Synaptic AMPA receptor subunit traffickingis independent of the C terminus in theGluR2-lacking mouseSandip Panicker, Keith Brown, and Roger A. Nicoll*

Departments of Cellular and Molecular Pharmacology and Physiology, University of California, San Francisco, CA 94143

Contributed by Roger A. Nicoll, November 30, 2007 (sent for review October 3, 2007)

Glutamate is the primary excitatory neurotransmitter in the brain,and �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptors mediate most fast synaptic transmission.AMPA receptors are tetrameric assemblies composed from fourpossible subunits (GluR1–4). In hippocampal pyramidal cells, AMPAreceptors are heteromeric receptors containing the GluR2 subunitand either GluR1 or GluR3. It is generally accepted that thetrafficking of GluR1/GluR2 receptors to synapses requires activity,whereas GluR2/GluR3 receptors traffic constitutively. It has beensuggested that the trafficking is governed by the cytoplasmic Ctermini of the subunits. Because the basis for this theory relied onthe introduction of unnatural, homomeric, calcium-permeableAMPA receptors, we have used the GluR2�/� knock out mouse todetermine whether the expression of mutated forms of GluR2 canrescue WT synaptic responses. We find that GluR2, lacking its entireC terminus, or a GluR2 chimera containing the C terminus of GluR1,is capable of trafficking to the synapse in the absence of activity.These findings suggest that the GluR2 C terminus is not requiredfor GluR2 synaptic insertion.

The vast majority of excitatory synapses in the mammaliancentral nervous system use the neurotransmitter glutamate.

Fast glutamatergic transmission at these synapses is predomi-nantly mediated by two different types of glutamatergic iono-tropic receptors: AMPA receptors and NMDA receptors. Be-cause NMDA receptors are blocked by magnesium ions atresting membrane potentials, AMPA receptors are responsiblefor most basal neurotransmission.

AMPA receptor subunits can form homomeric or, more often,heteromeric receptors (1, 2). Each subunit contains a largeextracellular N-terminal ligand-binding domain and four trans-membrane domains of which the second (M2) lines the pore ofthe channel. Of the four subunits, only GluR2 undergoes RNAediting of the critical glutamine 607 residue (Q607) to arginine(R607) in M2, making GluR2-containing AMPA receptorscalcium-impermeable and resistant to block by polyamines. Thelack of block by polyamines confers on GluR2-containing re-ceptors a linear current/voltage (I/V) relationship unlike GluR2-lacking AMPA receptors, which are strongly inwardly rectifyingat positive membrane potentials. Each subunit also contains adistinct cytoplasmic C-terminal tail (3–7). In general, GluR1 andGluR4 subunits have relatively longer tails than GluR2 orGluR3. These different C termini are proposed to be responsiblefor conferring specific trafficking properties to each AMPAreceptor subtype.

At the hippocampal CA3-CA1 (Schaffer collateral) synapse, theAMPA receptor subtypes that predominate are composed ofGluR1/2- and GluR2/3-containing receptors (8). The currentmodel of AMPA receptor trafficking at these synapses suggests thatAMPA receptors with a long-tailed subunit are excluded from thesynapse under basal conditions and require an activity-dependentsignal (i.e., long-term potentiation, or LTP) for synaptic insertion(9). In contrast, AMPA receptors that contain only short-tailedsubunits traffic to the synapse constitutively, independent of activity(9). In this ‘‘subunit rules’’ model, GluR1/2 receptors inserted into

the synapse after activity are then replaced over time in anactivity-independent manner with GluR2/3 receptors. Chimerastudies in which the tails of GluR1 and GluR2 were swappedsuggested that trafficking properties are conferred from the donorsubunit to the recipient, providing evidence that protein–proteininteractions in the C-tails are responsible for determining consti-tutive vs. activity-dependent trafficking (9). Two caveats regardingthis work are (i) the expressed subunits form nonphysiologichomomeric channels in the WT background, and (ii) the GluR2expression experiments used the unedited GluR2(Q) form, whichforms homomeric rectifying receptors for use as an electrophysio-logical tag. However, recent studies have shown that receptorassembly and trafficking are profoundly affected by the editing ofGluR2 (10, 11).

