Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy.

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.

The effects of Tityus bahiensis scorpion venom on the contractility of jejunum, vas deferens, and the aorta is differentially affected by tetrodotoxin.

The pharmacological effects of the crude venom of the scorpion Tityus serrulatus or its isolated toxins have been widely studied. However, few studies are available on Tityus bahiensis venom. We recently discovered that T. serrulaus venom leads to the release of tetrodotoxin-resistant acetylcholine. Thus, our objective was to verify whether T. bahiensis venom could have a similar action in the jejunum. Furthermore, we evaluated the possibility that this action occur in other tissues innervated by the autonomic nervous system. Thus, organ bath studies were conducted to evaluate the contractile and relaxant effects of venom on the jejunum, vas deferens and aorta of rats in the presence or absence of tetrodotoxin. We observed that jejunum, vas deferens and aorta contracted when the T. bahiensis venom was applied. In the jejunum, the venom reveals a contractile component resistant to tetrodotoxin. It also was able to relax pre-contracted preparations of jejunum and aorta but not vas deferens. Only in the aorta, the relaxation was resistant to tetrodotoxin. The effects of scorpion venoms are attributed to its action on ionic channels leading to neuronal depolarization and neurotransmitter release. Our results indicated that a similar mechanism is present in the observed effects of the venom. However, another mechanism must be present in the venom-induced contraction in the jejunum and relaxation in the aorta. Possible involvement of tetrodotoxin-resistant sodium channels or non-neuronal release of neurotransmitters is discussed. We emphasize that the study of the Tityus scorpion's venom, especially T. bahiensis, is of great importance because it can unveil unknown pharmacological and physiological mechanisms of excitable cells.

Effect of Tityus serrulatus scorpion venom on isolated jejunum: A very useful tool to study the interaction between neurons in the enteric nervous system.

Scorpion envenomation is a public health problem in tropical and subtropical areas. In Brazil, Tityus serrulatus is the biggest cause of accidents with venomous animals. Tityus serrulatus venom causes symptoms related to a great activation of the autonomic system attributed to a massive release of sympathetic and parasympathetic mediators. This effect is attributed to the presence of toxins acting in Na+ and K+ ion channels, leading to an increase in cell excitability. Although gastrointestinal symptoms, like diarrhoea and sialorrhea, is observed in moderate to severe cases, little attention is given in clinical reports. Gastrointestinal motility is controlled by the enteric nervous system which is composed of a wide variety of interconnected neurons that are influenced by the sympathetic and parasympathetic nervous systems. Thus, this work aimed to characterize the effects of Tityus serrulatus venom on sympathetic and parasympathetic neurotransmission of rat jejunum, as well as to investigate possibles effects on other neurons of the enteric nervous system. To this, we verify the effects of Tityus serrulatus venom on the contractility of isolated rat jejunum through organ-bath experiments. We observed that venom can induce both contraction and relaxation. The contraction was partially inhibited by atropine (1 µM) and by suramin (0.1 mM) through tetrodotoxin-resistant and sensitive mechanisms. The relaxation was completely inhibited by 3 µM propranolol and partially inhibited by 1 µM phentolamine. Suramin induced a slowing of relaxation curve. Tetrodotoxin completely inhibits the relaxation induced by Tityus serrulatus venom, but the contraction curves were only partially reduced in their initial portion. The final part of the curve was largely enhanced by Tetrodotoxin. Atropine blocks almost completely the contraction curve in the presence of Tetrodotoxin. These results indicate that Tityus serrulatus venom induces the release of both excitatory (predominantly acetylcholine) and inhibitory (mainly noradrenaline) neurotransmitters. The effects of Tityus serrulatus venom on organ contractility was quite complex and seem to derive from a diffuse and nonspecific release of mediators from autonomic and enteric nervous systems. Further investigation of venom action and its isolated toxins can reveal important aspects to deepen our knowledge about the enteric nervous system transmission and the interaction between excitatory and inhibitory mediators as well as the physiological role of Na+ and K+ ion channels in gut motility.

The analgesic effect of crotoxin on neuropathic pain is mediated by central muscarinic receptors and 5-lipoxygenase-derived mediators.

Crotoxin (CTX), a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg, i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). On the other hand, indomethacin (4 mg/kg, i.v.), naloxone (1 mg/kg, i.p.), and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of alpha-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin.

Intrahippocampal injection of TsTX-I, a beta-scorpion toxin, causes alterations in electroencephalographic recording and behavior in rats.

