Visual identification of synaptic boutons on living ganglion cells and of varicosities in postganglionic axons in the heart of the frog.

1. Parasympathetic neurons were studied in the transparent interatrial septum of the frog (Rana pipiens) with light- and electron-microscopic techniques. The aim was to identify visually cellular and subcellular details in a living preparation, especially synaptic boutons on ganglion cells and the varicosities in postganglionic axons supplying the muscles of the heart. 2. The interatrial septum contains the following nervous elements: unipolar parasympathetic ganglion cells, their preganglionic vagal innervation, postganglionic sympathetic axons and sensory fibres. These structures and the nuclei of their related Schwann cells can be viewed with various optical systems, especially differential interference contrast optics. The same neural elements identified in the live preparation can be sectioned for electron microscopy. 3. Most ganglion cells are innervated by a single presynaptic axon, terminating in up to 27 synaptic boutons which on the average cover about 3.0% of the surface of nerve cell bodies. A few scattered boutons also occur on the initial axonal portion of the ganglion cells. 4. Synaptic boutons on ganglion cells were recognized in the living unstained preparation. Their identify was confirmed by electron microscopy and by light microscopy combined with methylene blue, zinc iodide and osmium, and cholinesterase staining methods. 5. The terminal branches of postganglionic axons have numerous varicosities along their course. Some are as close as a few hundred angstroms (10 angstrom = 1 nm) to muscle fibres, others are many $\mu $m away. There are two types of varicosities: (i) those which contain predominantly granular vesicles characteristic of neurons releasing catecholamines, and (ii) those with predominantly agranular vesicles which belong to the cholinergic axons of septal ganglion cells. Regardless of their distance from muscle fibres, the cholinergic varicosities have the same fine structural features, including membrane thickenings, as synaptic boutons on the ganglion cells. These findings support earlier suggestions that the varicosities along postganglionic axons are a series of transmitter release sites. 6. Varicosities were observed in the live septum; their identity was confirmed by subsequent electron microscopy. Many live varicose axons were traced back to the vicinity of individual septal ganglion cells. Additional evidence that they belonged to a particular ganglion cell, and were therefore cholinergic, was obtained by injecting Procion yellow into the cell body and observing the neuron with a fluorescence microscope after the dye had spread into the axonal processes. Time lapse photography of up to 24 h showed no 'peristaltic' movement of varicosities. 7. Granular or agranular vesicles also occur along cylindrical axons within nerve bundles many $\mu $m away from muscle fibres. Like the vesicles in varicosities, they are clustered close to 'thickenings' in the surface membrane and belong to postganglionic nerve fibres. 8. Ganglion cells in isolated septa survive for 2 weeks or longer, still giving membrane potentials and impulses. Time lapse cinematography for up to 2 weeks after removing the septum showed that the organelles within the neurons were in motion and that a two-way traffic takes place between the cell body and axon, as commonly found in cultured neurons.