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Human Molecular Genetics Advance Access originally published online on May 4, 2006
Human Molecular Genetics 2006 15(12):1984-1994; doi:10.1093/hmg/ddl121
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Exaggerated behavioral phenotypes in Fmr1/Fxr2 double knockout mice reveal a functional genetic interaction between Fragile X-related proteins

Corinne M. Spencer1, Ekaterina Serysheva1, Lisa A. Yuva-Paylor1, Ben A. Oostra3, David L. Nelson1 and Richard Paylor1,2,*

1Department of Molecular and Human Genetics and 2Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA and 3Department of Clinical Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands

* To whom correspondence should be addressed at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel: +1 7137986124; Fax: +1 7137986521; Email: rpaylor{at}bcm.tmc.edu

Received March 31, 2006; Accepted May 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Individuals affected by Fragile X syndrome (FXS) experience cognitive impairment, hyperactivity, attention deficits, social anxiety and autistic-like behaviors. FXS results from the loss of expression of the Fragile X mental retardation (FMR1) gene, whose protein product FMRP is thought to play an important role in neuronal function and synaptic plasticity. Two paralogs of FMRP, FXR1P and FXR2P, have been identified, forming the Fragile X-related (FXR) family of proteins. Although the functions of FXR1P and FXR2P are not well understood, there are similarities among all three FXR proteins in gene structure, amino acid sequence, expression pattern and cellular functions. Mouse models have been described for loss of Fmrp, Fxr1p and Fxr2p, the mouse homologs of FMRP, FXR1P and FXR2P. In earlier studies, we found that Fmr1 knockout (KO) mice, which do not express Fmrp, and Fxr2 KO mice, which do not express Fxr2p, show similarities in some behavioral responses such as hyperactivity. To better understand the functional relationship between FMRP and FXR2P, we generated Fmr1 KO, Fxr2 KO, Fmr1/Fxr2 double KO and wild-type control mice as littermates on the same genetic background and examined them in several behavioral assays. Results show that Fmr1/Fxr2 double KO mice have exaggerated behavioral phenotypes in open-field activity, prepulse inhibition of acoustic startle response and contextual fear conditioning when compared with Fmr1 KO mice, Fxr2 KO mice or wild-type littermates. Our findings suggest that Fmr1 and Fxr2 genes contribute in a cooperative manner to pathways controlling locomotor activity, sensorimotor gating and cognitive processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fragile X syndrome (FXS) is the most prevalent inherited cause of mental retardation in humans, with an estimated frequency of one in 4000 males (1Go). In addition to cognitive impairment, the characteristic phenotype of FXS includes mild craniofacial anomalies, hyperextensible joints, macroorchidism, hyperactivity, attention deficits, social anxiety and autistic-like behaviors (2Go). FXS results from the loss of expression of the Fragile X mental retardation (FMR1) gene, which is located on the X-chromosome. The loss of gene expression occurs most commonly as a result of expansion and methylation of CGG repeats in the 5'-untranslated region of the FMR1 gene (3Go–5Go).

Although the function(s) of FMRP is still not completely understood, it is thought that FMRP regulates the intracellular transport and translation of RNAs. In neurons, some of this activity is involved in synaptic plasticity (6Go). FMRP contains four RNA-binding domains [two hnRNP K homology (KH) domains, an RGG box and a novel NDF motif in the N-terminal region] (7Go–11Go) and has been shown to bind ~4% of human fetal brain mRNA (12Go), including several mRNAs related to synaptic function (13Go–16Go). A large subset of the mRNAs binding to FMRP contains a G quartet motif, which is specifically recognized by the RGG box (13Go,14Go,17Go). The KH2 domain of FMRP interacts with ‘kissing complex’, or loop–loop pseudoknot RNAs to mediate the association of FMRP with actively translating polyribosomes (18Go), a function important in FXS given that a single amino acid substitution in the KH2 domain (I304N) abolishes the association with polyribosomes and results in a severe FXS phenotype (19Go,20Go). FMRP has also been shown to modulate translation of mRNAs in vitro and in vivo (21Go–23Go). Regulation of translation by FMRP can be modified by phosphorylation (24Go) and interaction with microRNAs (25Go) and non-translatable RNA polymerase III transcripts such as BC1 RNA (26Go). FMRP is highly expressed in adult brain and testis, organs that are most affected in FXS (27Go). In the brain, FMRP is mainly localized to the cytoplasm of neurons. The presence of both a nuclear localization signal (NLS) and a nuclear export signal (NES) suggests that FMRP can shuttle between the nucleus and cytoplasm (28Go). Subsequent immunogold studies showed that FMRP is localized within the nuclei and nuclear pores and throughout the dendrites of neurons (20Go).

Two autosomal paralogs of FMRP, FXR1P and FXR2P, have been identified (8Go,29Go). These three proteins make up the Fragile X-related (FXR) family of proteins; all three have been found in every vertebrate analyzed to date. The gene structure is highly conserved among family members and suggests that all three are derived from a common ancestral gene (30Go). Lower organisms such as Caenorhabditis elegans or Saccharomyces cerevisiae do not have any genes homologous to the FXR family, whereas the Drosophila genome has a single homologous gene, termed Drosophila FMR1-related gene or dfmr1 (also called dfxr) (31Go). The vertebrate FXR proteins are very similar, sharing 73–90% amino acid identity over the first half of the sequence. Like FMRP, FXR1P and FXR2P contain two KH domains, an RGG box and NLS and NES motifs. FXR1P and FXR2P bind RNA, associate with ribosomes and interact with FMRP (8Go,29Go,32Go). The FXR proteins show an overlapping tissue distribution (33Go,34Go) and have been isolated together in mRNPs from mouse brain (32Go). Although the functions of FXR1P and FXR2P are unknown at this time, it has been suggested, given similar structural motifs, tissue distribution and ability to interact, that these proteins have functions analogous to FMRP and may be capable of compensating, in part, for the loss of FMRP in Fragile X patients. Compensation by paralogs is a possible explanation for the relatively mild phenotype in FXS, especially in light of the proposed importance of FMRP in neuronal function and plasticity.

