Fragile X Syndrome (FXS) is a neurodevelopmental disorder that is caused by the loss of function mutation of the fragile X mental retardation 1 (Fmr1) gene on the X chromosome (reviewed in O’Donnell and Warren, 2002; Santoro et al., 2012; Online Mendelian Inheritance in Man ® [OMIM] 309550) resulting in lack of fragile X mental retardation protein (FMRP) expression (Fu et al., 1991; Pieretti et al., 1991). In turn, lack of FMRP results in a number of symptoms including disorders of intellectual development, attention deficit and hyperactivity, anxiety, epilepsy, as well as particular physical features such as an elongated face and macroorchidism (Hagerman, 1996; Turner et al., 1996; O’Donnell and Warren, 2002; Hatton et al., 2006; Sullivan et al., 2006; Scerif et al., 2007). Importantly, a large proportion of individuals (25–47%) affected by FXS display autistic behaviors or a co-morbid diagnosis of autism (Kaufmann et al., 2004; Hatton et al., 2006), making FXS the only clear genetically associated form of autism. Relevant to the present investigation, FXS patients display poorer performances as compared to developmentally matched participants on a number of different visual-spatial dependent tasks (Cornish et al., 1998, 1999; Kogan et al., 2004, 2009; MacLeod et al., 2010; Van der Molen et al., 2010).

In fragile X mental retardation 1 knock-out (Fmr1 KO) mice an exaggerated form of mGluR mediated long-term depression (LTD) has been documented in hippocampal neurons (Huber et al., 2002) evidenced by elevated levels of “LTD” proteins at basal states (Nosyreva and Huber, 2006; Osterweil et al., 2010) and by the internalization of AMPA receptors (Snyder et al., 2001). Following the identification of, and much research on, LTD in Fmr1 KO mice, the prevailing opinion is that Fmrp, which binds to approximately 4% of total brain mRNA (Brown et al., 2001; Darnell et al., 2011), acts as a translational suppressor of proteins in vivo, many of which are implicated in synaptic plasticity (Bassell and Warren, 2008; Darnell et al., 2011; Bhakar et al., 2012).

It has been hypothesized that in the absence of the translational suppression functions of Fmrp, abnormally elongated spines develop and are responsible for some of the clinical manifestations of FXS such as disorders of intellectual development and audiogenic seizures (Bear et al., 2004; Krueger and Bear, 2011). Thus, intervention with antagonists that selectively target mGluR-5 has been promising in that these agents can mitigate signaling and as a result correct some of the downstream effects that occur in the absence of Fmrp. Consistent with group I mGluR-signaling as mediating prolonged LTD in Fmr1 KO mice, one study employed small interfering RNA (siRNA) specific to the Fmr1 gene sequence to demonstrate that reductions of Fmrp in dendrites of hippocampal neurons lead to an increase in the internalization of the AMPAR subunit, GluR1 (Nakamoto et al., 2007). Treatment with 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-antagonist, rescued the abnormal AMPAR trafficking, an effect not found for NMDA receptors (NMDARs). In the absence of Fmrp and following 20 days of in vitro culturing, neurons from adult Fmr1 KO mice were classified as having excess filopodia (spines with a long and thin appearance) relative to wild-type cultured neurons that had a mushroom shaped appearance with a large spine head (de Vrij et al., 2008). Treatment of Fmr1 KO neurons with two different mGluR-5 antagonists (200 μM MPEP and 300 μM fenobam) for 4 h rescued the protrusion phenotype, restoring the spine/filopodia ratio in Fmr1 KO neurons to the levels observed in wild-type neurons (de Vrij et al., 2008). Consistent with this finding, other researchers have reversed hippocampal spine elongations by using alternative mGluR-5 antagonists such as Mavoglurant (AFQ056; Levenga et al., 2011). Regarding cortical neurons, in one study, daily administration of 20 mg/kg of MPEP over the course of a week ameliorated average spine length and density in adult Fmr1 KO mice without producing significant tolerance or toxicity effects (Su et al., 2011).

Arguably the strongest support for targeting mGluR- signaling with antagonists comes from research studies that cross-bred Fmr1 KO mice with Grm5 mutant mice that have a 50% reduction of mGluR-5 expression (rather than a complete KO which would negatively impact brain function and lead to death). This procedure rescued several phenotypic aspects of the FXS mouse model. In this regard, reduction of mGluR-5 expression in Fmr1 KO mice significantly reduced hippocampal LTD, rescued the increased density of long and thin spines, reduced the elevated basal protein synthesis rates and finally, reduced audiogenic seizures (Dölen et al., 2007).

