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. Author manuscript; available in PMC: 2009 Mar 27.
Published in final edited form as: Neuron. 2008 Mar 27;57(6):872–882. doi: 10.1016/j.neuron.2008.01.028

An Essential Role for PICK1 in NMDA Receptor-Dependent Bidirectional Synaptic Plasticity

Akira Terashima 2,3,1, Kenneth A Pelkey 4,1, Jong-Cheol Rah 2, Young Ho Suh 5, Katherine W Roche 5, Graham L Collingridge 3, Chris J McBain 4, John TR Isaac 2,3,*
PMCID: PMC2336895  NIHMSID: NIHMS44332  PMID: 18367088

Summary

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 over expression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR-dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

INTRODUCTION

A major cellular mechanism underlying activity-dependent plasticity of glutamatergic transmission is the regulated trafficking of AMPARs through coordinated protein-protein interactions between AMPAR subunits and a host of postsynaptic scaffolding molecules (Bredt and Nicoll, 2003; Collingridge et al., 2004; Groc and Choquet, 2006; Malinow and Malenka, 2002). One important class of interactions involves the PDZ domain-containing proteins, GRIP (glutamate receptor interacting protein), ABP (AMPA receptor-binding protein) and PICK1 (protein interacting with C-kinase 1), which bind the extreme C-termini of GluR2 and GluR3 subunits to regulate AMPAR trafficking and synaptic transmission (Collingridge et al., 2004; Dev et al., 1999; Dong et al., 1997; Srivastava et al., 1998; Xia et al., 1999). PICK1 is a good candidate as a potential bidirectional regulator of synaptic AMPAR trafficking (Dev and Henley, 2006; Sossa et al., 2006). PICK1 contains a single PDZ domain that interacts with several proteins including GluR2/3 subunits, PKCα and mGluR7 (Boudin et al., 2000; Dev et al., 2000; Dev et al., 1999; Staudinger et al., 1995; Xia et al., 1999). Moreover, PICK1 can dimerize via a coiled-coil/BAR domain enabling dimeric PICK1 to link other proteins such as PKC to GluR2 in a multi-protein complex (Chung et al., 2000; Perez et al., 2001). PICK1 also interacts with components of the SNARE-dependent membrane fusion machinery (Hanley et al., 2002), as well as with GRIP and membrane lipids via its coiled-coil/BAR domain to coordinate PKC-dependent trafficking of AMPARs (Jin et al., 2006; Lu and Ziff, 2005). Most intriguingly, the affinity of PICK1 for GluR2 exhibits a calcium sensitivity (Hanley and Henley, 2005) potentially allowing PICK1 to act as a calcium-sensor that orchestrates AMPAR trafficking events during LTP and LTD (Sossa et al., 2006).

PICK1 is required for several forms of synaptic plasticity in diverse areas of the CNS (Isaac et al., 2007). One of the best defined roles for PICK1 is in mGluR-dependent LTD at parallel fiber-Purkinje cell synapses in the cerebellum, expression of which requires GluR2-PICK1 interactions for PKC-dependent AMPAR internalization (Chung et al., 2003; Steinberg et al., 2006; Xia et al., 2000). Also in the cerebellum, at parallel fiber-stellate cell synapses, PICK1-GluR2 interactions mediate an activity-dependent switch of GluR2-lacking for GluR2-containing AMPARs in another form of LTD (Gardner et al., 2005; Liu and Cull-Candy, 2005; Liu and Cull-Candy, 2000). A similar PICK1-dependent switch in GluR2 AMPAR subunit composition is also observed during LTD in the ventral tegmental area (Bellone and Luscher, 2005; Bellone and Luscher, 2006).

In contrast to the established role of PICK1 in cerebellar plasticity, the function of PICK1 in hippocampal plasticity remains unclear. Initial studies revealed that PICK1 decreases surface GluR2 levels in cultured hippocampal neurons consistent with a potential role in hippocampal LTD (Chung et al., 2000; Perez et al., 2001). However, subsequent studies using acute infusion into CA1 pyramidal neurons of peptides that block PICK1 PDZ domain interactions are inconclusive, yielding conflicting results. One study supports a role for PICK1 in NMDAR-dependent hippocampal LTD (Kim et al., 2001), whereas other work found no role for PICK1 in this form of LTD (Daw et al., 2000; Duprat et al., 2003). Furthermore, the over expression of PICK1 in CA1 pyramidal neurons results not in LTD, but in synaptic potentiation. This is because PICK1 expression caused removal of GluR2-containing, calcium-impermeable AMPARs from synapses and their replacement with GluR2-lacking, calcium-permeable AMPARs, which have a higher single channel conductance (Terashima et al., 2004). Interestingly, a similar incorporation of GluR2-lacking AMPARs during NMDAR-dependent LTP at CA1 synapses has been observed (Plant et al., 2006). Moreover, the PICK1-mediated enhancement in synaptic strength requires PKC and CaMK (Terashima et al., 2004), providing further correlation with the mechanism of LTP (Malenka and Nicoll, 1999; Wikstrom et al., 2003). Together these observations raise the possibility that PICK1 may also participate in hippocampal LTP. Thus, the PICK1-GluR2 interaction may broadly serve to regulate the GluR2 content, and hence the major biophysical properties, of AMPARs at synapses allowing for bidirectional control of synaptic efficacy. Moreover, the PICK1-dependent regulation of AMPAR subunit composition may be an expression mechanism for several prominent, but seemingly disparate, forms of long-term synaptic plasticity.

