Modulation index predicts the effect of ethosuximide on developmental and epileptic encephalopathy with spike-and-wave activation in sleep.

PURPOSE: In developmental and epileptic encephalopathy with spike-and-wave activation in sleep (DEE-SWAS), the thalamocortical network is suggested to play an important role in the pathophysiology of the progression from focal epilepsy to DEE-SWAS. Ethosuximide (ESM) exerts effects by blocking T-type calcium channels in thalamic neurons. With the thalamocortical network in mind, we studied the prediction of ESM effectiveness in DEE-SWAS treatment using phase-amplitude coupling (PAC) analysis. METHODS: We retrospectively enrolled children with DEE-SWAS who had an electroencephalogram (EEG) recorded between January 2009 and September 2022 and were prescribed ESM at Okayama University Hospital. Only patients whose EEG showed continuous spike-and-wave during sleep were included. We extracted 5-min non-rapid eye movement sleep stage N2 segments from EEG recorded before starting ESM. We calculated the modulation index (MI) as the measure of PAC in pair combination comprising one of two fast oscillation types (gamma, 40-80 Hz; ripples, 80-150 Hz) and one of five slow-wave bands (delta, 0.5-1, 1-2, 2-3, and 3-4 Hz; theta, 4-8 Hz), and compared it between ESM responders and non-responders. RESULTS: We identified 20 children with a diagnosis of DEE-SWAS who took ESM. Fifteen were ESM responders. Regarding gamma oscillations, significant differences were seen only in MI with 0.5-1 Hz slow waves in the frontal pole and occipital regions. Regarding ripples, ESM responders had significantly higher MI in coupling with all slow waves in the frontal pole region, 0.5-1, 3-4, and 4-8 Hz slow waves in the frontal region, 3-4 Hz slow waves in the parietal region, 0.5-1, 2-3, 3-4, and 4-8 Hz slow waves in the occipital region, and 3-4 Hz slow waves in the anterior-temporal region. SIGNIFICANCE: High MI in a wider area of the brain may represent the epileptic network mediated by the thalamus in DEE-SWAS and may be a predictor of ESM effectiveness.

Thermosensitive hydrogel containing ethosuximide-loaded multivesicular liposomes attenuates age-related hearing loss in C57BL/6J mice.

Ethosuximide is the first drug reported to protect against age-related hearing loss, but its benefits are hampered by the pronounced side effects generated through systemic administration. We prepared a thermosensitive hydrogel containing ethosuximide-encapsulated multivesicular liposomes (ethosuximide-loaded MVLs-Gel) and evaluated its functional and histological effects on age-related hearing loss in C57BL/6J mice. The MVLs-Gel showed slow sustained-release characteristics up to over 120 h. After 8 weeks of treatment, compared to the oral systemic administration of ethosuximide, intratympanic ethosuximide-loaded MVLs-Gel injection dramatically reduced the loss of age-related spiral ganglion neurons in the apical turns of the mice (low-frequency regions, p < 0.05). Correspondingly, compared to the oral systemic administration group, the intratympanic ethosuximide-loaded MVLs-Gel injection group showed significantly lower auditory brainstem response threshold shifts at stimulus frequencies of 4, 8, and 16 kHz (low-and middle-frequency regions, p < 0.05). In conclusion, intratympanic ethosuximide-loaded MVLs-Gel injection can reach the apical turn of the cochlea, which is extremely difficult with oral systemic administration of the drug. The ethosuximide-loaded MVLs-Gel, as a novel intratympanic sustained-release drug delivery system, attenuated age-related hearing loss in C57BL/6J mice.

A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterized by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9-mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared with haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.

The effect of chronic treatment with sodium channel blocker lacosamide on early development of absence seizures in genetic absence epilepsy rats.

