The mortality and morbidity due to neurodevelopmental and mood disorders has remained largely unchanged over the last fifty years (T. R. Insel and E. M. Scolnick, 2006
). Given the difficulty in treating these diseases, a cross-disciplinary approach that identifies the developmental origin of disease and vulnerable populations is needed to create novel prevention and treatment strategies. The predisposition toward sex-biased disorders, including schizophrenia, autism, and affective disorders has been associated with stress-related fetal antecedents (A. J. Ward, 1991
; J. B. Watson et al., 1999
; C. A. Pardo and C. G. Eberhart, 2007
). However, the mechanism underlying this influence of prenatal stress (PS) remains unclear. In our studies, behavioral coping responses of male and female offspring exposed to stress across gestation were initially examined to determine if temporal specificity may underlie a sex-dependent vulnerability to prenatal stress.
Male offspring exposed to stress early in gestation (E-PS) exhibited maladaptive behaviors in both the tail suspension and forced swim tests. While female controls generally showed increased immobility when compared to males, prenatal stress did not further alter stress coping responses. Because of the existing sex-differences in these tests, a ceiling effect may have precluded prenatal stress from further altering female outcomes. Interestingly, our data correlate with the recent clinical findings that show stress early in pregnancy increased the presence of schizophrenia in males, supporting a sex-specific risk early in development (A. S. Khashan et al., 2008
). While our previous studies have reported effects of stress during additional gestational time points on parameters of learning and memory and energy balance regulation (B. R. Mueller and T. L. Bale, 2006
), our current results reveal a vulnerability of stress pathway programming specific to early pregnancy.
As the male E-PS increased immobility in the tail suspension and forced swim tests was suggestive of a depression-like phenotype, we also examined hedonic sensitivity in these mice using a sucrose preference test (J. L. Moreau, 1997
). E-PS males exhibited a diminished preference for a 1% sucrose solution, suggesting a diminished basal sensitivity to hedonic rewards. However, an acute stress exposure induced E-PS males to consume 100% more sucrose in the two hours following a sucrose deprivation compared to continuous access mice. The majority of the difference between control and E-PS 24-hour sucrose consumption resulted from this two-hour intake, indicative of stress-induced binging responses (W. J. Lynch and J. R. Taylor, 2005
; N. M. Avena et al., 2006
Given the depression-like phenotype of E-PS males, behavioral responses to an acute SSRI, citalopram, were measured. E-PS males exhibited a greater sensitivity to a sub-threshold dose of citalopram in a tail suspension test. As this test measures active coping behaviors that are responsive to acute changes in synaptic serotonin (5-HT), the greater sensitivity detected in E-PS males may indicate a dysregulation in 5-HT pathways. Further examination of the 5-HT system in these mice revealed reduced 5-HT transporter levels in the hippocampus and a trend for increased tryptophan hydroxylase-2 expression in the dorsal raphe. Thus, potential increased 5-HT output and decreased reuptake may underlie the increased sensitivity to acute SSRI treatment in these mice.
