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Motherhood and ovarian hormones influence hippocampus-dependent cognition and neurogenesis later in life Roes, Meighen Maria 2013

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 MOTHERHOOD AND OVARIAN HORMONES INFLUENCE HIPPOCAMPUS-DEPENDENT COGNITION AND NEUROGENESIS LATER IN LIFE  by MEIGHEN MARIA ROES B.Sc., Queen?s University, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Psychology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   August 2013 ? Meighen Maria Roes, 2013 ii  Abstract Age-related cognitive decline in women may be influenced by hormonal experiences over the lifespan including parity (pregnancy and motherhood) and menopause. Previous research implicates hippocampal neurogenesis in spatial learning and age-related cognitive decline and indicates that different regions of the hippocampus (dorsal and ventral) may contribute differentially to spatial working and reference memory. Therefore, the current study investigates influences of parity and ovarian hormones on hippocampus-dependent spatial working and reference memory and neurogenesis during middle age.  Multiparous and nulliparous middle-aged rats were either ovariectomized or received sham surgery and were injected with the DNA synthesis marker bromodeoxyuridine (BrdU). Rats were trained on working/reference (hidden platform moved every two days) and reference (hidden platform was stationary) memory versions of the Morris water maze on days 12-21 after BrdU injection. On day 22 rats were given a probe trial to assess memory retention. Multiparous rats had enhanced early working/reference memory acquisition compared to nulliparous rats and this was more prominent in ovariectomized rats.  In contrast, nulliparous females had better reference memory acquisition compared to multiparous rats and had enhanced spatial reference memory during the probe trial. Multiparous females had a larger ventral dentate gyrus and greater density of immature neurons compared to nulliparous females, whereas nulliparous females had greater density of older BrdU-labelled cells in the dentate gyrus compared to multiparous females. Depending on ovarian hormone status and parity, neurogenesis in the dorsal dentate gyrus correlated with measures of spatial reference learning, whereas neurogenesis in the ventral dentate gyrus correlated with spatial working/reference performance. Overall, results indicate multiparous rats have better spatial working memory performance whereas nulliparous rats have enhanced reference performance.  These results may reflect differences in neuroplasticity (with multiparous rats having more iii  immature neurons and nulliparous rats having greater survival of new neurons) and/or stress resilience differences between the parous groups.  Importantly, the influence of parity on spatial working and reference memory and acquisition was modified by ovarian hormone status. These results also suggest that the role of new neurons in cognition may be moderated by parity and ovarian status in middle age.      iv  Preface This manuscript was conceived of and planned by Meighen M. Roes and Dr. Liisa Galea with input from Dr. Cindy K. Barha. Meighen M. Roes, Cindy K. Barha, Carmen C. Chow, and Stephanie Lieblich carried out the experimental work. Meighen M. Roes carried out the statistical analysis and writing of the thesis with the feedback and supervision of Dr. Liisa Galea. This research was conducted with approval of the UBC Animal Care Committee (A12-0004; The effects of motherhood on Cognition, Depression, and Neurogenesis).                  v  Table of Contents Abstract ......................................................................................................................................... ii Preface .......................................................................................................................................... iv Table of Contents ........................................................................................................................... v List of Figures .............................................................................................................................. ix Acknowledgements ..................................................................................................................... xii INTRODUCTION ......................................................................................................................... 1 Hippocampal-dependent memory and aging ............................................................................... 1 Adult hippocampal neurogenesis declines with age .................................................................... 6 Neuroendocrine changes during aging ......................................................................................... 7 Estrogens, hippocampal neurogenesis, and cognition ................................................................. 9 METHODS ................................................................................................................................... 16 Animals ...................................................................................................................................... 16 Apparatus ................................................................................................................................... 16 Procedures .................................................................................................................................. 17 Ovariectomy and sham surgery.................................................................................................. 17 5-bromo-2-deoxyuridine (BrdU) administration ....................................................................... 18 Morris water maze training ........................................................................................................ 18 Working/reference spatial memory trials ................................................................................... 19 Reference spatial memory trials ................................................................................................. 19 Lavage ........................................................................................................................................ 19 Probe trial and perfusion ............................................................................................................ 20 Immunohistochemistry .............................................................................................................. 21 vi  BrdU immunohistochemistry ..................................................................................................... 21 Doublecortin immunohistochemistry ......................................................................................... 21 Cell counting .............................................................................................................................. 22 Data analyses ............................................................................................................................. 24 RESULTS ..................................................................................................................................... 27 Multiparous females had significantly shorter distances to locate the hidden platform than did nulliparous during early training on the working/reference spatial learning task and this was more pronounced in ovariectomized rats ...................................................................... 27 Nulliparous rats had smaller path distances to the hidden platform in the reference version of the water maze compared to multiparous rats. ..................................................................... 28 Ovariectomized, but not intact, nulliparous females spent more time in the target quadrant during the probe trial than did multiparous females. Rats in proestrus spent significantly less time in the target quadrant during the probe trial. ......................................................... 29 Multiparous females had a greater volume of the ventral region of the dentate gyrus compared to Nulliparous females. Ovariectomized females had smaller ventral hilar volumes compared to sham-operated rats ........................................................................................... 30 Nulliparous females had a greater density of BrdU-labelled cells in the granule cell layer compared to Multiparous females, regardless of ovarian hormone status. .......................... 31 Density of BrdU-labelled cells in the ventral granule cell layer correlated with working/reference memory performance in sham-operated rats.......................................... 31 In ovariectomized females, dorsal densities of BrdU-labeled cells in the GCL significantly negatively correlated with total distance in the reference memory version of the water maze ..................................................................................................................................... 32 vii  Density of BrdU-labelled cells in the granule cell layer of the hippocampus did not correlate with probe trial performance ................................................................................................ 32 Multiparous rats had a greater density of Type B and C DCX-expressing cells in the dentate gyrus than did multiparous rats. The densities of Type B and C DCX-expressing cells were greater in the dorsal DG than in the ventral DG .................................................................. 32 In sham-operated nulliparous rats, density of Type B DCX-expressing cells in the dorsal granule cell layer negatively correlated with working/reference memory performance at an early stage of training ........................................................................................................... 33 Densities of DCX-expressing cells did not correlate with total distance to reach hidden platform during reference spatial learning. .......................................................................... 33 Density of ventral Type B DCX-expressing cells positively correlated with probe trial performance in nulliparous ovariectomized females. Density of dorsal DCX-expressing cells negatively correlated with probe trial performance in multiparous sham-operated females. ................................................................................................................................ 34 The majority of sham-operated rats continued to cycle through estrous phases ....................... 34 DISCUSSION ............................................................................................................................... 49 Multiparous rats had better early spatial working/reference acquisition than nulliparous rats and ovariectomy enhanced this effect .................................................................................. 50 Rats in proestrus showed poorer spatial memory. Nulliparity was associated with better spatial memory in ovariectomized but not sham animals. ............................................................... 54 Multiparous rats had more immature neurons, while nulliparous rats had greater survival of older new neurons (BrdU-labelled cells) ............................................................................. 55 Multiparity resulted in larger ventral dentate gyrus volume ...................................................... 57 viii  BrdU-labelled cells in the ventral dentate gyrus are associated with better performance in working/reference MWM in sham rats. ............................................................................... 59 BrdU-labeled cells in the dorsal dentate gyrus correlated with better spatial reference performance in ovariectomized female rats ......................................................................... 60 The density of immature neurons in the dorsal dentate gyrus was related to better spatial reference memory in intact females while the density of immature neurons in the ventral dentate gyrus was related to better spatial memory only in ovariectomized nulliparous rats.61 CONCLUSIONS .......................................................................................................................... 63 REFERENCES ............................................................................................................................ 64    ix  List of Figures Figure 1. A) Experimental outline. MWM = Morris Water Maze. B) BrdU-labeled cells in the granul cell layer. C) Type A, B, and C DCX-labeled cells in the GCL. Scale bar = 10?m) ................................................................................................................................... 25 Figure 2. Mean distance (A) and latency (B) for multiparous and nulliparous females to reach the hidden platform in the working/reference version of the Morris Water Maze during Early (days 1-3) and Late (days 4-6) training. Nulliparous females had greater distance and latency to reach the hidden platform during early training, p?s < .04. C) and D): Nulliparous ovariectomized females had greater path lengths and latencies to reach the hidden platform during Early learning (days 1-3 of training; p = .013) compared to sham-operated nulliparous females, whereas multiparous sham and ovariectomized females performed equivalently, p = .79 .................................................... 34 Figure 3. Mean distance to reach the hidden platform in the reference version of the Morris Water Maze across training days. Nulliparous females had smaller path length and shorter latencies to reach the hidden platform compared to multiparous females (p = .05)........................................................................................................................................ 36 Figure 4. Mean percentage of time spent in the target quadrant in parity and ovarian hormone groups, controlling for estrous phase. Ovariectomized nulliparous rats more time in the target quadrant than did ovariectomized multiparous rats (p = .019), whereas intact multiparous and nulliparous rats spent equal proportions of time in the target quadrant (p = .45). ...................................................................................................... 37 Figure 5. Mean percentage of time spent in Quadrant 1 (target quadrant) and Quadrants 2-3 for non-proestrous sham females, proestrous sham females, and ovariectomized females. Rats in proestrus spent significantly less time in the target quadrant than rats x  in non-proestrus (p  = .031) and  marginally less time in the target quadrant than did ovariectomized rats. ............................................................................................................. 38 Figure 6. Mean dentate gyrus volume (mm3) in the dorsal and ventral regions of the dentate gyrus as a function of ovarian status (sham-operated, ovariectomized) and area (granule cell layer and hilus). In the hilus, sham-operated rats had significantly greater ventral volume (p = .002) than did ovariectomized rats ...................................................... 