Transcriptomic Alteration in the Brain and Gut of Offspring Following Prenatal Exposure to Corticosterone

Abstract

Maternal stress during pregnancy can profoundly affect offspring health, increasing the risk of psychiatric disorders, metabolic diseases, and gastrointestinal problems. In this study, the effects of high prenatal corticosterone exposure on gene expression in the brain and small intestine of rat offspring were investigated via RNA-sequencing analysis. Pregnant rats were divided into two groups: Corti.Moms were injected with corticosterone daily, while Nor.Moms were given saline injections. Their offspring were labeled as Corti.Pups and Nor.Pups, respectively. The brain tissue analysis of Corti.Pups showed that the expression levels of the genes linked to neurodegenerative conditions increased and enhanced mitochondrial biogenesis, possibly due to higher ATP demands. The genes associated with calcium signaling pathways, neuroactive ligand-receptor interactions, and IgA production were also upregulated in the small intestine of Corti.pups. Conversely, the genes related to protein digestion, absorption, and serotonergic and dopaminergic synaptic activities were downregulated. These findings revealed that gene expression patterns in both the brain and intestinal smooth muscle of offspring prenatally exposed to corticosterone were substantially altered. Thus, this study provided valuable insights into the effects of prenatal stress on neurodevelopment and gut function.

INTRODUCTION

Psychiatric disorders including attention deficit hyperactivity disorder (ADHD), autism spectrum disorder, bipolar disorder, depressive disorder, and schizophrenia, pose substantial challenges to health care systems, economies, and societies worldwide [1-4]. Studies have found that increased depressive symptoms in childhood behavioral issues in humans and animals are associated with maternal prenatal stresses, anxiety, or increased cortisol levels [5-11]. The hypothalamic-pituitary-adrenal (HPA) axis is essential for regulating a range of responses, particularly stress. During pregnancy, the placenta functions as a crucial mediator between maternal and fetal systems, considerably influencing fetal development and maternal physiology. The HPA axis undergoes remarkable adaptations in which glucocorticoids are implicated in the complex interplay [12-15]. This proposed mechanism suggests that changes in these hormonal pathways, particularly in glucocorticoid levels or signaling, because of maternal stress or other factors may influence fetal development and contribute to the relationship between prenatal conditions and later psychiatric or neurodevelopmental outcomes [16-18].

The intricate relationship between psychiatric disorders and gastrointestinal (GI) function is mediated through the brain–gut axis, which facilitates complex bidirectional communication [19-23]. During periods of stress or anxiety, this axis can substantially influence the GI motor function. In response to emotional changes, this interaction often manifests as physical symptoms such as diarrhea, dyspepsia, and abdominal discomfort [24-26]. The interplay between the central nervous system and the enteric nervous system (ENS) is also relevant in neurological conditions such as Parkinson’s disease [27-30]. Therefore, a holistic approach that can manage conditions such as inflammatory bowel disease (IBD) and psychiatric disorders should be developed by considering mental health and GI function to optimize patient care and outcomes.

In our previous research, prenatal exposure to increased cortisol levels in pregnant rats (Corti.Moms; corticosterone-injected pregnant rat) has caused their offspring to exhibit ADHD-like behaviors (Corti.Pups; offspring exposed to elevated maternal corticosterone during pregnancy), including hyperactivity, impulsivity, and inattention [31]. Based on this finding, our current study aimed to explore the changes in the transcriptome underlying these behavioral and cognitive alterations induced by maternal prenatal stress. We conducted RNA sequencing (RNA-seq) on the brain tissue and the smooth muscle of the small intestine of Corti.Pups to investigate the transcriptomic changes associated with prenatal corticosterone exposure. Thus, we could gain further insights into the molecular mechanisms of prenatal stress-induced behavioral alteration and may recommend future strategies for the early intervention and treatment of stress-related neurodevelopmental disorders such as ADHD or disorder in GI functions.