Here we directly test whether PICK1 participates in NMDAR-dependent bidirectional plasticity by investigating how gain or loss of PICK1 function affects hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1-induced increase in synaptic strength (Terashima et al., 2004) is NMDAR-dependent and occludes subsequent LTP. Moreover, acute loss of PICK1 function by shRNA-mediated knock-down of PICK1, or expression of a peptide, pep2-EVKI, that blocks PICK1 PDZ interactions, prevents LTP. In addition, hippocampal LTP is absent in slices from PICK1 KO mice. Further, we show there is a loss of LTD in neurons lacking PICK1 or in which PICK1 PDZ domain interactions are blocked. Together these findings demonstrate a central requirement for PICK1 in hippocampal NMDAR-dependent bidirectional plasticity and suggest a role for PICK1 in the activity-dependent regulation of GluR2 subunit AMPAR composition in CA1 pyramidal neurons.

RESULTS

PICK1 over expression causes an NMDAR-dependent potentiation of transmission that occludes LTP

PICK1 over expression in CA1 pyramidal neurons potentiates AMPAR-mediated synaptic transmission and causes the expression of GluR2-lacking AMPARs at synapses in a mechanism requiring PKC and CaMK (Terashima et al., 2004). Because activity-induced LTP exhibits similar requirements (Malenka and Nicoll, 1999; Plant et al., 2006; Wikstrom et al., 2003), we further explored the link between PICK1-dependent potentiation and LTP by investigating whether the effects of PICK1 over expression also depend upon NMDAR activation, another critical requirement for LTP induction (Bliss and Collingridge, 1993; Collingridge et al., 1983; Malenka and Nicoll, 1999). We used Sindbis virus to bicistronically express PICK1 and GFP in CA1 pyramidal neurons in acute hippocampal slices maintained in culture for 1–2 days, and compared AMPA- and NMDAR-mediated synaptic transmission in neighboring infected and control (uninfected) neurons (Figure 1A). As previously reported (Terashima et al., 2004), viral expression of PICK1 produced an increase in the amplitude of AMPAR-mediated EPSCs in CA1 pyramidal neurons, and an increase in inward rectification indicative of the expression of GluR2-lacking AMPARs (Figure 1B). However, incubation with D-AP5 (100 µM) during viral infection, prevented these PICK1-driven changes in AMPAR function (Figure 1C). Thus, NMDAR activation in the slice during viral over expression of PICK1 is required for the potentiation in AMPAR-mediated synaptic transmission and the change in GluR2 subunit composition.

Figure 1.

Figure 1

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PICK1 over expression causes an NMDAR-dependent increase in the AMPAR-mediated EPSC. (A) Fluorescence image of CA1 pyramidal neurons in an acute cultured hippocampal slice infected with Sindbis virus expressing GFP (left) and schematic (right) of the experimental approach showing sequential recordings from neighboring infected and uninfected (control) neurons. (B) AMPAR-mediated EPSC amplitude and rectification index in PICK1 over-expressing and neighboring uninfected control neurons (n = 13). (C) AMPAR-mediated EPSC amplitude (compared between PICK1 over expressing and in slice control neurons, same stimulation position and intensity) and rectification index for slices incubated in 100 µM D-AP5 during viral infection (n = 11).

We next studied whether viral over expression of PICK1 or blockade of PICK1 PDZ domain interactions affects LTP in CA1 pyramidal neurons. First we investigated whether the potentiation of AMPAR-mediated transmission caused by over expression of PICK1 occludes LTP. In hippocampal slices acutely maintained in culture for 1–2 days, viral over expression of PICK1 prevented LTP (Figure 2A, D); however, in control uninfected neurons LTP was reliably induced by the same pairing protocol (Figure 2B, D). In these experiments baseline AMPAR-mediated EPSCs in PICK1 over expressing neurons exhibited greater amplitude and increased inward rectification compared with control neurons (PICK1 over expression: AMPA:NMDA ratio (A:N) = 7.2 ± 1.3, rectification index (RI) = 5.6 ± 2.0, n = 6; uninfected controls: A:N = 2.14 ± 0.6, RI = 1.7 ± 0.5, n = 7; P < 0.05 for A:N and RI).

Figure 2.

Figure 2

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Over expression of PICK1 or expression of a peptide blocking the PICK1-GluR2 interaction (pep2-EVKI) prevents LTP. Sindbis virus expression in acute cultured hippocampal slices. (A–C) Example LTP experiments from a CA1 pyramidal neuron virally over expressing PICK1 (A), a control uninfected neuron (B) and a neuron virally expressing pep2-EVKI (C). (D) Pooled data for LTP experiments under these three conditions and also for neurons virally expressing GFP alone (PICK1: n = 7; un-infected: n = 18, pep2-EVKI: n = 7; GFP: n = 7). Black bar indicates LTP pairing protocol.

PICK1 PDZ domain interactions, including the PICK1-GluR2 interaction, are blocked by a peptide, pep2-EVKI (YNVYGIEEVKI). Pep2-EVKI, however, does not block interactions of the two other PDZ domain-containing proteins (GRIP and ABP) that also bind GluR2 & 3 (Daw et al., 2000; Li et al., 1999). We tested whether blocking the PICK1 PDZ interactions affects LTP using virally expressed pep2-EVKI and found that this manipulation also completely blocks LTP (Figure 2C, D). The lack of LTP in the neurons virally expressing constructs did not result from non-specific effects of viral infection because in interleaved control experiments, neurons virally expressing GFP alone exhibited robust LTP (Figure 2D). Therefore, PICK1 over expression potentiates AMPAR-mediated transmission and this occludes with LTP, while blockade of the PICK1 PDZ interactions prevents LTP.

