Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells

Abstract

Whole-cell membrane capacitance measurements are frequently used to monitor neuronal and nonneuronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization-induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles between 0.6 and 2 fF. For comparison, measurements of membrane capacitance were combined with amperometric recordings of catecholamine release during intracellular perfusion of chromaffin cells with high [Ca2+]. Crosscorrelation of both signals yielded a mean capacitance contribution of individual catecholaminergic vesicles of 1.3 fF. We suggest that depolarization-induced capacitance increases in mouse adrenal chromaffin cells mainly represent fusion of chromaffin granules.

Figure 2

Typical C_m measurement in chromaffin cells in slices. (A) Capacitance increase in response to a 10 ms long depolarization from the holding potential of −80 mV to 0 mV (center), which is flanked by preceding and after control measurements (no depolarization). After the depolarization the capacitance exhibits a decaying transient (ΔC_t) in addition to the stable elevation. The measuring sweeps were separated by 1–3 s (intervals indicated by dashed lines). The dc voltage protocol is displayed in the uppermost panel with the 10-ms constant voltage segments indicated by bold bars. Test and control ΔC_m values were estimated as the differences between C_m averages taken over the shaded time window after the constant voltage segments (long) with respect to averages over the preceding shaded window (short). (B and C) Membrane conductance (G_m) and series resistance (G_s) are shown to illustrate that there was no major crosstalk between C_m, G_m, and G_s lock-in estimates.

Figure 1

Analysis of amperometric spikes and single vesicle capacitance during secretion of isolated chromaffin cells. Cells were stimulated by intracellular perfusion with high [Ca²⁺] (internal solution 2) and bathed in Ringer’s solution (external solution 2). (A) The top graph shows the distribution of the current integrals (Q_amp) of the fastest amperometric spikes (risetime <1.5 ms). The Inset displays the smallest fast spike (peak amplitude ≈4 pA) detected and compares it it to a large one, recorded during the same experiment. The lower graph displays the  ³ histogram. A Gaussian fit (solid line) is superimposed. Its mean and SD were 54.2 × 10⁻⁶ and 18.7 × 10⁻⁶ C^1/3, respectively. (B) Time-locked ΔC_m (Upper) and amperometric current (Lower) averages for three groups. Top, left: spikes <23 pA; bottom, left: spikes >23 pA but <70 pA; right, top: spikes >70 pA. The Δc values were estimated as the y − difference of two parallel lines fitted to the average trace (up to 20 ms before the spike-peak, and from 20 ms after the spike-peak). (C) Plot of the Q_amp of the average spikes versus Δc.

Figure 3

Application of nonstationary fluctuation analysis to test and control ΔC_m. (A) Test ΔC_m values, here measured in response to repetitive 10-ms depolarizations (control ΔC_m data not shown). The x-axis indicates the experimental time. The first two ΔC_m values were excluded (nonequilibrium conditions). An analysis bin of four (shaded boxes) was moved forward point by point to calculate sample (group) means and variances. Whereas test means were calculated from the “raw” ΔC_m values (circles) we measured test variances after subtracting a fit to the ΔC_m values (solid line, representing the secretory rundown) from the “raw” ΔC_m values (corrected ΔC_m values: crosses). This was done to avoid contaminating variance attributable to the trend. No such correction was performed for estimation of control variances. (B) The sample means for test (circles) and control (bars from zero) ΔC_m. (C) Test (crosses) and control (bars from zero) sample variances.

Figure 4

Sample variances and means obtained for test ΔC_m are positively correlated. (A) A plot of the trend-corrected sample variances versus their corresponding means shown in Fig. 3 (10-ms depolarizations, bin-size = 4). Both measures were correlated for test ΔC_m (circles). The regression line had a slope of ≈1.0 fF. There was no correlation between variances and means in the case of control ΔC_m (squares). (B) Sample variances (trend-corrected) and means calculated from the same ΔC_m data with an analysis bin-size of 8. The larger bin slightly reduced the scatter, but again yielded a slope of the regression line of ≈1.0 fF. (C) An analogous plot for the analysis of an experiment where 100-ms depolarizations were repetitively applied (bin-size = 4, symbols as used above). The slope of the regression line fitted to the test data was 1.6 fF.