Serotonin (5-HT) is a neurotransmitter in the brain. Among the multiple roles of serotonin in the brain, the most intriguing is its developmental role that has been recognized in recent years (Gaspar et al., 2003; Sodhi and Sanders-Bush, 2004; Daws and Gould, 2011). Serotonin modulates multiple physiological functions such as gastrointestinal motility, cardiovascular function, memory and mood in adult humans. The role of serotonin in neuropsychiatric conditions, such as autism spectrum or anxiety–related disorders, led to the recognition of a role during development (Whitaker-Azmitia et al., 1996; Whitaker-Azmitia, 2001, 2005; Gaspar et al., 2003; Daws and Gould, 2011).

Embryonic and postnatal mammalian brain development is highly reliant on multiple factors, one of which is serotonin. During embryonic and postnatal development, serotonin modulates neurodevelopmental processes, such as neuron cell division, migration and differentiation, axonal and dendritic elaboration, connectivity and myelination (Gaspar et al., 2003). Serotonergic neurons are first detected in the 5-week-old human embryos exhibiting rapid growth and proliferation (Sundstrom et al., 1993). After 15 weeks, serotonin neuron somata clusters are seen in raphe nuclei. In rodents, serotonin neurons appear at ~10.5 days gestation and begin releasing serotonin at ~13 days (E13) (Takahashi et al., 1986). Postnatally, brain serotonin concentrations peak in the first week of life, only to decline thereafter reaching adult concentrations at ~postnatal day 15 (P15) (Daws and Gould, 2011). Serotonin neurons continue their axonal growth through postnatal life until P21 (Daws and Gould, 2011). Perturbed brain serotonin concentrations during critical windows of development have long-lasting persistent effects on brain function, contributing toward the subsequent development of altered behaviors resulting in anxiety/depression related conditions during adult life (Whitaker-Azmitia, 2001; Sodhi and Sanders-Bush, 2004).

Serotonin transporter (SERT or 5-HTT) is a major regulator of serotonin neurotransmission and it has been implicated in the etiology of autism spectrum disorders (ASDs) (Muller et al., 2016). In the mature animal, SERT is located on presynaptic serotonin nerve terminals and serotonin neuronal cell bodies (Daws and Gould, 2011). SERT is first expressed during prenatal rodent brain development at E12, and by E18 it is found in all subcellular neuronal compartments (Bruning et al., 1997; Daws and Gould, 2011). During the course of embryonic development, SERT density peaks with advancing gestation (Moll et al., 2000; Daws and Gould, 2011) and is reported to be highest at P14, declining to relatively stable concentrations in adolescent rodents. This temporal pattern of SERT follows that seen with serotonin by one postnatal week of age. During critical stages of brain development, serotonin concentrations can influence the expression or function of key regulators of serotonin neurotransmission, including SERT (Brummelte et al., 2017). SERT expression and function are influenced by a large and diverse array of intrinsic and extrinsic factors. Intrinsic factors include genetic variants, auto- and hetero-dimerization, cytokines and hormones. Extrinsic factors such as diet, environmental stressors and drugs can also have prominent effects on SERT expression and function. Less is known about how these factors might come into play during prenatal and postnatal critical phases of development (Daws and Gould, 2011; Brummelte et al., 2017).

Many epidemiological studies in the offspring exposed in-utero to the Dutch Famine (Lumey and Stein, 1997) or born with a low birth weight revealed anxiety disorders in the survivors as adults (Boyle et al., 2011). This led to creation of animal models with maternal calorie restriction, to determine the long-term impact on the adult brain. One such study performed by our group revealed that maternal and/or postnatal caloric restriction in rats caused hyperactivity in a subset of the adults (Tomi et al., 2013), However, in these studies we did not decipher any changes in adult brain SERT concentrations (Tomi et al., 2013). However, maternal calorie restriction demonstrated reduced serotonin and SERT concentrations in E19 mouse brains (Ye et al., 2021). In contrast, studies in mice reared on a high fat diet revealed depressive symptoms in the adult (Aslani et al., 2015), while in-utero exposure to a high fat diet led to a lower trend in E19 brain serotonin concentrations with no comparable change in SERT concentrations (Ye et al., 2021). Similar investigations are lacking during the postnatal phase of development when both serotonin and SERT in brain are known to reach peak concentrations.

