Maternal consumption of alcohol during pregnancy results in a broad constellation of outcomes known as fetal alcohol spectrum disorder (FASD) and includes central nervous system (CNS) deficits manifesting as cognitive, behavioral and emotional dysregulation (Moore and Xia, 2021). Alcohol can interact with CNS systems such as the hypothalamic–pituitary–adrenal (HPA) axis that regulate stress responses. Published data show that, as a consequence of alcohol consumption during pregnancy, increased maternal glucocorticoids lead to detrimental exposure levels to the fetus (O'Connor et al., 1998; Przybycien-Szymanska et al., 2011). Further, alcohol can alter the expression of corticotropin-releasing hormone (CRH), a factor that initiates HPA activation in response to alcohol or stress, and is released within several key brain regions involved in stress regulation such as the periventricular nucleus within the hypothalamus (O'Connor et al., 1998), the amygdala, and the hippocampus (Tsigos et al., 2000; Charney, 2004; Zhou and Fang, 2018). In addition to fetal exposure to deleterious levels of maternal stress hormones, maternal consumption of alcohol poses direct developmental risks to the fetus, as alcohol is capable of crossing the placenta to interact directly with ongoing fetal CNS maturation including HPA axis regulation (Gabriel et al., 1998). That is, prenatal alcohol exposure (PAE) results in elevated glucocorticoids in fetal blood and acts in brain, altering glucocorticoid receptor expression and signaling in the hypothalamus, amygdala and hippocampus (Caldwell et al., 2014; Lam et al., 2018; Lu et al., 2018; Raineki et al., 2019). Importantly, the consequence of elevated glucocorticoid levels during CNS maturation results in reduced CNS neural development and myelination (De Kloet et al., 1988). While the changes and the impact of initial biochemical factors/receptors resulting from direct alcohol exposure from maternal alcohol consumption differ from maternal stress, the downstream consequences of fetal exposure to elevated glucocorticoids (Gabriel et al., 1998; Lan et al., 2017) converge resulting in an enduring dysregulation of the brain stress-response and HPA axis in offspring (Rash et al., 2016; Gartstein and Skinner, 2018; McGowan and Matthews, 2018).

A large body of evidence demonstrates an extensive interface of the neuro-immune-endocrine systems, with bi-directional communication and regulation (Elenkov et al., 2000; Haddad et al., 2002) through the actions of common ligands including cytokines/chemokines and their receptors in brain regions such as, but not limited to, the hypothalamus, amygdala and hippocampus. While glucocorticoids inhibit the production of several potent proinflammatory cytokines including interleukin (IL)-1β (Páez Pereda et al., 1996; Goujon et al., 1997; Plagemann et al., 1998), conversely, brain proinflammatory cytokines induce HPA activation through multiple mechanisms including reducing glucocorticoid receptor sensitivity (Pariante et al., 1999; Daun et al., 2000). More recent studies have extended the role of the neuro-immune-endocrine interface in underlying CNS dysregulation as a consequence of PAE (Bodnar et al., 2016), emphasizing the critical influence that a variety of cytokines exert in the developing CNS, particularly in offspring following PAE.

Prenatal alcohol exposure negatively impacts the delicate balance of neuroimmune factors. Increases in glial (microglia and astrocytes) activation, proinflammatory cytokines IL-1β and tumor necrosis factor-a (TNF-α), and the chemokine known as C–C motif chemokine ligand-2 (CCL2) are observed in the brain from offspring with PAE, along with decreases in the anti-inflammatory cytokine, interleukin-10 (IL-10; Drew et al., 2015; Topper et al., 2015) and in the spinal cord following peripheral nerve damage (Noor et al., 2017, 2020; Sanchez et al., 2017, 2019). Additionally, PAE female rat pups at postnatal day (PND) 8 reveal IL-1β and TNF-α protein are decreased in the hypothalamus and increased in the hippocampus (Bodnar et al., 2016). However, it remains poorly understood how altered early-life neuroendocrine-immune function may impact factors that initiate the brain stress response system (i.e., CRH) in neonatal brain regions where glucocorticoid receptors are expressed.

