Impaired conditioned fear and enhanced long-term potentiation in Fmr2 knock-out mice

Abstract

FRAXE mental retardation results from expansion and methylation of a CCG trinucleotide repeat located in exon 1 of the X-linked FMR2 gene, which results in transcriptional silencing. The product of FMR2 is a member of a family of proteins rich in serine and proline, members of which have been associated with transcriptional activation. We have developed a murine Fmr2 gene knock-out model by replacing a fragment containing parts of exon 1 and intron 1 with the Escherichia coli lacZ gene, placing lacZ under control of the Fmr2 promoter. Expression of lacZ in the knock-out animals indicates that Fmr2 is expressed in several tissues, including brain, bone, cartilage, hair follicles, lung, tongue, tendons, salivary glands, and major blood vessels. In the CNS, Fmr2 expression begins at the time that cells in the neuroepithelium differentiate into neuroblasts. Mice lacking Fmr2 showed a delay-dependent conditioned fear impairment. Long-term potentiation (LTP) was found to be enhanced in hippocampal slices of Fmr2 knock-out compared with wild-type littermates. To our knowledge, this mouse knock-out is the first example of an animal model of human mental retardation with impaired learning and memory performance and increased LTP. Thus, although a number of studies have suggested that diminished LTP is associated with memory impairment, our data suggest that increased LTP may be a mechanism that leads to impaired cognitive processing as well.

Fig. 1.

Map of Fmr2 knock-out construct and corresponding genomic region. a, Map of mouseFmr2 targeting construct. The bold lineswith arrows at both ends indicate genomic DNA fragments, corresponding to the bold lines witharrows at both ends in b.b, Map of Fmr2 exon 1, promoter region and intron 1 genomic region. The fine line witharrows at both ends represents the 6.7 kbXbaI-digested Southern blot band detected by 1.5 kbSalI–XbaI right probe in wild-type mice.c, Map of Fmr2 knock-out mouse genomic region after homologous recombination with the Fmr2knock-out construct and Fmr2 promoter, exon 1 and intron 1 region. The fine line with arrows at both ends represents the 5.0 kb XbaI-digested Southern blot band detected by the 1.5 kbSalI–XbaI right probe in knock-out mice.

Fig. 2.

a, Southern blot analysis ofFmr2 knock-out mouse tail DNA after digestion withBamHI and hybridization with the 1.5 kbSalI–XbaI right probe (Fig. 1).Lanes 1, 6, Knock-out male mice; lane 2, wild type; lanes 3–5, heterozygote females; lane 7, bacteriophage λ HindIII marker.b, RT-PCR analysis of Fmr2 knock-out and wild-type mouse adult brain. Lane 1, 100 bp ladder;lane 2, knock-out mouse brain RNA; lane 3, knock-out brain cDNA; lane 4, wild-type mouse brain RNA; lane 5, wild-type mouse brain cDNA. Thetop wells are products obtained with primer pair mfmr2-1 and mfmr2-2 from Fmr2. The bottom wellscontain amplification products using a primer pair (ma8 and ma11) designed from the murine ortholog of the human gene AF5q31 as a control.

Fig. 3.

