Abstract

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and results from a loss of Fragile X mental retardation protein (FMRP). FMRP is important for mRNA shuttling and translational control and binds to proteins important for synaptic plasticity. Like many developmental disorders, FXS is associated with alterations in synaptic plasticity that may impair learning and memory processes in the brain. However, it remains unclear whether FMRP plays a ubiquitous role in synaptic plasticity in all brain regions. We report that a loss of FMRP leads to impairments in N-methyl-d-aspartate receptor (NMDAR)-dependent synaptic plasticity in the dentate gyrus (DG), but not in the cornu ammonis area 1 (CA1) subregion of the hippocampus of adult mice. DG-specific deficits are accompanied by a significant reduction in NMDAR GluN1, GluN2A, and GluN2B subunit levels and reduced serine 831 GluA1 phosphorylation specifically in this region. Importantly, we demonstrate that treatment with NMDAR co-agonists (glycine or d-serine) independently rescue impairments in NMDAR-dependent synaptic plasticity in the DG of the Fragile X mental retardation 1 (Fmr1) knockout mouse. These findings implicate the NMDAR in the pathophysiology of FXS and suggest that indirect agonists of the NMDAR may be a successful therapeutic intervention in FXS.

Introduction

Fragile X Syndrome (FXS), the most common form of inherited intellectual impairment (Bagni and Greenough 2005), is caused by a cytosine-guanine-guanine (CGG) trinucleotide repeat expansion that results in transcriptional repression of the Fragile X Mental Retardation 1 (Fmr1) gene and subsequent loss of Fragile X Mental Retardation Protein (FMRP) (Fu et al. 1991; Pieretti et al. 1991; Ashley et al. 1993). FMRP is highly expressed in the hippocampus (Feng et al. 1997), a brain region known to be involved in learning and memory (Scoville and Milner 1957; Bliss and Lomo 1973) and affected by FXS (Reiss et al. 1994; Jakala et al. 1997; Kates et al. 1997; Hoeft et al. 2008). Within neurons, FMRP plays a number of important roles including the trafficking of mRNA from the nucleus to the cytoplasm and distal postsynaptic sites (Zalfa et al. 2007; Bassell and Warren 2008) and the translational control of mRNA at polyribosomes (Ashley et al. 1993; Antar and Bassell 2003).

There is increasing evidence that FMRP also plays a critical role in synaptic plasticity, the leading neurobiological model of learning and memory. Initially, it was shown that there was a small but significant increase in type I metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in the cornu ammonis area 1 (CA1) of the Fmr1 knockout (KO) mouse, although behavioral phenotypes correlating with this type of synaptic plasticity in this brain region have been hard to elucidate (Bear et al. 2004; Huber et al. 2002; Hou et al. 2006; Nosyreva and Huber 2006; Luscher and Huber 2010; Sharma et al. 2010; Choi et al. 2011; Westmark et al. 2011). Recently, there has been increasing focus on N-methyl-d-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mouse model of FXS. While NMDAR-mediated plasticity has not been reliably shown to be affected by the loss of FMRP in the CA1, with researchers showing decreased TBS-induced LTP with intact NMDAR contribution (Lauterborn et al. 2007) or no deficits (Godfraind et al. 1996; Huber et al. 2002; Pilpel et al. 2009), 2 independent groups have recently shown deficits in the DG of the adult Fmr1 KO mouse (Eadie et al. 2010; Yun and Trommer 2011). Interestingly, these deficits correlate to clear alterations of the Fmr1 KO animal in a context discrimination task, a behavior thought to be mediated by NMDARs in the DG (McHugh et al. 2007; Eadie et al. 2010).

These studies indicate there may be abnormalities in NMDAR-mediated signaling in the hippocampus of the Fmr1 KO mouse. Interestingly, FMRP binds GluN1, GluN2A, and GluN2B mRNAs suggesting that the loss of FMRP may lead to dysregulated translation of these NMDAR subunits (Schutt et al. 2009; Edbauer et al. 2010). A previous study did indeed find reductions in the levels of GluN1, GluN2A, and GluN2B proteins in the medial prefrontal cortex of Fmr1 KO mice (Krueger et al. 2011), while another found increases in GluN1 in the neocortex of Fmr1 KO mice (Schutt et al. 2009). Investigations of the hippocampus have revealed increases in the levels of GluN1 and GluN2B (Schutt et al. 2009). However, it is unclear whether similar dysregulation of these NMDAR subunit levels are apparent in the DG of Fmr1 KO mice.

The current study examined the effects of how a loss of FMRP affects synaptic plasticity, NMDAR subunit expression, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation in the CA(1) and DG subregions of the hippocampus. We show that NMDAR-dependent LTP is reduced in the DG but not the CA1 of adult Fmr1 KO mice. The loss of FMRP also selectively alters NMDAR subunit levels and AMPAR serine 831 phosphorylation in the DG. Furthermore, we show that application of the NMDAR co-agonist glycine or d-serine rescues functional plasticity in the DG of the Fmr1 KO mouse despite the underlying molecular deficits. These results indicate that there are region specific differences in synaptic plasticity and protein levels in the adult hippocampus that can be rescued by NMDAR co-agonist treatment, solidifying the role of the NMDAR in altered neuronal functioning in FXS.

