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Sex and strategy use matters for pattern separation, adult neurogenesis and immediate early gene expression… Yagi, Shunya; Chow, Carmen; Lieblich, Stephanie E.; Galea, Liisa A.M. Jan 31, 2016

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1 Sex and strategy use matters for pattern separation, adult neurogenesis and immediate 1 early gene expression in the hippocampus 2 3 Shunya Yagi1, Carmen Chow2, Stephanie E. Lieblich2 and Liisa A.M. Galea1,2,3 4 Graduate Program in Neuroscience1, Department of Psychology2, Centre for Brain 5 Health3, University of British Columbia,Vancouver Canada 6 7 Sex differences in similar pattern discrimination and neurogenesis 8 9 36 text pages, 6 figures, and 4 tables 10 11 Author for correspondence: 12 Dr. Liisa Galea 13 Department of Psychology 14 University of British Columbia 15 2136 West Mall 16 Vancouver, BC 17 Canada, V6T 1Z4 18 Tel: +1 (604) 822 6536 19 Fax: +1 (604) 822 6923 20 Email: lgalea@psych.ubc.ca 21 22 Grant sponsor: NSERC (Natural Sciences and Engineering Research Council of Canada); Grant 23 number: RGPIN 203596-13 to LAMG. 24 25 26 Keywords: dentate gyrus, cognition, zif268, c-Fos, bromodeoxyuridine 27 28 Page 1 of 50 Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Published in: Yagi, S., Chow C,  Lieblich, SE, Galea, L.A.M. (2016) Sex and strategy use matters for pattern separation, adult neurogenesis and immediate early gene expression in the hippocampus. Hippocampus, 26, 87-101 2 Abstract 29 Adult neurogenesis in the dentate gyrus (DG) plays a crucial role for pattern 30 separation and there are sex differences in the regulation of neurogenesis. Although sex 31 differences, favoring males, in spatial navigation have been reported, it is not known 32 whether there are sex differences in pattern separation. The current study was designed 33 to determine whether there are sex differences in the ability for separating similar or 34 distinct patterns, learning strategy choice, adult neurogenesis and immediate early gene 35 (IEG) expression in the DG in response to pattern separation training. Male and female 36 Sprague-Dawley rats received a single injection of the DNA synthesis marker, 37 bromodeoxyuridine (BrdU) and were tested for the ability of separating spatial patterns 38 in a spatial pattern separation version of delayed nonmatching to place task using the 39 8-arm radial arm maze. Twenty eight days following BrdU injection, rats received a 40 probe trial to determine whether they were idiothetic or spatial strategy users. We found 41 that male spatial strategy users outperformed female spatial strategy users only when 42 separating similar, but not distinct, patterns. Furthermore male spatial strategy users had 43 greater neurogenesis in response to pattern separation training than all other groups. 44 Interestingly neurogenesis was positively correlated with performance on similar pattern 45 trials during pattern separation in female spatial strategy users but negatively correlated 46 with performance in male idiothetic strategy users. These results suggest that the 47 survival of new neurons may play an important positive role for pattern separation of 48 similar patterns in females. Furthermore, we found sex and strategy differences in IEG 49 expression in the CA1 and CA3 regions in response to pattern separation. These 50 findings emphasize the importance of studying biological sex on hippocampal function 51 and neural plasticity. 52 Page 2 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603 1. Introduction53 Pattern separation is defined as the process that renders the pattern of 54 information to be stored as distinct from each other during memory encoding and 55 storage, while pattern completion is the process to recover the stored pattern from a 56 degraded or partial retrieval cue during the time of recall and retrieval of memory (Mar, 57 1971). The hippocampus plays critical roles in pattern separation and pattern completion 58 (Marr, 1971) as the hippocampus separates overlapping inputs from the entorhinal 59 cortex by increasing neural sparseness at the level of the dentate gyrus before the 60 information is transferred to the CA3, and subsequently the CA1 subregions (Amaral 61 and Witter, 1989). Both human and rodent studies support the finding that pattern 62 separation occurs at the level of dentate gyrus (Gilbert et al., 2001; Yassa et al., 2010). 63 In particular adult neurogenesis in the dentate gyrus plays a critical role in pattern 64 separation, as disruption of hippocampal neurogenesis disrupted the ability of mice to 65 distinguish between similar patterns in multiple tests of pattern separation in female 66 mice (Clelland et al., 2009). Furthermore increased neurogenesis was commensurate 67 with greater ability of pattern separation in male mice (Chen et al 2012). These studies 68 collectively suggest that neurogenesis in the dentate gyrus is important for the 69 separation of similar patterns. 70 There are numerous examples of sex differences in hippocampal learning and 71 memory, favoring males. Meta-analyses indicate that in both humans and rodents, males 72 outperformed females in spatial tasks (Voyer et al., 1995; Jonasson, 2005). It is 73 important to note that sex differences favoring men generally appear only when greater 74 cognitive demand, and/or perhaps similar patterns, are needed to process extramaze 75 cues (Chamizo et al., 2011). Furthermore there are sex differences in hippocampal 76 Page 3 of 50 Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604 plasticity (Miranda et al., 1999; Dalla et al., 2009) including adult neurogenesis, for 77 example cell proliferation is higher in proestrus females compared to males (Tanapat et 78 al., 1999; reviewed in Galea et al., 2013). However, to date, there are no studies 79 examining sex differences in pattern separation ability and the relationship to 80 neurogenesis in the dentate gyrus. Studying sex differences in hippocampal function and 81 plasticity are important as they may lead us to better understand why women are more 82 vulnerable to neuropsychiatric and neurodegenerative disorders that negatively impact 83 cognition and target the hippocampus such as depression (Gutiérrez-Lobos et al., 2002) 84 and Alzheimer’s disease (Beinhoff et al., 2008). 85 Males and females can use different strategies to solve spatial navigation tasks, 86 which is seen in both humans (Dabbs et al., 1998; Lawton, 1994; Silverman and Choi, 87 2006) and rodents (Korol, 2004; Hawley et al., 2012; Grissom et al, 2013). Females 88 preferentially use striatum dependent (idiothetic) strategies, although this is dependent 89 on ovarian hormone status, while males preferentially use hippocampus dependent 90 (spatial) strategies to solve the same tasks (Williams et al., 1990; Galea and Kimura, 91 1993; Cherney et al., 2008; Grissom et al., 2013). This sex difference in strategy choice 92 may be attributed to different usage of hippocampal neurons. In order to measure the 93 amount of neuron activation in a specified brain area, immediate early gene (IEG) 94 expression, such as c-Fos and zif268, is often examined. IEGs are transcribed rapidly 95 after neuronal stimulation and encode transcription factors that modify gene expression 96 in response to the neuronal stimulation (Sheng and Greenberg, 1990), and play an 97 important role in neural plasticity and memory consolidation (Guzowski et al., 2001; 98 Jones et al., 2001). Male and female rats show different patterns of c-Fos expression in 99 the hippocampus during spatial learning (Méndez-López et al., 2009). Furthermore 100 Page 4 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859605 activation of adult-born granule neurons was associated with better memory retrieval in 101 the Morris water maze in female, but not in male, rats (Chow et al., 2013). Thus 102 collectively these studies suggest that there may be sex differences in activation of new 103 neurons with pattern separation. 104 Therefore, the aim of this study was to explore sex differences in pattern 105 separation ability, strategy choice, neurogenesis and immediate early gene expression in 106 the hippocampus in response to a pattern separation task. To examine this, male and 107 female rats were tested on their ability to separate similar and distinct patterns with a 108 spatial pattern separation version of delayed non-match to place (PSDNMP). We also 109 examined the neurogenesis levels in the hippocampus in response to pattern separation 110 training 27 d after an injection of the DNA synthesis marker, bromodeoxyuridine 111 (BrdU) and after a probe trial to measure cell survival and IEG expression in the 112 hippocampus. We hypothesized that males would outperform females only when 113 separating similar patterns. We also predicted that better performance during pattern 114 separation would be associated with greater cell survival and greater activation of new 115 neurons that would be accompanied by sex differences possibly related to strategy use. 116 117 118 Page 5 of 50 Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606 2. Methods119 2. 