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Sex differences in neurogenesis and activation of new neurons in response to spatial learning Chow, Carmen; Epp, Jonathan Richard; Lieblich, Stephanie E.; Barha, Cindy K.; Galea, Liisa A.M. Aug 1, 2013

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SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  1 Sex differences in neurogenesis and activation of new neurons in response to spatial learning Carmen Chow, Jonathan R. Epp1, Stephanie, E. Lieblich, Cindy K. Barha, Liisa A.M. Galea*Program in Neuroscience, Department of Psychology, Brain Research Centre, University of British Columbia, Vancouver, BC, Canada 1 JRE is now located at the Hospital for Sick Children, Toronto, ON, Canada *Published In: Chow C, Epp, JR, Lieblich SE, Barha,C.K., Galea, L.A.M. (2013) Sex differences in neurogenesis and activation of new neurons in response to spatial learning. Psychoneuroendocrinology, 38, 1236-1250.SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  2 ABSTRACT Adult hippocampal neurogenesis is often associated with hippocampus-dependent learning and memory. Throughout a new neuron’s development, it is differentially sensitive to factors that can influence its survival and functionality. Previous research shows that, spatial training that occurred 6 to 10 days after an injection of the DNA synthesis marker, bromodeoxyuridine (BrdU), increased cell survival in male rats. Because sex differences in spatial cognition and hippocampal neurogenesis have been reported, it is unclear whether spatial training would influence hippocampal neurogenesis in the same way in males and females. Therefore, this study examined sex differences in hippocampal neurogenesis following training in a spatial task. Male and female rats were trained in the spatial or cued version of the Morris water maze task 6 to 10 days after one injection of BrdU (200mg/kg). Twenty days following BrdU injection, all animals were given a probe trial and perfused. Males performed better in the spatial, but not cue, task than females. Spatial training increased BrdU-labeled cells relative to cue training only in males, but both males and females showed greater activation of new cells (BrdU co-labeled with immediate early gene product zif268) after spatial training compared to cue training. Furthermore, performance during spatial training was positively correlated with cell activation in females but not males. This study shows that while spatial training differentially regulates hippocampal neurogenesis in males and females, the activity of new neurons in response to spatial memory retrieval is similar. These findings highlight the importance of sex on neural plasticity and cognition. Keywords: adult neurogenesis, cell survival, cell activation, spatial learning, Morris Water Maze, sex differences, hippocampus, dentate gyrus, zif268, immediate early gene  SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  3 The hippocampus is one of two main areas that harbours continual neurogenesis in adulthood (Altman and Das, 1965). Adult neurogenesis exists in most mammalian species, including humans (Eriksson et al, 1998) and can be subdivided into cell proliferation, migration, differentiation, and maturation. Regulation of adult neurogenesis at any of these stages can lead to changes in the number of new neurons that are produced. The function of these new neurons in the hippocampus has been linked to hippocampus-dependent learning and memory (Leuner et al., 2006). Training on hippocampus-dependent tasks such as the spatial Morris water maze increases hippocampal neurogenesis (Gould et al, 1999). Furthermore exercise, which increases hippocampal neurogenesis, also facilitated spatial learning (van Praag et al, 1999), while irradiation, which decreases hippocampal neurogenesis, impaired long-term spatial memory (Snyder et al, 2005). Furthermore a partial reduction in hippocampal neurogenesis, via genetic knockdown, was sufficient to disrupt spatial learning and discrimination (Zhang et al, 2008; Clelland et al, 2009). Additionally, greater reductions in hippocampal neurogenesis impaired spatial memory and novel object recognition memory, whereas lesser reductions in hippocampal neurogenesis did not significantly affect performance compared to controls (Jessberger et al., 2009). These studies show that reducing neurogenesis to certain levels can impair hippocampus-dependent learning and memory. Exposure to hippocampus-dependent learning can also differentially regulate the survival of new hippocampal cells depending on the type of task (Leuner et al, 2006), quality of learning (Sisti et al, 2007; Epp et al., 2007), task difficulty (Epp et al, 2010), and/or the age of cells at the time of exposure and perfusion (Epp et al, 2007; Epp et al, 2011).  The ability of spatial training to promote hippocampal neurogenesis depends on the stage of development during which immature neurons are exposed to spatial training (Epp et al., 2007). Spatial training 6-10 days SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  4    after an injection of the DNA synthesis marker, bromodeoxyuridine (BrdU) increased cell survival, but spatial training on days 1-5 or 11-15 after BrdU injection either did not change (Epp et al, 2007) or decreased cell survival depending on the age of new neurons at the time of examination (Epp et al, 2011). Because the time window for incorporation of BrdU into dividing cells is just 2 hours (Nowakowski et al, 1989), this method allows the tracking of a group of similarly aged cells. Therefore, these data suggest that there is a critical period in new neuron development during which cell survival is most affected by spatial training.  Studies have also investigated whether training on hippocampus-dependent tasks activates new neurons. Cell activation can be quantified using immediate early genes (IEG) such as c-Fos, arc, or zif268, which are transiently expressed in response to neuronal activation and have a role in neural plasticity and memory consolidation (Guzowski et al, 2001; Jones et al, 2001). IEG expression in adult-born neurons is increased in response to exploration of a new environment (Ramirez-Amaya et al, 2006), re-exposure to a familiar environment (Tashiro et al, 2007), spatial learning (Jessberger and Kempermann, 2003; Snyder et al, 2009b), and memory retrieval (Snyder et al, 2005; Epp et al, 2011).  Thus far the majority of research in this area has been conducted in male animals. However there are significant sex differences in the regulation of neurogenesis in the adult dentate gyrus by factors such as stress, breeding season, and gonadal hormones (Falconer and Galea, 2003; Westenbroek et al, 2004; Galea and McEwen, 1999; Barker and Galea, 2008). Furthermore, sex differences in spatial performance exist across a wide variety of species, with males typically outperforming females (Galea et al, 1996; Gaulin and Fitzgerald, 1986). To our knowledge only one study has examined sex differences in the effects of training on a hippocampus-dependent task on hippocampal neurogenesis (Dalla et al., 2009). This study SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  5    showed that faster acquisition of trace eyeblink conditioning was correlated with a greater percent increase in cell survival in females compared to males. Intriguingly, sex differences in performance of the trace eyeblink conditioning task favours females, unlike performance in the Morris water maze task which typically favours males (Galea et al, 1996). To our knowledge, no study has examined the effect of spatial training on hippocampal neurogenesis and activation of new neurons in males and females.  