Termination of the presynaptic effect of carbacholine by tubocurarine]

The presynaptic action of carbacholine (Cch) was studied in experiments on the frog sartorius muscle neuromuscular preparation. Cch proved to decrease the quantum content of the end plate potential (EPP); this effect was induced by a direct Cch action on the motor nerve endings. d-tubocurarine decreased the sensitivity of the nerve endings to Cch significantly. Both d-tubocurarine and Cch had concurrent antagonistic interrelations in respect to their action on the quantum content of EPP. Atropine in low concentrations had no influence on the presynaptic effect of Cch. It was concluded that Cch decreased the quantum content of EPP through the N-cholinergic structures of the motor nerve endings.     

Transmitter release in proximal and distal parts of a nerve terminal of the frog sartorius muscle

Evoked synaptic potential were recorded extracellularly in experiments on a nervemuscle preparation of the frog sartorius muscle. A decrease in evoked transmitter release was found from the proximal to the distal parts of the nerve ending, due to a decrease in the probability of transmitter quantum release. The terminal portions of the synapse are less sensitive than the proximal parts to changes in Ca⁺⁺ concentration, they show less marked facilitation of transmitter release during paired and repetitive stimulation, and exhibit deeper and more rapidly developing depression. It is concluded that differences in transmitter release in the terminal parts of the synapse are due to the low reserves of transmitter and the lower premeability of the presynaptic membrane to Ca⁺⁺.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of presynaptic ryanodine receptors in regulation of the kinetics of the acetylcholine quantal release in the mouse neuromuscular junction

To determine the role of presynaptic ryanodine receptors in the regulation of the kinetics of neurotransmitter quantum secretion caused by a nerve impulse in the experiments on the mouse neuromuscular junction, temporal parameters of phase synchronous and asynchronous delayed release of acetylcholine under the conditions of ryanodine receptors block and rhythmic stimulation were examined. The analysis of histograms of synaptic delays of the uni-quantal end-plate currents registered within 50 ms after the onset of the presynaptic action potential showed that ryanodine receptor blockers ryanodine, TMB-8 and dantrolene reduced the intensity of both phase synchronous and delayed asynchronous release of the mediator. The proportion of quanta released synchronously increased at the expense of the reduction of quantum numbers forming the delayed asynchronous release, i.e., there was a redistribution of quanta between synchronous and asynchronous phases of secretion. A block of ryanodine receptors also reduced the fluorescence intensity of the specific fluorescent calcium-sensitive dye Fluo-3 AM, which indicates a decrease in the intracellular calcium ion concentration. Thus, the presynaptic ryanodine receptors control the intracellular content of calcium ions under repetitive stimulation of the nerve endings and contribute to the modulation of the time parameters of the evoked release of the neurotransmitter quanta by increasing the intensity of the delayed asynchronous release of neurotransmitters.     

Dynamic aspects of presynaptic calcium currents mediating synaptic transmission

Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers transmitter release. Direct recording of Ca2+ currents from the calyx of Held nerve terminal revealed that presynaptic VGCCs undergo various modulations via presynaptic G protein-coupled receptors (GPCRs), Ca2+-binding proteins and a developmental switch of their alpha1 subunits. Dynamic changes of presynaptic VGCCs alter synaptic efficacy, thereby contributing to a variety of modulations of the CNS function.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.     

Effect of sodium nitroprusside on the mediator release and functional properties of the postsynaptic membrane at the neuromuscular junction]

The effect of nitric oxide donor sodium nitroprusside on the end-plate currents was studied under two-electrode voltage-clamp condition at frog neuro-muscular junction. Sodium nitroprusside (10(-4) M) reduced to the half the amplitude of end-plate currents while did not change miniature end-plate currents indicating the presynaptic nature of end-plate depression. In keeping with such suggestion sodium nitroprusside essentially (to 33%) suppressed the frequency of miniature end-plate currents but did not affect the decay time constant and voltage-dependence of miniature end-plate decay. In contrast to another presynaptic inhibitors sodium nitroprusside rather reduced than increased the presynaptic facilitation and did not change postsynaptic potentials. Thus, nitric oxide is the powerful inhibitor of both evoked and spontaneous transmitter release and did not change postsynaptic potential.     

Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.     

The generation and spread of excitation in the motor nerve ending of the frog

In experiments on the frog cutaneous-pectoris muscle under the visual control the evoked responses of nerve endings were recorded in proximal, central and distal parts of the terminal. At the fixed position of the recording electrode the local iontophoretic application of TTX to different terminal parts and Ranvier's nodes was performed. It was concluded, that local currents at more proximal terminal parts provide the depolarization of the located parts and conduction of excitation to the nonexcitable parts. Inward currents at behind located and already excited parts are shortened and decreased by local currents from more distal parts during the propagation of excitation. It results in shortening of the action potential and decreasing of the transmitter release in more proximal parts. This effect disappeared along the nerve terminal due to decreasing of inward Na current at the end parts.     

Temperature Effect on Proximal to Distal Gradient of Quantal Release of Acetylcholine at Frog Endplate

The conduction velocity of the nerve terminal, mean quantal content, and release latencies of uniquantal endplate currents (EPCs) were recorded in proximal, central, and distal parts of the terminal by extracellular pipettes located 5, 50, and 100 mm from the end of myelinated nerve trunk. The spike conduction velocity, minimal latency, modal value of the latency histograms, and time interval during which 90% of EPCs released (P₉₀) at distal, central, and proximal part of the frog nerve terminal have different temperature dependency between 10° and 28°C. As shown by the size and time-course of reconstructed multiquantal EPCs, the secretion synchronization, which is greatest in distal parts, compensates at least partly for the progressive slowing of spike conduction velocity in the proximodistal direction, in particular at lower temperatures.     

The voltage-dependence of transmitter release

In this paper we summarize voltage clamp experiments characterizing transmission at the squid giant synapse. The overall goal of these experiments was to determine a synaptic transfer curve relating presynaptic Ca currents (ICa) to resultant postsynaptic responses. Here we focus on interpreting the phenomenon of transfer curve "hysteresis", which has been proposed to result from an intrinsic voltage-dependence of the transmitter release process. One potential problem in analyzing transfer curves comes from contamination of presynaptic Ca currents by outward currents. Linear leakage currents can be measured and taken into account, but after such corrections ICa measurements at positive potentials are still distorted by outward currents. The presence of residual outward currents at positive potentials results in a voltage-dependent bias in ICa measurement and probably contributes to transfer curve hysteresis. A pharmacological procedure which subtracts currents other than those flowing through Ca channels can be used to circumvent this bias in ICa measurement. Gradients in membrane potential along a nominally voltage clamped presynaptic terminal can allow inappropriate release of transmitter from poorly clamped regions of the terminal. Release from such regions may also contribute to transfer-curve hysteresis when standard voltage clamp methods are employed. A method of localized Ca application which restricts transmitter release to well-clamped presynaptic regions can be used to avoid this problem. Transfer curves measured using refined procedures for ICa measurement and suppression of voltage gradient effects on release exhibit little hysteresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime inAplysia

1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation.     

Structure, isolation, composition and reconstitution of the neuronal fusion pore

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.     

Role of Ca(2+) channels in short-term synaptic plasticity

Repetitive nerve activity induces various forms of short-term synaptic plasticity that have important computational roles in neuronal networks. Several forms of short-term plasticity are caused largely by changes in transmitter release, but the mechanisms that underlie these changes in the release process have been difficult to address. Recent studies of a giant synapse - the calyx of Held - have shed new light on this issue. Recordings of Ca(2+) currents or Ca(2+) concentrations at nerve terminals reveal that regulation of presynaptic Ca(2+) channels has a significant role in three important forms of short-term plasticity: short-term depression, facilitation and post-tetanic potentiation.     

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.     

Estimates of quantal content during 'chemical potentiation' of transmitter release.

