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.

The neurotoxic mechanism of Jack Bean Urease in insects involves the interplay between octopaminergic and dopaminergic pathways.

In the last decades, the entomotoxicity of JBU and its derived peptides became an object of study, due mainly to the ubiquitous interaction of these compounds with different species of insects and their potential as natural insecticides. In this work, we investigated the neurotoxic effects of JBU in Nauphoeta cinerea cockroaches by dissecting pharmacologically the monoaminergic pathways involved. Selective pharmacological modulators for monoaminergic pathways in in vivo and ex vivo experimental models were employed. Thus, the analysis of N. cinerea neurolocomotory behavior demonstrated that JBU (1.5 and 3 µg/g) induces a significant decrease in the exploratory activity. In these assays, pretreatment of animals with phentolamine, SCH23390 or reserpine, interfered significantly with the response of JBU. Using in vivo abductor metathoracic preparations JBU (1.5 µg/g) induced progressive neuromuscular blockade, in 120 min recordings. In this set of experiments, the previous treatment of the animals with phentolamine, SCH23390 or reserpine, completely inhibited JBU-induced neuromuscular blockade. The recordings of spontaneous compound neural action potentials in N. cinerea legs showed that JBU, only in the smallest dose, significantly decreased the number of potentials in 60 min recordings. When the animals were pretreated with phentolamine, SCH23390, or reserpine, but not with mianserin, there was a significant prevention of the JBU-inhibitory responses on the action potentials firing. Meanwhile, the treatment of the animals with mianserin did not affect JBU's inhibitory activity. The data presented in this work strongly suggest that the neurotoxic response of JBU in N. cinerea involves a cross talking between OCTOPAMIN-ergic and DOPAMIN-ergic nerve systems, but not the SEROTONIN-ergic neurotransmission. Further molecular biology studies with expression of insect receptors associated with voltage clamp techniques will help to discriminate the selectivity of JBU over the monoaminergic transmission.

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.

Dietary omega-3 fatty acids prevent neonatal seizure-induced early alterations in the hippocampal glutamatergic system and memory deficits in adulthood.

OBJECTIVE: We investigated the influence of dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) on glutamatergic system modulation after a single episode of neonatal seizures and their possible effects on seizure-induced long-lasting behavioral deficits. METHODS: Male Wistar rats receiving an omega-3 diet (n-3) or an n-3 deficient diet (D) from the prenatal period were subjected to a kainate-induced seizure model at P7. Glutamate transporter activity and immunocontents (GLT-1 and GLAST) were assessed in the hippocampus at 12, 24, and 48 h after the seizure episode. Fluorescence intensity for glial cells (GFAP) and neurons (NeuN) was assessed 24 h after seizure in the hippocampus. Behavioral analysis (elevated-plus maze and inhibitory avoidance memory task) was performed at 60 days of age. RESULTS: The D group showed a decrease in glutamate uptake 24 h after seizure. In this group only, the GLT1 content increased at 12 h, followed by a decrease at 24 h. GLAST increased up to 24 h after seizure. GFAP fluorescence was higher, and NeuN fluorescence decreased, in the D group independent of seizures. In adulthood, the D group presented memory deficits independent of seizures, but short-term memory (1.5 h after a training session) was abolished in the D group treated with kainate. SIGNIFICANCE: N-3 PUFA positively influenced the glutamatergic system during seizure and prevented seizure-related memory deficits in adulthood.

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.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

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.

Modulation of octopaminergic and cholinergic pathways induced by Caatinga tree Manilkara rufula chemical compounds in Nauphoeta cinerea cockroaches.

The entomotoxic potential of Manilkara rufula crude extract (CEMR) and its aqueous (AFMR) and methanolic (MFMR) fractions were evaluated against Nauphoeta cinerea cockroaches. The results point out to a direct modulation of octopaminergic and cholinergic pathways in insect nervous system. CEMR induced an anti-acetylcholinesterase (AChE) effect in cockroach brain homogenates. CEMR significantly decreased the cockroach heart rate in semi-isolated heart preparations. CEMR also caused a broad disturbance in the insect behavior by reducing the exploratory activity. The decreased antennae and leg grooming activities, by different doses of CEMR, mimicked those of phentolamine activity, a selective octopaminergic receptor antagonist. The lethargy induced by CEMR was accompanied by neuromuscular failure and by a decrease of sensilla spontaneous neural compound action potentials (SNCAP) firing in in vivo and ex vivo cockroach muscle-nerve preparations, respectively. AFMR was more effective in promoting neuromuscular paralysis than its methanolic counterpart, in the same dose. These data validate the entomotoxic activity of M. rufula. The phentolamine-like modulation induced in cockroaches is the result of a potential direct inhibition of octopaminergic receptors, combined to an anti-AChE activity. In addition, the modulation of CEMR on octopaminergic and cholinergic pathways is probably the result of a synergism between AFMR and MFMR chemical compounds. Further phytochemical investigation followed by a bio-guiding protocol will improve the molecular aspects of M. rufula pharmacology and toxicology to insects.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Anacardium microcarpum Promotes Neuroprotection Dependently of AKT and ERK Phosphorylation but Does Not Prevent Mitochondrial Damage by 6-OHDA.

