Reduced Cortical Synaptic Plasticity and GluR1 Expression Associated with Fragile X Mental Retardation Protein Deficiency

Molecular and Cellular Neuroscience 19, 138–151 (2002)

Reduced Cortical Synaptic Plasticity and GluR1Expression Associated with Fragile X MentalRetardation Protein Deficiency

Jianxue Li, Marc R. Pelletier, Jose-Luis Perez Velazquez,and Peter L. Carlen1

Division of Cellular and Molecular Biology, Toronto Western Research Institute,University of Toronto, Toronto, Ontario M5T 2S8, Canada

Lack of expression of the fragile X mental retardationprotein (FMRP), due to silencing of the FMR1 gene,causes the Fragile X syndrome. Although FMRP was char-acterized previously to be an RNA binding protein, little isknown about its function or the mechanisms underlyingthe Fragile X syndrome. Here we report that the �-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor sub-unit, GluR1, was decreased in the cortical synapses, butnot in the hippocampus or cerebellum, of FMR1 geneknockout mice. Reduced long-term potentiation (LTP)was also found in the cortex but not in the hippocampus.Another RNA binding protein, FXR; the N-methyl-D-aspar-tate receptor subunit, NR2; and other learning-relatedproteins including c-fos, synapsin, myelin proteolipid pro-tein, and cAMP response element binding protein werenot different between FMR1 gene knockout and wild-typemice. These findings suggest that the depressed corticalGluR1 expression and LTP associated with FMRP defi-ciency could contribute to the Fragile X phenotype.

INTRODUCTION

The Fragile X syndrome, the most common heredi-tary form of mental retardation, affecting 1:4000 males(Turner et al., 1996; Morton et al., 1997), is due to thefunctional absence of the fragile X mental retardationprotein (FMRP) encoded by the FMR1 gene (Pieretti etal., 1991; Hoogeveen and Oostra, 1997). The 5� untrans-lated region of the FMR1 gene contains a polymor-

1 To whom correspondence and reprint requests should be ad-dressed at the Division of Cellular and Molecular Biology, TorontoWestern Research Institute, University of Toronto, McL 12-413, 399
Bathurst Street, Toronto, ON, Canada M5T 2S8. Fax: (416) 603-5745.E-mail: [email protected].
138

phous CGG trinucleotide repeat (6–54 repeats undernormal conditions), which can be amplified to hun-dreds or thousands of copies leading to hypermethyl-ation of the CpG island and loss of FMR1 gene tran-scription in affected individuals (Pieretti et al., 1991;Sutcliffe et al., 1992; Hoogeveen and Oostra, 1997; Chiu-razzi et al., 1998).

Intellectual impairment, ranging from a mild learn-ing disability to profound mental retardation, is a majorclinical phenotype of the Fragile X syndrome (Reiss etal., 1995; Abrams et al., 1997). A pattern of behavioralfeatures, including hyperactivity, anxiety, seizures, andmild autism, is also seen (Mazzocco et al., 1997). Patientswith the Fragile X syndrome usually do not have de-tectable FMRP, due to inactivation of the FMR1 gene(Siomi et al., 1993). The FMRP is one of the RNA-binding proteins containing both K homology domainsand Arg-Gly-Gly peptides and associates with polyri-bosomes as a ribonucleoprotein particle. This proteinprobably plays an important role both in RNA trans-port from the nucleus to the cytoplasm and in thetranslation of mRNA to protein (Siomi et al., 1993; Ver-heij et al., 1995; Eberhart et al., 1996; Bardoni et al., 1997;Brown et al., 1998). While many biochemical and cellu-lar characteristics of the FMRP have been carefullystudied, the actual roles of this protein and the celltypes in which its expression is critical have not beenestablished. Therefore, the mechanisms whereby theFMRP deficiency leads to human cognitive and behav-ioral changes remain to be elucidated.

FMR1 gene-targeted mice, which do not possessFMR1 mRNA or protein and display unusual cognition

doi:10.1006/mcnMCN
01.1085, available online at http://www.idealibrary.com on e.20
and behavior, were constructed recently in an attemptto provide a suitable animal model to study the human

1044-7431/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

Fragile X syndrome (The Dutch–Belgian Fragile X Con-sortium, 1994; Kooy et al., 1996; Oostra and Hoogeveen,1997). Since the development of these knockout mice,several studies have been conducted to assess either thephysiological function of the FMR1 gene or the clinicalphenotype caused by the absence of FMRP (D’Hooge etal., 1997; Slegtenhorst-Eegdeman et al., 1998; Fisch et al.,1999; Paradee et al., 1999; Van Dam et al., 2000; Chenand Toth, 2001, 2001; Nimchinsky et al., 2001). Becausenormal cognitive and behavioral activities are basedupon normal synaptic function, we hypothesized thatchanges in cortical synaptic plasticity could in part ex-plain the cognitive dysfunction associated with FMR1gene inactivation, leading to the Fragile X syndrome. Toelucidate the relationship between FMRP deficiencyand the clinical phenotype, we have examined (1) nor-mal synaptic transmission and long-term potentiation(LTP) in cortical and hippocampal slices prepared fromFMR1 gene knockout mice and wild-type controls and(2) the expression of several learning-related proteinsincluding glutamate receptors in the cerebral cortex,hippocampus, and cerebellum.

RESULTS

Normal Synaptic Transmission in Cortex Did NotDiffer between Wild-Type and Knockout Mice

Because of the cytoarchitecture and the organizationof the synaptic connections in the cortex, synaptic re-sponses recorded extracellularly are complex and are ofsmall amplitude. Therefore, before assessing differ-ences in LTP between wild-type and knockout mice, wefirst pharmacologically characterized normal synaptictransmission in the cortical slices. As shown in Fig.1A, application of d-2-amino-5-phosphonopentanoicacid (D-AP5) (50 �M), to block N-methyl-d-aspartate(NMDA) receptor-mediated transmission, did not affectsignificantly the amplitude of the postsynaptic potential(PSP) evoked by the test pulse in cortical slices preparedeither from wild-type mice (107.2 � 3.5% of control) orfrom knockout mice (107.8 � 5.7% of control) (n � 4slices from four mice for each group); however,6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) diso-dium (10 �M), which blocks �-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate recep-tors, reduced the later occurring, large negativelydeflecting component of the response by 23.3 � 7.8 and20.8 � 5.0% of control in wild-type and knockout cor-tical slices, respectively. The earlier occurring nega-tively deflecting component of the response was unaf-

fected. The addition of tetrodotoxin (TTX) (1 �M)reduced the remaining, earlier occurring, negativelydeflecting component by 54.1 � 33.4 and 35.6 � 22.2%of control, respectively. This pharmacological analysissuggested that the first negatively deflecting compo-

FIG. 1. Normal synaptic transmission in cerebral cortex of FMR1gene knockout and wild-type mice. (A) Upper left, representativerecord of a postsynaptic potential (PSP) evoked by test pulse intensityin a cortical slice prepared from a wild-type mouse. Upper right, nochange in the PSP after 15 min application of AP5 (50 �M). Lower left,addition of CNQX for 15 min has reduced significantly the lateroccurring negatively deflecting component of the PSP but has noeffect on the earlier occurring negatively deflecting component.Lower right, addition of TTX (1 �M) reduced significantly the earlieroccurring negatively deflecting component. A similar pharmacologi-cal profile was observed also in cortical slices prepared from knockoutmice. (B) Representative PSPs evoked at four stimulation intensities inwild-type and knockout mice. Arrows and dotted lines denote themethod of amplitude measurement for small-amplitude monophasicand larger amplitude biphasic responses. Upward pointing unfilledtriangle denotes presynaptic volley. (C) Plot of I/O relation demon-strates no difference in normal synaptic transmission when knockoutmice (filled squares) were compared to wild-type mice (filled circles)(n � 4 slices from four mice for each group).