Therefore, the current study aimed to determine whether there are sex differences in the survival of new neurons after Morris water maze task training and whether there is differential activation in response to spatial memory retrieval. Adult male and female rats were trained in the Morris water maze task 6-10 days after BrdU injection and given a probe trial on day 20 to examine new cell activation via the IEG product zif268. We hypothesized that males would outperform females in acquisition of the Morris water maze task, would have higher levels of hippocampal neurogenesis in response to training and show greater activation of new neurons in response to memory retrieval compared to females.  METHODS Subjects Sixty-three Sprague Dawley rats (males: n = 29; females: n = 34) between 58-62 days old that were bred and raised in the Department of Psychology at the University of British Columbia were used in this study. All animals were pair-housed in polycarbonate bins (48 x 27 x 20cm) with a polyvinylchloride tube, paper towels, aspen chip bedding, and free access to food and water. The colony room was kept at a temperature of 20ºC with 50-70% humidity, and maintained on a 12/12h light-dark cycle (lights on at 0700). Animals were left undisturbed until handling began five days prior to the start of the experiment. All testing was carried out in accordance with the Canadian Council for Animal Care guidelines and was approved by the SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  6    animal care committee at the University of British Columbia. All efforts were made to reduce the number of animals used and to minimize their suffering. Apparatus The Morris water maze was a white circular pool that was 180cm in diameter and filled with water mixed with white tempura (non-toxic) paint to render it opaque. Large distal cues were placed on all four walls of the room surrounding the pool and remained constant throughout the study. A camera installed above the center of the pool was connected to a computer running ANY-maze (Stoelting, Wood Dale, IL, USA) in order to record measures of performance such as latency, swim distance and percentage of time spent in the quadrant with the platform.  Procedure  One intraperitoneal injection of 200mg/kg bromodeoxyuridine (BrdU; Sigma-Aldrich, Oakville, ON, Canada) was administered to all animals at the start of the experiment (day 0). Six days later, female and male rats were exposed to four trials per day for five consecutive days in either the spatial (n = 25; 11 males and 14 females) or cued (n = 24; 11 males and 13 females) version of the Morris water maze task (Figure 1A).  A separate set of rats served as cage controls and did not leave their home cage except for bi-weekly cage changing (n = 14; 7 males and 7 females). In the spatial task, the platform was submerged 2cm beneath the pool surface and remained in the northeast quadrant throughout training. In the cue task, the platform was raised 2cm above the water and the location of this platform changed after every trial to ensure that animals relied on the visible platform as a cue rather than extramaze cues. Training began at approximately the same time each day and spanned from approximately 9am to 3 pm.  Training occurred over five consecutive days, with four trials per day. Each trial ended when the animal SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  7    reached the platform or when 60s had elapsed. Animals that were unable to locate the hidden or visible platform within the allotted time were guided to the platform and left there for 10s before removal from the pool. The inter-trial interval was approximately 5 minutes. For each day, performance on the four trials was averaged to obtain a measure of performance per day on the water maze task as previously reported (Vorhees and Williams, 2006; Epp et al, 2011).  Because estrous cycle phase influences both hippocampal cell proliferation (Tanapat et al, 1999; Rummel et al, 2009) and spatial learning (Warren and Juraska, 1997), estrous cycles were monitored. Animals (including cage controls) were lavaged on the day of BrdU injection, every day immediately after training (between 1-3pm) and the probe trial (approximately 11am to 12pm).  Vaginal lavage was carried out by inserting a small glass dropper with water into the rat’s vagina and collecting a sample. The vaginal samples were transferred onto microscope slides and stained with Cresyl Violet (Sigma, Oakville, Canada). Cells were analyzed under 200X magnification. A rat was determined to be in proestrus if the majority (70% or more) of the cells were nucleated epithelial cells (Frye, 1995; Warren and Juraska, 1997; Tanapat et al, 1999).  After training, animals were returned to the colony rooms and remained undisturbed until ten days later (20 days after BrdU injection).  On this day animals received a 30-second probe trial, during which the platform was removed from the pool. Percentage of time spent in the target quadrant (that previously contained the hidden platform) was recorded during the probe trial. Ninety minutes after probe trial, animals were administered an overdose of sodium pentobarbitol and perfused transcardially with 0.9% saline followed by 4% formaldehyde (Sigma-Aldrich). Brains were extracted and post-fixed in 4% formaldehyde for 24 hours and then transferred to 30% sucrose (Fisher Scientific) until sectioning. Brains were sliced into 40 μm coronal sections using a Leica SM2000R microtome (Richmond Hill, Ontario, Canada). SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  8    Sections were collected in series of ten throughout the entire rostral-caudal extent of the hippocampus and stored in an antifreeze solution composed of ethylene glycol, glycerol and 0.1M PBS at -20°C. BrdU immunohistochemistry Free-floating tissue slices were rinsed with 0.1M TBS (pH 7.4) three times between each of the following steps. Tissue was incubated in 0.6% H2O2 for 30 minutes, transferred to 2N HCl and incubated at 37°C for 30 minutes. The tissue was rinsed with 0.1M borate buffer (pH 8.5) for 10 minutes. After blocking the tissue with TBS+ solution, consisting of 0.3% Triton-X 100 (Sigma), and 3% normal horse serum (Vector Laboratories; Burlingame, CA, USA) in 0.1M TBS, slices were incubated in a primary antibody solution 1:200 mouse anti-BrdU (Roche Diagnostics, Laval, QC, Canada) and TBS, for 48 hours at 4°C. Then tissue was incubated in secondary antibody solution containing 1:500 horse anti-mouse biotinylated IgG (Vector Laboratories, Burlington, Ontario) in TBS+ for 4 hours at room temperature. Tissue was incubated for 1.5 hours in advitin/biotinylated enzyme solution (ABC kit, Vector Laboratories). Tissue slices were then transferred to diaminobenzidine (DAB; Sigma) solution and incubated for 5 minutes, then rinsed repeatedly with TBS. The tissue was mounted onto microscope slides, counterstained with cresyl violet, dehydrated, cleared with xylene and cover-slipped with Permount (Fisher Scientific; Ottawa, ON, Canada). BrdU/NeuN double labeling immunohistochemistry Brain sections were rinsed three times with 0.1M PBS and left overnight at 4°C. The tissue was transferred to a primary antibody solution containing 1:250 mouse anti-NeuN (Millipore; MA, USA), 3% normal donkey serum (NDS; Vector Laboratories), and 0.3% Triton-X in 0.1M PBS and incubated for 24hrs at 4°C. Tissue was then incubated in a secondary SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  9 antibody solution containing 1: 200 donkey anti-mouse ALEXA 488 (Invitrogen, Burlington, ON, Canada) in 