Fipronil affects cockroach behavior and olfactory memory.

Fipronil (Fpl), an insecticide belonging to the class of phenylpyrazoles, is associated with the widespread mortality of pollinator insects worldwide. Based on studies carried out on residual concentrations of Fpl commonly found in the environment, in this study, we evaluated the sublethal effects of Fpl on behavior and other neurophysiological parameters using the cockroach Nauphoeta cinerea as a biological model. Sublethal doses of Fpl (0.1-0.001 µg g-1) increased the time spent grooming and caused dose-dependent inhibition of exploratory activity, partial neuromuscular blockade in vivo and irreversible negative cardiac chronotropism. Fpl also disrupted learning and olfactory memory formation at all doses tested. These results provide the first evidence that short-term exposure to sublethal concentrations of Fpl can significantly disrupt insect behavior and physiology, including olfactory memory. These findings have implications for current pesticide risk assessment and could be potentially useful in establishing a correlation with pesticide effects in other insects, such as honey bees.

Entomotoxic Activity of Prasiola crispa (Antarctic Algae) in Nauphoeta cinerea Cockroaches: Identification of Main Steroidal Compounds.

Prasiola crispa is a macroscopic green algae found in abundance in Antarctica ice free areas. Prasiola crispan-hexaneextract (HPC) induced insecticidal activity in Nauphoeta cinerea cockroaches after 24 h of exposure. The chemical analysis of HPC revealed the presence of the followingphytosterols: ß-sitosterol, campesterol and stigmasterol. The incubation of cockroach semi-isolated heart preparations with HPC caused a significant negative chronotropic activity in the heartbeats. HPC affected the insect neuromuscular function by inducing a complete inhibition of the cockroach leg-muscle twitch tension. When the isolated phytosterols were injected at in vivo cockroach neuromuscular preparations, there was a progressive inhibition of muscle twitches on the following order of potency: ß-sitosterol > campesterol > stigmasterol. HPC also provoked significant behavioral alterations, characterized by the increase or decrease of cockroach grooming activity, depending on the dose assayed. Altogether, the results presented here corroborate the insecticide potential of Prasiola crispa Antarctic algae. They also revealed the presence of phytosterols and the involvement of these steroidal compounds in the entomotoxic activity of the algae, potentially by modulating octopaminergic-cholinergic pathways. Further phytochemical-combined bioguided analysis of the HPC will unveil novel bioactive compounds that might be an accessory to the insecticide activity of the algae.

Modulation of dopaminergic neurotransmission induced by sublethal doses of the organophosphate trichlorfon in cockroaches.

Organophosphate (OP) insecticides have been used indiscriminately, based on their high dissipation rates and low residual levels in the environment. Despite the toxicity of OPs to beneficial insects is principally devoted to the acetylcholinesterase (AChE) inhibition, the physiological mechanisms underlying this activity remain poorly understood. Here we showed the pharmacological pathways that might be involved in severe alterations in the insect locomotion and grooming behaviors following sublethal administration of the OP Trichlorfon (Tn) (0.25, 0.5 and 1 µM) in Phoetalia pallida. Tn inhibited the acetylcholinesterase activity (46±6, 38±3 and 24±6 nmol NADPH/min/mg protein, n=3, p<0.05), respectively. Tn (1 µM) also increased the walking maintenance of animals (46±5 s; n=27; p<0.05). Tn caused a high increase in the time spent for this behavior (344±18 s/30 min, 388±18 s/30 min and 228±12 s/30 min, n=29-30, p<0.05, respectively). The previous treatment of the animals with different cholinergic modulators showed that pirenzepine>atropine>oxotremorine>d-tubocurarine>tropicamide>methoctramine induced a decrease on Tn (0.5 µM)-induced grooming increase, respectively in order of potency. Metoclopramide (0.4 µM), a DA-D2 selective inhibitor decreased the Tn-induced grooming activity (158±12 s/30 min; n=29; p<0.05). Nevertheless, the effect of the selective DA-D1 receptor blocker SCH 23390 (1.85 µM) on the Tn (0.5 µM)-induced grooming increase was significative and more intense than that of metoclopramide (54±6 s/30 min; n=30; p<0.05). Taken together the results suggest that a cross-talking between cholinergic M1/M3 and dopaminergic D1 receptors at the insect nervous system may play a role in the OP-mediated behavioral alterations.