The study of knockout (KO) mouse models is useful in developing a better understanding of the functions and interactions of the FXR proteins. Mouse models have been described for loss of Fmrp (35Go,36Go), Fxr2p (37Go) and Fxr1p (38Go). Fmr1 KO mice exhibit several of the physical and behavioral characteristics of the human FXS syndrome such as macroorchidism, hyperactivity, abnormal anxiety-related responses, abnormal sensorimotor gating and impaired motor coordination (35Go,36Go). Some investigators have observed mild learning deficits in hippocampal-dependent tasks, including contextual fear conditioning and Morris water maze (35Go,39Go–43Go), although this phenotype appears dependent upon genetic background (40Go,41Go). More recently, Fmr1 KO mice have been shown to be impaired in object recognition (44Go,45Go) and leverpress escape/avoidance (46Go), tasks that are thought to involve striatal- and/or cortical-dependent learning processes. Very recently, a conditional Fmr1 mouse model was developed and mice lacking Fmrp in Purkinje neurons in the cerebellum were generated (47Go). Koekkoek et al. (48Go) subjected these mice to morphological, electrophysiological and behavioral studies and found elongated spines and enhanced LTD induction at the parallel fiber synapses innervating the spines in Fmrp-deficient Purkinje cells. Both global and Purkinje-specific Fmrp null mice were also shown to display similar cerebellar deficits in eyeblink conditioning such as that seen in Fragile X patients (48Go).

Fxr2 KO mice exhibit hyperactivity in the open-field test, abnormal sensorimotor gating, impaired motor coordination, decreased response to heat stimulus and impaired learning and memory performance in both contextual fear conditioning and the Morris water task (37Go). Thus some of the behavioral phenotypes of the Fxr2 KO mice are similar to phenotypes of the Fmr1 KO mice. Although these results suggested that Fmr1 and Fxr2 have similar roles in some behaviors, a direct comparison between Fmr1 and Fxr2 KO mice was not appropriate because the studies were performed on mice of different genetic backgrounds. Fxr1 KO mice have physical abnormalities that have precluded behavioral testing as yet (38Go).

To better understand the functional relationship between FMRP and FXR2P, we used a breeding strategy to generate Fmr1 KO, Fxr2 KO, Fmr1/Fxr2 double KO and wild-type control mice as littermates on the same genetic background. The mice were subjected to a battery of behavioral assays to assess multiple domains of CNS function, including locomotor activity, anxiety-related responses, sensorimotor gating, motor coordination and skill learning, conditioned fear and analgesic-related responses. With the Fmr1/Fxr2 double KO mice, we expected to see exaggeration of behavioral phenotypes in which Fmrp and Fxr2p proteins have similar functions. Our findings suggest that both Fmr1 and Fxr2 genes contribute in a cooperative manner to pathways controlling locomotor activity, sensorimotor gating and learning and memory, but not to pathways involved in anxiety-like behavior, motor coordination and analgesic responses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice were bred to generate Fmr1 KO, Fxr2 KO, Fmr1/Fxr2 double KO and wild-type controls as littermates. All genotypes, including heterozygous Fxr2 mice, were generated in numbers close to the expected ratios. Fmr1/Fxr2 double KO mice exhibited reduced survival rates; out of 17 mice generated, only 10 survived to start testing at 8 weeks of age and one died during the test battery. Several of those that died were noticeably runted in size. Those that participated in the study appeared healthy. Fmr1/Fxr2 double KO mice were not assessed for macroorchidism or fertility. There was a main effect of genotype in the body weights of the mice tested [F(3,47)=5.518, P=0.002]. Fmr1/Fxr2 double KO mice weighed significantly less than Fmr1 KO mice (P=0.005) and WT control littermates (P=0.024), but did not differ significantly from Fxr2 KO mice (P=0.677) (data not shown). There was no effect of genotype in the ages of the mice at the start of testing [F(3,45)=0.260, P=0.854] (data not shown).

Locomotor activity in the open field
The open-field test assesses exploratory activity and anxiety-related responses in a novel arena. The test was performed for 30 min on 2 consecutive days in order to examine habituation to the novel environment. A few of the many measures obtained in the open-field test are presented in Figure 1. Significant overall differences between the genotypes were found in total distance traveled [F(3,47)=12.263, P<0.0005], time spent moving [F(3,47)=8.213, P<0.0005] and average speed of movement [F(3,47)=9.780, P<0.0005]. Post hoc comparisons determined that Fmr1/Fxr2 double KO mice traveled a greater total distance than Fmr1 KO, Fxr2 KO and WT littermates (P<0.0005) (Fig. 1A). Fmr1/Fxr2 double KO mice spent more time moving than Fmr1 KO (P=0.001), Fxr2 KO (P=0.006) and WT littermates (P<0.0005) (Fig. 1B). Finally, Fmr1/Fxr2 double KO mice traveled at a greater speed than Fmr1 KO (P<0.0005), Fxr2 KO (P<0.0005) and WT littermates (P=0.001) (Fig. 1C). There was no overall difference between the genotypes for rearing, as measured by vertical activity [F(3,47)=0.853, P=0.472] (Fig. 1D).