Materials and Methods

Animals

All experiments were carried out in accordance with international standards on animal welfare and guidelines set by the Canadian Council on Animal Care and the Animal Care Committee at the University of Victoria. Adult C57Bl/6J Fmr1 KO and wild-type (WT) littermate male mice were generated by breeding Fmr1 heterozygous females with either a WT or Fmr1 KO male mouse from our established breeding colony as described previously (Eadie et al. 2010). Experimenters were blinded to the group identity of all genotypes during the course of experimentation.

Genotyping

To identify Fmr1 KO animals, we used the polymerase chain reaction (PCR) to detect a neomycin cassette insertion into the Fmr1 gene as previously described (Eadie et al. 2010). The cycling parameters for PCR were denaturation for 5 min at 94 °C, followed by 35 cycles of 60 s at 94 °C, 90 s at 65 °C and 150 s at 72 °C. Primers M2 = 5′-ATCTAGTCATGCTATGGATATCAGC-3′ and N2 = 5′-GTGGGCTCTATGGCTTCTGAGG-3′ were used to probe for the Fmr1 KO allele (fragments amplified were 800 base pairs). Primers S1 = 5′-GTGGTTAGCTAAAGTGAGGATGAT-3′ and S2 = 5′-CAGGTTTGTTGGGATTAACAGATC-3′ were used to probe for the WT allele (amplified fragments were 465 base pairs). The lack of FMRP was confirmed by western blotting (data not shown).

Electrophysiology

Electrophysiological Preparation

To obtain live hippocampal slices, adult mice (2–4 months) were briefly anesthetized with isoflurane, immediately decapitated, and their brains removed directly into oxygenated (95% O2/5% CO2), ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaHPO4, 25 NaHCO3, 2 CaCl2, 1.3 MgCl2, and 10 dextrose at a pH of 7.3. Transverse hippocampal slices were made at 350 μm in continuously oxygenated ACSF maintained at 2.4 °C using a cooled Vibratome 1500 (Ted Pella, Inc., Redding, CA, USA). Slices were then incubated in oxygenated ACSF at 30 °C and were allowed to recover for a minimum of 1 h before being used for electrophysiology.

Electrophysiological recordings were obtained in oxygenated ACSF (2 mL/min) at 30 °C. Electrodes were placed under visual guidance using an Olympus BX51 upright microscope. A concentric bipolar stimulating electrode (FHC, Inc., Bowdoin, ME, USA) was placed in either the medial perforant path (DG recordings) or the Schaffer collateral commissural path (CA1 recordings). Evoked field excitatory postsynaptic potentials (fEPSPs) were obtained using a glass-recording electrode (1–2 MΩ) filled with ACSF and placed in the medial molecular layer (DG recordings) or the stratum radiatum (CA1 recordings). Field EPSPs were collected using an Axon MultiClamp 700B amplifier and pCLAMP software (Version 10; Molecular Devices, Sunnyvale, CA, USA). Slopes from individual fEPSP traces were calculated from the initial slope of the fEPSP relative to the slope of the 10 ms interval immediately preceding afferent stimulation. The current magnitude was delivered through a digital stimulus isolation amplifier (Getting Instruments, Inc., San Diego, CA, USA). Input–output experiments were conducted to measure fEPSPs in response to increasing current intensities (using an increasing pulse width from 30 to 300 µs with 30 s interstimulus intervals). For synaptic plasticity experiments, the intensity was set to elicit a fEPSP ∼50% of maximum fEPSP obtained, and single-pulse stimulation (15 s interstimulus interval) was applied to generate stable baseline responses (20 min). All data are represented as percentage change from the initial average baseline fEPSP slope, which was defined as the average slope obtained for the 20 min prior to conditioning stimulus (CS) application.

Synaptic Plasticity Induction Protocols

LTP was induced by applying 4 trains of 50 pulses at 100 Hz, 30 s apart (high-frequency stimulation, HFS). LTP in the DG was conducted in the presence of 5 μM bicuculline methiodide (BIC; Sigma-Aldrich, Oakville, ON, Canada) to block the inhibitory effects of the γ-aminobutyric acid receptor type A (GABAA) on synaptic plasticity in this region of the hippocampus. BIC was bath applied for a minimum of 10 min prior to and during the CS. Experiments assessing the role of the NMDAR in LTP were conducted in the presence of (2R)-amino-5-phosphonovaleric acid (APV; Sigma-Aldrich, Oakville, ON, Canada) in addition to BIC. APV (50 μM) was applied for a minimum of 5 min before and during the respective CS. Experiments assessing whether pharmacological agents could rescue the deficits in NMDAR-dependent LTP in the DG of Fmr1 KO animals utilized bath application of glycine (10 μM) or d-serine (7.5 μM) during their respective baselines and HFS conditioning protocols.

Molecular Studies

Microdissection and Lysate Preparation

A separate cohort of adult mice (2–4 months) was used to examine glutamatergic receptors important for synaptic plasticity. Mice were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL, USA), immediately decapitated, and the brain was then rapidly cooled, removed, and placed in cold TBS (0.1M). The DG was separated from the CA as described previously (Farmer et al. 2004; Hagihara et al. 2009; Patten et al. 2013). Subregions of the hippocampus were quickly isolated, frozen in liquid N2 in a microcentrifuge tube and stored at −80 °C until sonication. Sonication was performed in lysis buffer at 4 °C (20 mM Tris pH8, 137 mM NaCl, 0.1% NP-40, 10% Glycerol, 2 mM EDTA, 1X Halt™ phosphatase and protease inhibitors [ThermoScience, Rockford, IL, USA]) at 10 mL buffer/1 g of tissue. The samples were centrifuged at 14 000 g for 15 min at 4 °C in a microcentrifuge, and supernatants were eluted and stored at −80 °C until processing.