Beneficial effect of crotamine in the treatment of myasthenic rats.

INTRODUCTION: Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats. METHODS: We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program. RESULTS: During the in-situ electromyographic recording, neostigmine (17 µg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine. CONCLUSIONS: These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.

Dopaminergic modulation by quercetin: In silico and in vivo evidence using Caenorhabditis elegans as a model.

Quercetin is a flavonol widely distributed in plants and has various described biological functions. Several studies have reported on its ability to restore neuronal function in a wide variety of disease models, including animal models of neurodegenerative disorders such as Parkinson's disease. Quercetin per se can act as a neuroprotector/neuromodulator, especially in diseases related to impaired dopaminergic neurotransmission. However, little is known about how quercetin interacts with the dopaminergic machinery. Here we employed the nematode Caenorhabditis elegans to study this putative interaction. After observing behavioral modulation, mutant analysis and gene expression in C. elegans upon exposure to quercetin at a concentration that does not protect against MPTP, we constructed a homology-based dopamine transporter protein model to conduct a docking study. This led to suggestive evidence on how quercetin may act as a dopaminergic modulator by interacting with C. elegans' dopamine transporter and alter the nematode's exploratory behavior. Consistent with this model, quercetin controls C. elegans behavior in a way dependent on the presence of both the dopamine transporter (dat-1), which is up-regulated upon quercetin exposure, and the dopamine receptor 2 (dop-2), which appears to be mandatory for dat-1 up-regulation. Our data propose an interaction with the dopaminergic machinery that may help to establish the effects of quercetin as a neuromodulator.

In vitro and in vivo safety evaluation of Dipteryx alata Vogel extract.

BACKGROUND: Dipteryx alata Vogel popularly known as "baru" is an important commercial leguminous tree species from the Brazilian Cerrado, which possess medicinal properties, besides its fruits consumption by animals and humans. The use of the "naturally occurring plants" as herbal remedies and foods mainly from leaves, seeds, flowers and roots of plants or extracts require precautions before ensuring these are safe and efficacious. The objective of this study was to evaluate the safety of D. alata barks extract. METHODS: Vegetal drugs of D. alata barks were submitted to quality control assays and further to the safety assays under 1) in vitro parameter by Salmonella (Ames) mutagenicity, and 2) in vivo parameter on the pregnancy of rats. RESULTS: The extract was non-mutagenic to any of the assessed strains TA97a, TA98, TA100 and TA102 even after metabolic activation (+S9). All in vivo parameters (reproductive ability evaluation, physical development of rat offsprings, and neurobehavioral development assays) showed no changes related to control group. CONCLUSION: D. alata barks extract is neither mutagenic by the Ames test nor toxic in the pregnancy of rats, with no physical-neurobehavioral consequences on the rat offsprings development.

Modulation of the cholinergic pathway induced by skin secretion of Phyllomedusa iheringii Boulenger, 1885 in a vertebrate model.

Amphibians represent one of the main natural sources of bioactive molecules of interest to biotechnological research. The Phyllomedusidae family has several species occurring in Brazil and some studies demonstrate the biological potential of poisons of these species, however many still need to be characterized. Phyllomedusa iheringii is endemic in Brazilian and Uruguayan Pampa Biome and has little data in the literature regarding the action of its poison on experimental organisms. Thus, the present work evaluates the biological activity of P. iheringii secretion on the central and peripheral nervous system of a vertebrate model. The skin secretions of P. iheringii (SSPI) were collected through manual compression and electrical stimulation of the animal's bodies. The resulting content was used in neurobiological tests searching for modulatory effects on the main pathways involved in the neurotoxicity mechanism of vertebrates. SSPI affected the contraction force of the chick biventer cervicis muscle (Gallus gallus domesticus) at some concentrations used (5, 10, and 12 µg/mL). In slices from the cerebral cortex of G. gallus domesticus an increase in cell viability was observed after treatment with SSPI (10 µg/mL) and a neuroprotective effect when treated simultaneously with hydrogen peroxide (H2O2), Neostigmine (NEO) and Trichlorfon (TRI). The cholinergic pathway is possibly the main pathway modulated by SSPI since assays with the cerebral cortex and biventer cervicis muscle demonstrated the increased activity of the enzyme acetylcholinesterase (AChE) (SSPI 10 µg/mL and 12 µg/mL, respectively). SSPI (10 µg/mL) also prevented the modulation of NEO and TRI, two recognized anticholinesterase agents, in AChE activity in slices of the cerebral cortex. Therefore, our results have demonstrated the unpublished biotechnological potential of P. iheringii over the vertebrate model and its modulation on the nervous system, with apparent action on the cholinergic pathway.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system.