In this study, we used the GluR2 KO mouse (GluR2�/�) (12, 13)to test subunit-dependent AMPA receptor trafficking. Because ofthe lack of GluR2, all AMPA receptors in GluR2�/� have inwardlyrectifying I/Vs. By reintroducing GluR2(R) or GluR2(R) tail chi-meras into neurons of this mouse, we can examine the necessity ofdifferent regions of the GluR2 tail for synaptic incorporation bydetermining the I/V of synaptic responses. Any change fromcomplete inward rectification at positive potentials is an unambig-uous indication that the introduced GluR2 subunit trafficked to thesynapse. In addition, we can use the edited form of GluR2[GluR2(R)] and thereby avoid possible trafficking and assemblydefects associated with the unedited GluR2(Q) subunit. Finally,this mouse had been used to provide strong evidence that the locusof LTP expression is at the postsynaptic side of the CA3-CA1synapse (14). Thus, having both constitutive and activity-dependenttrafficking mechanisms in place, in addition to providing a defin-itive electrophysiological assay to determine receptor trafficking,makes the GluR2�/� mouse an excellent system for studyingAMPA receptor trafficking mechanisms.

ResultsGluR2�/� KO Characterization. To test the validity of GluR2�/� asa system for determining the rules for subunit trafficking, we firstfully characterized the synaptic responses in this mouse. AMPAreceptor excitatory postsynaptic currents (AMPA EPSCs),evoked by stimulating Schaffer collateral axons, were recordedwith patch pipettes and isolated pharmacologically (see Meth-ods). AMPA EPSCs from WT hippocampal CA1 pyramidal cellsin acute slices, have linear I/Vs (Fig. 1 A and B). In contrast,AMPA EPSCs from the GluR2�/� mouse are inwardly rectifyingat positive potentials because of pore block by internal poly-amines (Fig. 1 A and B) (12, 15).

Because the current/voltage relationship is linear in the WT

Author contributions: S.P. and R.A.N. designed research; S.P. and K.B. performed research;R.A.N. contributed new reagents/analytic tools; S.P. analyzed data; and S.P. and R.A.N.wrote the paper.

The authors declare no conflict of interest.

*To whom correspondence should be addressed. E-mail: nicoll@cmp.ucsf.edu.

© 2008 by The National Academy of Sciences of the USA

1032–1037 � PNAS � January 22, 2008 � vol. 105 � no. 3 www.pnas.org�cgi�doi�10.1073�pnas.0711313105

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mouse, this suggests that most, if not all, AMPA receptors containthe GluR2 subunit in the WT background (16, 17). We thereforechecked to see whether there was a reduction in the overall AMPAsignaling in GluR2�/�. To accomplish this, we compared the size ofthe AMPA receptor-mediated EPSC to the NMDA receptor-mediated EPSC at the synapse. In the presence of the GABAAreceptor blocker picrotoxin, AMPA and NMDA EPSCs weremeasured at �70 and �40 mV, respectively. To ensure that theNMDA-mediated EPSC at �40 mV was free of AMPA receptorcontamination, the current was measured 100 ms after stimulation.In WT cells, the AMPA/NMDA ratio was 2.37 � 0.13, comparedwith 1.12 � 0.08 in the KO (Fig. 1C). These values are in agreementwith those reported for GluR2�/� (12).

The above finding suggests that the loss of the GluR2 subunitresults in either a decrease in the relative number of AMPAreceptors to NMDA receptors at these synapses or in a greaternumber of synapses containing only NMDA receptors (silentsynapses) in the KO. To determine whether AMPA receptornumber at the synapse is reduced in GluR2�/�, we recordedminiature EPSCs (mEPSCs). mEPSCs recorded in the presenceof the sodium channel blocker tetrodotoxin were modestly, butsignificantly, decreased in GluR2�/� compared with the WTmouse (mean mEPSC amplitude: GluR2�/�, 8.6 � 0.4 pA, n �9; WT, 9.8 � 0.3 pA, n � 15; P � 0.05; Fig. 1D). In addition, thefrequency of mEPSCs, an indicator of functional synapses, wassubstantially reduced in GluR2�/� (mean mEPSC frequency:GluR2�/�, 0.10 � 0.01 Hz, n � 9; WT, 0.41 � 0.06 Hz, n � 15;P � 0.05; Fig. 1E), suggesting that, in addition to a reduction in

