Impaired long-term memory retention: Common ...

Behavioural Brain Research 252 (2013) 275–286

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Impaired long-term memory retention: Common denominator for acutely or genetically reduced hippocampal neurogenesis in adult mice Nada M.-B. Ben Abdallah a,e,∗ , Robert K. Filipkowski b , Martin Pruschy c , Piotr Jaholkowski d , Juergen Winkler a , Leszek Kaczmarek d , Hans-Peter Lipp e,∗∗ a

Department of Molecular Neurology, University Hospital Erlangen, Germany Department of Biological Psychology, University of Finance and Management in Warsaw, Poland c University Hospital of Zurich, Switzerland d Laboratory of Molecular Neurobiology, Nencki Institute, Warsaw, Poland e Anatomy Institute, University of Zurich, Switzerland b

h i g h l i g h t s • • • •

Decreased murine adult hippocampal neurogenesis does not alter spatial learning. Cranial irradiation and cD2 KO both similarly impair long-term memory retention. Cranial irradiation and cD2 KO differently influence locomotor activity and habituation. Possible neurogenesis-unspecific brain alterations following irradiation and cD2 KO.

a r t i c l e

i n f o

Article history: Received 19 October 2012 Received in revised form 13 May 2013 Accepted 18 May 2013 Available online 25 May 2013 Keywords: Hippocampus Neurogenesis Irradiation Cyclin D2 Long-term memory Anxiety

a b s t r a c t In adult rodents, decreasing hippocampal neurogenesis experimentally using different approaches often impairs performance in hippocampus-dependent processes. Nonetheless, functional relevance of adult neurogenesis is far from being unraveled, and deficits so far described in animal models often lack reproducibility. One hypothesis is that such differences might be the consequence of the extent of the methodological specificity used to alter neurogenesis rather than the extent to which adult neurogenesis is altered. To address this, we focused on cranial irradiation, the most widely used technique to impair hippocampal neurogenesis and consequentially induce hippocampus-dependent behavioral deficits. To investigate the specificity of the technique, we thus exposed 4–5 months old female cyclin D2 knockout mice, a model lacking physiological levels of olfactory and hippocampal neurogenesis, to an X-ray dose of 10 Gy, reported to specifically affect transiently amplifying precursors. After a recovery period of 1.5 months, behavioral tests were performed and probed for locomotor activity, habituation, anxiety, and spatial learning and memory. Spatial learning in the Morris water maze was intact in all experimental groups. Although spatial memory retention assessed 24 h following acquisition was also intact in all mice, irradiated wild type and cyclin D2 knockout mice displayed memory deficits one week after acquisition. In addition, we observed significant differences in tests addressing anxiety and locomotor activity dependent on the technique used to alter neurogenesis. Whereas irradiated mice were hyperactive regardless of their genotype, cyclin D2 knockout mice were hypoactive in most of the tests and displayed altered habituation. The present study emphasizes that different approaches aimed at decreasing adult hippocampal neurogenesis may result in distinct behavioral impairments related to locomotion and anxiety. In contrast, spatial long-term memory retention is consistently altered after both approaches suggesting a plausible implication of hippocampal neurogenesis in this cognitive process. © 2013 Elsevier B.V. All rights reserved.

Abbreviations: ANOVA, analysis of variance; cD2, cyclin d2; DG, dentate gyrus; Gy, gray; KO, knockout; SEM, standard error of the mean; TLX, nuclear orphan receptor tailless gene; WT, wild type. ∗ Corresponding author at: University Hospital Erlangen, Department of Molecular Neurology, Schwabachanlage 6, 91054 Erlangen, Germany. Tel.: +49 9131 853 3581; fax: +49 9131 853 6597. ∗∗ Corresponding author at: University of Zurich, Institute of Anatomy, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Tel.: +41 44 635 5330; fax: +41 44 635 5702. E-mail addresses: [email protected], nada ben [email protected] (N.M.-B. Ben Abdallah), [email protected] (H.-P. Lipp). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.05.034

276

N.M.-B. Ben Abdallah et al. / Behavioural Brain Research 252 (2013) 275–286

1. Introduction Within the adult mouse dentate gyrus (DG) the majority of newly born cells transforms into functioning neurons with characteristics indistinguishable from older mature granule neurons within seven to eight weeks after their birth [1–3]. Adult hippocampal neurogenesis is an actively regulated process, and is severely decreased by many factors including physiological aging [4–7], irradiation [8–10], pharmacological interventions [11,12], and genetic manipulations [13]. Although the exact function of newborn neurons is still not yet fully understood, loss-andgain of function studies suggested their implication in various hippocampus-dependent cognitive and mood processes including spatial memory, contextual conditioning and discrimination, pattern separation, and anxiety [14–18]. Nevertheless, these reports bear conflicting conclusions, to the extent that some failed to detect any effect of altered DG neurogenesis on some of these hippocampus-dependent processes [19]. The exact underpinnings of such discrepancies are not known, but may be reflected by the approach used to ablate DG neurogenesis and its specificity, the experimental species and/or strains investigated, the age of the subjects at the time of intervention, the post-intervention recovery period, and the adopted functional readout procedures. There are several methods available to ablate DG neurogenesis, of which administration of antimitotic drugs, exposure to irradiation, and mutational approaches are the most commonly used. Considering the known side effects of antimitotic drugs in rodents, including sickness, body weight loss, passiveness, and muscular atrophy [20,21], which all may interfere with behavioral performance, we focused on cranial irradiation in combination with genetically-induced alteration of DG neurogenesis for several reasons, bearing in mind its clinical application in treating systemic and intracranial neoplasms [22]. Compared to antimitotic drugs such as methylazoxymethanol and temozolomide, cranial irradiation is preferably utilized for its temporal and spatial specificity [23], as well as for its minimal overt side effects [21]. In addition, whole-brain irradiation of adult mice with doses ranging between 4 and 15 Grays (Gy) is sufficient to decrease hippocampal neurogenesis almost by 80% of sham-irradiated neurogenic hippocampus [24–26]. This dramatic decrease is the result of impaired cell proliferation with transiently amplifying progenitors being the most sensitive to irradiation [27]. However, despite the wide use of irradiation in studies addressing the functional importance of adult hippocampal neurogenesis, this technique has been met with several critiques questioning the extent of its effects on other regions juxtaposing the hippocampus shedding the light on whether this may have contributed in one way or the other to the behavioral output (s) previously reported [21]. We reasoned that if indeed whole-brain irradiation induces specific behavioral impairments solely dictated by decreases in adult hippocampal neurogenesis then exposing animals that constitutively lack adult hippocampal neurogenesis to high-energy X-rays should not lead to any behavioral changes previously associated with adult hippocampal neurogenesis. One likely model to serve the purpose of our reasoning and that bears virtually no detectable hippocampal neurogenesis at adult stages is the cyclin D2 knockout mouse model (cD2 KO). We have previously reported a complete lack of adult neurogenesis in both the olfactory bulb and the hippocampus of cD2 KO mice [28]. Within the DG, neurogenesis (BrdU+ DCX+ neurons) decreased by 90% compared to WT littermates [29], most likely due to decreased cell proliferation [13,29–31]. Indeed, cD2 is an important regulator of cell-cycle progression and the only D-cyclin member known to be expressed in adult neuronal precursors [13]. Additionally, external factors known to consistently increase the rate of hippocampal neurogenesis such as exposure to running wheels and enriched environment failed to induce any

