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Effects of chronic estradiol, progesterone and medroxyprogesterone acetate on hippocampal neurogenesis… Chan, Melissa; Chow, Carmen; Hamson, Dwayne K.; Lieblich, Stephanie E.; Galea, Liisa A.M. Jun 30, 2014

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1 Effects of chronic estradiol, progesterone and medroxyprogesterone acetate on hippocampal neurogenesis and adrenal mass in adult female rats.  Melissa Chan, Carmen Chow, Dwayne K. Hamson, Stephanie E. Lieblich, Liisa A.M. Galea Graduate Program in Neuroscience, Department of Psychology,  Brain Research Centre, University of British Columbia, Vancouver, BC, CANADA Corresponding author: Liisa Galea, Ph.D. Department of Psychology University of British Columbia 2136 West Mall Vancouver, BC V6T1Z4 email: Acknowledgements: This work was funded by a CIHR Operating grant to LAMG (MOP: 102568). Page 1 of 51 Published in: Chan, M., Chow, C., Hamson, D.K., Lieblich SE, Galea, L.A.M.  (2014). Effects of chronic estradiol, progesterone and medroxyprogesterone acetate on hippocampal neurogenesis and adrenal mass in adult female rats. Journal of Neuroendocrinology 26(6):386-99. 1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859602 Abstract Both natural estrogens and progesterone influence synaptic plasticity and neurogenesis within the female hippocampus. However, less is known of the impact of synthetic hormones on hippocampus structure and function.  There is some evidence that administration of the synthetic progestin, medroxyprogesterone acetate (MPA) is not as beneficial as natural progesterone and can attenuate estrogen-induced neuroprotection. Although estradiol’s effects have been well studied, little is known about the effects of natural and synthetic progestins alone and in combination with estradiol on adult neurogenesis in the female. In the present study, we investigated the effects of chronic estradiol, progesterone, MPA, and a co-administration of each progestin with estradiol on neurogenesis within the dentate gyrus of adult ovariectomized female rats. Twenty-four hours following a bromodeoxyuridine (BrdU; 200mg/kg) injection, female rats were repeatedly administered with either progesterone (1 or 4 mg), MPA (1 or 4 mg), estradiol benzoate (EB), progesterone or MPA in combination with EB (10µg), or vehicle for 21 days. Rats were perfused on day 22 and brain tissue was analyzed for number of BrdU-labeled and Ki67 (an endogenous marker of cell proliferation)-expressing cells. EB alone and MPA+EB significantly decreased neurogenesis and surviving BrdU-labeled cells in the dorsal region of the dentate gyrus, independent of any effects on cell proliferation. Furthermore, MPA (1mg and 4mg) and MPA+EB treated animals had significantly lower adrenal/body weight ratios, and reduced serum corticosterone (CORT) levels. In contrast, P+EB treated animals had significantly higher adrenal/body weight ratios and 1mg P, P+EB, and EB significantly increased CORT levels. The results of the current study demonstrate that different progestins alone and in combination with estradiol can differentially affect neurogenesis (via cell survival) and Page 2 of 51Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603 regulation of the HPA axis. These findings have implications for women using hormone replacement therapies with MPA for both neuroprotection and stress-related disorders. Page 3 of 51 Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604  The dentate gyrus of the hippocampus is a highly plastic area of the brain that retains the ability to generate new neurons during adulthood in most mammalian species (Eriksson et al., 1998; Christie and Cameron, 2006). There are several stages in hippocampal neurogenesis including cell proliferation, migration, differentiation and cell survival, and alterations to any of these stages (independently or dependently) could affect neurogenesis. Cell proliferation refers to the production of new cells from the division of progenitor cells. Cell survival is a measure of the number of these newly formed daughter cells that survive to maturity (Cameron et al., 1993). Newly-formed daughter cells can continue to proliferate or migrate from the subgranular zone to the granule cell layer, become neurons, and integrate themselves into the existing network to become functionally mature (Esposito et al., 2005; Liu et al., 2000). Increases in neurogenesis can occur with increases in cell proliferation independently of effects on new cell survival (for example chronic antidepressants: Malberg et al., 2000), increases in the percentage of new cells differentiating into neurons (differentiation; ie Uban et al, 2010) or increases in cell survival independent of any changes cell proliferation (for example, androgen effects: Hamson et al., 2013). For more detailed information, the reader is directed to reviews (Taupin, 2007; Leuner and Gould, 2010; Barha et al., 2009). Estradiol affects hippocampal neuroplasticity in the young adult female rodent in a variety of ways including altering hippocampal neurogenesis (Barha & Galea, 2010), dendritic spine density (Woolley, 1998), and cell death (Wise et al., 2001). Acute treatment with estradiol rapidly increases cell proliferation (Barha et al., 2009; Tanapat et al,1999), while chronic treatment with estradiol decreases neurogenesis, via cell survival, in the dentate gyrus of young adult female rats (Barker & Galea, 2008).  Research has focused on investigating the effects of Page 4 of 51Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859605 estradiol on hippocampal neurogenesis and there are surprisingly few studies on the effects of progesterone on neuroplasticity in the adult brain. Like estradiol, progesterone is naturally present in the male and female brain, with levels fluctuating across the estrous cycle in young adult females (Frye et al, 2000). Beyond its effects to promote female sexual behaviour and maintain pregnancy (Etgen, 1984; Sugimoto et al., 1997), progesterone also regulates mood, cognition, and neural plasticity (Frye, 2011; Paris et al., 2011; Liu et al, 2009). Through the binding of the classical steroid nuclear progesterone receptor (PR), progesterone can protect against different brain insults, including traumatic brain injury and ischemic strokes in male rodents (Sayeed & Stein, 2009). Progesterone also regulates adult hippocampal neurogenesis in young adult male rats, but curiously, few studies have examined the effects of progesterone in female rats. In rat hippocampal cell cultures, progesterone increased cell proliferation dose-dependently by increasing the expression of genes that promote mitosis and decreasing the expression of genes that repress cell proliferation (Liu et al., 2009). Additionally, progesterone may regulate neurogenesis via the membrane-bound progesterone receptor membrane component 1 (PGRMC1) rather than the classical PRs, as cells that expressed PCNA, an endogenous marker of cell proliferation, co-expressed PGRMC1 but not PRs (Liu et al, 2009). In young adult male rodents, subchronic progesterone upregulated the survival and proliferation of new neurons and decreased cell death in the hippocampus (Barha et al., 2011; Zhang et al., 2010). Treatment with progesterone once a day for 3 consecutive days significantly increased the survival of newly generated cells at 7, 28 and 56 days but not 2 days after hormone injection in young adult male mice (Zhang et al., 2010).  