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Ovarian hormones, but not fluoxetine, impart resilience within a chronic unpredictable stress model in… Mahmoud, Rand; Wainwright, Steven R.; Chaiton, Jessica A.; Lieblich, Stephanie E.; Galea, Liisa A.M. Aug 31, 2016

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1  Published in: Mahmoud R, Wainwright SR, Chaiton J, Lieblich SE, Galea LAM. (2016). Ovarian 1 hormones impart resilience against chronic unpredictable stress and modulate the effects of 2 fluoxetine on hippocampal plasticity in middle-aged female rats. Neuropharmacology, 107:278-293 3  4 Ovarian hormones, but not fluoxetine, impart resilience within a chronic unpredictable 5 stress model in middle-aged female rats   6 Rand Mahmouda,c, Steven R. Wainwrighta,c, Jessica A. Chaitonb, Stephanie E. Lieblichb, and 7 Liisa A.M. Galeab,c   8 a Graduate Program in Neuroscience, b Department of Psychology, c Centre for Brain Health, 9 University of British Columbia, Vancouver Canada  10   11  12  13  14  15 Corresponding author: 16 Dr. Liisa Galea 17 Department of Psychology 18 University of British Columbia 19 2136 West Mall 20 Vancouver, BC  21 Canada, V6T 1Z4  22 Tel: +1 (604) 822 6536  23 Fax: +1 (604) 822 6923  24 Email: lgalea@psych.ubc.ca 25 Keywords: long-term ovariectomy, chronic unpredictable stress, depressive-like behavior, 26 neurogenesis, antidepressant-efficacy  27  28  29  30  31  32  33 2   34  35  36 Highlights 37  Long-term ovariectomy (OVX) increased depressive like-behavior in chronically stressed rats 38  Long-term OVX altered basal and stress corticosterone levels in chronically stressed rats 39  Fluoxetine increased neurogenesis and PSA-NCAM in the ventral hippocampus in an ovarian 40 status dependent manner 41  Fluoxetine reduced microglial number in the ventral hippocampus of chronically stressed rats 42  43  44  45  46  47  48  49  50  51  52  53  54 3   55  56  57 ABSTRACT  58 Depression is more prevalent in women than in men, and women are at a heightened risk for 59 depression during the postpartum and perimenopause. There is also evidence to suggest that the 60 ovarian hormone milieu may dictate antidepressant efficacy. Thus, it is important to investigate 61 the role of ovarian hormones in the pathogenesis of depression and in the mechanisms that may 62 underlie antidepressant efficacy. In the present study, we used 10-month-old female Sprague-63 Dawley rats to examine the effects of long-term ovarian hormone deprivation on the 64 development of depressive-like endophenotypes after chronic stress, and on antidepressant 65 efficacy. Four months following ovariectomy (OVX) or sham surgery, all rats were subjected to 66 6 weeks of chronic unpredictable stress (CUS). During the last 3 weeks of CUS, rats received 67 daily injections of fluoxetine (5 mg/kg) or vehicle. All rats were assessed on measures of 68 anxiety- and depressive-like behavior, hypothalamic-pituitary-adrenal (HPA) negative feedback 69 inhibition, and on markers of neurogenesis and microglia in the dentate gyrus. Our findings 70 demonstrate that long-term ovarian hormone deprivation increased anxiety and depressive-like 71 behavior, as seen by increased immobility in the forced swim test and latency to feed in the 72 novelty suppressed feeding test, and decreased sucrose preference. Further, long-term OVX 73 resulted in impaired HPA negative feedback inhibition, as seen in the dexamethasone 74 suppression test. Fluoxetine treatment showed limited behavioral and neuroendocrine efficacy, 75 however it reduced microglial (Iba-1) expression and increased cell proliferation, neurogenesis 76 (via cell survival) and the expression of the polysialylated neuronal cell adhesion molecule 77 (PSA-NCAM) in the dentate gyrus, and those effects varied by region (dorsal, ventral) and 78 ovarian status. Taken together, our findings demonstrate that ovarian hormones may impart 79 resilience against the behavioral and neuroendocrine consequences of chronic unpredictable 80 stress, and may modulate the effects of fluoxetine on cell proliferation, neurogenesis, and PSA-81 NCAM in the middle-aged female.   82  83 4   84  85  86 1. INTRODUCTION  87 Women are more than twice as likely as men to develop depression (Gutiérrez-Lobos et al., 88 2002);  a disparity that is particularly striking during the reproductive years of women (i.e. 25-50 89 years; (Gutiérrez-Lobos et al., 2002). Notably, periods that involve dramatic fluctuations and/or 90 reductions in ovarian hormones, such as the postpartum and perimenopause, carry the highest 91 risk of developing depression in women (Cohen et al., 2006; Hendrick et al., 1998; Soares, 92 2014). Together, these data provide compelling evidence for a role of ovarian hormones in the 93 pathoetiology of depression.       94 Indeed, in healthy women reductions in ovarian hormones triggered sub-clinical 95 depression scores, and estradiol levels were negatively associated with depressive symptoms 96 (Frokjaer et al., 2015).  In addition, withdrawal from a hormone simulated pregnancy in women 97 with a history of postpartum depression increased depressive symptoms relative to women 98 without a history of postpartum depression (Bloch et al, 2000). Thus, ovarian hormone 99 fluctuations contribute to the development of depressive symptoms in women. Similarly in rats, 100 estradiol withdrawal after hormone simulated pregnancy leads to depressive-like phenotypes 101 (Galea et al., 2001; Green et al., 2009; Green and Galea, 2008). Importantly, in this model, 102 treatment with estradiol or an estrogen receptor (ER) β agonist prevented the development of 103 depressive-like behaviors (Galea et al., 2001; Green et al., 2009). Ovariectomy itself can increase 104 depressive and anxiety-like behaviors while treatment with estradiol or selective estrogen 105 receptor modulators (SERMs) can restore this effect (Bekku and Yoshimura, 2005; Li et al., 106 2014; Okada et al., 1997; Walf et al., 2004). Taken together, these findings corroborate the 107 notion that reductions in ovarian hormones may increase the risk for developing a depressive-108 like endophenotype. However, most of these studies using females did not utilize a model of 109 depression, and thus the face and construct validity of such studies is uncertain. The present 110 5  study aimed to fill this gap by investigating the role of ovarian hormones in the development of a 111 depressive-like endophenotype within an animal model of depression. 112 Ovarian hormones are also implicated in antidepressant efficacy (Thase et al., 2005). For 113 example, in postmenopausal women with depression, antidepressant treatment results in superior 114 outcomes when prescribed alongside hormone therapy than when given alone (Thase et al., 115 2005), suggesting that ovarian hormones may enhance antidepressant efficacy. Furthermore, in 116 women diagnosed with depression, estradiol has been given as either an adjunct therapy or a 117 standalone antidepressant, with some success (Ahokas et al., 2001; Moses-Kolko et al., 2009; 118 Rasgon et al., 2007). Similarly 17β-estradiol treatment in ovariectomized rats augments the 119 effects of the SSRI, sertraline, to reduce immobility in the forced swim test (Sell et al., 2008). 120 However, this latter study was not conducted in conjunction with an animal model of depression, 121 and thus it is unclear whether similar findings would be obtained from animals with a 122 depressive-like phenotype. Our current study aimed to determine whether ovarian hormones 123 influence antidepressant efficacy in an animal model of depression in middle-aged rodents.  124 The hippocampus exhibits compromised structural plasticity in depressed patients, as a 125 meta-analysis showed that untreated depression is associated with reduced hippocampal volume, 126 which is seen two years after diagnosis (McKinnon et al., 2009). Decreased hippocampal 127 neurogenesis is observed in post-mortem tissue of depressed individuals (Boldrini et al., 2012) 128 and in animal models of depression (Bessa et al., 2008; Green and Galea, 2008; Wainwright et 129 al., 2011). Conversely, antidepressant treatment restores the reductions in neurogenesis in 130 clinical depression and animal models (Bessa et al., 2008; Boldrini et al., 2012; Green and Galea, 131 2008). Notably, antidepressant use results in a greater increase in hippocampal volume in 132 depressed women than depressed men (Vakili et al., 2000), and the pro-neurogenic effects of 133 antidepressants are seen in the post-mortem tissue of women, but not men (Epp et al., 2013). 134 Thus, ovarian hormones may modulate the effects of antidepressants on neurogenesis in the 135 hippocampus.  