Differential effects of neonatal SSRI treatments on hypoxia-induced behavioral changes in male and female offspring

Accepted Manuscript Differential effects of neonatal SSRI treatments on hypoxia-induced behavioral changes in male and female offspring Reiko Nagano, Masatoshi Nagano, Akihito Nakai, Toshiyuki Takeshita, Hidenori Suzuki PII: DOI: Reference:

S0306-4522(17)30528-6 http://dx.doi.org/10.1016/j.neuroscience.2017.07.051 NSC 17928

To appear in:

Neuroscience

Received Date: Accepted Date:

31 October 2016 19 July 2017

Please cite this article as: R. Nagano, M. Nagano, A. Nakai, T. Takeshita, H. Suzuki, Differential effects of neonatal SSRI treatments on hypoxia-induced behavioral changes in male and female offspring, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.07.051

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Differential effects of neonatal SSRI treatments on hypoxia-induced behavioral changes in male and female offspring

Reiko Naganoa, Masatoshi Naganob*, Akihito Nakaia, Toshiyuki Takeshitaa, and Hidenori Suzukib

a

Department of Obstetrics and Gynecology, bDepartment of Pharmacology, Nippon Medical School,

1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan

E-mail: Reiko Nagano: [email protected] Masatoshi Nagano: [email protected] Akihito Nakai: [email protected] Toshiyuki Takeshita: [email protected] Hidenori Suzuki: [email protected]

*Corresponding author: Masatoshi Nagano Tel: +81 3 3 3822 2131; Fax: +81 3 5814 1684

Keywords: prenatal hypoxia, behavior, intervention, serotonin, dopamine

Abbreviations: 5-HIAA, 5-hydroxyindole acetic acid 5-HT, serotonin 1

CS, conditioned stimulus DA, dopamine DAT, dopamine transporter DOPAC, 3,4-dihydroxy-phenylacetic acid EPM, Elevated plus maze ESC, escitalopram FLX, fluoxetine FST, forced swim test GD, gestational day HPLC, high performance liquid chromatography L/D, Light/dark transition test NET, norepinephrine transporter OF, open field test PFC, prefrontal cortex PPI, prepulse inhibition test SERT, serotonin transporter SSRI, selective serotonin reuptake inhibitor UAO, uterine artery occlusion US, unconditioned stimulus

2

Abstract Prenatal hypoxia induced by transient intrauterine ischemia is a serious clinical problem, and at present, effective treatments are lacking. Currently, it is unknown how prenatal hypoxia affects behaviors in adulthood. Therefore, we developed a mouse model that mimics prenatal hypoxia in humans using uterine artery occlusion in late gestation. We examined whether prenatal hypoxia induces behavioral changes in adult male and female offspring by conducting a series of behavioral tests. In adulthood, longer immobility was observed in the forced swim test in males, whereas females showed decreased inhibition in the prepulse inhibition test. We then investigated the effects of two different selective serotonin reuptake inhibitors (SSRIs), fluoxetine (FLX) and escitalopram (ESC), on these behavioral changes. These drugs affect the neurodevelopmental process and have long-term neurological consequences. FLX treatment from postnatal day 3 (P3) to P21 ameliorated the behavioral changes in both male and female mice. In comparison, ESC treatment ameliorated the behavioral changes only in female mice. Neurochemical analysis revealed that dopamine was increased in the female hippocampus, but not in males. Thus, neonatal SSRI treatment decreases dopamine levels in the hippocampus in females selectively. Our findings suggest that prenatal hypoxia is a risk factor for behavioral abnormalities in adulthood, and that neonatal SSRI treatment might have clinical potential for alleviating these long-term behavioral deficits.

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Introduction Adverse events during the perinatal period, such as infection, stress, injury and exposure to drugs, can affect brain development in the offspring (Rees et al., 2011, Li et al., 2012, Bock et al., 2015, Cordeiro et al., 2015). We previously showed that prenatal glucocorticoid exposure induces behavioral and neurochemical changes in the offspring in adulthood (Nagano et al., 2008, 2012). Intrauterine hypoxia may also affect brain development, and occasionally occurs in clinical practice (Golan and Huleihel 2006). Transient intrauterine ischemia, induced by events such as fetal heart rate changes or placental abruption, sometimes occurs at the late stage of pregnancy. Because heartbeat changes are only detectable when fetal heart rates are monitored, it is possible that some babies suffer transient intrauterine hypoxia during late pregnancy that is not detected. Fetal monitoring is even less common when natural birthing is chosen. While intrauterine hypoxia/ischemia is a major clinical concern, it is unclear how this type of event during the prenatal period impacts fetal brain development or subsequent neurological changes in adulthood. Although many studies have examined the effect of prenatal hypoxia on behaviors at later stages in animal models (Foley et al., 2005, Golan and Huleihel 2006, Dubrovskaya and Zhuravin, 2010, Delcour et al., 2012, Sab et al., 2013), few studies have examined strategies to treat prenatal hypoxia-induced changes. Identifying effective interventions for late emerging symptoms is an important clinical need. We previously developed a rodent model to evaluate the effects of prenatal hypoxia using uterine artery occlusion (UAO) in late gestation, and we examined mitochondrial dysfunction in the fetal brain (Nakai et al., 2001), particularly as abnormal cerebral energy metabolism may affect fetal brain development. Therefore, in the present study, we used our UAO model to examine the effects of prenatal hypoxia on brain development. We also tested the effects of fluoxetine (FLX) and escitalopram (ESC) on the changes induced by the prenatal hypoxia. In addition to functioning as selective serotonin reuptake inhibitors (SSRIs), these drugs affect neurodevelopmental processes and 4

neuronal plasticity, producing long-term neurological effects (Maciag et al., 2006, Mostert et al., 2008, Alboni et al., 2010, Rayen et al., 2011). Furthermore, these drugs modulate functional deficits induced by brain ischemia and exert neuroprotective actions (Chang et al., 2006, Li et al., 2009, Lee et al., 2011, Espinera et al., 2013). We found that UAO had sex-specific effects, inducing differential changes in male and female offspring. Furthermore, neonatal FLX and ESC treatments produced differential effects on these changes in male and female offspring exposed to UAO.

