Rapid recruitment of NMDA receptor transport packets to nascent synapses

In the central nervous system, NMDA receptors mediate excitatory neurotransmissions and play important roles in synaptic plasticity. The regulation of NMDA receptor trafficking is critical for neural functions in the brain. Here, we directly visualized individual exocytic events of NMDA receptors in rat hippocampal neurons by total internal reflection fluorescence microscopy (TIRFM). We found that the constitutive exocytosis of NMDA receptors included both de novo exocytic and recycling events, which were regulated by different Rab proteins. We also identified the SNAP25-VAMP1-syntaxin4 complex mediating the constitutive exocytosis of NMDA receptors. Transient knockdown of each component of the SNARE complex interfered with surface delivery of NMDA receptors to both extrasynaptic and synaptic membranes. Our study uncovers the postsynaptic function of the SNAP25-VAMP1-syntaxin4 complex in mediating the constitutive exocytosis of NMDA receptors, suggesting that this SNARE complex is involved in excitatory synaptic transmission.tamate receptors, mediating excitatory neurotransmission in the brain and playing crucial roles in synaptogenesis, synaptic plasticity, and excitotoxicity. NMDA receptors consist of tetrameric combinations of the homologs subunits NR1, NR2A-D, and NR3A-B. In hippocampal neurons during synaptogenesis, most NMDA receptors are NR1-NR2B heteromers. As the neurons mature, the abundance of NR1-NR2A and NR1-NR2A-NR2B heteromers increased (1). Electrophysiological and immunocytochemical evidences showed that in mature neurons, NMDA receptors primarily clustered at excitatory synapses, which were localized on dendritic spines (2-8). To achieve this specific distribution, NMDA receptors undergo regulations in various trafficking steps, including de novo exocytosis, endocytosis, recycling, lateral movement between synaptic and extrasynaptic membranes, and stabilization by synaptic scaffolding proteins (1, 9).Recent evidences suggest that SNARE (soluble N-ethylmaleimidesensitive factor attachment receptor) proteins are involved in NMDA receptor trafficking (10-16). SNARE proteins are a large family of proteins mediating fusion events between target membranes and vesicles. SNARE family members are categorized as three subfamilies based on their localizations. tSNAREs (target SNAREs) are localized on target membranes and consist of SNAPs (synaptosome-associated proteins) and syntaxins. vSNAREs (vesicle SNAREs) are VAMPs (vesicle associated membrane proteins or synaptobrevin) and localized on the vesicles. The four-helix SNARE complex, formed by SNAP, syntaxin and VAMP, brings the target membrane and the vesicle to close proximity and allows their fusion (17). This process can be inhibited by clostridial neurotoxins that specifically cleave different SNARE proteins (18). In the central nervous system, the presynaptic function of SNARE complexes in mediating neurotransmitter release is extensively studied (19); however, the postsynaptic function of these complexes remain to be investigated.In the current st...

Section: Resultsmentioning

confidence: 92%

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confidence: 99%

Identification of the SNARE complex mediating the exocytosis of NMDA receptors

Huganir

2016

“…To visualize glutamatergic input synapses, we expressed a PSD-95:GFP fusion protein. PSD-95 is a scaffolding protein that localizes to the postsynaptic density of glutamatergic synapses (10) and has been extensively used as a postsynaptic marker of glutamatergic synapses (11)(12)(13)(14). PSD-95 is present in virtually all GC glutamatergic synapses, where it is restricted to clusters in the postsynaptic density (15), is already highly expressed at birth (16), and appears early during assembly of the postsynaptic density (15).…”

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confidence: 99%

Sequential development of synapses in dendritic domains during adult neurogenesis

