Spontaneous Transmitter Release Research Articles

Visualization of Cyclic AMP–Regulated Presynaptic Activity at Cerebellar Granule Cells

Adenylyl cyclase (AC) modulation of vesicular cycling was visualized at cultured cerebellar granule cell synapses using the sequential uptake of antibodies directed against the intraluminal domain of synaptotagmin I. Vesicle recycling due to spontaneous transmitter release in the absence of action potentials was increased by the AC/protein kinase A (PKA) activators forskolin and CPT–cAMP. These effects were blocked by the PKA inhibitor Rp–cAMPs. Cyclic AMP elevation also induced new cycling at previously silent sites. Activation of L-AP4–sensitive mGluR reduced the cAMP/PKA enhancement at preexisting synapses downstream of both AC and calcium channels. Modulation of the turnover and the number of vesicular release sites provide one mechanism that may underlie cAMP-dependent cerebellar long-term potentiation.

Amyotrophic lateral sclerosis: serum factors enhance spontaneous and evoked transmitter release at the neuromuscular junction.

Sera from 30 patients with sporadic amyotrophic lateral sclerosis (ALS) were tested to determine their effects at the neuromuscular junction. Spontaneous transmitter release was significantly increased, as evidenced by a 151% increase in MEPP frequency, by sera from 16 ALS patients. In addition, 16 patients' sera elevated EEP quantal content by an average of 89%. Eleven sera produced both effects. There was no consistent change in MEPP amplitude or time course, indicating the absence of a humoral effect on postjunctional ACh receptors or endplate membrane function. These results suggest that a portion of the sporadic ALS patient population possess serum factors that can alter presynaptic function of the motor nerve terminal. Evidence from the present experiments indicates that alterations at the neuromuscular junction are a result of a combination of increased Ca2+ influx into the cell and an independent increase in intracellular calcium concentration.

An Analysis of Low Level Doses of Cholinesterase Inhibitors in Cultured Neurons and Hippocampal Slices of Rats

Recent data indicate that the neurotoxic effects of organophosphate compounds, including those of the nerve agents VX and sarin, are not solely due to irreversible cholinesterase inhibition. in this study we applied the patch clamp technique to hippocampal neurons in culture and slices to investigate the effects of VX, sarin and huperzine A on transmitter release and the mechanisms related with such effects. the nerve agents VX and sarin at very low concentrations significantly reduced the evoked release of GABA and glutamate. This effect was dependent of the activation of muscarinic receptors. in the presence or absence of the Na* -channel blocker tetrodotoxin (TTX), VX increased the frequency of spontaneous glutamate and GABA-induced postsynaptic currents. the effect of VX on TTX-insensitive spontaneous currents appears to be unrelated to cholinesterase inhibition, because it could be detected even after cholinesterase was blocked by high concentrations of the nerve agent soman. the ability of the nerve gases to decrease evoked release of GABA and increase spontaneous transmitter release may underlie some of the neurotoxic effects of the compounds. Huperzine A did not affect spontaneous or evoked release of GABA and glutamate, suggesting that this compound may be a pure cholinesterase inhibitor and had no effect on postsynaptic GABAA or AMP A receptors

Spontaneous acetylcholine release in mammalian neuromuscular junctions.

Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter, release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 microM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, omega-conotoxin GVIA (omega-CgTx, 5 microM). On the other hand, nifedipine and omega-CgTx had no effect on K(+)-induced transmitter secretion. omega-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca(2+)-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by "bulk" intracellular Ca2+ concentration increases.

Pre- and postsynaptic modulation of spinal GABAergic neurotransmission by the neurosteroid, 5β-pregnan-3α-ol-20-one

The neuroactive steroid 5β-pregnan-3α-ol-20-one (5β3α) modulates GABA A receptor function by potentiating postsynaptic GABA currents. While much is now known about the postsynaptic action of neurosteroids, far less is known about how they affect neurotransmission. We have investigated the synaptic actions of 5β3α in a simple vertebrate model, the embryo of the clawed toad, Xenopus laevis, in which a known GABAergic pathway, activated by the rostral cement gland, terminates swimming when the animal contacts an obstruction. Cement gland stimulation evokes bicuculline-sensitive inhibitory postsynaptic potentials (IPSPs) in motorneurones that terminate swimming and which are greatly enhanced by the presence of (1–5 μM) 5β3α. In the presence of TTX, depolarising inhibitory potentials are recorded with KCl-filled microelectrodes reflecting the spontaneous release of transmitter. The majority are glycinergic with durations of 20–80 ms and are blocked by strychnine while the remainder are GABAergic with durations of 90–200 ms and are abolished by bicuculline. We show here that, in the presence of 5β3α, the spontaneous GABA IPSPs lengthen dramatically in some cases to over 500 ms, but the glycine potentials are unaffected. The steroid has no other detectable postsynaptic effects in that the range of amplitudes of GABA potentials is unaffected and there is no change in the resting membrane potential. However, 5β3α also caused a marked increase in the rate of occurrence of spontaneous GABA potentials. This suggests a novel presynaptic site of action in which the steroid enhances the probability of vesicular GABA release from GABA terminals.

