This study describes the effects of prenatal DEX treatment on gene expression in the PFC, a brain region implicated in ADHD and other psychiatric disorders, at neonate and adult life stages. Thus, prenatal DEX exposure in marmoset monkeys leads to alterations in GR and calcyon (a risk gene for ADHD) mRNA expression in the neonate PFC. Taken together with recent findings (see below), the present results suggest that in non-human primates antenatal DEX exposure might modify the early postnatal development and synaptic plasticity of PFC with possible functional consequences.
Studies in rodents have shown that the developing PFC is extremely sensitive to stress, possibly including elevated levels of stress hormones (i.e. glucocorticoids). For example, prenatal stress in rodents leads to lateralized changes in PFC DA function [25, 26], alterations of PFC neuronal structure (e.g. spine density and dendritic complexity; ), and long-lasting changes in some subtypes of dopamine and glutamate receptors , which are thought to underlie cognitive and emotional deficits observed in prenatally stressed animals . In humans, maternal stress during pregnancy is associated with an increased risk of developing ADHD-like symptoms [1, 2, 26]. Moreover, antenatal exposure to multiple courses of synthetic glucocorticoids is also associated with increased neurobehavioral problems (e.g. aggressive/destructive behaviour, increased distractibility, and hyperactivity) in children [5, 28, 29]. Taken together, these results suggest that fetal glucocorticoid overexposure leads to altered development and functioning of PFC, a critical region involved in cognition and emotional regulation.
Recently, we described the effects of prenatal DEX exposure on behavioral traits in juvenile and adolescent common marmosets. We showed for the first time that exposure of the fetus to DEX has a long-term postnatal impact on social development, skilled motor reaching/dexterity, motivational and learning functions . In the present study, we assessed whether the same treatment could affect expression of corticosteroid receptors and calcyon (a risk gene for ADHD; see below) in neonate and adult PFC. Prenatal DEX exposure, especially during late gestation, led to significant reductions in GR expression in the neonate PFC. The expression of MR mRNA was not significantly affected by prenatal DEX treatment, which is consistent with the fact that DEX has a low affinity for the MR and selectively binds to GR . A recent study showed that in the developing human hippocampus both GR and MR are expressed between 24 and 34 weeks of gestation . This is the time-window when synthetic glucocorticoids are administered to pregnant women at risk for preterm delivery. Interestingly, the same study did not find an effect of antenatal DEX treatment on GR or MR gene expression in the human hippocampus at the third trimester of gestation. Based upon previous work in the squirrel monkey  and rhesus monkey  demonstrating the relatively low expression of GR in hippocampus versus neocortex, and the present results, we postulate that, in primates, GR in neocortical areas are more important potential mediators of effects of antenatal glucocorticoid treatment than are GR in hippocampus.
One of the most interesting findings of the present study is the observation that prenatal DEX treatment induced a down-regulation of calcyon (a risk gene for ADHD) gene expression in the neonate PFC. Calcyon is a single transmembrane protein highly expressed in PFC [12, 14]. New evidence indicates a role for calcyon in clathrin mediated endocytosis, a critical component of synaptic plasticity . This is consistent with anatomical findings localizing calcyon to vesicular compartments in dendritic spines and axon terminals , two sites in the brain where clathrin mediated endocytosis is essential for efficient neurotransmission and plasticity associated with learning and memory . Support for a role of calcyon in the aetiology of ADHD comes primarily from genetic studies. In a recent genome-wide linkage study for loci influencing ADHD, the calcyon gene was found to coincide with one of the highest positive linkage sites identified at chromosome 10q26 . More recently, the inheritance of nine polymorphisms in the calcyon gene was examined with ADHD and their immediate families using the transmission disequilibrium test . This study reported evidence for excess transmission of the most common calcyon haplotype, designated C1. In addition, C1 was positively associated with both hyperactive/impulsive and inattentive symptoms, supporting the idea that variations in calcyon may contribute to both deficits in motor control and cognitive functions of the disorder. This notion is supported by recent animal studies demonstrating alterations in calcyon gene expression in a genetic rat model of ADHD  and ADHD-like phenotypes in calcyon over-expressing transgenic mice . In the current study we showed that calcyon is expressed during early postnatal development of the non-human primate PFC and that it is sensitive to fetal glucocorticoid overexposure. These observations have marked translational significance given the genetic link of calcyon to ADHD.
DEX is known to cause its effects by binding to the GR . Once activated, the GR subunits homodimerize and bind to DNA via glucocorticoid responsive elements (GREs) in the promoter region of target genes resulting in the regulation of gene expression . In addition, GR is also involved in crosstalk with other transcription factors, such as AP-1, CREB-binding protein, STAT5, and NFkB (see ). Crosstalk between GR and these transcription factors may occur either through composite response element, overlapping response element, or interaction between GR and transcription factors. We used bioinformatic tools (MatInspector, Genomatix Software GmbH) to search for the GRE consensus sequence (consisting of the palindromic binding site AGAACAnnnTGTTCT) and related elements in the human CALY promoter. This analysis revealed that the human CALY promoter does not contain a consensus sequence for the GRE (see Additional file 2), suggesting that antenatal DEX-mediated decreases in calcyon gene expression are not likely to be a direct effect of GR binding to the calcyon promoter. However, the CALY promoter contains consensus sequences for the binding of several transcription factors including Sp-1 (see Additional file 2). Interestingly, previous studies have shown that DEX can regulate gene transcription through a mechanism dependent on the Sp-1 transcription factor , and therefore could be a potential mechanism mediating antenatal DEX effects on calcyon gene expression.
In our previous studies in marmoset monkeys, we observed that the outcome of antenatal DEX exposure on behavioral traits was dependent upon the timing of administration i.e. EDEX versus LDEX [6, 19]. In the present study, we also observed that early and late DEX treatments differentially affected GR and calcyon mRNA levels in neonates, with early DEX affecting calcyon and late DEX affecting GR. We hypothesize that the different effects of early vs. late DEX treatment on gene expression and behavior could originate from the difference in the stage of brain maturation (e.g. GR expression and/or transcription factors interaction with GR) at the time of these two antenatal DEX treatments.
The absence of significant effects of antenatal DEX on either GR or calcyon gene expression in adult PFC suggests the presence of compensatory - and possibly but not necessarily protective - mechanisms acting during the protracted postnatal maturation of the primate PFC [39, 40]. However, it will be important to assess in future studies potential effects of repeated antenatal glucocorticoid exposure on PFC gene expression and cognitive-executive functions during the juvenile period when ADHD-symptoms usually manifest in children.