Anxiolytic effect of GABAergic neurons in the anterior cingulate cortex in a rat model of chronic inflammatory pain.

Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABAAR agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABAAR antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety.

The Anticonvulsant Effect of Hydroethanolic Leaf Extract of Calotropis procera (Ait) R. Br. (Apocynaceae).

A number of currently used drugs have been obtained from medicinal plants which are a major source of drugs. These drugs are either used in their pure form or modified to a semisynthetic drug. Drug discovery through natural product research has been fruitful over the years. Traditionally, Calotropis procera is used extensively in the management of epilepsy. This study is conducted to explore the anticonvulsant effect of a hydroethanolic leaf extract of Calotropis procera (CPE) in murine models. This effect was evaluated using picrotoxin-induced convulsions, strychnine-induced convulsions, and isoniazid- and pilocarpine-induced status epilepticus in mice of both sexes. The results showed that CPE (100-300 mg/kg) exhibited an anticonvulsant effect against strychnine-induced clonic seizures by significantly reducing the duration (p = 0.0068) and frequency (p = 0.0016) of convulsions. The extract (100-300 mg/kg) caused a profound dose-dependent delay in the onset of clonic convulsions induced by picrotoxin (p < 0.0001) and tonic convulsions (p < 0.0001) in mice. The duration of convulsions was reduced significantly also for both clonic and tonic (p < 0.0001) seizures as well. CPE (100-300 mg/kg), showed a profound anticonvulsant effect and reduced mortality in the pilocarpine-induced convulsions. ED50 (~0.1007) determined demonstrated that the extract was less potent than diazepam in reducing the duration and onset of convulsions but had comparable efficacies. Flumazenil-a GABAA receptor antagonist-did not reverse the onset or duration of convulsions produced by the extract in the picrotoxin-induced seizure model. In isoniazid-induced seizure, CPE (300 mg kg1, p.o.) significantly (p < 0.001) delayed the onset of seizure in mice and prolonged latency to death in animals. Overall, the hydroethanolic leaf extract of Calotropis procera possesses anticonvulsant properties.

Effects of Tempol on Epileptic Activity in Picrotoxin-Induced Epilepsy in Rats.

INTRODUCTION: Epilepsy is a common neurological disease, although its etiology and pathophysiology are not yet fully understood. Oxidative stress plays a key role in the pathogenesis of many neurological diseases, including epilepsy, and there have been many studies reporting that antiepileptic medicines with neuroprotective and antioxidant activity inhibit free oxygen radicals. This study evaluates the effects of tempol on epileptic activity through behavioral parameters in acute picrotoxin (Ptx) models. MATERIALS AND METHODS: This experimental study was conducted on 42 adult male Wistar Albino rats weighing 450-500 g. Ptx (2.5 mg/kg) was injected i.p. as a single dose and observed for one hour to establish the acute Ptx model. Following injection, the animals were observed for 30 min in glass observation cages measuring 35 cm x 35 cm x 35 cm. RESULTS: In picrotoxin-induced epilepsy, the total number of seizures and the total duration of seizures were decreased significantly with Ptx + tempol 100 mg/kg and Ptx + Tempol 150 mg/kg. The seizure phases were reduced significantly by Ptx + tempol 150 mg/kg (P < 0.05). CONCLUSION: Tempol 100 mg/kg and tempol 150 mg/kg are found to be effective in epilepsy models caused by Ptx, with tempol 150 mg/kg found especially to be more effective.

Anticonvulsant effects of the aqueous and methanol extracts from the stem bark of Psychotria camptopus Verdc. (Rubiacaea) in rats.

ETHNOPHARMACOLOGY RELEVANCE: The decoction from the stem bark of Psychotria camptopus (Rubiaceae) is used in the Cameroonian pharmacopoeia to treat neurological pathologies including epilepsy. AIM: The present work was undertaken to study the anticonvulsant properties of the aqueous (AE) and methanol (ME) extracts from the stem bark of P. camptopus in acute models of epileptic seizures in Wistar rats. METHOD: AE and ME were obtained by decoction and maceration of the stem bark powder in water and methanol, respectively. They were tested orally at the doses of 40, 80 and 120 mg/kg, on the latency of onset and duration of epileptic seizures induced by pentylene tetrazole (PTZ, 70 mg/kg, i.p.). The kinetic effect of both extracts at 120 mg/kg was evaluated. Their effects on diazepam (50 mg/kg) induced sleep and strychnine (STR, 2.5 mg/kg, i.p.) induced seizures were determined. ME was further tested on picrotoxin (PIC, 7.5 mg/kg, i.p.) and thiosemicarbazide (TSC, 50 mg/kg, i.p.) induced seizure models. The phytochemical composition of ME was assessed using LC-MS method, as well as its acute toxicity. RESULTS: AE and ME significantly (p < 0.001) reduced the duration of seizures in both PTZ and STR models. Their maximal effect was observed at 1 h after administration, though their effect at 120 mg/kg was maintained (p < 0.05) up to 24 h post-treatment. Both extracts significantly (p < 0.01) reduced sleep duration. ME significantly (p < 0.001) increased the latency of rat death on PIC-induced convulsions. In TSC rats, ME significantly (p < 0.001) delayed the latency to the first convulsion, and decreased the duration and frequency of convulsions. ME showed no acute toxicity while its phytochemical screening revealed the presence of two flavonoids (Rutin and Butin), two triterpenoid saponins (Psycotrianoside B and Bauerenone) and four alkaloids (10-Hydroxy-antirhine, 10-hydroxy-iso-deppeaninol, Emetine and Hodkinsine). In conclusion, AE and ME from the stem bark of P. camptopus have comparable anticonvulsant properties. The effect of ME is likely due to the presence of flavonoids and alkaloid and the activation of GABA pathway. These results further justify and support the use of P. camptopus in traditional medicine for the treatment of epilepsy.

