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. 2011 Apr;18(4):207–220. doi: 10.1101/lm.2043811

Fragile X mental retardation protein regulates heterosynaptic plasticity in the hippocampus

Steven A Connor 1, Charles A Hoeffer 2, Eric Klann 3, Peter V Nguyen 1,4,5,6
PMCID: PMC3072772  PMID: 21430043

Abstract

Silencing of a single gene, FMR1, is linked to a highly prevalent form of mental retardation, characterized by social and cognitive impairments, known as fragile X syndrome (FXS). The FMR1 gene encodes fragile X mental retardation protein (FMRP), which negatively regulates translation. Knockout of Fmr1 in mice results in enhanced long-term depression (LTD) induced by metabotropic glutamate receptor (mGluR) activation. Despite the evidence implicating FMRP in LTD, the role of FMRP in long-term potentiation (LTP) is less clear. Synaptic strength can be augmented heterosynaptically through the generation and sequestration of plasticity-related proteins, in a cell-wide manner. If heterosynaptic plasticity is altered in Fmr1 knockout (KO) mice, this may explain the cognitive deficits associated with FXS. We induced homosynaptic plasticity using the β-adrenergic receptor (β-AR) agonist, isoproterenol (ISO), which facilitated heterosynaptic LTP that was enhanced in Fmr1 KO mice relative to wild-type (WT) controls. To determine if enhanced heterosynaptic LTP in Fmr1 KO mouse hippocampus requires protein synthesis, we applied a translation inhibitor, emetine (EME). EME blocked homo- and heterosynaptic LTP in both genotypes. We also probed the roles of mTOR and ERK in boosting heterosynaptic LTP in Fmr1 KO mice. Although heterosynaptic LTP was blocked in both WT and KOs by inhibitors of mTOR and ERK, homosynaptic LTP was still enhanced following mTOR inhibition in slices from Fmr1 KO mice. Because mTOR will normally stimulate translation initiation, our results suggest that β-AR stimulation paired with derepression of translation results in enhanced heterosynaptic plasticity.

Fragile X syndrome (FXS), a leading cause of mental retardation (Hagerman et al. 2009), is linked to silencing of the FMR1 gene (Verkerk et al. 1991; Eichler et al. 1994; Feng et al. 1995). FXS is characterized by mild to severe cognitive deficits, including impaired learning and memory (Macleod et al. 2010) resulting from altered synaptic function (Huber et al. 2002; Bear et al. 2004; Hou et al. 2006; Zhang et al. 2009). The Fmr1 knockout (KO) mouse recapitulates the primary molecular pathology associated with FXS: a reduction in expression of the FMR1 protein product, fragile X mental retardation protein (FMRP) (Bakker et al. 1994). FMRP is an RNA binding protein that is implicated in processes critical for synaptic plasticity, including regulation of mRNA trafficking and translation (Corbin et al. 1997; Feng et al. 1997; Huber et al. 2002; Bear et al. 2004; Stefani et al. 2004; Weiler et al. 2004). Studies of the hippocampus, a brain structure involved in memory formation (Scoville and Milner 1957; Zola-Morgan et al. 1986), revealed enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in the absence of FMRP (Hou et al. 2006). mGluR-LTD is rendered insensitive to translation inhibitors in Fmr1 KO mouse hippocampus, suggestive of altered regulation of protein synthesis (Hou et al. 2006; Nosyreva and Huber 2006).

Despite the evidence implicating FMRP in mGluR-LTD, it is less clear whether FMRP significantly impacts the expression of long-term potentiation (LTP), an activity-induced increase in synaptic strength believed to underlie specific types of learning and memory (Bliss and Collingridge 1993; Godfraind et al. 1996; Paradee et al. 1999; Larson et al. 2005; Lauterborn et al. 2007).

β-Adrenergic receptors (β-ARs) are G protein-coupled receptors that enhance hippocampal homosynaptic LTP and long-term memory formation through translation regulation (Gelinas and Nguyen 2005; Gelinas et al. 2007). As β-ARs and mGluRs both couple to signaling cascades that regulate translation (Banko et al. 2006; Gelinas et al. 2007), we asked whether β-AR-dependent heterosynaptic LTP was altered in Fmr1 KO mouse hippocampus. Frey and Morris (1997) have shown that the induction of protein synthesis-dependent LTP at one synaptic pathway (S1), can facilitate the generation of heterosynaptic LTP at a second set of synapses (S2). However, to date, heterosynaptic processes have not been assessed in any mouse model of FXS. To test the hypothesis that β-AR-dependent heterosynaptic LTP is altered in Fmr1 KO mice, we induced heterosynaptic plasticity using a novel protocol consisting of β-AR activation paired with brief stimulation in one synaptic pathway followed later with milder stimulation at a neighboring pathway.

We report that β-AR-dependent heterosynaptic LTP is altered in the hippocampus of Fmr1 KO mice. Although homosynaptic LTP was similar to wild-type (WT) controls, heterosynaptic LTP was enhanced in Fmr1 KO mouse hippocampus. This promotion of heterosynaptic plasticity may contribute to cognitive abnormalities in FXS, such as impaired learning during periods of heightened attention or arousal that recruit the noradrenergic neuromodulatory system and engage β-ARs.

Results

Synaptic tagging induced by HFS alone is normal in Fmr1 knockout mouse hippocampus

Induction of translation-dependent LTP at one synaptic pathway can facilitate the induction of heterosynaptic LTP following the activity-dependent setting of “tags,” which capture plasticity-related proteins (PRPs) necessary for expression of LTP (Frey and Morris 1997; for review, see Barco et al. 2008; Frey and Frey 2008). Synaptic tagging and capture provides a mechanistic explanation for how translational and transcriptional products are appropriately targeted to previously active dendritic compartments, thereby maintaining synaptic specificity believed to be critical for cellular processes underlying memory formation (Morris 2006; Barco et al. 2008; Frey and Frey 2008). Regulated protein synthesis produces PRPs necessary for heterosynaptic transfer of LTP (Frey and Morris 1997). Knockout of Fmr1 reduces the expression of the translational repressor FMRP, which binds to several mRNAs that encode PRPs involved in synaptic plasticity (Hou et al. 2006; Muddashetty et al. 2007; Park et al. 2008; Schütt et al. 2009). Thus, we asked whether reduced expression of FMRP would alter synaptic tagging and capture of LTP, by using a stimulus protocol similar to one that is commonly implemented to probe tagging (Frey and Morris 1997).

Two stimulating electrodes were placed in CA1 straddling a recording electrode positioned in stratum radiatum. Independence of synaptic pathways was confirmed using paired-pulse facilitation both before and at the conclusion of the experiments. To determine if synaptic tagging was altered in FMR1 knockout mouse hippocampus we first established that synaptic tagging and capture could be induced in WT controls. A high-frequency stimulation protocol consisting of four trains of 100-Hz stimulation at 3-sec intertrain intervals was applied to one synaptic pathway (S1; homosynaptic), in WT hippocampal slices, which induced long-lasting (>2 h) LTP (Fig. 1A). The magnitude of LTP in S1 as assessed 120 min after high-frequency stimulation was 154 ± 10% of the baseline (n = 9). When the same stimulation protocol was applied to Fmr1 KO mouse hippocampal slices, long-lasting LTP was induced (fEPSPs were potentiated to 158 ± 8% of the baseline, 120 min after HFS), which was not significantly different from WT controls (P > 0.05) (Fig. 1C). Following a 30-min delay, one train of 100-Hz stimulation (1-sec duration) was applied to a second synaptic pathway (S2), which converged on the same group of post-synaptic cells. One train of 100-Hz stimulation normally induces decremental LTP in mouse hippocampal slices, which returns to baseline in <2 h (Duffy et al. 2001; Gelinas and Nguyen 2005). When preceded by multiple HFS trains of LTP homosynaptically, a single train of HFS applied heterosynaptically generated long-lasting LTP in both WT (mean fEPSP slope was 142 ± 8%) and Fmr1 KO (mean fEPSP slope was 144 ± 10%) (Fig. 1C) slices as assessed 2 h post-stimulation. Although, no significant differences were detected between KO and WT slices at the second synaptic pathway (P > 0.05) (Fig. 1C), these results suggest that in both WT and Fmr1 KO mouse hippocampi, 4 × 100-Hz stimulation generates long-lasting LTP, which can be captured at a second synaptic pathway in an activity-dependent manner.

Figure 1.

Figure 1.

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HFS-induced synaptic tagging and capture are not altered in Fmr1 knockout mice. (A) Application of 4 × 100-Hz stimulation to S1 (•) facilitates the induction of LTP by brief, HFS (1 × 100 Hz; □) at S2 (n = 9). (B) In Fmr1 KO slices, HFS-induced synaptic tagging and capture are similar to WT. Stimulation with repeated HFS at one synaptic pathway (4 × 100 Hz; •) induces long-lasting LTP, which can be captured heterosynaptically following a single train of HFS (1 × 100 Hz; □; n = 8) at a second set of synapses converging on the same post-synaptic cells. (C) Summary histogram comparing fEPSP slopes obtained in WT and KO slices 120 min after HFS at S1 and LFS at S2. No statistically significant differences were detected between WT and KO fEPSPs when homo- and heterosynaptic LTP were compared. Sample traces were taken 10 min after the commencement of the baseline recordings and 120 min after stimulation at S1 and S2. Results in C represent mean ± SEM.