0.1M PBS, for 18 hours at 4°C. Following three rinses with PBS, sections were rinsed with 4% formaldehyde, and washed twice in 0.9% NaCl. The tissue was incubated in 2N HCl for 30 minutes at 37°C and incubated in a BrdU primary antibody solution consisting of 1: 500 rat anti-BrdU (AbD Serotec; Raleigh, NC, USA), 3% NDS, and 0.3% Triton-X in 0.1M PBS for 24 hours at 4°C. Tissue was incubated in a secondary antibody solution containing 1:500 donkey anti-rat Cy3 (Jackson ImmunoResearch; PA, USA) in 0.1M PBS for 24 hours at 4°C. After rinsing with PBS, tissue was mounted onto microscope slides and cover-slipped with PVA DABCO. BrdU/zif268 double labeling immunohistochemistry The tissue was washed with 0.1M PBS and left to sit overnight at 4°C. The next day, slices were transferred to zif268 primary antibody solution made with 1: 1000 Rabbit anti-Egr-1 (Santa Cruz Biotechnologies; CA, USA), 3% NDS, and 0.3% Triton-X in 0.1M PBS and incubated for 24hours at 4°C. Then the tissue was incubated in secondary antibody solution, consisting of 1: 5000 Donkey anti-Rabbit ALEXA 488 (Invitrogen, Burlington, ON, Canada) in 0.1M PBS, for 18 hours at 4C. The tissue was fixed with 4% formaldehyde and rinsed twice in 0.9% NaCl. Following incubation in 2N HCl for 30 minutes at 37°C, slices were washed with PBS.  The BrdU primary antibody solution was prepared with 1: 500 mouse anti-BrdU (Roche), 3% NDS, and 10% Triton-X in 0.1M PBS and tissue was incubated in this solution for 24 hours at 4°C. The secondary antibody solution containing 1:250 Donkey anti-Mouse Cy3 (Jackson ImmunoResearch; PA, USA) in 0.1M PBS was used to incubate the tissue for 16 hours at 4°C. After rinsing with PBS, slices were mounted onto slides and cover-slipped with PVA DABCO. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  10    Cell counting All microscope slides were coded to ensure that counting was done by an experimenter blind to the group assignment of each animal. BrdU-labeled cells were counted using a Nikon E600 light microscope under a 1000x magnification (Figure 1B) while BrdU/NeuN and BrdU/zif268 positive cells were counted at 400x magnification (Figure 1E and 1H). For BrdU-labeled cells, every 10th section of the granule cell layer (GCL; including the subgranular zone) and hilus was counted separately and an estimate of total immunoreactive cells per region was obtained by multiplying the aggregate number of cells per region by 10 (Epp et al, 2007; Epp et al, 2011). Cells in the hilus were counted because hilar cells are generally considered ectopic and we were interested in determining whether cells in both areas were affected similarly. Area measurements for the GCL and hilus were obtained with digitized images and the software ImageJ (NIH). Volume estimates were calculated using Cavalieri’s principle (Gundersen and Jensen, 1987) by multiplying the summed areas of each region by the distance between sections (400μm). Density of BrdU-labeled cells in the GCL and hilus were calculated by dividing the sum of BrdU-labeled cells by volume of the corresponding region.  The percentages of BrdU/NeuN and BrdU/zif268 double-labeled cells were obtained by randomly selecting 50 or 100 BrdU-labeled cells, respectively, and determining the percentage of cells that coexpressed NeuN or zif268. We also noted whether labeled cells were located in the dorsal or ventral GCL using the criterion defined by Banasr and others (2006), with sections 6.20-3.70mm from the interaural line defined as dorsal and sections 3.70-2.28mm from the interaural line as ventral.    SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  11    Data analyses All analyses were conducted using Statistica (Statsoft Tulsa, OK). Swim distance and latency to reach the platform were each analyzed using repeated-measures analysis of variance (ANOVA), with training group (spatial, cue) and sex (male, female) as between-subject factors and training day (1 to 5) as the within-subject factor. Repeated-measures ANOVAs were used to analyze total number of BrdU-labeled cells, volume of GCL and hilus, and cell density with sex and training group (spatial, cue, cage controls) as the between subject factors and region (GCL, hilus) as the within-subject factor.  For percentage of cells co-expressing BrdU/NeuN or BrdU/zif268, repeated-measures ANOVAs were performed with dentate gyrus subregion (dorsal, ventral) as the within-subject factor and with sex and training group as between-subject factors. Pearson product-moment correlations were calculated to examine the relationship between spatial performance and density of BrdU-labeled cells or cells co-expressing BrdU and zif268.   To examine spatial training performance across the estrous cycle in female rats, an analysis of covariance (ANCOVA) was used, with estrous state (proestrus, non-proestrus) as the covariate, group (spatial, cue) as the between-subject factor, and training day (1 to 5) as the within-subject factor. For probe trial performance across the estrous cycle, an ANOVA was used with estrous state and training group as between-subject factor. Post-hoc tests were performed with the Neuman-Keuls procedure. A priori tests were subjected to Bonferoni corrections.   RESULTS Females swam greater distances and required more time than males to locate the hidden, but not visible, platform  SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  12 There was a significant sex by group interaction for swim distance (F(1,45) = 7.76, p < .008) and latency (F(1,45) = 7.45, p < .01) to reach the platform across training days (Figure 2A and B). Post-hoc analyses revealed that in the spatial training group, females swam longer distances (p < .001) and required more time (p < .001) than males to locate the hidden platform. There was also a significant interaction of day by group on distance travelled (F(4,180) = 11.22, p < .0001) and on latency to reach the platform (F(4,180) = 4.42, p < .002). Post-hoc tests revealed that for the spatial group, distance traveled during training decreased significantly between days 1 to 4 (p’s > .008), whereas for the cue group, swim distance decreased significantly only between days 1 to 3 (p’s > .02). To determine whether there were sex differences in the learning curves in the spatial task we also performed a trend analysis within the spatial group, on performance across the days and found that there was no significant sex difference for linear, quadratic, or cubic trend, for distance travelled across days (p’s > 0.60). There was, however, a significant quartic curve difference between males and females on distance swum across days (p = .005), indicating that there were significant differences in the learning curve between males and females.  For latency to reach the platform, post-hoc tests showed that in the spatial group, latency decreased significantly between days 1 to 4 (p's > .02), and for the cue group, latency was significantly decreased only between days 1 to 3 (p's > .002).  There were also main effects of day, sex, and group but no other significant effects for either distance or latency to reach the platform were found (p’s > 0.19).  Finally, to test for potential sex and/or group differences in motivation and locomotion, swim speed during training was analyzed. There was a trend for a sex difference favouring females (F(1, 45) = 3.51, p < .07), but no other significant main or interaction effects (p’s > .39). SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  13 Training performance (distance travelled) in females was analyzed using estrous state as a covariate across training days with training group as the between-subject factor.  