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Maternal postpartum corticosterone and fluoxetine differentially affect adult male and female offspring… Gobinath, Aarthi R.; Workman, Joanna L.; Chow, Carmen; Lieblich, Stephanie E.; Galea, Liisa A.M. Feb 29, 2016

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1  Published in Gobinath, A.R., Workman, J.L., Chow C, Lieblich, SE, Galea, L.A.M. (2016). Maternal 1 postpartum corticosterone and fluoxetine differentially affect adult male and female offspring on anxiety-2 like behavior, stress reactivity, and hippocampal neurogenesis. Neuropharmacology, 101:165-178.  3  4 Maternal postpartum corticosterone and fluoxetine differentially affect 5 adult male and female offspring on anxiety-like behavior, stress 6 reactivity, and hippocampal neurogenesis 7 Aarthi R. Gobinath1, Joanna L. Workman2†, Carmen Chow2, Stephanie E. Lieblich2, Liisa A.M. Galea1,2,3* 8  9 1Program in Neuroscience, 2Department of Psychology, and 3Brain Research Centre 10 University of British Columbia 11  12 *Corresponding Author: 13 2136 West Mall 14 Vancouver, BC V6T 1Z4 15 lgalea@psych.ubc.ca 16  17 †Present Address:  18 University at Albany, State University of New York 19 Department of Psychology 20 1400 Washington Ave. 21 Albany, NY 12222 22  23  24  25  26  27  28  29  30  31  32 2   33 Abstract 34 Postpartum depression (PPD) affects approximately 15% of mothers, disrupts maternal care, and 35 represents a form of early life adversity for the developing offspring. Intriguingly, male and female 36 offspring are differentially vulnerable to the effects of postpartum depression. Antidepressants, such as 37 fluoxetine, are commonly prescribed for treating postpartum depression. However, fluoxetine can reach 38 offspring via breast milk, raising serious concerns regarding the long-term consequences of infant 39 exposure to fluoxetine. The goal of this study was to examine the long-term effects of maternal 40 postpartum corticosterone (CORT, a model of postpartum stress/depression) and concurrent maternal 41 postpartum fluoxetine on behavioral, endocrine, and neural measures in adult male and female offspring. 42 Female Sprague-Dawley dams were treated daily with either CORT or oil and fluoxetine or saline from 43 postnatal days 2-23, and offspring were weaned and left undisturbed until adulthood. Here we show that 44 maternal postpartum fluoxetine increased anxiety-like behavior and impaired hypothalamic-pituitary-45 adrenal (HPA) axis negative feedback in adult male, but not female, offspring. Furthermore, maternal 46 postpartum fluoxetine increased the density of immature neurons (doublecortin-expressing) in the 47 hippocampus of adult male offspring but decreased the density of immature neurons in adult female 48 offspring. Maternal postpartum CORT blunted HPA axis negative feedback in males and tended to 49 increase density of immature neurons in males but decreased it in females. These results indicate that 50 maternal postpartum CORT and fluoxetine can have long-lasting effects on anxiety-like behavior, HPA 51 axis negative feedback, and adult hippocampal neurogenesis and that adult male and female offspring are 52 differentially affected by these maternal manipulations.  53 Keywords: postpartum corticosterone, fluoxetine, doublecortin, sex differences, hippocampus, anxiety,  54  55  56  57  58  59  60  61  62 3   63 1. Introduction  64 According to the DSM-5, perinatal depression is defined as depression during pregnancy and the 65 early postpartum. As with major depression, one of the most common treatments for perinatal depression 66 is pharmacological antidepressants, such as selective serotonin reuptake inhibitors (SSRIs; Oberlander et 67 al., 2006; Kim et al., 2014). As more women receive antidepressants to treat perinatal depression, the 68 population of children who have been exposed to antidepressants during the perinatal period also 69 increases (Oberlander et al., 2006). However, maternal SSRI use may be problematic as SSRIs such as 70 fluoxetine (Prozac) can cross the placental barrier (Hendrick et al., 2003) and pass into breast milk 71 (Wisner et al., 1996; Weissman et al., 2004), potentially affecting the developing offspring. Indeed, 72 perinatal SSRI exposure is associated with adverse outcomes in the infant such as reduced weight gain 73 (Chambers et al., 1999), levels of reelin required for normal brain development (Brummelte et al., 2013), 74 psychomotor scores during the first year (Santucci et al., 2014), increased hypertension (Chambers et al., 75 2006), cardiac defects (Malm et al., 2011), and risk for autism (Croen et al., 2011). However, the negative 76 effects of perinatal fluoxetine may outweigh the detrimental effects of untreated maternal depression on 77 child development. Specifically, children of mothers with postpartum depression (PPD) are more likely to 78 develop depression, anxiety, and attention deficits even long after the mother’s depression has remitted 79 (Pilowsky et al., 2006; Murray et al., 2011). Thus, the potential therapeutic effect of maternal SSRIs may 80 mitigate these negative effects on child development. In fact, maternal SSRI use is associated with 81 enhanced infant readiness to interact with their mother (3 mo infants; Weikum et al., 2013b), accelerated 82 perceptual development (6 mo and 10 mo infants; Weikum et al., 2012), and improved executive function 83 (6 yo; Weikum et al., 2013a).  However, it is unclear whether the effects of maternal fluoxetine are 84 advantageous in the long term or precede negative behavioral outcomes that emerge later in life. This 85 study aims to fill this gap. 86 Preclinical research investigating the long term effects of perinatal fluoxetine on emotional 87 behavior has yielded mixed results, likely due to methodological differences including timing and method 88 of administration. For example, direct administration of fluoxetine to pups during the postnatal period 89 increased anxiety-like behavior (Yu et al., 2014), while maternal exposure to fluoxetine (gestation and 90 lactation) resulted in no significant effect on anxiety-like behavior in adult offspring (Lisboa et al., 2007; 91 Francis-Oliveira et al., 2013). Additionally, direct administration of fluoxetine to pups during the 92 postnatal period decreased depressive-like behavior in adult rats (Mendes-da-Silva et al., 2002) whereas 93 maternal fluoxetine (gestation and postpartum) increased depressive-like behavior in adult female but not 94 male mice offspring (Lisboa et al., 2007). In addition, the current state of research examining neonatal 95 4  fluoxetine exposure is hindered by a general lack of preclinical research investigating maternal fluoxetine 96 exposure within a model of depression or PPD.  Because mothers typically use SSRIs to treat depression, 97 there is a need for preclinical research to address how maternal fluoxetine influences offspring within a 98 concurrent model of depression or stress in order to contribute valid conclusions regarding the use of 99 SSRIs to treat PPD. To this end, there are a few studies examining how gestational stress followed by 100 maternal postpartum fluoxetine normalizes immobility in the forced swim test in adolescent male and 101 female offspring (Rayen et al., 2011) as well as blunts serum corticosterone (CORT; primary 102 glucocorticoid in rats) levels in adolescent male, but not female, offspring (Pawluski et al., 2012c). 103 However, gestational stress did not result in a depressive phenotype in the dam in this study (Pawluski et 104 al., 2012b), so it is unclear whether these results can be interpreted as modeling maternal depression. 105 Moreover, it is unknown how modeling depression and antidepressant treatment occurring exclusively in 106 the postpartum affect offspring development. This is an important problem to investigate because 107 approximately 40% of perinatal depression arises solely in the postpartum period (Wisner et al., 2013) 108 and treatment and outcome for mother and child differ depending on the timing of depression onset 109 (Cooper & Murray, 1995). Thus, there is a need to study postpartum antidepressant treatment in animal 110 models of depression based on postpartum and antenatal depression, respectively. 