39 Figure 7. Mean dentate gyrus volume (mm3) in the dorsal and ventral regions of the dentate gyrus  as a function of reproductive experience (multiparous, nulliparous). Multiparous females had significantly greater ventral (p  = .0001) but not dorsal (p = .094) dentate gyrus volumes. ............................................................................................... 40 Figure 8. Mean density (cells/mm3) in the the dentate gyrus as a function of reproductive experience (Multiparous, Nulliparous) and dentate gyrus area (Granule cell layer, Hilus). Nulliparous females had significantly greater BrdU-labelled cell density in the GCL (p  <.001) but not the hilus (p = .89). .......................................................................... 41 Figure 9. Mean density (cells/mm3) of BrdU-labeled cells in the ventral region of the granule cell layer of sham-operated females, and total distance to reach the hidden platform across testing days in the Working/Reference version of the Morris Water Maze. Ventral density of BrdU-labeled cells was negatively correlated with total distance, r = -.58, p = .005. ................................................................................................... 42 Figure 10. Density (cells/mm3) of BrdU-labeled cells in the dorsal region of the granule cell layer and total distance for females to reach the hidden platform across testing days in the Reference version of the Morris Water Maze. A) In ovariectomized females, BrdU density in the dorsal GCL negatively correlated with total distance to reach the hidden platform during the Reference version of the Morris water maze, r = -.50, p = .025. B) xi  In sham-operated females, BrdU density in the dorsal GCL did not correlate with total distance, p = .40. .................................................................................................................. 43 Figure 11 A). Mean density of Type A, Type B, and Type C DCX-expressing cells in the dentate gyrus as a function of parity (multiparous, nulliparous). Multiparous females had significantly greater densities of Type B and C cells compared to nulliparous females (p?s < .001). *** signifies p < .001 B): Mean density of Type A, Type B, and Type C DCX-expressing cells in the dentate gyrus as a function of region (dorsal, ventral). The densities of Type B and C cells were greater in the dorsal dentate gyrus than in the ventral dentate gyrus (p?s < .001).. ..................................................................... 44 Figure 12. Density (cells/mm3) of DCX-expressing Type B cells in the dorsal region of the granule cell layer and total distance for sham-operated nulliparous females to reach the hidden platform across testing days in the Working/Reference version of the Morris Water Maze. In sham-operated nulliparous rats, the density of Type B cells in the Dorsal GCL significantly negatively correlated with total distance across the entire testing period, r = -.83, p = .003 ........................................................................................... 46 Figure 13. A) Density (cells/mm3) DCX-expressing cells in the dorsal region of the granule cell layer and percentage of time that ovariectomized nulliparous spent in the target quadrant during the probe trial. In sham-operated multiparous females, dorsal DCX densities significantly negatively correlated with probe trial performance (r = -.84, p = .037) B) Density (cells/mm3) of  Type B DCX-expressing cells in the ventral region of the granule cell layer and percentage of time that ovariectomized nulliparous spent in the target quadrant during the probe trial. In ovariectomized nulliparous rats, density of Type B cells in the ventral dentate gyrus significantly positively correlated with probe trial performance, r  = .71, p = .033. .......................................................................... 47 xii  Acknowledgements I?d like to thank my supervisor, Dr. Liisa Galea, for her insight, support, and mentorship. She has challenged me to become a better researcher and a more critical thinker while fostering a fascination in this field of study. Thanks for the talks, the chocolate, and the wisdom (and for cracking the whip once or twice!) I look forward to the years ahead! Thank you, too, to my wonderful labmates.  This project could not have been completed without the help of Dr. Cindy Barha. A special thank-you to Stephanie Lieblich and Carmen Chow, who have been incredible sources of knowledge and support for the last two years but especially these last few months. To friends, family, past and present labmates, and the people who keep me sane ? the Ahticles, Roeses, and the Maiers, the truly amazing people in the Speech lab and CPD lab at Queen?s, and the people I?ve had the pleasure of knowing at UBC: Thank you!1  INTRODUCTION Human aging, particularly after the sixth decade, is associated with a decline in certain aspects of cognitive function. Both in ?normal? aging and under pathological conditions such as Alzheimer's disease that are associated with severe and global deficits, there are significant inter-individual differences in the progression and extent of cognitive decline. Some individuals retain high cognitive performance even into the late decades of life (Berkman et al., 1993), calling into question the appropriateness of the concept of ?healthy? levels of cognitive decline and highlighting the importance of understanding individual and experiential predictors of cognitive health in later life. Even a modest decrease or delay of cognitive impairment could have significant implications for quality of life. At the population level the societal burden will grow as the population of individuals aged 65 and older doubles over the next decade (Fougere & Merette, 1999; Maxime & Marcel, 1999). This population increase will lead to increased numbers of cognitively impaired citizens, increased challenges to the health care system, and decreased independence and quality of life for the affected individuals. A number of factors, including cardiovascular health (Joosten et al., 2013; Knopman. et al., 2001), exercise level (Jak, 2012), and cognitive engagement (Kramer, Bherer, Colcombe, Dong, & Greenough, 2004) are associated with decreased risk for cognitive decline. However, the majority of variance accounting for individual differences in age-related cognitive decline remains unaccounted for. In thesis I will examine the contribution of ovarian hormones and reproductive experience on cognitive ability and neuroplasticity in female rodents. Hippocampal-dependent memory and aging   Different cognitive domains show greater relative decline with aging, and these include working memory, long-term (declarative) memory, and spatial learning (Kausler, 1994). In contrast, measures of vocabulary and semantic knowledge remain relatively unaffected until late life (Park et al., 2  2002). Changes in the structural integrity and function of the brain underlie cognitive declines in susceptible domains. The two domains I will concentrate on in this thesis are reference memory and working memory. Reference memory can be defined as long term memory for events or stimuli that stay stable over time and relies on the integrity of the hippocampus and caudate nucleus (Packard & White, 1990). On the other hand working memory can be defined as trial unique information to guide prospective action (A. Baddeley, 1992), requires storage and manipulation of changing information over a brief period of time and relies on the integrity of the hippocampus and prefrontal cortex(PFC). Intriguingly the hippocampus is involved in both aspects of memory. The hippocampus has long been of interest as a structure important in learning and memory since descriptions by Scoville and Milner of ?patient H.M.,? a man who lost the ability to form declarative memories after bilarteral removal of the medial temporal lobe, including the hippocampus, to alleviate his epileptic seizures (Squire, 2009). The hippocampus is particularly vulnerable in Alzheimer?s disease (Du et al., 2001; Jack et al., 1998) and aging (Driscoll et al., 2003; Driscoll et al., 2006; Rosenzweig & Barnes, 2003). Older humans and rodents show impaired performance of hippocampus-dependent learning, such as spatial learning (Driscoll et al., 2003; Lyons-Warren, Lillie, & Hershey, 2004; Moffat & Zonderman, 2001; Drapeau et al., 2003; Gallagher, Burwell, & Burchinal, 1993; Lindner, 1997).  Spatial learning and memory can be demonstrated in rodents using Morris water maze protocols that keep spatial information and targets constant over trials (Drapeau et al., 2003; Gallagher, Burwell, & Burchinal, 1993; Lindner, 1997).  Hippocampal lesions produce reference spatial memory deficits similar to those seen in aging (Kessels, de Haan, Kappelle, & Postma, 2001; Winocur, 1988). However, the functional role of the hippocampus in spatial reference memory seems to be restricted to activity of the dorsal hippocampus (Bannerman et al., 2004; Sahay & Hen, 2007), as lesions to the dorsal but not ventral hippocampus impair spatial reference acquisition in rodents (Pothuizen, Zhang, Jongen-R?lo, Feldon, & Yee, 2004).   3   Spatial working memory is another category of declarative memory affected by age (J. L. Bizon et al., 2009; Reuter-Lorenz et al., 2000; Winocur, 1992) and Alzheimer?s disease (A. D. Baddeley, Bressi, Della Sala, Logie, & Spinnler, 1991). The neural substrates of spatial working memory are somewhat different from those used during spatial reference memory, as they include a greater role of the PFC in addition to the ventral region of the hippocampus, from where hippocampal projections to the PFC originate (Bizon & Woods, 2009).  Lesions to the PFC and hippocampus reliably produce impairment in spatial working memory (Bannerman et al., 2004; Winocur & Moscovitch, 1990). However, the relative role of the dorsal and ventral hippocampus to working memory remains unclear. Whereas some findings indicate that lesions or disruptions of the ventral hippocampus impair spatial working memory (Levin, Christopher, Weaver, Moore, & Brucato, 1999; Wang & Cai, 2006), others have found effects only of dorsal lesions on spatial working memory (Bannerman et al., 2004; Pothuizen et al., 2004).  Positron emission tomography (PET) research indicates that aging is associated with decreased activation of the hippocampus during spatial working memory tasks, coupled by an increased activation of the prefrontal cortex (PFC), possibly indicating functional decrease in the hippocampus a degree of compensation by the PFC (Reuter-Lorenz et al., 2000).  Declines in both spatial working and reference memory are mediated in part by functional and structural changes in the hippocampus with age  (Winocur, 1988). The Morris water maze can target spatial working memory by changing the target spatial location across training trials (Lindner, Balch, & VanderMaelen, 1992; Vorhees & Williams, 2006).The involvement of ovarian hormones, parity, and hippocampal region (dorsal and ventral) to spatial reference and working memory in middle age has not been investigated but is the focus of this thesis.   The aging hippocampus  The hippocampus is highly affected during aging in both humans and rodent models. Human 4  imaging studies show decreases in both grey and white matter in the PFC and hippocampus with age (Raz et al., 2005; Raz, Rodrigue, Head, Kennedy, & Acker, 2004; Resnick, Pham, Kraut, Zonderman, & Davatzikos, 2003). Rodent research has indicated that the hippocampus of the aged rat has decreased structural (neuropil, synaptic density, neurogenesis) and electrophysiological (long-term potentiation) properties among other changes (Driscoll et al., 2006; Jessberger & Gage, 2008; Rosenzweig & Barnes, 2003). This thesis will focus on adult hippocampal neurogenesis, a form of hippocampal plasticity that decreases robustly with age and may contribute to age-related cognitive decline. adult hippocampal neurogenesis  Adult neurogenesis, the production of new neurons in adulthood, occurs in the dentate gyrus of the hippocampus of most mammalian species studied, including humans (Eriksson et al., 1998; Spalding et al., 2013). Neurogenesis is comprised of the processes of cell proliferation, differentiation, migration and survival (Barha, Barker, Brummelte, Epp, & Galea, 2009). Briefly, progenitor cells in the subgranular zone of the dentate gyrus give rise to daughter cells  that migrate into the granule cell layer (Barha et al., 2009). The majority of these cells differentiate into mature neurons, become morphologically similar to mature neurons already residing in the GCL and form functional synapses onto pyramidal cells in the CA3 region of the hippocampus (Gould, Tanapat, Rydel, & Hastings, 2000). The level of neurogenesis can be altered independently by influencing the rate of cell proliferation, differentiation, maturation and/or cell survival. The production of new cells can be identified by labelling DNA as it is synthesized during mitosis using exogenous markers such as 5-bromo-2-deoxyuridine (BrdU), a synthetic thymidine analog. BrdU becomes incorporated into the DNA of every cell undergoing DNA synthesis during the two hours after injection, and may be used to examine labelled cells at time points ranging from hours, weeks or even years later depending on the timeline of interest (Eriksson et al., 1998; Kempermann, Gast, Kronenberg, Yamaguchi, & Gage, 2003). To verify 5  that these new BrdU-labelled cells are indeed neurons, BrdU-labelled cells must be co-labelling with neuronal markers such as NeuN (von Bohlen und Halbach, 2011).  Endogenous markers such as Ki67 or doublecortin (DCX) can be used to assess numbers of proliferating cells and immature neurons, respectively (von Bohlen und Halbach, 2011).    There is evidence that new hippocampal neurons are involved in hippocampus-dependent learning and memory (Epp, Chow, & Galea, 2013). Studies that manipulate neurogenesis levels using irradiation or other ablation techniques find that adult neurogenesis is required for pattern separation (Clelland et al., 2009). Spatial learning can also manipulate neurogenesis levels in the hippocampus (Epp et al., 2013). Spatial reference learning increases cells survival dependent on age of cells at training (Epp, Haack, & Galea, 2011) and task difficulty (Epp, Haack, & Galea, 2010). The relationship between neurogenesis and learning may differ for reference and working spatial memory, as a number of findings indicated decreased survival of immature neurons with working memory training (Mohapel, Mundt-Petersen, Brundin, & Frielingsdorf, 2006; Xu et al., 2011). A single experimental study found that ablating neurogenesis caused an improvement of hippocampal-dependent working memory (Saxe et al., 2007). The exact role of adult-generated neurons in the function of the hippocampus remains to be fully elucidated. One of the more intriguing recent findings has been that new neurons are preferentially activated in response to spatial memory retrieval (Epp, Haack, et al., 2011; Kee, Teixeira, Wang, & Frankland, 2007).  Thus the activation of new neurons, identified by immediate early genes, in dorsal or ventral hippocampus in response to memory may be an important tool to understand the contribution of adult neurogenesis to learning and memory.   There may also be regional differences in the functional role of adult-generated neurons. The hippocampus seems to be functionally dissociated along a dorsal-ventral pathway, with the dorsal region important for spatial memory, while the ventral region is more important in stress regulation and 6  ?emotionality? (Bannerman et al., 2004; Fanselow & Dong, 2010).  