1. Animals 120 Thirty-six 8 week old Sprague Dawley rats (males: n = 19; females: n = 17), 121 were purchased from Charles River Canada (St-Constant, Quebec, Canada) for this 122 study. Rats were housed in same sex pairs for 2 weeks after arrival and single-housed 123 throughout the entire experiment. Males and females were housed in separate rooms to 124 avoid odor cues of opposite-sex conspecifics interfering with performance. Rats were 125 housed in opaque polyurethane bins (48 × 27 × 20 cm) with paper towels, 126 polyvinylchloride tube, cedar bedding, and free access to food and water, and 127 maintained under a 12 : 12 hour light/ dark cycle (lights on at 07:00 h). All animals 128 were handled every day for 2 minutes beginning one week after arrival. All experiments 129 were carried out in accordance with Canadian Council for Animal Care guidelines and 130 were approved by the animal care committee at the University of British Columbia. All 131 efforts were made to reduce the number of animals used and their suffering during all 132 procedures. 133 2. 2. Apparatus 134 The radial arm maze had 8 arms (53 cm long × 10 cm wide) and an octagonal 135 center platform (36 cm in diameter) and was set 80 cm above the floor in the center of a 136 dimly lit room. Metal gates were used to block entries to arms. Large extramaze cues 137 were placed on all four walls of the room and were not moved throughout the study. At 138 the end of each arm was a small cup securely glued to hold a sugar reward. 139 2. 3. Procedure 140 2. 3. 1 Experimental timeline 141 One intraperitoneal injection of bromodeoxyuridine (BrdU; 200mg/kg; 142 Page 6 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859607 Sigma-Aldrich, Oakville, ON, Canada) was administered to all animals at 11 weeks of 143 age (experimental Day1). Four days after BrdU injection, all animals were food 144 restricted and maintained at 87-92% of their original weight throughout the entire 145 experiment (Day4-27). One week after BrdU injection, all animals were habituated to 146 the radial arm maze for 2 days (Day 8, 9). Following habituation, rats were shaped for 3 147 consecutive days for 5 minutes each day (Day10-12). Following shaping, all rats were 148 tested in the spatial pattern separation version of delayed nonmatching to place radial 149 arm maze task for 14 days (Day 13-26), followed by a day of probe trial (Day 27) 150 (Figure 1A). 151 2. 3. 2. Habituation and shaping 152 During habituation, rats were placed on the center platform and allowed to 153 explore all arms freely for 10 minutes. During the first day of shaping, 3 quartered pieces 154 of a Froot Loop (Kellog’s) were placed along the length of each arm at equidistant 155 intervals and a quarter was placed in a cup (3 cm in diameter) at the end of each arm. 156 During the second and the third day of shaping, each arm was baited with a quarter of 157 Froot Loops placed in each cup. 158 2. 3. 3. Behavioral testing for spatial pattern separation 159 All testing began at approximately the same time every day at 08:00 h. Rats 160 received 4 trials a day for 14 consecutive days (56 trials total with 28 trials of each 161 separation – adjacent or separate). There was approximately 45 minutes interval 162 between trials for each rat. The orders of testing for each rat were randomized every day. 163 One trial consisted of a sample phase and a choice phase (40 seconds interval between 164 the two phases – see Figure 1B). Rats were tested in their ability to discriminate the 165 newly-opened arm during choice phases. During the sample phase, only a start arm and 166 Page 7 of 50 Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859608 a sample arm were open and all other arms were closed. A rat was placed on the start 167 arm and the rat was allowed to visit the sample arm and retrieve a quarter of Froot Loop 168 (reward). Rats were retrieved from the maze after eating the reward (max 10 seconds). 169 During the choice phase, all arms were blocked off except the start, sample and an 170 additional arm (correct arm). The additional/correct arm, but not the sample arm, was 171 baited during the choice phase. Rats that made incorrect choices (sample arm or start 172 arm) were permitted to self-correct and retrieve the reward from the additional arm. A 173 choice was defined as being made when the entire body, excluding the tail, had entered 174 the arm. Rats were retrieved from the maze after they ate the reward or after 60 seconds 175 had passed and returned to the colony room. During the interval between a sample 176 phase and a choice phase, the maze was rotated to minimize the ability of rats to utilize 177 intramaze cues such as odor. After the rotation, the location of the start and sample arms 178 relative to extramaze cues, but not the arms themselves, were held constant during each 179 trial. The radial arm maze was wiped with distilled water after each rat. 180 Two patterns of sample-correct arm pairs were used in this study, ADJACENT 181 and SEPARATE. Correct arms during ADJACENT trials were 45° away from the 182 sample arm and correct arms during SEPARATE trials were 135° away from the sample 183 arm. Start arms were located perpendicular to either the correct or sample arms (Figure 184 1B). Sample-correct-start arm combinations were pseudo-randomly chosen for each day 185 from the pool of possible combinations so that overlaps in the presentation of arms were 186 minimized both within each day and across the entire experiment. 187 2. 3. 4. Probe trial 188 On the last day, Day 27, rats received one testing trial in the morning between 189 08:00 and 10:00 h. Eighty minutes after the testing trial, rats received a probe trial to 190 Page 8 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859609 determine whether they relied on idiothetic cues or spatial (extramaze) cues to solve the 191 spatial pattern separation version of delayed non-match to place task. A probe trial 192 consisted of a sample phase and a choice phase with a 40 second interval between the 193 two phases. During a sample phase of the probe trial, the same rules as a sample phase 194 of testing trials were applied (Figure 1C). The start arm during sample phase was 195 perpendicular to the sample arm that was located to the right from the start arm. After 196 the sample phase, all arms were blocked off and the maze was rotated. A new start arm 197 was moved to a new location (unlike in the testing trials) and two choice arms were 198 opened after the rotation. The sample arm during choice phase was held the same 199 position as the sample phase, relative to the extramaze cues, and the correct arm was 200 located 135° away from the sample arm. The location of new start arm was 201 perpendicular to the correct arm that was located to the right from the new start arm. In 202 short, the orientation of start-sample arm pair during the sample phase was the same as 203 that of start-correct arm pair. If a rat chose the correct arm, they were categorized as a 204 spatial strategy user but if a rat chose the sample arm they were categorized as an 205 idiothetic strategy user (Figure 1C). Immediately after the probe trial, rats were perfused 206 (about 90 minutes after the testing trial). 207 208 2. 4. Tissue processing 209 Rats were administered an overdose of sodium pentobarbitol and perfused 210 transcardially with 60 ml of 0.9% saline followed by 120 ml of 4% paraformaldehyde 211 (Sigma-Aldrich). Brains were extracted and post-fixed in 4% paraformaldehyde 212 overnight, then transferred to 30% sucrose (Fisher Scientific) solution for 213 cryoprotection and remained in the solution until sectioning. Brains were sliced into 40 214 Page 9 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596010 µm coronal sections using a Leica SM2000R microtome (Richmond Hill, Ontario, 215 Canada). Sections were collected in series of ten throughout the entire rostral-caudal 216 extent of the hippocampus and stored in anti-freeze solution consisting of ethylene 217 glycol, glycerol and 0.1M PBS at -20°C. 218 2. 5. Immunohistochemistry 219 2. 5. 1. BrdU 220 Brain tissue slices were washed with 0.1 M TBS (pH 7.4) three times between 221 each of the following steps. Tissue was pretreated in 0.6% H2O2 for 30 minutes, 222 transferred to 2N HCl and incubated at 37 °C for 30 minutes. Then the tissue was rinsed 223 with 0.1 M borate buffer (pH 8.5) for 10 minutes. After blocking the tissue with TBS+ 224 solution, consisting of 0.3% Triton-X 100 (Sigma), and 3% normal horse serum (Vector 225 Laboratories; Burlingame, CA, USA) in 0.1 M TBS, slices were incubated in a primary 226 antibody solution 1:200 mouse anti-BrdU (Roche Diagnostics, Laval, QC, Canada) and 227 TBS, for 24 hours at 4 °C. This was followed by incubation with the secondary antibody 228 solution containing 1:200 horse anti-mouse biotinylated IgG (Vector Laboratories, 229 Burlington, ON, Canada) in TBS+ for 4 hours at room temperature. Tissue was 230 incubated for 1.5 hour in ABC solution (Vector Laboratories). Tissue slices were then 231 visualized with diaminobenzidine (DAB; Vector Laboratories) solution. The tissue was 232 mounted onto microscope slides, followed by counterstaining with cresyl violet, 233 dehydrated, cleared with xylene and cover-slipped with Permount (Fisher Scientific; 234 Ottawa, ON, Canada). 