139Reduced Cortical LTP and GluR1 in Fragile X Mice

nent of the cortical response was nonsynaptic and likelycomprised both presynaptic volley and antidromicspike. Therefore, we defined the later occurring, nega-tively deflecting component as the PSP, which is medi-ated largely by the activation of AMPA receptors. Theamplitude of the cortical PSPs evoked by weak stimu-lation was measured from the most negative peak to thebaseline, and the amplitude of the biphasic corticalPSPs evoked by stronger stimulation was measuredfrom the most negative, later occurring peak to the mostpositive peak of the response (Fig. 1B).

As shown in Fig. 1B, cortical PSPs recorded extra-cellularly and evoked with weak stimulation weresmall-amplitude, negatively deflecting responses. Asthe stimulation intensity increased, an earlier occurringnegatively deflecting component emerged and the am-plitude of the second negatively deflecting componentincreased progressively. PSPs evoked with the stron-gest stimulation intensities often were biphasic. A sum-mary of these data is presented in Fig. 1C, which illus-trates a superimposition of the input/output (I/O)relations, suggesting that there were no differences innormal synaptic transmission when knockout micewere compared to wild-type mice.

No Differences in Hippocampal, but SignificantlyReduced Cortical, LTP in FMR1 Gene KnockoutMice Compared to Wild-Type Mice

The persistent increase in synaptic efficacy (plasticity)of excitatory synaptic transmission characteristic of LTPhas been studied extensively and is considered to be arepresentative cellular model of learning and memory(Bliss and Collingridge, 1993; Lynch, 1998; McEachernand Shaw, 1999; Moser and Moser, 1999). We assessedfirst whether there were differences in LTP betweenknockout and wild-type mice in the hippocampus. Asseen in Figs. 2A and 2B, there were no differences inhippocampal LTP, measured 45 min after the tetanicstimulation, when wild-type mice (143.5 � 9.5% of con-trol, n � 9 slices from four mice) were compared toknockout mice (156.5 � 9.0% of control, n � 8 slicesfrom four mice). This observation is consistent withwhat has been published previously (Godfraind et al.,1996; Paradee et al., 1999).

In contrast, when LTP was assessed in the cortex, weobserved a significant reduction in the magnitude ofLTP in cortical slices prepared from knockout micecompared to wild-type mice. Representative records ofcortical PSPs before and after tetanic stimulation areshown in Fig. 2C. As seen in Fig. 2D, differences becamesignificant 10 min after the tetanization protocol (third

FIG. 2. Long-term potentiation (LTP) in cortex and hippocampus ofFMR1 gene knockout and wild-type mice. (A) Representative PSPsevoked by orthodromic stimulation of Schaffer collaterals and recordedin CA1 stratum radiatum in hippocampal slices prepared from wild-type(top) and knockout (bottom) mice. Numbers correspond to time pointsin (B) (1, prior to tetanic stimulation; 2, 45 min after tetanic stimulation).(B) Summary of time course of LTP in hippocampal slices from wild-type (filled circles, n � 9 slices from four mice) and knockout mice (filledsquares, n � 8 slices from four mice). Arrow denotes tetanic stimulation.(C) Representative PSPs evoked by stimulation of white matter andrecorded in layer IV/V in cortical slices prepared from wild-type (top)and knockout (bottom) mice. Calibration bars are the same as in (A).Numbers correspond to time points in (D) (1, prior to tetanic stimulation;2, 100 min after tetanic stimulation). (D) Summary of time course of LTPin cortical slices from wild-type (filled circles, n � 13 slices from six mice)and knockout mice (filled squares, n � 17 slices from six mice). Arrowsdenote tetanic stimulation. Asterisks denote P � 0.05 (applies to subse-quent figures).

140 Li et al.

tetanic stimulation) and remained significantly differ-ent for the duration of the experiment (100 min) atwhich point the amplitudes of PSPs from knockout andwild-type mice were 110.1 � 9.0% (n � 17 slices fromsix mice) and 172.4 � 10.6% (n � 13 slices from sixmice) of control, respectively.

FMRP Was Expressed in Various Brain Regionsbut Not in Heart and Liver

We quantified the expression of FMRP and anotherRNA binding protein, FXR, in brain (cerebral cortex,hippocampus, and cerebellum) as well as in heart andin liver from the wild-type control mice (n � 8 mice).As illustrated in Fig. 3A, Western blot analysis demon-strated that the level of FXR (69 kDa) was highest in theheart and lowest in the brain. On the other hand, FMRP(74 kDa) was higher in the cerebral cortex (0.126 � 0.035OD/�g protein) and hippocampus (0.085 � 0.022OD/�g protein) than in the cerebellum (0.052 � 0.019OD/�g protein) and was not detected in either theheart or the liver. These values are consistent with theessential role proposed for FMRP in cognition and inbehavior (Hinds et al., 1993).

FMR1 Gene Knockout Did Not Affect FXRExpression

There are three highly homologous genes in the Frag-ile X gene family, FMR1, FXR1, and FXR2. The FXR1 orFXR2 gene-encoded FXR protein, which possesses anamino acid sequence partly homologous with FMRP, isa cytoplasmic protein with the characteristics of anRNA binding protein (Siomi et al., 1995; Zhang et al.,1995). Because the FMR1 gene knockout mice did notexpress FMRP it was of particular interest to determineif the expression of the related protein, FXR, was af-fected also in these mice. Using an antibody that couldrecognize both FMRP and FXR, we carried out Westernblot analysis on brains from the FMR1 gene knockoutand wild-type mice. The results, illustrated in Fig. 3B,demonstrated that the level of FXR in the brains ofknockout mice (n � 8 mice) was not reduced comparedto wild-type controls (n � 8 mice), indicating that theexpression of FXR genes did not appear to be linked tothat of the FMR1 gene.

FMRP Deficiency Reduced GluR1, but Not NR2,Expression in Cerebral Cortex

Glutamate is the major excitatory neurotransmitter inthe central nervous system (CNS) (Greenamyre and

Porter, 1994). The glutamate receptors, such as AMPAand NMDA receptors that have been identified in themammalian brain, are classified into several subtypesbased upon pharmacological and electrophysiologicaldata (Hollmann and Heinemann, 1994; Dingledine et al.,1999). Both AMPA and NMDA receptors are multi-meric heteromers composed of distinct subunits, andthere is consensus concerning their critical roles inlearning and memory (Bliss and Collingridge, 1993;Danysz et al., 1995; Asztely and Gustafsson, 1996;McHugh et al., 1996; Tsien, 2000; Lu et al., 2001). There-fore, we were interested in assessing whether FMRPdeficiency was accompanied by alterations in the ex-

FIG. 3. Expression of FMRP and FXR in brain, heart, and liver fromFMR1 gene knockout and wild-type mice. Sample proteins preparedfrom cerebral cortex (cortex), hippocampus (hippoc), cerebellum(cerebe), heart, and liver were processed for Western blot analysis.The protein was separated by 10% SDS–polyacrylamide gel electro-phoresis and transferred to nitrocellulose membrane, which was in-cubated with mouse monoclonal antibody against both FMRP (74kDa) and FXR (69 kDa). Protein bands were visualized by incubationwith horseradish peroxidase-conjugated goat anti-mouse IgG fol-lowed by ECL Western blotting detection regents. (A) Tissue-specificdistribution of FMRP and FXR from wild-type mice. The loadedsample protein was 50 (a) or 10 �g (b) per lane. FMRP was found onlyin the brain and not in the heart and liver, while the expression of FXRwas much higher in the heart and liver than in the brain. (B) Brainfrom FMR1 gene knockout mice did not express any FMRP (100 �g ofprotein per lane); however, the expression of FXR was detected inboth wild-type mice (w) and knockout mice (k). (C) Expression ofFMRP was significantly greater in cerebral cortex compared to cere-bellum from wild-type mice (black column; n � 8 mice). Note, theexpression of FMRP in knockout mice was below the level of detec-tion (i.e., OD values were zero).


pression of representative types of these glutamate re-ceptors.