Western Blot and Analysis

Sample protein concentrations were determined using the bicinchoninic acid (BCA) assay (BCA Protein Assay Kit, Pierce, Rockford, IL, USA). Equal amounts of protein were separated on a 10% SDS-PAGE gel and transferred at 4 °C onto Polyvinylidene Fluoride (PVDF, Perkin Elmer, Boston, MA, USA) membranes. Membranes were probed with α-GluA1 (1:2500), α-GluN1 (1:500), α-GluN2A (1:5000) (Upstate [Millipore], Temecula, CA, USA), α-p831 GluA1 (1:800), α-p845 GluA1 (1:25 000) (Chemicon [Millipore]), α-GluN2B (1:2500; PhosphoSolutions, Aurora, CO, USA), α-PSD-95 (1:1000; StressMarq, Victoria, BC, Canada), or α-β-actin (1:20 000; loading control; Sigma) and were detected with the appropriate secondary antibody (goat α-rabbit IgG [H + L] horseradish peroxidase [HRP]-conjugate [Chemicon {Millipore}] or goat α-mouse IgG [H + L] HRP-conjugate [KPL, Gaithersburg, MD, USA]). Western blots were visualized by enhanced chemiluminescence (ECL; GE Healthcare, Buckinghanshire, UK) and quantified by densitometric analysis (Quantity One Software program; BioRad, Hercules, CA, USA). Protein levels were calculated as a ratio to the levels of the loading control (β-actin) and were expressed as a percentage of WT signal. The optical density for phosphoproteins were normalized to β-actin, then divided by the normalized total amount of protein for each corresponding sample and converted into a percentage of WT.

Statistical Analysis

Statistical analyses were carried out using Statistica 7.0 software (StatSoft, Tulsa, OK, USA). The statistical analysis used for each experiment is indicated in their corresponding figure legends. If the data violated the assumptions of homogeneity of variance or normality, the corresponding nonparametric test was utilized as appropriate. Statistical significance was set at P < 0.05. Data are presented as means ± standard error of the mean (SEM).

Results

Intact Input–Output Functions in the DG and CA1 of Fmr1 KO Mice

To determine whether Fmr1 KO animals had equivalent response to increasing stimulus intensities, synaptic input–output (IO) curves were constructed as described previously (Eadie et al. 2010). As shown in Figure 1A1, the slope of the fEPSP in the medial perforant path (MPP) of the dentate gyrus increased significantly with increasing current strength (WT: n = 12; KO: n = 11; repeated-measures ANOVA: F8,168 = 213.700, P < 0.001); however, it did so equivalently for both genotypes (repeated-measures ANOVA: F1,21 = 0.000, P = 0.833). Input/output curves were also generated for stratum radiatum evoked responses. As shown in Figure 1B1, increasing stimulus intensities produced equivalent increases in response size for both genotypes (WT n = 10, KO n = 10; repeated-measures ANOVA: F1,18 = 0.200, P = 0.684).

Figure 1.

NMDAR-dependent synaptic plasticity is decreased in the DG but intact in the CA1 of young adult male Fmr1 KO mice. (A1) Synaptic responses to single-pulse stimuli at predetermined incremental intensities in the DG of Fmr1 KO animals are not significantly different from those observed in WT animals (repeated-measures ANOVA: F1,21 = 0.000, P = 0.833). (A2) The magnitude of LTP (HFS applied at time 0) is significantly decreased in the DG of Fmr1 KO animals when compared with controls 50–60 min after conditioning stimulation (KO: 6.679 ± 5.878%, n = 6; WT: 69.808 ± 16.181%, n = 8; Student's t-test: t(12) = −3.284, P = 0.007). (B1) Synaptic responses to single-pulse stimuli at predetermined incremental intensities in the CA1 of Fmr1 KO animals are not different from those observed in WT animals (WT n = 10, KO n = 10; repeated-measures ANOVA: F1,18 = 0.200, P = 0.684). (B2) The magnitude of LTP (HFS applied at time 0) is similar in the CA1 of Fmr1 KO and WT animals when compared 50–60 min after conditioning stimulation (WT: 54.933 ± 13.366%, n = 8; KO: 75.480 ± 10.201%, n = 8; U(14) = 22.000, z = −1.050, P = 0.294). *Denotes a P value <0.05.

Figure 1.