235 2. 5. 2. Zif268 / c-Fos 236 Brain tissue was rinsed overnight with 0.1 M PBS at 4 °C. The tissue was 237 incubated in 0.6% H2O2 for 30 minutes and then incubated in primary antibody solution 238 Page 10 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596011 containing 1:1000 Rabbit anti-Erg-1 (Santa Cruz Biotechnologies; CA, USA) or 1:1000 239 anti-c-Fos (Santa Cruz Biotechnologies), 0.04% Triton-X, and 3% normal goat serum 240 (NGS; Vector Laboratories) in 0.1 M PBS for 24 hours at 4 °C. Following rinsing the 241 tissue four times, the tissue was incubated in secondary antibody solution consisting of 242 1:1000 goat anti-rabbit biotinylated IgG (Vector Laboratories, Burlington, ON, Canada) 243 in 0.1 M PBS for 24 hours at 4 °C. The tissue was then incubated in ABC solution 244 (Vector Laboratories) for 1 hour at room temperature. Tissue slices were then visualized 245 with diaminobenzidine (DAB; Vector Laboratories) solution and mounted onto 246 microscope slides, followed by dehydrated, cleared with xylene and cover-slipped with 247 Permount (Fisher Scientific; Ottawa, ON, Canada). 248 2. 5. 3. BrdU/NeuN double labelling 249 Brain tissue was prewashed three times with 0.1 M PBS and left overnight at 250 4 °C. The tissue was incubated in a primary antibody solution containing 1:250 mouse 251 anti-NeuN (Milli- pore; MA, USA), 0.3% Triton-X, and 3% normal donkey serum 252 (NDS; Vector Laboratories) in 0.1 M PBS for 24 hours at 4 °C. Tissue was incubated in 253 a secondary antibody solution containing 1:200 donkey anti-mouse ALEXA 488 254 (Invitrogen, Burlington, ON, Canada) in 0.1 M PBS, for 18 hours at 4 °C. After rinsing 255 three times with PBS, tissue was washed with 4% paraformaldehyde, and rinsed twice 256 in 0.9% NaCl, followed by incubation in 2N HCl for 30 minutes at 37 °C. Tissue was 257 then incubated in a BrdU primary antibody solution consisting of 1:500 rat anti-BrdU 258 (AbD Serotec; Raleigh, NC, USA), 3% NDS, and 0.3% Triton-X in 0.1 M PBS for 24 259 hours at 4 °C. Tissue was then incubated in a secondary antibody solution containing 260 1:500 donkey anti-rat Cy3 (Jackson ImmunoResearch; PA, USA) in 0.1 M PBS for 24 261 hours at 4 °C. Following three rinses with PBS, tissue was mounted onto microscope 262 Page 11 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596012 slides and cover-slipped with PVA DABCO. 263 2. 5. 4. BrdU/zif268 double labeling 264 The tissue was prewashed with 0.1 M TBS and left to sit overnight at 4 °C. The 265 next day, tissue was incubated in zif268 primary antibody solution made with 1:1000 266 Rabbit anti- Egr-1 (Santa Cruz Biotechnologies; CA, USA), 3% NDS, and 0.3% 267 Triton-X in 0.1 M TBS for 24 h ours at 4 °C. Then the sections were incubated in 268 secondary antibody solution, consisting of 1:500 Donkey anti-Rabbit ALEXA 488 269 (Invitrogen, Burlington, ON, Canada) in 0.1 M TBS, for 18 hours at 4 °C. The tissue 270 was then rinsed with 4% paraformaldehyde and washed twice in 0.9% NaCl. After 271 incubation in 2N HCl for 30 minutes at 37 °C, slices were incubated with BrdU primary 272 antibody solution consisting of 1:500 mouse anti-BrdU (Roche), 3% NDS, and 0.3% 273 Triton-X in 0.1 M TBS for 24 hours at 4 °C. Then the tissue was incubated with 274 secondary antibody solution consisting of 1:250 Donkey anti-Mouse Cy3 (Jackson 275 ImmunoResearch; PA, USA) in 0.1 M TBS for 16 hours at 4 °C. After three rinses with 276 TBS, slices were mounted onto slides and cover-slipped with PVA DABCO. 277 2. 6. Cell counting 278 All counting was conducted by an experimenter blind to the group assignment 279 of each animal using a Nikon E600 microscope. Immunoreactive cells were determined 280 to be in the dorsal or ventral DG using the criterion defined by Banasr et al. (2006), with 281 sections 6.20-4.00mm from the interaural line defined as dorsal and sections 282 4.00-2.28mm from the interaural line as ventral. Cells were counted separately in each 283 region because the dorsal hippocampus is associated with spatial learning and memory, 284 while the ventral hippocampus is associated with stress and anxiety-like responses 285 (Moser et al., 1993; Kjelstrup et al., 2002). 286 Page 12 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596013 BrdU-ir cells were counted under a 100x oil immersion objective lens (Figure 287 3B) using light microscopy. For BrdU-ir cells, every 10th section of the granule cell 288 layer (GCL) that includes the subgranular zone were counted. Total immunoreactive 289 cells per region were estimated by multiplying the aggregate number of cells per region 290 by 10 (Epp et al., 2007). Density of BrdU-ir cells was calculated by dividing the total 291 immunoreactive cells in the GCL by volume of the corresponding region. Volume 292 estimates of the dentate gyrus were calculated by multiplying the summed areas of the 293 dentate gyrus by distance between sections (400µm; using Cavalieri’s principle; 294 Gundersen and Jensen, 1987). Area measurements for the dentate gyrus were obtained 295 using digitized images on the software ImageJ (NIH). 296 The percentages of BrdU/NeuN and BrdU/zif268 double-labeled cells were 297 obtained by randomly selecting 50 BrdU-ir cells and calculating the percentage of cells 298 that coexpressed NeuN or zif268 under 400x magnification using a Nikon E600 299 epifluorescent microscope (Figure 3D-F). 300 2. 6. 3. zif268 and c-Fos expression 301 Optical density of zif268 and c-Fos expression in the dentate gyrus, CA1 and 302 CA3 were analyzed as an estimate of the proportion of immunoreactive cells in the 303 subregions. Images of the hippocampus were acquired at 40× magnification from three 304 sections from the dorsal hippocampus and three sections from the ventral hippocampus 305 on a Nikon E600 light microscope (see Figure for zif268: 4C-E; c-Fos: 4H-J). The 306 proportion of area that exhibited above-threshold zif268 and c-Fos immunoreactive 307 intensity in the corresponding subregions was obtained using ImageJ with digitized 308 images. The threshold was set to 2.5 times above the background gray levels (Hartig, 309 2013). The background gray levels were the mean gray values that were obtained from 310 Page 13 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596014 three randomly selected areas without immunoreactivity. The total value of optical 311 density for each brain was calculated by dividing the total immunoreactive areas by the 312 total area of the corresponding subregions on the three sections. 313 2. 7. Data analyses 314 All analyses were conducted using Statistica (Statsoft Tulsa, OK) and 315 significance level was set at α = 0.05. The percentage of correct choices during 316 ADJACENT and SEPARATE trials in the radial arm maze were each analyzed using 317 analysis of variance (ANOVA), with strategy choice (spatial, idiothetic) and sex (male, 318 female) as between-subject variables. Chi-square analysis was used for strategy choice 319 across sex and estrous cycle phase. Repeated-measures ANOVAs were used to 320 separately analyze the density of BrdU-ir cells, optical density of zif268 and c-Fos 321 expression, and volume of dentate gyrus with strategy choice and sex as between 322 subject factors and hippocampal subregion (dorsal, ventral) as within-subject factors. 323 For percentage of cells co-expressing BrdU/NeuN or BrdU/zif268, repeated-measures 324 ANOVAs were performed with sex and strategy choice as between-subject variables. 325 Pearson product-moment correlations were calculated to examine the relationship 326 between spatial pattern separation performance and density of BrdU/NeuN cells, zif268 327 expression, c-Fos expression or cells co-expressing BrdU/zif268. Post-hoc tests utilized 328 the Neuman-Keuls procedure. A priori comparisons were subjected to Bonferroni 329 corrections. 330 331 3. Results332 3. 1. Behavioral testing 333 3. 1. 1. Male spatial strategy users made more correct choices than females in 334 Page 14 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596015 ADJACENT trials, but not in SEPARATE trials 335 Males had significantly greater percentage of correct arm choices than females 336 for ADJACENT trials [main effect of sex: F( 1, 32 ) = 4.39, p = 0.044, all other main 337 and interaction effects: p > 0.15; see Figure 2A]. However a priori we expected there to 338 be a strategy difference and when broken down by strategy group, a priori tests revealed 339 that only male spatial strategy users chose significantly greater percentage of correct 340 choices than female spatial strategy users in ADJACENT trials (p = 0.011; see Figure 341 2B), but not in idiothetic strategy users (p = 0.69) (Figure 2C). 