Depolarization-Induced Ca2+ Entry Preferentially Evokes Release of Large Quanta in the Developing Xenopus Neuromuscular Junction (original) (raw)
Related papers
1997
The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (KCa) currents. The onset of KCa current preceded the peak of the action potential. The predominantly omega-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca2+ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. omega-CgTX also blocked KCa currents and transmitter release during an action potential, suggesting that Ca and KCa channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, KCa channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of KCa channels or the diffusion/removal of Ca2+ from sites of neurotransmitter release after an action potential.
The Journal of General Physiology
Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK~c~, on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK~c~) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with g~c~ not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK~c~ by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltagedependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gKtc~ by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK<ca~ was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (> 50 ms) suggest either that residual Zucker, R. S., and L. O. Lara-Estrella. 1983. Post°tetanic decay of evoked and spontaneous transmitter release and a residual calcium model of synaptic facilitation at crayfish neuromuscular junctions.
The Journal of physiology, 1989
1. The calcium dependence of spontaneous transmitter release from nerve terminals of different lengths was examined at neuromuscular junctions in frog muscle. Miniature endplate potential (MEPP) frequency was positively correlated with the endplate potential (EPP) quantal content and was dependent on external Ca2+. The higher the resting MEPP frequency in a 0.25 mM-Ca2+ Ringer solution, the greater the dependence on external Ca2+. MEPP frequency in all terminals dropped to approximately the same low level in a Ca2(+)-free Ringer solution containing EGTA. This suggests that terminals with higher release levels have a larger Ca2+ influx at rest. 2. Several tests were done to try to characterize the mode of Ca2+ entry into resting terminals. omega-Conotoxin (omega-CgTx) blocked evoked release and reduced MEPP frequency, but not as effectively as zero Ca2(+)-EGTA Ringer solution. Some component of Ca2+ influx thus appears to enter through channels insensitive to omega-CgTx. Tetrodotoxin...
Single Ca2+ entry and transmitter release systems at the neuromuscular synapse
Synapse, 1988
The possibility that there exists more than one voltage-gated Ca2+ channel system subserving quantal release of neurotransmitter at nerve endings was examined by measuring the sensitivity of quantal release to agents that block Ca2+ or Ba2+ entry, namely Cd2+, M2', neomycin, and bekanamycin. The results show equal effectiveness vs. release evoked by presynaptic action potentials, brief intense presynaptic depolarizations, or prolonged relatively mild depolarizations, from which it is concluded that the same channel system is involved in each case. In the presence of Ba2+ and no Ca2+, there occur essentially normal (but small) endplate potentials (epps), while in the presence of Ba2+ and Ca2+ epp amplitude and frequency of miniature epps co-modulate with stimulation frequency in a manner which corresponds to a residual Ba2+ model for augmentation. It is therefore also concluded that a single transmitter release system is responsible for normal phasic release and the asynchronous release that is mediated by Ba2+.
Neuroscience, 2001
AbstractÐUsing Xenopus nerve±muscle co-cultures, we have examined the contribution of calcium-activated potassium (K Ca ) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K Ca channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K Ca channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium in¯ux was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents.
Journal of Neuroscience, 2004
We examined the mechanism underlying increased quantal content after block of activity at the mouse neuromuscular junction in vivo. We found that, when quantal content was measured in solution containing normal extracellular calcium, block of activity had no effect on either quantal content or the response to repetitive stimulation. However, when quantal content was measured in low extracellular calcium, there was a large increase in quantal content after block of activity. The increase in quantal content was accompanied by increased depression during repetitive stimulation. The explanation for these findings was that there was a shift in the calcium dependence of release after block of activity that manifested as an increase in probability of release in low extracellular calcium. Block of presynaptic P/Q channels eliminated the increase in probability of release. We propose that activity-dependent regulation of presynaptic calcium entry may contribute to homeostatic regulation of quantal content.
The Journal of Physiology, 1995
The effects of the calcium channel blockers, funnel-web spider toxin (FTX), w-agatoxin IVA (w-Aga IVA) and w-conotoxin GVIA (w-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50= 0o02 #l ml-') and w-CgTX (1 #M) but was not affected by w-Aga IVA (0'5/M). When FTX (0 1 #l ml-') was assayed on spontaneous release either in normal Ringer solution or in low Ca2+-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by-2-fold in both conditions. 3. Presynaptic calcium currents (Ica), measured by the perineurial technique in the presence of 10 mm tetraethylammonium chloride (TEA) and 200 juM BaCl2 to block K+ currents, were blocked by w-CgTX (5 ,UM), partially blocked by FTX (1 1d ml-') and not affected by w-Aga IVA (0 5,UM). 4. The presynaptic calcium-activated potassium current (IK(ca)) measured by the perineurial technique in the presence of 0 F5jM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 juM). This current was also blocked by w-CgTX (5 FM) and by CdC12 (200 FM) but was not affected by FTX (1 Fl ml-'). The blockade by w-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two w-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a w-CgTXand FTX-sensitive population.
Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2015
Calcium entry into the nerve endings through voltage dependent calcium channels triggers a chain of events leading to exocytosis of neurotransmitter, providing the transmission of excitation through the synapse. In this regard, a significant role of calcium ions and presynaptic calcium channels in the modulation of secretion is evident. However, the question of the contribution of different types of voltage dependent cal cium channels in the calcium regulation parameters of the quantal secretion still remains unclear. The secre tion kinetics characterizes a degree of synchrony of the neurotransmitter release. In recent decades it is regarded as one of the important factors maintaining the effectiveness of the synaptic transmission. Since neuromuscular synapses of frogs and mice are classical objects of physiological and pharmacological studies, the results of which are summarized and extrapolated to other synapses, it is interesting to compare changes of the acetylcholine secretion in these synapses under different conditions of calcium entry into the nerve endings. In this review we discuss the data on the neuromuscular synapses of frogs and mice and analyze some aspects of calcium regulation and involvement of different types of voltage dependent calcium channels in the modulation of the acetylcholine secretion kinetics.