Alexandra Olimpio Siqueira Cunha

Inhibition of acute nociceptive responses in rats after i.c.v. injection of Thr6-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis

In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti‐nociceptive effects were characterized in well‐established pain induction models.

Neuroprotective Effects of Diazepam, Carbamazepine, Phenytoin and Ketamine after Pilocarpine-induced Status Epilepticus

Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/microl, 1 microl) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.

Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae).

Neural mechanisms underlying the onset and maintenance of epileptic seizures involve alterations in inhibitory and/or excitatory neurotransmitter pathways. Thus, the prospecting of novel molecules from natural products that target both inhibition and excitation systems has deserved interest in the rational design of new anticonvulsants. We isolated the alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine from the flowers of Erythrina mulungu and evaluated the action of these compounds against chemically induced seizures in rats. Our results showed that the administration of different doses of (+)-erythravine inhibited seizures evoked by bicuculline, pentylenetetrazole, and kainic acid at maximum of 80, 100, and 100%, respectively, whereas different doses of (+)-11-α-hydroxy-erythravine inhibited seizures at a maximum of 100% when induced by bicuculline, NMDA, and kainic acid, and, to a lesser extent, PTZ (60%). The analysis of mean latency to seizure onset of nonprotected animals, for specific doses of alkaloids, showed that (+)-erythravine increased latencies to seizures induced by bicuculline. Although (+)-erythravine exhibited very weak anticonvulsant action against seizures induced by NMDA, this alkaloid increased the latency in this assay. The increase in latency to onset of seizures promoted by (+)-11-α-hydroxy-erythravine reached a maximum of threefold in the bicuculline test. All animals were protected against death when treated with different doses of (+)-11-α-hydroxy-erythravine in the tests using the four chemical convulsants. Identical results were obtained when using (+)-erythravine in the tests of bicuculline, NMDA, and PTZ, and, to a lesser extent, kainic acid. Therefore, these data validate the anticonvulsant properties of the tested alkaloids, which is of relevance in consideration of the ethnopharmacological/biotechnological potential of E. mulungu.

Effects of microinjections of neurotoxin AvTx8, isolated from the social wasp Agelaia vicina (Hymenoptera, Vespidae) venom, on GABAergic nigrotectal pathways.

Several investigations have provided information that defensive behaviors evoked by stimulation of deep layers of the superior colliculus (dlSC) are subjected to inhibitory nigral modulation. This inhibition is made mainly through GABAergic neurons from substantia nigra, pars reticulata (SNpr), that sends outputs toward neural networks of the deep layers of the superior colliculus and dorsal periaqueductal gray matter involved with the organization of fear-like responses. In this work, we compared the effects of two GABAergic agonists, muscimol and baclofen, with the effect of neurotoxin AvTx8 (1567 Da), isolated from the venom of the social wasp Agelaia vicina, microinjected into SNpr of Rattus norvegicus (Wistar rats) prior to dlSC saline or bicuculline microinjections, considering that wasp venom has some influence on the uptake of GABA and/or glutamate neurotransmitters. GABA(A) receptor blockade in the dlSC evoked a vigorous escape behavior, expressed by rapid running, jumps and turns, as compared to control. These defensive reactions were maximized after the intranigral GABA(A) agonism with muscimol, but not after in situ GABA(B) agonism. Nigral microinjection of AvTx8 induced similar effects to those of baclofen, decreasing the intensity of behavioral defensive reactions caused by GABA(A) receptor blockade in the dorsal mesencephalon. These findings suggest that AvTx8 has some effects on GABAergic neurotransmission, increasing the activity of the inhibitory nigro-collicular pathways, causing an anti-panic (antiaversive) effect. Therefore, our work suggests AvTx8 as a novel pharmacological tool to study differences between the two types of GABAergic receptors and excitatory amino acid-mediated mechanisms in the brain and brainstem networks.

Anticonvulsant and anxiolytic activity of frPbaII, a novel GABA uptake inhibitor isolated from the venom of the social spider Parawixia bistriata (Araneidae: Araneae)

This study was aimed at determining the effects of FrPbAII (174 Da), a novel isolated component from Parawixia bistriata spider venom, in the CNS of Wistar rats. Considering that FrPbAII inhibits the high affinity GABAergic uptake in a dose-dependent manner, its anxiolytic and anticonvulsant effects were analyzed in well-established animal models. Injection of FrPbAII in the rat hippocampus induced a marked anxiolytic effect, increasing the occupancy in the open arms of the elevated plus maze (EC(50)=0.09 microg/microl) and increasing the time spent in the lit area of the light-dark apparatus (EC(50)=0.03 microg/microl). Anxiolytic effects were also observed considering the number of entries in the open arms of the EPM and in the lit compartment of the light-dark box. Interestingly, when microinjected bilaterally in the SNPr of freely moving rats, FrPbAII (0.6 microg/microl) effectively prevented seizures induced by the unilateral GABAergic blockade of Area tempestas (bicuculline, 0.75 microg/microl). This anticonvulsant effect was similar to that evoked by muscimol (0.1 microg/microl) and baclofen (0.6 microg/microl), but differed from that of the specific GAT1 inhibitor, nipecotic acid (0.7 microg/microl). This difference could be accounted either for the parallel action of FrPbAII over glycinergic transporters or to an inspecific activity on GABAergic transporters. Data from the present investigation might be pointing to a novel compound with interesting and yet unexplored pharmacological potential.

