The relationship of transmitter release and storage to fine structure in a sympathetic ganglion

Summary

Cat sympathetic ganglia were prepared for electron microscopy by perfusion fixation with glutaraldehyde in the presence of Mg⁺⁺. At resting boutons de passage the populations of synaptic vesicles were 6400 per bouton. The vesicle distributions displayed many of the features of spheres in close-packing. Calculations based on a vesicle close-packing hypothesis gave a figure of 8000 vesicles per bouton. In ganglia stimulated for 20 min at 20/s the vesicle populations were reduced to 25%, and to 28.5% in ganglia in which acetylcholine (ACh) synthesis was inhibited by hemicholinium (HC-3). The reduction was to 34% when stimulation was for 1 min. In ganglia stimulated for 20 min at 1/s and 4/s the vesicle populations were reduced to 44% and 46% respectively. Even following 1 min stimulation at 4/s over half the boutons showed significant loss of vesicles. ACh stores in ganglia are known not to be depleted by any of these procedures except stimulation in the presence of HC-3. The fraction of ganglionic ACh stores known to be released by stimulation for 1 min at 20/s or 4/s and by 20 min stimulation at 1/s is substantially less than the fraction of vesicles lost. The observations therefore were not readily accounted for by the vesicle theory of transmitter storage and release. They were consistent with the idea the ACh is stored in vesicles at rest, but that during maintained activity over half the bouton ACh is free in the cytoplasm. The concentration of cytoplasmic ACh was calculated to be approximately 50–150 mm 1⁻¹. Examination of the hypothesis that ACh may be released from this cytoplasmic pool during synaptic activation indicated an efflux of approximately 1.5–3.0 × 10⁻¹² M Ach cm⁻² synapsing membrane/impulse.