LTP is absent in CA1 pyramidal neurons lacking PICK1

Our over expression studies suggest a central role for PICK1 in hippocampal LTP, and thus predict that LTP should be absent in neurons lacking PICK1. To test this hypothesis, we first investigated whether acute knock-down of PICK1 using RNA interference affects LTP. Several different short hairpin RNA (shRNA) constructs directed against PICK1 were expressed in cultured hippocampal neurons using lentivirus and their effectiveness in knocking down endogenous PICK1 assayed immunocytochemically. Based on this characterization, we selected one shRNA (‘T197’), which was the most effective. Using lentiviral expression of shRNA and GFP, we found that the T197 PICK1 shRNA reduced endogenous PICK1 expression in cultured hippocampal neurons to levels below detection after seven days of infection, while virus expressing a scrambled control shRNA had no effect on PICK1 expression (Figure 3A). We then compared the effects of these two viruses on LTP. When CA1 pyramidal neurons in cultured hippocampal slices were infected for seven days with lentivirus (Figure 3B), we found that LTP was completely blocked by PICK1 shRNA, but reliably induced in cells expressing the control scrambled shRNA (Figure 3C–E).

Figure 3.

Figure 3

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Knock down of PICK1 using shRNA prevents LTP. (A) Examples of GFP fluorescence (top) and endogenous PICK1 staining (center; with overlay, bottom) from dissociated cultured hippocampal neurons infected with lentivirus expressing GFP and control scrambled shRNA (left) or PICK1 shRNA (right). Scale bar is 20 µm. (B) Two-photon fluorescence images of CA1 neurons in a one week cultured hippocampal slice expressing lentivirus shRNA and GFP at low magnification (top, showing two sites of injection of virus in the slice, scale bar 250 µm) and higher magnification (bottom, region boxed in top image, scale bar 50 µm). (C) EPSC amplitude vs. time for an example LTP experiment from a CA1 pyramidal neuron in a one week cultured slice that is expressing PICK1 shRNA. Inset top: example averaged EPSCs from baseline and following LTP induction (and superimposed). Black bar indicates the induction protocol. (D) As for B but for a neuron expressing control scrambled shRNA. (E) Pooled data for all LTP experiments on neurons expressing PICK1 shRNA (blue; n = 8) or control scrambled shRNA (black; n = 8).

To confirm the requirement for PICK1 in LTP revealed by our knock-down studies, we next determined whether there is an LTP deficit in PICK1 knockout (KO) mice (Gardner et al., 2005). We investigated this using whole-cell voltage-clamp recordings from CA1 pyramidal neurons in acute hippocampal slices from PICK1 KOs and in interleaved experiments, in slices from wild-type (WT) littermates. In slices from the WT littermates an LTP-inducing pairing-protocol yielded robust stable LTP (P < 0.01 baseline vs. 20 min post-pairing; Figure 4A, C). However, the same pairing-protocol produced no long-lasting potentiation in slices from PICK1 KOs (P = 0.26 baseline vs. 20 min post-pairing; P < 0.05 for KO vs. WT at 20 min post-pairing; Figure 4B, C). Importantly, this LTP deficit in PICK1 KOs did not appear to result from alterations in basal synaptic properties since no differences were observed between PICK1 KO and WT slices in the I-V relationships of AMPARs and NMDARs nor the AMPA to NMDA ratios (Figure 4D). Thus, it is unlikely that the LTP deficit in PICK1 KO mice is due to a disruption of NMDAR function, suggesting that PICK1 functions downstream of NMDAR activation.

Figure 4.

Figure 4

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Pairing-induced LTP is deficient in slices from PICK1 knock out mice. (A) Example LTP experiment using whole-cell voltage-clamp recording from a CA1 pyramidal neuron in an acute hippocampal slice from WT. Inset top: traces taken at the time points indicated by the letters (a–c; bars 100 pA/50 ms). (B) Example LTP experiment in a CA1 pyramidal neuron in a slice from a PICK1 KO (as for A; bars 100 pA/50 ms). (C) Pooled data for all whole-cell LTP experiments (PICK1 KO: n = 20 cells from 6 animals; WT littermates: n = 16 cells from 4 animals). (D) I–V analysis of EPSCs from PICK1 KOs (n = 4) and WT (n = 7), lower inset: AMPAR:NMDAR EPSC ratio from the same cells (bars 100 pA /20 ms).

Despite the lack of LTP in slices from PICK1 KO animals, a considerable amount of transient post-pairing potentiation was observed in many recordings (Figure 4B, C). The duration of this potentiation was similar to that of the transient incorporation of GluR2-lacking, inwardly rectifying AMPARs we reported during LTP expression (Plant et al., 2006). Therefore, in a subset of LTP experiments in slices from PICK1 KO animals we assayed for changes in AMPAR rectification during the transient potentiation. Interestingly, the transient potentiation was associated with an increase in rectification of the AMPAR-mediated component of EPSCs determined as the ratio of the initial slope of EPSCs obtained at holding potentials of −60 mV and +40mV (at 3–5 min post-pairing RI = 172 ± 20 % of baseline, P < 0.01, n = 5; at 15 – 20 min post-pairing RI = 130 ± 20 %, P = 0.33, n = 5). This change in the inward rectification of AMPAR-mediated EPSCs was dependent upon the presence of intracellular spermine because in interleaved experiments using a whole-cell solution lacking spermine, no change in rectification was observed during the transient potentiation (at 3–5 min post-pairing RI = 105 ± 11 % of baseline, n = 4; P < 0.05 spermine compared to no spermine). These findings are consistent with an initial incorporation of GluR2-lacking AMPARs following LTP induction in PICK1 KOs that is responsible for the transient potentiation. Further, the failure to maintain this potentiation suggests that the LTP deficit in PICK1 KOs may result, in part, from an inability to consolidate the initial potentiation because of disruption in the mechanism(s) responsible for replacement of the initially inserted GluR2-lacking AMPARs by GluR2-containing receptors. It is important to note that the transient potentiation observed in these whole-cell experiments is not due to presynaptic post-induction potentiation (i.e, post-tetanic potentiation or short-term potentiation) (Lauri et al., 2007), since the transient potentiation we observed in the PICK1 KO occurs in the absence of dramatic changes in presynaptic stimulation rates during induction and is associated with a postsynaptic change in inward rectification of the AMPAR-mediated EPSC.

