Co-Requirement of PICK1 Binding and PKC Phosphorylation for Stable Surface Expression of the Metabotropic Glutamate Receptor mGluR7

SUMMARY

The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release, and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a novel form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo.

INTRODUCTION

Metabotropic glutamate receptors (mGluR1–8) are widely expressed at excitatory synapses throughout the CNS. The mGluRs are divided into three groups based on sequence identity and pharmacological properties: group I mGluRs (mGluR1 and mGluR5), group II mGluRs (mGluR2 and mGluR3), and group III mGluRs (mGluR4, 6, 7, and 8) (Conn and Pin, 1997; Nakanishi, 1994; Pin and Bockaert, 1995; Pin and Duvoisin, 1995). Group III mGluRs predominantly localize at presynaptic terminals to regulate neurotransmitter release through Gi/o coupled signaling cascades (Chavis et al., 1998; Conn and Pin, 1997; Shigemoto et al., 1997; Shigemoto et al., 1996). The group III receptor mGluR7 is particularly intriguing as it has been shown to regulate synaptic efficacy at diverse locations throughout the CNS and also to rapidly undergo agonist-induced internalization (Ayala et al., 2007; Laezza et al., 1999; O'Connor et al., 1999; Pelkey et al., 2005; Pelkey et al., 2007; Perroy et al., 2002; Perroy et al., 2000; Schoepp, 2001). Thus, synaptic regulation by mGluR7 is dynamically modulated by acute changes in receptor surface expression on presynaptic terminals.

Consistent with a critical role for mGluR7 trafficking in defining synaptic regulation, recent studies have demonstrated that the polarity of synaptic plasticity at hippocampal mossy fiber-CA3 interneuron synapses is dictated by mGluR7 surface expression (Pelkey et al., 2005; Pelkey and McBain, 2007a; Pelkey and McBain, 2007b). At naïve mossy fiber –interneuron synapses, high frequency stimulation (HFS) elicits a presynaptic form of long-term depression (LTD) that relies on mGluR7 activation and subsequent inhibition of P/Q-type voltage gated Ca²⁺ channels (Pelkey et al., 2005; Pelkey et al., 2006). However, following agonist-induced internalization of mGluR7, the same HFS elicits presynaptically expressed long-term potentiation (LTP). Thus, in this system mGluR7 surface expression serves as a metaplastic switch controlling the sign of synaptic plasticity revealing a novel gating mechanism for plasticity through mGluR7 trafficking.

Rapid regulation of receptor surface expression by endocytosis is a mechanism shared by a variety of G-protein-coupled receptors (GPCRs) (Coutinho and Knopfel, 2002; Dhami and Ferguson, 2006; El Far and Betz, 2002; Thompson et al., 1993). The molecular events underlying the trafficking and recycling of many GPCRs are well characterized; however, studies on mGluRs are still in their infancy. We recently described several features of mGluR7 endocytosis (Lavezzari and Roche, 2007; Pelkey et al., 2005; Pelkey et al., 2007), including the post-endocytic trafficking route of mGluR7 following constitutive endocytosis (Lavezzari and Roche, 2007). We now show that a combination of PKC phosphorylation and protein-protein interactions regulate mGluR7 surface expression. In particular, the integrity of the mGluR7 PDZ-binding domain and phosphorylation of a specific residue within the mGluR7 C-terminus, S862, are critical for stabilizing receptor surface expression. Furthermore, PKC phosphorylation of S862 promotes binding to the synaptic PDZ-domain-containing protein PICK1 (protein interacting with C-kinase 1) and inhibits binding to calmodulin (CaM). Finally, using mice lacking PICK1, we demonstrate that PICK1 binding, coupled with PKC phosphorylation of S862, regulates mGluR7 surface expression in neurons. Thus, we define PKC phosphorylation and binding of PICK1 as molecular mechanisms necessary to stabilize mGluR7 on the surface of neurons for regulation of presynaptic function.

RESULTS

We recently demonstrated that mGluR7 undergoes endocytosis (Pelkey et al., 2005); however, little is known of the specific mechanisms regulating mGluR7 surface expression and intracellular trafficking. Previous studies have shown that the PDZ ligand at the extreme C-terminus of mGluR7 is critical for the synaptic localization of mGluR7 (Boudin et al., 2000). To test if the PDZ ligand also plays a role in mGluR7 endocytosis, we transfected primary hippocampal neurons with mGluR7 containing a myc tag in the extracellular N-terminal domain of the receptor, so that surface-expressed receptor could be specifically labeled. To monitor the constitutive trafficking of mGluR7, we labeled surface-expressed receptor with myc antibodies and allowed endocytosis at 37°C for 15 minutes. We visualized labeled surface receptors with secondary antibodies before permeabilization (red) and internalized receptors after permeabilization (green) and observed a marked increase in mGluR7 endocytosis with truncation of the distal C-terminus (mGluR7 Δ893 and mGluR7 Δ907) compared to WT mGluR7 (Figure 1). Truncation of the extreme C-terminus (mGluR7 Δ907) to remove the PDZ ligand, further truncation to disrupt the α–tubulin binding domain (mGluR7 Δ893) (Saugstad et al., 2002), or a single point mutation in the PDZ ligand (mGluR7 V914P), which disrupts binding to PICK1 (Dev et al., 2000; Hirbec et al., 2002; Perroy et al., 2002), all increased constitutive internalization of mGluR7, with trafficking of mGluR7 Δ907, mGluR7 Δ893, or mGluR7 V914P being indistinguishable.

Figure 1.

mGluR7 internalization is regulated by the PDZ binding domain. (A) Schematic diagram showing the cytoplasmic terminus of mGluR7a. S862 is indicated in red and the PDZ binding domain in bold italics. (B) Rat hippocampal neurons were transiently transfected with cDNA encoding mGluR7 containing an extracellular myc epitope tag. Neurons expressing mGluR7 wild-type (WT), mGluR7 Δ893, mGluR7 Δ907, or mGluR7 V914P were labeled with anti-myc monoclonal antibody, washed, and returned to conditioned media at 37°C for 15 minutes. The cells were fixed and incubated with Alexa 568-conjugated anti-mouse secondary antibody (red) to label surface-expressed receptors. After permeabilization, cells were incubated with Alexa 488-conjugated secondary antibody (green) to label the internalized receptors. Green fluorescence indicates internalization compared to total (green plus red). The lower panels show higher-magnification images of the individual processes boxed in the upper panels. Scale bar, 20 µm. (C) Summary histogram quantitating internalization of mGluR7 WT, mGluR7 Δ893, mGluR7 Δ907, and mGluR7 V914P in hippocampal neurons. The amount of internalization was measured using Improvision Volocity software. Data represent means ± SEM; ^*p<0.001 (n = 4).