Erysothrine, an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth: Evaluating its anticonvulsant and anxiolytic potential

In this study, we isolated the alkaloid erysothrine from the hydroalcoholic extract of flowers from E. mulungu and screened for its anticonvulsant and anxiolytic actions based on neuroethological and neurochemical experiments. Our results showed that the administration of erysothrine inhibited seizures evoked by bicuculline, PTZ, NMDA and most remarkably, kainic acid. Also, erysothrine induced an increase in the number of entries but not in the time spent in the open arms of the EPM. However, we did not notice any alterations in the light-dark choice or in the open-field tests. In preliminary neurochemistry tests, we also showed that erysothrine (0.001-10 μg/mL) did not alter the GABA or glutamate synaptossomal uptake and binding. Altogether, our results describe an alkaloid with anticonvulsant activity and mild anxiolytic activity that might be considered well tolerated as it does not alter the general behavior of the animals in the used doses.

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.

INHIBITION OF LONG-TERM POTENTIATION IN THE SCHAFFER-CA1 PATHWAY BY REPETITIVE HIGH-INTENSITY SOUND STIMULATION

High-intensity sound can induce seizures in susceptible animals. After repeated acoustic stimuli changes in behavioural seizure repertoire and epileptic EEG activity might be seen in recruited limbic and forebrain structures, a phenomenon known as audiogenic kindling. It is postulated that audiogenic kindling can produce synaptic plasticity events leading to the spread of epileptogenic activity to the limbic system. In order to test this hypothesis, we investigated if long-term potentiation (LTP) of hippocampal Schaffer-CA1 synapses and spatial navigation memory are altered by a repeated high-intensity sound stimulation (HISS) protocol, consisting of one-minute 120 dB broadband noise applied twice a day for 10 days, in normal Wistar rats and in audiogenic seizure-prone rats (Wistar Audiogenic Rats - WARs). After HISS all WARs exhibited midbrain seizures and 50% of these animals developed limbic recruitment, while only 26% of Wistar rats presented midbrain seizures and none of them had limbic recruitment. In naïve animals, LTP in hippocampal CA1 neurons was induced by 50- or 100-Hz high-frequency stimulation of Schaffer fibres in slices from both Wistar and WAR animals similarly. Surprisingly, HISS suppressed LTP in CA1 neurons in slices from Wistar rats that did not present any seizure, and inhibited LTP in slices from Wistar rats with only midbrain seizures. However HISS had no effect on LTP in CA1 neurons from slices of WARs. Interestingly HISS did not alter spatial navigation and memory in both strains. These findings show that repeated high-intensity sound stimulation prevent LTP of Schaffer-CA1 synapses from Wistar rats, without affecting spatial memory. This effect was not seen in hippocampi from audiogenic seizure-prone WARs. In WARs the link between auditory stimulation and hippocampal LTP seems to be disrupted which could be relevant for the susceptibility to seizures in this strain.

Anxiety: A Systematic Review of Neurobiology, Traditional Pharmaceuticals and Novel Alternatives from Medicinal Plants

Pathologic anxiety is a disproportional reaction of individuals to anticipation or misinterpretation of a potential danger, which affects individual social and personal life. Despite the advances already accomplished, further studies are still necessary in order to understand the mechanisms involved in anxiety. These may provide more effective and safer treatments to aid in the control of anxiety and improve patient quality of life. In this work, we review the current issue about anxiety disorders, covering general aspects such as basic epidemiology and classification, an overview of the pharmacological treatments employed and the current search for natural anxiolytics. Also, a compilation of data investigating the neurobiology that underlies anxiety disorders and a brief discussion evolving the most usual animal experimental models to study anxiety is presented.

Neurobiological activity of Parawixin 10, a novel anticonvulsant compound isolated from Parawixia bistriata spider venom (Araneidae: Araneae).

The neurobiological activity of Parawixin 10, isolated from Parawixia bistriata spider venom, was investigated. Cannulas were implanted in the lateral ventricles of Wistar rats (200-250 g, n=6-8 per group) to perform anticonvulsant and behavioral assays, and synaptosomes from cerebral cortices of male Wistar rats were used for neurochemical studies. The results indicate that pretreatment with Parawixin 10 prevents the onset of seizures induced with kainic acid, N-methyl-D-aspartate, and pentylenetetrazole in a dose-response manner. Lower doses of Parawixin 10 significantly increased the latency to onset of kainic acid-, pentylenetetrazole-, and N-methyl-D-aspartate-induced seizures. There were maximum increases of 79% in L-[(3)H]glutamine uptake and 40% in [(3)H]glycine uptake; [(3)H]GABA uptake did not change. The findings demonstrate that this novel compound from P. bistriata venom exerts a pharmacological effect on the glutamatergic and glycinergic systems.