342 Analyzing the percent correct responses in SEPARATE trials revealed a trend 343 for spatial strategy users to have more correct choices than idiothetic strategy users 344 (main effect of strategy, p<0.06) but no other significant effects (all p’s >0.57). 345 As the range of scores may influence whether or not correlations between 346 variable are seen we also examined the range of performance scores. The range in 347 performance scores across all ADJACENT trials and strategy users was similar in males 348 (47.6-95.7%, range =48.1) and females (44.4-88%, range =44.5). When we broke this 349 down by strategy use and ADJACENT trials versus SEPARATE trials, performance 350 scores of ADJACENT trials the range values were 22.37 in male spatial strategy users, 351 26.9 in female spatial strategy users, 48 in male idiothetic strategy users and 44 in 352 female strategy users. Performance scores of SEPARATE trials range 27.59 in male 353 spatial strategy users, 45.6 in female spatial strategy users, 21 in male idiothetic strategy 354 users and 38 in female idiothetic strategy users. 355 356 3. 1. 2. There were no significant sex or estrous cycle differences in the number of 357 strategy users 358 Page 15 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596016 There was no significant difference in the distribution of strategy choices 359 between males and females (χ2 = 0.139, p = 0.709), nor between proestrous and 360 non-proestrous females (χ2 = 0.131, p = 0.252; see Table 1). 361 3. 2. Males had a significantly larger dentate gyrus than females 362 Males had a larger dentate gyrus volume than females in both dorsal and 363 ventral regions despite the significant interaction [region by sex interaction: F(1,34) = 364 11.72, p = .0016, main effect of sex: F(1,34) = 51.41, p < .0001; main effect of region: 365 F(1.34) = 74.38, p <.0001; see Table 2]. Because there were sex differences in the 366 volume of dentate gyrus, cell density was used in order to directly compare the sexes 367 without volume being a confounding variable. 368 3. 3. Male spatial strategy users had greater neurogenesis in the dorsal dentate gyrus 369 than male idiothetic strategy users and females 370 Male spatial strategy users had greater density of BrdU-ir cells in the dorsal 371 DG compared to all other groups [all p’s <0.004; sex by strategy interaction: p = 0.031; 372 see Figure 3A also a main effect of region: F(1,26) =7.63, p =0 .011;]. A ‘neurogenesis 373 index’ was also calculated by multiplying the density of BrdU-ir cells by percentage of 374 BrdU-ir cells that also co-expressed NeuN (Snyder et al., 2009). Male spatial strategy 375 users had significantly greater BrdU/NeuN cell density than all other groups in the 376 dorsal dentate gyrus [all p’s <0.005; region by sex by strategy interaction: F(1,26) = 377 4.09, p = 0.05; main effect of region: F(1,26) =9.98, p < 0.004; sex by strategy 378 interaction p < 0.07; see Figure 3C]. 379 There was greater percentage of BrdU-ir cells co-expressing NeuN in the 380 dorsal compared to the ventral dentate gyrus [main effect of region: F(1,27) = 13.175, 381 p = 0.001] and a trend for males to have a greater percentage of BrdU/NeuN co-labelled 382 Page 16 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596017 cells than females (p = 0.061; see Table 3). 383 There were very few cells co-expressing BrdU/zif268 (see Table 4). There was 384 no significant difference between groups for the percentage of cells co-expressing 385 BrdU/zif268 (all p’s > 0.09). 386 3. 4. zif268 expression in the dorsal CA3 was greater in females than males 387 Females had significantly greater density of zif268 expression in the dorsal 388 CA3, but not in the ventral CA3, than males [sex by region: F(1, 31) = 12.79, p = 0.001; 389 see Figure 4A]. Zif268 expression in the dorsal CA1 was greater than in the ventral 390 CA1 [region: F(1,30) = 105.2, p < 0.001; see Figure 4B]. There were no other 391 significant main or interaction effects (p’s > .20 ). There were no significant main or 392 interaction effects in expression of zif268 in the dentate gyrus (p’s > 0.22). 393 3. 5. c-Fos expression in the CA1 region was greater in idiothetic strategy users than 394 spatial strategy users, while in the CA3 region males had greater expression in the 395 dorsal CA3 than females, and there was greater expression in the dorsal dentate gyrus 396 compared to ventral dentate gyrus 397 In the dentate gyrus, there was greater c-Fos expressing cell density in the 398 dorsal dentate gyrus than ventral dentate gyrus [main effect of region: F( 1, 32 ) = 14.7, 399 p < 0 .001]. For the CA3 region, males had greater expression of c-Fos in the dorsal 400 CA3 than all other groups but there were no other significant effects [sex by region: F( 1, 401 32 ) = 8.05, p < 0.001; main effects of sex and region: both p’s <0.001; see Figure 4F]. 402 In the CA1 region, idiothetic strategy users had greater c-Fos expression than spatial 403 strategy users [main effect of strategy: F(1,32) = 7.20, p < 0.011; see Figure 4G] and 404 greater c-Fos expression in ventral compared to dorsal CA1 [F(1,32) = 56.3, p < 0.0001] 405 but no other significant effects (p’s > 0.28). 406 Page 17 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596018 407 3. 6. Correlations: 408 3. 6. 1. Greater BrdU/NeuN cell density was associated with better performance during 409 a pattern separation task in spatial strategy users but not in idiothetic strategy users. 410 These correlations were stronger in females than in males. 411 Proportion of correct choices during ADJACENT trials was positively 412 correlated with BrdU/NeuN co-expressing cell density in the dorsal dentate gyrus [r(17) 413 = .571, p =0 .017 ] and in the ventral dentate gyrus [r(17) = .663, p = 0.004] in spatial 414 strategy users, but not in idiothetic strategy users (p’s > .44; see Figure 5). Proportion of 415 correct choices in SEPARATE trials and BrdU/NeuN co-expressing cell density in the 416 ventral dentate gyrus was positively correlated in spatial strategy users [r(17) = .600, p 417 = 0.011] and negatively correlated in idiothetic strategy users [r(14) = -.626, p =0 .017]. 418 When broken down by sex, in female rats, proportion of correct choices during 419 ADJACENT trials was positively correlated with BrdU/NeuN co-expressing cell 420 density in the ventral dentate gyrus [r(13) = .574, p = .040], but not in male rats [r(18) 421 = .627, p < 0.81]. When broken down by sex and strategy users, proportion of correct 422 choices during ADJACENT trials was positively correlated with BrdU/NeuN 423 co-expressing cell density in the ventral dentate gyrus in female spatial strategy users 424 [r(8) =0 .838, p =0.009; see Figure 5B], negatively correlated in male idiothetic strategy 425 users [r(9) = -0.657, p = 0.05; see Figure 5B] and a trend for a positive correlation in 426 male spatial strategy users [r(9) = 0.603, p = 0.086; see Figure 5B] but no other 427 significant correlation in other groups (p > .10). 428 3. 6. 3. Zif268 expression in the ventral CA3 and CA1 was negatively correlated to 429 performance of pattern separation in male spatial strategy and idiothetic strategy users 430 Page 18 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596019 but not in females. 431 Proportion of correct choices during ADJACENT trials was negatively 432 associated with zif268 expressing cell density in the ventral CA3 in spatial strategy 433 users [all spatial strategy users: r(20) = -.440, p = 0.05; see Figure 6A], and in both 434 males and females [all males: r(19) = -.446, p = 0.056; all females: r(17) = -.465, p = 435 0.06]. However, when broken down by sex and strategy, there were no significant 436 correlations (p’s > 0.12). 437 Zif268 expression in the ventral dentate gyrus was negatively correlated with 438 proportion of correct choices during ADJACENT trials in male idiothetic strategy users 439 [r(9) = -.725,  p = 0.027, See Figure 6B] and during SEPARATE trials in male spatial 440 strategy users [r(10) = -.648,  p = 0.043] but no significant correlations were seen in 441 females (p’s > 0.30). 442 Zif268 expression in the ventral CA1 was negatively correlated to proportion 443 of correct choices during ADJACENT trials in male idiothetic strategy users [r(9) = 444 -.745,  p = .021; see Figure 6C]. There was no other significant correlations between 445 zif268 expression in the CA1, DG or CA3 and performance in ADJACENT or 446 SEPARATE trials (p’s >.10). 447 3. 6. 4. Performance in SEPARATE trials was positively correlated with dorsal CA3 448 c-Fos expression in male spatial strategy users, but negatively correlated in dorsal CA1 449 c-Fos expression in male idiothetic and female spatial strategy users. Performance 450 during ADJACENT trials was positively correlated with dorsal CA1 c-Fos expression in 451 female spatial strategy users. 