Inhaled molecular hydrogen reduces hippocampal neuroinflammation, glial reactivity and ameliorates memory impairment during systemic inflammation.

Sepsis is associated with numerous physiological and biochemical abnormalities that result in a life-threatening condition. The involvement of the Central Nervous System (CNS) during sepsis has received considerable attention, especially the hippocampus which plays a key role in the learning and memory processes. The increased interest in this limbic region during systemic inflammation (SI) is related to the number of sepsis survivor patients who have cognitive impairments. A single injection of lipopolysaccharide (LPS)-induced systemic inflammation is the most commonly used murine endotoxemia model because it replicates several pathophysiological changes observed in severe sepsis. Molecular hydrogen (H2) has been used as an anti-inflammatory therapeutic strategy to prevent neuroinflammation. However, the mechanisms by which inhaled H2 mitigate memory loss during SI remains unknown. To understand how H2 acts in the hippocampus, the current study focused on specific mechanisms that may be involved in reducing neuroinflammation in rats during SI. We hypothesized that inhaled H2 decreases LPS-induced hippocampal pro-inflammatory cytokines surges and this effect is associated with reduced memory loss. Using different and integrative approaches, i.e., from hippocampal cells electrophysiology to animal behavior, we report that inhaled H2 decreased LPS-induced peripheral and hippocampal inflammation, decreased microglial and astrocytic activation, lessen memory loss without affecting long-term potentiation (LTP). To our knowledge, this is the first evidence showing that inhaled H2 reduces hippocampal microglial and glial cells inflammation, which may be associated with a reduced memory impairment induced by SI.

Modeling Hippocampal CA1 Gabaergic Synapses of Audiogenic Rats.

Wistar Audiogenic Rats (WARs) are genetically susceptible to sound-induced seizures that start in the brainstem and, in response to repetitive stimulation, spread to limbic areas, such as hippocampus. Analysis of the distribution of interevent intervals of GABAergic inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells showed a monoexponential trend in Wistar rats, suggestive of a homogeneous population of synapses, but a biexponential trend in WARs. Based on this, we hypothesize that there are two populations of GABAergic synaptic release sites in CA1 pyramidal neurons from WARs. To address this hypothesis, we used a well-established neuronal computational model of a CA1 pyramidal neuron previously developed to replicate physiological properties of these cells. Our simulations replicated the biexponential trend only when we decreased the release frequency of synaptic currents by a factor of six in at least 40% of distal synapses. Our results suggest that almost half of the GABAergic synapses of WARs have a drastically reduced spontaneous release frequency. The computational model was able to reproduce the temporal dynamics of GABAergic inhibition that could underlie susceptibility to the spread of seizures.

Inflammatory markers in the hippocampus after audiogenic kindling.

Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the audiogenic kindling protocol with a loud broad-band noise. We measured pro and anti-inflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure severity, we did not observe differences in the pro inflammatory cytokines IL-1ß, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate.

Alterations in brainstem auditory processing, the acoustic startle response and sensorimotor gating of startle in Wistar audiogenic rats (WAR), an animal model of reflex epilepsies.

While acute audiogenic seizures in response to acoustic stimulus appear as an alteration in sensory-motor processing in the brainstem, the repetition of the stimulus leads to the spread of epileptic activity to limbic structures. Here, we investigated whether animals of the Wistar Audiogenic Rat (WAR) strain, genetically selected by inbreeding for seizure susceptibility, would have alterations in their auditory response, assessed by the auditory brainstem responses (ABR) and sensory-motor gating, measured as pre-pulse inhibition (PPI), which could be related to their audiogenic seizures susceptibility or severity. We did not find differences between the amplitudes and latencies of ABR waves in response to clicks for WARs when compared to Wistars. Auditory gain and symmetry between ears were also similar. However, hearing thresholds in response to some tones were lower and amplitudes of wave II were larger in WARs. WARs had smaller acoustic startle reflex amplitudes and the percentages of startle inhibited by an acoustic prepulse were higher for WARs than for Wistars. However, no correlation was found between these alterations and brainstem-dependent seizure severity or limbic seizure frequency during audiogenic kindling. Our data show that while WARs present moderate alterations in primary auditory processing, the sensory motor gating measured in startle/PPI tests appears to be more drastically altered. The observed changes might be correlated with audiogenic seizure susceptibility but not seizures severity.