Behaviorally, Fmr1 KO mice of the hybrid strain C57Bl/6J X Friend Virus B NIH Jackson (FVB/NJ) displayed increased center square entries and duration during open field testing indicative of impulsivity and disinhibiton. Single intraperitoneal (i.p.) injection of either 10 or 30 mg/kg of MPEP rescued these deficits such that open field performance 30 min after injection was statistically indistinguishable from control mice (Yan et al., 2005).

Despite much progress with antagonism interventions, there remains a need for reliable and valid means of assessing improvement in patients receiving treatments, which are comparable to those used in animal studies. Typically human FXS studies attempting to assess progress in various cognitive domains have produced inconsistent findings as a result of outcome measures that are confounded by floor and ceiling effects (Berry-Kravis et al., 2006). We previously showed that Hebb-Williams (H-W) mazes are a viable visual-spatial assay for use with both FXS participants and KO mice. Both populations exhibit similar behavioral impairments (i.e., more errors than controls) (MacLeod et al., 2010). More recently, we demonstrated that Fmrp intact mice, but not Fmr1 KO mice, evidenced upregulations of post-synaptic density-95 (PSD-95) following completion of the H-W mazes (Gandhi et al., 2014). Given that PSD-95 has been hypothesized as a key protein ostensibly involved in both AMPAR regulation and dendritic spine structure (Keith and El-Husseini, 2008), our data suggests that PSD-95 is a good candidate protein in order to examine the effects of antagonism treatment in Fmr1 KO mice.

Thus, for the present study, we hypothesized that MPEP treatment of Fmr1 KO mice would result in reversal of the previously described deficit (i.e., significantly fewer errors) on the H-W mazes as well as a reversal of the PSD-95 protein deficit relative to saline treated controls. We also report results from a manipulation check (i.e., a marble burying assay) experiment that confirms that the MPEP treatment remains active throughout maze testing. Specifically, when MPEP is pharmacologically active, marble burying (a repetitive behavior) is significantly reduced without a corresponding decline in locomotor activity (Thomas et al., 2012).

A 2 × 7 × 5 mixed measures ANOVA was conducted to evaluate the effects of treatment (MPEP; saline) as the between-groups measure and repeated measures of both maze (seven levels) and trial (five levels) on the latency to complete the H-W mazes. There was a main effect for maze, F_{(5, 94)} = 3.01, p = 0.01, partial η² = 0.13, and for trial, F_{(3, 67)} = 60.12, p < 0.001, partial η² = 0.75, but not for treatment, F_{(1, 20)} = 1.45, p = 0.24, partial η² = 0.07, indicating that the latency to complete the mazes did not differ between MPEP and saline treated mice. There was also a significant interaction between treatment and maze, F_{(5, 94)} = 4.06, p = 0.003, partial η² = 0.17, as well as maze and trial F_{(9, 189)} = 1.89, p = 0.05, partial η² = 0.08. However, the interaction between treatment and trial F_{(3, 67)} = 1.07, p = 0.37, partial η² = 0.05 was not significant. Likewise, the three-way interaction between treatment, maze, and trial was not significant F_{(9, 189)} = 1.01, p = 0.44, partial η² = 0.05.

Bonferroni corrections were made to the α-level of 0.05 before exploring simple main effect analyses of treatment within maze, resulting in p < 0.007 (0.05/7 = 0.007) for significance. These analyses indicated that there were differences in the latencies between MPEP and saline treated mice on maze #9, F_{(1, 20)} = 5.08, p = 0.04, partial η² =0.20, maze #11, F_{(1, 20)} = 5.36, p = 0.03, partial η² = 0.21 and maze #12, F_{(1, 20)} = 6.08, p = 0.02, partial η² = 0.23. However, given the adjustment to guard against Type I error, these differences were deemed not statistically significant. Given the similar latencies to complete maze running between drug and vehicle groups, these findings are consistent with previous research indicating that MPEP treatment does not significantly reduce locomotor activity (Figure 2A).