Different induction protocols may engage distinct signaling requirements for LTP; therefore, we also examined whether there was a deficit in tetanus-induced LTP in PICK1 KO mice using two-pathway extracellular field potential recordings from hippocampal slices. Following a stable baseline period, a high-frequency stimulation protocol (HFS; two 100 Hz, 1 s tetani separated by 20 s) produced no significant long-lasting potentiation in slices from PICK1 KO mice (112 ± 10 %, n = 6; LTP pathway: baseline vs. 30 min after tetanic stimulation, P = 0.16, n = 6 animals; control pathway vs. LTP pathway 30 min after tetanic stimulation, P = 0.17, n = 6 animals; Figure 5A, B). Conversely, the same HFS protocol applied to WT littermate control slices, exhibited robust LTP in interleaved experiments (177 ± 10 %, n = 6; LTP pathway: baseline vs. 30 min after tetanic stimulation, P < 0.01, n = 6 animals; control pathway vs. LTP pathway 30 min after tetanic stimulation, P < 0.01, n = 6 animals; Figure 5C, D) and the level of potentiation measured at 30 min post-HFS was significantly greater in slices from WT animals compared to that in slices from PICK1 KOs (WT vs. PICK1 KO: P < 0.05). These findings further confirm that loss of PICK1 leads to a lack of LTP, revealing a requirement for PICK1 in NMDAR-dependent LTP. Moreover, the LTP deficit in both the whole-cell and field potential recording experiments demonstrates that the role of PICK1 in LTP is not limited to a single specific recording condition or induction protocol.

Figure 5.

Figure 5

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LTP is deficient in slices from PICK1 KO animals using extracellular field potential recordings. (A) Example two-pathway field potential experiment from the CA1 region in a slice from a PICK1 KO. Inset top: fEPSP traces taken at the times indicated by the letters (a, b), scale bars = 0.2 mV/ 5 ms. For this and subsequent panels, arrow indicates LTP induction (two, 1s, 100 Hz tetani, test intensity, 20s apart), open symbols represent control path, closed symbols are LTP path. (B) Pooled data for all extracellular field potential LTP experiments in PICK1 KO slices (n = 6 animals, all interleaved with WT litter mates shown in C, D). (C, D) LTP experiments from interleaved experiments on slices from WT littermates (n = 6 animals; as for A, B).

PICK1 is required for hippocampal NMDAR-dependent LTD

There is good evidence that PICK1 plays a central role in mGluR-dependent cerebellar LTD by mediating the internalization of GluR2-containing AMPARs (Steinberg et al., 2006; Xia et al., 2000). However, it is unclear if PICK1 plays a role in NMDAR-dependent LTD, a prominent form of LTD in the hippocampus that is also expressed in many regions of neocortex (Dudek and Bear, 1992; Kemp and Bashir, 2001; Malenka and Bear, 2004; Mulkey and Malenka, 1992). We therefore directly tested whether PICK1 is involved in hippocampal NMDAR-dependent LTD. In whole-cell recordings from CA1 pyramidal neurons we first compared LTD in slices from PICK1 KO and WT littermates using a low-frequency pairing induction protocol (LFP; 300 stimuli at 1Hz paired with a holding potential of −40 mV), which robustly triggers NMDAR-dependent LTD (Hjelmstad et al., 1997; Luthi et al., 1999). In slices from PICK1 KO mice, LTD was absent (Figure 6A; LTD pathway baseline vs. 30 min after pairing, P = 0.88), whereas in slices from WT litter mates, LTD was readily induced by the same LFP protocol (Figure 6B; LTD pathway baseline vs. 30 min after pairing, P < 0.0005; LTD pathway 30 min after pairing PICK1 KO vs. WT, P < 0.001). To confirm that loss of PICK1 causes an LTD deficit we next determined whether LTD was affected by acute knock-down of PICK1 using lentiviral-mediated expression of shRNA to knock down PICK1 in CA1 pyramidal cells in one week cultured hippocampal slices. In agreement with our findings on PICK1 KO mice, neurons in which PICK1 had been knocked down using the PICK1 shRNA failed to show LFP-induced LTD (Figure 6C), whereas neurons expressing control scrambled shRNA exhibited robust LTD (Figure 6D). Finally, we investigated whether viral expression of the PICK1 PDZ domain interaction blocking peptide, pep2-EVKI, using Sindbis virus-mediated expression in acute slice culture also prevents LTD. CA1 pyramidal neurons virally expressing pep2-EVKI failed to exhibit LTD (Figure 6E) while control uninfected neurons reliably yielded robust LTD (Figure 6F). Thus, using three independent loss-of-function approaches we show that PICK1 is required for hippocampal NMDAR-dependent LTD.

Figure 6.

Figure 6

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Hippocampal LTD requires PICK1. (A) Pooled data of EPSC amplitude vs. time for two-pathway whole-cell LTD experiments from CA1 pyramidal neurons in acute hippocampal slices from PICK1 KO (n = 7 animals, all interleaved with experiments on slices from WT litter mates shown in B). For this and panel B, inset top is example averaged EPSCs from baseline and following LTD induction in control (open symbols) and LTD (closed symbols) pathways. Black bar indicates the LTD induction protocol. (B) Pooled data for LTD experiments in slices from WT (n = 7 animals, interleaved with data in A). (C) Pooled data of EPSC amplitude vs. time for whole-cell LTD experiments from CA1 pyramidal neurons virally expressing PICK1 shRNA in 1 week cultured hippocampal slices (n = 8). (D) Pooled data of EPSC amplitude vs. time for whole-cell LTD experiments from CA1 pyramidal neurons virally expressing control scrambled shRNA in 1 week cultured hippocampal slices (n = 8). (E) Pooled data of EPSC amplitude vs. time for whole-cell LTD experiments from CA1 pyramidal neurons virally expressing pep2-EVKI in acute cultured hippocampal slices (n = 5). (F) Pooled data of EPSC amplitude vs. time for whole-cell LTD experiments from control uninfected CA1 pyramidal neurons in acute cultured hippocampal slices (n = 5).

