Figure 7 - uploaded by Guido A Zampighi
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The Synaptic Vesicle/Indentation Complex. Panel A shows a lateral view of an indentation with three rods associated with its wall (yellow) and one protruding into the cytoplasm (cyan). Panel B is a top view of the plasma membrane (P pre ) indicating that a complex
Source publication
We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that "rods" assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We...
Citations
... The proteins that connect synaptic vesicles to the plasma membrane can be visualized as filaments with electron-microscopic tomography (EMT) connecting vesicles to the presynaptic active zone (17)(18)(19)(20)(21)(22)(23)(24). Studies suggest that the SNARE complex begins to form when a vesicle and presynaptic membrane are within 8 nm of each other rendering them loosely docked (25)(26)(27). ...
... In the past, rapid freezing and freeze-substitution have been used to capture ultrastructural changes during synaptic transmission (19,21,23,(40)(41)(42)(43). Here microwave-enhanced aldehyde fixation of brain slices likely occurs over seconds rather than the millisecond timescale achieved with freezing (44). ...
Significance
Long-term potentiation (LTP), a form of synaptic plasticity important for learning and memory, results in an increased probability of release of neurotransmitter from presynaptic vesicles. Prior work showed total vesicle number was decreased following LTP, seemingly inconsistent with this increased probability of release. Presynaptic vesicles are tethered to the active zone via filaments composed of molecules engaged in docking, priming, and release processes. Here, electron-microscopic tomography revealed a higher density of docked vesicles at active zones. Tethering filaments on vesicles at the active zone were shorter, and their attachment sites were shifted closer to the active zone. These changes suggest more vesicles were docked and primed, which would increase the probability of release 2 h after induction of LTP.
... Thus, although hemifusion of synaptic vesicles is likely to occur at active zones prior to fusion, as a part of productive fusion pathways, the results shown here and from other studies using chemical fixation or high-pressure freezing [43][44][45]61] indicate that hemifused vesicles are not commonly observable, probably due to their instability, which facilitates full fusion with the presynaptic membrane. The occurrence and stability of hemifused synaptic vesicles may depend on synapse specific factors regulating the spatial relationship of synaptic vesicles with the presynaptic membrane at the active zone, such as multiple AZM macromolecules connected to docked synaptic vesicles, which may contain key proteins for fusion of the vesicles with the presynaptic membrane [34,35,45,47,49,50,[62][63][64]. Accordingly, docked synaptic vesicles are likely to constitute immediately release-ready vesicles in general [43,44]. ...
Synaptic vesicles dock on the presynaptic plasma membrane of axon terminals and become ready to fuse with the presynaptic membrane or primed. Fusion of the vesicle membrane and presynaptic membrane results in the formation of a pore between the membranes, through which the vesicle’s neurotransmitter is released into the synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked synaptic vesicles with the presynaptic membrane, however, the extent of the contact area between the membrane of docked synaptic vesicles and the presynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked synaptic vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused synaptic vesicles among the docked vesicles was briefly reported. Here, the spatial relationship of 81 synaptic vesicles with the presynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and presynaptic membrane, similar to the docked vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused vesicles were found to have large contact areas with the presynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked synaptic vesicles with the largest contact areas are most primed for fusion with the presynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the presynaptic membrane for synaptic transmission.
The transmission of neuronal information is propagated through synapses by neurotransmitters released from presynapses to postsynapses. Neurotransmitters released from the presynaptic vesicles activate receptors on the postsynaptic membrane. Glutamate acts as a major excitatory neurotransmitter for synaptic vesicles in the central nervous system. Determining the concentration of glutamate in single synaptic vesicles is essential for understanding the mechanisms of neuronal activation by glutamate in normal brain functions as well as in neurological diseases. However, it is difficult to detect and quantitatively measure the concentration of glutamate in single synaptic vesicles owing to their small size, i.e., ∼40 nm. In this study, to quantitatively evaluate the concentrations of the contents in small membrane-bound vesicles, we developed an optical trapping Raman spectroscopic system that analyzes the Raman spectra of small objects captured using optical trapping. Using artificial liposomes encapsulating glutamate that mimic synaptic vesicles, we investigated whether spontaneous Raman scattered light of glutamate can be detected from vesicles trapped at the focus using optical forces. A 575 nm laser beam was used to simultaneously perform the optical trapping of liposomes and the detection of the spontaneous Raman scattered light. The intensity of Raman scattered light that corresponds to lipid bilayers increased with time. This observation suggested that the number of liposomes increased at the focal point. The number of glutamate molecules in the trapped liposomes was estimated from the calibration curve of the Raman spectra of glutamate solutions with known concentration. This method can be used to measure the number of glutamate molecules encapsulated in synaptic vesicles in situ.
Neuromuscular junctions (NMJs) have long been studied as particularly accessible examples of chemical synapses. Nonetheless, some important features of neuromuscular transmission are still poorly understood. One of these is the low statistical variability of the number of transmitter quanta released from motor nerve terminals by successive nerve impulses. This variability is well-described by a binomial distribution, suggesting that the quanta released are drawn, at high probability, from a small subset of those in the terminals. However the nature of that subset remains unclear. In an effort to clarify what is understood, and what is not, about quantal release at NMJs, this review addresses the relationship between NMJ structure and function. After setting the biological context in which NMJs operate, key aspects of the variability of release and the structure of the motor nerve terminals are described. These descriptions are then used to explore the functional logic of motor nerve terminal organization and the structural basis of the low variability of release. This analysis supports the suggestion that the probability of release differs significantly at the different 'active zones' from which quanta are released. Finally, after a brief consideration of how release is maintained in the long term, a comparison is made of the features of NMJs with those of some well-studied neuronal synapses. An important conclusion is that NMJs share some important features with neuronal synapses, so continuing efforts to understand how motor nerve terminals work are likely to have much more general implications.
