LTP requires a reserve pool of glutamate receptors independent of subunit type

Summary Long-term potentiation (LTP) of synaptic transmission is thought to be a key cellular mechanism underlying memory formation. A widely accepted model posits that LTP requires the cytoplasmic tail of the AMPA receptor subunit GluA1. To find the minimum necessary requirement of the GluA1 C-tail for LTP in CA1 hippocampal pyramidal neurons, we used a single-cell molecular replacement strategy to replace all endogenous AMPA receptors with transfected subunits. In striking contrast to the prevailing model, we found no requirement of the GluA1 C-tail for LTP. In fact, replacement with the GluA2 subunit showed normal LTP, as did an artificially expressed kainate receptor not normally found at these synapses. The only conditions under which LTP was impaired were those with dramatically decreased AMPA receptor surface expression, indicating a requirement for a reserve pool of receptors. These results demonstrate the synapse’s remarkable flexibility to potentiate with a variety of glutamate receptor subtypes, requiring a fundamental change in our thinking with regard to the core molecular events underlying synaptic plasticity.


Introduction
Information storage in the brain is widely accepted to involve the rapid increase in synaptic strength between two neurons that can persist over long periods of time. This phenomenon, known as long-term potentiation (LTP), has been well described at glutamatergic synapses in the hippocampus, a region of the brain that is required for formation of new memories. At these CA1 synapses, LTP is expressed by the immediate increase in post-synaptic AMPA-Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms type glutamate receptors (AMPARs) following coincident activation of pre-and postsynaptic neurons. However, the exact mechanism of rapid AMPAR insertion during LTP is not fully understood.
AMPARs mediate the majority of fast, excitatory synaptic transmission in the brain. A functional AMPAR is a tetramer of individual subunit proteins, of which there are four unique isoforms, GluA1 -4 1,2 . In CA1 pyramidal neurons, which serve as a model for understanding LTP, most receptors exist as GluA1/A2 heteromers, with a minor contribution from GluA2/A3 receptors 3,4 . Over the past decade, a large body of research has focused on how individual AMPAR subunits are trafficked. A widely held model posits that GluA1/A2 receptors are excluded from synapses unless an LTP-stimulus is provided, whereas GluA2/A3 receptors traffic to the synapse constitutively. This difference in trafficking behavior is mediated by the cytoplasmic tails (C-tails) of the individual subunit proteins [5][6][7] . Supporting this model is the finding that LTP is impaired in GluA1 knock-out mice 8 , but normal in GluA2/A3 double knock-outs 9 . Based on these findings, a broad consensus has emerged that LTP is mediated by synaptic insertion of GluA1-containing receptors via its Ctail interactions [10][11][12][13][14][15][16][17] .
Despite the consensus that GluA1 is required for LTP, no single phosphorylation site or protein-protein interaction in the GluA1 C-tail has been shown to be absolutely necessary. Our goal was to find the minimum requirement of the GluA1 C-tail for LTP, and, if found, use that region to identify crucial protein interactions that mediate synaptic AMPAR potentiation. To accomplish this, we used a single-cell molecular replacement strategy to replace all endogenous AMPARs with transfected subunits 18,19 . Using this approach, we systematically mutated the GluA1 C-tail and examined the effects on three stages of AMPAR trafficking: surface expression, synaptic transmission, and LTP. Surprisingly, we failed to identify any region in the GluA1 C-tail that was essential either for basal synaptic incorporation or for LTP. In fact, homomeric GluA2 receptors exhibited normal LTP. Most surprisingly, hippocampal synapses in which AMPARs had been replaced with kainate-type glutamate receptors (KARs) also expressed normal LTP. Only manipulations that severely compromised the extrasynaptic surface pool of receptors showed defects in potentiation.