Neuromodulation of activity-dependent synaptic enhancement at crayfish neuromuscular junction

Action potential-evoked transmitter release is enhanced for many seconds after moderate-frequency stimulation (e.g. 15 Hz for 30 s) at the excitor motorneuron synapse of the crayfish dactyl opener muscle. Beginning about 1.5 s after a train, activity-dependent synaptic enhancement (ADSE) is dominated by a process termed augmentation (G.D. Bittner, D.A. Baxter, Synaptic plasticity at crayfish neuromuscular junctions: facilitation and augmentation, Synapse 7 (1991) 235–243 [4]; K.L. Magleby, Short-term changes in synaptic efficacy, in: G.M. Edelman, L.E. Gall, C.W. Maxwell (Eds.), Synaptic Function, John Wiley and Sons, New York, 1987, pp. 21–56; K.L. Magleby; J.E. Zengel, Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.) 257 (1976) 449–470) which decays approximately exponentially with a time constant of about 10 s at 16°C, reflecting the removal of Ca 2+ which accumulates during the train in presynaptic terminals (K.R. Delaney, D.W. Tank, R.S. Zucker, Serotonin-mediated enhancement of transmission at crayfish neuromuscular junction is independent of changes in calcium, J. Neurosci. 11 (1991) 2631–2643). Serotonin (5-HT, 1 μM) increases evoked and spontaneous transmitter release several-fold (D. Dixon, H.L. Atwood, Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode, J. Neurobiol. 16 (1985) 409–424; J. Dudel, Modulation of quantal synaptic release by serotonin and forskolin in crayfish motor nerve terminals, in: Modulation of Synaptic Transmission and Plasticity in Nervous Systems, G. Hertting, H.-C. Spatz (Eds.), Springer-Verlag, Berlin, 1988; S. Glusman, E.A. Kravitz, The action of serotonin on excitatory nerve terminals in lobster nerve–muscle preparations, J. Physiol. (Lond.) 325 (1982) 223–241). We found that ADSE persists about 2–3 times longer after moderate-frequency presynaptic stimulation in the presence of 5-HT. This slowing of the decay of ADSE by 5-HT was not accompanied by significant changes in the initial amplitude of activity-dependent components of enhancement 1.5 s after the train. Measurements of presynaptic [Ca 2+] indicated that the time course of Ca 2+ removal from the presynaptic terminals after trains was not altered by 5-HT. Changes in presynaptic action potential shape, resting membrane potential or postsynaptic impedance after trains cannot account for slower recovery of ADSE. Axonal injection of EDTA slows the removal of residual Ca 2+ and the decay of synaptic augmentation after trains of action potentials (K.R. Delaney, D.W. Tank, A quantitative measure of the dependence of short-term synaptic enhancement on presynaptic residual calcium, J. Neurosci. 14 (1994) 5885–5902), but has little or no effect on the 5-HT-induced persistence of ADSE. This also suggests that the time course of ADSE in the presence of 5-HT is not determined primarily by residual Ca 2+ removal kinetics. The slowing of ADSE recovery after trains by 5-HT reverses with washing in 5-HT-free saline along with the 5-HT-mediated enhancement of release.

