Effects of maternal stress and perinatal fluoxetine exposure on behavioral outcomes of adult male offspring
Introduction
Clinical studies show that maternal stress is linked to unfavourable pregnancy outcomes, such as reduced gestational length, lower birth weight, and impaired emotional and cognitive development in infants and children (Beydoun and Saftlas, 2008). Maternal stress increases a child’s risk for developing affective and psychiatric disorders, such as schizophrenia and autism (for a review see Talge et al., 2007, Kinney et al., 2008). Stress can precipitate a depressive episode and may accompany depression, which may occur during pregnancy (for a review see Monroe and Hadjiyannakis, 2002, Tennant, 2002). The prevalence of depression among pregnant women is 10–20% (Kitamura et al., 1993, Andersson et al., 2003, Marcus et al., 2003, Gavin et al., 2005), and can reach 51% in pregnant women of low socio-economic status (Hobfoll et al., 1995, McKee et al., 2001). Selective serotonin reuptake inhibitors (SSRIs) are the primary pharmacological treatment for depression (Mandrioli et al., 2012). SSRI use in pregnant women is on the rise, with 6.2% exposed to this medication (Cooper et al., 2007, Andrade et al., 2008). Fluoxetine (Flx) (brand name Prozac, Sarafem, Rapiflux) is one of the most commonly prescribed SSRIs, with 1.9–2.1% of women taking Flx at one point during pregnancy (Cooper et al., 2007, Andrade et al., 2008). In fact, Flx is one of the top 20 specific prescription medications most commonly taken by pregnant women (Mitchell et al., 2011).
Preclinical studies of gestational stress show a number of alterations in the anatomy, physiology, and behavior of the offspring. Some of the anatomical changes include decrease in hippocampal neurogenesis in males (Morley-Fletcher et al., 2011), alteration in serotonin 2A receptor and metabotropic glutamate receptor 2 expression in the brain (Holloway et al., 2013), and sex-dependent decrease in pyramidal cell and hippocampal glial cell density (Behan et al., 2011). Exposure to gestational stress has also been found to alter feedback inhibition of the hypothalamic–pituitary–adrenal axis function (Vallee et al., 1997, Dugovic et al., 1999, Vallee et al., 1999, Koenig et al., 2005, but see Behan et al., 2011) and to decrease brain-derived neurotrophic factor (BDNF) levels in the frontal cortex of adult offspring (Fumagalli et al., 2004, Matrisciano et al., 2012). Behavioral changes associated with perinatal stress have been extensively studied. However, studies have found contradictory results for every behavior, including learning and memory (Vallee et al., 1997, Stohr et al., 1998, Vallee et al., 1999, Lehmann et al., 2000, Nishio et al., 2001, Bustamante et al., 2010), exploratory behavior (Vallee et al., 1997, Miyagawa et al., 2011, Matrisciano et al., 2012), and depressive- and anxiety-like behaviors (Stohr et al., 1998, Bosch et al., 2006, Miyagawa et al., 2011, Morley-Fletcher et al., 2011).
Depression is often viewed as a stress-related disorder given the relationship between stressful life events and the onset of depressive episodes (Kessler, 1997). SSRIs are the most common medications used to treat depression and, pre-clinically, SSRI treatment has been found to reverse many of the adverse effects of chronic stress with a similar response/non-response profile as seen in the clinic (Levinstein and Samuels, 2014). That being said, there is very little work examining the interactive effects of stress and SSRI treatment during pregnancy on the outcome of the offspring. This is especially relevant because the number of women taking SSRI medication during pregnancy has increased dramatically over the last decade with four times as many pregnant women having received SSRI treatment in 2006 than 1994 (Andrade et al., 2008). Therefore, it is common for a human fetus to be exposed to a combination of maternal stress and SSRIs. Flx and its active metabolite norfluoxetine (NorFlx) cross the placental barrier (Kim et al., 2006), and are excreted in breast milk, resulting in plasma concentrations that can reach therapeutic levels in breastfed infants (Lester et al., 1993, Brent and Wisner, 1998). Given that serotonin modulates a multitude of developmental processes, availability of serotonin alters the organization of the brain, as well as behavior later in life (Nordquist and Oreland, 2010); therefore, it is important to understand long-term effects of perinatal exposure to Flx. Though a considerable amount of research has been conducted on the effects of perinatal Flx exposure (Alwan and Friedman, 2009), there has been a lack of focus on the outcomes of exposed individuals beyond the preschool age. To address this concern, many laboratories have turned to modeling the developmental effects of perinatal SSRI exposure in non-humans. SSRIs cross the placental barrier in several mammalian species, producing a comparable level of fetal exposure to that seen in humans (Morrison et al., 2005, Noorlander et al., 2008, Olivier et al., 2011). SSRIs cross the fetal blood–brain barrier of rodents, leading to detectable Flx and NorFlx levels in the blood and brain of pups (Ansorge et al., 2004, Noorlander et al., 2008, Capello et al., 2011, Olivier et al., 2011, Nagano et al., 2012, Kiryanova and Dyck, 2014).