Given PAE exerts strong influences on the brain neuroimmune-neuroendocrine interface, the goal of the current study was to examine whether PAE alters early-life transcriptional (mRNA) expression of CRH and arginine vasopressin (AVP), which play critical roles in initiating the HPA stress responses, in addition to basal and stress-induced neuroimmune factors generated by stimulation of the Toll-like Receptor 4 (TLR4). Activated TLR4 results in a proinflammatory cellular signaling cascade of cytokines, chemokines, and transcription factors (Chen and Jiang, 2013). The stress-integrative and responsive brain regions, the hypothalamus, amygdala and hippocampus, following maternal separation stress at PND10 are examined. Interestingly, growth arrest specific 5 (GAS5) is characterized to occupy the nuclear DNA-binding domain of the glucocorticoid receptor and FK506-binding protein 51 (FKBP51) binds to the glucocorticoid receptor in the cytoplasm resulting in reduced nuclear translocation of the glucocorticoid-glucocorticoid receptor complex (Davies et al., 2002; Petta et al., 2016). Consequently, GAS5 and FKBP51 may act to reduce glucocorticoid receptor signaling. Conversely, FKBP52 acts to promote glucocorticoid receptor high affinity for glucocorticoids, thus enhancing glucocorticoid nuclear translocation of the glucocorticoid receptor complex (Schiene-Fischer and Yu, 2001). Thus, we speculate that PAE reduces glucocorticoid receptor function and ultimately signaling through the dysregulated increase in GAS5, FKBP51, and FKBP52 in limbic regions critical for stress regulation. We hypothesized that PAE acts to sensitize stress neuropeptides, endogenous glucocorticoid receptor signaling regulators and TLR4- related and -independent proinflammatory factors in the hippocampus, amygdala and hypothalamus in response to an acute stressor during early postnatal life.

Accumulating evidence supports the adverse impact of PAE on CNS function that often goes unnoticed until a secondary challenge occurs after birth and well into adulthood, typically from direct immune activation or from direct peripheral tissue damage. Significant PAE-induced susceptibility of the CNS to dysregulation may involve sensitized inflammatory cytokines/chemokines and transcriptional factors (Drew et al., 2015; Bodnar et al., 2016, 2020; Terasaki and Schwarz, 2016; Noor et al., 2017, 2020; Sanchez et al., 2017, 2019). The current report extends these observations by demonstrating that a secondary challenge can include non-tissue damaging stressors occurring in early life, and supports that both neuroimmune and neuroendocrine dysregulation may underlie early programing that leads to adult CNS vulnerabilities. In support of prior studies, a striking demonstration is made in the current report that the amygdala reveals significant mRNA upregulation of the stress-associated factors CRH and AVP (Figure 1A; Table 2), disruptors of glucocorticoid receptor signaling GAS5 and FKBP5 (Figure 2B; Table 2), the microglial activation marker transmembrane protein 119 (TMEM119; Figure 3B; Table 2), and factors that initiate or are induced downstream of TLR4 activation (Figure 4B; Table 2) by PAE in offspring treated with a single session of maternal separation stress at PND10.

Other proinflammatory factors measured in the amygdala that are not necessarily dependent on TLR4 activation, but can impact TLR4 downstream effects and are critical for mediating aspects of innate immune proinflammatory progression are altered in females as a consequence of stress (TNF), or with disparate IL1R1 stress-response changes between SAC and PAE (Figure 5B and Table 3). IL-10, as an endogenous anti-inflammatory cytokine showing compensatory upregulation upon inflammatory cytokine signaling, is robustly increased in females with PAE that are also exposed to maternal separation stress (Figure 5B; Table 3).

In the hypothalamus, blunted CCL2 (in males), IL1R1 and IL-10 (in females) with strong trends toward blunted TNF levels as a consequence of PAE are observed (Figure 5C; Table 3). The extent of low hypothalamic mRNA neuroimmune factors under basal conditions and blunted in response to stress in offspring with PAE consistently extends to a large number of proinflammatory indicators, as shown by blunted glial activation (astrocytes and microglia; Figure 3C; Table 3), factors associated with the TLR4 signaling cascade (HMGB1, TLR4, NFKBIA, and NLRP3; Figure 4C; Table 3), that were revealed predominantly in female offspring. Even strong trends in reduced CCL2 expression are shown in females (Figure 5C). The observed data from hypothalamic tissue are remarkably consistent, and while speculative, suggest that the underlying hypothalamic process may involve an exaggerated stress hyporesponsive period. That is, during later life stressors, the hypothalamus may be reprogramed as less responsive to elevated circulating glucocorticoids, with diminished glucocorticoid receptor function until elevated glucocorticoid levels at longer-durations are achieved before the activation and downstream impact by glucocorticoids occur.