the number of AMPA receptors at synapses, a greater propor-tion of synapses in GluR2�/� are silent. Because the AMPAreceptor composition is fundamentally different in GluR2�/�

compared with the WT mouse, we also examined the kinetics ofthe mEPSCs. Surprisingly, the decay kinetics of mEPSCs was notdifferent in the two mice, suggesting that the GluR2 subunit isnot the determining factor of mEPSC decay (Fig. 1F).

A reduction in the AMPA/NMDA ratio concomitant with areduction in both the amplitude and the frequency of AMPAreceptor-mediated mEPSCs indicate that hippocampal basalsynaptic transmission is diminished in GluR2�/�. To furthercharacterize AMPA-dependent synaptic transmission, we per-formed extracellular field recordings in acute slices of GluR2�/�

and WT mice. The presynaptic fiber volley amplitude was usedas a measure of input strength and plotted against the size of theAMPA field EPSP. We found a large reduction in the AMPAfield potentials of GluR2�/� compared with WT littermates(�50%, Fig. 2A), consistent with previous studies (13, 15). Wealso checked the NMDA field EPSP (recorded in reducedextracellular Mg2�) and found that NMDA receptor transmis-sion was also reduced in GluR2�/� (�25%), although to a lesserextent than AMPA receptor transmission (Fig. 2B). The loss ofCA3 pyramidal cells in the GluR2 KO mouse (15) couldcontribute substantially to the diminished synaptic transmissiononto CA1 pyramidal cells.

Finally, we compared plasticity in both animals using a pairing

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Fig. 1. Electrophysiological characterization of the CA3-CA1 hippocampalsynapse of the GluR2�/� mouse. (A) Sample traces of AMPA receptor-mediatedsynaptic responses at �70, 0, and �40 mV in WT and GluR2�/� mice. (B)Current/voltage relationship of synaptic responses in WT (E) and KO (F) mice.(C) AMPA to NMDA ratio is reduced in the GluR2�/� mouse (n � 7, WT; n � 12,GluR2�/�; P � 0.05). (D) mEPSCs from GluR2�/� mice have reduced amplitude(n � 15, WT; n � 9, GluR2�/�; P � 0.05). (E) Reduced frequency (n � 15, WT; n �9, GluR2�/�; P � 0.05). (F) No change in decay kinetics compared with WT mice.

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Fig. 2. Decreased basal synaptic transmission in the GluR2�/� mouse. (A)Plotting fiber volley amplitude vs. fEPSP slope shows AMPA field potentials aredecreased �55% in the KO (F) compared with WT (E) mice (n � 17, WT; n �17, GluR2�/�; P � 0.01). (B) NMDA field potentials of GluR2�/� mice are slightlyreduced (�25%) compared with WT (n � 25, WT; n � 25, GluR2�/�; P � 0.05).(C) Whole-cell LTP is similar in GluR2�/� (F) and WT (E) mice (n � 6, WT; n �8, GluR2�/�; P � 0.05).

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protocol to induce LTP in whole-cell recordings from CA1pyramidal cells. As has been reported, the magnitude of LTP wasnot diminished in GluR2�/� (12, 14, 15), suggesting that AMPAreceptor trafficking mechanisms in the context of synapticplasticity are not perturbed in the KO (Fig. 2C).