Experimental procedures Animals and transient UAO C57BL/6J female mice at gestational day (GD) 13 were obtained from Japan SLC and housed individually. On GD 16, transient UAO was performed. Under anesthesia with 1.5% isoflurane, an incision was made in the abdomen, and the two uterine horns were exposed and kept moist with saline. The uterine vessels near the lower and upper ends of the right uterine horn were clipped with microvascular clips (201/A, Moria, Tokyo, Japan) for 30 min. The left uterine horn was kept intact. After uterine occlusion, both uterine horns were returned into the abdomen. During operation, core temperature was maintained at 37 °C with a heating pad. On GD 19 (full term), Cesarean section was performed. The fetuses in the right uterine horn were designated UAO mice. The fetuses in the left uterine horn were designated control (Cont) mice. By examining whether the pups could breathe spontaneously, the percentage of dead pups was determined to be 10.7% in the Cont group and 16.5% in the UAO group. Female ICR mice which had just given birth were separated from their own pups and used to foster the C57BL/6J pups (6 to 9 C57BL/6J pups to one ICR mother). ICR fosters were used because the rate of successful fostering in this strain (about 30%) is higher than that in C57BL/6J fosters (less than 10%). All mice were kept in a room with constant temperature (22 ± 1 °C) and under a regular light/dark cycle (lights on from 06:00 to 20:00), with free access to food and water. Both male and female offspring were subjected to examination. All experiments 5

were conducted in accordance with the National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (approval number 27-033).

Drug treatment Fluoxetine hydrochloride (FLX; LKT Laboratories, St. Paul, MN, USA) or escitalopram oxalate (ESC; Sigma-Aldrich, Tokyo, Japan) were administered to pups by daily subcutaneous injection (50 µg/kg, dissolved in saline) from postnatal day 3 (P3) to P21. The affinities of FLX for human monoamine transporters are as follows: serotonin transporter (SERT), 5.7 ± 0.6 nM; norepinephrine transporter (NET), 574 ± 29 nM; dopamine transporter (DAT), 5, 960 ± 989 nM. The affinities of ESC for the transporters are as follows: SERT, 2.5 ± 0.4 nM; NET, 6, 514 ± 423 nM; DAT, >100,000 nM (Owens et al., 2001).

Analysis of serotonin and dopamine levels Brain tissue concentrations of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA; a metabolite of 5-HT), dopamine (DA) and 3,4-dihydroxy-phenylacetic acid (DOPAC; a metabolite of DA) were measured using a high performance liquid chromatography (HPLC) system (Eicom, Kyoto, Japan), as described in our previous study (Nagano et al., 2012, Nakai et al., 2017). The prefrontal cortex and hippocampus, 1 week after the last behavioral test, were used for the analysis. Tissue protein quantification was performed using a protein assay system according to the manufacturer’s protocol (BioRad, USA).

Behavioral tests All behavioral tests were performed between 08:00 and 15:00 during postnatal weeks 8–10. All the apparatuses and analysis software were from O’Hara & Co. Ltd. (Tokyo, Japan). 6

Open field (OF) test. The OF test was performed before the other behavioral tests. The plastic open field chamber was 50 cm (length) × 50 cm (width) × 40 cm (height). The field was illuminated at 40 lux. The behavior was recorded by a CCD camera connected to a computer for 15 min. The traveled distance and percentage of time spent in the center of the field were measured automatically using Image OF software. The center of the field was defined as a central square of 30 cm × 30 cm. Light/dark transition (LD) test. The apparatus consisted of a cage (21 × 42 × 25 cm) divided into two sections of equal size by a partition with a door (4.5 × 3 cm). One chamber was brightly illuminated (600 lux), whereas the other chamber was dark (8 lux). Mice were placed in the dark chamber. After 5 s, the door was opened for 10 min and the mice were allowed to move freely between the two chambers. The time spent on each side and the latency of the first transition to the light chamber were recorded and analyzed automatically using Image LD software. Elevated plus-maze (EPM) test. The apparatus consisted of two open arms (25 × 5 cm) and two closed arms (25 × 5 cm) with 15-cm-high transparent walls. The arms and central square were elevated to a height of 55 cm above the floor. Arms of the same type were arranged diametrically opposite each other. Each mouse was placed in the central square of the maze (5 × 5 cm), facing one of the closed arms. Behavior was recorded during a 10-min test period. Time spent in the open arms was recorded and analyzed automatically using Image EP software. Prepulse inhibition (PPI) test. A startle reflex measurement system was used to measure startle response and PPI. The test session was started by placing a mouse in a plastic cylinder and leaving it undisturbed for 15 min for acclimation in a sound-attenuated chamber with a 70-dB background white noise. The startle response was recorded for 150 ms (with measurement of the response every 1 ms) beginning with the onset of the prepulse stimulus. A test session consisted of six trial types (startle stimulus only trial, and five types of PPI trials). The intensity of the startle stimulus was 120 dB. The prepulse sound (74, 78, 82, 86 or 90 dB) was presented 100 ms before the startle stimulus. Six blocks of the six trial types were presented in a pseudorandom order such that each trial type was 7

presented once within a block. The average inter-trial interval was 15 s (range: 10–20 s). The data for mice whose startle responses were too small (<0.5) were omitted from subsequent analysis. Forced swim test (FST). Plastic cylinders (20 cm in height, 10 cm in diameter) were filled with water at 25 °C up to a height of 11 cm. On the first day, mice were placed into the cylinders and allowed to acclimate for 15 min. Twenty-four hours later, the mice were placed into the same cylinders used the previous day, and their behavior was recorded for 6 min and analyzed automatically with Image TS software. Contextual and cued fear conditioning test. On the first day of the fear conditioning protocol, each mouse was placed in a transparent chamber (11 × 11 × 12 cm) inside a sound-attenuated chamber with white walls and allowed to explore freely for 3 min. A sound of 70 dB, 10 kHz, which served as the conditioned stimulus (CS), was presented for 20 s, followed by a mild foot shock (2 s, 0.3 mA), which served as the unconditioned stimulus (US). One more CS–US pairing was presented with a 2-min inter-stimulus interval. On the second day, contextual testing was conducted in the same chamber for 6 min. On the third day, cued testing with an altered context was conducted using a white opaque Plexiglas chamber (11 × 11 × 12 cm) inside a sound-attenuated chamber with black walls for 6 min. The CS was presented during the last 5 min. Data acquisition, stimulations (i.e. tones and shocks), and data analysis were performed automatically using Image FZ software. Images were captured at 2 frames per second. For each pair of successive frames, the amount of area (pixels) by which the mouse moved was measured. When this area was below 20 pixels, the behavior was judged as freezing. When the area equaled or exceeded the threshold, the behavior was considered non-freezing. Freezing that lasted less than 2 s was not included in subsequent analysis.