Further, as previously mentioned, other studies have shown that the dorsal hippocampus is important for spatial reference memory while the ventral hippocampus plays a larger role in spatial working memory (See Bannerman, 2004 for a review). Furthermore the septo-temporal location of new neurons and their involvement in spatial learning may change from ventral to dorsal hippocampus as training progresses (Ruediger, Spirig, Donato, & Caroni, 2012).  It may therefore be that changes in reference and working spatial memory during aging are related to region-specific changes in neurogenesis. Thus far changes in neurogenesis in different areas of the hippocampus with respect to aging and memory have not been studied. Adult hippocampal neurogenesis declines with age  In young adult rats, approximately 9 000 new cells are generated each day in the dentate gyrus,  more than 250 000 per month (Cameron & McKay, 2001), suggesting a turnover of approximately 6% of cells per month. However, aging is associated with an exponential decline in hippocampal neurogenesis (Epp, Barker, & Galea, 2009; Lazarov, Mattson, Peterson, Pimplikar, & van Praag, 2010), raising the possibility that age-related cognitive deficits may result from a suppression of neurogenesis. Although hippocampal neurogenesis declines with age (Ben Abdallah, Slomianka, Vyssotski, & Lipp, 2010; Bizon & Gallagher, 2003; Kuhn, Dickinson-Anson, & Gage, 1996; Seki & Arai, 1995), the neurons that are added to the dentate gyrus later in life display functional and morphological features equivalent to those of adult-generated neurons produced in early adulthood (Morgenstern, Lombardi, & Schinder, 2008). The mechanism of age-induced suppression in hippocampal neurogenesis remains unclear. Age does not affect the maturation or migration of newborn cells (Morgenstern et al., 2008; Rao, Hattiangady, Abdel-Rahman, Stanley, & Shetty, 2005) . Findings regarding the effects of age on differentiation of newborn cells are mixed. Rao et al (2005) found no effect of age on phenotype (glial or neuronal) of newborn cells, whereas Lichtenwalner et al. (2001) found a 60% reduction of 7  differentiation into neurons in senescent rats and Mcdonald and Wojtowicz (2005) report a 20% reduction in differentiation in middle-aged rats. The raw number of surviving new cells decreases with aging (Kempermann, Kuhn, & Gage, 1998; van Praag, Shubert, Zhao, & Gage, 2005). However, when reductions in proliferation are accounted for, the proportion newborn cells surviving to maturity does not differ as a function of age (Bondolfi, Ermini, Long, Ingram, & Jucker, 2004). Instead, reductions in neurogenesis with age seem to be driven by reductions in proliferation of neural progenitor cells that reliably occur with aging (Bondolfi et al., 2004; Kempermann et al., 1998; McDonald & Wojtowicz, 2005; Rao et al., 2005). Moreover, in rodents the reduction in neurogenesis with age does not seem to result from a depletion of the neural stem cell population (Hattiangady & Shetty, 2008). Nor does reduced neurogenesis result from increased length of the cell cycle (Olariu, Cleaver, & Cameron, 2007). These findings suggest that, rather than becoming aberrant, neurogenesis in the aged brain is simply downregulated and is a result of increased quiescence of the neural stem cell population. Indeed adrenalectomy and exercise increases cell proliferation in aged male rodents, indicating that it is possible to increase neurogenesis levels in older age (Cameron & McKay, 1999; van Praag et al., 2005).  Thus factors that increase proliferation of progenitor cells could greatly influence the maintenance of brain health, plasticity, and cognition with aging. In females, ovarian hormone levels and reproductive experience may represent two such factors.  Indeed recent research suggests that cell proliferation could be increased with different forms of estrogens in multiparous but not nulliparous rats (Barha & Galea 2011). Neuroendocrine changes during aging  Aging is a multifaceted process that involves dramatic changes in the endocrinology of the brain (Bishop, Lu, & Yankner, 2010) and circuitry of the hippocampus. Aging is characterized by an increase in circulating levels of glucocorticoids (Cameron & McKay, 1999)  and decreased steroid 8  responsivity in the brain (Sapolsky, Krey, & McEwen, 1984; Wilkinson, Peskind, & Raskind, 1997). Gonadal hormones, in contrast, decrease with aging in both women and men (Lamberts, vandenBeld, & vanderLely, 1997). The high levels of corticosteroids and diminishing levels of ovarian hormones as women age may be at least partially responsible for age-related declines in cognition and in levels of neurogenesis in the hippocampus. Adrenalectomized animals do not show typical decline in neurogenesis with age (Gould, Cameron, Daniels, Woolley, & McEwen, 1992), and estradiol application attenuates age-related cognitive decline in a number of species (Gibbs & Gabor, 2003), perhaps due, in part, to their regulation of adult hippocampal neurogenesis. Estrogens have been investigated as possible agents for improving cognitive aging in postmenopausal and surgically menopausal women with some evidence of efficacy (Sherwin, 2006).   A further age-related change in the female brain is an apparent alteration in the hippocampus' response to estrogens. In young animals, estrogen replacement increases dendritic spine in the CA1 region, but this does not occur in aged (22-23 month old) Sprague Dawley rats, likely due, in part, to age-related decrements in estrogen receptor levels (Adams et al., 2002). Furthermore, 17?-estradiol replacement restores ovariectomy-induced impairment in reference memory in younger rats, enhances performance in middle-aged females, but does not influence performance in aged females (Talboom, Williams, Baxley, West, & Bimonte-Nelson, 2008). In this study, the cognitive effects of ovariectomy also differed across age groups, with surgical ovarian hormone loss impairing cognition in young adult female rats and enhancing cognition in old age. Therefore it seems that age alters both the responsiveness of the hippocampus to estrogens and the impact of estrogens on cognition. Reproductive experience is another factor altering the impact of estrogens in later life. Middle-aged multiparous rats show decreased cell proliferation in the hippocampus in response to ovariectomy, whereas nulliparous females are unaffected by ovariectomy (Barha & Galea, 2011). The effects of reproductive experience on neurogenesis and cognition during aging will be discussed below.  9  Estrogens, hippocampal neurogenesis, and cognition   The hippocampus is rich in estrogen receptors (ERs) and thus, perhaps not surprisingly both the function and structure of the hippocampus are sensitive to ovarian hormones. Adult hippocampal neurogenesis is modulated by ovarian hormones, such as estradiol (Galea & McEwen, 1999; Ormerod, Lee, & Galea, 2003; Tanapat, Hastings, Reeves, & Gould, 1999). For example, deprivation ;of endogenous ovarian hormone via ovariectomy is associated with a short-term reduction in cell proliferation (Tanapat et al., 1999), whereas administration of estradiol increases cell proliferation and cell survival in a dose-,  duration- and sex-dependent way (Galea, 2008). Studies are just beginning to untangle the effects of different hormones on neurogenesis in the dorsal or ventral hippocampus (Brummelte & Galea, 2010; Chow, Epp, Lieblich, Barha, & Galea, 2012).   Ovarian hormones not only modulate neurogenesis in the hippocampus but also modulate hippocampus-dependent learning and memory across a variety of species from rodents to primates (Barha & Galea, 2010) and there is a curvilinear relationship between ovarian hormones and cognition.  For example, both ovariectomy and high levels of circulating ovarian hormones are associated with a decline in hippocampus-dependent cognition in humans and rodents (Galea, Kavaliers, Ossenkopp, & Hampson, 1995; Galea, Lee, Kostaras, Sidhu, & Barr, 2002; Gibbs & Johnson, 2008; Hampson, 1990; Hogervorst, Williams, Budge, Riedel, & Jolles, 2000; Holmes, Wide, & Galea, 2002; Warren & Juraska, 1997).  In addition, moderate levels of ovarian hormones are associated with improved hippocampus-dependent cognition (Barha, Dalton, & Galea, 2010; Hampson, 1990).  Ovarian hormone levels therefore are potent moderators of both hippocampal neurogenesis and learning and memory. Thus, declines in ovarian functioning and hormone levels with age may therefore contribute to age-related cognitive decline.    Consistent with this possibility, hormone replacement therapies (HRT) have been implicated as a possible therapeutic agent for ameliorating age-related cognitive decline in post-menopausal 10  women.  Studies have reached conflicting findings regarding the effects of HRTs on cognition (Sherwin, 2006). A particularly influential study on the topic was the Women?s Health Initiative Memory Study, a large-scale  randomized, double-blind, placebo-controlled clinical trial that found no benefit of Premarin, the most common HRT, on mild cognitive impairment or risk for dementia (Espeland et al., 2004; Shumaker et al., 2004). However, this study had a number of critical design flaws, including the long period (greater than 10 years) since menopause before the use of HRT, the advanced age of the participants, and the composition of HRT given (Harman et al., 2004; Sherwin, 2005).  Meta-analyses reveal that the HRTs composed primarily of 17?-estradiol show more beneficial impacts on cognition than do estrone-based HRTs, including Premarin (Hogervorst et al., 2000; Ryan, Scali, Carriere, Ritchie, & Ancelin, 2008). Furthermore animal research findings indicate that the effect of HRT on cognition and hippocampal plasticity decreases with age of the subject and time since cessation of ovarian functioning (Smith, Vedder, Nelson, Bredemann, & McMahon, 2010; Walf, Paris, & Frye, 2009). HRT studies that implement usage near the time of menopause show more positive cognitive outcomes (Daniel, 2006; Maki, 2006). These studies suggest that more investigation into how estrogens impact cognition is needed and that special care must be taken to examine type of cognitive functioning, length of time after cessation of ovarian function and type of HRT.    Aging, reproductive experience and neurogenesis Factors that increase estrogen levels and estrogen sensitivity during aging may have potential for alleviating age-associated cognitive decline in women, as estrogens may up-regulate proliferation of quiescent neuronal precursor populations and improve cognition. Reproductive experience may represent one such moderator and will be examined in this thesis. Parity is associated with later onset of menopause in women (Gold et al., 2001) and delayed cessation of estrous cycling in rats (Matt, Sarver, & Lu, 1987). Maternal brains may therefore be exposed to more estrogens over the lifespan. Moreover, 11  reproductive experience changes the way the brain responds to estrogens in later life (Barha & Galea, 2011). Cell proliferation in the hippocampus is up-regulated in response to estrogens (17?-estradiol, 17?-estradiol, and estrone) in middle-aged multiparous, but not nulliparous, female rats, indicating that parity may enhance hippocampal sensitivity to estrogens in later life. If this is the case, the multiparous brain may be more susceptible to the effects of hormone deprivation. Indeed, hippocampal cell proliferation is decreased by ovariectomy in older multiparous females, whereas cell proliferation is unaffected by ovariectomy in age-matched nulliparous females (Barha & Galea, 2011).  Though one must be careful not to assume enhanced estrogen sensitivity necessitates improved cognition, persisting changes in how the brain responds to estrogens with reproductive experience may have profound implication for healthy cognitive aging.   These persisting changes in estrogen exposure and estrogen sensitivity likely arise from the substantial neuroendocrine and behavioral alterations required for the mother to meet the survival needs of her offspring. Pregnancy and early motherhood are accompanied by vast endocrine changes in steroid and peptide hormones, such as oxytocin, estradiol and progesterone, that stimulate maternal behavior  (Rosenblatt, 1988), alter cognitive performance (Workman, Barha, & Galea, 2012) and induce changes in neural circuitry (Fleming, O'Day, & Kraemer, 1999). The neural and behavioral changes occurring during pregnancy and lactation reveal a large degree of plasticity in the maternal brain that result from both endocrine changes and exposure to offspring ( Macbeth & Luine, 2010; Pawluski, Brummelte, Barha, Crozier, & Galea, 2009). In humans, brain size decreases across pregnancy and returns to preconception size 20 weeks after delivery  (Oatridge et al., 2002). The hippocampus, though not typically considered to be part of the maternal circuit, underlies the maternal behaviors of pup retrieval and nest building. It is also one brain area particularly sensitive to pregnancy and parturition-related hormones (Pawluski et al., 2009). 12  Co-occurring with the hormonal changes of pregnancy are changes in the structure and function of the hippocampus. Spine density in the CA1 region increases during pregnancy and lactation compared to nulliparity (Kinsley et al., 2006). These changes in hippocampal morphology are likely due to pregnancy-related endocrine changes, as nulliparous females treated with hormone-simulated pregnancy undergo similar changes in spine density of hippocampal CA1 pyramidal cells (Kinsley et al., 2006). After weaning, multiparous rats show greater spine density in the basal region of CA1 pyramidal neurons compared to nulliparous rats (Pawluski & Galea, 2005). The alterations in hippocampal pyramidal cell morphology resulting from pregnancy and motherhood are accompanied by time- and parity-specific changes in hippocampal neurogenesis and cognition (for a review, see  Macbeth & Luine, 2010; Workman et al., 2012).  The immediate postpartum period is associated with a transient decrease in cell proliferation in both primiparous and multiparous females, whereas only primiparous females show decreased cell survival across the postpartum period (Pawluski & Galea, 2007). In addition to affecting neurogenesis in the hippocampus, pregnancy and mothering alter the electrophysiological properties of the hippocampus as long-term potentiation (LTP) is augmented along Schaffer collaterals in multiparous rats who have mothered for 3 days (Tomizawa et al., 2003).  Consistent with the existence of enduring effects of reproductive experience on cognition and brain health, a number of behavioral studies indicate persisting effects of parity and exposure to offspring on cognition and brain health. In humans, verbal memory and attention skills in postpartum women are equivalent to those of non-mothers 10-13 months after delivery (Crawley, Dennison, & Carter, 2003) but may be enhanced by 2 years postpartum (Buckwalter, Buckwalter, Bluestein, & Stanczyk, 2001).  