The role of the GluA1 C-tail in surface expression
AMPAR trafficking can be broken down into three distinct steps: surface expression, basal synaptic targeting, and activity-dependent synaptic insertion. Since GluA1 is normally abundantly expressed on the neuronal surface 3,20 , we first screened for surface expression of various GluA1 C-tail truncations in wild-type neurons using somatic outside-out patches in organotypic slice culture. Because overexpressed receptors form inwardly rectifying homomers 7 , whereas endogenous heteromeric receptors show linear current-voltage (I-V) relationships 21 , we can detect surface expression of the expressed subunits as an increase in surface rectification (Fig 1a). Overexpression of full-length GluA1 by biolistic transfection into CA1 pyramidal neurons increased the rectification by approximately 40% compared to wild-type controls (Fig. 1b), indicating the presence of surface homomers. In contrast, overexpressing a GluA1 subunit with a full C-tail truncation (ΔC) showed rectification similar to wild-type neurons (Fig. 1b), indicating an impairment in trafficking to the surface. However, a less severe truncation up to amino acid 824 (GluA1Δ824), which removes two serine phosphorylation sites and the PDZ-binding domain, increased rectification to a similar degree as full-length GluA1. Selective excision of the remaining membrane proximal region (ΔMPR), which contains a well-characterized binding site of the protein Band4.1N 22,23 , also significantly increased rectification (Fig. 1b). Combined, these two modified subunits represent complementary truncations of the entire C-tail, ruling out a necessary role for any single part of the C-tail for steady-state surface expression.
Because competition with endogenous receptors may have hindered GluA1ΔC trafficking, we wished to study surface expression in the absence of native AMPARs. To accomplish this, we used mice with the genes for GluA1, GluA2, and GluA3 flanked by loxP sites (Gria1-3 fl/fl ). A previous study has shown that expression of Cre into Gria1-3 fl/fl neurons results in a complete absence of AMPARs within 12-15 days 3 , providing an effective AMPAR-null background onto which mutant GluA1 subunits can be expressed. We confirmed that Cre expression eliminated all glutamate-evoked current from somatic outside-out patches of Gria1-3 fl/fl CA1 neurons, which can be rescued to control amplitudes by co-expression with full-length GluA1 (Fig. 1c,d), indicating full rescue of surface expression. Consistent with overexpression, molecular replacement with GluA1ΔC showed significantly decreased glutamate-evoked currents (Fig. 1c,d). This trafficking defect was not due to decreased association with TARPs, auxiliary subunits important for AMPAR trafficking 24,25 , as both full-length and GluA1ΔC subunits had KA/Glu ratios similar to control ( Supplementary Fig 1a). Also, both GluA1 and GluA1ΔC replacement subunits showed strong inward rectification, confirming the absence of endogenous receptors ( Supplementary Fig 1b). Since both GluA1ΔMPR and GluA1Δ824 showed normal surface trafficking, the GluA1ΔC subunit may be impaired owing to its severe truncation so close to the transmembrane region, which may inhibit proper protein folding.

Basal Synaptic Transmission does not require the GluA1 C-tail
Given the decreased surface expression caused by complete truncation of the GluA1 C-tail, we next examined whether it would also impair basal synaptic targeting. Similar to surface currents, we assessed baseline synaptic transmission by transfecting Gria1-3 fl/fl organotypic slice cultures with Cre and a replacement GluA1 subunit. After 17 days, we recorded evoked AMPAR excitatory post-synaptic currents (EPSCs) simultaneously from control and neighboring GluA1-replacement CA1 neurons. Similar to previously described results 19 , full-length GluA1 rescued AMPAR EPSC amplitudes to ~68% of control cells, while leaving NMDAR EPSCs unchanged (Fig. 2a,c). However, these results contrast with previous studies showing that GluA1 only traffics to synapses after an LTP stimulus 5,7 . For experimental exploration of this discrepancy, please refer to Supplementary Fig. 2. We also observed no change in paired-pulse ratio, indicating that GluA1 molecular replacement did not affect presynaptic release probability ( Supplementary Fig. 3a). Synaptic EPSCs were strongly inwardly-rectifying compared to control, confirming the absence of endogenous receptors (Fig. 2e, Supplementary Fig. 3b). Replacement with GluA1ΔC rescued AMPAR EPSCs to the same degree as full-length GluA1, and also had no effect on the NMDA EPCS (Fig. 2b,d). Replacement with GluA1Δ824 produced similar results ( Supplementary Fig. 3ce). This demonstrates that despite having dramatically decreased somatic expression owing to its severe truncation, GluA1ΔC manages to effectively rescue basal synaptic transmission.