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

In vitro antiophidian properties of Dipteryx alata Vogel bark extracts.

Extracts from Dipteryx alata bark obtained with different solvents (hexane, dichloromethane, ethyl acetate and methanol) were mixed in vitro with Bothrops jararacussu (Bjssu, 40 µg/mL) and Crotalus durissus terrificus (Cdt, 15 µg/mL) snake venoms, and applied to a mouse phrenic nerve-diaphragm preparation to evaluate the possible neutralization of venom effects. Cdt venom neurotoxic effect was not inhibited by any of the extracts, while the neurotoxic and myotoxic actions of Bjssu venom were decreased by the methanolic extract. This inhibition appears to be augmented by tannins. Dichloromethane bark extract inhibited ~40% of Bjssu venom effects and delayed blockade induced by Cdt. The methodology used to determine which extract was active allows inferring that: (i) phenolic acids and flavonoids contained in the methanolic extract plus tannins were responsible mostly for neutralization of Bjssu effects; (ii) terpenoids from the dichloromethane extract may participate in the anti-Cdt and anti-Bjssu venom effects; (iii) a given extract could not inhibit venoms from different species even if those belong to the same family, so it is improper to generalize a certain plant as antiophidian; (iv) different polarity extracts do not present the same inhibitory capability, thus demonstrating the need for characterizing both venom pharmacology and the phytochemistry of medicinal plant compounds.

Entomotoxicity of jaburetox: revisiting the neurotoxic mechanisms in insects.

Ureases are metalloenzymes that hydrolyze urea to ammonia and carbamate. The main urease isoforms present in the seeds of Canavalia ensiformis (jack bean urease - JBU and canatoxin) exert a variety of biological activities. The insecticidal activity of JBU is mediated, at least in part, by jaburetox (Jbtx), a recombinant peptide derived from the JBU amino acid sequence. In this article, we review the neurotoxicity of Jbtx in insects. The insecticidal activity of Jbtx has been investigated in a variety of insect orders and species, including Blattodea (the cockroaches Blatella germânica, Nauphoeta cinerea, Periplaneta americana e Phoetalia pallida), Bruchidae (Callosobruchus maculatus - cowpea weevil), Diptera (Aedes aegypti - mosquito), Hemiptera (Dysdercus peruvianus - cotton stainer bug; Oncopeltus fasciatus - large milkweed bug, and the kissing bugs Rhodnius prolixus and Triatoma infestans), Lepidoptera (Spodoptera frugiperda - fall army worm) and Orthoptera (Locusta migratoria - locust). In N. cinerea, the injection of Jbtx induces marked alteration of locomotor and grooming behavior, whereas in T. infestans Jbtx causes leg paralysis, an extension of the proboscis and abnormal antennal movements. Electromyographical analysis showed that Jbtx causes complete neuromuscular blockade in P. pallida. The same treatment in N. cinerea and L. migratoria causes a decrease in the action potential firing rate. Jbtx forms membrane pore-channels compatible with cations in bilipid membranes. A study using B. germanica voltage-gated sodium (Nav1.1) channels that were heterologously expressed in Xenopus laevis oocytes correlated the entomotoxicity of Jbtx with the activation of these channels. Taken together, these findings demonstrate the potential of this peptide as a natural pesticide.

The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 µM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin's action on acetylcholine release at mammalian neuromuscular junctions.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

The neuromuscular activity of paradoxin: a presynaptic neurotoxin from the venom of the inland taipan (Oxyuranus microlepidotus).

