No abstract

The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4°K from the living state . In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix . In the corneal endothelium, both random and crystalline arrangements of the connexons were observed . In the stomach and liver, the connexons were packed but not crystalline . Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min . In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random . Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation . The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance .When gap junctions are chemically fixed and subsequently analyzed with the freeze-fracturing technique, the clustering of their subunits (particles or connexons) in the plane of the membrane varies in different tissues and often in different cells of the same tissue: hexagonal packing and complete randomness are the extreme configurations, but a great variety of intermediate arrangements have been described (4,10,20) . The functional correlate of these variations is not known, but it seems unlikely that they could reflect differences in the resistance state ofthe junctions. In fact, it was shown that aldehyde fixation abolishes coupling, and therefore freeze-fractured gap junctions in fixed tissues are probably visualized in their highresistance state (3) . Nevertheless, differences in the degree of clustering of the connexons were observed in tissues exposed to uncoupling agents before fixation as compared with their untreated controls (12, 21, 22); thus, it was proposed that the switch to high resistance is accompanied by movement of the connexons in the plane of the membrane and their lateral association in a compact, crystalline lattice . There are exceptions, however, to this behavior, because uncoupling agents did not seem to affect the state of connexon aggregation in the gap junctions of the lens (11).These observations raise important questions on the structure of gap junctions in vivo, and on the time-course of crystallization and its precise relationships with uncoupling. Such questions, however, can only be answered if the structure of gap junctions can be analyzed and experimentally manipulated in absence of chemical fixation. For this reason, we used the technique of rapid-freezing to near 4°K (14, 28) on a variety of tissues. With this method, freezing...

The internal structure of the synaptic membranes in the inner plexiform layer (IPL) of the retina of monkeys and rabbits was studied with the freeze-fracturing technique. In ribbon synapses, the presynaptic active zone is characterized by an aggregate of P-face particles, images of synaptic vesicle exocytosis, and forming coated vesicles which occupy distinct, contiguous membrane domains from apex to base of the synaptic ridge. The postsynaptic membrane contains a prominent aggregate of homogeneous particles which remain associated with the E-face. In the presynaptic membrane of conventional synapses, images of synaptic vesicle exocytosis are intermingled with large P-face particles, whereas forming coated vesicles surround the active zone. Three types of internal organization characterize the postsynaptic membrane of conventional synapses. Usually, the postsynaptic membrane exhibits the same internal structure as the surrounding nonjunctional plasmalemma. A second, less common type of conventional synapse contains a loose aggregate of heterogeneous particles which remain associated with the P-face. Finally, synapses were exceptionally found which are macular in shape and contain an aggregate of E-free particles within the postsynaptic membrane. The freeze-fracture evidence suggests that the axonal endings of bipolar cells--or at least some of them--make excitatory synapses, whereas the vast majority of amacrine cell dendrites make inhibitory synapses. Additional specializations of the cell surface in the IPL include gap junctions, puncta adhaerentia, subsurface cisterns, and cell corner aggregates.

By comparison of electron micrographs with light microscopical specimens impregnated with the Gold technique, the large endings of the rod bipolar cells have been identified in the innermost region of the inner plexiform layer of the rabbit retina.The rod bipolar endings contain ribbons and synaptic vesicles, do not synapse with the perikaryon of the ganglion cells, are presynaptic to ganglion cell dendrites and to nerve processes which contain synaptic vesicles but lack ribbons. In these synaptic contacts a ribbon is closely associated with the presynaptic membrane and a dense web of fuzzy material is adherent to the cytoplasmic aspect of the postsynaptic membrane. Commonly, one of these synaptic contacts involves a rod bipolar ending and two postsynaptic processes. The postsynaptic process which is provided with synaptic vesicles is often, in turn, presynaptic to the same rod bipolar ending. This synaptic contact i s characterized by the presence of a cluster of vesicles closely related to the presynaptic membrane, whereas the postsynaptic membrane lacks a definite subsynaptic web.In the intermediate and scleral regions of the inner plexiform layer endings containing ribbons and synaptic vesicles show with neighboring nerve processes a synaptic pattern similar to the rod bipolar endings.Nerve processes containing synaptic vesicles but lacking ribbons are presynaptic to the perikaryon and dendrites of the ganglion cells; the synaptic contact shows a cluster of vesicles adherent to the presynaptic membrane.Bipolar cells are proposed as the source of the ribbon containing processes while amacrine cells are proposed as the source of the processes devoid of ribbons and presynaptic to both bipolar endings and ganglion cell dendrites and perikarya.