Brain Struct Funct DOI 10.1007/s00429-015-0991-1

ORIGINAL ARTICLE

Environmental enrichment rescues memory in mice deficient for the polysialytransferase ST8SiaIV Meike Zerwas • Ste´phanie Trouche • Kevin Richetin • Timothe´ Escude´ He´le`ne Halley • Rita Gerardy-Schahn • Laure Verret • Claire Rampon

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Received: 22 September 2014 / Accepted: 9 January 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract The neural cell adhesion molecule NCAM and its association with the polysialic acid (PSA) are believed to contribute to brain structural plasticity that underlies memory formation. Indeed, the attachment of long chains of PSA to the glycoprotein NCAM down-regulates its adhesive properties by altering cell–cell interactions. In the brain, the biosynthesis of PSA is catalyzed by two polysialyltransferases, which are differentially regulated during lifespan. One of them, ST8SiaIV (PST), is predominantly expressed during adulthood whereas the other one, ST8SiaII (STX), dominates during embryonic and postnatal development. To understand the role played by ST8SiaIV during learning and memory and its underlying hippocampal plasticity, we used knockout mice deleted for the enzyme ST8SiaIV (PST-ko mice). At adult age, PST-ko mice show a drastic reduction of PSA-NCAM expression M. Zerwas and S. Trouche contributed equally.

Electronic supplementary material The online version of this article (doi:10.1007/s00429-015-0991-1) contains supplementary material, which is available to authorized users. M. Zerwas  S. Trouche  K. Richetin  T. Escude´  H. Halley  L. Verret  C. Rampon Universite´ de Toulouse, UPS, Centre de Recherches sur la Cognition Animale, 118 route de Narbonne, 31062 Toulouse Cedex 9, France M. Zerwas  S. Trouche  K. Richetin  T. Escude´  H. Halley  L. Verret  C. Rampon (&) Centre de Recherches sur la Cognition Animale (CNRS UMR 5169) Universite´ Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex 4, France e-mail: [email protected] R. Gerardy-Schahn Institute for Cellular Chemistry, Hannover Medical School, 30626 Hannover, Germany

in the hippocampus and intact hippocampal adult neurogenesis. We found that these mice display impaired longterm but not short-term memory in both, spatial and nonspatial behavioral tasks. Remarkably, memory deficits of PST-ko mice were abolished by exposure to environmental enrichment that was also associated with an increased number of PSA-NCAM expressing new neurons in the dentate gyrus of these mice. Whether the presence of a larger pool of immature, likely plastic, new neurons favored the rescue of long-term memory in PST-ko mice remains to be determined. Our findings add new evidence to the role played by PSA in memory consolidation. They also suggest that PSA synthesized by PST critically controls the tempo of new neurons maturation in the adult hippocampus. Keywords Memory  Hippocampus  Neurogenesis  PSA-NCAM  Polysialylation  Polysialyltransferase ST8iaIV/PST

Introduction In the adult mammalian brain NCAM establishes cell–cell adhesion ensuring stabilization of neural circuits, while the attachment of the polysialic acid (PSA) to NCAM provides NCAM with anti-adhesion properties, allowing structural plasticity of neuronal circuits to occur. Thus, PSA-NCAM has been involved in circumstances requiring neuronal network remodeling such as activity-dependent synaptic plasticity and formation of long-term memory that relies on the conversion of a labile short-term memory into a longlasting stable trace (Rønn et al. 2000; Dityatev and Schachner 2003; Bonfanti 2006). This process of postacquisition stabilization of the trace within minutes or

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hours that follow learning is called synaptic consolidation of memory (Dudai 2004). PSA-NCAM expression in the hippocampus is modulated following a variety of behavioral tasks including spatial navigation (Murphy et al. 1996; Sandi et al. 2004; Venero et al. 2006), odor discrimination (Foley et al. 2003; Knafo et al. 2005), olfactory associative learning (Manrique et al. 2014), contextual fear conditioning (Merino et al. 2000; Sandi et al. 2003; LopezFernandez et al. 2007) or passive avoidance (Doyle et al. 1992; Foley et al. 2003). Conversely, pharmacological removal of PSA from NCAM in the hippocampus, leads to memory impairment in these tasks and prevents the induction and maintenance of synaptic plasticity in the hippocampus (Becker et al. 1996; Muller et al. 1996; Roman et al. 2004). In the mammalian brain, two enzymes, the polysialyltransferases (ST8SiaII or STX and ST8SiaIV or PST), are responsible for the addition of PSA on NCAM (Hildebrandt et al. 2007). These enzymes are expressed according to different spatial and temporal patterns among neural tissues (Bonfanti 2006). While STX predominantly regulates PSA-NCAM expression during embryonic, peri-natal and early post-natal development, PST is the major polysialyltransferase of the post-natal and adult brain (Angata et al. 1997; Hildebrandt et al. 1998; Ong et al. 1998). During adulthood, a massive reduction of PSA expression is observed brain-wide, except in neurogenic regions such as the dentate gyrus (DG) of the hippocampus, where PSA is transiently expressed by immature neurons (Seki and Arai 1993; Hildebrandt et al. 1998). To specifically examine the role of PSA addition on NCAM in memory processes and related neuronal plasticity, genetically modified mice depleted for either PST or STX have been generated (Eckhardt et al. 2000; Angata et al. 2004), which leave NCAM protein expression intact. Consistent with the prevalent expression of PST in the adult brain, PST-ko mice display normal PSA-NCAM expression throughout development and no morphological defects after birth. Adult PST-ko mice exhibit intact NCAM expression, but reduced PSA-NCAM contents in the hippocampus and impaired synaptic plasticity in the CA1 hippocampal region (Eckhardt et al. 2000). At the behavioral level, mice lacking PST are impaired in hippocampal-dependent spatial learning (Markram et al. 2007) and contextual memory (Senkov et al. 2006) but show no deficit in amygdala-dependent cued fear memory (Senkov et al. 2006; Markram et al. 2007). To further characterize the role of PSA during learning and memory, PST-ko mice were tested for spatial and nonspatial memory using the Morris water maze, the object location and recognition tasks. At the cellular level, basal PSA-NCAM expression was evaluated in the hippocampus of PST-deficient mice. A particular focus was put on the dentate gyrus where PSA-NCAM expressing cells are

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present during adulthood, reflecting ongoing adult neurogenesis (Seki and Arai 1993). Then, we asked whether exposure to enriched environment may alleviate memory deficits in PST-ko mice. Given that some of the behavioral tasks used in the present work are sensitive to adult neurogenesis modulation (Goodman et al. 2010; Bruel-Jungerman et al. 2005), we also examined the extent to which, neurogenesis-dependent processes may participate to memory improvement in PST-deficient mice exposed to environmental enrichment.

