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Exposure to Female Pheromones During Pregnancy Causes Postpartum Anxiety in Mice
C H A P T E R
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Exposure to Female Pheromones During Pregnancy Causes Postpartum Anxiety in Mice Caroline M. Larsen and David R. Grattan Contents I. Materials and Methods A. Subjects B. Anxiety testing C. Maternal behavior testing D. Serum prolactin levels in early pregnancy E. Neurogenesis F. Statistical analysis II. Results A. Exposure to unfamiliar female pheromones throughout pregnancy increased anxiety in postpartum mice B. Anxious postpartum females displayed impaired maternal behavior C. Exposure to female pheromones decreased serum prolactin levels in early pregnancy D. The role of suppressed prolactin in mediating pheromoneinduced postpartum anxiety E. Female pheromone exposure throughout pregnancy decreased neurogenesis on day 7 of pregnancy III. Discussion References
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Abstract The postpartum period is associated with an increased incidence of pathological anxiety, exerting a substantial burden on both the mother and the baby. We have shown that pharmacological suppression of prolactin in early pregnancy decreases maternal neurogenesis to cause postpartum anxiety. The present data demonstrate that physiological suppression of prolactin secretion through Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand Vitamins and Hormones, Volume 83 ISSN 0083-6729, DOI: 10.1016/S0083-6729(10)83005-5
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2010 Elsevier Inc. All rights reserved.
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exposure to unfamiliar female pheromones throughout pregnancy prevented the normal postpartum attenuation of anxiety in mice, resulting in high anxiety relative to postpartum controls. Female pheromone-exposed mice also showed severely impaired maternal behavior in an anxiogenic situation. Mice exposed to female pheromones had decreased serum prolactin levels in early pregnancy, resulting in an ablation of the normal increase of neurogenesis on day 7 of pregnancy. These data demonstrate that low serum prolactin levels in early pregnancy, whether induced pharmacologically or as a physiological consequence of exposure to unfamiliar female pheromones, result in failure to show the normal adaptive decrease in anxiety after birth. This provides new insight into possible mechanisms that might underlie postpartum anxiety in women. ß 2010 Elsevier Inc.
During the postpartum period, there is an increased incidence of dysfunctional mood disorders (Munk-Olsen et al., 2006; Stowe and Nemeroff, 1995), with pathological anxiety, the most common (Britton, 2005, 2007; Matthey et al., 2003; Wenzel et al., 2003). Postpartum anxiety can severely affect the mother–infant relationship (Manassis et al., 1994), and have longlasting adverse effects on cognitive, emotional, and other aspects of infant development (Barnett et al., 1991; O’Connor et al., 2002). Postpartum mood disorders are also associated with an increased incidence of child abuse (Fraser et al., 2000), and increased conflict in marital relationships leading to a higher rate of separation and divorce (Boyce and Stubbs, 1994; Lovestone and Kumar, 1993). Attempts to understand the mechanisms underlying postpartum mood disorders have been impeded by the lack of an animal model that induces anxiety or depression postpartum, while retaining the unique hormonal characteristics and social stresses of gestation (NIH, USA, PA-09-174, 2009). The anterior pituitary hormone prolactin is critically involved in a number of adaptive responses in the maternal brain (Grattan et al., 2008). We have recently shown that pharmacological suppression of prolactin very early in pregnancy in mice decreased adult neurogenesis in the maternal brain during pregnancy and induced anxiety postpartum, potentially providing a novel model of postpartum mood disorders. The aim of this study was to determine whether more subtle physiological changes in prolactin during pregnancy, such as might occur in response to stress or infection (Brunton et al., 2008), might also alter mood and behavior postpartum. Exposure to pheromones alters levels of prolactin and neurogenesis in virgin female mice (Larsen et al., 2008; Mak et al., 2007). Therefore, we hypothesized that exposure to pheromones during pregnancy might similarly influence prolactin secretion during pregnancy, thereby inducing changes in mood postpartum. We exposed pregnant mice to male, female, or no pheromones throughout pregnancy and subsequently assessed anxiety on day 2 postpartum on an elevated plus maze (EPM) and in the light–dark box (LDB), well-characterized models of rodent anxiety. To determine
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whether pheromone exposure would change essential postpartum behaviors, we also examined maternal behavior, both in the home cage and in a clean novel cage (as an anxiogenic challenge; Larsen and Grattan, 2010). Finally, to identify possible mechanisms of pheromone action, pheromoneinduced changes in prolactin secretion and levels of neurogenesis in the subventricular zone (SVZ) were analyzed.
I. Materials and Methods A. Subjects Maternally naive virgin or day 1 pregnant C57BL/6J mice (6–9 weeks old) were either housed in 32 16 18 cm individual cages or in 28 54 18 cm split cages with another male, or a female, allowing pheromonal, visual, and olfactory contact without permitting physical contact (Larsen et al., 2008; all groups n ¼ 6). Some females were paired with males, and were considered day 1 pregnant when mating was confirmed by sperm being present in a vaginal smear, at which time they were transferred into the cages, as above. Pheromones are bound to major urinary proteins, and hence placing urine-soaked bedding in with the female will expose her to pheromones without the confounding effects of a companion. To confirm that effects seen were due to pheromones, additional groups were exposed to urine-soaked sawdust obtained from another mouse for 23 days prior to behavioral testing (Larsen et al., 2008). Virgin female and day 1 pregnant control groups had 0.5 g of clean sawdust placed in the cage daily for 23 days. After initial studies revealed an effect of female pheromones on anxiety and maternal behavior, all subsequent studies were undertaken using only female pheromones (i.e., without the male pheromone group). Mice were housed under a 12-h light–dark cycle, with ad libitum access to food and water. The University of Otago Animal Ethics Committee approved all experiments.
