7104 • J. Neurosci., May 23, 2012 • 32(21):7103–7105 Jurado and Knafo • Journal ClubFigure 1. Principles of FRAP experiment with AMPARs. Left, Scheme illustrating photobleaching and recovery of a whole synapse (top, Full Bleaching) versus half of a synapse (bottom, PartialBleaching). Before the bleach event, fluorescent AMPARs can be viewed on the synaptic surface (A, green dots, baseline). Immediately after photobleaching, AMPARs are no longer fluorescent (B,gray dots, total bleaching) and then fluorescence gradually recovers (C, green and gray dots, recovery) as unbleached AMPARs move into the bleached area. Note that, under basal conditions, fullbleaching and partial bleaching result with the same recovery graph (right, blue line and dashed red line, respectively). When intrasynaptic mobility of AMPARs is increased (e.g., after glutamateapplication), there is a stronger increase in recovery following partial bleaching (right, solid red line).the dendritic tree and require LTP-like PSD-95, GKAP, Shank, and Homer, all of monomer assembly into filaments (withevents to efficiently enter into dendritic which are postsynaptic scaffolding pro- latrunculin) and stabilizing actin polymer-spines (Shi et al., 1999). Therefore, in pri- teins. A high RF between a scaffold protein ization (with jasplakinolide) transformedmary neurons, recombinant AMPARs at and AMPARs at individual spines indicated the AMPAR clusters into absolutely rigidsynapses can be viewed without preceding they had similar subsynaptic distribu- structures. This finding suggests that consti-manipulations. With these methods, the tions. The highest RF was found between tutive reshaping of the synaptic AMPARauthors demonstrated the use of FRAP AMPARs and PSD-95, although the C ter- clusters requires ongoing actin turnover.as a practical and reproducible method mini of AMPA receptor subunits do not di- Contrary to some predictions, acute appli-to study AMPARs repositioning within rectly bind to this scaffolding protein. cation of latrunculin did not increasethe PSD. Nevertheless, this tight colocalization may AMPAR loss from the synapse nor did it af- Kerr and Blanpied (2012) first aimed account for the crucial role PSD-95 has fect intrasynaptic receptor mobility, as dis-to elucidate whether, under basal condi- in controlling the number of synaptic covered by subdomain FRAP. These aretions, AMPARs diffuse laterally within the AMPARs (Schnell et al., 2002). important findings, because they suggestPSD of single spines. They found that the The immobility of receptors within the that actin treadmilling is not acutely neces-fluorescence recovery curve in synapses that PSD led Kerr and Blanpied (2012) to ex- sary for AMPAR synaptic retention or mo-were entirely photobleached (Fig. 1, Full amine whether the overall structure of bility, challenging the notion that actinBleaching) was similar to the curve of syn- individual AMPAR clusters is rigid over anchors AMPARs at synapses.apses in which only a subdomain was time. To this end, the authors per- To test whether AMPAR activationbleached (Fig. 1, Partial Bleaching), formed extended (1 h) time-lapse imag- promotes internal AMPAR repositioning,implying that no AMPAR exchange oc- ing of synaptic clusters composed of Kerr and Blanpied (2012) applied gluta-curred within the PSD. This is in agreement surface AMPARs. As expected from pre- mate to cultured neurons. This manipula-with previous studies demonstrating re- vious studies showing a substantial PSD tion induced a significant increase in thestricted diffusion of AMPARs within indi- flexibility (Blanpied et al., 2008), they intrasynaptic mobility of AMPARs thatvidual synapses (Tardin et al., 2003; Makino observed that individual AMPAR clus- became evident when only a subdomainand Malinow, 2009). Thus, the postsynaptic ters exhibit substantial and continuous of the spine was photobleached (Fig. 1).scaffolding matrix significantly restricts the changes in their morphology. In contrast This suggests that activated synapses in-redistribution of AMPARs within the syn- to the continuously dynamic structure of crease their exchange rate of receptorsapse. It is, however, possible that the overex- AMPAR clusters, SEP fluorescence inten- among different subdomains. These re-pression of AMPAR subunits (also leading sity was extremely stable over time. These sults are consistent with the notion thatto formation of homomeric receptors in results imply that the structural flexibility the PSD acts as a network that regulatesnonphysiological levels, instead of the natu- of AMPAR clusters is not accompanied by subsynaptic receptor distribution so re-ral heteromeric receptors) physically re- significant changes in the number of sur- ceptors can respond with high efficacy tostricts their own mobility. face receptors. glutamate release (Elias and Nicoll, 2007). Kerr and Blanpied (2012) hypothe- Actin, a cytoskeletal protein highly en- Does intrasynaptic receptor mobility in-sized that AMPAR distribution within the riched in dendritic spine heads, where it is crease during LTP as well? A hint for thisPSD depends on their association with thought to anchor AMPARs, was an obvi- question can be found in a recent studyspecific postsynaptic scaffold proteins. ous candidate for the control of the ob- (Makino and Malinow, 2009) using similarThey determined the degree of this asso- served reshaping of AMPAR clusters and approaches (i.e., expression of fluorescentciation by calculating the pixel-wise fluo- perhaps for AMPAR retention within the receptors in organotypic hippocampal slicesrescence correlation coefficient (RF) for PSD. Remarkably, both preventing actin combined with FRAP and glutamate un-
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