Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.
The voltage dependence and kinetic properties of stage 40 ciliary ganglion calcium currents were determined using short (10 ms) voltage steps. These properties aided the interpretation of the action potential‐evoked calcium current described below, and the comparison of our data with those observed in other preparations.
Three different natural action potential waveforms were modelled by a series of ramps to generate voltage clamp commands. Calcium currents evoked by these model action potentials were compared before and after alterations in the repolarization phase of each action potential.
Abrupt step repolarizations from various time points were used to estimate the time course of calcium current activation during each action potential. Calcium current evoked by fast action potentials (duration at half‐amplitude, 0·5 or 1·0 ms) did not reach maximal activation until the action potential had repolarized by 40‐50 %. In contrast, calcium current evoked by a slow action potential (duration at half‐amplitude, 2·2 ms) was maximally activated near the peak of the action potential.
Slowing the rate of repolarization of the action potential (broadening) from different times was used to examine effects on peak and total calcium influx. With all three waveforms tested, broadening consistently increased total calcium influx (integral). However, peak calcium current was either increased or decreased depending on the duration of the control action potential tested and the specific timing of the initiation of broadening the repolarization phase.
The opposite effects on peak calcium current observed with action potential broadening beginning at different time points in repolarization may provide a mechanism for the variable effects of potassium channel blockers on transmitter release magnitude.
The contribution of synaptic input to input resistance was examined in 208 developing genioglossal motoneurons in 3 postnatal age groups (5-7 day, 13-16 day, and 18-24 day) using sharp electrode recording in a slice preparation of the rat brain stem. High magnesium (Mg(2+); 6 mM) media generated significant increases (21-38%) in both the input resistance (R(n)) and the first time constant (tau(0)) that were reversible. A large percent of the conductance blocked by high Mg(2+) was also sensitive to tetrodotoxin (TTX). Little increase in resistance was attained by adding blockers of specific amino acid (glutamate, glycine, and GABA) transmission over that obtained with the high Mg(2+). Comparing across age groups, there was a significantly larger percent change in R(n) with the addition of high Mg(2+) at postnatal days 13 to 15 (P13-15; 36%) than that found at P5-6 (21%). Spontaneous postsynaptic potentials were sensitive to the combined application of glycine receptor antagonist, strychnine, and the GABA(A) receptor antagonist, bicuculline. Application of either 10 microM strychnine or bicuculline separately produced a reversible increase in both R(n) and tau(0). Addition of 10 microM bicuculline to a strychnine perfusate failed to further increase either R(n) or tau(0). The strychnine/bicuculline-sensitive component of the total synaptic conductance increased with age so that this form of neurotransmission constituted the majority (>60%) of the observed percent decrease in R(n) and tau(0) in the oldest age group. The proportion of change in tau(0) relative to R(n) following strychnine or high magnesium perfusate varied widely from cell to cell and from age to age without pattern. Based on a model from the literature, this pattern indicates a nonselective distribution of the blocked synaptic conductances over the cell body and dendrites. Taken together, the fast inhibitory synapses (glycine, GABA(A)) play a greater role in determining cell excitability in developing brain stem motoneurons as postnatal development progresses. These findings suggest that synaptically mediated conductances effect the membrane behavior of developing motoneurons.
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