Axo-dendritic synaptogenesis was examined in live hippocampal cell cultures using the fluorescent dyes DiO to label dendrites and FM 4-64 to label functional presynaptic boutons. As the first functional synaptic boutons appeared in these cultures, numerous filopodia (up to 10 micron long) were observed to extend transiently (mean lifetime 9.5 min) from dendritic shafts. With progressively increasing numbers of boutons, there were coincident decreases in numbers of transient filopodia and increases in numbers of stable dendritic spines. Dendritic filopodia were observed to initiate physical contacts with nearby axons. This sometimes resulted in filopodial stabilization and formation of functional presynaptic boutons. These findings suggest that dendritic filopodia may actively initiate synaptogenic contacts with nearby (5-10 micron) axons and thereafter evolve into dendritic spines.
Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.
Long term time-lapse imaging reveals that individual synapses undergo significant structural remodeling not only when driven by activity, but also when network activity is absent, raising questions about how reliably individual synapses maintain connections.
Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial.
Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.
The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.
The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.
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