B. Anxiety testing Day 2 postpartum or virgin females were assessed for anxiety on the EPM and the LDB, as previously described (Larsen and Grattan, 2010). All testing was carried out between 9.30 and 10.30 a.m. in the room where the animals were usually housed. To avoid habituation to stress, the animals were not handled prior to the experiment. Elevated plus maze (EPM): To assess anxiety, the mouse was placed on the central platform facing an open arm, and allowed to roam at will for 5 min. All arm entries and exits were recorded. Once a mouse had all 4 paws on an arm of the maze, it was counted as having entered that arm, while placing 2 paws out of an arm was
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counted as an exit. Light–dark box (LDB): The LDB had an enclosed dark box (18 27 27 cm; black perspex sides, bottom and top, approx 30 lux) separated by a partition with a hole (6 cm) from a light box (27 27 27 cm; clear perspex, with a transparent cover with ventilation holes in it; lit 56 cm from above with a 100 W light bulb, approx 753 lux). Mice were placed at the entrance to the dark box and allowed to roam at will for 5 min. All entries and exits to the light or dark box were recorded. Once a mouse had all 4 paws within a box it was counted as having entered that box, while placing 2 paws out of a box was counted as an exit. To further evaluate the role of prolactin in female pheromone-induced anxiety, pheromone-exposed pregnant mice were injected s.c. with prolactin (50 mg of purified ovine prolactin in saline) at 4.30 p.m. from day 1 to day 3 of pregnancy (to mimic the early pregnancy surges in prolactin).
C. Maternal behavior testing Day 2 postpartum mice had their pups removed from the home cage. Immediately, three foster pups were placed into the cage and maternal behaviors recorded. To assess maternal behavior in an anxiogenic situation, another group of postpartum day 2 females were placed into a clean novel cage into which three foster pups had been placed, and any behavior recorded (as described previously, Larsen and Grattan, 2010). Full maternal behavior was defined as being when the mouse had gathered all three pups to a nest and was crouching over them. Testing was continued for a maximum of 120 min. Testing was performed between 9.30 and 11.30 a.m.
D. Serum prolactin levels in early pregnancy Pregnant mice, either exposed to no pheromones or female pheromones, were sacrificed for collection of trunk blood every 4 h from 9 a.m. on day 1 of pregnancy to 9 p.m. on day 4 of pregnancy for measurement of prolactin by radioimmunoassay using NIH reagents, as described previously (Larsen et al., 2008). Sensitivity was 2 ng/ml, and all samples were run in a single assay, with an intra-assay % C.V. 2.5%.
E. Neurogenesis Virgin female mice, or day 7 pregnant mice exposed to female or no pheromones, were injected with bromodeoxyuridine (BrdU), as reported previously (Larsen et al., 2008). Briefly, BrdU (Sigma) was injected every 2 h from 5 a.m. to 3 p.m. (6 injections of 12 mg/100 g of body weight, dissolved in phosphate buffer). Mice were then perfused with 4% paraformaldehyde at 5 p.m. BrdU-labeled cells were detected using immunohistochemistry
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(mouse monoclonal anti-BrdU, 1:200, Dako, Carpinteria, CA). Dual label immunofluorescence histochemistry was performed to determine whether the BrdU-labeled cells were neurons using doublecortin as a marker (goat antidoublecortin, 1:100, Santa Cruz). For quantification, the SVZ was observed under 20 magnification, and BrdU-immunoreactive cells counted in a systematic manner after a random start, such that all labeled nuclei were counted in 1-in-6 serial sections (120 mm apart) for a total of 14 sections throughout the SVZ (approximately Bregma þ1.94–3.9 mm). Data are presented as the total counts collected, and are not corrected for the total number of sections.
F. Statistical analysis The data are expressed as mean SEM. The amount of time on the open arms is expressed as a percentage of total time on the open and closed arms (open time/(open þ closed time) 100). Data were compared by ANOVA and Fishers PLSD, P < 0.05 was used as the level of significance.
II. Results A. Exposure to unfamiliar female pheromones throughout pregnancy increased anxiety in postpartum mice As has been previously reported (Maestripieri and D’Amato, 1991; Larsen and Grattan, 2010), postpartum females spent significantly more time on the open arms of the EPM than virgin controls (Fig. 5.1A and B, þP < 0.05), indicative of reduced anxiety. In contrast, postpartum females exposed to female pheromones for the duration of pregnancy, either by split-cage housing with a virgin female (Fig. 5.1A, *P < 0.05), or by exposure to urine-soaked bedding (Fig. 5.1B, *P < 0.05), were significantly more anxious on the EPM compared to postpartum controls. In contrast, there was no effect of exposure to male pheromones on anxiety on the EPM in postpartum females (Fig. 5.1A and B). Data from the LDB showed a similar increase in postpartum anxiety in the female pheromone-exposed animals. Postpartum mice exposed to female pheromones had a significant increase in the amount of time spent in the dark chamber of the LDB, and a decreased latency to enter the dark chamber of the LDB compared to postpartum controls (Fig. 5.1C, *P < 0.05). The data show that female pheromone exposure for the duration of pregnancy induces postpartum anxiety.
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Figure 5.1 Female pheromone exposure throughout pregnancy increases anxiety. Elevated plus maze (EPM). Day 2 postpartum controls spent significantly more time on the open arms of an EPM compared to virgin mice (þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy, either by split-cage housing (A), or by exposure to urine-soaked bedding (B), spent significantly less time on the open arms of the EPM compared to day 2 postpartum controls (*P < 0.05), and therefore displayed a significant increase in anxiety. In contrast, exposure to male pheromones throughout pregnancy had no effect. Light–Dark Box (LDB). Postpartum controls had an increased latency to enter the dark chamber of the LDB, and spent significantly less time in the dark chamber of the LDB compared to virgin controls (C, þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy had a decreased latency to enter the dark chamber of the LDB, and spent significantly more time in the dark chamber of the LDB, displaying a significant increase in anxiety compared to postpartum controls (C, *P < 0.05).
