2. 7104 • J. Neurosci., May 23, 2012 • 32(21):7103–7105 Jurado and Knafo • Journal Club
Figure 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, Partial
Bleaching). 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, full
bleaching 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 glutamate
application), 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 (with
events 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) transformed
mary neurons, recombinant AMPARs at and AMPARs at individual spines indicated the AMPAR clusters into absolutely rigid
synapses 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 AMPAR
authors 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 increase
the 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 are
tions, AMPARs diffuse laterally within the AMPARs (Schnell et al., 2002). important findings, because they suggest
PSD 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 actin
Bleaching) 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 activation
bleached (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 the
stricted diffusion of AMPARs within indi- flexibility (Blanpied et al., 2008), they intrasynaptic mobility of AMPARs that
vidual synapses (Tardin et al., 2003; Makino observed that individual AMPAR clus- became evident when only a subdomain
and 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 receptors
apse. 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 that
to formation of homomeric receptors in results imply that the structural flexibility the PSD acts as a network that regulates
nonphysiological 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 to
stricts 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 this
PSD depends on their association with thought to anchor AMPARs, was an obvi- question can be found in a recent study
specific postsynaptic scaffold proteins. ous candidate for the control of the ob- (Makino and Malinow, 2009) using similar
They determined the degree of this asso- served reshaping of AMPAR clusters and approaches (i.e., expression of fluorescent
ciation by calculating the pixel-wise fluo- perhaps for AMPAR retention within the receptors in organotypic hippocampal slices
rescence correlation coefficient (RF) for PSD. Remarkably, both preventing actin combined with FRAP and glutamate un-
3. Jurado and Knafo • Journal Club J. Neurosci., May 23, 2012 • 32(21):7103–7105 • 7105
caging). Makino and Malinow (2009) References fluorescence recovery after photobleaching.
suggested that the mobility of SEP- Beattie EC, Carroll RC, Yu X, Morishita W, Ya- J Supramol Struct 5:565(417)-576(428).
suda H, von Zastrow M, Malenka RC (2000) Kerr JM, Blanpied TA (2012) Subsynaptic
GluA1 subunits is significantly decreased af-
Regulation of AMPA receptor endocytosis by AMPA receptor distribution is acutely regu-
ter LTP induction to preserve the recently lated by actin-driven reorganization of the
a signaling mechanism shared with LTD. Nat
requited receptors and maintain synaptic Neurosci 3:1291–1300. postsynaptic density. J Neurosci 32:658 – 673.
potentiation. Blanpied TA, Kerr JM, Ehlers MD (2008) Struc- Makino H, Malinow R (2009) AMPA receptor
In summary, the study of Kerr and tural plasticity with preserved topology in the incorporation into synapses during LTP: the
Blanpied (2012) represents an important postsynaptic protein network. Proc Natl Acad role of lateral movement and exocytosis. Neu-
advance in the study of the microscale Sci U S A 105:12587–12592. ron 64:381–390.
Broutman G, Baudry M (2001) Involvement of Man HY, Ju W, Ahmadian G, Wang YT (2000)
organization and dynamics of the post- the secretory pathway for AMPA receptors in Intracellular trafficking of AMPA receptors
synaptic membrane. Using FRAP on NMDA-induced potentiation in hippocam- in synaptic plasticity. Cell Mol Life Sci
subdomains of spines in dissociated neu- pus. J Neurosci 21:27–34. 57:1526 –1534.
rons, they determined that AMPARs are Dahan M, Levi S, Luccardini C, Rostaing P,
´ Park M, Penick EC, Edwards JG, Kauer JA,
relatively immobile within the PSD while Riveau B, Triller A (2003) Diffusion dynam- Ehlers MD (2004) Recycling endosomes
ics of glycine receptors revealed by single- supply AMPA receptors for LTP. Science
displaying an overall motion as clusters
quantum dot tracking. Science 302:442– 445. 305:1972–1975.
in a matrix that constantly reshapes in Ehlers MD (2000) Reinsertion or degradation Schnell E, Sizemore M, Karimzadegan S, Chen L,
an actin-dependent manner. Their con- of AMPA receptors determined by activity- Bredt DS, Nicoll RA (2002) Direct interac-
clusions challenge previous models for dependent endocytic sorting. Neuron 28: tions between PSD-95 and stargazin control
AMPA receptor positioning and anchor- 511–525.
synaptic AMPA receptor number. Proc Natl
ing at the postsynaptic density, and offer Elias GM, Nicoll RA (2007) Synaptic trafficking
Acad Sci U S A 99:13902–13907.
of glutamate receptors by MAGUK scaffold-
critical insight into the inner organization ing proteins. Trends Cell Biol 17:343–352.
Shi SH, Hayashi Y, Petralia RS, Zaman SH,
of living synapses. Undoubtedly, the final Wenthold RJ, Svoboda K, Malinow R (1999)
Gerges NZ, Backos DS, Rupasinghe CN, Spaller
picture of AMPAR trafficking will require Rapid spine delivery and redistribution of
MR, Esteban JA (2006) Dual role of the exo-
cyst in AMPA receptor targeting and insertion AMPA receptors after synaptic NMDA recep-
the combination of complementary imag- tor activation. Science 284:1811–1816.
ing techniques. In the next years, FRAP will into the postsynaptic membrane. EMBO J
25:1623–1634. Shi S, Hayashi Y, Esteban JA, Malinow R (2001)
probably be combined with the use of Gruenberg J (2001) The endocytic pathway: a Subunit-specific rules governing AMPA re-
photo-switchable fluorescent proteins (flu- mosaic of domains. Nat Rev Mol Cell Biol ceptor trafficking to synapses in hippocampal
orescent proteins that change their excita- 2:721–730. pyramidal neurons. Cell 105:331–343.
tion and emission spectra when exposed to Heynen AJ, Quinlan EM, Bae DC, Bear MF Tardin C, Cognet L, Bats C, Lounis B, Choquet D
(2000) Bidirectional, activity-dependent reg- (2003) Direct imaging of lateral movements
specific light) to explore receptor mobility,
ulation of glutamate receptors in the adult of AMPA receptors inside synapses. EMBO J
and with high spatial and temporal resolu- 22:4656 – 4665.
hippocampus in vivo. Neuron 28:527–536.
tion imaging, such as Photoactivated Local- Jacobson K, Derzko Z, Wu ES, Hou Y, Poste G Vandenberghe W, Bredt DS (2004) Early events
ization Microscopy and stochastic optical (1976) Measurement of the lateral mobility of in glutamate receptor trafficking. Curr Opin
reconstruction microscopy. cell surface components in single, living cells by Cell Biol 16:134 –139.