Relative Distribution of Ca2+ Channels at the Crayfish Inhibitory Neuromuscular Junction

2004 ◽  
Vol 92 (3) ◽  
pp. 1491-1500 ◽  
Author(s):  
Tariq N. Allana ◽  
Jen-Wei Lin
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Single Channel ◽  
Procambarus Clarkii ◽  
Physiological Saline ◽  
Synaptic Delay ◽  
Similar Amount ◽  
Relative Distribution ◽  
P Type

We investigated the Ca2+ channel-synaptic vesicle topography at the inhibitor of the crayfish ( Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca2+ channel block on transmitter release. Initial identification of Ca2+ channels revealed the presence of two classes, P and non-P-type with P-type channels governing ∼70% of the total Ca2+ influx. The remaining Ca2+ influx was completely blocked by Cd2+ but not by saturating concentrations of ω-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca2+ channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca2+]i around release sites. Specifically, addition of either [Mg2+]o, which decreases single-channel current, or ω-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca2+ influx block was <40%. Because non-P-type channels are able to compensate for blocked P-type channels, it suggests that these channels overlap considerably in their distribution. However, when Ca2+ influx was blocked by ∼50%, ω-Aga IVA increased delay significantly more than Mg2+, suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca2+ channels was further supported by the observations that non-P-type channels are unable to trigger release in physiological saline and EGTA preferentially prolongs synaptic delay dominated by non-P-type channels when transmitter release is evoked with broad action potentials. We therefore conclude that although non-P-type channels do not directly trigger release under physiological conditions, their distribution partially overlaps with P-type channels.

The Neuromuscular Junction Revisited: Ca2+ Channels and Transmitter Release in Cholinergic Neurones in Xenopus Nerve and Muscle Cell Culture

1990 ◽  
Vol 153 (1) ◽  
pp. 129-140 ◽  
Author(s):  
T. P. FENG ◽  
ZHENG-SHAN DAI
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Single Channel ◽  
Calcium Currents ◽  
Patch Clamps ◽  
Whole Cell ◽  
Voltage Gated ◽  
Cholinergic Neurones ◽  
Ca2 Channels ◽  
Evoked Transmitter Release

Although the entry of calcium ions into the presynaptic nerve terminals through voltage-gated Ca2+ channels is now universally recognized as playing an essential role in evoked transmitter release at the neuromuscular junction (NMJ), and indeed in chemical synapses generally, we have as yet very little direct knowledge of the Ca2+ channels of the presynaptic terminals. In this work, making use of cocultured nerve and muscle cells from Xenopus embryos, we studied the NMJ formed between the soma of identified cholinergic neurones and myoball, which allowed the use of patch-clamps on both the pre- and postsynaptic components. Both whole-cell and single-channel recordings of Ca2+ channels in the presynaptic cell were made. We found only one type of voltage-gated Ca2+ channel with highvoltage activation and slow inactivation characteristics, allowing its classification either as the L or the N type. The channels were susceptible to block by metenkephalin but not to block by nifedipine or to enhancement by Bay K 8644. This combination of pharmacological properties favours their classification as the N type. Preliminary observations on the correlation between calcium currents and transmitter release disclosed a strikingly rapid run-down of the evoked release with unchanged calcium currents and spontaneous release during whole-cell recording, indicating a specific wash-out effect on some link between calcium entry and evoked transmitter release.

scholarly journals Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction

2015 ◽  
Vol 113 (7) ◽  
pp. 2480-2489 ◽  
Author(s):  
Fujun Luo ◽  
Markus Dittrich ◽  
Soyoun Cho ◽  
Joel R. Stiles ◽  
Stephen D. Meriney
Keyword(s):  
Neuromuscular Junction ◽  
Calcium Channels ◽  
Calcium Channel ◽  
Calcium Ions ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Vesicle Fusion ◽  
Frog Neuromuscular Junction ◽  
Low Probability ◽  
Presynaptic Calcium

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Study of the Inhibitor of the Crayfish Neuromuscular Junction by Presynaptic Voltage Control

1997 ◽  
Vol 77 (1) ◽  
pp. 103-115 ◽  
Author(s):  
Andrey Vyshedskiy ◽  
Jen-Wei Lin
Keyword(s):  
Neuromuscular Junction ◽  
Muscle Fiber ◽  
Branch Point ◽  
Transmitter Release ◽  
Voltage Control ◽  
Synaptic Delay ◽  
Equilibrium Potential ◽  
Space Constant ◽  
Release Model ◽  
Crayfish Neuromuscular Junction