MATERIALS AND METHODS

Animal model production

Female Sprague-Dawley rats were used from animal facility at Jeju National University Medical School. The experimental protocol, as previously described, involved two groups of pregnant rats [31]. The 8~9 week old pregnant rats received daily subcutaneous injections of corticosterone at a dose of 20 mg/kg throughout their pregnancy referred as Corti.Moms. The control group of pregnant rats received daily injections of saline (0.9% NaCl) instead of corticosterone, referred as Nor.Moms. The offspring of the Nor.Moms and Corti.Moms were designated as Nor.Pups and Corti.Pups, respectively. Corticosterone was suspended in 0.9% NaCl supplemented with 0.1% DMSO and 0.1% Tween 80, was used for injections. Brain and small intestine samples were collected from all male and female pups after they were sacrificed, following a two-week period of maternal care. To isolate smooth muscle from small intestine, the muscle layer was separated by sharp dissection on sylgard plate contained with ice-cold physiological solution. For bulk RNA-seq, the brain and smooth muscle of small intestine samples were immediately placed into TRIzol reagent.

RNA-seq data processing

The kallisto tool [32] was used to quantify the genome-wide gene expression profile from the fastq files based on the rat cDNA sequences obtained from the Ensembl database. The edgeR tool [33] was applied to perform the differential expression analysis between the Nor.Pup and Corti.Pup samples. The “TMM” method embedded in the edgeR tool was used for the normalization of the sequencing reads along with likelihood ratio test being used to identify the differentially expressed genes. The genes with the fold change (FC) greater than 1.5 and false discovery rate (FDR) less than 0.1 were deemed differentially expressed. Pathway/gene ontology analysis was performed based on the list of the differentially expressed genes using the DAVID online tool [34].

RESULTS

The differential expression of genes in the brain and small intestinal smooth muscle between the Nor.Pups and Corti.Pups

Volcano plots provide a visual representation of gene expression changes, illustrating the log₂-fold change between Nor.Pups and Corti.Pups (Fig. 1). This analysis revealed a substantial proportion of genes differentially expressed in both rat brain and small intestinal smooth muscle. In the brain, 60 genes showed differential expression, with 24 upregulated and 36 downregulated in the Corti.Pup group. The small intestine smooth muscle exhibited more extensive changes, with 108 differentially expressed genes, including 48 upregulated and 60 downregulated in Corti.Pups.

Fig. 1.

Volcano plot of the differential gene expression between Nor.Pups and Corti.Pups in brain and small intestine, respectively. Pink dots indicate the upregulated genes, while light blue dots indicate the downregulated genes. The genes with false discovery rate (FDR) <0.1 and fold change (FC) in expression >1.5 were defined as differentially expressed.

Among the upregulated genes in the brain of Corti.Pups, several mitochondrial genes associated with the electron transport chain, specifically Complex I components, were identified (Fig. 2). These included Mt-nd1, Mt-4, Mt-nd4I, Mt-nd5, Mt-nd6. The upregulation of these genes suggests enhanced mitochondrial activity and energy production in the brain tissue of Corti.Pups. Additionally, Mt-atp8, which encodes a subunit of ATP synthase (Complex V), was also upregulated, further supporting increased mitochondrial function and ATP production.

Fig. 2.

Expression heatmap of the differentially expressed genes in rat brain between the Nor.Pups and Corti.Pups. Red represents higher expression while blue indicates lower expression.

Fig. 3 is a heatmap generated from gene expression data related to differentially expressed genes in the small intestinal smooth muscle between Corti.Pups and Nor.Pups. In the small intestine of Corti.Pups, downregulation of several important transporter genes was observed (Fig. 3). Notably, Slc1a1, which encodes a glutamate was downregulated. Its downregulation in the intestine may indicate altered amino acid transport dynamics. Similarly, Slc22a3, which encodes the organic cation transporter 3, showed decreased expression. Another significant finding was the downregulation of Scn1a in the small intestine. This gene encodes the voltage-gated sodium channel subunit NaV1.1, which is critical for the generation and propagation of action potentials in excitable cells. While Scn1a is primarily associated with neuronal function and epilepsy when mutated, its down regulation in intestinal smooth muscle could potentially affect the electrical activity and contractility of the tissue. The development of the ENS is a complex process that includes migration, proliferation, and differentiation of neural crest cells. This process leads to the formation of a specialized neural network in the gut that regulates GI functions. An important aspect of this development is axonogenesis, which refers to the growth of axons needed to connect to intestinal muscle and other targets. Among upregulated genes in small intestinal smooth muscle of Corti.Pups compared to Nor.Pups, Spon2, Cxcr4, and Hoxc6 were identified. They play a role in developing nervous systems. For example, spondins are known to be involved in cell adhesion and neurite outgrowth [35]. Cxcr4 signaling is essential for proper formation of neural circuits by influencing neuronal migration and axon guidance during development [36]. Hoxc6 is also known to influence neuronal differentiation and axon guidance in specific regions of the nervous system. Hoxc6 could potentially play a role in establishing the ENS, which is a complex network of neurons within the intestinal smooth muscle [37]. While Spon2, Cxcr4, and Hoxc6 have known roles in axonogenesis in general, whether or not they are involved in intestinal axonogenesis would require further research.