Materials and methods Subjects and housing conditions Male and female 5- to 6-month-old mice from three different genotypes (wild type ‘‘PST-wt’’, knockout ‘‘PSTko’’, and PST-heterozygous ‘‘PST-het’’) were obtained by interbreeding PST-heterozygous mice of the ST8SiaIV (PST) mouse line as previously described [(Eckhardt et al. 2000); backcrossed into the C56BL/6J background for 6 generations]. All mice were housed under controlled temperature (23 °C) and 12/12 h light/dark cycle. In standard conditions, mice from the same genotype were groupedcage (4). In environmentally enriched (EE) conditions, females from the three genotypes were grouped-cage (8) during 4 weeks in large cages (150 9 80 9 80 cm) containing toys, wooden blocks, climbing platforms, plastic tubes and small houses that were rearranged and renewed daily to stimulate animals’ exploratory behavior, as previously described (Verret et al. 2013). Water and food were provided ad libitum. Statement of animal rights All experiments were performed in strict accordance with the recommendations of the European Union (86/609/EEC) and the French National Committee (87/848). The Centre de Recherches sur la Cognition Animale (CRCA) has received French legal approval for experiments on living vertebrate animals (Arreˆte´ Pre´fectoral dated 9-02-2011) and this work was carried out in accordance with the Policies of the French Committee of Ethics. Animal surgery and experimentation conducted in this study were authorized by the French Direction of Veterinary Services to LV (#31–238, 2005) and CR (#31-11555521, 2002) and all efforts were made to improve animals’ welfare. Genotyping All mice were genotyped at weaning by polymerase chain reaction (PCR). Tail DNA was extracted by Chelex resin

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(BioRad, Hercules, CA, USA) and submitted to PCR in a buffer containing 1.5 U/30 ll Taq DNA Polymerase, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris–HCl pH = 8.3 (MP Biomedicals, Santa Ana, CA, USA), 200 lM dNTP (Carl Roth, Karlsruhe, Germany) and 0.2 lM of the corresponding primers. For PST, the forward primer of PSTwt and PST-ko was 50 -GAG CTC ACA ACG ACT CTC CGA GC-30 , and the reverse primers for PST-wt and PSTko were 50 -CTC AGT TCT GGC TAT TTC TTT TGT-30 and 50 -ACC GCG AGG CGG TTT TCT CCG GC-30 , respectively. The cycling conditions for the PCR were 95 °C for 5 min for the first cycle. This was followed by 35 cycles of 95 °C for 1 min, 56.2 °C for 1 min and 72 °C for 2 min, with a final extension of 72 °C for 7 min. PCR products were resolved on a 2 % agarose gel to identify the genotypes (PST was 533 bp for PST-wt and 730 bp for PST-ko). Behavior Mice raised in standard conditions were tested in the elevated plus maze to determine the animal’s level of anxietylike behavior. One week later, mice were tested for memory randomly in either object location task or novel object recognition task followed by spatial or cued Morris water maze. For the enriched environment experiment, mice were tested over the last 3 days of enriched housing. One group of mice was subjected to the spatial Morris water maze whereas a second group of mice underwent the novel object recognition test. All behavioral experiments were monitored by a video camera and behavior was analyzed using a video tracking software (Ethovision, Noldus, The Netherlands). Setups were cleaned thoroughly between each mouse. Anxiety-like behavior (elevated plus maze) The maze consisted of a plus-shaped track with two closed arms and two opened arms (28 9 10 9 20 cm for each arm) that extended from a central platform (10 9 10 cm). The maze was elevated 45 cm above the floor. Each trial began with the mice placed in the central zone facing one of the open arms and lasted 5 min (standard housing: PSTwt n = 22; PST-het n = 22; PST-ko n = 27; EE conditions: PST-wt n = 21; PST-het n = 18; PST-ko n = 20). For each mouse, the percentage of time spent in the open arms was scored for each minute of the 5 min session. Locomotor activity (open-field) The total distance moved in the open-field was measured during the familiarization session of the object location or recognition tasks (see below). Each animal (standard

housing: PST-wt n = 11; PST-het n = 16; PST-ko n = 15; EE conditions: PST-wt n = 9; PST-het n = 10; PST-ko n = 12) was allowed to explore during 10 min a circular open-field (50 cm diameter, 40 cm-high wall) containing a conspicuous striped pattern on a part of its wall. Object-location memory task We used a procedure similar to that described previously (Goodman et al. 2010). The day before exploration phase, the mouse was placed for 10 min in the empty open-field for familiarization. The next day, two identical objects were placed in the middle of the arena and mice were allowed to explore for 10 min. The time spent exploring the two objects was scored. Then, two independent groups of mice were tested for 10 min, either 20 min (PST-wt n = 8; PST-het n = 7; PST-ko n = 8) or 24 h (PST-wt n = 11; PST-het n = 7; PST-ko n = 8) later, when one of the objects (left or right counterbalanced) was moved to a new position. As an index of spatial memory, the percentage of time spent exploring the displaced object was calculated. Object-recognition memory task We used a procedure similar to that described previously (Goodman et al. 2010) except that no criteria of minimum exploration time was applied during acquisition. Procedure, equipment, and analyses were similar to the ones described for the novel object location test, but the pattern inside the arena was removed and a white curtain surrounding the arena was used to eliminate visual landmarks. The day after familiarization the mice were given 10 min to explore two identical objects placed in the arena. Mice were tested 4 h later and 24 h later (PST-wt n = 8; PSThet n = 11; PST-ko n = 8) and were exposed for each test to one familiar object and a novel object (left or right counterbalanced) for 10 min. At both delays, memory for the familiar object was evaluated by calculating the preference index for the novel object, expressed as a percentage of time spent exploring the novel object. Mice of the environmental enrichment experiment (EE-PST-wt n = 9; EE-PST-het n = 10; EE-PST-ko n = 12) were tested according to the same protocols. Morris water maze We used a massed training procedure similar to that described previously (Trouche et al. 2009) because it implies a fast acquisition phase distinctly followed by memory consolidation (Sargolini et al. 2003), allowing to distinguish both phases from each other. In the spatial