452 Greater c-Fos expression in the dorsal CA3 was associated with better 453 performance in SEPARATE trials in males [r(19) = 0.534, p = 0.019], in spatial strategy 454 Page 19 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596020 users [r(20) =0.533, p =0.016], and specifically in male spatial strategy users [r(10) 455 =0 .806, p =0.005]. There was no other significant correlations in any other groups in 456 the CA3 (p’s > .26) or in the dentate gyrus (all p’s >0.05). 457 In males, greater c-Fos expression in the dorsal CA1 was negatively correlated with 458 proportion of correct choices during SEPARATE trials in males [r(19) = -0.542, p = 459 0.017] and females [r(19) = -.577, p = 0.010]. When broken down by strategy users, 460 greater c-Fos expression in the dorsal CA1 was negatively correlated with proportion of 461 correct choices during SEPARATE trials in male idiothetic strategy users [r(9) = -.706, 462 p = 0.034 ] and in female spatial strategy users [r(9) = -.810, p = 0.008]. In ADJACENT 463 trials, c-Fos expression in the dorsal CA1 was negatively correlated with proportion of 464 correct trials [r(9) = -.775, p = 0.014; see Figure 6D], in female spatial strategy users 465 only. However, there were no significant correlations between c-Fos expression in the 466 dentate gyrus and performance (all p’s > 0.06). 467 4. Discussion468 In the present study we found sex differences in pattern separation performance, 469 neurogenesis in response to pattern separation training and in associations of 470 neurogenesis and activation with performance. While we found that males outperformed 471 females on similar patterns (during ADJACENT trials), there was no sex difference in 472 performance of distinct patterns (during SEPARATE trials) in the spatial pattern 473 separation version of delayed non-match to place radial arm maze task. This effect was 474 stratified by strategy users as the sex difference in performance in similar pattern 475 (ADJACENT) trials emerged only in the spatial strategy users, but not in idiothetic 476 strategy users. Furthermore, male spatial strategy users had greater neurogenesis in the 477 dorsal dentate gyrus than all other groups. In addition in the dorsal CA3 we saw sex 478 Page 20 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596021 differences in zif268 and c-Fos expression patterns with females exhibiting greater 479 expression of zif268 and males exhibiting greater expression of c-Fos. Furthermore, 480 c-Fos expression in the CA1 region was greater in idiothetic strategy users than spatial 481 strategy users. There were also regional differences with greater dorsal expression in 482 IEG expression in the CA1 and DG regions. Finally neurogenesis and IEG expression 483 were significantly correlated with performance stratified by sex and strategy use. 484 Greater levels of neurogenesis was associated with better performance during 485 ADJACENT trials in the female spatial strategy users, but worse performance during 486 ADJACENT trials in male idiothetic strategy users. Zif268 expression in the ventral 487 dentate gyrus and CA1 was negatively correlated to performance of pattern separation 488 in male idiothetic strategy users. c-Fos expression in the dorsal CA1 was negatively 489 correlated with pattern separation (SERPATE and ADJACENT) in female spatial 490 strategy users. Thus, sex differences in the patterns of IEG expression and correlations 491 of IEG expression with performance varies based on location, type of pattern separation 492 and strategy use. Together these results demonstrate that male spatial strategy users 493 show superior performance and greater neurogenesis in the dentate gyrus than females, 494 but exhibit fewer associations with IEG expression and performance. These findings 495 collectively suggest that there are sex differences that extend beyond performance level 496 and are reflected in the neural response to pattern separation training. 497 498 4. 1. Male spatial strategy users are better at separating similar patterns than female 499 strategy users 500 In the present study, males outperformed females during similar (ADJACENT) 501 pattern trials, but not during distinct (SEPARATE) pattern trials. Because extramaze 502 Page 21 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596022 cues overlap more in the ADJACENT condition versus the SEPARATE condition, this 503 results in more similar patterns in ADJACENT versus distinct patters in SEPARATE 504 arm pairs, thus greater pattern separation ability is needed in ADJACENT trials. These 505 results indicate that males perform better than females on the ability to separate similar, 506 but not distinct, patterns. This finding is consistent with previous studies showing that 507 sex differences favoring males in spatial learning are more prominent when spatial cues 508 overlap to greater degrees – indicating the need for better pattern separation under 509 similar conditions (van Haaren et al., 1987; Williams et al., 1990; Chamizo et al., 2011). 510 Furthermore, our findings that the sex differences were only observed in spatial strategy 511 users are somewhat consistent with Chow et al. (2013), who showed sex differences, 512 favoring males, in acquisition of the spatial Morris water maze only among 513 spatially-trained, but not cue-trained, rats. 514 In the present study we found no significant sex difference in strategy choice. 515 This finding is inconsistent with previous findings that males rely more on hippocampus 516 dependent spatial strategies while females rely more on striatum dependent response 517 strategies (Hawley et al., 2012). Nevertheless our findings are consistent with another 518 study demonstrating that both males and females do not show preference of strategy 519 choice when spatial and response strategies are equally effective to reach a goal location 520 (van Gerven et al., 2012). Indeed in our study spatial users were not more effective than 521 idiothetic users overall, indicating that both strategies were equally effective. However, 522 although not statistically significant more proestrous females were spatial strategy users 523 than idiothetic strategy users, which is consistent with previous studies that proestrous 524 females rely more on hippocampus dependent spatial strategies (Korol et al., 2004; 525 Rummel et al., 2010). Collectively these findings suggest that because sex differences in 526 Page 22 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596023 spatial learning and pattern separation were limited to hippocampal-based strategy users 527 that any sex differences in hippocampal neurogenesis may tightly link to spatial pattern 528 separation. 529 530 4. 2. Male spatial strategy users showed greater cell survival of adult-born neurons in 531 the dorsal dentate gyrus than all other groups. 532 In the present study, we found greater neurogenesis (27 day old neurons) in the 533 dorsal dentate gyrus of male spatial strategy users compared to all other groups. This 534 finding is partially consistent with a previous finding showing that males that had 535 undergone spatial training where more likely to show greater neurogenesis than females 536 that had undergone spatial training (Chow et al., 2013). The greater levels of 537 neurogenesis in male spatial strategy users could be due to either a consequence of the 538 pattern separation training or may be better related to better innate ability in these males. 