Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.