In rats, reproductive experience (pregnancy and motherhood) is associated with enhanced memory on a variety of hippocampus?dependent tasks including the  radial arm maze (Kinsley et al., 1999) (Pawluski, Vanderbyl, Ragan, & Galea, 2006; Pawluski, Walker, & Galea, 2006), and reference memory tasks (Kinsley et al., 1999) in the weeks following weaning. Importantly, 13  previous reproductive experience may affect reference and working memory differently. For example, adult multiparous rats with 21 days of mothering experience had enhanced working memory performance in the radial arm maze compared to nulliparous rats (Kinsley et al., 1999). Furthermore, while primiparous rats show enhanced working and reference memory, multiparous rats show enhanced working memory in the working?reference memory version of the radial arm maze compared to nulliparous age-matched controls (Pawluski, Walker, et al., 2006). These results suggest that the beneficial effects of parity on spatial cognition may be particularly strong for working memory.   Changes in cognition may extend well beyond the time of offspring weaning. Primiparous and multiparous middle-aged rats show enhanced learning and memory compared to nulliparous rats long after last exposure to pups when repeatedly tested in the dry land maze (Gatewood et al., 2005; Love et al., 2005), water maze (Lemaire et al., 2006), reversal learning tasks (Gatewood et al., 2005), and object recognition and object placement tasks (Macbeth, Scharfman, MacLusk, Gautreaux, & Luine, 2008). Multiparity continues to be associated with enhanced spatial learning and memory well into reproductive senescence (2 years of age), although these rats were repeatedly tested across the lifespan (Gatewood et al., 2005; Lemaire et al., 2006).  In addition, the enhancement in LTP associated with parity in young rats (Tomizawa et al., 2003) also persists across the lifespan, as 22-month old primiparous females (16 months after parturition) show the same enhancement of excitatory post-synaptic potentiation as seen in young mothers after weaning (Lemaire, Koehl, Le Moal, & Abrous, 2000). Finally, reproductive experience is associated with enduring changes in maternal behavioral responsiveness (Bridges, 1975; Scanlan, Byrnes, & Bridges, 2006). These persisting effects are likely mediated by changes in brain and perhaps hippocampal circuitry. Three months after weaning, multiparity is associated with increased brain derived neurotrophic factor (BDNF) a neurotrophic factor associated with learning and memory and hippocampal plasticity (Macbeth et al., 2008) and decreased 14  amyloid precursor protein (Gatewood et al., 2005) compared to nulliparous rats. Both amyloid precursor protein and BDNF levels are regulated by estradiol (Jaffe, Toranallerand, Greengard, & Gandy, 1994; Scharfman & MacLusky, 2006) and are thought to play roles in learning and memory during aging (Tapia-Arancibia, Aliaga, Silhol, & Arancibia, 2008; Turner, O'Connor, Tate, & Abraham, 2003). These persisting effects of BDNF and APP may be due to parity-induced changes in secreted levels of estradiol (Bridges & Byrnes, 2006) along with changes in sensitivity to estradiol, as parous rats are more sensitive to high doses of estradiol (Bridges & Byrnes, 2006).   In summary, parity is associated with persisting effects on hippocampal neurogenesis and cognition, and this may, in part, be due to changes in how the brain responds to estrogens. It is therefore possible that parity affects cognition later in life differently depending on a female's ovarian hormone levels or hormonal state (hormonally-intact vs. hormonally-deficient). This has implications for interventions for healthy cognitive aging: If the cognitive effects of hormone depletion (e.g. through surgical or natural menopause) are altered by a woman's childbearing experience, it may be appropriate to develop tailored hormone-replacement regimens depending on parity.  The current experiment  The aim of this study was to explore how ovarian hormone status and previous reproductive experience impact hippocampus-dependent spatial working and reference memory and hippocampal neurogenesis in middle-aged female rodents. To examine this, nulliparous and multiparous rats received either sham surgery or ovariectomy and were trained on a spatial working/reference version of the Morris water maze and a reference memory version of the Morris water maze. We hypothesized, based on previous findings that parity increases sensitivity of the hippocampus to estrogens, that parity and ovarian status would have interacting effects on hippocampal neurogenesis (looking at two 15  different young neuron populations) and performance on the working and reference versions of the Morris water maze. In addition, we hypothesized that working and reference memory performance would correlate with neurogenesis levels differentially in the dorsal and ventral areas and with parity and ovarian hormone status.    16  METHODS Animals  Forty-four female Sprague-Dawley rats weighing 250-350g were used in this study. Multiparous retired breeder rats (n = 22) and nulliparous rats (n = 22) were purchased from Harlan Laboratories (Indianapolis). Rats were approximately 8 months old at the time of their arrival at the Department of Psychology at the University of British Columbia. They were housed in pairs until they underwent surgery at approximately 12 months of age, and they were 13 months old at the time of behavioral testing.  Multiparous retired breeder rats had approximately 4-5 litters prior to shipping.  Rats were individually housed in opaque polyurethane bins (48 x 27 x 20 cm) with aspen chip bedding and were given Purina rat chow containing 0.1-0.15% isoflavones by weight (lab diet 5012) and tap water ad libitum. Rats were maintained under a 12h :12h light/dark cycle (lights on at 07:30h). Retired breeders and nulliparous rats were housed in separate colony rooms. Nulliparous and multiparous rats were assigned to either ovariectomy (n = 11 per parity group) or sham surgery (n = 11 per parity group). All testing was conducted in accordance with ethical guidelines set by the Canada Council for Animal Care and all procedures were approved by the University of British Columbia Animal Care Committee. All efforts were made to reduce the number and suffering of animals.  Apparatus  The Morris Water Maze was a white circular pool, 180cm in diameter, filled with water mixed with white tempura (non-toxic) paint to render it opaque. Large and distinct distal cues were placed on all four walls of the room surrounding the pool and remained constant throughout behavioral testing. The maze is conceptually divided into four quadrants defined by the intersection of the secants connecting the North and South, and East and West cardinal points of the pool. In the water maze task, the animal must use the distal cues to navigate to a hidden platform, submerged roughly 2 cm beneath 17  the pool surface in the center of a quadrant. A camera installed above the center of the pool was connected to a computer running ANY-maze 4.98 software (Stoelting, Wood Dale, IL, USA) in order to record measures such as latency to reach the hidden platform, swim distance, and percentage of time spent in the quadrant containing the hidden platform. Water temperature was room temperature during all trials.   Procedures See Figure 1 for experimental timeline.  Ovariectomy and sham surgery  Approximately four months after arriving in the facility, rats underwent bilateral ovariectomy or sham surgery (n = 11 nulliparous and n = 11 multiparous rats per ovarian status group). All rats were placed in an induction chamber and anesthetized with isofluorane, which was delivered at an induction flow rate of 5% in 1.5% oxygen. Rats were maintained at a surgical plane of anesthesia on a warming blanket using an isofluorane flow rate of 2.5-3% and were given an injection of Lactated Ringer's Solution (10 mL) to maintain fluid balance. Also administered were Ketoprofen (.5 mL/kg; Anafen, MERIAL Canada INc., Baie d'Urfe', Quebec, Canada) as a general nonsteroidal and anti-inflammatory anesthetic and Marcaine (0.10 mL; Bupivacaine, HOSPIRA Inc., Lake Forest, Illinois, Canada) as local anesthetic. Sham surgery consisted of skin and muscle incisions and subsequently sutured without damage to or manipulation of the ovaries. A topical antibacterial ointment was externally applied to the incision after suturing was complete to all rats (Flamazine, Smith & Nephew, St. Laurent, Quebec, Canada). Rats were weighed and monitored daily after surgery for 7 days or until incisions had healed. All animals were given a second dose of Ketoprofen (.5 mL/kg) on day 1 to minimize post-operative pain. Rats were handled for 5 minutes on days 5 and 6 of recovery to habituate the rats to the experimenters. This amount of handling is much less than the level that has previously been shown to 18  interfere with estradiol's influence on cognition (Bohacek & Daniel, 2007).   5-bromo-2-deoxyuridine (BrdU) administration  On day 0 of of the experiment, approximately one week after surgery, rats received a single i.p. injection of 5-bromo-2-deoxyuridine (BrdU: Sigma, St. Louis, MO) (200mg/kg). The BrdU solution was prepared to a concentration of 20mg/ml just prior to injection by dissolving BrdU in freshly prepared warm (< 40 ?C) 0.9% saline containing 0.7% 1N NaOH. BrdU is a thymidine analogue that incorporates itself into the DNA of dividing cells within two hours of administration, during the synthesis phase of the cell cycle (Packard, et al., 1973). Depending on the amount of time elapsed between injection and perfusion of the animal, BrdU can be used to assess cell proliferation or cell survival in the granular cell layer of the dentate gyrus (Taupin, 2007). In the current study rats were perfused 23 days after BrdU administration. Therefore, the number of BrdU immunoreactive (BrdU-ir) cells measures the survival of adult-generated hippocampal cells over a 23 day period after being born under absent or normal ovarian functioning in multiparous and nulliparous middle-aged rats. Morris water maze training  Training began at approximately the same time each day (9:30 - 10am) and occurred over 10 consecutive days with four trials per day. Each trial began by placing the animal in the pool at one of the four cardinal compass points. Start locations were never repeated within a training day, and the order of start locations was randomized for each training day but was the same for all animals. Each trial ended when the animal reached the platform or when 60s had elapsed. Animals that were unable to locate the hidden platform within the allotted time were guided to the platform by the experimenter. Once the animal had reached or been placed on the platform, it was left there for 10s before being removed from the pool. The inter-trial interval was approximately 10 minutes. The order in which animals were tested each day was varied to prevent time-of-day effects, and the pool was cleaned of 19  floating debris between trials to eliminate potential intra-maze cues. For each training day, an average of each performance measure (latency, distance to reach the hidden platform) was calculated obtain indicators of daily performance on the water maze. There were 10 consecutive days of water maze training, with the first six days employing a mixed working/reference memory version of the MWM and the last for standard reference memory version of the MWM.  Working/reference spatial memory trials   The first 6 days of training were designed to assess animals' working/reference memory performance. In the working/reference memory segment of training, the platform location was changed after every 2 days of training for 6 days. The platform was located in the NW quadrant on days 1 and 2 of training, the SE quadrant on days 3 and 4, and the SW quadrant on days 5 and 6 of training. The location of the platform changed after each second day to ensure that animals utilized working memory in navigating to the platform. However, given that the platform was in the same location on the second day this suggests there was also a reference memory component at least on the second day of testing.  Reference spatial memory trials  The last 4 days of training were designed to assess reference spatial memory performance, memory for constant stimuli and events.  In the reference version of the water maze task, the platform remained in the NE quadrant throughout training days 7 ? 10 of testing.  Lavage  In females with intact ovarian functioning, ovarian hormone levels fluctuate over the estrous cycle and influence both hippocampal plasticity (Tanapat et al., 1999) and spatial learning (Warren & Juraska, 1997).  Furthermore, the length, regularity, and nature of the estrous cycle is known to change with aging in rats (LeFevre & McClintock, 1988). Reproductively senescent rats enter a state of 20  persistent diestrus after either 1) intervals of regular and irregular cycling, or 2) after intervals of constant estrus and irregular cycling (Levefre & McClintock, 1988). Therefore, estrous cycles were monitored in our study. Animals were lavaged on the three days preceding and including BrdU injection, each day after water maze testing, and at the time of perfusion to verify cyclicity. Vaginal cells were collected by lavage (as per Byers, Wiles, Dunn, & Taft, 2012). The lavage sample was transferred onto microscope slides, stained with Cresyl Violet (Sigma), and left to dry. Slides were assessed under 400x magnification using an Olympus CX22 LED light microscope. A rat was determined to be in the proestrous stage if at least 70% of cells were nucleated epithelial cells (Byers et al., 2012). Vaginal lavage samples were qualitatively categorized for evidence of normal cycling, abnormal cycling (consecutive lavage-cycles varying in length or order), persistent diestrus (consecutive lavage-cycles consisting primarily of leukocyte-dense cell samples), or persistent estrus (consecutive lavage-cycles consisting primarily of cornified cells; Modified from Levefre & McClintock, 1988 to reflect the shorter sampling period in this study).  Probe trial and perfusion  On Day 23 of the experiment, approximately 24 hrs after the last reference spatial memory trial and 23 days after BrdU injection, animals received a 30-second probe trial, during which the platform was removed from the pool. Percentage of time spent in the target quadrant (the NE quadrant, which had contained the hidden platform during the reference version of the water maze task) was recorded. Ninety minutes after probe trial, animals were administered an overdose of sodium pentobarbitol and were perfused transcardially with 120 ml 0.9% saline followed by 60 ml 4% paraformaldehyde (Sigma-Aldrich). Brains were extracted and post-fixed in 4% paraformaldehyde overnight, transferred to 30% sucrose (Fisher Scientific) solution 24h later, and remained in sucrose solution until sectioning.  21  Immunohistochemistry  Brains were sliced using a Leica SM2000R microtome (Richmond Hill, Ontario, Canada)  into 40 ?m coronal sections. Sections were collected in series of every 10th section throughout the entire rostral-caudal extent of the hippocampus and stored in ethylene glycol at -20?C.  