The PDZ ligand on mGluR7 directly interacts with PICK1 (Boudin et al., 2000; Dev et al., 2000; Dev et al., 2001; El Far et al., 2000; Enz and Croci, 2003; Fagni et al., 2004; Hirbec et al., 2002; Perroy et al., 2002). Furthermore, PICK1 binds to PKCα (Staudinger et al., 1997; Staudinger et al., 1995) and modulates the phosphorylation of mGluR7 (Dev et al., 2000; Dev et al., 2001). Interestingly, mGluR7 contains a single PKC phosphorylation site within its C-terminus at S862 (Figure S1) (Airas et al., 2001; Sorensen et al., 2002). To test if phosphorylation of S862 specifically regulates mGluR7 trafficking and surface expression in concert with the PDZ ligand, we compared the endocytosis of mGluR7 phosphorylation mutants, mGluR7 S862A or mGluR7 S862E, with that of mGluR7 WT in hippocampal neurons. Similar to disruption of the PDZ ligand, we observed a pronounced increase in internalization with mutation of S862 to alanine to eliminate phosphorylation (Figures 2A and 2B). Conversely, we saw a decrease in endocytosis of mGluR7 S862E, a mutation, which mimics phosphorylation (Figures 2A and 2B). These findings suggest that PKC phosphorylation of mGluR7 inhibits endocytosis to stabilize cell surface receptors. Indeed, treatment of neurons expressing WT mGluR7 with the PKC inhibitor chelerythrine significantly increased receptor endocytosis to levels comparable with S862A mutation or PDZ ligand truncation (Figures 2C and 2D). Moreover, endocytosis of mGluR7 Δ893 was diminished following incubation with the phorbol ester TPA to activate PKC (Figures 2C and 2D). Importantly, neither phosphorylation mutant responded to further PKC stimulation or inhibition, indicating that S862 mediates all of the PKC effects on mGluR7 surface expression (Figures 2A and 2B). Taken together, these studies demonstrate that phosphorylation of S862 critically regulates endocytosis of mGluR7 in neurons.

Figure 2.

PKC phosphorylation of mGluR7 on S862 regulates receptor endocytosis. (A, C) Hippocampal neurons were transiently transfected with myc-tagged mGluR7 WT, mGluR7 S862A, mGluR7 S862E, or mGluR7 Δ893 as indicated. Neurons were labeled with anti-myc antibody, washed, and returned to conditioned media containing 1 µM TPA, 5 µM chelerythrine, or vehicle control at 37°C for 15 minutes. The cells were stained and images acquired as described in Figure 1. (B) Summary histograms quantitating internalization from panel A. Data represent means ± SEM; ^*p<0.01, ^**p<0.05, n.s. indicates p>0.05 (n = 4). Scale bar, 20 µm. (D) Summary histograms quantitating internalization from panel C. Data represent means ± SEM; ^*p<0.001. Scale bar, 20 µm. (E) Surface expression of mGluR7 and GluR2 was analyzed using a biotinylation assay in primary cortical neurons. Cultured neurons were treated with 1 µM TPA, 5 µM chelerythrine, or vehicle control at 37°C for 15 minutes. Biotinylated surface-expressed proteins were isolated with streptavidin-agarose beads, and visualized by immunoblotting using either mGluR7a or GluR2 antibodies, with α-tubulin used as a loading control. In addition, expression of mGluR7 and GluR2, as well as changes in mGluR7 phosphorylation, were evaluated by probing immunoblots of total cell lysates with mGluR7, GluR2, or mGluR7 S862 phosphorylation state-specific antibody (pS862). (F) Quantitation of the immunoblots was performed by measuring the band intensity of the biotinylated fraction compared with the intensity of the band representing total input. Graphs represent means ± SEM; ^*p < 0.05, ^**p < 0.01 (n = 4). Tubulin had no significant effects by TPA or chelerythrine (less than mean ± 5%, p > 0.5). (G) Quantitation of phosphorylation was presented by measuring the band intensity of the phosphorylated fraction compared with the intensity of the band representing total input. Graphs represent means ± SEM; ^*p < 0.01, ^**p < 0.01 (n = 4).

Thus far we had relied on the characterization of exogenous mGluR7 expressed in cultured hippocampal neurons. This approach enabled us to define sequences within mGluR7 that are essential for regulating receptor trafficking. However, studies of exogenous receptors expressed in cultured neurons may be confounded by protein overexpression and mistargeting. Therefore, we extended our studies to analyze endogenous mGluR7, specifically evaluating the role of PDZ domain proteins and PKC phosphorylation of mGluR7 in regulating mGluR7 trafficking in neurons. First, we measured mGluR7 surface expression using biotinylation of primary cortical cultures. The cultures were treated with the phorbol ester TPA to stimulate PKC activity, chelerythrine to inhibit PKC activity, or vehicle alone for 15 minutes at 37°C. Cell surface proteins were biotinylated, surface proteins isolated using streptavidin beads, and proteins detected by immunoblotting. These experiments revealed a 40% increase in mGluR7 surface expression following PKC activation, but a decrease in surface expression when PKC was inhibited (Figures 2E and 2F). In contrast, surface expression of the AMPA receptor subunit GluR2 was decreased upon PKC stimulation (Figures 2E and 2F), as has been described previously (Chung et al., 2000; Matsuda et al., 2000).

To examine the phosphorylation of endogenous mGluR7 on the PKC site S862, we generated a phosphorylation-state specific antibody. The S862 phospho-antibody specifically recognized mGluR7 when S862 was phosphorylated as demonstrated by comparing immunoblots of mGluR7 WT and mGluR7 S862A. Furthermore, immunoreactivity for mGluR7 S862 was significantly increased upon TPA treatment of heterologous cells expressing mGluR7 (Figure S1). Consistent with all of our observations in vitro and in transfected neurons, we found that TPA treatment dramatically increased mGluR7 S862 phosphorylation and led to a significant increase in mGluR7 surface expression (Figures 2E–2G).

We have previously reported robust agonist-induced endocytosis of mGluR7 (Pelkey et al., 2005). To extend these findings we next examined whether other changes in neuronal activity could modulate mGluR7 phosphorylation and trafficking. We treated cultured neurons with either KCl or NMDA to increase neuronal activity, and evaluated surface expression of mGluR7 using the biotinylation assay described above. Under both conditions, we observed a dramatic reduction in mGluR7 surface expression (~84% with NMDA; 61% KCl; Figure 3A). GluR2 surface expression was also diminished under these conditions, but to a lesser extent than mGluR7.

