Revisions submitted to Hippocampus on August 27, 2014 Prior high corticosterone exposure reduces activation of immature neurons in the ventral hippocampus in response to spatial and non-spatial memory

Joanna L. Workman, Melissa Y.T. Chan, Liisa A.M. Galea* Department of Psychology and the Centre for Brain Health University of British Columbia * Corresponding Author: 2136 West Mall Vancouver, BC V6T 1Z4 [email protected]

Text pages: 29 Figures: 10 Tables: 2

Key Words neurogenesis, stress, doublecortin, depression, water maze Acknowledgements We thank Dr. Cindy Barha, Carmen Chow, Curran Emeruwa, Olivia Hershorn, Stephanie Lieblich, and Sophia Solomon for technical assistance and Dr. Sarah Heimovics for helpful discussions of microscopy and data analysis. This research was supported by a grant from the Canadian Institutes of Health Research (CIHR) to LAMG. JLW is supported by a Postdoctoral Fellowship from CIHR.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/hipo.22375

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CORT & New Neuron Activation Abstract Chronic stress or chronically high glucocorticoids attenuate adult hippocampal neurogenesis by reducing cell proliferation, survival, and differentiation in male rodents. Neurons are still produced in the dentate gyrus during chronically high glucocorticoids, but it is not known whether these new neurons are appropriately activated in response to spatial memory. Thus, the goal of this study was to determine whether immature granule neurons generated during chronically high glucocorticoids (resulting in a depressive-like phenotype) are differentially activated by spatial memory retrieval. Male Sprague Dawley rats received either 40 mg/kg corticosterone (CORT) or vehicle for 18 d prior to behavioral testing. Rats were tested in the forced swim test (FST) and then tested in a spatial (hippocampus-dependent) or cued (hippocampus-independent) Morris Water Maze (MWM). Tissue was then processed for doublecortin (DCX) to identify immature neurons and zif268, an immediate early gene product. As expected, CORT increased depressive-like behavior (greater immobility in the FST) however, prior CORT modestly enhanced spatial learning and memory compared with oil. Prior CORT reduced the number of DCX-expressing cells and proportion of DCX-expressing cells colabeled for zif268, but only in the ventral hippocampus. Prior CORT shifted the proportion of cells in the ventral hippocampus away from postmitotic cells and toward immature, proliferative cells, likely indicates postmitotic cells were produced and matured under CORT conditions but proliferative cells were produced after high CORT exposure ceased. Compared with cue, spatial training slightly increased DCX-expressing cells and shifted cells toward the postmitotic stage in the ventral hippocampus. These data suggest that the effects of CORT and spatial training on immature neurons are more pronounced in the ventral hippocampus. Further, high CORT reduced activation of immature neurons, suggesting that exposure to high CORT may have long-term effects on cell integration or function.

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CORT & New Neuron Activation Introduction Many individuals with major depression display hypothalamic-pituitary adrenal (HPA) axis dysregulation in the form of high basal levels of cortisol, impaired HPA axis negative feedback, and a disrupted diurnal rhythm, all of which contribute to long-term exposure to excessive glucocorticoids (Ising et al., 2007; Parker et al., 2003). The hippocampus contains a high density of glucocorticoid receptors and plays a role in HPA axis negative feedback, factors which render it particularly sensitive to high glucocortocoids (Sapolsky et al., 1984; Sapolsky et al., 1983). Further, patients who have been depressed for at least 2 years present with smaller hippocampi compared with those without depression (Campbell et al., 2004; McKinnon et al., 2009; Sheline et al., 2003; Sheline et al., 1999; Sheline et al., 1996). Rodent studies have revealed that chronic stress and chronically high glucocorticoids alter numerous aspects of hippocampal structural plasticity including adult neurogenesis, dendritic remodeling, and spine density in a sexually-dimorphic manner (Bessa et al., 2009; Brummelte and Galea, 2010b; Galea et al., 1997; Sunanda et al., 1995; Watanabe et al., 1992b; Wong and Herbert, 2004; Wong and Herbert, 2006). Thus, changes in hippocampal volume may be due to reductions in one or all of these processes and may be related to chronically high glucocorticoids evident in major depression. Adult hippocampal neurogenesis is of particular interest and may constitute a target of antidepressant action. The dentate gyrus of the hippocampus undergoes the production of new neurons throughout life (Altman and Das, 1965; Cameron et al., 1993). Neurogenesis comprises a multistep process involving cell proliferation, migration, survival, and differentiation of dividing cells into neurons. Glucocorticoids regulate cell proliferation (Cameron and Gould, 1994; Gould et al., 1992), survival (Wong and Herbert, 2004), and differentiation (Wong and Herbert, 2006). Hippocampal neurogenesis is compromised in depression in humans as well as in rodent models of depression and administration of antidepressants reinstates neurogenesis (Bessa et al., 2009; Boldrini et al., 2012; Boldrini et al., 2009; Czeh et al., 2001; Epp et al., 2013a; Malberg et al., 2000). Further, adult-generated hippocampal neurons may contribute to HPA axis regulation (Snyder et al., 2011), indicating another pathway to which neurogenesis in the adult dentate gyrus may relate to depression. However, the generation of hippocampal neurons is merely suppressed, not eliminated during exposure to chronically high glucocorticoids. New neurons are functionally integrated into existing hippocampal circuits (Ramirez-Amaya et al., 2006) and factors during or close to the time of cell division can alter the subsequent integration of these neurons into existing circuits. For instance, both running and seizures increase hippocampal neurogenesis but have very different effects on the electrophysiological properties

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CORT & New Neuron Activation of these new neurons with exercise increasing but seizures reducing excitation (Jakubs et al., 2006). Furthermore, seizure-induced neurogenesis contributes to cognitive impairment following seizures (Jessberger et al., 2007). Thus the environment into which new neurons are produced matters. Zif268 is an immediate early gene that serves as a transcription factor that signifies activated cells and is required for long-term, hippocampus-dependent memory (Guzowski, 2002; Jones et al., 2001). Zif268 can be combined with markers of newly-generated neurons to determine the extent to which immature neurons are activated in response to a stimulus (Epp et al., 2011b; Snyder et al., 2009a; Snyder et al., 2012a; Tashiro et al., 2007). Additionally, memory performance is particularly sensitive to zif268, as mice heterozygous for zif268 are impaired in both long term spatial (Bozon et al., 2002) and recognition memory (Bozon et al., 2003). Zif268 protein and mRNA expression is also increased after induction of long-term potentiation (LTP) and is dependent on the NMDA receptor (Cole et al., 1989; Wisden et al., 1990). Further, activation of new neurons (examined by immediate early genes) is positively associated with spatial learning in females (Barha and Galea, 2013; Chow et al., 2013; McClure et al., 2013). Thus in the present study we were interested in whether new neurons produced during high glucocorticoid exposure would be differentially activated, via immediate early gene expression, in response to spatial memory retrieval. Prior research has shown that an acute stressor, or acute CORT administration enhances the activation of immature neurons (Kirby et al., 2013) but chronic CORT has not been investigated. Stress and glucocorticoids have task-, and time course-dependent effects on hippocampus-dependent learning (Conrad, 2010). For instance, acute stress facilitates hippocampus-dependent trace conditioning in males (Wood et al., 2001). Chronic stress, in contrast, impairs hippocampus-dependent spatial learning in males in the radial arm maze, but this impairment recovers after stress is discontinued (Luine et al., 1996; Luine et al., 1994). Chronic restraint stress and exogenous glucocorticoids also lead to retraction of apical dendrites in the CA3 region in males (Watanabe et al., 1992a; Watanabe et al., 1992b), which recover after termination of a stressor (Conrad et al., 1999) and thus parallel changes in spatial learning. Although restructuring of the CA3 region in response to chronic stress is associated with memory impairments, dendritic retraction may only indirectly be responsible for spatial memory impairments (Conrad, 2006). Specifically, CA3 apical dendritic retraction after stress may lead to heightened levels of glucocorticoids during tasks, which in turn impair hippocampaldependent learning. Anatomically, this region is of interest as new granule cells from the dentate

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CORT & New Neuron Activation gyrus rapidly extend axons to the CA3 region (Hastings and Gould, 1999) and new neurons also appear to buffer stress responses (Snyder et al., 2011). In the present experiment, we sought to determine whether new neurons generated during and potentially after a period of high corticosterone (CORT) exposure, would be less likely to express the protein product of the immediate early gene zif268 compared with neurons generated during normal concentrations of CORT in response to spatial memory. We administered high CORT (40mg/kg/day) for 18 d and tested rats in the forced swim test the last two days of CORT exposure confirm that our CORT protocol elicited a depressive-like phenotype. We then trained rats in the reference memory version of the Morris Water Maze (MWM) for 5 days. After 5 days of training, rats remained undisturbed for 5 days and then were tested in a probe trial to determine spatial memory performance and to elicit activation of neurons in response to spatial memory retrieval. We hypothesized that prior CORT would reduce activation of immature neurons after spatial memory retrieval. Methods Animals Thirty-four male Sprague-Dawley rats were obtained from our breeding colony at University of British Columbia (Vancouver, BC Canada) and were used in this study. Rats were weaned at ~21 d and housed with same-sex siblings until puberty. Rats were then pair housed with one sibling until the start of the study in cages (48 × 27 × 20 cm) with aspen chip bedding. Rats were given Purina rat chow and tap water ad libitum throughout the study. At 53 d of age, rats were single housed and handled for 5 min/day for 7 days. No siblings were assigned to the same treatment/training group and males from 13 litters were used. Colony rooms were maintained on a 12:12 light/dark cycle (lights on at 7:30 am) and temperature- and humiditycontrolled (21 ± 1 °C; 50% ± 10% respectively). All protocols were in accordance with ethical guidelines set by the Canada Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. Hormone Preparation and Treatment An emulsion of CORT (Sigma-Aldrich, St Louis, MO, USA) was prepared every 2 – 3 days by mixing CORT with ethanol and then adjusting to a final concentration of 40 mg/ml and 10% ethanol in sesame oil. Rats were randomly assigned to receive subcutaneous injections of CORT (n = 17, 40mg/kg/day in a volume of 1ml/kg) or vehicle (n = 17, in a volume of 1ml/kg). This dose of CORT was chosen because it elevates CORT concentrations, reliably induces a

