Miniature Endplate Currents Research Articles

Impaired Activity-Dependent Plasticity of Quantal Amplitude at the Neuromuscular Junction of Rab3A Deletion and Rab3A Earlybird Mutant Mice

Rab3A is a small GTPase associated with synaptic vesicles that is required for some forms of activity-dependent plasticity. It is thought to regulate the number of vesicles that fuse through an effect on docking, vesicle maturation, or mobilization. We recently showed that at the neuromuscular junction, loss of Rab3A led to an increase in the occurrence of miniature endplate currents (mepcs) with abnormally long half-widths (Wang et al., 2008). Here we show that such events are also increased after short-term activity blockade, and this process is not Rab3A-dependent. However, in the course of these experiments we discovered that the homeostatic increase in mepc amplitude after activity blockade is diminished in the Rab3A deletion mouse and abolished in the Rab3A Earlybird mouse which expresses a point mutant of Rab3A. We show that homeostatic plasticity at the neuromuscular junction does not depend on tumor necrosis factor α, is not accompanied by an increase in the levels of VAChT, the vesicular transporter for ACh, and confirm that there is no increase in ACh receptors at the junction, three characteristics distinct from that of CNS homeostatic plasticity. Activity blockade does not produce time course changes in mepcs that would be consistent with a fusion pore mechanism. We conclude that Rab3A is involved in a novel presynaptic mechanism to homeostatically regulate the amount of transmitter in a quantum.

Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions

Pre-natal alcohol exposure induces delays in fine and gross motor skills, and deficiencies in reflex development via mechanisms that remain to be elucidated. The purpose of the present study was to investigate the effect of embryonic ethanol exposure (16-hour exposure window with1.5%, 2% or 2.5% EtOH) on synaptic properties at the neuromuscular junction (NMJ) in 3 day post fertilization (dpf) zebrafish larvae. Immunohistochemical studies show that exposure of embryos to 2.5% ethanol for 16 h results in motor neuron axons that display abnormal branching patterns. Co-labelling embryos with pre-synaptic markers such as SV-2 or 3A10, and the post-synaptic marker, α-bungarotoxin, which irreversibly binds to nicotinic acetylcholine receptors (nAChRs), indicates that pre- and post-synaptic sites are properly aligned even when motor neuron axons display abnormal morphology. Miniature endplate currents (mEPCs) recorded from muscle fibers revealed the presence of two types of mEPCs that we dubbed fast and slow. Ethanol treated fish experienced significant changes in the frequencies of fast and slow mEPCs, and an increase in the rise time of slow mEPCs recorded from red muscle fibers. Additionally, embryonic exposure to ethanol resulted in a significant increase in the decay time of fast mEPCs recorded from white fibers. Mean mEPC amplitude was unaffected by ethanol treatment. Together, these results indicate that zebrafish embryos exposed to ethanol may experience altered synaptic properties at the NMJ.

Ca2+ Dependence of the Binomial Parameters p and n at the Mouse Neuromuscular Junction

The Ca(2+) dependence of synaptic quantal release is generally thought to be restricted to probability of vesicular release. However, some studies have suggested that the number of release sites (n) at the neuromuscular junction (NMJ) is also Ca(2+) dependent. In this study, we recorded endplate currents over a wide range of extracellular Ca(2+) concentrations and found the expected Ca(2+) dependency of release. A graphical technique was used to estimate p (probability of release) and n using standard binomial assumptions. The results suggested n was Ca(2+) dependent. The data were simulated using compound binomial statistics with variable n (Ca(2+) dependent) or fixed n (Ca(2+) independent). With fixed n, successful simulation of increasing Ca(2+) required that p increase abruptly at some sites from very low to high values. Successful simulation with variable n required the introduction of previously silent release sites (p = 0) with high values of p. Thus the success of both simulations required abrupt, large increases of p at a subset of release sites with initially low or zero p. Estimates of the time course of release obtained by deconvolving evoked endplate currents with average miniature endplate currents decreased slightly as Ca(2+) increased, thus arguing against sequential release of multiple quanta at higher Ca(2+) levels. Our results suggest that the apparent Ca(2+) dependence of n at the NMJ can be explained by an underlying Ca(2+) dependence of a spatially variable p such that p increases abruptly at a subset of sites as Ca(2+) is increased.