111 The hippocampus exhibits morphological alterations long after exposure to developmental stress 112 (reviewed in Korosi et al., 2012, reviewed in Loi et al., 2014). Although maternal depression does not 113 predict significant changes in hippocampal volume in children (Lupien et al., 2011), childhood 114 maltreatment (Chaney et al 2014) and low maternal bonding (Buss 2007) are associated with reduced 115 hippocampal volume in adulthood, which both may be present in PPD. Reduction in hippocampal volume 116 can be attributed to a number of factors such as lower levels of hippocampal neurogenesis. Broadly 117 speaking, stress reduces adult hippocampal neurogenesis depending on age at the time of stress exposure 118 and sex of the subject (Gobinath et al., 2014). For example, maternal deprivation diminished expression 119 of doublecortin (an endogenous protein expressed in immature neurons) in adult male but not female rat 120 offspring (Oomen et al., 2010; Oomen et al., 2011). Furthermore, adult hippocampal neurogenesis may 121 play an important role in the etiology of mood-related disorders such as depression (reviewed in 122 DeCarolis & Eisch, 2010; reviewed in Eisch & Petrik, 2012), as well as regulation of the hypothalamic-123 pituitary-adrenal (HPA) axis (Snyder et al., 2011). Despite evidence that antidepressants can normalize 124 HPA axis activity (Ising et al., 2007) and increase hippocampal neurogenesis (Malberg et al., 2000; 125 Santarelli et al., 2003, Boldrini et al., 2009; Epp et al., 2013), little is known about how maternal 126 fluoxetine affects HPA axis and adult neurogenesis in the hippocampus of offspring beyond the time they 127 are exposed to the drug. Maternal postpartum fluoxetine reversed the detrimental effects of prenatal stress 128 on hippocampal doublecortin expression in both male and female adolescent rat offspring (Rayen et al., 129 5  2011). However, by adulthood, maternal postpartum fluoxetine only diminished doublecortin expression 130 after prenatal stress exposure, particularly in adult male offspring (Rayen et al., 2014). Thus, hippocampal 131 neurogenesis represents a neurobiological intersection of developmental exposure to stress, 132 antidepressants, and adult behavioral outcomes and will be investigated in the present study. 133 We have previously shown that chronic CORT administered to the dam postpartum increases 134 maternal depressive-like behavior and diminishes maternal care (Brummelte et al., 2006; Brummelte et 135 al., 2010; Brummelte & Galea, 2010; Workman et al., 2013b; Workman et al., submitted). Interestingly, 136 maternal postpartum CORT decreases hippocampal cell proliferation in male offspring at weaning 137 (Brummelte et al., 2006) and increases anxiety-like behavior in adolescent male, but not female, offspring 138 (Brummelte et al., 2012). However, it is unclear whether these sex differences or effects on offspring 139 brain and behavior persist when the dam is exposed to concurrent maternal antidepressant exposure. The 140 present study investigates whether high levels of maternal postpartum CORT and concurrent fluoxetine 141 administered to dams differentially affect adult male and female offspring outcome at the behavioral 142 (anxiety- and depression-like behavior, locomotion), endocrine (HPA axis dysregulation), and neural 143 (doublecortin expression) levels. We hypothesized that maternal postpartum fluoxetine would negatively 144 affect behavior, HPA axis regulation, and hippocampal neurogenesis in the affected adult offspring. 145 Further, we expect that both sexes will be differentially affected by maternal postpartum fluoxetine and 146 CORT. 147 2. Materials and methods 148 2.1. Animals 149 Thirty-two adult female Sprague-Dawley rats (2 – 3 months old) and 16 adult male Sprague-150 Dawley rats (2 – 3 months old, Charles River) were initially housed in same-sex pairs in opaque 151 polyurethane bines (24 x 16 x 46 cm) with aspen chip bedding. Rats were maintained in a 12 h: 12 h 152 light/dark cycle (lights on at 7:00 a.m) and given rat chow (Jamieson's Pet Food Distributors Ltd, Delta, 153 BC, Canada) and tap water ad libitum. All protocols were in accordance with ethical guidelines set by 154 Canada Council for Animal Care and were approved by the University of British Columbia Animal Care 155 Committee.  156 2.2. Breeding Procedures 157 For breeding, males were single housed and two females and one male were paired daily between 158 5:00 and 7:00 pm. Females were vaginally lavaged each morning between 7:30 and 9:30 am and samples 159 6  were assessed for the presence of sperm. Upon identification of sperm, females were considered pregnant, 160 weighed, and single housed into clean cages with autoclaved paper towels and an enrichment tube.  161 One day after birth (birth day = postnatal day 0), all litters were culled to 5 males and 5 females. 162 If there were not enough males or females in one litter, pups were cross-fostered from a dam that gave 163 birth the same day. If there were not enough pups available to support a 5 male and 5 female litter, then 164 dams maintained a sex-skewed or smaller litters (this happened twice with both being in the CORT/saline 165 group). Dams were randomly assigned to one of four treatment groups: 1) CORT/fluoxetine; 2) 166 CORT/saline; 3) Oil/fluoxetine; 4) Oil/saline. Beginning on postpartum day 2, dams received two daily 167 injections of either subcutaneous CORT (40 mg/kg) or sesame oil (1 ml/kg) and intraperitoneal fluoxetine 168 (10 mg/kg) or saline (1 ml/kg) for 22 consecutive days. The effects of maternal postpartum CORT/saline 169 on depressive-like behavior were verified in the dam (Workman et al., 2013b; Workman et al., 170 submitted), and data investigating maternal outcome will be published separately (Workman et al., 2013b; 171 Workman et al., submitted). Dams received both injections in succession between 11 A.M. and 2 P.M. 172 Pups were weaned on postpartum day 24 and pair-housed with an unrelated, same-sex cage mate whose 173 mother received the same treatment. No more than 2 males and 2 females were taken from each litter for 174 the behavioral tests. Besides weekly cage changing, offspring remained undisturbed until behavioral 175 testing.  176 2.3. Drug preparation  177 An emulsion of CORT (Sigma-Aldrich, St. Louis, MO, USA) was prepared every 2-3 days by 178 mixing CORT with ethanol and then adjusting with sesame oil to yield a final concentration of 40 mg/ml 179 of CORT in oil with 10% ethanol. The dose was chosen because it reliably induces a depressive-like 180 phenotype in dams, impairs maternal care, and affects offspring development (Brummelte et al., 2006; 181 Brummelte et al., 2010; Brummelte & Galea, 2010; Brummelte et al., 2012; Workman et al., 2013a). 182 Fluoxetine (Sequoia Research Products, Pangbourne, UK) was prepared every 2-3 days by dissolving in 183 dimethyl sulfoxide (DMSO; Sigma Aldrich) and adjusting with 0.9% saline to yield a final concentration 184 of 10 mg/ml fluoxetine in saline with 10% DMSO. This dose of fluoxetine was chosen based on work 185 illustrating that this dose increased brain derived neurotrophic factor and cell proliferation in the 186 hippocampus and amygdala after 21 days of injections in both male and female rodents (Hodes et al., 187 2010). Control dams were given two vehicle injections: “oil” consisted of 10% ethanol in sesame oil to 188 control for the CORT injections, and “saline” consisted of 10% DMSO in 0.9% saline to control for the 189 fluoxetine injections. 190 For the dexamethasone suppression test, a solution of dexamethasone (Sigma Aldrich) was 191 prepared 1-2 days prior to the test by dissolving dexamethasone in propylene glycol and adjusted to yield 192 7  a final dose of 50 ug/kg dexamethasone in propylene glycol. This dose and timing of dexamethasone 193 injection were chosen based on previous studies (Cole et al., 2000).   194 2.4.1. Behavioral Testing  195 Beginning at postnatal day 65 ± 2, 6-10 male and female rats per group underwent behavioral 196 testing (elevated plus maze, open field test, forced swim test, and novelty suppressed feeding). Based on 197 the four maternal treatments described above, rats from each of the following groups (60 rats total) were 198 utilized: Adult male Oil/Saline offspring, n=8; Adult male Oil/Fluoxetine offspring, n=6; Adult male 199 CORT/Saline offspring, n=6; Adult male CORT/Fluoxetine offspring, n=10; Adult female Oil/Saline 200 offspring, n=9; Adult female Oil/Fluoxetine offspring, n=6; Adult female CORT/Saline offspring, n=6; 201 Adult female CORT/Fluoxetine offspring, n=9. Behavioral tests were conducted in the same order for all 202 the animals with 48 h between each test. Behavioral testing occurred at 9:00 A.M. each day under dim 203 light conditions (approximately 12 lux). Twenty-four h after the final behavioral test, rats underwent a 204 dexamethasone-suppression test under standard bright light conditions (approximately 180 lux). Seventy-205 two h after dexamethasone suppression test, all rats were perfused and brain tissue was collected. For an 206 overview of experimental procedures, refer to Figure 1.  