Figure 1211
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  		Figure 1. Exploratory activity in the open field. Fmr1/Fxr2 double KO mice were more active than Fmr1 KO mice, Fxr2 KO mice and WT littermates as assessed by (A) total distance traveled in centimeters (cm), (B) time spent moving in seconds (s) or (C) average speed of movement (cm/s), but show similar levels of (D) vertical activity. Unlike the other genotype groups, Fmr1/Fxr2 double KO mice do not show habituation across 10 min intervals over 2 days of testing in total distance, time spent moving or vertical activity. Values represent mean±SEM.

 
Significant genotypexinterval interactions were found in total distance [F(15,235)=1.703, P=0.051], time spent moving [F(15,235)=2.905, P<0.0005] and vertical activity [F(15,235)=3.300, P<0.0005], indicating that the genotype influenced these variables differently over the time intervals measured. Simple effects analysis of each genotype group indicated that Fmr1/Fxr2 double KO mice did not show significant habituation over the six 10 min intervals [total distance: F(5,45)=1.246, P=0.304; time spent moving: F(5,45)=1.648, P=0.167; vertical activity: F(5,45)=0.567, P=0.725]. Fmr1 KO mice [total distance: F(5,75)=7.588, P<0.0005; time spent moving: F(5,75)=15.606, P<0.0005; vertical activity: F(5,75)=4.222, P=0.002], Fxr2 KO mice [total distance: F(5,55)=3.184, P=0.014; time spent moving: F(5,55)=10.490, P<0.0005; vertical activity: F(5,55)=7.000, P<0.0005] and WT mice [total distance: F(5,60)=4.050, P=0.003; time spent moving: F(5,60)=11.651, P<0.0005; vertical activity: F(5,60)=14.081, P<0.0005] all showed significant habituation over the time course.

In summary, Fmr1Fxr2 double KO mice traveled farther, spent more time moving and moved at a faster average speed than their littermates, indicating hyperactivity. Furthermore, unlike their Fmr1 KO, Fxr2 KO and WT control littermates, Fmr1Fxr2 double KO mice did not show habituated responses during the six 10 min test intervals in total distance traveled, time spent moving and vertical activity.

Anxiety-related responses
In the open-field test, the center distance:total distance ratio provides a measure of the anxiety-related response to a brightly lit, open area (49Go). These data are shown in Figure 2A. There was no overall effect of genotype in this measure [F(3,47)=0.662, P=0.579], although there was a significant genotypexinterval interaction [F(15,235)=2.110, P=0.010] indicating that genotype was influencing how the mice responded across the time intervals. Simple effects analyses indicated that all of the genotypes showed significant effects of time interval [WT: F(5,60)=6.139, P<0.0005; Fmr1 KO: F(5,75)=5.344, P<0.0005; Fxr2 KO: F(5,55)=2.822, P=0.024; Fmr1/Fxr2 KO: F(5,45)=2.892, P=0.024]. However, the genotypes differed in the way they responded over the course of the test. WT and Fxr2 KO mice both showed a significant linear relationship between center distance ratio and time interval [WT: P=0.003; Fxr2 KO: P=0.001], whereas neither Fmr1 KO nor Fmr1/Fxr2 double KO mice showed linear relationships between center distance ratio and time interval (Fmr1 KO: P=0.499; Fmr1/Fxr2 KO: P=0.602). Regardless, it does not appear that the combination of Fmr1 and Fxr2 dramatically influenced anxiety-related responses in the open field.


Figure 1212
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  		Figure 2. Anxiety-related responses in the open-field and light–dark tests. Fmr1 KO mice, Fxr2 KO mice, Fmr1/Fxr2 double KO mice and their WT littermates show similar levels of responses as measured by (A) center distance/total distance ratio in the open-field test and (B) number of transitions between light and dark compartments during a 10 min test. Values represent mean±SEM.

 
The light–dark test is also used to assess anxiety-like behavior. For 10 min mice were allowed to explore an apparatus consisting of a large, open, brightly lit chamber and a small, dark, closed chamber. The number of transitions between the two chambers is a standard indicator of anxiety-like behavior in mice (50Go) and has been shown to be sensitive to anxiolytic drugs (51Go). There were no significant overall differences between the genotypes in light–dark transitions [F(3,48)=1.639, P=0.193], latency to first enter the dark compartment [F(3,48)=1.454, P=0.239] or total time spent in the dark compartment [F(3,48)=0.928, P=0.435]. Thus, the contributions of Fmr1 and Fxr2 do not combine to indicate a genetic interaction with respect to anxiety-related responses in the light–dark exploration test.

Rotarod test
Motor coordination and skill learning were evaluated in an accelerating rotarod test. Mice were placed onto a rotating rod that accelerated over 5 min and assessed for their ability to maintain balance and remain walking on top of the rod. They were given four trials per day for 2 consecutive days. As seen in Figure 3, mice of all genotypes improved significantly during training [F(7,336)=58.635, P<0.0005]. There was no overall difference between the genotypes [F(3,48)=2.033, P=0.122] and no significant genotypextrial interaction [F(21,336)=1.396, P=0.117], indicating no difference in training improvements between genotypes.