Presynaptically Silent Synapses: Spontaneously Active Terminals Without Stimulus-Evoked Release Demonstrated in Cortical Autapses

This study addresses the question of whether synapses that are capable of releasing transmitters spontaneously can also release them in an excitation-dependent manner. For this purpose, whole cell patch recordings were performed for a total of 48 excitatory solitary neurons in a microisland culture to observe excitatory autaptic currents elicited by spontaneous transmitter release as well as by somatic excitation. A somatic Na+-spike, induced in response to a short voltage step, evoked excitatory postsynaptic currents (EPSCs) of various amplitudes through the autapses; in some cases, no response was noticeable. To make sure that the recorded autaptic spontaneous EPSCs (sEPSCs) under a voltage clamp resulted from independent release of transmitters and were not associated with action potentials, sEPSCS in the presence and absence of tetrodotoxin (TTX) were compared in six cells. In the presence of TTX the evoked EPSCs were completely eliminated, whereas the sEPSCs were still observed and the amplitude distribution histograms were statistically not different from those recorded in the absence of TTX. A quantitative analysis of the sEPSCs (presumably miniature EPSCs) showed that the amplitude of stimulus-evoked EPSCs did not correlate with either the frequency or median amplitudes of the sEPSCs or the age of the culture. To identify whether the absence of stimulus-evoked response was caused either by conduction failure of excitation along the axons or by impairment of the release machinery that links the terminal depolarization to vesicle exocytosis, we examined whether high K+ and hypertonic solutions could facilitate the spontaneous release of transmitters. Although the hypertonic solution increased the spontaneous release in all cells tested (n = 18), the high K+ solution had a differential effect in increasing spontaneous release, i.e., the cells with larger evoked responses were more readily facilitated by the high K+ solution. Because the high K+ solution induced depolarization of presynaptic terminals, the present results indicated that the smaller evoked responses were due to the larger number of impaired or "silent" presynaptic terminals that were unable to link presynaptic depolarization to transmitter release. In summary, the present experiments provided evidence that at least some of the presynaptic terminals are silent in response to stimuli, while remaining spontaneously active at the same time. Because this phenomenon is due to the lack of sensitivity to depolarization at the terminals, these synaptic terminals seem incapable of linking terminal depolarization to transmitter release.

Presynaptic inhibition of elicited neurotransmitter release

Activation of presynaptic receptors for a variety of neurotransmitters and neuromodulators inhibits transmitter release at many synapses. Such presynaptic inhibition might serve as a means of adjusting synaptic strength or preventing excessive transmitter release, or both. Previous evidence showed that presynaptic modulators inhibit Ca 2+ channels and activate K + channels at neuronal somata. These modulators also inhibit spontaneous transmitter release by mechanisms downstream of Ca 2+ entry. The relative contribution of the above mechanisms to the inhibition of elicited release has been debated for a long time. Recent evidence at synapses where the relationship between transmitter release and presynaptic Ca 2+ influx has been well characterized suggests that inhibition of presynaptic voltage-dependent Ca 2+ channels plays the major role in presynaptic inhibition of elicited neurotransmitter release. In addition, modulation of the release machinery might contribute to inhibition of elicited release.

Multiple types of Ca2+ channels in mouse motor nerve terminals.

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either omega-agatoxin IVA [omega-Aga-IVA (0.3 microM), a blocker of P-type Ca2+ channels] or omega-conotoxin MVIIC [omega-CTx-MVIIC (2 microM), a P- and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 microM, a blocker of L-type Ca2+ channels) plus omega-conotoxin GVIA [omega-CTx-GVIA (2 microM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3,4-diaminopyridine (3,4-DAP, a K+-channel blocker) or high Ca2+ (10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3,4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both omega-Aga-IVA and omgea-CTx-MVIIC; omega-Aga-IVA exerted only a partial inhibitory effect on endplate potentials, and the omega-Aga-IVA-resistant component was further inhibited by omega-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 microM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by omega-CTx-MVIIC with further incomplete inhibition by omega-Aga-IVA. Cd2+ (300 microM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 microM) with omega-CTx-GVIA (2 microM) was without inhibitory effect. We conclude that multiple types of Ca2+ channels, i.e. omega-Aga-IVA-sensitive, omega-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.

Pharmacological Properties and Identification of Cardiotonic Principles from the Indian Snuff,Maquira sclerophylla, Ducke

The powdered bark obtained from Maquira sclerophylla Ducke is a snuff used by Amazonian natives. Pharmacological studies on rodents, guinea-pigs, rabbits and dogs revealed cardiovascular changes as the major effects produced by the polar phase of the bark ethanol extract. Oriented by this pharmacological activity a cardenolide fraction was chemically purified from the crude extract. Both crude extract and the purified fraction produced a positive inotropic effect in isolated heart preparations from guinea-pigs and rabbits, and inhibited the Na+, K+-ATPase activity of the enzyme isolated from bovine ventricles. The fraction yielded two major purified cardenolides identified as Maquiroside A (C30H46O8) and Cymarin (C30H44O9). Simultaneous intracellular recordings of the resting membrane potential (RMP) and miniature endplate potentials in rat diaphragm muscle fibres showed that the effect of the purified fraction predominated on the motor nerve terminals enhancing the spontaneous transmitter release, without major changes of RMP values. Comparatively, the effect of ouabain prevailed on the muscle fibre membrane. Considering that both effects depend on the Na+, K+-ATPase activity, the results indicated that the compounds derived from Maquira exert differential actions on this enzyme in the nerve and muscle membrane. These observations may explain the use of this toxic snuff in native festivities. © 1997 by John Wiley & Sons, Ltd.

