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Growth Defects and Impaired Cognitive–Behavioral Abilities in Mice with Knockout for Eif4h, a Gene Located in the Mouse Homolog of the Williams-Beuren Syndrome Critical Region

2012; Elsevier BV; Volume: 180; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2011.12.008

1525-2191

Simona Capossela, Luca Muzio, Alessandro Bertolo, Veronica Bianchi, Gabriele Dati, Linda Chaabane, Claudia Godi, Letterio S. Politi, Stefano Biffo, Patrizia D’Adamo, Antonello Mallamaci, Maria Pannese,

Congenital heart defects research

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome. Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome. Protein synthesis is an energy consuming process, tightly regulated in its three main steps: initiation, elongation, and termination. Control of translation is fundamental for fine-tuning of gene expression and plays a critical role in development, cellular growth, proliferation, differentiation, synaptic plasticity, memory and learning. In eukaryotes this process is mainly regulated at the level of initiation, when the mRNA is recruited to the small subunit of the ribosome, thanks to catalytic activities of eukaryotic translation initiation factors (eIFs) of the eIF4 families.1Gingras A.C. Raught B. Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.Annu Rev Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1758) Google Scholar, 2Hernandez G. Vazquez-Pianzola P. Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families.Mech Dev. 2005; 122: 865-876Crossref PubMed Scopus (107) Google Scholar, 3Sonenberg N. Hinnebusch A.G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets.Cell. 2009; 136: 731-745Abstract Full Text Full Text PDF PubMed Scopus (2232) Google Scholar In this step the 5′ cap structure (m7Gpppn) of the mRNAs is bound by eIF4F, a complex composed of three proteins: eIF4E the cap binding protein, eIF4G a large scaffolding protein, and eIF4A an ATP-dependent helicase able to unwind secondary structures in the mRNA 5′UTR, so facilitating ribosome binding. The ATPase and helicase activities of eIF4A are stimulated by eIF4B and eIF4H, two factors that bind RNA by virtue of their N-terminal RNA recognition motifs.4Richter N.J. Rogers Jr., G.W. Hensold J.O. Merrick W.C. Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H.J Biol Chem. 1999; 274: 35415-35424Crossref PubMed Scopus (57) Google Scholar, 5Rogers Jr., G.W. Richter N.J. Lima W.F. Merrick W.C. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.J Biol Chem. 2001; 276: 30914-30922Crossref PubMed Scopus (258) Google Scholar, 6Rogers Jr, G.W. Richter N.J. Merrick W.C. Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A.J Biol Chem. 1999; 274: 12236-12244Crossref PubMed Scopus (246) Google Scholar The human homolog of Eif4h, WBSCR1, is located in 7q11.23 within a region commonly deleted in patients affected by the Williams-Beuren syndrome (WBS; OMIM 194050). WBS is classically associated to heterozygous deletions spanning a region of 1.6 Mb, containing about 28 genes. Deletions formation is mediated by low-copy repeat elements flanking the Williams-Beuren syndrome critical region that may lead to nonallelic homologous recombination, if misaligned in meiosis.7Bayes M. Magano L.F. Rivera N. Flores R. Perez Jurado L.A. Mutational mechanisms of Williams-Beuren syndrome deletions.Am J Hum Genet. 2003; 73: 131-151Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 8Peoples R. Franke Y. Wang Y.K. Perez-Jurado L. Paperna T. Cisco M. Francke U. A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome–deletion region at 7q11.23.Am J Hum Genet. 2000; 66: 47-68Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar This syndrome is a complex neurodevelopmental disorder including cardiovascular defects, elfin-like face, infantile hypercalcemia, cerebral dysplasias, and a peculiar cognitive-behavioral profile,9Francke U. Williams-Beuren syndrome: genes and mechanisms.Hum Mol Genet. 1999; 8: 1947-1954Crossref PubMed Scopus (180) Google Scholar, 10Meyer-Lindenberg A. Mervis C.B. Berman K.F. Neural mechanisms in Williams syndrome: a unique window to genetic influences on cognition and behaviour.Nat Rev Neurosci. 2006; 7: 380-393Crossref PubMed Scopus (327) Google Scholar, 11Pober B.R. Williams-Beuren syndrome.N Engl J Med. 2010; 362: 239-252Crossref PubMed Scopus (578) Google Scholar, 12Tassabehji M. Williams-Beuren syndrome: a challenge for genotype-phenotype correlations.Hum Mol Genet. 