To confirm that heterosynaptic facilitation of LTP had occurred, two control experiments were conducted in Fmr1 KO mouse hippocampal slices in which each pathway was stimulated alone. Either 4 × 100 or 1 × 100 Hz alone was applied to S1, while monitoring heterosynaptic baseline responses at S2. Consistent with previous results (Frey and Morris 1997), application of multiple high-frequency trains (4 × 100 Hz) induced LTP (fEPSPs were 156 ± 14% 120 min after HFS at S1, n = 6) (data not shown) that did not transfer to a second (S2) synaptic pathway receiving baseline stimulation (1 pulse per minute; fEPSPs were 101 ± 4% at 120 min). Next, we applied brief, HFS (1 × 100 Hz, 1 sec) alone, which failed to induce long-lasting synaptic changes at either synaptic pathway (fEPSPs were 107 ± 9% of baseline at S1 and 98 ± 8% of baseline at S2 120 min post-stimulation; n = 4). Our results show that HFS-dependent synaptic tagging and capture of LTP in Fmr1 KO mouse hippocampus is intact and similar to slices from wild-type mouse.

Activation of β-ARs facilitates heterosynaptic transfer of LTP

β-AR activation in area CA1 of mouse hippocampal slices facilitates induction of homosynaptic LTP by stimulation protocols normally subthreshold for inducing persistent, translation-dependent LTP (Thomas et al. 1996; Gelinas and Nguyen 2005). As β-AR-dependent LTP requires de novo protein synthesis for its expression (Straube et al. 2003; Gelinas and Nguyen 2005; Gelinas et al. 2007), we wanted to determine if inducing β-AR-dependent LTP at one synaptic pathway would enhance LTP at a second, independent set of synapses in WT slices. To do this, we repeated the two-pathway regimen previously described. Briefly, two stimulating electrodes were placed in stratum radiatum on either side of a recording electrode and staggered relative to each other to stimulate two independent groups of synapses. Using a β-AR-dependent LTP protocol previously established (Gelinas and Nguyen 2005) consisting of 1 × 100 Hz for 1 sec paired with ISO, LTP was induced at S1 (Fig. 2A), which lasted >2 h (mean fEPSP slope was 144 ± 7% 120 min after HFS) (Fig. 2E). To test the idea that heterosynaptic LTP expression is facilitated by prior induction of β-AR-dependent LTP, we applied brief LFS (5 Hz, 10 sec) to a second set of synapses. We found that subthreshold LFS in S2 induced long-lasting (>2 h) LTP if it was preceded by the induction of β-AR-dependent LTP in S1 (mean fEPSP slopes were 119 ± 6% of baseline at S2, 2 h post-stimulation; n = 8) (Fig. 2A,E). To control the effects of ISO application, we ran a control experiment consisting of ISO application (1 µM) for 15 min followed 25 min later with LFS (5 Hz, 10 sec). WT slices treated with ISO did not exhibit long-lasting potentiation in response to either ISO alone (fEPSPs were 98 ± 9% of baseline at S1 90 min after ISO application, n = 6) (data not shown) or LFS applied 25 min later to S2 (fEPSPs were 102 ± 7% of baseline 90 min after stimulation). Thus, ISO alone is insufficient for facilitating the subsequent induction of LTP by LFS in WT hippocampal slices.

Figure 2.

Figure 2.

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β-AR-dependent heterosynaptic plasticity is enhanced in Fmr1 KO mice. (A) In WT slices, pairing one train of 100-Hz simulation with ISO application induces LTP (•), which facilitates LTP at a second synaptic pathway (□) following LFS (5 Hz, 10 sec; n = 8). LTP at S1 was significantly enhanced (denoted by *, E) relative to S1 controls treated with ISO alone (C) or 100 Hz alone (D). Heterosynaptic LTP (□) was similarly enhanced (*, as shown in F) relative to controls when preceded by ISO paired with 100 Hz homosynaptically. (B) Similar to WT, homosynaptic long-lasting LTP was generated in Fmr1 KO hippocampal slices when 100 Hz was applied with ISO (•). However, heterosynaptic LTP was significantly potentiated (denoted as +) relative to both controls (C,D) and WT slices treated with ISO + 100 Hz (A) 120 min after LFS at S2 (□; n = 8). (C) Application of ISO alone to Fmr1 KO slices prior to LFS induces a modest, short-lasting increase in basal synaptic response, which has no long-lasting effects on synaptic transmission (n = 7). (D) 100-Hz stimulation alone induces transient LTP, which returns to baseline in <2 h and does not facilitate heterosynaptic LTP following LFS at a second synaptic pathway in Fmr1 KO mouse slices (n = 8). Importantly, LFS (5 Hz, 10 sec) does not last more than 45 min. (E) Summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1. (F) Summary histogram comparing fEPSP slopes 120 min after LFS at S2. Sample traces were taken 10 min after commencement of baseline recordings and 120 min after S1 stimulation. Results in panels E and F represent mean ± SEM, P < 0.05.

Next, we tested the hypothesis that 1 × 100 Hz alone is capable of enhancing the subsequent induction of heterosynaptic LTP by LFS in WT slices. Wild-type hippocampal slices tetanized with 100 Hz at S1 followed 30 min later with 5 Hz at S2 failed to generate LTP at either synaptic pathway. fEPSPs had returned to baseline at S1 (108 ± 6%) 120 min after HFS and at S2 (97 ± 9%) 120 min after LFS (n = 6) (data not shown). Based on these results, we wanted to determine if β-AR-dependent heterosynaptic plasticity is altered in Fmr1 KO mice.

β-AR-dependent heterosynaptic LTP is enhanced in Fmr1 KO mice

Metabotropic glutamate receptors (mGluRs) co-regulate translation by engaging signaling cascades including the extracellular-signal regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways (Huber et al. 2000; Gallagher et al. 2004; Hou and Klann 2004; Banko et al. 2006; Volk et al. 2007; Ronesi and Huber 2008). In Fmr1 KO mice, deletion of FMRP decreases the requirement for protein synthesis in the expression of mGluR-LTD (Hou et al. 2006; Nosyreva and Huber 2006). Similar to mGluRs, β-ARs couple to the ERK and mTOR signaling cascades to engage cap-dependent translation and promote persistent, translation-dependent homosynaptic LTP (Banko et al. 2006; Gelinas et al. 2007). Therefore, we wanted to determine whether β-AR-mediated LTP is altered homosynaptically and heterosynaptically in the absence of FMRP.

We applied the β-AR-dependent heterosynaptic plasticity protocol to Fmr1 KO mouse hippocampal slices. Briefly, following a 20-min baseline, ISO was applied for 10 min, then paired with 1 × 100 Hz stimulation at S1 (homosynaptic) and maintained in ISO for an additional 5 min. When this protocol was applied to Fmr1 hippocampal slices, homosynaptic, long-lasting LTP was induced which was not significantly different from WT slices (mean fEPSP slopes were 152 ± 6% in KO; n = 8, and 147 ± 7% in WT, 120 min after stimulation; n = 8) (Fig. 2B,E). Thirty minutes after HFS in S1, low-frequency stimulation (5 Hz, 10 sec) was applied to a second, independent set of synapses converging on the same post-synaptic cells (S2). LFS applied heterosynaptically generated long-lasting LTP that was significantly enhanced in Fmr1 KO slices relative to WT controls (mean fEPSP slopes in KO = 139 ± 8%, WT = 119 ± 6%, as assessed 120 min post-LFS; P < 0.05) (Fig. 2F). These results suggest that heterosynaptic plasticity is enhanced in response to β-AR stimulation in Fmr1 KO mouse hippocampus.

To determine if β-AR activation alone is sufficient for enhancing heterosynaptic plasticity, ISO (1 µM) was applied to Fmr1 knockout hippocampal slices for 15 min. Application of ISO induced a modest, transient increase in fEPSPs at both synaptic pathways, which returned to baseline, consistent with previously reported data (Gelinas and Nguyen 2005). Sixty minutes after ISO application, the mean fEPSP slopes at S1 were 103 ± 5% of baseline (n = 7) (Fig. 2C). After a 25-min delay, low-frequency stimulation (5 Hz for 10 sec) was applied to S2, which induced short-lasting LTP that decayed to baseline in <1 h: mean fEPSP slopes were 105 ± 5% of baseline 90 min after 5 Hz stimulation (Fig. 2C,F). Next, we sought to determine if a single tetanus of 100 Hz (1-sec duration) to FMR1 KO mouse hippocampal slices could facilitate the expression of heterosynaptic LTP induced by LFS. Consistent with previous results, a 1 × 100-Hz stimulation induced transient LTP that returned to baseline in <2 h (mean fEPSP slopes were 104 ± 8% at S1 120 min after HFS, n = 8) (Fig. 2D). Following a 30-min delay, low-frequency stimulation (5 Hz, 10 sec) was applied to a second group of synapses converging on the same post-synaptic cells. Heterosynaptic low-frequency stimulation failed to induce long-lasting LTP (mean fEPSPs were 102 ± 6% at S2 when assessed 2 h after stimulation; n = 8) (Fig. 2D,F). Taken together, these data suggest that application of either ISO alone or 1 × 100 Hz stimulation without ISO, does not facilitate the future induction of heterosynaptic LTP in Fmr1 KO mouse hippocampal slices.