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CORT & New Neuron Activation depressive-like phenotype in both males and females, and reliably reduces hippocampal plasticity, including neurogenesis (Brummelte and Galea, 2010a; Brummelte and Galea, 2010b; Gregus et al., 2005; Johnson et al., 2006; Kalynchuk et al., 2004; Wong and Herbert, 2006; Workman et al., 2013). Injections starting at 60 d of age and continued for 18 consecutive days at ~1300 h. Body mass was taken every 2 – 3 days and doses were adjusted accordingly. See Figure 1 for timeline and experimental details. Behavioral Testing Forced Swim Test. The forced swim test (FST) was conducted during the final two days of injections (days 17 and 18). Rats were injected 1 hr prior to behavioral testing. The FST is widely used to assess antidepressant efficacy and increasingly used as a test for depressivelike behavior (Cryan et al., 2005; Galea et al., 2001; Porsolt et al., 1978; Porsolt et al., 1977). The apparatus consisted of a vertical glass cylinder (45 × 28 cm) filled to a depth of 30 cm with tap water at 25 ± 1 °C and water was changed between rats. In session 1, rats were placed in the water for 15 min. Twenty-four h after the first session, rats were tested again for 5 min. Fecal boli were counted in both sessions 1 and 2. All sessions were recorded and two observers who were blind to treatment conditions scored the sessions for active escape behaviors (swimming and climbing) and immobility (making only the movements necessary to keep the head above water). Total time engaged in each behavior was quantified using BEST Collection Software (Educational Consulting, Inc., Hobe Sound, FL, USA). Data from both observations were averaged and used for analysis. Behaviors in the FST on both the first and second day (time spent immobile, swimming, and climbing) were converted to percentages.

Morris Water Maze. Morris Water Maze (MWM) training commenced 24 hours after the last injection of CORT in order to assess only the chronic effects of CORT exposure and eliminate the potential confound of acute effects of high CORT (Coburn-Litvak et al., 2003). We trained rats in two forms of the Morris water maze: a cued version in which the platform is visible and moves each trial and a spatial version in which the platform remains hidden and fixed in one location for the duration of the experiment such that rats must rely on extra-maze cues to locate the platform. Lesions of the hippocampus impair spatial, but not cued, water maze performance (Morris et al., 1982) and spatial, but not cued, training increases neurogenesis in the hippocampus, depending on the timing between BrdU injections and training (Epp et al., 2007; Gould et al., 1999). The MWM is a circular pool, 180 cm in diameter and was filled with water to a depth of 33 cm. The water remained at room temperature throughout testing. Nontoxic white

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CORT & New Neuron Activation tempera paint was added to the water to render it opaque. Large black geometric shapes were fixed to the walls to serve as spatial cues. Rats were recorded in the pool using a camera mounted to ceiling and centered above the pool. The camera was connected to Anymaze tracking software (Stoelting Co; Wood Dale, IL, USA). Half of the rats in each group were trained on the spatial (hippocampus-dependent) version of the task and half were trained on the cue (hippocampus-independent) version of the task. In the spatial version of the task, a platform was located in the center of one quadrant of the pool, 2 cm below the surface of the water. Thus, rats that were spatial-trained were required to locate the platform based on the extramaze cues. In the cue version of the task, the top of the platform extended 2 cm above the surface of the water and moved randomly to the center of a new quadrant in each trial so that a spatial strategy could not be used. In either case, all rats were given one daily training session for 5 days with four trials per session. For each trial, rats were released from one of the four different cardinal compass points. Five days following the final training session all rats were given a single probe trial. During this trial the rats swam for 60 s in the same pool without an escape platform to assess retention of the platform location. We compared the amount of time spent in the quadrant of the pool that had previously contained the hidden platform (the target quadrant) as a measure of probe trial performance. Tissue Collection Approximately ninety minutes following the probe trial the rats were deeply anesthetized with an overdose of sodium pentobarbital and then transcardially perfused with 60 ml of 0.9% saline followed by 120 ml of 4% paraformaldehyde (PFA; Sigma) in 0.1 M phosphate buffered saline (PBS; Sigma). Blood was collected via cardiac puncture just prior to perfusion. Brains were extracted and placed in 4% PFA for 24 h and then transferred into a solution containing 30% sucrose (Sigma) in 0.1 M PB until they sank to the bottom. Brains were frozen rapidly and sectioned using a freezing microtome (Leica, Richmond Hill, ON, Canada) at 40 µm in a series of 10. Sections were immediately stored in antifreeze (ethylene glycol and glycerol; Sigma) and stored at -20 °C until processing. Corticosterone Radioimmunoassay Blood samples were stored overnight at 4 °C, centrifuged at 10g for 10 min and serum stored at -20 °C until radioimmunoassay. Total CORT was measured using a double antibody 125

I radioimmunoassay kit (MP Biomedicals, Solon, OH, USA). All reagents were halved and

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CORT & New Neuron Activation samples run in duplicate. The cross reactivity with other steroids is less than 0.4%. The intraassay coefficient of variation was 12.8%. Immunohistochemistry Free-floating sections were rinsed 4 x 10 min in TBS and incubated for 24 h 4 °C in a primary antibody solution containing a 1:500 dilution of goat anti-DCX (SC-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 1:1000 dilution of rabbit anti-zif268 (Egr-1 SC-189, Santa Cruz Biotechnology) with 4% normal donkey serum and 0.03% Triton-X in 0.1 M TBS. Following three rinses in TBS, tissue was then transferred to a secondary antibody solution containing 1:500 donkey anti-rabbit Cy3 (Jackson ImmunoResearch, West Grove, PA, USA) and 1:250 donkey anti-goat Alexa 488 (Invitrogen) in 0.1 M TBS. Tissue was incubated overnight at 4 °C, rinsed three times in 0.1 M TBS, mounted on glass slides and cover slipped with PVA-DABCO (Epp et al., 2011b).

Microscopy All analyses were conducted by an experimenter who was blind to treatment conditions using a Nikon E600 microscope equipped with epifluoresence. The dorsal and ventral hippocampus was classified within limits previously described (Brummelte and Galea, 2010b). We counted these regions separately because the dorsal hippocampus is more involved in spatial processing whereas the ventral hippocampus is more involved in stress and anxiety (Bannerman et al., 2004; Fanselow and Dong, 2010). Doublecortin/zif268 Colabeling. DCX is transiently expressed in immature neurons and can be used as a marker of adult neurogenesis (Brown et al., 2003). One hundred DCX-expressing cells were selected for quantification of double labeling (50 dorsal and 50 ventral) at 1000
magnification (Epp et al., 2011b). In order to be selected, DCX-expressing cells had to 1) be within the granule cell layer, 2) have at least 1 bifurcating dendrite, and 3) cells had to relatively well isolated (i.e., not overlapping other cell bodies and not clustered). No more than 10 cells were selected from a single section. Doublecortin counting. To quantify number of immature neurons in the present study, we selected every 20th section throughout the extent of the hippocampus. DCX-expressing cells were exhaustively counted in both the left and right hemispheres of each of these sections at 1000
magnification and multiplied by 20 to get an estimate of total number of cells. Both dorsal and ventral measures were counted separately.

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CORT & New Neuron Activation Maturity of doublecortin-expressing cells. In order to determine proportion of neurons at particular developmental stages, DCX-expressing cells were classified based on stage of maturation (Hamson et al., 2013; Plumpe et al., 2006). At 1000
magnification, fifty cells positively labeled for DCX (25 dorsal and 25 ventral) were randomly selected for classification and were considered in one of three developmental stages based on morphological attributes. Cells had to be relatively isolated (i.e., not clumped or overlapping) and had to lie within the granule cells layer. Cells were classified as 1) proliferative if they had no processes or short, plump processes; 2) intermediate if they had one thin, unbranching process; or 3) postmitotic if they had long and branching dendrites reaching the molecular layer. Zif268 optical density and GCL volume estimation. Every 10th section was selected for zif268 quantification and estimation of the volume of the granule cell layer (GCL) of the dentate gyrus. Photomicrographs were taken with an Olympus BX51 microscope using cellSens Standard (version 1.5, Olympus) with consistent gain and exposure for each photo. Using ImageJ software (version 1.38x NIH, Bethesda, MD USA), an experimenter blind to treatment conditions traced the GCL including the subgranular zone for each image. From this tracing, both optical density and GCL volume were quantified. Intensity of zif268 immunoreactivity was calculated as percent above background after obtaining mean gray values for background levels for each image. Briefly, in order to obtain mean gray background levels, 6 elliptical regions of interest were placed randomly on each image where immunoreactivity was not present (Heimovics et al., 2012). Gray levels for each of these areas were determined and averaged. GCL volumes were calculated by summing the area of the GCL from each section and multiplying by the distance between sections (Cavalieri’s Principle). Dorsal and ventral measures were averaged separately.