Bonferroni corrections were made to the α-level of 0.05 before exploring simple main effect analyses of trial within maze, resulting in p < 0.007 (0.05/7 = 0.007) for significance. These analyses indicated that there were differences in the latencies between trials on maze #2, F_{(4, 17)} = 7.64, p = 0.001, partial η² =0.64, maze #4, F_{(4, 17)} = 23.36, p = 0.000001, partial η² = 0.85, maze #5, F_{(4, 17)} = 6.22, p = 0.003, partial η² = 0.59, maze #8, F_{(4, 17)} = 11.05, p = 0.0001, partial η² =0.72, maze #9, F_{(4, 17)} = 11.26, p = 0.0001, partial η² = 0.73, maze #11, F_{(4, 17)} = 12.54, p = 0.0001, partial η² = 0.75, but not on maze #12, F_{(4, 17)} = 4.68, p = 0.01, partial η² = 0.52.

Pairwise comparisons on the latency data adjusted to control for the effects of comparing mean trial differences within each maze (α = 0.05/60 = 0.0008) showed that on maze #2, Fmr1 KO mice were significantly slower on trial 1 relative to completion times on trials 3 and 4. In addition they were significantly slower on trial 2 compared to trial 4. On maze #4, mice were slower on trial 1 compared to their completion times on trials 3, 4 and 5; whereas trial 2 took longer to complete than trial 4. On maze #5, mice took longer to complete trial 1 compared with run times on trials 4 and 5. Subsequently, mice completed maze #8 slower on trial 1 compared to trials 2, 3, 4 and 5, whereas trial 4 was completed faster than trial 2. During maze #9, latencies were again slower on trial 1 compared with trials 3, 4 and 5. Finally on maze #11, run times were quicker on trials 2, 3 and 5 relative to trial 1. Thus, despite some variability in the trial by maze interaction data, pairwise comparisons indicated latencies were longest for trial 1 and in general, tended to decrease with increased repetition, as would be expected if mice were learning the maze configuration and motivated to obtain the food reward.

Regarding error data, a 2 × 7 × 5 mixed measures ANOVA was conducted to evaluate the effects of treatment (MPEP; saline) as the between-groups measure and repeated measures of both maze (seven levels) and trial (five levels) on the number of errors committed on the H-W mazes. There was a main effect for treatment F_{(1, 20)} = 63.71, p < 0.001, partial η² =0.76, for maze, F_{(4, 84)} = 4.13, p = 0.004, partial η² = 0.17, indicating that the number of errors made on the mazes differed between MPEP and saline treated mice. There was also a main effect for trial, F_{(3, 65)} = 24.43, p < 0.001, partial η² = 0.55. There was a significant interaction between treatment and maze, F_{(4, 84)} = 2.82, p = 0.03, partial η² = 0.12, whereas the interaction between treatment and trial F_{(3, 65)} = 2.22, p = 0.09, partial η² = 0.10 approached significance. However, the interaction between maze and trial F_{(9, 188)} = 1.32, p = 0.23, partial η² = 0.06 was not significant. Likewise, the three-way interaction between treatment, maze, and trial was not significant F_{(9, 188)} = 0.94, p = 0.50, partial η² = 0.04.

Bonferroni corrections were made to the α-level of 0.05 before exploring simple main effect analyses of treatment within maze, resulting in p < 0.007 (0.05/7 = 0.007) for significance. These analyses indicated that there were significantly less errors committed by MPEP treated mice on maze #2, F_{(1, 20)} = 6.21, p = 0.02, partial η² = 0.24, maze #4, F_{(1, 20)} = 5.94, p = 0.02, partial η² = 0.23, maze #8, F_{(1, 20)} = 8.67, p = 0.007, partial η² = 0.30, maze #9, F_{(1, 20)} = 7.53, p = 0.01, partial η² = 0.27, maze #11, F_{(1, 20)} = 30.80, p = 0.00002, partial η² = 0.61, and maze #12, F_{(1, 20)} = 17.28, p = 0.0005, partial η² = 0.46, However, when adjustments were made to guard against Type I error, MPEP treated mice committed significantly fewer errors on only three mazes relative to saline controls (mazes #8, 11, 12). Combined, these data indicate that on several of the H-W mazes, MPEP administration results in significantly less errors than in Fmr1 KO mice treated with saline only (Figure 2B).