As illustrated in Fig. 4, Western blot analysis of theNMDA receptor subunits in wild-type mice (n � 8mice) indicated that the levels of NR2A and 2B (180kDa) or NR2C (140 kDa) were higher in cerebral cortex(0.105 � 0.018 OD/�g protein) than in hippocampus(0.090 � 0.017 OD/�g protein) or in cerebellum(0.078 � 0.012 OD/�g protein). There was no differencein the expression in brain of these NMDA receptorsubunits when FMR1 gene knockout mice were com-pared to wild-type controls (n � 8 mice for eachgroup).

Recent work has demonstrated an important functionspecifically for the AMPA receptor in synaptic plasticity(Musleh et al., 1997; Nayak et al., 1998; Zamanillo et al.,1999). Therefore, because of the significantly reducedmagnitude of LTP in cortical slices prepared fromFMR1 gene knockout mice, it was of interest to assessthe expression of the AMPA receptor subunit GluR1, in

brain regions from both knockout mice and wild-typemice. As illustrated in Fig. 5, the expression of GluR1(105 kDa) in wild-type mice (n � 8 mice) was signifi-cantly higher in cerebral cortex (0.66 � 0.10 OD/�gprotein) and hippocampus (0.62 � 0.08 OD/�g protein)compared to cerebellum (0.33 � 0.04 OD/�g protein).The GluR1 levels in hippocampus and cerebellum werenot different between the two groups of mice; however,most importantly, the expression of GluR1 was reducedsignificantly in cerebral cortex from FMR1 gene knock-out mice (0.38 � 0.06 OD/�g protein) compared towild-type controls (0.66 � 0.10 OD/�g protein) (n � 8mice for each group). Furthermore, differences in thecortical expression of GluR1 between both groups werealso visualized using immunohistochemistry, which ispresented in Fig. 7 and Table 1.

Other Learning-Related Proteins Were NotAffected in the Absence of FMRP

The reduced expression of GluR1 in the knockoutmice encouraged us to measure other learning-relatedproteins such as the immediate early transcription fac-tors cAMP response element binding protein (CREB)and c-fos, the structural protein myelin proteolipid pro-

FIG. 5. Expression of GluR1 in brain from FMR1 gene knockout andwild-type mice. Sample proteins (100 �g per lane) prepared from thecerebral cortex (cortex), hippocampus (hippoc), and cerebellum(cerebe) were processed for Western blot analysis as described pre-viously. The primary antibody was a rabbit polyclonal antibodyagainst GluR1 (105 kDa). (A) Representative example of Western blotanalysis for wild-type (w) and knockout (k) mice. (B) Summary ofthese experiments demonstrates regional differences in the expressionof GluR1. The expression of GluR1 in the cortex of FMR1 geneknockout (white column) mice was significantly reduced compared towild-type mice (black column) (n � 8 mice for each group).

FIG. 4. Expression of NR2A, 2B, and 2C in brain from FMR1 geneknockout and wild-type mice. Sample proteins (100 �g per lane)prepared from the cerebral cortex (cortex), hippocampus (hippoc),and cerebellum (cerebe) were processed for Western blot analysis asdescribed previously. Rabbit polyclonal antibody against bothNR2A � 2B (180 kDa) and NR2C (140 kDa) was used as the primaryantibody. The secondary antibody was the horseradish peroxidase-conjugated goat anti-rabbit IgG. (A) Representative result of Westernblot analysis demonstrates two distinct bands for NR2A � 2B andNR2C in the cerebral cortex, hippocampus, and cerebellum. (B) Thelevels of total NR2 (NR2A � 2B � 2C) did not differ when wild-typemice (black column) were compared to knockout mice (white column)(n � 8 mice for each group).

142 Li et al.

tein (MPP), and the vesicular trafficking protein synap-sin (Syn). Western blot analysis revealed that MPP, Syn,CREB, and c-fos were expressed in the cerebral cortex,hippocampus, and cerebellum and, as illustrated in Fig.6, that their levels in the FMR1 gene knockout micewere not different compared to levels in the wild-typecontrols. Immunohistochemical detection also showedthat c-fos and Syn were stained in nuclei and synapticterminals, respectively, in both cerebral cortex and hip-pocampus, and their expression was not different be-tween the two groups of mice. Representative examplesof the expression of the learning-related proteins we

assessed, visualized using conventional immunohisto-chemical techniques, are presented in Fig. 7. Quantifi-cation of the immunohistochemical data is summarizedin Table 1. Additionally, light microscopic examinationindicated that the brains (cerebral cortex, hippocampus,striatum, corpus callosum, and hypothalamus) ofknockout mice (n � 8 mice) were normal in appearance(Fig. 7). This is consistent with the absence of grossabnormalities in brains of FMR1 gene knockout micereported previously (The Dutch–Belgian Fragile X Con-sortium, 1994).

Reduced Levels of GluR1 in Cortical SynapticPlasma Membrane (SPM) from FMR1 GeneKnockout Mice Persisted Following LTP Induction

There is increasing evidence that AMPA receptors arenot restricted to synapses, but are also expressed extra-synaptically and even found in glia (Noda et al., 2000).Therefore, to compare the GluR1 levels in cortical syn-apses between the two groups of mice, SPMs wereprepared from cortical slices, and the levels of NR2,GluR1, and Syn were measured using Western blotanalysis. Consistent with the above finding in corticaltissue homogenate, a reduced expression of GluR1 wasalso observed in cortical SPM from the FMR1 geneknockout mice (data not shown).

To observe GluR1 levels in cortical SPMs followingLTP induction, cortical slices were exposed to LTP-inducing factor (25 mM tetraethylammonium; TEA) for

TABLE 1

Staining Grades of GluR1, c-fos, and Syn Proteins in Brain Sections from FMR1 Gene Knockout and Wild-Type Mice

GluR1 (%) c-fos (%) Syn (%)

Wild-type Knockout Wild-type Knockout Wild-type Knockout

Cerebral cortex�� 10 5* 14 12 5 6�� 34 25* 32 35 20 18� 39 37* 28 30 33 36� 17 33* 26 23 42 40

Hippocampus�� 14 12* 14 13 19 18�� 36 32* 30 27 21 24� 28 30* 33 35 40 38� 22 26* 23 25 20 20

Note. 200 cells from each section of cerebral cortex or hippocampus were counted and scored into four grades: �� (dark brown), ��(brown), � (light brown), and � (not stained). The data represent the average percentage (%) for each staining grade in 10 sequential sections(n � 5 for each group). Comparisons between groups were performed using the Ridit analysis (Sermeus and Delesie, 1996; Donaldson, 1998).Asterisk denotes P � 0.05, when the GluR1 staining in the cerebral cortex (staining grade 10, 34, 39, and 17%) from wild-type mice is comparedto that in knockout mice (staining grade 5, 25, 37, and 33%).

FIG. 6. Western blot analysis of four learning-related proteins incortex from FMR1 gene knockout and wild-type mice. Sample pro-teins (100 �g per lane) prepared from the cerebral cortex were pro-cessed for Western blot analysis as described previously. Four poly-clonal antibodies were used to detect Syn, c-fos, CREB, and MPP.There were no differences in the expressions of these learning-relatedproteins in the cortex when knockout mice (k) were compared towild-type mice (w).


7 min and then left in TEA-free artificial cerebral spinalfluid (ACSF) for 45 min. This protocol produces a noveland well-developed form of LTP in either cortex (Pel-letier and Hablitz, 1996) or hippocampus (Aniksztejnand Ben-Ari, 1991). As illustrated in Fig. 8, a signifi-cantly reduced level of GluR1 in SPMs, during TEA-induced LTP, was found in knockout mice (2.75 � 0.43OD/�g protein) compared to wild-type mice (4.30 �0.65 OD/�g protein). Ratio values of GluR1:Syn were0.58 and 1.12 in knockout and wild-type mice, respec-tively. Moreover, Fig. 8B shows that these proteins werehighly enriched in SPM (2–5 OD/�g protein) compared

to brain homogenate (0.1–0.8 OD/�g protein, Figs. 4Band 5B).