AIMS: TsTX-I scorpion toxin, also known as γ-toxin, is a ß-toxin which binds to site 4 of the sodium channel, shifting its activation potential. There are few studies about its pharmacological action in the central nervous system. The objective of this work was to determine the electroencephalographic, behavioral and histopathological effects of intrahippocampal injection of TsTX-I. MAIN METHODS: Rats were anesthetized and fitted with cannulae for injection into the hippocampus and with electrodes for cerebral recording. The animals were treated with Ringer solution, some doses of TsTX-I, DMSO 0.1% or veratridine. Behavioral and electrographic recordings were observed for 4 hours after the injection. After 7 days, the rats were perfused, and their brains removed for histological analysis. KEY FINDINGS: Increasing doses of the toxin evoked epileptic-like discharges, wet dog shakes, and in some cases hind limb paralysis and intense respiratory difficulty followed by death. The histopathological analysis demonstrated no cell loss. Animals injected with veratridine developed epileptiform activity in the electrographic recording and neuronal loss. SIGNIFICANCE: The results suggest that TsTX-I toxin may be responsible, at least in part, for the epileptic and behavioral effects observed with the crude venom, and although veratridine and TsTX-I act on Na-channel, the differences between them are remarkable, demonstrating that toxins can have different functional effects depending on the site of action in the channel. Thus, animal neurotoxins are often highly selective and may be useful for the identification of the sequence of events underlying neurotransmission.

Effects of long-term fluoxetine treatment on adrenergic plasticity in rat vas deferens

Chronic antidepressant administration increases neurotrophin levels in the central and peripheral nervous system, leading to an increase of neuronal sprouting, reestablishment of neural networks and neurotransmitter levels. Injured peripheral nerves regenerate at very slow rates. However, the recovery of the hypogastric nerve in rodents after injury is significantly improved with neurotrophin administration. Accordingly, our goal was to determine whether treatment with the antidepressant fluoxetine affects catecholamine levels and neuronal function, after surgical denervation of the rat vas deferens. Noradrenaline levels in the denervated vas deferens were higher in fluoxetine-treated animals than in the vehicle-treated group, as measured by high performance liquid chromatography. In functional studies of smooth muscle contraction, the responses induced by phenylephrine or ATP, as well as pre-synaptic α2-adrenoceptor reactivity, were not modified by chronic treatment with the antidepressant. However, the contraction mediated by neuronal release of noradrenaline induced by tyramine was increased on days 7 and 21 after denervation in rats treated with fluoxetine. These data indicate that fluoxetine can improve functional recovery after rat vas deferens denervation.

A administração crônica de antidepressivos aumenta os níveis de neurotrofinas no sistema nervoso central, levando a um aumento da arborização neuronal, restabelecendo a rede neural e os níveis de neurotransmissores. Lesões do sistema nervoso periférico mostram uma regeneração muito lenta. Entretanto, a recuperação após a lesão do nervo hipogástrico em roedores é significativamente melhorada após a administração de neurotrofinas. Nesse sentido, nosso objetivo foi verificar se o tratamento com o antidepressivo, fluoxetina, interfere nos níveis de catecolaminas e na função neuronal, após a desnervação cirúrgica do ducto deferente de rato. Nos vasos deferentes desnervados, os níveis de catecolaminas nos grupos tratados com fluoxetina foram maiores que no grupo veículo, quantificados em cromatografia líquida de alta eficiência (CLAE). Nos estudos funcionais, a contração da musculatura lisa induzida pela fenilefrina ou pelo ATP, assim como a reatividade pré-sináptica α2-adrenérgica, não foram modificadas com o tratamento crônico de fluoxetina. Contudo, nas contrações mediadas pela liberação neuronal de norepinefrina induzida por tiramina, observou-se aumento da contração nos dias 7 e 21 após a desnevação em ratos tratados com fluoxetina. Esses dados indicam que a fluoxetina pode melhorar a recuperação funcional do vaso deferente de rato após a desnervação.

Alteration of purinergic P2X4 and P2X7 receptor expression in rats with temporal-lobe epilepsy induced by pilocarpine.

SUMMARY: Although ATP and P2X receptor activity have been lately associated with epilepsy, little is known regarding their exact roles in epileptogenesis. Temporal-lobe epilepsy (TLE) in rat was induced by pilocarpine in order to study changes of hippocampal P2X(2), P2X(4) and P2X(7) receptor expression during acute, latent or chronic phases of epilepsy. During acute and chronic phases increased P2X(7) receptor expression was principally observed in glial cells and glutamatergic nerve terminals, suggesting participation of this receptor in the activation of inflammatory and excitotoxic processes during epileptogenesis. No significant alterations of hippocampal P2X(2) and P2X(4) receptor expression was noted during the acute or latent phase when compared to the control group, indicating that these receptors are not directly involved with the initiation of epilepsy. However, the reduction of hippocampal P2X(4) receptor immunostaining in the chronic phase could reflect neuronal loss or decreased GABAergic signaling.