BrdU immunohistochemistry  A series of hippocampal sections was rinsed with 0.1M TBS three times, incubated in 0.6% H2O2 for 30 minutes, and rinsed with TBS again. Tissue was then transferred to 2N HCl and incubated in a 37?C water bath at for 30 minutes to denature the DNA. Next, the tissue was rinsed in a 0.1M borate buffer solution of pH 8.5 for 10 minutes and subsequently rinsed with TBS to remove any background staining. To prevent non-specific binding of the secondary antibody, the tissue was blocked with a TBS+ solution, consisting of 0.3% Triton-X (Sigma) and 3% normal horse serum (Vector Laboratories; Burlingame, CA, USA) in 0.1M TBS. Slices were then incubated in a primary antibody solution, which contained 1:200 mouse anti-BrdU (Roche; Mississauga, ON, Canada) and TBS+, for 48 hours at 4?C. Tissue was then rinsed with TBS and incubated  for 4 hours at room temperature in biotinylated secondary antibody solution containing 1:500 horse anti-mouse IgG Biotynlated (Vector Laboratories) in TBS+. Excess antibodies were rinsed away with TBS and then tissue was incubated for 1.5 hours in an avidin-biotin complex dissolved in TBS, as per instructions in the ABC kit (Vector Laboratories). Brain sections were then transferred to a diaminobenzidine (DAB; Sigma) solution and incubated for 5 minutes in a dark room, then rinsed with TBS. The tissue was mounted onto glass microscope slides, dried overnight, counterstained with cresyl violet, and cover-slipped with Permount (Fisher Scientific; Ottawa, ON, Canada).  Doublecortin immunohistochemistry  An additional series of tissue was labeled for the immature neuronal marker doublecortin 22  (DCX) . Tissue was pretreated with 0.6% hydrogen peroxide for 30 minutes at room temperature after rinsing in 0.1 M PBS. It was then incubated at 4 ?C for 24 h in a primary antibody solution consisting of 1:1000 polyclonal goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 0.04% Triton-X, and 3% normal rabbit serum, dissolved in 0.1 M PBS. The tissue was washed in 0.1 M PBS and then incubated  for 24 hour at 4 ?C in a secondary antibody solution containing 1:500 biotinylated rabbit anti-goat (Vector Laboratories, Burlingame, CA, USA) in 0.1 M PBS. Tissue was then incubated for 4 hours at room temperature in an avidin-biotin solution containing 1:1000 avidin and 1:1000 biotin in 0.1M PBS, rinsed in PBS, and washed in 0.175 M sodium acetate buffer. Doublecortin-labeled cells were visualized by developing tissue for approximately 10 minutes in a DAB solution. Once staining was complete, tissue was mounted on glass slides, dehydrated, and coverslipped with Permount Cell counting  All microscope slides were coded to ensure that counting was done by an experimenter blind to the group assignment of each animal. BrdU-labeled cells and DCX-expressing cells were counted using a Nikon E600 light microscope under a 100x oil immersion objective lens. Every 20th section was counted. For both DCX-expressing and BrdU-labelled cells, cells were counted in both the granule cell layer (GCL; including the subgranular zone) and hilus (See Figures 1B and 1C). Cells in the hilus were counted because 1) mature granule cells in the hilus are generally considered ectopic, as adult-generated hippocampal neurons are born in the subgranular zone and migrate to the granule cell layer 2) to serve as a comparison for the granule cell layer to account for potential changes in the blood-brain barrier permeability caused by reproductive experience or ovariectomy; and 3) because new cells in the hilus give rise to a different population of cells than are produced and migrate into the granule cell layer (Barha, 2012).  Area measurements for the GCL and hilus were obtained with digitized images and the software ImageJ (NIH). Volume estimates of the dentate gyrus were calculated using 23  Cavalieri?s principle (Gundersen & Jensen, 1987) by multiplying the summed areas of each region and area by distance between sections (800?m). Cells were considered to be BrdU-labeled if they were intensely stained and exhibited medium-sized round or oval cell body (Cameron, Woolley, McEwen, & Gould, 1993). Density of BrdU-labeled and DCX-expressing cells in each area (GCL or hilus) and region (Dorsal or Ventral) were calculated by dividing the sum of labeled cells in the area or region by volume of the corresponding region/area. We noted whether immunoreactive cells were located in the dorsal or ventral GCL using the criterion defined by Banasr and colleagues (2006) which defines sections 6.20-3.70mm from the interaural line as dorsal and sections 3.70-2.28mm from the interaural line as ventral.  This was done because previous studies (Brummelte & Galea, 2010; Chow et al., 2012) have found differential effects of steroid hormones on neurogenesis in dorsal and ventral dentate gyrus regions. DCX-expressing cells were classified into stages of maturity based on Pl?mpe et al. (2006): Type A cells (Proliferative stage cells) included DCX-expressing cells with no processes or short, plump processes no longer than a cell width; Type B cells (Intermediate stage cells) had unbranched processes of intermediate length reaching no farther than the moleculayer; Type C cells (Postmitotic stage cells) had a more mature appearance, with dendritic branching in the molecular and/or granule cell layer. DCX was labeled separately from BrdU because past research (Epp, Scott, & Galea, 2011) has indicated that spatial learning can produce different effects on cell survival (BrdU-labeled cells) and immature neurons (doublecortin-labeled cells). In addition, in Sprague-Dawley rats, spatial learning increases the percentage of doublecortin-labeled cells that are activated during memory retrieval (Epp, Scott, et al., 2011), suggesting that cells at an immature maturation stage are particularly recruited by spatial learning in this strain.  24  Data analyses  All analyses were conducted using Statistica 9 (Statsoft Tulsa, OK). Swim distance and latency to reach the platform were each analyzed using separate repeated measures analysis of variance (ANOVA) for working and working/reference versions of the Morris Water Maze, with ovarian hormone status (OVX, sham) and parity (nulliparous, multiparous) as between-subjects variables and training day (1 to 6 for the working memory task, 1 to 4 for the reference task) as the within-subjects variable.  Probe trial performance was analysed using a repeated-measures ANOVA with Quadrant (1-4) as a within-subjects variable and parity and ovarian status as between-subjects variables. To examine probe trial performance across the estrous cycle in sham-operated rats, an analysis of variance was used, with estrous state (proestrus, non-proestrus) and parity (nulliparous, multiparous) as between-subjects variables. To further analyze probe trial performance, a repeated-measures ANOVA with a within-subjects factor of quadrant and between-subjects factors of estrous state (sham non-proestrous, sham proestrous, and non-cycling ovariectomized) and parity. Repeated-measures ANOVAs were used to analyze the volume of the dorsal and ventral GCL and hilus (within-subjects factors of region and area) as a function of ovarian hormone status and parity (between-subjects factors). A similar repeated-measures ANOVA was used to assess density of BrdU-labelled cells, using within-subjects variables of region (Dorsal, Ventral) and region (GCL, Hilus) and between-subjects variables of parity and ovarian status. A repeated-measures ANOVA with within-subjects factors of Cell Type (A, B, or C) and Region (Dorsal, Ventral) and between-subjects factors of parity and ovarian hormone status was used to assess density of DCX-expressing cells. Pearson product-moment correlations were conducted for each ovarian status/parity group to determine whether density of dorsal and ventral BrdU-labelled and DCX-expressing cells correlated with total distance to reach the hidden platform across days in the working memory and reference/working memory versions of the Morris water maze. Similar correlations were run to determine whether dorsal and ventral BrdU-labeled or DCX-expressing cell 25  populations correlated with percentage of time spent in the target quadrant during the probe trial. An independent-samples ANOVA was used to assess average number of completed estrous cycles during an 11 day sampling period using between-samples variables of parity and ovarian status.  Post-hoc tests were performed with the Neuman-Keuls procedure. A priori comparisons were subjected to Bonferroni correction.   26  Figure 1. A) Experimental outline. MWM = Morris Water Maze. B) BrdU-labeled cells in the granule cell layer. C) Type A, B, and C DCX-labeled cells in the GCL. Scale bar = 10?m  A) B)            C)              A B C 27  RESULTS Multiparous females had significantly shorter distances to locate the hidden platform than did nulliparous during early training on the working/reference spatial learning task and this was more pronounced in ovariectomized rats  Distance to reach the hidden platform decreased across training days (main effect of days: F(5,200) = 22.75, p < .001; See Figure 2A). Post-hoc analyses indicated that path length to reach the hidden platform decreased across days of working/reference memory training as expected  (Day 3 lower compared to Days 1 and 2 (p?s < .01) and between Day 3 and 4 (p = .008) and Days 5 and 6 (p = .002)). Latency to reach the hidden platform also decreased across training days, indicating that as the days progressed the latency to reach the platform decreased (main effect of days: F(5,200) = 25.68, p < .001). No significant parity or ovarian hormone effects were found for either distance or latency to reach the hidden platform in the working/reference memory task nor were there any significant interactions (all p?s > .14).  Because learning in this task was acquired relatively quickly we examined early or late learning in the working/reference spatial memory. Distance to reach the hidden platforms was compared across early training days (days 1-3) to later training days (days 4-6).   The repeated-measures ANOVA indicated a parity by training stage interaction (F(1,40) = 4.74, p = 0.035) and a main effect of training stage (F(1,40) = 761.7, p < 0.0001). Post-hoc analyses indicated that multiparous females took significantly less distance to reach the hidden platform in the early stage of training compared to nulliparous females (p = .023), whereas multiparous and nulliparous females performed equivalently at the later stage of training (p = .48;  see Figure 2B). There were no other significant main or interaction effects (p?s > .07). A priori we expected that ovarian hormone status would affect the relationship between learning and parity. A priori comparisons indicate that ovariectomy significantly impaired 28  early learning performance in nulliparous females (p < .001), but not in multiparous females (p = .79)    Very similar results were obtained for latency to reach the hidden platform as a repeated-measures ANOVA indicated a parity by training stage interaction (F(1,40) = 4.57, p = 0.039) and a main effect of training stage (F(1,40) = 63.16, p < 0.0001). Post-hoc analyses indicated that multiparous females took significantly less distance to reach the hidden platform in the early stage of training compared to nulliparous females (p = .034), whereas multiparous and nulliparous females performed equivalently at the later stage of training (p = .41;  see Figure 2).  There were no other significant main or interaction effects (p?s > .16).  A priori we expected that ovarian hormone status would affect the relationship between learning and parity.   A priori comparisons indicate that  ovariectomy significantly impaired early learning performance in nulliparous females (p = 0.013), but not in multiparous females (p = .79). Nulliparous rats had smaller path distances to the hidden platform in the reference version of the water maze compared to multiparous rats.   There was a main effect of parity on distance to reach the hidden platform in the reference version of the water maze (F(1,40) = 3.99, p = .05; see Figure 3). Nulliparous females had a shorter average path length to reach the hidden platform than did multiparous females. There was also a main effect of training day (main effect of day: F(3,120) = 6.56, p < .001), indicating that as the days progressed, path lengths were shortened.  There were no other significant main or interaction effects (all p?s > .22).  For latency to reach the hidden platform, there was a main effect of training day F(3,120) = 6.31, p < .001)  but no main effects of  Parity (F(1,40) = 1.60, p = .21) or Ovarian hormone status (F(1,40 = 0.75, p = .39) or interactions (p?s > .21).   29  Ovariectomized, but not intact, nulliparous females spent more time in the target quadrant during the probe trial than did multiparous females. Rats in proestrus spent significantly less time in the target quadrant during the probe trial.  All groups spent greater than chance percentages of probe trial time in the target quadrant (a priori Bonferroni-corrected single-sample t tests against a value of 25%, t(10)'s > 466.61, p's < .001). A repeated-measures ANOVA indicated that different percentages of time were spent in the four quadrants of the water maze during the probe trial (main effect of quadrant, F(3,120) = 12.89, p < .001). Post-hoc analysis indicated that the percentage of time was greater in the target quadrant (Quadrant 1) than in any other quadrant (p's < .001) and that the percentages of probe trial time spent in Quadrants 2, 3 and 4 did not differ significantly (p's > .41). There was a main effect of Parity (F(1,120) = 7.11, p = .011) and a trend towards a quadrant * parity * ovarian status interaction (F(3,20) = 2.29, p = .082) but all other main and interaction effects were not significant (all  p?s ? .19).  A priori we expected differences in probe trial performance between multiparous and nulliparous rats and hypothesized that ovarian status would interact with parity. A priori tests indicated that in ovariectomized females nulliparous rats spent more time in the target quadrant during the 30 second probe trial than did multiparous rats (p = 0.019) but this was not the case for sham rats (p = 0.45; See Figure 4A).  Because estrous phase has been found to affect probe trial performance, with proestrus being associated with changes in both cell proliferation and spatial memory performance (Warren & Juraska, 1997), a repeated-measures ANOVA was performed to investigate probe trial performance as a function of reproductive experience and estrous condition (proestrous, non-proestrous, non-cycling ovariectomized). A quadrant main effect (F(3,105) = 4.56, p = .005) and quadrant by estrous condition interaction emerged (F(6,105) = 2.33, p = .037; See Figure 5). Post-hoc analyses indicated that 30  proestrous shams spent significantly less time in the target quadrant than did non-proestrous shams (p = 0.031) and marginally less time than non-cycling ovariectomized rats (p  = 0.06). Non-proestrous shams and ovariectomized rats did not differ in proportion of time spent in the target quadrant (p = .72). The proportion of time spent in quadrants 2, 3 and 4 did not differ among estrous status groups (p's > .50). There was a main effect of parity such that nulliparous rats spent more time per quadrant than did multiparous rats, F(1,35) = 6.28, p = .017.  