2003; 12: R229-R237Crossref PubMed Scopus (150) Google Scholar and occurs at a frequency of approximately 1 in 7500 live births.13Stromme P. Bjornstad P.G. Ramstad K. Prevalence estimation of Williams syndrome.J Child Neurol. 2002; 17: 269-271Crossref PubMed Scopus (574) Google Scholar The mouse genomic region syntenic to the human Williams-Beuren syndrome critical region is on chromosome band 5G2, in reverse orientation with respect to the centromere.14DeSilva U. Elnitski L. Idol J.R. Doyle J.L. Gan W. Thomas J.W. Schwartz S. Dietrich N.L. Beckstrom-Sternberg S.M. McDowell J.C. Blakesley R.W. Bouffard G.G. Thomas P.J. Touchman J.W. Miller W. Green E.D. Generation and comparative analysis of approximately 3.3 Mb of mouse genomic sequence orthologous to the region of human chromosome 7q11.23 implicated in Williams syndrome.Genome Res. 2002; 12: 3-15Crossref PubMed Scopus (68) Google Scholar A number of loss-of-function mouse models for single genes of this region, as well as strains with large deletions spanning the critical region have been generated and characterized.15Osborne L.R. Animal models of Williams syndrome.Am J Med Genet C Semin Med Genet. 2010; 154C: 209-219Crossref PubMed Scopus (54) Google Scholar Whereas there is general consensus about link between Elastin (ELN) hemizygosity and occurrence of supravalvular aortic stenosis,16Ewart A.K. Morris C.A. Atkinson D. Jin W. Sternes K. Spallone P. Stock A.D. Leppert M. Keating M.T. Hemizygosity at the elastin locus in a developmental disorder Williams syndrome.Nat Genet. 1993; 5: 11-16Crossref PubMed Scopus (940) Google Scholar more controversial is the origin of the WBS cognitive-behavioral profile. We generated a mutant mouse line, starting from an embryonic stem cell gene-trap clone, carrying an inactivated Eif4h allele. Eif4h null mice displayed growth retardation and a generalized weight and volume decrease in the majority of organs and tissues analyzed. In vivo profiling by magnetic resonance imaging (MRI) revealed a smaller brain volume in null mice than in controls; the volumes were differently affected in the anterior and posterior brain regions, indicating altered brain morphology. Histological and cytological studies revealed a reduction in number and complexity of neurons. In addition, behavioral tests revealed severe impairments of fear-related associative learning and memory formation in the knockouts. These results demonstrated that Eif4h might contribute to some deficits associated to the Williams-Beuren syndrome. The gene trap embryonic stem cell line (Ex279, strain 129/Ola) was provided by BayGenomics (San Francisco, CA) and used to produce chimeric animals by blastocyst injection techniques. Southern blot hybridization was used to identify the targeted allele in embryonic stem cell clones. Genomic DNA was extracted, digested with HindIII (Promega, Madison, WI), fractionated by agarose gel electrophoresis and transferred to hybond N+ (Amersham; GE Healthcare, Little Chalfont, UK) using alkaline transfer. The blot was hybridized with a random-labeled probe. Germ-line chimeras were crossed to the C57BL/6 strain. Primary embryo fibroblasts (MEFs) were prepared from E14.5 embryos. In brief, embryos were dissociated by 0.05% Trypsin/0.53 mmol/L EDTA [Gibco (Life Technologies), Paisley, UK] at 37°C for 10 minutes and then treated with 200 U DNaseI (Tebu-Bio, Le-Perray-en-Yvelines, France). After filtering with a 70-μm cell strainer, fibroblasts were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco). MEFs were cultured at 9% CO2 at 37°C. For Western blot MEFs were collected at passage P3. Genotyping analysis of mice was performed on tail genomic DNA by PCR, using the following primers: forward 5′-GTAAACTTGAGGGTGAGGACGTGGGAGCCTGCA-3′; reverse wild type (WT) 5′-CATGAGCATGTTCTAACAAAGCCGTGTAGGTGG-3′; reverse knockout (KO) 5′-CCCAGACCTTGGGACCACCTCATCAGAAGCAG-3′; PCR products length: wild-type allele 250 bp; Eif4h(EX279) allele 231 bp. Mice were housed in a temperature-controlled room with a 12-hour light/dark cycle and they had free access to food and water. Body weight was recorded weekly, and food intake was measured every day, at 5 to 6 months of age, for 15 consecutive days. For fertility study, mice were 8 to 16 weeks old. Males were housed in one cage with two females each one. Every morning the females were checked for the presence of vaginal plug, an indication that sexual activity had taken place. For