Whereas in ovariectomized young adult female rats, a single injection of progesterone significantly increased cell proliferation in the dentate gyrus 24hrs after injection (Bali et al., 2012; Lui et al., 2010). Page 5 of 51 Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606 However there have been no studies examining the effects of chronic progesterone on hippocampal neurogenesis in the adult female rat and the present study aims to fill this gap. There are several synthetic forms of progesterone, with medroxyprogesterone acetate (MPA) being the most commonly used for hormone replacement therapies in the United States (Chisholm and Juraska, 2012). Though MPA and progesterone exert similar uterotrophic effects (Chisholm and Juraska, 2012), it is important to note that these two forms exhibit differential effects in the brain. Progesterone alone or in combination with estradiol protects primary hippocampal neurons from glutamate-induced cell death, whereas MPA does not (Nilsen &Brinton, 2002). Not only was MPA an ineffective neuroprotectant against glutamate toxicity, MPA also suppressed the neuroprotective influence of estradiol by blocking estradiol-induced increase in expression of the anti-apoptotic gene Bcl-2 (Nilsen & Brinton, 2002). In ovariectomized young adult female rats, acute progesterone exposure increased cell proliferation, while MPA reduced cell proliferation in vitro and had no significant effect in vivo (Liu et al, 2010). Interestingly, when co-administered with estradiol, progesterone and MPA both increased cell proliferation, although acute treatment with MPA and EB also increased apoptosis in ovariectomized young adult female rats (Liu et al, 2010), therefore many of these new cells may not reach maturity. There are equivocal findings with regards to MPA’s effects on the brain, with some studies showing benefits in the hippocampus and prefrontal cortex (Silva et al, 2000; Chisholm et al, 2012), and some showing adverse effects in the hippocampus (Nilsen and Brinton, 2002; Nilsen and Brinton, 2003; Irwin et al, 2011).   Altogether the evidence suggests that progesterone may have different effects on different forms of neuroplasticity than MPA. In addition to effects on neuroplasticity, progesterone and estradiol modulate the hypothalamic pituitary adrenal (HPA) axis, with high estradiol and progesterone being associated Page 6 of 51Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859607 with greater corticosterone release in response to stress (Viau and Meaney, 1991; Handa and Weiser, 2013). Progesterone itself is increased in response to stress (Kalil et al., 2013 ) and modulates the stress response,via allopregnanolone (Bitran et al, 1993; Frye et al, 2000). Because exposure to chronic glucocorticoids decreases neurogenesis in the hippocampus of adult young females (Brummelte and Galea, 2010), the effects of ovarian steroids on neurogenesis in the hippocampus may be modulated in part by their effects on HPA functioning. To our knowledge there has yet to be any study comparing the effects of chronic treatment with natural progesterone and the synthetic form, MPA on adult female hippocampal neurogenesis. Estrogens and progestins are often both present in hormone therapies and previous work has suggested that progesterone can interfere with estrogen-induced cell proliferation in ovariectomized young adult female rats (Tanapat et al., 2005). Females are exposed to sustained high levels of progesterone throughout pregnancy (Soldin et al, 2005) and high levels of various progestins, including MPA, when prescribed contraceptives (oral, intrauterine devices, and including progestin-only contraceptives) and hormone replacement therapy (Hersh et al, 2004; Mosher et al 2004;  Endrikat et al., 2011). Further, MPA suppresses adrenal gland activity in older women (Malik et al, 1996), which may affect the HPA axis, and subsequently, adult neurogenesis. Therefore, it is important to study how chronic progesterone and MPA affect the female brain with and without estradiol. We hypothesized that progesterone and MPA alone and in combination with estradiol will produce differential effects on neurogenesis and levels of corticosterone in the dentate gyrus of adult female rats. Methods Animals Page 7 of 51 Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859608 Forty-one adult female Sprague-Dawley rats weighing 250-300g were used. All animals were bred within our animal facility. All animals were pair-housed in opaque polyurethane bins (48 X 27 X 20 cm) with aspen chip bedding and fed Purina rat chow and tap water ad libitum. Rats were housed in a colony room with a temperature of 21 ± 1OC with lights maintained on a 12-hlight: 12-h dark cycle (lights on from 07:00 to 19:00 h).  All experiments were conducted in accordance with the ethical guidelines set forth by the Canada Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. Surgery Bilateral ovariectomies were performed on all females under isoflurane anesthesia.  A single injection of lactated ringer’s solution (100 ml/kg, s.c.) and an injection of a nonsteriodal anti-inflammatory analgesic (Anafen, MERIAL Canada Inc., Baie d’Urfé, Quebec, Canada; 5 ml/kg, s.c.) were administered preoperatively. Following surgery, animals were singly housed in aclean, opaque polyurethane bin and placed on a heating pad until full recovery from anesthetic was observed. A topical antibacterical ointment was applied on the external surface of the incision (Flamazine, Smith & Nephed, St Laurent, Quebec, Canada). Rats were monitored daily and given approximately 7 days to recover before any experimental manipulation was conducted. Three days following surgery animals were handled every day for three days for 5 minutes. Procedure All adult ovariectomized females were given a single intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU, 200mg/kg; Sigma, St. Louis, MO, USA).  BrdU was dissolved in 0.9% saline and 0.7% 1N NaOH. BrdU is a thymidine analogue that incorporates itself into the DNA of cells during the synthesis stage of the cell cycle within two hours after injection (Packard et al, 1973). Twenty-four hours later animals were randomly assigned to one of 8 treatment groups Page 8 of 51Journal of Neuroendocrinology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859609 (n=5-6/group) and given a subcutaneous (s.c.) injection of either: vehicle (0.10 ml sesame oil), progesterone (P; 1 mg or 4 mg/0.10ml sesame oil), MPA (1 mg or 4 mg/0.10ml sesame oil), EB (10µg/0.10ml sesame oil), P + EB  (4mg of P +10µg EB/ 0.10 ml sesame oil), or MPA + EB (4mg MPA+10µg EB/0.10 ml sesame oil). Animals received a single 0.10ml s.c. injection of hormone once a day (between 11:00h-12:00h) for 21 days (see Figure 1A). We gave a single injection of BrdU prior to hormone treatment to determine the effects of these hormones on the survival of BrdU-labeled cells independent of any effects on cell proliferation (Barker and Galea, 2008). BrdU is biologically active for just 2 hours after injection while the entire cell cycle resulting in daughter cells requires 24 hours (Cameron and McKay, 2001). Therefore, injecting BrdU 24 hours prior to treatment ensures that only effects on labeled post-mitotic cells are examined. Thus using this design we are examining the survival of newly labeled cells independent of any effects on cell proliferation. Doses of progesterone were chosen based on previous