We aimed to elucidate whether pre-gestational stress in maternal rats affects HPA axis activity and the serotonergic system in the brains of their foetuses. We examined 5-HT activity (5-HT, 5-HIAA and the ratio of 5-HIAA to 5-HT) and 5-HT1A receptor and SERT levels in brain regions of foetuses. The ratio of 5-HIAA to 5-HT and the level of SERT were significantly lower for foetuses in the CUS group than in the control group. Levels of 5-HT1A receptor were significantly lower in foetal hippocampuses in the CUS group than in the controls, with no significant difference in the hypothalamus. Finally, the level of SERT was lower in both the hippocampus and hypothalamus of foetuses in the CUS group than in the control group. The HPA axis and serotonergic system may be dysregulated in the foetuses of mothers exposed to CUS before pregnancy. From the results of this study, we extended our knowledge of pre-gestational stress-induced changes across several regions of the foetal brain and provided several novel findings about how pre-gestational stress can affect serotonergic systems in foetal rats with stressed mothers.
We successfully established a model of CUS in maternal rats. Body weight and behaviour findings for maternal rats generally agreed with our previous findings , so the CUS model was appropriate for our study.
Along with the reduced body weight of maternal rats with CUS, the reduced body weight of their foetuses suggests that growth retardation may relate to their mothers with CUS before pregnancy. However, brain weight did not differ between foetuses in the CUS group and control group. Therefore, the growth retardation of foetuses in the CUS group cannot explain all of our findings, which appears to relate to an imbalanced neuro-endocrine network after CUS in maternal rats and their foetuses.
The serum COR and CRH levels were higher in maternal rats with CUS before pregnancy than in control maternal rats. This finding may relate to the dysregulation of the HPA axis after exposure to chronic stress. This view is supported by previous studies [27, 28]. We found that pre-gestational CUS in mothers significantly increased COR and CRH levels in foetuses in the CUS group as compared with controls for both males and females, and significant positive correlations in serum COR as well as CRH levels were observed between maternal rats and their foetuses in our study. Therefore, chronic stress can not only lead to an imbalance in the neuro-endocrine network of maternal rats, but it can also affect the HPA axis of the foetus via the maternal-placental-foetal interface . Several studies have provided evidence that maternal stress causes elevated levels of circulating COR in the foetus . The increase in the serum COR level of foetus results from reduced maternal COR-binding globulin and impaired regulation of the maternal HPA axis . The HPA axis, impaired in maternal rats with CUS, may release stress hormones such as COR and CRH to enter the circulation of foetus directly through the placental barrier. On the other hand, the elevated level of circulating COR in maternal rats reduces the blood flow of the placenta, which decreases the supply of oxygen and nutrients to the foetus and results in a high level of COR in the foetus .
Early pregnancy is the most sensitive period in which stress is associated with neuro-behavioural changes in offspring . Imposing CUS on maternal rats for a short time before pregnancy may also affect offspring in the early embryonic environment and thus have a profound influence on the brain development of offspring . During the foetal lifespan, the brain undergoes rapid growth that is characterised by a high turnover of neuronal connections. Therefore, the foetal brain is especially vulnerable to hormones that may reach it in excess amounts from the maternal circulation as a result of stress . Peptides such as CRH and beta-endorphin and COR may impede the formation of correct neural connections and reduce plasticity in the developing foetal brain . Stress-induced levels of COR activate glucocorticoid receptors that are present in the rat hippocampus, hypothalamus, pituitary, cingulate cortex and amygdala from day 13 of gestation . The ability of the foetal HPA axis to respond to maternal stress was shown by increased expression of CRH mRNA in the foetal para-ventricular nucleus (PVN) on day 15 of gestation in rats . The abnormal HPA axis of the foetuses results in high levels of COR and CRH. High CRH levels affect γ-aminobutyric acid (GABA) levels and glutamate (Glu) neurons , thus leading to disturbed inhibitory amino acids (GABA) and excitatory amino acids (Glu), which may influence release of neurotransmitters such as 5-HT and down-regulate the sensitivity of 5-HT1A autoreceptors .
In this study, we showed that pre-gestational CUS induced decreased serotonergic activity in foetuses with decreased serotonergic ratios (5-HIAA to 5-HT) in the hippocampus and hypothalamus and decreased 5-HIAA levels in the hippocampus. Compared with control foetuses, both male and female foetuses in the CUS group showed increased 5-HT levels in the hippocampus and hypothalamus. Surprisingly, 5-HIAA levels were lower in the hippocampuses of foetuses in the CUS group than in control group, with no significant difference in levels in the hypothalamus of foetuses in the CUS group and controls for both males and females. Serotonergic ratio and serum COR and CRH levels in foetuses were significantly correlated. Decreased serotonergic status in the hippocampus and hypothalamus in foetuses in the CUS groups is probably directly or indirectly associated with changes in the HPA axis. By activating 5-HT1A receptors in the hippocampus, 5-HT causes hyper-polarisation , the suppression of hippocampal output and the subsequent disinhibition of HPA axis [39, 40]. However, decreased serotonergic activity in foetuses in the CUS group could have resulted from changes in serotonergic function mediated by altered COR [41–43] and CRH [44, 45] availability.