DISCUSSION

These data show that the expression of FMRP couldbe detected in cerebral cortex, hippocampus, and cere-bellum, but not in heart and liver from wild-type mice.In contrast, the FMR1 gene knockout resulted in theabsence of FMRP, but did not change the amount ofFXR, another RNA binding protein. The expressions of

FIG. 7. Histology and immunohistochemical detection of Syn, c-fos, and GluR1 in cerebral cortex and hippocampus from FMR1 gene knockoutand wild-type mice. Cryostat sections (10 �m) were incubated in affinity-purified rabbit polyclonal antibody against Syn, c-fos, or GluR1. Boundantibodies were visualized by incubation with a biotinylated goat anti-rabbit IgG, followed by a preformed avidin-biotinylated horseradishperoxidase macromolecular complex and diaminobenzidine tetrahydrochloride. Some sections were stained with cresyl violet for lightmicroscopic examination. No gross differences in morphology were observed in the cerebral cortex (A) and hippocampus (B) from knockoutmice. Syn staining in the cerebral cortex and hippocampus did not differ between wild-type (C) and knockout (D) mice. c-fos was stained innuclei in both the cerebral cortex (E, F) and the hippocampus (G, H), and its expression also did not differ between wild-type (E, G) and knockout(F, H) mice. Note, the GluR1 immunohistochemical staining in the cellular membrane of the cerebral cortex was reduced in knockout mice (J)compared to wild-type mice (I), while this expression in the hippocampus (K, L) showed no difference between the two groups.

144 Li et al.

the NMDA receptor subunits NR2A, NR2B, and NR2Cand other learning-related proteins such as c-fos, Syn,MPP, and CREB were not different between the twogroups. In contrast, the FMRP deficiency in knockoutmice was associated with a significant reduction in theexpression of the AMPA receptor subunit GluR1 both inthe cerebral cortex and in the cortical SPMs. Althoughthere were no changes in the hippocampal LTP, we didobserve a significant reduction in the cortical LTP in theknockout mice compared to the wild-type controls.

Studies of mice have shown that the FMRP is ex-pressed in all embryonic tissues, reaching the highestlevel by day 10 of development (Hinds et al., 1993). Inadult mice, FMRP has been found throughout the CNS,but is absent in adult heart, mature ovary, and testis.These observations are similar to our findings and sug-gest that FMRP is important in the development of alltypes of cells and, at adulthood, in nervous systemfunction. The FMR1 gene knockout mice that lackednormal FMRP and showed macroorchidism, learningdeficits, and hyperactivity were reminiscent of the hu-man Fragile X condition (The Dutch–Belgian Fragile XConsortium, 1994). Using this animal model wechecked the effects of FMRP deficiency on the synaptictransmission and plasticity and the expression of sev-

eral learning-related proteins including two glutamatereceptors: the NMDA receptor and the AMPA receptor.

Although the cellular processes underlying LTP arenot understood fully, there is evidence to suggest thatsome mechanisms of synaptic plasticity are common toboth hippocampus and cortex (Aniksztejn and Ben-Ari,1991; Pelletier and Hablitz, 1996). Mental retardation isthought, in part, to result from abnormalities of synap-tic structure and function, arising from anomalies in thedevelopment of the CNS (Nelson et al., 2001); however,previous reports do not support the hypothesis thatFMRP plays an important role in hippocampal LTP(Godfraind et al., 1996; Paradee et al., 1999) and that theLTP induction changes FMR1 mRNA levels in hip-pocampus (Valentine et al., 2000). To address this issue,we first assessed normal synaptic transmission, thenLTP in the cerebral cortex slices prepared from knock-out and wild-type mice. Consistent with the previousreports, our observations showed no differences in hip-pocampal LTP between both groups of mice. On theother hand, although there was no alternation in nor-mal synaptic transmission, demonstrated by similar I/Orelations between the two groups, we did observe asignificant reduction in the magnitude of cortical LTP inknockout mice compared to wild-type mice. A reduc-tion in cortical LTP, in the absence of differences innormal synaptic transmission, has been reported previ-ously in mice deficient for endothelial nitric oxide syn-thase (Haul et al., 1999). Our findings indicate that themental retardation derived from FMRP deficiencycould be associated with the depressed cortical synapticplasticity.

FMRP plays an important role in processes of proteinsynthesis including RNA transportation from nucleusto cytoplasm and the translation of mRNA to protein(Siomi et al., 1993; Eberhart et al., 1996; Bardoni et al.,1997; Brown et al., 1998). FMRP also seems to be one ofthe proteins produced at synapses in response tometabotropic glutamate receptor activation (Weiler andGreenough, 1993, 1999). Thus, we hypothesized that themental retardation due to the FMRP deficiency in theFragile X syndrome could be mediated by a diminutionof learning-related proteins such as glutamate receptorsin the CNS. All of the six proteins involved in thepresent study have been implicated in some aspects ofLTP and play important roles in synaptic structures orfunctions (Requeiro et al., 1996; Lanahan et al., 1997;Matthies al et., 1997; Dawson et al., 1999; Maren, 1999;Zamanillo et al., 1999). For example, MPP, essential forrapid and effective propagation of the action potentialwithin the axon, is a predominant integral membraneprotein in the mammalian CNS myelin (Requeiro et al.,

FIG. 8. GluR1, NR2, and Syn levels in cortical SPM following LTPinduction. Sample proteins (10 �g per lane for GluR1 or Syn and 20�g per lane for NR2) of cortical SPM treated with TEA were processedfor Western blot analysis as described previously. Rabbit polyclonalantibody against NR2 (1 �g/ml), GluR1 (1 �g/ml), or Syn (0.1 �g/ml)was used as the primary antibody. The secondary antibody was thehorseradish peroxidase-conjugated goat anti-rabbit IgG. (A) Repre-sentative result of Western blot analysis for wild-type (w) and knock-out (k) mice. (B) Summary of these SPM experiments demonstrates asignificant difference in GluR1 (not NR2 and Syn) levels betweenwild-type (black column) and knockout mice (white column) (n � 6mice for each group).


1996). Syn is a neuron-specific protein that serves as anexcellent marker for synaptic terminals and is believedto regulate neurotransmitter release through a phos-phorylation-dependent interaction with cytoskeletal el-ements (Edelmann et al., 1995). The transcription factorCREB is a nuclear phosphoprotein responsible for thetranscriptional activation of a number of differentgenes, and the transcription mediated by CREB is nec-essary for consolidation of long-term memory (Ginty,1997). Neuronal induction of c-fos, an immediate earlygene, occurs from calcium influx through both gluta-mate receptors and voltage-sensitive calcium channelsand has been shown to play a role in learning andmemory (Dragunow, 1996). Both the NMDA receptorsubunit NR2 and the AMPA receptor subunit GluR1mediate a number of neuronal processes such as exci-tatory synaptic transmission (Hollmann and Heine-mann, 1994; Wenthold et al., 1996) and memory forma-tion of inhibitory avoidance learning (Cammarota et al.,1998). Our observation of the significant reduction inGluR1 expression in FMR1 gene knockout mice sug-gests that the process whereby the FMRP deficiencyleads to abnormal brain development might be associ-ated with the reduced GluR1 activity. By studyingGluR1 activity-dependent changes in postsynapticallylocalized protein translation in the CNS, Weiler et al.(1997) and Weiler and Greenough (1999) have foundthat FMR1 mRNA and FMRP are rapidly synthesized atsynapses following stimulation of metabotropic GluR1and that Fragile X knockout mice, like human Fragile Xpatients, have excess numbers of long, thin, immature-appearing dendritic processes, suggesting an involve-ment of FMRP and GluR1 in the structural and func-tional maturation of the synapse.