Since a brain serotonin imbalance can alter emotions, it is known that more than 90% of serotonin in the body is synthesized in the gut by various serotonin-producing bacteria such as Streptococcus spp., Enterococcus spp., Escherichia spp., Lactobacillus plantarum, Klebsiella pneumonia, and Morganella morganii (Barandouzi et al., 2022). In addition, certain indigenous spore-forming microbes, specifically Clostridial species from the gut microbiota, produce metabolites that promote host serotonin biosynthesis in colonic enterochromaffin cells with changes in TPH1 and SERT expression, thereby impacting gastrointestinal motility and homeostasis (Yano et al., 2015). Further, Hsiao et al. demonstrated that the maternal microbiome also modulates fetal neurodevelopment (Vuong et al., 2020). In their studies, the maternal gut microbiome promoted fetal thalamocortical axonogenesis, likely by signaling of microbially modulated metabolites to neurons in the developing brain (Vuong et al., 2020). “Dysbiosis” of the maternal gut microbiome, in response to environmental changes such as infection, altered diet such as a high fat and high fructose diet during pregnancy, has been increasingly associated with abnormalities in the offspring’s brain function and behavior (Buffington et al., 2016). Thus altered gut microbiome can regulate the biosynthesis, release and reuptake of certain neurotransmitters including serotonin. This in turn can affect the host serotonin receptors and signaling pathways. Such disturbances in the gut microbiome related to early life exposures to certain diets may perturb the gut-brain axis toward affecting permanent axonal-circuitary. Such alterations can form the basis of perturbed neurobehaviors that span from heightened stress responses to full fledged presentations of neuropsychiatric disorders that become evident in the adult offspring.

Based on the collection of these prior observations, we hypothesized that both maternal calorie restriction and exposure to a high fat and high fructose diet during fetal and postnatal life would alter postnatal brain serotonin and/or SERT concentrations associated with a dysbiosis relevant to serotonin producing bacterial species. These changes would display a temporal pattern providing the gut-brain based mechanism for previously observed neuropsychiatric presentations in response to early dietary modifications.

In our present study, we have demonstrated an imbalance between brain serotonin and SERT concentrations early in life in response to two different maternal dietary modifications. Serotonin being necessary for normal brain development, these observed changes have the ability to affect brain plasticity and adaptation of the serotonergic neural networks during their formative stages of embryonic and postnatal development, with a lasting impact on adult neurobehavioral expression. In addition, to these early life changes, we have observed sex-specific changes as early as during the suckling phase of postnatal development. We and others have conducted many postnatal studies with an impact on the adult phenotype in the rat (Cha et al., 1987; Garg et al., 2010, 2012, 2013; Dai et al., 2012; Shin et al., 2012), a species that displays exemplary maternal instincts and nurturing capabilities.

To ensure nutritional consistency, it is imperative to maintain the same litter size throughout the suckling phase. The previously characterized rat model of maternal calorie restriction producing intra-uterine growth restriction (Dai et al., 2012; Garg et al., 2012; Shin et al., 2012) was instituted prenatally at a time in gestation when denovo synthesis of serotonin occurs in the brain (Gaspar et al., 2003). In the hyper-caloric group the rats received high fat + high fructose in their diet to reflect the consumption of high fat and high carbohydrates in the Western population. Soon after birth (P1-P2), the mothers in both the IUGR and the HFhf groups were placed on HFhf diet and compared to controls reared on a regular chow (RC) diet. These mothers’ transmitted their metabolic changes in response to dietary modifications via their milk to their suckling offspring (Nicholas and Hartmann, 1991). In response to intra-uterine exposure to a maternal caloric restricted environment, the IUGR offspring revealed no changes in the immediate postnatal brain serotonin and SERT concentrations, while the male HFhf offspring displayed a diminution in both molecules. These observations demonstrate differences in the maternal dietary impact upon embryonic (via the placenta) and postnatal (via mother’s milk) brain 5-HT-SERT axis.