136 There is also a growing recognition for the role of inflammation in depression and 137 chronic stress (Miller et al., 2009). Remarkably, microglial activation is increased in the 138 prefrontal cortex, insula and anterior cingulate cortex by approximately 30% in depressed 139 patients relative to controls (Setiawan et al., 2015). Increased inflammation and microglial 140 6  activation in the hippocampus are also evident in animal models of chronic stress (Frank et al., 141 2007; Kreisel et al., 2013a). Despite the well-established anti-inflammatory effects of ovarian 142 hormones in a variety of disease models (Habib and Beyer, 2015) for review), to our knowledge, 143 the influence of ovarian hormones on microglia in relation to chronic stress or SSRI exposure 144 has not been investigated, and thus the current study aimed to fill this gap. 145 Disturbances in the hypothalamic-pituitary-adrenal (HPA) axis are arguably the best 146 characterized endocrine markers of depression. Specifically, a meta-analysis showed that 147 depressed individuals display elevated levels of serum cortisol (Stetler and Miller, 2011), and at 148 least a subset of depressed individuals show impairments in the HPA negative feedback system 149 (Ising et al., 2007; Stetler and Miller, 2011). Antidepressant efficacy is more tightly linked to the 150 restoration of HPA function in women compared to men (Binder et al., 2009) and hypogonadal 151 women have impaired HPA negative feedback (Maes et al., 1992; Young et al., 1993). 152 Therefore, similar to the effects of ovarian hormones on the behavioral efficacy of 153 antidepressants, ovarian hormones may potentiate the efficacy of antidepressants to restore HPA 154 dysfunction.  155 In this study, we investigated whether ovarian hormones impart resilience against the 156 behavioral and HPA outcomes of chronic unpredictable stress in middle-aged female rats, and 157 whether ovarian hormones influence antidepressant efficacy using fluoxetine under stress. 158 Depressive-like phenotype was examined at the behavioral (forced swim test, novelty suppressed 159 feeding test, and sucrose preference test), endocrine (cortisol levels and HPA negative feedback 160 inhibition) and neural levels; examining both neuroplasticity (neurogenesis, PSA-NCAM), and 161 microglial expression in the dentate gyrus. We hypothesized that ovarian hormones would be 162 associated with resilience to the development of a depressive phenotype under chronic 163 unpredictable stress, and that antidepressant efficacy would be modulated by ovarian hormone 164 status in middle-aged female rats. 165 2. MATERIALS AND METHODS  166 2.1. Animals 167 Forty female Sprague-Dawley rats (Charles River Laboratories, Montreal, Canada), weighing 168 200–250g, were used in this study. Animals were pair-housed in a female-only colony room and 169 7  given ad-libitum access to food (Purina rat chow) and water. Colony rooms were temperature 170 and humidity controlled (21 ± 1°C; 50 ± 10%, respectively), and maintained on a 12-hour 171 light/dark cycle (lights on at 07:00). All procedures were approved by the Animal Care 172 Committee at the University of British Columbia, and performed in agreement with ethical 173 guidelines set by the Canadian Council on Animal Care. All efforts were made to reduce the 174 number of animals used and to minimize their suffering.  175 2.2. Surgery   176 Animals were randomly assigned to receive bilateral ovariectomy (OVX, n=20) or sham surgery 177 (Sham, n=20), which were performed at approximately five months of age, as we have done 178 previously (Barha et al., 2010). Ovarian hormone deprivation by ovariectomy was chosen as a 179 model of surgical menopause (Barha and Galea, 2013). Surgeries were performed under 180 isoflurane anesthesia, with ketamine (30mg/kg, Bimeda-MTC, Cambridge, ON), xylazine 181 (2mg/kg, Bayer HealthCare, Toronto, ON) and bupivacaine (applied locally; 4mg/kg, Hospira 182 Healthcare Corporation, Montreal, QC). Following recovery, animals were handled only during 183 cage cleaning procedures, and left otherwise undisturbed until the beginning of the chronic 184 unpredictable stress (CUS) procedure at approximately 9 months of age.   185 2.3. BrdU administration 186 To label dividing progenitor cells in the dentate gyrus and their progeny, all rats received two 187 intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU; 200mg/kg; Sigma–Aldrich, St. 188 Louis, MO, USA), given 8 hours apart, one day prior to the initiation of CUS. Two BrdU 189 injections were chosen due to the lower rates of cell proliferation in the hippocampus in middle-190 aged rodents (Kuhn et al., 1996; Rao et al., 2005). Further, the timing of BrdU administration 191 was chosen to delineate the effects of CUS (and subsequently antidepressant treatment) on the 192 survival of newly produced cells, independent of treatment effects on cell proliferation. 193 2.4. Chronic Unpredictable Stress (CUS)  194 A CUS paradigm was used as we have done previously (Wainwright et al., 2011), with some 195 modifications. Briefly, all animals were exposed to a variety of stressors, applied twice daily in a 196 semi-random order at unpredictable times for a duration of 6 weeks. We allowed for at least 2 197 hours between the two daily stressors. The stressors and their descriptions are listed in Table 1.   198 8  2.5. Drug preparation and treatment 199 The first 3 weeks of CUS were utilized to develop a depressive-like phenotype. During the last 3 200 weeks of CUS (timeline depicted in Figure 1), all animals received daily subcutaneous injections 201 of 5mg/kg Fluoxetine hydrochloride (Sequoia Research Products Ltd, Pangbourne, UK) or 202 vehicle (Veh). Fluoxetine was dissolved in 5% DMSO (Sigma–Aldrich, St. Louis, MO, USA) in 203 0.9% saline, and given at a dose of 5mg/kg. Vehicle treatment consisted of 5% DMSO in 0.9% 204 saline. Thus, we obtained four treatment groups (n=10 each), with drug and ovarian status as the 205 between-subject variables: (i) vehicle-treated sham-operated rats (Veh-Sham); (ii) fluoxetine-206 treated sham-operated rats (FLX-Sham); (iii) vehicle-treated ovariectomized rats (Veh-OVX); 207 and (iv) fluoxetine-treated ovariectomized rats (FLX-OVX). An intermediate dose of fluoxetine 208 at 5mg/kg was chosen to in order to avoid a possible ceiling effect, which may mask potential 209 differences between ovarian status groups in the response to treatment. This rationale was based 210 on prior work in ovariectomized middle-aged female rats showing that while 10mg/kg FLX 211 decreased immobility in the forced swim test without hormone replacement, a low dose of 212 1.5mg/kg FLX decreased immobility only when administered in conjunction with estradiol 213 replacement (Récamier-Carballo et al., 2012).214  215 Figure 1. (A) Timeline depicting the sequence of experimental events. Sham surgery or bilateral 216 ovariectomy (OVX) were performed at 5 months of age. Following a four month delay, rats were 217 subjected to 6 weeks of chronic unpredictable stress (CUS; 24 hours after bromodeoxyuridine 218 (BrdU) administration). Fluoxetine (FLX) or vehicle (Veh) treatment was administered daily for 219 the final 3 weeks of CUS. Behavioral and neuroendocrine testing were initited 24 hours after the 220 final day of CUS, in the order shown (one test per day), with the exception of the sucrose 221 preference test, which was administered weekly througout CUS (not shown). Ninety minutes 222 after the second session of FST, the animals were perfused and tissue was collected. D indicates 223 day of experiment, beginning at BrdU administration on D0. NSF= novelty suppressed feding, 224 DEX= dexamethasone suppression test. 225 2.6. Behavioral testing 226 4 months Surgery  DEX CUS FLX/Veh D0        D1                                   D22                                  D41         D42       D43       D44      D45              BrdU   FST #1 FST #2,  Perfusion  NSF 9  Behavioral testing occurred following the 6 weeks of CUS, with the exception of the sucrose 227 preference test, which was administered weekly (see Figure 1).   228 2.6.1. Sucrose preference test. Rats were acclimatized to the 1% sucrose solution and the two-229 bottle procedure by introducing two identical bottles to their home cage for 24 hours, each 230 containing 1% sucrose or tap water. After acclimatization, sucrose preference test was 231 administered once prior to CUS to obtain a baseline measure of sucrose preference, and weekly 232 thereafter until the termination of CUS (last test was on the last day of CUS). Briefly, the rats 233 were singly-housed and simultaneously deprived of food and water for 4 hours, then presented 234 with two bottles, one containing 1% sucrose and the other tap water. The test lasted 1 hour 235 beginning at the start of the dark-phase (19:00-20:00hrs), after which the rats were re-paired with 236 cage mates. All bottles were weighed before and after the test, and sucrose preference was 237 calculated using the formula: sucrose preference = (sucrose consumed/(sucrose + water 238 consumed)) x 100. The right-left placement of the sucrose and water bottles were counter-239 balanced for all animals between test days.  240 2.6.2. Novelty-Suppressed Feeding Test (NSF). The apparatus consisted of an open arena (60 x 241 60 x 50 cm) in which three pellets of a palatable food (Kellogg’s Froot Loops) were placed in the 242 center. Prior to the test, and to avoid neophobic reactions, the rats were acclimatized to the food 243 by introducing four Froot Loops pieces to the home cage for three consecutive days. On test day, 244 the food-deprived rats (18 hours) were individually placed in the arena, at a consistent corner of 245 the chamber, and the latency to start feeding was measured. The rats were then immediately 246 returned to the home cage, and the amount of Purina rat chow (in grams) consumed within the 247 first hour was measured. The NSF arena was thoroughly cleaned with 70% EtOH between 248 animals.    