reports in which a significant increase in 28-day-old BrdU+ cells within the dentate gyrus was observed following an injection of progesterone for 3 days (Zhang et al., 2010). Progesterone ranging from 1mg-4mg has also been previously found to be effective in preventing neuronal death after kainic acid treatment but MPA at these doses did not show the same attenuation (Ciriza et al., 2006). The progesterone doses were chosen to simulate both normal circulating levels (Ciriza et al, 2006) and supraphysiological levels, as that observed during pregnancy in female rats (Green and Galea, 2008). The estradiol dose produces serum levels of estradiol similar to that observed during the proestrous phase of the rat estrous cycle (Viau and Meaney, 1991). In order to maintain consistency and allow for comparisons between P and MPA, the dose of MPA was administered similarly to that of P. Page 9 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596010 On day 29, one day following the final hormone injection, rats were deeply anesthetized with sodium pentobarbital and then perfused with saline followed by 4% paraformaldehyde (Sigma–Aldrich). Brains were extracted and then post-fixed in 4% paraformaldehyde for 24 h at 4OC before being transferred to 30% sucrose (Fisher Scientific) diluted in phosphate-bufferedsaline (PBS) solution. Adrenals were also extracted and weighed. Serum from cardiac puncture at the time of perfusion was collected for analysis of corticosterone levels. Brains were sliced at 40-µm and collected into 10 series of coronal sections using a Leica SM2000R microtome (Richmond Hill, ON, Canada). Sections were collected throughout the entire rostral-caudal extent of the hippocampus and stored in antifreeze solution (0.1 M PBS, 30% ethylene glycol, and 20% glycerol) at -20OC.Immunohistochemistry BrdU Tissue was removed from antifreeze and rinsed three times with 0.1 M Tris-buffered saline (TBS; pH = 7.4) and left overnight. On the following day tissue was placed into nets in TBS for 10 minutes before being incubated in a 0.6% H2O2 solution for 30 minutes. After three sets of 10 minute TBS rinses, the tissue was transferred into a 2N hydrochloric acid solution and placed in a hot water bath (37OC) for 30 minutes.  Following three rinses of TBS, a 30 minute incubationwith a solution containing normal horse serum (NHS; Vector Laboratories; Burlingame, CA, USA) and 0.1% Triton-X (Sigma) in 0.1M TBS was done before tissue was incubated in a primary antibody solution consisting of 1:200 mouse anti-BrdU (Roche, Indianapolis, IN), 0.1% Triton X, 3% NHS in 0.1M TBS on a shaker for 20 hrs at 4OC. After three rinses in 0.1M TBSthe tissue was transferred to the secondary antibody consisting of  1:200 dilution of horse anti-mouse biotinylated antibody (Vector Laboratories, Burlington, ON, Canada),  0.1 % Triton X, Page 10 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596011 and NHS in 0.1M TBS for a 4 hr incubation period.  Following three sets of TBS rinses, the tissue was incubated in an avidin/biotinylated enzyme solution (ABC kit, Vector Laboratories) for 1.5 hrs. Visualization of BrdU-labeling was achieved through incubation of tissue in 0.02% diaminobenzidine (DAB) for 12 minutes. Tissue was then mounted on glass slides and cover-slipped with Permount (Fisher Scientific; Ottawa, ON, Canada). BrdU/NeuN To determine the phenotype of BrdU-labeled cells, staining for NeuN was conducted. Tissue was rinsed in 0.1M PBS three times for 10 minutes each and left overnight. The following day, tissue was incubated in a solution containing 1: 250 mouse anti-NeuN (Millipore, Temecula, CA) primary in 0.1M PBS, 3% normal donkey serum (NDS; Vector Laboratories), and 0.3% Triton-X at 4oC for 24 hrs. All tissue was then given three 10 minute washes of 0.1M PBS beforeincubation in a 1:200 dilution of donkey anti-mouse Alexa 488 (Invitrogen, Eugene, OR) secondary antibody in 0.1M PBS for 16-18 hrs at 4oC. Following three PBS rinses the tissue wasrinsed with a 4% paraformaldehyde solution for 10 minutes at room temperature, then incubated for 30 minutes in a 2N hydrochloric acid solution at 37oC. Labeling for BrdU was then carriedout  by rinsing tissue three times in PBS then incubation with the primary antibody solution at a dilution of 1:500 consisting of rat anti-BrdU (Roche, Indianapolis, IN), 0.1M PBS, 3% NDS, and 0.3% Triton-X for 24 hrs at 4OC. After three rinses with PBS, tissue was transferred to a solutionconsisting of a 1:500 dilution of donkey anti-rat Cy3 (Jackson ImmunoResearch, Westgrove, PA) and 0.1M PBS for 20 hrs at 4OC. Finally, tissue was rinsed three more times with PBS andthen mounted on glass slides and covers-lipped using PVA-DABCO. Ki67 Page 11 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596012 Tissue was removed from antifreeze and rinsed with 0.1M PBS three times, ten minutes each then left in the fridge overnight. The following day, the tissue was transferred to a 0.6% H2O2 solution for 30 minutes at room temperature. Following three rinses with PBS for ten minutes each,  the tissue was then incubated for 24 hrs at 4oC with the primary antibody solutioncontaining a 1:3000 Rabbit anti-Ki-67 monoclonal antibody (Vector, Burlingame, CA), 3% normal goat serum, and 0.3% Triton-X in 0.1 M PBS.  The tissue was then rinsed four times with PBS and incubated with the secondary antibody solution of biotinylated goat anti-rabbit (Vector, Burlingame, CA) at 1:500 for 24 hrs at 4oC. After rinsing four times with PBS, tissue was nextincubated with ABC solution as per kit instructions for 4 hrs at room temperature and then rinsed three times with PBS. Tissue was then rinsed two times with 0.175M sodium acetate for 2 minutes each rinse before being developed with DAB at a reaction time of 15 minutes.  Finally, the tissue was rinsed two times with sodium acetate for 2 minutes and three times with PBS for 10 minutes. Sections were then mounted on glass slides and cover-slipped using Permount. Cell Counting Counting of BrdU-labeled and Ki67-expressing cells was conducted under a 100 X objective and NeuN/BrdU-labeled cells was conducted under a 40 X objective, by an experimenter blind to the treatment conditions. Cells were counted separately for the granule cell layer (GCL; which included the subgranular zone (SGZ)) and the hilus. Counting of cells in the hilus was conducted in order to control for the possibility of effects due to changes in blood brain barrier permeability as cells in this region are not normally neurons and new neurons in this region are generally considered to be ectopic (Cameron et al., 1993). The SGZ was defined as approximately 50µm between the GCL and the hilus. The GCL and hilus of every 10th hippocampal slice was countedseparately and the total number of BrdU-labeled cells was estimated by taking the sum of the cell Page 12 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596013 counts per animal and multiplying by the sampling fraction of 10 as described previously (Epp et al., 2010). The percentage of BrdU/NeuN double labeled cells was determined by selecting 25 BrdU-labeled cells per brain from at least 5 sections and calculating the proportion of cells that were also expressing NeuN (Leuner et al., 2007; Snyder et al., 2009). Percentages of BrdU/NeuN co-labeled cells were also verified using a confocal microscope (Leica SP8) and viewed a 630 X objective. Images were captured with Leica Application Suite-Advanced Fluorescence (Leica, Ontario, Canada) and are seen in Figure 1D. The percentages of BrdU-labelled cells expressing NeuN is a common measure used to identify the differentiation of new cells into neurons (Hamson et al., 2013; Leuner et al., 2007; Snyder et al., 2009). The area of the GCL and hilus were measured separately using Image J (National Institutes of Health, Bethesda, MD, USA). To calculate the volume of each region, Cavalieri’s principle (Gundersen and Jensen, 1987) was applied in which the sum of the measured areas was multiplied by the distance between sections (400µm). Fig 1B-1D depicts a photomicrograph representation of BrdU-labeled cells, Ki67-expressing cells and a BrdU/NeuN-labeled cell in the SGZ of the dentate gyrus after 21 days of hormone treatment.   