the behavior analysis, 20 wild-type and 20 knockout mice were maintained on a reversed 12-hour dark/light cycle at 22°C to 25°C and tested at 2 to 4 months of age. For all of the experiments knockout and control animals were sex and age matched. The observers were blind to the genotype during data collection and data analysis. Experiments were done according to the animal protocols approved by the DIBIT Institutional Animal Care (Milan, Italy) and were approved by the National Ministry of Health. All experiments were performed in accordance with the guidelines established by the European Community Council Directive of November 24, 1986 on the use of animals in research (86/609/EEC). All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable results. Total RNA was extracted from tissue samples with Trizol Reagent (Invitrogen [Life Technologies], Paisley, UK) according to the manufactures instructions. After digestion with Dnase RNase-free (Promega, Madison, WI) for 30 minutes at 37°C, RNA was isolated by using RNeasy Mini kit (Qiagen, Hilden, Germany). cDNA synthesis was performed using ThermoScript RT-PCR System (Invitrogen) and Random Primers (Promega). The LightCycler 480 System (Roche, Basel, Switzerland) and SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, St. Louis, MO) were used. The primers used for the quantifications were: Eif4h forward 5′-GTCGCTTTCGAGATGGCCCTCCTCTGCGTGGC-3′, reverse 5′-CCTAAGCTCATTCTTGCTCCTTCTGAACCACTTC-3′; Cyln2 forward 5′-ACGGTATTCACCAGCCAGAC-3′, reverse 5′-CACATCTCCAAGGGGACAGT-3′; Limk1 forward 5′-TGCTCAAGTTCATCGGAGTG-3′, reverse 5-'-TTCATCGAATGGAGGTAGGC-3′; Gtf2I forward 5′-CAATCGGATGAGTGTGGATG-3′, reverse 5′-GGTTGCGAGGTCGTAATGTT-3′; Gtf2ird1 forward 5′-CCAGACAAGATCCCCTTCAA-3′, reverse 5′-GTCTTCTGGTGGGCTAGCTG-3′. β-actin was used as housekeeping gene: forward 5′-GACTCCTATGTGGGTGACGAGG-3′; reverse 5′-CATGGCTGGGGTGTTGAAGGTC-3′. The experiments were done in triplicate. A fold change in expression was calculated using the 2-ΔΔCt formula of the δ-delta Ct method. Adult mouse brains were dissected, fixed 30 minutes in 4% paraformaldehyde in PBS, included in 5% low melting agarose in PBS, and sectioned to 150 μm by using a vibroslicer. For in situ hybridization sections were fixed overnight in 4% paraformaldehyde at 4°C. Sense and antisense riboprobes for Eif4h and LacZ were prepared by in vitro transcription with DIG-11-UTP (DIG RNA Labeling Mix; Roche) by T3 or T7 polymerases (Promega). Sections were hybridized overnight at 60°C with the DIG-labeled riboprobes and visualized by alkaline phosphatase conjugated anti-DIG antibody. For X-gal staining sections were fixed 2 minutes in 4% paraformaldehyde and washed three times for 15 minutes in 0.02% PBS NP-40. They were stained overnight at room temperature in 1 mg/mL X-gal, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6, 2 mmol/L MgCl2, 0.01% sodium-deoxycholate, 0.02% NP-40 in PBS. After staining, sections were washed in PBS and postfixed for 5 minutes in 4% paraformaldehyde. Sections were analyzed and photographed using a Nikon SMZ 800 stereomicroscope (Objective P-Achro 0.5X; Zoom 2X) and a Nikon DS-L1 digital camera (Nikon Corporation, Tokyo, Japan). Subcellular fractions were prepared from wild-type brains, dissected free from bulbs, cerebellum, and brain stem. The pool of cortical and subcortical regions (H), was homogenized with ice-cold H buffer (320 mmol/L sucrose, 5 mmol/L HEPES-NaOH buffer, pH 7.4, 2 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1/1000 protease inhibitor), and was centrifuged for 10 minutes at 1000 × g The resulting pellet (P1) was resuspended in Homogenization Buffer, while the supernatant (S1) was collected and centrifuged for 15 minutes at 10,000 × g. The supernatant (S2) was centrifuged for 20 minutes at 75000 rpm in a Beckman TL100.2 rotor (Beckman Coulter, Brea, CA) to give the supernatant (S3) and the pellet (P3); while the pellet (P2) was resuspended in Homogenization Buffer and lysed with 9 volumes of HEPES 5 mmol/L – 1/1000 protease inhibitor. The suspension was kept on ice for 30 minutes and then was centrifuged for 20 minutes at 25000 rpm in a Beckman TL100.2 rotor to yield a lysate pellet (LP1) and a lysate supernatant (LS1). The lysate supernatant was collected and centrifuged for 20 minutes at 75000 rpm in a Beckman TL100.2 rotor, to obtain the supernatant (LS2) and the pellet (LP2). The pellets (P3, LP1, and LP2) were resuspended in HB. MEFs pellet and