Preclinical studies demonstrate that perinatal exposure to Flx has a number of short-term and long-term effects (for a review of the subject, see Kiryanova et al., 2013a). This exposure leads to increased aggression (Singh et al., 1998, Kiryanova and Dyck, 2014), alterations in circadian system (Kiryanova et al., 2013b), change in emotional memory (Olivier et al., 2011, Rebello et al., 2014), and increase or decrease in anxiety-like and depressive-like behaviors (Mendes-da-Silva et al., 2002, Lisboa et al., 2007, Noorlander et al., 2008, Karpova et al., 2009, Olivier et al., 2011, Kiryanova and Dyck, 2014, Rebello et al., 2014).
At this time, few studies examine combined effects of perinatal stress and Flx exposure on adult offspring outcomes. Rayen et al. (2013) demonstrated that Flx and stress have specific long-term effects on reproductive behavior and sexual brain differentiation of the adult offspring. Other studies demonstrate that while maternal exposure to stress and Flx can have distinct effects, treatment with Flx early in life can counteract some of the effects of maternal stress, including alterations in hippocampal morphology (Ishiwata et al., 2005, Rayen et al., 2015), spatial learning (Ishiwata et al., 2005), and sensitivity to post-operative pain (Knaepen et al., 2013). Behaviors in adults have not yet been examined aside from reproductive behavior and spatial memory.
The present study investigated the effects of prenatal maternal stress and the effects of perinatal maternal exposure to Flx, separately and in combination, on the behavior of the adult male offspring. This study sought to provide a more complete insight by conducting an in-depth analysis of the offspring’s cognitive ability, memory, aggression, anxiety, sensorimotor information processing, and exploratory and risk assessment behaviors. Both, stress and antidepressant treatments, can regulate brain BDNF levels in adulthood and during development (Fumagalli et al., 2004, Krishnan and Nestler, 2008, Karpova et al., 2009, Matrisciano et al., 2012). Furthermore, early stress and early exposure to Flx have the ability to affect offspring’s serotonin levels (Peters, 1990, Cabrera-Vera et al., 1997). Therefore, early-life alterations in brain serotonin levels and later-life changes in prefrontal cortex BDNF levels were also examined in this experiment.
Section snippets
Animals
Animals were kept on a 12:12-h light/dark (LD, 1500 lx/0 lx) and were provided ad libitum access to food (LabDiet Mouse Diet 9F, #5020) and water throughout the experiment with the exception of stress manipulation measures (see below). Animal treatment and husbandry were performed in accordance with the Canadian Council on Animal Care. All experimental procedures were approved by the Ethics Committee for Animal Research at the University of Calgary.
C57BL/6 breeders were obtained from the
Data analysis
Tests that involved a single measurement (i.e., open field, EPM, PPI) were analyzed using a two-way (Flx × stress) factorial analysis of variance (ANOVA). For tasks that involved repeated testing of the animals (e.g., passive avoidance, Morris water test, and levels of serotonin, Flx and NorFlx in different brain regions) a split plot ANOVA was computed. Tukey’s post hoc test for multiple comparisons was used to assess differences between individual treatment groups. Protected t-tests using the
Pup retrieval
In the pup-retrieving test, 88% of all dams were able to retrieve their first and last pup within the first 10 min. There was no difference in how long it took each group to approach the pups, latency to pick up the first pup, latency to retrieve the first and the last pup, or in the proportion of dams in each group that did not retrieve their first and last pup within 10 min of the test.
Assessment of maternal behavior
On-going Flx administration reduced the amount of time dams spent self-grooming out of nest (Flx: 1.72 ± .47% of
Open field
There was no effect of prenatal stress, Flx treatment, nor an interaction of the two, on time spent in the outer arena, speed, and distance traveled (see Table 3A).
Elevated plus
Mice perinatally exposed to Flx (FLX and PS + FLX) spent more time on the open arms of the EPM (perinatal Flx: 20.43 ± 2.3%time; no perinatal Flx: 10.46 ± 2.3% time; F(1,64) = 9.48, p = .003; Fig. 2A) and showed a greater number of head dips (perinatal Flx: 11.50 ± 1.1 head dips; no perinatal Flx: 8.0 ± 1.1 head dips; F(1,64) = 5.01, p = .029) when
Discussion
The results of this study demonstrate that prenatal exposure to chronic stress and perinatal exposure to Flx have a number of effects on the behavior of male offspring when assessed as adults. Maternal stress, exposure to Flx, and their combination did not affect cognitive abilities, social behavior, emotional memory, spatial memory, PPI, and startle response of the offspring. However, prenatal stress led to an increase in activity and reduced levels of BDNF in the frontal cortex, regardless of
Conclusion
In this paper we demonstrate that maternal exposure to stress and Flx have long-lasting, dissociable effects. These behavioral outcomes are not likely to be a result of changes to maternal behavior, as neither treatment affected maternal behavior of mouse dams in this study. We show that Flx affects some, but not all behaviors. Behaviors that are affected by Flx, such as an increase in aggression and a decrease in anxiety, are, not surprisingly, those that rely on serotonergic regulation.
Competing interests
The authors declare that no competing interests exist. The authors have full control of all primary data and agree to allow the journal to review their data if requested.
Acknowledgment of funding
This work was supported by NSERC Discovery and CIHR Operating grants to RHD, CIHR operating funds to MNH, an NSERC graduate scholarship to VK and a CIHR summer studentship to SI.
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