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version, mice from the three genotypes were trained to locate the hidden platform (‘‘target annulus’’) using distal landmarks during four blocks of three trials each (PST-wt n = 22; PST-het n = 20; PST-ko n = 21). Spatial memory was then tested during a 1 min-probe test without the platform held either immediately (PST-wt n = 12; PST-het n = 7; PST-ko n = 10) or 24 h (PST-wt n = 10; PST-het n = 13; PST-ko n = 11) after training. Another group of mice was tested after environmental enrichment (EE-PSTwt n = 12; EE-PST-het n = 8; EE-PST-ko n = 8). In the cued version of the water maze, distal landmarks were removed and a visual cue (a ball) was suspended 7 cm above the hidden platform, indicating its location. The training phase followed the procedure described above, except that the ball and platform changed location after each block of three trials (PST-wt n = 6; PST-het n = 6; PST-ko n = 8). Non-spatial memory was tested 24 h after training, during a 1 min-probe test, with no platform but the cue suspended in the quadrant opposing the location of the platform during the last training session. The mean swim speeds during familiarization and the mean distance swum to reach the platform during each training session were calculated. During the probe tests, numbers of annulus (virtual 12 cm-diameter circle) crossings in the four quadrants was calculated. For each mouse, the number of target annulus crossings was compared to the mean crossing number of the three other annuli. Histological experiments Injections of BrdU Animals were injected with 5-bromo-20 -deoxyuridine (BrdU, i.p. 100 mg/kg in 0.9 % NaCl, Fluka). Three injections were given in 4 h intervals over 1 day (PST-wt n = 8; PST-het n = 7; PST-ko n = 8), and occurred the day after been introduced into the enriched environment, for enriched animals (EE-PST-wt n = 5; EE-PST-het n = 5; EE-PST-ko n = 5). Mice were killed 28 days later, to assess long-term survival of adult-born BrdU-labeled cell. Stereotactic injections of GFP-expressing retroviral vector The Moloney-leukemia-derived retroviral vector pCMMPIRES2eGFP-WPRE expressing enhanced green fluorescent protein (MoMulV::GFP) (Roybon et al. 2009) was used to label adult-born neurons in the DG of PST-wt (n = 3) and PST-ko (n = 3) 3- to 4-month-old mice raised in standard conditions. As previously described (Krezymon et al. 2013) bilateral DG was targeted (antero-posterior -2.0 mm; lateral ±1.6 mm; ventral -2.5 mm; 0.5 ll of

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MoMulV::GFP per site, 100 nl/min of rate). Mice were killed 28 days later to assess the morphology of GFPlabeled neurons. Tissue preparation and sectioning Mice were deeply anesthetized with pentobarbital and perfused with 4 % paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were post-fixed in the same solution at 4 °C overnight and then cryoprotected in 30 % sucrose. Coronal 30 or 50 lm thick serial sections were cut using a cryostat and stored in cryoprotectant at -20 °C until use. Immunohistochemistry For BrdU and Ki67 staining, we used a protocol similar to that described previously (Goodman et al. 2010). Sections were rinsed in phosphate buffered saline with 0.25 % Triton-X 100 (PBS-T) and processed for immunostaining. All steps were performed at room temperature and under agitation unless specified otherwise. The 30 lm thick coronal sections were blocked with 5 % normal goat serum (NGS) for 1 h and were then incubated in either monoclonal rat anti-BrdU antibody (1:200; OBT-0030; Harlan Seralab, Loughborough, UK), rabbit anti-Ki67 antibody (1:1,000; NCL-Ki67p; Novocastra Laboratories, Newcastle upon Tyne, UK), or goat anti-DCX antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight or in mouse anti-Men B monoclonal anti-PSA-NCAM antibody (1:3,000; AbCys, Paris, France) at 4 °C for 48 h. Next, sections were incubated for 90 min in secondary antibody (Vector Laboratories, Burlingame, CA) either biotinylated goat anti-rat (1:250), biotinylated goat anti-rabbit (1:500), biotinylated rabbit anti-goat (1:500) or biotinylated goat anti-mouse (IgM isotype; 1:400) in PBST followed by 90 min in avidin–biotin-peroxidase complex (1:400; Elite kit, Vector Laboratories). Staining was visualized with 3,30 diaminobenzidine (DAB; Fluka) alone (for DCX, PSANCAM) or with 0.06 % nickel ammonium sulfate (for BrdU, Ki67). Sections were mounted on slides, counterstained with Nuclear Fast Red (Vector Laboratories) or hematoxylin, dehydrated through alcohols and coverslipped. For GFP fluorescent labeling, 50 lm thick coronal sections were incubated overnight in rabbit anti-GFP antibody (1:1,000; Torrey Pines Biolabs, Houston, TX, USA) in PBST with 0.1 % sodium azide (PBST-Az) containing 5 % normal goat serum. On the next day, sections were incubated with Alexa 488 donkey anti-rabbit (1:500; Invitrogen, Carlsbad, CA) in PBST before being counterstained with Hoechst and mounted onto slides, coverslipped using Mowiol and stored at 4 °C.

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Quantification and image analysis Quantification of immunolabeled cells and fibers An experimenter blind to the experimental conditions conducted cell quantifications of labeled cells. In both, housing and enriched conditions, 5–8 animals per genotype were analyzed for each marker (BrdU, PSA-NCAM, Ki67, DCX). Labeled (BrdU?) cells were counted bilaterally in the DG of 180 lm-spaced sections spanning the dorsal hippocampus (series of 1-in-6 sections). All other labeled cells (PSA-NCAM?, Ki67?, DCX?) were counted from 360 lm-spaced sections (series of 1-in-12 sections). GFP? cells were analyzed from 150 lm-spaced sections (series of 1-in-3 sections). The area comprising the granular cell layer (GCL) and subgranular zone (SGZ) of the DG was measured on each section with Mercator software (Explora Nova, La Rochelle, France). The sum of all surfaces sampled for each mouse was multiplied with the sections thickness to calculate the sectional volume. It was multiplied with spacing between sections to calculate the reference volume, which was neither affected by genotypes nor housing conditions. The total number of labeled cells per DG was given by the ratio of counted labeled cells per sectional volume multiplied by the reference volume. Analyses are based on cell numbers averaged over left and right DG, for each animal. Photographs were processed using Adobe Photoshop software CS5 (Adobe System). Three to five animals per genotype were analyzed to quantify PSA-NCAM immunoreactivity density in the hilus and CA3 region on two serial sections through the dorsal hippocampus. Digitized images were obtained with a digital camera (Optronics, Goleta, CA) on a light microscope (910 objective, BX51 Olympus, Essex, UK) using Mercator Pro stereology system (Explora Nova, La Rochelle, France). Integrated optical density (IOD) of PSA-NCAM immunostaining was measured using image analysis software ImageJ (open source, available on http:// rsbweb.nih.gov/ij/index.html) after systematically subtracting the background measured in the dorsal third ventricle for each section. Levels of PSA-NCAM immunoreactivity in individual mice were normalized against mean level of PST-wt or PST-wt-EE mice. Characterization of GFP-labeled cells The morphology and localization of GFP-labeled (GFP?) cells in the DG of mice were analyzed 28 days postinjection (dpi) of the viral vector as described before (Krezymon et al. 2013). In the DG, GFP? cells presenting a round soma, a conspicuous axonal projection extending toward the hilus and spiny dendrites reaching the outer