X-Gal staining of telencephalon or brains ofFmr2 knock-out mice. A, X-Gal staining of telencephalon of embryonic day 10.5. The ganglionic hillock is labeled.B, X-Gal staining of telencephalon of embryonic day 12.5. The wall of cerebra was divided into three zones: matrix zone at the ventricular lumen, intermediate zone, and marginal zone. The neuroblasts for the cerebral cortex migrate out of the inner matrix zone, where critical mitosis occurs, and enter the marginal zone, where they form the cortical plate. The neuroblasts in the cortical plate are no longer able to divide. C, X-Gal staining of cerebra (frontal cortex) at embryonic day 15. The neuroblasts and neuronal cells have not reached the outer one-third zone of cerebral cortex when neuroblasts migrate from inside matrix zone to outside zone, passing the neurons differentiated by neuroblasts migrating out early.D, X-Gal staining of the adult cerebellum. The most highly stained cells are Purkinje cells. E, X-Gal staining of adult brain, cut by coronal section. CA1, CA3, and dentate gyrus of hippocampus are strongly stained by X-Gal. The amygdala is also well stained. F, Enlargement of X-Gal staining of the hippocampus from D. G, Hematoxylin and eosin staining of adult brain by coronal section. No abnormalities are observed. H, Hematoxylin and eosin staining of adult cerebellum. These structures appear normal. GE, Ganglionic eminence; MZ, matrix zone; PP, preplate; IZ, intermediate zone; MaZ, marginal zone; CP, cortical plate; PC,Purkinje cell layer of cerebellum; AM, amygdala;DG, dentate gyrus of hippocampus.

Fig. 4.

Conditioned fear and hot plate analgesia test ofFmr2 knock-out mice. Knock-outs are represented byopen bars; normal controls are represented byfilled bars. A, First series of contextual and conditioned fear tests 24 hr after CS–US training.n (KO) = 14 males; n(WT) = 11 males. B, Second series of context and conditioned fear tests 24 hr after CS–US training. n (KO) = 11 males;n (WT) = 12 males.C, Third series of context and conditioned fear tests 30 min after CS–US training [n (KO) = 17 males; n (WT) = 18 males] and third series of context and conditioned fear tests 24 hr after CS–US training. D, Hot plate analgesia test ofFmr2 knock-out and normal controls. In the first batch,n (KO) = 14 males; n(WT) = 11 males. In the second batch,n (KO) = 11 males; n(WT) = 12 males.

Fig. 5.

Performance of Fmr2 knock-out and wild-type mice on the hidden platform version of the Morris water task. The escape latency in seconds (A) and swim distance in centimeters (B) to locate the hidden platform during training are shown. C, Number of platform crossings for knock-out and wild-type mice during the probe trial. n (KO) = 11 males;n (Wild-type) = 12 males. Data are plotted as the mean ± SEM.

Fig. 6.

Electrophysiological responses at Schaffer collateral synapses in area CA1 of hippocampus. A, Loss of Fmr2 had no effect on baseline synaptic transmission in stratum radiatum of the CA1 region of the hippocampus measured inFmr2 knock-out mice (open squares;n = 14, male) or wild-type mice (closed squares; n = 9, male). B, Paired-pulse facilitation was likewise unaffected inFmr2-knock-out (n = 14, male) compared with wild-type (n = 11, male) mice.

Fig. 7.

Enhanced LTP in Fmr2knock-out mice. A, Fmr2 knock-out hippocampal slices showed enhanced LTP compared with wild types after a modest LTP-inducing protocol consisting of a single set of tetani while maintaining slices at 25°C [60 min after tetanus: n(KO, male) = 9, 167 ± 9%; n(WT, male) = 14, 132 ± 6%;p = 0.003]. B, Enhanced LTP inFmr2 knock-out hippocampal slices is present after a single set of tetani stimulation while maintaining slices at 32°C [60 min after tetanus: n (KO, male) = 6, 170 ± 11%; n (WT, male) = 6, 150 ± 5%; p = 0.14]. C,Fmr2 knock-out mice maintain the enhanced LTP after three sets of HFS at 32°C [60 min after tetanus: n(KO, male) = 7, 244 ± 18%; n(WT, male) = 5, 189 ± 20%;p = 0.020]. D, In the presence of the NMDA receptor antagonist AP-5 (50 μm),Fmr2 knock-out mice showed enhanced NMDA-independent LTP compared with wild types after three trains of 200 Hz stimulation for 1 sec separated by 4 min at 32°C [60 min after tetanus: n (KO, male) = 6, 155 ± 8%; n (WT, male) = 6, 135 ± 4%; p = 0.038].