Regulation Of The Acetylcholine Receptor Function By Thyroid Hormones

The basic molecular mechanisms underlying the muscular weakness initiated early during thyroid dysfunction are poorly understood. In this work we have explored how the acute and chronic alterations of thyroid hormones (THs) affect the acetylcholine receptor (AChR) channel function in the neuromuscular junction (NMJ). Acute methimazole (MMI) treatment produced a significant (p<0.005) reduction in weight of euthyroid mice of 8.64%, induced by a transient hyperthyroid phase. This result is compatible with the loss of weight produced acutely by T3 (1μg/g-bw) of 4.11%. The resulting action of these acute treatments is a significantly (p<0.001) reduced muscular strength by MMI (58.0%) and T3 (52.9%) when compared to sham animals. The hypothyroidism induced by the chronic MMI treatment causes a reduced daily weight gain of 76.5%. Focal recordings of miniature endplate currents (MEPCs) in isolated NMJ of chronically MMI-treated mice revealed a significant (p<0.001) reduction in the frequency (57.7%), amplitude (59.3%), and decay time (66.7%). The electrophysiological results appear to be related to the membrane-lipid metabolism in particular cholesterol, since NMJ of hypothyroid mice treated with methyl-beta-cyclodextrin (MβCD) recover the MEPCs normal characteristics. This data also agrees with results where the MEPCs’ decay time 1.45 ± 0.06 ms (n=8) of fat euthyroid mice differs significantly (p<0.001) from those of slim animals 1.01 ± 0.08 ms (n=7). The relationship between weight (w) and decay time (τ) is well-described by the equation: τ = -0.00447w + 2.456. We propose that membrane cholesterol in the NMJ could be an important target for the in vivo regulation of synaptic activity during thyroid dysfunction. Importantly, the docking of cholesterol to the AChR occurs in the C418 vicinity, a highly relevant residue in the protein-lipid interface.NIH 5S06 GM050595 and G12 RR03035 to LVR

Different sensitivity of miniature endplate currents in rat external and internal intercostal muscles to the acetylcholinesterase inhibitor C-547 as compared with diaphragm and extensor digitorum longus

Derivative of 6-methyluracil, selective cholinesterase inhibitor C-547 potentiates miniature endplate currents (MEPCs) in rat external intercostal muscles (external ICM) more effectively than in internal intercostal muscles (internal ICM). Effect of the C-547 on intercostal muscles was compared with those on extensor digitorum longus (EDL) and diaphragm muscles. Half-effective concentrations for tau of MEPC decay arranged in increasing order were as follows: EDL, locomotor muscle, most sensitive = 1.3 nM, external ICM, inspiration muscle = 6.8 nM, diaphragm, main inspiration muscle = 28 nM, internal ICM, expiration muscle = 71 nM. External ICM might therefore be inhibited, similarly as the limb muscles, by nanomolar concentrations of the drug and do not participate in inspiration in the presence of the C-547. Moreover, internal ICM inhibition can hinder the expiration during exercise-induced fast breathing of C-547- treated experimental animals.

Triiodothyronine (T3) action on aquatic locomotor behavior during metamorphosis of the bullfrog Rana catesbeiana

Thyroid hormones--particularly triiodothyronine, T3--play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre- or pro-metamorphic stages. Exposure to T3 in the water induced a rapid (beginning within 5 min) and significant decrease (approximately 20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI<2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI> or =2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.

Topography and Affinity of Calcium Sensors of Exo- and Endocytosis in Motor Nerve Terminals

Measurements with extracellular microelectrode technique showed that depolarization of motor nerve terminals in frog cutaneous pectoris muscle with high-potassium solution (40 mM K(+)) increased frequency of miniature end-plate currents. Both fast intracellular calcium chelator BAPTA-AM and slow chelator EGTA-AM equally moderated the increase in the frequency of miniature end-plate currents. Intravital fluorescent microscopy with FM 1-43 dye showed that under conditions of stimulation of neurotransmitter exocytosis and secretion with high-potassium solution, internalization of the dye into newly-formed endocytotic synaptic vesicles proceeded both in the control and in the presence of EGTA-AM. In contrast, internalization of the dye was not observed in the presence of BAPTA-AM. It was concluded that asynchronous exocytosis of synaptic vesicles goes on in the active zones enriched with Ca-channels due to activation of high-affinity Ca-site in Ca-macrodomain. Endocytosis of vesicles is probably initiated by Ca-microdomain during activation of low-affinity Ca-site in the immediate proximity to the Ca channel.

