Receptor Lateral Diffusion During Synaptogenesis

The formation of a synapse is thought to be a multi-step process initiated shortly after initial axo-dendritic contact formation and requiring the clustering of numerous partners. Over the last decades, the glutamate synapse formation has retained a lot of attention and a model has then emerged on how pre- and postsynaptic elements are sequentially pull together57. First, cell adhesion molecules (e.g. cadherins) form complexes, tightening together the pre- and postsynaptic membranes (see Chapters 4-10). Since surface adhesion molecules are highly diffusive29'35, the clustering of these proteins within developing synapses is possibly due to lateral diffusion process. Second, dense-core vesicles (~80 nm) that could be precursors of the active zone are recruited to the adhesion site. Third, on the postsynaptic side, the scaffolding molecules are recruited, elaborating the PSD; the glutamate receptors being recruited later. In the following paragraphs we discuss the potential role of lateral diffusion of excitatory neurotransmitter receptor in this process.

7.1. Nicotinic Acetylcholine Receptor Lateral Diffusion During the Neuromuscular Junction Formation

During the neuromuscular junction formation, AChRs that are initially dispersed all over the plasma membrane become highly concentrated in the synaptic membrane. The clustering of AChRs within the synaptic cleft is mainly dependent on the extracellular glycoprotein agrin, which is released by motoneurons at the neuromuscular junction58. The actual model for the neuro-muscular junction formation is that ACh, released by the motoneuron, destabilizes synaptic AChRs, and an important role of agrin is to counteract such "anti-synaptogenic" effect of ACh58. As previously mentioned, the initial reports of neurotransmitter receptor lateral diffusion described the diffusion of AChRs within the plasma membrane of developing myotubes1,59-61. It was shown that approximately 50% of alpha-bungarotoxin sensitive AChRs are mobile, and the mobile fraction diffuse on average at 0.1-0.01 p.m2/s (comparable to average diffusion coefficient for other receptor types). The surface diffusion was modulated by temperature changes (e.g. switch from 35° to 22°C) consistent with the fact that receptor diffusion is due to the thermal motion of surrounding molecules12 (see Section 3). Interestingly, AChR lateral diffusion is higher in immature myotubes when compared to mature and well-interconnected ones. This led to a developmental model in which AChRs freely diffuse in the plasma membrane and are continuously "trapped" and stabilized by agrin at the site of developing synaptic contact12,60. It was even shown in vivo that AChRs diffuse laterally away and to the neuromuscular junction on an activity-dependent manner, suggesting that the perijunctional receptor pool is both a source and a sink for the junctional receptors62. Recently, using the genetically screening approach in C. elegans, it has been reported that the clustering of ionotropic AChRs at the synapse requires a protein, lev-1063, and the stabilization of synaptic AChRs relies on extracellular protein-protein interactions. In conclusion, it emerges that the neuromuscular junction formation relies on the synaptic clustering of laterally diffusive AChRs located in extra/perijunctional membrane. Moreover, within the synapse, AChRs are stabilized by both agrin and interactions with other proteins.