While an earnest attempt was made to measure protein levels of CRH, FKBP5, NLRP3 and IL-1β from the amygdala, only CRH protein from males revealed significant increases relative to controls without PAE (Table 4). Considering the pattern of CRH mRNA together with protein CRH in the male amygdala, the data suggest that protein levels retrieved from these samples are too low to achieve interpretable results. Moreover, due to loss of material, large discrepancies in samples sizes for various targets resulted, and the resultant data analyses are limited in this respect. No additional changes in protein levels were observed.

The amygdala is uniquely positioned to regulate responses to psychogenic stressors, as cortical association areas project to the amygdala, and in turn, the amygdala influences the function of critical stress-integrative brain areas such as the hippocampus and hypothalamus through direct amygdala-hippocampal (Stefanacci et al., 1996; Leal et al., 2017; Zheng et al., 2019) and amygdala-hypothalamic circuitry (Alexander et al., 2012). In addition, the amygdala is characterized as the fundamental brain area supporting fear-related behaviors such that a perceived threat is immediately processed as dangerous (Murison, 2016; Godoy et al., 2018) with amygdaloid activation being important for emotional retrieval and analysis of any given stressor. The amygdala can directly stimulate components of brain stress-response circuits via CRH neuropeptide release in (a) an autocrine manner within the amygdala and also (b) at more distant sites such as in the hippocampus (Tsigos et al., 2000). Notably, CRH peptidergic neurons in the amygdala are activated by glucocorticoids and upon activation, lead to the stimulation of the stress circuitry and fear/anxiety (Kolber et al., 2008). The amygdala-hippocampus circuit is bidirectional, and under normal conditions, the hippocampus can exert a tonic and evoked inhibitory effect on CRH neurons of the amygdala. Indeed, the hippocampus plays an important role in shutting off the HPA stress response [(Tsigos et al., 2000) for Review].

In light of this background concerning hippocampal influences on amygdala function, one aspect of the CNS stress-response system examined by the current report is the potential impact that PAE exerts on general amygdaloid responses to a single, acute stress (maternal separation). The current data demonstrate pronounced changes in mRNA expression of CRH and AVP, both of which are significantly elevated in the amygdala (Figure 1A). Unexpectedly, these changes are observed from tissue derived from females with PAE and not from males.

Under non-pathological conditions, the ventral hippocampus has been characterized to influence hypothalamic responses resulting in activation of hypothalamic subnuclei. The present data show that blunted AVP mRNA expression is observed in males (Figure 1B), which may be a consequence of dysregulated ventral hippocampal influences on hypothalamic subnuclei (e.g., lateral hypothalamic area) by PAE. However, meaningful mRNA changes of CRH in the hypothalamus were not observed in neither males nor females.

The current data also show that not only are stress-associated factors elevated in the amygdala following PND10 maternal separation, but also factors that disrupt glucocorticoid receptor signaling. Glucocorticoid receptors are a key effector element of the HPA stress response (De Kloet et al., 1988; Oakley and Cidlowski, 2013). Glucocorticoid receptors interact with factors that function to fine tune the HPA stress response, thereby regulating glucocorticoid receptor signaling or its sensitivity. GAS5 is a long non-coding RNA expressed throughout the body originally found to have tumor suppressor activity by arresting the growth cycle of stressed cells, but also promotes apoptosis and acts to remove micro-RNA, ultimately decreasing glucocorticoid-dependent transcription of genes; it essentially acts as a decoy glucocorticoid response element (GRE) within the cell nucleus (Petta et al., 2016; Yu and Hann, 2019). The current report reveals that transcriptional expression of GAS5 is elevated in the male hippocampus, and to a much greater degree, in the female amygdala of PAE offspring following maternal separation stress (Figures 2A,B). The data support the possibility that glucocorticoid receptor signaling is reduced only in PAE offspring, which is unmasked by the secondary challenge of an acute maternal separation stress in females.

Interestingly, prenatal arsenic exposure induced sex-specific differences in the expression of GAS5 mRNA bound to the glucocorticoid receptor in the developing telencephalon (gestational days 14–18) (Caldwell et al., 2018), which includes the neocortex and the hippocampus (Hebert and Fishell, 2008). Arsenic has been characterized to impact the glucocorticoid and HPA axis system (Tyler and Allan, 2014). The data by Caldwell and colleagues (Caldwell et al., 2018) demonstrate that arsenic-exposed female telencephalon revealed relatively lower bound GAS5-to-glucocorticoid receptor compared to males, suggesting that males undergo reduced control over telencephalic glucocorticoid signaling and/or regulation during this critical gestational period. That is, during late gestational periods, glucocorticoids facilitate fetal neuronal maturation, axon remodeling and cell survival (De Kloet et al., 1988). Note that in the current report, GAS5 in female hippocampus with PAE is significantly lower (Figure 2A; Table 3) compared to the opposite trend observed in male hippocampus. While speculative, reduced glucocorticoid receptor signaling during this late gestational period may impact postnatal corticolimbic development resulting altered responses to stress during later life, particularly in males.

