Recycling and refilling of transmitter quanta at the frog neuromuscular junction

Abstract

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.

Figure 1. Examples of MEPP recordings and the fitting of distribution functions to the data

A, MEPP recordings in gluconate Ringer solution from a preparation that had been previously soaked for 20 min in 30 mm K⁺ solution + 1 μM HC3. The examples were selected to illustrate the wide range of MEPP sizes. B, a cumulative plot of the data from a similar experiment (green circles), the fit to a single lognormal distribution (orange line), and the fit to two lognormal distributions (black line).

Figure 2. Estimates of the number of quanta released while stimulating quantal release in 30 mm K⁺ solution

The error bars show the ± 95 % confidence limits.

Figure 3. The properties of the quanta change when release is promoted in the presence of the inhibitors

A, the mean ∫MEPP becomes smaller in the presence of VES, NH₄⁺ or HC3. The results with all three inhibitors have been pooled. The error bars show the ± 95 % confidence limits. B, the CVs of the ∫MEPPs increase. Without inhibitor (○), with HC3 (□), and with NH₄⁺ (▵). The error bars show the +95 % confidence limits. C, changes in the CVs in the presence of VES; the error bars show the ± 95 % confidence limits.

Figure 4. The reproducibility at the same junctions of the determinations of the fraction of the ∫MEPPs that are in the sub-population with the larger mean

Each group of points falling along a vertical shows measurements from the same endplate. The preparation had been exposed to 30 mm K⁺ solution containing NH₄⁺, VES or HC3 for 15–20 min before the measurements were made in gluconate Ringer solution.

Figure 5. Changes in the sub-populations as quanta are released

A, the percentage of the preparations that have two sub-populations increases as more quanta are released. The numbers in parentheses show the total number of sets of ∫MEPPs analysed. With no inhibitor (□); with HC3, VES or NH₄⁺ (○). B, the fraction of the ∫MEPPs falling in the larger sub-population decreases exponentially as more quanta are released in the presence of HC3, NH₄⁺ or VES. For the exponential curve R² = 0.92. In parentheses are the number of examples in each of the points.

Figure 6. The mean sizes in the larger and smaller distributions change relatively little when more quanta are released

The inhibitors used were HC3 (black circles), NH₄⁺ (orange squares), and VES (purple triangles).

Figure 7. The number of quanta released from preparations soaked in 10 μM CCCP

A, the time course of the release from paired preparations from the same frog. The example shows the most substantial difference in the time course observed in the experiments. B, a scatter diagram of the release from the two preparations from the same frogs. Different protocols have been pooled in the figure, because they all illustrate the correlation in the numbers of quanta released by preparations from the same animal, R² = 0.68. Untreated preparations, preparations in which one muscle was kept in 30 mm K⁺ solution for 1 h before exposure to CCCP and preparations in which one muscle was in Ca²⁺-free gluconate Ringer solution containing 1 mm EGTA (○). Experiments in which the second preparation was exposed for 2 h to Ca²⁺-free gluconate Ringer solution containing 1 mm EGTA and 0.2 mm BAPTA AM before CCCP was added to the same solution. The first preparation was in Ca²⁺-free gluconate Ringer solution containing 1 mm EGTA throughout (□).

Figure 8. Estimates of the size of the store of releasable quanta

The error bars show the +95 % confidence limits. The numbers in parentheses below the the x-axis show the number of junctions from which the measurements were made. The values for NH₄⁺, HC3 and VES were calculated from data from individual junctions that had been treated in 30 mm K⁺ solution + inhibitor and showed two sub-populations. The CCCP values come from estimates of the total number of quanta released in the presence of 10 μM CCCP from the same batches of animals used in the inhibitor studies.

Figure 9. The recovery in ∫MEPP size at junctions in preparations pre-treated in 30 mm K⁺ solution and then placed in gluconate Ringer solution without NH₄⁺

A, mean ∫MEPP size (○) ± 95 % confidence limits, with the scale on the left vertical axis, and the fraction in the larger sub-population (□), with the scale on the right vertical axis. B, cumulative plots of the data from a second experiment. The time (min) in the NH₄⁺-free solution at which recording of the data in each set finished is shown beside the curves.

Figure 10. Quantal size does not increase following a second exposure to 30 mm K⁺ solution that does not contain NH₄⁺ to inhibit ACh accumulation

The results with two different K⁺ exposure times are shown. The error bars show the +95 % confidence limits.