The Journal of Neuroscience, May 23, 2012 • 32(21):7103–7105 • 7103Journal ClubEditor’s Note: These short, critical review...
7104 • J. Neurosci., May 23, 2012 • 32(21):7103–7105                                                                      ...
Jurado and Knafo • Journal Club                                                                                J. Neurosci...
Upcoming SlideShare
Loading in …5
×

Jurado and knafo jns

264 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
264
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
2
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Jurado and knafo jns

  1. 1. The Journal of Neuroscience, May 23, 2012 • 32(21):7103–7105 • 7103Journal ClubEditor’s Note: These short, critical reviews of recent papers in the Journal, written exclusively by graduate students or postdoctoralfellows, are intended to summarize the important findings of the paper and provide additional insight and commentary. For moreinformation on the format and purpose of the Journal Club, please see http://www.jneurosci.org/misc/ifa_features.shtml.Microscale AMPAR Reorganization and Dynamics of thePostsynaptic DensitySandra Jurado1 and Shira Knafo21 Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Palo Alto, California, 94304, and2 Centro de Biología Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Científicas/Universidad Autonoma de Madrid, 28049 Madrid, Spain ´Review of Kerr and BlanpiedAMPA-type receptors (AMPARs) are tutive trafficking that involves both exocytic ing (FRAP) (Jacobson et al., 1976) toglutamate-gated channels whose post- delivery from intracellular compartments quantify the dynamics of proteins and lip-synaptic activation convey the major (Gerges et al., 2006) and fast exchange with ids within a defined subcellular compart-depolarization in brain excitatory neu- surface extrasynaptic receptors through lat- ment. In this assay, fluorescent moleculesrotransmission. Trafficking of these recep- eral diffusion (Tardin et al., 2003). Still, are irreversibly photobleached in a smalltors to and from synapses is tightly regulated knowledge is lacking regarding the organi- area of the cell by a focused laser beam.in neurons and underlies long-lasting forms zation and regulation of AMPARs within Subsequent diffusion of surroundingof synaptic plasticity. For example, export of the postsynaptic density (PSD) and the nonbleached molecules into the bleachedAMPARs from the endoplasmic reticulum events triggering their repositioning. area leads to a total or partial recovery ofto the Golgi (Vandenberghe and Bredt, AMPAR trafficking can be evaluated fluorescence that is proportional to the2004) is suggested to contribute to the ex- by biochemical, electrophysiological, and mobility of a given molecule under differ-pression of certain types of synaptic plastic- imaging approaches. Synaptic delivery of ent experimental conditions (Fig. 1).ity (Broutman and Baudry, 2001). In endogenous AMPARs can be monitored However, with regular FRAP, it is techni-addition, endocytosis removes AMPARs by measuring levels of specific AMPAR cally challenging to pinpoint subcellularfrom synapses during LTD (Beattie et al., subunits in synaptoneurosomes (a frac- AMPAR movement within living syn-2000) and in response to other stimuli (Man tion of brain extracts enriched in synaptic apses with sufficient temporal and spatialet al., 2000). Internalized AMPARs can be elements) (Heynen et al., 2000). To track resolution.degraded in lysosomes or recycled back to specifically the movement of endogenous The study by Kerr and Blanpied (2012)the surface membrane (Ehlers, 2000; Gru- AMPARs on the cell surface, rapid time- overcame these technical limitations us-enberg, 2001). This AMPAR sorting is regu- lapse imaging of individual semiconductor ing high-resolution photobleaching of re-lated by synaptic activity (Ehlers, 2000) and quantum dots coupled to AMPAR antibod- combinant fluorescent receptors on theprovides the local intracellular pool of ies are performed (Dahan et al., 2003). surface of single spines. To view exclu-AMPARs needed for LTP expression (Park Overexpression of GluAl-GFP or GluA2 sively surface AMPARs (but not the re-et al., 2004). AMPARs also undergo consti- (R586Q)-GFP AMPAR subunits allows vi- ceptors in intracellular compartments) sualization of recombinant AMPARs to the authors used primary neurons ex- detect general distribution and movement. pressing GluA1 and GluA2 AMPAR sub-Received March 2, 2012; revised April 4, 2012; accepted April 4, 2012. This work was supported by a grant from the Spanish Ministry of Science This overexpression results in formation units fused to the pH-sensitive GFPand Innovation (SAF2010-15676 to S.K.). S.K. is the recipient of a “Ramon y ´ primarily of homomeric AMPARs that have [Super Ecliptic pHluorin (SEP)], whichCajal” contract from the Spanish Ministry of Science and Innovation. We different conductance properties than en- does not fluoresce in the acid environ-thank Prof. Jose A. Esteban for commenting on the manuscript. dogenous AMPARs (Shi et al., 2001). This ment of intracellular compartments. In Correspondence should be addressed to either of the following: Dr.Sandra Jurado, Nancy Pritzker Laboratory, Department of Psychiatry and “electrophysiological tagging” is a powerful addition, Kerr and Blanpied (2012) tookBehavioral Sciences, Stanford University School of Medicine, Palo Alto, CA tool to detect trafficking of AMPARs into advantage of the fact that in primary hip-94304, E-mail: sjurado@stanford.edu; or Dr. Shira Knafo, Centro de Bi- synapses, yet it does not identify movements pocampal neurons, GluA1-GFP subunitsología Molecular “Severo Ochoa,” Consejo Superior de Investigaciones of AMPARs within synapses. spontaneously concentrate at synapses.Científicas/Universidad Autonoma de Madrid, 28049 Madrid, Spain, ´E-mail: sknafo@cbm.uam.es. The past decades have witnessed a re- This is in contrast to neurons in organo- DOI:10.1523/JNEUROSCI.1048-12.2012 markable increase in the application of typic slice cultures in which GluA1-GFPCopyright©2012theauthors 0270-6474/12/327103-03$15.00/0 fluorescence recovery after photobleach- subunits distribute diffusely throughout
  2. 2. 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-
  3. 3. Jurado and Knafo • Journal Club J. Neurosci., May 23, 2012 • 32(21):7103–7105 • 7105caging). 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) SubsynapticGluA1 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. Natrequited 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: theBlanpied (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 receptorssynaptic membrane. Using FRAP on NMDA-induced potentiation in hippocam- in synaptic plasticity. Cell Mol Life Scisubdomains 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. Sciencedisplaying 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 controlAMPA receptor positioning and anchor- 511–525. synaptic AMPA receptor number. Proc Natling 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, Spallerpicture 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 hippocampalorescent 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 movementsspecific light) to explore receptor mobility, ulation of glutamate receptors in the adult of AMPA receptors inside synapses. EMBO Jand 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 eventsization Microscopy and stochastic optical (1976) Measurement of the lateral mobility of in glutamate receptor trafficking. Curr Opinreconstruction microscopy. cell surface components in single, living cells by Cell Biol 16:134 –139.

×