Altered inhibition is not the primary cellular mechanism mediating β-AR-dependent heterosynaptic facilitation in Fmr1 KO hippocampus

Alterations to GABAergic inhibition have previously been associated with neuropathologies observed in FXS rodent models (Burgard and Sarvey 1991; El Idrissi et al. 2005; Curia et al. 2009). Hippocampal tissue from adult male Fmr1 KO mice exhibits reduced expression of the GABA-A receptor β subunit, which is required for normal receptor function (El Idrissi et al. 2005). In addition, recent evidence suggests that ISO can reduce inhibitory drive within the hippocampus (Zsiros and Maccaferri 2008). Therefore, we investigated whether altered inhibitory circuit function could account for the enhanced heterosynaptic LTP observed in Fmr1 KO hippocampus. To examine the effects of altered inhibitory function, we preapplied the GABA-A receptor antagonist, bicuculline (BICU; 10 µM), for 20 min prior to, and overlapping with, ISO. If altered GABA-A receptor function is the mechanism responsible for enhanced heterosynaptic plasticity, then prior application of BICU should occlude the LTP enhancement generated by β-AR stimulation. First, Fmr1 KO mouse hippocampal slices were exposed to a 20-min preincubation in BICU. Subsequently, a cocktail consisting of ISO and BICU was added to the bath for 15 min. Ten minutes into ISO + BICU application, 1 × 100 Hz, 1-sec stimulation was applied to S1. This protocol generated long-lasting LTP that was potentiated to 164 ± 12% (n = 6) 2 h after stimulation in Fmr1 slices (Fig. 3B). To test if heterosynaptic plasticity was altered by BICU, 25 min after homosynaptic 100-Hz stimulation, 5-Hz, 10-sec stimulation was given to a second synaptic pathway. Heterosynaptic responses were potentiated to 171 ± 14%, of baseline 2 h after LFS (Fig. 3B). It should be noted that Fmr1 slices treated with BICU exhibited epileptiform activity following LTP induction. This activity dissipated after 2 h and did not appear to affect the stability or consistency of our fEPSPs.

Figure 3.

Figure 3.

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β-ARs facilitate heterosynaptic plasticity independent of altered GABA-A receptor function in Fmr1 KO hippocampus. (A) To determine if enhanced heterosynaptic plasticity is the result of β-AR-mediated disinhibition, wild-type hippocampal slices were preincubated with the GABA-A receptor antagonist, bicuculline (BICU). Subsequent induction of homosynaptic β-AR-dependent LTP (•) yielded long-lasting LTP that was not significantly different from the WT slices not exposed to BICU (C). Comparisons of heterosynaptic LTP in BICU-treated (□) and nontreated slices demonstrated a significant boosting of LTP in the presence of BICU (as denoted by *; D). (B) When BICU was added to the bath of Fmr1 KO slices, homosynaptic LTP (•) was slightly, but not significantly, enhanced relative to BICU naive slices. When heterosynaptic LTP (□) was compared, prior application of BICU significantly enhanced the magnitude of subsequently induced LTP relative to Fmr1 slices not treated with BICU (denoted by +). (C) Summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1. (D) Summary histogram comparing fEPSP slopes 120 min after LFS at S2. Sample traces were taken 10 min after commencement of baseline recordings and 120 min after S1 stimulation. Results in panels C and D represent mean ± SEM, P < 0.05.

Next, wild-type slices were treated with the same regimen. Preincubation with BICU followed by ISO paired with HFS induced long lasting LTP at S1 (fEPSPs were potentiated to 144 ± 10% 2 h after stimulation; n = 6) (Fig. 3A). Subsequent low-frequency stimulation (5 Hz) applied heterosynaptically generated long-lasting LTP, which was potentiated to 154 ± 11% at 2 h post-tetanus (Fig. 3A). When homosynaptic (S1) β-AR-dependent LTP in slices from WT (Fig. 2A) was compared to WT slices exposed to BICU (Fig. 3A) 2 h after stimulation, no significant differences were detected (P > 0.05) (Fig. 3C). Similarly, comparisons between homosynaptic LTP in Fmr1 KO slices in the presence or absence of BICU failed to reveal any significant differences (P > 0.05) (Fig. 3C). When heterosynaptic comparisons were conducted between fEPSPs 2 h after LFS in WT slices either exposed (Fig. 3A) or not exposed (Fig. 2A) to BICU, a significant enhancement of LTP in the presence of BICU was observed (P < 0.01) (Fig. 3D). Likewise, comparisons between heterosynaptic LTP in Fmr1 KO slices treated or not treated with BICU showed that BICU contributed to a significant increase in potentiation (P < 0.05) (Fig. 3D) 2 h after low-frequency stimulation. These results suggest that decreasing inhibition results in a further enhancement of heterosynaptic LTP in both genotypes and that stimulation of β-ARs engages cellular plasticity mechanisms that are not primarily dependent on altered GABAergic receptor function.

β-AR activation is required for isoproterenol-induced heterosynaptic plasticity

Genetic and pharmacological inhibition of β-ARs in area CA1 can interfere with expression of LTP and long-term memory formation (Winder et al. 1999; Ji et al. 2003). To test if β-ARs are required for heterosynaptic plasticity induced with isoproterenol, hippocampal slices from wild-type and Fmr1 knockout mice were pretreated with a β-AR antagonist, propranolol (PROP; 50 µM). In wild-type mice, following a 10-min baseline, PROP was bath-applied 20 min prior to, overlapping with, and 10 min after ISO application. When high-frequency stimulation was applied homosynaptically, LTP was induced, which decayed to baseline in <2 h (Fig. 4A) (mean fEPSP slopes were 101 ± 8%, 120 min after HFS, n = 9). When LFS (5 Hz, 10 sec) was applied at a second set of synapses 30 min after homosynaptic high-frequency stimulation, we found that heterosynaptic plasticity was also blocked (mean fEPSPs were 98 ± 6% 90 min after LFS at S2) (Fig. 4A). Similar to wild-type slices, prior application of propranolol prevented the induction of both homo- and heterosynaptic LTP (Fig. 4B) (mean fEPSP slopes were 104 ± 3% 120 min after HFS at S1 and 101 ± 5% 90 min after LFS at S2, n = 8) (Fig. 4C). Thus, heterosynaptic plasticity produced by ISO application requires β-AR activation.

Figure 4.

Figure 4.

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β-ARs are required for heterosynaptic facilitation of LTP. (A) Application of the β-AR antagonist propranolol during β-AR activation inhibited both homosynaptic (•) and heterosynaptic (□) LTP in WT slices (n = 9). (B) Similarly, in Fmr1 KO slices propranolol application overlapping with ISO impaired LTP at S1 (•) and S2 (□; n = 8). (C) A summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1 and 90 min after LFS at S2 demonstrates that propranolol blocked the maintenance of LTP both homo- (S1; black bars) and heterosynaptically (white bars) in both wild-type and Fmr1 KO genotypes. Sample traces were taken 10 min after commencement of baseline recordings and 120 and 90 min after stimulation. Results in C represent mean ± SEM.

Prior low-frequency stimulation can express LTP when B-AR-dependent LTP is generated heterosynaptically

The synaptic tagging hypothesis states that local molecular traces induced by synaptic activity serve as tags, which can sequester plasticity-related proteins generated within a discrete time window before or after tags are set (Frey and Morris 1997). In “weak before strong” stimulation experiments, the activity-dependent tag is set by mild stimulation prior to the generation of plasticity-related proteins by a later, stronger stimulus (Frey and Morris 1998). To determine if prior synaptic potentiation can be facilitated by the future induction of β-AR-dependent LTP we first applied low-frequency stimulation (5 Hz, 10 sec) to WT slices in pathway S1. Twenty-five minutes later, β-AR-dependent LTP was induced heterosynaptically (S2). Prior LFS induced long-lasting LTP (mean fEPSP slope was 122 ± 9% 120 min post-stimulation, n = 8) provided that LTP was induced heterosynaptically through pairing of ISO with high-frequency stimulation (1 × 100 Hz). (Fig. 5A) (fEPSPs at S2 were 144 ± 9% 90 min post-HFS). Similar to WT slices, low-frequency stimulation applied 25 min prior to the induction of heterosynaptic β-AR-dependent LTP induced homosynaptic LTP, which was still potentiated 2 h after LFS. Comparisons between slices from Fmr1 KO and WT mice revealed no significant differences in the magnitude of LTP either homosynaptically (Fig. 5B) (P > 0.05; mean fEPSP slope was 120 ± 8% at S1 in Fmr1 KOs, n = 8) or heterosynaptically (Fig. 5C) (P > 0.05; mean fEPSPs were 147 ± 9% 90 min after HFS in Fmr1 KOs). These data suggest that low-frequency stimulation initiates an activity-dependent molecular trace that lasts 25 min and can interact with plasticity products generated by a strong stimulus applied heterosynaptically at a future time point.

Figure 5.

Figure 5.

