One such example of an animal model in which normal fetal programming has been disrupted resulting in a phenotype of adult heightened stress sensitivity is prenatal stress. Stress experience during gestation is associated with an increased incidence of numerous neuropsychiatric disorders, including depression, anxiety, schizophrenia, and autism (6
). The mechanism through which fetal antecedents contribute to disease development is not known, though likely involves a complex interaction between maternal environment and genetics. As many of the diseases associated with prenatal stress exhibit a sex bias in presentation, elucidation of the mechanisms by which sex-specific susceptibility arises may provide critical insight into disease etiology.
While prenatal stress has been broadly associated with offspring disease, the developing nervous system is unlikely to show uniform vulnerability to perturbations over the course of gestation. We hypothesize that the impact on offspring would be dependent on the timing of stress insult, and therefore have examined outcomes specific to early, mid or late pregnancy stress. In confirmation of our hypothesis, we demonstrated that the influence of prenatal stress on hippocampal dependent learning and memory was specific to the timing of stress exposure and sex of the offspring (13
). However, we are just beginning to appreciate the importance of the timing of stress insult during pregnancy for its impact on stress pathway development that may underlie disease predisposition. In support of such a timing specificity to the effects of stress on long-term outcome in neurodevelopmental disorders, a recent clinical study revealed an association between maternal stress experience only during the first trimester of pregnancy with an increased risk of schizophrenia in males (38
). Studies in guinea pigs have demonstrated that the timing of prenatal stress insult as well as offspring estrous cycle stage during testing as adults was critical in behavioral outcome (39
). Further, these studies found that the timing of pregnancy stress on behavioral and physiological stress responsivity was dependent on offspring sex, supporting the need for mechanistic examination of parameters altering programming of stress circuitry during critical periods of sexually dimorphic brain development (40
). While rodent models provide a controlled environment in which pregnancy manipulations can be conducted, it is important to note the disparity in the timing of rodent and human brain development when considering effects of fetal antecedents. However, influences of early gestational perturbations affecting developmental programming in humans and rodents may contribute to mechanisms whereby changes in maternal hormones or placental gene expression patterns may impact the developing embryo throughout gestation. Therefore, recent studies from our lab have examined the temporal specific outcomes of stress during pregnancy on long-term programming of offspring stress physiological and behavioral sensitivity.
In our studies, male offspring that had been exposed to early prenatal stress (days 1–7; E-PS) exhibited maladaptive behaviors in both the tail suspension and forced swim tests with elevated levels of immobility, likened to stress-induced learned helplessness. No effect of E-PS was detected in females in these tests (17
). As the increased immobility in the tail suspension and forced swim tests by E-PS males was suggestive of a depression-like phenotype, we further examined anhedonia-like behaviors using a sucrose preference test. E-PS males exhibited a diminished preference for a 1% sucrose solution compared to controls. Presentation of increasing sucrose concentrations of 5% or 10% ameliorated this effect and revealed a diminished basal sensitivity to hedonic rewards in E-PS males. Further, following an acute stress, E-PS males consumed significantly more of the 10% sucrose than controls if access had been restricted prior to the stress, suggesting that stress-induced binge-like behaviors in E-PS males are another hallmark of stress pathway dysregulation found in neuropsychiatric disorders (17
Given the phenotype of these E-PS males, we examined behavioral responses to an acute administration of a selective serotonin reuptake inhibitor (SSRI), citalopram to determine if alterations in serotonergic signaling might be present. E-PS males exhibited a greater sensitivity to a low dose of citalopram with decreased immobile time and an increased latency to first immobile bout (17
). As this test measures active coping behaviors that are responsive to acute changes in synaptic serotonin, the greater sensitivity in E-PS males suggested a possible alteration in serotonin neurocircuitry. We detected reduced serotonin transporter levels in the CA1 region of the hippocampus and a trend for increased tryptophan hydroxylase-2, the rate-limiting enzyme in 5-HT production, in the dorsal raphe (17
). Thus, increased serotonin output and decreased re-uptake may underlie the increased sensitivity to a lower dose of SSRI.