We separated counts by the dorsal and ventral regions of the dentate gyrus because of different putative functional aspects of these regions. The dorsal hippocampus is important for spatial memory while ventral hippocampus is important for stress and anxiety (Moser et al, 1993; Fanselow and Dong, 2010). Region-specific effects may provide more insight into progesterone and MPA’s behavioural effects. Corticosterone Assay Progesterone and estradiol can influence corticosterone levels (Viau and Meaney, 1991; Carey et al, 1995) and because corticosterone negatively regulates hippocampal neurogenesis (Brummelte Page 13 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596014 and Galea, 2010), we examined corticosterone levels. Serum levels of corticosterone were assayed for all animals using blood collected from the right atrium during perfusion on day 22. Whole blood samples were then stored at 4oC for 24 hrs and then spun in a rotating centrifuge(10 000 rpm for 10 min). Serum was then collected and stored at -20oC. Serum corticosteronesample were assayed in duplicates using a commercially available 125I radioimmunoassay kit forcorticosterone (MP Biomedicals, Costa Mesa, CA). The standard curve ED50 was 153.5ng/ml, and the lower limit of detection was 7.7ng/ml. The average intra-assay coefficient of variation was less than 10%. Data Analyses All statistical analyses were performed using the program Statistica (StatSoft, Inc., USA). Total numbers of BrdU-labeled and Ki67-expressing cells were analyzed using a repeated measures analysis of variance (ANOVA) with within-subjects factors of region (GCL+SGZ and hilus) and dorsoventral axis (dorsal, ventral), and a between subjects factor of hormone treatment (oil control, 1 mg P, 4 mg P, 1 mg MPA, 4 mg MPA, EB, P+EB, and MPA+EB). Serum corticosterone levels, changes in body weight and adrenal/body weight were analyzed using an ANOVA with hormone treatment (oil control, 1 mg P, 4 mg P, 1 mg MPA, 4 mg MPA, EB, P+EB, and MPA+EB) as a between subjects factor. Post-hoc analyses used the Newman-Keuls procedure or a priori tests where appropriate. All statistical tests used a significance criterion of α = 0.05. Results Page 14 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596015 EB and MPA+EB decreased the survival of BrdU-labeled cells in the dorsal GCL Chronic treatment with EB and MPA+EB resulted in significantly fewer BrdU-labeled cells in the GCL relative to oil-treated controls (p =.019, .0003, respectively) but not the hilus (all p’s > .94; interaction effect of hormone treatment by region (F7,26 = 3.06, p =.017; see Figure 2A). Indeed, the MPA+EB group had significantly fewer BrdU-labeled cells than all other groups (all p’s < .024) except the EB group (p = .11), while the EB group had significantly fewer BrdU-labeled cells only when compared to the oil and the P groups (1mg P: p = .054; 4mg P: p = .003). There was also a significant region x dorsoventral region interaction (F1,26 = 14.63, p = 0.0007). Post-hoc analyses revealed that the dorsal GCL had a significantly greater number of BrdU-labeled cells compared to the ventral region (p < .002), but significantly fewer BrdU-labeled cells in the dorsal hilus versus the ventral hilus (p < .04). There were also significant main effects of region and hormone treatment interaction (both p’s =.005). Most BrdU-labeled cells co-expressed NeuN and chronic hormone treatment did not significantly affect the percentage of new neurons Analysis of percentage of BrdU-labeled cells that co-expressed the neuronal marker, NeuN, revealed no significant effect of hormone treatment on the percentage of BrdU/NeuN labeled cells (p =.76; see Table 1).  The majority of BrdU-labeled cells co-expressed NeuN, indicating that these new cells were neurons. Because BrdU does not exclusively label neurons (Taupin, 2007), it is unclear from the number of BrdU-labeled cells data alone how hormone treatment affected cells that specifically differentiated into neurons (as determined by BrdU/NeuN co-labeling). Therefore, we estimated the number of BrdU-labeled cells that differentiated into neurons by calculating the Page 15 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596016 ‘neurogenesis index’, which was done by multiplying the percentage of BrdU/NeuN labeled cells by the number of BrdU-labeled cells (van Praag et al, 1999; Kempermann et al, 2002; Brown et al, 2003; Hamson et al, 2013).  Post-hoc tests revealed that the number of new neurons was significantly reduced in the MPA+EB treated groups compared to oil-treated controls (p = .04; main effect of hormone treatment (F7,23 = 3.98, p = .005;  see Figure 2B). There was also a significant main effect of dorsoventral axis with more new neurons found in the dorsal compared to the ventral area (p =.04). Chronic hormone treatment had no significant effects on cell proliferation or the volume of the dentate gyrus Statistical analyses revealed that the dorsal GCL had significantly more Ki67-expressing cells than the ventral GCL (p < .001; significant region x dorsoventral region interaction (F1,32 = 8.73, p = .005), but no significant differences between the dorsal and ventral hilus on the number of Ki67-expressing cells was seen (p = .62). There was a significant main effect of region but no other significant main effects or interactions (p’s >.26) were found (see Figure 2C). As expected, the hilar volume was significantly greater than the GCL volume (F1,26 = 1199.94, p < .001; see Table 2) but there were no other significant main or interaction effects found (p’s > .53). MPA and MPA+EB decreased while P(1mg), EB, and P+EB increased serum corticosterone concentrations Analysis of corticosterone concentration in serum collected at time of perfusion revealed that the MPA-treated groups (1mg, 4mg, MPA +EB) had significantly lower corticosterone concentrations compared to the oil-treated controls (all p’s< .05) while 1mg P, EB, and P+EB Page 16 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596017 produced significantly higher corticosterone concentrations than oil-treated controls (all p’s < .02; significant main effect of hormone treatment (F7,27=22.08, p < .001; see Figure 3A)). MPA and MPA+EB have lower adrenal/body weight ratios while EB and P+EB treated animals had higher adrenal/body weight ratios All MPA groups (1mg MPA, 4mg MPA, and MPA+EB) had significantly lower adrenal to body weight ratios compared to oil-treated controls (p’s < .001; a significant main effect of hormone treatment (F7, 33  = 32.59, p < .001); see Figure 3B). In contrast, EB and P+EB treated groups had significantly larger adrenal to body weight ratios (all p’s < .04). Not surprisingly, adrenal/body weight ratio was positively correlated with the concentration of serum corticosterone on day of perfusion (r(35) = 0.78, p <.001; see Figure 3C), with larger adrenal/body weight ratio associated with a greater concentration of corticosterone. Serum CORT levels were negatively correlated with ventral dentate gyrus measures of neurogenesis in the EB and P+EB groups.  