Data analysis Values were expressed as means ± SEM. Student’s t-test was used to compare body weights, behavioral measures and monoamine levels in the Cont and UAO groups. The t-test with Welch’s 8

correction was employed for comparing monoamine levels when the variances were not equal. Two-way analysis of variance (ANOVA) was used to compare behavioral measures observed in the time-course analyses, and to compare the Cont-saline, Cont-FLX, Cont-ESC, UAO-saline, UAO-FLX and UAO-ESC groups. Dunnett’s test was used for post hoc comparisons after two-way ANOVA. Statistical significance was set at p < 0.05.

Results Hypoxia by UAO in late pregnancy reduces birth weight in only male offspring All the mice born by Cesarean section were weighed immediately before being given to the ICR foster mothers. Because the birth weight differed among litters (dependent on the mother), the birth weights of UAO mice were compared with the mean weight of Cont littermates of the same sex. In male offspring, the mean birth weight in the UAO group was significantly reduced to 96.4 ± 1.52% (n = 42) of the Cont birth weight (100 ± 0.6%, n = 44; p = 0.033). The mean birth weight of females in the UAO group was not affected (UAO [99.6 ± 1.46%, n = 42] vs. Cont [100 ± 0.66%, n = 30], p = 0.814). However, this difference in body weight was transient. At 8 weeks, just before starting behavioral tests, there were no differences in body weight in males (Cont [23.3 ± 0.33 g, n = 22] vs. UAO [23.3 ± 0.44 g, n = 27], p = 0.980) or females (Cont [18.5 ± 0.29 g, n = 22] vs. UAO [18.4 ± 0.26 g, n = 25], p = 0.628).

Effects of UAO on behaviors OF test. Abnormalities in posture and gait were not observed in male or female UAO offspring during the test. There were no significant differences in the total distance traveled between the Cont and UAO groups in either sex (males: Cont, 5,272 ± 385 cm vs. UAO, 5,219 ± 270 cm, p = 0.909; females: Cont, 5,116 ± 385 cm vs. UAO, 5,785 ± 295 cm, p = 0.175) (Fig. 1A, F). Neither male nor female mice exhibited motor functional deficits. No differences were observed between Cont and 9

UAO mice in the time spent in the center of the field in either sex (males: Cont, 110.1 ± 14.4 s vs. UAO, 88.3 ± 12.5 s, p = 0.265; females: Cont, 90.2 ± 9.5 s vs. UAO, 87.5 ± 9.3 s, p = 0.847) (Fig. 1B, G). LD test. There were no significant differences in the time spent in the light chamber between Cont and UAO mice in either sex (males: Cont, 214.6 ± 13.8 s vs. UAO, 233.3 ± 10.5 s, p = 0.290; females: Cont, 235.3 ± 4.6 s vs. UAO, 225.0 ± 10.0 s, p = 0.406) (Fig. 1C, H). EPM test. There were no significant differences in the time spent in the open arms between Cont and UAO mice in either sex (males: Cont, 64.4 ± 10.8 s vs. UAO, 69.1 ± 14.8 s, p = 0.806; females: Cont, 35.0 ± 8.6 s vs. UAO, 60.5 ± 8.7 s, p = 0.054) (Fig. 1D, I). A non-significant tendency for increased time spent in the open arms was observed in female UAO mice. Together, the results from the OF, L/D and EPM tests suggest that UAO does not lead to anxiety-like behavior in male or female offspring. FST. In male mice, there was a significant difference between the Cont and UAO groups in the FST. Significantly longer immobility was observed in UAO offspring (two-way ANOVA; males: F1, 19 = 6.387, p = 0.021; females: F1, 20 = 0.288, p = 0.597) (Fig. 1E, J). Longer immobility in the FST is an indicator of depression-like behavior. Therefore, UAO may induce depression-like behavior only in male offspring.

Effects of UAO on sensorimotor gating In the startle response to the 120-dB stimulus, there were no significant differences between the Cont and UAO groups in either sex (males: Cont, 3.18 ± 0.36 vs. UAO, 3.11 ± 0.26, p = 0.880; females: Cont, 2.07 ± 0.28 vs. UAO, 1.59 ± 0.20, p = 0.170) (Fig. 2A, C). However, inhibition of the startle response induced by prepulse presentation was significantly reduced only in female UAO offspring (two-way ANOVA; males: F1, 20 = 0.037, p = 0.850; females: F1, 20 = 6.138, p = 0.022) (Fig. 2B, D). Although there was a trend for suppressed inhibition at all prepulse levels, significant suppression 10

was only detected at 78 dB (78-dB prepulse: Cont, 20.8 ± 5.8% vs. UAO, −8.9 ± 7.8% s; p < 0.05) (Fig. 2D). These findings suggest that UAO perturbs sensorimotor gating only in female offspring.

Effects of UAO on learning and memory Fear conditioning test. There were no significant differences between the Cont and UAO groups in either sex in either the contextual test (two-way ANOVA; males: F1, 20 = 0.002, p = 0.963; females: F1, 21 = 2.102, p = 0.162) or the sound-cued test (males: F1, 20 = 0.045, p = 0.834; females: F1, 21 = 0.020, p = 0.888) (Fig. 3). These results suggest that UAO does not affect learning and memory functions, in which the hippocampus and amygdala play key roles. Furthermore, the results of the PPI and fear conditioning tests suggest that UAO offspring do not have a hearing impairment.