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Heterosynaptic facilitation of LTP is still observed when LFS precedes ISO paired with HFS at a second synaptic pathway. (A) Application of LFS (5 Hz, 10 sec: S1; •) 25 min prior to ISO application paired with HFS, still shows heterosynaptic facilitation (S2; □) (n = 8). (B) Similar to WT, prior LFS (S1; •) in Fmr1 KO hippocampal slices allows for the transfer of LTP induced by β-AR activation paired with HFS at a second synaptic pathway (S2; □; n = 8). Comparisons between WT and Fmr1 KO mouse genotypes (C) 120 min after LFS at S1 or HFS paired with ISO at S2 failed to demonstrate significant differences in mean fEPSP slopes. These results suggest that application of the low-frequency “weak” stimulus homosynaptically can capture subsequently induced LTP heterosynaptically regardless of genotype. (C) Summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1 (black bars) and LFS at S2 (white bars). Sample traces were taken 10 min after commencement of baseline recordings and 120 min after stimulation. Results in C represent mean ± SEM.

mGluR activation is not required for enhanced heterosynaptic LTP in Fmr1 KO mouse hippocampus

mGluR-dependent LTD is enhanced in Fmr1 KO mouse hippocampus. Normally, mGluR LTD requires protein synthesis for maintenance of GluR internalization (Nosyreva and Huber 2006; Nakamoto et al. 2007). In Fmr1 KO mouse hippocampus, mGluR LTD does not require translation, suggestive of an increase in basal protein synthesis (Hou et al. 2006; Nosyreva and Huber 2006). It is unclear whether heterosynaptic LTP in Fmr1 KO mouse hippocampus requires mGluR activation. To determine whether mGluRs are required for heterosynaptic facilitation of LTP by β-ARs in Fmr1 mouse hippocampal slices, we used the mGluR5 antagonist, MPEP (10 µM). Application of MPEP 20 min prior to and overlapping with β-AR stimulation paired with 100 Hz tetanus in S1, did not affect LTP expression in either synaptic pathway: mean fEPSP slopes in S1 were 147 ± 8% 2 h after tetanization in Fmr1 KO slices (n = 6) (Fig. 6A). When compared to WT controls not exposed to MPEP (144 ± 6%, n = 8) (Fig. 6B) there was no significant difference in homosynaptic LTP (P > 0.05). However, hippocampal slices from Fmr1 KO mice, treated with MPEP, still displayed heterosynaptic LTP that was enhanced relative to WT slices not exposed to MPEP: mean fEPSP slopes in KOs were 144 ± 8% of baseline, which is significantly elevated relative to wild-type slices (mean fEPSPs were 118 ± 6% of baseline), when compared 120 min after stimulation (P < 0.05) (Fig. 6C). Thus, the enhancement of heterosynaptic LTP in slices from Fmr1 KO mice does not appear to require mGluR-5 activation.

Figure 6.

Figure 6.

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MPEP does not block the β-AR-dependent enhancement of heterosynaptic plasticity in Fmr1 KO mice. (A) Induction of β-AR-dependent LTP at one synaptic pathway (S1; •) facilitates the subsequent expression of heterosynaptic LTP (S2; □) in WT slices (n = 8 ). (B) The same protocol applied to Fmr1 KO mouse hippocampal slices in the presence of the mGluR5 antagonist MPEP, induces heterosynaptic plasticity (S2; □) that is still enhanced relative to WT controls (n = 6). (C) A summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1 (black bars) revealed no significant differences in homosynaptic LTP. Heterosynaptic LTP (S2; white bars) was significantly elevated (*) 120 min after LFS in Fmr1 KO slices treated with MPEP when compared to WT slices not treated with MPEP. Sample traces were taken 10 min after commencement of baseline recordings and 120 min after HFS. Results in C represent mean ± SEM, P < 0.05.

β-AR-dependent heterosynaptic LTP requires translation in Fmr1 KO mice

Protein synthesis critically involved in the generation of LTP and long-term memory formation. Results from Auerbach and Bear (2010) suggest that priming of LTP by prior activation of mGluRs, involves a reduced requirement for translation in Fmr1 KO mice, consistent with enhanced synthesis of PRPs. Previously, cap-dependent translation was shown to be a critical target for regulation during β-AR-induced synaptic plasticity (Gelinas et al. 2007). As β-ARs and mGluRs both engage cap-dependent translation, we asked whether β-AR activation in Fmr1 knock-out mice renders LTP immune to protein synthesis inhibition. To test this hypothesis, we preincubated slices in a translational inhibitor, emetine (EME). Homosynaptic LTP elicited by pairing β-AR activation with 1 × 100 Hz high-frequency stimulation in S1 decayed in the presence of EME. Mean fEPSP slopes were 115 ± 10% in KO (n = 8) and 107 ± 13% in WT (n = 7), P > 0.05, 120 min after HFS (Fig. 7A,B). When compared to homosynaptic LTP induced with β-ARs in WT controls (mean fEPSP slopes were 144 ± 9% 120 min after HFS, n = 6) (data not shown), Tukey-Kramer post hoc tests revealed that emetine significantly inhibited LTP in both WTs and KOs (P < 0.05). Bath application of emetine overlapping with ISO likewise inhibited LTP heterosynaptically when 5-Hz, 10-sec LFS was applied 30 min later in S2 relative to EME-free control slices (Fig. 7A,B) (mean fEPSP slopes in the presence of EME were 106 ± 6% in KO (n = 8) and 104 ± 10% in WT (n = 7), P > 0.05, 90 min after LFS) (Fig. 7C). When compared to heterosynaptic LTP induced with β-AR stimulation in WT controls (127 ± 7%, 90 min after LFS) (data not shown), Tukey-Kramer post hoc tests revealed that heterosynaptic LTP in both wild-type and Fmr1 knockout slices (P < 0.05) (Fig. 7D) decayed in the presence of EME. Thus, when compared 2 h post-stimulation, Fmr1 KO mouse slices exhibited significant decreases in potentiation at both S1 and S2, which was similar to WT slices exposed to EME.

Figure 7.

Figure 7.

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Knockout of Fmr1 does not confer immunity to translation inhibition. (A) WT slices treated with EME during β-AR activation did not express LTP at S1 (•) or S2 (□; n = 7) when compared to β-AR-dependent LTP induced in WT slices without EME. (B) Similar to WT, EME blocked both homo (•) and heterosynaptic (□) plasticity in Fmr1 KO slices. Comparisons were made with β-AR-dependent heterosynaptic plasticity induced in WT slices (data not shown, n = 6). (C) Summary histogram comparing fEPSP slopes obtained 120 min after HFS at S1 (black bars) demonstrates a significant (*) enhancement of LTP in WT slices not exposed to EME relative to WT and Fmr1 slices treated with EME. (D) Similarly, LTP in both WT and Fmr1 KO slices treated with EME was significantly (*) less 90 min after LFS at S2 (white bars) than compared to WT slices without EME. Sample traces were taken 10 min after commencement of baseline recordings and 120 min after HFS. Results in C represent mean ± SEM, P < 0.05.

Knockout of Fmr1 confers immunity to inhibition of mTOR but not ERK

β-ARs recruit the mammalian target of rapamycin (mTOR) signaling pathway to facilitate LTP maintenance through translation initiation (Gelinas et al. 2007). We assessed whether heterosynaptic LTP was affected by an mTOR inhibitor, rapamycin, in Fmr1 knockout mouse hippocampus. Wild-type slices treated with rapamycin (RAP) demonstrated reduced homo- and heterosynaptic plasticity. Application of RAP 30 min prior to, overlapping with, and 10 min after ISO paired with high-frequency stimulation inhibited the expression of homosynaptic LTP as assessed 2 h after HFS (mean fEPSP slopes in S1 were 108 ± 12%). Subsequent (30 min post-HFS) stimulation with low-frequency stimulation at S2 generated decremental LTP which was 108 ± 9% 90 min post-LFS (Fig. 8A) (n = 9). Interestingly, Fmr1 KO mouse slices treated with RAP demonstrated intact homosynaptic LTP. Homosynaptic LTP was significantly enhanced (mean fEPSP slope was 140 ± 9% in S1, 120 min post-stimulation) in rapamycin-treated slices (Fig. 8B,E) (n = 6, P < 0.02) relative to wild-type slices exposed to RAP. However, inhibition of mTOR still attenuated LTP heterosynaptically in Fmr1 mouse slices. Mean fEPSP slope at S2 90 min after low-frequency stimulation was 115 ± 9% (P > 0.05 relative to WT) (Fig. 8F). To control for potential synaptic crosstalk generated by heterosynaptic LFS, the RAP experiments were repeated in Fmr1 KO slices in the absence of LFS in S2. Consistent with our previous results, RAP failed to inhibit homosynaptic LTP (fEPSPs were 137 ± 12% in S1 120 min after HFS, P < 0.05 compared to WT slices exposed to RAP) (data not shown). Stimulation of a second control pathway showed that RAP did not adversely affect baseline synaptic responses (fEPSPs were 96 ± 10% 2 h after RAP washout at S2). These results support previous research (Sharma et al. 2010), which found that mTOR signaling was intact and enhanced in the absence of FMRP.

Figure 8.

Figure 8.

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Inhibition of mTOR does not block β-AR-dependent homosynaptic LTP in Fmr1 KO mice. (A) Application of rapamycin completely blocked homo- (•) and heterosynaptic (□) plasticity in WT slices (n = 9). (B) In Fmr1 KO mice, co-application of rapamycin and ISO blocked the heterosynaptic facilitation of LTP at S2 (□). However, LTP at S1 (•) was not blocked by mTOR inhibition and was significantly elevated (as denoted by the *) relative to WT slices exposed to RAP (n = 6). (C) In WT slices, application of the MEK inhibitor PD98059 prevented the expression of LTP at both S1 (•) and S2 (□; n = 6). (D) Similar to WT, inhibition of MEK in Fmr1 hippocampal slices blocked LTP expression at both synaptic pathways (S1: •, S2: □; n = 7). (E) Summary histogram comparing fEPSP slopes obtained 120 after HFS at S1 (black bars) shows that inhibition of mTOR failed to prevent homosynaptic LTP in Fmr1 KO hippocampal slices. (F) Heterosynaptic LTP (white bars) was inhibited in both genotypes by treatment with either RAP or PD98059. Sample traces were taken 10 min after commencement of baseline recordings and 120 after stimulation. Results in E and F represent mean ± SEM, P < 0.05.