GluR2 Synaptic Insertion in GluR2�/� Pyramidal Cells. To assesswhether introducing GluR2 constructs into GluR2�/� neuronswas a valid strategy for determining regions of the GluR2 C-tailrequired for constitutive trafficking, we introduced the full-length GluR2 subunit into hippocampal pyramidal cells ofGluR2�/� mice. Using biolistics as a method of gene delivery, weshot cultured organotypic hippocampal slice preparations madefrom GluR2�/� with microprojectiles coated with DNA encod-ing GFP-tagged GluR2. Simultaneous dual whole-cell record-ings were then performed on a cell transfected with GFP-GluR2and a neighboring untransfected cell. GluR2 introduction intoGluR2�/� pyramidal cells did not change the amplitude of theNMDA EPSC (recorded at �40 mV) or the AMPA EPSC(recorded at �70 mV) compared with untransfected neighbor-ing cells (Figs. 3 A and B). However, GluR2-containing AMPAreceptors clearly were incorporated into the synapse, appearingto completely replace the GluR2-lacking receptors, because thesynaptic I/Vs of the GluR2-transfected KO cells were linear (Fig.3C). Because the single-channel conductance of GluR2-lackingreceptors is different from GluR2-containing receptors, it isdifficult to determine in what ratio the receptors were swapped.Regardless, the linear I/V indicates that these synapses containonly GluR2-containing receptors.

Synaptic incorporation of introduced GluR2 in GluR2�/� cellsconfirmed that GluR2-trafficking mechanisms are still in placein the KO. Therefore, to test whether the GluR2 tail is requiredfor synaptic incorporation of AMPA receptors, we deleted theentire region of the GluR2 C-tail, from amino acid V827onwards (GluR2�C, Fig. 4A). Similar to full-length GluR2,GluR2�C expression did not result in a change in the amplitudeof either AMPA or NMDA receptor transmission (Fig. 4 A andB). Surprisingly, GluR2�C did traffic to the synapse just as wellas the full-length GluR2, because the synaptic I/Vs of cellstransfected with this construct were also linear (Fig. 4C). Onemight argue that because all AMPA receptors in the GluR2�/�

mouse are calcium-permeable, the calcium flux through thesereceptors is sufficient to engage the activity-dependent mecha-nism responsible for inserting the introduced GluR2 C-tail-lacking receptors into the synapse. To address this possibility, weadded kynurenic acid, an antagonist of both AMPA and NMDAreceptors, immediately after transfection. Although this treat-ment blocks all excitatory transmission (18), it did not preventthe linearization of the I/V (Fig. 4D).

Finally, we made a GluR2 construct in which the C-tail ofGluR2 was replaced with that of GluR1 (GluR2/R1). Specifi-cally, this GFP-GluR2/R1 chimera contains amino acids 823–889 of GluR1 fused to the body of GluR2 (amino acids 1–826,Fig. 5A). Based on the lack of GFP fluorescence, Shi et al. (9)have reported that this construct is excluded from spines.Moreover, when the critical arginine at position 607 was mutatedto glutamine in this construct [GluR2(Q)], which creates arectifying homomeric receptor, no change in rectification wasobserved at the synapse, further suggesting that this constructwas not delivered to the synapse (9). When we transfectedGluR2/R1 into GluR2�/� pyramidal cells, AMPA and NMDAreceptor transmission appeared to be unaltered when comparedwith untransfected neighboring cells (Fig. 5 A and B). However,cells expressing GluR2/R1, similar to both GluR2 full-length andGluR2�C constructs, exhibited synaptic currents with linear I/Vrelationships, indicating that it trafficked and incorporated intothe synapse (Fig. 5C). In two cells cultured in the presence ofkynurenic acid, GluR2/R1 still linearized the I/V (RI � 0.71 �

0.05), suggesting an activity-independent mechanism of synapticincorporation.

DiscussionIn this study, we introduced different tail constructs of GluR2into GluR2�/�hippocampal pyramidal cells to test the necessityof the GluR2 tail in synaptic delivery. By using GluR2�/�, wetook advantage of the unique electrophysiological properties ofthe AMPA receptors in this mouse that allow us to detect thesynaptic presence of our introduced GluR2 constructs. In addi-tion, studies in GluR2�/� mouse have contributed importantly toestablishing that the site of expression of LTP at the Schaffercollateral synapse is postsynaptic (14), a process that most likelyrequires the synaptic trafficking of AMPA receptors (3–7). Wefind that GluR2 constructs that entirely lack the C-terminal tailor contain the C-terminal tail of GluR1 are trafficked tosynapses in an activity-independent manner. The implication ofthese findings in the regulation of synaptic AMPA receptors isdiscussed below.