B. Anxious postpartum females displayed impaired maternal behavior To determine whether pheromone-induced postpartum anxiety affected maternal behavior, maternal behavior was examined using a pup retrieval paradigm in pheromone-exposed mice. As reported previously, in the
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Figure 5.2 Female pheromone exposure throughout pregnancy inhibits postpartum maternal behavior in an anxiogenic situation. Maternal behavior in a home cage. Day 2 postpartum mice were significantly faster to express maternal behavior to foster pups compared to virgin controls (A, þP < 0.05). Exposure to female pheromones had no effect, either by split-cage housing (data not shown), or by exposure to urine-soaked bedding (A), while mice exposed to male pheromones for the duration of pregnancy expressed maternal behavior more rapidly to foster pups compared to all other groups (A, *P < 0.05). Maternal behavior in a novel cage. Both day 2 postpartum controls, and male pheromone-exposed females, were significantly faster to express maternal behavior to foster pups than virgin controls (B and C, þP < 0.05). Day 2 postpartum mice exposed to female pheromones for the duration of the pregnancy displayed significantly impaired maternal behavior to foster pups in an anxiogenic situation compared to both the male pheromone-exposed mice and the individually housed controls (B and C, *P < 0.05).
home cage, postpartum females were significantly faster to express maternal behavior compared to virgin controls (Fig. 5.2A, þP < 0.05), and females exposed to male pheromones throughout pregnancy were significantly faster to express maternal behavior than postpartum controls (Fig. 5.2A, *P < 0.05; Larsen et al., 2008). Maternal behavior in mice exposed to
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female pheromones was not significantly different compared to postpartum controls when tested in the home cage (Fig. 5.2A). In the anxiogenic situation of a novel cage, all groups tested took significantly longer to express full maternal behavior compared to that in the home cage. Both day 2 postpartum control mice and male pheromone-exposed females remained significantly faster to express full maternal behavior compared to virgin controls, yet, there was now no significant difference between these two groups (Fig. 5.2B and C, þP < 0.05). In contrast, however, female pheromone-exposed mice placed in an anxiogenic situation (novel cage) displayed dramatically impaired maternal behavior to foster pups (Fig. 5.2B and C, *P < 0.05). The female pheromone-exposed mice investigated the pups occasionally, but did not gather them to their nest, or sit with the pups. Instead, they made a nest some distance away from the pups and either took significantly longer to express full maternal behavior or, in the majority of animals, did not express full maternal behavior at all within the designated 120-min observation period.
C. Exposure to female pheromones decreased serum prolactin levels in early pregnancy To test the hypothesis that changes in prolactin secretion during pregnancy might underlie the pheromone-induced alterations in mood postpartum, levels of serum prolactin during early pregnancy were measured by radioimmunoassay. Although the previously described pattern of twice daily prolactin surges in early pregnancy was maintained (Fig. 5.3, þP < 0.05; Larsen and Grattan, 2010), serum prolactin levels in pregnant mice exposed to female pheromones were significantly lower than those of controls at many time points on days 1–3 of pregnancy (Fig. 5.3, *P < 0.05; # urinesoaked sawdust added at 9 a.m. daily). From day 5 of pregnancy there were not any significant differences between the two groups.
D. The role of suppressed prolactin in mediating pheromoneinduced postpartum anxiety To test whether the decreased serum prolactin in early pregnancy induced by exposure to female pheromones was specifically responsible for inducing postpartum anxiety, we injected pheromone-exposed mice with prolactin at 5 p.m. daily from day 1 to day 3 of pregnancy. Animals were subsequently tested for anxiety on day 2 postpartum, approximately 3 weeks after hormone treatment. Remarkably, restoring normal prolactin levels in early pregnancy completely prevented the increased anxiety seen in postpartum mice exposed to female pheromones throughout pregnancy (Fig. 5.4). This implies that prolactin in early pregnancy is specifically
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Figure 5.3 Serum prolactin levels from day 1 to day 2 of pregnancy. Pregnant mice had a twice daily surge in serum prolactin levels with an afternoon peak just prior to lights off at 6 p.m. and a smaller nocturnal peak approximately 1 h before lights on (þP < 0.05). Pregnant mice had urine-soaked sawdust placed in the cage at 9 a.m. daily throughout pregnancy (#P < 0.05). Pregnant mice exposed to female pheromones showed significantly decreased serum prolactin levels at many time points from day 1 to 3 of pregnancy, compared to pregnant controls (*P < 0.05). From day 5 of pregnancy there were no significant differences between the groups (data not shown).
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Figure 5.4 Restoration of prolactin prevents the female pheromone-induced increase in anxiety postpartum. Day 2 postpartum controls spent significantly more time on the open arms of an EPM compared to virgin mice (þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy by exposure to urine-soaked bedding spent significantly less time on the open arms of the EPM compared to day 2 postpartum controls (*P < 0.05), and therefore displayed a significant increase in anxiety. Female pheromone-exposed pregnant mice injected with prolactin from day 1 to day 3 of pregnancy had no significant differences in anxiety compared to postpartum controls.
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required for adaptive responses that occur in the maternal brain during pregnancy and become evident as behavioral changes postpartum.