No GluA1 C-tail domains are required for LTP
To assess how GluA1 C-tail truncations affect LTP, we transfected Cre and GluA1 into the hippocampus of ~E15.5 Gria1-3 fl/fl mouse embryos by electroporation. Like biolistic transfection, this results in sparse expression of transfected cells. Electroporation of Cre alone results in complete absence of an AMPAR EPSC by p10 with no effect on NMDAR EPSCs, and no AMPAR EPSCs appearing following an LTP stimulus ( Supplementary Fig.  4a-c). In p17-20 acute hippocampal slices, we induced LTP after recording stable (3-5 minute) baseline AMPAR EPSCs simultaneously from control and GluA1-replacement neurons. We found that replacement with full-length GluA1 exhibited normal LTP (Fig. 3a), confirming that the GluA1 subunit is sufficient. To avoid the confounding effect of decreased surface expression seen by GluA1ΔC, we next assessed the competence of GluA1Δ824 and GluA1ΔMPR subunits, which represent overlapping truncations of the entire C-tail. Both expressed LTP comparable to control (Fig. 3b,c), as did neurons replaced with a truncated GluA1Δ824 subunit with S816A and S818A (GluA1Δ824-AA) mutations to specifically prevent Band4.1N binding 26 ( Supplementary Fig. 5a,b). We also found that expressing GluA1ΔC with GluA2 to produce more natural GluA1/A2 heteromers was able to rescue the surface trafficking defect of GluA1ΔC (Supplementary Fig. 6a-d), and show synaptic responses similar to controls (Supplementary Fig. 6e-h). Finally, LTP was fully rescued by replacement with GluA1ΔC/GluA2 (Fig. 3d). Combined, these data show that the GluA1 C-tail is not required for LTP.

GluA2 is sufficient for LTP
Given that no individual portion of the GluA1 C-tail was necessary for LTP, we hypothesized that expression of an alternative AMPAR subunit might also rescue LTP. GluA2 is another such subunit with limited C-tail homology to GluA1 15 that is normally highly expressed in CA1 neurons, but is ineffective at forming homomers and trafficking to the cell surface 3,20 . This is attributable to Q/R RNA editing in the pore of the receptor, which severely limits channel permeability and may make formation of homomers energetically unfavorable 27 . Expression of un-edited GluA2(Q) resulted in abundant appearance of homomers on the neuronal surface, as observed by increased rectification (Fig. 4a). Like GluA1ΔC, GluA2(Q)ΔC also showed impaired surface expression ( Supplementary Fig. 7d). Similarly, both full-length GluA2(Q) (Fig. 4b) and GluA2(Q)ΔC ( Supplementary Fig. 7b) trafficked to the synapse, arguing against any necessary role for the GluA2 C-tail in synaptic targeting, in agreement with prior experiments 21 . NMDA EPSCs and paired-pulse ratio remain unchanged in these replacement neurons, and complete replacement of endogenous receptors was confirmed by synaptic rectification (Supplementary Fig. 7a,b). Moreover, LTP in Gria1-3 fl/fl neurons that expressed only GluA2(Q) was indistinguishable from control cells (Fig. 4c), despite lacking any of the intracellular phosphorylation sites and protein-protein binding sites of GluA1. Similarly intact synaptic targeting and LTP was seen in a GluA2(Q) truncation that lacks the majority of its C-tail and known protein-interaction sites ( Supplementary Fig. 7c-e).