Parkinson's disease is a degenerative and progressive illness characterized by the degeneration of dopaminergic neurons. 6-hydroxydopamine (6-OHDA) is a widespread model for induction of molecular and behavioral alterations similar to Parkinson and has contributed for testing of compounds with neuroprotective potential. The Brazilian plant Anacardium microcarpum is used in folk medicine for treatment of several illnesses; however, the knowledge about toxicology and biological effects for this plant is very rare. The neuroprotective effect from hydroalcoholic extract and methanolic and acetate fraction of A. microcarpum on 6-OHDA-induced damage on chicken brain slices was investigated in this study. 6-OHDA decreased cellular viability measured by MTT reduction assay, induced lipid peroxidation by HPLC, stimulated Glutathione-S-Transferase and Thioredoxin Reductase activity, and decreased Glutathione Peroxidase activity and the total content of thiols containing compounds. The methanolic fraction of A. microcarpum presented the better neuroprotective effects in 6-OHDA-induced damage in relation with hydroalcoholic and acetate fraction. The presence of AKT and ERK1/2 pharmacological inhibitors blocked the protective effect of methanolic fraction suggesting the involvement of survival pathways in the neuroprotection by the plant. The plant did not prevent 6-OHDA autoxidation or 6-OHDA-induced mitochondrial dysfunction. Thus, the neuroprotective effect of the methanolic fraction of A. microcarpum appears to be attributed in part to chelating properties of extract toward reactive species and is dependent on ERK1/2 and AKT phosphorylation. This study contributes to the understanding of biochemical mechanisms implied in neuroprotective effects of the vegetal species A. microcarpum.

Entomotoxic activity of Rhinella icterica (Spix, 1824) toad skin secretion in Nauphoeta cinerea cockroaches: An octopamine-like modulation.

Rhinella icterica is a poisonous toad whose toxic secretion has never been studied against entomotoxic potential. Sublethal doses of Rhinella icterica toxic secretion (RITS) were assayed in Nauphoeta cinerea cockroaches, in order to understand the physiological and behavioral parameters, over the insect central and peripheral nervous system. RITS (10 µg/g) injections, induced behavioral impairment as evidenced by a significant decrease (38 ±â€¯14%) in the distance traveled (p < .05), followed by an increase (90 ±â€¯6%) of immobile episodes (p < .001, n = 28, respectively). In cockroaches semi-isolated heart preparations, RITS (16 µg/200 µl) induced a significant irreversible dose-dependent negative chronotropism, reaching ~40% decrease in heart rate in 20 min incubation. In in vivo cockroach neuromuscular preparations, RITS (20, 50 and 100 µg/g of animal weight) induced a time-dependent inhibition of twitch tension that was complete for 20 µg/g, in 120 min recordings. RITS (10 µg/g) also induced a significant increase in the insect leg grooming activity (128 ±â€¯10%, n = 29, p < .01), but not in the antennae counterparts. The RITS increase in leg grooming activity was prevented in 90% by the pretreatment of cockroaches with phentolamine (0.1 µg/g). The electrophysiological recordings of spontaneous neural compound action potentials showed that RITS (20 µg/g) induced a significant increase in the number of events, as well as in the rise time and duration of the potentials. In conclusion, RITS showed to be entomotoxic, being the neuromuscular failure and cardiotoxic activity considered the main deleterious effects. The disturbance of the cockroaches' behavior together with the electrophysiological alterations, may unveil the presence of some toxic components present in the poison with inherent biotechnological potentials.

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.

Methylprednisolone as a memory enhancer in rats: Effects on aversive memory, long-term potentiation and calcium influx.

It is well recognized that stress or glucocorticoids hormones treatment can modulate memory performance in both directions, either impairing or enhancing it. Despite the high number of studies aiming at explaining the effects of glucocorticoids on memory, this has not yet been completely elucidated. Here, we demonstrate that a low daily dose of methylprednisolone (MP, 5mg/kg, i.p.) administered for 10-days favors aversive memory persistence in adult rats, without any effect on the exploring behavior, locomotor activity, anxiety levels and pain perception. Enhanced performance on the inhibitory avoidance task was correlated with long-term potentiation (LTP), a phenomenon that was strengthen in hippocampal slices of rats injected with MP (5mg/kg) during 10days. Additionally, in vitro incubation with MP (30-300µM) concentration-dependently increased intracellular [Ca2+]i in cultured hippocampal neurons depolarized by KCl (35mM). In conclusion, a low daily dose of MP for 10days may promote aversive memory persistence in rats.