Kelsch

Lin²,

Lois³

2008

126

178

During the process of integration into brain circuits, new neurons develop both input and output synapses with their appropriate targets. The vast majority of neurons in the mammalian brain are generated before birth and integrate into immature circuits while these are being assembled. In contrast, adult-generated neurons face an additional challenge as they integrate into a mature, fully functional circuit. Here, we examined how synapses of a single neuronal type, the granule cell in the olfactory bulb, develop during their integration into the immature circuit of the newborn and the fully mature circuit of the adult rat. We used a genetic method to label pre and postsynaptic sites in granule neurons and observed a stereotypical development of synapses in specific dendritic domains. In adult-generated neurons, synapses appeared sequentially in different dendritic domains with glutamatergic input synapses that developed first at the proximal dendritic domain, followed several days later by the development of inputoutput synapses in the distal domain and additional input synapses in the basal domain. In contrast, for neurons generated in neonatal animals, input and input-output synapses appeared simultaneously in the proximal and distal domains, respectively, followed by the later appearance of input synapses to the basal domain. The sequential formation of synapses in adult-born neurons, with input synapses appearing before output synapses, may represent a cellular mechanism to minimize the disruption caused by the integration of new neurons into a mature circuit in the adult brain.bulb ͉ dendrite I ntegration of new neurons continues throughout life in the adult mammalian olfactory bulb (OB) (1, 2). During the process of integration into brain circuits, new neurons develop both input and output synapses with their appropriate targets. Whereas the majority of neurons in the olfactory bulb integrate into an immature circuit while it is being assembled, neurons generated in adulthood face an additional challenge as they integrate into a mature, fully functional circuit. In particular, the formation of synapses by a new neuron in a functioning circuit may interfere with circuit operation and, thus, it could result in maladaptive behaviors. Additionally, it is still not known whether new neurons integrating into the neonatal and adult olfactory system have the same or different functions in the circuit and, therefore, adult-and neonatal-generated neurons could employ different modes of integration. To compare how new neurons are added to neonatal and adult circuits, we examined the pattern of synapse development of a single neuronal type, the granule cell (GC) in the olfactory bulb, during its integration into the immature circuit of the newborn and the mature circuit of the adult rat.The majority of neurons added to the OB of adult rats are GC neurons. GCs are axonless inhibitory interneurons that have both a basal dendrite and an apical dendrite (Fig. 1A). The apical dendrite can be divided into an unbranched segm...

“…Likewise, synaptic vesicles traffic rapidly along axons before stabilizing at dendritic contact sites (6), suggesting that a dendrite-associated signal regulates presynaptic stability. At glutamatergic synapses, AMPA and NMDA receptors are recruited to the postsynaptic density in 1 h or less (7,8), raising the possibility that they may play an important role in regulating synapse stability.Electrophysiological and immunohistochemical experiments suggest that a large percentage of young synapses contain NMDA receptors but lack AMPA receptors (9, 10). AMPA receptors are recruited gradually to postsynaptic sites, resulting in an increase in the AMPA/NMDA ratio at these synapses (11-13).…”

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confidence: 99%

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confidence: 99%

Regulation of synaptic stability by AMPA receptor reverse signaling

Ripley

Otto

Tiglio

et al. 2010

The establishment of neuronal circuits relies on the stabilization of functionally appropriate connections and the elimination of inappropriate ones. Here we report that postsynaptic AMPA receptors play a critical role in regulating the stability of glutamatergic synapses. Removal of surface AMPA receptors leads to a decrease in the number and stability of excitatory presynaptic inputs, whereas overexpression increases synapse number and stability. Furthermore, overexpression of AMPA receptors along with Neuroligin-1 in 293T cells is sufficient to stabilize presynaptic inputs from cortical neurons onto heterologous cells. The stabilization of presynaptic inputs by AMPA receptors is not dependent on receptor-mediated current and instead relies on structural interactions mediated by the N-terminal domain of the glutamate receptor 2 (GluR2) subunit. These observations indicate that transsynaptic signaling mediated by the extracellular domain of GluR2 regulates the stability of presynaptic terminals.GluR2 N-terminal domain | Presynaptic stabilization | Presynaptic input dynamics T he development of neural circuits is characterized by exuberant synapse formation followed by elimination of inappropriate connections (1-4). Although there is considerable evidence that activity-dependent mechanisms are involved in synapse elimination, the mechanisms responsible for regulating and maintaining stable synapses are largely unknown.Synapse formation is a dynamic process and involves the rapid recruitment of several synaptic proteins to sites of axo-dendritic contact. Transport packets containing essential components of the presynaptic active zone are highly motile before they reach synaptic sites (5). Likewise, synaptic vesicles traffic rapidly along axons before stabilizing at dendritic contact sites (6), suggesting that a dendrite-associated signal regulates presynaptic stability. At glutamatergic synapses, AMPA and NMDA receptors are recruited to the postsynaptic density in 1 h or less (7,8), raising the possibility that they may play an important role in regulating synapse stability.Electrophysiological and immunohistochemical experiments suggest that a large percentage of young synapses contain NMDA receptors but lack AMPA receptors (9, 10). AMPA receptors are recruited gradually to postsynaptic sites, resulting in an increase in the AMPA/NMDA ratio at these synapses (11-13). The increase in the AMPA/NMDA ratio correlates with the stabilization of dendritic spines.Synapse formation requires the precise apposition of presynaptic and postsynaptic elements underlying the need for bidirectional signaling. Key postsynaptic proteins such as neuroligins, synaptic cell-adhesion molecules (SynCAMs), Ephrin type B (EphB) receptors, netrin G ligands, and leucine-rich repeat transmembrane (LRRTM) proteins signal transsynaptically to induce recruitment of presynaptic components (14)(15)(16)(17)(18)(19)(20). Postsynaptic adhesion molecules also are involved in the functional maturation of presynaptic inputs (21-23). Recent work demons...