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 depolarizationinduced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles between 0.6 and 2 fF. Measurements of membrane capacitance (C m ) are used to study Ca 2ϩ -regulated exocytosis with high time resolution (reviewed in refs. 1 and 2). In many cases the vesicles are too small to be resolved as individual fusion events in whole-cell capacitance recordings. This is true also for chromaffin granules (3, 4) and even more for small synaptic-like microvesicles (SLMVs), which are present in neuroendocrine cells (reviewed in ref. 5). Capacitance recordings from single chromaffin cells in mouse adrenal slices have revealed a very fast exocytic component in response to membrane depolarization (ref. 6, time constant 7 ms). Does this fast C m rise represent fusion of chromaffin granules, SMLVs, or both? The uncertainty about the type of small fusing vesicles is circumvented if the release itself can be independently detected, for example by amperometry (7,8), provided that only one vesicle type contains oxidizable substances. However, recently catecholamine storage was suggested not only for chromaffin granules but also for SLMVs in chromaffin cells (9). Moreover, in contrast to C m measurements, the carbon-fiber microelectrodes detect release only from a variable portion of the cell. Thus, if exocytosis preferentially takes place at certain regions of the plasma membrane, the amperometrically detected release might not be representative for the secretion of the whole cell. Therefore, even combined C m and amperometric measurements do not necessarily answer the question of how many vesicles of which kind are released during a secretory response.We stimulated secretion in isolated mouse chromaffin cells by intracellular perfusion with high [Ca 2ϩ ] and observed only one population of catecholaminergic vesicles in amperometric recordings, most likely chromaffin granules. Averaging of the simultaneously recorded C m traces-time-locked at the peak of amperometric spikes-revealed corresponding C m step increases similar to the mean single-vesicle capacitance estimated from fluctuations in depolarization-induced capacitance increases in mouse chromaffin cells in slices. From this comparison we conclude that the fast secretory component in mouse chromaffin cells in slices mainly represents release of chromaffin granules.

Probing the Mechanism of Exocytosis at the Hair Cell Ribbon Synapse

“…Analysis of such fluctuations can reveal the apparent size of elementary fusion events (C app ). If vesicles fuse independent of each other this estimate is identical to the average vesicle capacitance, as in the case of chromaffin granules (Moser and Neher, 1997a). However, if release of vesicles was coordinated (statistically dependent) the apparent elementary size would be larger than the capacitance of a single vesicle.…”

“…Five series of these nonoverlapping windows were used. Furthermore, for correct variance estimation, we removed the trend resulting from the rundown (Moser and Neher, 1997a). The trend was described by a Double-exponential fit to the C m values or by forward-reverse low-pass filtering of the C m values (filtfilt function in Matlab).…”

“…This loss of depolarizationinduced C m increments argues strongly against the presence of a nonexocytic ⌬C m as observed in cells with high expression levels of voltage-gated sodium channels (Horrigan and Bookman, 1994;Moser and Neher, 1997b) or ion transporter molecules (Santos-Sacchi, 1991). We would expect these depolarization-evoked movements of gating charges in the bilayer to be much more stable over time than exocytosis (Santos-Sacchi, 1991;Horrigan and Bookman, 1994;Moser and Neher, 1997a). The mechanism of the exocytic rundown is at present unknown.…”

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca2+-dependence

“…Since chromaYn granules cannot be detected as single fusion events due to the low signal-to-noise ratio of the recording apparatus, the single quantal size can be estimated only by non-stationary Xuctuation analysis of repeated C events, as described by Moser and Neher (1997) (Fig. 5a, b).…”

A New Platform to Study the Molecular Mechanisms of Exocytosis

“…The equations were propagated in time, and the concentrations of the intermediates and the final products were saved as vectors. To quantitatively reconstruct the experimental signal, the fusion vector (the sum of reactions 10 and 15; see above), was multiplied by the capacitance of a single vesicle (1.25 fF, for mouse chromaffin cells) (Moser and Neher, 1997). Thus, the fusion process was reconstructed in discrete quanta, each representing the fusion of one vesicle.…”