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W Van der Kloot

Publications and source records attributed to W Van der Kloot.

At least 19 recordsLinked to original sources
PubMed2001

Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction.

We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.

Acetylcholine↗
PubMed2000

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.

Animals↗
PubMed1999

Examining the timing of miniature endplate potential releases at the frog and mouse neuromuscular junctions for deviations from Poisson expectations.

The intervals between miniature endplate potentials (MEPPs) were measured at frog and mouse neuromuscular junctions in several different solutions. Data sets with monotonic trends in MEPP frequency were discarded. The remaining data sets had between 283 and 5024 MEPPs. The fit to the exponential distribution, which describes a Poisson process, was tested with Sherman's statistic. If the intervals are not distributed exponentially, this statistic indicates whether they deviate because they are more concentrated or more diffuse. In 6 of 20 data sets from the frog and in 3 of 7 data sets from the mouse the intervals between MEPPs were not distributed exponentially. Some of these were clustered, while others were diffuse. In one frog data set release was periodic. In all data sets releases appeared to be independent, judging from the integrated power spectra. One data set may show fractal behavior. Calculations of the approximate entropy gave no indication of an underlying regularity, so there is no evidence for a chaotic process. The lack of fit to the exponential distribution due to either concentrated or diffuse interval distributions is mimicked by a model in which release is Poisson, but with a mean rate that randomly shifts to a different level.

Action Potentials↗
PubMed1998

Calcitonin gene-related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions.

1. Calcitonin gene-related peptide (CGRP) is found in dense-cored vesicles in the motor nerve terminal. 2. Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (-)-vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3. The hCGRP effect was not blocked by fragment 8-37, an antagonist of one class of CGRP receptor. 4. hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5. CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.

Acetylcholine↗
PubMed1997

Making quantal analysis easier and more accurate.

Problems inherent in applying quantal analysis to synapses are discussed. These include the dispersion in time of the evoked quantal releases and the skew to the right in the distribution of the sizes of the spontaneous miniature potentials. To circumvent some of the problems, quantal analysis was performed at the neuromuscular junction by dividing each individual endplate current (EPC) by the mean miniature endplate current (MEPC) or each individual time integral of the endplate current (integral of EPC) by the mean integral of MEPC. The results were evaluated by comparing the resulting distributions to the predictions of Poisson's law, based on the number of stimuli not followed by a response. The quantal distributions obtained from the integral of MEPCs by this 'point-by-point' were in excellent agreement with these predictions. This method may make quantal analysis easier and more reliable.

Animals↗
PubMed1997

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by omega-conotoxin.

Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567-589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (FMEPP) in solutions containing elevated levels of K+ and Ca2+. Carbachol did not decrease FMEPP in hypertonic solutions or in solutions containing the Ca2+ ionophore ionomycin and Ca2+. We conclude that the nicotinic agonists decrease Ca2+ influx through voltage-gated Ca2+ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca2+ channels, omega-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by omega-conotoxin MVIIC, a blocker of P-type and certain other Ca2+channels. The Ca2+ channel targeted by the nicotinic agonists appears to be of the N-type.

Animals↗
PubMed1996

Localizing quantal currents along frog neuromuscular junctions.

1. We spatially localized the origins of quantal currents by recording simultaneously with two intracellular electrodes and employing the prediction of the one-dimensional cable equations that the time integrals of the resulting voltage changes fall off exponentially with distance. 2. Miniature endplate potentials (MEPPs) were more frequent near the centre of the endplate. In contrast to some work using other methods, we did not find MEPPs originating at the margins of the endplate to be strikingly smaller. 3. Spontaneous MEPPs and uniquantal endplate potentials (EPPs) were released over the same length of endplate and with the same relative probabilities at different regions. 4. Nicotinic agonists decreased evoked quantal output, but did not change the length over which uniquantal EPPs were generated. We conclude they do not block nerve conduction in the terminals. 5. Data sets were obtained with an extracellular electrode and two intracellular electrodes. The extracellular electrode was invariably near the centre of the region in which congruous MEPPs appeared to be generated. However, the range in the calculated positions of the synchronous MEPPs was as long as 0.8 mm. Therefore, it may be possible that extracellular electrodes have a longer recording range than commonly assumed.

Animals↗
PubMed1996

Monoethylcholine as a false transmitter precursor at the frog and mouse neuromuscular junctions.

