Rescue strategy and rationale
Because both the human FMR1 and GRM5 genes have functional homologues in the mouse (Fmr1 and Grm5), we were able to generate Fmr1 knockout mice with reduced expression of mGluR5, the major Gp1 mGluR in the forebrain. By crossing two mutant lines, the functional relationship between two protein products can be examined; genetic “rescue” occurs when single mutant phenotypes are attenuated in the double mutant. The power of this approach in the murine model is two-fold: (1) it is a precise and selective method to reduce mGluR5 function, (2) it permits analysis of diverse phenotypes across many developmental time points, using a variety of experimental methods both in vitro and in vivo. In addition, unlike simpler genetically modifiable organisms, endophenotypes identified in this mammalian model can serve not only to establish genetic interaction, but may also bear direct relation to the phenotype in humans with the disease.
mutant mice (Consortium, 1994
) were crossed with Grm5
mutant mice (Lu et al., 1997
) to produce Fmr1
knockout animals with a selective reduction in mGluR5 expression (Figure S2
). To increase the therapeutic relevance, we concentrated on animals with a 50% reduction in mGluR5 rather than a complete knockout (which impairs brain function (Jia et al., 1998
; Lu et al., 1997
)). Littermates with 4 different genotypes were created in our cross: Wild type [Fmr1
(+/−)], and the knockout/heterozygote cross [Fmr1
(+/−)]; these animals are termed WT, KO, HT, and CR, respectively. In all crossings, animals were on the C57Bl/6J clonal background.
The key question that we address in this study if a reduction of mGluR5 expression will correct diverse fragile X mutant phenotypes, as predicted by the mGluR theory (Figure S1
). Our genetic rescue strategy rests on the assumption that the FMRP-regulated “readout” of mGluR5 activation is modulated by Grm5
gene dosage. One FMRP-regulated consequence of mGluR5 activation is hippocampal long-term synaptic depression (LTD), which is approximately doubled in the KO (Huber et al., 2002
). It had already been established that there is a significant effect of mGluR5 expression level on LTD in the C57Bl/6J WT background (Huber et al., 2001
), and we confirmed in the present study that a 50% reduction in mGluR5 protein expression also significantly reduces LTD in the Fmr1
KO background (Figure S2
). We therefore went on to examine diverse phenotypes with relevance to the human disorder, including experience-dependent cortical development, hippocampus-dependent memory, altered body growth, seizure, and postpubertal macroorchidism. All analyses of these mice were performed “blind”, without experimenter knowledge of the genotype. Note that in each experiment, three outcomes were possible: the reduced Grm5
gene dosage could ameliorate, exacerbate, or have no effect on Fmr1
Altered ocular dominance plasticity in Fmr1 KO mice is rescued by reducing mGluR5 expression
Ocular dominance (OD) plasticity in visual cortex, elicited by temporary monocular deprivation (MD), is the classic example of how experience modifies the brain during critical periods of development. Here, we use this paradigm to study the interaction of genes and environment in a disease model.
Visually evoked potentials (VEPs) were recorded in visual cortex of awake mice (), as described previously (Frenkel and Bear, 2004
). We initially assessed absolute levels of visual responsiveness across genotypes on postnatal day (P) 28 and found no difference (). Additional mice were studied before and after MD begun on P28. Previous studies using the chronic VEP method have shown how visual responses evolve during the course of MD (Figure S3
). Closure of the contralateral eyelid initially causes depression of responses to the deprived- (contralateral-) eye (apparent at 3 d. MD), followed by potentiation of nondeprived- (ipsilateral-) eye responses (apparent by 7 d. MD) (Frenkel and Bear, 2004
). Because they are recorded chronically, changes in VEPs for each animal can be conveniently described by two values: the fractional change from baseline in contralateral-eye response, and the fractional change from baseline in the ipsilateral-eye response. For reference, average effects (± SEM) of 3 and 7 days of MD in WT mice from a previous study (Frenkel and Bear, 2004
) appear in Figure S3
Genetic rescue of OD plasticity phenotype in FXS
In the current study we also found that the response to 3 d MD in WT mice was dominated by deprived-eye depression, as expected. In KO littermates, however, the response to brief MD was characterized by substantial open-eye potentiation, reminiscent of what happens in WT mice after longer periods of MD. On the other hand, the HT mice showed a “hypoplastic” response to MD, as they lacked significant deprived-eye depression. However, crossing the two mutant mice resulted in a phenotype very similar to WT that was again dominated by deprived-eye depression ().
Plots of the average (± SEM) fractional changes after 3 d MD in the 4 genotypes are shown in . The KO mice displayed increased plasticity compared to the WT (MANOVA WT:KO, P = 0.011); HT mice displayed diminished plasticity compared to WT (MANOVA WT:HT, P = 0.013); CR mice showed a rescue of the KO phenotype and were not significantly different from WT (MANOVA WT:CR, P = 0.8268, KO:CR P = 0.037, HT:CRS P = 0.161).