However, while lower, presumably protective GAS5 mRNA levels are demonstrated in the female hippocampus from PAE, a striking elevation of GAS5 in the female amygdala with PAE is revealed upon stress exposure (Figure 2B). The data are additionally striking in that no significant alterations are observed in the male amygdala of any glucocorticoid receptor signaling regulator examined in the current report (GAS5, FKBP5, and FKBP4), suggesting that adverse stress responses between males and females are regulated by different corticolimbic areas. Prior reports have demonstrated sex differences in glucocorticoid dysregulation involving hippocampal pathways, and a discussion of the data considers the possibility that PAE impacts glucocorticoid signaling in different pathways of the female brain (Caldwell et al., 2018). Our current data support and extend the differential impact of PAE between male and female limbic pathways, and especially following stress.

Glucocorticoid receptors are present in the cytoplasm in a complex with other proteins including chaperone proteins and FKBP51 (encoded by FKBP5) (Schiene-Fischer and Yu, 2001; Oakley and Cidlowski, 2013) that ultimately reduces glucocorticoid receptor signaling (Zannas et al., 2016). In further support that PAE dysregulates glucocorticoid receptor signaling, elevated levels of FKBP5 are observed in the hippocampus and, again to a greater magnitude, in the amygdala following stress (Figures 2A,B; Table 3). Significantly increased FKBP5 may act to further reduce the affinity of glucocorticoid receptor-glucocorticoid ligand binding, as has been previously considered (Schiene-Fischer and Yu, 2001). An additional mechanism by which FKBP51 may alter glucocorticoid receptor signaling is via FKBP51’s direct interaction with the IKK complex (scaffold and isomerase activity) that ultimately increases NF-kB activation (Romano et al., 2015). NF-kB is a pivotal transcription factor that regulates the expression of several potent proinflammatory cytokines such as IL-1β, which is increased in the hippocampus and amygdala following stress and reduces glucocorticoid receptor signaling (Sugama et al., 2011; Wohleb et al., 2011).

The current study additionally examined the transcriptional expression of FKBP52, encoded by the FKBP4 gene, as it promotes high affinity binding of glucocorticoid receptors with glucocorticoids through interactions with microtubule proteins and thus facilitates the nuclear translocation of this complex (Schiene-Fischer and Yu, 2001; Davies et al., 2002). While FKBP51 and FKBP52 compete for binding to the glucocorticoid receptor in the cytoplasm, evidence demonstrates that in the presence of both FKBP52 and FKBP51, the glucocorticoid receptor preferentially associates with FKBP51 (Barent et al., 1998). The data from the current report demonstrate that elevated transcriptional expression of FKBP4 is present in male hippocampus from offspring with PAE and maternal separation stress (Figure 2A; Table 3), and to a large degree in female amygdala from offspring with PAE and stress exposure (Figure 2B; Table 3). Given that elevated FKBP51 binds glucocorticoid receptors reducing glucocorticoid ligand binding and preferential glucocorticoid receptor-FKBP51 occurs in the presence of FKBP52, an examination of the FKBP5:FKBP4 ratio was additionally performed. The data show that significantly elevated FKBP5:FKBP4 is present only in the hypothalamus of PAE female offspring, further supporting the possibility that PAE reduces glucocorticoid receptor signaling. Together, the observation that increased GAS5 with concurrent elevations of FKBP5 in the hippocampus, and to a much greater degree, in the amygdala of PAE offspring following maternal separation stress support the possibility that PAE dysregulates CRH/AVP actions and glucocorticoid receptor signaling in the amygdaloid-hippocampal circuit response to early life stress.