detectable changes in cD2 KO mice [28], advancing this mouse model as constitutively and consistently devoid of physiological detectable levels of hippocampal neurogenesis. We therefore exposed a group of adult wild type (WT) and cD2 KO mice to whole brain X-irradiation (10 Gy), and, after a sixweek period during which radiation-induced inflammation is due to subside, we compared their behavioral performance to shamirradiated WT and cD2 KO littermates. Such approach permitted thus the standardization of the experimental setup, by controlling for strain, gender, and age, among other factors otherwise leading to experimental variability, and allowing as such a direct evaluation of two different approaches frequently used to decrease hippocampal neurogenesis in rodents. We particularly characterized behavior in paradigms for locomotor activity, anxiety, and cognitive tasks, with specific focus on hippocampal-dependent tasks. Our methodological approach aimed firstly at highlighting the specificity of whole-brain irradiation in the context of adult hippocampal neurogenesis studies, and secondly at highlighting common and distinct alterations using two different methods both ensuing decreased hippocampal neurogenesis, thus addressing the specificity of each method. In parallel, our approach allowed us to address in parallel the role of adult hippocampal neurogenesis in primarily hippocampus-dependent processes. 2. Materials and methods 2.1. Animals In total, 28 cD2++ and cD2−/− female mice, all aged between 4 and 5 months, were used in the present study. In particular, 14 adult C57BL/6J X BALB/c mice devoid of the cD2 gene and 14 adult WT littermates [13,28]. The cD2 KO mice were obtained in the 129X1/SvJ background and crossed once with the C57BL/6J strain, then kept and bred continuously with each other as heterozygotes for >15 generations [13]. These mice were then crossed once with BALB/cAnNCrl mice, then kept and bred continuously with each other as heterozygotes for >10 generations. Their homozygous progeny [−/− (cD2 KO) and ++ (WT)], that were always littermates derived from several breeding pairs, were used in this study. Unlike male mice that show high levels of aggression when group-housed, females are tolerant to group housing, and were thus particularly chosen for our study due to the requirement of social housing for the recovery period to avoid long-term isolation stress and its deleterious effects on hippocampal neurogenesis [32], and the intellicage experiments where mice were required to be housed in large groups. Mice were kept in social groups of approximately four mice in standard mouse cages, in a temperature- and humidity-controlled room, under a reversed 11/13 h light and dark cycle. All mice had free access to laboratory animal diet and water. Handling and testing of the mice were performed during the dark cycle. All experimental procedures were conducted in accordance with the Swiss animal welfare guidelines and approved by the cantonal veterinarian office of Zurich, Switzerland. 2.2. Radiation procedure Prior to irradiation, all mice (sham and irradiated) were anaesthetized with an intraperitoneal injection of combined Ketamine (50 mg/kg Narketan® 10, Vétoquinol, Bern, Switzerland) and Xylazin (10 mg/kg, Streuli, Uznach, Switzerland). Irradiation was performed using an irradiation device (RT100 Philips). In order to ensure comparable radiation exposure between animals, anaesthetized mice were fitted in a radiation custom-made frame that allows the exclusive irradiation of the brain, while eyes, ears and body remain shielded with a 6 mm thick plate of lead. Irradiated mice received a single dose of 10 Gy at a rate of 1.75 Gy/min emitted through a 20 cm × 10 cm field size. Exposure to such X-ray dose has previously been shown to induce long-term reduction of DG cell proliferation and neurogenesis in adult C57BL/6J mice [24,26]. Sham-irradiated WT and cD2 KO mice were handled and anaesthetized similarly to irradiated mice only did not receive any X-rays. Mice were thus assigned to four groups consisting of sham-irradiated WT and cD2 KO mice, and irradiated WT (WT-10 Gy) and cD2 KO (cD2 KO-10 Gy) littermates (with 7 animals per group). 2.3. Behavioral testing 2.3.1. Experimental timeline After exposure to irradiation, all mice, kept in their social groups, were given approximately six weeks of recovery period, after which newborn neurons with a potential to integrate into the existing circuitry should be absent in the irradiated mice [24,26]. Moreover, this recovery period is suggested to allow radiation-induced inflammation to subside [27,33]. During this time, the body weight was monitored

N.M.-B. Ben Abdallah et al. / Behavioural Brain Research 252 (2013) 275–286 every 2–3 days. Thereafter, except for the intellicage experiments, mice were housed in single cages one week prior to the start of behavioral testing which consisted of automated monitoring of home cage activity in the ActiviscopeTM system, followed by burrowing test, open field, elevated null maze, Morris water maze. 2.3.2. Video tracking During exploration tests as well as during the Morris water maze, a video camera was suspended directly above the testing arenas, and video tracks of the animals were recorded using a computer-assisted Noldus Ethovision 3.0 system. Data of the experiments were then transferred to public domain software Wintrack 2.4.50109 for further analyses (www.dpwolfer.ch/wintrack; [34,35]). 2.3.3. Activiscope Mice were kept in individual cages in one single rack and the assessment of daily activity was carried out by means of an automated system that used small passive infrared sensors positioned on top of each cage (ActiviscopeTM , NewBehavior AG, Zurich, Switzerland, http://www.newbehavior.com). The system was operated continuously throughout one week. The sensors detected every movement of the mice and transmitted the signals through an interface to a recording computer with dedicated software. No movements were detected by the sensors when mice were sleeping, inactive, or performed moderate self-grooming. An additional sensor was installed in proximity of the experimental rack containing the cages, for recording human activity in the animal room. Scores were processed and counts were cumulated over one-hour bins in order to produce a 24 h profile. The access of the personnel to the animal room was not restricted and followed the routine schedule (between 08:00 and 11:00 a.m.). 2.3.4. Burrowing test Burrowing is a spontaneous species specific behavior displayed particularly by rodents and considered to be mediated by the hippocampus and associated to some extent with anxiety and possibly depression [36–38]. In mice, the burrowing test proved to be sensitive to strain differences [39], where both C57BL/6J and BALB/c are good burrowers. To evaluate the effect of irradiation and/or knockout of cD2 gene on burrowing behavior, mice were housed separately in a large standard home cage (40 × 24.5 × 15 cm) devoid from food and equipped only with a cardboard box, mouse bedding, and a bottle of water. During the test, a gray plastic tube (diameter 6.3 cm and length 18.2 cm) filled with 350 g of the usual food pellets was introduced into the home cage of each mouse, at 10 a.m. (during the active phase). The amount of food pellets displaced from the tube (i.e., burrowed) was calculated 5 h and 24 h after presenting the tubes by subtracting the weight of pellets remaining in the tube from the initial pellets’ weight. 2.3.5. Open field test The open field test is designed to assess the locomotor activity, exploration, as well as habituation of rodents when forced to explore a new environment. Exploration permits mice to collect information in a new environment, and from which information about emotional behavior related to anxiety could be inferred. The arena of the open field (Fig. 2) consists of a plastic platform placed horizontally at the bottom of a circular pool (150 cm in diameter, 50 cm high), surrounded by opaque walls (35 cm high). The arena is dimly illuminated by four 40 W bulbs. The test consists of two sessions of 10 min each, performed on two consecutive days. At the beginning of each session, a mouse is released close to the sidewall into the open field. The arena is cleaned with 70% ethanol after each animal to avoid any olfactory interference. Recorded tracks are segmented into three motion states: (1) progression episodes, which correspond to long-distance locomotion (>5 cm) and a > 8.5 cm/s velocity. (2) Resting episodes which last 2 s or longer, and include periods of immobility as well as grooming. (3) Scanning episodes which correlate to exploratory behaviors such as brief stopping, sniffing, stretch-attend postures, rearing, and leaning against the open field wall. To assess exploration, the arena is virtually divided into three concentric zones: (1) exploration zone located in the center and composing 50% of the arena surface, (2) wall or home zone corresponding to 18% of the arena, and (3) intermediate zone in between the first two occupying 32% of the total arena. Parameters measured in the open field test are mentioned in more details in Madani et al. (2003; [34]), and include path turtuosity, percent of time spent in each zone, percent of time of each motion state (resting, scanning, and progressing), and the number of fecal boli after each session. Additionally, stereotypy index inferred from the pattern of locomotion is measured. Briefly, a sequence of visits to 5 × 5 cm quadratic tiles is scanned for repeated patterns. Each repetition increments the stereotypy counts of all involved tiles. Stereotypy counts of all tiles are summed and reflect the stereotypical pattern of locomotion. 2.3.6. Elevated O-maze Response to aversive environments is an important aspect of the behavioral investigation of mutant mice and can reflect altered emotional behaviors such as anxiety which has been linked to altered hippocampal function. One such test is the elevated O-maze test which is a modified version of the elevated plus-maze test. For the elevated O-maze (46 cm in diameter), the arena consists of a 5.5 cm wide circular plastic runway, elevated 40 cm above the floor (Fig. 3A; [34]. The maze is divided into two sectors protected by 16 cm high walls (closed sectors), which are