OBJECTIVE: Lacosamide (LCM) is a new generation antiepileptic drug that affects the slow inactivation of voltage-gated sodium channels. We studied whether chronic LCM treatment prior to onset of absence seizures was able to prevent/reduce the development of absence seizures in GAERS rats, a well-validated animal model of absence epilepsy and epileptogenesis. Drug effects on the duration, mean duration, number and spectral characteristics of spike-wave discharges (SWDs) were measured both 1 and 2 months after treatment withdrawal and compared with the ethosuximide (ETX) that has anti-epileptogenic activity in GAERS. Furthermore, the acute effects of LCM on SWDs in adult GAERS were evaluated. METHODS: GAERS rats were administered either with LCM (10 mg/kg/day or 30 mg/kg/day, i.p) or ETX (25 mg/kg/day, i.p) or saline (%0.9 NaCl) until PN60 for 40 consecutive days starting from PN20. Animals were stereotaxically implanted with cortical screw electrodes under ketamine/xylazine anesthesia at PN53. Following recovery period, EEG were recorded at PN60 (last day of drug administration)- 61-62, PN90-91-92 and PN120-121-122 time periods for 3 consecutive days. RESULTS: The chronic treatment with both LCM and ETX led to an ∼50% reduction in the development of spontaneous absence seizures in GAERS at PN90 and PN120 after the treatment withdrawal at PN60. The spectral analysis of EEG data revealed significant slowing of the peak frequency of SWDs in LCM treated animals at PN62. CONCLUSION: These results confirm that chronic LCM treatment modifies the development of absence seizures in GAERS and suggest that LCM exerts beneficial effects on absence seizure epileptogenesis.

Sensitivity of unilateral- versus bilateral-onset spike-wave discharges to ethosuximide and carbamazepine in the fluid percussion injury rat model of traumatic brain injury.

Unilateral-onset spike-wave discharges (SWDs) following fluid percussion injury (FPI) in rats have been used for nearly two decades as a model for complex partial seizures in human posttraumatic epilepsy (PTE). This study determined if SWDs with a unilateral versus bilateral cortical onset differed. In this experiment, 2-mo-old rats received severe FPI (3 atm) or sham surgery and were instrumented for chronic video-electrocorticography (ECoG) recording (up to 9 mo). The antiseizure drug, carbamazepine (CBZ), and the antiabsence drug, ethosuximide (ETX), were administered separately to determine if they selectively suppressed unilateral- versus bilateral-onset SWDs, respectively. SWDs did not significantly differ between FPI and sham rats on any measured parameter (wave-shape, frequency spectrum, duration, or age-related progression), including unilateral (∼17%) versus bilateral (∼83%) onsets. SWDs with a unilateral onset preferentially originated ipsilateral to the craniotomy in both FPI and sham rats, suggesting that the unilateral-onset SWDs were related to surgical injury and not specifically to FPI. ETX profoundly suppressed SWDs with either unilateral or bilateral onsets, and CBZ had no effect on either type of SWD. These results suggest that SWDs with either a unilateral or bilateral onset have a pharmacosensitivity similar to absence seizures and are very different from the complex partial seizures of PTE. Therefore, SWDs with a unilateral onset after FPI are not a model of the complex partial seizures that occur in PTE, and their use for finding new treatments for PTE could be counterproductive, particularly if their close similarity to normal brain oscillations is not acknowledged.NEW & NOTEWORTHY Unilateral-onset spike-wave discharges (SWDs) in rats have been used to model complex partial seizures in human posttraumatic epilepsy (PTE), compared to bilateral-onset SWDs thought to reflect human absence seizures. Here, we show that both unilateral- and bilateral-onset SWDs following traumatic brain injury are suppressed by the antiabsence drug ethosuximide and are unaffected by the antiseizure drug carbamazepine. We propose that unilateral-onset SWDs are not useful for studying mechanisms of, or treatments for, PTE.

Ethosuximide inhibits acute histamine- and chloroquine-induced scratching behavior in mice.

We have recently reported that the Cav3.2 T-type calcium channel which is well known for its key role in pain signalling, also mediates a critical function in the transmission of itch/pruritus. Here, we evaluated the effect of the clinically used anti-seizure medication ethosuximide, a well known inhibitor of T-type calcium channels, on male and female mice subjected to histaminergic- and non-histaminergic itch. When delivered intraperitoneally ethosuximide significantly reduced scratching behavior of mice of both sexes in response to subcutaneous injection of either histamine or chloroquine. When co-delivered subcutaneously together with either pruritogenic agent ethosuximide was also effective in inhibiting scratching responses in both male and female animals. Overall, our results are consistent with an important role of Cav3.2 T-type calcium channels in modulating histamine-dependent and histamine-independent itch transmission in the primary sensory pathway. Our findings also suggest that ethosuximide could be explored further as a possible therapeutic for the treatment of itch.

First evidence of altered microbiota and intestinal damage and their link to absence epilepsy in a genetic animal model, the WAG/Rij rat.