Multiparous females had a greater volume of the ventral region of the dentate gyrus compared to Nulliparous females. Ovariectomized females had smaller ventral hilar volumes compared to sham-operated rats  A repeated-measures ANOVA on region and area volumes revealed an area by region by ovarian status interaction (F(1,39) = 5.03, p = .03.) Unpacking this interaction with post-hoc tests revealed that ovariectomized and sham-operated rats were equivalent in dorsal (p = 0.91) and ventral (p = 0.73) dentate gyrus volumes. However, in the hilus, sham-operated rats had significantly greater ventral volume (p < 0.001) than did ovariectomized rats, while there was no significant difference in the dorsal hilar volume (p = .66; see Figure 6).  In addition, a parity by region interaction emerged (F(1,39) = 5.97, p = 0.019) and an ovarian status by region interaction (F(1, 39) = 4.18, p = 0.048). Post-hoc tests revealed that multiparous females had significantly greater ventral (p  = 0.0001) but not dorsal (p = 0.094) dentate gyrus volumes (See Figure 7). There were also main effects of region and area and an interaction of area and region but no other significant main or interaction effects (all p?s> 0.09).    31  Nulliparous females had a greater density of BrdU-labelled cells in the granule cell layer compared to Multiparous females, regardless of ovarian hormone status.   Because dentate gyrus volume was influenced by parity and ovarian status, the density rather than total number of BrdU-labelled cells in the dentate gyrus was used. A repeated-measures ANOVA on the density of BrdU-labelled cells indicated a significant main effect of parity (F(1,37) = 19.21, p <0.001) and a parity by area interaction ( F(1,37) = 16.06, p < 0.001). Post-hoc analysis indicated that nulliparous rats had significantly greater density of BrdU-labelled cells in the GCL (p <0.001) but not the hilus (p = 0.89). There were no other significant main or interaction effects (all p?s > 0.16). (See Figure 8). Density of BrdU-labelled cells in the ventral granule cell layer correlated with working/reference memory performance in sham-operated rats  Pearson product-moment correlations run separately for each ovarian status/parity group revealed specific correlations of BrdU-labelled cells to performance measures in the working/reference memory version of MWM. In sham-operated females, ventral densities of BrdU-labeled cells in the GCL significantly negatively correlated with total distance to reach the platform (r  = -.58, p = .005), indicating greater density of ventral BrdU-labelled cells was related to better performance. This effect was significant in both sham-operated multiparous and nulliparous rats (rnull = -.74, p = .01; rmult = -.64, p = .034; See Figure 9). There were no correlations between BrdU-labeled cell density in the GCL and total distance for either ovariectomized nulliparous or multiparous females, p?s > .24.     32  In ovariectomized females, dorsal densities of BrdU-labeled cells in the GCL significantly negatively correlated with total distance in the reference memory version of the water maze  Pearson product-moment correlations run separately for each ovarian status/parity group revealed specific correlations of BrdU-labelled cells to performance measures in the reference memory version of MWM. In ovariectomized females, dorsal densities of BrdU-labeled cells in the GCL significantly negatively correlated with total distance to reach the platform (r  = -.50, p = .025), indicating greater dorsal BrdU-labelled cell density was related to better reference memory learning. The effect did not reach significant when ovariectomized multiparous and nulliparous rats were assessed separately, p?s > .32; See Figure 10. No other correlations were significant (p?s > .13).  Density of BrdU-labelled cells in the granule cell layer of the hippocampus did not correlate with probe trial performance  BrdU-labeled cell density in the granule cell layer did not correlate with proportion of time spent in the target quadrant during the probe trial, p = .71. Nor did the density of BrdU-labelled cells in the hilus correlate with probe trial performance p = .61. Neither dorsal nor ventral BrdU-labelled cell density in the granule cell layer correlated with probe trial performance in any parity or ovarian status group (p?s > .39).  Multiparous rats had a greater density of Type B and C DCX-expressing cells in the dentate gyrus than did multiparous rats. The densities of Type B and C DCX-expressing cells were greater in the dorsal DG than in the ventral DG  A repeated-measures ANOVA on the density of DCX-expressing cells revealed a parity by cell type interaction (F(2,60) = 3.18, p = 0.048). Post-hoc analyses indicated that multiparous females had a greater density of Type B (p < 0.001) and Type C tells (p < 0.001) than nulliparous females  but had the same density of Type A cells (p  = 0.41) as nulliparous females (See Figure 11A). In addition, a 33  region by cell type interaction emerged, F(2,60) = 11.29, p < 0.001. The densities of Type B and Type C cells were greater in the dorsal dentate gyrus than in the ventral dentate gyrus (p?s < 0.001), whereas the density of Type A cells did not differ between the dorsal and ventral regions (p = 0.074) (See Figure 11B). There was no effect of ovarian status on DCX-expressing cell density, regardless of cell type or region (Ovarian status main effect: F(1,30) = 0.71, p  = 0.41; ovarian status*cell type interaction: F(2,60) = 1.36, p = 0.26; ovarian status*cell type* region: F(2,60) = 0.08, p = 0.10.) Main effects of region and parity emerged (p?s ? 0.011).  All other interactions and main effects were non-significant, p?s ? 0.18). In sham-operated nulliparous rats, density of Type B DCX-expressing cells in the dorsal granule cell layer negatively correlated with working/reference memory performance at an early stage of training  Pearson product-moment correlations run separately for each ovarian status/parity group revealed specific correlations of DCX-expressing cell types to performance measures in the reference memory version of MWM. In sham-operated nulliparous rats, the density of Type B cells in the Dorsal GCL significantly negatively correlated with total distance across the entire testing period, r = -.83, p = .003 (See Figure 12) and with total distance during the Early stage of training, r = -.63, p = .05. No other correlations were significant, p?s > .07. Densities of DCX-expressing cells did not correlate with total distance to reach hidden platform during reference spatial learning.   The densities of Type A, B, and C DCX-expressing cells did not correlate with total distance (summed across training days 1-4) to reach the hidden platform in the reference version of the Morris Water Maze for any group, p?s > .15. 34  Density of ventral Type B DCX-expressing cells positively correlated with probe trial performance in nulliparous ovariectomized females. Density of dorsal DCX-expressing cells negatively correlated with probe trial performance in multiparous sham-operated females.   Pearson product-moment correlations run separately for each ovarian status/parity group revealed specific correlations of DCX-expressing cell types to percentage of time spent in the target quadrant during the probe trial. In sham-operated multiparous females, dorsal DCX densities significantly negatively correlated with probe trial performance (r = -.84, p = .037; Figure 13A). In ovariectomized nulliparous rats, density of Type B cells in the ventral dentate gyrus significantly positively correlated with probe trial performance, r  = .71, p = .033  (See Figure 13B) No other correlations were significant, p?s > .05. The majority of sham-operated rats continued to cycle through estrous phases  Vaginal lavage from all sham-operated rats taken during water maze training days and probe day (11 days total) were qualitatively assessed to determine whether females continued to cycle through estrous phases and to identify signs of reproductive senescence. No females had lavage samples consistent with having lapsed into continuous diestrus. One multiparous female had lavage samples consistent with having lapsed into continuous estrous. Five multiparous females and six nulliparous females showed signs of irregular cycling. The remaining multiparous and nulliparous females (n's = 5 and 4, respectively) showed smears consistent with normal cycling.  The number of estrous cycle phases completed in the 11 days of sampling was compared between multiparous and nulliparous females. Of the cycling females, nulliparous females had a lower average number of completed cycles in the sampling period (Mmultiparous = 2.56 cycles, SEmultiparous = 0.24; Mnulliparous = 2.10 cycles, SE = 0.23) than did multiparous females, but this did not reach statistical significance (p = .19).  35  Figure 2. A and B: Mean distance (A) and latency (B) for multiparous and nulliparous females to reach the hidden platform in the working/reference version of the Morris Water Maze during Early (days 1-3) and Late (days 4-6) training. Nulliparous females had greater distance and latency to reach the hidden platform during early training, p?s < .04. * indicates significant within-stage comparison, p < .05.       36  Figure 2. C and D: Mean distance for multiparous (C) and nulliparous (D) females to reach the hidden platform in the working/reference version of the Morris Water Maze during Early and Late training, as a function of ovarian status. Nulliparous ovariectomized females had greater path lengths to reach the hidden platform during Early learning (p = .013) compared to sham-operated nulliparous females, whereas multiparous sham and ovariectomized females performed equivalently, p = .79. * indicates significant within-stage comparison, p < .05.      37  Figure 3. Mean distance to reach the hidden platform in the reference version of the Morris Water Maze across training days. Nulliparous females had smaller path length and shorter latencies to reach the hidden platform compared to multiparous females (p = .05).   38  Figure 4. Mean percentage of time spent in the target quadrant in parity and ovarian hormone groups, controlling for estrous phase. Ovariectomized nulliparous rats more time in the target quadrant than did ovariectomized multiparous rats (p = 0.019), whereas intact multiparous and nulliparous rats spent equal proportions of time in the target quadrant (p = 0.45).         39  Figure 5. Mean percentage of time spent in Quadrant 1 (target quadrant) and Quadrants 2-4 for non-proestrous sham females, proestrous sham females, and ovariectomized females. Rats in proestrus spent significantly less time in the target quadrant than rats in non-proestrus (p  = 0.031) and  marginally less time in the target quadrant than did ovariectomized rats. * indicates significant p < 0.05 difference from proestrous sham; # indicates p < 0.10 trend towards difference from proestrous sham.    40  Figure 6. Mean dentate gyrus volume (mm3) in the dorsal and ventral regions of the dentate gyrus as a function of ovarian status (sham-operated, ovariectomized) and area (granule cell layer and hilus). In the hilus, sham-operated rats had significantly greater ventral volume (p = 0.002) than did ovariectomized rats. Dorsal volumes did not differ between ovarian status groups regardless of area (p?s ? 0.75).  ** indicates significant effect of ovariectomy within region and area, p  < .01.       41  Figure 7. Mean dentate gyrus volume (mm3) in the dorsal and ventral regions of the dentate gyrus  as a function of reproductive experience (multiparous, nulliparous). Multiparous females had significantly greater ventral (p  ?0 .0001) but not dorsal (p =0.094) dentate gyrus volumes. *** indicates a significant effect of parity within region, p < 0.0001.        42  Figure 8. Mean density (cells/mm3) in the the dentate gyrus as a function of reproductive experience (Multiparous, Nulliparous) and dentate gyrus area (Granule cell layer, Hilus). Nulliparous females had significantly greater BrdU-labelled cell density in the GCL (p  <.001) but not the hilus (p = .89). ** indicates a significant effect of parity within area p < .01.        43  Figure 9. Mean density (cells/mm3) of BrdU-labeled cells in the ventral region of the granule cell layer of sham-operated females, and total distance to reach the hidden platform across testing days in the Working/Reference version of the Morris Water Maze. Ventral density of BrdU-labeled cells was negatively correlated with total distance, r = -.58, p = .005.     44  Figure 10.  Density (cells/mm3) of BrdU-labeled cells in the dorsal region of the granule cell layer and total distance for females to reach the hidden platform across testing days in the Reference version of the Morris Water Maze. A) In ovariectomized females, BrdU density in the dorsal GCL negatively correlated with total distance to reach the hidden platform during the Reference version of the Morris water maze, r = -.50, p = .025. B) In sham-operated females, BrdU density in the dorsal GCL did not correlate with total distance, p = .40.      A          B     45  Figure 11 A). Mean density of Type A, Type B, and Type C DCX-expressing cells in the dentate gyrus as a function of parity (multiparous, nulliparous). Multiparous females had significantly greater densities of Type B and C cells compared to nulliparous females (p?s < .001). *** signifies a significant  p < .001 parity effect within cell type.            46  Figure 11 B). Mean density of Type A, Type B, and Type C DCX-expressing cells in the dentate gyrus as a function of region (dorsal, ventral). The densities of Type B and C cells were greater in the dorsal dentate gyrus than in the ventral dentate gyrus (p?s < .001). ). *** signifies a significant  p < .001 region effect within cell type.     47  Figure 12. Density (cells/mm3) of DCX-expressing Type B cells in the dorsal region of the granule cell layer and total distance for sham-operated nulliparous females to reach the hidden platform across testing days in the Working/Reference version of the Morris Water Maze. In sham-operated nulliparous rats, the density of Type B cells in the Dorsal GCL significantly negatively correlated with total distance across the entire testing period, r = -.83, p = .003    48  Figure 13. A) Density (cells/mm3) DCX-expressing cells in the dorsal region of the granule cell layer and percentage of time that ovariectomized nulliparous spent in the target quadrant during the probe trial. In sham-operated multiparous females, dorsal DCX densities significantly negatively correlated with probe trial performance (r = -.84, p = .037) B) Density (cells/mm3) of  Type B DCX-expressing cells in the ventral region of the granule cell layer and percentage of time that ovariectomized nulliparous spent in the target quadrant during the probe trial. In ovariectomized nulliparous rats, density of Type B cells in the ventral dentate gyrus significantly positively correlated with probe trial performance, r  = .71, p = .033.    