Characterization of GluR2�/� revealed that the AMPA fieldpotentials and the AMPA/NMDA ratio are reduced by �50%compared with WT (Figs. 1C and 2 A). NMDA field potentials

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Fig. 3. GFP-GluR2 transfection into pyramidal cells of GluR2�/� organotypichippocampal slice cultures rescues synaptic currents at positive potentials. (A)Sample traces of simultaneous dual whole-cell recordings from a GFP-GluR2-transfected cell (green trace) and an untransfected neighboring cell (blacktrace) show similar AMPA (taken at �70 mV) and NMDA currents (taken at�40 mV). (Inset) Schematic of the GFP-GluR2 construct. (B) Bar graphs showingthat neither AMPA (n � 17, P � 0.29; paired t test) nor NMDA (n � 11, P � 0.61;paired t test) components changed after transfection. (C) Isolated AMPAreceptor-mediated synaptic currents at �70 mV and �40 mV from a GFP-GluR2-transfected cell and an untransfected neighbor illustrates that theintroduced GFP-GluR2 construct has trafficked into the synapse. Bar graphshows that the rectification index is almost linear in the GFP-GluR2-transfected cells (n � 13, GFP-GluR2; n � 12, untransfected; P � 0.05).

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were also reduced (Fig. 2B), indicating that the loss of AMPAreceptors is greater than the AMPA/NMDA ratio would suggest.Furthermore, we found a slight reduction in mEPSC amplitudeand a large reduction in frequency in the KO compared with WTcells (Fig. 1E). Considering that the single-channel conductanceof GluR2-lacking receptors is higher than that of GluR2-containing receptors (19), the overall AMPA receptor numberat the synapse of the GluR2�/� mouse is most likely less than the40% loss of GluR1 measure with ImmunoGold labeling (20). Wedo not have an explanation for this seeming quantitative differ-

ence, but it should be kept in mind that the loss of CA3 neuronsin this mouse (15) would contribute significantly to the impairedtransmission. The loss of AMPA receptors is not due to aninability of GluR2-lacking neurons to insert receptors into thesynapse, because the LTP machinery appears to be intact in theabsence of GluR2 (12, 14, 15) (Fig. 2C). Nevertheless, the severeloss of synaptic AMPA receptors is an indication that neurons inGluR2�/� have difficulty delivering AMPA receptors to thesynapse, which is consistent with a role for GluR2 in constitutivetrafficking of AMPA receptors to the synapse (21). We reasonedthat by reintroducing GluR2 into the cell, we would restore thecell’s native complement of AMPA receptors (GluR1/2 andGluR2/3 receptors), allowing us to track how they traffic.

The subunit-rules model of AMPA receptor trafficking isbased largely on the overexpression of GFP-tagged AMPAreceptor subunits in WT hippocampal pyramidal cells. Theexpressed subunits form homomeric channels, which, unlike theheteromeric endogenous AMPA receptors, generate rectifyingcurrents. With this electrophysiological tag, one can monitor theappearance of these exogenous receptors at the synapse (9).Based on these studies it was proposed that receptors containingGluR1, either homomeric or in association with GluR2, requireactivity to traffic to synapses, whereas receptors containingGluR2, either homomeric [GluR2(Q)] or in association withGluR3, traffic to synapses constitutively in an activity-independent manner. Chimeric experiments in which the tails of