Statistical Analyses Body mass throughout the study was analyzed using a repeated-measures ANOVA with day as the within-subjects factor and treatment as the between-subjects factor. Percent time immobile, swimming and climbing, and latency to immobility were each analyzed using repeated-measures ANOVA with day (FST 1, FST 2) as the within-subjects factor and hormone treatment (oil, CORT) as the between-subjects factor. In the water maze, distance (m) to reach the platform and swim speed (m/s) were analyzed using repeated-measures ANOVA with day of training as the within-subjects factor and hormone treatment and type of water maze training (cue, spatial) as between-subjects factors. Time in target quadrant was analyzed using factorial ANOVA with hormone treatment and type of water maze training as between-subjects factors.

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CORT & New Neuron Activation Because perfusion blood samples were collected and assayed for CORT (and thus resulted in a delay between time of probe trial and blood collection), we analyzed corticosterone concentrations using an ANCOVA with time between probe trial and perfusion as a covariate and treatment and training as the between-subjects variables. Total DCX+ cells, zif268 expression, GCL volume, and the percentage of DCX/zif268 co-expressing cells were each analyzed using repeated-measures ANOVA with hippocampal region (dorsal, ventral) as the within-subjects factor and hormone treatment and water maze training as between-subjects factors. Proportion of DCX-expressing cells at 3 stages of maturation were analyzed with hippocampal region and stage (proliferative, intermediate, postmitotic) as within-subjects factors and treatment and training as between-subjects factors. Post hoc comparisons were conducted using Newman-Keuls. Planned comparisons based on a priori hypotheses were subjected to a Bonferroni correction. Pearson correlations were conducted between water maze performance and density of DCX-expressing cells, proportion of double-labeled cells, and CORT concentrations. Effects were considered significant where P ≤ 0.05. Trends are discussed where P ≤ 0.07. Results CORT exposure induced body mass loss over time Among CORT-treated rats, body mass reduced over time, whereas for oil-treated rats, body mass increased over time (day by treatment interaction: F8,240 = 347.43, P < 0.001; data not shown). On all days assessed except the first, CORT-treated rats had lower body masses compared with oil-treated rats (Ps < 0.007). At perfusion, CORT-treated rats still had lower body mass compared with oil-treated rats (P < 0.001). However, body mass for CORT-treated rats at perfusion was greater than the 6 preceding measurements prior to water maze training (Ps < 0.001). CORT exposure increased immobility and reduced climbing on day 2 of the forced swim test. Both hormone treatment and day of training significantly altered latency to immobility (F1,32 = 6.22, P = 0.018, F1,32 = 20.29, P < 0.001, respectively), but did not significantly interact (P = 0.36). Specifically, CORT treatment reduced the latency to immobility compared with oil treatment (P = 0.018; Figure 2A). Latencies to immobility were also shorter on the second day of training compared with the first (P < 0.001). Compared with oil treatment, CORT treatment significantly increased percent time immobile on the second day of training (P = 0.001; treatment by day interaction: F1,32 = 4.998, P = 0.033; Figure 2B). Additionally, among oil-treated

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CORT & New Neuron Activation rats, percent of time spent immobile was lower on the second day of training compared with the first (P < 0.001). CORT treatment tended to increase percent time swimming relative to oil treatment, although this comparison did not reach significance (P = 0.067). Compared with oil treatment, CORT treatment significantly reduced percent time climbing on the second day (P < 0.001; hormone treatment by day interaction: F1,32 = 7.57, P = 0.01, Figure 2B). Additionally, among oil-treated rats, percent time climbing was higher on the second day compared with the first (P < 0.001). Finally, day of testing significantly altered fecal boli in the FST: rats had fewer fecal boli on the second day compared with the first (F1,32 = 5.86, P = 0.021; data not shown) but there was no significant effect of hormone treatment and no significant interaction (Ps > 0.67). Prior CORT treatment enhanced spatial performance and reduced swim speed. Prior CORT treatment reduced distance to platform compared with oil treatment regardless of type of training (day by CORT interaction: F4,120 = 2.53, P = 0.044; Figure 3A) and cue-trained rats had shorter distances to the platform over time (day by training interaction: F4,120 = 4.037, P = 0.004) but there was no significant three-way interaction. Planned comparisons, however, revealed that among spatial-trained rats, CORT-treated rats had shorter distances to the platform compared with oil-treated rats on the second day of training (P = 0.002). Rats trained in the spatial version of the water maze traveled faster than those tested in the cue version regardless of hormone treatment (day by training interaction: F4,120 = 17.11, P < 0.001). Specifically, compared with cue-trained rats, spatial-trained rats swam faster on all days (Ps < 0.001) except for the first day (P = 0.31). Additionally, among cue-trained rats, swim speed slowed early in the water maze, with rats traveling more slowly on days 3 – 5 compared with days 1 and 2 (Ps < 0.001). However, for spatial-trained rats, swim speed remained relatively stable across days with the exception of day 5, in which rats traveled more slowly compared with all previous days (Ps < 0.04). CORT-treated rats were also slower than oiltreated rats regardless of training or day (main effect of treatment: F1,30 = 23.56, P < 0.001). Regardless of day of training, treatment and training tended to interact but did not reach statistical significance (P = 0.056). There were no other significant interactions or main effects. Time spent in the quadrant that had previously contained the hidden platform is an index of spatial memory (Vorhees and Williams, 2006). Prior CORT treatment increased the percentage of time spent in target quadrant during the probe trial (main effect of treatment: F1,29 = 7.015, P = 0.013; Figure 3B). Planned comparisons revealed that this effect was only in spatial-trained rats (P = 0.022) but not cue-trained rats (P = 0.19).

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Prior CORT exposure and type of water maze training altered CORT concentrations. Spatial training significantly decreased CORT concentrations following the probe trial in oil-treated rats (P = 0.042; treatment by training interaction: F1,29 = 5.21, P = 0.049; Figure 4). Type of water maze training did not significantly alter CORT concentrations in CORT-treated rats (P = 0.97). The covariate of time between probe trial and perfusion did not account for a significant proportion of variability (P = 0.37). Prior CORT treatment and water maze training interacted to alter GCL volume but did not affect zif268 intensity Regardless of hormone treatment, spatial-trained rats had greater ventral (P = 0.004) but not dorsal GCL volume compared with cue-trained rats (P = 0.99; region by training interaction: F1,29 = 5.31, P = 0.029; Table 1). Cue-trained rats had smaller ventral compared with dorsal GCL volume (P = 0.001). Regardless of region, spatial trained rats had larger GCL volume compared with cue-trained rats among oil- (P = 0.011) but not CORT-treated rats (P = 0.5; treatment by training interaction: F1,29 = 8.08, P = 0.008; Table 1).

Because there were

significant differences between groups in GCL volume we analyzed density as well as total number of DCX-expressing cells. Zif268 expression was significantly greater in the dorsal compared with ventral hippocampus (main effect of region: F1,29 = 245.29, P < 0.001; Table 2) but there were no other significant main effects or interactions (Ps > 0.37).

Prior CORT exposure reduced total number and density of DCX-expressing cells in the ventral hippocampus Regardless of water maze training, prior CORT treatment significantly reduced total number of DCX-expressing cells in the ventral hippocampus (P < 0.002) and tended to reduce total DCX-expressing cells in the dorsal hippocampus (P = 0.09; treatment by region interaction: F1,29 = 23.5, P < 0.001; Figure 5A). A priori, we expected spatial training to increase total number of DCX-expressing cells compared with cue training in the oil-treated rats and planned comparisons revealed that spatial-trained, oil-treated rats had more DCX-expressing cells in the ventral (P = 0.005) and a trend for dorsal hippocampus (P = 0.06, one-tailed; See also Figure 6 for representative photomicrographs of DCX-expressing cells in the ventral dentate gyrus from