Dysregulation of the serotonin system has been classically linked to the etiology of affective disorders. Corticotropin-releasing factor (CRF) is a known regulator of 5-HT neurotransmission and a key determinant of stress responsivity (R. J. Valentino et al., 1991
; T. L. Bale and W. W. Vale, 2004
; H. Tan et al., 2004
). Therefore, dysregulation of CRF and stress pathway components could be an upstream contributor to abnormalities in 5-HT transmission and a stress-sensitive phenotype. In the CeA, E-PS males showed a significant increase in CRF expression. As an additional indication of increased stress responsiveness, E-PS males also exhibited higher corticosterone levels following an acute restraint stress, and decreased hippocampal glucocorticoid receptors (GR). While previous studies have reported similar changes in GR levels following stress experienced late in gestation, our results indicate an earlier developmental window during which stress pathways display a sex-specific vulnerability (M. S. Cratty et al., 1995
; M. Darnaudery and S. Maccari, 2007
The long-term impact of prenatal stress on regulation of central gene expression may occur via effects on the epigenome. In our examination of the methylation pattern of CRF, we report reductions in methylation at specific cytosines within the identified CRF promoter CpG island in both the hypothalamus and CeA that may contribute to the increased CRF detected in these mice. In our examination of the exon 17
region of the GR promoter, E-PS males showed a site-specific increase in methylation that occurred in the region containing the consensus sequence of NGFI-A where methylation status has previously been linked to early life experience and decreased expression (I. C. Weaver et al., 2004b
; I. C. Weaver et al., 2004a
As the fetal brain is not present during this period of early stress, we hypothesized that sex-specific changes in fetal development and long-term sensitivity may occur through actions on the placenta as it is derived from the developing blastocyst and undergoes critical development during this period of E-PS (M. Hemberger, 2007
). The placenta is the intermediary by which the maternal hormonal milieu influences the developing embryo, and therefore is poised to be influenced by perturbations occurring early in pregnancy that could affect fetal development throughout pregnancy by altering 1) nutrient and oxygen transport, 2) inflammatory responses, and 3) epigenetic programming machinery. To examine this hypothesis, we examined effects of E-PS on male and female placental gene expression patterns utilizing a focused PCR Array for growth factors and nutrient transport genes. E-PS male placentas exhibited significant increases
in PPARα, IGFBP-1, GLUT4, and HIF3α. Surprisingly, E-PS female placentas showed a reduction
in PPARα and IGFBP-1 expression. As glucocorticoids increase expression of PPARα (T. Lemberger et al., 1994
), and PPARα in turn increases expression of IGFBP-1 (T. Degenhardt et al., 2006
), our results support a potential mechanism whereby maternal stress could directly affect placental gene expression patterns. Reductions in growth factors have been linked to affective and neurodevelopmental disorders (J. E. Malberg et al., 2007
). Thus, an elevation in placental IGFBP-1, and a consequent decrease in available growth factors during critical developmental periods may play a role in male fetal programming.
Alterations in oxygen and nutrient availability have been associated with inflammation (T. Altay et al., 2004
), and as recent studies have shown an important link between inflammatory events and the onset of major depression (R. Dantzer et al., 2008
) additional comparisons of placental inflammatory markers were conducted. While no significant differences were detected for levels of TNFα and IL-6 at this time point, it is interesting that male placentas showed a directional change suggestive of possible inflammation following the E-PS that may be more robust at different pregnancy time points.
To further investigate potential mechanisms underlying the sex-specific effect of E-PS on placenta gene expression, we examined DNA methylation machinery in male and female placentas. Interestingly, expression of DNMT1, the enzyme responsible for methylation maintenance, was significantly lower in male control placentas as compared to female control placentas, providing support for our hypothesis that the influence of E-PS may begin at the level of the placenta (H. Leonhardt et al., 1992
). In addition, while E-PS appeared to increase DNMT1 expression in both sexes, this change was only significant in females. As previous studies have shown that the maintenance of methylation patterns are critical in neurodevelopment, and perturbations in placental DNA methylation patterns are strong predictors of fetal changes, a stress-induced elevation of DNMT1 may indicate that females are able to circumvent the effects of stress by strengthening the maintenance of methylation during this perturbation (R. M. Rivera et al., 2008
; H. Wang et al., 2008
These changes in placental gene expression related to growth factors, nutrient transport, and epigenetics may impact the developing central nervous system. As a gross marker of brain development we compared neurofilament-1 (NF-1) levels from postnatal day 1 male and female offspring. E-PS males but not females showed a significant reduction in NF-1, supporting a possible delay in brain development. As neuronal abnormalities and delayed development have been associated with major depressive disorder and schizophrenia, such changes in central NF-1 may be predictive of future disease (A. Reines et al., 2004
; J. J. Miguel-Hidalgo et al., 2005
). No effects of E-PS were detected in NF-1 levels in adult males, indicating that E-PS males eventually catch up with control males.
In summary, as it is unlikely that any one specific gene produces the difference in behaviors detected between control and E-PS males, an array of genetic, hormonal, and epigenetic factors were examined in these studies. Our results support the hypothesis that E-PS dysregulation of CRF and stress pathway programming may be a key contributor to the stress-sensitive phenotype detected in these male mice, including the increased immobility in the tail suspension and forced swim tests, heightened HPA axis responsivity, and influences on the 5-HT system. These studies provide critical mechanistic insight regarding the underlying programming of sex-biased neurodevelopmental disorders.