Since the KO and HT mutations affected OD plasticity in opposite directions, one could question whether the CR phenotype reflects rescue or the simple addition of two independent effects. However, a compound phenotype would be the absence of deprived-eye depression (the effect of reducing mGluR5) and an exaggeration of open-eye potentiation (the effect of reducing FMRP). Instead, we observe a phenotype in the CR mice that is significantly different from KO mice, and not significantly different from WT. Thus, reducing mGluR5 by 50% corrects the defect in plasticity caused by the absence of FMRP.
Density of dendritic spines on cortical pyramidal neurons is increased in Fmr1 KO and rescued by reducing mGluR5 expression
Abnormalities in dendritic spines, the major targets of excitatory synapses in the brain, have long been associated with various forms of human mental retardation, including FXS. The increased spine density phenotype observed in humans has been recapitulated in the Fmr1
KO mouse (reviewed by (Grossman et al., 2006
). Because one protein synthesis-dependent consequence of activating Gp1 mGluRs on cortical neurons in vitro
is an increase in the density of long, thin spines (Vanderklish and Edelman, 2002
), we hypothesized that FMRP and mGluR5 antagonistically regulate dendritic spine density in vivo
We chose to examine this question in layer 3 pyramidal neurons of binocular visual cortex at P30, since we had established that OD plasticity at this age was altered in the Fmr1 KO mice. Dendritic spine density was analyzed separately in apical and basal branches across the four genotypes, using the Golgi-Cox silver staining method (). We observed a highly significant increase in total dendritic spine density in the KO, readily apparent as a rightward shift in the cumulative probability histogram (). Reducing mGluR5 expression had no effect on spine density in the HT mice, but the fragile X phenotype was completely rescued in the CR mice (Apical, Kruskal-Wallis test P < 0.0001; Kolmogorov-Smirnov test WT:KO P < 0.0001; WT:HT P = 0.3920; CR:WT P = 0.4407; CR:KO P < 0.0001; Basal, Kruskal-Wallis test P < 0.0001; Kolmogorov-Smirnov test WT:KO P < 0.0001; WT:HT P > 0.9999; CR:WT P > 0.9999; CR:KO P < 0.0001).
Genetic rescue of dendritic spine phenotype in FXS
We also performed a segmental analysis of spine density across the four genotypes. Consistent with previous observations, we observed an inverted U shaped distribution of synapses in both apical and basal branches across all genotypes. However, as shown in , the density of spines was uniformly increased in the Fmr1 KO and rescued in the CR (Repeated measures ANOVA: apical distance P < 0.0001, apical distance* genotype P < 0.0001, apical genotype P < 0.0001, basal distance P < 0.0001, basal distance*genotype P = 0.0181, basal genotype P < 0.0001; ANOVA genotype: apical, basal, 10–100, in 10 μm segments P < 0.0001; unpaired t-tests apical, basal, 10–100, in 10 μm segments WT:KO P < 0.05, WT:HT P > 0.05, WT:CR P > 0.05, KO:CR P < 0.05). These results suggest that neither the Fmr1 KO phenotype, nor the rescue by selective reduction in gene dosage in the CR, reflects a redistribution of synapses within the segment.
Increased basal protein synthesis in hippocampus of Fmr1 KO mice is rescued by reducing mGluR5 expression
A previous study reported an elevated basal rate of in vivo
protein synthesis in the hippocampus of Fmr1
KO mice (Qin et al., 2005
). We asked if this difference could also be observed in hippocampal slices in vitro
by examining the incorporation of 35
S-methionine/cysteine into new protein. We observed a significant effect of genotype on protein synthesis (). The increased protein synthesis seen in KO hippocampus was prevented by selective reduction in mGluR5 gene dosage.
Genetic rescue of protein synthesis phenotype in FXS
Electrophoretic separation of radiolabeled translation products () suggests that increased protein synthesis in the KO is not limited to one or few predominant protein species, but rather extends across a broad range of resolved molecular weights. Because the rate of protein synthesis was unaffected in the HT mice relative to WT, the rescue in the CR mice is unambiguous, and does not simply reflect an offsetting decrease in synthesis of a separate pool of proteins.
Inhibitory avoidance extinction is exaggerated in Fmr1 KO mice and rescued by reducing mGluR5 expression
Although humans with FXS show mental retardation in the moderate to severe range, prior studies of cognitive performance in Fmr1
KO mice on the C57Bl/6J clonal background have revealed only subtle deficits (Bernardet and Crusio, 2006
). Consistent with these observations, we found that acquisition of one-trial inhibitory avoidance (IA), a hippocampus-dependent memory, did not differ from normal in the Fmr1
KO mice. However, we were inspired to additionally investigate IA extinction
(IAE) by a recent report that this process requires protein synthesis in the hippocampus (Power et al., 2006
). We discovered that IAE is exaggerated in the Fmr1
KO mouse, and that this phenotype is corrected by reducing expression of mGluR5.