NMDAR-dependent synaptic plasticity is decreased in the DG but intact in the CA1 of young adult male Fmr1 KO mice. (A1) Synaptic responses to single-pulse stimuli at predetermined incremental intensities in the DG of Fmr1 KO animals are not significantly different from those observed in WT animals (repeated-measures ANOVA: F1,21 = 0.000, P = 0.833). (A2) The magnitude of LTP (HFS applied at time 0) is significantly decreased in the DG of Fmr1 KO animals when compared with controls 50–60 min after conditioning stimulation (KO: 6.679 ± 5.878%, n = 6; WT: 69.808 ± 16.181%, n = 8; Student's t-test: t(12) = −3.284, P = 0.007). (B1) Synaptic responses to single-pulse stimuli at predetermined incremental intensities in the CA1 of Fmr1 KO animals are not different from those observed in WT animals (WT n = 10, KO n = 10; repeated-measures ANOVA: F1,18 = 0.200, P = 0.684). (B2) The magnitude of LTP (HFS applied at time 0) is similar in the CA1 of Fmr1 KO and WT animals when compared 50–60 min after conditioning stimulation (WT: 54.933 ± 13.366%, n = 8; KO: 75.480 ± 10.201%, n = 8; U(14) = 22.000, z = −1.050, P = 0.294). *Denotes a P value <0.05.

Reduced NMDAR-Dependent Synaptic Plasticity in the DG of Fmr1 KO Mice

Given that IO functions were intact in the Fmr1 KO mouse, we next investigated NMDAR-dependent synaptic plasticity in the DG of the Fmr1 KO mouse. The induction of LTP in Fmr1 KO animals was significantly less than that produced in WT animals (KO: 6.679 ± 5.878%, n = 6; WT: 69.808 ± 16.181%, n = 8; Student's t-test: t(12) = −3.284, P = 0.007; Fig. 1A2). This conditioning stimulus (CS) induced an NMDAR-dependent form of LTP, as this same CS failed to induce LTP in the presence of the NMDAR antagonist APV (50 μM) in separate slices (WT: 3.512 ± 6.704%, n = 4; KO: 3.738 ± 2.441%, n = 5; Mann–whitney U-test: U(7) = 10.000, z = 0.000, P = 1.000; data not shown).

NMDAR Synaptic Plasticity is Intact in the CA1 of Fmr1 KO Mice

To determine whether there were similar deficits in NMDAR-dependent plasticity in the CA1 region of the hippocampus, a high-frequency stimulation (HFS) was utilized. Unlike the DG, application of HFS in the CA1 subfield produced equivalent LTP in both WT and Fmr1 KO slices (WT: 54.933 ± 13.366%, n = 8; KO: 75.480 ± 10.201%, n = 8; U(14) = 22.000, z = −1.050, P = 0.294; Fig. 1B2).

NMDAR Subunit Levels are Reduced in the DG of Young Adult Fmr1 KO Mice

We next examined whether the reductions in NMDAR-dependent LTP in the DG were due to a reduction in GluN1 levels specific to this hippocampal subregion of Fmr1 KO mice. Compared with WT mice, Fmr1 KO mice had significantly lower levels of GluN1 specifically in the DG, indicating for the first time that the expression of NMDARs is decreased in the DG (WT: n = 6, KO: n = 6, t(10) = 2.49, P = 0.034), but not in the CA of young adult Fmr1 KO mice (WT: n = 7, KO: n = 6, t(11) = 0.498, P = 0.628; Fig. 2A,B).

Figure 2.

GluN1, GluN2A, and GluN2B subunit levels are decreased in the DG but not the CA region of Fmr1 KO mice. Western blots were performed with 25 μg of total protein. (A,C, and E) Significant decrease in GluN1, GluN2A and GluN2B subunit levels in the DG but not in the CA of Fmr1 KO mice. GluN1: WT-DG: 100 ± 14.9%, KO-DG: 61.5 ± 5.03%, P = 0.034; WT-CA: 100 ± 17.0%, KO-CA: 89.8 ± 9.73%, P = 0.628. GluN2A: WT-DG: 100 ± 9.18%, KO-DG: 74.4 ± 2.81%, P = 0.031; WT-CA: 100 ± 7.59%, KO-CA: 99.3 ± 12.7%, P = 0.964. GluN2B: WT-DG: 100 ± 14.8%, KO-DG: 56.0 ± 7.49%, P = 0.021; WT-CA: 100 ± 8.08%, KO-CA: 103 ± 17.1%, P = 0.894. (B, D, and F) Representative western blots for GluN1, GluN2A, GluN2B, and their respective β-actin in the DG and CA of WT and Fmr1 KO mice. Data are expressed as percent mean ± SEM. Number of samples are denoted in their respective bars. *Denotes a P value <0.05.

Figure 2.

GluN1, GluN2A, and GluN2B subunit levels are decreased in the DG but not the CA region of Fmr1 KO mice. Western blots were performed with 25 μg of total protein. (A,C, and E) Significant decrease in GluN1, GluN2A and GluN2B subunit levels in the DG but not in the CA of Fmr1 KO mice. GluN1: WT-DG: 100 ± 14.9%, KO-DG: 61.5 ± 5.03%, P = 0.034; WT-CA: 100 ± 17.0%, KO-CA: 89.8 ± 9.73%, P = 0.628. GluN2A: WT-DG: 100 ± 9.18%, KO-DG: 74.4 ± 2.81%, P = 0.031; WT-CA: 100 ± 7.59%, KO-CA: 99.3 ± 12.7%, P = 0.964. GluN2B: WT-DG: 100 ± 14.8%, KO-DG: 56.0 ± 7.49%, P = 0.021; WT-CA: 100 ± 8.08%, KO-CA: 103 ± 17.1%, P = 0.894. (B, D, and F) Representative western blots for GluN1, GluN2A, GluN2B, and their respective β-actin in the DG and CA of WT and Fmr1 KO mice. Data are expressed as percent mean ± SEM. Number of samples are denoted in their respective bars. *Denotes a P value <0.05.

