Rate Of Quantal Release Research Articles

Increased asynchronous GABA release causes more inhibition in human epileptic brain?

Increased asynchronous GABA release causes more inhibition in human epileptic brain?

Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (Pr) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRPcum), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRPcum after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in Pr. Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.

Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons

We employed optical detection of the lipophylic dye FM1-43 and focal recordings of quantal release to investigate how synapsin affects vesicle cycling at the neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye employing high K + stimulation, which presumably involves the recycling pool of vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons, while in Syn KO the dye was redistributed towards the center of the bouton. When endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was significantly enhanced in WT boutons, and the entire boutons were stained, suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same loading paradigm produced fainter staining and significantly faster destaining. When the axon was stimulated electrically, a distinct difference in dye loading patterns was observed in WT boutons at different stimulation frequencies: a low stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a higher frequency (10 Hz) the dye was redistributed towards the center of the bouton and the fluorescence intensity was significantly increased. This difference in staining patterns was essentially disrupted in Syn KO boutons, although synapsin did not affect the rate of quantal release. Stimulation of the nerve in the presence of bafilomycin, the blocker of the transmitter uptake, produced significantly stronger depression in Syn KO boutons. These results, taken together, suggest that synapsin maintains the reserve pool of vesicles and segregation between the recycling and reserve pools, and that it mediates mobilization of the reserve pool during intense stimulation.

Efficiency of Synaptic Transmission of Single-Photon Events from Rod Photoreceptor to Rod Bipolar Dendrite

A rod transmits absorption of a single photon by what appears to be a small reduction in the small number of quanta of neurotransmitter ( Q count) that it releases within the integration period (∼0.1 s) of a rod bipolar dendrite. Due to the quantal and stochastic nature of release, discrete distributions of Q count for darkness versus one isomerization of rhodopsin (R*) overlap. We suggested that release must be regular to narrow these distributions, reduce overlap, reduce the rate of false positives, and increase transmission efficiency (the fraction of R* events that are identified as light). Unsurprisingly, higher quantal release rates ( Q rates) yield higher efficiencies. Focusing here on the effect of small changes in Q rate, we find that a slightly higher Q rate yields greatly reduced efficiency, due to a necessarily fixed quantal-count threshold. To stabilize efficiency in the face of drift in Q rate, the dendrite needs to regulate the biochemical realization of its quantal-count threshold with respect to its Q count. These considerations reveal the mathematical role of calcium-based negative feedback and suggest a helpful role for spontaneous R*. In addition, to stabilize efficiency in the face of drift in degree of regularity, efficiency should be ≈50%, similar to measurements.

Calmodulin Mediates Rapid Recruitment of Fast-Releasing Synaptic Vesicles at a Calyx-Type Synapse

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca 2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca 2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held.

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

Recycling and refilling of transmitter quanta at the frog neuromuscular junction.

1. Fluorescent dyes have been used at the frog neuromuscular junction to label synaptic vesicular membrane. Retrieved membrane is reformed into vesicles, which are released along with pre-existing vesicles. Consequently, if vesicular refilling with acetylcholine (ACh) is depressed by inhibitors, two sizes of quanta should be released: normal and smaller. As recycling continues the fraction of smaller size quanta should increase exponentially. 2. We enhanced the rate of quantal release by elevating the K+ concentration. The principal inhibitors were (-)-vesamicol (VES), hemicholinium-3 (HC3), and NH4+. Quantal size measurements were fitted to one and to two cumulative lognormal probability distribution functions. When two fitted better, the statistical significance assessment took into account the three additional parameters used in calculating the fit. 3. After recycling in the presence of inhibitor, many sets were fitted better by two lognormal functions. As recycling continued, the fraction of the miniature endplate potential voltage-time integrals ( MEPPs) in the larger sub-population decreased exponentially. 4. The size of the releasable pool was estimated by counting the quanta released by carbonyl cyanide m-chlorophenylhydrazone (CCCP). This was compared to pool sizes calculated from the inhibitor experiments. The two estimates of pool size were indistinguishable, with mean values ranging from about 170,000 to 270,000. 5. With all of the treatments tested, the means of the sizes in the smaller sub-population of MEPPs were about 1/3 those of the larger sub-populations. 6. Recycling synaptic vesicles appear to be incorporated into the releasable pool from which they have roughly the same probability of release as the pre-existing vesicles.