There was a significant day by group interaction (F(4,80) = 4.92, p < .001) and a main effect of group and day (p’s < 0.026), but no significant effect of the estrous cycle covariate (p’s > 0.21).  Post-hoc tests showed that distance to reach the platform was greater in the spatial compared to cue group on each training day (p’s > .001). We also analyzed distance to reach the platform across each trial (trial 1 to 4) on the first and final day (day 1 and 5) in order to determine whether there were preexisting sex differences on the first day of training and whether performance was equivalent on the first and last trial of training in the spatial group. There was a significant interaction of sex by day by trial (p < .013) and main effects of sex, day, and trial (p’s < .03). Post-hoc comparisons revealed that males outperformed females for distance travelled only on day 1 trial 4 (p = 0.006) but not on day 1 trial 1 (p = 0.93) or on day 5 (trial 1: p = 0.08 and trial 4: p = 0.73; Figures 2E and 2F). This indicates that there were no preexisting (or lasting) sex differences in performance on the first or last trial of training in the spatial group.  Additionally, we examined the change in swim distance between the first and final day of training as another measure of performance, and no significant main effects or interactions were found (all p’s > .37). However, we also compared differences in performance (distance to reach the platform) on the first half (Days 1-3) versus the second half (Days 3-5) of training. A priori tests showed that, in the spatial group, females showed a significantly greater change in swim distance in the first compared to the second half of training (p < .003), while males exhibited a trend for a difference between the two time points (p = .07). These findings, along with the SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  14 findings from the trend analysis, show that males and females significantly differed in their distance swum curves across training. Spatial-trained animals spent a greater percentage of time in the target quadrant than cue-trained animals during the probe trial   There was a significant effect of group on time spent in the target quadrant during the probe trial (F(1,36) = 46.55, p < .0001) with spatial-trained rats spending more time in the target quadrant than cue-trained rats (Figure 2G). No other significant main or interaction effects were found (p’s > 0.70).  Proestrous females in the spatial group spent a significantly greater percentage of time in the target quadrant than non-proestrous females during the probe trial. For the probe trial, there was a main effect for estrous cycle status on percentage of time spent in the target quadrant (F(1, 15) = 6.17, p = .025) and a main effect of group (F(1, 15) = 8.08, p = .012); Figure 2H), with proestrous females in the spatial group spending a significantly greater percentage of time in the target quadrant relative to non-proestrous females in the spatial (p = .005), but no differences were found in the cue groups (p’s  < .52).  Proestrous females in the spatial group exhibited a trend to spend a greater percentage of time in the target quadrant than males in the spatial group (p < .08). To determine whether the estrous cycle difference in probe trial performance was due to differences during training, we compared performance (distance to platform) during training for females that were in proestrus with those who were not in proestrus on the day of the probe trial. As expected, there was a main effect of training day (F(4, 92) = 24.42, p < .001), but no significant differences in performance across the five days of training for proestrous and non-SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  15 proestrous females (p's > .22), showing that there were no significant differences in performance prior to the probe trial. Males had larger dentate gyrus volumes than females As expected, the dentate gyrus volume in males was significantly larger than females (main effect of sex: F(1,48) = 12.55, p < .0001; region by sex interaction: F(1,48) = 4.26, p < .044; Table 1). Despite the interaction, post-hoc tests revealed that there were sex differences with males having both larger GCL and hilus volumes (p’s < 0.04). No other significant main or interaction effects in dentate gyrus volume (p’s > .40) were found.  Because there were sex differences in dentate gyrus volume BrdU-labeled cell densities were used so that direct comparisons between males and females could be made. Males trained in the spatial task but not the cue task showed significantly greater cell survival compared to females Spatial-trained animals had a greater density of BrdU-labeled cells in the GCL, but not hilus, than cued-trained or cage-control rats (Group by region: F(2,45)= 3.22, p < .049; post-hoc tests: all p’s < .002). A priori we wanted to determine whether there were sex differences in BrdU-labeling after spatial learning, and we found that spatial-trained males had significantly greater BrdU-labeled cell density than cue-trained and cage-control males (p’s < .007) but there were no significant differences between any of the female groups (p’s > .36).  Furthermore, density of BrdU-labeled cells was higher in spatial-trained males than spatial-trained females (p < .010) but there were no sex differences in BrdU-labeled cell density for cue-trained (p > .4) or cage-control animals (p > .6; Figure 3A). As expected, there was also a main effect of region (p < .001) and group (p < .05) but no other significant effects were found. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  16 We also analyzed the total number of BrdU-labeled cells and found males had a significantly higher number of BrdU-labeled cells in the GCL (F(1, 45) = 9.56, p < .003; Figure 3C), but not the hilus (p’s > .59; Figure 3D), than females. Similarly, as we expected sex differences in cell survival after training, an a priori analyses on total cell counts showed that males in the spatial group had significant more BrdU-labeled cells compared to females in the spatial, cue, and control groups (p’s < .001) and also compared to males in the cue and control groups (p’s <.02).  Spatial performance was not significantly correlated with cell survival Spatial performance was quantified by total distance swum during training or by the change in swim distance between the first and last training days (days 1 and 5). In the spatial group, no significant correlations were found between density of BrdU-labeled cells in the GCL and total swim distance or change in swim distance for males or females (p’s > .15). Likewise, in the cue group, there were no significant correlations between density of BrdU-labeled cells in the GCL and the total swim distance or change in swim distance for males or females (p’s > .20). No significant correlations were found in the hilus (p's > .05). The majority of BrdU-labeled cells co-expressed NeuN The majority of BrdU-labeled cells were colabeled with NeuN (Table 2).  There was a significantly greater proportion of new neurons in the dorsal compared to ventral hippocampus (main effect of region (dorsal, ventral): F(1,33) = 63.38, p < .001). However, there were no significant sex or group main or interaction effects in proportion of BrdU cells co-expressing NeuN (p’s  > .2). Spatial-trained rats had significantly greater percentage of BrdU/zif268 co-expression than cue-trained rats and greater activation was found in the dorsal compared to ventral GCL SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  17 Spatial-trained rats had greater activation of BrdU-labeled cells in response to spatial memory retrieval compared to cue-trained rats (F(1,28) = 20.52, p < .023; Figures 4A and 4B).  