Energy Metabolism and Redox State in Brains of Wistar Audiogenic Rats, a Genetic Model of Epilepsy.

The Wistar Audiogenic Rat (WAR) strain is a genetic model of epilepsy, specifically brainstem-dependent tonic-clonic seizures, triggered by acute auditory stimulation. Chronic audiogenic seizures (audiogenic kindling) mimic temporal lobe epilepsy, with significant participation of the hippocampus, amygdala, and cortex. The objective of the present study was to characterize the mitochondrial energy metabolism in hippocampus and cortex of WAR and verify its relationship with seizure severity. Hippocampus of WAR naïve (no seizures) presented higher oxygen consumption in respiratory states related to the maximum capacities of phosphorylation and electron transfer system, elevated mitochondrial density, lower GSH/GSSG and catalase activity, and higher protein carbonyl and lactate contents, compared with their Wistar counterparts. Audiogenic kindling had no adding functional effect in WAR, but in Wistar, it induced the same alterations observed in the audiogenic strain. In the cortex, WAR naïve presented elevated mitochondrial density, lower GSH/GSSG and catalase activity, and higher protein carbonyl levels. Chronic acoustic stimulation in Wistar induced the same alterations in cortex and hippocampus. Mainly in the hippocampus, WAR naïve presented elevated mRNA expression of glucose, lactate and excitatory amino acids transporters, several glycolytic enzymes, lactate dehydrogenase, and Na+/K+ ATPase in neurons and in astrocytes. In vivo treatment with mitochondrial uncoupler 2,4-dinitrophenol (DNP) or N-acetylcysteine (NAC) in WAR had no effect on mitochondrial metabolism, but lowered oxidative stress. Unlike DNP, NAC downregulated all enzyme genes involved in glucose and lactate uptake, and metabolism in neurons and astrocytes. Additionally, it was able to reduce brainstem seizure severity in WAR. In conclusion, in WAR naïve animals, both cerebral cortex and hippocampus display elevated mitochondrial density and/or activity associated with oxidative damage, glucose and lactate metabolism pathways upregulation, and increased Na+/K+ ATPase mRNA expression. Only in vivo treatment with NAC was able to reduce seizure severity of kindled WARs, possibly via down regulation of glucose/lactate metabolism. Taken together, our results are a clear contribution to the field of mitochondrial metabolism associated to epileptic seizures.

Assessment of neuropharmacological potential of low molecular weight components extracted from Rhinella schneideri toad poison.

BACKGROUND: Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. METHODS: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. RESULTS: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 µg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. CONCLUSIONS: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.

Assessment of neuropharmacological potential of low molecular weight components extracted from Rhinella schneideri toad poison

Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. Methods: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. Results: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 μg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. Conclusions: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.(AU)

Parawixin2 Protects Hippocampal Cells in Experimental Temporal Lobe Epilepsy.