E. Female pheromone exposure throughout pregnancy decreased neurogenesis on day 7 of pregnancy One known action of prolactin in the brain during early pregnancy is an increase in neurogenesis in the SVZ of the maternal brain (Larsen and Grattan, 2010; Shingo et al., 2003), and we have previously shown that pharmacological suppression of prolactin during early pregnancy led to a decrease in neurogenesis in this region (Larsen and Grattan, 2010). Therefore, we examined whether this was also affected by exposure to female pheromones during pregnancy. Decreased serum prolactin in early pregnancy, induced by exposure to female pheromones, significantly reduced levels of neurogenesis in the SVZ on day 7 of pregnancy compared to postpartum controls, reducing the numbers of BrdU-labeled cells to that of a virgin nonpregnant mouse (Fig. 5.5A, *P < 0.05). There were no significant differences in the percentage of BrdU-labeled cells in the SVZ that were double labeled with doublecortin, a neuronal marker, between any of the groups (Fig. 5.5B).
III. Discussion Day 2 postpartum mice displayed decreased anxiety on the EPM compared to virgin mice, consistent with previous experimental findings (Larsen and Grattan, 2010; Maestripieri and D’Amato, 1991). In contrast, female pheromone exposure throughout pregnancy significantly increased anxiety on day 2 postpartum. In women, postpartum anxiety can impair maternal responsiveness to, and care of the infant (Barnett et al., 1991; Fraser et al., 2000; O’Connor et al., 2002). In a similar manner, mice exposed to female pheromones for the duration of pregnancy also displayed dramatically impaired responsiveness to, and an extreme lack of care for pups when placed in an anxiogenic situation. The combined syndrome of an increase in anxiety together with impairment of maternal behavior suggests that exposing mice to female pheromones throughout pregnancy provides a novel model for studying postpartum mood disorders. Importantly, this approach is completely noninvasive, and retains the unique characteristics of gestation. We have previously shown that pharmacological suppression of serum prolactin levels, solely in early pregnancy, can initiate increased postpartum anxiety over 2 weeks later (Larsen and Grattan, 2010). The results shown here indicate that postpartum anxiety caused by female pheromone exposure is also induced by low prolactin during early pregnancy. Moreover,
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Figure 5.5 Female pheromone exposure throughout pregnancy decrease neurogenesis on day 7 of pregnancy. (A). Day 7 pregnant mice had a significant increase in the number of BrdU-labeled cells in the subventricular zone (SVZ) compared to virgin controls (þP < 0.05). Day 7 pregnant females exposed to female pheromones for 7 days showed a significant decrease in BrdU-labeled cells in the SVZ compared to controls (*P < 0.05). Micrographs show representative images from the SVZ, with black staining representing BrdU-labeled cells (Lat. V.: lateral ventricle). (B) There were no significant differences between pheromone-exposed or control mice, in the percentage of BrdU-labeled cells that coexpressed Dcx. Representative merged images immunoreactive for BrdU (green) and Dcx (red) in the lateral ventricle of the SVZ in each group are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)
reminiscent of the pharmacological suppression of prolactin, injections of prolactin to restore serum prolactin levels in early pregnancy in pheromoneexposed mice completely prevented the pheromone-induced increase in anxiety. This suggests that the pheromone-induced decrease in prolactin in early pregnancy is the mechanism underlying the induction of postpartum anxiety. In our previous work using pharmacological suppression of prolactin, we identified a potential role for the increase in SVZ neurogenesis on mood
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postpartum (Larsen and Grattan, 2010). In the present study, serum prolactin levels in mice exposed to female pheromones during pregnancy were significantly lower than in pregnant controls, but they were still higher than basal levels seen in virgin mice, and higher than the levels seen in mice when prolactin was suppressed pharmacologically (Larsen and Grattan, 2010). Nevertheless, the decrease in prolactin induced by female pheromone exposure was sufficient to completely suppress the pregnancy-induced increase in neurogenesis in the SVZ. These data support the hypothesis that increased neurogenesis in the SVZ is important for adaptive changes in mood postpartum. Extensive work has linked impaired neurogenesis in other brain regions to the development of mood disorders (Duman et al., 2001), but how the increase in SVZ neurogenesis during pregnancy moderates mood postpartum remains unclear. The implication of our data is that any physiological or pathophysiological factor that might influence prolactin levels or neurogenesis during pregnancy could lead to development of mood disorders. Previous studies examining the hormonal regulation of mood during the peripartum period have predominantly focused on acute changes in hormones at that time, and no clear role of hormones in the control of mood has emerged. The present data support the hypothesis that levels of hormones in early pregnancy are crucial for euthymic mood postpartum. Hence, factors influencing hormones early in pregnancy, such as stress, nutrition, or medication, might have an important and previously unsuspected impact on mood in the postpartum period.