Bonferroni corrections were made to the α-level of 0.05 before exploring simple main effect analyses of treatment within trial, resulting in p < 0.01 (0.05/5 = 0.01) for significance. MPEP administration resulted in significantly fewer errors on trial 1, F_{(1, 20)} = 54.95, p = 0.0000004, partial η² = 0.73, trial 2, F_{(1, 20)} = 17.16, p = 0.001, partial η² = 0.46, trial 4, F_{(1, 20)} = 21.62, p = 0.0002, partial η² = 0.52, and trial 5, F_{(1, 20)} = 25.97, p = 0.0001, partial η² = 0.57. Unexpectedly, treatment had no effect on the mean errors observed on trial 3, F_{(1, 20)} = 4.43, p = 0.05, partial η² = 0.18. Thus, MPEP treatment reduces errors committed on four out of five trials with the biggest impact (as reflected by effect size) occurring on the first trial (Figure 3).

Several Mann-Whitney U-tests were conducted to evaluate whether MPEP remained at physiologically active levels during the experiments. Bonferroni corrections were made to the α-level of 0.05 before performing these tests, resulting in p < 0.007 (0.05/7 = 0.007) for significance. The results of the Mann-Whitney U-tests were in the expected direction and significant such that MPEP treated mice were found to bury significantly more marbles than saline treated controls for maze #2, z = −3.306, p = 0.001 (MPEP average rank = 6.95; Saline = 16.95), maze #4, z = −3.31, p = 0.001 (MPEP average rank = 6.95; Saline = 16.05), maze #8, z = −3.30, p = 0.001 (MPEP average rank = 6.95; Saline = 16.05), maze #9, z = −2.74, p = 0.006 (MPEP average rank = 7.73; Saline = 15.27), maze #11, z = −3.34, p = 0.001 (MPEP average rank = 6.95; Saline = 16.05), and maze #12, z = −3.56, p < 0.001 (MPEP average rank = 6.59; Saline = 16.41). With the adjusted level of α, the number of marbles buried was not statistically different between MPEP and saline treated mice for maze #5, z = −2.58, p = 0.01 (MPEP average rank = 7.95; Saline = 15.05). Taken together, the marble burying assay following the completion of each of the H-W test mazes confirmed that the MPEP treatment was physiologically active during the test phases (Figure 4).

Independent sample t-tests were performed to evaluate the hypothesis that mGluR-5 antagonist treatment could selectively rescue hippocampal PSD-95 protein levels. Hippocampal β-tubulin levels were also measured because this housekeeping protein was not expected to vary with treatment condition. Bonferroni corrections were made to the α-level of 0.05 before performing these tests, resulting in p < 0.025 (α = 0.05/2 = 0.025) for significance. The t-tests indicated that PSD-95 levels were significantly higher in MPEP treated mice compared with vehicle condition, t₍₂₀₎ = 3.00, p = 0.007, 95% CI [0.064, 3.56], whereas there were no differences in β-tubulin levels between MPEP and vehicle treated mice, t₍₂₀₎ = 0.851, p = 0.40, 95% CI [−0.80, 1.89]. The effect size as reflected by, η², indicated that 31% of the variance in PSD-95 levels was accounted for by whether or not mice received MPEP/vehicle treatment whereas only 0.03% of the variance in β-tubulin levels was accounted for the treatment. These data suggest that mGluR-5 antagonism has an augmenting affect on the levels of the scaffolding protein PSD-95 (Figure 5).

Similar to the group data from Gandhi et al. (2014; that comprised the entire sample of animals) a correlation of all Fmr1 KO mice, irrespective of treatment, indicated there was a negative association between PSD-95 levels and mean total errors on the H-W mazes, r₍₂₀₎ = −0.40, p = 0.03, r² = 0.16 (Figure 6). This association was not evident when examining the correlation between β-tubulin levels and mean total errors from Fmr1 KO mice, r₍₂₀₎ = −0.26, p = 0.12, r² = 0.06. Within treatment groups, there were no relationships between the PSD-95 levels of MPEP treated mice and mean total errors, r₍₉₎ = −0.042, p = 0.45, r² = 0.01, nor saline treated mice and mean total errors, r₍₉₎ = 0.28, p = 0.21, r² = 0.08. In addition, after adjusting the α-levels to control for repeated tests, (0.05/4 = 0.012) only the initial correlation consisting of the entire sample of Fmr1 KO mice trended towards significance. Collectively, these data confirm that as PSD-95 levels increase, mean errors on the H-W mazes decrease and vice versa.