Figure 1213
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  		Figure 3. Motor skill learning and coordination was assessed in the accelerating rotarod test. Time spent walking on top of the rotarod (s) during eight trials is shown for Fmr1 KO mice, Fxr2 KO mice, Fmr1/Fxr2 double KO mice and their WT littermates. There were no significant differences between genotypes. Values represent mean±SEM.

 
Acoustic startle and prepulse inhibition of the acoustic startle response
Prepulse inhibition (PPI) of the acoustic startle response was used to assess sensorimotor gating. In normal animals, a weak non-startling sound presented immediately before a startling sound will suppress the startle response. The maximum startle responses and PPI data are shown in Figure 4. Mice that do not exhibit a startle response are eliminated from the analysis because it is not possible to show an inhibition of this response in these animals. In the present study, one Fmr1 KO mouse and two WT control mice did not show startle responses and their data were not included in the analysis. In the remaining subjects, a significant effect of genotype was observed in the maximum response to the 120 dB startling stimulus [F(3,45)=7.009, P=0.001]. Fmr1 KO, Fxr2 KO and Fmr1/Fxr2 double KO mice exhibited significantly lower responses than their WT littermates (P=0.001, P=0.039 and P=0.002) (Fig. 4A). For PPI, there was a main effect of prepulse level [F(4,180)=77.506, P<0.0005] as expected. As the prepulse level increases, normally there is greater suppression of the startle response. There was also an overall effect of genotype [F(3,45)=3.218, P=0.031]. Fmr1/Fxr2 double KO mice showed significantly less PPI than WT control mice (P=0.019) (Fig. 4B). There was no genotypexprepulse level interaction [F(12,180)=0.610, P=0.832].


Figure 1214
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  		Figure 4. Sensorimotor gating was measured by PPI of acoustic startle response. (A) Maximum startle response to 120 dB white noise sound burst is shown. Fmr1 KO mice, Fxr2 KO mice and Fmr1/Fxr2 double KO mice had significantly lower responses than their WT littermates. Values represent mean±SEM. (B) Inhibition of the acoustic startle response was determined with five prepulse levels (74, 78, 82, 86 and 90 dB). As there was no genotypexprepulse level interaction, the average PPI data (±SEM) are presented for simplicity. Fmr1/Fxr2 double KO mice showed a significant impairment in PPI compared with WT littermates.

 
Conditioned fear
Cognitive function was assessed using a conditioned fear test, a Pavlovian learning and memory paradigm in which mice were trained to associate both contextual cues and an auditory cue [conditioned stimulus (CS)] with an aversive unconditioned stimulus (US), a mild foot shock. Freezing behavior is used as an index of fear. Levels of freezing for the contextual and auditory cue-based fear tests, performed 24 h after training, are shown in Figure 5.


Figure 1215
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  		Figure 5. Contextual and cued (CS) fear conditioning. Fmr1/Fxr2 double KO mice showed significantly less freezing during the context test than Fmr1 KO, Fxr2 KO and WT littermates, but showed similar levels of freezing in the cued CS test. Values represent mean±SEM.

 
In the context test, mice were placed back into the chamber in which they had received two mild foot shocks and their freezing levels were measured for 5 min. There was an overall main effect of genotype [F(3,47)=7.623, P<0.0005]. Post hoc comparisons indicated that Fmr1/Fxr2 double KO mice showed less freezing in the context test than Fmr1 KO mice (P=0.001), Fxr2 KO mice (P=0.002) and WT controls (P=0.001), indicating a significant impairment in associating contextual cues with the foot shocks. There were no other significant differences between the genotypes (P>0.05).

In the auditory CS test, mice were placed into a novel chamber. After 3 min (pre-CS phase), the auditory cue, which had immediately preceded the foot shock during training, came on for an additional 3 min (during-CS phase). Freezing levels for the CS test represent the difference between the pre-CS and during-CS phases. There was an overall main effect of genotype in the CS test [F(3,47)=2.794, P=0.050]. The post hoc comparisons indicated that there were no significant differences between genotypes (P>0.05), although the Fmr1/Fxr2 double KO mice showed a tendency to freeze less than Fmr1 KO mice (P=0.078) and Fxr2 KO mice (P=0.064). Fmr1/Fxr2 double KO mice did not differ from WT controls (P=0.516) in the CS test.

Hotplate
The hotplate test provides an assessment of sensitivity to painful stimuli, which provides useful information to properly interpret the results of fear conditioning tests. Mice were placed on the hotplate (55°C) and the latency to show their first hindlimb response was recorded (Fig. 6). There was a main effect of genotype [F(3,47)=3.403, P=0.025]. Fxr2 KO mice showed a higher latency to first hind limb response than WT controls (P=0.023), indicating that Fxr2 KO mice are less sensitive to painful stimuli. Fmr1/Fxr2 double KO mice did not differ significantly from WT control mice (P=0.131). This suggests that the impairment in the Fmr1/Fxr2 double KO mice in fear conditioning is not likely due to decreased sensitivity to the foot shock. Fmr1/Fxr2 double KO mice also did not differ significantly from Fxr2 KO mice (P=0.959) or Fmr1 KO mice (P=0.711), indicating a lack of interaction between Fmr1 and Fxr2 genes in hotplate sensitivity.