249 2.6.3. Forced Swim Test (FST). FST was conducted as we previously described (Wainwright et 250 al., 2011). Briefly, each rat was subjected to two FST sessions in a vertical glass cylinder (45 x 251 28cm) filled with clean water (25 ± 0.5 °C) to a depth of 30 cm. Rats were individually placed in 252 the cylinder for 15 minutes in the first session, and 5 minutes in the second session, which 253 occurred 24 hours later. The test was videotaped and behavior was subsequently scored using the 254 BEST Collection Software (Educational Consulting, Hobe Sound, FL, USA), by an observer 255 blinded to treatment condition. Time spent in each of three distinct behaviors was scored: (1) 256 10  Immobility- floating with only those movements necessary to maintain the head above water; (2) 257 Swimming- active paddling movements of forelimbs and/or hindlimbs; and (3) Struggling- 258 vigorous climbing-like movements, with forelimbs surfacing above the water.   259 2.7. Blood sampling for basal corticosterone quantification 260 To assess changes in basal corticosterone levels throughout CUS, blood samples were collected 261 every 10 days via the tail-vein, wherein the first sample was collected prior to the first stressor on 262 day 1 of CUS. The procedure was consistently performed between 07:00-07:45hrs, and 263 completed within 2 minutes of touching the home cage to avoid acute stress-induced increases in 264 corticosterone. Blood samples were allowed to clot over night at 4°C, then centrifuged at 10 g for 265 15 minutes and serum aliquots were stored at -20°C until processing.   266 2.8. Dexamethasone Suppression Test  267 To evaluate the integrity of the glucocorticoid-dependent negative-feedback function of the HPA 268 axis, the Dexamethasone suppression test (DEX) was administered 48 hours after the termination 269 of CUS (see Figure 1). DEX was administered between 8:00-10:30hrs to all rats in order to avoid 270 major circadian fluctuations in corticosterone. In short, 3 hours following the administration of 271 Dexamethasone (in propylene glycol; i.p.; 100 ug/kg; Sigma) , blood was collected via the tail 272 vein immediately before and after 30 minutes of restraint stress, and again 30 minutes after the 273 termination of restraint (60 minutes after the first collection). Rats were left undisturbed in their 274 home cages during recovery from restraint. Blood samples were allowed to clot over night at 275 4°C, then centrifuged at 10 g for 15 minutes and serum aliquots were stored at -20°C until 276 processing.   277 2.9. Estrous cycle phase determination   278 In order to account for potential effects of estrous cycle phase on behavior and cell proliferation, 279 vaginal cells were collected by lavage on experimental days 42-45 (Figure 1), estrous cycle 280 phase was determined as previously described (Brummelte and Galea, 2010). Given the animals 281 were in middle age at the beginning of CUS, the analysis of vaginal cells was also intended to 282 determine if any rats in the sham group had reached persistent anestrous, however all rats were 283 found to be cycling.  284 2.10. Tissue collection  285 11  Ninety minutes following session two of the FST, animals were deeply anesthetized with sodium 286 pentobarbital and blood was collected via cardiac puncture. Animals were then transcardially 287 perfused with cold 0.9% saline followed by 4% paraformaldehyde (PFA, Sigma–Aldrich) in 288 0.1M phosphate buffer (PB). Brains were extracted immediately and post-fixed in 4% PFA for 289 24 hours, then transferred to 30% sucrose solution in 0.1M PB (pH 7.4) and stored at 4 °C until 290 sectioning. Brains were sliced in 40μm coronal sections using a Leica SM2000R microtome 291 (Richmond Hill, ON, Canada), and were stored at -20 °C in a cryoprotective medium (0.1M 292 PBS, 30% ethylene glycol and 20% glycerol; Sigma) until processing. Adrenals, thymus and 293 ovaries (in sham-operated rats) were also extracted and weighed.  Blood samples were allowed 294 to clot over night at 4°C, then centrifuged at 10 g for 15 minutes and serum aliquots were stored 295 at -20°C until processing.   296 2.11. Immunohistochemistry   297 All staining was performed on free-floating brain sections, and unless otherwise specified, 298 incubation periods were conducted at room temperature and on a rotator.  299 2.11.1. BrdU. Sections were rinsed 3x10 minutes with 0.1M Tris-phosphate buffer (TBS; pH 300 7.4) between each of the following steps. After a 30 min incubation in 0.6% hydrogen peroxide 301 (H2O2), sections were transferred to 2N HCL for 30 minute at 37 °C to denature DNA. Sections 302 were then incubated in 0.1M Borate buffer for 10 min then blocked in TBS+ (containing 0.1% 303 Triton X and 3% normal donkey serum (NDS)) for 30 minute. Sections were then transferred to 304 the primary antibody solution (1:200 mouse monoclonal antibody against BrdU, Roche 305 Diagnostics, Laval, Quebec, Canada) in TBS+ at 4 °C for 20 hours, and then the secondary 306 antibody solution for 4 hours (1:200 anti-mouse IgG biotinylated, Vector Laboratories, 307 Burlington, Ontario, Canada) in TBS+. Next, tissue was incubated in avidin-biotin complex for 308 1.5 hours (ABC; Elite kit; 1:50; Vector Laboratories). Immunoreactants were visualized by a 309 peroxidase-diaminobenzidine (DAB) reaction (Vector Laboratories) for 7 minutes. Finally, 310 sections were mounted on glass slides and allowed to dry, then counterstained with cresyl violet, 311 dehydrated in increasing graded ethanol, defatted with xylenes, and cover-slipped with 312 Permount.  313  2.11.2. Ki67. Sections were rinsed 3x10 minutes with 0.1M phosphate buffered saline (PBS; pH 314 7.4) between each of the following steps. After a 25 minutes incubation in 0.3% H2O2, sections 315 12  were transferred to a primary antibody solution for 16 hours, containing 1:1000 Rabbit anti-Ki67 316 (Vector Laboratories) monoclonal antibody solution in 0.1M PBS, 1% Normal Goat Serum 317 (NGS, Vector Laboratories), and 0.5% Triton X. Afterwards, sections were incubated in a 318 secondary antibody solution for 1 hour, containing 1:200 biotinylated Goat anti-rabbit IgG 319 (Vector Laboratories, in 0.1M PBS), then in ABC (Vector Laboratories) for 40 min. Finally 320 immunoreactivity was visualized with a 5 minutes DAB reaction (Vector Laboratories) and 321 sections were mounted on glass slides and allowed to dry. Tissue was dehydrated in increasing 322 graded ethanol, defatted with xylenes, and cover-slipped with Permount.  323 2.11.3. BrdU/NeuN. Sections were rinsed 3x10 minutes with 0.1M PBS (pH 7.4) between each 324 of the following steps. Sections were incubated for 24 hours at 4 °C in a primary antibody 325 solution containing 1:250 mouse anti-NeuN (Chemicon/Millipore), 3% Triton-X + 3% Normal 326 donkey Serum (NDS) in 0.1M PBS. Tissue was then transferred to a secondary antibody solution 327 for 18 hours at 4°C containing 1:200 donkey anti-mouse Alexa 488 (Invitrogen, Eugene, OR) in 328 0.1M PBS. Next, sections were incubated in 4% PFA for 10 minutes, then washed 2x10 minutes 329 in 0.9% NaCl. To denature DNA, sections were subsequently incubated in 2N HCl at 37 °C for 330 30 minutes. Sections were then transferred to a primary antibody solution for 24 hours at 4°C, 331 containing 1:500 Rat anti-BrdU (AbD Serotech, Oxford, UK) in 0.1M PBS + 3%NDS + 3% 332 Triton-x. Next, sections were incubated in 1:500 Donkey anti-rat Cy3 (Jackson 333 ImmunoResearch, Westgrove, PA) in 0.1M PBS for 24 hours at 4°C. Sections were mounted on 334 glass slides and coverslipped with PVA-DABCO. 335 2.11.4. PSA-NCAM. Sections were rinsed 5x10 min with 0.1M TBS (pH 7.4) between each of 336 the following steps. Sections were incubated in 0.3% H2O2 for 20 minutes, then transferred to a 337 primary antibody solution at 4°C for 48 hours, containing 1:1000 mouse anti-PSA-NCAM 338 (chemicon/Millipore) in 0.1M PBS + 0.3% Triton-X + 3% Normal horse Serum (NHS). Next, 339 sections were incubated in a secondary antibody solution containing 1:250 horse anti-mouse IgG 340 biotinylated (Vector) in 0.1M PBS for 24 hours at 4°C. Sections were then incubated in ABC for 341 2 hours, and immunoreactivity was visualized with a 5 minute DAB reaction (Vector 342 Laboratories). Sections were finally mounted on glass slides and allowed to dry, then dehydrated 343 in increasing graded ethanol, defatted with xylenes, and cover-slipped with Permount.  344 13  2.11.5. Iba-1. Sections were rinsed 3x10 minutes with 0.1M PBS (pH 7.4) between each of the 345 following steps. Following a 25 minute incubation in 0.3% H2O2, sections were blocked for 1 346 hour with 10% normal goat serum (NGS) in 0.5% Triton X in 0.1 M PBS. Next, sections were 347 incubated in a primary antibody solution for 18 hours, containing 1:1000 anti-Iba-1 (Wako, 348 Osaka, Japan) in 10% NGS and 0.4% Triton X in 0.1M PBS. Afterwards, sections were 349 transferred to a secondary antibody solution of 1:500 biotinylated anti-rabbit (Vector 350 Laboratories) in 0.4% Triton-X and 2.5% NGS in PBS. Then, sections were incubated in ABC 351 (Vector Laboratories) in 0.4% Triton-X in PBS for 1 hour, and immunoreactants were visualized 352 by a Ni-DAB reaction (Vector Laboratories) for 7 minutes. Finally, sections were mounted on 353 glass slides and allowed to dry, then counterstained with cresyl violet, dehydrated in increasing 354 graded ethanol, defatted with xylenes, and cover-slipped with Permount.  