Because the ERK pathway cooperates with mTOR to regulate translation-dependent, β-AR-mediated LTP in the mouse hippocampus (Banko et al. 2006; Gelinas et al. 2007, 2008), we asked whether homo- and heterosynaptic LTP in Fmr1 KO mouse hippocampus were affected by inhibition of MEK, which our group has previously established as a pharmacological blocker of β-AR-dependent synaptic enhancement (Gelinas and Nguyen 2005). In WT slices, induction of LTP was blocked homosynaptically (mean fEPSP slope was 116 ± 11%, 120 min after HFS) and heterosynaptically (mean fEPSP slope was 105 ± 7%, 90 min post-LFS) (Fig. 8C,E) by a MEK inhibitor, PD98059 (n = 6) when it was applied 30 min before as well as overlapping with and 10 min after ISO + HFS at S1. In slices from Fmr1 KO mice, LTP was also blocked by PD98059: mean fEPSP slopes were 103 ± 8% at S1 and 98 ± 6% at S2 (Fig. 8D,E) (n = 7) of baseline 120 and 90 min post-stimulation, respectively.

Discussion

Fragile X syndrome is characterized by behavioral (Hagerman and Hagerman 2002; Spencer et al. 2005; Moon et al. 2006) and cognitive (Tsiouris and Brown 2004; Macleod et al. 2010) abnormalities that have been linked to altered metabotropic receptor signaling and translation regulation (Huber et al. 2002; Bear et al. 2004; Kelleher and Bear 2008; for review, see O'Donnell and Warren 2002; Pfeiffer and Huber 2009). The noradrenergic system, acting through metabotropic adrenergic receptors, regulates multiple processes including sleep, attention, learning, memory, and arousal (Gelinas and Nguyen 2007; Berridge and Waterhouse 2003). β-ARs also mediate synaptic plasticity involved in encoding the association of disparate events over time (Shapiro et al. 2006). Heterosynaptic facilitation provides a mechanism for associating stimuli over time by integrating synaptic events following induction of persistent synaptic plasticity (Barco et al. 2008; Frey and Frey 2008).

A previously defined heterosynaptic protocol known as “synaptic tagging and capture” is commonly initiated by applying HFS (4 × 100 Hz) at one pathway (S1), which generates plasticity-related proteins (PRPs) that can be captured by activity-dependent tags set at a second synaptic pathway (S2) (Frey and Morris 1997). Neuromodulators gate both protein synthesis and activation of second messengers that can support this type of heterosynaptic plasticity (Gelinas and Nguyen 2005; Navakkode et al. 2007). Application of a cAMP phosphodiesterase inhibitor, rolipram, enabled the heterosynaptic transformation of translation-independent LTP induced by one 100-Hz train into translation-dependent LTP, suggestive of a cAMP-mediated increase in plasticity products (Navakkode et al. 2004). Noradrenergic receptors can facilitate the generation and regulation of plasticity proteins capable of prolonging LTP (Winder et al. 1999; Straube et al. 2003; Gelinas and Nguyen 2005) and boosting long-term memory (Cahill et al. 1994; Hu et al. 2007; Kemp and Manahan-Vaughan 2008). Interestingly, prior exposure of rats to novel experiences engages the noradrenergic system (Kitchigina et al. 1997) to promote the consolidation of short-term memory into protein synthesis-dependent long-term memory, that is likely to involve “behavioral tagging” (Moncada and Viola 2007; Ballarini et al. 2009). Taken together, these results suggest that β-AR activation engages intracellular pathways critical for heterosynaptic facilitation of LTP. Herein, we describe a novel heterosynaptic plasticity protocol that relies upon activation of β-ARs for its induction. We found that pairing β-AR activation with high-frequency stimulation induces LTP that could be transferred to a second synaptic pathway provided that stimulation sufficient for generating a molecular activity trace was applied.

How does β-AR activation facilitate heterosynaptic plasticity in Fmr1 KO mouse hippocampus? One of the core phenotypes of FXS models is enhanced epileptogenesis as a result of dysregulated inhibitory circuit function (Burgard and Sarvey 1991; El Idrissi et al. 2005; Curia et al. 2009). This fact, coupled with the ability of isoproterenol to depress inhibitory interneurons, suggests that altered inhibitory circuit function may be responsible for enhanced heterosynaptic LTP in Fmr1 KO mice. However, we found that disinhibition through application of the GABA-A receptor antagonist bicuculline failed to occlude the effects of β-AR activation. Indeed, consistent with previous data (Burgard and Sarvey 1991), co-application of ISO and BICU resulted in further enhancement of heterosynaptic LTP (additionally, there was a trend for enhanced homosynaptic LTP in Fmr1 KO slices, but this failed to reach statistical significance). Our results do not support the idea that altered GABAergic inhibition could account for enhanced heterosynaptic LTP in Fmr1 mice.

The mGluR theory of FXS states that stimulation of mGluR1/5 leads to enhanced synthesis of LTD-related plasticity proteins, which are normally repressed through FMRP-dependent mRNA regulation (Bear et al. 2004; Pfeiffer and Huber 2007). The enhancement of heterosynaptic LTP in Fmr1 KO mice observed following our β-AR-dependent heterosynaptic protocol did not require mGluR activation, as application of the mGluR5 antagonist MPEP had no effect on the magnitude of LTP at either synaptic pathway in Fmr1 KO slices. The mGluR theory has been extended to include signaling molecules that affect cap-dependent translation, including ERK and mTOR (Hou et al. 2006; Volk et al. 2007; Sharma et al. 2010). Importantly, β-AR activation has been linked to regulation of cap-dependent translation through ERK and mTOR pathways, both of which are dysregulated in Fmr1 KO mouse hippocampus (Hou et al. 2006; Kim et al. 2008; Sharma et al. 2010). These pathways are critical for coupling mGluRs and β-ARs to protein synthesis (Banko et al. 2006; Gelinas et al. 2007) through interactions with eIF4E, a translation initiation factor required for eIF4F initiation complex formation and translation initiation (Costa-Mattioli et al. 2009; Richter and Klann 2009). Our data suggest that activation of the ERK pathway is required for homosynaptic LTP and for heterosynaptic facilitation of LTP in both WT and Fmr1 KO mouse hippocampus. In contrast, activation of mTOR is required for heterosynaptic LTP, but not for homosynaptic LTP, in Fmr1 KO mouse hippocampus. Although the mTOR inhibitor rapamycin prevented the expression of heterosynaptic LTP, homosynaptic LTP was still intact. Up-regulated mTOR signaling may lower the threshold for initiation of protein synthesis by elevating the levels of “free” eIF4E (Sharma et al. 2010), thereby boosting heterosynaptic plasticity.

As inhibition of protein synthesis, but not inhibition of mTOR, blocked heterosynaptic facilitation of LTP, decreased levels of FMRP appear to affect processes upstream of mTOR-mediated translation regulation. Previous results suggest that in Fmr1 KO mice, mTOR signaling is elevated, which may facilitate mGluR-dependent LTD (Hoeffer and Klann 2010; Sharma et al. 2010). Hyperphosphorylation of eIF4E-binding proteins (4E-BPs) by mTOR regulates translation initiation by increasing levels of unbound eIF4E. Once dissociated from the 4E-BPs, eIF4E is able to complex with other initiation factors to engage translation (Klann et al. 2004; Richter and Klann 2009). Dysregulation of the PI3K-mTOR pathway in the absence of FMRP has recently been reported (Sharma et al. 2010) and could provide a mechanism for enhanced β-AR-dependent synaptic facilitation (Fig. 9A). As β-AR-dependent synaptic plasticity was partially immune to mTOR inhibition, our results suggest that, in a broader perspective, dysregulation of mTOR signaling may be an important cause of altered synaptic plasticity, which both mGluRs and β-ARs critically modulate.

Figure 9.

Figure 9.

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(A) Increased mTOR signaling in the absence of FMRP enhances heterosynaptic plasticity. In wild-type mouse hippocampus, FMRP represses mTOR activity (left side). The mTOR signaling cascade can regulate protein synthesis mechanisms necessary for heterosynaptic transfer of LTP. Without the repressive effects of FMRP (right side), mTOR activity is elevated resulting in dysregulated translation and exaggerated heterosynaptic plasticity. (B) Activation or β-ARs confers sensitivity to protein synthesis inhibition. (a) This schematic diagram represents the sensitivity of mouse hippocampal synaptic plasticity to protein synthesis inhibition. In wild-type mice, the fulcrum is centered, indicative of situations in which the ability of synapses to undergo translation-dependent changes is modifiable. (b) β-AR activation shifts the fulcrum to the left through up-regulation of protein synthesis, thereby increasing the sensitivity of synaptic changes (LTP) to translation inhibition. (c) In Fmr1 knockout mouse hippocampus, the fulcrum is shifted to the right under basal conditions, rendering certain forms of synaptic plasticity (mGluR-dependent LTD) insensitive to inhibition of protein synthesis. (d) Activation of β-ARs enhances activation of signaling cascades that regulate translation, resetting the sensitivity to protein synthesis inhibition (shifting the fulcrum to the left) in Fmr1 knockout mouse hippocampus, thereby restoring the translation-dependence of synaptic plasticity.

Interestingly, β-AR-dependent heterosynaptic facilitation in Fmr1 mouse hippocampus is blocked by a protein synthesis inhibitor, in contrast to mGluR-LTD, which is immune to translational inhibition (Nosyreva and Huber 2006). Evidence suggests that FMRP may act as a brake on cap-dependent translation, evident from the increased basal eIF4F initiation complex formation observed in Fmr1 KO mice (Sharma et al. 2010). FMRP suppresses translation through interaction with cytoplasmic FMRP-interacting protein (CYFIP1), which binds to eIF4E, thereby preventing formation of the eIF4F initiation complex (Napoli et al. 2008). Our data suggest that translation is still required for heterosynaptic plasticity mediated by β-ARs in both WT and Fmr1 KO mice. These results contrast with mGluR-LTD (Nosyreva and Huber 2006) and mGluR-primed LTP (Auerbach and Bear 2010) in Fmr1 knockout mice, which persist in the presence of translation inhibitors. The disparity in these data could be the result of a higher demand for plasticity proteins in heterosynaptic plasticity protocols relative to single pathway experiments. Enhanced basal protein synthesis may be sufficient for overcoming translational inhibition using protocols that do not exhaust the availability of PRPs. Indeed, previous research has described a form of intersynaptic competition for plasticity proteins under conditions of reduced protein synthesis (Fonseca et al. 2004). When plasticity protein synthesis is limited, the maintenance of LTP at one synaptic pathway occurs at the expense of a second previously potentiated synaptic pathway, in a process known as “competitive maintenance” (Fonseca et al. 2004). Thus, even in circumstances of enhanced basal protein synthesis, the sharing of plasticity proteins required for heterosynaptic facilitation of synaptic changes may require additional translation to maintain LTP in two pathways.

