A new class of peptides from wasp venom: a pathway to antiepileptic/neuroprotective drugs.

The ability of venom-derived peptides to disrupt physiological processes in mammals provides an exciting source for pharmacological development. Our research group has identified a new class of neuroactive peptides from the venom of a Brazilian social wasp, Polybia occidentalis, with the potential pharmacological profile to treat epilepsies. The study was divided into five phases: Phase 1 concerned the extraction, isolation and purification of Occidentalin-1202(n) from the crude venom, followed by the synthesis of an identical analogue peptide, named Occidentalin-1202(s). In Phase 2, we described the effects of both peptides in two acute models of epilepsy-kainic acid and pentylenetetrazole-induced model of seizures-and measured estimated ED50 and therapeutic index values, electroencephalographic studies and C-fos evaluation. Phase 3 was a compilation of advanced tests performed with Occidentalin-1202(s) only, reporting histopathological features and its performance in the pilocarpine-induced status epilepticus. After the determination of the antiepileptic activity of Occidentalin-1202(s), Phase 4 consisted of evaluating its potential adverse effects, after chronic administration, on motor coordination (Rotarod) and cognitive impairment (Morris water maze) tests. Finally, in Phase 5, we proposed a mechanism of action using computational models with kainate receptors. The new peptide was able to cross the blood-brain barrier and showed potent antiseizure effects in acute (kainic acid and pentylenetetrazole) and chronic (temporal lobe epilepsy model induced by pilocarpine) models. Motor and cognitive behaviour were not adversely affected, and a potential neuroprotective effect was observed. Occidentalin-1202 can be a potent blocker of the kainate receptor, as assessed by computational analysis, preventing glutamate and kainic acid from binding to the receptor's active site. Occidentalin-1202 is a peptide with promising applicability to treat epilepsy and can be considered an interesting drug model for the development of new medicines.

Sex-specific differences in KCC2 localisation and inhibitory synaptic transmission in the rat hippocampus.

Sexual differentiation of the brain is influenced by testosterone and its metabolites during the perinatal period, when many aspects of brain development, including the maturation of GABAergic transmission, occur. Whether and how testosterone signaling during the perinatal period affects GABAergic transmission is unclear. Here, we analyzed GABAergic circuit functional markers in male, female, testosterone-treated female, and testosterone-insensitive male rats after the first postnatal week and in young adults. In the hippocampus, mRNA levels of proteins associated with GABA signaling were not significantly affected at postnatal day (P) 7 or P40. Conversely, membrane protein levels of KCC2, which are critical for determining inhibition strength, were significantly higher in females compared to males and testosterone-treated females at P7. Further, female and testosterone-insensitive male rats at P7 showed higher levels of the neurotrophin BDNF, which is a powerful regulator of neuronal function, including GABAergic transmission. Finally, spontaneous GABAergic currents in hippocampal CA1 pyramidal cells were more frequent in females and testosterone-insensitive males at P40. Overall, these results show that perinatal testosterone levels modulate GABAergic circuit function, suggesting a critical role of perinatal sex hormones in regulating network excitability in the adult hippocampus.

Cannabinoids in Audiogenic Seizures: From Neuronal Networks to Future Perspectives for Epilepsy Treatment.

Cannabinoids and Cannabis-derived compounds have been receiving especial attention in the epilepsy research scenario. Pharmacological modulation of endocannabinoid system's components, like cannabinoid type 1 receptors (CB1R) and their bindings, are associated with seizures in preclinical models. CB1R expression and functionality were altered in humans and preclinical models of seizures. Additionally, Cannabis-derived compounds, like cannabidiol (CBD), present anticonvulsant activity in humans and in a great variety of animal models. Audiogenic seizures (AS) are induced in genetically susceptible animals by high-intensity sound stimulation. Audiogenic strains, like the Genetically Epilepsy Prone Rats, Wistar Audiogenic Rats, and Krushinsky-Molodkina, are useful tools to study epilepsy. In audiogenic susceptible animals, acute acoustic stimulation induces brainstem-dependent wild running and tonic-clonic seizures. However, during the chronic protocol of AS, the audiogenic kindling (AuK), limbic and cortical structures are recruited, and the initially brainstem-dependent seizures give rise to limbic seizures. The present study reviewed the effects of pharmacological modulation of the endocannabinoid system in audiogenic seizure susceptibility and expression. The effects of Cannabis-derived compounds in audiogenic seizures were also reviewed, with especial attention to CBD. CB1R activation, as well Cannabis-derived compounds, induced anticonvulsant effects against audiogenic seizures, but the effects of cannabinoids modulation and Cannabis-derived compounds still need to be verified in chronic audiogenic seizures. The effects of cannabinoids and Cannabis-derived compounds should be further investigated not only in audiogenic seizures, but also in epilepsy related comorbidities present in audiogenic strains, like anxiety, and depression.

Loss of Brain-Derived Neurotrophic Factor Mediates Inhibition of Hippocampal Long-Term Potentiation by High-Intensity Sound.