Adult mice of all four genotypes were given IA training, followed at 6 and 24 hours by IAE training (). For each animal, we measured the latency to enter the dark side of the box on the first trial (baseline), the latency 6 hours later (post-acquisition) to assess IA memory, and again at 24 and 48 hours (post-extinction 1 and 2, respectively) to assess IAE. As shown in , animals of all four genotypes showed both significant IA acquisition at 6 h and extinction by 48 h. A global statistical test suggested that the pattern of learning across time varied across genotypes (repeated measures ANOVA genotype *time P = 0.0239). As shown in , these differences are likely due to extinction rather than acquisition of inhibitory avoidance. At 6 h, there was no difference across genotypes in latency to enter (6 h ANOVA P = 0.1525); however, at 24 h KO mice showed significantly shorter latencies, suggesting exaggerated extinction in the absence of FMRP. This phenotype was rescued by selective reduction in mGluR5 gene dosage in the CR mice (24 h ANOVA P = 0.0013; t-tests: WT:KO P < 0.0001,WT:HT P = 0.8251, WT:CR P = 0.1156, KO:CR P = 0.0132).
Genetic rescue of behavioral learning and memory phenotype in FXS
Because the primary aim of this study is to examine genetic interaction between Fmr1 and Grm5, we also performed a multivariate analysis which takes into consideration both acquisition and extinction as they vary across genotypes. As shown in , KO animals showed significant difference in 24 h latency (memory retention) as it varied with 6 h latency (memory acquisition), and this difference was rescued by the selective reduction in mGluR5 gene dosage in the CR mice (MANOVA for genotype 6:24 P = 0.0054, MANOVA WT:KO P = 0.0005, WT:HT 0.0785, KO:CR P = 0.0490, WT:CR P = 0.1863). The difference in retention across all genotypes was not significant by 48 h. Regardless of whether this KO phenotype reflects exaggerated extinction or diminished stability of the formed memory, there clearly is a significant genetic interaction between Fmr1 and Grm5: The Fmr1 KO phenotype is rescued by the selective reduction in mGluR5 expression.
Audiogenic seizures and accelerated body growth in Fmr1 KO mice are rescued by reducing mGluR5 expression
Consistent with neurological findings in fragile X patients, previous studies have shown increased seizure susceptibility in the Fmr1
KO mouse, using both in vitro
and in vivo
epilepsy models (Bernardet and Crusio, 2006
). We employed the audiogenic seizure (AGS) paradigm, which shows a robust phenotype in Fmr1
KO mice and exhibits developmental changes consistent with epilepsy in human FXS (Yan et al., 2005
). Because C57Bl/6 WT mice are normally seizure resistant (Robertson, 1980
), seizures in the KO mice are a specific consequence of the absence of FMRP. As shown in Table S1
, significant differences in AGS susceptibility were observed across the four genotypes examined. WT and HT mice showed zero incidences of AGS, as expected, whereas 72% of the KO mice had a seizure in response to the tone (Mann-Whitney U test WT:KO P < 0.0001). This mutant phenotype was significantly attenuated in the CR mice (Mann-Whitney U test CR:KO P = 0.028). Thus chronic reduction of mGluR5 gene dosage in KO mice produced a substantial rescue of the seizure phenotype that is caused specifically by the lack of FMRP.
Children with FXS show accelerated prepubescent growth (Loesch et al., 1995
). We discovered that this phenotype is recapitulated in the KO mouse, and is rescued by reducing mGluR5 gene dosage (Figure S4
). At weaning (P20-21), animals from all four genotypes had similar body weights, but by P26 KO mice showed a slight (10%) but significant increase in body weight as compared to WT animals at the same age. This difference was not observed in either the HT or CR mice (ANOVA P = 0.048, post-hoc t-tests WT:KO P = 0.017, KO:CR P = 0.004, CR:WT P = 0.818). The WT:KO body weight difference was maximal at P30 (~15%) when it was again rescued by a reduction in mGluR5 gene dosage in the CR mice (ANOVA P = 0.005, post hoc t-tests WT:KO P = 0.020, KO:CR P = 0.001, CR:WT P = 0.555). As in humans, the KO growth increase in mice was no longer apparent after adolescence (P45).
Macroorchidism in Fmr1 KO mice is not rescued by reducing mGluR5 expression
Children with FXS (and KO mice) have dysmorphic features, including postadolescent macroorchidism. Testes express Gp1 mGluRs (Storto et al., 2001
), so we wondered if this phenotype might also be rescued in our CR mice. Postadolescent testicular weight was increased by 24% in KO mice compared to WT (P < 0.0004; t-test); however there was no rescue of this phenotype in the CR mice (Figure S5
). To investigate if the absence of rescue was a matter of gene dosage, we generated KO mice that had a complete absence of mGluR5 (Fmr1
KO, dKO). Again, however, there was no rescue of the testicular phenotype.