molecular layer were classified as neuron-like cells. These morphological parameters were characterized using an Olympus BX-51 microscope equipped with Mercator software (Explora Nova). The localization of each GFP? neuron-like cell was then evaluated by virtually dividing the GCL into inner, middle and outer layers and each GFP? cell was assigned to one of these layers or to the SGZ. Dendritic morphology of 28 dpi GFP? cells was analyzed from at least 6 sections per mouse (around 10 cells per mouse) from confocal acquisition of z-series of 50–75 optical sections at 0.5 lm intervals, with a 409 oil lens, digital zoom of 1.7, with a TCS SP5 (Leica Microsystem) system. Three-dimensional reconstructions of GFP? neuron-like cells were analyzed using Imaris XT (Bitplane, Zurich, Switzerland). Length of primary dendrite and maximal dendritic length were calculated automatically from 3D reconstruction. Statistical analysis The results are expressed as mean ± SEM. GraphPad Prism 6 software was used for data analysis. Differences between groups were assessed by ANOVAs, or a repeated measure ANOVA when appropriate. For object location and object recognition tests, the Wilcoxon signed rank test (WSRT) was applied to detect a difference to 50 % i.e. the chance level. Post hoc multiple comparisons using Tukey’s Honestly Significant Distance (HSD) test were performed when allowed. For all comparisons, values of p \ 0.05 were considered significant.

Results Altered PSA-NCAM expression pattern in the hippocampus of PST-ko mice To evaluate the impact of the polysialyltransferase PST deletion on overall polysialylation of NCAM in the hippocampus, we examined PSA-NCAM immunostaining in the DG of three groups of mice housed in standard conditions (Fig. 1a, b): control wild type (PST-wt), PST-heterozygous (PST-het) and PST-knockout (PST-ko). All groups exhibited similar numbers of PSA-NCAM-immunoreactive (PSA-NCAM?) cells (PST-wt: 2,929 ± 79; PST-het: 3,268 ± 296; PST-ko: 2,752 ± 226; F(2,12) = 1.737, p = 0.217; Fig. 1c) located in the innermost region of the granule cell layer and subgranular zone of the DG, where the adult neural progenitor cells lie (Fig. 1a, b). Such pattern of PSA immunoreactivity (IR) in the DG is typical of PSA-expression on the soma of immature neurons, and reflects the neurogenic activity of

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Fig. 1 PSA-NCAM expression in the hippocampus of PST-wt (a) and PST-ko (b) mice raised in standard conditions. Insets below (a) and (b) show PSA-NCAM immunoreactive cells in the subgranular layer of the dentate gyrus (DG) (left), labeled mossy fibers in the hilus region (middle) and the CA3 (right), on sections counterstained with hematoxylin. Scale bars 150 lm and 40 lm for insets. Graphs

illustrating the effect of PST deletion on the total number of PSANCAM-labeled cells in the DG (c). Quantification of PSA-NCAM immunoreactivity (IR) in fibers traveling in the hilus (d) and CA3 region (e) of mice from the three genotypes. PST-wt wild type, PSTko PST-knockout, PST-het PST-heterozygous. Data are expressed as group mean ± SEM; *p \ 0.05, ***p \ 0.001

this brain region (Seki and Arai 1993; Schuster et al. 2001). However, in PST-ko mice, we observed the near absence of PSA-IR in the mossy fibers traveling in the hilus (O.D. measurements of PSA-NCAM expression: PST-wt: 1.00 ± 0.03; PST-het: 0.57 ± 0.04; PST-ko: 0.41 ± 0.03) towards CA3 (PST-wt: 1.00 ± 0.04; PST-het: 0.52 ± 0.08; PST-ko: 0.44 ± 0.05), contrasting with the robust PSA-NCAM labeling of fibers observed in PST-wt mice (genotype effect in hilus: F(2,8) = 104.2, p \ 0.001; PST-wt vs PST-het, PST-ko p \ 0.001; PST-het vs PST-ko p \ 0.05; in CA3: F(2,8) = 37.15, p \ 0.001; PST-wt vs PST-het, PST-ko p \ 0.001; Fig. 1d, e). These findings are consistent with previous description of PSA expression in PST-ko mice (Markram et al. 2007; Nacher et al. 2010) and confirm the different pattern of PSA expression in the hippocampus of PST-ko mice compared to their PST-wt littermates.

Intact adult hippocampal neurogenesis in PST-ko mice

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Adult hippocampal neurogenesis was examined in mice from all three genotypes. We first evaluated proliferation, and differentiation towards the neuronal fate of newborn cells with specific markers (Ki67 and doublecortin DCX, respectively, Online Resources 1a, c). Numbers of Ki67? and DCX? cells remained the same among the three genotypes (Ki67: PST-wt: 483.6 ± 36.3; PST-het: 350.6 ± 47.0; PST-ko: 424.7 ± 46.9; and DCX: PST-wt: 2,536.4 ± 207.2; PST-het: 2,376.6 ± 338.6; PST-ko: 2,511.8 ± 278.3; Online Resources 1b, 1d), indicating that neither new cell proliferation nor neuronal differentiation was affected by PST deletion. Next, the survival of adultgenerated cells in the hippocampus was evaluated by quantifying BrdU-immunoreactive (BrdU?) cells in the DG 28 days after BrdU injection (Online Resource 1e).

