Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories

The basal forebrain complex, which includes the nucleus basalis magnocellularis (NBM), provides widespread cholinergic and γ-aminobutyric acid-containing projections throughout the brain, including the insular and pyriform cortices. A number of studies have implicated the cholinergic neurons in the mediation of learning and memory processes. However, the role of basal forebrain activity in information retrieval mechanisms is less known. The aim of the present study is to evaluate the effects of reversible inactivation of the NBM by tetrodotoxin (TTX, a voltage-sensitive sodium channel blocker) during the acquisition and retrieval of conditioned taste aversion (CTA) and to measure acetylcholine (ACh) release during TTX inactivation in the insular cortex, by means of the microdialysis technique in free-moving rats. Bilateral infusion of TTX in the NBM was performed 30 min before the presentation of gustative stimuli, in either the CTA acquisition trial or retrieval trial. At the same time, levels of extracellular ACh release were measured in the insular cortex. The behavioral results showed significant impairment in CTA acquisition when the TTX was infused in the NBM, whereas retrieval was not affected when the treatment was given during the test trial. Biochemical results showed that TTX infusion into the NBM produced a marked decrease in cortical ACh release as compared with the controls during consumption of saccharin in the acquisition trial. Depleted ACh levels were found during the test trial in all groups except in the group that received TTX during acquisition. These results suggest a cholinergic-dependent process during acquisition, but not during memory retrieval, and that NBM-mediated cholinergic cortical release may play an important role in early stages of learning, but not during recall of aversive memories.

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.

Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.

Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-four minutes after an intraperitoneal injection of saline or glucose (100, 250, or 1000 mg/kg), rats were tested in a four-arm cross maze for spontaneous alternation behavior combined with microdialysis collection. Glucose at 250 mg/kg, but not 100 or 1000 mg/kg, produced an increase in spontaneous alternation scores (69.5%) and ACh output (121.5% versus baseline) compared to alternation scores (44.7%) and ACh output (58.9% versus baseline) of saline controls. The glucose-induced increase in alternation scores and ACh output was not secondary to changes in locomotor activity. Saline and glucose (100-1000 mg/kg) treatment had no effect on hippocampal ACh output when rats remained in the holding chamber. These findings suggest that glucose may enhance memory by directly or indirectly increasing the release of ACh. The results also indicate that hippocampal ACh release is increased in rats performing a spatial task. Moreover, because glucose enhanced ACh output only during behavioral testing, circulating glucose may modulate ACh release only under conditions in which cholinergic cells are activated.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals. (C) 1999 IBRO. Published by Elsevier Science Ltd.

Evidence of muscarinic acetylcholine receptors in the retinal centrifugal system of the chick

In this study we characterize the presence of muscarinic acetylcholine receptors (mAChR) in the isthmo-optic nucleus (ION) of chicks by immunohistochemistry with the M35 antibody. Some M35-immunoreactive fibers were observed emerging from the retinal optic nerve insertion, suggesting that they could be centrifugal fibers. Indeed, intraocular injections of cholera toxin B (CTb), a retrograde tracer, and double-labeling with M35 and CTb in the ION confirmed this hypothesis. The presence of M35-immunoreactive cells and the possible mAChR expression in ION and ectopic neuron cells in the chick brain strongly suggest the existence of such a cholinergic system in this nucleus and that acetylcholine release from amacrine cells may mediate interactions between retinal cells and ION terminals.

Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle.

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20-80% rise time (tr) of approximately 80 micros at 22 degrees C, shorter than any previously reported values, and tr variability (SD) with an upper limit of 25-30 micros. Extracellular electrode pressure can increase tr and its variability by 2- to 3-fold. Using Monte Carlo simulations, we modeled passive acetylcholine diffusion through a vesicle fusion pore expanding radially at 25 nm x ms(-1) (rapid, from endplate omega figure appearance) or 0.275 nm x ms(-1) (slow, from mast cell exocytosis). Simulated mEPCs obtained with rapid expansion reproduced tr and the overall shape of our experimental mEPCs, and were similar to simulated mEPCs obtained with instant acetylcholine release. We conclude that passive transmitter diffusion, coupled with rapid expansion of the fusion pore, is sufficient to explain the time course of experimentally measured synaptic currents with trs of less than 100 micros.