Specific Muscle Contacts Induce Increased Transmitter Release and Neuritic Arborization in Motoneuronal Cultures

Identified buccal motoneuron 19 (B19), isolated from the nervous system ofHelisomaand plated into cell culture, is selective in synapse formation and requires contact with appropriate muscle targets before acquiring secretory competence. Action potential-regulated transmitter release was elevated specifically at neuritic arbors in contact with appropriate muscle fibers. In contrast, contact between B19 and inappropriate muscle targets failed to induce changes in presynaptic secretory properties. Spontaneous transmitter release rate was elevated globally across neuritic arbors of B19 in appropriate muscle cocultures, including arbors without direct muscle contact. In addition, dual contacts formed between B19 and two different muscle targets resulted in significant elevations in excitation–secretion coupling only at neuritic sites of appropriate muscle contact and not at sites of contact with mismatched targets. Image analyses of presynaptic neuronal architecture revealed that appropriate, but not novel, muscle targets elicited increased arborization of neurites at sites of neuron–muscle contact. In contrast to results with neuron B19, secretory properties of buccal neuron 5 (B5), a neuron capable of forming inappropriate chemical synapses with a number of novel targets in culture, were not enhanced by inappropriate muscle contact. We conclude that muscle targets vary in their ability to induce presynaptic modifications (e.g., excitation–secretion coupling, spontaneous transmitter release, and neuritic arborization) in identified motoneurons ofHelisoma.These results imply that separate molecular pathways exist which control each of these events during synaptic differentiation. The differential potency of muscle targets for induction of presynaptic changes indicates the existence of a cellular mechanism of target recognition. We hypothesize that such a mechanism underlies the ability of neuron B19 to discriminate between potential postsynaptic partners in cell culture.

The effects of the muscle relaxant, CS-722, on synaptic activity of cultured neurones.

1. The pharmacological properties of the centrally acting muscle relaxant, CS-722, were studied in cultured hippocampal cells and dorsal root ganglion cells of the rat using the whole-cell variation of the patch clamp technique. 2. CS-722 inhibited the occurrence of spontaneous excitatory and inhibitory postsynaptic currents in hippocampal neurones at concentrations of 100-300 microM, but had no effect on postsynaptic currents evoked by the application of glycine, gamma-aminobutyric acid, glutamate or N-methyl-D-aspartate. 3. CS-722 reduced voltage-gated sodium currents, while shifting the sodium channel inactivation curve to more negative membrane potentials. This effect is similar to that reported for local anaesthetics. Voltage-gated potassium currents were decreased by CS-722 by approximately 20%, whereas voltage-activated calcium currents were inhibited by about 25%. 4. CS-722 inhibited evoked inhibitory postsynaptic currents. However, the spontaneous quantal release of inhibitory transmitter was not affected. 5. The inhibitory effect of CS-722 on spontaneous inhibitory postsynaptic currents and excitatory postsynaptic currents in hippocampal cultures probably results from an inhibition of both sodium and calcium currents. This inhibitory effect is likely to be amplified in polysynaptic neuronal circuits.

Synaptic Modulation by Neurotrophic Factors: Differential and Synergistic Effects of Brain-Derived Neurotrophic Factor and Ciliary Neurotrophic Factor