Effect of early neonatal treatment with SSRIs (FLX or ESC) on UAO-induced abnormal behaviors in offspring We next examined the effects of FLX and ESC on UAO-induced differential behavioral changes in male and female offspring to evaluate whether these drugs can ameliorate these deficits. In male offspring, neonatal FLX did not affect the total distance traveled in the OF in either the Cont or UAO group, while ESC increased total distance traveled in the UAO group (two-way ANOVA; interaction: F2, 53 = 2.155, p = 0.126; drug: F2, 53 = 8.980, p < 0.001; hypoxia: F1, 53 = 0.477, p = 0.492; Cont-saline, 5,470 ± 314 cm vs. UAO-ESC, 6,890 ± 456 cm, p = 0.043) (Fig. 4A). Neither SSRI affected the time spent in the center (two-way ANOVA; interaction: F2, 53 = 1.758, p = 0.182; drug: F2, 53 = 1.222, p = 0.303; hypoxia: F1, 53 = 1.322, p = 0.255) (Fig. 4B). In the FST, FLX suppressed the UAO-induced increase in immobility time, although it induced longer immobility in Cont offspring (two-way ANOVA; interaction: F2, 53 = 8.181, p < 0.001; drug: F2, 53 = 0.526, p = 0.594; hypoxia: F1, 53 = 2.009, p = 0.162; Cont-saline, 13.9 ± 2.0% vs. UAO-saline, 28.0 ± 3.9%, p = 0.040; Cont-saline vs. UAO-FLX, 18.4 ± 2.3%, p = 0.842; Cont-saline vs. Cont-FLX, 31.0 ± 5.0%, p 11

= 0.009) (Fig. 4C). In comparison, neonatal ESC failed to suppress the UAO-induced increase in immobility time, and it did not induce longer immobility in Cont offspring (Cont-saline vs. UAO-ESC, 28.7 ± 4.4%, p = 0.029) (Fig. 4C). In female offspring, neonatal FLX and ESC (individually) did not affect total distance or center time in the OF in either Cont or UAO offspring (total distance: two-way ANOVA; interaction: F2, 56 = 3.194, p = 0.049; drug: F2, 56 = 1.362, p = 0.265; hypoxia: F1, 56 = 0.347, p = 0.558, Fig. 5A; center time: interaction: F2, 56 = 0.112, p = 0.894; drug: F2, 56 = 2.021, p = 0.142; hypoxia: F1, 56 = 0.033, p = 0.856, Fig. 5B). Neonatal FLX suppressed the startle response in Cont offspring, in contrast to ESC, which suppressed the response in UAO offspring (two-way ANOVA; interaction: F2, 56 = 5.429, p = 0.007; drug: F2, 56 = 1.954, p = 0.151; hypoxia: F1, 56 = 4.175, p = 0.046; Cont-saline, 2.4 ± 0.3 vs. Cont-FLX, 1.4 ± 0.1, p = 0.012; Cont-saline vs. UAO-ESC, 1.4 ± 0.1, p = 0.005) (Fig. 5C). In the PPI test, both FLX and ESC effectively attenuated the UAO-induced reduction in inhibition, especially with the 78-dB prepulse (two-way ANOVA; interaction: F2, 56 = 7.596, p = 0.001; drug: F2, 56

= 4.935, p = 0.011; hypoxia: F1, 56 = 1.616, p = 0.209; Cont-saline, 22.3 ± 5.5% vs. UAO-saline,

−1.32 ± 3.1%, p = 0.002; Cont-saline vs. UAO-FLX, 24.2 ± 4.8%, p = 0.998; Cont-saline vs. UAO-ESC, 24.1 ± 3.7%, p = 0.999) (Fig. 5D).

Effect of UAO and neonatal SSRI treatment on 5-HT and DA levels in the brain Our findings above indicate that UAO induces behavioral abnormalities in both male and female offspring, and that neonatal SSRI treatment ameliorates these abnormalities. Therefore, it is possible that changes in brain monoamine levels occur in the offspring, and accordingly, we next examined the levels of 5-HT, DA and their metabolites in brain regions that are considered to be involved in the observed behavioral changes (Table 1). In the prefrontal cortex (PFC), which is an important region for executive functioning and the regulation of emotional behaviors (Moghaddam and Homayoun, 2008), levels of 5-HT, DA and their primary metabolites, 5-HIAA and DOPAC, tended to be reduced 12

in the UAO group compared with the Cont group in males and somewhat in females, but the differences were not significant. In contrast, in the hippocampus, a region associated with PPI (Bast and Feldon, 2003), levels of DA and DOPAC were highly and significantly increased only in female offspring (DA, p = 0.016; DOPAC, p < 0.001). There were no significant differences in 5-HT levels in either sex. We further examined the effects of SSRIs on monoamine levels in the PFC in males. Although the differences were not significant, the effect of UAO seemed larger in the PFC than in the hippocampus. Again, 5-HT, DA and their primary metabolites tended to be reduced in the UAO-saline group, although the differences were not significant. We then assessed the effects of SSRI treatment. Significant drug effects were observed for 5-HT levels, but there were no significant differences among the groups (two-way ANOVA; interaction: F2, 44 = 2.005, p = 0.147; drug: F2, 44 = 3.442, p = 0.041; hypoxia: F1, 44 = 0.049, p = 0.825) (Table 2). However, in animals subjected to UAO, both FLX and ESC had a tendency to increase 5-HT levels. Because significant changes were observed in the hippocampus (Table 1), we further examined the effects of SSRIs on the female hippocampus (Table 3). Although a significant difference was not observed in a comparison of the six groups, DA levels tended to be increased in the UAO-saline group (Cont-saline, 100.0 ± 10.0 vs. UAO-saline, 174.7 ± 29.1%, p = 0.057). Moreover, a significant drug effect was found, and both SSRIs tended to suppress the UAO-induced increase in DA levels (two-way ANOVA; interaction: F2, 46 = 2.609, p = 0.085; drug: F2, 46 = 3.346, p = 0.044; hypoxia: F1, 46

= 2.601, p = 0.114; Cont-saline vs. UAO-FLX, 120.9 ± 23.6%, p = 0.934; Cont-saline vs.

UAO-ESC, 69.8 ± 20.0%, p = 0.773).