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Again, no difference in BrdU? cell numbers was found between genotypes (PST-wt: 87.5 ± 27.6; PST-het: 88.9 ± 16.1; PST-ko: 105.3 ± 15.9; Online Resource 1f), indicating that new cell survival was not affected by the absence of PST. By reducing interactions between cells, PSA-NCAM may facilitate plastic processes in the adult brain such as neuritic growth, spine and synapse remodeling (Bonfanti 2006; Gascon et al. 2007). Conversely, non-polysialylated NCAM enables interactions that induce neuronal differentiation and neurite outgrowth (Seidenfaden et al. 2006). Therefore, we sought to investigate whether PST deletion affects neuronal migration or morphological development of newborn cells within the DG. Using a retrovirus expressing the GFP to label adult-born cells in PST-wt and PST-ko mice, we quantified the migration and dendritic arborization of 28-day-old GFP? cells that expressed a neuronal morphology (see methods; Online Resource 2a). We found that the migration of GFP? neuron-like cells within the GCL did not vary between PST-wt and PST-ko mice. In both genotypes, the majority of 28-day-old GFP? neurons was found within the inner third of the GCL (PSTwt 55.0 ± 8.2 %; PST-ko 59.7 ± 3.9 %; Online Resource 2b), indicating that the deletion of PST did not affect the positioning of new neurons within the DG. Then, we evaluated the morphological complexity of mature adultborn granule neurons in PST-ko and PST-wt mice. We quantified the dendritic arborization of 28-day-old GFP? neurons from confocal images. The maximal dendritic length of GFP? neurons, a rough index of neuronal morphology, was similar in both genotypes (PST-wt 185.9 ± 7.3 %; PST-ko 181.6 ± 12.6 %, p [ 0.05; Online Resource 2c). Furthermore, the primary dendritic branch of GFP? neurons, which corresponds to the distance between the soma and first dendritic branching point, reached similar length in both genotypes (PST-wt 25.9 ± 3.3 %; PSTko 28.5 ± 3.6 %, p [ 0.05; Online Resource 2d). Altogether our data indicate that the lack of PST has no major impact on adult neurogenesis, suggesting that new neurons might achieve correct functional integration into the hippocampal network. Normal anxiety phenotype and locomotion in PST-ko mice To examine whether the reduction or lack of PSA expression in the hippocampus of PST-het and -ko mice induces cognitive alterations, we tested home-cage (standard) housed mice in different behavioral tasks. We first evaluated anxiety and locomotor activity in mice of the three genotypes (Online Resource 3) in the elevated plus maze and open-field apparatus. No difference between genotypes was found in the time spent in the open arms

(F(2,68) = 0.553, p = 0.578) of the elevated plus maze and distance moved in the empty open-field (F(2,39) = 0.966, p = 0.390, Online Resources 3a, b). In contrast to our data, Calandreau and colleagues reported enhanced anxiety-like behavior and intact locomotor activity of PST-ko mice (Calandreau et al. 2010). Whether such discrepancy is related to the use of mice of different age and sex or different behavioral protocol, remains to be determined. However, our data indicate that anxiety-related behavior and locomotor activity were neither affected by partial nor by full PST deletion, ruling out any putative impact of differential anxiety or reactivity across genotypes on the subsequent cognitive tests. PST-ko display long-term memory deficits in object tasks The object location task evaluates the ability of mice to discriminate a novel from a familiar spatial location. During exploration phase, the percentage of time spent exploring each object (right object: PST-wt: 52.05 ± 1.62 %; PST-het: 48.90 ± 0.86 %; PST-ko: 53.11 ± 1.91 % F(2,46) = 1.718, p = 0.191) and the average total time spent exploring both objects (PST-wt: 39.2 ± 6.5; PST-het: 54.4 ± 12.0; PST-ko: 38.0 ± 10.0; F(2,46) = 0.890, p = 0.417) were not different across genotypes. At different delays (20 min and 24 h) after the testing phase, one of the objects was moved to a new location in the arena and spatial memory was evaluated. When tested 20 min after exploration phase (Fig. 2a), mice of all genotypes significantly spent more time exploring the displaced object than the stable one when compared to chance level (50 %) (exploratory preference index of PSTwt: 67.6 ± 3.2 %, PST-het: 73.6 ± 2.6 %, PST-ko: 70.7 ± 5.7 %; PST-wt: p \ 0.01, PST-het, PST-ko: p \ 0.05, index vs chance). All groups exhibited the same level of performance at this delay (F(2,20) = 0.502, p = 0.613). However, PST-ko mice showed no preference for the displaced object when tested 24 h after the exploration phase (Fig. 2b, PST-ko: 51 ± 3.0 %; p = 1.0 index vs chance), in contrast to the two other groups (PST-wt: 67.5 ± 2.2 %; PST-het: 63.8 ± 4.5 %; PST-wt: p \ 0.001, PST-het: p \ 0.05, index vs chance). Thus, PST-ko mice exhibit long-term spatial memory deficit compared to the two other groups (F(2,23) = 8.030, p \ 0.01, post hoc: PST-wt vs PST-ko p \ 0.01, PST-het vs PST-ko p \ 0.05) and the partial presence of the PST gene is sufficient to ensure long-term memory processes in this task. We next aimed at evaluating whether the memory deficit observed in PST-ko could be generalized to the non-spatial memory version of the object task. The object recognition task assesses the ability of mice to discriminate between a

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location tasks, respectively. Our findings imply that PSA biosynthesis by PST is required, during a crucial time window lying between 4 and 24 h after training, for the consolidation of non-spatial and spatial memories. PST-ko mice show long-term memory deficits in the spatial version of the Morris water maze

Fig. 2 PST deletion impaired long-term memory in object location and novel object recognition tasks. All mice showed similar performances in the object location task at short delay (a), but PSTko were impaired at longer retention time (b). Mice from all genotypes performed well in the object recognition task at short retention time (c), but at longer delay PST-ko mice showed altered performances (d). Data are expressed as the group mean (±SEM) preference index (% of time exploring the displaced (a, b) or novel (c, d) object, related to the total exploration time for both objects). Horizontal dotted lines represent equal exploration of the two objects (*p \ 0.05, **p \ 0.01 index vs chance level (50 %) WSRT, # p \ 0.05; ##p \ 0.01 between genotypes, Tukey’s HSD post hoc)

novel and a familiar object. During exploration phase, mice of all genotypes spent the same amount of time exploring each object (PST-wt: 51.1 ± 2.8 %, PST-het: 50.3 ± 0.9 % PST-ko: 50.1 ± 3.1 %). The average time spent exploring the objects was not different across genotypes (F(2,24) = 0.048), indicating that the objects elicited the same exploratory interest for all genotypes. Recognition memory was tested 4 h and 24 h after the exploration phase, when a new object replaced one of the familiar objects. No memory deficit was observed after 4 h (preference index in PST-wt: 68.4 ± 2.6 %; PST-het: 68.3 ± 4.1 %; PST-ko: 67.4 ± 7.0 %; no difference between groups F(2,24) = 0.012, p = 0.99, Fig. 2c). However, PST-ko mice showed memory impairments at 24 h (PST-wt: 67.1 ± 4.4 %, PST-het: 61.5 ± 3.6 %, PST-ko: 47.0 ± 2.5 %; F(2,24) = 7.237, p = 0.003, post hoc: PST-wt vs PST-ko p \ 0.01, PST-het vs PST-ko p \ 0.05; Fig. 2d). Altogether, we found that PST deletion has no effect on memory at short retention time, but impairs non-spatial and spatial memory at longer delays in object recognition and