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Fig. 4. GFP-GluR2�C transfection rescues synaptic currents at positive po-tentials in GluR2�/� pyramidal cells. (A) Sample traces of simultaneous whole-cell recordings from a cell transfected with GFP-GluR2�C (green trace) and anuntransfected neighbor (black trace) show that AMPA and NMDA currents areequivalent. (Inset) Schematic of the GFP-GluR2�C construct. (B) Bar graphsshow that the AMPA (n � 18, P � 0.93; paired t test) and NMDA (n � 15, P �0.50; paired t test) currents are unchanged after GFP-GluR2�C transfection. (C)AMPA-isolated currents at �70 mV and �40 mV of a GFP-GluR2�C-transfectedcell and an untransfected neighbor show that GFP-GluR2�C inserts into thesynapse. Bar graph shows that the rectification index is close to linear in theGFP-GluR2�C-transfected cell (n � 14, GFP-GluR2�C; n � 12, untransfected;P � 0.05). (D) Sample traces of simultaneous whole-cell recordings from a celltransfected with GFP-GluR2�C (green trace) and an untransfected neighbor(black trace) from GluR2�/� slices incubated in the presence of 10 mMkynurenic acid. The rectification index shown in the bar graph (n � 3) is similarto that observed in the absence of the blocker (see C).

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Fig. 5. GFP-GluR2/R1 traffics to the synapse in GluR2�/� cells in the absenceof activity. (A) Sample traces of simultaneous whole-cell recordings from a celltransfected with GFP-GluR2/R1 (green trace) and an untransfected neighbor(black trace) show that AMPA and NMDA currents are unchanged. (Inset)GFP-GluR2/R1 chimera, including contribution of amino acid stretch from eachsubunit. (B) Bar graphs show that AMPA (n � 13, P � 0.67; paired t test) andNMDA (n � 10, P � 0.65; paired t test) synaptic transmission is unchanged inthe GFP-GluR2/R1-transfected cell. (C) Sample traces of the isolated AMPAcurrent at �70 mV and �40 mV from a GFP-GluR2/R1-transfected cell and anuntransfected neighbor. Bar graph shows that the rectification index forGFP-GluR2/R1 is almost linear (n � 11, GFP-GluR2/R1; n � 12, untransfected;P � 0.05).

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GluR1 and GluR2 were swapped provided evidence that thecarboxyl tails of the receptors determine differential receptortrafficking. Thus, a receptor containing the GluR1 C terminus,either in the form of a homomeric receptor or in association withGluR2, requires activity to traffic to the synapse, whereasconstitutive trafficking requires an intact GluR2 C terminus.One possible confound in the study of Shi et al. (9) is that theyused GluR2(Q), an unedited form of GluR2 that is not found inneurons in appreciable amounts, so that they could monitorrectification changes associated with the unedited homomericreceptor. However, it has recently been shown that the editing ofGluR2(Q) to GluR2(R) profoundly influences receptor assem-bly (11) and trafficking (10). Thus, we reasoned that expressingGluR2(R) in the GluR2�/� background would overcome thesepotential pitfalls. Specifically, we would anticipate that theintroduced GluR2(R) would heteromerize with endogenousGluR1 and/or GluR3 subunits restoring the receptor types thatare natively found in the cell. It is unlikely that homomericGluR2(R) receptors, if formed, would contribute to the synapticcurrents, because their single-channel conductance is extremelylow (femtosiemens) (19).

Expression of the full-length GluR2(R) had no effect on theamplitude of the AMPA EPSC recorded at �70 mV, butrestored the WT linear I/V. This demonstrates that GluR2(R)-containing receptors replace the GluR2-lacking synaptic recep-tors. The reintroduction of GluR2(R) did not restore synapsesback to WT-sized responses, which could be due to a number ofreasons that are independent of GluR2, including synapsenumber, synapse size, or the loss of Schaffer collateral axons inGluR2�/�, which is known to occur in this mouse (15).

We found that GluR2(R) subunits entirely lacking the Cterminus trafficked to the synapse just as well as WT GluR2(R)in GluR2�/� cells. Consistent with this is the report that expres-sion of GluR2(�863Y)-GFP, a mutation that prevents PDZinteractions, can traffic to the synapse in GluR2�/� cells. How-ever, this mutant construct did not reach synapses in WT cells(22). In addition, we find that a GluR2 subunit containing the Cterminus of GluR1 also traffics to the synapse as well as WTGluR2(R). Presumably these subunits are assembling primarilywith endogenous GluR1 and indicate that in the completeabsence of the C terminus of GluR2, AMPA receptors can trafficconstitutively to the synapse. This finding is not easily reconciledwith the subunit rules model discussed above.