OBJECTIVE: A large number of studies have highlighted the important role of the gut microbiota in the pathophysiology of neurological disorders, suggesting that its manipulation might serve as a treatment strategy. We hypothesized that the gut microbiota participates in absence seizure development and maintenance in the WAG/Rij rat model and tested this hypothesis by evaluating potential gut microbiota and intestinal alterations in the model, as well as measuring the impact of microbiota manipulation using fecal microbiota transplantation (FMT). METHODS: Initially, gut microbiota composition and intestinal histology of WAG/Rij rats (a well-recognized genetic model of absence epilepsy) were studied at 1, 4, and 8 months of age in comparison to nonepileptic Wistar rats. Subsequently, in a second set of experiments, at 6 months of age, untreated Wistar or WAG/Rij rats treated with ethosuximide (ETH) were used as gut microbiota donors for FMT in WAG/Rij rats, and electroencephalographic (EEG) recordings were obtained over 4 weeks. At the end of FMT, stool and gut samples were collected, absence seizures were measured on EEG recordings, and microbiota analysis and histopathological examinations were performed. RESULTS: Gut microbiota analysis showed differences in beta diversity and specific phylotypes at all ages considered and significant variances in the Bacteroidetes/Firmicutes ratio between Wistar and WAG/Rij rats. FMT, from both Wistar and ETH-treated WAG/Rij donors to WAG/Rij rats, significantly decreased the number and duration of seizures. Histological results indicated that WAG/Rij rats were characterized by intestinal villi disruption and inflammatory infiltrates already at 1 month of age, before seizure occurrence; FMT partially restored intestinal morphology while also significantly modifying gut microbiota and concomitantly reducing absence seizures. SIGNIFICANCE: Our results demonstrate for the first time that the gut microbiota is modified and contributes to seizure occurrence in a genetic animal model of absence epilepsy and that its manipulation may be a suitable therapeutic target for absence seizure management.

Efficacy of anticonvulsant ethosuximide for major depressive disorder: a randomized, placebo-control clinical trial.

Results of a preclinical study suggested that the anticonvulsant drug ethosuximide may elicit ketamine-like rapid-acting antidepressant actions. We evaluated the antidepressant efficacy of ethosuximide versus placebo in non-medicated adult patients with major depressive disorder (MDD). This randomized, double-blind, placebo-controlled trial included patients at three mental health centers in China. Eighty eligible adults (aged 18-65 years) met the DSM-5 criteria for MDD. Patients in the acute single study received three doses (500, 1000, or 1500 mg) of ethosuximide or placebo. Patients in the repeated study received ethosuximide (1500 mg/day) or placebo for 2 weeks. The Hamilton Depression Rating Scale (HAM-D), the Montgomery-Åsberg Depression Rating Scale (MADRS), and the Hamilton Anxiety Rating Scale were used to assess antidepressant and antianxiety responses to ethosuximide. No significant reductions in depression and anxiety rating scale scores were observed after a single oral administration of ethosuximide, in comparison with placebo. Furthermore, patients receiving ethosuximide for 2 weeks did not show reductions in depression and anxiety rating scale scores. There were no serious adverse events. Responses to the study's primary and secondary outcome measures, the clinician-rated HAM-D and MADRS, showed no change from baseline to the end of treatment, with either ethosuximide or placebo. These results suggest that ethosuximide does not produce ketamine-like robust antidepressant actions in adult patients with MDD.

Anticonvulsant activity of Valeriana edulis roots and valepotriates on the pentylenetetrazole-induced seizures in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: For many centuries, Mexican Valerian (Valeriana edulis ssp. procera) has been an important plant in folk medicine. It has been considered useful to control epilepsy; however, electroencephalographic evidence of its anticonvulsant activity is missing in literature. AIM OF THE STUDY: In the present study, in situ electroencephalographic (EEG) analysis was performed along with administration of a crude ethanol extract of V. edulis and its valepotriate fraction on the pentylenetetrazole (PTZ)-induced convulsive behavior in rats. MATERIALS AND METHODS: Experiments were performed using male Wistar rats with nail-shaped electrodes implanted in the frontal and parietal cortices for EEG recording. All animals received a single dose of PTZ (35 mg/kg, i.p.) to test the anticonvulsant activity of V. edulis crude extract and valepotriate fraction (100 mg/kg, i.p.) 15 and/or 30 min after administration. EEG recordings were obtained from the cortices and were evaluated to assess ictal behavior over 60-75 min. Chromatographic analysis of the valepotriate fraction and in silico predictions of pharmacodynamic properties were also explored. The latency, frequency and duration of seizures evaluated using EEG recordings from the frontal and parietal cortices of rats showed significant changes demonstrating the inhibition of paroxystic activity. RESULTS: The spectral analysis confirmed the reduction of excitatory activity induced by V. edulis extract, which was improved in the presence of the valepotriate fraction as compared to that induced by ethosuximide (a reference anticonvulsant drug). The presence of valepotriates such as: isodihydrovaltrate (18.99%), homovaltrate (13.51%), 10-acetoxy-valtrathydrin (4%) and valtrate (1.34%) was identified by chromatographic analysis. Whereas, not only GABAA receptor participation but also the cannabinoid CB2 receptor was found to be likely involved in the anticonvulsant mechanism of action after in silico prediction. CONCLUSIONS: Our data support the anticonvulsant properties attributed to this plant in folk medicine, due to the presence of valepotriates.