Figure 1216
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  		Figure 6. Analgesia-related responses in the hotplate test. Fmr1/Fxr2 double KO mice showed similar latencies to exhibit a hind limb response as their littermates. Fxr2 KO mice took significantly longer than WT littermates to display a hind limb response. Values represent mean±SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
FMRP, FXR1P and FXR2P are members of the FXR family of proteins, sharing similar gene structures and amino acid sequences and having partially overlapping patterns of tissue expression. All three paralogs bind RNA, associate with polyribosomes and can interact with each other to form homo- and hetero-multimers in vitro (8Go,29Go,32Go). However, the physiological or behavioral consequences of these interactions were not known. In order to better understand how FMRP and FXR2P might function together, we generated Fmr1/Fxr2 double KO mice and examined them in a battery of behavioral tests similar to our previous studies with Fmr1 KO mice (36Go) and with Fxr2 KO mice (37Go). Our present results, summarized in Table 1, show that Fmr1/Fxr2 double KO mice exhibit a few exaggerated behavioral phenotypes relative to Fmr1 KO mice and Fxr2 KO mice, indicating a functional genetic or epistatic interaction between these genes and suggesting that FMRP and FXR2P have common functions in some behaviors. This study does not elucidate the mechanism by which these genes interact; indeed, the nature of their interaction is not necessarily at the level of intracellular function, but may reflect parallel extracellular pathways that contribute to the ultimate behavioral outcome. Additional studies will be necessary to understand exactly how Fmr1 and Fxr2 interact to influence locomotor activity, sensorimotor gating and cognitive processes.


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  		Table 1. Summary of behavioral responses of Fmr1 KO, Fxr2 KO and Fmr1/Fxr2 KO mice compared with wild-type littermates
 
Although it is premature to rule out subtle effects on gene expression, it appears that Fmrp and Fxr2p modulate each other's function by a mechanism other than direct regulation of gene expression. FXR1P and FXR2P levels are not altered in FXS patients (33Go) or in Fmr1 KO mice (34Go) when compared with normal controls. Similarly, the expression of Fmrp and Fxr1p are not altered in Fxr2 KO mice (37Go). Despite the possibility that the population of FXS patients may have a normal range of FXR2P levels, our study in mouse models suggests that the level of FXR2 gene expression in these individuals may affect their behavioral phenotype and warrants further investigation.

There is evidence at the molecular level to suggest that FXR proteins may be able to compensate for the loss of another family member. FXR proteins show considerable functional overlap at the level of translational regulation and RNA-binding specificity. Brown et al. (13Go) found that ~50% of mRNAs found in FMRP–mRNP complexes from normal mouse brain did not exhibit abnormal polyribosomal shifts in Fmr1 KO mouse brain, suggesting that many RNAs were translated normally in the absence of FMRP. Miyashiro et al. (15Go), who identified RNAs binding FMRP through antibody-positioned RNA amplification (APRA), found that several of these RNAs exhibited normal mRNA abundance, protein abundance and mRNA subcellular localization in Fmr1 KO mouse brain. Also, using antibodies with minimal cross-reactivity, they discovered a 50% overlap in RNA cargoes associated with FMRP and FXR1P. Thus, FXR proteins may not only have common functions in RNA binding and translational control, but also be able to substitute for one another. The physiological relevance of particular combinations of RNA, FXR proteins and other proteins in RNA–protein complexes remains to be determined. This study provides supportive, but not conclusive, evidence for compensation by FXR paralogs. The exaggerated behavioral phenotypes seen in the absence of both Fmr1 and Fxr2 suggests that the presence of Fxr2 is responsible for the milder behavioral phenotype of the Fmr1 KO mice and, conversely, that the presence of Fmr1 is responsible for the milder behavioral phenotype of the Fxr2 KO mice. However, compensation by FXR paralogs would be more conclusively proven if there was additional evidence that the over-expression of one paralog ameliorates or corrects behavioral abnormalities resulting from the loss of expression of another paralog. In addition, the possibility remains that Fxr1p is playing a compensatory role in the Fmr1/Fxr2 double KO mice. Creation of Fmr1/Fxr1/Fxr2 triple KO mice will potentially be informative about the functions and interactions of all three FXR proteins. However, given problems with early postnatal death with Fxr1 KO mice (38Go), a triple KO mouse will likely require the development of a conditional Fxr1 KO mouse model.

In addition to overlapping intracellular functions, previous studies suggested that FMRP and FXR2P might have similar functions in some behavioral responses (35Go–37Go). For example, both Fmr1 KO mice and Fxr2 KO mice exhibited hyperactivity in the open field. Our prediction that Fmr1/Fxr2 double KO mice would exhibit increased hyperactivity relative to Fmr1 KO mice and Fxr2 KO mice was true, indicating that both Fmr1 and Fxr2 genes contribute to exploratory/locomotor activity in a cooperative manner. Our results also show the importance of placing genetic mutations on a common genetic background and comparing littermates comprising all of the genotypes of interest. For example, Fmr1 KO mice on a mixed 129/Ola and C57BL/6 genetic background had previously shown enhanced PPI (52Go,53Go) (R. Paylor, unpublished data), whereas Fxr2 KO mice, also on a mixed genetic background, had impaired PPI (37Go). We had predicted that combining the mutations would yield normal PPI (37Go), but that was not the case when both mutations were analyzed in littermates on a common genetic background.