207 2.4.2. Elevated plus maze   208 The elevated plus maze was used to evaluate anxiety-like behavior in male and female offspring. 209 Briefly, the apparatus consists of two open arms bisected by two closed arms (arm length: 50 cm; arm 210 width: 10 cm; arm wall height: 40 cm). Rats were placed into the center of the apparatus, facing the open 211 arm. Each test session lasted 5 min and was video recorded. The apparatus was cleaned using a 15% 212 vinegar solution between each testing session to remove any odors or waste. The numbers of entries (all 213 four paws entering an arm) into the open arm and closed arm as well as time (in seconds) spent in the 214 open arm and closed arm were analyzed. Ratio of time spent in the closed arm versus the open arms and 215 center was used as an index of anxiety as previously described (Brummelte et al., 2012).     216 Figure 1: Timeline. CORT: corticosterone; DEX: dexamethasone; FLX: fluoxetine; PN: postnatal day 8  2.4.3. Open field test 217 The open field test was used to assess general locomotor activity as previously described 218 (Brummelte et al., 2006). The apparatus, a 90 x 90 x 40 cm square arena divided into 16 squares of equal 219 dimension, was placed in a dimly lit room. Rats were placed in the apparatus facing the same corner and 220 video recorded for 10 min. The apparatus was cleaned using a 15% vinegar solution between each testing 221 session to remove any odors or waste. A line crossing was defined as all four paws crossing a gridline 222 (Brummelte et al., 2006). Total number of line crossings was used as an index of general locomotion. 223 2.4.4. Forced swim test 224 Approximately 48 h after open field test, rats were tested in the forced swim test to assess 225 depressive-like behavior. A glass cylindrical tank (45 x 28 cm) filled to a depth of approximately 30 cm 226 of tap water 25 ± 1ºC. For the first session, rats were placed into the water for 15 min. The second session 227 took place 24 h later and rats were placed into the water for 5 min. Water was replaced between each rat. 228 An observer blind to treatment conditions scored the sessions for percent time spent swimming, climbing, 229 or immobile using BEST Collection Software (Educational Consulting, Inc., Hobe Sound, FL, USA).  230 2.4.5. Novelty suppressed feeding 231 Approximately 48 h after forced swim test, rats were tested for anxiety-like behavior in the 232 novelty suppressed feeding paradigm. In this test, rats must resolve an anxiogenic conflict of entering the 233 center of arena to access a morsel of chow after being food deprived (Bodnoff et al., 1989:, Santarelli et 234 al., 2003; Bessa et al., 2009; Leuner et al., 2010). Food was removed from rats’ cages 16 h prior to testing 235 to incite motivation to consume food during the test. Each rat was placed in a square arena (60 x 60 cm) 236 facing the right corner. Latency to feed was recorded in seconds as an index of anxiety-like behavior. The 237 trial was terminated either after the rat began to eat or after 10 min if the rat did not eat. Lab chow was 238 added to the cages after testing, and food consumption was measured in each cage 1 h after test to assess 239 whether feeding behavior was altered by maternal postpartum CORT or fluoxetine.  240 2.4.6. Dexamethasone Suppression Test 241 Approximately 48 h after novelty suppressed feeding, rats were tested for HPA axis negative 242 feedback using the dexamethasone suppression test. Dexamethasone was administered to all rats 243 subcutaneously 90 min prior to a 30 min restraint stressor. Tail blood samples were collected at the 244 beginning of restraint (t=0), the end of restraint (t=30), and 1 h after cessation of restraint (t=90).  245 2.5. Tissue Collection 246 9   Approximately 72 h after dexamethasone suppression test, rats were weighed and then given an 247 overdose of Euthanyl. Rats were perfused with 60 ml cold 0.9% saline followed by 120 ml cold 4% 248 paraformaldehyde. Brains were extracted and postfixed using 4% paraformaldehyde overnight at 4ºC. 249 Brains were then transferred to 30% sucrose in phosphate buffer at 4ºC until they sank to the bottom. 250 Brains were rapidly frozen with dry ice and sectioned using a freezing microtome (Leica, Richmond Hill, 251 ON, Canada) at 40 µm and collected in series of 10. Sections were stored in antifreeze (ethylene 252 glycol/glycerol; Sigma) and stored at -20ºC until processing.  253 2.6. Corticosterone Assay 254 Blood samples were stored overnight at 4ºC to allow blood to clot completely. Blood was then 255 centrifuged at 10,000 g for 15 min. The serum was collected and stored at -20 ºC until radioimmunoassay. 256 Total CORT (bound and free) was measured using the ImmuChem Double Antibody 125I 257 radioimmunoassay Kit (MP Biomedicals, Solon, OH, USA). The antiserum cross-reacts 100% with 258 CORT, 0.34% with deoxycorticosterone, 0.05% with cortisol, and does not cross-react with 259 dexamethasone (<0.01%). All reagents were halved and samples run in duplicate. 260 2.7. Doublecortin Immunohistochemistry 261  Sections were rinsed 5 x 10 min in 0.1 M phosphate buffered saline (PBS), treated with 0.3% 262 hydrogen peroxide in dH2O for 30 min, and incubated at 4 ºC in primary antibody solution: 1:1000, goat 263 anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 0.04% Triton-X in PBS and 264 3% normal rabbit serum for 24 h. Sections were then rinsed 5 x 10 min in 0.1 M PBS and transferred to a 265 secondary antibody solution with 1:500, rabbit anti-goat (Vector Laboratories, Burlington, ON, Canada) 266 in 0.1 M PBS for 24 h at 4ºC. Then, sections were washed 5 x 10 min in 0.1 M PBS and incubated in 267 ABC complex (ABC Elite Kit; 1:1000; Vector) for 4 h. Sections were then washed in 0.175 M sodium 268 acetate buffer 2 x 2 min. Finally, sections were developed using diaminobenzidine in the presence of 269 nickel (DAB Peroxidase Substrate Kit, Vector), mounted on slides, and dried. Sections were then 270 counterstained with cresyl violet, dehydrated, and coverslipped with Permount (Fisher).  271 Doublecortin-expressing cells were quantified in 3 dorsal sections (-2.76 mm to -4.68mm below 272 bregma) and 3 ventral sections (-5.52 mm to -6.60 mm below bregma) using the 40x objective using an 273 Olympus CX22LED brightfield microscope. Areas of these sections were quantified using ImageJ (NIH, 274 Bethesda, MD, USA) and used for density calculations (number of cells per mm2). To determine the 275 maturity of doublecortin-expressing cells, 100 cells positively labeled for doublecortin were randomly 276 selected in the ventral hippocampus because ventral hippocampus is associated with stress regulation and 277 affective behaviors (reviewed in Fanselow & Dong, 2010). Two hundred cells positively labeled for 278 10  doublecortin (100 dorsal and 100 ventral) were randomly selected and categorized as either proliferative 279 (no process or short process), intermediate (medium process with no branching), or post-mitotic (strong 280 dendrite branching in the molecular layer or delicate dendritic tree branching present in the granule cell 281 layer) based on previously published criteria (Plümpe et al., 2006; Workman et al., 2015, see Figure 7A-282 C). 