Monoethylcholine (MECH) enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endplate channels for about half of the average duration observed where they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quantum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, an inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhibitor, hemicholinium-3, is present or when extracellular Na+ (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We conclude that quanta released spontaneously and following nerve stimulation probably come from the same pool. The distribution of t1/2's during the incorporation of MECH can be accounted for in the framework of recent studies of the recycling of synaptic vesicles. We conclude that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.

Animals↗
PubMed1996

Spontaneous and uniquantal-evoked endplate currents in normal frogs are indistinguishable.

1. A recent paper concludes that the shapes of spontaneous and uniquantal-evoked signals are different. The signals were recorded with extracellular electrodes, often in the presence of neostigmine. Differences were reported between the voltage-time integrals, and between the decay times of spontaneous and evoked signals. 2. These results disagree with earlier studies using the two-electrode voltage clamp technique. 3. We recorded miniature-endplate currents (MEPCs) and uniquantal-endplate currents (u-EPCs) in a low-Mg(2+)-Ca2+ solution, sometimes with neostigmine present. Evoked quantal outputs were estimated by the method of failures, so we could reject the appropriate number of the largest evoked releases. The twenty-nine experiments showed that there were no consistent differences between the current-time integrals or half-decay times (t1/2), regardless of whether or not neostigmine was present. 4. When recording simultaneously with intracellular and extracellular electrodes, on average about 25% of the miniatures were seen in both recordings. On average, 63% of the endplate potentials were also seen in both recordings. Extracellular recording may not give the precise localization generally assumed. 5. We again conclude that quanta released by nerve stimulation and spontaneously are indistinguishable at normal frog neuromuscular junctions.

Animals↗
PubMed1996

Statistics for studying quanta at synapses: resampling and confidence limits on histograms.

This paper describes some statistical methods for working with data on quantal sizes. Since quantal sizes often do not fit to normal probability distribution functions, statistics based on the normal distribution are inappropriate. Resampling methods can be used to determine confidence limits and to test whether two sets of data differ by chance. Some hypotheses about the nature of quanta are based on apparent peaks and valleys in histograms. Confidence limits can be placed on the bins in the histogram by using the Kolomorogov-Smirnov statistic or by resampling. The confidence limits should assist in the evaluation of the significance of the valleys.

Animals↗
PubMed1996

Accounting for the shapes and size distributions of miniature endplate currents.

The current model does not account adequately for the characteristics of miniature endplate currents (MEPCs). We do not understand their relatively slow rise, the shape of their rise, their variable and sometimes prolonged decay, and the correlation between amplitude and decay time. If we assume that ACh is released from the vesicle through a pore and that the vesicle enlarges as it takes on additional transmitter, the predictions are more like MEPCs. However, previous measurements showed that after quantal size was increased the vesicles in the terminal were not enlarged. This need not be a problem, because some of the ACh is added to vesicles positioned at the active zones, a process known as second-stage loading. By using the false transmitter precursor monoethylcholine we provide additional evidence for second-stage loading. The distribution of quantal sizes at the junction usually does not follow a normal probability distribution; it is skewed to the right. The skew can be accounted for by a model incorporating second-stage loading in which the vesicles are released randomly, without regard to their ACh content. If the vesicles increase in size when they contain more transmitter, only vesicles at the active zone need swell.

Acetylcholine↗
PubMed1996

Transmitter packaging at frog neuromuscular junctions exposed to anticholinesterases; the role of second-stage acetylcholine loading.