Several of the phenotypes found in our previous studies of Fmr1 KO mice (36Go) and Fxr2 KO mice (37Go) were not evident in this study. For example, although both Fmr1 KO mice and Fxr2 KO mice exhibited hyperactivity in the open-field test in the original studies, these mice were not hyperactive relative to WT controls in the present study. In addition, the decreased anxiety-related responses previously observed in Fmr1 KO mice (36Go) were not reproduced in this study. There are several possible explanations for the differences between studies. First, as discussed above, the genetic backgrounds were not the same. However, we have found that the Fmr1 KO mice on a C57BL/6 (N12) background continued to exhibit hyperactivity and decreased anxiety-like behavior in the open field (54Go). The mice for the present study are also on a C57BL/6 background because both lines of mice had been backcrossed to the C57BL/6 strain for at least 10 generations. However, each line still carries a small amount of genetic material from the 129/Ola ES cells; therefore, combining these two lines, which likely have different sets of genetic contributions from 129/Ola, will increase genetic variation and have unpredictable consequences on behavior. Secondly, the genotype of the mother was different in all three studies. There are many examples of genetic mutations in mice that resulted in abnormal maternal behavior (55Go–58Go). Although the dams in all three studies appeared to exhibit normal maternal behavior, subtle differences cannot be ruled out and may contribute to significant differences in the behavioral phenotypes of their offspring. For example, rodent pups that receive higher levels of maternal licking and grooming later show increased exploratory activity, maze learning and response to novelty as adults (59Go–61Go). Although less is known about the contribution of paternal care (62Go,63Go), we use stable breeding pairs so the fathers, which were different genotypes across the three studies, may also have influenced the prenatal or early postnatal environment of their offspring. Thirdly, the Fmr1 KO and Fxr2 KO mice may have been influenced by the abnormal behavior of their Fmr1/Fxr2 double KO littermates. Observing mice in their homecages, the presence of a hyperactive or circling mouse sometimes inhibits the activity of their cagemates. The number of mice in this study was not sufficient to determine a difference between those mice that had Fmr1/Fxr2 double KO littermates or cagemates and those that did not. However, cagemate compositions were generated in a completely blind fashion with respect to genotype and were randomly mixed across the cages tested. Thus, our studies illustrate the importance of comparing all genotypes of interest as littermates, so they have the same genetic and maternal background and they experience similar littermate and cagemate behavior.

The types of behaviors in which the Fmr1/Fxr2 double KO mice exhibit exaggerated behaviors are relevant to individuals with FXS. Among many behavioral symptoms, FXS patients exhibit hyperactivity, cognitive impairment (2Go) and have impaired PPI (53Go). Individuals with FXS are also known to become hyperaroused in environments with excessive sensory stimuli and to habituate poorly to sensory stimuli (2Go). We examined exploratory behavior in the open field over 2 days in an attempt to model this behavior and discovered that the Fmr1/Fxr2 double KO mice did not show normal habituation in activity over the two 30 min sessions. In previous studies, Fmr1 KO mice and Fxr2 KO mice were hyperactive, but did show normal habituation during a single 30 min session (36Go,37Go). Under normal conditions, exploratory activity in a novel environment decreases over time and repeated exposures, i.e. as the environment becomes more familiar. The lack of habituation in the Fmr1/Fxr2 double KO mice suggests that they were hyperaroused in the novel environment and/or they were not becoming familiar with the environment. As the test was done over 2 days, it is possible that the Fmr1/Fxr2 double KO mice did not recognize the open field from the previous day, an idea supported by the inability of these mice to display normal freezing responses in the context in which they had received two foot shocks in a fear conditioning paradigm. The hippocampus, which detects and responds to novelty (64Go,65Go), has been shown to be involved in both behavioral habituation to a novel environment (66Go) and contextual fear conditioning (67Go,68Go).

The impaired responses in Fmr1/Fxr2 KO mice in conditioned fear could be a consequence of their hyperactivity or differences in sensory abilities. It is unlikely that sensory differences explain the fear conditioning impairment. First, all of the mice, including the Fmr1/Fxr2 double KO mice, showed a response to the two foot shocks (e.g. run, jump or vocalization). Secondly, the Fmr1/Fxr2 KO mice did not differ from WT littermates in their responses in the hotplate test. Thirdly, the Fmr1/Fxr2 KO mice did acquire a normal conditioned response to the auditory cue. The normal cued fear response also suggests that hyperactivity did not play a significant role in preventing freezing behavior during cued fear conditioning.

The lack of a profound cognitive impairment was one of the aspects about the behavioral phenotype of the original Fmr1 KO mouse that was of great concern to many researchers in the field. In addition to the possibility of compensation by paralogs, the obvious explanations for the mild phenotype were the appropriateness of mouse genetic background (i.e. strain) and the particular learning and memory tests applied. Individuals with FXS vary greatly in cognitive ability, with IQ levels ranging from mild to profound impairment. Thus, it is reasonable to predict that genetic background would play a significant role in the expression of a cognitive phenotype in mice. Indeed, studies have reported differences in cognitive response based on strain background (40Go,41Go). Currently, we are systematically evaluating the effect of genetic background on multiple behaviors including cognitive function to better understand the impact of background genes on the expression of behavioral responses in our mouse models of FXS.

Cognitive function encompasses many types of learning and memory. Thus, the type of behavioral test used to assess cognition can affect the ability to detect cognitive impairment in mice. Many of the cognitive tests first performed with Fmr1 KO mice were hippocampal-dependent tasks, including contextual fear conditioning and Morris water maze (35Go,39Go–43Go). More recently, tasks that tap into non-hippocampal (e.g. striatal, cortical and/or cerebellar) processes have been performed in these mice, and investigators are finding more profound impairments than previously detected (44Go,46Go,48Go). Thus, it is evident that in addition to the interacting effects of the Fxr2 gene, genetic background and test type affect cognitive ability in Fmr1 KO mice. Other learning and memory tests now need to be done such as the Morris water maze and leverpress avoidance to better understand the nature of the learning impairment.