Further investigation of the predominant hippocampal GluN2 subunits, GluN2A and GluN2B (Wenzel et al. 1997; Al-Hallaq et al. 2007) indicated that GluN2A levels were decreased in the DG of Fmr1 KO mice when compared with WT mice (WT: n = 7, KO: n = 6, t(11) = 2.48, P = 0.031), but not in the CA region (WT: n = 6, KO: n = 7, t(11) = 0.046, P = 0.964; Fig. 2C,D). Similarly, GluN2B levels were reduced in the DG (WT: n = 7, KO: n = 7, t(12) = 2.65, P = 0.021), but not the CA of Fmr1 KO mice (WT: n = 6, KO: n = 7, t(11) = −0.136, P = 0.894; Fig. 2E,F).

Phosphorylation of AMPAR GluA1 at Serine 831 is Reduced in the DG of Fmr1 KO Mice

Ca2+ entry via NMDARs can lead to phosphorylation of serine 831 (S831) and S845 of the GluA1 subunit of the AMPAR. As phosphorylation at these serine residues are critical for synaptic insertion of the GluA1 receptor (Thomas and Huganir 2004; Qin et al. 2005; Zhu et al. 2011), we examined whether a reduction in NMDARs reduces either the level, or the basal phosphorylation, of GluA1-containing AMPARs. In comparison to WT mice, Fmr1 KO mice had significantly lower amounts of total GluA1 (i.e., phosphorylated and unphosphorylated forms) in the CA (WT: n = 7, KO: n = 7, t(12) = 2.23; P = 0.045), whereas no significant changes were detected in the DG (WT: n = 6, KO: n = 7, t(11) = 0.704; P = 0.496; Fig. 3A,B).

Figure 3.

GluA1 subunit phosphorylation of the AMPAR is reduced in the DG, while total GluA1 is decreased in the CA of Fmr1 KO mice. Western blots were performed with 25 μg of total protein. (A, C, and E) Significant decrease in p831, p831/GluA1, and p845 in the DG, but GluA1 is significantly decreased in the CA of Fmr1 KO mice. GluA1: WT-DG: 100 ± 7.37%, KO-DG: 93.0 ± 6.66%, P = 0.496; WT-CA: 100 ± 2.41%, KO-CA: 93.5 ± 1.65%, P = 0.045. p831: WT-DG: 100 ± 4.20%, KO-DG: 63.7 ± 7.30%, P = 0.001; WT-CA: 100 ± 5.41%, KO-CA: 106 ± 6.96%, P = 0.502. p831/GluA1: WT-DG: 100 ± 3.42%, KO-DG: 65.8 ± 7.21%, P = 0.002; WT-CA: 100 ± 6.01%, KO-CA: 110 ± 6.72%, P = 0.298. p845: WT-DG: 100 ± 7.71%, KO-DG: 79.4 ± 1.27%, P = 0.034; WT-CA: 100 ± 4.62%, KO-CA: 101 ± 6.96%, P = 0.870. p845/GluA1: WT-DG: 100 ± 6.85%, KO-DG: 80.6 ± 8.88%, P = 0.110; Mann–Whitney U-test: WT-CA: 100 ± 2.27%, KO-CA: 114 ± 7.50%, P = 0.181. (B, D, and F) Representative western blots for total GluA1, GluA1 phosphorylation at S831 and GluA1 phosphorylation at S845 and their respective β-actin in the DG and CA of WT and Fmr1 KO mice. Data are expressed as percent mean ± SEM. Number of samples are denoted in their respective bars. *denotes a P value <0.05, and **denotes a P value <0.01.

Figure 3.

GluA1 subunit phosphorylation of the AMPAR is reduced in the DG, while total GluA1 is decreased in the CA of Fmr1 KO mice. Western blots were performed with 25 μg of total protein. (A, C, and E) Significant decrease in p831, p831/GluA1, and p845 in the DG, but GluA1 is significantly decreased in the CA of Fmr1 KO mice. GluA1: WT-DG: 100 ± 7.37%, KO-DG: 93.0 ± 6.66%, P = 0.496; WT-CA: 100 ± 2.41%, KO-CA: 93.5 ± 1.65%, P = 0.045. p831: WT-DG: 100 ± 4.20%, KO-DG: 63.7 ± 7.30%, P = 0.001; WT-CA: 100 ± 5.41%, KO-CA: 106 ± 6.96%, P = 0.502. p831/GluA1: WT-DG: 100 ± 3.42%, KO-DG: 65.8 ± 7.21%, P = 0.002; WT-CA: 100 ± 6.01%, KO-CA: 110 ± 6.72%, P = 0.298. p845: WT-DG: 100 ± 7.71%, KO-DG: 79.4 ± 1.27%, P = 0.034; WT-CA: 100 ± 4.62%, KO-CA: 101 ± 6.96%, P = 0.870. p845/GluA1: WT-DG: 100 ± 6.85%, KO-DG: 80.6 ± 8.88%, P = 0.110; Mann–Whitney U-test: WT-CA: 100 ± 2.27%, KO-CA: 114 ± 7.50%, P = 0.181. (B, D, and F) Representative western blots for total GluA1, GluA1 phosphorylation at S831 and GluA1 phosphorylation at S845 and their respective β-actin in the DG and CA of WT and Fmr1 KO mice. Data are expressed as percent mean ± SEM. Number of samples are denoted in their respective bars. *denotes a P value <0.05, and **denotes a P value <0.01.