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CORT & New Neuron Activation oil- and CORT treated rats). There were main effects of region and treatment but no other significant main effects or interactions (Ps > 0.18). Regardless of water maze training, CORT-treated rats had a lower density of DCXexpressing cells in the ventral (P = 0.002) but not dorsal hippocampus (P = 0.53; treatment by region interaction: F1,29 = 5.6, P = 0.025; Figure 5B). Additionally, oil-treated rats also had a greater density of cells in the ventral compared with dorsal hippocampus (P < 0.001). Cuetreated rats had a greater density of cells in the ventral compared with dorsal hippocampus (P < 0.001; training by region interaction: F1,29 = 5.45, P = 0.027). Further, cue-trained rats also had greater ventral DCX cell density compared with spatial-trained rats (P = 0.019). Prior CORT exposure reduces DCX/zif268 colabeling and reduces number of DCX-expressing cells per section in the ventral hippocampus Regardless of training, prior CORT treatment significantly reduced the proportion of DCX-expressing cells that were colabeled for zif268 in the ventral (P = 0.002) but not dorsal hippocampus (P = 0.15; treatment by region interaction: F1,28 = 5.87, P = 0.022; Figure 5C). Further, in oil-treated rats, there was a greater proportion of colabeled immature neurons in the ventral compared with dorsal hippocampus (P = 0.008) but not in CORT-treated rats (P = 0.65). There were no other significant main effects or interactions (Ps > 0.1). See Figure 7 for representative photomicrographs of DCX and zif268 co-expression. Prior CORT and training altered proportions of DCX-expressing cells at particular developmental stages Prior CORT increased proportion of proliferative cells (P = 0.01) and intermediate cells (P = 0.03) but decreased proportion of postmitotic cells (P < 0.001; stage by treatment interaction: F2,58 = 10.6, P < 0.001; Figure 8). Additionally, both CORT- and oil-treated groups had a smaller proportion of proliferative cells compared with both intermediate and postmitotic cells (Ps < 0.001). However, oil-treated rats had a greater proportion of postmitotic cells compared with intermediate cells (P < 0.001) but CORT-treated rats did not (P = 0.97). Both cue- and spatial-trained rats had a smaller proportion of proliferative cells compared with intermediate and with postmitotic cells in both the dorsal and ventral hippocampus (Ps ≤ 0.001; region by stage by training interaction: F2,58 = 5.2, P < 0.01). For both cue- and spatial-trained rats, the dorsal hippocampus contained a greater proportion of postmitotic cells compared with intermediate cells (Ps ≤ 0.001). The ventral hippocampus contained a greater proportion of postmitotic cells compared with intermediate cells for spatial-

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CORT & New Neuron Activation trained rats (P < 0.001) but not for cue-trained rats (P = 0.3). Among spatial-trained rats, proportions of cells at each particular stage were similar in the dorsal compared with the ventral hippocampus (Ps > 0.47). Among cue-trained rats, proportions of proliferative and intermediate cells were similar in the dorsal compared with the ventral hippocampus (proliferative: P = 0.54, intermediate: P = 0.085) but the ventral hippocampus contained a smaller proportion of postmitotic cells compared with the dorsal hippocampus (P = 0.019). In the ventral hippocampus, spatial-trained rats had a smaller proportion of intermediate cells (P = 0.004) and a greater proportion of postmitotic cells compared with cue-trained rats (P = 0.003). See figure 9 for representative photomicrographs of DCX-expressing cells at each developmental stage. Correlations Among spatial-trained, CORT-treated rats, time in target quadrant was negatively correlated with CORT concentrations (r = -0.81, P = 0.009) but not oil-treated, spatial trained rats (Ps > 0.41; Figure 10A). DCX-expressing cell density in the ventral hippocampus was negatively correlated with CORT concentrations oil-treated, cue-trained rats (r = -0.92, P = 0.001) but not other groups and not the dorsal hippocampus (Ps > 0.06). There were no significant correlations between percent of DCX/zif268 cells and time spent in target quadrant for any groups (Ps > 0.3). Among spatial-trained, oil-treated rats, total distance traveled during water maze training positively correlated with DCX-expressing cell density in the ventral hippocampus (r = 0.72, P = 0.029) but not in the dorsal hippocampus (P > 0.16). Among spatial-trained, CORT-treated rats, DCX-expressing cell density in the dorsal and ventral hippocampus did not correlate with time in target quadrant or with total distance traveled (Ps > 0.69). Among spatial-trained, oil-treated rats, time in target quadrant in the probe trial negatively correlated with DCX-expressing cell density (r = -0.89, P = 0.003, Figure 10B) but did not significantly correlate with cell density in the dorsal hippocampus (Ps = 0.28). Time in target quadrant among cue-trained rats did not significantly correlate with any DCX or zif268 measure (Ps > 0.17). Discussion Here, we demonstrate that prior CORT exposure slightly enhances spatial performance but reduces the number of immature neurons in the dentate gyrus of male rats. Prior CORT also decreased the proportion of activated new neurons in the ventral but not dorsal hippocampus in response to both spatial and cued training. Thus, prior CORT treatment may reduce the

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CORT & New Neuron Activation integration of newborn neurons into hippocampal circuits. Additionally, the ventral hippocampus was more sensitive to the effects of CORT than the dorsal hippocampus, as CORT decreased the total number and density of DCX-expressing cells and proportion of new neurons that were activated in the ventral, but not dorsal, dentate gyrus. Prior CORT shifted the proportion of immature neurons toward more immature, proliferative and away from postmitotic neurons, which may indicate that after CORT treatment ceases, hippocampal neurogenesis recovers, which is consistent with the homeostatic theory of neurogenesis regulation (Meltzer et al., 2005). Furthermore, DCX-expressing cells negatively correlated with spatial memory such that fewer cells were associated with a longer time spent in the target quadrant, but only for oiltreated, spatial-trained rats. In CORT-treated rats trained in the spatial MWM, CORT concentrations were correlated with spatial memory such that lower CORT was associated with more time spent in the target quadrant. These data indicate that environment into which a neuron is born alters the developmental trajectory and the activation of new neurons in the MWM.

Chronic CORT treatment increased depression-like behavior In order to confirm that 18 days of CORT exposure would increase depressive-like behavior, we tested rats in the forced swim test during the final two days of injections. Our data indicate that 18 d of exogenous high CORT yields a depression-like behavioral phenotype (i.e., increased immobility) and are consistent with previous research in adult male rats (Gregus et al., 2005; Johnson et al., 2006; Kalynchuk et al., 2004; Marks et al., 2009). CORT treatment also reduced the latency to immobility (i.e., CORT-treated rats attained immobility earlier in the FST compared with oil-treated rats), consistent with depressive-like behavior. Further, CORT treatment significantly reduced the time spent climbing, without significantly altering swimming behavior. Prior studies (including those from our laboratory) are equivocal in regard to the effects of CORT on swimming vs. climbing behavior. In some cases, high CORT reduced swimming but not climbing in either males, naïve females, or postpartum females (Brummelte and Galea, 2010a; Gregus et al., 2005; Johnson et al., 2006; Kalynchuk et al., 2004; Marks et al., 2009) whereas in other studies high CORT reduced climbing but not swimming in postpartum females (Brummelte et al., 2006). Finally, we also previously reported that high CORT reduced both swimming and climbing in postpartum females (Workman et al., 2013). Swimming and climbing may be dependent upon serotonergic and noradrenergic signaling, respectively (Detke et al., 1995) and long term exposure to glucocorticoids is capable of altering

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CORT & New Neuron Activation aspects of both serotonergic (Crayton et al., 1996; Karten et al., 1999) and noradrengergic neurotransmitter systems (de Villiers et al., 1992; Fan et al., 2014). Prior chronic CORT treatment slightly enhanced spatial performance Rats that had received CORT treatment prior to water maze training had shorter distances to reach the hidden platform on day 2 of training. CORT-treated rats also traveled more slowly in the water maze compared with oil-treated rats. This suggests that prior CORT administration that has ceased before training may subtly enhance spatial learning despite a reduction in swim speed. Prior CORT treatment also facilitated memory for location of the hidden platform as CORT-treated rats spent more time in the target quadrant compared with oiltreated rats, but only if they were spatial-trained. Exposure to chronic stressors may impair, facilitate, or have no effect on water maze performance (Conrad, 2010), although a majority of studies suggest that chronic stressors typically impair spatial performance of male rats in the water maze. Consistent with our study, rats that underwent a delay between chronic stress and testing had better spatial reference and working memory compared with rats that received no delay and better reference memory compared with unstressed rats (Hoffman et al., 2011). This suggests that spatial memory not only recovers once a stressor concludes, but also rebounds, which is consistent with results showing a rebound in dendritic complexity following chronic restraint stress (Luine et al., 1994). Stress-induced changes in spatial performance are in part due to changes in glucocorticoids. Although numerous studies support that chronic stress or CORT exposure impairs spatial learning and memory (Coburn-Litvak et al., 2003), other studies suggest that there are factors that modify the effects of glucocorticoids on spatial learning and memory such as timing of stressor, task difficulty, and intensity of the stressor. For instance, suppression of acute glucocorticoids during training prevents stress-induced impairments in spatial memory (Wright et al., 2006). It is important to note that in our study CORT treatment stopped the day prior to water maze training and was not administered during the training so previous chronic CORT administration may have served to suppress endogenous HPA axis responses early during water maze training once the exogenous CORT was no longer administered. Indeed, prior studies demonstrate that long-term exposure to CORT reduces relative adrenal mass (Coburn-Litvak et al., 2003; Magarinos et al., 1998), which could lead to lower stress-induced concentrations of glucocorticoids early in the water maze and permit enhanced performance. Divergent effects of CORT on spatial memory, however, may also rely on the aversive nature of the task (Conrad, 2010). The water maze elicits robust increases in CORT but rodents habituate