Regulation of quantal shape by Rab3A: evidence for a fusion pore‐dependent mechanism

The function of Rab3A, a small GTPase located on synaptic vesicles, is not well understood. Studies in the Rab3A(-/-) mouse support a role in activity-dependent plasticity, but have not reported any effects on spontaneously occurring miniature synaptic currents, except that there is a decrease in resting frequency at the neuromuscular junction. Therefore we were surprised to find an increase in the occurrence of mEPCs with abnormally long half-widths at the neuromuscular junctions of Rab3A(-/-) mice. The abnormal miniature endplate currents (mEPCs), which have significantly greater charge than the average mEPCs for the same fibres, could arise from larger vesicles. However, the type of mEPC most increased in Rab3A(-/-) mice has a slow rise, which suggests it is not the result of full collapse fusion. To test if the slow mEPCs increased after loss of Rab3A could be due to malfunctioning fusion pores, we used carbon fibre amperometry to record pre-spike feet, which have been shown to correspond to the initial opening of a narrow fusion pore, in adrenal chromaffin cells of wild-type and Rab3A(-/-) mice. We found that small amplitude pre-spike feet with abnormally long durations were increased in Rab3A(-/-) cells. The correspondence between mEPC and amperometric data supports our interpretation that slow rising, long half-width mEPCs are caused by reduced diameter fusion pores that remain open longer. These data could be explained by a direct action of Rab3A on the fusion pore, or by Rab3A-dependent control of vesicles with unusual fusion pore characteristics.

Temperature dependency of miniature end plate currents from the extraocular muscle of Antarctic teleost fishes

The effects of temperature on the decay phase of post-synaptic currents were examined to determine the extent of temperature compensation in the inferior oblique extraocular muscles of four Antarctic fishes (Trematomus bernacchii, Trematomus pennellii, Trematomus hansoni, and Pagothenia borchgrevinki, Family Nototheniidae). At ambient temperatures, different fish produce miniature end plate currents (MEPCs), which vary in duration and rate of decay. Low temperatures normally prolong MEPCs, however, Antarctic fishes were found to produce MEPCs of significantly shorter duration than predicted (based on back-extrapolation of temperate fish data to sub-zero temperatures). Notothenioid decay time constants were approximately 500 μs faster than their temperate counterparts, extrapolated to −2°C, suggesting that the difference is consistent with temperature compensation in the neural-systems of Antarctic fish. Results presented here conform the hypothesis that post-synaptic MEPCs of Antarctic fish exhibit temperature compensation, an adaptive feature that has permitted the successful radiation throughout the Southern Ocean.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04±0.06 ms in wild-type junctions and 5.4 ms±0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.

Minimal levels of cold-adaptation in postsynaptic currents of a subantarctic teleost, Notothenia rossii (Perciformes: Notothenioidei: Nototheniidae)

As a part of the ICEFISH04 project on the RVIB Nathaniel B. Palmer, miniature end plate currents (MEPCs) were recorded from the extraocular muscles of Notothenia rossii captured at King Edward Point, South Georgia. A total of 1,176 MEPCs were recorded from the inferior oblique extraocular muscles of four specimens, over a temperature range of 1–12°C. The MEPCs were normal in form, with a rapid quasi-linear increase in inward current (typically <500 μs), followed by a slower exponential decay of the inward current to baseline. Exponential decay rates were calculated for individual MEPCs by linear regression of the log-transformed data, and converted to exponential time constants (τ). Only those MEPCs that fit the exponential model well, with r 2 ≥ 0.95 (or in some cases r 2 ≥ 0.99) were used for further calculations. At temperatures between 1 and 2°C, τ ranged from about 2,000 to 4,000 μs, similar to values extrapolated for temperate teleosts at the same temperature, but significantly longer than τ from MEPCs of high-latitude Antarctic nototheniids. Between 11 and 12°C, τ values for the N. rossii MEPCS were mainly between 1,100 and 1,700 μs, giving a Q 10 of 2.05. An Arrhenius plot and linear regression were used to describe the effect of changing temperature on the decay phase of the N. rossii MEPCs: −ln τ = 27.887−6078/K, yielding an Arrhenius temperature coefficient (μ or apparent E a) of −50.5 ± 2.9 (95% CL) kJ mol−1 deg−1. When compared with other nototheniids, these results showed that the neuromuscular junctions of N. rossii are compensated for low temperature, but not to the same degree as those of high Antarctic species.

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca 2+ ions with other bivalent cations (Sr 2+, Ba 2+, Cd 2+, Mg 2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca 2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca 2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca 2+ from intracellular stores. The effect of extracellular Ca 2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.

