Mice deficient in collapsin response mediator protein-1 exhibit impaired long-term potentiation and impaired spatial learning and memory

Abstract

Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. We generated viable and fertile CRMP-1 knock-out (CRMP-1(-/-)) mice with no evidence of gross abnormality in the major organs. CRMP-1(-/-) mice exhibited intense microtubule-associated protein 2 (MAP2) staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 (growth-associated protein-43) and PSD95 (postsynaptic density-95) (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knock-out mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1(-/-) mice showed a normal synapsin I labeling pattern in CA1 and normal paired-pulse facilitation. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory.

Figure 1.

Generation and characterization of CRMP-1^−/− mice. A, The genomic structure of CRMP-1 and the targeted alleles. The CRMP-1 exons 2–6 (filled boxes) are depicted along with restriction enzyme sites (A, AflII; Ap, ApaI; B, BamHI; C, ClaI; H, HindIII; N, NcoI; X, XhoI; R, EcoRI). The wild-type allele (+) targeted by vector I generated the allele (“t”) harboring a loxP (filled triangle)-flanked HPRT (hatched box) in intron 5. Cre excision generated the one-loxP allele. Subsequent transfection of the one-loxP-containing ES cells with vector II generated the floxed allele (fl). Subsequent excision by Cre deleted the HPRT and exons 4 and 5 [the null allele (−)]. Open bars, Probes A and B for Southern blotting. Primers (2U, 3U, 3D, 4U, 4D, 5D, and 6D) for genotyping by PCR. C* in vector I, Deleted ClaI site. B, Southern blotting of recombinant ES cell clones digested by AflII and AflII plus BamHI and detected by probes A and B. DNA in each lane, 5–10 μg. Only probe A results are shown. Lane 1, Wild-type ES cells (showing 8.3 kb “+” allele). Wild-type allele is omitted in the following descriptions. Lane 2, Vector I targeted clone (10.8 kb; “t” allele). Lanes 3–5, DNA from ES cells of lane 2 after cre-excision and successful vector II recombination (lane 3, 9.8 kb floxed “fl” allele), unsuccessful vector II recombination in cis (lane 4, 6.8 kb “loxP” allele), and successful vector II recombination in trans (lane 5, a loxP allele plus a t* allele denoting the clone with the loxP-flanked HPRT fragment in intron 3 of the wild-type allele of lane 4). Lane 6, ES cell clone in lane 7 after Cre transfection to generate the CRMP-1^−/− allele, the (−) allele. Lane 7, ES cell clone in lane 3 by AflII. C, Southern blotting of AflII-digested tail DNA from pups derived from ES cell clones of lane 6 of panel B. +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1^−/− mice. Both probes A and B gave the same pattern. D, Northern blotting of 20 μg of total RNA from P7 mouse brains and kidneys, using exons 1–11 CRMP-1 cDNA and a GAPDH probe. M, RNA size maker (MBI Fermentas, Hanover, MD). E, RT-PCR analysis. Total RNA (P7) of brains and PCR primers 2U and 6D were used. The 582 and 356 bp bands represent the wild-type and knock-out alleles, respectively. F, Western blot analysis of CRMPs in whole-brain extracts from CRMP-1^+/+ and CRMP-1^−/− mice at P1. Tissue extract samples (15 μg) were loaded in each lane. The membrane was first reacted with anti-CRMP-1 antibody (in-house), followed by anti-CRMP-2, anti-CRMP-4, and then anti-actin antibodies (internal control) with stripping and washing steps in between each antibody reaction. Rat P1 brain, Positive control. Lanes +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1 knock-out mice, respectively. Multiple CRMP bands were attributable to phosphorylation events.

Figure 2.

Histology and neural morphology of CRMP-1^+/+ and CRMP-1^−/− mice. A, Cresyl violet staining of the hippocampus of wild-type CRMP-1^+/+ (+/+; a–e) and CRMP-1^−/− (−/−; a′–e′) brains at P7. No difference between wild-type and knock-out brains was observed at low (dorsal, a vs a′; ventral, d vs d′) or high magnification in any of the subregions of hippocampus [CA1 (b, b′), A3 (c, c′), and DG (e, e′)]. B, Comparisons of DiI tracing were conducted in P1 of CRMP-1^+/+ (left) and CRMP-1^−/− (right) mice using fluorescence microscopy. DiI crystals were placed in CA3, and the contralateral hippocampus was analyzed. Nuclei were counterstained with SYTOX Green. cc, Corpus callosum.

Figure 3.

CRMP-1 expression in adult CRMP-1^+/+ and CRMP-1^−/− mouse brains. A, Paraffin-embedded sections from CRMP-1^+/+ and CRMP-1^−/− mice (8–12 weeks of age) were labeled with anti-CRMP-1 (green) followed by DAPI (4′,6′-diamidino-2-phenylindole) nuclear counterstain (blue) and analyzed by confocal microscopy. CRMP-1 was identified predominantly in hippocampal CA1 pyramidal cells and Purkinje cells (arrow) in the cerebellum. Positive neurofilament labeling (green) of serial sections served as a validation of the appropriateness of the CRMP-1^−/− brain samples. Co, Cortex; Hi, hippocampus; Ce, cerebellum; p, pyramidal cell layer; d, apical dendritic layer; ML, molecular layer; WM, white matter; Pur, Purkinje cell. Scale bar, 50 μm (unless otherwise specified). Neurofilament labeling, which served as a positive control, was indistinguishable between the two genotypes. B, Semiquantitative Western blot analysis. Total protein extracts of cortex, hippocampus, and cerebellum from three 8- to 12-week-old mice were immunoblotted with anti-CRMP-1 polyclonal antibody. Relative protein levels (% α-actin) were quantified by densitometer scanning and normalized to corresponding α-actin loading controls. Results are presented as mean ± SEM. *p < 0.01 versus wild-type hippocampus; **p < 0.005 versus wild-type hippocampus.