A B 49  DISCUSSION  The present results illustrate that spatial working/reference and reference learning are affected differently by parity and ovarian hormone levels in middle age, that performance was correlated with neurogenesis in different hippocampal regions and that hippocampal neurogenesis was differentially regulated by parity in middle age. Specifically, nulliparity was associated better spatial reference learning and memory, whereas multiparity was associated with better initial spatial working memory performance particularly after ovariectomy. Parity groups also differed in levels of neurogenesis and regional dentate volumes, as multiparous females had larger ventral dentate volumes, greater numbers of immature neurons, and fewer older new neurons (BrdU-labeled cells) than nulliparous females. Furthermore, cell survival (BrdU-labelled cells) and immature neurons (DCX-expressing cells) correlated with both spatial reference and working memory performance differently depending on dorsal/ventral hippocampal region and ovarian status. Cell survival and immature neurons in the dorsal dentate gyrus correlated with spatial reference learning whereas BrdU-labelled and immature neurons in the ventral dentate gyrus correlated with spatial working?reference memory learning. However, whether these correlations emerged depended on hormonal status and parity, indicating endocrine and experience modulation of the relationship between new neurons and different forms of hippocampus-dependent learning. This study is the first to show that multiparity and ovarian hormones differentially affect working and reference memory in middle age. These findings also add to the growing literature regarding the different roles of ventral and dorsal hippocampus in spatial working and reference learning and implicate dorsal and ventral hippocampal neurogenesis as a possible mechanism through which parity and ovarian status might affect hippocampus-depended cognition in middle age.    50  Multiparous rats had better early spatial working/reference acquisition than nulliparous rats and ovariectomy enhanced this effect  Multiparous rats had better acquisition during the first three days of training in the working/reference memory version of the water maze than nulliparous rats. This is consistent with past findings that multiparity is associated with better working memory in older rats (Gatewood et al., 2005; Love et al., 2005). Further the finding that ovariectomy impaired early spatial working/reference performance in nulliparous rats is consistent with a large literature indicating that ovarian hormones modulate spatial learning and that ovarian hormone deprivation causes declines in cognition (Galea et al., 1995; Hampson, 1995; Gibbs & Johnson, 2008; Hampson, 1990; Hogervorst, Williams, Budge, Riedel, & Jolles, 2000; Warren & Juraska, 1997; Barha & Galea, 2010). However, ovariectomy did not significantly affect early acquisition of the working/reference memory version of the MWM in multiparous females, suggesting that multiparity protects against the detrimental effects of hormone deficiency on working/reference memory in middle age. One might expect that, because of heightened sensitivity to estrogens (Barha & Galea, 2011), multiparity might confer a greater cognitive advantage under hormone-intact conditions.  However, the results indicate at least in initial acquisition, multiparous rats were resilient to the ovariectomy induced deficits in early working/reference memory acquisition seen in the nulliparous rats.  A possible explanation for multiparous females? resilience to ovariectomy-induced spatial working/reference memory deficits lies in the unique stress-related demands of this task. First it is important to note that the working/reference memory version of the water maze was the first test conducted in these female rats and as such stress levels may be expected to be higher upon initial testing.  Once the task and task demands are familiar one would expect stress levels to decline (as discussed in H?lscher (1999). Spatial working memory is highly sensitive to the effects of stress 51  (Diamond, Fleshner, Ingersoll, & Rose, 1996), and sensitivity of spatial working memory to chronic stress increases with task difficulty (Diamond, Park, Heman, & Rose, 1999). Male rodents exposed to chronic stress show increased numbers of working memory errors in the radial arm maze (Diamond et al., 1996; Lupien, Gillin, & Hauger, 1999; Nishimura, Endo, & Kimura, 1999) In humans, working memory capacity is negatively correlated with level of life stress (Klein & Boals, 2001) and is more sensitive than (declarative) long-term memory to the acute effects of corticosteroids (Lupien et al., 1999). The stress burden of the working memory task in the current study was likely augmented by the fact that this task was presented first in the training sequence and task demands were therefore novel. Parous rats are more resilient than are nulliparous females to the effects of stressors on stress-related behaviors (Wartella et al., 2003). Furthermore, primiparous rats are protected against the effects of acute stress on learning (trace eyeblink conditioning) during the postpartum/lactation period  (Leuner & Shors, 2006) and long after weaning (Maeng & Shors 2012). Ovarian hormones, too, modulate HPA axis activity in humans and in rodents (Carey, Deterd, de Koning, Helmerhorst, & de Kloet, 1995; De Leo, la Marca, Talluri, D'Antona, & Morgante, 1998; Viau & Meaney, 1991; Young, Altemus, Parkison, & Shastry, 2001). Ovariectomy is associated with increased anxiety-like behavior (Bowman, Ferguson, & Luine, 2002; Mora, Dussaubat, & D?az-V?liz, 1996). Perhaps the interacting effects of parity and ovarian status seen in the current findings, then, are driven by heightened stress reactivity in the nulliparous ovariectomized rats.  This explanation could also account for the fact that group differences in working/reference performance were also observed only at the early stage of training, when stress demands were presumably the highest. This also suggests that the impairments in nulliparous rats may be due not to working memory deficits per se but to heightened stress responsivity. Under conditions of ovarian hormone deficiency, multiparity may be associated with better performance on stress-sensitive tasks.  These results could indicate that multiple previous reproductive experiences may confer resilience to stress- age-, menopause-, or oorphorectomy-related 52  hormone declines on working memory Nulliparous rats outperformed multiparous rats in the reference version of the water maze  The finding that middle-aged nulliparous rats outperformed multiparous rats in the reference version of the water maze was partially surprising, as previous studies using spatial mazes have reported advantages for older parous females (Gatewood et al., 2005; Kinsley et al., 1999; Lemaire et al., 2006; Pawluski, Walker, et al., 2006). However, a number of differences between the present study and previous studies likely contributed to the different findings. First, all of the studies that assessed cognition in middle-age parous rats (Gatewood et al., 2005; Lemaire et al., 2000; Love et al., 2005) used repeated testing procedures to assess cognition longitudinally, making comparisons with the present study difficult. In addition, the relationship between reference spatial memory and parity may change with age, perhaps accounting for differences with studies using younger females (Kinsley et al., 1999; Pawluski, Vanderbyl, et al., 2006). Love (2005) et al found that in early adulthood and middle age, parous Long-Evans rats were significantly faster than nulliparous rats in a reference memory task at 5 and 13 months of age. However, by 17 and 22 months, this difference had disappeared and the mean difference now favored nulliparous females. It is difficult to compare Love's study with our own due to 1) strain differences (which can affect rate of reproductive aging (Wu, Zelinski, Ingram, & Ottinger, 2005)); 2) the repeated-testing protocol; 3) task differences which include measuring spatial learning using latency rather than distance to reach a target location, resulting in possible contamination of measures of cognition by differences in motoric ability and motivation. However, collectively, these studies suggest that the relationship between parity and reference spatial memory may change with age, and in the present study, some of the parous rats which were already showing signs of irregular cycling may have been at an age in which the parity advantage was reversing. The finding that multiparity is associated with poor reference performance in middle age is perhaps not 53  surprising when considered in the context of findings from human research. A number of studies indicate that previous reproductive experience puts women at greater risk for Alzheimer?s and cognitive impairment (Beeri et al., 2009; Colucci et al., 2006; McLay, Maki, & Lyketsos, 2003; Ptok, Barkow, & Heun, 2002; Sobow & Kloszewska, 2004).   An additional possible explanation for the discrepancy of our results with previous findings may have to do with the extent and nature of reproductive experience in our parous rats. Multiparity affects both neurogenesis and cognition differently from primiparity. For example, young biparous (but termed multiparous) rats (2 previous pregnancy and mothering experiences) show the same levels of cell survival as nulliparous rats during the postpartum period, whereas primiparous rats show decreased cell survival during this time period (Pawluski & Galea, 2007). Furthermore, degree of multiparity is likely important to cognitive outcome as the previous Pawluski et al., studies (2006; 2006) use biparous rats who were defined as having had two previous reproductive experiences during young adulthood. The multiparous rats in the present study were retired breeders and experienced 4-5 previous births and mothering experiences over a period spanning young and middle adulthood. The cognitive and neural differences between lower and higher parity have yet to be fully described by the literature.  However, findings from human research suggest that the effects of higher parity may be qualitatively different from those of lower parity, as women with 3 or more (but not 1 or 2) pregnancy experiences are at a significantly higher risk of developing Alzheimer's disease (Colucci et al., 2006).  Furthermore the number of previous pregnancies has a cumulative effect on memory impairment during pregnancy and the early postpartum (Glynn, 2012). Such findings highlight the importance of not generalizing across difference levels of parity. In addition, the effects of age at parity on cognition and brain health are currently unexplored by the literature, and so it is possible that the experience of parity early or later in adulthood contributed to the deficits in reference spatial memory seen in multiparous females in the current study.  54   Finally, it is also possible that what manifested as a disadvantage for multiparous females during the reference memory task was not poorer reference learning per se, but rather was a cost of having learned the demands of the previous working/reference task well. Perhaps multiparous females? poorer performance during the reference task arose from applying previously-successful strategies (e.g. look in new locations on odd days and old locations on even days) during the reference task, where this strategy-shifting was now associated with a cost to performance.  Consistent with this possibility, the largest parity difference was seen on day 3 of reference testing, when the platform location would have been changed in the working/reference task. However, Workman et al. (2013) found no persisting effects of reproductive experience on strategy set shifting ability in non-spatial tasks, suggesting that a superior strategy-shifting ability in multiparous females was not likely a cost to them in subsequent reference-memory testing. Future studies assessing parity effects on reference memory in isolation (or counterbalancing reference and working memory task order) may help to shed light on whether carry-over effects may have contributed to the current study?s pattern of findings. Rats in proestrus showed poorer spatial memory. Nulliparity was associated with better spatial memory in ovariectomized but not sham animals.  The finding that proestrus resulted in poorer spatial memory for the platform location is consistent with past findings that spatial learning and probe trial performance is negatively impacted by high endogenous levels of estradiol (Chow et al., 2012; Warren & Juraska, 1997). High levels of estrogens, particularly estradiol, can impair spatial reference memory (Galea et al., 2002; Holmes et al., 2002; Wide, Hanratty, Ting, & Galea, 2004). However it is less clear why ovariectomized nulliparous females had better spatial memory than ovariectomized multiparous females. This may have reflected nulliparous females' superior performance during the acquisition of the spatial location during reference spatial learning. However, this does fully account for the probe trial findings, as the same 55  effect did not hold in sham-operated nulliparous females, who also performed well during acquisition. It is interesting to note that the same group of animals (nulliparous ovariectomized females) that performed poorly on the working/reference version the water maze had enhanced performance relative to their sham-operated controls in the reference version of the watermaze. If stress responsitivity was indeed responsible for the ovariectomized nulliparous rats' poor performance in early working/reference memory training, altered stress responsivity may also have been responsible for their subsequently enhanced probe trial performance. Consistent with this interpretation, previous research indicates that women with high HPA reactivity to a social stressor perform better on a subsequent declarative memory retrieval test (Domes, Heinrichs, Reichwald, & Hautzinger, 2002) and that glucocorticoids can have opposing effects on memory consolidation and retrieval (Roozendaal, 2002). This suggests that nulliparous ovariectomized females' heightened stress response during early water maze conferred an advantage in memory retrieval. Alternatively, it may be that higher hormone levels were detrimental spatial memory in nulliparous rats but not multiparous rats.  Multiparous rats had more immature neurons, while nulliparous rats had greater survival of older new neurons (BrdU-labelled cells) Multiparous females had significantly more immature neurons than did nulliparous females, and this reached significance for immature cells at the intermediate and postmitotic stages (see Figure 1 for a photomicrograph of these stages). In contrast, nulliparous females had significantly greater numbers of BrdU-labeled cells surviving over the 23 day period since BrdU injection. The different levels of BrdU-labeled and DCX-expressing cells in multiparous and nulliparous females could reflect a difference in the maturation rates of new neurons in multiparous and nulliparous females. It may be that new neurons mature more slowly in multiparous females, leading to the larger pool of immature neurons seen in the current study.  It is important to note that we did not verify the phenotype (neuronal 56  or glial) of the BrdU-labeled cells in the present study (although this is planned in the near future). Phenotyping could be accomplished by double-labeling cells with BrdU and NeuN, a neuronal specific nuclear marker and once done it will be possible to determine whether there are different maturation levels of new neurons in the dentate gyrus.  