The number of quantal transmitter packets (m), released from motor nerve terminals in response to a single stimulus, has been estimated from the ratio of the amplitudes of endplate currents (e.p.c.) to spontaneous miniature endplate currents (m.e.p.c.), in voltage-clamped endplates of the frog. At 6 degrees C, the average value of m at normal nerve-muscle junctions was about 300. If allowance is made for the temporal dispersion of quantal transmitter release during the e.p.c., this value is increased by about 30%. After treatment with diaminopyridine or tetraethylammonium, transmitter release in response to a nerve stimulus is greatly enhanced and values of m exceeding 10(4) are frequently found. Moreover, the duration of the e.p.c. becomes much longer than that of the m.e.p.cs. The number of packets then liberated during the e.p.c. is much larger than the number of 'active zones' of the endplate and may even exceed the total number of vesicles lined up in twin-files adjacent to the presynaptic membrane.     

Muscarinic agonists do not alter the process of spontaneous quantum secretion of the mediator from motor nerve endings in the frog

The action of carbacholine (Cch) and muscarinic agonists--methylfurmethide, oxotremorine, metacholine, L- and D-F-2268 isomers on spontaneous quantum transmitter release was investigated in experiments on the frog sartorius muscle of R. temporaria. Cch decreased the frequency of miniature and plate potentials (mEEP) by 48%. The muscarinic agonists at a wide concentration range had no effect on mEEP frequency both in normal ionic medium and with potassium concentration increase to 10 mmol/l. The data obtained confirm the assumption that the inhibitory action of Cch on spontaneous transmitter release is unrelated to its muscarinic activity. It was concluded that muscarinic cholinoreceptors controlling spontaneous transmitter release are absent in the frog motor nerve terminals.     

Increased density of fluorescent adrenergic fibers around the middle cerebral arteries of stroke-prone spontaneously hypertensive rats.

The distribution of fluorescent adrenergic nerve fibers in the proximal portion (horizontal segment, Hs) and the three distal portions (major branches) of the middle cerebral arteries (MCA) was examined in stroke-prone spontaneously hypertensive rats (SHRSP) aged 10, 30, 60, 90, and 180 days, by the glyoxylic acid method. The results were compared with those in age-matched normotensive Wistar Kyoto (WKY) rats. While the distribution pattern of fluorescent nerve fibers in the proximal portion of WKY rats changed from a straight linear arrangement at 10 and 30 days of age to a network-like arrangement after 60 days, those from SHRSP showed a constant meshwork pattern throughout the entire examination period. In the distal portions of the MCA of both SHRSP and WKY rats at all ages examined, fluorescent nerve fibers formed a coarse network. The distribution densities of adrenergic nerve fibers in the proximal and distal portions of the MCA of SHRSP were significantly higher (P less than 0.01 and 0.05) than those of WKY rats at all ages examined, except in the proximal portion at 90 and 180 days of age. The difference in nerve fiber density between SHRSP and WKY rats reached a peak at 30 days of age in both proximal and distal portions, and then gradually decreased with age. The present study suggests that sympathetic hyperinnervation is an important factor in the development of hypertension, and is involved in its maintenance in SHRSP.     

Depolarisation-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Factors Determining the Magnitude of the Response

Abstract: The sequence of molecular events linking depolarisation-dependent calcium influx to the release of neurotransmitters from nerve terminals is unknown; however, calcium-stimulated protein phosphorylation may play a role. In this study the incorporation of phosphate into proteins was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. The rate and relative incorporation of label into individual phosphoproteins depended on the prelabelling time and buffer concentrations of calcium and phosphate. After prelabelling for 45 min, depolarisation caused a >20% increase in the labelling of 10 phosphoproteins, and this initial increase was maximal with 41 mM K⁺ for 5 s, or 30 μ M veratridine for 15 s, in the presence of 1 mM calcium. Both agents also led to an initial dephosphorylation of four phosphoproteins. Depolarisation for 5 min led to a significant decrease in the labelling of all phosphoproteins. All of the depolarisation-stimulated changes in protein phosphorylation were calcium-dependent. The depolarisation conditions found to optimally alter the phosphorylation of synaptosomal proteins find many parallels in studies on calcium uptake and neurotransmitter release. However, the uniform responses of such a large number of phosphoproteins to the multitude of depolarisation conditions studied suggest that the changes could equally well relate to recovery events such as biosynthesis of neurotransmitters and regulation of intraterminal metabolic activity.