Response of spike-wave discharges in aged APP/PS1 Alzheimer model mice to antiepileptic, metabolic and cholinergic drugs.

Epileptic nonconvulsive spike-wave discharges (SWDs) are commonly seen in amyloid plaque bearing transgenic mice but only rarely in their wild-type littermates. To shed light on their possible treatment options, we assessed the effect of drugs with variable and known mechanisms of action on the occurrence of SWDs in aged APPswe/PS1dE9 mice. The treatments included prototypic antiepileptic drugs (ethosuximide and levetiracetam), donepezil as the typical Alzheimer drug and atropine as an antagonistic effect, GABAB antagonist CGP-35348, and alternate energy substrates beta-hydroxybutyrate (BHB), pyruvate and lactate on the occurrence of SWDs in aged APPswe/PS1dE9 mice. All agents were administered by single intraperitoneal injections at doses earlier documented to be effective and response was assessed by recording 3 h of video-EEG. Atropine at 25 mg/kg significantly decreased SWD occurrence in all behavioral states, and also resulted in altered frequency composition of SWDs and general EEG slowing during sleep. Ethosuximide at 200 mg/kg and levetiracetam at 75 mg/kg effectively suppressed SWDs only during a period of mixed behavioral states, but levetiracetam also increased SWDs in sleep. BHB at 1 g/kg decreased SWDs in sleep, while both pyruvate and lactate at the same dose tended to increase SWD number and total duration. Unexpectantly, donepezil at 0.3 mg/kg CGP-35348 at 100 mg/kg had no effect on SWDs. These findings call for re-evaluation of some prevailing theories on neural circuit alternations that underlie SWD generation and show the utility of APP/PS1 mice for testing potential new treatments for nonconvulsive epileptic activity related to Alzheimer pathology.

Long-term vigabatrin treatment modifies pentylenetetrazole-induced seizures in mice: focused on GABA brain concentration.

BACKGROUND: The goal of our study was to examine the long-term effect of vigabatrin (VGB), a γ-aminobutyric acid aminotransferase (GABA-AT) inhibitor on clonazepam (CLO), ethosuximide (ETX) and valproate (VPA) anticonvulsive activity against pentylenetetrazole (PTZ)-induced seizures in mice. METHODS: VGB was administered for 3 and 7 days. Convulsions were evoked by PTZ at its CD97 (99 mg/kg). The influence of CLO, ETX and VPA alone or in combination with VGB on motor performance and long-term memory was analyzed. γ-aminobutyric acid (GABA) concentration in mice brain and plasma as well as glutamate decarboxylase (GAD) activity was measured. RESULTS: After 3 days of treatment, VGB in doses up to 500 mg/kg increased PTZ-induced seizure threshold, whereas after 7 days VGB (at the dose of 125 mg/kg) inhibited clonic seizures in experimental mice. 7 days of VGB administration did not change the protective effect of CLO, ETX and VPA against PTZ-induced seizures. 7 days of VGB treatment at a subthreshold dose of 75 mg/kg decreased TD50 of ETX and CLO in the chimney test, but did not affect TD50 value for VPA. 7 days of VGB administration in combination with AEDs did not affect long-term memory in mice. VGB after 3 days or 7 days of administration increased brain GABA concentration. GAD activity was decreased after 3 and 7 days of VGB administration. CONCLUSIONS: The presented results confirm anticonvulsive activity of VGB through GABA metabolism alteration and suggest care when combining VGB with ETX or CLO in the therapy.