539 It is difficult to tease these explanations apart with the paradigm we have used. In the 540 latter explanation, subjects with greater adult neurogenesis may be able to recruit new 541 neurons to perform pattern separation or may be better at hippocampus-dependent 542 strategy compared to striatum-dependent strategies. Consistent with this explanation, a 543 previous study showed that inactivation of hippocampus results in shifts from spatial 544 strategy to response strategy (Packard and McGaugh, 1996). However it is also possible 545 that 14 testing days using a spatial strategy enhanced the survival of new neurons in the 546 dorsal dentate gyrus in males. This is partially consistent with studies showing that 547 exposure to spatial training 6-10 days after a BrdU injection enhances survival of 548 immature neurons in male but not in female rats (Chow et al. 2013) in the Morris water 549 maze. More studies have been done in males and spatial training 6-10 (Epp et al., 2007) 550 Page 23 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596024 or 7-14 days (Gould et al., 1999) after a BrdU injection increases neurogenesis in male 551 rats. However, there are studies that failed to find enhancement of cell survival by 552 spatial training in either males or females (Van der Borght et al., 2005; Mohapel et al., 553 2006; Epp et al., 2007; Barha and Galea., 2013). These inconsistencies may be due to 554 the use of different tasks, food restriction requirements, different timing of BrdU to 555 training, and/or duration of spatial learning (see Epp et al., 2013 for review). For 556 instance, there is no significant effect of spatial learning on survival of BrdU-ir cells 557 when rats received spatial training in the Morris water maze either 1-5, 7-9, 7-11 or 558 11-15 days after a BrdU injection (Epp et al., 2007; Mohapel et al., 2006; Van der 559 Borght et al., 2005). Furthermore, BrdU-ir cell survival decreases with longer exposure 560 to spatial learning in the Morris water maze 1-14 days after a BrdU injection in male 561 rats (Mohapel et al., 2006), and in the radial arm maze 1-33 days after a BrdU injection 562 in ovariectomized female rats (Barha and Galea., 2013). This reduction with prolonged 563 duration of spatial learning may be due to task associated stress and task difficulty. It 564 has been reported that chronically high levels of corticosterone or chronic stress reduces 565 cell survival (Pham et al., 2003; Brummelte and Galea., 2010) and increasing difficulty 566 in spatial learning tasks also reduces cell survival (Epp et al., 2010). In the present study, 567 rats were exposed to two weeks of spatial pattern separation task in the radial arm maze 568 and experienced three weeks of food restriction. It is possible that two weeks of spatial 569 pattern separation task could rescue the reduction of cell survival due to food restriction 570 and/or stress of task performance only in male spatial strategy users, but not in the other 571 groups. It is also possible that male spatial strategy users are more likely to have higher 572 levels of neurogenesis innately. However, in another study, hippocampus-dependent 573 strategy users were more likely to show lower levels of cell proliferation and no 574 Page 24 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596025 significant differences were observed in neurogenesis levels (Epp and Galea, 2009), 575 contrary to the findings of the present study. With the current design of this experiment 576 it is not possible to distinguish between the two possible explanations of why male 577 strategy users have greater levels of neurogenesis (innate difference or difference as a 578 result of testing). However it is clear that males that use a spatial strategy not only 579 perform better when distinguishing between similar patterns but also have greater levels 580 of neurogenesis in the dentate gyrus than females or males that use an idiothetic strategy. 581 In the present study, we found greater discrepancies for range of performance scores 582 during SEPARATE trials between males and females but not during ADJACENT trials. 583 Greater spread of the performance scores may influence correlation probabilities 584 between performance during SEPARATE trials and neurogenesis or IEG expression, 585 however there were very few correlations involving SEPARATE trials. 586 587 4. 3. There were very few BrdU labeled cells co-expressing zif268. 588 In the present study, we found very few (1.5%) cells that co-expressed BrdU 589 and zif268, partially inconsistent with previous studies which showed 2-7% co-labelling 590 (Snyder et al., 2009; Chow et al., 2013; McClure et al., 2013). This inconsistency may 591 be because of age of neurons, type of task, or presence of food restriction. In the present 592 study we examined 27 d old BrdU-ir cells after food restriction in the pattern separation 593 task while after water maze training Chow et al. (2013) and McClure et al. (2013) 594 examined zif268 expression in 20 day old BrdU-ir cells and Snyder et al. (2009) 595 examined zif268 expression in 14-28 day old BrdU-ir cells in rats. It is also possible 596 that new neurons are recruited at different times for pattern separation and we were too 597 late for these BrdU-ir cells to be recruited for pattern separation. A recent study is 598 Page 25 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596026 consistent with this interpretation as only young adult-born neurons are recruited for 599 pattern separation while 6 weeks or older adult-born and developmental-born neurons 600 do not play a role for pattern separation in mice (Nakashiba et al., 2012). Maturation of 601 adult-born neurons differs between mice and rats, faster in rats compared to mice 602 (Snyder et al., 2009). Therefore, 4 weeks old dentate granule neurons in rats in the 603 present study may not play a large role for pattern separation. More research examining 604 shorter timelines for BrdU/zif268 co-expression after a pattern separation task are 605 needed to determine the timing of when new neurons are activated in response to pattern 606 separation perforamnce. 607 608 4. 4. Adult neurogenesis in the ventral dentate gyrus was tightly linked to the ability to 609 separate similar patterns in female spatial strategy users. 610 In the present study, we found a significant positive correlation for spatial 611 strategy users showing better performance on similar pattern trials (ADJACENT) being 612 associated with greater neurogenesis in the dorsal DG. However when broken down by 613 sex, correlations only remained for the ventral DG as we found that proportion of 614 correct choices during ADJACENT trials was positively correlated with neurogenesis in 615 female spatial strategy users and negatively correlated with neurogenesis in male 616 idiothetic strategy users. The dorsal hippocampus is thought to be more responsible for 617 spatial learning while the ventral hippocampus is thought to be more responsible for 618 stress and anxiety (Moser et al., 1993; Kjelstrup et al., 2002). However, our results 619 suggest that ventral dentate gyrus may play an important role for spatial pattern 620 separation in females. Studies demonstrate that the ventral hippocampus plays a crucial 621 role for spatial working memory (Moser et al, 1993; Nott and Levin, 2006), during early 622 Page 26 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596027 stages of a goal oriented task in males (Ruediger et al., 2012) and for pattern separation 623 using odor discrimination (Weeden et al., 2012). It is also possible that the greater cell 624 survival of adult-born dentate granule neurons in the ventral dentate gyrus contributes to 625 mitigating anxiety during spatial testing and enhances performance in spatial strategy 626 users. The radial arm maze task and food restriction can be a source of stress in rodents 627 (Bodnoff et al., 1995; Hennessy, 1991), and greater neurogenesis in the ventral dentate 628 gyrus may rescue impairment driven by task related stress. These studies suggest that 629 adult neurogenesis in the ventral dentate gyrus may play a role for spatial pattern 630 separation either directly or indirectly in females. Further studies are necessary to 631 determine contributions of adult-born neurons in the dorsal and ventral dentate gyrus to 632 spatial pattern separation in both males and females. 633 634 4. 5. Zif268 activation in the dentate gyrus in male striatum-dependent learners, and 635 c-Fos activation in the CA1 region in female hippocampus-dependent learners may 636 interfere with pattern separation. 637 In the present study, greater zif268 expression in the ventral dentate gyrus and 638 in the CA1 region was associated with poorer performance of pattern separation only in 639 male idiothetic strategy users. This result suggests that striatum-dependent idiothetic 640 strategy users may recruit hippocampal neurons in a different way from 641 hippocampus-dependent spatial strategy users.  Greater zif268 activation in the 642 hippocampus may interfere with performance of spatial pattern separation in male 643 idiothetic strategy users because they rely more on striatal neurons and greater 644 activation of hippocampal neurons may induce greater noise during competition 645 between the two regions. In the present study, there was no association between 646 Page 27 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596028 behavioral performance and IEG expression in the dentate gyrus in 647 hippocampus-dependent spatial learners. This result partially supports the interpretation 648 that only dentate granule cells in a particular age play a role for pattern separation 649 (Nakashiba et al., 2012). We also found that greater c-Fos expression in the CA1 region 650 was associated with poorer performance in spatial pattern separation in female spatial 651 strategy users. This suggests that greater c-Fos activation may interfere with spatial 652 pattern separation in female spatial strategy users. Thus future studies need to determine 653 interactions among hippocampal subregions during pattern separation and pattern 654 completion in both males and females. 