277

separated from one another by two bare sectors surrounded by no walls (open sectors). Typically, mice at the beginning of the test tend to spend more time in the safe sectors, and less in the open sectors. This tendency will subsequently decrease throughout the session reflecting habituation. During the test, mice were released in one of the closed sectors and were observed and data were recorded for a total of 10 min. During the analysis, we defined three zones corresponding to exploration zone (open sectors, composing 39% of the arena surface), home zone (closed sectors, corresponding to 28% of the arena’s surface), and intermediate zone (transition between closed and open sectors, covering about 33% of the arena). Head dips and body stretches were registered manually and by the video-tracking system, and depending on the location from where they are performed, were classified as protected (from intermediate zone) or unprotected (from open sectors). Measures of overall activity included number of closed sector entries, total distance moved and time spent resting. As for anxiety-related measures, we determined entries to and time spent in open versus closed sectors, and the number of fecal boli. 2.3.7. Morris water maze Hippocampal dependent spatial learning and memory were assessed using a modified version of the reference water maze paradigm [40]. Lesions to the hippocampus are known to disrupt place navigation [41]. The paradigm is additionally sensitive to genetic changes which reduce behavioral flexibility, disrupt exploratory behavior or affect motivation. The arena consists of a circular pool (Fig. 4A; 150 cm in diameter, 50 cm high), virtually divided into four quadrants corresponding to the different possible locations of the hidden platform. Mice were thus assigned to different groups depending on the location of the platform they have to acquire (target) in order to avoid a location preference bias. The remaining three quadrants corresponded to control quadrants. To evaluate spatial learning, mice were introduced from random locations into the water maze and had to locate a submerged square platform (14 × 14 cm) which was placed in the quadrant corresponding to their assigned group. The water was made opaque by adding 1l of milk, and was held at a temperature of 24 ◦ C. The room was illuminated indirectly by four 40 W bulbs, and its walls provided extramaze cues including posters and shelves, in addition to the rack holding the animals’ cages, which all served as spatial cues to locate the hidden platform. After successfully locating the platform, mice were allowed to stay on it for 15 s. However, if a mouse failed to find the platform after 120 s, it was directed to and placed on it by the experimenter. Mice were trained to find the platform over four consecutive days, with six trials per day at approximately 30 min intertribal interval, and 120 s maximum time per trial (acquisition phase). Long-term memory was assessed during probe trial sessions, when the hidden platform was removed, and the same mice were returned to the pool at 24 h, 1, 2, and 3 weeks after training, and were allowed to swim freely for 120 s. The amount of time spent in the quadrant where the platform was previously located (target quadrant) relative to the averaged values from the other three quadrants (control quadrants) as well as the number of target crossings were used to evaluate long-term memory. 2.3.8. Intellicage The Intellicage system consists of four operant chambers that fit into the corners of a standard rat housing-cage that contained sleeping shelters and food made accessible ad libitum (NewBehavior AG; http://www.newbehavior.com; [42–44]). Mice were assigned to two different cage groups (14 mice of all experimental groups per cage), and were individually tagged by subcutaneous transponders that can be identified through antennas located at the entry of each operant chamber. Moreover, each operant chamber contains two sides operated with automatically controlled sliding doors that are equipped with a nose-poke sensor/recorder, permitting access to drinking bottles at each nose-poke action. The system is controlled by a microcomputer recording individual visits, nose-pokes, tube-lickings, as well as delivering reward (consisting of access to drinking water after a nose-poke) or aversive stimulus (consisting of air puffs delivered through air valves located within the chambers) based on programmed schedules to which each mouse is assigned. Activity was assessed during an initial adaptation session by using as an index of the total number of visits and nose-pokes. Moreover, we assessed the activity at the initial introduction into the Intellicage during the first hour, and extracted the latency to perform a first visit with a nose-poke action, that would reflect reactivity to novelty. Learning was evaluated in subsequent place learning modules. Mice were given the task to locate the only correct corner out of all four corners. An initial place learning session of about three days was followed by the same place learning only this time each incorrect visit initiated the delivery of an aversive stimulus consisting of an air-puff. It has been postulated that learning this task is dependent on an intact hippocampus [45]. Place learning schedule was followed by a reversal place learning during which the correct corner assigned to each mouse was relocated to the opposite corner, previously counted as incorrect. Learning chance level corresponded to 25% in both place learning and reversal place learning. To evaluate learning capacities, the percentage of correct visits out of the total number of visits was calculated. 2.3.9. Statistical analysis In all statistical comparisons, one- or two-way ANOVA was performed. Differences between different experimental groups were considered significant when the P-value was 0.05). During a period of one month following irradiation, we measured body weights regularly every three days and did not observe any differences between our experimental groups (data not shown). However, body weights steadily increased over the one-month period in both WT and cD2 KO mice regardless of irradiation (F(1,264) = 6.8; P < 0.001). Body weights as well as brain weights were both significantly decreased in cD2 KO mice compared to WT mice at the time of perfusion when mice were around 30 weeks of age (Fig. 1B; body weight: F(1,18) = 14.1; P < 0.001; Fig. 1C; brain weight: F(1,18) = 72; P < 0.001). Irradiation did not alter body or brain weights at any time points (data not shown). 3.1.2. Activity When initially placed in the Activiscope to measure their activity and rhythmic cycle, irradiated mice displayed reduced activity compared to sham-irradiated mice during the first two days (Factorial ANOVA; day 1: F(1,23) = 4.1; P < 0.05; day 2: F(1,23) = 13.1;

P < 0.001), with no effect of genotype (P > 0.05). This reflects acute alteration of reactivity to novel contexts. However, all mice displayed comparable activity in the Activiscope cages. Despite the body weights differences, evaluation of overall average activity (Fig. 1D) revealed no differences between WT and cD2 KO mice with and without irradiation (P > 0.5). Furthermore, all experimental groups showed undisturbed circadian rhythm as well (P > 0.05), with significantly lower activity during the light phase and increased activity during the dark phase. Observation of animals in their home cages did not reveal any overall health problems, suggesting that all mice were suitable for further behavioral experiments. 3.2. Burrowing test Irradiation did not alter burrowing behavior (Fig. 1E) neither in WT nor in cD2 KO mice at either 5 h or 24 h time points (F(1,23) = 1.1; P > 0.05). Moreover, both WT and cD2 KO mice displaced equal amounts of food pellets from the tubes after 5 h regardless of irradiation (F(1,23) = 0.01; P > 0.05). Interestingly, sham-irradiated cD2 KO mice displayed decreased burrowing behavior compared to their WT littermates 24 h after the initial introduction of the burrow tube (F(1,12) = 4.7; P < 0.05). Irradiated cD2 KO mice however did not show the same difference compared to WT littermates (F(1,11) = 0.7; P > 0.05).