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Larsen, C. M., and Grattan, D. R. (2010). Prolactin-induced mitogenesis in the subventricular zone of the maternal brain during early pregnancy is essential for normal postpartum behavioral responses in the mother. Endocrinology 151, 3805–3814. Larsen, C. M., Kokay, I. C., and Grattan, D. R. (2008). Male pheromones initiate prolactininduced neurogenesis and advance maternal behavior in female mice. Horm. Behav. 53, 509–517. Lovestone, S., and Kumar, R. (1993). Postnatal psychiatric illness: The impact on partners. Br. J. Psychiatry 163, 210–216. Maestripieri, D., and D’Amato, F. R. (1991). Anxiety and maternal aggression in house mice (Mus musculus): A look at interindividual variability. J. Comp. Psychol. 105, 295–301. Mak, G. K., Enwere, E. K., Gregg, C., Pakarainen, T., Poutanen, M., Huhtaniemi, I., and Weiss, S. (2007). Male pheromone-stimulated neurogenesis in the adult female brain: Possible role in mating behavior. Nat. Neurosci. 10, 1003–1011. Manassis, K., Bradley, S., Goldberg, S., Hood, J., and Swinson, R. P. (1994). Attachment in mothers with anxiety disorders and their children. J. Am. Acad. Child Adolesc. Psychiatry 33, 1106–1113. Matthey, S., Barnett, B., Howie, P., and Kavanagh, D. J. (2003). Diagnosing postpartum depression in mothers and fathers: Whatever happened to anxiety? J. Affect. Disord. 74, 139–147. Munk-Olsen, T., Laursen, T. M., Pedersen, C. B., Mors, O., and Mortensen, P. B. (2006). New parents and mental disorders: A population-based register study. JAMA 296, 2582–2589. NIH (USA, PA-09-174, 2009). Womens Mental Health in Pregnancy and the Postpartum Period. O’Connor, T. G., Heron, J., Golding, J., Beveridge, M., and Glover, V. (2002). Maternal antenatal anxiety and children’s behavioural/emotional problems at 4 years. Report from the Avon Longitudinal Study of Parents and Children. Br. J. Psychiatry 180, 502–508. Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross, J. C., and Weiss, S. (2003). Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 299, 117–120. Stowe, Z. N., and Nemeroff, C. B. (1995). Women at risk for postpartum-onset major depression. Am. J. Obstet. Gynecol. 173, 639–645. Wenzel, A., Haugen, E. N., Jackson, L. C., and Robinson, K. (2003). Prevalence of generalized anxiety at eight weeks postpartum. Arch. Womens Ment. Health 6, 43–49.
F I V E
Exposure to Female Pheromones During Pregnancy Causes Postpartum Anxiety in Mice Caroline M. Larsen and David R. Grattan Contents I. Materials and Methods A. Subjects B. Anxiety testing C. Maternal behavior testing D. Serum prolactin levels in early pregnancy E. Neurogenesis F. Statistical analysis II. Results A. Exposure to unfamiliar female pheromones throughout pregnancy increased anxiety in postpartum mice B. Anxious postpartum females displayed impaired maternal behavior C. Exposure to female pheromones decreased serum prolactin levels in early pregnancy D. The role of suppressed prolactin in mediating pheromoneinduced postpartum anxiety E. Female pheromone exposure throughout pregnancy decreased neurogenesis on day 7 of pregnancy III. Discussion References
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Abstract The postpartum period is associated with an increased incidence of pathological anxiety, exerting a substantial burden on both the mother and the baby. We have shown that pharmacological suppression of prolactin in early pregnancy decreases maternal neurogenesis to cause postpartum anxiety. The present data demonstrate that physiological suppression of prolactin secretion through Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand Vitamins and Hormones, Volume 83 ISSN 0083-6729, DOI: 10.1016/S0083-6729(10)83005-5
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exposure to unfamiliar female pheromones throughout pregnancy prevented the normal postpartum attenuation of anxiety in mice, resulting in high anxiety relative to postpartum controls. Female pheromone-exposed mice also showed severely impaired maternal behavior in an anxiogenic situation. Mice exposed to female pheromones had decreased serum prolactin levels in early pregnancy, resulting in an ablation of the normal increase of neurogenesis on day 7 of pregnancy. These data demonstrate that low serum prolactin levels in early pregnancy, whether induced pharmacologically or as a physiological consequence of exposure to unfamiliar female pheromones, result in failure to show the normal adaptive decrease in anxiety after birth. This provides new insight into possible mechanisms that might underlie postpartum anxiety in women. ß 2010 Elsevier Inc.
During the postpartum period, there is an increased incidence of dysfunctional mood disorders (Munk-Olsen et al., 2006; Stowe and Nemeroff, 1995), with pathological anxiety, the most common (Britton, 2005, 2007; Matthey et al., 2003; Wenzel et al., 2003). Postpartum anxiety can severely affect the mother–infant relationship (Manassis et al., 1994), and have longlasting adverse effects on cognitive, emotional, and other aspects of infant development (Barnett et al., 1991; O’Connor et al., 2002). Postpartum mood disorders are also associated with an increased incidence of child abuse (Fraser et al., 2000), and increased conflict in marital relationships leading to a higher rate of separation and divorce (Boyce and Stubbs, 1994; Lovestone and Kumar, 1993). Attempts to understand the mechanisms underlying postpartum mood disorders have been impeded by the lack of an animal model that induces anxiety or depression postpartum, while retaining the unique hormonal characteristics and social stresses of gestation (NIH, USA, PA-09-174, 2009). The anterior pituitary hormone prolactin is critically involved in a number of adaptive responses in the maternal brain (Grattan et al., 2008). We have recently shown that pharmacological suppression of prolactin very early in pregnancy in mice decreased adult neurogenesis in the maternal brain during pregnancy and induced anxiety postpartum, potentially providing a novel model of postpartum mood disorders. The aim of this study was to determine whether more subtle physiological changes in prolactin during pregnancy, such as might occur in response to stress or infection (Brunton et al., 2008), might also alter mood and behavior postpartum. Exposure to pheromones alters levels of prolactin and neurogenesis in virgin female mice (Larsen et al., 2008; Mak et al., 2007). Therefore, we hypothesized that exposure to pheromones during pregnancy might similarly influence prolactin secretion during pregnancy, thereby inducing changes in mood postpartum. We exposed pregnant mice to male, female, or no pheromones throughout pregnancy and subsequently assessed anxiety on day 2 postpartum on an elevated plus maze (EPM) and in the light–dark box (LDB), well-characterized models of rodent anxiety. To determine
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whether pheromone exposure would change essential postpartum behaviors, we also examined maternal behavior, both in the home cage and in a clean novel cage (as an anxiogenic challenge; Larsen and Grattan, 2010). Finally, to identify possible mechanisms of pheromone action, pheromoneinduced changes in prolactin secretion and levels of neurogenesis in the subventricular zone (SVZ) were analyzed.