Epilepsy is considered as one of the major disabling neuropathologies. Almost one third of adult patients with temporal lobe epilepsy (TLE) do not respond to current antiepileptic drugs (AEDs). Additionally, most AEDs do not have neuroprotective effects against the inherent neurodegenerative process underlying the hippocampal sclerosis on TLE. Dysfunctions in the GABAergic neurotransmission may contribute not only to the onset of epileptic activity but also constitute an important system for therapeutic approaches. Therefore, molecules that enhance GABA inhibitory effects could open novel avenues for the understanding of epileptic plasticity and for drug development. Parawixin2, a compound isolated from Parawixia bistriata spider venom, inhibits both GABA and glycine uptake and has an anticonvulsant effect against a wide range of chemoconvulsants. The neuroprotective potential of Parawixin2 was analyzed in a model of TLE induced by a long-lasting Status Epilepticus (SE), and its efficiency was compared to well-known neuroprotective drugs, such as riluzole and nipecotic acid. Neuroprotection was assessed through histological markers for cell density (Nissl), astrocytic reactivity (GFAP) and cell death labeling (TUNEL), which were performed 24 h and 72 h after SE. Parawixin2 treatment resulted in neuroprotective effects in a dose dependent manner at 24 h and 72 h after SE, as well as reduced reactive astrocytes and apoptotic cell death. Based on these findings, Parawixin2 has a great potential to be used as a tool for neuroscience research and as a probe to the development of novel GABAergic neuroprotective agents.

Intrinsic and synaptic properties of hippocampal CA1 pyramidal neurons of the Wistar Audiogenic Rat (WAR) strain, a genetic model of epilepsy.

Despite the many studies focusing on epilepsy, a lot of the basic mechanisms underlying seizure susceptibility are mainly unclear. Here, we studied cellular electrical excitability, as well as excitatory and inhibitory synaptic neurotransmission of CA1 pyramidal neurons from the dorsal hippocampus of a genetic model of epilepsy, the Wistar Audiogenic Rat (WARs) in which limbic seizures appear after repeated audiogenic stimulation. We examined intrinsic properties of neurons, as well as EPSCs evoked by Schaffer-collateral stimulation in slices from WARs and Wistar parental strain. We also analyzed spontaneous IPSCs and quantal miniature inhibitory events. Our data show that even in the absence of previous seizures, GABAergic neurotransmission is reduced in the dorsal hippocampus of WARs. We observed a decrease in the frequency of IPSCs and mIPSCs. Moreover, mIPSCs of WARs had faster rise times, indicating that they probably arise from more proximal synapses. Finally, intrinsic membrane properties, firing and excitatory neurotransmission mediated by both NMDA and non-NMDA receptors are similar to the parental strain. Since GABAergic inhibition towards CA1 pyramidal neurons is reduced in WARs, the inhibitory network could be ineffective to prevent the seizure-dependent spread of hyperexcitation. These functional changes could make these animals more susceptible to the limbic seizures observed during the audiogenic kindling.

Long-term high-intensity sound stimulation inhibits h current (Ih ) in CA1 pyramidal neurons.

Afferent neurotransmission to hippocampal pyramidal cells can lead to long-term changes to their intrinsic membrane properties and affect many ion currents. One of the most plastic neuronal currents is the hyperpolarization-activated cationic current (Ih ), which changes in CA1 pyramidal cells in response to many types of physiological and pathological processes, including auditory stimulation. Recently, we demonstrated that long-term potentiation (LTP) in rat hippocampal Schaffer-CA1 synapses is depressed by high-intensity sound stimulation. Here, we investigated whether a long-term high-intensity sound stimulation could affect intrinsic membrane properties of rat CA1 pyramidal neurons. Our results showed that Ih is depressed by long-term high-intensity sound exposure (1 min of 110 dB sound, applied two times per day for 10 days). This resulted in a decreased resting membrane potential, increased membrane input resistance and time constant, and decreased action potential threshold. In addition, CA1 pyramidal neurons from sound-exposed animals fired more action potentials than neurons from control animals; however, this effect was not caused by a decreased Ih . On the other hand, a single episode (1 min) of 110 dB sound stimulation which also inhibits hippocampal LTP did not affect Ih and firing in pyramidal neurons, suggesting that effects on Ih are long-term responses to high-intensity sound exposure. Our results show that prolonged exposure to high-intensity sound affects intrinsic membrane properties of hippocampal pyramidal neurons, mainly by decreasing the amplitude of Ih .

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices.