LTP requires a reserve pool of AMPARs
Previous studies have shown that LTP is impaired in mice with constitutive deletion of GluA1 8 , but not GluA2 or GluA3 9 , demonstrating that GluA1 is both necessary and sufficient for LTP. These findings appear to be at odds with our data showing that GluA2(Q) homomers readily express LTP. We therefore reexamined the requirement for GluA1 in single-cell conditional knockouts and found that conditional deletion of GluA1 alone did indeed impair LTP (Fig. 5a). Furthermore, deletion of GluA2 or 3 separately ( Supplementary Fig. 8a,b) or in combination (Fig. 5b) had no effect. How can this data be reconciled with our previous experiments? One profound difference between deleting GluA1 and deleting GluA2/A3 is that in the former condition there is an absence of extrasynaptic receptors 3,8,20 , whereas in the latter condition this pool remains entirely intact 3 . Also, unlike endogenous GluA2, our replacement GluA2(Q) showed abundant surface expression. We reasoned then that perhaps it is the depletion of this pool that accounts for the loss of LTP in the GluA1 KO. To test this possibility, we returned to the extreme C-tail truncations of both GluA1 and GluA2(Q), in which surface expression is impaired, but synaptic targeting is maintained (Fig. 1b,c; Supplementary Fig. 7d). Indeed, LTP was substantially impaired in both GluA1ΔC and GluA2(Q)ΔC replacement neurons (Fig. 5c,d). These findings suggest that the minimum requirement for LTP is a reserve pool of extrasynaptic AMPARs, regardless of the subunit type.

GluK1 is sufficient for mediating LTP
Having failed to identify any specific domains in the C-tails that are important for LTP, we wondered whether other domains in the AMPAR are required. In search of a null condition to conduct domain-swapping experiments, we turned to kainate receptors (KARs), a separate class of fast, ionotropic glutamate receptor which differs in fundamental ways from AMPARs. They bind to different auxiliary subunits and have no sequence homology in their C-tails 28 . We therefore set out to replace all endogenous AMPARs with KARs at CA1 synapses. CA1 pyramidal neurons do not express synaptic KARs, as shown by the absence of synaptic currents in the presence of the AMPAR-selective antagonist GYKI ( Supplementary Fig. 9a). However, co-expression of the KAR subunit GluK1 with the auxiliary subunit Neto2 29,30 in wild type CA1 neurons generated a GYKI-resistant current that was blocked by NBQX, an antagonist that blocks both KARs and AMPARs ( Supplementary Fig. 9a), This indicates that overexpressed KARs are capable of being targeted to the synapse and contribute to EPSCs. To examine KAR currents in isolation, we co-expressed Cre with GluK1 and Neto2 in Gria1-3 fl/fl CA1 neurons. In this case, we recorded a population of pure KARs on the surface that desensitize to glutamate even in the presence of cyclothiazide and are completely blocked by ACET, a highly specific GluK1 antagonist 31 (Fig. 6a). Furthermore, in this AMPAR-null background, these neurons exhibit EPSCs that are entirely blocked by ACET, while the EPSCs in neighboring controls neurons are unaffected (Fig. 6b), further demonstrating that exogenously expressed KARs are capable of targeting to synapses. As with AMPAR replacement, NMDA EPSCs were unaffected ( Supplementary Fig. 9b). Finally, we tested whether neurons expressing only KARs could express LTP. To our surprise, we found that the KAR EPSC showed potentiation indistinguishable from that recorded simultaneously from neighboring control neurons (Fig. 6c). To ensure that the EPSC in the KAR expressing neuron was, in fact, mediated entirely by KARs, we applied ACET at the end of the experiments and found that it abolished the EPSC, but had no effect on neighboring control neurons (Fig. 6c). We also wished to confirm that LTP mediated by KARs, which are also Ca 2+ -permeable, was not induced by a fundamentally different mechanism than wild-type LTP. We therefore tried inducing LTP in the presence of NMDAR-antagonist APV, and saw no significant potentiation ( Supplementary Fig. 9d,e). These experiments demonstrate that even neurons completely lacking AMPARs can undergo LTP, so long as they are provided with an alternative fast, ionotropic glutamate receptor.