The ability of central glutamatergic synapses tochange their strength in response to the intensity ofsynaptic input, which occurs, for example, in LTP, isthought to provide a cellular basis for memory forma-tion and learning (Derkach et al., 1999). There is awealth of evidence demonstrating a relation betweenNMDA receptors and synaptic plasticity, including theconcept that NMDA receptors are necessary for theinduction of associative LTP (Collingridge et al., 1983).More recently, several studies have elucidated a role forthe AMPA receptor in LTP induction and the significantcoordination of the AMPA receptor subunit GluR1 andLTP in memory formation and learning. In 1995, bothLiao et al. and Isaac et al. showed that following an LTPinduction protocol, previously “silent” synapses ac-quire AMPA-type responses. These data suggested thatAMPA receptors could be inserted into the postsynapticmembrane following LTP induction and also could ex-

plain LTP by a solely postsynaptic mechanism. Asso-ciative LTP is absent in CA3 to CA1 synapses fromadult GluR1 gene knockout mice (Zamanillo et al., 1999),while the increased synthesis of GluR1 is a postsynapticmechanism maintaining late-phase LTP (Nayak et al.,1998). Moreover, the subcellular localization of GluR1 isimportant to LTP induction, and Desmond and Wein-berg (1998) showed that insertion of GluR1 protein intothe postsynaptic membrane of previously silent syn-apses contributes to LTP. Tetanic synaptic stimulationinduces a rapid delivery of GluR1 into dendritic spinesand this postsynaptic trafficking event may contributeto the enhanced AMPA receptor-mediated transmissionobserved during LTP and activity-dependent synapticmaturation (Shi et al., 1999). Finally, LTP is also associ-ated with structural modifications of GluR1. Inductionof LTP increases the 32P labeling of the AMPA receptor(Barria et al., 1997). Phosphorylation of Ser-831 in GluR1provides a postsynaptic molecular mechanism for LTP(Derkach et al., 1999) and for synaptic plasticity (Barriaet al., 1997). Our findings from the FMR1 gene knockoutmice show that the FMRP deficiency reduces specifi-cally both the cerebral cortical LTP and the expressionof GluR1 in cerebral cortex or cortical SPM exposed toLTP-enhancing factor, further supporting the signifi-cant relationship between GluR1 and LTP.

The unchanged hippocampal LTP could reflect thenormal level of GluR1 expression in hippocampus fromFMR1 gene knockout mice compared to wild-type mice,but mechanisms that could account for the differentialregulations of GluR1 and LTP between cortex and hip-pocampus remain to be explored. Consistent with ourfindings in the cerebral cortex of the FRM1 gene knock-out mice, Greenough et al. (2001) reported that, also inthe somatosensory cortical region containing the barrel-like cell arrangements that process whisker informa-tion, the FMRP deficiency impairs normal dendrite re-gression, suggesting that FMRP may be required fornormal cerebral cortical development. Nimchinsky et al.(2001) showed, in layer V neurons in the barrel cortex,that dendritic spines in the intact brains of FMR1 knock-out mice were abnormally long in early postnatal de-velopment.

Taken together, the reduced GluR1 expression (im-paired postsynaptic activity) and the reduced LTP (im-paired synaptic plasticity), occurring in the cerebralcortex of the FMR1 gene knockout mice, are potentialcandidate mechanisms underlying the cognitive andbehavioral impairments observed in the Fragile X syn-drome.

146 Li et al.

EXPERIMENTAL METHODS

Animal Model

Male mice with the FMR1 gene knockout (C57BL/6J,The Jackson Laboratory, Bar Harbor, ME) were gener-ated by homologous recombination of a targeting vec-tor into the mouse germ line using embryonic stem celltechnology (The Dutch–Belgian Fragile X Consortium,1994). These knockouts (8–10 weeks of age), which hadbeen shown to be negative for both full-length FMR1mRNA and normal FMRP in their testes and brains,were used to explore the effects of FMRP deficiency onsynaptic plasticity and learning-related protein expres-sion. Male wild-type littermates of the knockouts wereused as normal controls. The use of animals in theseexperiments was in accordance with the guidelines es-tablished by the Canadian Institute of Health Researchand the University of Toronto. All efforts were made tominimize the number of animals used and their suffering.

Electrophysiology

Wild-type and knockout mice were anesthetized withHalothane (Halocarbon Laboratories, River Edge, NJ)and then decapitated. The brain was removed rapidlyand placed for approximately 1 min in ice-cold, oxy-genated (95% O2/5% CO2), ACSF containing (in mM)126 NaCl, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, 2 MgSO4, 26NaHCO3, and 10 glucose (pH 7.4; 300 � 5 mOsm). Ablock of brain was fixed to an aluminum chuck usingcyanoacrylate glue and then coronal frontal (somato-sensory) cortex and hippocampal slices (400 �m) wereprepared with a Vibratome. The methods for prepara-tion of cortical brain slices and evoking postsynapticresponses have been described previously (Pelletier andHablitz, 1996; Sutor and Hablitz, 1989). Briefly, coronalsections (400–500 �m) from the frontal neocortex wereprepared using a Vibratome. Neocortical slices rostralto the caudate were used in the experiments. After anincubation period of at least 1 h at room temperature(20–22°C), slices were transferred to an interface-typechamber (PDMI-2; Harvard Apparatus, South Natick,MA).

In cortical slices, synaptic responses were recordedextracellularly with NaCl-filled (150 mM) borosilicateglass pipettes located in layer IV/V and were evokedvia a bipolar stimulating electrode (enamel-insulatednichrome wire; 125 �m diameter) positioned directlybelow the recording pipette either in layer VI or in thewhite matter at the base of the cerebral cortex. In hip-pocampal slices, the recording pipette was positioned

in the stratum radiatum and synaptic responses wereevoked via a stimulating electrode positioned in thestratum radiatum to orthodromically stimulate theSchaffer collaterals. I/O relations were determined byvarying the amplitude of 100-�s duration pulses with 5to 10 intensities, which produced synaptic responsesranging from threshold to maximal. A stimulating in-tensity producing a PSP in the middle of the I/O rela-tion, typically 1 to 2 mV in amplitude, was selected asthe test pulse intensity and then stimulation was deliv-ered at 0.03 Hz. Tetanic stimulation was delivered aftera stable baseline was established, which we defined asresponses varying not more than �10% for 20 min, andtypically required approximately 30–40 min. Hip-pocampal LTP was produced by a single tetanic train ofpulses (100 Hz, 1 s duration) at maximal intensity. Incortex, LTP was induced by repeating a tetanic train ofpulses (200 Hz, 1 s duration) three times with an intertraininterval of 10 min. LTP was assessed by measuring theamplitude of the PSP at least 45 min after the tetanizationprotocol. Experiments were conducted at 34°C. Synap-tic responses were recorded using an Axopatch 200Bamplifier (normal I-clamp mode) and filtered at 1–3kHz. Data were stored via a 12-bit D/A interface (Digi-data 1200) and analyzed using pCLAMP version 6.0.3software (Axon Instruments, Foster City, CA).

Chemicals used in electrophysiological recordings in-cluded the NMDA receptor antagonist D-AP5 (Tocris,Ballwin, MO), the AMPA receptor antagonist CNQXdisodium (Tocris), and the sodium channel blocker TTX(Alomone Laboratories, Jerusalem, Israel). They weredissolved in water, stored as stock solutions (50 mM, 10mM, and 100 �M, respectively), and bath applied.

SPM Preparation

SPM was prepared from mouse cerebral cortex ac-cording to modifications of the published procedures(Monahan and Michel, 1987; Lai et al., 1999). Briefly, thesomatosensory cortex was removed and homogenizedin 0.32 M sucrose, 0.05 mM CaCl2, 0.25 mM phenyl-methylsulfonyl fluoride, and 0.2 mM Hepes (pH 7.4).The homogenate was centrifuged at 1000g for 5 min,and the supernatant was saved and kept on ice. The P1

pellet was suspended in the same sucrose solution andcentrifuged again at 1000g for 5 min. The combinedsupernatants were then centrifuged at 38,900g for 20min. The P2 pellet was lysed osmotically in 1 mMTris-acetate (pH 8.0), layered on 10 ml of 1.2 M sucrosein 1 mM Hepes, and centrifuged at 230,000g for 40 min.The gradient interface was collected, diluted, and lay-ered on 10 ml of 0.9 M sucrose in 1 mM Hepes. Follow-


ing centrifugation again at 230,000g for 40 min, the P3

pellet (SPM) was washed twice by centrifugation with 1mM Tris-acetate (pH 7.4) and stored at a concentrationof 5 mg/ml in 0.32 M sucrose, 0.5 mM EDTA, 1 mMMgSO4, and 5 mM Tris-acetate (�80°C).