Role of dorsal raphe nucleus 5-HT(1A) and 5-HT(2) receptors in tonic immobility modulation in guinea pigs

Tonic immobility (TI) is an innate defensive behavior characterized by a state of physical inactivity and diminished responsiveness to environmental stimuli. Behavioral adaptations to changes in the external and internal milieu involve complex neuronal network activity and a large number of chemical neurotransmitters. The TI response is thought to be influenced by serotonin (5-HT) activity in the central nervous system (CNS) of vertebrates, but the neuronal groups involved in the mechanisms underlying this behavior are poorly understood. Owing to its extensive afferents and efferents, the dorsal raphe nucleus (DRN) has been implicated in a great variety of physiological and behavioral functions. in the current study, we investigated the influence of serotonergic 5-HT(1A) and 5-HT(2) receptor activity within the DRN on the modulation of TI behavior in the guinea pig. Microinjection of a 5-HT(1A) receptor agonist (8-OH-DPAT, 0.01 and 0.1 mu g) decreased TI behavior, an effect blocked by pretreatment with WAY-100635 (0.033 mu g), a 5-HT(1A) antagonist. In contrast, activation of 5-HT(2) receptors within the DRN (alpha-methyl-5-HT, 0.5 mu g) increased the TI duration, and this effect could be reversed by pretreatment with an ineffective dose (0.01 mu g) of ketanserine. Since the 5-HT(1A) and 5-HT(2) agonists decreased and increased, respectively, the duration of TI, different serotonin receptor subtypes may play distinct roles in the modulation of TI in the guinea pig. (C) 2009 Elsevier B.V. All rights reserved.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurotics of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS), or the desensitising agonist, alpha,beta -methylene ATP. In contrast. EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 muM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 muM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gammaS, reversibly depressed EPSPs amplitude, The inhibitory effects of ATP and ATP gammaS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurotics from rat submandibular ganglia. focal application of ATP (100 LM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 muM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurotics to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Distribuição dos receptores da adenosina no plexo mioentérico – músculo longitudinal do íleo de rato: implicações funcionais

A acetilcolina (ACh) é o neurotransmissor mais importante no controlo da motilidade gastrointestinal. A libertação de ACh dos neurónios entéricos é regulada por receptores neuronais específicos (De Man et al., 2003). Estudos prévios demonstraram que a adenosina exerce um papel duplo na libertação de ACh dos neurónios entéricos através da activação dos receptores inibitórios A1 e facilitatórios A2A (Duarte-Araújo et al., 2004). O potencial terapêutico dos compostos relacionados com a adenosina no controlo da motilidade e da inflamação intestinal, levou-nos a investigar o papel dos receptores com baixa afinidade para a adenosina, A2B e A3, na libertação de acetilcolina induzida por estimulação eléctrica nos neurónios mioentéricos. Estudos de imunolocalização mostraram que os receptores A2B exibem um padrão de distribuição semelhante ao do marcador de células gliais (GFAP). No que respeita aos receptores A1 e A3, estes encontram-se distribuídos principalmente nos corpos celulares dos neurónios ganglionares mioentéricos, enquanto os receptores A2A estão localizados predominantemente nos terminais nervosos colinérgicos. Neste trabalho mostrou-se que a modulação da libertação de ACh-[3H] (usando os antagonistas selectivos DPCPX, ZM241385 e MRS1191) é balanceada através da activação tónica dos receptores inibitórios (A1) e facilitatórios (A2A e A3) pela adenosina endógena. O antagonista selectivo dos receptores A2B, PSB603, não foi capaz de modificar o efeito inibitório da NECA (análogo da adenosina com afinidade para receptores A2). O efeito facilitatório do agonista dos receptores A3, 2-Cl-IB MECA (1-10 nM), foi atenuado pelo MRS1191 e pelo ZM241385, os quais bloqueiam respectivamente os receptores A3 e A2A. Contrariamente à 2-Cl-IB MECA, a activação dos receptores A2A pelo CGS21680C, atenuou a facilitação da libertação de ACh induzida pela activação dos receptores nicotínicos numa situação em que a geração do potencial de acção neuronal foi bloqueada pela tetrodotoxina. A localização diferencial dos receptores excitatórios A3 e A2A ao longo dos neurónios mioentéricos explica porque razão a estimulação dos receptores A3 (com 2-Cl-IB MECA) localizados nos corpos celulares dos neurónios mioentéricos exerce um efeito sinérgico com os receptores facilitatórios A2A dos terminais nervosos no sentido de aumentarem a libertação de ACh. Os resultados apresentados consolidam e expandem a compreensão actual da distribuição e função dos receptores da adenosina no plexo mioentérico do íleo de rato, e devem ser tidos em consideração para a interpretação de dados relativos às implicações fisiopatológicas da adenosina nos transtornos da motilidade intestinal.