279

Fig. 2. Performance of the mice in the open field during two 10 min sessions over two consecutive days. Overall, all mice displayed a remarkable preference to the home wall zone compared to the exploratory center, as observed in the percentage of total time spent in the home zone compared to the open sectors (A), and the increased percentage of vertical movement performed from the home zone (B). cD2 KO mice displayed a prominent stereotypical pattern as shown by the stereotypy index (C). We observed also a significant tendency toward more zone visits in cD2 KO mice (D). Moreover, KOs traveled longer distances (E), and spent more time progressing (F). Finally, a depiction of how habituation is altered in cD2 KO mice is shown over four time bins of 5 min each in (G) with increased distance traveled while moving and (H) with decreased vertical movements performed from the home zones. Error bars correspond to standard errors of the mean. ‘˛’ represents comparisons to WT-sham mice and corresponds to P < 0.05; ‘ˇ’ represents comparisons to wall zone measures and corresponds to P < 0.05.

3.3. Open field test In the open field, we investigated the locomotor and exploratory activities of mice and inferred from these measures their anxietylike behaviors. All mice displayed zone preference throughout the test in the open field with more time spent in the wall zone and less time in the center (Fig. 2A; F(1,48) = 400; P < 0.001), and with exploration focalized mainly in the wall zone as reflected by the percentage of vertical movements in the different zones of the arena, i.e., wall, transition, and center (Fig. 2B; F(1,48) = 216.4; P < 0.001). We observed an overall tendency of all cD2 KO mice to display a repetitive “stereotypic” pattern of locomotion in the arena more often than their WT littermates (Fig. 2C; F(1,24) = 4.8; P < 0.05). In addition, the number of visits to the three zones of the arena was significantly higher in cD2 KO mice (Fig. 2D; F(1,24) = 7.2; P < 0.05).

Further analysis using ANOVA post hoc test (Bonferroni/Dunn) revealed significant differences between cD2 KO mice and WT mice (P < 0.05), while no radiation effect was observed (P > 0.05). Also, cD2 KO mice traveled longer distances while progressing (Fig. 2E; F(1,24) = 7.5; P < 0.05) and spent more time progressing compared to their WT littermates (Fig. 2F; F(1,24) = 6.8; P < 0.05). The distance traveled while in the intermediate zone was longer in cD2 KO mice than in WT mice (F(1,24) = 4.5; P < 0.05), regardless of exposure to irradiation. We analyzed habituation over the two days of the open field test, and observed no significant differences between experimental groups, with all animals displaying intact adaptation (data not shown). However, when looking at the within-session habituation over 5 min bins each day, cD2 KO mice increased the percentage of time spent progressing in the arena (F(1,24) = 6.8; P < 0.05) and decreased the number of vertical movements in the wall zone

280


Fig. 3. Performance on the elevated O-maze during a single 10 min session. (A) Picture of the elevated O-maze with delineation of the respective zones and sectors. Irradiated mice display significantly increased speed while progressing compared to their sham-irradiated counterparts (B). Similarly to the open field, all mice displayed a significant preference to the home protected zone compared to the exploratory open sectors, as observed in the percentage of visits to zones (C), and the reduced percentage of head dips performed from the open sectors (D). Interestingly, KO mice displayed a significantly decreased latency to perform a first progressive movement (E). On the other hand, irradiation decreased the latency to make the first vertical movement (F), and first head dip (G) in both WT and KO mice. The total path traveled while progressing on the arena was also significantly increased in irradiated mice (H). Error bars correspond to standard errors of the mean. ‘˛’ represents comparisons to WT-sham mice and corresponds to P < 0.05; ‘ˇ’ represents comparisons to home zone measures and corresponds to P < 0.05.

(Fig. 2H; F(1,39) = 14.9, P < 0.001). We also observed a significant increase in both progression time and distance over 5 min time bins from day 1 to day 2 of the test in cD2 KO mice, both irradiated and sham-irradiated (Fig. 2G; P < 0.05). In contrast, all WT mice showed habituation to the arena as observed on day 2 between first and second 5 min time bins (P < 0.05). In addition, when comparing the amount of fecal boli separately on test days 1 and 2, we did not observe any significant differences between any of the experimental groups (P > 0.05). However, when comparing the amount of boli between days 1 and 2, another measure reflecting habituation, only WT mice displayed a significant increase in boli numbers, both sham-irradiated (F(1,5) = 11; P < 0.05) and irradiated (F(1,6) = 8.6; P < 0.05). In contrast, no significant increase over days was observed in sham-irradiated cD2 KO mice (F(1,7) = 3.9; P > 0.05) and irradiated cD2 KO mice (F(1,6) = 0.4; P > 0.05). We did not observe an effect of irradiation on any of the variables measured in the open field test neither in WT nor in cD2 KO mice (data not shown).

3.4. Elevated O-maze A 10-min session in the elevated O-maze (Fig. 3A) is sufficient to reflect locomotor/exploratory activity, anxiety-like behaviors, and habituation. We first looked at performance of mice overall the 10min session, and observed no changes in the number of fecal boli between any of the experimental groups (data not shown). All mice, regardless of genotype and irradiation, displayed a zone preference with more specific increase in the velocity of movement in the transition zone versus significantly reduced speed of movement when performing exploratory behaviors or scanning compared to when progressing on the arena (Fig. 3A; F(1,19) = 570; P < 0.001). Significantly increased movement speed was further observed in irradiated WT and irradiated cD2 KO mice when compared to their sham-irradiated counterparts (Fig. 3A; F(1,19) = 6; P < 0.05). Similar results were observed in the percentage of visits to zones (Fig. 3C; P < 0.001) and the percentage of head dips in zones (Fig. 3D; P < 0.001) where all mice showed preference to the home


281

Fig. 4. Performance of the mice in the water maze task during the acquisition phase. Measures are averages of all 6 trials per each day. (A) Schematic depiction of the water maze arena and the paradigm of task acquisition and memory retention. (B) Latency (s) to find the hidden platform. (B) Distance (m) moved to find the platform. (C) Cumulative search error (m × s) corresponding to the average distance to goal multiplied by time to goal. (D) Percentage of time spent close to the walls. Error bars correspond to standard errors of the mean.

zone compared to exploration zones. Interestingly, irradiated WT and irradiated cD2 KO made more visits to the exploration zone (displayed by higher percentage of their total visits) compared to sham-irradiated mice (Fig. 3C; P < 0.05). When looking in particular on activity and exploration, we noted that both irradiated and sham-irradiated cD2 KO mice were the first group to show a progressive locomotion on the arena (Fig. 3E; F(1,19) = 6; P < 0.05), with no significant irradiation effect. Irradiation on the other hand had a strong effect on performance on the elevated O-maze arena, independently of genotype. Both WT and cD2 KO irradiated mice performed their first vertical movement in the intermediate zone after a significantly shorter latency (Fig. 3F; F(1,19) = 8.4; P < 0.01) in comparison with shamirradiated mice. Although there were no significant changes in the number of head dips, irradiated WT and irradiated cD2 KO mice made their first head dip from the intermediate zone significantly earlier than their sham-irradiated WT and cD2 KO littermates

(Fig. 3G; F(1,19) = 6.6; P < 0.05). In addition, they progressed over longer distances (Fig. 3H; F(1,19) = 9.1; P < 0.01), and at higher velocity (F(1,19) = 5.4; P < 0.05). Irradiated mice also tended to spend more time moving on the arena (F(1,19) = 4.1; P = 0.05), and spent their resting episodes at a close distance to open sectors of the arena (F(1,19) = 4.7; P < 0.05). We used factorial ANOVA to compare between performances of irradiated and sham-irradiated cD2 KO mice in an attempt to evaluate behavioral alterations that may be induced via brain modifications other than impaired DG neurogenesis. Irradiation of cD2 KO mice resulted in significant alterations in their performance on the elevated O-maze, when compared to their sham-irradiated cD2 KO counterparts, with an increase in numbers of excursions between zones (P < 0.05), decreased latencies to first visible move to the exploration zone (open sector) (P < 0.01), first visible vertical movement (P < 0.05), and increased velocity while moving on the arena, though not statistically significant (P = 0.05).