Temporally and spatially restricted translation gates persistent synaptic plasticity by generating proteins necessary for long-term modifications of synaptic structure and function. Fmr1 knockout mice exhibit alterations in translation regulation (Nimchinsky et al. 2001). Enhanced protein synthesis has been demonstrated in the hippocampus of Fmr1 KO mice (Qin et al. 2005; Dölen and Bear 2008) with several of the dysregulated mRNAs encoding proteins involved in LTP, LTD, and long-term memory (GluR1, Arc, PSD-95, MAP1B) (Hou et al. 2006; Muddashetty et al. 2007; Park et al. 2008; Schütt et al. 2009). As β-ARs couple to both the mTOR and ERK signaling cascades, which can co-regulate translation, we propose that activation of β-ARs resets the sensitivity of long-term synaptic changes to translational inhibition (Fig. 9B). Previous data have demonstrated an abnormally rapid ERK dephosphorylation in response to mGluR1/5 stimulation in synaptoneurosomes isolated from Fmr1 knockout mouse cortical tissue (Kim et al. 2008). Thus, β-AR activation may compensate for down-regulated ERK activity observed in Fmr1 knockouts, thereby re-establishing the homeostasis between ERK and mTOR signaling necessary for regulated translation. As a result, the sensitivity of long-term synaptic modifications to translational control would be reset to levels similar to that observed in wild-type mice. The impact of β-AR-mediated tagging and enhanced heterosynaptic plasticity on cognition in Fmr1 knockout mice remains to be determined.

Overall, our data show an enhancement of heterosynaptic plasticity in Fmr1 knockout mouse hippocampus following the induction of homosynaptic β-AR-dependent LTP. This form of synaptic plasticity requires translation, suggestive of a β-AR-mediated re-establishment of sensitivity to protein synthesis inhibition. Given that β-AR antagonists are already being used clinically with positive results (Rosenberg 2007), β-ARs and their downstream effectors are logical targets for the pharmacologic treatment of fragile X syndrome (Restivo et al. 2005; Kelley et al. 2008). Increased heterosynaptic plasticity in response to noradrenergic system activation may promote hyperconsolidation of synaptic changes across multiple neural networks that would normally be repressed when FMRP is present. Synaptic hyperconsolidation could stem from a lowered threshold for heterosynaptic plasticity, thereby compromising the network- and synapse-specific changes in synaptic weights that are believed to be necessary for normal learning to occur. This may lead to the cognitive impairments observed in fragile X syndrome.

Materials and Methods

Animals

C57BL/6 congenic Fmr1 KO mice and their WT littermates (3–4 mo) were used for all experiments. Animals were socially housed with food and water available ad libitum and were maintained on a 12 h on/off light cycle. Animals were housed at the University of Alberta using guidelines approved by the Canadian Council on Animal Care. Fmr1 KO mice and their WT littermates were generated in harem crosses using hemizygous Fmr1 mutant or WT males crossed to heterozygote Fmr1 (+/−) females. Mice were weaned between 21 and 28 d and genotyped using standard methods using primer sequences and PCR instructions supplied by Jax Labs (http://jaxmice.jax.org/protocolsdb).

Electrophysiology

After cervical dislocation and decapitation, transverse hippocampal slices (400 µm in thickness) were prepared as described by Nguyen and Kandel (1997). Slices were maintained in an interface chamber at 28°C, which is a consistently used temperature for recording field responses on extended (hours) time scales (Gelinas and Nguyen 2005; Young et al. 2006; Gelinas et al. 2007). Slices were perfused at 1–2 mL/min with artificial cerebrospinal fluid (ACSF) composed of (mM): 124 NaCl, 4.4 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 26.2 NaHCO3, 2.5 CaCl2, and 10 glucose, aerated with 95% O2 and 5% CO2. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded with a glass microelectrode filled with ACSF (resistances, 2–3 MΩ) and positioned in the stratum radiatum of area CA1. fEPSPs were elicited by using two bipolar nickel–chromium electrodes placed on either side of the recording electrode in stratum radiatum to stimulate two separate sets of inputs converging onto the same postsynaptic population of neurons. Stimulating electrodes were staggered within stratum radiatum relative to each other to maximize independence of synaptic pathways. For simplicity, “S1” refers to the first or homosynaptic pathway and “S2” refers to the second or heterosynaptic pathway. The independence of the two pathways was confirmed by the absence of interpathway paired-pulse facilitation, elicited by successive stimulation through the two electrodes at 75-, 100-, 150- and 200-ms intervals. Interpathway paired-pulse facilitation was assessed during baseline acquisition and at the conclusion of experiments. Stimulation intensity (0.08-msec pulse duration) was adjusted to evoke fEPSP amplitudes that were 40% of maximal size (Woo and Nguyen 2003; Gelinas and Nguyen 2007). Subsequent fEPSPs were elicited at the rate of once per minute at this “test” stimulation intensity, with S1 stimulation preceding S2 stimulation by 200 msec.

Our first series of experiments were conducted using a standard “synaptic tagging and capture” protocol (Frey and Morris 1997) in which repeated high-frequency stimulation (HFS) was induced by applying four trains (4 × 100-Hz, 1-sec duration at test strength, 3-sec interval) to S1. After 30 min, one train of HFS (1 × 100-Hz, 1-sec duration) was applied to the second set of synaptic inputs (S2) (Woo and Nguyen 2003; Young et al. 2006). β-AR-dependent LTP was induced by applying one train of HFS (100-Hz, 1-sec duration at test strength) following a 10-min application of the β-AR agonist, isoproterenol (ISO; 1 µM) (Gelinas and Nguyen 2005). Post-HFS, ISO was applied for an additional 5 min. Thirty minutes after HFS at S1, low-frequency stimulation (LFS: 5-Hz, 10-sec duration) was applied to S2. All experiments were performed blind to genotype.

Drugs

The β-AR agonist, R (−)-isoproterenol (+)-bitartrate (ISO, Sigma) was prepared daily as concentrated stock solutions at 1 mM, in distilled water. The β-AR antagonist (±)-propranolol hydrochloride (PROP; 50 µM; Sigma) was also prepared daily in distilled water as a 50-mM stock solution. The GABA-A receptor antagonist, bicuculline (BICU; 10 µM; Sigma) was prepared as a 1-mM stock solution and was used to assay for altered GABAergic inhibition. The mGluR5 antagonist, 6-methyl-2-(phenylethynyl) pyridine (MPEP, 10 µM; Sigma) was prepared as a 10-mM stock solution and perfused for 20 min prior to application of ISO. A translation inhibitor, emetine (EME; Sigma), was dissolved to a stock concentration of 20 mM in distilled water and perfused at 20 µM, 20 min prior to ISO application. At lower concentrations than 20 µM, EME blocked protein synthesis by >80% in hippocampal slices (Stanton and Sarvey 1984). A MEK inhibitor, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 98059) (50 µM; Sigma), was prepared in DMSO at a concentration of 10 mM. An mTOR inhibitor, rapamycin (Rap; 1 µM; Sigma), was dissolved in DMSO to make stock solutions at 1 mM. PD98059 and Rap were perfused for 30 min prior to commencing experiments, overlapping with ISO application, and 10 min following ISO. All drug experiments were performed under dimmed light conditions due to photosensitivity of the drugs. Drug experiments were interleaved with drug-free controls.

Data analysis

The initial slope of the fEPSP was measured as an index of synaptic strength (Johnston and Wu 1995). The average “baseline” slope values were acquired for 20 min before experimental protocols were applied. fEPSP slopes were measured at either 120 or 90 min after LFS or HFS for comparisons of LTP. Student's t test was used for statistical comparisons of mean fEPSP slopes between two groups, with a significance level of P < 0.05 (denoted with an asterisk on the graphs). One-way ANOVA and Tukey–Kramer post hoc tests were done for comparison of more than two groups to determine which groups were significantly different from the others. The Welch correction was applied in cases in which the SDs of compared groups was significantly different. All values shown are mean ± SEM, with n = number of slices.

Acknowledgments

Special thanks to Sabyasachi Maity for his technical assistance. This research was supported by grants from the Canadian Institutes of Health Research (to P.V.N.), the National Institutes of Health, and the FRAXA Research Foundation (to E.K.). Salary support was received from a graduate scholarship (to S.A.C.) from the Natural Sciences and Engineering Research Council of Canada and a Scientist Award (to P.V.N.) from the Alberta Heritage Foundation for Medical Research.