Fig. 3.

Expression heatmap of the differentially expressed genes in rat small intestinal smooth muscle between the Nor.Pups and Corti.Pups. Red represents higher expression while blue indicates lower expression.

Comparison of signaling pathways between the Nor.Pups and Corti.Pups

Based on the lists of the differentially expressed genes, we further performed gene ontology/pathway analyses. The differentially expressed genes in the brains of the Corti.Pups (compared to Nor.Pups) revealed significant associations with several Gene ontology Biological Process (GOBP) terms. Fig. 4 represents the results of GOBP term enrichment analysis, highlighting biological processes significantly enriched in the brain and small intestine when comparing Corti.Pups to Nor.Pups. In the brain of Corti.Pups, enriched GOBP terms are associated with mitochondrial electron transport, ATP synthesis, response to hypoxia/hyperoxia, glucose homeostasis, cell proliferation, and DNA replication. Many of the top GOBP terms are directly linked to mitochondrial function and energy production, reflecting the brain’s high energy demands. Additionally, terms such as response to hypoxia and response to hyperoxia indicate the brain’s sensitivity to oxygen level fluctuations and its active response to oxidative stress. Overall, the dominant themes in the brain of Corti.Pups are energy metabolism and stress responses, particularly those related to oxygen and oxidative stress. In the small intestinal smooth muscle of Corti.Pups, enriched GOBP terms include processes related to axonogenesis, brain development, cell morphogenesis, cellular response to growth factor stimulus, locomotory behavior, inner ear development, neuron differentiation, neuron projection development, response to nutrient levels, and response to xenobiotic stimulus. A notable feature is the presence of GOBP terms associated with neuronal development, reflecting the critical role of the ENS. In addition, we also tried a more stringent FDR cutoff of 0.05 to prioritize the differentially expressed genes. Gene ontology analysis reveals that the GOBP terms associated with new gene lists largely mirror the findings when a cutoff of 0.1 being used (Supplementary Fig. S1). These findings underscore the importance of neuronal growth, cell renewal, and responses to external stimuli such as nutrients, growth factors, and xenobiotics in the small intestine of Corti.Pups.

Fig. 4.

The top ten GOBP terms associated with the differentially expressed genes in rat brains and intestinal smooth muscle. The vertical dash line indicates the p-value level of 0.05.

Fig. 5 represents a comparative analysis of KEGG pathway enrichment in the brain and small intestine of Corti.Pups relative to Nor.Pups. In the brain of Corti.Pups, pathways associated with neurodegenerative disease show the strongest enrichment, including Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. This indicates significant dysregulation of genes involved in these disorders. The enrichment of oxidative phosphorylation pathways in the brain data is particularly noteworthy. This suggests potential alterations in mitochondrial function and energy metabolism, which are critical for normal neuronal activity and brain development [38]. Dysregulation of oxidative phosphorylation has been linked to the onset of cognitive decline and may be an early marker of neurodegeneration [39]. Additionally, the enrichment of pathways related to chemical carcinogenesis implies that genes involved in response to environmental toxins or cellular stress may also be affected in the brain of Corti.Pups. This could indicate altered neuroprotective mechanisms or increased susceptibility to cellular damage. On the other hand, the differentially expressed genes in rat small intestine showed significant enrichment in pathways such as calcium signaling pathway, neuroactive ligand−receptor interaction, intestinal immune network for IgA production, among others (Fig. 5 and Supplementary Table S1). Primarily enriched for pathways related to digestion, immune function, and cell signaling in Corti.Pups compared to Nor.Pups. In the small intestine, the enriched calcium signaling pathway likely modulates intestinal motility, secretion digestive enzymes, and nutrient absorption, as calcium signaling governs smooth muscle contraction, hormone secretion, and enzyme activation [40, 41]. The enriched neuroactive ligand-receptor interaction pathway suggests a significant role for neurotransmitters and neuropeptides in the intestinal environment. These interactions are particularly relevant because the ENS, the gut’s intrinsic nervous system, uses these signals to regulate digestive processes [42, 43]. These findings highlight prenatal corticosterone treatment effects on energy metabolism and neurological disorders in the brain, while the small intestine exhibited alteration in calcium signaling and immune pathways.