Vyshedskiy, Andrey and Jen-Wei Lin. Study of the inhibitor ofthe crayfish neuromuscular junction by presynaptic voltage control. J. Neurophysiol. 77: 103–115, 1997. The inhibitor of the crayfish opener muscle was investigated by a presynaptic voltage control method. Two microelectrodes were inserted into the inhibitor and the amplitude and duration of presynaptic depolarization were controlled by a voltage-clamp amplifier. The inhibitory postsynaptic potential (IPSP) was measured from a muscle fiber located near the presynaptic voltage electrode. Nonlinear summation of IPSP amplitudes was corrected after chloride equilibrium potential was measured. With the use of 5-ms presynaptic pulses, the depolarization-release coupling (D-R) curve constructed from IPSP peak amplitudes (IPSPcor) had a threshold of about −35 mV and reached its maximal level at −5 to −10 mV. Depolarization beyond the maximum led to a suppression of neurotransmitter release. When transmitter release during a presynaptic pulse was completely suppressed, IPSPs activated by tail current could be identified with an average synaptic delay of 2.5 ms. Transmitter secretion triggered by a calcium current activated during the 5-ms pulses (IPSPon) was also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5-ms pulses. D-R coupling plots measured from IPSPon exhibited a more pronounced suppression than that obtained from IPSPcor. The effect of presynaptic pulse duration on the level of transmitter release was analyzed. Transmitter release increased with increasing duration and was nearly saturated by 20-ms pulses depolarized to 0 mV. The following conditions were identified as necessary to obtain a consistent D-R curve with a clear suppression: 1) small animals, 3.8 cm head to tail, 2) 15°C, 3) 40 mM tetraethylammonium and 1 mM 4-aminopyridine, 4) an extracellular calcium concentration of ≤10 mM. In addition, a consistent correlation was found among the branching pattern of the inhibitor, the placement of the presynaptic electrode, and the characteristics of the D-R curves. An ideal presynaptic electrode configuration involved placing the voltage electrode in a secondary branch, ∼100 μm from the main branch point, and placing the current electrode at the branch point. Postsynaptically, optimal recordings were obtained from muscle fibers innervated by a single branch of the inhibitor that originated from a point near the presynaptic voltage electrode. A cable-release model was constructed to evaluate the relationship between the shape of the D-R coupling curves and the space constants of the presynaptic terminals. A comparison between the model and the D-R coupling curves suggested that the space constant of an inhibitor branch on a muscle fiber is ≥8 times longer than its actual length. Therefore the upper limit estimate of the space constant of a typical preparation is ∼3 mm. Results reported here outline morphological and physiological conditions needed to achieve optimal control of the presynaptic branch of the crayfish inhibitor. The cable-release model quantitatively defines the extent of presynaptic voltage control.

Synaptic vesicle recruitment for release explored by Monte Carlo simulation at the crayfish neuromuscular junction

1999 ◽  
Vol 77 (9) ◽  
pp. 634-650 ◽  
Author(s):  
K M Kennedy ◽  
S T Piper ◽  
H L Atwood
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Neuromuscular Junction ◽  
Calcium Channels ◽  
Calcium Channel ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
Single Channel ◽  
Separation Distance

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca2+-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.Key words: synapse, Monte Carlo simulation, synaptic vesicle, active zone, vesicle recruitment, crayfish, calcium, calcium buffer.

The Lateral Giant Fiber to Motor Giant Fiber Synapse in Crayfish

1972 ◽  
Vol 30 ◽  
pp. 170-171
Author(s):  
Charles A. Stirling
Keyword(s):  
Synaptic Vesicles ◽  
Procambarus Clarkii ◽  
Synaptic Contact ◽  
Giant Fiber ◽  
Synaptic Junctions

The lateral giant (LG) to motor giant (MoG) synapses in crayfish (Procambarus clarkii) abdominal ganglia are the classic electrotonic synapses. They have previously been described as having synaptic vesicles and as having them on both the pre- and postsynaptic sides of symmetrical synaptic junctions. This positioning of vesicles would make these very atypical synapses, but in the present work on the crayfish Astacus pallipes the motor giant has never been found to contain any type of vesicle at its synapses with the lateral giant fiber.The lateral to motor giant fiber synapses all occur on short branches off the main giant fibers. Closely associated with these giant fiber synapses are two small presynaptic nerves which make synaptic contact with both of the giant fibers and with their small branches.

Real-time measurement of transmitter release from single synaptic vesicles

Nature ◽  
1995 ◽  
Vol 377 (6544) ◽  
pp. 62-65 ◽  
Author(s):  
Dieter Bruns ◽  
Reinhard Jahn
Keyword(s):  
Real Time ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Time Measurement ◽  
Real Time Measurement

Physiological effects of two FMRFamide-related peptides from the crayfish Procambarus clarkii

1995 ◽  
Vol 198 (1) ◽  
pp. 109-116
Author(s):  
M Skerrett ◽  
A Peaire ◽  
P Quigley ◽  
A Mercier
Keyword(s):  
Transmitter Release ◽  
Procambarus Clarkii ◽  
Neuromuscular Junctions ◽  
Input Resistance ◽  
Physiological Effects ◽  
Synaptic Current ◽  
Extensor Muscles ◽  
Neuromuscular Systems ◽  
Dose Dependent ◽  
Measurable Change

The present study examined the effects of two recently identified neuropeptides on crayfish hearts and on neuromuscular junctions of the crayfish deep abdominal extensor muscles. The two peptides, referred to as NF1 (Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2) and DF2 (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH2), increased the rate and amplitude of spontaneous cardiac contractions and increased the amplitude of excitatory junctional potentials (EJPs) in the deep extensors. Both effects were dose-dependent, but threshold and EC50 values for the cardiac effects were at least 10 times lower than for the deep extensor effects. The heart responded equally well to three sequential applications of peptide in any given preparation, but the responses of the deep extensors appeared to decline with successive peptide applications. The results support the hypothesis that these two neuropeptides act as neurohormones to modulate the cardiac and neuromuscular systems in crayfish. Quantal synaptic current recordings from the deep extensor muscles indicate that both peptides increase the number of quanta of transmitter released from synaptic terminals. Neither peptide elicited a measurable change in the size of quantal synaptic currents. NF1 caused a small increase in muscle cell input resistance, while DF2 did not alter input resistance. These data suggest that DF2 increases EJP amplitudes primarily by increasing transmitter release, while the increase elicited by NF1 appears to involve presynaptic and postsynaptic mechanisms.

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