Identification of significant protein markers by mass spectrometry after co-treatment of cells with different drugs: An in vitro survey platform.

RATIONALE: Understanding drug-drug interactions and predicting the side effects induced by polypharmacy are difficult because there are few suitable platforms that can predict drug-drug interactions and possible side effects. Hence, developing a platform to identify significant protein markers of drug-drug interactions and their associated side effects is necessary to avoid adverse effects. METHODS: Human liver cells were treated with ethosuximide in combination with cimetidine, ketotifen, metformin, metronidazole, or phenytoin. After sample preparation and extraction, mitochondrial proteins from liver cells were isolated and digested with trypsin. Then, peptide solutions were detected using a nano ultra-performance liquid chromatographic system combined with tandem mass spectrometry. The Ingenuity Pathway Analysis tool was used to simulate drug-drug interactions and identify protein markers associated with drug-induced adverse effects. RESULTS: Several protein markers were identified by the proposed method after liver cells were co-treated with ethosuximide and other drugs. Several of these protein markers have previously been reported in the literature, indicating that the proposed platform is workable. CONCLUSIONS: Using the proposed in vitro platform, significant protein markers of drug-drug interactions could be identified by mass spectrometry. This workflow can then help predict indicators of drug-drug interactions and associated adverse effects for increased safety in clinical prescriptions.

Evaluation of the impact of compound C11 a new anticonvulsant candidate on cognitive functions and hippocampal neurogenesis in mouse brain.

Searching for the new and effective anticonvulsants in our previous study we developed a new hybrid compound C-11 derived from 2-(2,5-dioxopyrrolidin-1-yl) propanamide. C11 revealed high efficacy in acute animal seizure models such as the maximal electroshock model (MES), the pentylenetetrazole model (PTZ) and the 6 Hz (6 Hz, 32 mA) seizure model, as well as in the kindling model of epilepsy induced by repeated injection of PTZ in mice. In the aim of further in vivo C11 characterization, in the current studies we evaluated its influence on cognitive functions, neurodegeneration and neurogenesis process in mice after chronical treatment. All experiments were performed on 6 weeks old male C57/BL mice. The following drugs were used: C11, levetiracetam (LEV), ethosuximide (ETS) and lacosamide (LCM). We analyzed proliferation, migration and differentiation of newborn cells as well as neurodegenerative changes in a mouse brain after long-term treatment with aforementioned AEDs. Additionally, we evaluated changes in learning and memory functions in response to chronic C11, LEV, LCM and ETS treatment. C11 as well as LEV and ETS did not disturb the proliferation of newborn cells compared to the control mice, whereas LCM treatment significantly decreased it. Chronic AEDs therapy did not induce significant neurodegenerative changes. Behavioral studies with using Morris Water Maze test did not indicate any disturbances in the spatial learning and memory after C11 as well as LEV and ETS treatment in comparison to the control group except LCM mice where significant dysfunctions in time, distance and direct swim to the platform were observed. Interestingly, results obtained from in vivo MRI spectroscopy showed a statistically significant increase of one of the neurometabolites- N-acetyloaspartate (NAA) for LCM and LEV mice. A new hybrid compound C11 in contrast to LCM has no negative impact on the process of neurogenesis and neurodegeneration in the mouse hippocampus. Furthermore, chronic treatment with C11 turned out to have no negative impact on cognitive functions of treated mice, which, is certainly of great importance for further more advanced preclinical and especially clinical trials.

Ethosuximide improves chronic pain-induced anxiety- and depression-like behaviors.

Chronic pain is a heavy burden disease. Current treatments are generally weakly effective or associated with adverse effects. New therapeutic approaches are therefore needed. Recent studies have suggested T-type calcium channels as an attractive target for the treatment of chronic pain. In this perspective, it was decided to perform a preclinical evaluation of the efficacy of ethosuximide, a T-type channel blocker used clinically as an antiepileptic, as a novel pharmacological treatment for chronic pain. Assessment of the effect of ethosuximide was thus made in both nociception and pain-related comorbidities as anxiety and depression are frequently encountered in chronic pain patients. Our results show that such symptoms occurred in three animal models of chronic pain designed to reflect traumatic neuropathic, chemotherapy-induced neuropathic and inflammatory pain conditions. Administration of ethosuximide reduced both chronic pain and comorbidities with a marked intensity ranging from partial reduction to a complete suppression of symptoms. These results make ethosuximide, and more broadly the inhibition of T-type calcium channels, a new strategy for the management of uncontrolled chronic pain, likely to improve not only pain but also the accompanying anxiety and depression.