FXS is a debilitating mental, physical, and behavioral condition that occurs due to lack of expression of the Fragile X Mental Retardation 1 protein (FMRP; reviewed in Santoro et al., 2012). The altered expression results in a number of characteristic symptoms including disorders of intellectual development and frequently co-morbid autism spectrum disorder. Visual-spatial impairment is part of the cognitive profile in FXS and was the focus of the present investigation. Despite a common finding in the research literature that hippocampal lesions impair performance on tasks of spatial navigation and learning (Morris et al., 1982; Sutherland et al., 1982; Jarrard, 1993; Hock and Bunsey, 1998; Lee and Kesner, 2003; Clark et al., 2005; Okada and Okaichi, 2009), inconsistent results have been reported when testing Fmr1 KO mice. These differences may be a function of variability in the background strain used or the assays employed. In the present study we employed maze learning tasks, the H-W mazes, previously shown to be sensitive to detecting dorsal hippocampal deficits (Shore et al., 2001; Rogers and Kesner, 2006) including in a murine model of FXS, Fmr1 KO mice. Concomitant with greater errors committed by Fmr1 KO as compared to wild type mice (MacLeod et al., 2010), PSD-95, a hippocampal protein involved in synaptic plasticity and a target of Fmrp, is selectively upregulated in wild type but not KO mice (Gandhi et al., 2014). We demonstrate here that a selective antagonist for mGluR-5, MPEP, reverses both behavioral deficits in Fmr1 KO mice, as evidenced by fewer errors in treated vs. saline treated animals on most H-W maze problems, as well as the molecular deficit of interest, that is, PSD-95 levels. These results provide support for the importance of mGluR-5 signaling generally, and PSD-95, in particular, in the pathophysiology of FXS and autism spectrum disorder.

Although the molecular mechanisms of synapse modifications at dendritic spines are unknown, one perspective is that certain scaffolding proteins maintain the long-term transmission efficiency of a synapse (Ehrlich and Malinow, 2004; McCormack et al., 2006). In this scenario, scaffolding proteins are thought to serve as placeholders or slot proteins for receptors such as AMPARs. PSD-95 has been proposed to possess many qualities of a slot protein (Schnell et al., 2002) because it is more stable than other post-synaptic density (PSD) proteins such as CaMKIIα, CaMKIIβ, GluR2 or Stargazin, consistent with a role in regulating the PSD (Sturgill et al., 2009). Levels of PSD-95 were reported to be redistributed to dendrites in the visual cortex following eye opening in litters of rodents, and these changes lasted upwards of 6 h and were contingent on sustained environmental experience (Yoshii et al., 2003). Moreover, changes in the sizes of individual PSDs over days were associated with changes in PSD-95 retention times and PSD-95 increased with developmental age and dropped sharply following sensory deprivation (Gray et al., 2006). Importantly, in FXS, there is evidence that PSD-95 is dysregulated. Specifically, increased translational levels were observed during basal states in Fmr1 KO as compared to wild-type mice as well as relatively low protein levels following stimulus induction in this genotype. PSD-95 mRNA transcripts were also found to selectively deteriorate in the hippocampus but not in the cortex or cerebellum of Fmr1 KO mice (Todd et al., 2003; Muddashetty et al., 2007; Zhu et al., 2011).

Pharmacological treatments blocking mGluR-5 receptors can stabilize basal protein translation levels and this approach has been hypothesized as a means of ameliorating some of the core symptoms of FXS, including disorders in intellectual development (Dölen and Bear, 2005; Bhakar et al., 2012). In studies using drosophila KO (dfmr1) and Fmr1 KO murine models, the use of mGluR-5 antagonists has been successful in correcting many features of FXS including elevated and inappropriately expressed protein levels at basal states, decreasing frequency of audiogenic seizures, reversing excessive AMPA internalization, reducing the number of abnormally thin dendritic spines, and reversing behavioral/learning deficits (McBride et al., 2005; Yan et al., 2005; Nakamoto et al., 2007; de Vrij et al., 2008; Pan et al., 2008; Choi et al., 2010; Osterweil et al., 2010; Levenga et al., 2011; Su et al., 2011; Tauber et al., 2011). Despite the rescue of many phenotypic features of FXS, the identification of the specific proteins underlying these functions remains to be elucidated. Theoretically, the stabilization of PSD-95 protein in Fmr1 KO mice would allow for improved local regulation during periods of synaptic plasticity while learning the H-W mazes.