655 656 5. Conclusion657 We found sex differences in pattern separation ability for similar patterns and 658 adult neurogenesis in the dentate gyrus in a learning strategy dependent manner. These 659 findings highlight the importance of biological sex on hippocampal function and neural 660 plasticity. There were significant associations between adult neurogenesis and 661 performance of pattern separation stratified by sex and strategy use. Our findings of low 662 activation of new neurons, despite these new neurons being required for pattern 663 separation (Clelland et al, 2009) suggest that there may be a specific timeline for 664 adult-born neurons to be recruited during pattern separation task. Our findings further 665 emphasize the need for more research in examining the underlying mechanisms for sex 666 differences in hippocampus function and the time/age-dependent role of adult-born 667 neurons in pattern separation. It is vital to study sex differences in hippocampal 668 plasticity in response to hippocampus-dependent training as these findings may provide 669 important information for understanding the mechanisms for sex differences in severity 670 Page 28 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596029 and incidence of cognitive impairment in neuropsychiatric and neurodegenerative 671 disorders. 672 673 Acknowledgements. We would like to thank Alice Chan, Anne Cheng and Lucille 674 Hoover for their assistance with this work. This research was funded by NSERC 675 Discovery Grant (RGPIN 203596-13) to LAMG. 676 677 Page 29 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596030 References 678 Abraham WC, Mason SE, Demmer J, Williams JM, Richardson CL, Tate WP, Lawlor P 679 A, Dragunow M. 1993. 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Sex Roles 30:765–779. 782 Luine V, Villegas M, Martinez C, McEwen BS. 1994. Repeated stress causes reversible 783 impairments of spatial memory performance. Brain Res 639:167–170. 784 Marr D. 1971. Simple Memory: A Theory for Archicortex. Philos Trans R Soc London 785 Ser B, Biol Sci 262:24–80. 786 Page 33 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596034 McClure RES, Barha CK, Galea LAM. 2013. 17β-Estradiol, but not estrone, increases 787 the survival and activation of new neurons in the hippocampus in response to 788 spatial memory in adult female rats. Horm Behav 63:144–57. 789 Méndez-López M, Méndez M, López L, Arias JL. 2009. Sexually dimorphic c-Fos 790 expression following spatial working memory in young and adult rats. Physiol 791 Behav 98:307–17. 792 Miranda P, Williams CL, Einstein G. 1999. Granule cells in aging rats are sexually 793 dimorphic in their response to estradiol. J Neurosci 19:3316–3325. 794 Mohapel P, Mundt-Petersen K, Brundin P, Frielingsdorf H. 2006. Working memory 795 training decreases hippocampal neurogenesis. Neuroscience 142:609–613. 796 Moser E, Moser MB, Andersen P. 1993. Spatial learning impairment parallels the 797 magnitude of dorsal hippocampal lesions, but is hardly present following ventral 798 lesions. J Neurosci 13:3916–3925. 799 Nakashiba T, Cushman JD, Pelkey KA, Renaudineau S, Buhl DL, McHugh TJ, Barrera 800 VR, Chittajallu R, Iwamoto KS, McBain CJ, Fanselow MS, Tonegawa S. 2012. 801 Young dentate granule cells mediate pattern separation, whereas old granule cells 802 facilitate pattern completion. Cell 149:188–201. 803 Nott A, Levin ED. 2006. Dorsal hippocampal α7 and α4β2 nicotinic receptors and 804 memory. Brain Res 1081:72–78. 805 Packard MG, McGaugh JL. 1996. Inactivation of hippocampus or caudate nucleus with 806 lidocaine differentially affects expression of place and response learning. 807 Neurobiol Learn Mem 65:65–72. 808 Pham K, Nacher J, Hof PR, McEwen BS. 2003. Repeated restraint stress suppresses 809 neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate 810 gyrus. Eur J Neurosci 17:879–886. 811 Ruediger S, Spirig D, Donato F, Caroni P. 2012. Goal-oriented searching mediated by 812 ventral hippocampus early in trial-and-error learning. Nat Neurosci 15:1563–1571. 813 Page 34 of 50Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596035 Rummel J, Epp JR, Galea LAM. 2010. Estradiol does not influence strategy choice but 814 place strategy choice is associated with increased cell proliferation in the 815 hippocampus of female rats. Horm Behav 58:582–90. 816 Sheng M, Greenberg ME. 1990. The regulation and function of c-fos and other 817 immediate early genes in the nervous system. Neuron 4:477–485. 818 Silverman I, Choi J. 2006. Non-Euclidean navigational strategies of women: 819 compensatory response or evolved dimorphism? Evol Psychol 4:75–84. 820 Snyder JS, Choe JS, Clifford MA, Jeurling SI, Hurley P, Brown A, Kamhi JF, Cameron 821 H a. 2009. Adult-born hippocampal neurons are more numerous, faster maturing, 822 and more involved in behavior in rats than in mice. J Neurosci 29:14484–14495. 823 Tanapat P, Hastings NB, Gould E. 2005. Ovarian steroids influence cell proliferation in 824 the dentate gyrus of the adult female rat in a dose- and time-dependent manner. J 825 Comp Neurol 481:252–65. 826 Van der Borght K, Wallinga AE, Luiten PGM, Eggen BJL, Van der Zee EA. 2005. 827 Morris water maze learning in two rat strains increases the expression of the 828 polysialylated form of the neural cell adhesion molecule in the dentate gyrus but 829 has no effect on hippocampal neurogenesis. Behav Neurosci 119:926–932. 830 Van Gerven DJH, Schneider AN, Wuitchik DM, Skelton RW. 2012. Direct 831 measurement of spontaneous strategy selection in a virtual morris water maze 832 shows females choose an allocentric strategy at least as often as males do. Behav 833 Neurosci 126:465–478. 834 Van Haaren F, Wouters M, Van De Poll NE. 1987. Absence of behavioral differences 835 between male and female rats in different radial-maze procedures. Physiol Behav 836 39:409–412. 837 Voyer D, Voyer S, Bryden MP. 1995. Magnitude of sex differences in spatial abilities: 838 A meta-analysis and consideration of critical variables. Psychol Bull 117:250–270. 839 Weeden CSS, Hu NJ, Ho LUN, Kesner RP. 2014. The role of the ventral dentate gyrus 840 in olfactory pattern separation. Hippocampus 24:553–559. 841 Page 35 of 50 Hippocampus12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596036 Williams CL, Barnett AM, Meek WH. 1990. Organizational Effects of Early Gonadal 842 Secretions on Sexual Differentiation in Spatial Memory. Behav Neurosci 104:84–843 97. 844 Wisden W, Errington ML, Williams S, Dunnett SB, Waters C, Hitchcock D, Evan G, 845 Bliss TV, Hunt SP. 1990. Differential expression of immediate early genes in the 846 hippocampus and spinal cord. Neuron 4:603–614. 847 Yassa M a., Lacy JW, Stark SM, Albert MS, Gallagher M, Stark CEL. 2011. Pattern 848 separation deficits associated with increased hippocampal CA3 and dentate gyrus 849 activity in nondemented older adults. Hippocampus 21:968–979. 850 Page 36 of 50Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Table 1  The number of subjects within each strategy Strategy Estrus state Spatial Idiothetic Male - 10 9 Female Proestrus 4 1 Non-proestrus 6 6 There were no significant differences in strategy choice. Page 37 of 50 Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Table 2  Mean (+SEM) volume of the dorsal and ventral GCL Volume (mm3) N Dorsal Ventral Female Spatial 10 1.56 ± .088 1.98 ± .105 Female Idiothetic 9 1.43 ± .149 1.88 ± .082 Male Spatial 10 1.90 ± .096 2.90 ± .180 Male Idiothetic 7 1.87 ± .138 2.87 ± .130 Males had significantly greater dentate gyrus volume than females (p's < 0.004) Page 38 of 50Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Table 3  Mean (+SEM) percentage of cells co-expressing BrdU and NeuN in the GCL in female and male rats BrdU/NeuN co-expressing cells (%) N Dorsal Ventral Female Spatial 8 82.75 ± 3.34 74.00 ± 2.70Female Idiothetic 5 84.40 ± 4.12 74.00 ± 4.34Male Spatial 9 86.67 ± 1.05 81.56 ± 2.10 Male Idiothetic 9 82.67 ± 2.05 80.00 ± 2.67 There was greater percentage of BrdU-ir cells co-expressing NeuN in the dorsal compared to the ventral dentate gyrus but no significant differences between groups. Page 39 of 50 Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Table 4  Mean (+SEM) percentage of cells co-expressing BrdU and zif268 in the GCL in female versus male rats. BrdU/zif268 co-expressing cells (%) N Dorsal Ventral Female Spatial 8 0.00 ± 0.00 0.00 ± 0.00 Female Idiothetic 5 0.80 ± 0.80 0.00 ± 0.00 Male Spatial 9 0.22 ± 0.22 0.44 ± 0.44 Male Idiothetic 9 0.44 ± 0.44 1.56 ± 0.80 No significant differences between groups in percentage of BrdU/zif268 co-expressing cells were found. Page 40 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859601 FIGURES 1 Figure 1. Experimental design to examine spatial pattern separation for similar 2 (ADJACENT) and distinct (SEPARATE) patterns. (A) The experimental timeline. (B) 3 Pattern separation was tested with delayed non-match to place RAM task with two 4 separation patterns, ADJACENT and SEPARATE. (C) Probe trial to determine whether 5 a spatial or idiothetic strategy was used. 6 7 Figure 2. (A) Mean (+SEM) percentage of correct choices during ADJACENT and 8 SEPARATE trials in females versus males with data from the spatial and idiothetic 9 strategy users combined, and (B and C) shown separately with strategy users; (B) with 10 spatial strategy users and (C) with idiothetic strategy users. Males had significantly 11 greater percentage of correct choices than females during ADJACENT trials but not 12 during SEPARATE trials. Male spatial strategy users had significantly greater 13 percentage of correct choices than female spatial strategy users during ADJACENT 14 trials. * indicates p < 0.05. 