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CORT & New Neuron Activation by day 3 of training (Aguilar-Valles et al., 2005). Indeed in the present study we found that rats that were spatial-trained also had lower CORT concentrations 90 min after the probe trial compared with rats that underwent cue training. However, rats previously treated with CORT did not alter CORT concentrations after water maze training dependent upon type for training. These data suggest that spatial learning may buffer stress responses in aversive circumstances in oil-treated controls, but that prior CORT treatment may block beneficial effects of learning on HPA axis responses. Prior CORT exposure reduced the activation of immature neurons in the ventral hippocampus Prior CORT treatment significantly suppressed zif268 expression in ventral immature granule neurons, but not in GCL as a whole, in response to memory retrieval. These data suggest that new neurons produced during a period of high glucocorticoids are less likely to be activated in response to relevant stimuli. Because spatial-trained rats did not differ significantly from cue-trained rats in their percent of new neurons activated, we cannot conclude that CORT diminishes activation only in response to spatial memory retrieval. Rather, CORT may diminish immature neuron activation in response to exploration or navigation without a need for spatial strategy. Indeed, exploration of familiar contexts (Tashiro et al., 2007) as well as acute stress (Schoenfeld et al., 2013) enhance activation in newly generated granule cells. It is important to note that these activated immature neurons were all postmitotic. Given that DCX expression lasts up to 21 days after a cell divides (Brown et al., 2003) cells reach the postmitotic stage between 3 days and several weeks (Plumpe et al., 2006), indicating that the majority of these DCX/zif268-expressing neurons were likely generated under levels of high CORT. Many possible mechanisms may explain why immature postmitotic neurons are less activated after chronic CORT treatment. First, CORT may impair or delay formation of synaptic connections between new cells and existing circuits. If this is the case, then changes in activation would be driven by altered input a cell receives. Second, CORT may alter the cellular machinery during the time of division that leads to long term changes in the transcription of immediate early genes in response to relevant stimuli. Finally, it is possible that the hippocampal microenvironment may be persistently disrupted after chronic CORT exposure that can alter the activation of immature neurons (Jakubs et al., 2006). Future research is necessary to understand to the factors that may alter how new cells are functionally integrated in hippocampal circuits. Further, it is unclear the extent to which changes in activation after chronic CORT contribute to hippocampal-dependent cognition. Percent of DCX/zif268 colabeled cells did not significantly correlate with performance in the probe trial, suggesting these new cells are not

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CORT & New Neuron Activation solely recruited in response to spatial memory retrieval, but may be generally recruited during either aversive tasks or exploration. Prior research has shown that acute cold-water swim stress selectively activates young granule cells in the ventral, but not the dorsal dentate gyrus (Schoenfeld et al., 2013). Indeed, the ventral hippocampus is preferentially involved in stress, anxiety, and fear (Degroot and Treit, 2004; Hunsaker and Kesner, 2008; Kjelstrup et al., 2002; Maren and Holt, 2004; Trivedi and Coover, 2004) compared to the dorsal hippocampus Given this distinction, tasks that access anxiety-like behaviors and that are neurogenesis-dependent may reveal more about the functional consequences of diminished activation of newly generated neurons. For instance, novelty suppressed feeding (NSF) involves placing foodrestricted rodents in a novel context with chow or highly palatable food placed in the center of the arena. NSF is a neurogenesis-dependent task (David et al., 2009; Santarelli et al., 2003), relies on the ventral hippocampus (Bannerman et al., 1999; Burns et al., 1996; McHugh et al., 2004), and assesses anxiety-like behavior (Bodnoff et al., 1989). Thus, NSF may reveal more about the behavioral consequences of CORT-induced impairments in activation of new neurons in the ventral dentate gyrus. Collectively, our data suggest that the ventral hippocampus may be more sensitive to or that it may recover more slowly after high, chronic CORT. This is consistent with previous research showing that the ventral hippocampus (or temporal pole) matures more slowly than the dorsal hippocampus (or septal pole) (Snyder et al., 2012b). Future studies should further address this possibility by determining whether longer treatments of CORT, or spatial tasks temporally closer to CORT exposure, reveal changes in activation in the dorsal hippocampus. However, we did show that DCX-expression alone was correlated with spatial memory in the oil-treated groups. Our data are consistent with prior research indicating that the ventral hippocampus is activated during MWM training (Snyder et al., 2009b), which suggests that although the ventral hippocampus may be less involved in spatial navigation, it responds to tasks that require spatial processing or that have an aversive component. This is consistent with previous research showing that although the dorsal hippocampus may encode precise spatial information, the ventral hippocampus may also contribute to general aspects of spatial processing (de Hoz et al., 2003; Ferbinteanu et al., 2003; Loureiro et al., 2012; Ruediger et al., 2012; Tanti and Belzung, 2013). Alternatively, the ventral hippocampus may be activated due to the aversive nature of water maze training. Finally, zif268 immunoreactivity in the dentate gyrus was similar among all groups, which is consistent with prior research showing that zif286 mRNA expression does not vary as a consequence of spatial or cued water maze training (Guzowski et al., 2001). Our data suggest

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CORT & New Neuron Activation that prior CORT selectively reduces the activation of immature neurons in the ventral hippocampus in response after both cued and spatial training in the MWM. Prior CORT exposure reduced neurogenesis in the ventral hippocampus and altered maturation of immature neurons Our data indicate that chronic high CORT for 18 days, with a 10-day lapse between the final injection and tissue collection, yielded fewer DCX-expressing cells in the ventral, but not the dorsal dentate gyrus. This is consistent with prior research indicates that 18 d of the same dose of CORT is sufficient to suppress neurogenesis in male rats even after a 9-day delay between final dose and tissue collection, although this study did not investigate hippocampal regions separately (Wong and Herbert, 2006). When analyzed by region, 21 d of the same dose of CORT (without a delay between CORT administration and tissue collection) suppresses neurogenesis in both the dorsal and ventral hippocampus of male rats (Brummelte and Galea, 2010b). In females in the same study, however, CORT did not suppress neurogenesis in the dorsal dentate gyrus (Brummelte and Galea, 2010b). Two possibilities may explain why CORT only suppressed DCX-expressing cells in the ventral dentate gyrus in our study. First, dorsal hippocampal neurogenesis may recover rapidly after chronic high CORT due to the delay between final injection and tissue collection. If this is true, then prior CORT-treated rats should have a greater proportion of DCX-expressing cells at proliferative and intermediate stages compared with oil-treated rats. Indeed, we found that prior CORT-treated rats had a greater proportion of proliferative and intermediate cells and a lower proportion of postmitotic cells compared with oil-treated rats regardless of hippocampal region. These data suggest that after CORT treatment ceases, dorsal hippocampal cell proliferation may rebound after the cessation of glucocorticoids, yielding a relative increase in cell proliferation once CORT levels return to baseline. This could represent evidence for circuit homeostasis in neurogenesis (Meltzer et al., 2005). That is, after period of time wherein neurogensis is suppressed, homeostatic mechanisms drive upregulation of cell proliferation to compensate for previous suppression of neurogenesis. Additionally, our data are also consistent with a previous study demonstrating that high CORT shifts the proportion of new neurons toward more immature developmental stages (Lussier et al., 2013). Second, it is also possible that ventral hippocampal neurogenesis is more sensitive to high glucocorticoids and that perhaps 18 d of high CORT exposure was not sufficient to reduce neurogenesis to the same extent in dorsal as in the ventral hippocampus, at least in our study. Indeed, ventral hippocampal neurogenesis is differentially sensitive to acute stress (Kirby et al., 2013) and has

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CORT & New Neuron Activation greater implications for mood and anxiety disorders compared with the dorsal hippocampus (Kheirbek and Hen, 2011). Our findings suggest that CORT slows the maturation rate of DCX-expressing cells evidenced by a reduction in proportion of more mature phenotypes. However, it is important to note that DCX-expressing cells are a heterogenous population of immature neurons and DCX is expressed between 1-21 days after production (Brown et al., 2003). Thus our data also show that the timing of CORT exposure affects maturation rate of these cells, with the more immature DCX-expressing cells not exposed to high CORT and the more mature DCX-expressing cells produced and maturing during high CORT conditions. We think this explanation makes the most sense in light of our results that show that prior CORT increases the percentage of immature DCX-expressing cells but decreases the percentage of more mature DCX-expressing cells (as described above). We found that spatial training increased the total number of DCX-expressing cells in the ventral dentate gyrus, suppressed the proportion of DCX-expressing cells at an intermediate stage, and increased the proportion of postmitotic cells in the ventral hippocampus. These data are consistent with a body of research showing that hippocampus-dependent learning facilitates survival of immature neurons at critical periods in new neuron development (Epp et al., 2013b; Epp et al., 2011a; Epp et al., 2007; Gould et al., 1999). Specifically, prior research from our laboratory has shown that spatial training enhances cell survival when BrdU-positive cells are 610 days old during training, but not 1-5 or 11-15 days old at the time of spatial training (Epp et al., 2007) and in DCX-expressing cells in SD male rats . In this study, we used DCX as a marker of immature neurons, which are expressed in immature neurons up to approximately 21 days after production (Brown et al., 2003; Snyder et al., 2009a). Thus, the precise age of the DCXexpressing cells in this study will vary and future experiments using thymidine analogues, such as BrdU, could more precisely examine the timeline of activation of new neurons. BrdU-positive cells that are 6 – 10 days old during water maze training are less likely to be activated (as assessed using c-fos expression) than BrdU-positive cells that are 11 – 15 days old during spatial memory retrieval in adult male Sprague-Dawley rats (Epp et al., 2011a). Furthermore, Sprague-Dawley rats are more likely to show activation (co-expression with c-fos) in DCXexpressing rather than 6 – 10 day old BrdU-positive cells (Epp et al., 2011b). New neurons that are 6 – 15 days old are well within the time window of DCX expression, which collectively suggests that our study included neurons from at least two developmentally heterogeneous cell populations. Finally, it should be noted that rats have faster maturation timelines than mice for

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CORT & New Neuron Activation neurogenesis (Snyder et al., 2009a) so comparing studies across mice and rats will certainly yield different timing of activation of new neurons in response to memory retrieval. DCX-expressing cells and CORT concentrations were negatively correlated with spatial performance DCX-expressing cells in the ventral hippocampus correlated with spatial performance in oil-treated, spatial-trained rats only. In all cases, greater ventral cell density inversely related to spatial performance as ventral DCX cell density positively correlated with distance traveled in the water maze and negatively with time spent in target quadrant. Lower CORT concentrations were associated with more time in the target quadrant during the probe trial, but only in CORT-treated, spatial-trained rats. It is possible that higher endogenous CORT concentrations may interfere with recall of previously learned spatial information. It is also possible that poorer acquisition during training (and subsequently poor performance in the retrieval task) may elevate CORT concentrations.