Antibodies

Mouse monoclonal antibody against FMRP (Chemi-con, Temecula, CA) was generated with a fusion pro-tein that was localized to the N-terminal half of thehuman full-length FMRP. This antibody also cross-re-acts with a novel and homologous protein, FXR, incases of high expression of the latter protein (manufac-turer’s manual). Affinity-purified rabbit polyclonal IgGantibodies were all obtained from Calbiochem (Cam-bridge, MA). One antibody against AMPA receptorsubunit GluR1 could detect either a single band of 105kDa in Western blots or a GluR1 signal on the cellularmembrane in immunohistochemical sections. This anti-GluR1 antibody was generated with a synthetic peptidecorresponding to amino acids 271–285 of the GluR1.Another antibody against the NMDA receptor subunit,NR2C, was a pan antibody that was generated with a17-kDa fusion protein constructed from the N-terminalregion (amino acids 25–130) of NR2C. This anti-NR2Cantibody could recognize the 180-kDa NR2A and NR2Bas well as the 140-kDa NR2C in Western blots. Theother antibodies used were anti-c-fos, anti-Syn, anti-MPP, and anti-CREB.

Sample protein prepared from cerebral cortex ho-mogenate of the wild-type mice was processed for ex-ploring both titration curves for primary antibodies andstandard curves for densitometry. According to themanufacturer’s recommendation and our titrationcurve, the concentrations of primary antibodies used inWestern blot analysis were 1 �l/ml for FMRP/FXR orCREB; 1 �g/ml for NMDA receptor subunit NR2,AMPA receptor subunit GluR1, or c-fos; 0.1 �g/ml forSyn; and 5 �l/ml for MPP. The standard curve fordensitometry indicated also that 100 �g/lane of tissuehomogenate protein loaded on sodium dodecyl sulfate(SDS) polyacrylamide gel could produce a signal withinthe quantitative range of antibody response. In addi-tion, the concentrations of primary antibodies used inimmunohistochemical detection were 2.5 �g/ml forGluR1, 0.4 �g/ml for Syn, and 2 �g/ml for c-fos.

Western Blot Analysis

Cerebral cortex, hippocampus, and cerebellum frommouse brains were homogenized separately in chilled

phosphate-buffered saline (PBS), and a sample fromeach homogenate was allocated for protein determina-tion using a total protein assay kit (Sigma Diagnostics,St. Louis, MO). Samples (tissue homogenate or SPM)were dissolved in SDS sample buffer and denatured for3 min at 95°C prior to loading. The sample proteinswere, along with rainbow molecular weight markers(Amersham Pharmacia Biotech, Piscataway, NJ), sepa-rated by 10% SDS–polyacrylamide gel electrophoresis(Bio-Rad, Hercules, CA) and transferred from gel tonitrocellulose membrane (Schleicher & Schuell, Keene,NH). The gel was then stained with Gelcode blue stainreagent (Pierce, Rockford, IL) to control for proteintransfer. The membrane was blocked with 5% fat-freemilk at room temperature for 30 min, rinsed brieflywith TBST buffer (10 mM Tris, 150 mM sodium chlo-ride, 0.05% Tween 20), and then incubated overnight at4°C with antibodies against FMRP, FXR, GluR1, NR2,c-fos, Syn, MPP, or CREB. After being washed withTBST buffer four times, 15 min each, the membrane wasincubated with horseradish peroxidase-conjugated goatanti-mouse or anti-rabbit IgG (H�L) (Promega, Madi-son, WI) for 1 h and reacted with ECL Western blottingdetection reagents (Amersham Pharmacia Biotech) for 3min. A high-performance autoradiography film (Amer-sham Life Science, Buckinghamshire, England) wastaped onto the Saran-Wrapped membrane in a darkroom for 10 s to 1 min and then developed to visualizethe antibody binding. Quantity One software (Bio-Rad)was used for the quantitative analyses of protein bandson the film. Using both band density and band area,Quantity One software could calculate the integratedintensity of the band (OD value). We constructed astandard curve in each experiment by using a knownprotein concentration of mouse cortex, which permittedquantification of the results relative to the standardtissue and between-group comparisons. Actin or MPPwas used as a control protein on the same blot toeliminate loading differences.

Immunohistochemical Detection

To visualize both histological structure and immuno-histochemical signal, blocks of brains from knockoutand wild-type mice were immersed quickly in isopen-tane (�80°C) for 30 min, placed in a cryostat chamber,and mounted on cutting chucks using OCT embeddingmedium. The specimens were left in the cryostat for 10min to equilibrate the temperature with the prechilledmicrotome (�20°C) and then trimmed to a trapezoidshape using a prechilled razor blade. Once the speci-men blocks were cut such that the structure of interest

148 Li et al.

could be seen within the cut face, sections were col-lected. Cryostat sections (10 �m) from the brains wereprocessed for immunohistochemistry. The sectionswere prefixed with 4% chilled paraformaldehyde inPBS for 5 min and blocked with goat serum in PBS for20 min at room temperature to suppress nonspecificbinding of immunoglobin. After being rinsed with threechanges of PBS, the sections were incubated overnightat 4°C in affinity-purified rabbit polyclonal antibodiesagainst c-fos, Syn, and GluR1. Specifically bound anti-bodies were visualized at room temperature by incuba-tion with a biotinylated goat anti-rabbit IgG (Calbio-chem) followed by a preformed avidin-biotinylatedhorseradish peroxidase macromolecular complex anddiaminobenzidine tetrahydrochloride. Some sectionswere also stained with hematoxylin or cresyl violet forbasic histological examination.

Statistics

Comparisons between groups were performed usingthe Student t test for electrophysiological data andWestern blotting data and the Ridit analysis (Sermeusand Delesie, 1996; Donaldson, 1998) for ordinal data ofimmunohistochemistry, either paired or unpaired asrequired. Results are presented as means � SD anddifferences were considered significant at P � 0.05.

ACKNOWLEDGMENTS

This work was supported by grants from the Fragile X ResearchFoundation of Canada to J.L. and J.L.P.V. and the Canadian Institutesof Health Research to P.L.C. We thank Frank Vidic and Li Liu fortechnical assistance.

REFERENCES

Abrams, M. T., Doheny, K. F., Mazzocco, M. M., Knight, S. J., Baum-gardner, T. L., Freund, L. S., Davies, K. E., and Reiss, A. L. (1997).Cognitive behavioral, and neuroanatomical assessment of two un-related male children expressing FRAXE. Am. J. Med. Genet. 74:73–81.

Aniksztejn, L., and Ben-Ari, Y. (1991). Novel form of long-term po-tentiation produced by a K channel blocker in the hippocampus.Nature 349: 67–69.

Asztely, F., and Gustafsson, B. (1996). Ionotropic glutamate receptors.Their possible role in the expression of hippocampal synaptic plas-ticity. Mol. Neurobiol. 12: 1–11.

Bardoni, B., Sittler, A., Shen, Y., and Mandel, J. L. (1997). Analysis ofdomains affecting intracellular localization of the FMRP protein.Neurobiol. Dis. 4: 329–336.

Barria, A., Muller, D., Derkach, V., Griffith, L. C., and Soderling, T. R.(1997). Regulatory phosphorylation of AMPA-type glutamate re-

ceptors by CaM-KII during long-term potentiation. Science 276:2042–2045.

Bliss, T. V. P., and Collingridge, G. L. (1993). A synaptic model ofmemory: Long-term potentiation in the hippocampus. Nature 361:31–39.

Brown, V., Small, K., Lakkis, L., Feng, Y., Gunter, C., Wilkinson, K. D.,and Warren, S. T. (1998). Purified recombinant Fmrp exhibits se-lective RNA binding as an intrinsic property of the fragile X mentalretardation protein. J. Biol. Chem. 273: 15521–15527.