Discussion Our present study shows that intrauterine hypoxia induced by UAO in late gestation (GD16) produces differential changes in behaviors and monoamine levels in both male and female offspring 13

in adulthood. Moreover, neonatal SSRI treatment suppresses these UAO-induced changes. UAO led to a lower birth weight, but only in male offspring. A likely reason for the change in birth weight is growth restriction caused by reduced placental blood flow. However, the reason why the lower birth weight was observed only in male offspring is unknown. The difference in the birth weight between male and female offspring may be one of the causes of the differential behavioral outcomes observed in adulthood. In adulthood, UAO induced longer immobility in the FST in male offspring, and it impaired PPI in female offspring. Learning and memory impairment has been reported in many rodent studies of prenatal hypoxia (Cai et al., 1999, Foley et al., 2005, Golan and Huleihel 2006, Dubrovskaya and Zhuravin, 2010, Delcour et al., 2012), suggesting that the hippocampus is particularly vulnerable to hypoxia (Schmidt-Kastner 2015). Although we examined learning and memory in the fear conditioning test in this study, we did not detect any impairment. This discrepancy between our current findings and previous studies could be due to differences in timing or the duration of hypoxia (Golan and Huleihel 2006). Furthermore, some studies employed complete clamping of four uterine vessels (Cai et al., 1999, Sab et al., 2013), and another subjected only low birth weight offspring to further behavioral analysis (Delcour et al., 2012). Such differences in hypoxic conditions may cause differential abnormalities in the offspring. In this study, we adopted a unilateral two-vessel occlusion of the uterine horn, and we did not clip the vessels too tight, in an effort to minimize vessel lesions and the inflammatory response. UAO also differentially affected DA levels in the hippocampus in the two sexes, increasing it significantly only in adult females. Many studies showed that there are sex differences in behavioral outcomes induced by prenatal (Tashima et al., 2001, Wang et al., 2013) or postnatal (Hill et al., 2011, Sanches et al., 2013, Smith et al., 2014, Waddell et al., 2016) hypoxia. The mechanisms underlying the sex differences are not fully understood, but could, in part, stem from differences in the hypothalamic–pituitary–adrenal axis (Wang et al., 2013) and sex steroid levels. Testosterone might 14

be detrimental to neuronal survival by increasing the neurotoxicity of glutamate (Yang et al., 2002). In comparison, estrogen may ameliorate the ischemia-induced brain damage by impacting neuronal function (Waddell et al., 2016). Progesterone might also be neuroprotective by affecting the cell death pathway (Espinosa-García at al., 2013). Brain developmental differences might also contribute to the observed dissimilarities between the sexes, particularly as they have been observed in animal models of schizophrenia (Piontkewitz et al., 2012). Based on these previous observations, it is not surprising that there are sex differences in the outcomes induced by perinatal hypoxia. Further studies are needed to clarify the mechanisms underlying these sex differences. We also examined the ability of SSRIs, FLX and ESC, given neonatally, to ameliorate the behavioral changes in the offspring of mothers subjected to UAO. We chose these SSRIs because of the following reasons: (1) FLX has long-term effects on neurological disorders and has been shown to facilitate recovery from ischemia-induced neurological deficits after stroke (Chang et al., 2006, Li et al., 2009, Chollet et al., 2011, Li et al., 2012). Citalopram also has such actions (Maciag et al., 2006, Alboni et al., 2010, Lee et al., 2011, Espinera et al., 2013); (2) UAO induced longer immobility in the FST in male offspring. The FST is the most commonly used test to assess depressive behavior and examine the effect of antidepressant drugs. Both FLX and ESC have antidepressive effects in the FST (Wolak et al., 2015) and have demonstrated efficacy for post-stroke depression (Chollet et al., 2011); and (3) In female offspring, UAO reduced PPI. Reduced PPI is associated with increased DA transmission (Bast and Feldon, 2003). 5-HT and DA have opposing effects in some neural systems (Boureau and Dayan, 2011). Therefore, we conjectured that upregulation of 5-HT levels may counteract the increased DA and ameliorate the reduction in PPI in female UAO mice. In male mice, neonatal FLX treatment suppressed the UAO-induced lengthening of immobility in the FST, while it induced longer immobility in Cont offspring. In contrast, neonatal ESC failed to suppress the change, and had no effect on Cont offspring. These findings suggest that changes in 15

brain 5-HT levels in the neonatal stage are not likely the cause of this behavioral deficit. Both SSRIs are likely to have increased synaptic 5-HT levels, at least during treatment. However, FLX and ESC had opposing effects, including those on Cont offspring. Neonatal FLX treatment induced longer immobility in the FST in Cont offspring. FLX is used for the treatment of psychiatric disorders, such as anxiety and depression. Thus, the finding that it induces a depressive change in control mice is difficult to explain. However, it is in agreement with previous studies showing that neonatal FLX treatment induces depression-like behaviors in normal adult mice (Ansorge et al., 2004) and rats (Boulle et al., 2016). A recent report of human subjects found a correlation between low birth weight and the incidence of depressive symptoms in adolescence (Machado et al., 2015). Consistent with the report, we detected longer immobility in the FST only in male UAO offspring, whose birth weight was significantly lower than that of Cont offspring. Furthermore, 5-HT levels in the PFC in adulthood were not related to immobility in the FST. Interestingly, while both FLX and ESC tended to increase 5-HT levels in the PFC in UAO offspring, the effects on immobility were opposing. The effect of FLX observed here might be independent of its blockade of 5-HT transporters (SERTs), particularly as it was different from that of ESC. In the OF, neonatal ESC significantly increased the distance traveled in UAO males, but only slightly in UAO females. In line with this observation, postnatal citalopram, from P8 to P21, has been reported to increase locomotor activity in rats (Maciag et al., 2006). In female offspring, we observed an impairment in PPI. It is unlikely that the female UAO offspring had an auditory abnormality, because UAO female offspring responded to the softest sound (70 dB) in the fear conditioning test. Some reports have examined the relationship between schizophrenia and brain hypoxia during development (Schmidt-Kastner et al. 2012, Dela Cruz et al., 2014) and the relationship between hypoxia and dysregulation of the DA system (Laplante et al., 2012, Pagida et al., 2013, Dela Cruz et al., 2014). PPI impairment is one of the typical symptoms of 16