DCX expression occurs in the 1-21 days after the production of the new neurons, with maximal DCX expression occurring at 4-7 days (Brown et al., 2003). In the current study the oldest DCX-expressing cells would therefore have been born 2 days after testing began. Although only the younger (DCX-expressing) population was generated under learning conditions, the parity difference in immature neuron density was unlikely to have resulted from a ?cohort? effect. Spatial learning is known to increase neurogenesis in males but not females (Chow et al., 2013) and so the increase in DCX-expressing cell density we see in multiparous females was unlikely to reflect an increased neurogenic response to learning. In contrast, the finding that the number of BrdU-labelled cells in the dentate gyrus was lower in multiparous females compared to nulliparous rats could partly be a result of learning, as spatial learning can also decrease neurogenesis if learning is given at later timepoints after BrdU exposure (see Epp et al., (2011)).  In any case, it may be that parity influences the effects of learning on neurogenesis depending on how old new neurons are at the time of exposure to learning.   Regardless of parity or ovarian hormone status, more Type B and Type C (intermediate and postmitotic) immature neurons were found in the dorsal dentate gyrus than in the ventral dentate gyrus. Jinno (2003) similarly found that a greater number of doublecortin-expressing cells in the dorsal compared to the ventral dentate gyrus of male naive mice. These findings further suggest that the greater immature neuron density in the dorsal dentate gyrus in the current study was not a result of preferential up-regulation of neurogenesis in the dorsal dentate gyrus. Further studies would be required to assess the effects of parity and ovarian status on neurogenesis independent of learning.  The finding that nulliparous females had greater density of BrdU-labeled cells but multiparous 57  females had greater density of immature neurons is particularly intriguing in light of findings regarding activity of mature and immature neurons in response to spatial learning. Snyder (2009, 2011) measured cell activity in mature and new adult-generated neurons using Fos, an immediate early gene. Cells were assumed to be mature if found in granule cell layer (comprised mainly of mature neurons) and immature if found in the subgranular zone (where proliferation occurs) or expressing PSA-NCAM, an endogenous marker expressed during neuronal development and migration. He found that in the dorsal/septal pole, activity was mainly found in the mature neuron population. In contrast, activity in the ventral dentate gyrus was greatest in the population of new adult-generated neurons. These results were found both in adult (Snyder et al., 2009) and middle-aged (Snyder et al., 2011) male rats and suggest that mature and immature neurons may have specific, regional functions in response to spatial learning.  Multiparity resulted in larger ventral dentate gyrus volume  Ventral dentate gyrus volumes were approximately 30 percent larger in multiparous females than in nulliparous females, while dorsal volumes were not significantly affected by parity. The dorsal and ventral dentate gyrus seem to be functionally distinct, with the ventral hippocampus primarily associated with stress/anxiety and working memory, and the dorsal hippocampus playing a larger role in reference memory (Bannerman et al., 2004; Cerqueira, Almeida, & Sousa, 2008; Laroche, Davis, & Jay, 2000). The ventral hippocampus' role in spatial working memory stems from its functional connection to the PFC, an area required for and activated during working memory (reviewed in Fuster, 1984; Goldman-Rakic, 1987). The ventral, but not dorsal, hippocampus connects to the prefrontal cortex via a unidirectional, ipsilateral glutamatergic projection from the CA1 and subiculum regions to the medioprefrontal and orbitofrontal cortices (Rosene and Van Hoesen, 1977, Cavada et al., 1983; Goldman-Rakic et al., 1984; Jay et al., 1989; Jay and Witter, 1991; Barbas and Blatt, 1995; Carmichael 58  and Price, 1995; Sesack et al., 1989; Jay et al., 1992; Reviewed in Laroch and Cerqueira). This hippocampal-prefrontal cortex pathway is plastic, demonstrating LTP (Jay, Burette, & Laroche, 1996) and undergoing synaptic depression during delays in spatial working memory tasks (Burette, Jay, & Laroche, 2000). Floresco et al. (1997) found that disconnection lesions of this pathway produce impairments in spatial delayed nonmatching to sample task in the radial arm maze, suggesting a role of the hippocampal-prefrontal cortex connection in spatial working memory. In the present study, the finding that multiparous females had larger ventral volumes may help to explain their enhanced early spatial working memory.  In addition to a larger role in spatial working memory, the ventral hippocampus distinguishes itself from the dorsal hippocampus in its unique role in stress and anxiety. Lesion studies in rodents substantiate this dissociation, as lesions of the ventral hippocampus affect anxiety and have no effect on spatial learning (Kjelstrup et al., 2002; Moser, Moser, & Andersen, 1993), whereas lesions of the dorsal hippocampus affect reference spatial learning and memory (Moser et al., 1993; Moser, Moser, Forrest, Andersen, & Morris, 1995). As previously discussed, spatial working memory is impaired by chronic stress, particularly in male rodents (Diamond et al., 1996; Lupien, Gillin, & Hauger, 1999; Nishimura, Endo, & Kimura, 1999; Klein & Boals, 2001). The PFC is affected by stress and glucocorticoids, particularly in areas receiving limbic (including hippocampal) afferent connections (Swanson and Cowan, 1977). Furthermore, chronic stress impairs the development of long-term potentiation within the hippocampal-prefrontal circuit (Cerqueira et al., 2008; Jay et al., 2004; Rocher, Spedding, Munoz, & Jay, 2004). The ventral hippocampus also appears to be particularly sensitive to the effects of stress, as chronic mild stress decreases cell proliferation in the ventral but not dorsal dentate gyrus (Jayatissa, Bisgaard, Tingstr?m, Papp, & Wiborg, 2006). Both the PFC and the hippocampus have roles in regulating the HPA axis (Jacobson & Sapolsky, 1991; Sullivan & Gratton, 2002) and so stress-induced damage to either of areas may have feed-forward effects on stress, cognition, and the integrity of the 59  circuit.  However, as previously discussed, multiparous rats are resilient to the effects of (at least acute) stress on behavior and learning and memory (Leuner & Shors, 2006; Maeng & Shors, 2012). Therefore, both the larger ventral dentate gyrus volumes and the enhanced early working/reference spatial performance seen in multiparous females may stem in part from enhanced stress resilience.  The contribution of neurogenesis to the relationships between stress responsivity, spatial working memory, and the ventral hippocampus/prefrontal circuitry has yet to be fully described by the literature. Stress and elevated levels of glucocorticoids impair spatial working memory and suppress hippocampal neurogenesis in a sex-dependent manner (Brummelte & Galea, 2010; (Lin et al., 2009). The effects of stress and elevated glucocorticoids on neurogenesis seem to be particularly robust in the ventral dentate gyrus and in females (Jayatissa, Bisgaard, Tingstr?m, Papp, & Wiborg, 2006). This pattern of findings might suggest a beneficial role of new neurons in the ventral dentate gyrus in stress regulation and/or spatial working memory. However, the relationship between new neurons and working spatial memory is not straightforward, it seems: Ablating hippocampal neurogenesis has been found to enhance, rather than impair, spatial working memory in male mice (Saxe et al., 2007), and working memory training itself may decrease hippocampal neurogenesis, at least under a long training protocol (Xu et al., 2011). Other plastic changes, such as a resilience to stress- or glucocorticoid-induced atrophy of dendrites of hippocampal pyramidal cells, could also be moderated by parity and have caused the ventral dentate gyrus volume difference observed in multiparous females. Future studies can investigate the relative contribution of neurogenesis and other forms of hippocampal plasticity to the effects of parity on ventral dentate volume and spatial working memory. BrdU-labelled cells in the ventral dentate gyrus are associated with better performance in working/reference MWM in sham rats.  The finding that BrdU-labeled cell survival in the ventral dentate gyrus correlates with 60  working/reference memory is consistent with a role of the ventral dentate gyrus in both stress regulation and working memory. A positive correlation between cell survival and working/reference performance could indicate a functional role of neurogenesis in stress regulation and/or spatial working memory. If so, the finding that certain groups (nulliparous ovariectomized females) had increased cell survival without enhanced performance is puzzling.  Alternatively, the positive correlation between cell survival and working/reference performance could reflect a role of stress resilience as a third variable enhancing both working memory performance and new neuron survival in the ventral dentate gyrus. Assessing the activation of these new neurons to spatial learning and memory, rather than the raw number of surviving cells, may help to clarify the nature of the relationship between new neurons in the ventral dentate gyrus and spatial working/reference memory. Activation of cells in could be assessed using markers for immediate early genes (IEGs) such as zif268, which are rapidly and transiently transcribed in response to spatial learning and memory.  That the correlation between cell survival in the ventral dentate gyrus and working/reference spatial performance existed in only in sham-operated rats is intriguing and indicates that ovarian status modulates the relationship between cell survival and working memory performance.  Future studies investigating the activation of these surviving new neurons may help to elucidate the role of these cells and their relationship to stress, learning and memory, and ovarian hormones.   BrdU-labeled cells in the dorsal dentate gyrus correlated with better spatial reference performance in ovariectomized female rats  In contrast to the findings regarding working/reference performance and  the survival of BrdU-positive cells in the ventral dentate gyrus, spatial reference performance was  correlated with greater BrdU-labeled cell survival in the dorsal dentate gyrus of ovariectomized females. The finding that dorsal, but not ventral levels of BrdU-labeled cells correlated with spatial reference performance is 61  consistent with a functional dissociation between the dorsal and ventral hippocampus, wherein the dorsal region is involved in spatial reference memory and the ventral region is involved in spatial working memory. It is curious that the correlation was specific to ovariectomized rats, indicating a moderating role of ovarian hormone levels on the relationship of cell survival to spatial reference memory. It is also curious that ovarian hormones' effect on the cell survival-cognition relationship depends on the type of spatial ability assessed: Whereas ovariectomy abolished the correlation between cell survival and working memory performance, ovariectomy seem to be required for a correlation between survival and reference memory to emerge. This pattern of results suggests that cell survival relates to cognitive performance differentially based on region (dorsal or ventral), hormone status, and the type of cognition being assessed. The density of immature neurons in the dorsal dentate gyrus was related to better spatial reference memory in intact females while the density of immature neurons in the ventral dentate gyrus was related to better spatial memory only in ovariectomized nulliparous rats.   Greater cell density of intermediate immature (Type B) neurons in the dorsal dentate gyrus was related to better spatial reference performance in hormonally-intact nulliparous rats. This finding is generally consistent with a role of the dorsal dentate gyrus, and perhaps neurogenesis in this region, in spatial reference performance. However, the functional role of these immature cells in spatial reference performance remains unclear, as the correlation between intermediate immature neurons and reference memory was specific to a single parity and ovarian hormone status group.  Spatial memory positively correlated with immature neurons depending on ovarian status and parity. In nulliparous ovariectomized rats greater density of ventral intermediate immature neurons correlated with better spatial memory.  In contrast, density of dorsal immature neurons negatively correlated with probe trial performance in sham-operated multiparous females. As multiparous females 62  had greater average numbers of dorsal doublecortin-expressing cells in the dentate gyrus but did not perform more poorly as a group during the probe trial, this correlation is difficult to interpret without further information about how these new cells are responding to spatial learning and memory. It could be that in multiparous sham-operated females, cells are being created and subsequently fail to be activated by appropriate stimuli. Future studies studying IEG activation in immature neuron populations may help to elucidate the role of these cells in spatial learning and memory, and the influence of parity and hormone status.   63  CONCLUSIONS  This study provides evidence that parity and hormone status have interacting effects on hippocampal-dependent cognition and neurogenesis in middle-aged females. Multiparous rats had enhanced initial working?reference memory compared to nulliparous rats while nulliparous rats had enhanced spatial reference memory performance compared to multiparous rats. These effects on spatial cognition were also affected by ovariectomy, with ovariectomy enhancing spatial reference memory in nulliparous rats but impairing early working?reference memory acquisition. The findings also suggest that multiparous rats were resilient to the effects of ovariectomy on spatial working?reference memory acquisition, perhaps due to reduced stress responsivity compared to nulliparous rats. Multiparous rats had a larger ventral dentate gyrus, an area highly sensitive to the effects of stress and implicated in spatial working memory. Furthermore multiparous rats had larger number of immature neurons in the hippocampus, while nulliparous rats had larger number of older BrdU-labelled cells, possibly indicating greater survival of new neurons in nulliparous rats. Neurogenesis in the dorsal hippocampus correlated with spatial reference performance, whereas neurogenesis in the ventral hippocampus correlated with spatial working memory learning and these associations were moderated by ovarian status and parity. This may indicate the functional role of neurogenesis depends on reproductive experience, hormone exposure and type of cognitive task.  The exact nature of the role of new neurons in spatial working and reference memory abilities in middle age may be further elucidated by investigating how these new neurons are activated in response to spatial learning. The present study is an important step in understanding more about how hormonal events in a female's life impact learning, memory and neurogenesis in the dentate gyrus of the hippocampus in middle age.    64  REFERENCES Adams, M. M., Fink, S. E., Shah, R. A., Janssen, W. G., Hayashi, S., Milner, T. A., . . . Morrison, J. H. (2002). 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