Cammarota, M., Bernabeu, R., Levi De Stein, M., Izquierdo, I., andMedina, J. H. (1998). Learning-specific, time-dependent increases inhippocampal Ca2�/calmodulin-dependent protein kinase II activityand AMPA GluR1 subunit immunoreactivity. Eur. J. Neurosci. 10:2669–2676.

Chen, L., and Toth, M. (2001). Fragile X mice develop sensory hyper-reactivity to auditory stimuli. Neuroscience 103: 1043–1050.

Chiurazzi, P., Pomponi, M. G., Willemsen, R., Oostra, B. A., and Neri,G. (1998). In vitro reactivation of the FMR1 gene involved in fragileX syndrome. Hum. Mol. Genet. 7: 109–113.

Collingridge, G. L., Kehl, S. J., and McLennan, H. (1983). Excitatoryamino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334: 33–46.

Danysz, W. W., Zajaczkowski, W. W., and Parsons, C. G. C. (1995).Modulation of learning processes by ionotropic glutamate receptorligands. Behav. Pharmacol. 6: 455–474.

Dawson, G. R., Seabrook, G. R., Zheng, H., Smith, D. W., Graham, S.,O’Dowd, G., Bowery, B. J., Boyce, S., Trumbauer, M. E., Chen, H. Y.,van der Ploeg, L. H., and Sirinathsinghji, D. J. (1999). Age-relatedcognitive deficits, impaired long-term potentiation and reduction insynaptic marker density in mice lacking the beta-amyloid precursorprotein. Neuroscience 90: 1–13.

Derkach, V., Barria, A., and Soderling, T. R. (1999). Ca2�/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc.Natl. Acad. Sci. USA 96: 3269–3274.

Desmond, N. L., and Weinberg, R. J. (1998). Enhanced expression ofAMPA receptor protein at perforated axospinous synapses. Neuro-Report 9: 857–860.

D’Hooge, R., Nagels, G., Franck, F., Bakker, C. E., Reyniers, E., Storm,K., Kooy, R. F., Oostra, B. A., Willems, P. J., and De Deyn, P. P.(1997). Mildly impaired water maze performance in male Fmr1knockout mice. Neuroscience 76: 367–376.

Dingledine, R., Borges, K., Bowie, D., and Traynelis, S. F. (1999). Theglutamate receptor ion channels. Pharmacol. Rev. 51: 7–61.

Donaldson, G. W. (1998). Ridit scores for analysis and interpretationof ordinal pain data. Eur. J. Pain 2: 221–227.

Dragunow, M. (1996). A role for immediate-early transcription factorsin learning and memory. Behav. Genet. 26: 293–299.

The Dutch–Belgian Fragile X Consortium (1994). FMR1 knockoutmice: A model to study fragile X mental retardation. Cell 78: 23–33.

Eberhart, D. E., Malterm, H. E., Feng, Y., and Warren, S. T. (1996). Thefragile X mental retardation protein is a ribonucleoprotein contain-ing both nuclear localization and nuclear export signals. Hum. Mol.Genet. 5: 1083–1091.

Edelmann, L., Hanson, P. L., Chapman, E. R., and Jahn, R. (1995).Synaptobrevin binding to synaptophysin: A potential mechanismfor controlling the exocytotic fusion machine. EMBO J. 14: 224–231.

Fisch, G. S., Hao, H. K., Bakker, C., and Oostra, B. A. (1999). Learningand memory in the FMR1 knockout mouse. Am. J. Med. Genet. 84:277–282.


Ginty, D. D. (1997). Calcium regulation of gene expression: Isn’t thatspatial. Neuron 18: 183–186.

Godfraind, J. M., Reyniers, E., De Boulle, K., D’Hooge, R., De Deyn,P. P., Bakker, C. E., Oostra, B. A., Kooy, R. F., and Willems, P. J.(1996). Long-term potentiation in the hippocampus of fragile Xknockout mice. Am. J. Med. Genet. 64: 246–251.

Greenamyre, J. T., and Porter, R. H. (1994). Anatomy and physiologyof glutamate in the CNS. Neurology 44: S7–S13.

Greenough, W. T., Klintsova, A. Y., Irwin, S. A., Galvez, R., Bates,K. E., and Weiler, I. J. (2001). Synaptic regulation of synthesis andthe fragile X protein. Proc. Natl. Acad. Sci. USA 98: 7101–7106.

Haul, S., Godecke, A., Schrader, J., Haas, H. L., and Luhmann, H. J.(1999). Impairment of neocortical long-term potentiation in micedeficient of endothelial nitric oxide synthase. J. Neurophysiol. 61:494–497.

Hinds, H. L., Ashley, C. T., Sutcliffe, J. S., Nelson, D. L., Warren, S. T.,Housman, D. E., and Schalling, M. (1993). Tissue specific expressionof FMR-1 provides evidence for a functional role in fragile X syn-drome. Nat. Genet. 3: 36–43.

Hollmann, M., and Heinemann, S. (1994). Cloned glutamate recep-tors. Annu. Rev. Neurosci. 17: 31–108.

Hoogeveen, A. T., and Oostra, B. A. (1997). The fragile X syndrome.J. Inherit. Metab. Dis. 20: 139–151.

Isaac, J. T., Nicoll, R. A., and Malenka, R. C. (1995). Evidence for silentsynapses: Implications for the expression of LTP. Neuron 15: 427–434.

Kooy, R. F., D’Hooge, R., Reyniers, E., Bakker, C. E., Nagels, G., DeBoulle, K., Storm, K., Clincke, G., De Deyn, P. P., Oostra, B. A., andWillems, P. J. (1996). Transgenic mouse model for the fragile Xsyndrome. Am. J. Med. Genet. 64: 241–245.

Lai, S.-L., Ling, S.-C., Kuo, L.-H., Shu, Y.-C., Chow, W.-Y., and Chang,Y.-C. (1999). Characterization of granular particles isolated frompostsynaptic densities. J. Neurochem. 71: 1694–1701.

Lanahan, A., Lyford, G., Stevenson, G. S., Worley, P. F., and Barnes,C. A. (1997). Selective alteration of long-term potentiation-inducedtranscriptional response in hippocampus of aged, memory-im-paired rats. J. Neurosci. 17: 2876–2885.

Liao, D., Hessler, N. A., and Malinow, R. (1995). Activation ofpostsynaptically silent synapses during pairing-induced LTP inCA1 region of hippocampal slice. Nature 375: 400–404.

Lu, W., Man, H., Ju, W., Trimble, W. S., MacDonald, J. F., and Wang,Y. T. (2001). Activation of synaptic NMDA receptors induces mem-brane insertion of new AMPA receptors and LTP in cultured hip-pocampal neurons. Neuron 29: 243–254.

Lynch, G. (1998). Memory and the brain: Unexpected chemistries anda new pharmacology. Neurobiol. Learn. Memory 70: 82–100.

Maren, S. (1999). Long-term potentiation in the amygdala: A mecha-nism for emotional learning and memory. Trends Neurosci. 22: 561–567.

Matthies, H., Schulz, S., Thiemann, W., Siemer, H., Schmidt, H., Krug,M., and Hollt, V. (1997). Design of a multiple slice interface cham-ber and application for resolving the temporal pattern of CREBphosphorylation in hippocampal long-term potentiation. J. Neuro-sci. Methods 78: 173–179.

Mazzocco, M. M., Kates, W. R., Baumgardner, T. L., Freund, L. S., andReiss, A. L. (1997). Autistic behaviors among girls with fragile Xsyndrome. J. Autism Dev. Disord. 27: 415–435.

McEachern, J. C., and Shaw, C. A. (1999). The plasticity–pathologycontinuum: Defining a role for the LTP phenomenon. J. Neurosci.Res. 58: 42–61.

McHugh, T. J., Blum, K. I., Tsien, J. Z., Tonegawa, S., and Wilson,

M. A. (1996). Impaired hippocampal representation of space inCA1-specific NMDAR1 knockout mice. Cell 87: 1339–1349.

Monahan, J. B., and Michel, J. (1987). Identification and characteriza-tion of an N-methyl-d-aspartate-specific l-[3H]glutamate recogni-tion site in synaptic plasma membranes. J. Neurochem. 48: 1699–1708.