Figure 4.

Distribution of neuronal markers in and organization of the hippocampal region of the wild-type (A–I) and CRMP-1^−/− (a–i) mice. Representative images from the analysis of several mice are shown (8–12 weeks; n = 3 and 4 for wild-type and knock-out mice, respectively). Confocal microscopy images of MAP2 (A–C, a–c); PSD95, postsynaptic density specific marker (D–F, d–f); and GAP-43 (G–I, g–i) immunoreactivity in area CA1. Higher magnification of the stratum pyramidal (B, b, E, e, H, h) and stratum radiatum (C, c, F, f, I, i) of the CA1 area were shown in parallel for the respective lower magnification (A, a, D, d, G, g). Low magnifications were generated from projections of 8 serial optical 1.05 μm sections and higher magnifications were 22 optical 0.3 μm sections. The arbitrary MAP2 fluorescence levels in the proximal and distal portions of the dendrites were quantified by the MetaMorph software (Molecular Devices) and expressed as relative values by normalization to value of the proximal part. The MAP2 intensity (distal/proximal part of the dendrite) in CRMP-1^−/− (0.87 ± 0.03; n = 4) and wild-type (1.07 ± 0.08; n = 3) sections differed (p = 0.019). p, Pyramidal layer; d, apical dendritic layer.

Figure 5.

Double immunohistochemical examination of axon formation (A) and Western blotting of GAP-43, MAP2, PSD95, and synapsin I levels in wild-type and CRMP-1^−/− mouse brain regions (B, C). A, Wild-type and mutant mouse hippocampal sections were incubated with anti-Tau-1 (1:50 dilution; Millipore; MAB3420) and anti-synapsin I (1:50; Calbiochem; 574777) antibodies to reveal axonal cytoarchitecture. FITC-conjugated goat anti-mouse IgG (1:500) and rhodamine-conjugated goat anti-rabbit IgG (1:500) secondary antibodies were used to reveal Tau-1 and synapsin I, respectively. Double channel images were captured by confocal microscope (Nikon EZ-C1) of a single optical (0.3 μm) section. B, Representative patterns of Western blot analysis showing GAP-43, MAP2 (MAP2a and MAP2b), PSD95, synapsin I, and actin protein levels in isolated cerebral cortex, hippocampus, and cerebellum. C, Quantification of MAP2, GAP-43, PSD95, and synapsin I protein levels in B were normalized to α-actin (n = 3 per genotype). Data are presented as means ± SEM. *p < 0.05; **p < 0.001. +/+, Wild-type mice; −/−, CRMP-1^−/− mice. p, Pyramidal layer; d, apical dendritic layer; WT, wild type; KO, knock-out.

Figure 6.

Attenuated LTP in area CA1 of CRMP-1^+/− and CRMP-1^−/− mice. A, Strong tetanus stimulation (twice at 100 Hz for 1 s each separated by 20 s) in the stratum radiatum layer of CA1 induced LTP. Note that induction and maintenance of LTP was inhibited in CRMP-1^+/− and CRMP-1^−/− mice. Data are presented as means ± SEM (CRMP-1^+/+, n = 26 from 11 mice; CRMP-1^+/−, n = 10 from 6 mice; CRMP-1^−/−, n = 18 from 8 mice). B, PPF was also examined in area CA1 (0.01 Hz stimulation with ISIs of 20, 50, 80, 100, 200, 300, 400, and 500 ms with electrodes placed in the outer or inner molecular layer of the piriform cortex). PPF ratio was calculated by dividing the amplitude of the second fEPSP by the amplitude of the first fEPSP. PPF was not affected in CRMP-1^+/− or CRMP-1^−/− mice. n, Number of slices; N, number of mice.

Figure 7.

Impaired spatial learning and memory performance in CRMP-1^−/− mice. Young adult (8–12 weeks of age) (A, C) and aged (5–10 months) (B, D) mice were tested in the Morris water maze. A, B, Latency to find the hidden platform by young adult (A) and aged (B) wild-type (CRMP-1^+/+) and CRMP-1^−/− mice. CRMP-1^−/− mice showed a delayed escape latency for the daily training trials compared with CRMP-1^+/+ mice (one-way ANOVA followed by post hoc Scheffé's test; ANOVA, p < 0.01 for A; ANOVA, p < 0.001, and post hoc, *p < 0.05 for B). C, D, Probe tests for retention in adult (C) and aged (D) mice. CRMP-1^+/+ mice spent more time in the target than in a nontarget quadrant (*p < 0.005), whereas CRMP-1^−/− mice showed no difference in the target and nontarget quadrant. D, Averaged time spent in the target quadrant was 16.65 and 6.55 s for CRMP-1^+/+ and CRMP-1^−/− mice, respectively (**p < 0.0001). Error bars indicate SEM.