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We next evaluated whether long-term memory deficits in PST-ko mice are observable during navigation in a complex environment that requires flexible use of spatial information. In the hippocampal-dependent Morris water maze task (spatial version), no genotype effect on swim speed during familiarization was found (F(2,60) = 0.637, p = 0.532). Mice of all genotypes learned to locate the platform (session effect F(3,180) = 22.61, p \ 0.001; Fig. 3a) but control mice reached better performances than PST-ko after completion of spatial training (F(2,60) = 3.060, p = 0.054; PST-wt vs PST-ko p \ 0.05; Fig. 3a). Spatial memory was tested during a probe test held either immediately or 24 h after the last training trial. During immediate probe test, mice of all genotypes crossed preferentially the target annulus where the platform was located during training (PST-wt p \ 0.05; PST-het, PSTko p \ 0.01; Fig. 3b), indicating that short-term spatial memory of animals was not altered. Indicative of memory precision, numbers of target annulus crossings were not significantly different between genotypes (F(2,26) = 1.902, p = 0.169; Fig. 3b). However, 24 h after training, PST-ko mice did not cross preferentially the target annulus, in contrast to PST-wt and PST-het mice (PST-wt p \ 0.0001, PST-het p \ 0.001, PST-ko p = 0.121; Fig. 3c). Thus, our data suggest that the presence of the PST gene is required for the consolidation of spatial long-term memory. Next we examined whether the PST gene is required in the non-spatial (cued) version of the Morris water maze. Mice of all groups rapidly learned to locate the cued platform (F(3,51) = 11.13, p \ 0.001; Fig. 3d) and remembered the platform location when tested 24 h after (annulus crossings target vs others: PST-wt p \ 0.05; PSThet and PST-ko p \ 0.01; Fig. 3e). Performances were similar between genotypes (F(2,17) = 1.058, p = 0.369). These findings indicate that PST is not required for the cued version of the water maze, which relies on hippocampal-independent processes. Enrichment restores recognition memory and spatial memory in PST-ko mice Exposure to environmental enrichment (EE) exerts a beneficial effect on behavioral performance of wild-type rodents and efficiently attenuates memory deficits in several mouse models of central nervous system disorders and

Brain Struct Funct Fig. 3 PST deletion impairs spatial memory in the Morris water maze. a All mice learnt to locate the hidden platform in the water maze (one-way ANOVA with repeated measures, ***p \ 0.001) and showed intact immediate spatial memory (b). However, 24 h after training, PST-ko mice displayed impaired long-term memory unlike PST-wt and PST-het mice (c). In contrast, non-spatial (cued) learning (d) and memory (e) in the water maze were not affected by PST deletion. Probe test performances (b, c, e) are expressed as mean numbers of annulus crossings ± SEM (*p \ 0.05, **p \ 0.01, ***p \ 0.001 target annulus vs mean of other annuli)

during aging (Laviola et al. 2008). Here we asked whether exposure to EE may alleviate memory deficits of PST-ko mice. Therefore, naive adult animals from the three genotypes were exposed to EE during 4 weeks (Fig. 4a). First, we verified that mice of all genotypes still displayed similar anxiety phenotype and locomotor activity after EE. In the elevated plus maze, the percentage of time spent in the open arms over time (F(2,28) = 0.553, p = 0.578; Online Resource 4a) and the distance moved in the openfield (F(2,39) = 0.966, p = 0.390; Online Resource 4b) were similar between genotypes after EE. Then, a first group of enriched-housed mice was tested in the novel object recognition task. It is to note that our EE protocol did not affect exploratory behavior in this task as no change in time exploring the objects, neither during acquisition nor during the retention tests was found (data not shown). At both 4 h and 24 h retention delays, all genotypes explored preferentially the novel object compared to the familiar one (Fig. 4b, 4 h delay, F(2,28) = 0.024 p = 0.976, preference index vs chance (50 %): EE-PST-wt: 65.7 ± 4.8 % p \ 0.01; EE-PST-het: 64.3 ± 2.4 % p \ 0.01; EE-PST-ko: 65.5 ± 5.7 % p \ 0.05; Fig. 4c 24 h delay: F(2,28) = 0.451, p = 0.642; vs 50 %: EE-PST-wt: p \ 0.01; EE-PST-het: p \ 0.05; EE-PST-ko: p \ 0.05). Moreover, mice from the three genotypes exhibited the same level of preference for the

novel object (Fig. 4b 4 h-delay: F(2,28) = 0.024, p = 0.976; Fig. 4c 24 h-delay F(2,28) = 0.451, p = 0.642). Although standard-housed PST-ko mice display long-term recognition memory impairments (Fig. 2d), long-term recognition memory of enriched-housed PST-ko mice does not differ from that of EE-PST-wt mice. A second group of EE-mice from all three genotypes was trained in the spatial Morris water maze as previously described, and tested 24 h later, when long-term memory deficits in the standard-housed PST-ko were observed. All EE-mice learnt to locate the position of the hidden platform (session effect F(3,75) = 22.57, p \ 0.001, Fig. 4d) and remembered it 24 h later during a 1 min probe test (EEPST-wt: p \ 0.01; EE-PST-het: p \ 0.01; EE-PST-ko: p \ 0.05; Fig. 4e) in a similar manner (F(2,25) = 0.481, p = 0.624, Fig. 4e). In conclusion, EE has a beneficial effect on both spatial and non-spatial forms of long-term memory of PST-ko mice. Environmental enrichment increases PSA-NCAM expression in new hippocampal neurons of PST-ko mice We next analyzed the effects of 4-weeks enriched environment on PSA-NCAM expression in the DG of the three genotypes. We previously found that in standard housed