Figure 3.

Trafficking of mGluR7 is dependent on neuronal activation or agonist treatment. (A) Activity-dependent trafficking of mGluR7 and GluR2. Primary cortical neurons were treated with 20 mM KCl for 5 minutes or 50 µM NMDA for 10 minutes at 37°C. Surface biotinylation assay, immunoblotting, and quantitative analysis of immunoblots were performed as described above. Graphs represent means ± SEM; ^*p < 0.001 (n = 4), ^**p < 0.01 (n = 5), ^***p < 0.001 (n = 4). Tubulin had no significant effects by KCl or NMDA (less than mean ± 5%, p > 0.5). (B) Hippocampal neurons were transiently transfected with myc-tagged mGluR7 WT, mGluR7 S862A, or mGluR7 S862E as indicated. Neurons were labeled with anti-myc antibody, washed, and returned to conditioned media containing 20 mM KCl, 400 µM L-AP4, or vehicle control at 37°C for 15 minutes. The cells were stained and images acquired as described in Figure 1. (C) Summary histograms quantitating internalization from Panel B are included. Data represent means ± SEM; ^*p<0.01, ^**p<0.05, n.s. indicates p>0.05 (n = 4). Scale bar, 20 µm.

Importantly, there was a significant reduction in mGluR7 phosphorylation on S862 after neuronal activation (Figure 3A). Thus synaptic activity rapidly modulates phosphorylation and trafficking of endogenous mGluR7.

To determine if activity-induced mGluR7 trafficking relies on changes in S862 phosphorylation, we next compared L-AP4- and KCl-driven endocytosis of WT mGluR7 with that of mGluR7 S862A, and mGluR7 S862E mutants using our fluorescence-based trafficking assay. Similar to our findings with PKC inhibition, S862A and S862E mutations fully occluded and blocked activity-induced mGluR7 internalization respectively (Figures 3B and 3C). In addition to L-AP4 and KCl treatment NMDA also failed to alter the trafficking of mGluR7 S862A or S862E mutants (data not shown). Considered together these data support a central role for S862 phosphorylation not only in constitutive mGluR7 cycling but also in rapid activity-induced changes in receptor trafficking.

CaM binds to the mGluR7 C-terminus, and in vitro this binding is negatively regulated by PKC phosphorylation of S862 (Airas et al., 2001; Nakajima et al., 1999). As our data thus far indicate that mGluR7 surface expression and endocytosis are regulated by PKC phosphorylation of S862, we next investigated potential roles of CaM binding. We first examined the effects of PKC phosphorylation of mGluR7 S862 on CaM vs. PICK1 binding. We incubated GST fusion proteins of the mGluR7 C-terminal domain with rat brain homogenates and found that both CaM and PICK1 bound to mGluR7 by immunoblotting (Figure 4A). As expected from previous reports, we observed a decrease in CaM binding to the phosphomimetic mutant mGluR7 S862E, However, unexpectedly, this mutation also generated a robust increase in PICK1 binding to mGluR7. The increase in binding was specific to the phosphomimetic glutamic acid mutation, because mutation to alanine, S862A, did not enhance the mGluR7-PICK1 interaction (Figure 4A).

Figure 4.

Phosphorylation of S862 regulates the interaction of mGluR7 with PICK1 and CaM. (A) Total rat brain extract was subjected to a pull-down assay using GST fusion proteins containing the mGluR7 cytoplasmic domain (WT, S862A, or S862E). After washing, bound proteins were analyzed by SDS-PAGE and immunoblotting using PICK1 or CaM antibodies. Graphs represent means ± SEM; ^*p < 0.01 (n = 4). ^**p < 0.01 (n = 3). (B) HeLa cells were co-transfected with FLAG-tagged PICK1 and myc-tagged mGluR7 (WT, S862A, or S862E). After treatment with 1 µM TPA or vehicle control at 37°C for 15 minutes, cells were solubilized and immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted with either myc or FLAG antibodies, and total cell lysates were also probed with myc antibody. Summary histogram quantitating binding of mGluR7 to PICK1 is shown, with graphs representing means ± SEM; ^*p < 0.05, ^**p < 0.05 (n = 3). (C) HeLa cells were transfected with myc-tagged mGluR7 (WT, S862A, or S862E) and CaM. After treatment with 1 µM TPA or vehicle control at 37°C for 15 minutes, cells were solubilized and immunoprecipitated with anti-myc antibody. Summary histogram quantitating binding of CaM to mGluR7 is shown. Graphs represent means ± SEM; ^*p < 0.05, ^**p < 0.01 (n = 3).

To verify that phosphorylation of mGluR7 at S862 increased mGluR7-PICK1 binding using a different technique, we co-expressed FLAG-tagged PICK1 and myc-tagged mGluR7 in HeLa cells. We performed immunoprecipitations with FLAG antibody to isolate PICK1 and probed immunoblots for mGluR7. Consistent with the GST pull-down assay using brain homogenate described above, the S862E mutation in mGluR7 increased receptor-PICK1 binding (Figure 4B). The mutation to a negatively charged residue was critical, as introduction of the S862A mutation caused a significant decrease in PICK1 binding to mGluR7. Phorbol ester treatment of heterologous cells expressing mGluR7 also increased the PICK1-mGluR7 interaction as detected by co-immunoprecipitations, providing additional evidence that PKC phosphorylation of mGluR7 modulates receptor binding to PICK1 (Figure 4B). We also coexpressed full-length CaM and mGluR7 in HeLa cells, immunoprecipitated mGluR7, and evaluated CaM binding and S862 phosphorylation by immunoblotting. We found that CaM bound less efficiently to phosphorylated mGluR7 following PKC activation with TPA (Figure 4C) as observed previously in GST pull down assays (Airas et al., 2001; Nakajima et al., 1999; Sorensen et al., 2002). Similarly, less CaM co-immunoprecipitated with mGluR7 containing the phosphomimetic mutation, S862E (Figure 4C).