Discussion
Using a single-cell molecular replacement approach that gave us complete control over the complement of expressed AMPA receptors, we found no requirement for the GluA1 C-tail for basal synaptic transmission or for LTP. In fact, we found no requirement for the GluA1 subunit generally, as both GluA2(Q), another AMPAR subunit, and GluK1, an entirely separate class of glutamate receptor, exhibited normal levels of LTP. Previous studies that have implicated the GluA1 C-tail in LTP demonstrated phenotypes with a largely normal initial stage of potentiation, followed by a gradual decrease in EPSC amplitude towards baseline 5,6,28,32 . The most compelling of these studies demonstrate impaired LTP in mice with posphonull knock-in mutations of two key phosphorylation sites, and complete absence of long-term depression (LTD) 32 . Another study with phosphomimetic knock-in mutations also demonstrated a decreased threshold for LTP induction 33 . Given these findings, we cannot rule out the possibility that the C-tails play some modulatory effect on synaptic plasticity. In the present experiments, however, we saw immediately impaired potentiation in GluA1 conditional KO cells and with GluA1ΔC and GluA2ΔC replacement, which more closely mimics the absence of LTP seen with pharmacological blockade of NMDA receptors. With all three of these manipulations, there was a profound decrease in the pool of extrasynaptic receptors, indicating that the main requirement for LTP is an adequate reserve pool of glutamate receptors. Another equally plausible model is that AMPAR-containing recycling endosomes are required for LTP and conditions that deplete the surface receptor pool also deplete this pool 34 (see Supplementary Discussion).
Fundamentally, our results suggest that synapses can cluster a broad variety of receptors following LTP, shifting the focus of LTP expression from the receptor subunits to the synapse itself and specifically the post-synaptic density (PSD). Our data suggests a model in which AMPARs freely diffuse on the neuronal surface, and are trapped at the PSD for use in synaptic transmission 35 . LTP, then, can be understood as an immediate increase in the ability of the PSD to trap receptors that relies on a reserve pool of freely diffusing surface receptors. This model is consistent with evidence from 2-photon glutamate uncaging experiments, which show an immediate increase in the volume of post-synaptic spines following LTP induction [36][37][38] , suggesting significant alterations to the synapse and PSD. Despite this shift of focus, research on AMPARs and their auxiliary subunits, such as TARPs, remain important for identifying LTP-related PSD proteins. In the absence of a role for the GluA1 C-tail, the question remains exactly which specific interactions cluster AMPARs at the synapse both basally and during plasticity. Identification of these

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Mouse Genetics
Animals were housed according to the IACUC guidelines at the University of California, San Francisco. Mice with the Gria1 fl/fl , Gria2 fl/fl , and Gria3 fl/fl (Gria1-3 fl/fl ) were generated and genotyped as previously described 3 .

Experimental Constructs
Flip-isoform GluA1, GluA2(Q), and Cre:mCherry were cloned into the pFUGW expression plasmid by PCR and In-Fusion® HD Cloning System (Invitrogen). pFUGW-GluA1 and GluA2(Q) co-expressed with GFP behind an internal ribosomal entry site (IRES). GluA1 and GluA2(Q) truncations were generated by overlapping extension PCR. GluA1ΔC ended in amino acid 812, with the last 4 amino-acids being EFCY. GluA1Δ824 ended in amino acid 824, with the sequence MKGF. GluAΔ824-AA contained the C-tail sequence EFCYKSRAEAKRMKGF. GluA1ΔMPR had the following amino acids excised from the C-tail: KSRSESKRMKGFC, with the rest of the C-tail intact. GluA2(Q)ΔC also truncated to amino acids EFCY, and GluA2(Q)Δ847 ended in amino acids MKGF. GluK1 and Neto2 were cloned into the pCAGGs expression plasmid with GFP and mCherry, respectively, coexpressed behind an IRES.