There were no significant correlations between the number of BrdU-labeled cells, the neurogenesis index (number of BrdU-labeled cells with a neuronal phenotype), and number of Ki67-expressing cells on any of these measures with serum CORT levels (p’s > .08) or adrenal/body weight ratios (p’s > .13) when all groups were considered. Breaking down the correlations by group, we found that serum CORT levels were negatively correlated with ventral Ki67-expressing cells in the hilus (r = -0.96, p = 0.043) in the P+EB group and with BrdU-labeled cells in the ventral GCL (r = -0.89, p = 0.038) in the EB group only. These correlations indicate that higher serum CORT was related to lower cell proliferation and BrdU-labeled cell survival in the ventral dentate gyrus only in P+EB and EB groups, respectively.  However Page 17 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596018 neither of these correlations was significant when controls for multiple correlations were conducted. MPA treatment and EB alone or in combination with P or MPA significantly decreased the percent change in body weight Body weights were taken at days 7, 13, 20, and 29 of hormone administration; therefore percent changes in body weight over days 7-13, 13-20, and 20-29 were calculated: [(weight2 – weight1)/weight1]*100. There was a main effect of hormone treatment (F7,33 = 69.76, p <.001) and day (F2,66 = 3.75, p =.029) on percentage of body weight change. No significant hormone treatment x day interaction was found (p = .47). Post-hoc analyses revealed that rats treated with MPA (1 and 4mg), EB, P+EB, and MPA+EB had significantly less weight gain than oil controls (p’s < .001; see Table 3). Additionally, the body weight changes for the MPA (1mg and 4mg), EB, P+EB, and MPA+EB groups were all significantly different from each other (all p’s < .03).  Discussion Chronic estradiol benzoate reduced BrdU-labeled cell survival, but had no significant effect on cell proliferation, consistent with previous literature in young adult female rats (Barker and Galea, 2008; Tanapat et al, 2005, respectively). Interestingly, while there were no significant effects of MPA or progesterone alone, the combination of MPA with EB significantly reduced the survival of the number of BrdU-labeled cells relative to oil-treated controls, while progesterone partially reversed the EB-induced suppression in BrdU-labeled cell survival. Synthetic and natural progestins differentially affected adrenal/ body weight ratio and serum CORT levels. Specifically, females treated with MPA alone or in combination with EB had significantly reduced adrenal/ body weight ratios, while the opposite effect occurred in the EB Page 18 of 51Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer Review Only19  and P+EB groups. The synthetic progestin, MPA and the natural progestin, progesterone, also had opposing effects on serum CORT concentrations, with MPA decreasing and progesterone, estradiol benzoate, or P+EB increasing CORT concentrations. Thus, MPA alone or co-administered with EB may suppress the HPA axis, as seen in human patients (Malik et al, 1996), while estradiol or progesterone alone or co-administered has the opposite effect. Furthermore serum CORT levels were negatively related to ventral Ki67-expressing cells in the P+EB group and to ventral BrdU-labeled cells in the EB group, indicating that CORT levels were associated with ventral measures of cell proliferation and survival in the EB-treated groups only and not when given with MPA. These results show that different types of progesterone, alone or in combination with EB, differentially regulate adult hippocampal neurogenesis and the HPA axis in female rodents.   21-day administration of EB and MPA+EB, but not MPA alone, significantly reduced neurogenesis in the hippocampus via BrdU-labeled cell survival   In the present study, chronic MPA+EB significantly reduced neurogenesis via a reduction in the number of surviving BrdU-labeled cells in young adult female rats.  However, MPA alone had no significant effect on adult neurogenesis. This finding is somewhat consistent with short-term studies that have shown that MPA by itself had no significant effect on apoptosis in vitro (Nilsen and Brinton, 2002; Singh, 2001), but co-administration of MPA and EB increased apoptosis in young adult female rats (Liu et al, 2010).  Curiously, in short-term studies, MPA counteracted estradiol-induced upregulation of pro-survival proteins such as CREB, Bcl-2, and Akt; however in our study, MPA did not counteract the effects of EB, as EB by itself also reduced the survival of BrdU-labeled cells. Indeed, when we examined the neurogenesis index Page 19 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596020 (BrdU-labeled cells that co-expressed NeuN) only MPA+EB reduced the survival of new neurons, whereas EB alone did not, suggesting that MPA+EB has some different effects than either MPA or EB alone. Possible discrepancies between past findings and the present study may be due to the fact that we used chronic (21 days) treatment with MPA and EB while previous studies examined the effects of MPA and EB over much shorter periods. The finding that chronic MPA+EB reduced neurogenesis suggests that these hormones either alter cell differentiation, downregulate cell proliferation, or increase the apoptosis of adult-born neurons. The first two scenarios are unlikely, as we found no significant differences in the percentage of BrdU/NeuN co-labeled cells, nor did we find any effect of chronic hormone treatment on the number of Ki67-expressing cells. The final option, which seems most likely, may be related to the effects of MPA and estradiol on mitochondrial function (Nilsen and Brinton, 2004). Further, low ovarian hormone conditions induced for two weeks by ovariectomy increase the risk of oxidative stress in neurons (Irwin et al, 2011), which is associated with a reduction in the number of neural progenitor cells in the hippocampus, an inhibition in cell proliferation and apoptosis (Limoli et al, 2006; Lin and Beal, 2006). Acute estradiol may rescue new neurons in ovariectomized young adult animals via upregulation of certain antioxidant proteins, which counteracts oxidative stress (Irwin et al, 2011). When estradiol is administered in conjunction with MPA, however, the antioxidant effects of estradiol are blocked and in some cases, decreased in young adult female rats (Irwin et al, 2011). Therefore, increased oxidative stress induced by ovariectomy combined with a reduction in antioxidant defense could have resulted in a reduction in the number of BrdU-labeled cells via cell survival in the present study. Although the site of action for MPA+EB is still unclear, MPA has a greater affinity for the classical progesterone receptors (PRs) than progesterone (Sitruk-Ware, 2008). However, to Page 20 of 51Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960For Peer Review Only21 date most studies have not found a significant contribution of this receptor for cell survival in vitro or in young adult female rats (Liu et al, 2009, Liu et al, 2010, Bali et al, 2012). However PRs have been implicated in neurogenesis in young adult male mice (Zhang et al, 2010). Although it is currently unknown whether MPA acts on the membrane-bound PGRMC-1,  acute in vitro studies showed that MPA counteracted estradiol’s anti-apoptotic effects by blocking a part of the MAPK/ERK pathway (Nilsen and Brinton, 2002; Nilsen and Brinton, 2003), suggesting that MPA’s apoptotic effects may be due to activation of the non-classical progesterone receptors. Partially contrary to our study, Liu and colleagues (2010) reported acute MPA reduced cell proliferation, but increased apoptosis, in young adult SD female rats in vitro. However, it is important to note that the dose of MPA, time of exposure, and use of in vitro versus in vivo differed between the two studies.  