The inland taipan is the world's most venomous snake. However, little is known about the neuromuscular activity of the venom or paradoxin (PDX), a presynaptic neurotoxin from the venom. Venom (10microg/ml) and PDX (65nM) abolished indirect twitches of the chick biventer cervicis and mouse phrenic nerve diaphragm preparations. The time to 90% inhibition by PDX was significantly increased by replacing Ca(2+) (2.5mM) in the physiological solution with Sr(2+) (10mM). In the biventer cervicis muscle, venom (10microg/ml), but not PDX (65nM), significantly inhibited responses to ACh (1mM) and carbachol (20microM), but not KCl (40mM). In the mouse diaphragm (low Ca(2+); room temperature), the inhibitory effect of PDX (6.5nM) was delayed and a transient increase (746+/-64%; n=5) of contractions observed. In intracellular recording experiments using the mouse hemidiaphragm, PDX (6.5-65nM) significantly increased quantal content and miniature endplate potential frequency prior to blocking evoked release of acetylcholine. In extracellular recording experiments using the mouse triangularis sterni, PDX (2.2-65nM) significantly inhibited the voltage-dependent K(+), but not Na(+), waveform. In patch clamp experiments using B82 mouse fibroblasts stably transfected with rKv 1.2, PDX (22nM; n=3) had no significant effect on currents evoked by 10mV step depolarisations from -60 to +20mV. PDX exhibits all the pharmacology associated with beta-neurotoxins, and appears to be one of the most potent, if not the most potent beta-neurotoxin yet discovered.

Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 µg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential.

Central and peripheral neurotoxicity induced by the Jack Bean Urease (JBU) in Nauphoeta cinerea cockroaches.

BACKGROUND: Ureases of Canavalia ensiformis are natural insecticides with a still elusive entomotoxic mode of action. We have investigated the mechanisms involved in the neurotoxicity induced by Jack Bean Urease (JBU) in Nauphoeta cinerea (Olivier). METHODS: To carry out this study we have employed biochemical and neurophysiological analysis of different cockroach organ systems. RESULTS AND CONCLUSIONS: The injection of the insects with JBU (0.75-6µg/g animal), although not lethal within 24h, caused significant inhibition of the brain acetylcholinesterase activity (60±5%, p<0.05, n=6). JBU (1.5µg/200µL), acetylcholine (0.3µg/200µL) or neostigmine (0.22µg/200µL), induced a positive cardiac chronotropism (∼25%) in the cockroaches (p<0.05, n=9). JBU (6µg/g) increased the insects' grooming activity (137±7%), similarly to octopamine (15µg/g) (p<0.05, n=30, respectively). Pretreating the insects with phentolamine (0.1µg/g) prevented the JBU- or octopamine-induced increase of grooming activity. JBU (6µg/g) caused 65±9% neuromuscular blockade in the cockroaches, an effect prevented by bicuculline (5µg/g) (p<0.05, n=6). JBU (6µg/g) decreased the frequency whilst increasing the amplitude of the spontaneous neural compound action potentials (1425±52.60min-1, controls 1.102±0.032mV, p<0.05, n=6, respectively). Altogether the results indicate that JBU induces behavioral alterations in Nauphoeta cinerea cockroaches probably by interfering with the cholinergic neurotransmission. The neuromuscular blocking activity of JBU suggests an interplay between acetylcholine and GABA signaling. GENERAL SIGNIFICANCE: The search for novel natural molecules with insecticide potential has become a necessity more than an alternative. Understanding the mode of action of candidate molecules is a crucial step towards the development of new bioinsecticides. The present study focused on the neurotoxicity of Canavalia ensiformis urease, a natural insecticide, in cockroaches and revealed interferences on the cholinergic, octopaminergic and GABA-ergic pathways as part of its entomotoxic mode of action.

Draft Plastid and Mitochondrial Genome Sequences from Antarctic Alga Prasiola crispa.

The organelle genomes of the Antarctic alga Prasiola crispa (Lightfoot) Kützing have been sequenced. The plastid and mitochondrial genomes have a total length of 196,502 bp and 89,819 bp, respectively. These genomes have 19 putative photosynthesis-related genes and 17 oxidative metabolism-related genes, respectively.

Neuromuscular activity of Micrurus laticollaris (Squamata: Elapidae) venom in vitro.

In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1-30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1-10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.

Presynaptic activity of an isolated fraction from Rhinella schneideri poison

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.