Extracellular application of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) to developing neuromuscular junctions in Xenopus nerve-muscle cultures resulted in an increase in the frequency of spontaneous synaptic currents (SSCs) and in the amplitude of nerve-evoked synaptic currents. Analyses of the amplitude and time course of the SSCs suggest that these effects are attributable to elevation of presynaptic transmitter release. The actions of these two factors on the transmitter secretion process, however, are distinctly different. Fura-2 Ca2+ imaging showed that an increase in presynaptic cytosolic Ca2+ ([Ca2+]i) accompanied the synaptic potentiation by BDNF, whereas no change in [Ca2+]i was observed during synaptic potentiation by CNTF. Removing external Ca2+ also abolished the potentiating effect of BDNF but did not influence the CNTF effect. Moreover, the two factors exerted different effects on the short-term synaptic plasticity. Paired-pulse facilitation normally found at these synapses was reduced by BDNF but unaffected by CNTF; CNTF, but not BDNF, reduced the extent of synaptic depression during high-frequency tetanic stimulation. Finally, the potentiation effect of BDNF and CNTF on spontaneous transmitter release was additive when both factors were applied together to the synapse at saturating concentrations (100 ng/ml) and was highly synergistic when low doses (1 and 10 ng/ml) of both factors were used. These results suggest that because of their differential effects on the secretory machinery, BDNF and CNTF may act cooperatively in modulating the development and functioning of synapses.

Single-domain/bound calcium hypothesis of transmitter release and facilitation.

1. We describe a model of transmitter release that is based on the finding that release can be gated during the opening of individual Ca2+ channels, suggesting that the release site can be activated by the Ca2+ domain under a single channel. In this model each release site contains four independent Ca2+ binding sites or gates with unbinding kinetics graded from slow to fast and affinities ranging from high to low. All four gates must be bound for release to occur. Thus synaptic dynamics are governed by the kinetics of Ca2+ binding and unbinding from release sites, not Ca2+ diffusion. 2. Fast facilitation occurs when an action potential invades a terminal with one or more ions remaining bound to the release sites. Residual free Ca2+ is not necessary for facilitation with this mechanism, but if present it would enhance facilitation by binding to high-affinity gates between pulses. 3. This model can account for key features of release. These include fourth-power cooperativity with regard to external Ca2+; a release time course that is virtually independent of an increase in quantal content; an inverse relation between external Ca2+ and the degree of facilitation; and a steplike increase in facilitation with increasing stimulus frequency, with each step corresponding to a unitary decline in the Ca2+ cooperativity. 4. Facilitation of single-channel-based secretion is shown to be robust even if channel opening is stochastic. Spontaneous release of transmitter, assumed to be due in part to spontaneous Ca2+ channel openings, is shown to be elevated during and after a train of impulses. 5. An extension of the model to include multiple Ca2+ channels per release site demonstrates that one role of overlapping Ca2+ domains may be to accentuate depolarization-evoked release relative to spontaneous release.

Long-lasting synaptic facilitation induced by serotonin in superficial dorsal horn neurones of the rat spinal cord.

1. Modulatory actions of serotonin (5-hydroxytryptamine, 5-HT) on excitatory postsynaptic currents (EPSCs) were studied with whole-cell recordings from superficial dorsal horn (SDH) neurones in neonatal rat spinal cord slices. In one-third of SDH neurones, 5-HT induced a sustained potentiation of evoked EPSCs lasting for more than 30 min after wash-out. This potentiation was often preceded by a transient suppression of EPSCs. 2. Serotonin differentially modulated the frequency of miniature EPSCs recorded in the presence of tetrodotoxin (TTX) according to the SDH neurones, producing a transient suppression, a transient facilitation or a long-lasting facilitation. 3. The 5-HT1A-receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) suppressed the amplitude of evoked EPSCs and frequency of miniature EPSCs in a reversible manner. In contrast, the 5-HT2-receptor agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and alpha-methyl-5-HT induced long-lasting potentiations of EPSC amplitude and miniature EPSC frequency. 4. Neither the mean amplitude nor the kinetics of miniature EPSCs were affected by 5-HT during the sustained facilitation of miniature EPSC frequency, suggesting that the facilitatory effect of 5-HT was presynaptically mediated. The 5-HT-induced long-lasting facilitation of miniature EPSC frequency was observed also in Ca(2+)-free, Mg2+ solution. 5. The long-lasting facilitation of evoked EPSC amplitude and miniature EPSC frequency by 5-HT was mimicked by the phorbol ester, phorbol 12,13-dibutyrate (PDBu), and blocked reversibly by the protein kinase C (PKC) inhibitor, calphostin C. Forskolin applied together with 3-isobutyl-1-methylxanthine (IBMX) had no effect on the evoked EPSCs. 6. We conclude that serotonin can induce a long-lasting facilitation of evoked EPSCs and spontaneous release of excitatory transmitter at SDH synapses of rat spinal cord. Our results suggest that intracellular PKC linked to the 5-HT2 receptor may mediate this effect by directly activating the exocytotic machinery.

Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion.

We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [Ca2+] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavioral abnormalities and male sterility.