Dual role of T-type calcium channels in anxiety-related behavior.

T-type calcium channels are low voltage activated calcium channels that are widely expressed in various brain regions including stress-responsive regions. These channels regulate the diverse functions of the central nervous system, and modulation of these channels is shown to modulate the anxiety. Studies have described that modulation of T-type calcium channels may either aggravate or ameliorate anxiety-related behavior, suggesting the dual role of these channels. The studies employing animals with overexpression of T-type calcium channels reported their anxiety-inducing role. Therefore, the blockade of these channels using various pharmacological agents such as ethosuximide, plant extracts of linalool or rosemary, and corticotropin-releasing factor (CRF) is reported to ameliorate anxiety. On the contrary, knockout of the gene encoding these channels predisposes the rodents to anxiety-related disorders, suggesting the anxiety-attenuating role of these channels. It may be possible that these channels in normal or basal state attenuate anxiety, whereas activation of these channels in stressful condition may produce anxiety. The present review describes the dual role of T-type calcium channels in anxiety-related behavior in both preclinical and clinical studies.

A statistical analysis plan for a randomized clinical trial to evaluate the efficacy and safety of ethosuximide in patients with treatment-resistant depression.

BACKGROUND AND OBJECTIVE: A recent striking advance in the treatment of depression has been the finding of rapid antidepressant effects in over 70% of patients with treatment-resistant depression (TRD) using ketamine. However, the potential risk of addiction may limit its clinical use. Recent research revealed that blockade of N-methyl-D-aspartate receptor (NMDAR) dependent bursting activity in the lateral habenula (LHb) could mediate the fast antidepressant effects of ketamine. Further, LHb bursting plays an important role in the pathophysiology of depression that requires both NMDARs and low-voltage-sensitive T-type calcium channels (T-VSCCs). Ethosuximide, which is used to treat absence seizures, is a T-VSCCs inhibitor, may be a novel drug candidate for depression. The objective of this clinical trial is to investigate the efficacy and safety of ethosuximide in patients with TRD. DESIGN: The study is a single center, randomized, double-blind, placebo-controlled, parallel-group, two-stage clinical trial. Forty patients with TRD will be randomly assigned to Group A (treatment group) or Group B (control group). In the first stage ethosuximide or placebo will be given for 2 weeks. In the second stage, escitalopram (or another antidepressant if escitalopram has been used before) will be given for the next 4 weeks for all trial patients to ensure effective treatment. The primary outcome measure is the Montgomery-Åsberg Depression Rating Scale (MADRS) scores. Secondary outcome measures include the Quick Inventory of Depressive Symptomatology-Self Report score, Hamilton Anxiety Rating Scale scores, individual scores of MADRS, and Young Mania Rating Scale scores. All these scales are measured at baseline and at each treatment visit. Two-way repeated measures analysis of variance is used to analyze the study outcomes. DISCUSSION: A statistical analysis plan is employed to enhance the transparency of the clinical trial and reduce the risks of outcome reporting bias and data-driven results.

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells.

BACKGROUND: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. METHODS: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. RESULTS: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. CONCLUSIONS: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa.

Opening of T-type Ca2+ channels and activation of HCN channels contribute in stress adaptation in cold water immersion stress-subjected mice.

AIM: The present study was designed to investigate the possible role of T-type Ca2+ channels and HCN channels in the development of stress adaptation in cold-water immersion stress-subjected mice. MATERIAL AND METHODS: The mice were subjected to cold-water immersion stress by placing them individually in a water tank (depth = 15.5 cm; temperature = 15 ±â€¯2 °C) for 5 min. The mice were subjected to single episode of cold-water immersion stress for inducing acute stress; while for inducing stress adaptation, mice were subjected to repeated episodes of homotypic stressor (5 min) for 5 consecutive days. Animals were administered with ethosuximide (100 and 200 mg/kg, i.p.) and ivabradine (5 and 10 mg/kg, i.p.) before subjecting them to stress for five days. The stress-related behavioral alterations were assessed using the actophotometer, the hole board, the open field and the social interaction tests. The plasma corticosterone levels were quantified as a biochemical parameter of hypothalamic-pituitary-adrenal (HPA) axis activation. RESULTS: Acute stress altered the behavioral and biochemical parameters of the animals. However, repeated stress significantly restored the behavioral and biochemical alterations signifying the development of adaptation. Administration of ethosuximide and ivabradine abolished the restoration of behavioral and biochemical changes in the animals subjected to repeated stress. CONCLUSION: The ethosuximide and ivabradine mediated attenuation of stress adaptation demonstrates that the opening of T-type Ca2+ channels and activation of HCN channels are involved in inducing stress adaptation in repeated stress-subjected animals.