schizophrenia. Moreover, it is reported that, in the hippocampus, dopaminergic stimulation results in reduced PPI, whereas decreased DA transmission may enhance PPI (Bast and Feldon, 2003). These observations suggest that increased DA levels in the hippocampus observed in this study might affect sensorimotor functioning and diminish PPI in female offspring. However, it remains unclear why DA upregulation in the hippocampus and PPI impairment were observed only in females. Laplante and colleagues (2012) reported sex differences in DA levels induced by perinatal anoxia in the rat brain. These investigators observed a tendency for decreased DA in the PFC and significantly increased DA in the amygdala in females. In this study, we also observed a trend towards decreased DA in the PFC in female UAO mice. However, the mechanisms underlying the sex and regional-specific changes in DA levels remain unclear. Our findings suggest a long-term impact on sex-dependent brain development. In contrast to male offspring, both FLX and ESC suppressed the reduction in PPI in female UAO offspring. FLX reduced the amplitude in Cont mice, while ESC reduced it in UAO mice. However, the amplitude of the startle response did not seem to relate to PPI levels here, and the mechanisms by which neonatal SSRI treatments affect the startle response is not clear. A previous study reported that chronic FLX treatment reduces the startle response in adult male rats (Raz and Berger, 2010), and that perinatal chronic citalopram treatment increases the startle response (Sprowles et al., 2016). It is possible that the differential effects of the SSRIs on the acoustic startle response are not related to the blockade of SERTs. As discussed above, if increased hippocampal DA levels are responsible for the PPI reduction in UAO mice, the reduction in DA levels produced by neonatal SSRIs may be responsible for the amelioration of the PPI impairment. Chronic treatment with both FLX and citalopram has been shown to reduce levels of tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of DA, in the rat substantia nigra (MacGillivray et al., 2011). Thus, it is possible that both SSRIs reduce DA synthesis in the substantia nigra, thereby lowering DA levels in the hippocampus. Both FLX and ESC might have the same function, other than the blockade of SERTs, in females. 17

Further research is needed to fully understand the mechanisms by which neonatal SSRIs suppress the abnormal behaviors induced by prenatal hypoxia. Importantly, our current findings suggest that SSRIs might have the potential to prevent abnormalities in newborns induced by prenatal hypoxia. However, neonatal SSRI treatment had unexpected and sex-specific effects in offspring, which need to be addressed in future research. SSRIs might be particularly useful in cases where intrauterine hypoxia is detected, as in cases when fetal heart rate is monitored, and when placental abruption occurs in humans. It is important to note that SSRIs showed efficacy despite being administered several days later than the prenatal hypoxic event, indicating that the neonatal period is a critically important window for intervention. Future studies using lower doses, shorter treatment durations and a combination of SSRIs will help optimize intervention strategies and identify potential complications and side effects. Investigating the mechanisms underlying the effects of FLX and citalopram may advance the development of more effective drugs and help adapt therapeutic strategies according to sex.

Acknowledgements This study was supported by Grants-in-aid for Science Research (C) [project no. 17K10085 to M.N.] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Authorship Contributions Participated in research design: R. Nagano, M. Nagano, Nakai and Suzuki; Conducted experiments: R. Nagano and M. Nagano; Developing animal models: M. Nagano and Nakai; Performed data analysis: M. Nagano; Wrote or contributed to the writing of the manuscript: R. Nagano, M. Nagano, Nakai, Takeshita and Suzuki.

Conflict of interest 18

All authors report no conflicts of interest.

19

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25

Table 1 Effects of uterine artery occlusion (UAO) on monoamine levels in the brain. male Mean ± SEM

female t-test t value P value

Mean ± SEM

t-test

Cont

UAO

t value

P value

0.187

100±8.76

89.2±2.78

1.177

0.264

1.156

0.266

100±7.64

97.8±4.79

0.245

0.809

0.23±0.01

0.539

0.601

0.25±0.00

0.27±0.02

1.166

0.259

100±9.42

88.0±5.78

1.048

0.311

100±13.7

71.4±4.38

1.988

0.073

DOPAC

100±8.92

89.2±5.34

1.010

0.329

100±13.7

93.8±8.83

0.3832

0.707

DOPAC/DA

0.51±0.02

0.51±0.03

0.183

0.857

0.38±0.04

0.56±0.04 *

2.524

0.022

5-HT

100±6.03

98.5±8.65

0.143

0.889

100±3.89

106±5.29

0.932

0.366

5-HIAA

100±8.03

99.9±7.37

0.007

0.994

100±4.93

115±4.56 *

2.246

0.04

5-HIAA/5-HT

0.48±0.04

0.49±0.03

0.226

0.824

0.46±0.02

0.52±0.02

1.956

0.069

DA

100±17.8

72.9±8.99

1.305

0.211

100±8.29

232±41.8 *

3.09

0.016

DOPAC

100±9.04

123±18.9

1.175

0.258

100±4.88

161±10.5 **

5.426

<0.001

DOPAC/DA

0.66±0.11

1.06±0.25

1.505

0.153

0.55±0.06

0.42±0.06

1.521

0.149

%

Cont

UAO

5-HT

100±3.26

91.8±4.95

1.384

5-HIAA

100±7.29

88.6±6.51

5-HIAA/5-HT

0.23±0.01

DA

PFC

HIP

Monoamine levels are expressed as a % of the Cont value and statistical analyses were performed on these % values. *p < 0.05; **p < 0.01 vs. Cont. (male PFC: Cont, n = 9; UAO, n = 8; female PFC: Cont, n = 10; UAO, n = 10; male hippocampus (HIP): Cont, n = 9; UAO, n = 8; female HIP: Cont, n = 9; UAO, n = 8). Control values in the PFC (male: 4.6 ± 0.2 ng 5-HT/mg tissue protein, 1.2 ± 0.1 ng 5-HIAA/mg, 480 ± 48 pg DA/mg, 270 ± 24 pg DOPAC/mg; females: 4.9 ± 0.4 ng 5-HT/mg, 1.3 ± 0.1 ng 5-HIAA/mg, 550 ± 73 pg DA/mg, 270 ± 24 pg DOPAC/mg). Control values in the HIP (male: 4.6 ± 0.3 ng 5-HT/mg, 2.4 ± 0.2 ng 5-HIAA/mg, 270 ± 49 pg DA/mg, 160 ± 15 pg DOPAC/mg; females: 5.0 ± 0.2 ng 5-HT/mg, 2.5 ± 0.1 ng 5-HIAA/mg, 200 ± 18 pg DA/mg, 110 ± 6 pg DOPAC/mg).