Conclusions Our data indicate that prior chronic CORT exposure reduced neurogenesis and the activation of new neurons in the ventral dentate gyrus but did not significantly alter activation of the dentate gyrus of adult male rats. Chronic CORT may impair the functional connectivity or the internal cellular processes of immature neurons. Further, prior CORT altered the maturity of new neurons, shifting the development away from postmitotic cells to more immature, proliferative cells, which may be a compensatory increase in response to reduced proliferation during CORT exposure. Additionally, spatial training increased neurogenesis and, increased the proportion of new neurons that were in the postmitotic stage of development but reduced the proportion in the intermediate stage in the ventral dentate gyrus. These data suggest that newly generated neurons in this region are sensitive to spatial training.

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CORT & New Neuron Activation Figure Captions Figure 1: Experimental timeline indicating sequence of events and samples sizes. CORT or oil injections were administered for 18 d. FST was conducted the last two days of injections. MWM training began the day after the final injection and lasted for 5 days. Five days after the last training day, a probe trial was conducted and blood and brains were collected ~90 min after probe. Figure 2: Mean + SEM of behaviors assessed on the second day of the forced swim test (FST). A) CORT significantly reduced the latency to immobility (* main effect of treatment, P = 0.018). B) CORT significantly increased percent time spent immobile (* P = 0.001) and decreased percent time spent climbing (* P < 0.001). Figure 3: A) Mean ± SEM distance in meters to the platform in the water maze. Among spatialtrained rats, prior CORT treatment significantly reduced distance to the platform on day 2 of training (* P = 0.014). Cue-trained rats had significantly shorter distances to the platform on each day of training compared with spatial-trained rats († P < 0.001). B) Mean + SEM time spent in target quadrant during the probe trial. Prior CORT exposure significantly increased time in target quadrant (§ P = 0.013) but only in rats trained in the spatial water maze (* P = 0.022). Figure 4: Mean + SEM ng/ml serum corticosterone. Prior CORT treatment and type of training interacted to alter corticosterone concentrations 90 min after the probe trial. Specifically, in oiltreated

rats,

spatial

training

was

associated

with

significantly

lower

corticosterone

concentrations compared with cue training (* P = 0.041).

Figure 5: A) Mean + SEM total number of DCX-expressing cells. Prior CORT treatment significantly reduced number of DCX-expressing cells in the ventral hippocampus (§ P < 0.001). Planned comparisons revealed that oil-treated, spatial trained rats had more DCX-expressing cells in the ventral hippocampus (*P = 0.005) and tended to have more in the dorsal hippocampus (P = 0.06) compared with oil-treated, cue-trained rats. B) Mean + SEM density of DCX-expressing cells. Comparied with oil treatment, prior CORT treatment significantly reduced density of DCX-expressing cells in the ventral hippocampus (§ P = 0.027). C) Mean + SEM percent of DCX-expressing cells colabeled with zif268. Prior CORT treatment reduced percent of DCX-expressing cells colabeled with zif268 in the ventral hippocampus (§ P = 0.001). In oil-

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CORT & New Neuron Activation treated rats, the ventral hippocampus contained a greater percentage of colabled cells compared with the dorsal hippocampus (@ P = 0.022).

Figure 6: Representative photomicrographs of DCX-expressing cells in the ventral dentate gyrus (suprapyramidal blade). A) Depicts an oil-treated rat whereas B) depicts a CORT-treated rat. Images were taken at 200x. Scale bar = 100 µm. gcl = granule cell layer. Figure 7: Representative images of dorsal hippocampal sections showing DCX- (A & B), zif268labeled tissue (C & D) and the overlay (E & F). A, C & E) Images were taken at 40x. Scale bar represents 400 µm. Inset represents images on the right (B, D & F, respectively). B, D & F) Images depict a positively labeled cell. F) Indicates double-labeled cell. Images were taken at 1000x. Scale bar represents 10 µm. Figure 8: Mean + SEM proportions of DCX-expressing cells at particular developmental stages. A) CORT exposure significantly increased proportion of proliferative cells regardless of hippocampal region or MWM training (‡ P = 0.01). B) CORT treatment also significantly increased the proportion of intermediate cells regardless of hippocampal region or MWM training (‡ P = 0.035). Additionally, regardless of CORT treatment, spatial training significantly reduced the proportion of intermediate cells, but only in the ventral hippocampus (# P = 0.004). C) CORT treatment reduced the proportion of postmitotic cells regardless of hippocampal region or MWM training (‡ P < 0.001). Additionally, compared with spatial-trained rats, cue-trained rats had a lower proportion of postmitotic cells in the ventral hippocampus, regardless of CORT treatment (# P = 0.003).

Figure 9: Photomicrographs depicting DCX-expressing cells at A) proliferative, B) intermediate and C) postmitotic developmental stages. Images were taken at 600x. Scale bar = 20 µm. gcl = granule cell layer. Figure 10: Pearson’s correlations among rats trained in the spatial MWM. A) CORT concentrations negatively correlated with time spent in target quadrant in the probe trial in CORT-treated rats (P = 0.009) but not in oil-treated rats (P = 0.86). B) Time in target quadrant negatively correlated with ventral DCX cell density in oil- (P = 0.003) but not CORT-treated rats (P = 0.89).

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CORT & New Neuron Activation References Aguilar-Valles A, Sanchez E, de Gortari P, Balderas I, Ramirez-Amaya V, Bermudez-Rattoni F, Joseph-Bravo P. 2005. Analysis of the stress response in rats trained in the water-maze: differential expression of corticotropin-releasing hormone, CRH-R1, glucocorticoid receptors and brain-derived neurotrophic factor in limbic regions. Neuroendocrinology 82(5-6):306-19. Altman J, Das GD. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124(3):319-35. Bannerman DM, Rawlins JN, McHugh SB, Deacon RM, Yee BK, Bast T, Zhang WN, Pothuizen HH, Feldon J. 2004. Regional dissociations within the hippocampus--memory and anxiety. Neurosci Biobehav Rev 28(3):273-83. Bannerman DM, Yee BK, Good MA, Heupel MJ, Iversen SD, Rawlins JN. 1999. Double dissociation of function within the hippocampus: a comparison of dorsal, ventral, and complete hippocampal cytotoxic lesions. Behav Neurosci 113(6):1170-88. Barha CK, Galea LA. 2013. The hormone therapy, Premarin, impairs hippocampus-dependent spatial learning and memory and reduces activation of new granule neurons in response to memory in female rats. Neurobiol Aging 34(3):986-1004. Bessa JM, Ferreira D, Melo I, Marques F, Cerqueira JJ, Palha JA, Almeida OF, Sousa N. 2009. The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling. Molecular Psychiatry 14(8):764-73, 739. Bodnoff SR, Suranyi-Cadotte B, Quirion R, Meaney MJ. 1989. A comparison of the effects of diazepam versus several typical and atypical anti-depressant drugs in an animal model of anxiety. Psychopharmacology (Berl) 97(2):277-9. Boldrini M, Hen R, Underwood MD, Rosoklija GB, Dwork AJ, Mann JJ, Arango V. 2012. Hippocampal angiogenesis and progenitor cell proliferation are increased with antidepressant use in major depression. Biol Psychiatry 72(7):562-71. Boldrini M, Underwood MD, Hen R, Rosoklija GB, Dwork AJ, John Mann J, Arango V. 2009. Antidepressants increase neural progenitor cells in the human hippocampus. Neuropsychopharmacology 34(11):2376-89. Bozon B, Davis S, Laroche S. 2002. Regulated transcription of the immediate-early gene Zif268: mechanisms and gene dosage-dependent function in synaptic plasticity and memory formation. Hippocampus 12(5):570-7. Bozon B, Kelly A, Josselyn SA, Silva AJ, Davis S, Laroche S. 2003. MAPK, CREB and zif268 are all required for the consolidation of recognition memory. Philos Trans R Soc Lond B Biol Sci 358(1432):805-14. Brown JP, Couillard-Despres S, Cooper-Kuhn CM, Winkler J, Aigner L, Kuhn HG. 2003. Transient expression of doublecortin during adult neurogenesis. J Comp Neurol 467(1):1-10. Brummelte S, Galea L. 2010a. Chronic corticosterone during pregnancy and postpartum affects maternal care, cell proliferation and depressive-like behavior in the dam. Horm Behav 58(5):769-79. Brummelte S, Galea L. 2010b. Chronic high corticosterone reduces neurogenesis in the dentate gyrus of adult male and female rats. Neuroscience 168(3):680-90. Brummelte S, Pawluski JL, Galea LA. 2006. High post-partum levels of corticosterone given to dams influence postnatal hippocampal cell proliferation and behavior of offspring: A model of post-partum stress and possible depression. Horm Behav 50(3):370-82. Burns LH, Annett L, Kelley AE, Everitt BJ, Robbins TW. 1996. Effects of lesions to amygdala, ventral subiculum, medial prefrontal cortex, and nucleus accumbens on the reaction to novelty: implication for limbic-striatal interactions. Behav Neurosci 110(1):60-73.