Our unexpected findings that phosphorylation of S862 had a profound effect on PICK1 binding in addition to CaM binding (Figures 4A–4C), led us to directly compare conditions that might affect binding of CaM or PICK1 to mGluR7. Using the GST-mGluR7 C-terminal fusion protein, we conducted a pull down assay, incubating fusion protein with whole brain lysate in the presence of Ca²⁺ or EGTA. We observed robust binding of CaM to mGluR7 in the presence of Ca²⁺ (Figure 5A). In sharp contrast, there was an increase in PICK1 binding and no CaM binding in the presence of EGTA (Figure 5A). These findings supported differential binding of CaM and PICK1 under different Ca²⁺ conditions. To specifically examine the effect of CaM binding on the PICK1-mGluR7 interaction, we used several approaches. We first performed pull-down assays in the presence of CaM inhibitors and found corresponding increases in PICK1 binding (Figure 5B). In addition, we observed decreasing amounts of PICK1 binding in the GST-mGluR7 C-terminal fusion protein pull down assay with increasing amounts of CaM added to the binding buffer, consistent with CaM competing with PICK1 for mGluR7 binding (Figure 5C). We find that CaM decreases PICK1 binding in the presence or absence of PKC (Figure 5C). Finally, to test if the mechanism by which S862 phosphorylation regulates PICK1 binding was solely dependent on CaM binding, we examined PICK1 binding to mGluR7 in the presence of the Ca²⁺ chelator EGTA, which eliminates CaM binding to mGluR7. We found that in the presence of EGTA, there was no longer an increase in PICK1 binding to the mGluR7 C-terminus containing the phosphomimetic mutation (S862E) compared to mGluR7 S862A, the non-phosphorylatable mutant (Figure 5D). These data support a role for S862 phosphorylation in facilitating PICK1 binding, not based on phosphorylation, but by dissociating CaM from the mGluR7 C-terminal domain.

Figure 5.

CaM regulates mGluR7 trafficking by competing with PICK1 binding to S862 phosphorylated region of mGluR7. (A) Ca²⁺-dependence of CaM and PICK1 binding was evaluated using a GST pull down assay as described for Figure 4. Total rat brain lysates were incubated with wild-type GST-mGluR7 C-terminal fusion protein containing either 2 mM calcium or 5 mM EGTA, and immunoblots were probed with PICK1 or CaM antibodies. Graphs represent means ± SEM; ^*p < 0.01 (n = 3). (B) Effect of CaM antagonists on PICK1 binding to mGluR7. W-7 (100 µM) or calmidazolium (CMZ, 10 µM) was applied prior to and during a GST pull down assay as described above. Graphs represent means ± SEM; ^*p < 0.05 (n = 3). (C) A CaM competition assay was performed by incubating GST-mGluR7 C-terminal fusion proteins phosphorylated by PKC enzyme in vitro with rat brain lysate including different concentrations of purified CaM. Summary histogram quantitating binding of PICK1 to GST-mGluR7 C-terminal is shown, with graphs representing means ± SEM; ^*p<0.01, ^**p<0.05, ^***p<0.01 (n = 5). (D) A CaM competition assay was performed using GST-mGluR7 S862A or S862E with rat brain lysate combined with purified CaM or EGTA as indicated. Summary histograms quantitating binding of PICK1 or CaM to GST-mGluR7 C-terminal are shown, with graphs representing means ± SEM; ^*p<0.01, n.s. indicates p>0.05 (n = 3). (E) Myc-tagged mGluR7 was co-transfected with CaM WT or CaM₁₂₃₄ in rat hippocampal neurons. Surface expression and endocytosis were evaluated as described in Figure 1 with surface seen in red and internalization in green. Effect of CMZ (5 µM) on mGluR7 trafficking is also presented. Graphs represent means ± SEM; ^*p <0.001, n.s. indicates p>0.05 (n = 3). Scale bar, 20 µm.

The newly discovered interplay between PKC phosphorylation at S862 and CaM vs. PICK1 binding to mGluR7 further prompted us to evaluate a potential role for CaM in mGluR7 internalization. We measured the constitutive endocytosis of mGluR7 expressed in hippocampal neurons and inhibited CaM activity in two ways: cultures were treated with the CaM inhibitor calmidazolium (CMZ) or a dominant negative form of CaM (CaM₁₂₃₄), in which aspartate has been mutated to alanine in each of the four EF hands, rendering CaM unable to bind to Ca²⁺ (Geiser et al., 1991; Peterson et al., 1999; Pitt et al., 2001; Putkey et al., 1989), was co-expressed with mGluR7. In both cases, the inhibition of CaM depressed mGluR7 internalization (Figure 5E), consistent with wild-type CaM acting to facilitate endocytosis of mGluR7.

There are many neuronal PDZ domain-containing proteins, and PDZ-dependent interactions are often found to be promiscuous. Therefore, we used PICK1 KO mice to specifically evaluate the role of PICK1 in regulating PKC-dependent mGluR7 trafficking and phosphorylation (Gardner et al., 2005; Steinberg et al., 2006). Primary cortical cultures were prepared from WT or PICK1-deficient mice at P0–P1 and at 14 DIV we performed biotinylation assays as described above to evaluate surface levels of native mGluR7 in the cultured neurons. We found that overall surface levels of mGluR7 were similar in vehicle treated WT and KO neurons (Figures 6A and 6B) confirming that mGluR7 is trafficked to the plasma membrane in the absence of PICK1 interactions (Boudin et al., 2000). However, whereas TPA treatment significantly increased the surface expression of mGluR7 in WT cultures, this regulation was lost in PICK1 KO neurons (Figures 6A and 6B). Furthermore, TPA produced significantly less mGluR7 S862 phosphorylation in KO cultures compared to WT cultures (Figures 6A and 6C). These results were in sharp contrast to the regulation of GluR2 surface expression, which was reduced in PICK1 KO neurons and upon TPA stimulation indicating target protein-specific roles for PICK1 and PKC phosphorylation in receptor trafficking (Figures 6A and 6B). Thus, although PICK1 is not essential for membrane insertion of mGluR7, it does regulate PKC-mediated phosphorylation, and hence surface stabilization, of the receptor.

Figure 6.

PICK1 specifically regulates S862 phosphorylation and surface expression of mGluR7 as revealed in PICK1 knockout mice. (A) Surface expression of mGluR7 was analyzed using a biotinylation assay in primary cortical neurons from PICK1 knockout mice. Neurons were treated with 1 µM TPA or vehicle control at 37°C for 15 minutes. Biotinylated surface-expressed receptors were isolated with streptavidin-agarose beads and revealed by immunoblotting using either mGluR7a or GluR2 antibody. Total cell lysates were also probed using the indicated antibodies. TPA-induced phosphorylation of mGluR7 was revealed by immunoblotting cell lysates using the phosphorylation state-specific pS862 antibody. (B, C) For quantitation of the immunoblots, surface-expressed receptors or phosphorylated mGluR7 was determined, and the normalized results for each condition were expressed relative to total receptor expression. Graphs represent means ± SEM; ^*p < 0.01 (n = 6), ^**p < 0.05 (n = 5), and ^***p < 0.001 (n = 6).