26

Table 2 Effects of neonatal SSRIs on monoamine levels in the male PFC. male

PFC

Hypoxia effect

Drug effect

Interaction

%

Cont-saline

UAO-saline

Cont-FLX

UAO-FLX

Cont-ESC

UAO-ESC

F value P value F value P value F value P value

5-HT

100±2.72

89.1±7.04

106±7.82

105±3.53

88.2±3.13

97.5±4.98

0.049

0.825

3.442

0.041

2.005

0.147

5-HIAA

100±4.17

90.6±10.3

118±10.8

105±5.24

91.6±4.75

98.8±7.05

0.717

0.402

3.019

0.059

1.019

0.370

5-HIAA/5-HT

0.25±0.02

0.26±0.04

0.29±0.03

0.26±0.02

0.30±0.01

0.29±0.01

0.194

0.662

1.281

0.288

0.535

0.589

DA

100±21.5

61.4±19.3

133±47.8

155±51.0

75.4±6.52

201±93.6

0.765

0.386

1.003

0.375

1.369

0.265

DOPAC

100±11.3

68.8±14.9

91.4±28.1

124±14.5

92.1±7.85

94.0±9.16

0.004

0.950

1.251

0.296

2.266

0.116

DOPAC/DA

0.59±0.07

0.48±0.05

0.70±0.23

0.63±0.17

1.04±0.19

0.86±0.189

0.883

0.353

3.719

0.032

0.068

0.935

Monoamine levels are expressed as % of the Cont value and statistical analyses were performed on these % values (Cont-saline, n = 10; UAO-saline, n = 9; Cont-FLX, n = 7; UAO-FLX, n = 8; Cont-ESC, n = 7; UAO-ESC, n = 9). Control values in the male PFC (4.0 ± 0.1 ng 5-HT/mg, 1.1 ± 0.1 ng 5-HIAA/mg, 630 ± 247 pg DA/mg, 280 ± 39.5 pg DOPAC/mg).

Table 3 Effects of neonatal SSRIs on monoamine levels in the female hippocampus. female %

Cont-saline

UAO-saline

Cont-FLX

Hypoxia effect

UAO-FLX

Cont-ESC

Drug effect

Interaction

UAO-ESC

F value P value F value P value F value P value

5-HT

100±1.99

111±8.86

95.3±8.61

101±9.76

99.3±10.6

101±7.86

0.799

0.376

0.411

0.666

0.139

5-HIAA

100±4.08

102±7.69

107±6.70

96.4±8.23

122±7.14

99.4±5.80

0.240

0.627

1.240

0.299

0.209

0.871 0.812

5-HIAA/5-HT

0.61±0.03

0.57±0.02

0.57±0.03

0.50±0.03

0.65±0.04

0.62±0.02

2.162

0.148

8.132

<0.001

0.993

0.378

HIP DA

100±10.0

175±29.1

84.9±13.0

121±23.6

94.3±24.9

69.8±20.0

2.601

0.114

3.346

0.044

2.609

0.085

DOPAC

100±7.38

99.8±9.08

103±8.76

89.8±17.7

145±26.2

118±13.4

1.371

0.248

3.665

0.033

0.468

0.629

DOPAC/DA

1.10±0.18

0.70±0.14

1.60±0.14

0.93±0.16

1.44±0.32

1.53±0.23

4.310

0.044

4.690

0.014

1.857

0.168

Monoamine levels are expressed as % of the Cont value and statistical analyses were performed on these % values (Cont-saline, n = 9; UAO-saline, n = 10; Cont-FLX, n = 8; UAO-FLX, n = 9; Cont-ESC, n = 7; UAO-ESC, n = 9). Control values in the female HIP (4.5 ± 0.2 ng 5-HT/mg, 3.0 ± 0.2 ng 5-HIAA/mg, 152 ± 21.5 pg DA/mg, 233 ± 30.5 pg DOPAC/mg).

27

Figure Legends Fig. 1. Effects of uterine artery occlusion (UAO) on emotional behaviors in male and female offspring. Upper panels (A–E) represent male offspring. Lower panels (F–J) represent female offspring. (A, B, F, G) Open field (OF) tests. (C, H) Light/Dark (LD) transition test. (D, I) Elevated plus maze (EPM) test. (E, J) Forced swim test (FST). Control (Cont) offspring, open bars and circles; UAO offspring, gray bars and circles. Data represent the mean ± SEM. *p < 0.05, vs. Cont offspring. The numbers in parentheses indicate the number of mice tested and their litters (mice/litters) in each group.

Fig. 2. Effects of UAO on sensorimotor gating in male and female offspring examined by the acoustic startle response and prepulse inhibition (PPI). Upper panels (A, B) represent male offspring. Lower panels (C, D) represent female offspring. (A, C) Acoustic startle response to 120-dB startle stimuli. (B, D) PPI tests. Cont offspring, open bars; UAO offspring, gray bars. Data represent the mean ± SEM. *p < 0.05, vs. Cont offspring. The numbers in parentheses indicate the number of mice tested and their litters (mice/litters) in each group.

Fig. 3. Effects of UAO on learning and memory in male and female offspring examined with the acoustic fear conditioning test. Upper panels (A, B) represent male offspring. Lower panels (C, D) represent female offspring. (A, C) Context exposure test on the 2nd day. (B, D) Sound cue exposure test on the 3rd day. The cue sound was presented after 1 min of acclimation. Cont offspring, open circles; UAO offspring, gray circles. Data represent the mean ± SEM. *p < 0.05, vs. Cont offspring. The numbers in parentheses indicate the number of mice tested and their litters (mice/litters) in each group.