Our Western blot analyses following completion of the H-W mazes revealed that MPEP treated mice had statistically higher PSD-95 protein levels. This effect was specific to PSD-95 since levels of the control protein (β-tubulin) remained unchanged across treatment conditions. Thus, our findings suggests that PSD-95 protein deficits can be rescued by targeting mGluR-5 receptors. An additional implication pertains to the broader question of “when” it is appropriate to intervene with pharmacological treatment. As FXS is a developmental disorder, the vast majority of animal model studies have targeted intervention at the embryonic stages or very early in post-natal life. Conceptually, it is of great interest to determine if the FXS phenotype can be corrected after symptom onset. If not, it would suggest that a critical therapeutic window has been missed and argue against the idea that the symptoms of FXS are caused by ongoing irregularities of synaptic signaling (Michalon et al., 2012). This question was addressed in a study examining Fmr1 KO mice aged 4–5 weeks with anatomically developed and highly plastic brains, corresponding to young adults. Specifically, treatment with an mGluR-5 inhibitor corrected learning and memory deficits in an inhibitory avoidance paradigm, improved dendritic spine abnormalities, and ameliorated elevated Extracellular signal-regulated kinase (ERK) and mTOR kinase activation (pathways previously shown to underlie the pathophysiology of FXS). Our data, which suggest reversal of molecular and behavioral deficits, are consistent with these findings (Michalon et al., 2012). In addition, since the Fmr1 KO mice in our study were 12 weeks or older before beginning behavioral testing, our findings further demonstrate that a model of the FXS phenotype can be corrected in aged mice roughly corresponding to adulthood.

The behavioral data from the H-W mazes were analyzed according to two dependent variables of interest, latency and error. Regarding the former, analyses of the treatment by maze interaction indicated that there were differences in the latency between MPEP and saline treated mice on several mazes. Owing to high levels of variability in the runs times, faster completion times by MPEP treated mice were not statistically different from controls. However, the similar latency to complete mazes between drug and vehicle groups indicates that our data are consistent with previous research demonstrating that MPEP treatment does not adversely affect locomotor activity (Yan et al., 2005; Silverman et al., 2010; Mehta et al., 2011; Thomas et al., 2012). Collapsed across treatment, we also observed that latency of maze completion was longest for trial 1 and generalizing across mazes, tended to decrease with increased repetition. Thus, both groups of mice were capable of improving their latency performance with increased exposure to the mazes.

Consistent with our hypothesis, on mazes deemed more challenging (#8, 9, 11, 12; Shore et al., 2001), MPEP treated mice made significantly fewer errors (i.e., #8, 11, 12). When examining the behavioral performance of the mazes deemed more difficult, on maze #8, saline treated mice continued to explore previously unsuccessful routes towards the goal box whereas MPEP treated mice demonstrated a reduction in errors over trials. Counterintuitively, there were no differences between drug and saline treated mice on maze #9, which may reflect the variability in the data set or a lack of difficulty of this maze for this background strain. Qualitatively, on mazes #11 and #12, saline treated controls committed more perseverative errors, circling isolated and removed barriers from the goal box, thereby getting stuck in unsuccessful “loops”. That MPEP treated mice did not commit such responses suggests that MPEP treatment may correct perseveration, a common cognitive feature of FXS (Hooper et al., 2008).

Finally, the treatment by trial interaction data revealed that MPEP treated mice made significantly fewer errors on trials #1, 2, 4, and 5 relative to controls. Given that the largest effect size occurred on the first trial, this suggests that MPEP may also have corrected impulsive responding, which is another feature that is commonly observed in FXS (Hagerman, 2002).

The pharmacological efficacy of MPEP was confirmed with a marble burying assay immediately following the test phase in order to validate our findings. Marble burying, a repetitive behavior, has been shown to be decreased following the administration of Grp I mGluR antagonists (Spooren et al., 2000; Thomas et al., 2012). In the present investigation, MPEP treated mice buried significantly fewer marbles than controls after the completion of all mazes (thus confirming drug efficacy), with the exception of #5. It is unclear why fewer marbles relative to controls were buried here, however as there were no error differences between treatment groups for this maze; interpretation of our findings is not affected by this result.