7.2. Focus on the Glutamatergic Synapse Maturation

How glutamatergic receptors are incorporated and stabilized into the nascent synapse has been a subject of large interest. Several cellular pathways for glutamate receptor trafficking during development have been proposed. In the developing brain, AMPARs are expressed and are functional at the surface of neuronal progenitors64-66 before synaptogenesis takes place. Migrating and unconnected neurons express both surface AMPARs and NMDARs67 and it has been proposed that these receptors are themselves involved in the early step of synaptogenesis by regulating dendritic filopodia movements68. How and when are these receptors recruited in the developing synapse? Schematically, the scaffold molecules seem to be recruited first in the postsynaptic side approximately 30 min after the formation of the presynaptic bouton, and then both AMPARs and NMDARs are recruited, suggesting that the presence of scaffold protein is necessary to cluster and retain the receptors57. The recruitment of NMDARs was found to be progressive, not stepwise as shown for presynaptic markers, indicating a constant accumulation of individual receptors into the nascent synapse69. These results have suggested that the incorporation of glutamate receptors within the postsynaptic membrane relies more on random lateral diffusion of AMPARs from extrasynaptic membrane to synapse rather than the insertion of receptor-containing vesicles. Consistently, during in vitro development, the diffusion of extrasynaptic AMPARs is high at the surface of hippocampal neurons35 (Figure 15.3A,B). Within the synaptic area, AMPAR lateral diffusion markedly decreases over development, indicating that the number of AMPARs that exchange between the extrasynaptic and synaptic membrane is higher early one. Such developmental change can be due to several factors, including the formation of actin skeleton beneath the plasma membrane and the development of spines70. Regarding the NR1-containing NMDARs, they also diffuse laterally although at a lower level than AMPARs. Moreover, their surface exchange rate between extrasynaptic and synaptic membrane is consistently high in immature neurons36. To what extent these in vitro results apply to the in vivo situation remains unknown. However, there is now evidence that lateral diffusion of glutamate receptors plays a role in vivo in the Drosophila neuromuscular formation. Indeed, new glutamate synapses were found to form de novo, and the recruitment of GluR-IIA (related to the mammalian non-NMDAR type) to the newly formed PSDs occurs preferentially via surface diffusion of extrasynaptic receptors71. Thus, experimental evidences point toward a critical role of glutamate receptor lateral diffusion in synapse formation, although as for the neuromuscular junction, direct evidence for such a role is still lacking.

Figure 15.3. Lateral Diffusion of AMPARs and NMDARs During

Synaptogenesis. (A)

Lateral diffusion of extrasynaptic GluR2-contain-ing AMPARs at the surface of developing hippocampal neurons (from 2 to 14 days in vitro, d.i.v.). Adapted, with permission, from ref. 35. (B) Schematic representation of the lateral diffusion (double head arrows) of surface glutamate receptors in immature (left panel) and mature (right panel) neurons. The synaptic area is represented by the circle (broken line).

Immature neuron

Immature neuron

Mature neuron

Over the last decade, there have been some controversies about the insertion timing of AMPARs and NMDARs within developing synapses72,73. It emerges that, as mentioned above, already from the initial steps of synapse formation both NMDARs and AMPARs are present in the membrane, but the AMP A- and not NMD A-signalling is highly unstable. Indeed, AMPA signalling depends on whether spontaneous or evoked (test frequencies: 0.05-1 Hz) AMPAR currents are assessed74-76. In spontaneous conditions AMPAR as much as NMDAR contribute to synaptic activity whereas after evoked synaptic activity only NMDAR synaptic activity is recorded76. This stimulation-induced AMPA "silencing" was found in approximately half of the synapses, and was only observed in the neonatal hippocampus76, consistent with the previously reported age dependent decrease of AMPA silent synapses in the developing hippocampus77. Thus, although both receptors are present in developing synapses, AMPARs are prone to be quickly removed from the postsynaptic membrane by synaptic stimulation. It is interesting to note that such effect is similar to the ACh-induced destabilization of AChRs in the neuromuscular junction58, as if this process of neurotransmitter-induced receptor removal is a common feature of developing synapses. By which pathway are AMPARs removed from the postsynaptic membrane after synaptic stimulation? As already mentioned, AMPARs and NMDARs can undergo endocytosis from the plasma membrane to an intracellular pool. Since the endocytotic rate of glutamate receptors is high at early stages of development45,46,78, the labile behaviour of AMPARs in immature synapses may be partly due to its high cycling rate. Consistently, AMPARs are more internalized over time than NMDARs17'55'79'80. When the lateral diffusion of AMPARs and NMDARs was compared during synaptogenesis, AMPAR lateral diffusion was higher than that of NMDARs17, indicating that AMPARs are also less stable than NMDARs within the plasma membrane. Furthermore, changes in neuronal activity affected only AMPAR surface diffusion17. Thus, AMPAR signalling is highly unstable in immature synapse and this behaviour is possibly due to high lateral diffusion of AMPARs. Future experiments in which electrophysiological and SPT recordings will be coupled will certainly help to better understand this process.

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