Fig. 4. Effects of neonatal fluoxetine (FLX) or escitalopram (ESC) treatment on UAO-induced 28

behavioral changes in male offspring. (A, B) OF tests. (C) FST on the 2nd day. Cont offspring, open bars; UAO offspring, gray bars. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, vs. Cont-saline offspring. The numbers in parentheses indicate the number of mice tested and their litters (mice/litters) in each group.

Fig. 5. Effects of neonatal FLX or ESC treatment on UAO-induced behavioral changes in female offspring. (A, B) OF tests. (C) Acoustic startle response to 120-dB startle stimuli. (D) PPI tests. Two-way ANOVA at each prepulse level (74 dB: interaction: F2, 56 = 3.225, p = 0.047; drug: F2, 56 = 2.101, p = 0.132; hypoxia: F1, 56 = 0.074, p = 0.786; 78 dB: interaction: F2, 56 = 7.596, p = 0.001; drug: F2, 56 = 4.935, p = 0.011; hypoxia: F1, 56 = 1.616, p = 0.209; 82 dB: interaction: F2, 56 = 5.313, p = 0.008; drug: F2, 56 = 0.759, p = 0.473; hypoxia: F1, 56 = 0.136, p = 0.714; 86 dB: interaction: F2, 56 = 3.916, p = 0.026; drug: F2,

56

= 0.639, p = 0.532; hypoxia: F1,

56

= 0.008, p = 0.928; 90 dB:

interaction: F2, 56 = 1.725, p = 0.1875; drug: F2, 56 = 1.410, p = 0.253; hypoxia: F1, 56 = 0.044, p = 0.835). Cont offspring, open bars; UAO offspring, gray bars. Data represent the mean ± SEM. *p < 0.05, vs. Cont-saline offspring. The numbers in parentheses indicate the number of mice tested and their litters (mice/litters) in each group (In the PPI test, sample numbers were equal to those for the startle response in (C)).

29

2000 1000 0

G 80 60 40 20 Cont UAO (10/6) (13/6)

80

50

60 40 20

250 200 150 100 50 Cont UAO (10/6) (13/7)

UAO (11/6)

40

*

30 20 10

Cont (10/6)

0

0 Cont UAO (10/6) (11/6)

I L/D

0

0

60

Cont UAO (10/6) (11/6)

Light time (sec)

Total distance (cm)

Center time (sec)

Fig.1.

0

OF 100

Cont UAO (10/6) (13/7)

50

H

OF 7000 6000 5000 4000 3000 2000 1000 0

100

Cont UAO (10/6) (12/6)

Cont UAO (10/6) (12/6)

F

150

FST on 2nd day

100

% immobility

3000

200

E EPM

1

J EPM 70 60 50 40 30 20 10 0

2

3

4

min

5

6

FST on 2nd day

60

% immobility

4000

Light time (sec)

5000

L/D 250

140 120 100 80 60 40 20 0

Center time (sec)

Total distance (cm)

6000

D

OF

Open arm time (sec)

C

OF

Open arm time (sec)

B

A

Cont (10/6)

50 40 30 20

UAO (12/6)

10 0

Cont UAO (10/6) (13/7)

1

2

3

4

min

5

6

A

Startle response

B

PPI

60 50 40 30 20 10 0 -10

% inhibition

Startle amplitude

4 3 2 1 0

74

Cont UAO (10/6) (12/6)

Startle response

60 50 40 30 20 10 0 -10 -20

2

1

0 Cont UAO (10/6) (12/7)

Fig.2.

78

82

86

90

86

90

prepulse (dB)

D

% inhibition

Startle amplitude

C

Cont (10/6) UAO (12/6)

PPI Cont (10/6) UAO (12/7)

* * 74

78

82

prepulse (dB)

A

contextual at 2nd day

B 60

UAO (11/5)

40 30 20 10

% immobility

% immobility

50

50 40 30 20 10

Cont (11/5)

0 1

2

C

3

4

min

5

1

6

UAO (13/7)

0

Fig.3.

3

4

min

5

6

50 40 30 20 10

Sound cued

0 2

4

min

sound cued at 3rd day

% immobility

% immobility

30

1

3

60

40

10

2

D

Cont (10/5)

20

Sound cued

0

contextual at 2nd day 50

sound cued at 3rd day

5

6

1

2

3

4

min

5

6

Sound cue d

B

8000 6000

FLX

*

4000 2000 0

Cont UAO Cont UAO Cont UAO (10/6)(10/6) (10/6)(12/7) (7/4)(10/5)

Fig.4.

C

OF

ESC

saline 140 120 100 80 60 40 20 0

FLX

FST on 2nd day

ESC

saline 40

% immobility

OF saline

Center time (sec)

Total distance (cm)

A

30

FLX

* **

ESC

*

20 10

Cont UAO Cont UAO Cont UAO (10/6)(10/6) (10/6)(12/7) (7/4)(10/5)

0

Cont UAO Cont UAO Cont UAO (10/6)(10/6) (10/6)(12/7) (7/4)(10/5)

B

OF FLX

8000 6000 4000 2000 0

Cont UAO Cont UAO Cont UAO (11/6)(12/6) (9/6)(13/7) (7/6)(10/6)

D

74dB

160 140 120 100 80 60 40 20 0

% inhibition

FLX

Startle response

ESC

saline

FLX

ESC

3 2

*

**

1

Cont UAO Cont UAO Cont UAO (11/6)(12/6) (9/6)(13/7) (7/6)(10/6)

82dB

78dB

60 50 40 30 20 10 0 -10

C

OF saline

ESC

Startle amplitude

saline

Center time (sec)

Total distance (cm)

A

0

Cont UAO Cont UAO Cont UAO (11/6)(12/6) (9/6)(13/7) (7/6) (10/6)

86dB

90dB

** saline

FLX

ESC

Fig.5.

saline

FLX

ESC

saline

FLX

ESC

saline

FLX

ESC

saline

FLX

ESC

Highlights    

Prenatal hypoxia produced differential behavioral changes in male and female offspring. Neonatal fluoxetine, an SSRI, ameliorated the behavioral changes in both males and females. Neonatal escitalopram, an SSRI, ameliorated the behavioral changes only in females. Both SSRIs suppressed the increase in dopamine levels induced by prenatal hypoxia in the female hippocampus.

32