DISCUSSION

Here we show a critical requirement for PICK1 in hippocampal NMDAR-dependent LTP and LTD. We find that the potentiation of AMPAR-mediated synaptic transmission and the change in GluR2 AMPAR subunit composition, caused by PICK1 over expression requires NMDAR activity and occludes subsequent LTP. Blockade of PICK1 PDZ interactions prevents both LTP and LTD, and both forms of synaptic plasticity are absent in CA1 pyramidal neurons lacking PICK1. Thus we demonstrate that PICK1 is required for hippocampal NMDAR-dependent LTP and LTD, prominent forms of long-term synaptic plasticity in the hippocampus and neocortex that are strongly implicated in learning and memory, development and disease.

For PICK1 deletion studies we used two different approaches, shRNA-mediated knock-down of PICK1 and genetic knock out. A recent study shows that shRNA can produce off-target effects on synaptic function that are dependent on the species of shRNA used, but not related to the protein that is knocked-down (Alvarez et al., 2006). Thus, the lack of LTP and LTD in both the shRNA and KO models provides important complementary results establishing a requirement for PICK1 in bidirectional NMDAR-dependent long-term synaptic plasticity. Additionally, the similar findings with both approaches alleviate concerns that plasticity deficits resulted from developmental defects in the chronically PICK1-deficient mice or culture artifacts in the acutely PICK1-deficient neurons in cultured slices.

Role of PICK1 in LTP

Our study directly demonstrates a central requirement for PICK1 in the mechanism of hippocampal LTP. We show that PICK1 over expression potentiates transmission in an NMDAR-dependent manner that occludes LTP, while blockade or deletion of PICK1 prevents LTP. Together, these findings indicate that PICK1 is a critical mediator of LTP rather than a participant in an indirect modulatory pathway. The mechanistic role(s) of PICK1 in hippocampal LTP remains to be fully elucidated; however, our results combined with previous studies provide some insight. Our previous work indicates that PICK1 can regulate the GluR2 subunit composition of AMPARs and that a change in the GluR2 content of AMPARs can occur during LTP (Terashima et al., 2004; Plant et al., 2006). Our present results showing a lack of LTP in neurons in which PICK1 has been knocked down, or in which PICK1 PDZ domain interactions are blocked by peptide expression, suggest that PICK1 plays a role in the induction or initial expression of LTP. One possibility in this regard is that PICK1 acts to retain GluR2-containing AMPARs to enable the initial incorporation of GluR2-lacking AMPARs that can underlie the earliest phase of LTP expression (Plant et al., 2006). Evidence for such a retention role for PICK1 in maintaining an intracellular pool of GluR2-containing AMPARs has recently been provided (Ho et al., 2007; Jin et al., 2006; Lin and Huganir, 2007) and may itself be regulated by additional novel PICK1 interactions (Cao et al., 2007).

Experiments on the PICK1 KO, however, indicate that a component of the initial phase of LTP expression can occur in the absence of PICK1. In slices from PICK1 KO mice, we find that LTP induction protocols produce a transient potentiation. The presence of this transient potentiation may be the result of a partial compensation in the knock out, since it is not observed in neurons in which PICK1 has been acutely knocked down. The transient potentiation in neurons in the PICK1 KO is associated with an increase in inward rectification of the AMPAR-mediated EPSC indicating that the potentiation is mediated by GluR2-lacking AMPARs. This finding suggests a possible additional role for PICK1 in LTP mediating a switch from GluR2-lacking to GluR2-containing AMPARs during LTP expression. This role is consistent with the previously proposed idea that PICK1 regulates the recruitment of GluR2-containing AMPARs to hippocampal synapses (Daw et al., 2000). Furthermore, such a mechanism is similar to that reported for the expression of cerebellar stellate cell plasticity in which PICK1 is required for the activity- and calcium-dependent switch of GluR2-lacking AMPARs to GluR2-containing AMPARs (Gardner et al., 2005; Liu and Cull-Candy, 2002; Liu and Cull-Candy, 2005; Liu and Cull-Candy, 2000).

A related issue of interest is how synaptic potentiation remains constant during LTP when expression involves changes in AMPAR subunit composition from GluR2-lacking to GluR2-containing receptors. Since the GluR2-lacking AMPARs have a higher single channel conductance compared with their GluR2-containing counterparts (Isaac et al 2007), a simple one-to-one exchange of GluR2-containing for GluR2-lacking receptors would result in a reduction of potentiation during LTP expression. For LTP induced by protocols such as pairing, which do not include dramatic changes in stimulation frequency (that can lead to transient presynaptic potentiation), little or no decrement in LTP is typically observed (as for example shown in the present data). This suggests that GluR2-lacking AMPARs must be replaced by more GluR2-containing AMPARs during LTP expression to maintain the increased synaptic strength. This idea argues for a homeostasis of increased synaptic strength, rather than the creation of a set number of ‘slots’ for AMPARs at synapses during LTP.