Effects of high-potassium solutions and caffeine on synaptic vesicle exoendocytosis processes in the frog neuromuscular junction

Studies on frog skin-pectoris muscle preparations using vital fluorescent microscopy showed that stimulation of transmitter secretion using high-potassium solutions with the endocytosis marker FM 1-43 induced bright spots in all motor nerve terminals, these representing accumulations of vesicles undergoing the exoendocytic cycle in the active zones of nerve endings. Stimulation of transmitter secretion with caffeine evoked bright spots only in some nerve terminals and only in some parts of the terminals. In summer, the number of bright spots on stimulation of transmitter secretion by caffeine increased sharply. Extracellular recording of spontaneous synaptic signals showed that high-potassium solutions, like caffeine, produced dose-dependent increases in the frequency of miniature endplate currents. However, while high-potassium solutions always increased the frequency, this occurred with caffeine in only a proportion of experiments. This leads to the conclusion that exoendocytosis processes can occur both because of the influx of Ca(2+) ions into nerve endings as a result of depolarization (high-potassium solutions) and because of the release of Ca(2+) ions from the endoplasmic reticulum (caffeine). The possible spatial localization of the endoplasmic reticulum in nerve endings is discussed. The endoplasmic reticulum is suggested to have a role in synapse remodeling processes.

Effect of Calcium on Spontaneous Quantal Transmitter Secretion from ACh-Loaded Myocytes

Spontaneous secretions occur in both neurons and non-neuronal cells, and calcium is important for these secretion processes. However, the detailed roles of calcium on the secretions have not yet been identified. In the present study, cultured Xenopus myocytes loaded with exogenous acetylcholine (ACh) into the cytoplasm in the absence of extracellular Ca 2+ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents. Analysis of the frequencies, amplitudes, and time courses of these currents suggests that similar cellular mechanisms are involved in the secretions of ACh in normal medium and Ca 2+-free solution. Various doses of ryanodine were used to regulate the intracellular Ca 2+ to different levels. The spontaneous ACh secretion from myocytes in Ca 2+-free medium was decreased by reducing intracellular Ca 2+ levels and enhanced by increasing cytosolic Ca 2+ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca 2+ while Ca 2+ in the sarcoplasmic reticulum plays a crucial role in the secretions.

Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia'

The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eight patients with congenital myasthenic syndromes affecting primarily proximal limb muscles ['limb-girdle myasthenia' (LGM)]. All had moderate to severe weakness of the proximal muscles, without short-term clinical fatigability but with marked variation in strength over periods of weeks or months, with little or no facial weakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showed decrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increased jitter and blocking was detected by SFEMG, confirming the presence of impaired neuromuscular transmission. None of the patients had serum antibodies against acetylcholine receptors (AChRs). Two of the patients had similarly affected siblings. Intracellular recording from isolated nerve-muscle preparations revealed that the quantal content (the number of ACh quanta released per nerve impulse) was only approximately 50% of that in controls. However, the quantal size (amplitude of miniature end-plate currents) and the kinetic properties of synaptic potentials and currents were similar to control values. The area of synaptic contact and extent of post-synaptic folding were approximately 50% of control values. Thus, the quantal content per unit area of synaptic contact was normal. The number of AChRs per NMJ was also reduced to approximately 50% of normal, so the local AChR density was normal. Immunolabelling studies revealed qualitatively normal distributions and abundance of each of 14 proteins normally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane and post-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the following proteins: AChR subunits, rapsyn, ColQ, ChAT or muscle-specific kinase. Response of these patients to treatment was varied: few showed long-term improvement with pyridostigmine and some even deteriorated with treatments, while others had intolerable side-effects. Several patients showed improvement with 3,4-diaminopyridine, but this was generally only transient. Ephedrine was helpful in half of the patients. We conclude that impaired neuromuscular transmission in these LGM patients results from structural abnormalities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immediate events of neuromuscular transmission.

Novel β subunit mutation causes a slow-channel syndrome by enhancing activation and decreasing the rate of agonist dissociation

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V → F substitution in the M1 domain of the β subunit (βV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation βV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the βV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the βV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the βV229F mutation on the miniature endplate currents and thus are direct evidence that the βV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Two-component action of ATP on transmitter release from the motor nerve endings at the neuromuscular junction