283 2.8. Data Analyses  284  Data collected from the elevated plus maze test, open field test, and novelty suppressed feeding 285 task were analyzed using ANOVA with sex, maternal postpartum CORT, and maternal postpartum 286 fluoxetine as between-subjects factors. Behavior in the elevated plus maze was analyzed using repeated 287 measures ANOVA with arm of maze (closed and open arm) as the within-subjects factor. Behavior in 288 open field test was analyzed using repeated measures ANOVA with area of maze (center, periphery) as 289 within-subjects factor. Behavior in the forced swim test was analyzed using repeated measures ANOVA 290 with behavior (percent time climbing, swimming, and immobile) as the within-subjects factor. CORT 291 concentrations from the dexamethasone suppression test were analyzed using repeated measures ANOVA 292 with time (t=0, beginning of restraint; t=30, end of restraint; t=90, 1 h after restraint ended) as the within-293 subjects factor. The density of doublecortin-expressing cells was analyzed using repeated measures 294 ANOVA with region (dorsal, ventral) as the within-subjects factor. Morphology of doublecortin-295 expressing cells was analyzed using repeated measures ANOVA with region (dorsal, ventral) and type of 296 cell (proliferative, intermediate, post-mitotic) as the within-subjects factor. Post hoc comparisons used 297 Newman-Keuls. Because we had hypotheses that there would be interactions between sex, CORT, and 298 fluoxetine, a priori comparisons were subjected to Bonferroni corrections. All data were analyzed using 299 Statistica software (v. 9, StatSoft, Inc., Tulsa, OK, USA). All effects were considered statistically 300 significant if p ≤ 0.05, trends are discussed if p <0.10. 301 3. Results 302 3.1. Maternal postpartum fluoxetine increased anxiety-like behavior in the elevated plus and novelty 303 suppressed feeding task in adult male, but not female, offspring  304  In the elevated plus maze, maternal postpartum fluoxetine increased the ratio of time spent in the 305 closed arms versus open arms + center in comparison to maternal postpartum saline in adult male (a 306 priori; p=0.023), but not female offspring (p=0.946; figure 2A).  Overall males had a higher ratio of time 307 spent in the closed arms versus open arms + center compared to females (main effect of sex; p=0.027). 308 There was a trend for maternal postpartum fluoxetine to increase ratio of time spent in closed arms versus 309 open arms + center compared to maternal saline in adult male, but not female, offspring (interaction 310 11  between sex and fluoxetine; F(1, 52)=2.78; p=0.099) but no other significant main or interaction effects 311 (all p’s > 0.10). Males spent more time in the closed arms in comparison to females (interaction between 312 arm of maze and sex; F(1, 52)=867.7; p=0.02; Table 1). There were no other significant main or 313 interaction effects for time in open and closed arms (all p’s>0.14). Females had more arm entries into 314 closed arms in comparison to males (interaction between arm of maze and sex; F(1, 52)=13.21; p<0.001) 315 regardless of maternal postpartum CORT or fluoxetine.  Maternal postpartum CORT increased closed 316 arm entries compared to maternal postpartum oil (interaction between arm, maternal postpartum CORT, 317 maternal postpartum FLX; F(1, 52)=4.76; p=0.034; Table 1) within saline exposed offspring (p=0.05) but 318 not fluoxetine exposed offspring (p=0.83). There were no other significant main or interaction effects for 319 arm entries (all p’s>0.11).  320 12  321  322   Mean percent time in the open arms ± SEM Mean percent time in the closed arms ± SEM Mean open arm entries ± SEM Mean closed arm entries ± SEM Maternal OIL/SAL 3.04 ± 0.95 86.92 ± 2.73* 2.50 ± 0.73 8.63 ± 0.68 Maternal CORT/SAL 3.94 ± 2.31 83.44 ± 4.22* 1.83 ± 0.40 10.67 ± 1.38* Maternal OIL/FLX 3.22 ± 1.42 92.44 ± 2.37* 1.17 ± 0.48 8.33 ± 0.56 Maternal CORT/FLX 6.47 ± 3.11 86.80 ± 3.95* 1.60 ± 0.40 9.30 ± 0.79 Maternal OIL/SAL 9.33 ± 3.23 72.26 ± 4.50 2.00 ± 0.55 10.44 ± 0.85 Maternal CORT/SAL 4.50 ± 1.61 82.83 ± 3.13 1.17 ± 0.40 12.17 ± 0.95* Maternal OIL/FLX 4.06 ± 1.33 78.94 ± 2.51 1.33 ± 0.49 12.17 ± 1.35 Maternal CORT/FLX 6.89 ± 3.11 79.70 ± 4.56 1.44 ± 0.34 11.00 ± 0.41 Table 1. Mean ± SEM of additional variables in the elevated plus maze. Males overall spent more time in the closed arms and made fewer closed arm entries (p<0.05). Maternal CORT/saline increased closed arm entries in comparison to maternal oil/saline (p<0.05). CORT: corticosterone; FLX: fluoxetine; SAL: saline.   Figure 2. Anxiety-like behavior as measured by (A) ratio of time spent in closed arms compared to center and open arms (mean + SEM)  in elevated plus maze and (B) latency to feed (mean + SEM)  in novelty suppressed feeding task. Maternal postpartum FLX increased ratio of time spent in the closed arms versus open arms and center of elevated plus maze and increased latency to feed in novelty suppressed feeding task in comparison to maternal postpartum saline in adult male offspring only. Dashed line in (B) represents end of test session (600 seconds). * denotes p<0.05. n=6-10/group/sex. CORT: corticosterone. FLX: fluoxetine. SAL: saline. 13       323 324 In the novelty suppressed feeding task, maternal postpartum fluoxetine increased latency to feed 325 compared with maternal postpartum saline in adult male (a priori; p=0.023), but not female offspring 326 (p=0.801; figure 2B). Females had longer latencies to feed than males (main effect of sex; p<0.001) and 327 there was a trend for maternal postpartum fluoxetine to increase latency to feed in comparison to maternal 328 postpartum saline in adult males only (interaction between maternal fluoxetine and sex; F(1,52)=3.08; 329 p=0.085). There were no other significant main or interaction effects (p’s > 0.086). Lastly, males ate more 330 than females within an hour of returning to their home cage (main effect of sex; p<0.001; Table 2).  331 3.2. Maternal postpartum CORT increased total locomotor activity and peripheral crossings in adult male, 332 but not female, offspring in the open field test. Maternal CORT/fluoxetine decreased peripheral crossings. 333  Maternal postpartum CORT increased total crossings in the open field test compared to maternal 334 postpartum oil in adult male (a priori; p=0.002), but not female offspring (p=0.383; Figure 3A). Females 335 made more total crossings than males (main effect of sex; p<0.001) and maternal postpartum CORT 336 increased total crossings in comparison to maternal postpartum oil controls (main effect of maternal 337 postpartum CORT; p=0.003). There were no other significant main or interaction effects were present for 338 total crossings (all p’s > 0.077).  339  Males* Females Maternal OIL/SAL 23.00 ± 3.65 8.40 ± 1.97 Maternal CORT/SAL 22.00 ± 5.69 7.67 ± 0.67 Maternal OIL/FLX 17.33 ± 1.45 4.33 ± 2.33 Maternal CORT/FLX 18.50 ± 0.65 8.40 ± 0.93 Table 2. Mean (± SEM) food consumption per cage 1 h after novelty suppressed feeding task. Males overall ate more than females within an hour of being returned to their home cage (*p<0.001). CORT: corticosterone; FLX: fluoxetine; SAL: saline.   14   Maternal postpartum CORT increased peripheral crossings in comparison to maternal postpartum 340 oil in adult males (p<0.0001) but not adult females (p=0.40; interaction between area, sex, and maternal 341 CORT; F(1, 52)=4.28; p=0.04; figure 3B). Furthermore maternal postpartum fluoxetine decreased 342 peripheral crossings in comparison to maternal postpartum saline (interaction between area, maternal 343 CORT and maternal fluoxetine; F(1, 52)=4.73; p=0.034; figure 3B) only within the CORT-exposed 344 offspring (p=0.032) but not oil-exposed offspring (p=0.24). There were no significant differences in  345 Figure 3. Locomotor behavior as measured by total crossings (mean + SEM) in open field test (n=6-10/group/sex). Maternal postpartum CORT increased ambulation in adult male offspring only (A). Maternal postpartum oil-exposed males had fewer peripheral crossings in comparison to maternal postpartum CORT-exposed males and oil-exposed females. Maternal postpartum CORT/fluoxetine diminished peripheral crossings in comparison maternal postpartum CORT/saline (B). There were no significant effects on center crossings (see inset in B).   * denotes p<0.05. CORT: corticosterone. FLX: fluoxetine. SAL: Saline. A. B.   Mean percent time in the periphery ± SEM (*) Mean percent time in the center ± SEM Maternal OIL/SAL 96.17 ± 1.34 3.83 ± 0.47 Maternal CORT/SAL 95.29 ± 3.29 4.71 ± 1.34 Maternal OIL/FLX 97.45 ± 0.66 2.55 ± 0.66 Maternal CORT/FLX 94.30 ± 0.86 5.69 ± 0.86 Maternal OIL/SAL 94.79 ± 0.91 5.21 ± 0.91 Maternal CORT/SAL 94.86 ± 0.64 5.14 ± 0.64 Maternal OIL/FLX 96.01 ± 1.09 3.99 ± 1.09 Maternal CORT/FLX 96.20 ± 0.62 3.80 ± 0.62  Table 3. All animals spent more time in the periphery than in the center of the open field (*p<0.001). CORT: corticosterone; FLX: fluoxetine; SAL: saline. 15  center crossings (p’s>0.22).  