I. Materials and Methods A. Subjects Maternally naive virgin or day 1 pregnant C57BL/6J mice (6–9 weeks old) were either housed in 32 16 18 cm individual cages or in 28 54 18 cm split cages with another male, or a female, allowing pheromonal, visual, and olfactory contact without permitting physical contact (Larsen et al., 2008; all groups n ¼ 6). Some females were paired with males, and were considered day 1 pregnant when mating was confirmed by sperm being present in a vaginal smear, at which time they were transferred into the cages, as above. Pheromones are bound to major urinary proteins, and hence placing urine-soaked bedding in with the female will expose her to pheromones without the confounding effects of a companion. To confirm that effects seen were due to pheromones, additional groups were exposed to urine-soaked sawdust obtained from another mouse for 23 days prior to behavioral testing (Larsen et al., 2008). Virgin female and day 1 pregnant control groups had 0.5 g of clean sawdust placed in the cage daily for 23 days. After initial studies revealed an effect of female pheromones on anxiety and maternal behavior, all subsequent studies were undertaken using only female pheromones (i.e., without the male pheromone group). Mice were housed under a 12-h light–dark cycle, with ad libitum access to food and water. The University of Otago Animal Ethics Committee approved all experiments.
B. Anxiety testing Day 2 postpartum or virgin females were assessed for anxiety on the EPM and the LDB, as previously described (Larsen and Grattan, 2010). All testing was carried out between 9.30 and 10.30 a.m. in the room where the animals were usually housed. To avoid habituation to stress, the animals were not handled prior to the experiment. Elevated plus maze (EPM): To assess anxiety, the mouse was placed on the central platform facing an open arm, and allowed to roam at will for 5 min. All arm entries and exits were recorded. Once a mouse had all 4 paws on an arm of the maze, it was counted as having entered that arm, while placing 2 paws out of an arm was
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counted as an exit. Light–dark box (LDB): The LDB had an enclosed dark box (18 27 27 cm; black perspex sides, bottom and top, approx 30 lux) separated by a partition with a hole (6 cm) from a light box (27 27 27 cm; clear perspex, with a transparent cover with ventilation holes in it; lit 56 cm from above with a 100 W light bulb, approx 753 lux). Mice were placed at the entrance to the dark box and allowed to roam at will for 5 min. All entries and exits to the light or dark box were recorded. Once a mouse had all 4 paws within a box it was counted as having entered that box, while placing 2 paws out of a box was counted as an exit. To further evaluate the role of prolactin in female pheromone-induced anxiety, pheromone-exposed pregnant mice were injected s.c. with prolactin (50 mg of purified ovine prolactin in saline) at 4.30 p.m. from day 1 to day 3 of pregnancy (to mimic the early pregnancy surges in prolactin).
C. Maternal behavior testing Day 2 postpartum mice had their pups removed from the home cage. Immediately, three foster pups were placed into the cage and maternal behaviors recorded. To assess maternal behavior in an anxiogenic situation, another group of postpartum day 2 females were placed into a clean novel cage into which three foster pups had been placed, and any behavior recorded (as described previously, Larsen and Grattan, 2010). Full maternal behavior was defined as being when the mouse had gathered all three pups to a nest and was crouching over them. Testing was continued for a maximum of 120 min. Testing was performed between 9.30 and 11.30 a.m.
D. Serum prolactin levels in early pregnancy Pregnant mice, either exposed to no pheromones or female pheromones, were sacrificed for collection of trunk blood every 4 h from 9 a.m. on day 1 of pregnancy to 9 p.m. on day 4 of pregnancy for measurement of prolactin by radioimmunoassay using NIH reagents, as described previously (Larsen et al., 2008). Sensitivity was 2 ng/ml, and all samples were run in a single assay, with an intra-assay % C.V. 2.5%.
E. Neurogenesis Virgin female mice, or day 7 pregnant mice exposed to female or no pheromones, were injected with bromodeoxyuridine (BrdU), as reported previously (Larsen et al., 2008). Briefly, BrdU (Sigma) was injected every 2 h from 5 a.m. to 3 p.m. (6 injections of 12 mg/100 g of body weight, dissolved in phosphate buffer). Mice were then perfused with 4% paraformaldehyde at 5 p.m. BrdU-labeled cells were detected using immunohistochemistry
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(mouse monoclonal anti-BrdU, 1:200, Dako, Carpinteria, CA). Dual label immunofluorescence histochemistry was performed to determine whether the BrdU-labeled cells were neurons using doublecortin as a marker (goat antidoublecortin, 1:100, Santa Cruz). For quantification, the SVZ was observed under 20 magnification, and BrdU-immunoreactive cells counted in a systematic manner after a random start, such that all labeled nuclei were counted in 1-in-6 serial sections (120 mm apart) for a total of 14 sections throughout the SVZ (approximately Bregma þ1.94–3.9 mm). Data are presented as the total counts collected, and are not corrected for the total number of sections.
F. Statistical analysis The data are expressed as mean SEM. The amount of time on the open arms is expressed as a percentage of total time on the open and closed arms (open time/(open þ closed time) 100). Data were compared by ANOVA and Fishers PLSD, P < 0.05 was used as the level of significance.
II. Results A. Exposure to unfamiliar female pheromones throughout pregnancy increased anxiety in postpartum mice As has been previously reported (Maestripieri and D’Amato, 1991; Larsen and Grattan, 2010), postpartum females spent significantly more time on the open arms of the EPM than virgin controls (Fig. 5.1A and B, þP < 0.05), indicative of reduced anxiety. In contrast, postpartum females exposed to female pheromones for the duration of pregnancy, either by split-cage housing with a virgin female (Fig. 5.1A, *P < 0.05), or by exposure to urine-soaked bedding (Fig. 5.1B, *P < 0.05), were significantly more anxious on the EPM compared to postpartum controls. In contrast, there was no effect of exposure to male pheromones on anxiety on the EPM in postpartum females (Fig. 5.1A and B). Data from the LDB showed a similar increase in postpartum anxiety in the female pheromone-exposed animals. Postpartum mice exposed to female pheromones had a significant increase in the amount of time spent in the dark chamber of the LDB, and a decreased latency to enter the dark chamber of the LDB compared to postpartum controls (Fig. 5.1C, *P < 0.05). The data show that female pheromone exposure for the duration of pregnancy induces postpartum anxiety.