Evidence is mounting that adverse CNS function from PAE offspring is due to elevated expression of brain proinflammatory cytokines, chemokines, and increased activation of related immune receptors and glia (Drew and Kane, 2014; Drew et al., 2015; Noor and Milligan, 2018). Further, the possibility that dysregulated neuroimmune function may adversely impact the brain stress-response and neuroendocrine system has been emerging for a number of years (Weinberg, 1989; Zhang et al., 2005; Hellemans et al., 2008, 2010; Bodnar et al., 2016; Raineki et al., 2017; Lam et al., 2018). Given cytokines are now characterized to act as important contributors to healthy brain development (Smith et al., 2007; Jakovcevski et al., 2009; Stephan et al., 2012), alterations in the levels and regulation of a number of immune-related factors can wreak havoc on brain function, including the brain stress-response system. The current report extends this prior work demonstrating that significantly increased amygdaloid microglial activation is present in female offspring with PAE following maternal separation stress (Figure 3B; Table 3), suggesting that the consistent interaction of PAE and early life stress on glucocorticoid signaling blockers, and proinflammatory signaling factors may be mediated by microglial activation. However, not all brain areas examined demonstrated consistent microglial activation, as blunted hypothalamic microglial activation is observed in female PAE offspring (Figure 3C; Tables 2, 3), with no such changes occurring in male PAE offspring.

Activated TLR4 results in the synthesis of a number of proinflammatory factors even following non-tissue damaging stressors. For example, non-physiological stress (i.e., maternal separation) can induce the release of classic immune danger-associated molecular patterns (DAMPs) such as the high-mobility group box 1 (HMGB1) independently of cell necrosis (Fleshner et al., 2017; Johnson et al., 2019). HMGB1 released from neurons, glia and immune cells activates TLR4. Following TLR4 activation by DAMPs, intracellular signaling mediated by myeloid differentiation factor 88 (MyD88) that ultimately induces activation of the NFkB transcription factor that initiates the synthesis of a number of proinflammatory cytokines including tumor necrosis factor-α (TNF-α, encoded by TNF), and precursor proteins pro-IL-1β (encoded by IL1B), pro-caspase-1 and the inflammasome, Nod-like receptor family pyrin domain containing 3 (NLRP3). Activated NLRP3 interacts with pro-caspase-1 to form the NLRP3 inflammasome protein complex, allowing for caspase-1 activation and consequent conversion of pro-IL-1β to mature, active IL-1β (Liu et al., 2017). Termination of active NFkB transcription is based on NFkB-dependent gene transcription of I kappa B-alpha (IkBa), an inhibitory subunit encoded by the NFKBIA gene (Bottero et al., 2003). Thus, quantification of NFkB activity is best captured by assessing gene synthesis encoding the protein IκB-α because induction of NFkB is reliably followed by NFKBIA mRNA synthesis, and a reduction in NFkB transcriptional activity is reflected in a reduction in NFKBIA mRNA. Notably, stabilization and accumulation of NFKBIA does not occur, which allows for the real time examination of NFKBIA mRNA levels to indirectly assess NFkB activity (Bottero et al., 2003).

In support of this background, the data from the current report reveals a striking increase in factors characterized to be induced following TLR4 activation predominantly in amygdala of female PAE offspring in response to maternal separation stress (Figure 4B; Table 3). Compared to prenatal control exposed offspring, reliable increases in mRNA for HMGB1, TLR4, NFKBIA, NLRP3 as well as IL1B are observed in the amygdala of offspring with PAE in response to stress, suggesting a primed proinflammatory state is created by PAE that is triggered by the acute stressor. It is notable that the significant alterations in these factors are observed predominantly in females rather than males. However, significant TLR4 and NLRP3 mRNA increases are observed in hippocampus of male PAE offspring regardless of stress exposure (Figure 4A; Table 3), while no changes in TLR4-associated factors were present in the male amygdala, consistent with a lack of astrocyte and microglial activation. Further, the hypothalamus from male offspring with PAE reveals blunted astrocyte activation as well as a non-significant trend toward blunted microglial activation. Combined, the data suggest that during early postnatal life, stress-induced increases in TLR4-related factors are observed different limbic structures between males and females, with some factors altered in the male hippocampus, and a large majority of these factors altered in female hippocampus and amygdala. One possibility may be that with regard to neuroimmune dysregulation, males may have greater protection against stress-induced neuroimmune dysregulation, but may be more vulnerable to stress-induced glucocorticoid receptor insensitivity.