Animals spent a higher percent time in the periphery of the open field than in 346 the center (main effect of area; p<0.0001). There were no other statistically significant main or interaction 347 effects for percent time in periphery or center (all p’s>0.09; Table 3).  348  349 3.3. Maternal postpartum fluoxetine increased time spent swimming in the forced swim test in both adult 350 male and female offspring 351  Maternal postpartum fluoxetine increased time spent swimming compared with maternal 352 postpartum saline, regardless of maternal postpartum CORT during day 2 of the forced swim test 353 (interaction between maternal fluoxetine and behavior type; F(2, 104)=4.497; p=0.013; Figure 4).  There 354 were no other significant main or interaction effects on any other forced swim test behaviors (all p’s > 355 0.146). To determine if this effect on swimming behavior was affected by day, we further analyzed 356 percent time swimming with a repeated measures ANOVA using day (day 1, day 2) as a within factor. 357 Maternal postpartum fluoxetine increased percent time spent swimming regardless of day, sex or maternal 358 postpartum CORT (main effect of fluoxetine; F(1,52)=5.721, p=0.02; Figure 4B). Additionally, animals 359 had a higher percent swimming on day 2 than day 1(main effect of day; F(1, 52)=176.75, p<0.0001; 360 Figure 4B). 361  362  363  364 Figure 4. Percent time spent swimming, climbing, and immobile (mean + SEM) in forced swim test in both males and females (n=6-10/group/sex). * denotes p<0.05. CORT: corticosterone. FLX: fluoxetine. FST: forced swim test. N.S.: non-significant effect. SAL: saline.  16  3.4. Maternal postpartum fluoxetine impaired HPA axis negative feedback only in adult male offspring in 365 the dexamethasone suppression test. Maternal postpartum CORT enhanced HPA axis negative feedback 366 in both adult male and female offspring. 367  Male and female offspring were analyzed separately, due to the well-established sex differences 368 in HPA axis regulation (reviewed in Viau, 2002). In adult male offspring, maternal postpartum fluoxetine 369 exaggerated male offspring CORT release at t=30 in comparison to maternal saline male controls 370 (interaction between time and maternal fluoxetine; F(2,22)=8.05; p=0.002; Figure 5A). Furthermore, in 371 adult male offspring, maternal postpartum CORT blunted serum CORT release at t=30 in comparison to 372 maternal postpartum oil male controls (interaction between time and maternal CORT; F(2,22)=4.74; 373 p=0.019; Figure 5B). Similarly, in adult female offspring, a priori comparisons revealed that maternal 374 postpartum CORT blunted serum CORT release at t=30 in comparison to maternal postpartum oil in the 375 adult female offspring (p=0.014). No other significant main or interaction effects were present in the 376 female offspring (all p’s > 0.10).  377   Mean percent time climbing ± SEM Mean percent time immobility ± SEM Mean percent time swimming ± SEM  Maternal OIL/SAL 29.22±2.09 34.26±4.00 36.52±3.82 Maternal CORT/SAL 30.63±4.26 36.54±3.98 32.84±5.02 Maternal OIL/FLX 22.99±3.21 43.63±4.01 33.38±5.08* Maternal CORT/FLX 19.33±1.90 33.70±4.90 46.97±5.28* Maternal OIL/SAL 25.16±3.46 33.72±5.31 44.12±5.88 Maternal CORT/SAL 34.66±7.55 27.65±3.88 37.69±7.18 Maternal OIL/FLX 31.10±2.00 18.14±4.98 50.75±4.31* Maternal CORT/FLX 28.51±1.82 28.85±5.37 42.65±5.53*  Table 4. Maternal postpartum fluoxetine increased percent time swimming in comparison to maternal postpartum saline during day 1 of the forced swim test (*p<0.001). CORT: corticosterone; FLX: fluoxetine; SAL: saline. 17  378 3.5. Maternal postpartum fluoxetine and maternal postpartum CORT increased the density of 379 doublecortin-expressing cells in dorsal hippocampus but not ventral hippocampus in adult male offspring. 380 Males had a higher proportion of proliferative doublecortin-expressing cells in comparison to females381  Maternal postpartum fluoxetine increased the density of dorsal, but not ventral, doublecortin-382 expressing neurons compared to maternal saline in adult males (interaction between region, sex, and 383 maternal postpartum fluoxetine; F(1, 51)=3.97; p=0.05; Figure 6A). However this effect was driven by 384 the male offspring also exposed to maternal postpartum CORT/fluoxetine (a priori; p=0.01) but not in 385 maternal oil/fluoxetine group (p=0.79). Intriguingly, the opposite effect was seen in females such that 386 maternal postpartum fluoxetine tended to decrease the density of dorsal doublecortin-expressing 387 immature neurons in adult females compared to maternal postpartum saline controls (p=0.07; Figure 6A). 388 Maternal postpartum CORT increased density of dorsal doublecortin-expressing cells in male offspring in 389 comparison to maternal CORT-exposed female offspring (p<0.001) and to maternal postpartum oil 390 control males (a priori: p=0.023, interaction between region, sex, and maternal CORT; F(1,51)=4.367; 391 p=0.042; Figure 6B). Maternal postpartum CORT diminished the density of doublecortin-expressing cells 392 in the dorsal hippocampus in the adult females in comparison to maternal postpartum oil (p=0.017). There 393 was also significant interaction between region and maternal CORT (p=0.033), main effects of region (p 394 <0.001) and sex (p=0.033) but no other significant main or interaction effects (all p’s>0.21).  395 Figure 5. Serum CORT (mean ± SEM) during dexamethasone suppression tests in males (A-C) and females (D). Maternal postpartum FLX exaggerated CORT release after restraint stress in comparison to maternal postpartum SAL in adult male offspring (A). Maternal postpartum CORT blunted serum CORT release after restraint stress in comparison to maternal postpartum oil in adult male offspring (B). All four maternal experimental groups are displayed for the male offspring (C) and female offspring (D). Only t=30 was greater than all other time points. Solid black line represents 30 min of restraint stress. * denotes p<0.05. n=6-10/group/sex. CORT: corticosterone. FLX: fluoxetine. SAL: saline.  18    396 A. B. C. D. E. Figure 6. Maternal postpartum FLX increased density of dorsal doublecortin-expressing cells (mean + SEM) in the adult male offspring compared to maternal saline in the maternal CORT group only (p<0.01). However maternal postpartum FLX tended to decrease the density of doublecortin-expressing cells in the adult female offspring in the dorsal hippocampus compared to controls (p<0.07) (A). Maternal postpartum CORT increased the density of doublecortin expression in males (p<0.02) but decrease it in the females (p<0.017) in comparison to maternal postpartum oil. There was no significant effect of either sex or maternal postpartum FLX in the ventral hippocampus (see inset). * denotes p<0.05, # denotes p<0.10. B, representative photomicrograph of adult male offspring exposed to maternal postpartum saline; C, representative photomicrograph of adult male offspring exposed to maternal postpartum fluoxetine, scale bar = 100 µm; D, representative photomicrographs of dorsal hippocampus, scale bar = 100 µm; E, representative photomicrographs of ventral hippocampus CORT: corticosterone. DCX+: doublecortin-expressing; SAL: saline. FLX: fluoxetine. 19   397 398  We also examined the phenotype of the doublecortin-expressing cells in both the dorsal and 399 ventral dentate gyrus. Males and females had significantly more proliferative doublecortin-expressing 400 cells in comparison to females, regardless of region (p=0.01; interaction between sex and type of cell; 401 F(2, 100)=274.3; p=0.05; figure 7D). There was a trend for doublecortin morphology to differ based on 402 region (F(2, 100)=2.69; p=0.07; figure 7D) and a main effect of doublecortin morphology with more 403 proliferative cells compared to the other two types of cells and more intermediate cells than post-mitotic 404 cells (all p<0.0002) but no other significant effects (p>0.4) .  405 3.6. Maternal postpartum CORT/fluoxetine diminished body mass of adult male offspring  406  In adult male offspring only, maternal postpartum Oil/fluoxetine increased body mass in 407 comparison to maternal postpartum Oil/Saline whereas maternal postpartum CORT/fluoxetine diminished 408 mass in comparison to maternal postpartum CORT alone or fluoxetine alone (interaction between sex, 409 CORT, and fluoxetine; F(1, 52)=5.120; p=0.028; Table 5). As expected, adult males weighed more than 410 the adult females (main effect of sex; p<0.001). No other significant main or interaction effects were 411 present (all p’s > 0.081).  