Further results with the ‘uniquantal EPSP’ hypothesis

The hypothesis of Geisler (Brain Res. 212 (1981) 198–201), in which the different spontaneous-rate classes of primary auditory neurons were accounted for by the different sizes of uniquantal EPSPs relative to the gap between resting membrane and threshold potentials, was represented with an expanded model which included relative refractory effects. The spike rates generated by the expanded model, when plotted vs. estimated sound level, are qualitatively similar to those of experimentally obtained rate-level curves. The hypothesis is also consistent with recent ultrastructural data which suggest that average quantal-release rates for any particular primary auditory neuron are inversely related to its spontaneous rate. The model's recovery processes following spike generation (hazard functions) are also similar to those observed experimentally.

Veratridine-induced high-frequency asynchronous release of inhibitory transmitter quanta in crayfish nerve-muscle synapses superfused with normal and low-calcium saline.

Crayfish fibres of opener muscles were voltage clamped to E = -80 mV membrane potential (T = 19-22 degrees C), and veratridine (10-100 mumol/l) was added to the superfusate. Within 30-60 s this caused large fluctuations of the clamp current due to vigorous asynchronous quantal release from the inhibitory nerve terminals along the muscle fibre. Excitatory postsynaptic receptors were previously desensitized by application of 5 mmol/l glutamate. Current fluctuations were evaluated by means of the noise analysis technique. Typically, 100 mumol/l veratridine increased instantaneously the quantal release rate n from n less than 1 quantum/s to n congruent to 10,000 quanta/s. Thereafter, n declined exponentially with a time constant of congruent to 70 s. On average, about 500,000 inhibitory quanta could be liberated in this way from the terminals on a single muscle fibre of congruent to 1 mm length. Serotonin (1 mumol/l) facilitated the effect of lower veratridine concentrations (1-10 mumol/l). In opener muscles veratridine-induced asynchronous quantal release showed little dependence on the bath concentration of Ca2+. The opposite was found for fibres of the superficial abdominal extensor muscle. Beside postsynaptic current fluctuations, veratridine elicited slowly changing average postsynaptic DC-currents which could be explained partly by superposition of individual inhibitory quantal currents. These DC-currents suggest that beside inhibitory quantal release another factor activates inhibitory postsynaptic receptors after application of veratridine.

Inhibitory effect of intraterminal lithium on asynchronous release of excitatory quanta induced by veratridine in nerve-muscle synapses of crayfish.

Crayfish muscle fibres were voltage-clamped at E = -80 mV membrane potential and superfused for about 10 min with Li+ saline (Na+ replaced by Li+) which contained picrotoxin to block inhibitory post-synaptic currents. Addition of veratridine (100 mumol/l) caused intense fluctuations in the voltage clamp current within 20-60 s due to vigorous asynchronous quantal release of excitatory transmitter from the nerve terminals distributed over the muscle fibre surface. Most likely, this quantal release resulted from loading the nerve terminals with Li+ via voltage-gated Na+ channels activated by veratridine. However, in the presence of Li+ quantal release was only transient; the quantal release rate, ñ, attained a maximum of congruent to 10,000 quanta/s and then declined exponentially with tau congruent to 10 to 20 s. Removal of Li+ and reapplication of normal Na+ increased ñ a second time. The amount of quanta released in the presence of Na+ was about an order of magnitude larger than that released previously in the presence of Li+. In preparations pretreated with Li+ superfusate for t greater than 45 min no marked quantal release could be elicited by veratridine. The experiments suggest an inhibitory effect of intraterminal Li+ on the quantal release process.

Differential effect of intraterminal sodium on spontaneous quantal release of transmitter in two neuromuscular junctions of crayfish

Nerve terminals on the superficial abdominal extensor muscle and the claw opener muscle of small crayfish were loaded with sodium by bath application of 100 μmol/l veratridine in superfusates where normal Ca 2+ was removed (low-Ca 2+ superfusate). In both preparations this caused an increase in spontaneous quantal release of excitatory and inhibitory transmitter which was evaluated by means of the noise analysis technique. About 2.5 min after application of veratridine, when spontaneous quantal release had largely ceased, the normal Ca 2+ concentration was reestablished. This increased transiently the quantal release rate a second time. However, release activated by Ca 2+ application was much more vigorous at the superficial abdominal extensor muscle than at the claw opener. At the superficial abdominal extensor muscle on average about 8% of the total number of quanta could be released in low Ca 2+ and 92% in normal Ca 2+ superfusate, while at the claw opener 75% of the quanta were released in low Ca 2+ and 25% in normal Ca 2+ superfusate. The experiments suggest that intraterminal sodium has a differential effect in the terminals of the two preparations. Possibly, the intraterminal source from which Na + may liberate Ca 2+ is more restricted in the superficial abdominal extensor muscle than in the opener muscle of the claw.

Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.