282


To evaluate habituation, we divided the analysis of the session into four time bins of 150 s each. Repeated measures ANOVA reflected no significant effects of irradiation or genotype on any of the measures, and all mice displayed normal habituation to the arena throughout the session, particularly showing decreases in their number of vertical movements (P < 0.001). In contrast, irradiated WT and irradiated cD2 KO mice displayed significantly increased total path length while progressing throughout the 150 s time bins (P < 0.01). 3.5. Morris water maze During the learning acquisition phase (Fig. 4A), mice searched for the hidden platform at similar speeds (data not shown). All mice acquired the water maze task equally well regardless of treatment and genotype (Fig. 4B; F3,69 = 1.8; P > 0.05), with a significant decrease in search latencies over consecutive days (P < 0.001) as well as the path swam to find the hidden platform (Fig. 4C; F3,69 = 36.0; P < 0.001). No genotype or irradiation effect was observed in the cumulative search error index and the percentage of time spent close to walls (Fig. 4D and E, respectively; P > 0.05 for both). During the first probe trial on day five (Fig. 4A), 24 h after the last acquisition trial, all mice displayed a significant preference for the trained quadrant in comparison with the three remaining control quadrants (65%; P < 0.001), with no genotype or irradiation effects (Fig. 5A; F3,23 = 1.3; P > 0.05). However, irradiated cD2 KO mice made significantly less crossings over the trained quadrant compared to their WT littermates (Fig. 5B; F1,23 = 8.1; P < 0.01). In addition, cD2 KO mice displayed a lower swim speed (P < 0.05). We also observed more crossings of the left quadrant adjacent to the trained quadrant, made by irradiated mice (P < 0.05), thus reflecting a decreased spatial search accuracy. We evaluated spatial memory retention one week after the last acquisition trial in the same animals, and found significant group differences, with the irradiated WT as well as both irradiated and sham-irradiated cD2 KO mice displaying less preference to the target quadrant than their sham-irradiated WT littermates (Fig. 5A and B; F3,23 = 5.0; P < 0.01). In addition, they made fewer crossings over the target quadrant (Fig. 5B; Pgenotype < 0.05; Pirradiation < 0.001; Pgenotype X irradiation < 0.05). All cD2 KO mice and irradiated WT mice were at a bigger distance from the trained goal than their nonirradiated WT littermates during most of the search time (P < 0.05 and >0.05, respectively). In the subsequent probe trials (two and three weeks after acquisition), sham-irradiated WT mice reduced their preference for the trained goal, corresponding to extinction of acquired long-term memory. We did not however observe any genotype or treatment effects (P > 0.05). Two weeks after acquisition however, sham-irradiated cD2 KO mice spent more percentage of their time swimming close to the walls (P < 0.05). In the last probe trial, three weeks after the acquisition phase, genotype and treatment had equally affected the activity of the mice (P < 0.05). Fig. 5C depicts in details the deficits in long-term memory retention, at a 30 s time bins within each probe trial. It shows that deficits are most remarkable during the one-week probe trial at 60 s, 90 s, and 120 s where irradiated WT and cD2 KO as well as sham-irradiated cD2 KO mice showed less preference to the trained quadrant compared to sham-irradiated WT mice (P < 0.01). This impairment was also observed during the first 30 s of the two-week probe trial (Fig. 5C; P < 0.05). 3.6. Intellicage In an attempt to evaluate activity and learning capabilities in a semi-naturalistic environment that mitigates experimenters’ interventions and testing-induced stress often occurring in

Fig. 5. Probe trials at 24 h, 1, 2, and 3 weeks after the acquisition phase. Significant preference to the trained quadrant was observed in all experimental groups, with the percentage of time spent in the trained (target) quadrant significantly higher than chance level (25%; dashed line) and from the time spent in control quadrants (control) (A), and the target crossings significantly higher than the number of crossings of control quadrants (B). However, cD2 KO mice and irradiated WT mice displayed significantly less preference on probe trial one-week in comparison to their sham-WT littermates. (C) Percentage time in quadrant over two-minute trials are divided into four time and reveals a significant differences in the accuracy to the trained quadrant in irradiated WT and both irradiated and sham-irradiated KO mice. Error bars correspond to standard errors of the mean. ‘˛’ represents comparisons to WT-sham mice and corresponds to P < 0.05; ‘ˇ’ represents comparisons to control locations measures and corresponds to P < 0.05.

afore-employed tests, we designed a set of paradigms to be performed in the Intellicage system. During a habituation session of three days, mice displayed comparable activity as shown by the total number of visits to the operant chambers (Pgenotype > 0.05, Pirradiation > 0.05). However, cD2 KO mice made significantly less visits to the Intellicage corners during the first 60 min after the initial introduction into the system (Fig. 6A; F(1,24) = 8.7; P < 0.01), regardless of exposure to irradiation (F(1,24) = 0.2; P > 0.05), with no interaction between genotype and irradiation (P > 0.05). This reflects a certain novelty-induced hypoactivity in cD2 KO mice. Moreover, we observed a significant effect of genotype and irradiation on the latency to make the first visit with a nose-poke, with all


283

the performance, and all mice learned the task throughout training (F(1,24) = 42.0; P < 0.001). 4. Discussion We investigated the relevance of new DG neurons in hippocampal function by combining whole brain irradiation and knockout of cD2 to alter adult neurogenesis. By irradiating cD2 KO mice that are constitutively devoid of adult neurogenesis, we could additionally address radiation-related functional sequelae that are mediated by brain modifications independently of DG neurogenesis. We report that mice with significantly decreased DG neurogenesis induced either by irradiation or knockout of cD2 are able to adequately learn different hippocampus-dependent tasks, as evidenced by their successful acquisition of the water maze paradigm, and the place and reversal place learning in the Intellicage. We also note that reduction of DG neurogenesis either acutely by irradiation or chronically by constitutive ablation of cD2 leads to comparable hippocampus-dependent memory deficits in the Morris water maze, reflected by less preference to the trained quadrant during probe trials separated by over one-week from acquisition. 4.1. Behavioral impairments induced by cranial irradiation independently of DG neurogenesis

Fig. 6. Activity and learning in the Intellicage. (A) The total number of visits during the first hour of initial adaptation session showing a significantly reduced activity of KO mice. (B) The latency to perform a nose-poke was significantly longer in KO mice. (C) The total number of correct visits in non-punished and punished sessions of the corner place learning schedule. Note a significant genotype effect during the first day of the session, only upon the use of air punishment where KO mice perform significantly poorer. Error bars correspond to standard errors of the mean. ‘˛’ represents comparisons to WT-sham mice and corresponds to P < 0.05.

cD2 KO mice exhibiting longer latency (Fig. 6B; F(1,24) = 6.5; P < 0.05) while all irradiated mice scored shorter latencies (F(1,24) = 0.03; P < 0.05). Mice had to acquire place learning during corner place learning and reversal place learning schedules. In the first phase of place learning, mice could make errors without receiving any air puffs as punishment; in the second phase, every committed error was followed by an immediate air puff. We did not observe any genotype or irradiation effects on the percent of correct visits out of total visits overall the entire session (P > 0.05). However delivering air-puffs at each error improved significantly the performance of all mice (Category for air-puff: F(1,24) = 37.3; P < 0.001). Interestingly, during the first day of place learning paradigm with air puff, cD2 KO mice did not improve their performance compared to (Fig. 6C; F(1,24) = 10.0; P < 0.01), and showed an altered place learning compared to WT littermates regardless of irradiation. During the reversal place learning session, when the correct corner was now at the opposite side, we did not observe any group effect on