Liu et al. (2010) used a higher dose of MPA than was used in the current study, and the past study used acute rather than chronic exposure to MPA, as in our study. Further studies could examine the time-dependent effects of MPA alone and in combination with estradiol on neurogenesis in the female dentate gyrus. In the present experiment we found that estradiol benzoate decreased neurogenesis when BrdU was given before treatment indicating estradiol decreased survival of new neurons independent of effects on cell proliferation consistent with a past study (Barker and Galea, 2008). However if estradiol is administered prior to BrdU, so that the new cells are produced and survive with estradiol, estradiol serves to increase neurogenesis (McClure et al., 2013). This is likely due to the effects of acute estradiol to increase cell proliferation (Tanapat et al., 2005; Barha et al, 2009). Intriguingly, when new neurons are produced and survive under high estradiol conditions we found a positive correlation between learning and activation of new Page 21 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596022 neurons (McClure et al., 2013). Thus, depending on the timing between BrdU and estradiol treatment, the effects on neurogenesis can be very different. Chronic progesterone alone did not significantly alter neurogenesis in adult females rats but partially reversed EB-induced reduction in BrdU-labeled cell survival In the current study we found that neither dose of progesterone, nor the combination of estradiol benzoate and progesterone, affected cell proliferation (Ki67-expressing cells) compared to controls in adult female rats. Further we found that chronic estradiol alone decreased the survival of BrdU-labeled cells but there was no significant effect on the survival of BrdU-labeled cells seen in the P+EB group compared to controls. These findings taken together with those of Tanapat and colleagues (2005) suggest that both acute and chronic progesterone counteract estradiol’s effects on BrdU-labeled cell survival. The finding that P+EB did not significantly affect cell proliferation is consistent with previous results (Tanapat et al, 2005). The fact that chronic progesterone did not significantly alter neurogenesis differs from previous literature using short-term progesterone treatment in male rodents and hippocampal neural progenitor cells derived from female rats (Liu et al, 2009; Zhang et al, 2010; Bali et al, 2012). Zhang and colleagues (2010) showed that short-term progesterone treatment in male mice increased the survival of BrdU-labeled in the hippocampus but only when treatment was administered 0-2 or 5-7 days after BrdU injection in young adult male mice. However, it is unclear whether a similar timeline exists in female rodents. Liu and colleagues (2009) showed that treating progenitor cells from the dentate gyrus of adult female rats with progesterone, dose-dependently increased cell proliferation via the promotion of cell cycle entry, a mechanism mediated by the membrane-bound receptor PGRMC1. Additionally, the induction of cell cycle entry began after 4 hours of progesterone exposure but stopped at 24 hours, suggesting that Page 22 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596023 progesterone may only stimulate cell proliferation for a short period of time in female rats. Indeed in the current study we found no evidence of 21 d of progesterone to significantly affect cell proliferation. More recently, Bali and colleagues (2012) showed that, in young adult female rats, acute progesterone not only increased the number of BrdU-labeled cells 30 hours later, but also increased the number of immature cells. These differences on the survival of BrdU-labeled cells between acute and chronic treatments of progesterone suggest that the neurogenic effects of progesterone are time-dependent, and that the timing of progesterone’s action in the dentate gyrus may be different in males and females.  As mentioned above, the membrane-bound receptor PGRMC-1 has been implicated as a mechanism for acute exposure of progesterone’s effects on neurogenesis. Liu and colleagues (2009) found that knocking down PGRMC-1 blocked progesterone’s proliferative effects. Bali and colleagues (2012) found that the induction of PGRMC-1 by acute estradiol and progesterone (together or separately) was observed in the CA1, CA3, and the dentate gyrus in adult female rats.  However, short-term estradiol exposure induced PR expression only in the CA1 region, and progesterone administration with or without estradiol induced expression of PR only in the CA1 and CA3 regions, with no effects of either hormone to induce PR in the dentate gyrus. Therefore, the literature suggests that the acute effects of estradiol and progesterone are exerted mainly through PGRMC-1 in the dentate gyrus. Intriguingly, one study using male mice found that the progesterone receptor antagonist RU-486 partially blocked progesterone’s effects on cell proliferation and cell survival (Zhang et al, 2010). Estradiol alone or with progesterone also induced hippocampal PR expression in female, but not male, Wistar rats (Guerra-Araiza et al, 2002), and interestingly, PR expression in the hippocampus did not vary over the estrous cycle in Sprague-Dawley rats (Guerra-Araiza et al, 2003). However, given species (rats vs mice; Snyder Page 23 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596024 et al, 2009), strain (Wistar vs Sprague Dawley; Epp et al, 2010), and sex (Galea et al, in press) differences in neurogenesis, further testing remains to be done to confirm the role of classical progesterone receptors in regulating neurogenesis in female rats. Both progesterone and estradiol can be synthesized in the brain (Naftolin et al, 1996; Schumacher et al, 2004). While Fester and colleagues (2006) showed that cell proliferation in hippocampal slices from 5-day old rats was dependent on local estradiol production, it is unclear how adult neurogenesis is affected by local sources of ovarian hormones. Short-term OVX reduced cell proliferation in the adult female hippocampus (Tanapat et al 1999), while long-term OVX had no significant effects on cell proliferation (Tanapat et al, 1999; Green and Galea, 2008), suggesting a compensatory mechanism that may involve neuroactive steroids. However, there is no evidence for measurable levels of estradiol in the hippocampus of  long-term gonadectomized adult male and female rats (Barker and Galea, 2009), suggesting that neurosteroid production may be limited to younger ages, different areas of the brain, or affected by gonadectomy. Furthermore, as shown by Barker and Galea (2008) and