Further examination at P21 after having been exposed to the same postnatal dietary changes for a longer period of time, led to a persistent diminution of brain serotonin concentrations in both the HFhf male and female offspring, while the SERT concentrations were the same as control concentrations. This misalignment between serotonin-SERT may reflect lower synaptic availability of serotonin. In contrast, the IUGR offspring that was reared by a mother switched to a regular chow diet, demonstrated higher than normal in male and no change in female brain serotonin concentrations. While no differences were observed with SERT in the males, a reduction in SERT was evident in the female IUGR/HFhf group versus the other three groups. Thus, at this postnatal age, these changes set the stage for availability of higher serotonin concentrations in the synaptic cleft in both sexes via differing mechanisms.

In addition to these postnatal brain serotonin-SERT changes, significant alterations in the gut microbial constituents were seen at this developmental stage. These changes consist of a significant decrease in Bacteroides species within both the male and female sexes in certain groups exposed to maternal dietary modifications. Clostridium species were observed to increase in the three male groups while Streptococcus species increased in the IUGR/HFhf group alone versus the controls. Clostridium species is involved in serotonin production (Kuley et al., 2011) as well. Brain SERT concentrations which affect the availability of serotonin were also associated with changes in specific gut microbial genera in a sex-specific manner. Such changes in serotonin or other metabolite producing or regulating microbes can lead to a disequilibrium of the ecosystem of the host gut. This in turn may contribute toward dysregulation locally in serotonin or other metabolite production. Such perturbations have the propensity of altering host processes that impact circulatory concentrations and in some cases the neural networks including that of serotonin. These findings within the postnatal gut microbiome secondary to maternal dietary changes, along with the dysregulation of brain serotonin-SERT system, gives credence to the concept of the gut microbiome-brain axis. In fact changes in the maternal gut microbiome in mice have been demonstrated to influence prenatal neurodevelopment (Vuong et al., 2020, 2021). Our correlative observations between specific gut microbiota and brain serotonin/SERT concentrations, albeit descriptive, may support the influence of the gut microbiome during the critical window of prenatal and postnatal serotonergic neurodevelopment.

While we did not assess all the dietary groups, limited follow-up long term in the 180d adult male and female offspring temporally demonstrated a lack of persistence of these early life changes in brain serotonin in the HFhf and SERT concentrations in the IUGR-HFhf and HFhf fed groups when compared to respective controls (unpublished). A similar finding was also seen in the IUGR/RC male and female offspring in our previous study (Tomi et al., 2013). Thus it appears that even if the adult brain concentrations of serotonin and SERT may normalize despite ongoing exposure to the respective dietary modifications, the perturbations induced during the critical embryonic/postnatal window of plasticity in development may alter multiple aspects of neural wiring that may have long lasting functional effects upon the brain (Daws and Gould, 2011).

While in this study we did not undertake neurobehavioral studies, many other investigators have studied the neurobehavior of the adult IUGR offspring or high fat exposed offspring, and observed anxiety and hyperactivity in the former (Lahti et al., 2010; Mikaelsson et al., 2013; Tomi et al., 2013) and depressive behaviors in the latter (Contu and Hawkes, 2017). While there may be multiple factors contributing toward this ultimate neurobehavioral phenotype, our studies demonstrate a major role of maternal diet upon the chiseling of this ultimate phenotype. In addition, despite the normalization of either serotonin or SERT in the adult brain, various other perturbations related to activation of enhancers/modifiers, suppression of endogenous inhibitors, or other epigenetic factors can alter the serotonin-SERT pathway resulting in altered availability of serotonin for neurotransmission (Daws and Gould, 2011; Brummelte et al., 2017). Our observations where maternal dietary modifications perturb the 5-HT-SERT balance is reminiscent of the impact of drugs such as selective serotonin reuptake inhibitors (SSRI) that increase the availability of synaptic serotonin for neurotransmission. Maternal SSRIs can easily cross the placenta and alter fetal brain serotonin availability (Olivier et al., 2013), akin to what we observed in the postnatal IUGR offspring.

