The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes

Abstract

Cryo-electron microscopy of vitreous section makes it possible to observe cells and tissues at high resolution in a close-to-native state. The specimen remains hydrated; chemical fixation and staining are fully avoided. There is minimal molecular aggregation and the density observed in the image corresponds to the density in the object. Accordingly, organotypic hippocampal rat slices were vitrified under high pressure and controlled cryoprotection conditions, cryosectioned at a final thickness of Ϸ70 nm and observed below ؊170°C in a transmission electron microscope. The general aspect of the tissue compares with previous electron microscopy observations. The detailed analysis of the synapse reveals that the density of material in the synaptic cleft is high, even higher than in the cytoplasm, and that it is organized in 8.2-nm periodic transcleft complexes. Previously undescribed structures of presynaptic and postsynaptic elements are also described.Cryo-electron microscopy of vitreous section ͉ high-pressure freezing ͉ hippocampus S ynapses of the central nervous system (CNS) play a key role in neuronal information processing. Their physiology and structural organization have been extensively characterized (1, 2). The presynaptic compartment contains synaptic vesicles (SVs) filled with neurotransmitters. There is an intensive tethering and fusion activity between SVs and the presynaptic membrane. The postsynaptic membrane is covered with neurotransmitter receptors, which detect variations in neurotransmitter concentration. Postsynaptic submembrane cytoplasm is occupied by a complex network of proteins, the postsynaptic density, which modulates the strength of synaptic transmission. The space between both cells is the synaptic cleft. It contains neurotransmitter receptors and various adhesion molecules, such as N-cadherin and neural cell adhesion molecule. Their function in synapse formation and regulation is only beginning to be understood, and their precise spatial organization remains unclear (3).Resolving the molecular structure of the synapse is difficult because, on one hand, high resolution x-ray diffraction data are obtained outside their cellular context (4, 5). On the other hand, conventional electron microscopy generally fails to resolve individual proteins in their natural environment because the preparation relies on chemical fixation, dehydration, resinembedding, and heavy-metal staining and, thus, produces aggregation artifacts and artificial contrast, resulting in a limited resolution and a difficulty of interpreting the nature of the observed structures (6, 7). Freeze substitution and freeze etching, by which the specimen is immobilized by freezing and dehydrated at low temperature before being embedded in resin or replicated, were developed to reduce preparation artifacts but, even under these conditions, aggregation cannot be completely avoided. Depending on the protocols used for specimen preparation, the synaptic cleft presents different aspects. A dense central plaque was observed in some s...

Organization of the core structure of the postsynaptic density

“…Structures as small as 3-4 nm in diameter within 1-to 1.5-nm-thick virtual sections can be segmented. Dimensions of structures such as actin filaments (7 nm wide) (data not shown), synaptic vesicles (40 nm in diameter), and synaptic cleft (25 nm wide) are in close agreement with dimensions determined by cryo-EM and other methods (12)(13)(14).…”

The optimal height of the synaptic cleft

“…Physiological experiments that would test this hypothesis directly are likely to be difficult because the rigid cleft structure appears to withstand various experimental manipulations, including physical separation of the synaptic membrane fraction or even the solubilization of the neighboring membranes (21). Indeed, electron micrographs obtained with different tissue fixation methods suggest a relatively constant average distance between pre-and postsynaptic membranes ranging between 15 and 25 nm (19,22). The molecular basis of this architecture appears to rely on cadherin-type adhesion molecules that provide transcellular structural scaffolding connecting pre-and postsynaptic membranes (20,21).…”

“…Remarkably, in electron micrographs of synaptosomes (a preparation obtained using strong mechanical forces of centrifugal separation), the distance between the apposing synaptic membranes is indistinguishable from that in the intact neuropil. Across many synaptic types, this distance lies within a relatively narrow range of 15-25 nm (19), which appears to be determined by the rigid structure of the intracleft protein scaffolding (20)(21)(22). However, the adaptive purpose for the synaptic cleft height to be within this range is not clear: in theory, narrower cleft should provide for chemical synaptic transmission that is more efficient.…”

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…In the latter case, the authors report that it took ~7 minutes from removal of the brain to HPF of dissected hippocampal slices. Zuber et al, (2005) used organotypic rat hippocampal slices in cryo-protectant to study synapse structure in vitreous ice sections examined at low temperature without any fixation, staining or embedding. The method provides a more near-to-native ultrastructure and produces images sometimes containing new features but the technique is difficult, requires the use of an expensive cryo-electron microscope as well as a HPF machine and is presently limited to thin sections of 70-100 nm thick.…”