Neuronal Transfection
Sparse biolistic transfections of organotypic slice cultures were performed as previously described 3 . Briefly, 80 ug total of mixed plasmid DNA was coated on 1 uM-diameter gold particles in 0.5 mM spermidine, precipitated with 0.1 mM CaCl 2 , and washed 4x in pure ethanol. The gold particles were coated onto PVC tubing, dried using ultra-pure N 2 gas, and stored at 4 degrees in desiccant. DNA-coated bullets were shot with a Helios Gene Gun (BioRad). Cre expression was confirmed by mCherry epifluorescence, and replacement AMPA/KAR subunits confirmed by GFP epifluorescence.
For in-utero electroporations, ~E15.5 pregnantGria1-3 fl/fl mice were anesthetized with 2.5% isoflurane in 0 2 and injected with buprenorphine for analgesic. Embryos within the uterus were temporarily removed from the abdomen and injected with 2 μl of mixed plasmid DNA into the left ventricle via a beveled micropipette. pFUGW-Cre:mCherry was typically diluted to approximately 0.5 μg/μl in 2-3 μg/μl of the replacement pFUGW AMPAR or pCAGGS GluK1 plasmid. Each embryo was electroporated with 5x50 msecond, 35 volt pulses. The positive electrode was placed in the lower right hemisphere and the negative electrode placed in the upper left hemisphere. Following electroporation, the embryos were sutured into the abdomen, and sacrificed on p17-20 for LTP recording. For further detail on electroporation, please see Navaroo-Quirogo et al 42 .

Electrophysiology
Voltage-clamp recordings were taken from CA1 pyramidal neurons in either acute hippocampal slices or organotypic slice cultures. For acute slices, 300 μM transverse slices were cut using a Microslicer™ DTK-Zero1 (Ted Pella, Inc.) in chilled high sucrose cutting solution containing (in mM): 2.5 KCl, 7 MgSO 4 , 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 7 glucose, 210 sucrose, 1.3 ascorbic acid, 3 sodium pyruvate. The slices were then incubated for 30 minutes at 34 degrees in artificial cerebral spinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 1 NaH 2 PO 4 , 26.2 NaHCO 3 , and 11 glucose. For acute slices, 2.5 mM CaCl 2 and 1.3 mM MgSO 4 were added to the aCSF, and for organotypic slice cultures 4 mM CaCl 2 and MgSO 4 were added. The aCSF was bubbled with 95% O 2 and 5% CO 2 to maintain pH, and the acute slices allowed to recover at room temperature for 45 minutes to 1 hour. Cultured slices were prepared as previously described 39 , and recorded between 7-24 DIV depending on the experiment. During recording, slices were transferred to a perfusion stage on an Olympus BX51WI upright microscope and perfused at 2.5 ml/min. with aCSF containing 0.1 mM pictrotoxin for acute slices experiments, and 0.01 mM gabazine, and 2-5 μM 2-Cl-adenosine for organotypic slice cultures. Synaptic responses were evoked by stimulating with a monopolar glass electrode filled with aCSF in stratum radiatum of CA1.
To ensure stable recording, membrane holding current, input resistance, and pipette series resistance were monitored throughout recording. Data was gathered through a MultiClamp 700B amplifier (Axon Instruments), filtered at 2 kHz, digitized at 10 kHz.

Whole-cell synaptic recordings and LTP
Simultaneous dual whole-cell recordings were made between GFP and/or mCherry positive with amplitudes taken 100 msec following stimulation to avoid contamination by AMPA receptor current. Paired-pulse ratios of AMPAR EPSCs were taken by stimulating twice at a 40 ms interval. To examine AMPA receptor rectification, 0.1 mM D-AP5 was washed in to block NMDA receptors. LTP was induced by stimulating at 2 Hz for 90 sec while clamping the cell at 0 mV, after recording a stable 3-5 minute baseline, but not more than 6 minutes after breaking into the cell. To minimize run-up of baseline responses during LTP, slices were stimulated for ~10 minutes prior to breaking in, and both cells held cell-attached for 2-5 minutes before breaking into the whole cell. Prior to breaking in, stimulation intensity was calibrated just below the threshold required to elicit an action potential from the wild-type control neuron. Rectification was calculated as the ratio of the slopes of the lines connecting AMPA EPSC amplitude from 0 to +40 mV and from −70 mV to 0 mV. This calculation can be taken as follows: R.I. = 7(I 40 -I 0 )/4(I 0 -I 70 ) where I x represent EPSC amplitude at x mV.

Outside-out patches
Outside-out patches were taken from CA1 cells by obtaining whole-cell access to CA1 pyramidal neurons at −70 mV with a 4-5 MΩ patch pipette, then slowly pulling the pipette away from the soma until a high-resistance seal reformed.