Although mGluR7 reaches the surface in neurons lacking PICK1, it is possible mGluR7 function is disrupted (Boudin et al., 2000; Perroy et al., 2002). Thus, in a final series of experiments we compared mGluR7-dependent mossy fiber-interneuron plasticity in acute hippocampal slices from PICK1 WT and KO mice (Figure 7). At naïve calcium-permeable AMPAR containing WT mossy fiber-stratum lucidum interneuron (MF-SLIN) synapses HFS produces robust presynaptically expressed LTD (Figure 7A). This presynaptic form of LTD results from mGluR7-triggered inhibition of P/Q-type voltage-gated Ca²⁺ channels and proceeds completely independent of postsynaptic changes in AMPAR subunit composition or function (Lei and McBain, 2002, 2004; Pelkey et al., 2005, 2006). Remarkably, slices from PICK1 KO mice lacked activity-induced LTD and, in fact, displayed a transient post-tetanic potentiation (PTP) following HFS (Figure 7A; p<0.01 for WT vs. KO at 15 minutes post-HFS). Moreover, chemically-induced LTD by transient mGluR7 activation with brief agonist (L-AP4, 400 µM) exposure was absent in slices from PICK1 KO mice (Figure 7B, p<0.05 for KO vs. WT at 10 minutes washout of L-AP4). Importantly, acute synaptic depression during L-AP4 treatment was comparable between slices from KO and WT mice consistent with efficient surface expression of mGluR7 in the absence of PICK1 (Figure 6). The LTD deficits in PICK1 KO mice likely reflect combined deficits in mGluR7 activation due to mislocalization of surface mGluR7 away from the active zone of presynaptic terminals (Boudin et al., 2000) and coupling of mGluR7 to downstream signaling cascades necessary for LTD expression (see DISCUSSION). Alternatively, the lack of MF-SLIN LTD in PICK1 KOs could result from alterations in postsynaptic Ca²⁺-permeable AMPA receptors since PICK1 affects GluR2 content (Ho et al., 2007; Terashima et al., 2004) and postsynaptic calcium permeable-AMPA receptor signaling participates in the induction of presynaptic MF-SLIN LTD (Lei and McBain, 2004; Pelkey et al., 2006). However, AMPA receptor-mediated current-voltage relationships of MF-SLIN synapses exhibited similar degrees of polyamine-dependent inward rectification in slices from PICK1 WT and KO mice (rectification indices: 0.34 ± 0.18 and 0.38 ± 0.14 for WT and KO respectively, n = 3 for each; p>0.5) indicating that loss of PICK1 did not affect AMPA receptor composition at these connections. Based on these findings we conclude that presynaptic PICK1-mGluR7 interactions are essential for NMDAR-independent MF-SLIN LTD.

Figure 7.

Loss of mGluR7-mediated LTD in PICK1 knockout mice. (A) Normalized group data plots showing the effects of HFS (arrow) on MF-SLIN synaptic transmission in slices from PICK1 knockout mice (KO, black squares, n = 5) and wild-type controls (WT, grey circles, n = 7). Plotted are the 1 minute running average EPSC amplitudes normalized to responses obtained during the baseline recording period prior to HFS. Traces above and below are the average of 20 consecutive EPSCs obtained in a representative recording from a KO or WT slice respectively acquired at the times indicated (bars 20 ms/100 pA). Sensitivity of transmission to the group II mGluR agonist DCGIV applied during the period indicated by the horizontal bar during each recording confirmed MF origin of the evoked EPSCs (see EXPERIMENTAL PROCEDURES). (B) Similar to A but for recordings in which slices were perfused with the group III mGluR agonist L-AP4 for mGluR7 activation rather than subjected to HFS (KO, n = 8; WT, n = 4; bars upper 20 ms/25 pA, lower 20 ms/100 pA).

DISCUSSION

For many GPCRs, the mechanisms underlying endocytosis and recycling are well described. For example, receptor phosphorylation and binding to intracellular regulatory proteins play a key role in the endocytosis/recycling of the β₂-adrenergic receptor (Cao et al., 1999; Dhami and Ferguson, 2006; Ferguson et al., 1996). In the present study, we have elucidated several key molecular mechanisms regulating mGluR7 trafficking and surface expression by focusing on PKC phosphorylation of mGluR7 and receptor binding to the regulatory proteins PICK1 and CaM. We find that treating neurons with TPA to activate PKC increases surface expression of mGluR7 via phosphorylation of a critical residue, S862, on the mGluR7 C-terminus. However, increases in neuronal activity rapidly trigger dephosphorylation of S862 and a subsequent reduction in mGluR7 surface expression. Therefore, S862 phosphorylation and receptor stability on the cell surface are dynamically regulated under a variety of conditions, including changes in neuronal activity or directly stimulating kinase activity.

To define which protein-protein interactions directly regulated mGluR7 surface expression, we examined the trafficking of truncated mGluR7. We found that elimination of the PDZ ligand on mGluR7 dramatically reduces surface expression. PICK1 is a PDZ domain-containing protein that interacts directly with mGluR7 (Boudin et al., 2000; Dev et al., 2000; Dev et al., 2001; El Far et al., 2000; Enz and Croci, 2003; Fagni et al., 2004; Hirbec et al., 2002; Perroy et al., 2002), binds to PKC (Staudinger et al., 1997; Staudinger et al., 1995), supports the synaptic localization of mGluR7 (Boudin et al., 2000; Dev et al., 2001) and participates in mGluR7-mediated regulation of synaptic transmission (Bockaert et al., 2003; El Far and Betz, 2002; Fagni et al., 2004; Perez et al., 2001; Perroy et al., 2002). Therefore this protein seemed a logical candidate for an mGluR7 binding protein that could regulate receptor phosphorylation and surface expression. Our initial working hypothesis was that PICK1 binding to mGluR7 would recruit PKCα, another PICK1 binding protein, to the receptor and promote S862 phosphorylation and augment mGluR7 surface expression. Therefore, we performed an extensive analysis of PICK1 binding to mGluR7 and explored the potential interplay between PICK1 and S862 phosphorylation. Our findings revealed an unexpected enhancement of PICK1 binding to mGluR7 via PKC phosphorylation of S862 that suggested a cooperative model in which both PKC phosphorylation and PICK1 binding worked together to increase mGluR7 surface expression.