It is unlikely that high neuronal activity is driving receptorsinto the GluR2�/� synapse, considering that the overall gluta-matergic synaptic transmission is greatly reduced in the mouse(Figs. 1 and 2). Another explanation could be that because allAMPA receptors in GluR2�/� are calcium-permeable, the cal-cium flux through these receptors is sufficient to engage theactivity-dependent mechanism responsible for inserting the in-troduced GluR2 C tail-lacking receptors (GluR2�C and GluR2/R1) into the synapse. This is also unlikely because the magnitudeof LTP in the KO was identical to WT mice, suggesting that LTPhad not occurred in the KO cells. Furthermore, the addition ofkynurenic acid, an ionotropic glutamatergic receptor antagonistthat blocks both AMPA and NMDA receptors into the culturemedium immediately after transfection of the slices did notprevent the insertion of either GluR2�C or GluR2/R1. Theseresults suggest that neither hyperactivity of the slice nor calciumpermeation through glutamatergic receptors was responsible fortrafficking these constructs into the synapse.

The C terminus of GluR2 is distinct from that of the othersubunits in that it contains a binding site for NSF, an ATPasethat facilitates membrane fusion thought to be responsible forconferring the constitutive trafficking property on GluR2 (23–25). Intracellular introduction of a 10 amino acid peptidesequence (pep2m) that blocks the interaction of NSF and GluR2causes a rundown of AMPA receptor-mediated EPSCs, suggest-

ing that the NSF/GluR2 interaction is necessary to maintainsynaptic transmission (23, 26–28). However, the EPSC canrecover fully if synaptic activation is halted (29), suggesting thatan NSF-independent mechanism traffics AMPA receptors in theabsence of synaptic activity. It is possible that in the presentstudy, the AMPA receptors formed on reintroduction of GluR2subunits lacking the NSF-binding domain use this mechanism.

Based on our results, we propose a revised model of AMPAreceptor trafficking wherein the C terminus of GluR2 is notinvolved in the constitutive trafficking of AMPA receptors to thesynapse, nor does the C terminus of GluR1 mediate a restrictivesignal on synaptic delivery of AMPA receptors. Based on theseresults, the protein complex targeting AMPA receptors to thesynapse is not likely to rely on accessory proteins that selectivelyinteract with the GluR2 subunit, but rather on a protein part-ner(s) that may have similar affinity for all GluR subunits.

A main difference between our study and previous work is thatwe introduced heteromeric receptors to the GluR2 KO, whereasothers introduced homomeric channels to WT synapses. How-ever, to explain our positive results while accepting the hypoth-esis that there are differences in the trafficking of GluR1- versusGluR2-containing receptors requires also accepting that a fun-damental reorganization of the AMPA receptor traffickingmechanisms have occurred in GluR2�/�. This seems quiteunlikely given that LTP is normal, suggesting that the basictrafficking mechanisms remain intact.

MethodsMolecular Biology. The GluR2 subunit and GluR2 subunit constructs weretagged with enhanced GFP after amino acid S24 in the N-terminal domain,after the putative signal peptide sequence of GluR2. These constructs werethen cloned into the pCI-neo mammalian expression vector (Promega) be-tween the XbaI (5) and NotI (3) sites. Amino acids V827–I862 of GluR2 weredeleted to make GFP-GluR2�C by using a PCR-based mutation strategy. Sim-ilarly, an overlap PCR-based strategy was used to fuse the carboxyl tail ofGluR1 (amino acids G824–L892) to the end of the fourth transmembranedomain of GluR2 (after amino acid K826) to make GFP-GluR2/R1. All constructsmade by using PCR were validated by DNA sequencing (ElimBio).