Fig. 5.

The top ten KEGG pathways associated with the differentially expressed genes in rat brains and intestinal smooth muscle. The vertical dash line indicates the p-value level of 0.05.

DISCUSSION

During pregnancy, numerous factors, including maternal diet, maternal stress, depression, environmental toxins, and medication use, can influence neurodevelopmental and metabolic processes in the developing fetus [31, 44-48]. A previous study established remarkable links between these prenatal exposures and various outcomes, including an increased risk of ADHD and obesity/overweight among offspring [49, 50]. One of the major stress hormones released from maternal psychological stress is cortisol, which can be transferred to the fetus [14, 18, 51, 52]. Cortisol is a key regulator of stress-induced depression, and corticosterone injections in animal models have been shown to elevated plasma cortisol levels, resulting in neuroendocrine dysregulation and depressive-like symptoms [53]. Typically, corticosterone is administered to pregnant rats to explore potential behavioral changes in their offspring that could be linked to neuropsychiatric disorders such as depression or autism. Surprisingly, in our previous study, rather than displaying the depressive-like behaviors commonly observed in adult models, the offspring of corticosterone-treated mothers exhibited hyperactivity, reduced anxiety, and impulsive behaviors [31]. To further explore this unexpected finding, the current study examines how maternal corticosterone exposure influences the transcriptome during early brain development and its potential impact on intestinal smooth muscle function via the brain-gut axis. Our transcriptomic analysis of the brain of Corti.Pups reveled several interesting findings such as a concerning pattern of the gene expression related to neurodegenerative diseases. Specifically, the genes associated with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease were upregulated. The increased expression of neurodegeneration-related genes suggested that maternal stress during pregnancy, as modeled prenatal corticosterone exposure, might potentially increase the offspring’s susceptibility to these disorders later in life.

Neurological conditions are characterized by progressive neuronal loss and cognitive decline, which are often coupled with metabolic dysfunctions that affect glucose metabolism and mitochondrial biogenesis [54-60]. Notably, our transcriptomic analysis revealed that the mitochondrial-related genes in the brain of Corti.Pups were significantly upregulated compared with those of Nor.Pups. This finding suggested that prenatal stress might influence cellular energy metabolism. Specifically, we observed that the expression levels of several key mitochondrial genes in the brain tissue of Corti.Pups increased; these genes included those encoding NADH dehydrogenase subunits; Mt−nd1, Mt−nd4, Mt−nd4l, Mt−nd5, Mt−nd6, mitochondrial cytochrome b, and ATP synthase subunits; and Mt-atp6 and Mt-atp8. They are crucial components of the mitochondrial respiratory chain, which is essential for ATP production and overall energy metabolism [61-64]. Their upregulation suggests a potential compensatory response to increased energy demands or mitochondrial dysfunction induced by prenatal stress exposure. Neurons are dependent on the mitochondria for ATP production because of their high energy and oxygen consumption [65, 66]. Thus, they are more vulnerable to any kind of mitochondrial damage and dysfunction, which may trigger various neurodegenerative disorders [67, 68]. Their energy needs can be satisfied by enhancing mitochondrial function, potentially improving their survival and function [59, 60]. The increased mitochondrial gene expression observed in our study could indicate enhanced mitochondrial biogenesis, which was possibly triggered by the increased ATP demand. The compensatory upregulation of mitochondrial function is a vital adaptive response to increased energy demands, metabolic stress, and mitochondrial dysfunction. Cells can maintain energy homeostasis and support overall function by enhancing mitochondrial biogenesis, improving phosphorylation efficiency, and upregulating antioxidant defenses [69, 70].