replicated in our current study, chronic estradiol reduced cell survival in females relative to females that were ovariectomized without hormone treatment. Taken together, there is likely an age and sex-dependent effect of local and peripheral estradiol on adult hippocampal neurogenesis. MPA and MPA+EB had different effects on adrenal/body weight ratios and serum CORT levels compared to P, EB, and P+EB Adrenal size is influenced by HPA axis activity and is used as a functional indicator of the HPA axis (Rubin et al, 1995). In the present study, MPA alone and in combination with estradiol benzoate reduced adrenal/body weight ratio, while estradiol benzoate and P+EB Page 24 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596025 produced the opposite effect, which is consistent with previous findings (Malik et al, 1996; Di Carlo et al, 1984; Young et al, 2001). These findings suggest MPA and progesterone differentially interact with estradiol to regulate HPA activity. In the present study, we found that chronic MPA alone or in combination with estradiol decreased, while low progesterone, estradiol alone or in combination with progesterone increased, serum corticosterone levels. This is consistent with previous findings that show an acute high dose of estradiol alone or combined with progesterone reduced mineralocorticoid receptor activity and increased serum corticosterone and adrenocorticotrophic hormone (ACTH) levels (Carey et al, 1995), and a chronic high dose of MPA suppressed adrenal activity in older women (Malik et al, 1996). Additionally, CORT levels fluctuate over the estrous cycle, with basal levels peaking during the proestrous phase (Viau and Meaney, 1991). Progesterone does show a high affinity for mineralocorticoid receptors (Rupprecht et al 1993), which regulates basal HPA activity. It appears likely that there is a dose dependent response of progesterone, as both in the present study and that of Carey and colleagues (1995), the higher dose of progesterone did not significantly increase CORT levels, but in the present study, the 1mg of progesterone did increase CORT levels.  Clearly, estradiol, progesterone, and MPA differentially regulate the HPA axis. Estradiol modulates both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activity and regulates negative feedback (Viau and Meaney, 1991; Carey et al, 1995).  Although progesterone does not appear to influence GR activity (Carey et al, 1995; Young et al, 2001), RU486, a progesterone and glucocorticoid receptor antagonist, disrupts cortisol-mediated negative feedback in men and women (Gaillard et al, 1984; Kling et al, 1993). Taken together, these studies suggest that the absence, rather than presence, of progesterone, regulates GR function. While MPA does bind to GRs (Sitruk-Ware, 2008), its Page 25 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596026 exact effects are unknown. Furthermore, progesterone has high affinity for MRs (Rupprecht et al 1993) and can antagonize estradiol’s downregulation of MR transcription (Patchev and Almeida, 1996; Carey et al, 1995), but at the high dose has no apparent effects on HPA activity in female rats (Carey et al, 1995). MPA, however, has negligible affinity for MRs, and is therefore an unlikely mechanism for any effects it may have on the HPA axis (Africander et al, 2013).  Future experiments should examine how progesterone and MPA’s effects on MRs and GRs might regulate HPA function. Given these hormone treatment effects on HPA function, it is perhaps surprising in the present experiment why cell proliferation was not significantly affected by hormone treatment. In male rats, an increase in CORT via acute or chronic stressors reduces cell proliferation and new neuron survival (Shors et al, 2007; Westenbroek et al, 2004; Hillerer et al, 2013). However in females, elevated CORT or exposure to stress does not always result in a reduction in cell proliferation. For example while chronic social isolation decreased neurogenesis in males, it increased neurogenesis in the hippocampus of female rats (Westenbroek et al, 2004). After exposure to acute stress, male rats showed a rapid suppression in cell proliferation but female rats showed no significant effect on cell proliferation in the hippocampus (Falconer and Galea, 2003). Female rats given 10mg/kg of CORT for 21 days showed no significant changes in cell proliferation in the hippocampus (Brummelte and Galea, 2010). It is not clear in the present experiment how long CORT was altered with hormone treatment as blood was not sampled throughout the experiment.  However, findings from the present study and past findings together suggest that manipulations of HPA activity do not always result in suppressed cell proliferation in the hippocampus of adult female rats. Page 26 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596027 Interestingly, although not statistically significant after correcting for multiple correlations, we found that CORT levels were negatively correlated with cell proliferation (Ki67-expressing cells) in the P+EB group and cell survival (BrdU-labeled cells) in the EB group. More specifically, this correlation was found only in the ventral region of the dentate gyrus, which is consistent with the idea that the ventral hippocampus is associated with the regulation of stress (Fanselow and Dong, 2010).Additionally, progesterone metabolizes to allopregnanolone, which has been implicated in regulating stress and cell proliferation (Bitran et al, 1993). On the other hand, MPA does not metabolize to allopregnanolone (Bernadi et al, 2006), which may explain the difference in mechanism for regulating neurogenesis and basal HPA activity. These results suggests either that neurogenesis is influenced by P+EB and EB via their chronic effects on the HPA axis, or that alterations in neurogenesis regulate the changes to HPA axis functioning. Furthermore, our findings shows that regulation of neurogenesis by MPA or MPA+EB may not be via the HPA axis but mainly through apoptosis, as described earlier. Functional implications of reduced neurogenesis in the hippocampus In the present study we found that both chronic estradiol benzoate and estradiol benzoate with medroxyprogesterone acetate significantly decreased neurogenesis in the hippocampus of adult female rats. While it is tempting to speculate on the effects of reduced neurogenesis in these cases, it is important to keep in mind that a decrease in neurogenesis is not always detrimental, just as an increase is not always beneficial. Depending on the task, ablating neurogenesis could benefit, impair, or have no effect on learning and memory in male rats and mice (Shors et al, 2002; Snyder et al, 2005; Saxe et al, 2006; Saxe et al, 2007). In addition, seizures, traumatic brain injury and Alzheimer’s disease can increase neurogenesis but are conditions that are associated with impaired cognition (reviewed in Bizon et al, 2004). Seizure Page 27 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596028 activity can stimulate neurogenesis in rats, and neurons produced under these conditions are ectopic, show abnormal morphology and overly-excitable (Jessberger et al, 2007a). Indeed seizures result in impaired performance in an object recognition task that was reversed by inhibiting seizure-induced neurogenesis (Jessberger et al, 20007b). Furthermore while both exercise and chemically-induced seizures increase neurogenesis, the environment that a new neuron is born into matters as the electrophysiological properties of new neurons created under these two conditions significantly differed (Jakubs et al., 2006). In adult female rats, while Premarin increased neurogenesis in the dentate gyrus neurogenesis levels were correlated with poorer performance in the radial arm maze (Barha and Galea, 2013). Thus, at least in some instances, enhancing neurogenesis may not be related to better learning and memory. Therefore, our finding that estradiol reduced neurogenesis, and that P+EB reversed this decrease, could have different behavioural implications. Conclusions The present study found that chronic MPA and progesterone differentially interacted with estradiol to alter hippocampal neurogenesis in the female rodent. Previously, it was found that acute progesterone increased hippocampal neurogenesis in male rodents (Zhang et al, 2010) and neural progenitor cells from female rats (Liu et al, 2009).  