A prominent current theory for activity-dependent potentiation of central synapses posits the initial recruitment of GluR1-containing AMPARs followed by their subsequent replacement with GluR2-containing AMPARs (Hayashi et al., 2000; Malinow and Malenka, 2002; Shi et al., 2001). Our present findings are consistent with this model in which the GluR1-containing AMPARs would be GluR1 homomers that are initially incorporated during LTP and then are replaced by GluR1/2 heteromers in a PICK1-dependent fashion. This sequence provides a resolution to the findings that CaMKII is critically required for LTP (Malenka and Nicoll, 1999) and regulates GluR1 homomers, yet cannot regulate GluR2-containing AMPARs (Oh and Derkach, 2005). Moreover, recent studies suggest that Kalirin-7 and cGKII selectively interact with GuR1 and are involved in LTP (Serulle et al., 2007; Xie et al., 2007), providing potential specific chaperones for GluR2-lacking receptors. Thus, our findings on the role of PICK1 and the regulation of GluR2-content of AMPARs in LTP are consistent with and expand upon current models of the expression mechanism of LTP. Complete elucidation of the involvement of PICK1 in the induction and expression mechanisms of LTP will be of great importance in understanding how GluR2-lacking AMPARs are regulated during synaptic plasticity, and may also provide mechanistic insight into the pathological regulation of GluR2-lacking AMPARs that occurs in disease (Cull-Candy et al., 2006).

In the present study we have demonstrated a role of PICK1 in LTP in hippocampal slices from two-three week old animals; however, it is possible that PICK1 may not be required for some forms of LTP, for example, induced by different protocols or at different developmental stages. Accumulating evidence demonstrates the co-existence of multiple forms of LTP that can be induced by different patterns of activity or at different stages of development (Palmer et al., 2004). For example, the GluR1 (GluRA) knock out mouse lacks adult hippocampal LTP induced by typical tetanus or low-frequency pairing protocols (Zamanillo et al., 1999), but exhibits LTP following a theta-burst pairing protocol (Hoffman et al., 2002). In addition, the GluR1 knock out exhibits LTP, albeit of diminished size, in young mice (Jensen et al., 2003). Thus, development and induction protocol influence the expression mechanism utilized for LTP with respect to the requirement for GluR1. Interestingly, in the GluR1 knock out, theta-burst pairing induced LTP exhibits a delayed time course of potentiation (Hoffman et al., 2002) suggesting a prominent role for GluR1 during the initial expression, consistent with the transient incorporation of GluR1 homomers at conditioned synapses during the early phase of LTP (Plant et al., 2006). Of particular relevance to the present study are the recent findings indicating that LTP can be expressed without an alteration in GluR2 AMPAR subunit composition (Adesnik and Nicoll, 2007; Gray et al., 2007) or with an increase (rather than decrease) in GluR2 content (Bagal et al., 2005). However, other work demonstrates a role for incorporation of GluR2-lacking AMPARs during LTP (Lu et al., 2007; Plant et al., 2006). Moreover, a previous study shows that LTP can be expressed by a mechanism involving an increase in single channel conductance, or with no increase in single channel conductance, under identical induction conditions (Benke et al., 1998). These two different mechanisms for the expression of LTP may reflect the incorporation of high conductance GluR2-lacking AMPARs in one case and incorporation of lower conductance GluR2-containing AMPARs in the other mechanism. Thus, taken together, these studies indicate that LTP can involve the incorporation of GluR2-lacking AMPARs, but this is not the only mechanism for expression, even at the same age and under very similar experimental conditions. Therefore, it will be of great importance to determine how development and distinct activity patterns influence the utilization of different mechanisms for LTP expression.

Role of PICK1 in LTD

There is compelling evidence that PICK1 plays a central role in cerebellar LTD. This form of LTD is dependent on the activation of mGluR1 and PKC, is expressed as a reduction in surface GluR2-containing AMPARs, and involves a mechanism requiring the PICK1-GluR2 interaction and the PKC-dependent phosphorylation of serine 880 on GluR2 (Chung et al., 2003; Ito, 2002; Steinberg et al., 2006; Xia et al., 2000). In contrast, the role for PICK1 in hippocampal LTD has until now remained controversial. There is good evidence that PICK1 regulates AMPAR surface expression in cultured hippocampal neurons (Chung et al., 2000; Hanley and Henley, 2005; Perez et al., 2001; Sossa et al., 2006; Terashima et al., 2004); however, these studies did not directly test a role for PICK1 in hippocampal synaptically-induced LTD. Indeed, only two studies have attempted to directly test the requirement for PICK1 in hippocampal LTD, and these provide conflicting results. Both studies used acute intracellular application of peptides to block PICK1 PDZ domain interactions; in one study there was no effect of this manipulation on LTD (Daw et al., 2000), while in the other work, a partial block of LTD was observed (Kim et al., 2001). However, functional analyses based primarily on the acute infusion of a peptide that competitively inhibits PICK1 PDZ domain interactions can be difficult to interpret since the peptide blocks PICK1 PDZ domain interactions with all binding partners (GluR2/3, PKC, mGluR7 etc.) and may also block interactions of other similar PDZ domain-containing proteins (Sheng and Sala, 2001). Moreover, the effects of the peptide block can only be observed for the relatively short period of time that a whole-cell recording can be maintained (< 2 hrs) and so effects over longer time scales cannot be readily assessed. Thus, the present work in which PICK1 function is specifically manipulated in CA1 pyramidal neurons in hippocampal slices over longer time scales is the first direct test of the requirement for PICK1 in hippocampal LTD. We show that acute knock down of PICK1 or genetic deletion of PICK1, as well as viral expression of pep2-EVKI, prevents hippocampal NMDAR-dependent LTD, the predominant form of LTD in juvenile hippocampus (Dudek and Bear, 1992; Kemp et al., 2000; Malenka and Bear, 2004; Mulkey and Malenka, 1992). However, despite being necessary for LTD, PICK1 does not mimic or occlude LTD (A. Terashima and J. Isaac, unpublished observations), which is in contrast to its ability to mimic LTP. It is interesting to note that the previous work in which pep2-EVKI, or similar peptides, were acutely infused into CA1 neurons resulted in no block or only a partial reduction in LTD (Daw et al., 2000; Duprat et al., 2003; Kim et al., 2001). In the present study we show that viral expression of pep2-EVKI prevents LTD. The difference in the results between the present and previous studies is likely due to the different duration of peptide expression, indicating that prolonged blockade of PICK1 PDZ domain interactions has additional effects not observed during the acute infusion experiments.