Neurological conditions can remarkably disrupt the bidirectional communication between the gut and the brain, thereby changing intestinal sensitivity, motility, secretion, and permeability. As a result, a wide range of neurological and GI complications occur, emphasizing the intricate relationship between these systems [71-73]. For instance, patients with irritable bowel syndrome and IBD often suffer from psychological comorbidities, and the underlying mechanisms of these conditions involve complex interactions between the central nervous system, peripheral neurons, gut microbiota, and mucosal immune activation [74-76]. Our analysis of the small intestine of Corti.Pups compared with that of Nor.Pups revealed that the increased expression in calcium signaling and neuroactive ligand-receptor interactions could indicate that gut motility and sensitivity were altered. The upregulation of the IgA production network might imply that the intestinal immunity changed. Conversely, the decreased expression in protein digestion and absorption genes might indicate that nutrient processing capabilities were reduced. The downregulation of serotonergic and dopaminergic synapses could indicate that the gut–brain axis and ENS function were altered. Collectively, these changes highlighted the complex effect of prenatal stress on intestinal development and function. Serotonin, a major neurotransmitter in the ENS, can stimulate both excitatory and inhibitory enteric neurons, affecting motility patterns. It promotes peristalsis by stimulating intrinsic primary afferent neurons and can increase intestinal secretions and motility. Dopamine generally elicits an inhibitor effect on GI motility. These neurotransmitters, along with the ENS, modulate gut function [77-79]. These findings suggest that prenatal exposure to corticosterone may substantially influence intestinal function, potentially altering signaling mechanisms, neuronal communication, and immune responses in the gut of the offspring.

Calcium dynamics are crucial for the proper functioning of intestinal smooth muscle. In the small intestinal smooth muscle of Corti.Pups, Atp2b2, which encodes plasma membrane calcium ATPase isoform 2 (PMCA2), plays a critical role in maintain these dynamics by transporting cytosolic calcium ions across the plasma membrane [80]. This process contributes helps regulate intracellular calcium levels, indirectly influencing motility and peristalsis [81]. In the colon, nitric oxide (NO), released by nitrergic neurons, serves as another key regulator of smooth muscle contraction and colonic migrating contractions through its interaction with interstitial cells of Cajal (ICCs) [82, 83]. Nitrergic neurons are essential for sustained smooth muscle relaxation, which is critical for propulsion and storage activities. Their inhibitory regulation ensure proper colonic motility and signal transmission. Interestingly, a reduced expression of Nos1 (neuronal nitric oxide synthase) was observed in the small intestinal smooth muscle of Cori.Pups. Nos1, a primary source of NO in the ENS, acts as an important inhibitory neurotransmitter [84, 85]. The decreased expression of Nos1 may impair local NO production, potentially contributing to motility disorders in the GI tract. Additionally, differences in the expression of Myo16 and Myo19 were noted. While distinct in function, both genes are critical for cellular processes. Myo16 primarily operates in neuronal cells, where it regulates cytoskeleton dynamics and synaptic organization. It localizes to postsynaptic dendritic spines and modulates actin dynamics to influence synaptic structure and function. In contrast, Myo19, a mitochondrial motor protein, is involved in mitochondrial distribution and homeostasis. Together, these proteins are essential for maintaining cellular functionality. The synaptotagmin family members Syt13 and Syt15 also exhibited differential expression. Syt13 is a multifunctional protein involved in pancreatic endocrine development, vesicle trafficking via calcium-independent mechanisms, and neuroendocrine signaling. It also has potential implications in cancer biology [86, 87]. Unlike most synaptotagmins that rely on calcium binding for their function, Sys13 lacks calcium sensitivity due to structural differences in its C2A and C2B domains [88]. Its expression in enteroendocrine cells suggests a role in regulatory secretory pathways [87]. On the other hand, Syt15 likely contributes to secretory vesicle trafficking and exocytosis, particularly in non-neuronal tissues. It may also act as a calcium sensor for membrane fusion processes; however, its precise role requires further investigation [89].

Our findings reveal significant transcriptomic changes in the brains of offspring exposed to maternal corticosterone, particularly in upregulated neurodegenerative processes, energy metabolism, and stress response mechanisms. These results emphasize the importance of further investigating these pathways, as they may uncover potential therapeutic targets or early biomarkers for neurodegenerative diseases. Moreover, the observed impact on differentially expressed genes in intestinal smooth muscle could provide valuable insights into ENS development and GI function through the brain-gut axis. This highlights the interconnectedness of neural and digestive systems and opens avenues for exploring their mutual influences. It’s important to note that our study, like many bioinformatics analyses, faces inherent challenges in translating computational predictions into biological systems. While large-scale gene expression analyses serve as powerful tools for generating hypotheses, experimental validation remains crucial.