412 Figure 7. Examples of doublecortin-expressing cells at the proliferative (A), intermediate (B), and post-mitotic stage (C). All offspring expressed a greater proportion of proliferative doublecortin-expressing cells in comparison to intermediate or post-mitotic cells. There was a trend for males to express more proliferative doublecortin-expressing cells than females in the dorsal hippocampus. * denotes p<0.05; # denotes p<0.10. DCX: doublecortin A. B. C. D. 20    413 Table 5. Mean (± SEM) body mass (g). Maternal postpartum FLX alone increased body mass in comparison to maternal postpartum saline in the adult male offspring. Additionally, maternal postpartum CORT/FLX significantly diminished body mass in adult male offspring in comparison to maternal postpartum CORT or FLX alone. * denotes p<0.05. n=6-10/group/sex. CORT: corticosterone; FLX: fluoxetine; SAL: saline.    Males Females Maternal OIL/SAL 503.75 ± 10.95 309.89 ± 9.38 Maternal CORT/SAL 533.50 ± 11.05 283.50 ± 17.23 Maternal OIL/FLX 554.17 ± 18.30* 298.33 ± 11.20 Maternal CORT/FLX 486.20 ± 15.63* 283.56 ± 3.88  21  3.7. Density of doublecortin expression in ventral hippocampus was positively correlated with percent 414 time spent in closed arms of the elevated plus maze in maternal postpartum CORT/fluoxetine male 415 offspring.  416 Among the maternal postpartum CORT/fluoxetine-exposed male offspring, time spent in the 417 closed arms of the elevated plus maze was positively associated with density of ventral hippocampus 418 doublecortin expression (r=0.832; p=0.01; Figure 8). All other variables were either not significant after 419 correcting for multiple correlations or when outliers in correlations were removed. 420 4. Discussion  421  Here we show that maternal exposure to fluoxetine during the postpartum period can have long-422 lasting effects on anxiety-like behavior, HPA axis negative feedback, and hippocampal neurogenesis in 423 adult offspring. In adult male offspring, maternal postpartum fluoxetine increased anxiety-like behavior in 424 the elevated plus maze and novelty suppressed feeding test and density of doublecortin-expressing cells in 425 the dorsal hippocampus. Maternal postpartum fluoxetine also impaired HPA axis negative feedback in 426 males. Perhaps not surprising, both adult male and female offspring from maternal postpartum fluoxetine-427 treated dams exhibited increased swimming behavior in the forced swim test, indicative of enhanced 428 serotoninergic tone (Detke et al., 1995). Maternal postpartum CORT enhanced HPA axis negative 429 feedback, increased locomotor behavior and increased hippocampal doublecortin-expressing cells in adult 430 male offspring. Perhaps the most striking finding in our study is that the majority of effects of maternal 431 postpartum CORT and fluoxetine were seen in adult male offspring. This is consistent with many studies 432 that indicate that males may be more susceptible to perturbations during early development (Stevenson et 433 Table 3. Mean (± SEM) body mass (g). Maternal postpartum FLX alone increased body mass in comparison to maternal postpartum saline in the adult male offspring. Additionally, maternal postpartum CORT/FLX significantly diminished body mass in adult male offspring in comparison to maternal postpartum CORT or FLX alone. * denotes p<0.05. n=6-10/group/sex. CORT: corticosterone; FLX: fluoxetine; SAL: saline.    Males Females Maternal OIL/SAL 503.75 ± 10.95 309.89 ± 9.38 Maternal CORT/SAL 533.50 ± 11.05 283.50 ± 17.23 Maternal OIL/FLX 554.17 ± 18.30* 298.33 ± 11.20 Maternal CORT/FLX 486.20 ± 15.63* 283.56 ± 3.88                                                   Figure 8 – Density of doublecortin-expressing cells in ventral hippocampus was positively associated with percent time in the closed arm of the elevated plus maze in male offspring of CORT/fluoxetine-treated dams.  22  al., 2000; Kent et al., 2002). Collectively, these data reveal that maternal postpartum fluoxetine has long-434 lasting effects on anxiety-like behaviors, the HPA axis, and neuroplasticity in male offspring. 435 4.1. Maternal postpartum fluoxetine increased anxiety-like behavior in adult male offspring, but not 436 female offspring, regardless of maternal postpartum CORT exposure 437 Maternal postpartum fluoxetine increased anxiety-like behavior in adult male but not female 438 offspring in the elevated plus maze and the novelty suppressed feeding tests. This increase in anxiety-like 439 behavior was not due to differences in locomotor activity as maternal postpartum fluoxetine did not affect 440 total crossings in the open field test in either adult male or female offspring. This is in line with similar 441 studies showing that either prenatal fluoxetine exposure (Olivier et al., 2011) or direct administration of 442 fluoxetine to mice pups (postnatal days 2-21; Yu et al., 2014) increases latency to feed in the novelty 443 suppressed feeding test. However, our results are the first to show that fluoxetine increases anxiety-like 444 behavior in adult male offspring when administered to nursing dams, even with concurrent CORT 445 exposure (a model of postpartum stress/depression). In women, maternal postpartum fluoxetine increases 446 breast milk concentration of both fluoxetine and its active metabolite norfluoxetine (Wisner et al., 1996). 447 Therefore, it is possible that in nursing offspring, maternal fluoxetine exposes the developing brain to 448 high levels of serotonin and subsequently disturbs development of the serotonin system. Indeed, 449 developmental disturbances to the serotonin system, such as genetically knocking out the serotonin 450 transporter or the 5HT-1a receptor, are associated with increased anxiety-like behavior (Lira et al., 2003; 451 Lo Iacono & Gross, 2008, respectively), which is consistent with our findings. The relationship between 452 perinatal exposure to fluoxetine and anxiety-like behavior may be related to abnormal activity of the 453 serotonin reuptake transporter and 5-HT1a receptor, both of which are implicated in the etiology of 454 anxiety (SERT: Sen et al., 2004; 5-HT1a: Heisler et al., 1998; Ramboz et al., 1998). Although we did not 455 find an effect of maternal postpartum fluoxetine in adult female offspring in elevated plus maze, possible 456 effects of maternal postpartum fluoxetine on anxiety-like behavior in the novelty suppressed feeding test 457 could have been obscured by a ceiling effect, as most females did not feed in the 10 minute trial. Further 458 studies need to optimize this test for female rats by food depriving for longer, extending the length of the 459 trial, or offering more palatable food (Machado et al., 2013). Moreover, in adult mice, females metabolize 460 fluoxetine faster than males (Hodes et al., 2010; McNamara et al., 2010). Given this sex difference in 461 fluoxetine exposure due to metabolism, it is likely that developmental fluoxetine exposure had a more 462 potent effect on the males than in the females, resulting in larger effects of developmental fluoxetine in 463 males than females. Finally, our results are also consistent with previous work with this model of PPD 464 that have found that maternal postpartum CORT does not increase anxiety-like behavior in adult male or 465 female offspring (Brummelte et al., 2006).   466 23  It should be noted that lower doses of maternal fluoxetine have been shown to not significantly 467 affect anxiety-like behavior in either male or female offspring (7.5 mg/kg/day: Lisboa et al., 2007; 5 468 mg/kg/day: Francis-Oliveira et al., 2013). Additionally, differences in timing of fluoxetine administration 469 may contribute to differences in anxiety-related outcomes as both Lisboa et al., 2007 and Francis-Oliveira 470 et al., 2013 exposed dams during gestation and postpartum whereas the current study exposed dams only 471 in the postpartum. These lower doses of fluoxetine may not be sufficient to alter offspring development, 472 or there may be differences in offspring outcome if dams are treated with fluoxetine throughout gestation 473 as well as postpartum. Furthermore, higher doses of maternal fluoxetine (25 mg/kg/day) during mid-474 gestation (prenatal day 15) through postpartum (postnatal day 12) decreased anxiety-like behavior in adult 475 male (Kiryanova & Dyck, 2014) and female mice (McAllister et al., 2012). Together, this highlights the 476 importance of dose and timing of fluoxetine as crucial methodological factors when evaluating effects of 477 maternal fluoxetine on offspring outcome (reviewed in Kiryanova et al., 2013). 