Increased physiological stress responsivity is a hallmark of affective disorders and is also present in many other neuropsychiatric diseases (36
). In examination of the HPA stress axis in our studies, we found that E-PS males also showed a significant increase in stress-induced corticosterone levels. We analyzed expression of genes important in stress pathway modulation in these mice and detected a significant increase in CRF expression in the central nucleus of the amygdala and a decrease in hippocampal glucocorticoid receptor expression in E-PS males (17
). Glucocorticoid receptors in the hippocampus provide negative feedback for the HPA stress axis. While previous studies examining effects of prenatal stress have reported similar biochemical and physiological changes in offspring exposed to stress late in gestation, our results determined a wider developmental window during which stress neurocircuitry is vulnerable (42
One potential mechanism whereby stress can influence fetal development and programming is through epigenetics, including histone modification and DNA methylation (43
). Further, epigenetic analyses have been applied to the examination of the long-term outcomes attributed to disparate levels of maternal care that impact DNA methylation of specific genes (44
). While recent emphasis has been placed on the involvement of epigenetic machinery in the programming of early development, the influence of fetal antecedents on the epigenome remains unknown. In our studies, DNA isolated from the specific brain regions where gene expression changes had been detected in adult E-PS males showed reductions in methylation at specific points within the cytosine and guanine rich region of the CRF promoter in close proximity to the cAMP response element (CRE) and glucocorticoid response element (GRE) (17
). This reduction in promoter methylation corresponds with the increased CRF expression detected in this brain region. Similarly, we also detected a site-specific increase in methylation of the GR promoter in E-PS males that correlated with the decreased GR expression in the hippocampus from these mice (17
). These changes in gene methylation patterns detected in adult brains may contribute to the long-term alterations detected in expression of these genes important in regulation of stress responsivity.
Although the specific mechanism whereby maternal stress contributes to disease risk via effects on epigenetic programming remains unclear, several key contributors have been suggested involving changes in the maternal hormonal milieu. Elevated glucocorticoids in utero
can alter early life ‘programming’ (43
). However, the placenta inactivates a significant percentage of maternal glucocorticoids via 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), protecting the fetus through the late stages of pregnancy (43
). Despite the placenta’s critical role in regulating the exchange of hormones, nutrients, and waste products between the maternal and fetal circulatory systems, very little is known regarding either the effects of maternal stress or the influence of fetal sex on placental function. Further, the placenta is a candidate tissue for mechanistic investigation as it is derived from the blastocyst and undergoes critical development during this period of early prenatal stress used in our studies (47
). We examined effects of stress early in pregnancy on male and female placental gene expression patterns through analysis of a focused PCR array. E-PS male placentas exhibited significant increases
in peroxisome proliferator-activated receptor alpha (PPARα), insulin-like growth factor binding protein-1 (IGFBP-1), glucose transporter 4 (GLUT4), and hypoxia inducible factor-3a (HIF3a) compared to control male levels (17
). Surprisingly, E-PS female placentas showed a reduction
in PPARα and IGFBP-1 as compared to control female levels. Expression of PPARα, a transcription factor that regulates cellular metabolism and differentiation, is increased by glucocorticoids (48
). Further, PPARα directly increases expression of IGFBP-1 (49
). Reductions in growth factors are linked to depression and neurodevelopmental disorders (50
). Thus, an elevation in placental IGFBP-1 and consequent decrease in available growth factors during critical developmental periods may play a role in male fetal programming.
These studies reveal a sex-specific effect of stress during early pregnancy on the programming of long-term dysregulated stress neurocircuitry resulting in an adult organism with heightened stress sensitivity and maladaptive behavioral stress responsivity. Examination of sex differences and temporal specificity in these studies provided critical mechanistic information regarding the underlying developmental origin of male-biased neurodevelopmental disorders such as schizophrenia. Interestingly, these results support current findings in clinical evaluation of sex differences in schizophrenic patients. Imaging studies have revealed dysregulation of the human neuroendocrine system with fetal antecedents coinciding with brain sexual differentiation (51
). Further, fMRI analyses in male and female schizophrenics confirmed a disruption of the normal sexually dimorphic ratio of orbitofrontal cortex to amygdala where male schizophrenia patients showed a feminization of these brain regions (52
). Future investigation into the underlying mechanisms whereby disruptions in normal sexual differentiation of the brain occurs may provide insight as to the timing of pregnancy stress and the specific genes targeted during this period of re-programming (5