355 2.12. Microscopy and cell counting   356 An experimenter blinded to treatment condition completed all immunohistochemical 357 quantification under a Nikon E600 microscope equipped with epifluoresence. Representative 358 photomicrographs are depicted in Figure 2. BrdU- and Ki67-immunoreactive (ir) cells were 359 counted under 1000x magnification in every 10th section of the hippocampus along the rostral-360 caudal axis. To obtain an estimate of total immunoreactive cells in the dentate gyrus, raw 361 numbers were multiplied by 10 (Hamson et al., 2013). BrdU- and Ki67-ir cells in the hilus were 362 counted separately from those in the granule cell layer (GCL) and subgranular zone (SGZ, band 363 approximately 50μm between the GCL and hilus). Counts were segregated in this way as cells in 364 the hilus are considered ectopic and give rise to a distinct population of cells (Cameron et al., 365 1993). Furthermore, cells were quantified separately in the dorsal and ventral regions of the 366 hippocampus, according to previously established coordinates (Banasr et al., 2004). The dorsal 367 hippocampus is thought to be functionally distinct from the ventral hippocampus with the former 368 being more important for spatial learning, and the latter being more important for stress and 369 affect (Fanselow and Dong, 2010). To determine the proportion of BrdU-ir cells of neuronal 370 phenotype, BrdU-ir cells in every 10th section of the hippocampus were examined for co-371 expression of NeuN under 400x magnification. An exhaustive count of PSA-NCAM and Iba-1-ir 372 cells was completed in four hippocampal sections per animal, two sections each from the dorsal 373 and ventral regions of the hippocampus (Kondratiuk et al., 2015), with approximately the 374 following respective coordinates: (Bregma -3.12, -3.48, 6.00, and -6.36).  PSA-NCAM-ir cells 375 14  were counted in the GCL only, whereas Iba-1-ir cells were counted (separately) in the 376 GCL+SGZ and within an approximately 50μm band of the molecular layer (ML). Dentate gyrus 377 areas (GCL and Hilus) were measured using digitized images of the sections and the software 378 ImageJ (NIH).  379 2.13. Morphological analysis of Iba-1 immunoreactive cells  380 An experimenter blinded to treatment condition performed morphological analysis of Iba-1 381 immunoreactive cells in the GCL+SGZ. Specifically, using the measure feature of the NIS-382 Elements Basic Research software (Nikon) and under a Nikon E600 microscope, the number and 383 length of processes were measured in 20 randomly selected cells from each animal, including 10 384 cells each in the dorsal and ventral regions of the hippocampus. The average length of processes 385 was calculated for each cell, using the total length and number of processes for that cell, and 386 subsequently an average value was taken for each animal, following previously established 387 methods (Nemeth et al., 2014).  388 15   389 Figure 2. Representative photomicrographs of the granule cell layer (GCL) in the dentate gyrus, 390 showing (A) BrdU-immunoreactive (ir; red), (B) NeuN-ir (green) (C) BrdU/NeuN-ir (merged 391 image), (D) Ki67-ir, (E) BrdU-ir. (F) Iba-1 and (G) PSA-NCAM-ir cells. Images A-C, F-G 392 viewed at 400x magnification, and D,E at 1000x magnification.  393 2.14. Determination of serum hormone levels  394 Radioimmunoassay kits were used according to the manufacturers’ instructions to quantify 395 serum corticosterone levels (Corticosterone, double-antibody RIA, MP Biomedicals, Solon, OH) 396 and serum 17β-estradiol levels (Estradiol, Ultra-sensitive RIA, Beckman Coulter, Mississauga, 397 Ontario). Samples were run in duplicates and the inter- and intra-assay coefficients were below 398 10%. 399 2.15. Statistical analyses 400 A B C C D E F G 16  All statistical tests were performed using Statistica software (Tulsa, OK). Behavioral tests 401 (Forced swim test, Novelty Suppressed Feeding, Sucrose Preference Test), serum 17β-estradiol 402 levels and organ mass were each analyzed using factorial analysis of variance (ANOVA) with 403 ovarian status (Sham, OVX) and drug treatment (Veh, FLX) as the between-subject factors. 404 Percent sucrose preference and percent change in body mass were analyzed using a repeated-405 measures ANOVA with ovarian status (Sham, OVX) and drug treatment (Veh, FLX) as the 406 between subjects variables, and weeks as the within-subject variable. Serum corticosterone 407 (CORT) concentrations (basal or after DEX challenge) were each analyzed using a repeated-408 measures ANOVA with day (1, 10, 20, 30, 40) or time (0, 30, 60 min) as the within-subject 409 variable, with ovarian status (Sham, OVX) and drug treatment (Veh, FLX) as the between-410 subjects variables. Ki67, BrdU-ir, Iba-1-ir and PSA-NCAM-ir cells, and the percentage of 411 BrdU/NeuN colabeled cells were each analyzed using a repeated-measures ANOVA with 412 ovarian status (Sham, OVX) and drug treatment (Veh, FLX) as the between-subjects variables 413 and with hippocampal region (dorsal, ventral) and dentate gyrus area (GCL+SGZ, hilus) or 414 (GCL+SGZ, ML) as the within-subject variables. A priori we expected differences between 415 ovarian status groups on antidepressant efficacy. Post-hoc tests utilized Newman-Keul’s 416 comparisons and any a priori comparisons were subjected to Bonferroni correction (α=0.05). 417 Unless otherwise stated adding estrous cycle phase as a covariate did not significantly alter the 418 results. 419 3. RESULTS  420 3.1. Ovariectomy increased immobility and decreased swimming and struggling behavior 421 in the forced swim test  422 As predicted, OVX increased immobility in the FST (Figure 3A; F(1, 36) = 15.998, p = 0.0003, 423 main effect of ovarian status). Interestingly, fluoxetine treatment did not significantly alter 424 immobility in either group (p= 0.20), and there was no treatment by ovarian status interaction 425 (p=0.83). Similarly, OVX decreased swimming and struggling in the FST (Figure 3B,C; F(1, 36) 426 = 10.401, p < 0.003, F(1, 36)=4.6799, p=.037, respectively; main effect of ovarian status), but 427 there were no other significant effects (all p’s > 0.2). When analyzing with estrous phase as a 428 covariate, there was a significant behaviour by ovarian status interaction (F(2,70)=3.71, 429 17  p=0.029), in which OVX increased immobility (p=<0.0002),  decreased swimming (p<0.003), 430 but had no significant effect on struggling (p>0.3).  431 3.2. Ovariectomy increased anxiety-like behavior in the novelty suppressed feeding test 432 As expected, OVX increased latency to feed in the NSF (Figure 3D; F(1, 36)= 18.502, p< 433 0.0001, main effect of ovarian status), but there were no other significant effects or interactions 434 (all p’s >0.2). A priori comparisons revealed only a trend for fluoxetine to increase latency to 435 feed in Sham rats (p=0.09). Importantly, the groups did not differ on food consumption in the 436 hour following NSF (all p’s > 0.1).  437 3.3. Ovariectomy increased anhedonia-like behavior in the sucrose preference test 438 Because drug treatment was initiated after 3 weeks of CUS, we ran a repeated measures ANOVA 439 on sucrose preference with only weeks 4-6 of CUS as within subject variable. This revealed a 440 significant CUS week by ovarian status interaction (F(2, 72)=5.098, p=0.009), in which as 441 expected, OVX rats had a significantly lower sucrose preference on week 4 (p<0.0004; Figure 442 3E). Further, there was a significant CUS week by drug treatment interaction (F(2, 72)=3.393, 443 p=.039), such that fluoxetine treatment significantly increased sucrose preference on week 6 of 444 CUS (p < 0.025) but not any other week (all p’s > 0.4), regardless of ovarian status (Figure 3F). 445 There were also significant main effects (ovarian status, and CUS week; all p’s < 0.008), but no 446 other significant interactions (all p’s > 0.08). Importantly, the groups did not differ significantly 447 on baseline sucrose preference prior to CUS (all p’s >0.05).   448 18   449 Figure 3. Effects of ovariectomy (OVX) and/or fluoxetine (FLX) treatment on behavior in the 450 forced swim test (A-C), novelty suppressed feeding (D), and sucrose preference (E-F). In 451 comparison to sham controls, OVX significantly increased immobility (A), and reduced 452 swimming (B) and struggling (C) in the forced swim test, but fluoxetine treatment did not have a 453 significant effect (A-C). (D) OVX significantly increased latency to feed in the novelty 454 suppressed feeding test, and there was no significant effect of fluoxetine treatment (p=0.09 for 455 FLX to increase latency to feed in Sham rats). (E-F) Effects of OVX and FLX treatment on 456 preference for sucrose. OVX significantly decreased percentage of sucrose preference on week 4 457 of CUS, in comparison to sham controls (E). Fluoxetine significantly increased % sucrose 458 preference on week 6 of CUS, relative to vehicle treatment (F). * Indicates p < 0.05, ** indicates 459 19  p <0.0004, and *** indicates p <0.0001 in relation to Sham controls. # Indicates p<0.025 in 460 comparison to vehicle treatment. Veh= vehicle, FLX=fluoxetine. Data is represented in mean 461 values + standard error of the mean (SEM).  