478 4.2. Maternal postpartum fluoxetine increased serotonin-mediated behavior (swimming) in the forced 479 swim test in both adult male and female offspring 480 In the present study, maternal postpartum fluoxetine increased percent time spent swimming, but 481 not percent time spent immobile or climbing, in the forced swim test in both adult male and female 482 offspring. Increased swimming behavior is indicative of increased serotonin activity (Detke et al., 1995). 483 Thus, our findings suggest that maternal postpartum fluoxetine increased serotonin-mediated behavior in 484 both adult male and female offspring. This may not be surprising given the aforementioned evidence that 485 maternal postpartum fluoxetine increases milk concentration of fluoxetine (Wisner et al., 1996). It should 486 be noted that another study did not find a significant effect on swimming behavior after maternal 487 postpartum fluoxetine (Rayen et al., 2011). However, there were differences between studies in terms of 488 dose and administration (Rayen et al., 2011: 5 mg/kg via osmotic mini-pump) as well as age at testing 489 (Rayen et al., 2011: adolescence). Maternal postpartum fluoxetine did not significantly alter immobility, 490 which is inconsistent with studies showing that maternal fluoxetine (7.5 mg/kg/day) increased immobility 491 in adult female but not male mice offspring (Lisboa et al., 2007). However, dose and species differences 492 could account for this discrepancy, as forced swim test outcomes differ between mice and rats (Slattery & 493 Cryan, 2012). Our results confirm previous work with this model of PPD in which maternal postpartum 494 CORT did not significantly affect depressive-like behavior of adult offspring in the forced swim test 495 (Brummelte et al., 2006; Brummelte et al., 2012). Our findings show that maternal postpartum fluoxetine 496 exerts enduring changes in serotonin-related behavior, which may manifest from disturbances to the 497 developing serotonin system following developmental exposure to fluoxetine.  498 24  4.3. Maternal postpartum fluoxetine impaired HPA negative feedback whereas maternal postpartum 499 CORT enhanced HPA negative feedback in adult male offspring 500  In adult male offspring, maternal postpartum fluoxetine exaggerated stress-induced increase in 501 serum CORT concentrations whereas maternal postpartum CORT blunted stress-induced increase in 502 serum CORT concentrations in the dexamethasone suppression test. To our knowledge, no studies have 503 examined the effects of maternal fluoxetine on HPA axis negative feedback in offspring. One study found 504 maternal postpartum fluoxetine blunted serum CORT in adolescent male but not female rat offspring 505 although samples were collected at the time of perfusion (Pawluski et al., 2012c), complicating whether 506 this reflects a basal or stress-induced measure as anesthetics can rapidly increase CORT levels (Wu et al, 507 2015). Clinical findings indicate that prenatal fluoxetine increased corticosteroid-binding globulin levels 508 in neonates (Pawluski et al., 2012a) and blunted evening levels of serum cortisol in 3 month old  infants 509 (Oberlander et al., 2008). Developmental fluoxetine also may alter HPA axis negative feedback by 510 affecting limbic structures that regulate HPA axis activity. For instance, maternal postpartum fluoxetine 511 diminished hippocampal glucocorticoid receptor density in adolescent male but not female rat offspring 512 (Pawluski et al., 2012c). Additionally, maternal fluoxetine during gestation and lactation enhanced 513 activation (Fos expression) in the basolateral amygdala and medial amygdala after restraint stress in adult 514 female but not male rat offspring (Francis-Oliveira et al., 2013). Both the hippocampus and amygdala are 515 sources of limbic control over the HPA axis (reviewed in Herman & Cullinan, 1997) and could therefore 516 contribute to differences in HPA axis negative feedback. Sex differences in stress circuits may underlie 517 these effects of maternal fluoxetine on HPA axis in males. Although we did not find an effect of maternal 518 postpartum fluoxetine on adult female HPA axis activity, it is possible that our dose of dexamethasone 519 was not sufficient to elicit group differences in CORT concentrations. Basal and stress-induced activity of 520 the HPA axis are generally higher in females compared with males and as seen in our data (compare 521 Figure 5C with 5D; reviewed in Goel et al, 2014). Thus, a higher dose of dexamethasone for females may 522 be necessary to optimally assess HPA axis negative feedback (Osborn et al., 1996).  523 Interestingly, maternal postpartum CORT blunted serum CORT concentrations in adult male and 524 female offspring. Previous work with using this model showed that after 1 h of restraint, maternal 525 postpartum CORT did not significantly alter serum CORT concentrations in either adult male or female 526 offspring (Brummelte et al., 2006; Brummelte et al., 2012). This suggests that maternal postpartum 527 CORT results in developmental disturbance specific to negative feedback of the HPA axis. This may be 528 related to the fact the maternal postpartum CORT results in increased brain and serum CORT content in 529 the offspring (Brummelte et al., 2010). Alternatively, maternal postpartum CORT could indirectly affect 530 the developing HPA axis via diminished quality of maternal care (Brummelte et al., 2006; Brummelte et 531 25  al., 2012). Indeed, maternal separation (a similar model of maternal stress/neglect) blunted HPA axis 532 activity in juvenile (Litvin et al., 2010) and adolescent male rats (Ogawa et al., 1994). Additionally, 533 clinical evidence suggests that children under conditions of extreme parental neglect in Romanian 534 orphanages exhibit blunted diurnal cortisol release (Carlson & Earls, 1997). Thus, early life adversity, 535 such as maternal postpartum CORT, can induce permanent disruptions to the HPA axis of both male and 536 female offspring. 537 4.4. Maternal postpartum fluoxetine increased density of doublecortin-expressing cells in the dorsal 538 hippocampus of adult male offspring   539  Maternal postpartum fluoxetine increased density of doublecortin-expressing cells in the dorsal 540 dentate gyrus in adult male offspring. A prior study also showed that maternal postpartum fluoxetine 541 slightly increased density of doublecortin-expressing cells in adult male offspring and decreased it in the 542 adult female offspring (Rayen et al., 2014). However, our results suggest that after behavioral testing, 543 maternal postpartum fluoxetine stimulates doublecortin expression in the dorsal (but not ventral) dentate 544 gyrus, and only in the adult male offspring. This difference might be attributed to a higher dose of 545 fluoxetine (10 mg/kg) than Rayen et al., 2014 (5 mg/kg). Regardless, maternal fluoxetine appears to 546 increase doublecortin expression in adult males although this may be mitigated by prenatal stress (Rayen 547 et al., 2014), but not by maternal postpartum CORT. Indeed, the increase immature neurons due to 548 maternal fluoxetine was only evident in the male offspring of CORT-treated dams. Although the exact 549 mechanism of how maternal fluoxetine influences adult hippocampal neurogenesis in the offspring is not 550 well understood, there are many possible explanations: serotonergic influences, changes in maternal care, 551 or increased environmental enrichment via behavioral testing.  552  One explanation for how maternal postpartum fluoxetine could have disrupted adult offspring 553 hippocampal neurogenesis is that fluoxetine present in the milk directly affected serotoninergic regulation 554 of hippocampal neurogenesis. Indeed, direct administration of fluoxetine to pups enhanced CA1 555 hippocampal dendritic spine density (Zheng et al., 2011) and hippocampal brain derived neurotrophic 556 factor content in adult male mice (Karpova et al., 2009). This enhanced hippocampal plasticity with 557 fluoxetine exposure is in line with findings that adult exposure to chronic fluoxetine administration 558 stimulates hippocampal neurogenesis in adult male rats (Malberg et al., 2000; Huang & Herbert, 2006; 559 David et al., 2009). Thus, our findings that maternal fluoxetine enhances adult hippocampal neurogenesis 560 parallel findings from studies directly exposing pups to fluoxetine. Direct administration of fluoxetine to 561 pups also diminished serotonin terminals in the dentate gyrus in adult male rats (Silva et al., 2010). This 562 supports the possibility that early exposure to fluoxetine itself from the dam may disrupts serotonergic 563 regulation of hippocampal neurogenesis in the offspring.  