Furthermore there were significantly greater numbers of BrdU-labeled cells in the dorsal GCL that co-expressed the IEG product zif268 compared to the ventral GCL (F(1,28) = 39.89, p < .001).  No other significant main or interaction effects were found (p’s > .28) and there were no significant main or interaction effects of estrous cycle on activation of BrdU-labeled cells (p’s > .06), which may have been due to low sample size especially for proestrous females in the cue group (n = 1; proestrous females in the spatial group and non-proestrous females: n = 4-5), as some animals were excluded due to lack of BrdU labeling in the GCL. In females, better spatial learning performance was correlated with more cell activation in the dorsal GCL Total swim distance was negatively correlated with the percentage of BrdU/zif268 co-labeled cells in the GCL (dorsal and ventral combined) for animals in the spatial (r(19) = -.49, p = .027) but not cue group (p = .63; Figures 4C and 4D). This shows that more activation in new neurons was associated with better overall performance during acquisition in the water maze task, a relationship that was significant in spatial-trained females (r(8) = -.82, p = .007) but not males (r(10) = .14, p = .68). Additionally, when broken down by GCL region (dorsal or ventral), this significant negative correlation was driven by the relationship in the dorsal GCL in spatial-trained females (r(8) = -.73, p = .02; Figure 4F) rather than spatial-trained males (r(10) = .12, p = .73; Figure 4E). No significant correlations were found in the ventral GCL for either males (r(10) = .10, p = .77) or females (r(8) = -.47, p = .20) in the spatial group. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  18 DISCUSSION Males outperformed females during acquisition of the spatial Morris water maze task; however, no sex differences were found in memory retention during the probe trial, which is consistent with previous findings (e.g. Galea et al., 1996). Females in the proestrous stage showed better memory during the probe trial than non-proestrous females, which is inconsistent with studies where water maze training occurred in one day (Warren and Juraska, 1997; Berry et al, 1997), but somewhat consistent to past studies showing proestrous rats are more likely to use a spatial strategy (Korol et al., 1994; Rummel et al., 2010) or in studies using exogenous estradiol (Packard and Teather, 1997; Chen et al, 2002). In the present study, consistent with past literature, males trained on the spatial task 6-10 days after BrdU injection showed greater new neuron survival in the dentate gyrus than males trained on a cued task or cage controls (Epp et al, 2007; Epp et al., 2011; Gould et al., 1999). Furthermore we found that spatial-trained females did not show the same change in density of BrdU-labeled cells compared to cued-trained females, suggesting that the enhancement in cell survival is related to spatial training rather than memory retention in males. Furthermore we saw that spatial-trained rats, regardless of sex, had greater activation of new neurons in response to spatial retrieval memory compared to cue-trained rats. In addition, activation of new neurons was negatively correlated with total swim distance during spatial training and this correlation was stronger in the dorsal GCL in female rats. Therefore, better spatial performance during acquisition predicted higher levels of new cell activation in female, but not male, rats. This demonstration of the relationship between sex differences in spatial learning, cell survival (neurogenesis), and cell activation suggests that spatial training differentially affects hippocampal neurogenesis in males and females. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  19 Sex differences in spatial training performance may be linked to differences in cell survival In the spatial-trained groups, males swam shorter distances and latencies to reach the hidden platform than females, but there were no significant sex differences in distance or latency to reach the visible platform in the cue-trained groups. The sex difference in spatial training was not evident on the first day of training, suggesting there were no pre-existing sex differences in performance.  Furthermore the sex difference in spatial training performance was not attributable to motivational or motoric differences as we found no significant differences between the sexes in swim speed. These findings coupled together with the finding that sexes differed in the quartic learning curves and rate of change in distance swum suggests that there were sex differences in learning strategy, which is consistent with previous studies (Beiko et al, 2004; Williams et al, 1990; Galea et al, 1996; Galea and Kimura, 1993; Grön et al, 2000). Intriguingly, we found that spatial, but not cue, training increased neurogenesis in the hippocampus of males but not females. Results from previous literature show that increases in hippocampal neurogenesis after spatial learning (Gould et al, 1999) is dependent on factors such as task difficulty (Epp et al, 2010) and quality of learning (Epp et al, 2007; Sisti et al, 2007). Therefore, enhancement of neurogenesis with spatial training in males, but not females, suggests that sex differences in learning strategy, which affects task difficulty and learning quality, may have differentially altered hippocampal involvement during spatial training. Support for this supposition extends from the spatial strategy literature. Sex differences in spatial learning strategies have been well documented in both humans and rodents (Galea and Kimura, 1993; Willams et al., 1990). In general, males focus more on spatial and geometric cues in the environment, which is a hippocampus-dependent strategy, while females rely more on landmark cues, which activates other systems such as the striatum (Willams et al., 1990; Miranda et al, 2006). SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  20 However, a recent study showed that males do not necessarily choose a place strategy more often than females, and that females favoured a place strategy when both place and cue strategies were equally effective in an ―ambiguous‖ water maze task (van Gerven et al, 2012). Intriguingly, among place strategy users, males had significantly better spatial performance than females, suggesting that although males may not favour this type of strategy, they use it more efficiently than females (van Gerven et al, 2012). Therefore, in a task such as the one used in our study, where a place strategy is better suited for efficient task completion, males performed better because they could utilize place learning more easily. Therefore, although the potential sex difference in strategy choice did not affect mastery of the spatial task in the current study, use of less hippocampus-dependent strategies may have contributed to less neurogenesis in females, as seen previously using a different testing paradigm (Rummel et al, 2010). It is possible that learning strategy choice, or differences in how the strategies are executed, may influence task difficulty, as both choosing a more efficient strategy and using it more effectively would make learning less difficult, and vice versa. Because our study and previous research have demonstrated that males perform better than females during spatial training, one may infer that males employ a more efficient strategy than females. Epp and colleagues (2010) showed that increasing difficulty of the spatial water maze task reduced cell survival in male rats. Perhaps this can be generalized to females such that choosing a less efficient strategy, or having more difficulty using the place strategy, rendered the spatial task more difficult, thus down-regulating the survival of new neurons. All together, our study and the current literature suggest that learning strategy influences hippocampal activation and task difficulty, which in turn affects cell survival. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  21 Dalla and colleagues (2009) were the first to show that sex differences in hippocampus-dependent learning produce sex differences in neurogenesis. Females acquired the trace eyeblink conditioning task faster than males and showed greater increases in cell survival. Because all animals performed similarly by the end of training, the initial phase of learning appears to have a greater impact on cell survival. We showed a similar pattern of results using a hippocampus-dependent task that favours learning in males, which resulted in increased neurogenesis in males but not females. However, in our study, spatial performance was not significantly correlated with cell survival, which is consistent with Epp and colleagues (2011), and may have been due to the fact that we used a different learning task than Dalla and colleagues (2009). Ablation studies showed that new hippocampal neurons have different roles in spatial learning versus trace conditioning (Snyder et al, 2005; Shors et al, 2001); therefore it is possible that quality of spatial learning has a smaller influence on cell survival than other factors such as learning efficiency. Taken together, both studies support the idea that acquisition rate, perhaps as a function of learning strategy choice or execution, can influence cell survival depending on the degree to which the strategy engages the hippocampus. Intriguingly, the use of hippocampus-dependent learning strategies appears to have sexually-dimorphic effects on cell proliferation. Males that favored a spatial strategy in the Morris water maze task showed lower levels of cell proliferation compared to males that chose a cue strategy, whereas the opposite was true in females (Epp and Galea, 2009; Rummel et al, 2010). Spatial memory retrieval increased activation of 20-day-old new neurons in both males and females In the present study we found no significant sex differences in cell activation, as assessed by expression of the IEG product zif268 by new neurons in response to spatial memory retrieval, SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  22 which is consistent with the lack of sex differences observed in probe trial performance. However we did find greater activation of 20 day old neurons in the dorsal relative to the ventral GCL, consistent with previous research showing that, in general, cells in the dorsal dentate gyrus are more active compared to the ventral region (Snyder et al, 2009b) and that the dorsal GCL may be more important for spatial navigation (Moser et al, 1993). Additionally, in both the dorsal and ventral GCL, spatial-trained animals had significantly greater levels of cell activation compared to cue-trained animals, which is also consistent with past findings (Epp et al., 2011; Kee et al, 2007). To our knowledge, only one other study has directly examined the activation of immature neurons across the dorsal-ventral axis of the dentate gyrus in response to spatial learning. Snyder and colleagues (2009b) found that, in male rats, new (4 weeks old) neurons in the ventral dentate were more likely to be activated by spatial training than the dorsal dentate, which is somewhat inconsistent with our finding that new neurons were activated in both the dorsal and ventral dentate in response to the probe trial. This discrepancy may have been due to differences in the time point at which new neuron activation was examined, the method of training, that activation was characterized after spatial training versus after the probe trial (spatial memory retrieval), or the use of different markers to identify immature neurons. Snyder and colleagues (2009b) trained all rats in 16 trials on one day and examined new neuron activation in response to spatial learning, while we examined cell activation in response to retrieval of long-term spatial memory after 20 trials of learning spread out over 5 days. The ventral hippocampus is thought to be more important for stress and anxiety while the dorsal hippocampus is thought to be important for spatial memory (Moser et al, 1993; Kjelstrup et al, 2002). Although the Morris water maze has been shown to be initially stressful to rodents by increasing levels of circulating corticosterone SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  23 levels, by day 3 of testing corticosterone levels are decreased (Aguilar-Valles et al., 2005). This study suggests that the rats in the Snyder et al (2009b) study may have had higher levels of corticosterone (given that all training was done in one day) while the levels of corticosterone in our study may have habituated over the five days of training. Alternatively, the use of different markers to identify immature neurons or in the timing between training and tissue examination may have also contributed to differences between the studies. Snyder and colleagues (2009b) quantified activation of immature neurons using PSA-NCAM/c-fos. PSA-NCAM is an endogenous protein expressed in developing neurons for 2 to 4 weeks (Seki, 2002). In our study, the exogenous marker BrdU, which is incorporated into dividing cells within a 2-hour period (Nowakowski et al, 1989), was used; therefore our sample of activated new neurons would have been more specifically aged at 3 weeks. It is possible that activated neurons in the study by Snyder and others (2009b) predominantly contained neurons younger than 3 weeks of age. Taken together, results from both studies suggest that the shift in activation patterns of new neurons across the dorsal-ventral axis of the dentate gyrus may occur between 2 to 4 weeks after neuronal birth.  Further research examining the region-specific activation of young neurons over different memory retention periods may resolve the differences between these studies. Differences in water maze training procedures may differentially alter learning and neurogenesis in males and females Exposure to novel environments such as the water maze can be a source of stress in rodents (Hennessy, 1991). Stress can impair learning (Bodnoff et al, 1995) as well as neurogenesis in male rodents (Westenbroek et al, 2004; Brummelte & Galea, 2010). Additionally, Beiko and colleagues (2004) showed that females that were naive to the water maze had higher serum CORT levels and performed more poorly than males. If animals received SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  24 pre-training, however, sex differences were eliminated and CORT levels were reduced relative to naive animals, although female CORT levels were still elevated compared to males. It is possible that in our study, elevated stress levels contributed to a slower rate of spatial learning in females and subsequently resulted in lower rates of cell survival relative to males. However, it is important to keep in mind that while acute or chronic stress suppresses neurogenesis in the hippocampus of males, stress does not always lead to a reduction in neurogenesis in the hippocampus of females (Westenbroek et al, 2004; Falconer and Galea, 1993).  