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.

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed.

DOMESTIC BUFFALOES ARE DIVIDED INTO TWO GROUP BASED ON CYTOGENETIC CHARACTERISTICS AND HABITATS: the "river buffaloes" with 2n = 50 and the "swamp buffaloes", 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 cross-breed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.

Dietary omega-3 deficiency reduces BDNF content and activation NMDA receptor and Fyn in dorsal hippocampus: implications on persistence of long-term memory in rats.

Omega-3 (n-3) fatty acids are important for adequate brain function and cognition. The aim of the present study was to evaluate how n-3 fatty acids influence the persistence of long-term memory (LTM) in an aversive memory task and to explore the putative mechanism involved. Female rats received isocaloric diets that included n-3 (n-3 group) or not (D group). The adult litters were subjected to an inhibitory avoidance task (0.7 mA, 1.0 seconds foot shock) to elicit persistent LTM. Twelve hours after the training session, the fatty acid profile and the brain derived neurotrophic factor (BDNF) content of the dorsal hippocampus were assessed. In addition, we measured the activation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor and the SRC family protein Fyn. Despite pronounced learning in both groups, the persistence of LTM was abolished in the D group 7 days after the training session. We also observed that the D group presented reductions in hippocampal DHA (22:6 n-3) and BDNF content. Twelve hours after the training session, the D group showed decreased NR2B and Fyn phosphorylation in the dorsal hippocampus, with no change in the total content of these proteins. Further, there was a decrease in the interaction of Fyn with NR2B in the D group, as observed by co-immunoprecipitation. Taken together, these data suggest that n-3 fatty acids influence the persistence of LTM by maintaining adequate levels of DHA and BDNF as well as by influencing the activation of NR2B and Fyn during the period of memory formation.

In vitro antiophidian mechanisms of Hypericum brasiliense choisy standardized extract: quercetin-dependent neuroprotection.

The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 µg/mL) or crotoxin (1 µg/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 µg/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 µg/mL) decreased cell viability. HBE (100 µg/mL) inhibited significantly the facilitatory action of crotamine (1 µg/mL) and was partially active against the neuromuscular blockade of crotoxin (1 µg/mL) (data not shown). Quercetin (10 µg/mL) mimicked the neuromuscular protection of HBE (100 µg/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 µg/mL) and crotamine (1 µg/mL). HBE (100 µg/mL) and quercetin (10 µg/mL) also increased cell viability in mice brain slices. Quercetin (10 µg/mL) was more effective than HBE (100 µg/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 µg/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.

Short-term alterations in hippocampal glutamate transport system caused by one-single neonatal seizure episode: implications on behavioral performance in adulthood.

Impairment in the activity and expression of glutamate transporters has been found in experimental models of epilepsy in adult animals. However, there are few studies investigating alterations on glutamate transporters caused by epilepsy in newborn animals, especially in the early periods after seizures. In this study, alterations in the hippocampal glutamate transporters activity and immunocontent were investigated in neonatal rats (7 days old) submitted to kainate-induced seizures model. Glutamate uptake, glutamate transporters (GLT-1, GLAST, EAAC1) and glutamine synthetase (GS) were assessed in hippocampal slices obtained 12 h, 24 h, 48 h, 72 h and 60 days after seizures. Immunoreactivity for hippocampal GFAP, NeuN and DAPI were assessed 24 h after seizure. Behavioral analysis (elevated-plus maze and inhibitory avoidance task) was also investigated in the adult animals (60 days old). The decrease on glutamate uptake was observed in hippocampal slices obtained 24 h after seizures. The immunocontent of GLT-1 increased at 12 h and decreased at 24 h (+62% and -20%, respectively), while GLAST increased up to 48 h after seizures. No alterations were observed for EAAC1 and GS. It should be mentioned that there were no long-term changes in tested glutamate transporters at 60 days after kainate treatment. GFAP immunoreactivity increased in all hippocampal subfields (CA1, CA3 and dentate gyrus) with no alterations in NeuN and DAPI staining. In the adulthood, kainate-treated rats showed anxiety-related behavior and lower performance in the inhibitory avoidance task. Our findings indicate that acute modifications on hippocampal glutamate transporters triggered by a single convulsive event in early life may play a role in the behavioral alterations observed in adulthood.

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: relevance for behavior alterations.

Essential omega-3 polyunsaturated fatty acids (omega3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary omega3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with omega3 (omega3 group) or a omega3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (alphaCaMKII) were evaluated. Additionally, hippocampal [(3)H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the omega3 group, although the difference disappeared at 21 days of life (except for alphaCaMKII, which content normalized at 60 days old). The same pattern was found for [(3)H]glutamate binding, whereas [(3)H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary omega3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary omega3 deficiency is discussed.