Exposure to noise produces cognitive and emotional disorders, and recent studies have shown that auditory stimulation or deprivation affects hippocampal function. Previously, we showed that exposure to high-intensity sound (110 dB, 1 min) strongly inhibits Schaffer-CA1 long-term potentiation (LTP). Here we investigated possible mechanisms involved in this effect. We found that exposure to 110 dB sound activates c-fos expression in hippocampal CA1 and CA3 neurons. Although sound stimulation did not affect glutamatergic or GABAergic neurotransmission in CA1, it did depress the level of brain-derived neurotrophic factor (BDNF), which is involved in promoting hippocampal synaptic plasticity. Moreover, perfusion of slices with BDNF rescued LTP in animals exposed to sound stimulation, whereas BDNF did not affect LTP in sham-stimulated rats. Furthermore, LM22A4, a TrkB receptor agonist, also rescued LTP from sound-stimulated animals. Our results indicate that depression of hippocampal BDNF mediates the inhibition of LTP produced by high-intensity sound stimulation.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids.

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Ceftriaxone pretreatment confers neuroprotection in rats with acute glaucoma and reduces the score of seizures induced by pentylenotetrazole in mice.

ß-Lactam antibiotics such as ceftriaxone, are potent stimulators of the expression of l-glutamate transporter GLT-1 and may exert neuroprotective effects when chronically used in rats and mice. In this study, we used two animal models to test the neurological effect of subchronic treatment with ceftriaxone: experimental acute glaucoma in Wistar rats and induction of acute seizures with pentylenetetrazole in mice. We also assessed the performance of mice in the rotarod to calculate therapeutic indexes and exploratory activity in the open field. Our results showed that subchronic use of ceftriaxone was neuroprotective in both models, reducing injury in acute ischemia and ischemia/reperfusion in specific layers of retina and leading to a decrease in the seizure severity score. In behavioral experiments, we observed that ceftriaxone increased hyperactivity followed by a decrease in exploratory behavior in the open field, and there was no motor impairment in the rotarod test. We conclude that ceftriaxone may be useful as a tool in the development of new neuroprotective drugs targeting diseases which present a possible dysfunction in the balance of glutamatergic neurotransmission.

Increased hippocampal GABAergic inhibition after long-term high-intensity sound exposure.

Exposure to loud sounds is related to harmful mental and systemic effects. The hippocampal function can be affected to either high-intensity sound exposure or long-term sound deprivation. We previously showed that hippocampal long-term potentiation (LTP) is inhibited after ten days of daily exposure to 2 minutes of high-intensity noise (110 dB), in the hippocampi of Wistar rats. Here we investigated how the glutamatergic and GABAergic neurotransmission mediated by ionotropic receptors is affected by the same protocol of high-intensity sound exposure. We found that while the glutamatergic transmission both by AMPA/kainate and NMDA receptors in the Schaffer-CA1 synapses is unaffected by long-term exposure to high-intensity sound, the amplitude of the inhibitory GABAergic currents is potentiated, but not the frequency of both spontaneous and miniature currents. We conclude that after prolonged exposure to short periods of high-intensity sound, GABAergic transmission is potentiated in the hippocampal CA1 pyramidal neurons. This effect could be an essential factor for the reduced LTP in the hippocampi of these animals after high-intensity sound exposure. We conclude that prolonged exposure to high- intensity sound could affect hippocampal inhibitory transmission and consequently, its function.

Parawixin2, a novel non-selective GABA uptake inhibitor from Parawixia bistriata spider venom, inhibits pentylenetetrazole-induced chemical kindling in rats.

The aims of the present work were to investigate the effects of the repeated administration of Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII), a novel GABA and glycine uptake inhibitor, in rats submitted to PTZ-induced kindling. Wistar rats were randomly divided in groups (n=6-8) for different treatments. Systemic injections of PTZ were administered every 48 h in the dose of 33 mg/kg; i.p., that is sufficient to induce fully kindled seizures in saline i.c.v. treated rats in a short period of time (28 days). Treatments in two types of positive controls (diazepam - DZP and nipecotic acid - NA groups) consisted in daily systemic injections of DZP (2mg/kg; i.p.) or i.c.v. injections of NA (12 µg/µL), while in experimental groups in daily i.c.v. injections of different doses of Parawixin2 (0.15; 0.075; 0.015 µg/µL). Seizures were analyzed using the Lamberty & Klitgaard score and kindling was considered as established after at least three consecutive seizures of score 4 or 5. Cumulative seizure scores for each group were analyzed using repeated measures of ANOVA followed by Tukey test. PTZ induced 4 and 5-score seizures after 12 injections in saline treated rats, whereas daily injection of Parawixin2 inhibited the onset of seizures in a dose dependent manner. Also, the challenging administration of PTZ did not raise seizure score in animals treated with the highest dose of Parawixin2 or those treated with DZP or NA. These findings together with previous data from our laboratory show that Parawixin2 could be a useful probe to design new antiepileptic drugs.