subcellular fractions were lysated in ice-cold radioimmunoprecipitation assay buffer (50 mmol/L TrisHCl ph7.4, 1% Triton, 0.2% sodium-deoxycholate, 0.2% SDS, 1 mmol/L EDTA, 1 mmol/L PMSF). Protein content was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). Briefly, proteins were separated by SDS-polyacrylamide gel and transferred to Hybond ECL membranes (Amersham) for 1 hour at 300 mA. The membranes were blocked for 1 hour with 5% blocking agent (milk) in Tris-buffered saline containing 0.05% Tween-20 (TBST). Membranes were incubated overnight at 4°C with primary antibodies: eIF4H (Cell Signaling, Danvers, MA) diluted 1:1000 in 5% BSA-TBST; and ERK 2 (K-23) (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:5000, β-Tubulin (Sigma) diluted 1:1000, synaptophysin (p38)17Valtorta F. Jahn R. Fesce R. Greengard P. Ceccarelli B. Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion.J Cell Biol. 1988; 107: 2717-2727Crossref PubMed Scopus (104) Google Scholar diluted 1:8000, in 5% milk-TBST. Incubation with primary antibody β-actin (Sigma), diluted 1:10,000 in TBST was performed for 2 hours at room temperature. Secondary antibodies were diluted in 5% milk-TBST and incubated for 1 hour at room temperature. Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Cell Signaling) diluted 1:2000 was used for eIF4H; HRP-conjugated anti-mouse antibody (Amersham) diluted 1:20,000 for β-actin, and HRP-conjugated anti-rabbit and anti-mouse antibody (Amersham) diluted 1:5000 for others primary antibodies. Detection was performed with ECL plus Western blotting detection kit (Amersham). Computed tomography (CT) scans were performed on a human-grade 64-channel multislice apparatus (Light Speed VCT; GE Healthcare, Barrington, IL). The imaging protocol included a biplanar scout and a helical volumetric CT acquisition with coverage of the whole body, with a tube speed rotation of 0.5 seconds, 0.625-mm slice thickness, and 0.3 mm/sec table motion, 120 KV, 680 mA, reconstruction field of view of 17 cm, and matrix of 512 × 512. CT images were filtered with both the standard parenchyma and the high-resolution bone algorithms. On a dedicated workstation (Advantage 4.4; GE Healthcare) the total body, skeletal, fat and muscle volumes (in cubic centimeters) of each mouse were measured after applying an automatic segmentation (bone threshold >160 Hounsfield Unit; density range from −190 to −10 H.U. for fat; density range from 10 to 65 H.U. for muscle). For each mouse, skull anteroposterior, nasal-zygomatic, and latero-lateral distances were measured (in centimeters) on the reformatted images. Brain imaging was performed at 6 months of age on a 7T-MRI scanner (Pharmascan, Bruker, Ettlingen, Germany). High-resolution coronal sections were acquired along the brain of mice maintained under anesthesia with flurane gas mixed with O2. A multislice multiecho (MSME) sequence (TR = 3465 ms and TE = 18 and 44 ms) with a slice thickness of 0.85 mm was used to generate 15 contiguous T2-weighted images with a plane spatial resolution of 86 × 93 μm2. For neuroanatomical analysis, the area of the measured regions was manually traced using Image J (National Institutes of Health, Bethesda, MD). The total volume of each structure was calculated by multiplying the sum of the measured areas by the slice thickness. Immunohistochemistry experiments were realized on mice that were transcardially perfused with 4% paraformaldehyde (PFA). Brains were then removed, postfixed overnight in 4% PFA, and cryoprotected in 30% sucrose before freezing in OCT. The 10-μm cryosections were cut and stored at −80°C. Cryosections were washed in PBS, incubated in methanol-3% H2O2 to block endogenous peroxidase activity, rehydrated, and incubated for 1 hour at room temperature with blocking mix (PBS 1X/FBS 10%/BSA 1 mg/mL/Triton ×100 0.1%). Sections were then incubated at 4°C overnight with the following primary antibodies: NeuN (Millipore, Billerica MA), myelin binding protein (Abcam, Cambridge, MA) diluted 1:500 in blocking mix. A biotin conjugated goat anti-mouse IgG secondary antibody (Vector Laboratories, Peterborough, UK) was used 1:200 for 2 hours and then sections were further incubated with avidin–biotin complex Vectastain Elite ABC Kit (Vector Laboratories) accordingly manufacture's recommendation. Signals were revealed by incubating sections in amino-9-ethyl carbazole (AEC, Sigma). Four sections, 240 μm spaced each other, were used for cell counting. Images were taken by using Olympus microscope (Tokyo, Japan), magnification ×20. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, adult brain cryosections (10 μm) were post fixed in 4% paraformaldehyde, incubated in methanol-3% H2O2 to block endogenous peroxidase activity, and permeabilized with proteinase K 10 mg/mL for 15 minutes at room temperature. Slices were incubated with TUNEL transferase buffer for 15 minutes before adding the following reagents: 0.01 mmol/L biotin 16 dUTP, 1.25 mmol/L CoCl2 and 80 U of terminal transferase (Roche). Reaction was incubated 1 hour at 37°C. Sections were incubated with avidin–biotin complex Vectastain Elite ABC Kit (Vector Laboratories) for 1 hour and stained with amino-9-ethyl carbazole (AEC, Sigma) and H2O2. Positive controls were obtained by incubating slices in 3U/mL DNase for 15 minutes at room temperature. An FD rapid Golgi staining kit (FD Neurotechnologies, Baltimore, MD) was used to stain 60 μm-thick brain coronal sections according to the manufacture's recommendation. Sholl analysis was done on deep cortical neurons of the posterior cortical plate. Briefly, neuron images were captured at magnification ×20 and concentric circles were applied by using ImageJ software, number of intersections of dendrites, at increasing distances from the soma, were counted on each circle. Number of dendritic spines was calculated by counting the total number of spines divided by the dendritic length (10 μm). Only completely impregnated dendrites within the tissue sections were used for spine counts, as previously described.18Centonze D. Muzio L. Rossi S. Cavasinni F. De Chiara V. Bergami A. Musella A. D'Amelio M. Cavallucci V. Martorana A. Bergamaschi A. Cencioni M.T. Diamantini A. Butti E. Comi G. Bernardi G. Cecconi F. Battistini L. Furlan R. Martino G. Inflammation triggers synaptic alteration and degeneration in experimental autoimmune encephalomyelitis.J Neurosci. 2009; 29: 3442-3452Crossref PubMed Scopus (289) Google Scholar The rota-rod is an apparatus composed by a horizontal rotating rod (diameter approximately 3 cm) on which the mouse has to keep its balance. Five mice were simultaneously placed on the rota-rod apparatus, separated by large disks. In the accelerating rota-rod test (test 1) each mouse was subjected to five consecutive trials, with an interval of 30 minutes; 5 minutes is the maximum time for each trial. Mice were placed every trial on the rota-rod apparatus with the rod rotating at 4 rpm (rotations/minute) during the first minute, then the rotation speed is increased every 30 seconds by 4 rpm reaching the maximum speed of 36 rpm. A trial ended for a mouse when it fell down or when 5 minutes (300 seconds) were completed. In the constant rota-rod test of 5 minutes (test 2) all mice were tested for five consecutive trials at a constant speed, which is the average of speed reached from all mice on the accelerating test. A trial ended for a mouse when it fell down or when 5 minutes (300 seconds) were completed. Additionally, mice were tested on the rota-rod apparatus for five consecutive trials of 10 minutes each (test 3). Each trial consisted of 5 minutes in accelerating rotation speed every 30 seconds by 4 rpm, followed by 5 minutes at maximum constant speed. A trial ended for a mouse when it fell down or when 10 minutes (600 seconds) were completed. The latency to fall off the rod is taken as the dependent variable for every trial. A 20 × 30-cm lit chamber with transparent perspex walls (20-cm high) and open top was connected to a 20 × 15 × 20-cm plastic dark box that was completely closed except for the 7.5 × 7.5-cm door connecting it to the lit chamber. Illumination was by direct room light (500 lx). Each mouse was released in the middle of the lit compartment and observed for 5 minutes. The time spent in the dark compartment, the distance traveled, and the speed were measured. Frames of nonreflective aluminum (37-cm high) were used to partition a round open field arena (diameter of 150-cm and 35-cm high walls) into four squares 50 × 50-cm arenas, allowing for concurrent observation of four animals. Illumination in the room was by indirect diffuse room light (4 × 40-W bulbs, 12 lx). The novel object was a 50 mL Falcon tube positioned vertically in the center of the arena. Each animal was observed for 30 minutes in the empty arena as pre-exposure. The novel object was then introduced, and observation continued for another 30 minutes. For time course