CONTEXT: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals. OBJECTIVE: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats. MATERIALS AND METHODS: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 µM). RESULTS: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 µM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes. DISCUSSION AND CONCLUSION: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.

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.

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.

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.

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.

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.

Glutamate binding is altered in hippocampus and cortex of Wistar rats after pilocarpine-induced Status Epilepticus.

Several evidences have pointed to biochemical alterations in some brain structures after experimental Status Epilepticus (SE). Thus, the effects of pilocarpine-induced SE on the glutamate binding in the hippocampus and cortex of Wistar rats were evaluated. Groups of animals were submitted to a 3h SE induced by intrahippocampal microinjection of pilocarpine, which was interrupted by the administration of sodium thiopental. Two weeks later the animals were sacrificed and had their cerebral cortices and hippocampi removed in order to perform the binding experiments. The results show that the pilocarpine-induced SE provoked an increase in 2.5-fold in the B(max) values for glutamate binding in the cortex, but not in the hippocampus. Moreover, we observed a 4-fold increase for the Kd values in the hippocampus and a 2-fold increase in the cortex. These findings might indicate that the epileptogenesis involves alterations in the glutamate receptors that are not restricted to the limbic system. Moreover, changes in these receptors are not exclusively of number, but rather involve the affinity for their ligands.

Neurotoxins from invertebrates as anticonvulsants: from basic research to therapeutic application.

Invertebrate venoms have attracted considerable interest as a potential source of bioactive substances, especially neurotoxins. These molecules have proved to be extremely useful tools for the understanding of synaptic transmission events, and they have contributed to the design of novel drugs for the treatment of neurological disorders and pain. In this context, as epilepsy involves neuronal substrates, which are sites of action of many neurotoxins; venoms may be particularly useful for antiepileptic drug (AED) research. Epilepsy is a chronic disease whose treatment consists of controlling seizures with antiepileptics that very often induce strong undesirable side effects that may limit treatment. Here, we review the vast, but yet unexplored, world of neurotoxins from invertebrates used as probes in pharmacological screening for novel and less toxic antiepileptics. We briefly review (1) the molecular basis of epilepsy, as well as the sites of action of commonly used anticonvulsants (we bring a comprehensive review of the elements from invertebrate venoms which are mostly studied in neuroscience research and may be useful for drug development); (2) peptides from conus snails; (3) peptides and polyamine toxins from spiders and wasps; and (4) peptides from scorpions.

Neuropharmacological profile of FrPbAII, purified from the venom of the social spider Parawixia bistriata (Araneae, Araneidae), in Wistar rats.

The aims of the present study were to investigate the anticonvulsant activity and behavioral toxicity of FrPbAII using freely moving Wistar rats. Moreover, the effectiveness of this compound against chemical convulsants was compared to that of the inhibitor of the GABAergic uptake, nipecotic acid. Our results show that FrPbAII was effective against seizures induced by the i.c.v. injection of pilocarpine (ED(50) = 0.05 microg/animal), picrotoxin (ED(50) = 0.02 microg/animal), kainic acid (ED(50) = 0.2 microg/animal) and the systemic administration of PTZ (ED(50) = 0.03 microg/animal). The anticonvulsant effect of FrPbAII differed from that of nipecotic acid in potency, as the doses needed to block the seizures were more than 10 folds lower. Toxicity assays revealed that in the rotarod, the toxic dose of the FrPbAII is 1.33 microg/animal, and the therapeutic indexes were calculated for each convulsant. Furthermore, the spontaneous locomotor activity of treated animals was not altered when compared to control animals but differed from the animals treated with nipecotic acid. Still, FrPbAII did not induce changes in any of the behavioral parameters analyzed. Finally, when tested for cognitive impairments in the Morris water maze, the i.c.v. injection of FrPbAII did not alter escape latencies of treated animals. These findings indicate that the novel GABA uptake inhibitor is a potent anticonvulsant with mild side-effects when administered to Wistar rats.

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.