The 5-HT1A receptor is a crucial pre-synaptic autoreceptor in regulating the activity of 5-HT neurons and a post-synaptic receptor in the limbic system, cortex, hypothalamus and other parts of the brain . Therefore, the regulation of 5-HT1A receptor can have an important role in psychological diseases. The hippocampus is a major limbic target of the brainstem serotonergic neurons that modulate emotion, cognitive and behaviour through post-synaptic 5-HT1A receptors . Serotonergic neurons in the raphe nucleus are among the earliest to appear in the developing central nervous system. The hippocampus accepts the neurotransmission of 5-HT neurons from the dorsal raphe nucleus and median raphe nucleus . The hippocampus also contains high levels of COR receptors in central brain areas. It is extremely vulnerable to excessive COR , which results in injury and may lead to decreased 5-HT1A receptor expression. Thus, pre-gestational stress may cause abnormal development of the serotonergic neurons in the hippocampus of foetuses by changing levels of stress hormones such as COR and CRH. We found lower 5-HT1A mRNA expression in the hippocampus of foetuses in the CUS group than in the control group and significant correlations between 5-HT1A and serum COR as well as CRH levels, which can support the above view. It appears that hippocampal serotonin is critical for programming of HPA function; however, it is possible that ascending fibres innervating the hypothalamus also play a role in this process. Many hypothalamic functions are regulated by the serotonergic system, which modulate the function of hypothalamus through dendrites of the 5-HT1A autoreceptor in cell bodies and the 5-HT1B autoreceptor at the nerve ends . We found no difference between male or female foetuses in the CUS group and control group in level of 5-HT1A receptor in the hypothalamus, which suggests that the effect of pre-gestational stress on the hypothalamus might have a closer relationship with the 5-HT1B autoreceptor at the nerve ends.
We found a lower ratio of 5-HIAA to 5-HT and 5-HT1A mRNA expression in the hippocampus of both male and female foetuses in the CUS group than in the control group and lower levels of SERT in both the hippocampus and hypothalamus of foetuses in the CUS group, which suggests a possible link between the three factors. SERT is present throughout foetal development and is expressed at high levels at the time of neurogenesis and rapid brain growth. Serotonin levels within the synaptic cleft are regulated by the activity of the SERT protein, which actively re-uptakes serotonin into the pre-synaptic terminal and therefore regulates the duration of serotonin activity on its own autoreceptors . In foetal rats, the main bilateral bundles of the ascending serotonergic axons pass through the mesencephalon, and a ventral branch leaves the medial forebrain bundle in the diencephalon to innervate the hypothalamus . This pathway forms the direct synaptic connections between serotonergic neurons and the CRH-containing cells of the hypothalamic PVN . In this connection, serotonin can activate HPA function through its excitatory effects on the PVN in adult animals, and the same likely occurs in the peri-natal period . Our findings of the significant correlations between the SERT level and CRH levels in foetuses may support this view. In foetuses of the CUS group, which SERT was down-regulated in the hippocampus and hypothalamus, the serotonergic ratio (5-HIAA/5-HT) was also down-regulated in the same region. Indeed, SERT gene promoter variations conferred a blunted COR response in rhesus monkeys exposed to pre-natal stress . Therefore, COR-mediated changes in SERT expression would likely have a significant impact on the metabolism of serotonin in the developing brain.
In our experiment, the impaired HPA axis of the foetuses of maternal rats exposed to CUS before pregnancy results in high levels of COR and CRH. High COR and CRH levels can alter serotonin-mediated neurotransmission and may directly affect serotonin levels pre-synaptically, in the synaptic cleft and post-synaptically [36, 37]. Late pregnancy is known to be an important period for synaptic formation of serotonergic neurons in rats . Pre-natal exposure to dexamethasone during late pregnancy in rats decreases serotonin turnover in the cerebral cortex, hypothalamus and hippocampus . Exposure to COR during pregnancy leads to developmental changes in central serotonergic systems, as well as other brain monoaminergic systems, including the noradrenergic and dopaminergic pathways . As serotonergic functions can be modified by COR and CRH, programmed changes in HPA function that are independent of serotonin will have a permanent effect on the serotonergic system. However, the relationship between dopamine (DA) or norepinephrine (NE) and 5-HT systems in the regulation of HPA axis activity are complex and not fully understood, and this requires further elucidation in our future experimental studies.