Comparison of the toxicokinetics of the convulsants picrotoxinin and tetramethylenedisulfotetramine (TETS) in mice.

Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 µM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2ß3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.

Potential Mechanisms Involved in the Anticonvulsant Effect of Methanol Extract of Pyrenancantha staudtii in Mice.

OBJECTIVE: To determine the potential effect of Pyrenancantha staudtii extract on experimentally induced seizures in mice and to evaluate the role of benzodiazepines, naloxone, and serotonin within these pathways. METHODS: Animal behaviours were evaluated using open field, hexobarbitone-induced sleep model, and anticonvulsant activity using picrotoxin-, or strychnine-, or isoniazid-induced convulsions. Attempt to understand the mode of action of the anticonvulsant activity of the plant, three notable antagonists (flumazenil, 3 mg/kg; naloxone 5 mg/kg, i.p., and cyproheptadine, 4 mg/kg, i.p) were used. RESULTS: The results revealed a significant (p < 0.05) reduction in the frequency of rearing and grooming episodes compared with the control. The extract of P. staudtii potentiates the sleeping time of hexobarbitone-induced hypnosis in a dose-related manner. P. staudtii stem bark extracts significantly (p<0.05) prolonged the onset of a seizure and attenuated the duration of seizure in a dose-dependent manner in picrotoxin- and or isoniazid-induced seizures. While, P. staudtii stem bark extract at all doses (100, 200, and 400 mg kg-1) though significantly prolonged the onset of action, but did not confer any significant changes on the duration, as well as mortality in this strychnine-induced seizure model. However, the anticonvulsant activity of the methanolic extract of P. staudtii was significantly reversed following intraperitoneal pre-treatment with flumazenil (GABA receptor antagonist) and naloxone (opioid receptor antagonist) but not cyproheptadine (5-HT2 receptor antagonist) in picrotoxin-induced convulsion. CONCLUSION: The data obtained suggest that methanol extract of P. staudtii possessed significant anticonvulsant effect, thereby confirming the traditional uses of P. staudtii in the treatment of epilepsy; mechanisms of which could involve the interaction with GABAergic and or opioidergic system.

Susceptibility of larval zebrafish to the seizurogenic activity of GABA type A receptor antagonists.

Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABAAR) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABAAR antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABAAR antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABAAR antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABAAR positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABAAR antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABAAR antagonism.

Interleukin-10 inhibits interleukin-1ß production and inflammasome activation of microglia in epileptic seizures.

BACKGROUND: Microglia are important for secreting chemical mediators of inflammatory responses in the central nervous system. Interleukin (IL)-10 and IL-1ß secreted by glial cells support neuronal functions, but the related mechanisms remain vague. Our goal was to demonstrate the efficacy of IL-10 in suppressing IL-1ß and in inflammasome activation in mice with epileptic seizure based on an epileptic-seizure mouse model. METHODS: In this study, mice in which epileptic seizures were induced by administering picrotoxin (PTX) were used as a case group, and mice injected with saline were employed as the control group. The expression of nucleic acids, cytokines, or signaling pathways was detected by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting. RESULTS: Our results demonstrated that IL-10 inhibits IL-1ß production through two distinct mechanisms: (1) Treatment with lipopolysaccharides (LPS) results in IL-10 overexpression in microglia and reduced NLRP3 inflammasome activity, thus inhibiting caspase-1-related IL-1ß maturation; (2) next, autocrine IL-10 was found to subsequently promote signal transducer and activator of transcription-3 (STAT-3), reducing amounts of pro-IL-1ß. CONCLUSIONS: Our results indicate that IL-10 is potentially effective in the treatment of inflammation encephalopathy, and suggest the potential usefulness of IL-10 for treating autoimmune or inflammatory ailments.

Human iPSC-derived neuronal models for in vitro neurotoxicity assessment.

Neurotoxicity testing still relies on ethically debated, expensive and time consuming in vivo experiments, which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds, such as picrotoxin (PTX) and endosulfan, and the neurotoxicant methylmercury (MeHg). However, the chemical-induced effects on different parameters for neuronal activity, such as mean spike rate (MSR) and mean burst rate (MBR), may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening.

Microglia response in retina and optic nerve in chronic experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common rodent model for multiple sclerosis (MS). Yet, the long-term consequences for retina and optic nerve (ON) are unknown. C57BL/6 mice were immunized with an encephalitogenic peptide (MOG35-55) and the controls received the carriers or PBS. Clinical symptoms started at day 8, peaked at day 14, and were prevalent until day 60. They correlated with infiltration and demyelination of the ON. In MOG-immunized animals more microglia cells in the ONs and retinas were detected at day 60. Additionally, retinal ganglion cell (RGC) loss was combined with an increased macroglia response. At this late stage, an increased number of microglia was associated with axonal damage in the ON and in the retina with RGC loss. Whether glial activation contributes to repair mechanisms or adversely affects the number of RGCs is currently unclear.