Suppression of potassium currents by cyanide on the mouse motor nerve terminals

NaCn at low concentrations markedly depressed the potassium currents in the motor nerve terminal of mouse triangularis sterni neuromuscular junction pretreated with potassium channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA) or glucose-free medium. Neither azide nor dinitrophenol nor ouabain mimicked the effect of cyanide. This inhibitory effect of cyanide on nerve terminal spikes was correlated to its dramatic increase in spontaneous transmitter release under glucose-free condition. These results suggest that the effect of cyanide on the electrogenesis of nerve terminals is due to the direct suppression of ATP-sensitive K + current since the effect was antagonized by ATP-sensitive K + channels opener diazoxide and this may modulate the transmitter release.

Actions of banana tree extract on smooth and cardiac muscles and in the anesthetized rat.

Pygmy tribesmen of central and southern Africa have been reported to use the juice of the banana plant stem for hunting animals and in tribal warfare (Heymer, 1974). Wooden arrows or darts may be given a poisonous tip by driving them into the plant stem for 1–2 days. An animal hit by one of these weapons becomes paralyzed but its flesh is safe to eat. An extract prepared from the juice augments, then blocks contractions of both directly or indirectly stimulated skeletal muscles (Singh and Dryden, 1985, 1990). In higher concentrations, the extract induces a sustained muscle contracture which is slowly reversed by removal of the extract. It also increases the rate of spontaneous transmitter release, as measured by the frequency of miniature endplate potentials (Singh and Dryden, 1985). The effect of the extract has now been tested on smooth and cardiac muscle preparations and in the anesthetized rat to assess whether the presumed incapacitation of the animal by the extract-tipped arrows and darts of the Pygny tribesmen was due to paralysis of limb skeletal muscles and the diaphragm or by a collapse of its cardiovascular activity.KeywordsTibialis Anterior MuscleDevelop TensionLimb Skeletal MuscleBanana StemLangendorff PreparationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Quantal components of spontaneous excitatory junction potentials at visualised varicosities

The electrical signs of spontaneous transmitter release were recorded with an extracellular electrode from single visualized sympathetic varicosities on the mouse vas deferens. Ultrastructural examination of these varicosities with the electron microscope showed that they formed close-contacts with smooth muscle cells. Amplitude-frequency histograms of the spontaneous excitatory junction potentials (SEJPs) were constructed in order to determine the statistical nature of spontaneous transmitter release from a varicosity. SEJP histograms often possessed several peaks. Statistical tests showed that these were separate modes in the histograms indicating that the SEJPs were composed of subunits. The SEJP histograms were described by a mixture of distributions in which the components were identified as quanta and there was a Poisson release of quanta. The second mode in the SEJP histograms was sometimes twice that of the first mode but generally greater, suggesting a potentiation of the effects of one quantum released nearly simultaneously with another quantum. The components in the SEJP histograms were well fitted by either a Gaussian or a gamma distribution indicating that the quantum of transmitter could be described as a Gaussian or gamma variate.

Inhibition by riluzole of glycinergic postsynaptic currents in rat hypoglossal motoneurones.

1. Riluzole has been shown to have beneficial effects in motoneurone disease, yet its effect on motoneurones is not known. To address this question, we investigated synaptic modulation by riluzole in hypoglossal motoneurones by recording glycinergic inhibitory postsynaptic currents evoked by stimulation of nearby single interneurones. 2. Glycinergic inhibitory postsynaptic currents were evoked by electrical stimulation of single interneurones and were recorded from visually identified hypoglossal motoneurones. Riluzole (10 microM) inhibited mean amplitude of evoked glycinergic inhibitory postsynaptic currents by 87%. 3. We found that riluzole suppressed sodium currents in brainstem interneurones by 23.8%. Riluzole did not modulate barium currents through voltage-activated calcium channels (98% of control). Therefore, the effect of riluzole on synaptic transmission may be mediated, in part, by stabilizing presynaptic neurones through inhibition of voltage-activated sodium currents. 4. In the presence of tetrodotoxin (0.5 microM), riluzole reduced the frequency (1.2 Hz in control to 0.6 Hz in riluzole) of spontaneous transmitter release recorded in motoneurones. 5. Riluzole was found to have no effect on mean miniature inhibitory postsynaptic current amplitude, therefore the reduction in spontaneous transmitter release cannot be due to an action on postsynaptic glycine receptors. 6. We conclude that riluzole inhibits synaptic transmission presynaptically, independent of a reduction in the excitation of presynaptic neurones.