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Figure 5.1 Female pheromone exposure throughout pregnancy increases anxiety. Elevated plus maze (EPM). Day 2 postpartum controls spent significantly more time on the open arms of an EPM compared to virgin mice (þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy, either by split-cage housing (A), or by exposure to urine-soaked bedding (B), spent significantly less time on the open arms of the EPM compared to day 2 postpartum controls (*P < 0.05), and therefore displayed a significant increase in anxiety. In contrast, exposure to male pheromones throughout pregnancy had no effect. Light–Dark Box (LDB). Postpartum controls had an increased latency to enter the dark chamber of the LDB, and spent significantly less time in the dark chamber of the LDB compared to virgin controls (C, þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy had a decreased latency to enter the dark chamber of the LDB, and spent significantly more time in the dark chamber of the LDB, displaying a significant increase in anxiety compared to postpartum controls (C, *P < 0.05).
B. Anxious postpartum females displayed impaired maternal behavior To determine whether pheromone-induced postpartum anxiety affected maternal behavior, maternal behavior was examined using a pup retrieval paradigm in pheromone-exposed mice. As reported previously, in the
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Figure 5.2 Female pheromone exposure throughout pregnancy inhibits postpartum maternal behavior in an anxiogenic situation. Maternal behavior in a home cage. Day 2 postpartum mice were significantly faster to express maternal behavior to foster pups compared to virgin controls (A, þP < 0.05). Exposure to female pheromones had no effect, either by split-cage housing (data not shown), or by exposure to urine-soaked bedding (A), while mice exposed to male pheromones for the duration of pregnancy expressed maternal behavior more rapidly to foster pups compared to all other groups (A, *P < 0.05). Maternal behavior in a novel cage. Both day 2 postpartum controls, and male pheromone-exposed females, were significantly faster to express maternal behavior to foster pups than virgin controls (B and C, þP < 0.05). Day 2 postpartum mice exposed to female pheromones for the duration of the pregnancy displayed significantly impaired maternal behavior to foster pups in an anxiogenic situation compared to both the male pheromone-exposed mice and the individually housed controls (B and C, *P < 0.05).
home cage, postpartum females were significantly faster to express maternal behavior compared to virgin controls (Fig. 5.2A, þP < 0.05), and females exposed to male pheromones throughout pregnancy were significantly faster to express maternal behavior than postpartum controls (Fig. 5.2A, *P < 0.05; Larsen et al., 2008). Maternal behavior in mice exposed to
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female pheromones was not significantly different compared to postpartum controls when tested in the home cage (Fig. 5.2A). In the anxiogenic situation of a novel cage, all groups tested took significantly longer to express full maternal behavior compared to that in the home cage. Both day 2 postpartum control mice and male pheromone-exposed females remained significantly faster to express full maternal behavior compared to virgin controls, yet, there was now no significant difference between these two groups (Fig. 5.2B and C, þP < 0.05). In contrast, however, female pheromone-exposed mice placed in an anxiogenic situation (novel cage) displayed dramatically impaired maternal behavior to foster pups (Fig. 5.2B and C, *P < 0.05). The female pheromone-exposed mice investigated the pups occasionally, but did not gather them to their nest, or sit with the pups. Instead, they made a nest some distance away from the pups and either took significantly longer to express full maternal behavior or, in the majority of animals, did not express full maternal behavior at all within the designated 120-min observation period.
C. Exposure to female pheromones decreased serum prolactin levels in early pregnancy To test the hypothesis that changes in prolactin secretion during pregnancy might underlie the pheromone-induced alterations in mood postpartum, levels of serum prolactin during early pregnancy were measured by radioimmunoassay. Although the previously described pattern of twice daily prolactin surges in early pregnancy was maintained (Fig. 5.3, þP < 0.05; Larsen and Grattan, 2010), serum prolactin levels in pregnant mice exposed to female pheromones were significantly lower than those of controls at many time points on days 1–3 of pregnancy (Fig. 5.3, *P < 0.05; # urinesoaked sawdust added at 9 a.m. daily). From day 5 of pregnancy there were not any significant differences between the two groups.
D. The role of suppressed prolactin in mediating pheromoneinduced postpartum anxiety To test whether the decreased serum prolactin in early pregnancy induced by exposure to female pheromones was specifically responsible for inducing postpartum anxiety, we injected pheromone-exposed mice with prolactin at 5 p.m. daily from day 1 to day 3 of pregnancy. Animals were subsequently tested for anxiety on day 2 postpartum, approximately 3 weeks after hormone treatment. Remarkably, restoring normal prolactin levels in early pregnancy completely prevented the increased anxiety seen in postpartum mice exposed to female pheromones throughout pregnancy (Fig. 5.4). This implies that prolactin in early pregnancy is specifically
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Figure 5.3 Serum prolactin levels from day 1 to day 2 of pregnancy. Pregnant mice had a twice daily surge in serum prolactin levels with an afternoon peak just prior to lights off at 6 p.m. and a smaller nocturnal peak approximately 1 h before lights on (þP < 0.05). Pregnant mice had urine-soaked sawdust placed in the cage at 9 a.m. daily throughout pregnancy (#P < 0.05). Pregnant mice exposed to female pheromones showed significantly decreased serum prolactin levels at many time points from day 1 to 3 of pregnancy, compared to pregnant controls (*P < 0.05). From day 5 of pregnancy there were no significant differences between the groups (data not shown).