Our primary question was to explore the behavioral alterations induced by irradiation that are not related to DG neurogenesis and that might be a confounding factor in the context of exploring the functional relevance of DG neurogenesis using this technique. Irradiated WT mice exhibited hyperactivity in the elevated null maze and at the beginning of both sessions of the open field. No previous reports exist on any effects of irradiation on spontaneous motor activity [23,46], which might be resulting from discrepancies in experimental design, e.g., the species or mouse strain used, as well as the radiation protocol adopted. Nonetheless, we may suggest that such activity changes result from other neural systems affected by this technique. On the other hand, we observed a significant change in exploratory parameters in the elevated O-maze, where both irradiated WT and irradiated cD2 KO mice displayed shorter latencies to explore the arena and moved at longer distances. Since sham-irradiated cD2 KO mice did not display such behavior, we suggest that this may be the result of irradiationinduced brain alterations unrelated to hippocampal neurogenesis. In contrast, we observed alteration of habituation to novel environment in the cD2 KO mice but not irradiated WT mice. Moreover, cD2 KO mice exhibited hypoactivity when placed in a novel environment. Although activity normalizes when allowed longer time of exploration, this initial activity impairment is seemingly a feature specific for the cD2 KO model, since other mouse models where neurogenesis was genetically ablated do not show similar hypoactivity [47], nor those in which DG neurogenesis was altered by other means [25,48,49]. 4.2. Cognitive outcome consequential to alterations of DG neurogenesis Spatial long-term memory and neurogenesis cellular markers are concomitantly increased in both adult and senescent mice housed in an enriched environment [50] or with access to a running wheel [51]. A substantial number of reports have suggested that decreased DG neurogenesis following low doses of radiation is associated with a variety of hippocampus-dependent learning and memory impairments [26,52]. Other cognitive processes implicating DG neurogenesis include contextual fear conditioning [53,54] and spatial memory retention [52,55,56].

284


When administered at low doses, irradiation affects almost exclusively transiently amplifying precursors [27]. Accordingly, cranial exposure to low X-ray doses is reported to decrease cell proliferation in the DG by 95% at doses exceeding 1 Gy [27], in the absence of any obvious histological or physiological changes [22,57,58]. Previous studies also reported a long-term radiationinduced reduction of neurogenesis [27,33]. Using a single dose of 10 Gy, reported to result in significant decrease of adult DG neurogenesis exceeding 75% and persisting for over 3–4 months after irradiation in adult mice [24,26], we noted intact learning skills in irradiated WT mice compared to their sham-irradiated littermates, in various paradigms, including spatial learning in the water maze, and place learning in the Intellicage. We could therefore reproduce previous findings suggesting that DG neurogenesis does not play an essential role in spatial learning in the water maze task. In addition, we noted a radiation-induced effect on the extent of long-term memory retention in the spatial reference memory paradigm of the water maze. This is supported by previous findings where memory retention was altered in irradiated rats [56], mice [26,48], and gerbils [55]. Based on accumulating evidence reporting a relation between these cognitive changes and altered DG neurogenesis, we suggest that our results are related to a significant reduction in DG neurogenesis. This has been also suggested in an earlier study where cell proliferation and neuronal differentiation were abolished in rats by the cytostatic drug methotrexate, resulting in longer latency to cross the trained goal during probe trials compared to controls [49]. Gene targeting is another valid new approach used to alter adult neurogenesis. Genetically modified mice including transgenic and KO models identified several genes specifically involved in the process of adult neurogenesis [59]. The cD2 KO mice are one such model that additionally bears architectural changes including smaller olfactory bulb, hippocampus, cerebellum, and sensory cortex [13]. These mice are characterized by almost complete absence of newly born neurons in both the DG and the olfactory bulb [13]. We found that cD2 KO mice could learn adequately hippocampal and non-hippocampal paradigms, including the spatial reference memory task in the water maze, and the place and reversal place learning in the Intellicage. Indeed we and others have previously reported intact learning abilities in cD2 KO mice in various hippocampus-dependent paradigms including the Morris water maze [29], the trace fear conditioning [29] and the Barnes maze [60]. The lack of impairment in spatial learning despite the dramatic decrease of DG neurogenesis could only be explained by the fact that adult DG neurogenesis could be dispensable in spatial learning processes. In no way could we be uncertain of the long-term irreversible decrease of neurogenesis in cD2 KO mice despite the extensive behavioral training. Indeed it has been shown that activity and novelty induced by physical exercise and environmental enrichment, respectively, do not restore DG neurogenesis [13]. In terms of memory retention, we observed that cD2 KO mice exhibit significantly lower preference for the trained target, as soon as 24 h after acquisition, slightly earlier than irradiated WT mice. We have not observed such impairments in our previous study [29]. Such discrepancy may be the result of several factors. For example, here we used females solely whereas in 2009 we used both males and females. We also have only tested memory retention on one probe trial administered 24 h after the last acquisition trial. Zhang et al. have used a TLX conditional KO mouse model in which the nuclear orphan receptor tailless gene was deleted in adult neural stem cells, leading to advanced reduction of hippocampal cell proliferation and neurogenesis. They reported that memory retention is only affected 12 h after the acquisition phase but not three weeks after [47]. Despite their interpretation that only shortterm memory is affected, their findings are in agreement with ours, since we noted reduced crossing of the trained goal in the cD2 KO

mice as early as 24 h after acquisition, but no deficits were observed after two weeks, at which time the WT controls displayed memory extinction. We should also note that the TLX mouse model displays a pronounced reduction but not a full absence of DG neurogenesis, suggesting that perhaps the remaining newborn neurons may be sufficient for preserving intact cognitive processing such as longterm memory. Moreover, while we subjected mice to the probe trials at the end of the acquisition phase, Zhang et al. subjected their animals to probe trials that were intercalated between acquisition trials. Our data go in line with another study in which ablation of newly formed neurons in tamoxifen-treated line 4/NSE-DTA mice induced spatial memory impairment in the water maze one week after the acquisition phase and not 24 h later [61]. 4.3. Beneficial or deleterious effect of hippocampal neurogenesis alterations on long-term memory formation? On the one hand, the high preference the mice displayed for the trained platform position in the water maze (65%) exceeds that of any other study including our observations in C57BL/6J males of the same age as the mice from the present study (40%; unpublished observations). It is therefore important to acknowledge the role of the mice genetical background (129X1/SvJ/C57BL/6J x BALB/c) in the observed data. Earlier, Kempermann et al. suggested an influence of genetic background on DG neurogenesis, showing that different mouse inbred strains display distinct rates of proliferation, survival, and differentiation of new cells in the DG [62]. On the other hand, realizing that irradiated and cD2 KO mice showed a certain preference for the trained goal in all probe trials suggests that these mice are capable of accurate memory. We ought to consider alternatively the fact that they searched less in the trained zone as a reflection of a better learning flexibility. Flexibility is normally required in the process of acquiring new information, and this might be translated by initiating the search in other quadrant in the probe trials of the water maze. This cannot be confirmed by our data, therefore we cannot judge if differences observed in the one-week probe trial reflect a loss (altered long-term memory) or gain (behavioral flexibility) of cognitive abilities. However, the absence of hippocampal neurogenesis has been already reported beneficial in the performance on the spatial radial maze working memory task [47]. 5. Conclusion The controversy surrounding the role of DG neurogenesis in hippocampal functions is apparently a consequence of various factors including the technique adapted to alter neurogenesis and its specificity, the species and/or strains investigated, the gender, and the age at which experimental subjects are tested. The latter factor is particularly important knowing that DG neurogenesis dramatically decreases with age [4,5,7], and that this may be associated with hippocampus-related behavioral deficits [63]. Previous studies have used mice at younger ages, i.e., 1–2 months [23,26,55], when neurogenesis in the hippocampus is in a phase of transition from early postnatal to adult neurogenesis and is thus at a higher level [4]. At the time of irradiation, our mice were around 4–5 months of age. First, our previous investigation on age effect on neurogenesis in the hippocampus have shown that between the ages of 4 and 6 months there are no changes in cell proliferation or neurogenesis in general in C57BL/6 mice [4], excluding therefore any interferences with our behavioral observations. Second and most importantly, in humans, the susceptibility to radiationinduced neurobehavioral changes is correlated with age, with older adults (>40 years) exhibiting a more rapid neurobehavioral decline after irradiation than younger adults [64,65]. On the other hand,