Bidirectional regulation of synaptic strength by PICK1

How can PICK1 dually participate in both hippocampal LTP and LTD? Recent work demonstrates that PICK1 has a calcium binding domain at its N-terminus, the occupancy of which biphasically regulates PICK1-GluR2 interactions: a rise in free calcium from rest to 15 µM increases the affinity of PICK1 for GluR2 ~5 fold, whereas further increases in free calcium decrease the affinity of this interaction (Hanley and Henley, 2005). Such bimodal calcium sensitivity could endow PICK1 with the ability to differentially regulate AMPAR trafficking for both LTD and LTP since both phenomena rely on increased intracellular calcium, but to different levels. The relatively small rise in calcium during LTD induction may promote PICK1-mediated internalization of GluR2 containing AMPARs, while larger more rapid calcium changes associated with LTP induction could trigger release of GluR2-contianing AMPARs from intracellular reserve pools for eventual incorporation at conditioned synapses to maintain potentiation. Indeed previous work suggests a role for PICK1 in mediating a release of AMPARs from intracellular pools (Daw et al., 2000). In addition, studies on cultured hippocampal neurons support the idea that PICK1 is a calcium sensor for synaptic plasticity since the N-terminal calcium-binding domain of PICK1 is required for NMDA-induced AMPAR internalization, a form of LTD in culture (Hanley and Henley, 2005). Moreover, PICK1 can differentially regulate AMPAR surface expression in culture depending on the level of NMDAR activation, with large calcium rises producing a PICK1-dependent increase in surface AMPARs, and smaller calcium rises causing a PICK1-dependent decrease in surface AMPARs (Hanley and Henley, 2005; Sossa et al., 2006). Considered together, the evidence supports a model in which PICK1 functions as a calcium sensor capable of transducing distinct spatiotemporal calcium profiles into appropriate AMPAR trafficking events to yield bidirectional plasticity of synaptic efficacy.

EXPERIMENTAL PROCEDURES

Constructs and viruses

Sindbis viruses used were as previously described (Terashima et al., 2004). shRNA species to specifically knock down PICK1 were identified and packaged into lentivirus, commercially (America Pharma Source, Frederick, MD, USA). In addition, a scrambled shRNA control lentivirus was also provided. The expression of shRNA was driven by the human U6 promoter and all viruses also coded for GFP as a marker. For lentivirus infection of neurons, the virus was concentrated to ~1×107 particles/ml by ultracentrifugation at 112700 g, 90 min, 4°C. Efficiency in knocking down endogenous PICK1 expression in cultured dissociated hippocampal neurons was tested immunocytochemically as follows. Low density hippocampal cultures were prepared from E18 rats using standard techniques (Roche and Huganir, 1995). At DIV 5–7, neurons were infected with lentivirus and cultured for a further seven days. Neurons were then fixed with 4% paraformaldehyde / 4% sucrose in PBS for 15 min, permeabilized, and incubated with 10% normal goat serum for 1 hour. The neurons were then incubated with anti-PICK1 primary antibody (Affinity BioReagents) followed by Alexa 568-conjugated (red) anti-rabbit secondary antibody (Molecular probes). Images were collected with a 63x oil-immersion objective on a Zeiss LSM510 confocal microscope both for green (GFP) and red (PICK1) channel. Series of optical sections were collected at intervals of 0.4 µm and maximal projections are shown. The immunocytochemical analysis showed that knock down of endogenous PICK1 in hippocampal neurons was effectively achieved by the T197 shRNA species (T197) when there was a high level of viral expression (as determined by high levels of GFP fluorescence) for at least four days, necessitating infecting neurons seven days prior to experimental analysis.

Acute hippocampal slice culture and viral infection

For all Sindbis virus expression experiments hippocampal slices maintained in culture for one to two days (‘acute slice culture’) were used. Slices were prepared and cultured as described previously (Terashima et al., 2004). Briefly, transverse hippocampal slices (300 µm thick) were prepared from Wistar rat pups (10 – 12 days old) in a modified extracellular solution (mM): 4 KCl, 26 NaHCO3, 1 CaCl2, 5 MgSO4, 10 glucose, 5 ascorbic acid, 248 sucrose, saturated with 95% O2/ 5% CO2. Following recovery for 30 – 60 min at 27 °C, Sindbis virus was pressure ejected into a region of the CA1 pyramidal cell layer and slices were placed in a standard sterile culture medium and incubated at 35 °C for 20 – 48 hours before use.

One week slice culture and viral infection

For all synaptic physiology lentivirus shRNA expression experiments, one week hippocampal slice cultures were used because it takes seven days for lentivirus-mediated shRNA to effectively knock down PICK1 expression. Hippocampal slices (350 µm thick) were prepared from rats aged P7 using a McIllwain tissue chopper. Following 1 hour recovery after slicing, lentivirus was pressure ejected into part of the CA1 cell body layer and slices were and explanted onto a membrane (Millicell-CM, 0.4 µm pore size) placed in 1 ml of MEM (GIBCO no. 61100–061) containing 2.5 mM glutamine, 30 mM Hepes, 5 mM NaHCO3, 30 mM d-glucose, 0.5 mM l-ascorbate, 2mM CaCl2, 2.5 mM MgSO4, 1 µg insulin, and 20% horse serum (Musleh et al., 1997). Slices were incubated at 35 °C for seven days before use.