Overall, our correlational findings are inconclusive and merit further investigation. Although we replicated a negative correlation between PSD-95 levels and mean errors for the entire sample of mice, as found in our previous study (Gandhi et al., 2014), we did not demonstrate a statistically significant relationship within the treatment groups. We suspect that larger sample sizes of mice will provide the necessary power to allow us to characterize this relationship appropriately.

Whether pharmacological studies of mGluR-5 antagonists in mouse models of FXS will translate into effective treatments for human patients remains to be determined. To date, only two studies have been completed in patients affected by FXS. A pilot study was conducted to determine pharmacokinetics and side effects of a single dose trial of the mGluR-5 antagonist, fenobam, to 12 male and female FXS patients (Berry-Kravis et al., 2009). Pre/post outcome measures included prepulse inhibition (PPI) and the continuous performance test (CPT) to assess sensory gating, attention and inhibition. The results indicated there were no adverse reactions to the fenobam administration and PPI improved by at least 20% in half of the sample relative to baseline. By comparison, performance on the CPT did not improve although this finding was attributable to ceiling effects. The other study employing an mGluR-5 antagonist was conducted using AFQ056 in 30 male FXS patients ranging in age from 18–35 (Jacquemont et al., 2011). These researchers initially did not find any improvement in behavioral symptoms of FXS following treatment as assessed by the Aberrant Behavior Checklist-Community Edition (ABC-C). However, a subset of the patients who had the full Fmr1 promoter methylation and no detectable Fmr1 mRNA improved significantly more on the ABC-C and the Repetitive Behavior Scale following treatment compared with placebo. Since those patients with partial promoter methylation did not show behavioral improvement following AFQ056 treatment, the authors posited that mGluR-5 antagonism might be better suited for FXS patients with full methylation at the Fmr1 promoter. mGluR-5 antagonists are not the only receptor mechanism/molecular target under investigation. FXS is a complex neurodevelopmental disorder and Fmrp regulates signaling by other receptors as well. Therefore, antagonism of Group I mGluR signaling is not likely to produce beneficial therapeutic effects for every patient. Moreover, there are other aspects of the FXS phenotype that are unrelated to mGluR function. Other research has focused on other targets and agents such as GABA-A and B receptors, ampakines, brain derived neurotrophic factor (BDNF), aripiprazole, lithium and intracellular signaling pathways via phosphatase and kinase inhibitors. In all likelihood, patients will display varied outcomes to different targeted treatments based on interplay between genetics, intracellular neuronal pathways, and synaptic function (Gross et al., 2012).

FXS is the most common single gene disorder associated with autism spectrum disorder (Hagerman et al., 2010) and there are numerous commonalities between FXS and autistic spectrum disorder. Similar to FXS, many autistic patients suffer from seizure disorder and cognitive impairment (Canitano, 2007). There is also delayed language acquisition and repetitive behaviors (Hagerman, 1996) and 25–47% of FXS patients have a diagnosis of autism (Kaufmann et al., 2004; Hatton et al., 2006). Models of FXS are potentially advantageous to autism because Fmrp controls the translation of plasticity proteins implicated in autism such as neuroligins and SHANK proteins (Darnell et al., 2011). Moreover, low levels of FMRP relative to controls have been reported in autism spectrum disorder (Fatemi and Folsom, 2011). Using a Black and Tan BRachyury TBR T+ Itpr3tf/J (BTBR) murine model of autism, one study reported that MPEP treatment ameliorated repetitive self-grooming behavior without significant sedating side effects (Silverman et al., 2010). Likewise, in a valproic acid (VPA) murine model of autism, MPEP reduced excessive self-grooming as well as marble burying behavior (Mehta et al., 2011). Although further study is needed, preliminarily, MPEP appears to be a suitable pharmacological intervention for both FXS and autistic disorder. Our findings indicate that MPEP treatment can reverse PSD-95 protein deficits and errors on more complicated H-W test mazes. Given the significant phenotypic overlap between FXS and autism as well as the lack of a viable behavioral assay to test symptoms improvement in the autism field, the use of the H-W mazes with murine models of autism spectrum disorder appears promising and warrants further investigation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.