Phosphorylation of S862 has previously been shown to inhibit CaM binding to the mGluR7 C-terminus in vitro (Airas et al., 2001; Nakajima et al., 1999). Furthermore, CaM binding disrupts mGluR7 coupling to G-protein βγ subunits and inhibits S862 phosphorylation in vitro (El Far et al., 2001; Nakajima et al., 1999; O'Connor et al., 1999; Sorensen et al., 2002). We therefore examined the binding of CaM to mGluR7 in detail using both co-immunoprecipitation of full-length proteins expressed in heterologous cells and GST-fusion protein pull down assays from rat brain homogenate. In both cases, we detected robust binding of CaM to wild-type mGluR7 that was inhibited by PKC phosphorylation or by introducing a phosphomimetic mutation (S862E) into the mGluR7 C-terminal domain. Because phosphorylation of S862 promoted PICK1 binding to receptor, but inhibited CaM binding, we performed a competition assay. We found that CaM binding decreased the amount of PICK1 interacting with the mGluR7 C-terminus. Therefore, PICK1 and CaM binding are differentially regulated by PKC phosphorylation.

By directly comparing the different conditions under which CaM or PICK1 binding to mGluR7 can occur, we discovered a profound Ca²⁺ dependence for CaM binding. Whereas, both CaM and PICK1 interact in a competitive manner with mGluR7 in the presence of Ca²⁺, EGTA treatment abolished CaM binding and PICK1 binding was augmented. It was quite provocative that phosphorylation of S862 could regulate PICK1 binding to mGluR7 because S862 is approximately 50 amino acids away from the PDZ binding domain. We therefore explored the molecular basis by which S862 phosphorylation influences PICK1 binding. It seemed unlikely that phosphorylated S862 was a secondary binding site for PICK1 because we, and others, have shown that disruption of the PDZ binding domain on mGluR7 eliminates PICK1 binding (data not shown) (Boudin et al., 2000; Dev et al., 2000; El Far et al., 2000; Enz and Croci, 2003; Hirbec et al., 2002; Perroy et al., 2002). A more likely hypothesis was that S862 phosphorylation affects PICK1 binding due to a conformational change or binding to an intermediate protein such as CaM. To address this question more directly, we evaluated PICK1 binding to mGluR7 S862E in the presence of EGTA to chelate Ca²⁺ and eliminate CaM binding. Under these conditions, there was no difference in PICK1 binding to mGluR7 S862A or mGluR7 S862E consistent with CaM binding being a key factor in modulating PICK1 binding, and not S862 phosphorylaton per se.

To specifically evaluate the role of PICK1 in regulating mGluR7 trafficking and mGluR7-dependent plasticity, we characterized PICK1 KO mice. In cultured neurons derived from PICK1 KO mice, we found that PKC-dependent increases in S862 phosphorylation and mGluR7 surface expression were dramatically impaired. These findings confirmed a key role for PICK1 in regulating dynamic changes in mGluR7 surface expression. However, we did not observe any change in the steady state amount of mGluR7 expressed on the cell surface between WT and KO littermates, which may reflect compensatory mechanisms to increase receptor insertion rates or adoption of alternative mechanisms to stabilize surface receptors such as binding to other scaffolding proteins (Hirbec et al., 2002). Despite similar mGluR7 surface expression in WT and KO neurons, it is likely that presynaptic active zone targeting is impaired in the absence of PICK1 (Boudin et al., 2000). Given the extremely low affinity of mGluR7 for glutamate, mistargeting of surface receptor could dramatically impair synaptically driven receptor activation. Indeed such a deficit in synaptic localization of mGluR7 could explain the failure to observe any HFS-induced depression of MF-SLIN synapses in PICK1 KO mice. Consistent with this interpretation, the transient PTP observed in KO mice is reminiscent of that observed in slices from WT mice when HFS is given in the presence of an mGluR7 antagonist (Pelkey et al., 2005). However, the LTD deficit in PICK1 KO mice cannot solely be attributed to mistargeting since chemical LTD induced by L-AP4 treatment, which circumvents the need of synaptic localization for efficient receptor activation, is also absent in PICK1 KOs. Thus, it seems that PICK1 binding to mGluR7 is also necessary for efficient coupling to downstream targets for LTD induction (see also Perroy et al., 2002). The transient L-AP4-induced depression observed in PICK1 KOs suggests that signals generated by mGluR7 activation still have access to presynaptic release sites indicating that the PICK1 interaction mediates active zone but not axonal targeting of the receptor. Consistently, disruption of the PICK1-mGluR7 interaction prevents active zone but not axonal targeting (Boudin et al., 2000). This proximity to the synapse without residing in the active zone may explain why HFS-induced PTP in PICK1 KOs is not consolidated into LTP as the surface expressed axonal receptors may still be able to sequester proteins necessary for activity induced LTP at MF-SLIN synapses (Pelkey et al., 2005; Pelkey and McBain, 2007b).

PICK1 has been extensively characterized as an AMPA receptor binding protein, which regulates receptor trafficking and synaptic plasticity (Chung et al., 2003; Daw et al., 2000; Gardner et al., 2005; Hanley, 2006; Hanley et al., 2002; Kim et al., 2001; Liu and Cull-Candy, 2005; Lu and Ziff, 2005; Matsuda et al., 2000; Steinberg et al., 2006; Terashima et al., 2004; Xia et al., 2000). These studies focus on postsynaptic roles for PICK1. For example, it has been shown that PICK1 is a key regulator of postsynaptic GluR2 AMPA receptor endocytosis in cerebellar Purkinje cells (Steinberg et al., 2006; Xia et al., 2000) and in hippocampus (Kim et al., 2001). There are also studies demonstrating that PICK1 regulates the synaptic localization and function of the presynaptic receptor mGluR7 (Perroy et al., 2002). Based on our current study, we conclude that PICK1 plays a role in stabilizing mGluR7 on the cell surface. Therefore it is clear that PICK1 plays essential roles in both pre- and postsynaptic trafficking of receptors. However, the direct roles of PICK1 in receptor endo/exocytosis appear to be complicated and receptor subtype-specific. Moreover, it is possible that PICK1 participates in bidirectional regulation of receptor trafficking (Sossa et al., 2006; Terashima et al., 2008). For example, a recent study shows that PICK1 mediates both hippocampal LTP and LTD (Terashima et al., 2008). The many studies demonstrating that PICK1 regulates AMPA receptors, kainate receptors, and mGluRs at both pre- and postsynaptic sites illustrate the diversity in PICK1 regulation of excitatory synapses.

Consistent with PICK1 playing a diverse role in regulating presynaptic and postsynaptic glutamate receptors, PICK1 has a broad localization. For example, in cultured hippocampal neurons, most of endogenous or exogenous PICK1-containing clusters localize to excitatory synapses in both dendrites and axons. Indeed, significant amounts of PICK1 clusters colocalize postsynaptically with AMPA and/or NMDA receptors, and also presynaptically with mGluR7 (Boudin and Craig, 2001; Boudin et al., 2000; Dev et al., 2000; Perez et al., 2001; Xia et al., 1999). Furthermore, mGluR7 has been identified as a component of the active zone (Kinoshita et al., 1998; Ohishi et al., 1995; Shigemoto et al., 1997; Shigemoto et al., 1996). Therefore, considerable evidence supports the localization of PICK1 on both sides of the synaptic cleft consistent with a broad role in coordinating PKC regulation pre- and postsynaptically.