1. This investigation was undertaken to explore an unexpected effect of vesamicol, an agent that inhibits active acetylcholine (ACh) uptake into isolated synaptic vesicles. Previous studies at the neuromuscular junction showed that vesamicol makes miniature end-plate currents (MEPCs) smaller only after tens of thousands of quanta have been released. Inhibiting acetylcholinesterase (AChE) makes the MEPCs larger than normal. Our unexpected finding was that with the AChE inhibitor present, adding 2 microM (-)-vesamicol decreases the size of the MEPCs by approximately 30%. The decrease was apparent within 15-30 min, during which only a few thousand quanta had been released. 2. Experimental tests showed that the (-)-vesamicol treatment is unlikely to be acting postsynaptically. For example, it did not slow the rise of MEPCs, which would occur if the endplate receptors were blocked. 3. When AChE was inhibited, three treatments expected to block active choline (Ch) uptake into the presynaptic terminals decreased MEPC size: 1) elevating extracellular K+ to diminish the Na+ electrochemical gradient required for Ch uptake; 2) replacing extracellular Na+ with methylamine+; and 3) adding hemicholinium-3 (HC-3), an inhibitor of the Ch transporter. These treatments did not act by reactivating AChE, blocking the endplate ACh receptor, or by enhancing the desensitization of the ACh receptor. 4. Previous evidence suggests that synaptic vesicles are formed and partially filled with ACh in the cytoplasm and then receive additional ACh when they attach to the active zones, a process that is called second-stage loading. We conclude that the MEPCs are becoming smaller when second-stage loading is blocked by (-)-vesamicol or when the supply of ACh in the cytoplasm of the motor nerve terminal is depleted. 5. To follow the time course of second-stage loading, we used the false transmitter precursor monoethylcholine (MECh). It enters the terminal and is transformed into acetylmonoethylcholine (AMECh). When 200 microM MECh was placed in the extracellular solution and the AChE was inhibited, MEPC size was significantly smaller after 10 min. MEPC size increased once again over a period of time when MECh was removed from the extracellular solution and replaced with Ch. 6. We conclude that at the neuromuscular junction second-stage loading is responsible for loading a significant fraction of the ACh into the quanta.

Acetylcholine↗
PubMed1995

The relationship between quantal content and delayed quantal release.

Following an endplate potential there is a period of elevated spontaneous quantal release, known as delayed release. We have estimated the relationship between evoked quantal release and delayed release in two ways. First, in a solution in which the Na+ was replaced with methylamine, the number of delayed release evoked by electronic depolarization of the nerve terminal changed little over a 30-fold range of output. Second, we varied [Ca2+]out stepwise in Na solution with added Mg2+ and measured delayed release and EPP amplitude at single junctions. Over much of the range, elevating the EPP amplitude many-fold increased delayed release only slightly, if at all. Our interpretation is that the effects of residual Ca2+ are not proportional to the amount of Ca2+ entering to trigger quantal release.

Action Potentials↗
PubMed1995

The rise times of miniature endplate currents suggest that acetylcholine may be released over a period of time.

Models of miniature endplate currents predict 20-80% rise times of 100 microseconds or less. These predictions are substantially less than most of the rise times recorded in the literature. New measurements were made of rise times at the frog neuromuscular junction using extracellular recording. The mean 20-80% rise time was 250 microseconds. Rise times were variable; at 20 degrees C, 95% of them fell in a range from 140 to 460 microseconds. The most questionable assumption in the models is that the acetylcholine (ACh) is released instantaneously. Modifying the model, so that ACh diffuses from the vesicle through a pore, lengthens the rise time to observed levels. It has been proposed that ACh is released from the vesicle in exchange for Na+. However, the rise times of miniature endplate currents recorded in solutions in which the Na+ is replaced by sucrose are in the normal range. The Q10 for the rise of miniature endplate currents is approximately 2, which is consistent with the models and with temperature effects on pore formation in mast cells.

Acetylcholine↗
PubMed1994

Anandamide, a naturally-occurring agonist of the cannabinoid receptor, blocks adenylate cyclase at the frog neuromuscular junction.

Anandamide (arachydonylethanolamide) is a naturally-occurring ligand of the canabinoid receptor. When anandamide binds to its receptor, adenylate cyclase is inhibited. At the frog neuromuscular junction, anandamide lessened the increase in quantal size produced by pretreatment in hypertonic solution. It did not alter the increases in quantal size produced by insulin or by a permeable agonist of cAMP. It was known that hypertonic treatment increases quantal size by way of the cAMP-protein kinase A pathway. Anandamide had no effect on miniature endplate potential frequency (fmepp) in untreated preparations. After fmepp was increased in the presence of a permeable cAMP agonist, anandamide brought fmepp back to resting levels. The conclusions are that the motor nerve terminal has a cannabinoid receptor. The binding of anandamide to this receptor seems to block adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1994

The timing of channel opening during miniature endplate currents at the frog and mouse neuromuscular junctions: effects of fasciculin-2, other anti-cholinesterases and vesamicol.

Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the "tail" that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 microM (-)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (-)-vesamicol.

Acetylcholine↗