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

Coral snakes, the New World Elapidae, are included in the genera Micniroides and Micrurus. The genus Mlcrurus comprises nearly all coral snake species and those which are responsible for human snake-bite accidents. The following generalizations concerning the effects induced by their venoms, and their venom-properties can be made. Coral snake venoms are neurotoxic, producing loss of muscle strenght and death by respiratory paralysis. Local edema and necrosis are not induced nor blood coagulation or hemorrhages. Proteolysis activity is absent or of very low grade. They display phospholipase A2 activity. Nephrotoxic effects are not evoked. The main toxins from elapid venoms are postsynaptic and presynaptic neurotoxins and cardiotoxins. Phospholipases A2 endowed with myonecrotic or cardiotoxin-like properties are important toxic components from some elapid venoms. The mode of action of Micrurus frontalis, M. lemniscatus, M. corallinus and M. fulvius venoms has been investigated in isolated muscle preparations and is here discussed. It is shown that while M. frontalis and M. lemniscatus venoms must contain only neurotoxins that act at the cholinergic end-plate receptor (postsynaptic neurotoxins), M. corallinus venom also inhibits evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like effect) and M. fulvius induces muscle fiber membrane depolarization (cardiotoxin-like effect). The effects produced by M. corallinus and M. fulvius venoms in vivo in dogs and M. frontalis venom in dogs and monkeys are also reported.

OnabotulinumtoxinA and multiple sclerosis.

Lower urinary tract dysfunction is present in two of three patients with multiple sclerosis five years after the diagnosis. Most frequent symptoms are related to neurogenic detrusor overactivity, often associated with detrusor-sphincter dyssynergia. From the end of the 1990s, there is growing evidence that neurogenic detrusor overactivity can be effectively managed by intradetrusorial injections of botulinum toxin type A. This treatment has shown, in different randomised placebo-controlled trials, to be safe and effective on clinical and urodynamic parameters with significant improvement in quality of life. The median duration of effect is in mean nine months. The vast majority of studies have been conducted with onabotulinumtoxinA. The dose of onabotulinumtoxinA commonly used to treat neurogenic detrusor overactivity in patients with multiple sclerosis is 200 UI, even if in selected patients lower doses can be preferred. To be considered eligible for treatment, all patients should accept and be instructed to perform clean intermittent self-catheterisation, since the risk of increased post-void residual volume and/or urinary retention after injection is high, especially with 200 UI of onabotulinumtoxinA. However, quality of life and patient satisfaction seem not to be affected by the need of intermittent catheterisation. The risk of urinary infection after the procedure is to be kept in mind, mainly in patients with multiple sclerosis, so that adequate antibiotic prophylaxis is highly recommended.