Phosphorylation of existing AMPARs at S831 of the GluA1 subunit occurs through the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or protein kinase C (PKC) during NMDAR-mediated LTP. Phosphorylation at this GluA1 site increases the ionic conductance of the channel and the number of AMPARs at the postsynaptic membrane (Derkach et al. 1999; Snyder et al. 2000). Conversely, NMDAR-mediated LTD occurs when protein phosphatases (PP) dephosphorylate S845 on the GluA1 subunit of the AMPAR. This causes internalization of AMPARs, thereby decreasing the amount of receptors present at the postsynaptic membrane (Lee et al. 1998, 2000, 2003; Ehlers 2000; Man et al. 2007). We first investigated S831 GluA1 phosphorylation to determine whether it is impacted by a decrease in NMDAR protein levels. S831 phosphorylation was significantly decreased in the DG (WT: n = 7, KO: n = 7, t(12) = 4.31, P = 0.001), but not in the CA (WT: n = 7, KO: n = 6, t(11) = −0.694, P = 0.502; Fig. 3C,D) of Fmr1 KO mice. Similarly, the ratio of p831/GluA1 was significantly decreased in the DG (WT: n = 6, KO: n = 7, t(11) = 4.05, P = 0.002), but not in the CA (WT: n = 7, KO: n = 6, t(11) = −1.09, P = 0.298; Fig. 3C,D) of Fmr1 KO mice. We next investigated S845 GluA1 phosphorylation to determine whether it is also affected by a decrease in NMDAR protein levels. Phosphorylated S845 was also significantly decreased in the DG (WT: n = 7, KO: n = 6, t(11) = 2.43, P = 0.034), but not in the CA (WT: n = 7, KO: n = 7, t(12) = −0.167, P = 0.870; Fig. 3E,F) of Fmr1 KO mice. However, the ratio of p845/GluA1 showed no significant differences between WT and Fmr1 KO mice in the DG (WT: n = 7, KO: n = 7, t(12) = 1.72, P = 0.110) or CA (WT: n = 6, KO: n = 7, Mann–Whitney U-test: U(11) = 11.0, Z = −1.43, P = 0.181; Fig. 3E,F) regions of the hippocampus.

Glycine and d-serine Normalize Synaptic Plasticity in Fmr1 KO Mice

A decrease in NMDAR subunit protein levels may explain a decrease in NMDAR-dependent synaptic plasticity and AMPAR phosphorylation. If an overall decrease in NMDAR protein levels at the postsynaptic membrane is the main factor contributing to NMDAR-dependent synaptic plasticity deficits, an obvious way to alleviate these impairments would be to augment the functionality of the reduced number of NMDARs present. We chose to utilize glycine, an endogenous co-agonist of the NMDAR, to augment NMDAR function during HFS-induced LTP. A 2-way ANOVA with Tukey's postcomparative test on the effects of glycine revealed a significant effect of treatment (F1,1 = 7.4, P = 0.011) with glycine having a significant difference from no treatment (Tukey's post hoc P = 0.021; Fig. 4A1). There was also a significant interaction effect between genotype and treatment (F1,30 = 10.24, P = 0.003), whereby Fmr1 KO slices had a significantly lower level of LTP than WT slices (Tukey's post hoc P = 0.017) and a lower level of LTP than Fmr1 KO slices with glycine (Tukey's post hoc P = 0.002) or WT slices with glycine (Tukey's post hoc P = 0.037; Fig. 4A1). Importantly, there was no significant difference between WT slices with and without glycine (Tukey's post hoc P = 0.985) and WT slices without glycine compared with Fmr1 KO slices with glycine (Tukey's post hoc P = 0.819), indicating a complete normalization of Fmr1 KO LTP to WT levels with application of glycine.

Figure 4.

NMDAR-dependent synaptic plasticity is normalized by application of glycine or D-serine in the DG of young adult male Fmr1 KO mice. (A1) The magnitude of LTP (HFS applied at time 0) in Fmr1 KO slices is normalized to WT levels by bath application of glycine in the DG when the fEPSP is examined 50–60 min after conditioning stimulation (2-way ANOVA: post hoc P > 0.05). Application of glycine does not alter LTP in WT slices (2-way ANOVA: post hoc P > 0.05). (A2) The magnitude of LTP (HFS applied at time 0) in Fmr1 KO slices is also normalized to WT levels by application of D-serine in the DG when the fEPSP is examined 50–60 min after conditioning stimulation (Kruskal–Wallis ANOVA: P > 0.05). Application of D-serine does not alter LTP in WT slices (Kruskal–Wallis ANOVA: P > 0.05). *denotes a P value <0.05. LTP in the dentate gyrus as shown in Figure 1A2 has been re-plotted in this figure for comparative purposes with the application of glycine and D-serine to WT and Fmr1 KO slices.

Figure 4.