Electrophysiology

Whole-cell patch-clamp recordings were made from CA1 pyramidal neurons using standard techniques (Pelkey et al., 2005; Terashima et al., 2004) either in acute slices prepared from 2–3 week old mice, or in cultured rat hippocampal slices. Experiments on knock out mice and WT littermates were performed blind to genotype. For recordings in cultured slices, infected neurons were identified as those expressing GFP, and near-by non-infected neurons were used for in-slice controls (Terashima et al., 2004). The extracellular solution during recordings was as follows (mM): 125 NaCl, 3.25 KCl, 1.25 NaHPO4, 25 NaHCO3, 2.5 CaCl2, 1.3 MgSO4 (or 1.5 mM MgCl2), 10–15 glucose, 0.1 picrotoxin (or 0.005 bicuculline), saturated with 95% O2/ 5% CO2.

For extracellular field potential recordings from mouse slices, a patch electrode filled with extracellular solution was placed in stratum radiatum of CA1 to record field EPSPs and responses were evoked by electrical stimulation of axons in stratum radiatum at a frequency of 0.1 Hz. No GABAA receptor antagonists were included in the extracellular solution. Two independent pathways were monitored, only one of which received the LTP induction protocol of two, 100 Hz, 1 s tenani (at 20 s interval).

For whole-cell recordings from CA1 pyramidal neurons in acute slices, the following intracellular solution was used (mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 1–5 QX-314, 0.1 spermine, pH 7.2, 285–290 mOsm. For the acute slice LTD experiments EPSCs were evoked by electrical stimulation of axons in stratum radiatum at a frequency of 0.2 Hz and recorded at a holding potential of −70 mV. LTD was induced at one of two independent pathways by pairing 300 stimuli at 1 Hz with a holding potential of −40 mV. For the acute slice LTP experiments EPSCs were evoked at a frequency of 0.5 Hz at a holding potential of −60 mV. LTP was induced by pairing 50–100 stimuli at 0.5 or 1 Hz with a holding potential of −10 mV. In a subset of acute slice LTP experiments an intracellular solution of the following composition was used: 100 Cs-gluconate, 5 CsCl, 0.6 EGTA, 5 MgCl2, 8 NaCl, 2 ATP2Na, 0.3 GTPNa, 40 HEPES, 0.1 spermine, and 1 QX-314, pH 7.2–7.3, 285–290 mOsm. Similar results were obtained for these LTP experiments with both intracellular solutions, and thus the data were pooled.

For whole-cell recordings from CA1 pyramidal neurons in acute cultured slices, the following intracellular solution was used (mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 1–5 QX-314, 0.1 spermine, pH 7.2, 285–290 mOsm‥ EPSCs were evoked by electrical stimulation of axons in stratum radiatum at a frequency of 0.2 Hz at a holding potential of −70 mV. LTP was induced by pairing 100 stimuli at 1 Hz with a holding potential of 0 mV. LTD was induced by pairing 300 stimuli at 1 Hz with a holding potential of −40 mV.

For whole-cell recordings from CA1 pyramidal neurons in one week cultured slices the same solutions as for acute cultured slices were used except that Na-phosphocreatine (0.6 mM) was included in the intracellular solution, and for the extracellular solution the concentration of Ca2+ and Mg2+ were both 4 mM and 2 µM 2-chloroadenosine was included. Only neurons expressing high levels of GFP were recorded from indicating a high level of shRNA expression. LTP was induced by pairing 3 Hz stimulation with a holding potential of 0 mV for 3 min (Boehm et al., 2006), and LTD by pairing 300 stimuli at 1 Hz with a holding potential of −10 mV (Rial Verde et al., 2006).

Data were collected using Axopatch 200B or Multicalmp 700A amplifiers (Axon Instruments), filtered at 5 KHz and digitised at 10 KHz. EPSC amplitude, DC current, input resistance and series resistance were continuously monitored on-line. Recordings were terminated if series resistance deviated by more than 20 %.

Electrophysiology analysis

Changes in AMPAR rectification in the experiments using viral over expression were estimated as previously described (Terashima et al., 2004); briefly, three averaged EPSCs (each an average of 3 consecutive single EPSCs) were collected at −70 mV, interleaved with two averaged EPSCs (each an average of 3 consecutive single EPSCs) collected at +40 mV. The rectification index is expressed as EPSC −70 / EPSC+40. In the viral expression experiments in Figure 1, the amplitude of the AMPA EPSC at −70 mV in infected cells is compared to that in neighbouring un-infected cells in the same slices with the same stimulus position and intensity, as previously described (Terashima et al., 2004).

For analysis of the I–V relationship of EPSCs in WT and PICK1 KO animals (Figure 4D), the AMPA component of the EPSC was estimated using a slope measurement from EPSC onset to 1.4 ms. The NMDA component was measured as the amplitude at 75 ms after EPSC onset. The AMPA:NMDA ratio was calculated as the ratio of the peak of the EPSC at −60 mV (AMPA) and the amplitude of the NMDA component measured at 75 ms after EPSC onset at +40 mV. For a subset of LTP experiments using whole-cell recordings from the PICK1 KOs averaged EPSCs (5 individual events) collected at −60 mV and +40 mV were used to measure RIs by determining the ratio of the initial slope (onset to 1.4 ms) of the EPSCs obtained at holding potentials of −60 and +40 mVs. The RI values obtained within 3–5 minutes post-pairing are expressed as a percentage of control RI values obtained prior to pairing during the baseline recording period for spermine-containing and spermine-free intracellular solutions. In the figures for presentation purposes the stimulus artefacts have been truncated or subtracted. Statistical analysis was performed using the Student’s t-test (paired or unpaired as appropriate), P < 0.05 was considered significant. In the figures * indicates P < 0.05, ** P < 0.01, *** P < 0.005.

Acknowledgements

Supported by the Wellcome Trust (A.T., G.L.C., J.T.R.I.), NINDS Intramural Program (J.T.R.I., K.W.R.) and NICHD Intramural Program (K.A.P., C.J.M.). We are very grateful to Dr. R. Huganir for providing the PICK1 KO mice.

Footnotes

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