Organization and dynamics of AMPA receptors inside synapses—nano-organization of AMPA receptors and main synaptic scaffolding proteins revealed by super-resolution imaging
Graphical abstract
Introduction
Historically, new developments in microscopy have triggered novel concepts in biology. This is particularly evident in the field of neuroscience, where neuronal cells are composed of thin and dense structures ranging from micrometers to tens of nanometers in size.
This relationship between imaging techniques and neuroscience started in the early 20th century with the controversy between Ramon y Cajal and Golgi over the structure of the neuronal network. Only new silver-based cellular labeling developed by Golgi allowed Cajal to demonstrate with microscopy (i) that neurons are independent and delimitated cells and (ii) that the junctions between neurons, called synapses, are physically separated. In the 1950s, the intimate details of synaptic structure were finally revealed by applying electron microscopy to neurons [1]. Subsequently, the discovery of the electrical component of synaptic transmission by patch and voltage clamp techniques and the development of molecular biology completed the current vision of the electrochemical synapse: the pre-synaptic element triggers fast calcium dependent release of neurotransmitter vesicles, and the post-synapse accumulates ionic and metabotropic receptors of these neurotransmitters. This static vision of the synapse with neurotransmitter receptors anchored at the post-synapse was sufficient to sketch a functional model of the synapse.
In the 1970s, new imaging techniques capable of capturing live cell dynamics, such as fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT), added the idea that these membrane proteins are not static at the surface but show a fast diffusive behavior [2•]. The principle of the SPT technique relies on sparse labeling of individual proteins and follows their movement in real-time. Analysis of this motion allows determining protein behavior in different subcellular environments, reflecting interactions with various molecular complexes. In the 1990s, improvements to imaging setups, charge-coupled device (CCD) cameras, image analysis and engineering of smaller and photostable fluorescent probes like quantum dots (QD), allowed more efficient particle detection [3]. While traditional bulk microscopy techniques were limited in resolution to about 250 nm due to diffraction, SPT imaging enabled monitoring protein motion with nanometric localization using dedicated image analysis [4, 5, 6, 7].
The application of SPT techniques to neuroscience gave access to a better understanding of the role of various proteins in synaptic transmission. The first studies focused particularly on the family of glutamate receptors, which are most notably composed of the NMDA receptors responsible for calcium entry inside the synapse and the AMPA receptors responsible for fast synaptic transmission [8]. These experiments revealed that AMPA receptors present two different motions, alternating between states of transient immobility and high mobility. The main conclusions were that (i) AMPA receptors containing the GluA2 subunit stop reversibly at the synaptic site, (ii) during neuronal maturation, the stationary periods of these receptors increase in frequency and length and (iii) a rise in calcium favors local receptor immobilization.
These princeps single particle tracking experiments paved way to neurotransmitter receptor mobility characterization, ushering in a new vision of the synapse by identifying components of molecular complexes and unpredicted roles of receptor motion. Nonexhaustively, applications of the SPT technique demonstrated that (i) AMPARs are attached to the postsynaptic density (PSD), and more particularly to a primary scaffolding protein PSD95, through their main associated protein named stargazin [9], (ii) the postsynaptic adhesion protein neuroligin tends to aggregate AMPARs through PSD95 scaffolding protein recruitment [10, 11], and (iii) Ncadherin, a pre-synaptic adhesion protein, can transsynaptically interact with the extracellular domain of AMPARs to limit their diffusion [12] (Fig. 1a). Additional QD-based SPT experiments coupled with electrophysiology explained why synapses can sustain a stimulation frequency higher than predicted by showing that synaptic receptors exchange rapidly within tens of milliseconds [10].
More generally, the incorporation of membrane receptor motion into the synaptic paradigm further tuned the understanding of synaptic transmission. These QD-based SPT experiments have now been extended to a large spectrum of studies, demonstrating various relationships between protein motion and physiology [13, 14, 15, 16].
Section snippets
Super-resolution microscopy and first applications to studying postsynaptic molecule organization
Traditional single particle techniques, based on non-renewed low probe concentration, provided between ten and several hundred trajectories per cell. This is insufficient to gather enough temporal and spatial information to map the entire protein organization, which is essential to deciphering its exact physiological role. During the last 15 years, new super-resolution light microscopy techniques have been developed to circumvent the diffraction limit. Amongst them, stimulated emission
AMPAR are organized in nanodomains
The various different imaging methodologies used in these three studies converged to the common conclusion that AMPARs are organized in clusters much smaller than the PSD (Fig. 2a). These AMPAR nanodomains were visible with all used super-resolution techniques as well as with electron microscopy, exhibiting a homogeneous size distribution sharply centered around a 80 nm half-maximum. Such a well-defined size could reflect a specific molecular organization of a subarea of the post-synaptic
PSD95 is assembled in nanoclusters
To explore such local potentiation of a PSD subarea, various groups investigated the organization of PSD95, one of the main scaffolding proteins, either in its ensemble [44, 46•] or exclusively its palmitoylated form [45•]. They all led to the similar conclusion that PSD95 covers the entire postsynaptic density but, similarly to AMPARs, is over-concentrated in subdomains measuring around 150 nm in diameter (Fig. 3). PSD95 organization, observed with either sptPALM, dSTORM or STED techniques,
PSD95/AMPAR nanoscale colocalization and implications to neurophysiology
The nanoscale colocalization of PSD95 and AMPAR was investigated in basal conditions and as a function of synaptic activity by multicolor super-resolution microscopy. Interestingly, the expected PSD95/AMPAR nanocluster colocalization was not conclusively demonstrated. While Nair et al. and Fukata et al. could not demonstrate a full colocalization between nanodomains of endogenous AMPARs and endogenous PSD95 clusters (Fig. 3), MacGillavry et al. observed a strong overlap between AMPAR clusters
Conclusion
In this review, we demonstrated that advances in microscopy have imparted new conceptual steps to the understanding of synaptic function. Application of super-resolution microscopy techniques has revealed that PSD95 accumulates in sub-PSD clusters, which reorganize and resize during synaptic activity and strongly concentrate AMPARs. AMPARs also concentrate inside the synapse in nanodomains of about 80 nm in diameter, partially colocalized with PSD nanoclusters. This unexpected nanoscale
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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