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CORT & New Neuron Activation Cameron HA, Gould E. 1994. Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience 61(2):203-9. Cameron HA, Woolley CS, McEwen BS, Gould E. 1993. Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56(2):337-44. Campbell S, Marriott M, Nahmias C, MacQueen GM. 2004. Lower hippocampal volume in patients suffering from depression: a meta-analysis. Am J Psychiatry 161(4):598-607. Chow C, Epp JR, Lieblich SE, Barha CK, Galea LA. 2013. Sex differences in neurogenesis and activation of new neurons in response to spatial learning and memory. Psychoneuroendocrinology 38(8):1236-50. Coburn-Litvak PS, Pothakos K, Tata DA, McCloskey DP, Anderson BJ. 2003. Chronic administration of corticosterone impairs spatial reference memory before spatial working memory in rats. Neurobiol Learn Mem 80(1):11-23. Cole AJ, Saffen DW, Baraban JM, Worley PF. 1989. Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340(6233):474-6. Conrad CD. 2006. What is the functional significance of chronic stress-induced CA3 dendritic retraction within the hippocampus? Behav Cogn Neurosci Rev 5(1):41-60. Conrad CD. 2010. A critical review of chronic stress effects on spatial learning and memory. Prog Neuropsychopharmacol Biol Psychiatry 34(5):742-55. Conrad CD, LeDoux JE, Magarinos AM, McEwen BS. 1999. Repeated restraint stress facilitates fear conditioning independently of causing hippocampal CA3 dendritic atrophy. Behav Neurosci 113(5):902-13. Crayton JW, Joshi I, Gulati A, Arora RC, Wolf WA. 1996. Effect of corticosterone on serotonin and catecholamine receptors and uptake sites in rat frontal cortex. Brain Res 728(2):260-2. Cryan JF, Valentino RJ, Lucki I. 2005. Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test. Neurosci Biobehav Rev 29(45):547-69. Czeh B, Michaelis T, Watanabe T, Frahm J, de Biurrun G, van Kampen M, Bartolomucci A, Fuchs E. 2001. Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc Natl Acad Sci U S A 98(22):12796-801. David DJ, Samuels BA, Rainer Q, Wang JW, Marsteller D, Mendez I, Drew M, Craig DA, Guiard BP, Guilloux JP and others. 2009. Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression. Neuron 62(4):479-93. de Hoz L, Knox J, Morris RG. 2003. Longitudinal axis of the hippocampus: both septal and temporal poles of the hippocampus support water maze spatial learning depending on the training protocol. Hippocampus 13(5):587-603. de Villiers AS, Russell VA, Taljaard JJ. 1992. Effect of corticosterone on noradrenergic nuclei in the pons-medulla and [3H]NA release from terminals in hippocampal slices. Neurochem Res 17(3):273-80. Degroot A, Treit D. 2004. Anxiety is functionally segregated within the septo-hippocampal system. Brain Res 1001(1-2):60-71. Detke MJ, Rickels M, Lucki I. 1995. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology (Berl) 121(1):66-72. Epp JR, Beasley CL, Galea LA. 2013a. Increased hippocampal neurogenesis and p21 expression in depression: dependent on antidepressants, sex, age, and antipsychotic exposure. Neuropsychopharmacology 38(11):2297-306. Epp JR, Chow C, Galea LA. 2013b. Hippocampus-dependent learning influences hippocampal neurogenesis. Front Neurosci 7:57.

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CORT & New Neuron Activation Epp JR, Haack AK, Galea LA. 2011a. Activation and survival of immature neurons in the dentate gyrus with spatial memory is dependent on time of exposure to spatial learning and age of cells at examination. Neurobiol Learn Mem 95(3):316-25. Epp JR, Scott NA, Galea LA. 2011b. Strain differences in neurogenesis and activation of new neurons in the dentate gyrus in response to spatial learning. Neuroscience 172:342-54. Epp JR, Spritzer MD, Galea LA. 2007. Hippocampus-dependent learning promotes survival of new neurons in the dentate gyrus at a specific time during cell maturation. Neuroscience 149(2):273-85. Fan Y, Chen P, Li Y, Cui K, Noel DM, Cummins ED, Peterson DJ, Brown RW, Zhu MY. 2014. Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine beta-hydroxylase in rat locus coeruleus and its terminal regions. J Neurochem 128(3):445-58. Fanselow MS, Dong HW. 2010. Are the dorsal and ventral hippocampus functionally distinct structures? Neuron 65(1):7-19. Ferbinteanu J, Ray C, McDonald RJ. 2003. Both dorsal and ventral hippocampus contribute to spatial learning in Long-Evans rats. Neurosci Lett 345(2):131-5. Galea LA, McEwen BS, Tanapat P, Deak T, Spencer RL, Dhabhar FS. 1997. Sex differences in dendritic atrophy of CA3 pyramidal neurons in response to chronic restraint stress. Neuroscience 81(3):689-97. Galea LA, Wide JK, Barr AM. 2001. Estradiol alleviates depressive-like symptoms in a novel animal model of post-partum depression. Behav Brain Res 122(1):1-9. Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ. 1999. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 2(3):260-5. Gould E, Cameron HA, Daniels DC, Woolley CS, McEwen BS. 1992. Adrenal hormones suppress cell division in the adult rat dentate gyrus. J Neurosci 12(9):3642-50. Gregus A, Wintink AJ, Davis AC, Kalynchuk LE. 2005. Effect of repeated corticosterone injections and restraint stress on anxiety and depression-like behavior in male rats. Behav Brain Res 156(1):105-14. Guzowski JF. 2002. Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 12(1):86-104. Guzowski JF, Setlow B, Wagner EK, McGaugh JL. 2001. Experience-dependent gene expression in the rat hippocampus after spatial learning: a comparison of the immediate-early genes Arc, c-fos, and zif268. J Neurosci 21(14):5089-98. Hamson DK, Wainwright SR, Taylor JR, Jones BA, Watson NV, Galea LA. 2013. Androgens increase survival of adult-born neurons in the dentate gyrus by an androgen receptor-dependent mechanism in male rats. Endocrinology 154(9):3294-304. Hastings NB, Gould E. 1999. Rapid extension of axons into the CA3 region by adult-generated granule cells. J Comp Neurol 413(1):146-54. Heimovics SA, Prior NH, Maddison CJ, Soma KK. 2012. Rapid and widespread effects of 17betaestradiol on intracellular signaling in the male songbird brain: a seasonal comparison. Endocrinology 153(3):1364-76. Hoffman AN, Krigbaum A, Ortiz JB, Mika A, Hutchinson KM, Bimonte-Nelson HA, Conrad CD. 2011. Recovery after chronic stress within spatial reference and working memory domains: correspondence with hippocampal morphology. Eur J Neurosci 34(6):1023-30. Hunsaker MR, Kesner RP. 2008. Dissociations across the dorsal-ventral axis of CA3 and CA1 for encoding and retrieval of contextual and auditory-cued fear. Neurobiol Learn Mem 89(1):61-9. Ising M, Horstmann S, Kloiber S, Lucae S, Binder EB, Kern N, Kunzel HE, Pfennig A, Uhr M, Holsboer F. 2007. Combined dexamethasone/corticotropin releasing hormone test predicts