564 26  Another alternative explanation for how maternal postpartum fluoxetine could have disrupted 565 adult offspring hippocampal neurogenesis is that fluoxetine indirectly affected offspring hippocampal 566 development via alterations in maternal care. In a complimentary study maternal postpartum fluoxetine 567 reversed CORT-induced reductions in maternal care (Workman et al., 2013b; Workman et al., submitted). 568 Therefore, it is possible that the positive effect of maternal postpartum fluoxetine on maternal care 569 resulted in enhanced neurogenesis. This is in line with findings that higher maternal care (licking and 570 grooming) increases hippocampal plasticity in adult offspring whereas lower maternal care reduces it (Liu 571 et al, 2000, Bredy et al., 2003; Champagne et al., 2008). Interestingly, males and females were 572 differentially affected by maternal fluoxetine exposure. Given that there are sex differences in the amount 573 of maternal care pups receive (Moore & Morelli, 1979), it is possible that maternal fluoxetine further 574 skewed the amount of attention male and female pups receive and may explain the opposing effects of 575 maternal fluoxetine on adult offspring hippocampal neurogenesis. 576 Another reason for maternal fluoxetine to selectively increase the density of immature neurons in 577 males is that behavioral testing constituted exploration of a variety of different apparatuses over 10 days 578 and may have an enriching component. Doublecortin is expressed for up to 21 days after the cell divides 579 in rats (Brown et al., 2003), and it is possible that behavioral testing altered doublecortin expression. 580 Environmental enrichment increases doublecortin expression in adult male and female rodents (Leal-581 Galicia et al., 2007; Ramirez-Rodriguez et al., 2014). Interestingly, environmental enrichment increased 582 number of early immature neurons expressing doublecortin in adult male mice selectively in the septal 583 (dorsal) region of the hippocampus (Tanti et al., 2013). This is consistent with our results that maternal 584 postpartum fluoxetine increased doublecortin expression exclusively in the dorsal hippocampus. 585 Therefore, it is possible that maternal postpartum fluoxetine in combination with the enrichment present 586 in the battery of behavioral tests increased neurogenesis specifically in the male offspring.  Alternatively, 587 it is possible that the stress of multiple behavioral and neuroendocrine tests diminished doublecortin 588 expression, except in the male subjects exposed to maternal postpartum fluoxetine. Generally, stress 589 reduces hippocampal neurogenesis but is sex- and stressor-dependent (reviewed in Gobinath et al., 2014). 590 In adult male rodents, fluoxetine can reverse the stress-induced reduction in hippocampal neurogenesis 591 (Malberg & Duman, 2003; reviewed in Warner-Schmidt & Duman, 2006). Therefore, it is possible that 592 maternal postpartum fluoxetine buffered against the stress of multiple behavioral tests in the adult male 593 offspring but not in the other experimental conditions.  594  We also found that maternal postpartum CORT increased density of doublecortin-expressing cells 595 in adult male offspring and diminished the density of doublecortin-expressing cells in adult female 596 offspring. As in the case of maternal postpartum fluoxetine, it is possible that this effect of maternal 597 27  postpartum CORT can be explained by increased environmental enrichment or stress resulting from 598 behavioral testing. Developmental stress is typically associated with detrimental effects on hippocampal 599 plasticity (reviewed in Gobinath et al., 2014). However, early life stress in the form of maternal 600 deprivation (Oomen et al., 2010) or low maternal care (Champagne et al., 2008; Bagot et al., 2009) 601 enhanced long term potentiation of adult-born granule cells in the dentate gyrus only in the presence of 602 CORT.  This suggests that early life adversity could promote hippocampal plasticity under mildly 603 stressful conditions, such as multiple behavioral tests. Moreover, there are sex differences in how early 604 life adversity affects the adult hippocampus (reviewed in Gobinath et al., 2014), which may explain the 605 opposing effects of maternal postpartum CORT on offspring doublecortin expression in this study.  606  We did not find a significant effect of maternal postpartum CORT or fluoxetine on doublecortin-607 expression in either males or females in the ventral hippocampus. This was surprising because we 608 observed significant effects of these maternal treatments on anxiety-like behavior and HPA axis negative 609 feedback regulation, and both affective behavior and HPA axis function are associated with ventral 610 hippocampus activity (reviewed in Fanselow & Dong, 2010).  However, we did observe that among the 611 males exposed to both maternal CORT and fluoxetine, doublecortin expression selectively in the ventral 612 hippocampus was correlated with increased anxiety-like behavior in the elevated plus maze. This suggests 613 that developmental exposure to CORT and fluoxetine may have long-term effects on the association 614 between immature neurons of the ventral hippocampus and neural circuits underlying anxiety-like 615 behavior.  616 4.5. Maternal CORT and concurrent fluoxetine during the postpartum period attenuated body mass in 617 adult male offspring  618  Our results indicate that maternal postpartum CORT and concurrent fluoxetine diminished body 619 mass only in adult male offspring, consistent with a study using prenatal dexamethasone followed by 620 maternal postpartum fluoxetine in adult males (Nagano et al., 2012). This is consistent with findings that 621 maternal fluoxetine diminished body mass in neonatal rats (da Silva et al., 1999) and infants (Chambers et 622 al., 1999). However, maternal postpartum fluoxetine alone increased body mass in adult male offspring.  623 Perinatal exposure to SSRIs (including fluoxetine) is also associated with being overweight in boys but 624 not girls (7 y; Grzeskowiak et al., 2013). Collectively, this suggests that maternal SSRI use can impact 625 body mass of offspring differently depending on developmental time point and whether exposure 626 occurred exclusively during the prenatal, postnatal, or both periods of development.  627 5. Conclusions  628 28  Our data indicate that maternal postpartum fluoxetine can have long-lasting effects on behavioral, 629 endocrine, and neural outcomes of adult offspring in a sex-specific manner. Specifically, adult male 630 offspring were more vulnerable to the effect of maternal postpartum fluoxetine than the female offspring 631 with regards to anxiety-like behavior, HPA axis negative feedback regulation, and hippocampal 632 neurogenesis. However, both adult male and female offspring exhibited more serotonin-dependent 633 behavior in the forced swim test. Finally, maternal postpartum CORT was associated with blunted HPA 634 activity in adult male and female offspring. Collectively, these findings bear implications for treating 635 mothers with pharmacological antidepressants and highlight the importance of studying the consequences 636 of maternal pharmacology on both male and female offspring. 637 CONFLICTS OF INTEREST 638  The authors have nothing to declare.  639 ACKNOWLEDGMENTS  640  The authors would like to thank Lucille Hoover, Nikki Kitay, Robin Richardson, and Cara Tweed 641 for their assistance and contributions throughout the experiment. This work was funded by a CIHR 642 operating grant to LAMG and Coast Capital funding to JLW and LAMG. 643 REFERENCES 644 Azak, S. (2012). Maternal depression and sex differences shape the infants' trajectories of 645 cognitive development. 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