A major goal in seeking modifier or epistatic genes, manipulating genetic background and using new behavioral assays is to develop mouse models with robust phenotypes that can be used to elucidate Fmr1 gene function and mechanisms underlying FXS and also to provide a means to test new therapies for behavioral problems in individuals with FXS. Affecting 70–100% of males with FXS, hyperactivity and cognitive impairment are significant problems for these individuals and their families (2Go). It is clear from the present results that both Fmr1 and Fxr2 genes cooperatively contribute to these behaviors in our mouse model. The exaggerated behaviors in the Fmr1/Fxr2 double KO mouse model will enable easier screening for pharmacological agents that can ameliorate these behaviors. Furthermore, the finding that Fxr2 interacts with Fmr1 gene function at the behavioral level supports the possibility that a therapeutic approach to upregulate FXR2P levels could compensate for the loss of FMRP and normalize locomotor activity levels and/or cognitive ability in FXS patients. Further studies are needed to evaluate the potential of this approach.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Subjects were derived from a two-step breeding process. First, crosses between Fmr1 KO (Fmr1–/y) mice (36Go) and Fxr2 KO (Fxr2–/–) mice (37Go), both on C57BL/6 genetic backgrounds, were used to generate Fmr1+/–Fxr2+/– females and Fxr2+/– males. These mice were bred to obtain 52 male littermates representing all of the genotypes analyzed in this study (16 Fmr1–/y, 12 Fxr2–/–, 10 Fmr1–/yFxr2–/– and 14 Fmr1+/yFxr2+/+ wild-type control mice). Mice were housed two to five per cage in a room with a 12 h light:dark cycle (lights on at 6 AM, off at 6 PM) with access to food and water ad libitum. All housing and behavioral testing procedures were approved by the Baylor College of Medicine Animal Care and Use Committee and followed the NIH Guidelines, ‘Guide for the care and use of laboratory animals’.

Genotyping
Animals were genotyped by standard PCR techniques. For detection of the Fmr1 WT allele (527 bp product), PCR was performed on DNA from tails with primers Fmr1_S1 (5'-GTGGTTAGCTAAAGTGAGGATGAT-3') and Fmr1_S2 (5'-CAGGTTTGTTGGGATTAACAGATC-3'). The Fmr1 KO allele (501 bp product) was detected by PCR with the Fmr1_S1 primer and primer Fmr1_N2 (5'-TGGGCTCTATGGCTTCTGA-3') which binds to a Neo cassette that replaced exon 5 of the Fmr1 gene (36Go). Cycle conditions were identical for both S1/S2 and S1/N2 combinations: 2 min at 94°C, 34x (30 s at 94°C, 30 s at 55°C, 60 s at 72°C) and 10 min at 72°C using standard PCR reagents. For detection of the Fxr2 WT allele, PCR was performed with primers Fxr2_407 (5'-GAGCCAACTGCATCTTTCTCAAC-3') and Fxr2_336 (5'-GTGAGAACAGCAGTCAGACTTACC-3'). The Fxr2 KO allele was detected by PCR with the Fxr2_407 primer and primer Fxr2_NeoR (5'-CTTGCTCCTGCCGAGAAAGTA-3') which binds to a Neo cassette that replaced exon 7 of the Fxr2 gene (37Go). Cycle conditions were identical for both 407/336 and 407/Neo combinations: 2 min at 94°C, 35x (30 s at 94°C, 30 s at 60°C, 60 s at 72°C) and 10 min at 72°C.

Behavioral testing
Male mice were subjected to a rapid version of the test battery originally described by Crawley and Paylor (69Go,70Go). Tests were performed in the following order with 1–3 days between tests: open-field, light–dark, acoustic startle/PPI, rotarod, conditioned fear and hotplate. Behavioral testing was performed between 8 AM and 3 PM. At the start of testing, the mice were approximately 8 weeks of age. There were no statistical differences in test age among the genotypes. Experiments were conducted by an experimenter who was blind to the genotypes of the mice.

Open-field test
Mice were placed into the center of a clear Plexiglas (40 cmx40 cmx30 cm) open-field arena and allowed to explore for 30 min on each of 2 consecutive days. Bright, overhead lighting provided ~800 lux of illumination inside the arenas. White noise was present at ~55 dB inside the arenas. Data were collected in 2 min intervals by a computer-operated Digiscan optical animal activity system (RXYZCM, Accuscan Electronics) (16Go). Average speed was calculated by dividing the total distance by time spent moving. The center distance was divided by the total distance to obtain a center distance: total distance ratio, which is used as a measure of anxiety-like behavior (36Go). Because of experimenter error, data for one wild-type control mouse were missing for the first day of testing. Open-field activity data were pooled into six 10 min intervals and analyzed using a two-way (genotypexinterval) analysis of variance (ANOVA) with repeated measures.

Light–dark exploration
One or 2 days after the open-field test, anxiety-related responses were assessed in the light–dark box. The apparatus is a Plexiglas chamber (44 cmx21 cmx21 cm) divided unequally into two chambers by a black partition containing a small opening. The larger chamber is twice the size of the smaller chamber, has clear walls and an open top and is brightly illuminated (800 lux). The small chamber is enclosed on all sides by black walls except for the small opening between the chambers. White noise was present in the room at ~55 dB in the test chamber. Mice were placed into the illuminated side and allowed to explore freely for 10 min. Mice were scored for the number and latency of entries and time spent in each compartment using a hand-held computer (Psion Workabout mx, Psion Teklogix) with the OBSERVER® program (Noldus Information Technologies). An entry was defined as the mouse placing all four feet into the zone. Data were analyzed by one-way (genotype) ANOVA.