s / Journal of Physiology Paris 99 (2006) 245–278 255 evoked synaptic responses, muscle twitch and tetanic tensions. In addition, complementary information was obtained by confocal laser scanning microscopical examination of the endplate region of the various transgenic muscles in order to determine the characteristics of muscle nicotinic ACh receptor clustering. The results obtained indicate that focally recorded miniature endplate currents (MEPC) exhibit a prolonged decay time in all transgenic mice examined with the exception of PRiMA / mutant muscles in which MEPCs were not affected. Complementary morphological examination revealed that there is an important remodeling and different clustering of muscle nicotinic ACh receptors at junctions lacking AChE activity in which MEPCs were prolonged. These results will be discussed in terms of physiological adaptation or compensation for the absence of AChE activity, and in relation to the expression of AChE and BChE at the neuromuscular junction. Supported by the AFM and by ACI # 0220530. doi:10.1016/j.jphysparis.2005.12.035 Camp, S., Zhang, L., Marquez, M., et al., 2004. In: Inestrosa, N.C., Campos, E.O. (Eds.), Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects. p. 43. Feng, G., Krejci, E., Molgo, J., Cunningham, J.M., Massoulie, J., Sanes, J.R., 1999. Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function. J. Cell. Biol. 144, 1349–1360. Minic, J., Chatonnet, A., Krejci, E., Molgo, J., 2003. Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions. Br. J. Pharmacol. 138, 177–187. Perrier, A.L., Massoulie, J., Krejci, E., 2002. PRiMA: the membrane anchor of acetylcholinesterase in the brain. Neuron 33 (2), 275–285. Xie, W., Stribley, J.A., Chatonnet, A., Wilder, P.J., Rizzino, A., McComb, R.D., Taylor, P., Hinriches, S.H., Lockridge, O., 2000. Postnatal developmental delay and supersensitivity to organophosphate in genetargeted mice lacking acetylcholinesterase. J. Pharmacol. Exp. Ther. 293, 896–902. The possible contribution of mast cells in the methylmercury-evoked effects Elizabeth E. Graevskaya Biophysical Department, Biological Faculty, Lomonosov Moscow State University, Vorobevy Gory, Moscow 119992, Russia E-mail address: graevsk@biophys.msu.ru Methylmercury (MeHg) is a well-established significant environmental neurotoxic agent, known as the cause of Minamata disease (IPCS, 1990). As it is known MeHg penetrates the blood-brain barrier to cause severe neurological symptoms in human beings and experimental animals, destructs neurons and glial cells, and disrupts neuronal migration. At the same time, increasing evidence indicates that neuroimmune interactions, including neuroinflammatory processes, may involve also mast cells. Mast cells and their transmitter content play a role in triggering an immune response in the brain. In our study we tried to define the possible contribution of mast cells in the development of methylmercury-evoked effects. As a model we used rat peritoneal mast cells, because it is easier to get the requisite amount of mast cells for measuring modifications of transmitter release. We investigated the effects of MeHg on mast cell degranulation induced by non-immunological stimuli (the selective inductor of histamine release): the compound 48/80 and the calcium ionophore A23187, both in vivo and in vitro. Eight, 12 and 15 days after administration of MeHg (10 mg/kg/day) we observed that the calcium ionophore A23187and 48/80-induced histamine release gradually decreased. Mast cells from rat exposed 24 or 72 h to a single dose of MeHg (20 mg/kg) showed a significant inhibition of the A23187-stimulated histamine secretion, but no change in the compound 48/ 80-stimulated release. In vitro incubation of peritoneal mast cells with MeHg alone (10 –10 6 M) did not induce mast cell degranulation, but modified the activation of mast cells by compound 48/80 and calcium ionophore A23187. We observed a facilitation of stimulated secretion by prior incubation with low dose of MeHg (10 8 M) and an inhibition at 10 6 M MeHg. These results show that MeHg treatment can modify mast cell function in vivo and in vitro and provide insight into the understanding what role this cell has in the pathogenesis of Minamata disease-connected disorders. doi:10.1016/j.jphysparis.2005.12.036 International Programm of Chemical Safety (IPCS), 1990. Environmental health criteria 101, methylmercury. World Health Organization, Geneva, p. 144. Effect of delta sleep inducing peptide on NMDA and GABA evoked ion currents in cortical, cerebellar and hippocampal

Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate

Glutamate toxicity in traumatic brain injury, ischemia, and Huntington’s disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 μM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 μM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 μM N-benzyloxylcarbonyl-Asp(OCH 3)-Glu(OCH 3)-Val-Asp(OCH 3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na + channels, N-methyl- d-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na + channels, N-methyl- d-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na + channels, N-methyl- d-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.

Different sensitivity of miniature endplate currents of the rat extensor digitorum longus, soleus and diaphragm muscles to a novel acetylcholinesterase inhibitor C-547

A novel derivative of 6-methyluracil, C-547, increased the amplitude and prolonged the duration of miniature endplate currents (MEPCs) which is typical for acetylcholinesterase inhibition. In the soleus and extensor digitorum longus significant potentiation was detected at nanomolar concentrations. In contrast, in the diaphragm muscle, the increase in the amplitudes of the MEPCs and the decay time constant appeared only when the concentration of C-547 was elevated to 1 x 10(-7) M. Possible consequences for the exploitation of this drug, which can selectively inhibit AChE in particular synapses, are discussed.