462 3.4. Fluoxetine treatment significantly decreased body mass in OVX groups 463 Fluoxetine treatment significantly reduced percent change in body mass in OVX but not sham 464 rats from week 4 of CUS onwards when compared to weeks 1-3 (all p’s<0.03; week by drug 465 treatment by ovarian status interaction: F(5, 180)=2.723, p=0.021; Figure 4A). Additionally, on 466 week 6, the % change in body mass is significantly greater in fluoxetine-treated OVX rats when 467 compared to vehicle-treated OVX rats (p=0.047). Furthermore, there were also significant main 468 effects (ovarian status and CUS week, all p’s< 0.01), and a CUS week by drug interaction 469 (p>0.00001), but no other significant main effects or interactions (all p’s >0.15).  470 3.5. Ovariectomy impaired glucocorticoid-dependent negative feedback on the HPA axis 471 OVX rats had significantly higher post-stress (DEX 30) CORT concentration compared to 472 SHAM rats (p <0.0001; time by ovarian status interaction; Figure 4B, F(2, 62)=7.63, p=0.0001)). 473 Importantly however, there were no differences in serum CORT concentrations at baseline (DEX 474 0, p=0.74) or after recovery from stress (DEX 60, p=0.94). Fluoxetine reduced CORT levels at 475 the 30 minute time point significantly only in Sham (p=0.023) but not OVX (p=0.33) rats. There 476 was also a significant main effect of ovarian status (p=0.005), and a trend towards a drug 477 treatment effect with fluoxetine attenuating CORT concentrations (p=0.097).   478 3.6. Sham rats showed an initial increase in percent change in basal CORT that was 479 reduced by ovariectomy  480 OVX rats showed no significant percent change in baseline CORT across all weeks tested while 481 the percent change was significantly lower than Sham groups across the first twenty days (Figure 482 4D, F(3, 87)=3.83, p=0.013, time by ovarian status interaction). In Sham rats, the percent change 483 in CORT was significantly reduced by day 40 in comparison to days 10 and 20. There was also a 484 significant main effect of time (p=0.017) and ovarian status (p=0.0006), but no other significant 485 effects.  486 3.7. Ovariectomy reduced relative adrenal mass and modulated the effect of fluoxetine 487 treatment on relative adrenal mass. 488 20  OVX significantly reduced relative adrenal mass (Figure 4C, F(1,35)=5.90, p<0.02, ovarian 489 status by drug treatment interaction). Interestingly, post-hoc tests revealed only weak trends for 490 fluoxetine to decrease relative adrenal mass in Sham rats (p=0.096) and to increase relative 491 adrenal mass in OVX rats (p=0.093). There was also a significant main effect of ovarian status 492 (F(1,35)=61.53, p<0.0001) but no other significant effects. Additionally, the groups did not 493 differ on thymus/body mass (all p’s > 0.15).  494 3.8. Ovariectomy decreased serum 17β-estradiol levels 495 As expected, OVX significantly decreased serum estradiol in samples collected prior to 496 perfusion (Figure 4D, F(1,36)=18.88, p=0.0001, main effect of ovarian status). There were no 497 other significant effects (all p’s >0.4).  498 21   499 Figure 4. (A) Percent change in body mass across weeks of chronic unpredictable stress (CUS). 500 Fluoxetine treatment significantly reduced body mass in ovariectomized (OVX) but not sham 501 rats from week 4-6 of CUS when compared to weeks 1-3; * indicates p<0.03. Fluoxetine 502 treatment significantly decreased body mass in OVX rats on week 6, relative to vehicle 503 treatment; # indicates p<0.05. (B) Corticosterone (CORT) concentrations in the dexamethasone 504 (DEX) suppression test. OVX rats had significantly higher post-stress CORT concentration 505 relative to SHAM rats (DEX 30, p=0.000114). Serum CORT concentrations at baseline (DEX 0), 506 22  or after recovery from stress (DEX 60) did not differ between groups. Fluoxetine significantly 507 reduced post-stress CORT in sham (p=0.023) but not OVX (p=0.33) rats. (C) OVX significantly 508 reduced adrenal/body mass ratio; * indicates p<0.02. (D) 17β-estradiol concentrations from 509 serum taken on perfusion day. OVX significantly decreased serum 17β-estradiol relative to 510 shams; * denotes p=0.00011. (E) Percent change in basal serum CORT was significantly lower 511 in OVX groups compared to Shams across the first twenty days p=0.013. OVX=ovariectomy, 512 Veh= vehicle, FLX= fluoxetine. Data is represented in mean values ± SEM.  513 3.9. Ovarian status and fluoxetine treatment did not significantly affect dentate gyrus 514 volume  515 Apart from the expected volume differences between the GCL and hilus (F(3, 102)=323.84, 516 p<0.00001, main effect of region), there were no significant differences between ovarian status 517 or drug treatment groups (all p’s >0.3); thus the number of immunoreactive cells, instead of 518 density, was used in all analyses.  519 3.10. Fluoxetine treatment increased the number of Ki67-ir cells in the GCL; dependent on 520 hippocampal region and ovarian status 521 Fluoxetine treatment significantly increased the number of Ki67-ir cells in both the dorsal and 522 ventral GCL+SGZ (p’s< 0.008) but not in the hilus (p’s >0.94; Figure 5A, F(1, 36)=9.004, 523 p<0.005, region by area by treatment interaction). Furthermore, when compared to sham groups, 524 OVX groups had a significantly higher number of Ki67-ir cells in the GCL+SGZ (p<0.006) but 525 not in the hilus (p=0.96; area by ovarian status interaction F(1, 36)=4.261, p=0.046). A priori, we 526 expected differences in the effects of fluoxetine treatment based on ovarian status, and 527 comparisons revealed that when compared to vehicle, fluoxetine treatment significantly 528 increased the number of Ki67-ir cells in both the dorsal and ventral GCL+SGZ in OVX rats (p’s 529 <0.0002), but only in the ventral GCL+SGZ in Sham rats (ventral p=0.00017, dorsal p=0.96). In 530 order to account for the effects of estrous cycle phase on cell proliferation (Tanapat et al., 1999), 531 the number of Ki67-ir cells were analyzed with estrous phase as a covariate and did not change 532 the main findings, furthermore there were no significant effects of the covariate (all p's>0.78). 533 3.11. Fluoxetine increased the number of PSA-NCAM-ir cells in the ventral GCL in an 534 ovarian status-dependent manner 535 There was a strong trend for fluoxetine treatment to increase the number of PSA-NCAM-ir cells 536 in the ventral but not dorsal GCL+SGZ (F(1, 33)=3.884, p=0.057, region by treatment 537 interaction;  Figure 5B). A priori comparisons indicated that fluoxetine significantly increased 538 23  the number of PSA-NCAM-ir cells in the ventral GCL+SGZ of OVX animals (p=0.0025), but 539 not in any other group or region (all p’s>0.1). There was a significant main effect of region 540 (F(3,108)=101.16, p=0.0000), a trend for a main effect of fluoxetine treatment (p=0.084) but no 541 other significant effects (all p’s>0.3).  542 3.12. Fluoxetine treatment increased the number of BrdU-ir cells in the dorsal GCL in an 543 ovarian status-dependent manner 544 Interestingly, fluoxetine treatment significantly increased the number of BrdU-ir cells in the 545 dorsal GCL of OVX rats (Figure 5C, F(1, 34)=7.4185, p=0.01, region by ovarian status by 546 treatment interaction). As expected, there was a main effect of region and of area on BrdU-ir (all 547 p’s<0.0001). There were no other significant main effects or interactions (all p’s > 0.1). 548 Additionally, there were no group differences in the percentage of BrdU/NeuN colabeled cells, 549 such that the average percentage of colabeled cells was above 75% for all groups (Table 2; all 550 p’s> 0.75).  551 3.13. Fluoxetine treatment decreased the number of Iba-1-immunoreactive cells in the 552 ventral GCL, but had no effect on the average length of microglial processes 553 Fluoxetine treatment significantly decreased the number of Iba-1-ir cells in the GCL+SGZ 554 (p=0.0011; Figure 5D) but not the ML (p=0.55; F(1, 35)=4.56, p=.04 area by treatment 555 interaction), and there was a trend towards a main effect of drug treatment ( p<0.06). Planned 556 comparisons revealed that fluoxetine decreased the number of Iba-1-ir cells in the ventral 557 (p=0.000008, p=0.008 in sham and OVX, respectively) but not dorsal GCL+SGZ (p=0.016, p= 558 0.39, in sham and OVX, respectively). There were main effects of region and of area (all 559 p’s<0.0001), but no other significant effects (all p’s>0.1). The average length of processes was 560 significantly higher in the ventral relative to the dorsal GCL+SGZ, regardless of ovarian status 561 (Table 3; F(1, 35)=6.75, p=0.014, main effect of region). There were no other significant main 562 effects or interactions (all p’s>0.19).  563 24   564 Figure 5. (A) Fluoxetine increased Ki67-ir in the dorsal and ventral granule cell layer (GCL) and 565 subgranular zone (SGZ) in ovariectomized (OVX) rats, and in the ventral GCL+SGZ in sham 566 rats, relative to vehicle treatment. (B) Fluoxetine increased PSA-NCAM-ir in the ventral 567 GCL+SGZ in OVX rats, compared to vehicle treatment. (C) Fluoxetine increased BrdU-ir in the 568 dorsal GCL+SGZ in OVX rats, compared to vehicle treatment. (D) Fluoxetine treatment 569 significantly decreased Iba-1-ir in the ventral GCL, relative to vehicle treatment. Data is 570 represented in mean values +SEM. Veh= vehicle, FLX= fluoxetine.  * indicates p <0.01, ** 571 indicates p <0.003, *** indicates p<0.0002, and # indicates p <0.000001, in relation to vehicle 572 treatment.  573 3.14. Higher levels of estradiol are associated with reduced immobility in the forced swim 574 test 575 25  Interestingly, serum estradiol was negatively correlated with percent immobility in FST in 576 fluoxetine (r = -0.6472, p = 0.043; Figure 6A) but not vehicle treated rats (r = 0.0982, p = 0.787; 577 Figure 6B).  578  579 Figure 6. Correlation of serum estradiol concentrations with percent time spent immobile in the 580 forced swim test (FST) in Sham rats. Serum estradiol was negatively correlated with percent 581 immobility in FST in fluoxetine treated rats ((A) r = -0.6472, p = 0.043) but not vehicle treated 582 rats ((B) r = 0.0982, p = 0.787). If the outlier (serum estradiol=25.8 pg/ml) is removed from the 583 analysis in (A), the correlation between serum estradiol and percent time spent immobile is 584 strengthened (r= -0.818, p=0.007).   585 4. DISCUSSION  586 Here, we report that under conditions of chronic stress, long-term ovarian hormone deprivation 587 increased the vulnerability of middle-aged female rats to develop a depressive-like behavioral 588 and endocrine phenotype. Interestingly, we found fluoxetine treatment showed weak behavioral 589 and endocrine efficacy; fluoxetine treatment increased sucrose preference only in the last week 590 of CUS and improved HPA negative feedback in Sham but not ovariectomized rats. Fluoxetine 591 treatment was also associated with significant changes in neurogenesis, PSA-NCAM and 592 microglia expression, in a region-specific and ovarian status-dependent manner. To our 593 knowledge, this is the first report to integrate the behavioral, neurogenic, and neuroendocrine 594 consequences of long-term ovarian hormone deprivation in tandem with chronic stress and 595 antidepressant treatment. A schematic summary of findings is included in figure 7.    596 4.1. Ovarian hormones imparted resilience against the development of anxiety- and 597 depressive-like behaviors under chronic stress conditions  598 A B 26  Long-term ovarian hormone deprivation markedly increased the percent time spent immobile 599 and decreased the percent time spent swimming in the FST, compared to sham controls. This 600 indicates that under conditions of chronic stress, ovarian hormones may afford resilience against 601 the development of behavioral despair or depressive-like behavior. Our findings are in partial 602 agreement with a study indicating that long, but not short, term ovarian hormone deprivation 603 increased immobility in the FST in mice subjected to CUS (Lagunas et al., 2010a). However, 604 much of the previous literature on the role of ovarian hormones in depressive-like behavior is 605 comprised of studies in which animals are not subjected to any prior stress manipulations or 606 model of depression (Li et al., 2014; Rachman et al., 1998; Récamier-Carballo et al., 2012). 607 While these studies demonstrate that short term ovariectomy alone (1-5 weeks) increased 608 immobility in the FST, long-term ovariectomy (3 - 15 months) does not alter immobility in the 609 FST (de Chaves et al., 2009; Estrada-Camarena et al., 2011). Together these past findings 610 suggest that the increase in depressive-like behavior as a result of ovariectomy alone is 611 transitory, and taken together with our current data, this indicates that long-term ovarian 612 hormone deprivation results in a state of enhanced vulnerability to depressive-like 613 endophenotypes in the face of chronic stress.  614 In a similar manner, in the present study we found that long-term ovarian hormone 615 deprivation increased anxiety-like behavior in the novelty suppressed feeding test, in comparison 616 with sham controls. This is in concert with the previously established anxiolytic effects of 617 estradiol and estrogen receptor agonists (Lund et al., 2005; Walf and Frye, 2005), and with 618 studies indicating enhanced anxiety-like behavior with longer periods of ovarian hormone 619 depletion (Lagunas et al., 2010b; Picazo et al., 2006). Interestingly, we observe a trend for 620 fluoxetine to increase latency to feed in the sham but not OVX group. This is not completely 621 surprising given previous reports suggesting anxiogenic effects of fluoxetine treatment (Belzung 622 et al., 2001; Leuner et al., 2004; Silva et al., 1999). Interestingly, one study found that chronic 623 fluoxetine treatment increased anxiety-like behavior in female but not male rats (Leuner et al., 624 2004). This sex difference coupled with our current data suggests that ovarian hormones may be 625 implicated in the anxiogenic effects of fluoxetine, however this requires further investigation.  626 In line with our other behavioral findings, sucrose preference was also significantly 627 reduced by long-term ovariectomy. However, because there were no differences in basal sucrose 628 preference prior to CUS, it appears that long-term ovariectomy does not itself produce 629 27  anhedonia-like behavior, but rather increases the susceptibility to anhedonia-like behavior under 630 chronic stress. Importantly, this effect was not seen at all time points, however this may not be 631 surprising given that sucrose anhedonia is less robust in female compared to male rodents 632 (Grippo et al., 2005; Dalla et al., 2005, 2008; Kamper et al., 2009). Regardless of ovarian status, 633 fluoxetine treatment resulted in a significant increase in sucrose preference after the last week of 634 CUS, but had no significant effect on behavior in any other test (NSF and FST). This indicates 635 that in middle-aged female rats, a 5mg/kg dose of fluoxetine may be efficacious in alleviating 636 some but not all depressive-like behavioral phenotypes. Our findings are somewhat inconsistent 637 with another study showing that non-efficacious doses of fluoxetine and estradiol, when 638 administered in concert, act synergistically to reduce immobility in the FST in ovariectomized 639 middle-aged female rats (Récamier-Carballo et al., 2012). However, important differences 640 between the study by Recamier-Carballo et al. and ours may explain the inconsistencies in 641 findings. These differences include the length of ovarian hormone deprivation (4 months in the 642 current study versus 3 weeks in Recamier-Carballo et al., 2012), treatment regimen (3 weeks of 643 fluoxetine treatment in our study versus 3 injections within 24 hours in Recamier-Carballo et al., 644 2012), and condition (rats were exposed to chronic stress in our study but were stress-naive in 645 Recamier-Carballo et al., 2012). Our data is consistent with previous studies in which estradiol is 646 shown to increase resilience; for example, estradiol replacement in ovariectomized rats reduced 647 the incidence of helplessness after inescapable shock relative to vehicle treatment (Bredemann 648 and McMahon, 2014). In addition, estradiol increases cognitive resilience in the face of repeated 649 stress (Wei et al., 2013). Collectively, findings from our study and others show that ovarian 650 hormone deprivation may result in a state of enhanced vulnerability to the deleterious effects of 651 chronic stress compared to sham controls.  652 It is important to note that it is not possible to ascertain the reason behind the lack of 653 behavioural efficacy of fluoxetine treatment in the FST or NSF from the current data. In order to 654 address this caveat, future work in middle-aged female rats should compare the response to 655 additional doses of fluoxetine, and/or other SSRIs. However, it is also important to note that 656 although SSRIs are the first-line pharmacological treatment for major depression, only a third of 657 patients will respond to these drugs following initial treatment (Kornstein et al., 2013; Trivedi et 658 al., 2006). Even in patients that are initially responsive to treatment, up to 57% will have 659 depressive symptoms return due to a loss of drug efficacy. Thus, our findings are at least 660 28  somewhat consistent with the clinical literature, and perhaps it is not surprising that we see 661 limited behavioral efficacy with SSRIs in this report.  662 4.2. 17β-estradiol was associated with a facilitated antidepressant-like effect of fluoxetine to 663 decrease immobility in the forced swim test    664 Higher levels of serum 17β-estradiol were positively correlated with reduced immobility in the 665 FST only in the fluoxetine treated sham group (OVX fluoxetine group had undetectable estradiol 666 levels). Because the same relationship was not seen in the vehicle treated sham group, this 667 suggests that in chronically stressed female rats, higher levels of endogenous 17β-estradiol 668 facilitate the effect of fluoxetine to reduce immobility in the FST. Our findings are in line with a 669 previous report demonstrating that sub-optimal doses of 17β-estradiol and fluoxetine 670 administered to OVX rats act synergistically to reduce immobility in the FST (Récamier-671 Carballo et al., 2012). Our findings also parallel the human literature, which indicates that in 672 post-menopausal women with depression, SSRIs are more efficacious in alleviating symptoms 673 when prescribed in conjunction with hormone therapy (HT; Thase 2005), however other studies 674 have failed to show this effect (Kornstein et al., 2013). The mechanisms that underlie the ovarian 675 hormone-mediated enhancement of antidepressant efficacy are not clearly delineated. However, 676 estradiol increases serotonergic tone via enhanced serotonin synthesis (Bethea et al., 2000), 677 reduced degradation (Gundlah et al., 2002), decreased autoreceptor-mediated inhibition (Lu and 678 Bethea, 2002), and serotonin transporter levels  (Frokjaer et al., 2015); thus such mechanisms 679 may be at play in the mediation of antidepressant effects.  