramadol protected the animals against seizures following high dose of tramadol mediated, at least in part, by Norbin up regulation, and NR1 and GluR1 down regulation.

Central deficiency of norepinephrine synthesis and norepinephrinergic neurotransmission contributes to seizure-induced respiratory arrest.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of mortality in patients with intractable epilepsy. However, the pathogenesis of SUDEP seems to be poorly understood. Our previous findings showed that the incidence of seizure-induced respiratory arrest (S-IRA) was markedly reduced by atomoxetine in a murine SUDEP model. Because the central norepinephrine α-1 receptor (NEα-1R) plays a vital role in regulating respiratory function, we hypothesized that the suppression of S-IRA by atomoxetine was mediated by NE/NEα-1R interactions that can be reversed by NEα-1R antagonism. We examined whether atomoxetine-mediated suppression of S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) in DBA/1 mice can be reversed by intraperitoneal (IP) and intracerebroventricular (ICV) administration of prazosin, a selective antagonist of NEα-1R. The content and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme for NE synthesis, in the lower brainstem was measured by ELISA. Electroencephalograms (EEG) were obtained from using the PTZ-evoked SUDEP model. In our models, atomoxetine-mediated suppression of S-IRA evoked by either acoustic stimulation or PTZ was significantly reversed by low doses of IP and ICV prazosin. Neither repetitive acoustic stimulation nor S-IRA reduced TH levels in lower brainstem. However, the enzyme activity of TH levels in lower brainstem was significantly increased by mechanical ventilation with DBA/1 mice, which makes the dying DBA/1 mice suffering from S-IRA and SUDEP recover. EEG data showed that although the protective effect of atomoxetine was reversed by prazosin, neither drug suppressed EEG activity. These data suggest that deficient synthesis of NE and norepinephrinergic neurotransmission contributed to S-IRA and that the NEα-1R is a potential therapeutic target for the prevention of SUDEP.

Monoterpenes as a perspective for the treatment of seizures: A Systematic Review.

BACKGROUND: Epilepsy affects more than 65 million people worldwide. Treatment for epileptic seizures is ineffective and has many adverse effects. For this reason, the search for new therapeutic options capable of filling these limitations is necessary. HYPOTHESIS/PURPOSE: In this sense, natural products, such as monoterpenes, have been indicated as a new option to control neurological disorders such as epilepsy. STUDY DESIGN: Therefore, the objective of this study was to review the monoterpenes that have anticonvulsive activity in animal models. METHODS: The searches were performed in the PubMed, Web of Science and Scopus databases in September, 2020 and compiled studies using monoterpenes as an alternative to seizure. Two independent reviewers performed the study selection, data extraction and methodological quality assessment using the Syrcle tool. RESULTS: 51 articles that described the anticonvulsant activity of 35 monoterpenes were selected with action on the main pharmacological target, including GABAA receptors, glutamate, calcium channels, sodium and potassium. In addition, these compounds are capable of reducing neuronal inflammation and oxidative stress caused by seizure. CONCLUSION: These compounds stand out as a promising alternative for acting through different pharmacological mechanisms, which may not only reduce seizure, but also promote neuroprotective effect by reducing toxicity in brain regions. However, further studies are needed to determine the mechanism of action and safety assessment of these compounds.