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CORT & New Neuron Activation treatment response in major depression - a potential biomarker? Biol Psychiatry 62(1):4754. Jakubs K, Nanobashvili A, Bonde S, Ekdahl CT, Kokaia Z, Kokaia M, Lindvall O. 2006. Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability. Neuron 52(6):1047-59. Jessberger S, Nakashima K, Clemenson GD, Jr., Mejia E, Mathews E, Ure K, Ogawa S, Sinton CM, Gage FH, Hsieh J. 2007. Epigenetic modulation of seizure-induced neurogenesis and cognitive decline. J Neurosci 27(22):5967-75. Johnson SA, Fournier NM, Kalynchuk LE. 2006. Effect of different doses of corticosterone on depression-like behavior and HPA axis responses to a novel stressor. Behav Brain Res 168(2):280-8. Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, Charnay P, Bozon B, Laroche S, Davis S. 2001. A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat Neurosci 4(3):289-96. Kalynchuk LE, Gregus A, Boudreau D, Perrot-Sinal TS. 2004. Corticosterone increases depressionlike behavior, with some effects on predator odor-induced defensive behavior, in male and female rats. Behav Neurosci 118(6):1365-77. Karten YJ, Nair SM, van Essen L, Sibug R, Joels M. 1999. Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons. Proc Natl Acad Sci U S A 96(23):13456-61. Kheirbek MA, Hen R. 2011. Dorsal vs ventral hippocampal neurogenesis: implications for cognition and mood. Neuropsychopharmacology 36(1):373-4. Kirby ED, Muroy SE, Sun WG, Covarrubias D, Leong MJ, Barchas LA, Kaufer D. 2013. Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2. Elife 2:e00362. Kjelstrup KG, Tuvnes FA, Steffenach HA, Murison R, Moser EI, Moser MB. 2002. Reduced fear expression after lesions of the ventral hippocampus. Proc Natl Acad Sci U S A 99(16):1082530. Loureiro M, Lecourtier L, Engeln M, Lopez J, Cosquer B, Geiger K, Kelche C, Cassel JC, Pereira de Vasconcelos A. 2012. The ventral hippocampus is necessary for expressing a spatial memory. Brain Struct Funct 217(1):93-106. Luine V, Martinez C, Villegas M, Magarinos AM, McEwen BS. 1996. Restraint stress reversibly enhances spatial memory performance. Physiol Behav 59(1):27-32. Luine V, Villegas M, Martinez C, McEwen BS. 1994. Repeated stress causes reversible impairments of spatial memory performance. Brain Res 639(1):167-70. Lussier AL, Lebedeva K, Fenton EY, Guskjolen A, Caruncho HJ, Kalynchuk LE. 2013. The progressive development of depression-like behavior in corticosterone-treated rats is paralleled by slowed granule cell maturation and decreased reelin expression in the adult dentate gyrus. Neuropharmacology 71:174-83. Magarinos AM, Orchinik M, McEwen BS. 1998. Morphological changes in the hippocampal CA3 region induced by non-invasive glucocorticoid administration: a paradox. Brain Res 809(2):314-8. Malberg JE, Eisch AJ, Nestler EJ, Duman RS. 2000. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 20(24):9104-10. Maren S, Holt WG. 2004. Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus. Behav Neurosci 118(1):97-110. Marks W, Fournier NM, Kalynchuk LE. 2009. Repeated exposure to corticosterone increases depression-like behavior in two different versions of the forced swim test without altering nonspecific locomotor activity or muscle strength. Physiol Behav 98(1-2):67-72.

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CORT & New Neuron Activation McClure RE, Barha CK, Galea LA. 2013. 17beta-Estradiol, but not estrone, increases the survival and activation of new neurons in the hippocampus in response to spatial memory in adult female rats. Horm Behav 63(1):144-57. McHugh SB, Deacon RM, Rawlins JN, Bannerman DM. 2004. Amygdala and ventral hippocampus contribute differentially to mechanisms of fear and anxiety. Behav Neurosci 118(1):63-78. McKinnon MC, Yucel K, Nazarov A, MacQueen GM. 2009. A meta-analysis examining clinical predictors of hippocampal volume in patients with major depressive disorder. J Psychiatry Neurosci 34(1):41-54. Meltzer LA, Yabaluri R, Deisseroth K. 2005. A role for circuit homeostasis in adult neurogenesis. Trends Neurosci 28(12):653-60. Morris RG, Garrud P, Rawlins JN, O'Keefe J. 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297(5868):681-3. Parker KJ, Schatzberg AF, Lyons DM. 2003. Neuroendocrine aspects of hypercortisolism in major depression. Horm Behav 43(1):60-6. Plumpe T, Ehninger D, Steiner B, Klempin F, Jessberger S, Brandt M, Romer B, Rodriguez GR, Kronenberg G, Kempermann G. 2006. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation. BMC Neurosci 7:77. Porsolt RD, Anton G, Blavet N, Jalfre M. 1978. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 47(4):379-91. Porsolt RD, Le Pichon M, Jalfre M. 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266(5604):730-2. Ramirez-Amaya V, Marrone DF, Gage FH, Worley PF, Barnes CA. 2006. Integration of new neurons into functional neural networks. J Neurosci 26(47):12237-41. Ruediger S, Spirig D, Donato F, Caroni P. 2012. Goal-oriented searching mediated by ventral hippocampus early in trial-and-error learning. Nat Neurosci 15(11):1563-71. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O and others. 2003. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301(5634):805-9. Sapolsky RM, Krey LC, McEwen BS. 1984. Glucocorticoid-sensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proceedings of the National Academy of Sciences 81(19):6174-7. Sapolsky RM, McEwen BS, Rainbow TC. 1983. Quantitative autoradiography of [3H]corticosterone receptors in rat brain. Brain Res 271(2):331-4. Schoenfeld TJ, Rada P, Pieruzzini PR, Hsueh B, Gould E. 2013. Physical exercise prevents stressinduced activation of granule neurons and enhances local inhibitory mechanisms in the dentate gyrus. J Neurosci 33(18):7770-7. Sheline YI, Gado MH, Kraemer HC. 2003. Untreated depression and hippocampal volume loss. Am J Psychiatry 160(8):1516-8. Sheline YI, Sanghavi M, Mintun MA, Gado MH. 1999. Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J Neurosci 19(12):5034-43. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW. 1996. Hippocampal atrophy in recurrent major depression. Proceedings of the National Academy of Sciences 93(9):390813. Snyder JS, Choe JS, Clifford MA, Jeurling SI, Hurley P, Brown A, Kamhi JF, Cameron HA. 2009a. Adultborn hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice. J Neurosci 29(46):14484-95.

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CORT & New Neuron Activation Snyder JS, Clifford MA, Jeurling SI, Cameron HA. 2012a. Complementary activation of hippocampalcortical subregions and immature neurons following chronic training in single and multiple context versions of the water maze. Behav Brain Res 227(2):330-9. Snyder JS, Ferrante SC, Cameron HA. 2012b. Late maturation of adult-born neurons in the temporal dentate gyrus. PLoS One 7(11):e48757. Snyder JS, Radik R, Wojtowicz JM, Cameron HA. 2009b. Anatomical gradients of adult neurogenesis and activity: young neurons in the ventral dentate gyrus are activated by water maze training. Hippocampus 19(4):360-70. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA. 2011. Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature 476(7361):458-61. Sunanda, Rao MS, Raju TR. 1995. Effect of chronic restraint stress on dendritic spines and excrescences of hippocampal CA3 pyramidal neurons--a quantitative study. Brain Res 694(1-2):312-7. Tanti A, Belzung C. 2013. Neurogenesis along the septo-temporal axis of the hippocampus: are depression and the action of antidepressants region-specific? Neuroscience 252:234-52. Tashiro A, Makino H, Gage FH. 2007. Experience-specific functional modification of the dentate gyrus through adult neurogenesis: a critical period during an immature stage. J Neurosci 27(12):3252-9. Trivedi MA, Coover GD. 2004. Lesions of the ventral hippocampus, but not the dorsal hippocampus, impair conditioned fear expression and inhibitory avoidance on the elevated T-maze. Neurobiol Learn Mem 81(3):172-84. Vorhees CV, Williams MT. 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848-58. Watanabe Y, Gould E, Cameron HA, Daniels DC, McEwen BS. 1992a. Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons. Hippocampus 2(4):431-5. Watanabe Y, Gould E, McEwen BS. 1992b. Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 588(2):341-5. Wisden W, Errington ML, Williams S, Dunnett SB, Waters C, Hitchcock D, Evan G, Bliss TV, Hunt SP. 1990. Differential expression of immediate early genes in the hippocampus and spinal cord. Neuron 4(4):603-14. Wong EY, Herbert J. 2004. The corticoid environment: a determining factor for neural progenitors' survival in the adult hippocampus. Eur J Neurosci 20(10):2491-8. Wong EY, Herbert J. 2006. Raised circulating corticosterone inhibits neuronal differentiation of progenitor cells in the adult hippocampus. Neuroscience 137(1):83-92. Wood GE, Beylin AV, Shors TJ. 2001. The contribution of adrenal and reproductive hormones to the opposing effects of stress on trace conditioning in males versus females. Behav Neurosci 115(1):175-87. Workman JL, Brummelte S, Galea LA. 2013. Postpartum corticosterone administration reduces dendritic complexity and increases the density of mushroom spines of hippocampal CA3 arbors in dams. J Neuroendocrinol. Wright RL, Lightner EN, Harman JS, Meijer OC, Conrad CD. 2006. Attenuating corticosterone levels on the day of memory assessment prevents chronic stress-induced impairments in spatial memory. Eur J Neurosci 24(2):595-605.

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1057x793mm (72 x 72 DPI)

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Table 1. Granule cell layer volume (mm3) Cue

Spatial

Oil

CORT

Oil

CORT

Dorsal

1.68 ± 0.07

1.94 ± 0.08

1.88 ± 0.07c

1.73 ± 0.07

Ventral

1.18 ± 0.17a

1.46 ± 0.19a

1.92 ± 0.16b,c

1.49 ± 0.16b

a

significantly less than dorsal (within cue comparison)

b

significantly greater than cue group (within region comparison)

c

significantly greater than cue group (within oil comparison)

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Table 2. Zif268 optical density Cue

Spatial

Oil

CORT

Oil

CORT

Dorsala

12.47 ± 1.74

12.71 ± 1.86

12.45 ±1.64

10.81 ± 1.64

Ventral

8.76 ± 1.36

9.02 ± 1.45

7.16 ± 1.28

8.00 ± 1.28

a

significantly greater than ventral

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