we found evidence for behavioral changes in our mouse models relating to activity and exploration, both of which are prerequisites for behavioral performances. These findings highlight the need to cautiously analyze behavioral data from neurogenesis studies, and mandate the need to find additional activity-independent paradigms. Our findings also highlight the inter-laboratory disparities in behavioral findings, despite efforts of standardizations. Indeed, it has been reported that there remains enormous differences in behavioral experiments across laboratories, arising primarily from idiosyncratic features of the paradigms and/or laboratories in which testings are performed [66]. This is highlighted in our findings that cD2 KO mice display altered activity in the open field, which is in disagreement with our previous findings performed at another laboratory [29]. We have herein dissociated some of the induced behavioral outcome of adult DG neurogenesis alteration from those arising from changes affecting extrahippocampal regions. Our present findings however are still in agreement with most previous studies suggesting in such that adult neurogenesis, particularly in the hippocampus, is indeed causally implicated in cognitive processes, at least those of long-term memory formation. We have also reported differences in the behavioral performances of both our mouse models in tasks presumably related to normal hippocampal function. This by itself is of large interest, suggesting that these model-dependent behavioral discrepancies might be mediated by dissociated changes at the cellular level in relation to adult DG neurogenesis. Our results suggest a role of DG neurogenesis in several hippocampus-related processes, primarily related to long-term memory retention, as evidenced by the data from the water maze. Such role is also supported by previous studies in rats and mice [49,56], though contrasted by several reports where no impairments were observed (for review see [67]). We thus consolidate the notion that DG neurogenesis is required for long-term memory formation. Additionally, we highlight behavioral deficits in both models, which, we suggest, might be independent of alterations in DG neurogenesis. These conclusions however are captive of our experimental setup and parameters, and further follow up plausibly under different conditions may be required to confirm our present results. Acknowledgments This study was supported by the Swiss National Science Foundation and the NCCR (Neural Plasticity and Repair), and Adalbert Raps Foundation. We thank Lazslo Szarras for constructing the radiation mouse box, Dinh-Van Vuong for his excellent assistance during the radiation procedure at the University hospital Zurich, Inger Drescher and Rosemarie Lang for recording the body weights of mice during a four week period following irradiation, and Beni Nissan for animal care. We also would like to thank David P. Wolfer, Hans Welzl, and Lutz Slomianka for their scientific comments and editing of the manuscript. References [1] Deng W, Saxe MD, Gallina IS, Gage FH. Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2009;29:13532–42. [2] Ming GL, Song H. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron 2011;70:687–702. [3] Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neurogenesis. Cell 2008;132:645–60. [4] Ben Abdallah NM, Slomianka L, Vyssotski AL, Lipp HP. Early age-related changes in adult hippocampal neurogenesis in C57 mice. Neurobiology of Aging 2010;31:151–61.

285

[5] Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 1996;16:2027–33. [6] Rao MS, Hattiangady B, Shetty AK. The window and mechanisms of major agerelated decline in the production of new neurons within the dentate gyrus of the hippocampus. Aging Cell 2006;5:545–58. [7] Seki T, Arai Y. Age-related production of new granule cells in the adult dentate gyrus. Neuroreport 1995;6:2479–82. [8] Ben Abdallah NM, Slomianka L, Lipp HP. Reversible effect of X-irradiation on proliferation, neurogenesis, and cell death in the dentate gyrus of adult mice. Hippocampus 2007;17:1230–40. [9] Monje ML, Palmer T. Radiation injury and neurogenesis. Current Opinion in Neurology 2003;16:129–34. [10] Kalm M, Karlsson N, Nilsson MK, Blomgren K. Loss of hippocampal neurogenesis, increased novelty-induced activity, decreased home cage activity, and impaired reversal learning one year after irradiation of the young mouse brain. Experimental Neurology 2013. [11] Garthe A, Behr J, Kempermann G. Adult-generated hippocampal neurons allow the flexible use of spatially precise learning strategies. PloS ONE 2009;4:e5464. [12] Shors TJ, Townsend DA, Zhao M, Kozorovitskiy Y, Gould E. Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus 2002;12:578–84. [13] Kowalczyk A, Filipkowski RK, Rylski M, Wilczynski GM, Konopacki FA, Jaworski J, et al. The critical role of cyclin D2 in adult neurogenesis. The Journal of Cell Biology 2004;167:209–13. [14] Fuss J, Ben Abdallah NM, Vogt MA, Touma C, Pacifici PG, Palme R, et al. Voluntary exercise induces anxiety-like behavior in adult C57BL/6J mice correlating with hippocampal neurogenesis. Hippocampus 2010;20:364–76. [15] Kheirbek MA, Klemenhagen KC, Sahay A, Hen R. Neurogenesis and generalization: a new approach to stratify and treat anxiety disorders. Nature Neuroscience 2012;15:1613–20. [16] Sahay A, Wilson DA, Hen R. Pattern separation: a common function for new neurons in hippocampus and olfactory bulb. Neuron 2011;70:582–8. [17] Sahay A, Scobie KN, Hill AS, O’Carroll CM, Kheirbek MA, Burghardt NS, et al. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature 2011;472:466–70. [18] Saxe MD, Malleret G, Vronskaya S, Mendez I, Garcia AD, Sofroniew MV, et al. Paradoxical influence of hippocampal neurogenesis on working memory. Proceedings of the National Academy of Sciences of the United States of America 2007;104:4642–6. [19] Leuner B, Waddell J, Gould E, Shors TJ. Temporal discontiguity is neither necessary nor sufficient for learning-induced effects on adult neurogenesis. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2006;26:13437–42. [20] Dupret D, Montaron MF, Drapeau E, Aurousseau C, Le Moal M, Piazza PV, et al. Methylazoxymethanol acetate does not fully block cell genesis in the young and aged dentate gyrus. The European Journal of Neuroscience 2005;22:778–83. [21] Wojtowicz JM. Irradiation as an experimental tool in studies of adult neurogenesis. Hippocampus 2006;16:261–6. [22] Crossen JR, Garwood D, Glatstein E, Neuwelt EA. Neurobehavioral sequelae of cranial irradiation in adults: a review of radiation-induced encephalopathy. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 1994;12:627–42. [23] Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, et al. Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proceedings of the National Academy of Sciences of the United States of America 2006;103:17501–6. [24] Fuss J, Ben Abdallah NM, Hensley FW, Weber KJ, Hellweg R, Gass P. Deletion of running-induced hippocampal neurogenesis by irradiation prevents development of an anxious phenotype in mice. PloS ONE 2010;5. [25] Meshi D, Drew MR, Saxe M, Ansorge MS, David D, Santarelli L, et al. Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nature Neuroscience 2006;9:729–31. [26] Raber J, Rola R, LeFevour A, Morhardt D, Curley J, Mizumatsu S, et al. Radiationinduced cognitive impairments are associated with changes in indicators of hippocampal neurogenesis. Radiation Research 2004;162:39–47. [27] Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR. Extreme sensitivity of adult neurogenesis to low doses of X-irradiation. Cancer Research 2003;63:4021–7. [28] Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, et al. Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 1996;384:470–4. [29] Jaholkowski P, Kiryk A, Jedynak P, Ben Abdallah NM, Knapska E, Kowalczyk A, et al. New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning. Learning and Memory 2009;16:439–51. [30] Ansorg A, Witte OW, Urbach A. Age-dependent kinetics of dentate gyrus neurogenesis in the absence of cyclin D2. BMC Neuroscience 2012;13:46. [31] Jedynak P, Jaholkowski P, Wozniak G, Sandi C, Kaczmarek L, Filipkowski RK. Lack of cyclin D2 impairing adult brain neurogenesis alters hippocampal-dependent behavioral tasks without reducing learning ability. Behavioural Brain Research 2012;227:159–66. [32] Evans J, Sun Y, McGregor A, Connor B. Allopregnanolone regulates neurogenesis and depressive/anxiety-like behaviour in a social isolation rodent model of chronic stress. Neuropharmacology 2012;63:1315–26.