Slice Culture Transfection and Electrophysiology. Organotypic hippocampalslice cultures made from P6-P9 GluR2�/� mice were made as described (30).Methods conform to the University of California Institutional Animal Care andUse Committee guidelines. For experiments in which 10 mM kynurenic acidwas added to the slice culture media, the pH of the media was brought backto 7.3 after addition of kynurenic acid. Biolistic transfection (Bio-Rad) wasused to introduce GFP-GluR2 constructs into pyramidal cells of GluR2�/� slicecultures as described (31). Whole-cell recordings from slice cultures wereperformed 3–7 days after transfection. For whole-cell electrophysiologicalrecordings, slices were perfused in artificial cerebrospinal fluid (aCSF) con-taining the following: 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl2, 4 mM MgSO4,1 mM NaH2PO4, 26.2 mM NaHCO3, 11 mM D-glucose, 0.1 mM picrotoxin, 0.02mM bicuculline methiodide, and 0.01 mM 2-chloroadenosine (to suppressepileptiform activity), and bubbled with 5% CO2/95% O2. aCSF osmolarity was297–305 mOsm. To isolate AMPA receptor-mediated currents, 100 �M D-APVwas added to the perfusate. Patch electrodes (3–5 M) were filled with thefollowing internal solution: 110 mM CsMeSO4, 15 mM CsCl, 10 mM Hepes, 2.5mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM EGTA, 0.1 mM sperminetetrahydrochloride, and 5 mM QX-314 chloride (pH � 7.3), �290 mOsm.Simultaneous whole-cell recordings were made by first patching a transfectedcell, followed by an untransfected neighboring cell (identifying cells with UVfluorescence and guided by 40� DIC optics). Cells were broken into immedi-ately after achieving G patch resistances on both cells. Synaptic stimulationwas induced by placing a monopolar electrode filled with aCSF in the stratumradiatum to stimulate Schaffer collateral axons (0.2-Hz stimulation frequency)by delivering a 100-�s current injection of varying strength. Twenty to 50synaptic responses were recorded and averaged at membrane potentials of�70, 0, and �40 mV. Stimulus artifacts have been blanked for clarity. Therectification index was then calculated by using the following formula: RI �[(I�40 � I0)/(I0 � I�70)] � 7/4.

Acute Slice Electrophysiology. Transverse hippocampal slices were preparedfor whole-cell (300 �m) or field potential (400 �m) recordings from

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GluR2�/� and WT mice between postnatal day (P)14 and P28 as described(32). Whole-cell recordings were performed by using the same aCSF andinternal solutions as described above. Current/voltage relationships forsynaptic AMPA currents were done in the presence of 100 �M D-APV (Fig.1 A and B). The AMPA component of the AMPA/NMDA ratios was taken at�70 mV. The NMDA component was determined by taking the current at�40 mV 100 ms after the stimulation artifact. mEPSCs were recorded in thepresence of 500 nM tetrodotoxin. LTP experiments were also performed inthe same aCSF and internal solutions described above, although the inter-nal solution lacked 10 mM EGTA. fEPSP recordings were made by placing astimulating electrode filled with aCSF in stratum radiatum �150 –200 �maway from a recording electrode (�3–5 M) filled with aCSF also in thestratum radiatum. Care was taken to ensure that the latitude of stimulationand recording within the radiatum was similar between WT and GluR2�/�

slices. Input– output relationships were generated in this way for bothAMPA receptor-mediated fields and NMDA receptor-mediated fields, thelatter in the presence of 10 �M CNQX. Stimulation frequency for fEPSP was0.1 Hz. Input– output relationships were determined in similar aCSF asdescribed above, except CaCl2 concentration was 2.5 mM, and MgSO4

concentration was 1.3 mM (AMPA fEPSP experiments) or 0.1 mM (NMDAfEPSP experiments). All data in this study are reported as mean � SEM.Statistics performed in this study were done by using Student’s t test(except for mEPSC analysis in which the Kolmogorov–Smirnov test wasused), and statistical significance was determined as P � 0.05.

ACKNOWLEDGMENTS. We thank Dr. Z. Jia for the GluR2�/� mice and membersof the R.A.N. laboratory for their advice and comments on the manuscript.R.A.N. was supported by grants from the National Institutes of Health.

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