Acoustic startle and PPI of acoustic startle
Acoustic startle responses were measured using the SR-Lab System (San Diego Instruments, San Diego, CA, USA) as previously described (69Go). Mice were placed in the Plexiglas cylinder and left undisturbed with background white noise (~70 dB) for 5 min prior to beginning the test session, which consisted of seven trial types. During one trial type, no stimulus was presented to measure baseline movement in the cylinder. Another trial type was a 40 ms, 120 dB sound burst used as the startle stimulus. Five different 20 ms prepulse sounds (74, 78, 82, 86 or 90 dB) were presented 100 ms before the startle stimulus. Each trial type was presented six times, once per block of seven trials in pseudorandom order. The inter-trial interval ranged from 10 to 20 s. The startle response was recorded every 1 ms during a 65 ms period that followed the onset of the startle stimulus. The maximum startle amplitude during this period was used as the dependent variable. Percent PPI of the startle response was calculated as 100–[(startle response on acoustic prepulse plus startle stimulus trials/startle response alone trials)x100]. Acoustic response amplitude data were analyzed using an one-way ANOVA. PPI data were analyzed using a two-way (genotypexprepulse sound level) ANOVA with repeated measures. Three mice (two WTWT and one Fmr1 KO) did not meet our criterion for minimum startle response to the 120 dB sound stimulus (100) and therefore their data were not included in the analysis.

Rotarod test
Motor coordination and skill learning were tested 1–3 days after acoustic startle/PPI using an accelerating rotarod (UGO Basile, Varese, Italy). Mice were placed on a rotating drum, which accelerated from 4 to 40 r.p.m. over a 5 min period. Time spent walking on top of the rod before falling off the rod or hanging on and riding completely around the rod was recorded. Mice were given four trials on 2 consecutive days with a maximum time of 300 s (5 min) and a 30–60 min inter-trial rest interval. Rotarod data were analyzed using a two-way (genotypextrial) ANOVA with repeated measures.

Pavlovian conditioned fear
Freezing behavior in a conditioned fear paradigm was measured as described previously (37Go). The test chamber (26 cmx22 cmx18 cm high) had clear Plexiglas sides and a grid floor bottom that was used to deliver a mild foot shock. The chamber was placed inside a sound-attenuated chamber (Med Associates, internal dimensions: 56 cmx38 cmx36 cm) that had a window through which mice could be observed without disturbance. On the training day, mice were placed into the test chamber and allowed to explore for 2 min. The CS (a white noise 80 dB sound) was presented for 30 s and followed immediately by a mild foot shock (2 s, 0.7 mA) that served as the US. After 2 min, the mice received a second CS–US pairing. The FreezeFrame monitor system (San Diego Instruments, USA) was used to control the timing of CS and US presentations. Freezing behavior was measured manually by an experimenter who was blind to the genotypes using a sampling procedure in which freezing was determined every 10 s. Percent freezing was determined by the percentage of 10 s intervals in which freezing was observed. During the conditioning procedure, responses to the foot shock, typically run, jump or vocalize, were also recorded. In the present study, all of the mice responded to the foot shock.

Mice were tested for contextual and cued fear conditioning 24 h after conditioning. For the context test, mice were placed back into the original test chamber for 5 min and freezing behavior was recorded every 10 s. One to two hours later, mice were tested for responses to the auditory CS in a new environment. For the CS test, black Plexiglas inserts were placed on the sides and floor of the chamber to alter the shape, texture and color of the chamber. Vanilla extract was placed in the chamber behind the insert to alter the odor. Transfer cages were altered (paper towels instead of bedding) and red house lights replaced the normal white house lights. Mice were placed into this new chamber and freezing was recorded for 3 min during this ‘pre-CS’ phase. The auditory CS was then presented for another 3 min and freezing was recorded as described. Data for the CS test were calculated as the percent freezing during the CS minus percent freezing in the pre-CS phase. Context and CS test data were analyzed using an one-way ANOVA. One Fmr1/Fxr2 KO mouse died during fear conditioning; therefore, its data were not available.

Hotplate test
Mice were tested for analgesia-related responses using a hotplate apparatus (Columbus Instruments, Columbus, OH, USA). The hotplate was preheated to 55±3°C and then mice were placed one at a time onto the hotplate. The time to first show a hind limb response was recorded. Typical responses are licking or shaking the hindpaw, or jumping. Mice were immediately removed after showing a response.

Statistical analyses
All data were analyzed using one-way (genotype) or two-way (genotypexrepeated measure such as time interval) ANOVA with the SPSS statistical software package (SPSS, Chicago, IL, USA). Post hoc comparisons were made using Tukey's HSD following a significant interaction or main effect of genotype. The level of significance was set at P≤0.05.


   ACKNOWLEDGEMENTS
 
This research was supported by FRAXA Research Foundation, the Baylor Fragile X Research Center (HD24064-S1), NICHD grants HD38038, HD29256 and the Administrative and Neurobehavioral Cores of the Baylor Mental Retardation and Developmental Disabilities Research Center (HD24064).

Conflict of Interest statement. None declared.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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