We have now identified a novel interplay between PKC phosphorylation and PICK1 binding in the regulation of mGluR7 surface expression. Our results support a model in which PKC phosphorylation of mGluR7 on S862, together with PICK1 binding, stabilizes mGluR7 on the plasma membrane (Figure 8). PKC phosphorylation of mGluR7 S862 reduces receptor endocytosis. However, in the absence of PICK1, TPA-stimulated increases in mGluR7 phosphorylation and surface expression are inhibited. This model is consistent with our experiments, but also with previous reports that PICK1 regulates synaptic expression and functional properties of mGluR7 (Bockaert et al., 2003; Boudin et al., 2000; El Far and Betz, 2002; Fagni et al., 2004; Perroy et al., 2002). If PICK1 binding is disrupted, then mGluR7 would be destabilized from the cell surface and unable to cluster and function at synapses. Generation of mice expressing mGluR7 with mutated S862 will be an important approach to examine the precise role of PKC phosphorylation and PICK1 binding on receptor trafficking. Such animal models will ultimately allow a direct way to examine the role of S862 on mGluR7-dependent synaptic plasticity.

Figure 8.

Model of PICK1 and S862-dependent regulation of mGluR7 surface expression. Activated PKCα (1) is recruited to either PICK1 (2) or mGluR7 (3), and PICK1 binding and/or S862 phosphorylation promotes surface expression of mGluR7 (4). CaM competes with PICK1 binding to mGluR7, inhibits PKC phosphorylation of mGluR7 S862, and promotes receptor endocytosis (5). The PICK1-PKCα complex facilitates and stabilizes mGluR7 surface expression (6). Neuronal activation or agonist treatment leads to S862 de-phosphorylation and induces internalization of mGluR7 (7). Following dissociation from PICK1, mGluR7 is internalized (8).

EXPERIMENTAL PROCEDURES

Cells, Antibodies, and DNA Constructs

HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM containing 10% fetal bovine serum, 1% L-glutamine, and 0.1% gentamicin. Primary hippocampal or cortical neurons were prepared from E18 Sprague-Dawley rats (Harlan, Indianapolis, IN) or postnatal day 0 (P0) to P1 mouse pups as previously described (Roche and Huganir, 1995). The neurons were grown in serum-free Neurobasal media (Invitrogen, Carlsbad, CA) with glutamine and B-27 supplement (Invitrogen). The use and care of animals used in this study followed the guidelines of the NIH Animal Research Advisory Committee.

Rabbit phosphorylation state-specific antibodies recognizing phosphorylated S862 of mGluR7 were generated by Quality Controlled Biochemicals (Hopkinton, MA). Rabbits were immunized with a synthetic phosphopeptide Ac-QKRKR(pS)FKAVC-amide corresponding to amino acids 857–866 of mGluR7. Sera were collected and affinity-purified using the antigen phosphopeptide. Anti-mGluR7a antibody was purchased from Chemicon International (Temecula, CA). Mouse anti-myc (9E10), rabbit anti-myc, rabbit anti-flag, and mouse anti-α tubulin antibodies were purchased from Sigma. Rabbit anti-PICK1 polyclonal antibody was purchased from Santa Cruz Biotechnology and Affinity BioReagents. Anti-CaM antibody was purchased from Upstate Biotechnology (Lake Placid, NY). All secondary antibodies for immunofluorescence were obtained from Molecular Probes (Eugene, OR).

The mGluR7 cDNA containing a myc epitope inserted into the extracellular N-terminus (myc-mGluR7a) was provided by Dr. Laurent Fagni (Institute of Functional Genomics, Montpellier, France). CaM and CaM₁₂₃₄ expression plasmids were provided by Dr. Geoffrey S. Pitt (Department of Medicine, Duke University Medical Center, Durham, NC). Myc-mGluR7a S862A, myc-mGluR7a S862E, myc-mGluR7a Δ893, myc-mGluR7a Δ907, and myc-mGluR7a V914P were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. To generate glutathione S-transferase (GST)-fusion constructs, the cytoplasmic regions of mGluR7a (amino acids 851–915), mGluR7a S862A, and mGluR7a S862E were amplified by PCR using synthetic primers that include flanking EcoRI or Xho I recognition sequences and subcloned into the GST fusion vector pGEX4T-1 (Amersham Biosciences). All constructs were verified by DNA sequencing.

Internalization and Immunofluorescence in Neurons

Receptor internalization was evaluated using a fluorescence-based antibody uptake assay described in detail elsewhere (Lavezzari et al., 2004; Lavezzari and Roche, 2007; Pelkey et al., 2005; Pelkey et al., 2007). Briefly, transfected neurons (12–14 DIV) were incubated with anti-myc antibody (9E10) for 45 min at room temperature to label surface-expressed receptor, washed, and returned to conditioned media with 1 µM phorbol-12-myristate-13-acetate (TPA), 5 µM chelerythrine, or DMSO as a control for 15 min at 37°C. Cells were washed and fixed with 4% paraformaldehyde/4% sucrose in PBS for 15 min, and were incubated for 30 min with Alexa 568-conjugated (red) anti-mouse secondary antibody to label the surface population of receptors. Following removal of the red secondary antibody, cells were permeabilized and incubated with 10% normal goat serum for 30 min, then incubated with Alexa 488-conjugated (green) anti-mouse secondary antibody to label the internalized population of receptors. The cells were washed and mounted (ProLong Antifade Kit, Molecular Probes) and imaged with a 63× or 40× objective on a Zeiss LSM 510 confocal microscope. Serial optical sections collected at 0.34 µm intervals were used to create maximum projection images shown as Figures. Internalization was assessed in at least three independent experiments (from at least three independent hippocampal preparations) for each condition. The amount of internalization was quantitated by counting green voxels not colocalized with red voxels using Volocity 2 software (Improvision) or MetaMorph 6.0 software (Universal Imaging Corp., Downingtown, PA). Data are presented as percentage of surface fraction (red) as compared with total fraction (green + red). Significance was determined using a Student’s unpaired t test.