Indeed, while chronic restraint stress can impair spatial learning in males, it can facilitate learning in females (Luine et al, 1994; Bowman et al, 2001). Thus these studies suggest that while stress may influence learning and neurogenesis it does so in a sexually-dimorphic manner. While it is difficult to know, from our present study alone, if the stress of water maze training is related to the effects on hippocampal neurogenesis, we did find that the density of BrdU-labeled cells was not significantly different between females trained in either the spatial or cue task, or from cage controls. This suggests that stress did not play a large role on cell survival in our study. However, Ehninger and Kempermann (2006) showed that water maze-related stress reduced neurogenesis compared to controls in female mice. It is difficult to know whether the discrepancy between our study and the study of Ehninger and Kempermann (2006) is due to inter-species differences, parameters of the Morris water maze training or due to differences in stress susceptibility between the two groups of subjects. Further studies on this issue may provide more definitive answers. Other aspects of water maze training may also have influenced learning and indirectly affected cell survival. Epp and colleagues (2010) observed that increasing task difficulty by reducing the number of extramaze cues increased latency for platform location and decreased SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  25 cell survival in males. Additionally, Roof and Stein (1999) found that manifestation of sex differences in spatial learning varied with slight alterations to task parameters. Releasing animals at different points of the maze between trials impaired learning in females. When the release points were constant between trials, no sex differences were observed. Furthermore, when release points were different between trials but the experimenter remained in the same location when animals were released, females learned as quickly as males. In our study, both the release points and experimenter location varied across trials, which may have prevented females from using their preferred strategy, as altering the position of landmark cues impairs spatial learning in females but not males (Suzuki et al, 1980; Williams & Meck, 1991). Therefore, sex differences in sensitivity to water maze task parameters may have influenced learning and indirectly affected cell survival. Sex differences in learning strategies may influence activation of new neurons in response to spatial memory In the present study, better spatial learning was associated with greater activation of 20-day-old neurons only in females, suggesting either that these new neurons are more excitable in females than in males or that new neurons mature faster in females. Both explanations are plausible, as estradiol influences both neuronal activity and phosphorylation of cyclic AMP response element binding protein, which regulates cell development (Smith and McMahon, 2006; Lee et al, 2004). However, given that we found no sex differences in proportion of cells expressing a mature phenotype, new neurons reach similar levels of maturity by day 20 in males and females. Thus it is more likely that 20-day old neurons were more excitable in response to spatial memory retrieval in females than in males. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  26 While there is evidence that female rodents (and humans) use less hippocampus-dependent strategies (Galea et al, 1996; Galea and Kimura, 1993; Grön et al, 2000), there may be more overall activation in the hippocampus in females during spatial training compared to males (Méndez-López et al, 2009).  There is also evidence that estradiol increases neuronal excitability and potentiates LTP in rodents (Smith and McMahon, 2006) and intriguingly, higher ovarian hormones increase overall brain activity during spatial tasks in women (Dietrich et al, 2001). As mentioned earlier, females may experience greater difficulty in spatial learning due to their choice of learning strategy. Coupled with the fact that task difficulty increases hippocampal involvement (Beylin et al, 2001) while also down-regulating neurogenesis (Epp et al, 2010) it is possible that, in females, immature neurons receive more activation during acquisition and were more responsive to excitatory input. Further research is needed to determine whether maturation and activation of adult-born neurons follow a similar time course in males and females as well as how task difficulty and/or how estradiol alters activation of new neurons. Sex differences in stress, learning, neurogenesis, and new neuron activation Previous studies have shown that females experience more stress during the initial phases of water maze training compared to males, which could negatively influence spatial learning (e.g. Perrot-Sinal et al, 1996; Beiko et al, 2004) and neurogenesis (Ehninger and Kempermann, 2006; Falconer and Galea, 1993). Although the relationship between stress and cell survival is unclear in our study, it is unlikely that sex differences in stress could have influenced our findings on new neuron activation. The dorsal hippocampus has a more important role in spatial learning and memory, whereas the ventral hippocampus regulates stress and emotional responses (Moser et al, 1993; Kjelstrup et al, 2002), and we observed only a significant relationship between spatial SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  27 performance and new neuron activation in the dorsal GCL. If new neuron activation in females was a result of stress, we may have expected to see a negative correlation between BrdU/zif268 expression in the ventral GCL and spatial performance. Furthermore, spatial-trained females did not show significantly more in BrdU/zif268 expression in the ventral GCL compared to males, suggesting again that activation of new neurons during the probe trial was due more to retrieval of spatial memories rather than stress. Conclusion The purpose of this study was to determine how the survival and activation of adult-born hippocampal neurons were affected by spatial training and whether or not these processes were mediated by sex. Males performed better during spatial Morris water maze training compared to females. However, males and females showed similar levels of spatial memory retention, indicating equivalent mastery of the spatial task.  Spatial training enhanced the survival of new neurons in the dentate gyrus of males, but not females. Although no sex differences in cell activation were found, activation of new neurons appears to be regulated differently between the sexes. 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Direct measurement of spontaneous strategy selection in a virtual Morris water maze shows females choose an allocentric strategy at least as often as males do. Behav. Neurosci. 126: 465-478. SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  33     59. van Praag, H., Christie, B.R., Seinowski, T.J., Gage, F.H., 1999. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc. Natl. Acad. Sci. U.S.A. 96, 13427-13431.   60. Vorhees, C.V. and Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 1, 848-858.  61. Warren, S.G., Juraska, J.M., 1997. Spatial and nonspatial learning across the rat estrous  cycle. Behav. Neurosci. 111, 259-266.  62. Westenbroek, C., Den Boer, J.A., Veenhuis, M., Ter Horst, G.J., 2004. Chronic stress and social housing differentially affect neurogenesis in male and female rats. Brain Res. Bull. 64, 303-308.  63. Williams, C.L., Barnett, A.M., Meck, W.H., 1990. Organizational effects of early gonadal secretions on sexual differentiation in spatial memory. Behav. Neurosci. 104, 84-97.  