NMDAR-dependent synaptic plasticity is normalized by application of glycine or D-serine in the DG of young adult male Fmr1 KO mice. (A1) The magnitude of LTP (HFS applied at time 0) in Fmr1 KO slices is normalized to WT levels by bath application of glycine in the DG when the fEPSP is examined 50–60 min after conditioning stimulation (2-way ANOVA: post hoc P > 0.05). Application of glycine does not alter LTP in WT slices (2-way ANOVA: post hoc P > 0.05). (A2) The magnitude of LTP (HFS applied at time 0) in Fmr1 KO slices is also normalized to WT levels by application of D-serine in the DG when the fEPSP is examined 50–60 min after conditioning stimulation (Kruskal–Wallis ANOVA: P > 0.05). Application of D-serine does not alter LTP in WT slices (Kruskal–Wallis ANOVA: P > 0.05). *denotes a P value <0.05. LTP in the dentate gyrus as shown in Figure 1A2 has been re-plotted in this figure for comparative purposes with the application of glycine and D-serine to WT and Fmr1 KO slices.

As glycine normalized LTP from Fmr1 KO animals, we next chose to examine whether an NMDAR co-agonist with a higher binding affinity to synaptic NMDARs (Papouin et al. 2012) may also be able to rescue LTP in Fmr1 KO slices. A Kruskal-Wallis ANOVA was utilized to examine the effects of d-serine on LTP induced by HFS. There was found to be a significant effect of the application of d-serine on LTP (Kruskal–Wallis ANOVA; H = 14.13, n = 33, P = 0.003) with Fmr1 KO slices revealing a significantly lower level of LTP than WT slices (P = 0.006) and a lower level of LTP than Fmr1 KO slices with d-serine (P = 0.032) or WT slices with d-serine (P = 0.004; Fig. 4A2). Importantly, similar to the results with glycine, there was no significant difference between WT slices with and without d-serine (P = 1.000) and WT slices without d-serine when compared with Fmr1 KO slices with d-serine (P = 1.000), indicating a complete normalization of Fmr1 KO LTP to WT levels with application of d-serine.

Discussion

FMRP is involved in transporting and stabilizing key molecules involved in the maintenance of synaptic structure and function, including the NMDAR subunits GluN1, GluN2B (Schutt et al. 2009), and GluN2A (Edbauer et al. 2010). Here, we show for the first time that reduced NMDAR-dependent synaptic plasticity in the DG is accompanied by a decrease in the levels of GluN1, GluN2A, and GluN2B subunits of the NMDAR and in the levels of GluA1 phosphorylation on S831 of the AMPAR specifically in this hippocampal region of Fmr1 KO mice. Importantly, we show that application of the NMDAR co-agonists glycine and d-serine restore the DG-specific deficits in NMDAR-dependent LTP in Fmr1 KO slices to WT levels.

NMDAR Subunits are Reduced in the DG of Fmr1 KO Mice

We and others have shown that NMDAR-dependent LTP and LTD are reduced in the DG, with a corresponding reduction in the NMDAR/AMPAR current ratio in DG granule cells (Eadie et al. 2010; Yun and Trommer 2011). The current study indicates that significant reductions in NMDAR protein levels (GluN1) (Fig. 2A) underlie impairments in synaptic plasticity in the DG (Fig. 1A2). In addition, both GluN2A and GluN2B subunits were reduced specifically in the DG (Fig. 2C,E). Although the specific roles of GluN2A and GluN2B in LTP are still debated, there is considerable evidence that both subunits play important roles in both LTP and LTD in the DG (Vasuta et al. 2007). In contrast to our results, a previous study found increases in GluN1 and GluN2B in PSD fractions from adult Fmr1 KO whole hippocampal homogenates (Schutt et al. 2009). The discrepancy between this study and ours may be due to subregion-specific differences in the levels of NMDAR proteins in the hippocampus, as our study examined protein levels in the 2 main subregions of the hippocampus. In addition, Schutt et al. (2009) utilized PSD fractions to investigate synaptically located NMDARs. As extrasynaptic NMDARs can also contribute to synaptic signaling (Harris and Stevens 1989), the investigation of total NMDAR levels may better assess total NMDAR dysfunction in Fmr1 KO animals.

GluA1 Phosphorylation is Reduced in the DG of Fmr1 KO Mice

As AMPAR phosphorylation can be directly affected by NMDAR signaling, the basal phosphorylation state of the GluA1 subunit of the AMPAR was studied using western blotting. We found that Fmr1 KO animals exhibited a decrease in the level of S831 phosphorylation that was specific to the DG (Fig. 3C). Since S831 on the GluA1 subunits of the AMPAR will become phosphorylated during NMDAR-dependent LTP, leading to increased conductance and insertion into the postsynaptic membrane (Sweatt et al. 1998; Derkach et al. 1999; Ehlers 2000), a decrease in S831 phosphorylation may negatively impact synaptic plasticity in the DG of Fmr1 KO mice.

In contrast to S831 phosphorylation, phosphorylation of S845/GluA1-containing AMPARs was not altered in either hippocampal region in Fmr1 KO mice (Fig. 3E). Phosphorylation at S845 is necessary for basal AMPAR-mediated synaptic responses (Greengard et al. 1991; Wang et al. 1991; Rosenmund et al. 1994), supporting our findings of normal basal synaptic responses in both the DG and the CA1 of Fmr1 KO mice (Fig. 1A1,B1). Our results for both S831 and S845 are in accordance with Hu et al. (2008), who found similar levels of phosphorylated S831and phosphorylated S845 in the CA1 of Fmr1 KO animals as our study at a similar time point in their circadian cycle.