The pathophysiology of PAE that is mediated by proinflammatory factors that can influence TLR4 activation and downstream cellular signaling. These factors include TNFα, CCL2, IL-1R1 alterations, and compensatory production of the and the anti-inflammatory cytokine, IL-10 (Noor and Milligan, 2018). Brain TNFα and CCL2 are upregulated as a consequence of TLR4 activation following alcohol exposure (Alfonso-Loeches et al., 2010; Montesinos et al., 2016; Pascual et al., 2017), and other recent reports suggest that PAE leads to suppressed anti-inflammatory IL-10 expression in peripheral and central nervous tissue (Noor et al., 2017; Sanchez et al., 2019; Noor et al., 2020). Notably, IL-10 is well-characterized to limit the actions of IL-1β, TNFα and CCL2, in addition to a number of other proinflammatory factors (Foey et al., 1998). The data from the current report demonstrate elevated IL1R1 mRNA is unmasked in the hippocampus of female offspring with PAE due to a stressor, in support of IL-1R1 brain elevations from inflammatory insults (Andre et al., 2005). Hippocampal IL10 mRNA elevations are observed only in stressed PAE male offspring (Figure 5A; Tables 2, 3), suggesting that IL-10 in males may be one mechanism conferring protection from stress-induced neuroimmune alterations.

Sex-specific outcomes as a consequence of PAE have been documented demonstrating sexually dimorphic effects of activity-dependent hippocampal glucocorticoid receptor protein levels (Caldwell et al., 2018), as well as HPA activity in PAE offspring (Weinberg et al., 2008; Lam et al., 2018; Lussier et al., 2021). While the inflammatory effects between PAE and/or stress on placental cytokines are similar (Ruffaner-Hanson et al., 2022), the levels of these inflammatory factors are distinctly different in male versus female fetuses (Terasaki and Schwarz, 2016). While speculative, the current data demonstrating consistent elevations in stress-associated and immune-related factors in the amygdala of PAE females, and blunted hypothalamic expression of many of these same markers suggests that PAE predisposes females to dysregulated limbic stress responses (e.g., CRH, AVP, GAS5, FKBP5 and FKBP4) as a consequence of altered glial and TLR4-assoicated factors. Interestingly, females often demonstrate an increased incidence of immune disorders and increased brain proinflammatory cytokine gene activation in animal models also expressing depressive-like behaviors (Whitacre, 2001; Tonelli et al., 2008). Thus, the sensitized female limbic pathways from PAE due to underlying alterations in the stress and neuroimmune axis may be of particular relevance to understanding the development of mood disorders later in life in individuals with an FASD (Hellemans et al., 2008; Hagan et al., 2016; van Bodegom et al., 2017; Wang et al., 2019). If dysregulated limbic cytokine expression is ultimately found to be critical in the neuropathology of mood disorders, the intriguing possibility emerges that treatment strategies can be identified that target re-balancing the brain cytokine milieu in individuals living with FASD.

In conclusion, our sex-specific findings of robustly elevated transcriptional expression of key stress-related neuropeptides as well as TLR4 signaling pathway factors in the female amygdala that is unmasked by early postnatal maternal separation stress provides new insight into how PAE may remodel developing brain areas responsible for integrating the stress response during early and adult life. The data further reveal that PAE per se, is insufficient to alter basal transcription of stress-related neuropeptides and TLR4 neuroimmune factors, but instead, changes in gene expression are unmasked only following stressor exposure. Thus, together the results support our hypothesis that PAE sensitizes key stress-integrative limbic circuits such that significant exaggerated responses in the amygdala and blunted responses in the hypothalamus are unmasked due to early life stress. Prior work demonstrates a unique cytokine profile such as IL-1β and TNFα, from PAE occurs in the hippocampus, prefrontal cortex and the hypothalamus depending on early postnatal age, with blunted patterns of expression observed in the hypothalamus (Bodnar et al., 2016). This work suggests that even subtle changes in brain cytokine expression levels may act to alter the course of normal brain development. It is possible that in the PAE brain, even minor stressors during early postnatal life may be sufficient to exert enduring developmental changes.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by University of New Mexico Animal Care and Use Committee.

CR–H performed all the real-time PCRs and statistical analysis, and prepared the first draft of the manuscript. AF-O performed the protein ELISAs and statistical analyses. MS participated in tissue collection. KC and AA contributed to conception and design of the study, generated the mouse model of prenatal alcohol exposure, and collected tissue. EM contributed to conception and design of the study, participated in collection of tissues, assisted in statistical analyses, and prepared the manuscript. DS and CV supported the generation of the mouse model of prenatal alcohol exposure. SN contributed to tissue collection, real-time PCR data collection, statistical analysis and edited the manuscript. All authors contributed to the article and approved the submitted version.

The experiments conducted to generate this manuscript were funded by the NIH NIAAA 2P50 AA022534, and R01-AA025967.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.