Biotinylation Assay of Surface-Expressed Receptors

Primary cultured cortical neurons (14 DIV) were treated with 1 µM TPA, 5 µM chelerythrine, or DMSO as a control, and washed three times with ice-cold PBS containing 1 mM MgCl₂ and 0.1 mM CaCl₂ (PBS+). Cells were incubated with 1 mg/ml EZ-Link Sulfo-NHS-LC-biotin (Pierce) in PBS+ for 20 min at 4°C with gentle agitation. Cells were washed four times with ice-cold quenching buffer (50 mM glycine in PBS+). The cells were lysed in lysis buffer [50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2% Triton X-100, EDTA-free complete protease inhibitor (Roche, Indianapolis, IN)] and incubated for 30 min at 4°C. The lysates were centrifuged at 20,000 × g for 20 min. Supernatant was then incubated with 60 µl of 50% slurry of streptavidin-Sepharose beads (Pierce) for 2 hours at 4°C. Beads were washed four times in lysis buffer, and eluted and analyzed by immunoblotting with the relevant antibodies as indicated. The data were quantified by measuring surface receptor to total receptor band intensity ratios using ImageJ software and normalizing to DMSO-or vehicle-treated control cultures from at least three independent experiments.

GST Fusion Protein Production, Immunoprecipitation, and Immunoblotting

GST fusion proteins were purified according to the protocol recommended by the manufacturer (Amersham Biosciences). P30 Sprague-Dawley rat whole brain was homogenized with a Dounce homogenizer in PBS containing EDTA-free complete protease inhibitor. After addition of Triton X-100 to a final concentration of 1%, the lysates were incubated for 1 hour at 4°C, and centrifuged at 20,000 × g for 20 min, and supernatant was collected. For GST pull-down experiments, the supernatant was incubated with GST or GST-mGluR7a cytoplasmic regions bound to glutathione Sepharose 4B beads for 4 hours at 4 °C. The beads were then washed four times with PBS with 1% Triton X-100 at 4 °C and analyzed by immunoblotting with specific antibodies. For a CaM competition assay, GST-mGluR7 C-terminal fusion proteins bound to glutathione Sepharose 4B were phosphorylated by PKC enzyme in vitro, washed, and incubated with 300–500 µg of rat brain lysates with different concentrations of purified CaM (1µg of CaM corresponds to approximately 0.7 µM) for 2 hours at 4 °C. The beads were then washed four times with PBS with 1% Triton X-100 at 4 °C and analyzed by immunoblotting. For immunoprecipitation, cells were solubilized in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, EDTA-free complete protease inhibitor) for 30 minutes at 4 °C. Insoluble material was removed by centrifugation at 20,000 × g for 20 minutes. The supernatant was incubated with antibody-bound protein A or G beads (Sigma) for 2 hours at 4 °C and washed four times with lysis buffer. Precipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblotting with the relevant antibodies as indicated. All immunoprecipitation experiments were performed at least three times.

Electrophysiology

Hippocampal slices (300–350 µm thick) were prepared from 2–3 week old (range P12–22) C57BL/6 or PICK1^−/− and PICK1^+/+ (129/C57BL/6 hybrid genetic background) mice as described previously (Pelkey et al., 2005). Briefly, mice were anesthetized with isoflurane, and the brain was dissected in ice-cold saline solution (in mM): 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 5.0 MgCl₂, and 10 glucose, saturated with 95% O₂ and 5% CO₂, pH 7.4. Transverse slices were cut using a VT-1000S vibratome (Leica Microsystems, Bannockburn, IL) and incubated in the above solution at 35°C (30 min) following which they were kept at room temperature until use.

Slices were transferred to a recording chamber and perfused (3–5 ml/minute, 32–35°C) with extracellular solution (in mM): 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 2.5 CaCl₂, 1.5 MgCl₂, 10 glucose, and 0.005 bicuculline methobromide saturated with 95%O₂/5%CO₂, pH 7.4. DL-AP5 (50–100 µM) was added to the perfusate ensuring that only NMDAR-independent presynaptic MF-SLIN LTD was assayed (Lei and McBain, 2002); however, the presynaptic NMDAR-independent form of MF-SLIN LTD predominates in the mouse due to the high incidence of calcium permeable-AMPA receptor containing synapses (Lei and McBain, 2002; Lei and McBain, 2004; Pelkey et al., 2006). SLINs were visually identified within the first 100 µm of slices using a 40x objective and IR-DIC video microscopy (Zeiss Axioskop). Only interneurons with cell somata localized within or on the border of stratum lucidum and radiatum were included. Whole-cell recordings were performed using a multiclamp 700A amplifier (Axon Instruments, Foster City, CA) in voltage-clamp mode at a holding potential of −60mV. Recording electrodes (3–5MΩ) pulled from borosilicate glass (WPI, Sarosota, FL) were filled with (in mM): 100 Cs-gluconate, 5 CsCl, 0.6 EGTA, 5 MgCl₂, 8 NaCl, 2 Na₂ATP, 0.3 GTPNa, 40 HEPES, 0.1 spermine, and 1 QX-314, pH 7.2–7.3, 290 mOsm. Biocytin (0.2%) was routinely added to the recording electrode solution to allow post hoc morphological processing of recorded cells as previously described (Pelkey et al., 2005). Uncompensated series resistance, 8–20 MΩ, was rigorously monitored by delivery of −5mV voltage steps and recordings were discontinued following changes of >15%. MF-SLIN responses were evoked at 0.33 Hz by low intensity stimulation (150 µs/10–30 µA) in the dentate gyrus, or stratum lucidum, via a constant current isolation unit (A360, WPI) connected to a patch electrode filled with oxygenated extracellular solution. MF origin of EPSCs was confirmed by perfusion with DCGIV or L-AP4 (Tocris) both of which selectively depress MF release (Pelkey et al., 2005). HFS consisted of 100 Hz stimulation for 1s given 3 times at an interval of 10 s. Data acquisition (filtered at 3 kHz, digitized at 20 kHz) and analysis were performed using a PC equipped with pClamp 9.0 software (Axon Instruments). EPSC amplitudes were measured during a 1–1.5 msec window around the peak of the waveform, then for each recording EPSC amplitudes were binned as 1 minute running averages and normalized to the average amplitude observed during the baseline recording period (3 to 5 minutes) prior to HFS or L-AP4 application. Group data are presented as means ± SEM with paired or unpaired t-tests used to assess statistical significance as appropriate. Rectification index (RI) was generated from averaged (10–20 traces) EPSC amplitudes at a series of holding potentials between −60 and +40 mV. EPSC amplitudes recorded at negative holding potentials (from −60 to −20 mV) were fit by a linear regression and the RI was defined as the ratio of the actual current amplitude at +40 mV to the predicted linear value at +40 mV.