Open field behavior and intra-nucleus accumbens dopamine release in vivo in virgin and lactating rats

In adult female mammals, reproductive experience (e.g., mating, pregnancy, parturition, and lactation) has long-term behavioral, endocrine, and neurochemical implications. This experience causes behavioral and neurochemical changes that involve several brain areas important for the expression of maternal behavior. The present study showed that lactating rats exhibited reduced general locomotor activity in the open field test compared with virgin animals. Our hypothesis was that nucleus accumbens dopamine, which regulates maternal behavior in lactating rats, is also involved in the low expression of maternal locomotion in the open field test observed during the early stages of lactation and reflects decreased motivation. Initially we compared open field behavior in virgin and lactating rats to confirm our previous data. Thus, the in vivo release of dopamine in the nucleus accumbens in virgin and lactating female rats was measured. Perfusate concentrations of extracellular dopamine and its metabolites showed no differences between virgin and lactating rats. Thus, the reduced general activity observed in lactating rats might not be related to intra-nucleus accumbens dopamine control.

Absence of oxytocin in the central nervous system of the snake Bothrops jararaca.

We used four complementary techniques to investigate the presence of oxytocin peptide in the hypophysis and brain of the snake Bothrops jararaca. A high-pressure liquid chromatographic analysis failed to show oxytocin in extracts of hypophysial and brain tissues but provided estimative values of the amounts of vasotocin (12 ng/mg hypophysis and 0.5 ng/mg brain) and mesotocin (500 pg/mg hypophysis and 8 pg/mg brain). Western blots with a polyclonal anti-oxytocin antibody failed to detect oxytocin in both tissues but detected compounds with higher molecular weight than oxytocin, as well as a relatively weak cross-reactivity with mesotocin. The reverse transcription-polymerase chain reaction analysis failed to detect the expression of oxytocin gene transcript, but detected a transcript related to the mesotocin-neurophysin precursor in both tissues. Immunohistochemistry with the same anti-oxytocin antibody detected strong staining in the neurohypophysis and in few fibers in the inner zone of the median eminence, which was not abolished by pre-adsorption of this antibody with oxytocin, vasopressin, vasotocin or mesotocin and might not be attributed to oxytocin. In conclusion, our data demonstrate the absence of oxytocin in the central nervous system of the snake B. jararaca and underline the pitfalls that can result from the use of a single technique to investigate the presence of peptides in tissues.

Efeitos da toxina TsTX-I do escorpiäo Titus serrulatus sobre a neurotransmissäo simpática no ducto deferente de rato e célula cromafim bovina / Effects of toxin TsTX-I from the scorpion Titus serrulatus on the sympathetic neurotransmisson of rat vas deferens and bovine chromaffin cells

1. Contraçao induzida por TsTX-I ou veratridina no ducto deferente de rato: Tanto a TsTX-I como a veratridina induzem contraçao no ducto deferente de rato. A contraçao eliciada pela TsTX-I se inicia rapidamente após a aplicaçao da toxina atingindo um pico máximo ao redor de 1-2 minutos. Após atingir o máximo o órgao inicia uma relaxamento gradual e quase completo. Em contraste, a contraçao provocada pela veratridina possui um inicio mais lento e segue contraindo até o final do período experimental. Os efeitos das duas toxinas sao irreversíveis. A TsTX-I é cerca de 200 vezes mais potente que a veratridina em induzir contraçao no ducto deferente de rato, apesar de apresentarem a mesma eficácia. 2. Liberaçao de catecolaminas na célula cromafim bovina As duas toxinas sao capazes de liberar catecolaminas das células cromafins bovinas. Novamente, a TsTX-I é cerca de 200 vezes mais potente que a veratridina. Entretanto, na célula cromafim bovina a TsTX-I é muito menos eficaz que a veratridina em liberar noradrenalina e adrenalina (nas doses áximas, a veratridina libera cerca de 10 vezes ma is catecolaminas que a TsTX-I. 3. Efeitos da TsTX-I sobre as correntes de sódio na célula cromafim bovina A TsTX-I produz um deslocamneto da dependência de voltagem para a ativaçäo dos canais de sódio para potenciais mais negativos, além de reduzir a corrente de sódio que flui por esses canais. 4. Efeitos da concentraçäo de cálcio extracelular Quando altas concentraçöes de cálcio extracelular säo utilizadas, observando-se um sinergismo entre a TsTX-I e a veratridina. Esse sinergismo foi observado tanto no ducto deferente de rato(avaliado pela contraçäo desse órgäo) como na célula cromafim bovina (avaliado através da secreçäo de catecolaminas, captaçäo de 45Ca2+ e das oscilaçöes de cálcio intracelular. 5. Efeitos de bloquadores de canais de cálcio sensíveis à voltagem Na célula cromafim bovina, as oscilacöes de cálcio intracelular induzida por TsTX-I e veratridina utilizadas em associaçäo foram prevenidas pelo bloqueio dos canais de cálcio sensíveis à voltagem. No ducto de rato, o bloqueio de canais dos tipos N e P/Q näo afetou a contraçäo induzida por TsTX-I, mas o bloqueio de canais de cálcio do tipo N produziu uma pequena depressäo da contraçäo induzida pela veratridina. 6. Participaçäo dos componentes adrenérgico e purinéegico na contraçäo induzida por TsTX-I em ducto deferente de rato. Verificamos que aTsTX-I parece causar a liberaçäo preferencial de ATP ...(au)