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Fig. 4 Exposure to environmental enrichment enables the formation of spatial and non-spatial memory in PST-ko mice. Photography illustrating an enriched environment (EE) (a). Performances of EEPST-wt, EE-PST-ko and EE-PST-het mice in the object recognition task at short (b) and long (c) retention delays. Data are expressed as the group mean (±SEM) preference index (in %). Horizontal dotted lines represent chance level (50 %) (*p \ 0.05, **p \ 0.01, index vs

50 %). Distance swam by enriched animals from all genotypes to reach the platform during training in the spatial water maze (d). Enrichment-restored memory performances of PST-ko mice in the 24 h probe test (e). Data are expressed as mean numbers of annulus crossings (±SEM). (*p \ 0.05, **p \ 0.01, target annulus vs mean of other annuli)

conditions, all groups exhibited similar numbers of PSANCAM? cells in the DG (Fig. 1c). Strikingly after EE, numbers of PSA-NCAM? cells were significantly higher in EE-PST-ko mice compared to EE-PST-wt and EE-PSThet mice (EE-PST-wt: 3,094 ± 160; EE-PST-het: 3,579 ± 134; EE-PST-ko; 4,857 ± 286, F(2,12) = 19.75, p = 0.0002; Fig. 5a–c). While PSA expression was considerably reduced in the hilus of PST-ko and PST-het in standard conditions (Fig. 1d), enriched conditions brought it back to similar levels that in EE-PST-wt mice (EE-PSTwt: 1.00 ± 0.10; EE-PST-het: 0.82 ± 0.13; EE-PST-ko; 0.68 ± 0.14, F(2,8) = 1.708, p = 0.299, see Fig. 5d). In CA3, only PSA expression in EE-PST-het, but not EEPST-ko mice, reached the level of EE-PST-wt animals (EE-PST-wt: 1.00 ± 0.09; EE-PST-het: 0.93 ± 0.11; EEPST-ko: 0.40 ± 0.03, F(2,8) = 17.42, p = 0.0012, Fig. 5e).

beneficial effect of EE in long-term memory observed in PST-ko EE-mice is associated with an increased number of new hippocampal neurons. Mice from all three genotypes were housed in enriched cages for 4 weeks, and the next day after enrichment had started, mice received 3 injections of BrdU at 4 h intervals. We quantified and compared the number of surviving BrdU-labeled (BrdU?) cells in the DG of mice from the three genotypes housed in enriched conditions (Fig. 6a). No difference in BrdU? cells numbers was found between genotypes (EE-PST-wt: 200.8 ± 23.6; EE-PST-het: 208.9 ± 40.5; EE-PST-ko: 174.9 ± 28.8) (Fig. 6b). When we compared the number of BrdU? cells between standard versus enriched conditions, we found that survival of BrdU? cells is increased by exposure to enriched environment (F(5,32) = 4.824, p = 0.0021; Fig. 6b). The comparison with their corresponding standard-housed genotype revealed that PST-wt and PST-het enriched mice experienced over a twofold increase of their BrdU? cell number following EE, while this increase remained limited in the case of PST-ko mice (standard vs enriched conditions: PST-wt, PST-het: p \ 0.05; PST-ko: ns). These data suggest that increased survival of adult-generated cells might not be determinant in the rescue of memory deficits in PST-ko mice.

Enrichment-induced enhancement of new cell survival is attenuated in PST-ko mice One of the most indisputable effect of enrichment on adult hippocampal neurogenesis is the increase of new neurons survival following EE (Kempermann et al. 1997; BruelJungerman et al. 2005). We next asked whether the

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Fig. 5 Enrichment-induced increased expression of PSA-NCAM in PST-ko mice. Photographs of PSA-NCAM expression in the hippocampus of EE-PST-wt (a) and EE-PST-ko (b) mice after exposure to enriched environment. Insets show PSA-NCAM immunoreactive cells in the granular layer of the DG (left), labeled mossy fibers in the hilus (middle) and CA3 region (right). Scale bars 150 lm, 40 lm for

inset. Quantification of PSA-NCAM-labeled cells in the DG of enriched mice from all genotypes (c). PSA-NCAM immunoreactivity (IR) level was quantified in fibers traveling in the hilus (d) and CA3 region (e) of enriched mice from the three genotypes (mean values ± SEM) (**p \ 0.01, ***p \ 0.001)

Fig. 6 Effect of environmental enrichment (EE) on the survival of BrdU-labeled cells. Photomicrographs depicting a BrdU-immunoreactive (?) (a) cell (arrowhead) in the DG of PST-ko mice (scale bar 20 lm). Quantification of 28-day-old BrdU-labeled cells in the DG of

standard-housed and enriched mice from all three genotypes, showing increased long-term survival of BrdU? cells after enrichment (b) (*p \ 0.05 standard vs enriched for each genotype)

Discussion

overcome by exposure to environmental enrichment, which also leads to the enhancement of the population of immature, PSA-NCAM expressing new neurons in the dentate gyrus. Our findings suggest that such larger pool of highly plastic new neurons might contribute to memory improvements after enrichment.

Using a genetic approach to address the question of PSANCAM contribution to memory processes, we report that mice deficient for PST have impaired long-term memory. Furthermore, we show that these cognitive deficits can be

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PST is required for long-term consolidation of spatial and non-spatial memories We report that mice deficient for PST have impaired longterm spatial memory in two tasks that critically rely on the hippocampus: the Morris water maze and the object location task (Morris et al. 1982; Ennaceur et al. 1997; Mumby et al. 2002). In both tests, PST-ko mice were able to learn and remember the task when tested shortly after training, but they failed to perform correctly at a longer retention delay. Our data are in line with previous reports showing that disruption of NCAM-mediated interactions by removal of PSA in the hippocampus induces spatial memory deficits in the water maze (Becker et al. 1996; Venero et al. 2006). Interestingly, using PST-ko mice, Markram and colleagues showed that the mice performed like PST-wt during initial training in the water maze, but their performances were altered during the last days of the training procedure and during reversal learning, two events that involve memory consolidation and reconsolidation (Markram et al. 2007). Several studies have reported a transient increase of PSANCAM expression in the hippocampus 10–24 h after acquisition of the spatial water maze task (Murphy et al. 1996; Murphy and Regan 1999; Sandi et al. 2003, 2004; Van der Borght et al. 2005; Venero et al. 2006), suggesting that memory consolidation critically depends on PSANCAM mediated plasticity. Supporting this idea, infusion of PSA mimetic peptides in the hippocampus during memory consolidation was found to facilitate the formation of long-term spatial memory (Florian et al. 2006). In the present work, we precisely used a massed spatial training protocol in the water maze, which implies a fast acquisition phase distinctly followed by memory consolidation (Sargolini et al. 2003). To strengthen our findings in the water maze, we used another test of spatial memory, the object location task, during which acquisition is limited to the time of exposure to the objects and distinctly followed by memory consolidation. We also report intact learning and short-term memory in this task, but impaired long-term memory. Altogether, these protocols allowed us to demonstrate that the polysialylation of NCAM is required for memory consolidation and critically relies on PST activity. Noticeably, the partial presence of the PST gene in PST-het mice was always sufficient to ensure establishment of longterm spatial memory, despite reduced basal PSA-NCAM expression. We also evaluated the contribution of PST to non-spatial forms of memory. PST-ko mice showed intact long-term memory in the cued version of the Morris water maze, which highly relies on the striatum, while they displayed impaired long-term recognition memory, which depend on intact perirhinal cortex at short and long delays (Barker and Warburton 2011). Recently, Kro¨cher et colleagues also