However, we found that chronic progesterone had no significant effects on cell proliferation or cell survival. This disparity shows that the effects of progesterone may either be time-dependent or sex-specific. Interestingly, MPA and progesterone alone or together with estradiol had differential effects on the HPA axis, as quantified by adrenal/body weight ratios and serum corticosterone levels. The mechanism for these effects may be a point of interest for further investigation. This study highlights the different effects of synthetic and natural hormone formulations in the female brain and that their Page 28 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596029 effects may be similar or dissimilar depending on the tissue of interest (uterus versus brain and adrenals). It is important to understand the implications of synthetic formulations of ovarian hormones available in oral contraceptives and hormonal replacement therapies to further our knowledge of brain health in women. Page 29 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596030 Acknowledgements We would like to thank Alice Chan, Anne Cheng, and Lucille Hoover for their assistance with this work. 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Jakubs K, Nanobashvili A, Bonde S, Ekdahl CT, Kokaia Z, Kokaia M, Lindvall O.Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability. Neuron 2006; 52: 1047-1059. Page 42 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596044 Table 1: Mean (±SEM) percentage of cells co-expressing BrdU and NeuN in the GCL after 21 days of hormone treatment Hormone Treatment BrdU/NeuN labeled cells (%) Oil 80.21±2.08 1mg P 79.17±2.40 4mg P 83.33±2.40 1mg MPA 79.17±0.00 4mg MPA 80.83±1.02 EB 80.00±2.43 P+EB 82.64±1.90 MPA+EB 78.13±1.99 No significant differences in percentage of BrdU/NeuN co-labeled cells were found between groups.   Page 44 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596045 Table 2: Mean (±SEM) total volume of GCL in female rats following 21 days of hormone treatment Volume (mm3)Hormone Treatment GCL Hilus Oil 1.14 + .09 mm33.02 + .24 mm31mg P 1.12 + .09 mm33.06 + .21 mm34mg P 1mg MPA 1.26 + .04 mm3 1.13 + .03 mm33.41 + .13 mm3 3.03 + .09 mm34mg MPA 1.28 + .05 mm33.02 + .11 mm3EB 1.13 + .12 mm33.08 + .39 mm3P+EB 1.20 + .06 mm3 3.26 + .18 mm3MPA+EB 1.13 + .02 mm32.73 + .08 mm3No significant differences in GCL or hilus volumes were found between groups. Page 45 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596046 Table 3: Mean (+ SEM) percent change in body weight in female rats between days 7-13, days 13-20, and days 20-29.  Percent change in body weight (%) Hormone Treatment Days 7-13 Days 13-20 Days 20-29 Oil 6.56 + 0.85 8.70 + 0.58 5.29 + 0.74 1mg P 6.79 + 0.55 8.37 + 0.37 4.05 + 0.60 4mg P 4.69 + 4.04 11.86 + 4.72 4.51 + 1.00 1mg MPA 1.42 + 1.20 6.18 + 0.57 4.10 + 0.87 4mg MPA 5.19 + 2.31 0.81 + 2.72 0.89 + 0.52 EB -2.13 + 1.48 -0.05 + 1.18 1.09 + 1.02 P+EB -1.44 + 1.42 2.16 + 1.25 1.65 + 0.92 MPA+EB -4.80 + 2.48 0.54 + 2.88 -1.55 + 0.38 Animals treated with MPA (1 and 4mg), EB, P+EB, and MPA+EB had significantly less weight gain than oil controls (p’s < .001). Page 46 of 51Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596047 Figure captions Figure 1. A. Experimental timeline. B. BrdU-labeled cell and C. Ki67-expressing cells in the GCL of the dentate gyrus viewed at 1000x magnification under a light microscope. Scale bar = 20µm. Arrows point to individual cells. D. Fluorescent labeling of cells labeled with NeuN (green) and BrdU (red). A merged image depicts co-labeling of BrdU and NeuN. Cells viewed at 630x magnification under a confocal microscope. E. Picture of the anatomical regions that were analyzed, viewed at 40x magnification under a light microscope. The granule cell layer (GCL), subgranular zone (SGZ), and the hilus are depicted. Figure 2. (A) Mean number of BrdU-labeled cells in the granule cell layer and hilus. There were significantly reduced numbers of BrdU-labeled cells in the dorsal GCL for females chronically treated with EB and MPA+EB (* indicates p = .019). (B) Mean number of BrdU-labeled cells with a neuronal phenotype (the neurogenesis index, BrdU*percentage of BrdU/NeuN cells) for all treatment groups. MPA+EB significantly reduced the number of new neurons compared to controls (* indicates p = .005). No significant differences were found in the hilus. Error bars represent + SEM.   (C) Mean number of Ki67-expressing cells in the GCL+SGZ and hilus. There were no significant differences between any groups in the number of Ki67-expressing cells and treatment. Error bars represent + SEM. Figure 3. (A) Mean concentrations of serum corticosterone on perfusion day for each treatment group. Compared to oil-treated controls, animals treated with MPA (1mg or 4mg) and MPA+EB had significantly lower levels of serum corticosterone, whereas treatment with 1mg P, EB, and P+EB significantly increased serum corticosterone concentrations on perfusion day. (B) Mean adrenal/body weight ratios for each treatment group. Treatment with MPA (1mg or 4mg) and MPA+EB produced significantly lower adrenal/body weight ratios, while EB and P+EB treated Page 47 of 51 Journal of Neuroendocrinology12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596048 animals had significantly higher adrenal/body weight ratios (* indicates p < .05 compared to oil-treated controls). Higher adrenal/ body weight ratios were correlated with greater serum levels of corticosterone (r(35) = 0.78, p <.001). Error bars represent + SEM. Page 48 of 51Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960C D A B E Page 49 of 51 Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Treatmentoil 1 P 4 P 1 MPA 4 MPA EB P+EB MPA+EBNumber of BrdU/NeuN co-labeled cells010002000300040005000RegionGCL HilusKi67-expressing cells020004000600080001000012000oil 1mg P 4mg P 1mg MPA 4mg MPA EB P+EB MPA+EB * A B C Page 50 of 51Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Treatmentoil 1 P 4 P 1 MPA 4 MPA EB P+EB MPA+EBAdrenal weight/Body weight0.000000.000050.000100.000150.000200.000250.00030Treatmentoil 1 P 4 P 1 MPA 4 MPA EB P+EB MPA+EBCorticosterone concentration050100150200250300Adrenal weight/body weight0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 0.00030Corticosterone concentration-1000100200300400*A B C r(35) = 0.78, p < 0.001 ** *** * ***Page 51 of 51 Journal of Neuroendocrinology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


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