Cognitive impairment in the WAG/Rij rat absence model is secondary to absence seizures and depressive-like behavior.

Neuropsychiatric comorbidities are common in patients with epilepsy, remaining still an urgent unmet clinical need. Therefore, the management of epileptic disorders should not only be restricted to the achievement of seizure-freedom but must also be able to counteract its related comorbidities. Experimental animal models of epilepsy represent a valid tool not only to study epilepsy but also its associated comorbidities. The WAG/Rij rat is a well-established genetically-based model of absence epilepsy with depressive-like comorbidity, in which learning and memory impairment was also recently reported. Aim of this study was to clarify whether this cognitive decline is secondary or not to absence seizures and/or depressive-like behavior. The behavioral performance of untreated and ethosuximide-treated (300 mg/kg/day; 17 days) WAG/Rij rats at 6 and 12 months of age were assessed in several tests: forced swimming test, objects recognition test, social recognition test, Morris water maze and passive avoidance. According to our results, it seems that cognitive impairment in this strain, similarly to depressive-like behavior, is secondary to the occurrence of absence seizures, which might be necessary for the expression of cognitive impairment. Furthermore, our results suggest an age-dependent impairment of cognitive performance in WAG/Rij rats, which could be linked to the age-dependent increase of spike wave discharges. Consistently, it is possible that absence seizures, depressive-like behavior and cognitive deficit may arise independently and separately in lifetime from the same underlying network disease, as previously suggested for the behavioral features associated with other epileptic syndromes.

Spontaneous Recurrent Absence Seizure-like Events in Wild-Caught Rats.

Absence epilepsy is a heritable human neurological disorder characterized by brief nonconvulsive seizures with behavioral arrest, moderate-to-severe loss of consciousness (absence), and distinct spike-wave discharges (SWDs) in the EEG and electrocorticogram (ECoG). Genetic models of this disorder have been created by selectively inbreeding rats for absence seizure-like events with similar electrical and behavioral characteristics. However, these events are also common in outbred laboratory rats, raising concerns about whether SWD/immobility accurately reflects absence epilepsy as opposed to "normal" rodent behavior. We hypothesized that, if SWD/immobility models absence seizures, it would not exist in wild-caught rats due to the pressures of natural selection. To test this hypothesis, we compared chronic video/electrocorticogram recordings from male and female wild-caught (Brown-Norway [BN]) rats to recordings from laboratory outbred BN, outbred Long-Evans, and inbred WAG/Rij rats (i.e., a model of absence epilepsy). Wild-caught BN rats displayed absence-like SWD/immobility events that were highly similar to outbred BN rats in terms of spike-wave morphology, frequency, diurnal rhythmicity, associated immobility, and sensitivity to the anti-absence drug, ethosuximide; however, SWD bursts were less frequent and of shorter duration in wild-caught and outbred BN rats than the outbred Long-Evans and inbred WAG/Rij strains. We conclude that SWD/immobility in rats does not represent absence seizures, although they appear to have many similarities. In wild rats, SWD/immobility appears to represent normal brain activity that does not reduce survival in natural environments, a conclusion that logically extends to outbred laboratory rats and possibly to those that have been inbred to model absence epilepsy.SIGNIFICANCE STATEMENT Spike-wave discharges (SWDs), behavioral arrest, and diminished consciousness are cardinal signs of seizures in human absence epilepsy and are used to model this disorder in inbred rats. These characteristics, however, are routinely found in outbred laboratory rats, leading to debate on whether SWD/immobility is a valid model of absence seizures. The SWD/immobility events in wild-caught rats appear equivalent to those found in outbred and inbred rat strains, except for lower incidence and shorter durations. Our results indicate that the electrophysiological and behavioral characteristics of events underlying hypothetical absence epilepsy in rodent models are found in wild rats captured in their natural environment. Other criteria beyond observation of SWDs and associated immobility are required to objectively establish absence epilepsy in rat models.