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reported impaired long-term recognition memory in PSTko mice but in contrast to our data, they observed recognition memory impairment at a short delay (2 h) (Kro¨cher et al. 2013). Potentially increasing task difficulty, the exploration of the objects during acquisition was limited to 5 min in their paradigm, against 10 min in ours. Although further investigations are needed to understand these discrepancies, one explanation might arise from a recent study comparing transient polysialylation in the DG of individual rats after spatial learning, and showing a stronger PSANCAM expression in animals exhibiting greatest difficulty in task acquisition (Sandi et al. 2004). This suggests the need for a substantial hippocampal PSA response for the consolidation of more difficult tasks, as it might be the case between different protocols of object recognition task or between the cued water maze and the object recognition tasks. Thus, our findings provide further support to the involvement of PSA-NCAM synthetized by PST in the process that sustain spatial and non-spatial long-term memory. They also reveal that learning and short-term memory can occur without PST. Environmental enrichment enhances hippocampal PSA-NCAM expression and rescues memory deficits in PST-ko mice Numerous studies indicate that exposure to environmental enrichment can significantly attenuate memory deficits in genetically modified mice (Laviola et al. 2008; Verret et al. 2013). We therefore asked whether enrichment could alleviate long-term memory impairments in PST-ko mice. Remarkably, deficits in spatial and non-spatial forms of long-term memory were rescued by exposure to enrichment, indicating that long-term memory consolidation was made functional upon environmental enrichment. It is well documented that exposure to EE triggers a large range of morphological, biochemical, and physiological changes that facilitates hippocampus-dependent plasticity and learning in enriched animals (Rampon and Tsien 2000, 1993; Nithianantharajah and Hannan 2006). Although the present work focused on cellular changes in the dentate gyrus, it is obvious that several other mechanisms may account for the beneficial effects of EE. For instance, alterations of the synaptic plasticity in hippocampal CA1 region have been described in PST-ko mice (Eckhardt et al. 2000) whilst evidence indicate that deficits of CA1 longterm potentiation (LTP) can be restored by enriched environment (Yang et al. 2007; Morelli et al. 2014). These data raise the possibility not addressed in the present work that EE might have modulated CA1 synaptic plasticity in PSTKO mice, and thereby contributed to the improvement of memory performances, independently of adult neurogenesis. Remarkable effects of enriched experience also include

Brain Struct Funct

structural changes in the hippocampus. We examined the impact of enrichment on PSA-NCAM expression in this structure, in the absence of PST. Strikingly, the number of PSA expressing cells in the DG of PST-ko mice after enrichment, largely overpassed that of other genotypes. Moreover, the density of PSA-labeled fibers in the hilus of PST-ko mice reached PST-wt levels after environmental enrichment. In the absence of PST, PSA expression can only reflect the activity of STX in PST-ko mice. Such expression of PSA located in the somatic and axonal compartments of DG cells is characteristic of STX activity in immature neurons (Seki and Arai 1999; Schuster et al. 2001; Nacher et al. 2010) suggesting that PSA-labeled cells in the subgranular layer of the DG are in fact adult-generated neuroblasts. Interestingly, our findings after enrichment seem to refer to those of Nacher et al. (2010) reporting higher numbers of immature DCX-expressing new neurons in 3-month-old PST-ko mice compared to other genotypes. In contrast, we found that PST-ko mice raised in standard conditions, displayed intact adult hippocampal neurogenesis, including proliferation, differentiation, migration and survival. It is likely that differences existing between genotypes at 3 months of age weakened with age, due to age-related reduction of adult neurogenesis (Kuhn et al. 1996). These differences were revealed in 5- to 6-month-old PST-ko mice after the stimulation of neurogenic processes following environmental enrichment. The increase of PSA-labeled cells in the DG of PST-ko mice might be explained by the hypothesis of Nacher et al. (2010) suggesting that the maturation of new neurons might occur at a slower pace in PST-ko compared to PSTwt mice, leading to an accumulation of immature PSANCAM expressing neurons in the subgranular layer, in the total absence of PST. In the adult DG, PSA-NCAM expression corresponds to the time period when postmitotic neuroblasts extend their processes and migrate. After new neurons reach their final destination in the granular cell layer and develop into mature granule cells, PSA-NCAM expression is down-regulated (Seki et al. 2007). Our results suggest that PST activity might critically determine the timely maturation of new neurons in the adult hippocampus. During adult neurogenesis, new neurons expressing PSA-NCAM display unique functional properties such as enhanced synaptic plasticity and lower threshold for the induction of glutamatergic potentiation (Schmidt-Hieber et al. 2004). These properties make them more prone to be recruited upon hippocampal activation (Mongiat and Schinder 2011). Further investigations shall determine whether and to which extent, the accumulation of immature and highly plastic neurons in the DG of PSTko mice after enrichment supports the beneficial effect of enrichment on cognitive performances in these mice.

Acknowledgments This work was supported by grants from the EU FrameWorkProgram 6 LSHM-CT-2005-512012 (Integrated project PROMEMORIA) to C.R., by the CNRS and Toulouse University. We thank L. Roybon at Lund University, Sweden for graciously providing the GFP retroviral vector and M. Alonzo, F. Zaidi at Toulouse University 3 for their technical support. We also thank the ABC facility from ANEXPLO for housing mice. Conflict of interest

The authors declare no conflict of interest.

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