680 4.3. Ovarian hormones imparted resilience against glucocorticoid-dependent negative 681 feedback impairment  682 Coinciding with our behavioral results, long-term OVX rats that had undergone CUS 683 exhibited impaired HPA negative feedback compared to the sham rats in the dexamethasone 684 suppression test. This finding signifies that ovarian hormones may protect against HPA negative 685 feedback impairment in the context of chronic stress. In general, ovarian hormones potentiate 686 acute stress-induced activation of the HPA axis (Burgess and Handa, 1992) and impair HPA 687 negative feedback inhibition (Weiser and Handa, 2009). These findings may initially seem 688 contradictory to our current data in which the DEX suppression of CORT release is reduced in 689 OVX rats. However, a critical consideration is that in the aforementioned studies, the subjects 690 29  were not previously exposed to chronic stress. Thus, these findings coupled with our own 691 suggest that the role of ovarian hormones to regulate HPA axis negative feedback may be 692 drastically affected by chronic stress. Intriguingly, our findings translate to a study of pre- and 693 post- menopausal women, all of whom were diagnosed with depression, in which resistance to 694 DEX suppression was found in a significantly higher proportion of postmenopausal women as 695 compared to premenopausal women (Young et al., 1993). Furthermore our data are also 696 consistent with the finding that estradiol improved HPA negative feedback function after DEX 697 suppression test in women made hypogonadal (Lee et al., 2012). Lastly, we found that fluoxetine 698 was able to improve HPA negative feedback inhibition but only in Sham and not ovariectomized 699 controls. To our knowledge there are no studies examining antidepressant efficacy in depressed 700 women on HPA negative feedback regulation with differing estradiol levels. Taken together, this 701 suggests that ovarian hormones may protect against HPA axis negative feedback dysregulation in 702 women with depression, and similarly as a result of chronic stress in female rodents.  703 In the present study, in OVX rats, basal levels of serum CORT were reduced, but the 704 percent change in basal CORT did not change throughout CUS, while it was increased in Sham 705 controls. This finding is in concert with prior research indicating that ovarian hormones exert 706 stimulatory effects on basal HPA axis activity (Goel et al., 2014) for review).  707 4.4. Fluoxetine treatment increased neurogenesis and PSA-NCAM in a region specific and 708 ovarian status-dependent manner  709 In the present study we found no significant differences in levels of cell proliferation (Ki67 710 expression), neurogenesis (BrdU/NeuN), or PSA-NCAM expression in the dentate gyrus of 711 vehicle treated sham and long-term OVX middle-aged rats. These findings are in accordance 712 with previous literature showing long-term ovariectomy (3-4 weeks) results in no significant 713 change in neurogenesis (Tanapat et al., 1999) or PSA-NCAM expression (Banasr et al., 2001), 714 unlike short-term (1 week) ovariectomy in young adult rats. Our work extends these findings to 715 middle aged females. The return of neurogenesis levels following longer periods of ovariectomy 716 is unlikely related to the de novo synthesis of estrogens in the brain, as after three weeks of 717 ovariectomy, levels of estradiol in the hippocampus of adult female rats are not detectable 718 (Barker and Galea, 2009), and hippocampal estradiol correlates with plasma estradiol (Kato et 719 al., 2013). Thus, alternative mechanisms may be at play, such as ovariectomy-induced reductions 720 30  in basal corticosterone (Goel et al., 2014), which may be linked to the normalization of 721 neurogenesis levels after long term ovariectomy. Together, our results indicate that in middle 722 age, long-term deprivation of ovarian hormones does not alter neurogenesis or PSA-NCAM 723 expression in the dentate gyrus. 724 Fluoxetine treatment increased cell proliferation, neurogenesis, and the expression of 725 PSA-NCAM within the dentate gyrus. These findings are in line with prior reports indicating that 726 chronic treatment with fluoxetine upregulates the same markers (Guirado et al., 2012; Malberg et 727 al., 2000). While in older age, fluoxetine does not increase neurogenesis in the dentate gyrus 728 (Couillard-Despres, 2009), our findings are consistent with others showing that fluoxetine 729 upregulated PSA-NCAM in middle-aged male rats (Guirado et al., 2012). Interestingly, our 730 current data shows that fluoxetine treatment, when initiated after BrdU administration, increased 731 adult hippocampal neurogenesis, unlike what is seen in stress-naïve, young adult male rats 732 (Malberg et al., 2000). Indeed, our finding is consistent with another study using young adult 733 female rats (Vega-Rivera et al., 2015), perhaps indicating a sex difference in the response of 734 fluoxetine administration on survival of BrdU-labelled cells. Interestingly, the fluoxetine-735 mediated upregulation of cell proliferation, neurogenesis, and PSA-NCAM was seen despite its 736 lack of behavioral efficacy in the majority of tests, suggesting that these neural changes are not 737 necessarily synonymous with behavioral efficacy, partially consistent with the existing literature 738 in young adult male rats (Bessa et al., 2008; Santarelli, 2003). The 5mg/kg dose of fluoxetine 739 utilized in this study has been previously shown to increase cell proliferation in adult female rats 740 relative to a higher dose of 10mg/kg, but not compared to vehicle treatment, and the study did 741 not examine dorsal versus ventral dentate gyrus (Pawluski et al., 2014). The same study found 742 this differential effect of dose on cell proliferation only when fluoxetine was administered via 743 osmotic minipump, but not orally via wafer cookie (Pawluski et al., 2014). Future studies should 744 consider the effects fluoxetine administration route and dose on cell proliferation in different 745 regions of the hippocampus.     746 Somewhat surprisingly, the effect of fluoxetine treatment to enhance cell proliferation 747 (Ki67-ir cells) was seen in both the dorsal and ventral hippocampus of OVX rats, but only in the 748 ventral hippocampus of sham rats. Similarly fluoxetine-mediated enhancement of neurogenesis 749 and PSA-NCAM was exclusive to OVX rats, in the dorsal and ventral hippocampus, 750 respectively. Thus contrary to our initial expectations, the potential for plasticity in response to 751 31  fluoxetine treatment is not reduced with long-term ovarian hormone deprivation, but rather 752 appears to be enhanced. The mechanism for this unexpected finding is not clear but may be due 753 to HPA-HPG interactions; because ovarian steroids have stimulatory effects on the HPA axis 754 (Viau and Meaney, 1991) the higher levels of basal cortisol in sham rats may interact with 755 fluoxetine to prevent the upregulation of cell proliferation that is seen in ovariectomized rats. 756 Interestingly, fluoxetine has been shown to increase the neurosteroid allopregnanolone in the 757 female rat brain (Fry et al., 2014), therefore it would be interesting to investigate potential 758 modulatory effects of ovarian hormones and subsequent effects on hippocampal neurogenesis. 759 These findings are also in partial contrast to those showing that an acute dose of estradiol with a 760 suboptimal dose of fluoxetine can increase cell proliferation in stress-naïve female Wistar rats 761 (Vega-Rivera et al., 2015). Considering that in the present study there were no significant 762 differences in the effects of fluoxetine on behavior between OVX and sham groups, the 763 functional consequences of this differential upregulation of neuroplasticity based on ovarian 764 hormone milieu remain elusive. A region specific influence of fluoxetine in CUS exposed Sham 765 rats on neurogenesis is perhaps not surprising given that CUS decreases neurogenesis in the 766 ventral hippocampus and is reversed by chronic antidepressant treatment (Tanti and Belzung, 767 2013).   