analysis, the total observation time was portioned into six periods of 10 minutes. The distance to the object and the locomotor activity (as distance traveled) were measured. The arena was the same of novelty test. Twenty-four hours before testing, a plastic home box (12 × 8 × 4 cm with opening of 8 × 4 cm) was placed in the home cage of each test mouse. The next day the home box was placed in a corner of the arena, at 5 cm from the nearest walls, with the opening facing away from the wall. The mice were introduced into the arena and observed for 30 minutes. For time course analysis, the total observation time was portioned into three periods of 10 minutes. The percentage of the time spent inside the home box, the distance traveled and the speed were measured. The standard hidden-platform version of the Morris water maze was done as previously described.19Lipp H.P. Wolfer D.P. Genetically modified mice and cognition.Curr Opin Neurobiol. 1998; 8: 272-280Crossref PubMed Scopus (186) Google Scholar, 20Morris R.G. Garrud P. Rawlins J.N. O'Keefe J. Place navigation impaired in rats with hippocampal lesions.Nature. 1982; 297: 681-683Crossref PubMed Scopus (5067) Google Scholar, 21Morris R. Developments of a water-maze procedure for studying spatial learning in the rat.J Neurosci Methods. 1984; 11: 47-60Crossref PubMed Scopus (5750) Google Scholar Mice were trained in a circular pool (150-cm diameter and 50-cm height) according to standardized protocols.22Wolfer D.P. Lipp H.P. A new computer program for detailed off-line analysis of swimming navigation in the Morris water maze.J Neurosci Methods. 1992; 41: 65-74Crossref PubMed Scopus (72) Google Scholar The wire-mesh platform was 14 × 14 cm. In the hidden-platform version of the water maze, mice had to locate a hidden platform in a fixed position. The test included an acquisition phase (18 trials, six/day, intertrial time 30 to 40 minutes) followed by a reversal phase during which the platform was moved to the opposite position (12 trials, six/day). The first 30 seconds of trial 19 (first reversal trial) were considered as a probe trial. For the analysis the trials were averaged in blocks of two trials. The following measures were calculated: escape latency, swimming speed, floating, and wall hugging time. Spatial selectivity during the probe trial was quantified using the following parameters: percentage of time in the trained quadrant, percentage of time in a circular target zone comprising one-eighth of the pool surface and the annulus crossings. Behavioral assessment of visual acuity was performed in a modifying form of the water maze. Mice were trained in a circular pool (150-cm diameter and 50-cm height) with a 14 × 14-cm wire-mesh platform located in a fixed position with a flag that was visible by the mice. The test was composed by six trials of 2 minutes each. The dependent variable considered was the ability to reach the platform. For exploration tests and water maze test, animals were video-tracked using the EthoVision 2.3 system (Noldus Information Technology, Wageningen, The Netherlands) using an image frequency of 4.2/second. Raw data were transferred to Wintrack 2.4 (http://www.dpwolfer.ch/wintrack)22Wolfer D.P. Lipp H.P. A new computer program for detailed off-line analysis of swimming navigation in the Morris water maze.J Neurosci Methods. 1992; 41: 65-74Crossref PubMed Scopus (72) Google Scholar for off-line analysis. Statistical computations were done using Statview 5.0 (SAS Institute, Cary, NC). Auditory fear conditioning was performed by placing the mice in an opaque conditioning chamber (L × W × H: 25 × 17 × 23 cm) with a grid floor through which scrambled foot shocks could be delivered as unconditioned stimuli (US; 0.26 mA average intensity). The chamber was placed into a dimly lit (< 5 lux) sound attenuating box (background noise level 55 dB), and a speaker on top of the chamber allowed to deliver sounds as conditioning stimuli (CS; 2000Hz). The day before conditioning all mice were pre-exposed to the test chamber for 10 minutes. Animals were either submitted to a delay or trace fear conditioning session. Both sessions consisted of 1 minute adaptation period followed by five identical CS-US conditioning trials with 60 second intertrial intervals (ITI). In the delay fear conditioning each trial started with the presentation of the CS (15 seconds), and the US was presented during the last 2 seconds of the CS presentation. Trace fear co