References

  1. Auerbach BD, Bear MF 2010. Loss of the fragile X mental retardation protein decouples metabotropic glutamate receptor dependent priming of long-term potentiation from protein synthesis. J Neurophysiol 104: 1047–1051 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bakker CE, Verheij C, Willemsen R, van der Helm R, Oerlemans F, Vermey M, Bygrave A, Hoogeveen AT, Oostra BA, Reyniers E, et al. 1994. Fmr1 knockout mice: A model to study fragile X mental retardation, The Dutch-Belgian Fragile X Consortium. Cell 78: 23–33 [PubMed] [Google Scholar]
  3. Ballarini F, Moncada D, Martinez MC, Alen N, Viola H 2009. Behavioral tagging is a general mechanism of long-term memory formation. Proc Natl Acad Sci 106: 14599–14604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banko JL, Hou L, Poulin F, Sonenberg N, Klann E 2006. Regulation of eukaryotic initiation factor 4E by converging signaling pathways during metabotropic glutamate receptor-dependent long-term depression. J Neurosci 26: 2167–2173 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barco A, Lopez de Armentia M, Alarcon JM 2008. Synapse-specific stabilization of plasticity processes: The synaptic tagging and capture hypothesis revisited 10 years later. Neurosci Biobehav Rev 32: 831–851 [DOI] [PubMed] [Google Scholar]
  6. Bear MF, Huber KM, Warren ST 2004. The mGluR theory of fragile X mental retardation. Trends Neurosci 27: 370–377 [DOI] [PubMed] [Google Scholar]
  7. Berridge CW, Waterhouse BD 2003. The locus coeruleus-noradrenergic system: Modulation of behavioral state and state-dependent cognitive processes. Brain Res Brain Res Rev 42: 33–84 [DOI] [PubMed] [Google Scholar]
  8. Bliss TVP, Collingridge GL 1993. A synaptic model of memory: Long-term potentiation in the hippocampus. Nature 361: 31–39 [DOI] [PubMed] [Google Scholar]
  9. Burgard EC, Sarvey JM 1991. Long-lasting potentiation and epileptiform activity produced by GABAB receptor activation in the dentate gyrus of rat hippocampal slice. J Neurosci 11: 1198–1209 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cahill L, Prins B, Weber M, McGaugh JL 1994. β-Adrenergic activation and memory for emotional events. Nature 371: 702–704 [DOI] [PubMed] [Google Scholar]
  11. Corbin F, Bouillon M, Fortin A, Morin S, Rousseau F, Khandjian EW 1997. The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes. Hum Mol Genet 6: 1465–1472 [DOI] [PubMed] [Google Scholar]
  12. Costa-Mattioli M, Sossin WS, Klann E, Sonenberg N 2009. Translational control of long-lasting synaptic plasticity and memory. Neuron 61: 10–26 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Curia G, Papouin T, Séguéla P, Avoli M 2009. Downregulation of tonic GABAergic inhibition in a mouse model of fragile X syndrome. Cereb Cortex 19: 1515–1520 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dölen G, Bear MF 2008. Role for metabotropic glutamate receptor 5 (mGluR5) in the pathogenesis of fragile X syndrome. J Physiol 586: 1503–1508 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duffy SN, Craddock KJ, Abel T, Nguyen PV 2001. Environmental enrichment modifies the PKA-dependence of hippocampal LTP and improves hippocampus-dependent memory. Learn Mem 8: 26–34 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eichler EE, Holden JJ, Popovich BW, Reiss AL, Snow K, Thibodeau SN, Richards CS, Ward PA, Nelson DL 1994. Length of uninterrupted CGG repeats determines instability in the FMR1 gene. Nat Genet 8: 88–94 [DOI] [PubMed] [Google Scholar]
  17. El Idrissi A, Ding XH, Scalia J, Trenkner E, Brown WT, Dobkin C 2005. Decreased GABA(A) receptor expression in the seizure-prone fragile X mouse. Neurosci Lett 377: 141–146 [DOI] [PubMed] [Google Scholar]
  18. Feng Y, Zhang F, Lokey LK, Chastain JL, Lakkis L, Eberhart D, Warren ST 1995. Translational suppression by trinucleotide repeat expansion at FMR1. Science 268: 731–734 [DOI] [PubMed] [Google Scholar]
  19. Feng Y, Gutekunst CA, Eberhart DE, Yi H, Warren ST, Hersch SM 1997. Fragile X mental retardation protein: Nucleocytoplasmic shuttling and association with somatodendritic ribosomes. J Neurosci 17: 1539–1547 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fonseca R, Nägerl UV, Morris RG, Bonhoeffer T 2004. Competing for memory: Hippocampal LTP under regimes of reduced protein synthesis. Neuron 44: 1011–1020 [DOI] [PubMed] [Google Scholar]
  21. Frey S, Frey JU 2008. ‘Synaptic tagging’ and ‘cross-tagging’ and related associative reinforcement processes of functional plasticity as the cellular basis for memory formation. Prog Brain Res 169: 117–143 [DOI] [PubMed] [Google Scholar]
  22. Frey U, Morris RG 1997. Synaptic tagging and long-term potentiation. Nature 38: 533–536 [DOI] [PubMed] [Google Scholar]
  23. Frey U, Morris RG 1998. Weak before strong: Dissociating synaptic tagging and plasticity-factor accounts of late-LTP. Neuropharmacology 37: 545–552 [DOI] [PubMed] [Google Scholar]
  24. Gallagher SM, Daly CA, Bear MF, Huber KM 2004. Extracellular signal-regulated protein kinase activation is required for metabotropic glutamate receptor-dependent long-term depression in hippocampal area CA1. J Neurosci 24: 4859–4864 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gelinas JN, Nguyen PV 2005. β-Adrenergic receptor activation facilitates induction of a protein synthesis-dependent late phase of long-term potentiation. J Neurosci 25: 3294–3303 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gelinas JN, Nguyen PV 2007. Neuromodulation of hippocampal synaptic plasticity, learning, and memory by noradrenaline. Cent Nerv Syst Agents Med Chem 7: 17–33 [Google Scholar]
  27. Gelinas JN, Banko JL, Hou L, Sonenberg N, Weeber EJ, Klann E, Nguyen PV 2007. ERK and mTOR signaling couple β-adrenergic receptors to translation initiation machinery to gate induction of protein synthesis-dependent long-term potentiation. J Biol Chem 282: 27527–27535 [DOI] [PubMed] [Google Scholar]
  28. Gelinas JN, Tenorio G, Lemon N, Abel T, Nguyen PV 2008. β-Adrenergic receptor activation during distinct patterns of stimulation critically modulates the PKA-dependence of LTP in the mouse hippocampus. Learn Mem 15: 281–289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Godfraind JM, Reyniers E, De Boulle K, D'Hooge R, De Deyn PP, Bakker CE, Oostra BA, Kooy RF, Willems PJ 1996. Long-term potentiation in the hippocampus of fragile X knockout mice. Am J Med Genet 64: 246–251 [DOI] [PubMed] [Google Scholar]
  30. Hagerman RJ, Hagerman PJ 2002. Fragile X syndrome: diagnosis, treatment, and research, 3rd ed The Johns Hopkins University Press, Baltimore, MD [Google Scholar]
  31. Hagerman RJ, Berry-Kravis E, Kaufmann WE, Ono MY, Tartaglia N, Lachiewicz A, Kronk R, Delahunty C, Hessl D, Visootsak J, et al. 2009. Advances in the treatment of fragile X syndrome. Pediatrics 123: 378–390 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hoeffer CA, Klann E 2010. mTOR signaling: At the crossroads of plasticity, memory and disease. Trends Neurosci 33: 67–75 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hou L, Klann E 2004. Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression. J Neurosci 24: 6352–6361 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hou L, Antion MD, Hu D, Spencer CM, Paylor R, Klann E 2006. Dynamic translational and proteasomal regulation of fragile X mental retardation protein controls mGluR-dependent long-term depression. Neuron 51: 441–454 [DOI] [PubMed] [Google Scholar]
  35. Hu H, Real E, Takamiya K, Kang MG, Ledoux J, Huganir RL, Malinow R 2007. Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking. Cell 131: 160–173 [DOI] [PubMed] [Google Scholar]
  36. Huber KM, Kayser MS, Bear MF 2000. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288: 1254–1257 [DOI] [PubMed] [Google Scholar]
  37. Huber KM, Gallagher SM, Warren ST, Bear MF 2002. Altered synaptic plasticity in a mouse model of fragile X mental retardation. Proc Natl Acad Sci 99: 7746–7750 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ji J-Z, Wang X-M, Li B-M 2003. Deficit in long-term contextual fear memory induced by blockade of β-adrenoceptors in hippocampal CA1 region. Eur J Neurosci 17: 1947–1952 [DOI] [PubMed] [Google Scholar]
  39. Johnston D, Wu SM-S 1995. Foundations of cellular neurophysiology. MIT Press, Cambridge, MA [Google Scholar]
  40. Kelleher RJ 3rd, Bear MF 2008. The autistic neuron: Troubled translation? Cell 135: 401–406 [DOI] [PubMed] [Google Scholar]
  41. Kelley DJ, Bhattacharyya A, Lahvis GP, Yin JCP, Malter J, Davidson RJ 2008. The cyclic AMP phenotype of fragile X and autism. Neurosci Biobehav Rev 32: 1533–1543 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kemp A, Manahan-Vaughan D 2008. β-Adrenoreceptors comprise a critical element in learning-facilitated long-term plasticity. Cereb Cortex 18: 1326–1334 [DOI] [PubMed] [Google Scholar]
  43. Kim SH, Markham JA, Weiler IJ, Greenough WT 2008. Aberrant early-phase ERK inactivation impedes neuronal function in fragile X syndrome. Proc Natl Acad Sci 105: 4429–4434 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kitchigina V, Vankov A, Harley C, Sara SJ 1997. Novelty-elicited, noradrenaline-dependent enhancement of excitability in the dentate gyrus. Eur J Neurosci 9: 41–47 [DOI] [PubMed] [Google Scholar]
  45. Klann E, Antion MD, Banko JL, Hou L 2004. Synaptic plasticity and translation initiation. Learn Mem 11: 365–372 [DOI] [PubMed] [Google Scholar]