64. Zhang, C.L., Zou, Y., He, W., Gage, F.H., Evans, R.M., 2008. A role for adult TLX positive neural stem cells in learning and behavior. Nature.  451, 1004-1007.   SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  34    Figure Captions Figure 1: (A) Experimental outline. MWM = Morris water maze (B) BrdU-labeled cells in the GCL of the dentate gyrus viewed at 1000x magnification. (C) BrdU-labeled cells in the GCL. (D) Cells in the GCL labeled with the neuronal marker NeuN. (E) Merged image with the arrow pointing to cells co-labeled with BrdU (red) and NeuN (green). Images in figures C to E captured at 400x magnification. (F) BrdU-labeled cells in the GCL. (G) Cells expressing the IEG product zif268 in the GCL. (H) Merged image showing co-expression of BrdU (red) and zif268 (green). Images in figures F to H were captured at 600x magnification. Scale bar = 10µm.  GCL = granule cell layer Figure 2: (A) Mean distance to the hidden platform in males and females of the spatial-trained group. Females swam significantly greater distances before reaching the hidden platform compared to males (p < .001). (B) Mean distance to the visible platform in males and females of the cue-trained group. There were no significant sex differences in the cue-trained group. (C) Mean latency to locate the hidden platform in males and females of the spatial group. Females swam significantly longer times before reaching the hidden platform compared to males (p < .001). (D) Mean latency to locate the visible platform in males and females of the cue group. (E, F) Mean distance to the hidden platform on the first and final trials of training days 1 (E) and 5 (F). Spatial-trained females swam significantly greater distances than males on the final trial of day 1 (p < .01) and showed a trend toward significance on first trial of day 5 (p = .09) but  there were no significant sex differences on the first trial of day 1 or the last trial of day 5, indicating no preexisting sex differences. (G) Mean percentage of time spent in the target quadrant in spatial and cue-trained groups. Spatial-trained groups spent more time in the target quadrant than cue-trained groups (* indicates p < .001). (H) Mean percentage of time spent in the target SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  35 quadrant for proestrous versus non-proestrous females. For proestous females in the cue group, n = 3, and n = 5-6 for proestrous females in the spatial group and non-proestrous females. Females in proestrous spent significantly more time in the target quadrant than non-proestrous females (* indicates  p < .005). Error bars represent +  SEM. Figure 3: (A, B) Mean density of BrdU-labeled cells in the granule cell layer (GCL) (A) and hilus (B) in males and females. (C, D) Mean number of BrdU-labeled cells in the GCL (C) and hilus (D) in males and females Males in the spatial group showed greater BrdU-labeled cell density and counts in the GCL than all other groups (* indicates p < .01). No significant differences were found in the hilus. * indicates p < .05. Error bars represent + SEM. Figure 4: (A, B) Mean percentage of cells co-expressing BrdU and the immediate early gene product zif268 in the dorsal and ventral GCL for animals in the spatial versus cue group with data from males and females combined (A) and shown separately (B). The dorsal GCL had significantly greater levels of BrdU/zif268 co-labeling compared to the ventral region (* indicates p < .001) and spatial trained animals had significantly increased level of cell activation compared to the cue group (p < .022). Error bars represent + SEM. (C,D) Correlation between total swim distance during spatial (C) and cue (D) training and percentage of cells co-expressing BrdU/zif268 in the granule cell layer (GCL) for both males and females. There was a significant negative correlation between total swim distance and BrdU/zif268 co-expression for the spatial group (r(19) = -.49, p = .027). (E,F) Correlation between total swim distance during spatial training and percentage of cells co-expressing BrdU/zif268 in the dorsal GCL for males (E) and females (F). In females, there was a significantly negative correlation between total swim distance and percentage of BrdU/zif268 co-expressing cells in the dorsal GCL (r(8) = -.82, p = .007). Table 1: Mean (+ SEM) volume of the GCL and hilus in male and female rats   Volume (mm3)  N GCL Hilus Male – Spatial 10 1.02 + .07 mm3 2.16 + .05 mm3 Male – Cue  9 1.05 + .09 mm3 2.34 + .08 mm3  Male - Control Female – Spatial 7 10 1.03 + .06 mm3 .90 + .08 mm3 2.18 + .09 mm3 1.96 + .14 mm3 Female - Cue 11 .88 + .04 mm3 1.94 + .11 mm3 Female - Control 7 .86 + .05 mm3 1.82 + .16 mm3 Males, regardless of group, had significantly greater GCL and hilus volume than females, regardless of group (p’s < .04).   Table(s)Table 2: Mean (+SEM) percentage of cells co-expressing BrdU and NeuN in the GCL in male versus female rats.  BrdU/NeuN labeled cells (%)  N Dorsal Ventral Dorsal and ventral Male - Spatial 10 87.59 + 1.89 76.17 + 1.55 82.10 + 1.32 Male – Cue 9 86.40 + 1.66 77.67 + 2.63 81.71 + 1.90 Female - Spatial 10 87.70 + 1.73 79.42 + 3.00 83.38 + 2.25 Female - Cue 8 82.15 + 3.41 73.88 + 2.49 77.76 +  2.65 No significant differences between groups in percentage of BrdU/NeuN co-labeled cells were found.  SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  1                        B C D E F G A H Figure(s)SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  2 Day0 1 2 3 4 5Distance (m)05101520 MaleFemaleHidden platform (Spatial)Day0 1 2 3 4 5Distance (m)05101520 MaleFemaleVisible platform (Cue)Day 1 Trials0 1 2 3 4Distance (m)0510152025MaleFemaleDay 5 Trials0 1 2 3 4Distance (m)0510152025 MaleFemaleSexMale FemalePercentage time (%)010203040SpatialCueEstrous stageProestrus Non-proestrusPercentage time (%)010203040Spatial Cue Day0 1 2 3 4 5Latency (s)102030405060 MaleFemaleDay0 1 2 3 4 5Latency (s)0102030405060 MaleFemaleE F A B C D * *G *H *SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  3    A                                 GCL B                                     Hilus  C D SexMale FemaleBrdU-labeled cells01000200030004000500060007000Spatial Cue Control SexMale FemaleBrdU-labeled cells01000200030004000500060007000SpatialCueControl          SexMale FemaleBrdU-labeled cells (/mm3)01000200030004000500060007000Spatial Cue Control SexMale FemaleBrdU-labeled cells (/mm3)01000200030004000500060007000 SpatialCueControl* * SEX DIFFERENCES IN NEUROGENESIS AND SPATIAL LEARNING  4 A B GCL regionDorsal VentralPercentage BrdU/zif268 (%)0123456Male-Spatial Male-Cue Female-Spatial Female-Cue C D E F GCL regionDorsal VentralPercentage BrdU/zif268 (%)0123456SpatialCueSpatialTotal swim distance (m)0 20 40 60 80Percentage BrdU/zif268 (%)0246810 CueTotal swim distance (m)0 20 40 60 80Percentage BrdU/zif268 (%)0246810Female - spatial (dorsal GCL)Total swim distance (m)0 20 40 60 80Percentage BrdU/zif268 (%)0246810Male - spatial (dorsal GCL)Total swim distance (m)0 20 40 60 80Percentage BrdU/zif268 (%)0246810********r(19) = -.49, p = .027 r(11) = .15, p = .63 r(10) = .12, p = .73 r(8) = -.73, p = .02 Funding body agreements and policies Financial support for this research was provided by the Canadian Institutes of Health Research. *Role of the Funding SourceContributors Carmen Chow performed the experiment and wrote the first draft of the manuscript. Carmen Chow, Jonathan Epp, Stephanie Lieblich, and Liisa Galea designed the study and wrote the protocol. . Stephanie Lieblich and Cindy Barha assisted with the immunohistochemistry portion of the experiment. All authors contributed to and have approved the final manuscript. *Contributors

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