Our study also revealed reduced amounts of total GluA1 in the CA, but normal levels in the DG. The observed decrease in CA levels of GluA1 supports previous studies that have shown GluA1 mRNA to co-immunoprecipitate with FMRP, implicating FMRP as a translational regulator of GluA1 (Muddashetty et al. 2007; Schutt et al. 2009; Soden and Chen 2010). While AMPAR subunit expression in the Fmr1 KO mouse has been investigated in several previous publications (Li et al. 2002; Giuffrida et al. 2005; Nosyreva and Huber 2006; Muddashetty et al. 2007; Hu et al. 2008), studies focusing on the hippocampus have not found differences in whole hippocampal homogenates of adult mice (Nosyreva and Huber 2006) or CA1 homogenates of juveniles (Hu et al. 2008).

Despite these previous studies, ours is the first study to differentiate between the DG and CA regions in the adult Fmr1 KO mouse. Our observed decrease in GluA1 subunits in the CA region may be indicative of less GluA1-containing AMPARs in the intracellular pool but not of altered synaptic functioning, as direct measures of the amount of depolarization in response to increasing current intensities (IO curves) were not altered in the CA1 region of the Fmr1 KO animal (Fig. 1B1). Due to the limitations of whole-region homogenates, we were not able to discriminate between intracellular pools of AMPARs and those already at the postsynaptic membrane. Further studies may warrant the use of synaptoneurosomes to more clearly differentiate between these 2 possibilities.

Glycine and d-serine Normalize Synaptic Plasticity in Fmr1 KO Mice

Given that reduced NMDAR subunit levels in the DG may underlie impairments in NMDAR-dependent LTP and S831 phosphorylation of GluA1-containing AMPARs in Fmr1 KO mice, we investigated whether increasing the functionality of existing NMDARs at the postsynaptic membrane could reverse the NMDAR-dependent LTP deficits seen in Fmr1 KO mice. The NMDAR co-agonists glycine and d-serine increase the open channel frequency of the NMDAR (Johnson and Ascher 1987; Iwayama et al. 2006), thus indirectly increasing the action of endogenous glutamate and allowing for an increase in the functionality of any NMDARs located at the postsynaptic membrane. Excitingly, the application of either glycine or d-serine was able to increase LTP in Fmr1 KO animals to WT levels without changing control levels of WT LTP (Fig. 4A1,A2). Thus, glycine or d-serine both specifically alleviated synaptic plasticity impairments in Fmr1 KO slices.

Although direct agonism of the NMDAR can cause excitotoxicity (Papouin et al. 2012), administration of glycine and d-serine can influence the NMDAR without leading to toxic effects (Coyle et al. 2003). In addition, application of co-agonists of the NMDAR such as glycine and d-serine have been shown to be promising in schizophrenia (Tsai et al. 1998; Heresco-Levy et al. 1999; Javitt et al. 1999), another model of brain dysfunction that has underlying NMDAR hypofunction (Javitt and Zukin 1991; Krystal et al. 1994; Goff and Coyle 2001; Coyle et al. 2003). Interestingly, d-serine has been found to gate GluN2A-containing NMDARs at the synapse whereas glycine gates GluN2B-containing extrasynaptic receptors in the hippocampus (Papouin et al. 2012). Our results suggest that augmentation of either synaptic or extrasynaptic NMDARs has the capacity to normalize the levels of LTP in the Fmr1 KO-DG, pointing to a global deficit of NMDARs in the postsynaptic membrane as a causative agent of impaired neuronal function.

The present study reveals a region specific loss of NMDAR subunits and a reduced level of S831 phosphorylation on GluA1 receptors in the DG of Fmr1 KO mice. Both deficits provide a basis for the loss in bidirectional NMDAR-dependent synaptic plasticity that occurs in Fmr1 KO animals (Eadie et al. 2010; Yun and Trommer 2011). These deficits were subregion specific, affecting the DG but not the CA region of the hippocampus, indicating that the loss of FMRP differentially affects separate regions of the hippocampus. This may indicate that FMRP plays a special role in regulating NMDAR signaling in developing neurons (Philpot et al. 2001; Hu et al. 2008), and that these deficits persist in regions of the brain which continue to show neurogenic behavior in adulthood, such as the DG (Altman and Das 1965). DG-specific alterations in NMDAR levels and phosphorylation of GluA1 subunits may together play a critical role in synaptic plasticity and learning and memory impairments observed in Fmr1 KO mice. Importantly, deficits in synaptic functioning in the DG of Fmr1 KO animals can be rescued by augmenting the functionality of existing NMDARs through the application of glycine or d-serine. This raises an exciting prospect of pharmacological manipulation of NMDARs in the quest to normalize cognitive functioning in FXS.

Funding

This work was supported by grants from the Canadian Institutes for Health Research (CIHR MOP 125888) and Fragile-X Research, University of Victoria Fellowship to C.A.B. and N.-M.M.

Notes

We thank M. Vetrici, E. Wiebe, J. Graham, J. Gil-Mohapel, P. Brocardo, A. Ashley, and J. Chiu for assistance in this project. Conflict of Interest: None declared.

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Author notes

C. A. Bostrom and N.-M. Majaess contributed equally to this work