  46. Larson J, Jessen RE, Kim D, Fine AK, du Hoffmann J 2005. Age-dependent and selective impairment of long-term potentiation in the anterior piriform cortex of mice lacking the fragile X mental retardation protein. J Neurosci 25: 9460–9469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Lauterborn JC, Rex CS, Kramár E, Chen LY, Pandyarajan V, Lynch G, Gall CM 2007. Brain-derived neurotrophic factor rescues synaptic plasticity in a mouse model of fragile X syndrome. J Neurosci 27: 10685–10694 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Macleod LS, Kogan CS, Collin CA, Berry-Kravis E, Messier C, Gandhi R 2010. A comparative study of the performance of individuals with fragile X syndrome and Fmr1 knockout mice on Hebb-Williams mazes. Genes Brain Behav 9: 53–64 [DOI] [PubMed] [Google Scholar]
  49. Moncada D, Viola H 2007. Induction of long-term memory by exposure to novelty requires protein synthesis: Evidence for a behavioral tagging. J Neurosci 27: 7476–7481 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Moon J, Beaudin AE, Verosky S, Driscoll LL, Weiskopf M, Levitsky DA, Crnic LS, Strupp BJ 2006. Attentional dysfunction, impulsivity, and resistance to change in a mouse model of fragile X syndrome. Behav Neurosci 120: 1367–1379 [DOI] [PubMed] [Google Scholar]
  51. Morris RG 2006. Elements of a neurobiological theory of hippocampal function: The role of synaptic plasticity, synaptic tagging and schemas. Eur J Neurosci 23: 2829–2846 [DOI] [PubMed] [Google Scholar]
  52. Muddashetty RS, Kelić S, Gross C, Xu M, Bassell GJ 2007. Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome. J Neurosci 27: 5338–5348 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Nakamoto M, Nalavadi V, Epstein MP, Narayanan U, Bassell GJ, Warren ST 2007. Fragile X mental retardation protein deficiency leads to excessive mGluR5-dependent internalization of AMPA receptors. Proc Natl Acad Sci 104: 15537–15542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Napoli I, Mercaldo V, Boyl PP, Eleuteri B, Zalfa F, De Rubeis S, Di Marino D, Mohr E, Massimi M, Falconi M, et al. 2008. The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP. Cell 134: 1042–1054 [DOI] [PubMed] [Google Scholar]
  55. Navakkode S, Sajikumar S, Frey JU 2004. The type IV-specific phosphodiesterase inhibitor rolipram and its effect on hippocampal long-term potentiation and synaptic tagging. J Neurosci 24: 7740–7744 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Navakkode S, Sajikumar S, Frey JU 2007. Synergistic requirements for the induction of dopaminergic D1/D5-receptor-mediated LTP in hippocampal slices of rat CA1 in vitro. Neuropharmacology 52: 1547–1554 [DOI] [PubMed] [Google Scholar]
  57. Nguyen PV, Kandel ER 1997. Brief θ-burst stimulation induces a transcription-dependent late phase of LTP requiring cAMP in area CA1 of the mouse hippocampus. Learn Mem 4: 230–243 [DOI] [PubMed] [Google Scholar]
  58. Nimchinsky EA, Oberlander AM, Svoboda K 2001. Abnormal development of dendritic spines in Fmr1 knock-out mice. J Neurosci 21: 5139–5146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Nosyreva ED, Huber KM 2006. Metabotropic receptor-dependent long-term depression persists in the absence of protein synthesis in the mouse model of fragile X syndrome. J Neurophysiol 95: 3291–3295 [DOI] [PubMed] [Google Scholar]
  60. O'Donnell WT, Warren ST 2002. A decade of molecular studies of fragile X syndrome. Annu Rev Neurosci 25: 315–338 [DOI] [PubMed] [Google Scholar]
  61. Paradee W, Melikian HE, Rasmussen DL, Kenneson A, Conn PJ, Warren ST 1999. Fragile X mouse: Strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience 94: 185–192 [DOI] [PubMed] [Google Scholar]
  62. Park S, Park JM, Kim S, Kim JA, Shepherd JD, Smith-Hicks CL, Chowdhury S, Kaufmann W, Kuhl D, Ryazanov AG, et al. 2008. Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD. Neuron 59: 70–83 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Pfeiffer BE, Huber KM 2007. Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation. J Neurosci 27: 3120–3130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Pfeiffer BE, Huber KM 2009. The state of synapses in fragile X syndrome. Neuroscientist 15: 549–567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Qin M, Kang J, Burlin TV, Jiang C, Smith CB 2005. Postadolescent changes in regional cerebral protein synthesis: An in vivo study in the FMR1 null mouse. J Neurosci 25: 5087–5095 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Restivo L, Ferrari F, Passino E, Sgobio C, Bock J, Oostra BA, Bagni C, Ammassari-Teule M 2005. Enriched environment promotes behavioral and morphological recovery in a mouse model for the fragile X syndrome. Proc Natl Acad Sci 102: 11557–11562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Richter JD, Klann E 2009. Making synaptic plasticity and memory last: Mechanisms of translational regulation. Genes Dev 23: 1–11 [DOI] [PubMed] [Google Scholar]
  68. Ronesi JA, Huber KM 2008. Homer interactions are necessary for metabotropic glutamate receptor-induced long-term depression and translational activation. J Neurosci 28: 543–547 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Rosenberg LB 2007. Necessary forgetting: On the use of propranolol in post-traumatic stress disorder management. Am J Bioeth 7: 27–28 [DOI] [PubMed] [Google Scholar]
  70. Schütt J, Falley K, Richter D, Kreienkamp HJ, Kindler S 2009. Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities. J Biol Chem 284: 25479–25487 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Scoville WB, Milner B 1957. Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry 20: 11–21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Shapiro ML, Kennedy PJ, Ferbinteanu J 2006. Representing episodes in the mammalian brain. Curr Opin Neurobiol 16: 701–709 [DOI] [PubMed] [Google Scholar]
  73. Sharma A, Hoeffer CA, Takayasu Y, Miyawaki T, McBride SM, Klann E, Zukin RS 2010. Dysregulation of mTOR signaling in fragile X syndrome. J Neurosci 30: 694–702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Spencer CM, Alekseyenko O, Serysheva E, Yuva-Paylor LA, Paylor R 2005. Altered anxiety-related and social behaviors in the Fmr1 knockout mouse model of fragile X syndrome. Genes Brain Behav 4: 420–430 [DOI] [PubMed] [Google Scholar]
  75. Stanton PK, Sarvey JM 1984. Blockade of long-term potentiation in rat hippocampal CA1 region by inhibitors of protein synthesis. J Neurosci 4: 3080–3088 [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Stefani G, Fraser CE, Darnell JC, Darnell RB 2004. Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. J Neurosci 24: 7272–7276 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Straube T, Korz V, Balschun D, Frey JU 2003. Requirement of β-adrenergic receptor activation and protein synthesis for LTP-reinforcement by novelty in rat dentate gyrus. J Physiol 552: 953–960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Thomas MJ, Moody TD, Makhinson M, O'Dell TJ 1996. Activity-dependent β-adrenergic modulation of low frequency stimulation induced LTP in the hippocampal CA1 region. Neuron 17: 475–482 [DOI] [PubMed] [Google Scholar]
  79. Tsiouris JA, Brown WT 2004. Neuropsychiatric symptoms of fragile X syndrome: Pathophysiology and pharmacotherapy. CNS Drugs 18: 687–703 [DOI] [PubMed] [Google Scholar]
  80. Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, et al. 1991. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65: 905–914 [DOI] [PubMed] [Google Scholar]
  81. Volk LJ, Pfeiffer BE, Gibson JR, Huber KM 2007. Multiple Gq-coupled receptors converge on a common protein synthesis-dependent long-term depression that is affected in fragile X syndrome mental retardation. J Neurosci 27: 11624–11634 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Weiler IJ, Spangler CC, Klintsova AY, Grossman AW, Kim SH, Bertaina-Anglade V, Khaliq H, de Vries FE, Lambers FA, Hatia F, et al. 2004. Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses. Proc Natl Acad Sci 101: 17504–17509 [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Winder DG, Martin KC, Muzzio IA, Rohrer D, Chruscinski A, Kobilka B, Kandel ER 1999. ERK plays a regulatory role in induction of LTP by θ frequency stimulation and its modulation by β-adrenergic receptors. Neuron 24: 715–726 [DOI] [PubMed] [Google Scholar]
  84. Woo NH, Nguyen PV 2003. Protein synthesis is required for synaptic immunity to depotentiation. J Neurosci 23: 1125–1132 [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Young JZ, Isiegas C, Abel T, Nguyen PV 2006. Metaplasticity of late-LTP: A critical role for PKA in synaptic tagging. Eur J Neurosci 23: 1125–1132 [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Zhang J, Hou L, Klann E, Nelson DL 2009. Altered hippocampal synaptic plasticity in the FMR1 gene family knockout mouse models. J Neurophysiol 101: 2572–2580 [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Zola-Morgan S, Squire LR, Amaral DG 1986. Human amnesia and the medial temporal region: Enduring memory impairment following a bilateral lesion limited to field CA1 of the hippocampus. J Neurosci 6: 2950–2967 [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Zsiros V, Maccaferri G 2008. Noradrenergic modulation of electrical coupling in GABAergic networks of the hippocampus. J Neurosci 28: 1804–1815 [DOI] [PMC free article] [PubMed] [Google Scholar]

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