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Figure 5.4 Restoration of prolactin prevents the female pheromone-induced increase in anxiety postpartum. Day 2 postpartum controls spent significantly more time on the open arms of an EPM compared to virgin mice (þP < 0.05). Mice exposed to female pheromones for the duration of pregnancy by exposure to urine-soaked bedding spent significantly less time on the open arms of the EPM compared to day 2 postpartum controls (*P < 0.05), and therefore displayed a significant increase in anxiety. Female pheromone-exposed pregnant mice injected with prolactin from day 1 to day 3 of pregnancy had no significant differences in anxiety compared to postpartum controls.
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required for adaptive responses that occur in the maternal brain during pregnancy and become evident as behavioral changes postpartum.
E. Female pheromone exposure throughout pregnancy decreased neurogenesis on day 7 of pregnancy One known action of prolactin in the brain during early pregnancy is an increase in neurogenesis in the SVZ of the maternal brain (Larsen and Grattan, 2010; Shingo et al., 2003), and we have previously shown that pharmacological suppression of prolactin during early pregnancy led to a decrease in neurogenesis in this region (Larsen and Grattan, 2010). Therefore, we examined whether this was also affected by exposure to female pheromones during pregnancy. Decreased serum prolactin in early pregnancy, induced by exposure to female pheromones, significantly reduced levels of neurogenesis in the SVZ on day 7 of pregnancy compared to postpartum controls, reducing the numbers of BrdU-labeled cells to that of a virgin nonpregnant mouse (Fig. 5.5A, *P < 0.05). There were no significant differences in the percentage of BrdU-labeled cells in the SVZ that were double labeled with doublecortin, a neuronal marker, between any of the groups (Fig. 5.5B).
III. Discussion Day 2 postpartum mice displayed decreased anxiety on the EPM compared to virgin mice, consistent with previous experimental findings (Larsen and Grattan, 2010; Maestripieri and D’Amato, 1991). In contrast, female pheromone exposure throughout pregnancy significantly increased anxiety on day 2 postpartum. In women, postpartum anxiety can impair maternal responsiveness to, and care of the infant (Barnett et al., 1991; Fraser et al., 2000; O’Connor et al., 2002). In a similar manner, mice exposed to female pheromones for the duration of pregnancy also displayed dramatically impaired responsiveness to, and an extreme lack of care for pups when placed in an anxiogenic situation. The combined syndrome of an increase in anxiety together with impairment of maternal behavior suggests that exposing mice to female pheromones throughout pregnancy provides a novel model for studying postpartum mood disorders. Importantly, this approach is completely noninvasive, and retains the unique characteristics of gestation. We have previously shown that pharmacological suppression of serum prolactin levels, solely in early pregnancy, can initiate increased postpartum anxiety over 2 weeks later (Larsen and Grattan, 2010). The results shown here indicate that postpartum anxiety caused by female pheromone exposure is also induced by low prolactin during early pregnancy. Moreover,
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Figure 5.5 Female pheromone exposure throughout pregnancy decrease neurogenesis on day 7 of pregnancy. (A). Day 7 pregnant mice had a significant increase in the number of BrdU-labeled cells in the subventricular zone (SVZ) compared to virgin controls (þP < 0.05). Day 7 pregnant females exposed to female pheromones for 7 days showed a significant decrease in BrdU-labeled cells in the SVZ compared to controls (*P < 0.05). Micrographs show representative images from the SVZ, with black staining representing BrdU-labeled cells (Lat. V.: lateral ventricle). (B) There were no significant differences between pheromone-exposed or control mice, in the percentage of BrdU-labeled cells that coexpressed Dcx. Representative merged images immunoreactive for BrdU (green) and Dcx (red) in the lateral ventricle of the SVZ in each group are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)
reminiscent of the pharmacological suppression of prolactin, injections of prolactin to restore serum prolactin levels in early pregnancy in pheromoneexposed mice completely prevented the pheromone-induced increase in anxiety. This suggests that the pheromone-induced decrease in prolactin in early pregnancy is the mechanism underlying the induction of postpartum anxiety. In our previous work using pharmacological suppression of prolactin, we identified a potential role for the increase in SVZ neurogenesis on mood
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postpartum (Larsen and Grattan, 2010). In the present study, serum prolactin levels in mice exposed to female pheromones during pregnancy were significantly lower than in pregnant controls, but they were still higher than basal levels seen in virgin mice, and higher than the levels seen in mice when prolactin was suppressed pharmacologically (Larsen and Grattan, 2010). Nevertheless, the decrease in prolactin induced by female pheromone exposure was sufficient to completely suppress the pregnancy-induced increase in neurogenesis in the SVZ. These data support the hypothesis that increased neurogenesis in the SVZ is important for adaptive changes in mood postpartum. Extensive work has linked impaired neurogenesis in other brain regions to the development of mood disorders (Duman et al., 2001), but how the increase in SVZ neurogenesis during pregnancy moderates mood postpartum remains unclear. The implication of our data is that any physiological or pathophysiological factor that might influence prolactin levels or neurogenesis during pregnancy could lead to development of mood disorders. Previous studies examining the hormonal regulation of mood during the peripartum period have predominantly focused on acute changes in hormones at that time, and no clear role of hormones in the control of mood has emerged. The present data support the hypothesis that levels of hormones in early pregnancy are crucial for euthymic mood postpartum. Hence, factors influencing hormones early in pregnancy, such as stress, nutrition, or medication, might have an important and previously unsuspected impact on mood in the postpartum period.
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