768 Interestingly, fluoxetine treatment improved HPA negative feedback regulation in sham 769 rats, but had more profound pro-neurogenic effects in OVX rats. This is somewhat inconsistent 770 with previous research in young adult male rodents, in which newly generated hippocampal 771 neurons were shown to be required for antidepressants to improve HPA axis regulation (Surget et 772 al., 2011), and data indicating that hippocampal neurogenesis may be important for HPA 773 negative feedback regulation (Snyder et al., 2011). This discrepancy is interesting, as it suggests 774 that the relationship between hippocampal neurogenesis and HPA axis function may differ 775 between males and females. Indeed, the fact that fluoxetine only increased cell proliferation in 776 the ventral, but not dorsal hippocampus in sham animals, unlike what occurred in OVX animals, 777 may explain the reason for better efficacy of fluoxetine in Sham rats in the DEX suppression test. 778 In addition, it is also possible that the fluoxetine-induced increase in neurogenesis in the dorsal 779 hippocampus of OVX rats may have interfered with the efficacy of fluoxetine. Finally, it is 780 important to note that it is not without precedent that neurogenesis may have different functions 781 or links to behavior/stress in males versus females, as sex differences in neurogenesis have been 782 32  noted in response to spatial learning (Chow et al., 2013; Yagi et al., 2015), trace eyeblink 783 conditioning (Dalla et al., 2009), acute and chronic stress (Falconer and Galea, 2003; 784 Westenbroek et al., 2004), adolescent stress exposure (Barha et al., 2011) and chronic estradiol 785 treatment (Barker and Galea, 2008).  786 4.5. Fluoxetine treatment reduced microglial number in the ventral dentate gyrus but had 787 no effect on microglial morphology 788 Regardless of ovarian status, fluoxetine treatment significantly reduced the number of Iba-1-ir 789 microglial cells in the ventral dentate gyrus, partially consistent with previous findings showing 790 imipramine treatment reverses the initial increase and subsequent decline in microglial number 791 as a result of chronic unpredictable stress (Kreisel et al., 2013b).  However, our data provide no 792 evidence for alterations in microglial activation with fluoxetine treatment, as there was no effect 793 of fluoxetine on the number or length of processes in Iba-1-ir cells. Thus, our findings suggest 794 that SSRI treatment may be associated with a reduction in microglia-mediated 795 neuroinflammation, however this requires further investigation as microglial function depends on 796 its activational state (Kettenmann et al., 2011) and levels of pro- versus anti-inflammatory 797 cytokines. Importantly, our findings are in partial agreement with human literature suggesting 798 that antidepressant treatment is associated with a reduction of peripheral inflammation, as 799 measured by reduced pro-inflammatory cytokines such as IL-6 (Basterzi et al., 2005) and TNF- α 800 (Tuglu et al., 2003), and confirmed by a meta-analysis (Eller et al., 2008). Taken together with 801 our present findings, this suggests that antidepressants may exert their effects, at least in part, 802 through the amelioration of microglia-mediated neuroinflammation in the hippocampus. 803 Our findings reveal no basal differences in the number or morphology of Iba-1-ir 804 microglial cells between OVX and sham groups. This is somewhat surprising considering that 805 ovarian hormones inhibit microglial proliferation in vivo (Ganter et al., 1992), and can directly 806 alter microglial function and morphology both in vivo and in vitro (Habib and Beyer, 2015). Our 807 findings are in contrast with a study of middle-aged female rats, in which Iba-1 mRNA 808 expression in the hippocampus was significantly increased when measured approximately 40 809 days after ovariectomy (Sárvári et al., 2014). However, in our present study, the period of 810 ovarian hormone deprivation was substantially longer (4 months), and thus compensatory 811 mechanisms may be at play to return baseline levels of Iba-1 expression.  812 33  4.6. The physiological effects of fluoxetine were dependent on ovarian status  813 Fluoxetine treatment reduces body weight or attenuates weight gain in rodents (McGuirk et al., 814 1992). In the present study, fluoxetine reduced body weight in OVX rats only. Although the 815 functional outcome of fluoxetine-induced weight reduction is unknown, ovarian hormones 816 appear to protect against this effect.  817 5. CONCLUSIONS  818 In summary, ovarian hormones afforded resilience against the development of a depressive-like 819 phenotype in middle-aged female rats exposed to chronic unpredictable stress. Long-term 820 ovariectomy increased depressive-, and anxiety-like behaviors in the forced swim, novelty 821 suppressed feeding, and sucrose preference tests after exposure to chronic unpredictable stress. 822 Further, long-term ovariectomy resulted in significant impairments in the HPA negative 823 feedback system, as demonstrated by the dexamethasone suppression test. Thus, our behavioral 824 and endocrine findings converge to suggest that ovarian hormones are key determinants of stress 825 resilience. With the exception of the sucrose preference test and HPA dysregulation, chronic 826 fluoxetine treatment at 5mg/kg did not produce anti-depressant-like effects, regardless of ovarian 827 status. However, higher levels of endogenous estradiol were associated with a facilitation of 828 fluoxetine to decrease immobility in the forced swim test. Despite its limited efficacy, fluoxetine 829 treatment was associated with reduced microglial expression in the ventral dentate gyrus, and 830 increased cell proliferation, neurogenesis, and PSA-NCAM expression; effects that were varied 831 by ovarian status and region. Our findings reinforce the need for considering the role of ovarian 832 hormones in stress-resilience, depression, antidepressant efficacy, and the neural correlates of 833 antidepressant use in females.  834 Role of the funding source  835 Financial support for this research was provided by an operating grant from the Canadian 836 Institutes for Health Research (MOP142308) to LAMG. The funding source had no involvement 837 in study design; in the collection, analysis and interpretation of data; in the writing of the report; 838 and in the decision to submit the paper for publication.  839  Acknowledgements 840 34  The authors thank Carmen Chow, Lucille Hoover, Anne Cheng, and Alice Chan for their 841 assistance with this work.   842  843  844  845  846 Figure 7. Schematic summary of behavioral, endocrine, and neural findings. (A) The effect of 847 long-term ovariectomy in comparison to sham controls. (B) The effect of fluoxetine treatment 848 compared to vehicle treatment in each ovarian hormone condition. OVX: ovariectomy, FLX: 849 fluoxetine, Veh: vehicle, FST: Forced Swim Test, NSF: Novelty Suppresses Feeding, SPT: 850 Sucrose Preference Test, DEX= dexamethasone.  indicates significant increase,  indicates 851 significant decrease, = indicates no significant change. * indicates significance only in week 4 of 852 chronic unpredictable stress, ** indicates significance only in week 6 of chronic unpredictable 853 stress.  854  855  856  857  858  859 35  Table 1.  Stressors included in the Chronic Unpredictable Stress paradigm. 860 Stressor Duration Description Wet bedding 2 hours 200 ml of tap water in home cage, rats moved to a dry and clean cage afterwards  Food and water deprivation 4 hours Rats deprived of food and water, always prior to the sucrose preference test Tail pinch 5 mins  Plastic clothespin placed at base of tail  Cage tilt  2 hours Home cage tilted at 45°  Elevated platform 5 mins Rats placed on a plexiglass platform (20x20 cm) at 90 cm above ground.  Continuous lighting  36 hours Colony lights on for 36 hours  Restraint 1 hours Rats restrained in well ventilated plexiglass tubes   Social isolation 18 hours Rats individually housed from 15:00-10:00   Soiled cage 2 hours Rat pairs placed in cage soiled by other rats. Rats moved to a clean cage afterwards  White noise  1 hour Rats exposed to 80 dB of white noise  Strobing light 1 hour Strobing light at 1flash/second in an otherwise dark room, during light cycle Tail bleed 2 mins Blood sample taken via tail vein nick  Food deprivation 16 hour  Rats deprived of food but not water Two stressors were presented daily, in a pseudo-random order, for a duration of 6 weeks. 861   862 36  Table 2.  Mean (±SEM) percentage of BrdU/NeuN colabeled cells in the granule cell layer of the 863 dentate gyrus of all groups. 864 Ovarian status  Drug treatment  % of BrdU/NeuN colabeled cells  Sham Veh 77.50 ± 6.77  FLX 75.85 ± 4.90 OVX Veh 75.35 ± 7.08  FLX 77.54 ± 4.65 The groups did not significantly differ on the average percentage of BrdU/NeuN colabeled cells 865 (all p’s>0.75). Veh= vehicle, FLX=fluoxetine, OVX=ovariectomy. 866 Table 3.  Mean (±SEM) length of processes of Iba-1 immunoreactive cells in the granule cell 867 layer of the dorsal and ventral dentate gyrus of all groups. 868 Ovarian status  Drug treatment  Average length of processes   Dorsal  Ventral  Sham Veh 149.22 ± 4.76 158.73 ± 4.33  FLX 152.06 ± 5.05 157.63 ± 4.91 OVX Veh 154.15 ± 4.11 162.01 ± 6.18  FLX 159.40 ± 2.65 162.59 ± 4.01 The average length of processes was significantly higher in the ventral dentate gyrus relative to 869 the dorsal dentate gyrus (p=0.014), but there were no other significant main effects or 870 interactions (all p’s>0.19). Veh= vehicle, FLX=fluoxetine, OVX=ovariectomy.  871 37  References 872  873 Ahokas, A., Kaukoranta, J., Wahlbeck, K., Aito, M., 2001. Estrogen deficiency in severe 874 postpartum depression: successful treatment with sublingual physiologic 17beta-estradiol: a 875 preliminary study. J Clin Psychiatry 62, 332–336. 876 Banasr, M., Hery, M., Brezun, J.M., Daszuta, A., 2001. 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