Ultrastructure of the Cholinergic Synapse Revealed by Quick-Freezing

Abstract

This abstract outlines the use of quick-freezing to elucidate the ultrastructure of the cholinergic synapse and to capture the structural changes that underlie synaptic transmission. The machine developed for this purpose (Heuser, J.E., et al., Cold Spring Harbor Symp. quant. Biol., 40, 17–24, 1976), is designed to stimulate nerve-muscle preparations, record the resulting end-plate potentials, and at precisely timed intervals thereafter to freeze the tissues almost instantaneously by plunging them onto a pure copper block cooled to 4°K with liquid helium. High speed resistance and capacitance measurements at the time of impact indicate that the surface of the muscle freezes within one msec. Quick-freezing of the frog neuromuscular junction has been used to reconstruct the sequence of events that occurs during presynaptic nerve secretion. First, it has established that transmitter release is brought about by exocytosis of synaptic vesicles, by showing that such exocytosis occurs at exactly the same time as transmitter release, and that one synaptic vesicle opens for each quantum that is discharged (Fig. 1) (Heuser, J.E., et al., J. Cell Biol., in press). Second, quick-freezing has shown that discharged vesicle membrane begins to be retrieved from the plasma membrane within a few seconds, by a specialized form of endo-cytosis known as “coated vesicle” formation. These sorts of timing experiments add further evidence in favor of the original idea of Heuser and Reese (J. Cell Biol., 57, 315–344, 1973) that synaptic vesicle membrane is recycled and used over again to make a new generation of synaptic vesicles (Heuser, J.E. and Reese, T.S., in The Nervous System, Handbook of hysiology, Vol. 1, Ed. E. Kandel, Amer. Physiol. Soc., Chapter 8, 1977). Fig. 1 Standard electron microscopy view of a frog neuromuscular junction that was frozen so fast that ice crystals are invisible, and then was OsO4 fixed while still frozen, to obtain optimum preservation of membrane specializations and synaptic vesicles that characterize the cholinergic synapse. The magnified image shows what we take to be synaptice vesicle exocytosis caught by quick-freezing a nerve just at the moment it secreted acetylcholine in response to one nerve impulse. ×40,000. The method of quick-freezing has also been used to stabilize the molecular architecture of the postsynaptic membrane at the cholinergic synapse (Heuser J.E. and Salpeter, S.R., J. Cell Biol., in press), The acetylcholine receptors have been visualized by etching water away from the surface of frozen electrocytes, the postsynaptic cells in the Torpedo electric organ (Fig. 2). The view thus obtained has illustrated that the acetylcholine receptors are arranged in a highly ordered lattice (Fig, 3). The forces which stabilize this lattice are unknown, but deep-etching reveals other aspects of the matrix around the postsynaptic membrane which may be involved. For one, it reveals the presence of a dense filamentous network in the cytoplasm beneath the postsynaptic membrane, which attaches to it periodically (Figs. 2, 3) and could possibly serve a role in immobilizing the receptors. Also, deepetching reveals the structure of the basal lamina in the synaptic cleft (Figs. 1–3) and indicates its important function in cholinergic transmission, which is that of binding acetylcholinesterase and interconnecting the pre- and postsynaptic membranes. Fig. 2 View of a trumpet-shaped invagination of the postsynaptic membrane in the fish electrocyte cholinergic synapse, analogous to the muscle of folds at the neurornuscular junction (Boxed in Fig. 1). Before replication with platinum and carbon, this freeze fracture was freeze-dried for 3 min at −100°C to remove water and bring structures inside and outside the membrane into view: the filamentous net work in the cytoplasm beneath, and the white “barbed-wire” looking basal lamina in the synaptic cleft above, Note: the basal lamina characteristically extends down into the center of the invagination, as it does at the neuromuscular folds in Fig. 1. × 120,000. Fig. 3 Panoramic view of the postsynaptic membrane revealed by deep etching, rotary shadowing, and photographic reversal (Heuser, J.E. and Salpeter, S.R., J. Cell Biol., in press). With this technique, the platinum deposits which coat every surface irregularity look white, producing a 3-D perspective like that obtained from scanning electron microscopy. To the left of this figure, the lace-like basal lamina lies above the membrane and obscures it from view; and below, it assumes a ring-like appearance as it extends down into a dark post-synaptic invagination. Here and there, strands of the basal lamina extend out and attach to the membrane with claw-like feet (arrow). In the center of the figure, the basal lamina has been broken away t o reveal the true external surface of the postsynaptic membrane with its characteristic clusters and linear arrays of “donuts” which are thought to be the acetylcholine receptor molecules. To the right, the membrane has been cleaved clear through, and deepetching has revealed the underlying “trabecular meshwork” of cytoplasmic filaments which gird this membrane from beneath. × 175,000.