Genetic mutations of the serotonin receptor isoforms (5-HT1B and 5-HT1D) have led to changes in fetal neural circuitry causing aberrant neuro-behavior (Bonnin et al., 2007; Bonnin and Levitt, 2011). Non-genetic environmental alterations in either serotonin or SERT or both impact the synaptic serotonin availability and function. Particularly during embryonic development, serotonin availability is necessary for neurogenesis, migration and axonal development (Gaspar et al., 2003). During the postnatal stage, when brain serotonin and SERT surge, increasing dendritic-axonal networks and thereby neural connections and circuitry are established along with ongoing pruning (Daubert and Condron, 2010; Lesch and Waider, 2012). We have shown that dietary environments can cause an early 5-HT-SERT imbalance with the possibility of deranging neural/axonal development and circuitry. These derangements come with the propensity of long-term implications that adversely affect plasticity and adult neuro-behavior (Shah et al., 2018). More recently, mutations of SERT have been implicated in autism spectrum disorders (Muller et al., 2016; Siemann et al., 2017), and circulating maternal serotonin concentrations have been associated with ASD in their offspring (Montgomery et al., 2018). However, serotonin fails to cross the placenta (Bonnin et al., 2011), suggesting other indirect effects of these changes having implications for the developing embryo. It is known that males predominate in developing ASD (Loomes et al., 2017), while females have a higher incidence of anxiety (McLean et al., 2011). We have observed a sex-specific effect of changes in serotonin, with the male predominating in particular. However, longer duration of exposure to dietary modifications end up affecting both the males and females as observed at P21. It is important to decipher the mechanism of action between diet and 5-HT-SERT pathway. While one of the mechanisms tying the dietary modifications to the neural serotonin-SERT connectivity may involve the gut microbiome as the intermediary, other dietary modified enhancers/inhibitors or epigenetic/nutrigenomics may also contribute requiring future mechanistic investigations (Daws and Gould, 2011; Brummelte et al., 2017). Further the impact of the X versus the Y chromosome upon these mediating molecular mechanisms needs unraveling.

In conclusion, we have demonstrated that prenatal maternal dietary modifications (either low or high calories) alter postnatal brain serotonin and/or SERT concentrations in a sex-specific manner, with the male offspring being primarily affected during the immediate early postnatal stage. With ongoing exposure to the altered dietary environment, both males and females are ultimately affected. The IUGR offspring created in response to maternal caloric restriction demonstrates a reduction in serotonin with concomitant diminution of SERT which can be restituted with a normal postnatal diet, only to overshoot the normal with a prenatal to postnatal nutritional mismatch, namely postnatal exposure to a high fat and high fructose diet. In contrast, a high fat and high fructose diet prenatally and postnatally creating a nutritional match led to a reduction in serotonin with no change in SERT, particularly in the male offspring. Thus reduced calorie intake by mothers can increase serotonin availability, while increased calorie intake decrease serotonin availability during this critical window of development. These changes may underlie the development of anxiety and hyperactivity in the adult IUGR offspring (Lahti et al., 2010; Mikaelsson et al., 2013) and depression in the offspring exposed to early high fat and high fructose nutritional environment (Contu and Hawkes, 2017). In all cases, the molecular mechanisms tying early introduction of dietary modifications with the developing offspring’s brain neurotransmitters is of significant importance necessary for unraveling the pathogenesis of neurodevelopmental and mental health disorders. The resulting gut dysbiosis induced by the maternal dietary modifications likely underlies the brain-gut axis reliant changes seen in the developing postnatal brain. Future mechanistic studies are warranted toward unraveling mechanisms required for developing targeted therapeutic measures.

The datasets presented in this study can be found in online repositories. This data can be found in the Gene Expression Omnibus accessible through GEO Series accession number GSE220146.

The animal study was approved by UCLA Animal Research Committee. The study was conducted in accordance with the local legislation and institutional requirements.

XY: Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. SG: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – review & editing. BS: Investigation, Methodology, Supervision, Validation, Writing – review & editing. AG: Methodology, Writing – review & editing. LM: Methodology, Writing – review & editing. JJ: Data curation, Software, Validation, Writing – review & editing. SD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.