1. Quantal transmitter release was examined at nerve terminals of the excitatory motor axon of the crayfish opener muscle. The magnitude of synaptic currents, recorded with macro-patch electrodes at a nerve terminal, served as a measure of quantal size. Transmitter release was initiated by pulses of depolarizing current applied intracellularly to the axonal terminals after application of tetrodotoxin. Quantal release was altered by a variety of methods and the resulting quantal output and quantal size were measured. 2. Amplitude distributions of quantal events were obtained during experimental manipulations which altered the rate of quantal release by up to 25-fold. These manipulations consisted of: varying pulse amplitude or pulse duration; facilitating the release by prolonged depolarization; and application of a potassium channel blocker, 4-aminopyridine. 3. The amplitude of quantal events is impervious to marked changes in presynaptic depolarization and is not affected by experimental procedures which promote accumulation of calcium ions in the terminals. The vesicular mechanism of release, in which transmitter substance is prepackaged in vesicles which individually undergo exocytosis at a release zone, could account for the observed results.

The effects of the antibiotic, primycin, on spontaneous transmitter release at the neuromuscular junction.

The effects of primycin, a potent ionophore in biological membranes, have been studied at the neuromuscular junction of the garter snake. Primycin in concentrations greater than 2 X 10(-7)M produced a time- and concentration-dependent depolarization of twitch muscle fibres. Primycin (10(-7)-5 X 10(-7)M) produced an increased rate of quantal release of acetylcholine, which was not maintained, and a slight reduction in quantal size. Time to onset and to peak effect of primycin were concentration-dependent whereas maximum frequency was not. Absence of extracellular Ca2+ produced a significant delay in the time to onset and to peak effect of primycin, but did not affect the peak miniature endplate potential (m.e.p.p.) frequency. Following 60 min exposure to primycin (5 X 10(-7)M), introduction of a high concentration of potassium (20 mM) produced no further increase in spontaneous release. In cut muscle preparations, exposure to primycin (10(-7)-5 X 10(-7)M) reduced peak endplate current (e.p.c.) amplitude until nerve stimulation resulted in failures or the release of one or two quanta. E.p.c. amplitude was not restored with prolonged washing. The effects of primycin on the nerve terminal are considered to be consistent with its ability to increase the permeability of membranes to calcium ions resulting in an influx of extracellular calcium, an efflux of mitochondrial calcium and eventual depletion of synaptic vesicles.

Neurotransmitter release and its facilitation in crayfish muscle. V. Basis for synapse differentiation of the fast and slow type in one axon.

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from synaptic spots on crayfish opener muscle fibers. Synapses on the proximal fiber bundle were characterized as fast, with a relatively high quantal-release rate m of 0.2-5 and a low twin-pulse facilitation Fs of 1.1-3, at 13.5 mM [Ca]o and low (0.5/s) repetition rate. Under the same conditions, distal "slow" synapses had a release rate m of 0.02-0.4 and a facilitation Fs of 2-4. When the [Ca]o was varied between 1.7 and 27 mM, release and facilitation were much less affected in proximal, fast synapses than in distal, slow ones. The average maximal slope of the log release to log [Ca]o relation was 1.5 in proximal, and 3.1 in distal synapses, while the average maximal facilitation Fs was 2.5 in proximal and 4.7 in distal synapses, respectively. Assuming saturation kinetics for entry of Ca into the terminal and release of transmitter, possible variations of parameters generating the fast-slow differentiation were explored. Excluding a number of possibilities, it was found that in addition to a higher maximal release level, fast synapses seem to have a higher resting [Ca]i and/or a lower cooperativity of the release mechanism, as compared to slow synapses.

Anion blockers inhibit impulse-evoked quantal transmitter release at the crayfish neuromuscular junction.

1. The effects of the anion-transport blocking reagents Probenecid, 4,4'diisothiocyanostilbene-2,2'-disulphonate (DIDS), and pyridoxal phosphate on the number of quanta released (m) and the time course of the probability of quantal release, alpha(t), following an action potential were studied at single synaptic sites on the opener muscle in the crayfish leg.2. Low concentrations (10-50 muM) of DIDS or Probenecid produced decreases in m to about 30-50% of the initial value; at higher concentrations (100 muM or greater), evoked release failed. Much greater concentrations (500 muM) of pyridoxal phosphate were necessary to produce decreases in m.3. Synaptic depression in 50 muM-Probenecid was reversible by removing the agent; after treatment with 50 muM-DIDS, however, m partially regained the initial value upon removal of the drug.4. The time course of alpha(t) after an action potential was unaffected by any of the reagents.5. It is concluded that the reagents decrease the number of quanta evoked by a nerve impulse by decreasing the magnitude, but not the time course, of the average rate of quantal release following an action potential; it is suggested that nerves and other secretory cells may share a common mechanism during exocytosis that is subject to modification by these agents.

Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of D-tubocurarine.

1. Neuromuscular transmission in the presence and absence of D-tubocurarine was examined in cut muscle preparations of the rat diaphragm using intracellular recording techniques. The method of localizing the end-plates is described and evidence is presented indicating that end-plate potential (e.p.p.) recordings were made close to the end-plate region and that reliable estimates of the quantum content of e.p.p.s could be made.2. During repetitive nerve stimulation at rates between 2 and 10 Hz, e.p.p. amplitudes declined in response to the first 6-8 stimuli to reach a plateau level which was maintained for 1500 stimuli. At higher rates of stimulation some 500-700 stimuli were needed before a plateau level of amplitude was reached. It was demonstrated that these amplitude changes reflected similar changes in the quantal release of ACh.3. Tubocurarine in a concentration of 4 x 10(-8) g/ml. had no effect on quantal release but did reduce quantal size. In the presence of tubocurarine, 8 x 10(-8) g/ml., small reductions in the quantum content of the first e.p.p. in response to a train of stimuli and the maintained quantal release were observed. In the presence of tubocurarine 4 x 10(-7) g/ml., the quantum content of the first e.p.p. and the maintained release were further reduced, to the level previously described for curarized preparations. At this concentration a significant effect of tubocurarine upon the presynaptic measurements could be detected (t test). Studies on single junctions revealed that the post- and presynaptic effects of tubocurarine developed with different time courses.4. Neuromuscular preparations in vivo have been reported to be much more resistant to maintatined stimulation than curarized neuromuscular preparations in vitro. It is suggested that this disparity is explained by the presynaptic action of tubocurarine.5. From the quantal release rates of the present investigation and reports of ACh release/stimulus/end-plate in previous investigations it can be calculated that a quantum of ACh contains between 12,000 and 21,000 molecules.

The kinetics of transmitter release at the frog neuromuscular junction.

1. Fluctuations in the latency of focally recorded end-plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0.5 msec at 11 degrees C, and increases with decreasing temperature with a Q(10) of at least 4 over the range 1-12 degrees C.3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non-linear manner.

A study of the mechanism of quantal transmitter release at a chemical synapse.

1. The nerve-muscle preparation of the cutaneous pectoris of the frog has been used to study quantal transmitter release.2. When the osmotic pressure of the external solution is raised 1.5-2 fold, the frequency of miniature end-plate potentials (m.e.p.p.s) rises by 1.5-2 orders of magnitude. This effect is independent of the presence of Ca(2+) ions and of the nature of the substances by which the osmotic pressure has been increased.3. In Ca(2+) free hypertonic solution the nerve impulse still invades the nerve terminals but does not alter the frequency of the m.e.p.p.s.4. The arrival of the impulse in the terminals causes an immediate increase in the rate of quantal release, provided divalent cations are present whose passage through the axon membrane is facilitated by excitation (Ca(2+), Sr(2+), Ba(2+)).5. Divalent cations which penetrate only slightly (Mg(2+), Be(2+)) lower the frequency of m.e.p.p.s and suppress the end-plate potential (e.p.p.) evoked by an impulse, in the presence of Ca(2+) ions. Be(2+) is a more effective inhibitor than Mg(2+).6. In Ca(2+) free solutions, adding Mg(2+) causes an increase in the frequency of m.e.p.p.s evoked by depolarization of the nerve endings or by treatment with ethanol.7. The trivalent cation La(3+) is more effective than divalent cations are in increasing the frequency of m.e.p.p.s. The tetravalent cation Th(4+) also raises the m.e.p.p. frequency.8. The observations summarized in paragraphs 2-7 indicate that the frequency of m.e.p.p.s at a constant temperature depends only on the concentration of uni-, di- and trivalent cations inside the nerve ending. It is suggested that the internal cation concentration influences the adhesion between synaptic vesicles and the membrane of the nerve ending.9. For a model experiment, artificial phospholipid membranes have been used to study the effect of uni-, di-, tri- and tetravalent cations on the adhesion process. At pH 7-7.4, the time required for adhesion to take place decreases with increasing cation concentration in the bath. Ca(2+) ions are 100-1000 times more effective than K(+) ions; La(3+) and Th(4+) ions are still more effective. The ;adhesion time' decreases when the pH is lowered; it increases greatly with lowering of temperature.10. The hypothesis is put forward that the mutual adhesion of artificial vesicles made of phospholipid membranes, and the adhesion between synaptic vesicles and the membrane of the nerve ending arise by a common mechanism. In both cases, the important factor is the influence of cations on the electric double layer at the membrane surface.