15 16 Figure 3. (A) Mean (+SEM) density of BrdU-ir cells in the dentate gyrus and (B) 17 photomicrograph of BrdU-ir cell stained with DAB in the granule cell layer (GCL). 18 Page 41 of 50 Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859602 Male spatial strategy users had significantly greater density of BrdU-ir cells in the 19 dorsal dentate gyrus than the other groups. (C) Mean (+SEM) density of BrdU/NeuN 20 coexpressing cells in the dentate gyrus. Male spatial strategy users had significantly 21 greater density of BrdU/NeuN coexpressing cells in the dorsal dentate gyrus than the 22 other groups. (D-F) photomicrographs of cells labeled with the fluorescent neuronal 23 marker NeuN (green) (D), BrdU (red) (E), and merged image indicating a 24 double-labelled cell (F). Images were captured at 1000× magnification (B) and 400× 25 magnification (D-F). White arrows indicate immune-reactive cells. * indicates p < 0.05. 26 27 Figure 4. (A) Mean (+SEM) density of zif268 expressing cells in the CA3 in all females 28 versus all males. Females had significantly increased zif268 expression in the dorsal 29 CA3 than males. (B) Mean (+SEM) density of zif268 expressing cells in the CA1. 30 Dorsal CA1 had significantly greater zif268 expression compared to the ventral CA1 in 31 all groups. (C-E) Photomicrographs of zif268 immunoreactive cells in the CA1 (C), in 32 the CA3 (D) and in the dentate gyrus (DG) (E). (F) Mean (±SEM) density of c-Fos 33 expressing cells in the CA3 in all females versus all males. Males had significantly 34 increased c-Fos expression in the dorsal CA3 than females. (G) Mean (+SEM) density 35 of c-Fos expressing cells in the CA1. Idiothetic strategy users had significantly greater 36 Page 42 of 50Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603 expression of c-Fos in the dorsal CA1 than spatial strategy users, and greater c-Fos 37 expression in the ventral than dorsal CA1 in all groups. (H)-(J) Photomicrographs of 38 c-Fos immunoreactive cells in the CA1 (H), in the CA3 (I) and in the DG (J). Images 39 were captured at 200× magnification.* indicates p < 0.05. 40 41 Figure 5. Correlations between performance during a spatial pattern separation task and 42 neurogenesis. (A and B) Correlation in spatial strategy users with data from both males 43 (blue) and females (red) between proportion of correct choices during ADJACENT 44 trials and density of BrdU/NeuN coexpressing cells in the dorsal (A) and ventral dentate 45 gyrus (B). (C) Correlation between proportion of correct choices during ADJACENT 46 trials and density of BrdU/NeuN coexpressing cells in the ventral dentate gyrus in 47 idiothetic strategy users with data from both males (blue) and females (red). 48 49 Figure 6. Correlations between performance during a spatial pattern separation task and 50 immediate early gene expression. (A) Correlation in spatial strategy users with data 51 from both males (blue) and females (red) between proportion of correct choices during 52 ADJACENT trials and density of zif268 expressing cells in the ventral CA3. (B and C) 53 Correlation between proportion of correct choices during ADJACENT trials and zif268 54 Page 43 of 50 Hippocampus1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604 expressing cell density in the ventral dentate gyrus (B), and in the ventral CA1(C) in 55 male idiothetic strategy users. (D) Correlation between proportion of correct choices 56 during ADJACENT trials and c-Fos expressing cell density in the dorsal CA1 in female 57 spatial strategy users. 58 59 60 Page 44 of 50Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 1. Experimental design to examine spatial pattern separation for similar (ADJACENT) and distinct (SEPARATE) patterns. (A) The experimental timeline. (B) Pattern separation was tested with delayed non-match to place RAM task with two separation patterns, ADJACENT and SEPARATE. (C) Probe trial to determine whether a spatial or idiothetic strategy was used.  172x184mm (96 x 96 DPI)  Page 45 of 50 Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 2. (A) Mean (+SEM) percentage of correct choices during ADJACENT and SEPARATE trials in females versus males with data from the spatial and idiothetic strategy users combined, and (B and C) shown separately with strategy users; (B) with spatial strategy users and (C) with idiothetic strategy users. Males had significantly greater percentage of correct choices than females during ADJACENT trials but not during SEPARATE trials. Male spatial strategy users had significantly greater percentage of correct choices than female spatial strategy users during ADJACENT trials. * indicates p < 0.05.  177x172mm (96 x 96 DPI)  Page 46 of 50Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 3. (A) Mean (+SEM) density of BrdU-ir cells in the dentate gyrus and (B) photomicrograph of BrdU-ir cell stained with DAB in the granule cell layer (GCL). Male spatial strategy users had significantly greater density of BrdU-ir cells in the dorsal dentate gyrus than the other groups. (C) Mean (+SEM) density of BrdU/NeuN coexpressing cells in the dentate gyrus. Male spatial strategy users had significantly greater density of BrdU/NeuN coexpressing cells in the dorsal dentate gyrus than the other groups. (D-F) photomicrographs of cells labeled with the fluorescent neuronal marker NeuN (green) (D), BrdU (red) (E), and merged image indicating a double-labelled cell (F). Images were captured at 1000× magnification (B) and 400× magnification (D-F). White arrows indicate immune-reactive cells. * indicates p < 0.05.  174x224mm (96 x 96 DPI)  Page 47 of 50 Hippocampus123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 4. (A) Mean (+SEM) density of zif268 expressing cells in the CA3 in all females versus all males. Females had significantly increased zif268 expression in the dorsal CA3 than males. (B) Mean (+SEM) density of zif268 expressing cells in the CA1. Dorsal CA1 had significantly greater zif268 expression compared to the ventral CA1 in all groups. (C-E) Photomicrographs of zif268 immunoreactive cells in the CA1 (C), in the CA3 (D) and in the dentate gyrus (DG) (E). (F) Mean (±SEM) density of c-Fos expressing cells in the CA3 in all females versus all males. Males had significantly increased c-Fos expression in the dorsal CA3 than females. (G) Mean (+SEM) density of c-Fos expressing cells in the CA1. Idiothetic strategy users had significantly greater expression of c-Fos in the dorsal CA1 than spatial strategy users, and greater c-Fos expression in the ventral than dorsal CA1 in all groups. (H)-(J) Photomicrographs of c-Fos immunoreactive cells in the CA1 (H), in the CA3 (I) and in the DG (J). Images were captured at 200× magnification.* indicates p < 0.05.  171x237mm (96 x 96 DPI)  Page 48 of 50123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 5. Correlations between performance during a spatial pattern separation task and neurogenesis. (A and B) Correlation in spatial strategy users with data from both males (blue) and females (red) between proportion of correct choices during ADJACENT trials and density of BrdU/NeuN coexpressing cells in the dorsal (A) and ventral dentate gyrus (B). (C) Correlation between proportion of correct choices during ADJACENT trials and density of BrdU/NeuN coexpressing cells in the ventral dentate gyrus in idiothetic strategy users with data from both males (blue) and females (red).  173x170mm (96 x 96 DPI)  Page 50 of 50123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer ReviewFigure 6. Correlations between performance during a spatial pattern separation task and immediate early gene expression. (A) Correlation in spatial strategy users with data from both males (blue) and females (red) between proportion of correct choices during ADJACENT trials and density of zif268 expressing cells in the ventral CA3. (B and C) Correlation between proportion of correct choices during ADJACENT trials and zif268 expressing cell density in the ventral dentate gyrus (B), and in the ventral CA1(C) in male idiothetic strategy users. (D) Correlation between proportion of correct choices during ADJACENT trials and c-Fos expressing cell density in the dorsal CA1 in female spatial strategy users.  171x170mm (96 x 96 DPI)  Page 51 of 50123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

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