Using the Monte Carlo method, spontaneous fast excitatory postsynaptic currents (mEPSCs) at a hippocampal synapse were simulated by releasing 150-20,000 glutamate molecules from a point source centred 15 nm above a rectangular grid of 14 x 14 alpha-amino-3-hydroxy-methyl-isoxazole (AMPA) receptors and assuming the channel kinetics to be as reported by Jonas et al. [J Physiol (Lond) 472:615; 1993]. The relationship between the amplitudes of mEPSCs and their time constants of decay is positive, but not pronounced in physiological conditions (except when the number of molecules released is very high). It increases as desensitization is reduced and becomes highly pronounced when it is eliminated. mEPSCs are prolonged with repeated opening of AMPA channels due to enhancement of two concentration-dependent processes: (1) binding of glutamate molecules by AMPA receptors, and (2) occupancy of both activatable bound states. In contrast, the time constant of decay of the patch currents evoked by a short glutamate pulse is independent of glutamate concentration and current amplitude in control conditions, and only moderately concentration dependent in the absence of desensitization. The fast application protocol thus fails to reproduce synaptic currents reliably when there is repeated binding of glutamate molecules to AMPA receptors. During an mEPSC, the occupancy of desensitized states increases rapidly and it strongly depends on the number of glutamate molecules released. Desensitization reaches its maximum after an mEPSC decays to very low levels, and recovers very slowly (from tens to hundreds of milliseconds), and in a concentration-dependent manner. In conclusion, under physiological conditions the desensitization of AMPA receptors plays a major role in shaping the time course of mEPSCs by minimizing the repeated opening of AMPA channels.

Using the Monte Carlo technique, Ca2+ dynamics were simulated in the absence and presence of vesicles to gain better insight into what governs quantal release. A vesicle, represented as a flat, infinitely thin surface, was positioned parallel to the plasma membrane at a chosen distance from the locus of Ca2+ entry. Because vesicles act as important diffusion barriers after the synchronous opening of Ca2+ channels (as occurs during evoked release), [Ca2+] close to the plasma membrane reaches higher levels than it would in the absence of vesicles. The rise in [Ca2+] is greater under larger vesicles close to the plasma membrane, which thus have a higher probability of release. The power-law relationship between the [Ca2+] and the probability of release, and the cubic relationship between the vesicular diameter and its volume can make this relationship very steep. In contrast, when release occurs owing to fluctuations of [Ca2+]--as a result of Ca2+ release from an internal store or asynchronous opening of Ca2+ channels (during spontaneous release)--the effect of vesicles as diffusion barriers is less pronounced and vesicles of different sizes should have a similar probability of release. Since the preferential release of large vesicles depends on how the Ca2+ needed for secretion is raised (synchronously versus asynchronously), the quantal size of evoked and spontaneous release should differ. The main factors influencing the preferential release of large vesicles are the distance between vesicles and the plasma membrane, the concentration of Ca2+ buffers, and single-channel Ca2+ flux. Vesicles also have a pronounced effect on Ca2+ binding to buffers and on the spatio-temporal distribution of bound buffers. The greater the vesicular size and the closer their position to the plasma membrane, the more fixed buffers will be bound near the plasma membrane because of limited diffusion of Ca2+. Since bound fixed buffers act as "memory elements", such a change in their spatial distribution will further enhance the probability of release of large vesicles during stimulation.

Changes in quantal size and in the parameters of release were examined in chromaffin cells using amperometric recordings during and following various stimuli that induce secretion. As a general rule, a greater quantal content was associated with a greater quantal size. Following a short depolarizing pulse (0.5-2 s; 100 mV from a holding potential of -80 mV), the mean value of quantal size increased by 54% over several seconds before gradually (over tens of seconds) returning toward the control value, whilst its variability rose by 62%. The changes observed following 30-s applications of high extracellular K+ (50 mM) were more modest. A rapid application of short depolarizing pulses (2 s every 10-20 s; 100 mV from a holding potential of -80 mV) also led, at least initially, to greater quantal content and quantal size. Mean quantal size rose initially by 68%, but decreased subsequently to 31% below pre-stimulation levels. In whole-clamped cells, the frequency of quantal release can rise abruptly, probably owing to a mechanical disturbance that makes the membrane leaky to Ca2+. In such cases, a marked rise in quantal content (>ten-fold) was paralleled by an almost as dramatic (up to ten-fold) rise in quantal size and an important, although less pronounced and slower, rise in its variability (up to four-fold). The return toward control values of mean quantal size occurred over several minutes, whilst its variability decayed more slowly. The release parameters were evaluated directly from the number of events to avoid a large and time-dependent contribution of the amplitude variability of spontaneous amperometric current spikes (minis). In general, the greater probability of release contributed more than the greater size of the immediately available store to the increase in quantal output. In conclusion, quantal size was found to be highly labile. Its change can alter strongly the facilitation and depression of evoked quantal output and probably occurs due to a preferential release of large vesicles that are more efficient barriers to Ca2+ diffusion when Ca2+ rises rapidly following a synchronous opening of several Ca2+ channels. When intracellular Ca2+ levels rise slowly to threshold levels for secretion, as during an asynchronous and generally spontaneous release, vesicles are less effective diffusion barriers and quantal size changes less.

No abstract

Nanopores with irregularities are promising tools for distinguishing nano-size objects by their shape, but the forces on the object that critically influence its axial and rotational movement are unclear. The physics of the situation was described using the Poisson-Nernst-Planck and Navier-Stokes equations. With uniformly charged object the axial Coulomb and dielectric pressure (which opposes it and is surprisingly important), control the object's axial movement and rotation. Even without external pressure the hydrodynamic pressure is significant (negative at its upper and positive at its lower surface), but its total value is almost zero. If the object is charged only on the upper surface the axial upper Coulomb pressure is near zero close to the center, but negative near its end (the pressure is zero at the lower surface). The total axial dielectric pressure, which is largely dominated by the pressure at the upper surface, is positive along the length of the object becoming pronounced near its end. The axial hydrodynamic pressure is negative and significant at the upper surface (zero at the lower surface), diminishes in value near the object's end, critically influencing its axial movement, which becomes much faster. At its end the axial dielectric pressure prevails, and controls its rotation.