Morton, J. E., Bundey, S., Webb, T. P., MacDonald, F., Rindl, P. M.,and Bullock, S. (1997). Fragile X syndrome is less common thanpreviously estimated. J. Med. Genet. 34: 1–5.

Moser, E. I., and Moser, M. B. (1999). Is learning blocked by saturationof synaptic weights in the hippocampus? Neurosci. Biobehav. Rev. 23:661–672.

Musleh, W., Bi, X., Tocco, G., Yaghoubi, S., and Baudry, M. (1997).Glycine-induced long-term potentiation is associated with struc-tural and functional modifications of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors. Proc. Natl. Acad. Sci.USA 94: 9451–9456.

Nayak, A., Zastrow, D. J., Lickteig, R., Zahniser, N. R., and Browning,M. D. (1998). Maintenance of late-phase LTP is accompanied byPKA-dependent increase in AMPA receptor synthesis. Nature 394:680–683.

Nelson, K. B., Grether, J. K., Croen, L. A., Dambrosia, J. M., Dickens,B. F., Jelliffe, L. L., Hansen, R. L., and Phillips, T. M. (2001). Neu-ropeptides and neurotrophins in neonatal blood of children withautism or mental retardation. Ann. Neurol. 49: 597–606.

Nimchinsky, E. A., Oberlander, A. M., and Svobods, K. (2001). Ab-normal development of dendritic spines in FMR1 knock-out mice.J. Neurosci. 15: 5139–5146.

Noda, M., Nakanish, H., Nabekura, J., and Akaike, N. (2000). AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia.J. Neurosci. 20: 251–258.

Oostra, B. A., and Hoogeveen, A. T. (1997). Animal model for fragileX syndrome. Ann. Med. 29: 563–567.

Paradee, W., Melikian, H. E., Rasmussen, D. L., Kenneson, A., Conn,P. J., and Warren, S. T. (1999). Fragile X mouse: Strain effects ofknockout phenotype and evidence suggesting deficient amygdalafunction. Neuroscience 94: 185–192.

Pelletier, M. R., and Hablitz, J. J. (1996). Tetraethylammonium-in-duced synaptic plasticity in rat neocortex. Cereb. Cortex 6: 771–780.

Pieretti, M., Zhang, F., Fu, Y.-H., Warren, S. T., Oostra, B. A., Caskey,C. T., and Nelson, D. L. (1991). Absence of expression of the FMR-1gene in fragile X syndrome. Cell 66: 817–822.

Regueiro, P., Monreal, J., Diaz, R. S., and Sierra, F. (1996). Preparationof giant myelin vesicles and proteoliposomes to register ionic chan-nels. J. Neurochem. 67: 2146–2154.

Reiss, A. L., Freund, L. S., Baumgardner, T. L., Abrams, M. T., andDenckla, M. B. (1995). Contribution of the FMR1 gene mutation tohuman intellectual dysfunction. Nat. Genet. 11: 331–334.

Sermeus, W., and Delesie, L. (1996). Ridit analysis on ordinal data.West. J. Nurs. Res. 18: 351–359.

Shi, S. H., Hayashi, Y., Petralia, R. S., Zaman, S. H., Wenthold, R. J.,Svoboda, K., and Malinow, R. (1999). Rapid spine delivery andredistribution of AMPA receptors after synaptic NMDA receptoractivation. Science 284: 1811–1816.

Siomi, H., Siomi, M. C., Nussbaum, R. L., and Dreyfuss, G. (1993). Theprotein product of the fragile X gene, FMR1, has characteristics ofan RNA-binding protein. Cell 74: 291–298.

Siomi, M. C., Siomi, H., Sauer, W. H., Srinivasan, S., Nussbaum, R. L.,and Dreyfuss, G. (1995). FXR1, an autosomal homolog of the fragileX mental retardation gene. EMBO J. 14: 2401–2408.

Slegtenhorst-Eegdeman, K. E., de Rooij, D. G., Verhoef-Post, M., vande Kant, H. J., Bakker, C. E., Oostra, B. A., Grootegoed, J. A., and

150 Li et al.

Themmen, A. P. (1998). Macroorchidism in FMR1 knockout mice iscaused by increased Sertoli cell proliferation during testicular de-velopment. Endocrinology 139: 156–162.

Sutcliffe, J. S., Nelson, D. L., Zhang, F., Pieretti, M., Caskey, C. T.,Saxe, D., and Warren, S. T. (1992). DNA methylation repressesFMR-1 transcription in fragile X syndrome. Hum. Mol. Genet. 1:397–400.

Sutor, B., and Hablitz, J. J. (1989). EPSPs in rat neocortical neurons invitro. I. Electrophysiological evidence for two distinct EPSPs.J. Neurophysiol. 61: 607–620.

Tsien, J. Z. (2000). Linking Hebb’s coincidence-detection to memoryformation. Curr. Opin. Neurobiol. 10: 266–273.

Turner, G., Webb, T., Wake, S., and Robinson, H. (1996). Prevalence offragile X syndrome. Am. J. Med. Genet. 64: 196–197.

Valentine, G., Chakravarty, S., Sarvey, J., Bramham, C., and Herken-ham, M. (2000). Fragile X (fmr1) mRNA expression is differentiallyregulated in two adult models of activity-dependent gene expres-sion. Brain Res. Mol. Brain Res. 75: 337–341.

Van Dam, D., D’Hooge, R., Hauben, E., Reyniers, E., Gantois, I.,Bakker, C. E., Oostra, B. A., Kooy, R. F., and De Deyn, P. P. (2000).Spatial learning, contextual fear conditioning and conditioned emo-tional response in Fmr1 knockout mice. Behav. Brain Res. 117: 127–136.

Verheij, C., de Graaff, E., Bakker, C. E., Willemsen, R., Willems, P. J.,Meijer, N., Galjaard, H., Reuser, A. J., Oostra, B. A., and Hoogeveen,

A. T. (1995). Characterization of FMR1 proteins isolated from dif-ferent tissues. Hum. Mol. Genet. 4: 895–901.

Weiler, I. J., and Greenough, W. T. (1993). Metabotropic glutamatereceptor triggers postsynaptic protein synthesis. Proc. Natl. Acad.Sci. USA 90: 7168–7171.

Weiler, I. J., and Greenough, W. T. (1999). Synaptic synthesis of thefragile X protein: Possible involvement in synapse maturation andelimination. Am. J. Med. Genet. 83: 248–252.

Weiler, I. J., Irwin, S. A., Klintsova, A. Y., Spence, C. M., Brazelton,A. D., Miyashiro, K., Comery, T. A., Patel, B., Eberwine, J., andGreenough, W. T. (1997). Fragile X mental retardation protein istranslated near synapses in response to neurotransmitter activation.Proc. Natl. Acad. Sci. USA 94: 5395–5400.

Wenthold, R. J., Petralia, R. S., Blahos, J. I. I., and Niedzielski, A. S.(1996). Evidence for multiple AMPA receptor complexes in hip-pocampal CA1/CA2 neurons. J. Neurosci. 16: 1982–1989.

Zamanillo, D., Sprengel, R., Hvalby, O., Jensen, V., Burnashev, N.,Rozov, A., Kaiser, K. M., Koster, H. J., Borchardt, T., Worley, P.,Lubke, J., Frotscher, M., Kelly, P. H., Sommer, B., Andersen, P.,Seeburg, P. H., and Sakmann, B. (1999). Importance of AMPAreceptors for hippocampal synaptic plasticity but not for spatiallearning. Science 284: 1805–1811.

Zhang, Y., O’Connor, J. P., Siomi, M. C., Srinivasan, S., Dutra, A.,Nussbaum, R. L., and Dreyfuss, G. (1995). The fragile X mentalretardation syndrome protein interacts with novel homologs FXR1and FXR2. EMBO J. 14: 5358–5366.

Received September 5, 2001Revised November 26, 2001Accepted December 3, 2001


Documents

Reduced Cortical Synaptic Plasticity and GluR1 Expression Associated with Fragile X Mental Retardation Protein Deficiency