286


[33] Monje M. Cranial radiation therapy and damage to hippocampal neurogenesis. Developmental Disabilities Research Reviews 2008;14:238–42. [34] Madani R, Kozlov S, Akhmedov A, Cinelli P, Kinter J, Lipp HP, et al. Impaired explorative behavior and neophobia in genetically modified mice lacking or overexpressing the extracellular serine protease inhibitor neuroserpin. Molecular and Cellular Neurosciences 2003;23:473–94. [35] Wolfer DP, Madani R, Valenti P, Lipp HP. Extended analysis of path data from mutant mice using the public domain software Wintrack. Physiology and Behavior 2001;73:745–53. [36] Deacon RM. Digging and marble burying in mice: simple methods for in vivo identification of biological impacts. Nature Protocols 2006;1:122–4. [37] Deacon RM. Burrowing in rodents: a sensitive method for detecting behavioral dysfunction. Nature Protocols 2006;1:118–21. [38] Deacon RM, Rawlins JN. Hippocampal lesions, species-typical behaviours and anxiety in mice. Behavioural Brain Research 2005;156:241–9. [39] Contet C, Rawlins JN, Deacon RM. A comparison of 129S2/SvHsd and C57BL/6JOlaHsd mice on a test battery assessing sensorimotor, affective and cognitive behaviours: implications for the study of genetically modified mice. Behavioural Brain Research 2001;124:33–46. [40] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods 1984;11:47–60. [41] Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982;297:681–3. [42] Galsworthy MJ, Amrein I, Kuptsov PA, Poletaeva II, Zinn P, Rau A, et al. A comparison of wild-caught wood mice and bank voles in the Intellicage: assessing exploration, daily activity patterns and place learning paradigms. Behavioural Brain Research 2005;157:211–7. [43] Knapska E, Walasek G, Nikolaev E, Neuhausser-Wespy F, Lipp HP, Kaczmarek L, et al. Differential involvement of the central amygdala in appetitive versus aversive learning. Learning and Memory 2006;13:192–200. [44] Onishchenko N, Tamm C, Vahter M, Hokfelt T, Johnson JA, Johnson DA, et al. Developmental exposure to methylmercury alters learning and induces depression-like behavior in male mice. Toxicological Sciences: An Official Journal of the Society of Toxicology 2007;97:428–37. [45] Voikar V, Colacicco G, Gruber O, Vannoni E, Lipp HP, Wolfer DP. Conditioned response suppression in the IntelliCage: assessment of mouse strain differences and effects of hippocampal and striatal lesions on acquisition and retention of memory. Behavioural Brain Research 2010;213:304–12. [46] Madsen TM, Kristjansen PE, Bolwig TG, Wortwein G. Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat. Neuroscience 2003;119:635–42. [47] Zhang CL, Zou Y, He W, Gage FH, Evans RM. A role for adult TLX-positive neural stem cells in learning and behaviour. Nature 2008;451:1004–7. [48] Rola R, F2ishman K, Baure J, Rosi S, Lamborn KR, Obenaus A, et al. Hippocampal neurogenesis and neuroinflammation after cranial irradiation with (56)Fe particles. Radiation Research 2008;169:626–32. [49] Seigers R, Schagen SB, Beerling W, Boogerd W, van Tellingen O, van Dam FS, et al. Long-lasting suppression of hippocampal cell proliferation and impaired cognitive performance by methotrexate in the rat. Behavioural Brain Research 2008;186:168–75.

[50] Kempermann G, Kuhn HG, Gage FH. Experience-induced neurogenesis in the senescent dentate gyrus. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 1998;18:3206–12. [51] van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature Neuroscience 1999;2:266–70. [52] Fan Y, Liu Z, Weinstein PR, Fike JR, Liu J. Environmental enrichment enhances neurogenesis and improves functional outcome after cranial irradiation. The European Journal of Neuroscience 2007;25:38–46. [53] Shen L, Nam HS, Song P, Moore H, Anderson SA. FoxG1 haploinsufficiency results in impaired neurogenesis in the postnatal hippocampus and contextual memory deficits. Hippocampus 2006;16:875–90. [54] Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S. Inhibition of neurogenesis interferes with hippocampus-dependent memory function. Hippocampus 2006;16:296–304. [55] Raber J, Fan Y, Matsumori Y, Liu Z, Weinstein PR, Fike JR, et al. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Annals of Neurology 2004;55:381–9. [56] Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM. A role for adult neurogenesis in spatial long-term memory. Neuroscience 2005;130:843–52. [57] Abayomi OK. Pathogenesis of irradiation-induced cognitive dysfunction. Acta Oncologica 1996;35:659–63. [58] Roman DD, Sperduto PW. Neuropsychological effects of cranial radiation: current knowledge and future directions. International Journal of Radiation Oncology, Biology, Physics 1995;31:983–98. [59] Filipkowski RK, Kiryk A, Kowalczyk A, Kaczmarek L. Genetic models to study adult neurogenesis. Acta Biochimica Polonica 2005;52:359–72. [60] Urbach A, Robakiewicz I, Baum E, Kaczmarek L, Witte OW, Filipkowski RK. Cyclin D2 knockout mice with depleted adult neurogenesis learn Barnes maze task. Behavioral Neuroscience 2013;127:1–8. [61] Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M, et al. Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nature Neuroscience 2008;11:1153–61. [62] Kempermann G, Gage FH. Genetic influence on phenotypic differentiation in adult hippocampal neurogenesis. Brain Research Developmental Brain Research 2002;134:1–12. [63] van Praag H, Shubert T, Zhao C, Gage FH. Exercise enhances learning and hippocampal neurogenesis in aged mice. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2005;25:8680–5. [64] Armstrong CL, Corn BW, Ruffer JE, Pruitt AA, Mollman JE, Phillips PC. Radiotherapeutic effects on brain function: double dissociation of memory systems. Neuropsychiatry, Neuropsychology, and Behavioral Neurology 2000;13:101–11. [65] Stylopoulos LA, George AE, de Leon MJ, Miller JD, Foo SH, Hiesiger E, et al. Longitudinal CT study of parenchymal brain changes in glioma survivors. AJNR American Journal of Neuroradiology 1988;9:517–22. [66] Crabbe JC, Wahlsten D, Dudek BC. Genetics of mouse behavior: interactions with laboratory environment. Science 1999;284:1670–2. [67] Leuner B, Gould E, Shors TJ. Is there a link between adult neurogenesis and learning. Hippocampus 2006;16:216–24.