Compelling evidence suggests that early life challenge, as a result of maternal immune activation (MIA), can have long-lasting, negative effects on neurochemistry, brain excitability, and behavior [4, 5, 22, 43]. Epidemiological studies show that prenatal/neonatal immune activation such as infection, malnutrition, stress, and obstetric complications can increase the susceptibility of individuals to schizophrenia, autism spectrum disorders (ASD), and epilepsy [7, 22, 27]. The literature reveals that infection-induced MIA has profound effects on developing neural circuits with studies suggesting that certain gestational windows are more vulnerable than others (e.g., early versus late pregnancy) to developmentally initiated functional deficits following MIA [22, 29]. Animal models have been developed to study the link between behavioral discrepancies and cellular, molecular, and morphological alterations in the offspring’s brain after prenatal exposure to an immune modulating agents such as polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) which mimic viral or bacterial infections, respectively [1, 6, 17, 22]. In rodents, early-life exposure to LPS induces the production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis alpha (TNFα) both in the periphery and the brain [35]. Additionally, MIA induced with LPS challenge has produced abnormalities such as decreased expression of Reelin (structural marker), increased immunoreactivity for glial fibrillary acidic protein (GFAP), activation of microglial cells, long-term changes in GABAergic function, and decreases in neocortical and hippocampal thickness [12, 15, 26, 32]. Exposure of pregnant dams to other inflammatory agents during gestation also results in deficits that include other hippocampal abnormalities, such as neuronal loss, astrogliosis, and changes in neurotransmitter receptor expression [1, 34, 36]. Several studies have focused on the developing hippocampus, cortex, and mid-brain, but surprisingly, research on MIA-induced amygdalar changes is limited [22]. The amygdala, a limbic structure, is the center for the control of emotions, and disturbances to this component of the limbic system can result in behavioral deficits as seen with anxiety, fear, learning and memory impairments, and also neurodevelopmental disorders such as ASD [20]. Moreover, human MRI studies and in vivo animal studies support the link between developmental disorders and amygdalar involvement [19, 20, 38]. Therefore, the first aim of this study was to investigate the effects of LPS-induced MIA on murine amygdalar development with regard to the cellular, molecular, and cytoarchitectural changes that may occur over key developmental stages.

Inflammation during pregnancy (MIA) can affect several aspects of neurodevelopment, with disturbances that may initiate a series of events eventually interrupting neuronal and glial functions. MIA can induce a “cytokine storm” in the fetal brain, which has been shown to have negative effects on brain structure, both biochemically and structurally. Studies inducing MIA via LPS challenge have shown that this results in increased microglial activation with alterations in morphology from “resting” ramified to an “activated” amoeboid cell type [5]. These changes may lead to downstream effects on the cytoarchitecture of the amygdala as seen at P14, a period known to be important for brain maturation [11]. The present in vivo study demonstrates that an acute maternal LPS injection at E12 upregulates pro-inflammatory cytokines and toll-like receptors in the fetal CNS that subsequently increases glial cell immunoreactivity, possibly leading to a developmental delay and/or cell loss in the postnatal amygdala.

TLRs are expressed on most immune-cell types, in particular on microglia, which increase TLR expression in response to inflammatory stimuli. TLR-4 is a known receptor for LPS, and here, after MIA induction, TLR-4 was upregulated in fetal CNS tissue and remained upregulated in late postnatal ages suggestive of glial activation. Moreover, TLR-2 that is also known to be elevated after inflammation was upregulated at prenatal/neonatal and postnatal developmental ages. In addition, COX-2, iNOS, and CCL2 were also significantly upregulated at P40 which may further suggest prolonged glial “activation” state. Taken together, an acute bacterial challenge during a critical window of amygdalar development maybe sufficient to induce inflammatory mediators and TLRs in the developing CNS that may persist and affect the later development of the amygdala. However, it should be taken into consideration that other CNS cells such as astrocytes and/or infiltrating immune cells (monocytes, macrophages, etc.) can also produce pro-inflammatory cytokines. During development, the BBB is not fully formed, and infiltration from the periphery is highly likely. While our study examined gene expression in whole tissue lysates, isolation of microglial cells using other technologies such as flow cytometry might help characterizing immune cells involved in MIA effects in future studies.

Our data suggests a particular effect at P14 on the central nucleus (Ce) of the amygdala that had lower neuronal numbers and corresponding volume reduction compared to saline-treated controls. Astrogliosis, which is an indicator of cell damage and inflammation, was most evident in the amygdala at the same developmental stage. The reduction in Ce volume and cell numbers in the LPS group at P14 may indicate a developmental delay at this time point, considering that amygdalar development and growth continues postnatally [14], because both the volume and cell numbers in the Ce reached adult levels as in saline-treated offspring at P40. Studies in rodents have shown that postnatal day 14 (P14) is a crucial period for brain maturation [36] and synaptic pruning during this postnatal development is known to play a vital role [31]. Synaptic pruning is particularly efficient at P14 in mice, and alterations to the microglial functions induced by an inflammatory event during this time or earlier may contribute to synaptic abnormalities seen in some neurodevelopmental disorders [42]. Adolescence is an important time of neurobehavioral maturation during which limbic structures such as the hippocampus, amygdala, and prefrontal cortex undergo maturation [8, 23]. It has been shown that alteration to the microglial environment during early life impairs synaptic elimination and negatively impacts on functional behaviors [42]. After maternal LPS, fetal brain IL-1β mRNA gene expression levels were significantly elevated as soon as 4 h postinjection. The elevated pro-inflammatory milieu early in development may “activate” glial cells such that in later postnatal stages, they display activated phenotypes as indicated by increased CCL2, COX-2, and iNOS with TLR-2 and TLR-4 expression gene expression and also increased Iba-1 and GFAP immunoreactivity and morphological changes. This is in agreement with studies showing that prenatal exposure to LPS induces elevated IL-1β expression and is associated with postnatal cell death and gliosis [33].

Interestingly, CD36-TLR-4 signaling is implicated in microglial inflammatory response that promotes neurotoxicity [37]. Our data showed that CD36 is elevated at prenatal stages, which coincides with increased expression of TLR-4. This elevated CD36/TLR-4 expression prenatally may enhance the inflammatory profile and thus microglial priming in the developing brains after MIA. The transient upregulation of the anti-inflammatory cytokine IL-10 at E16 might have contributed to the temporary decline in TLRs and some pro-inflammatory mediators in neonatal MIA offspring and at early postnatal stages, but postnatal IL-10 levels remained unaffected. While analyses of further anti-inflammatory markers may allow a more comprehensive picture of MIA-induced signaling, the current results with upregulation of IL-1β at P7 and the pro-inflammatory molecular profile found at P40 support the notion that a single prenatal LPS injection can cause long-term alterations in the forebrain including upregulation of TLRs, potentially permanently altering the brain’s responsivity to infectious agents and inflammation. Elevated expression of inflammatory molecules may combine together to have a contributing negative effect on neurons in the postnatal amygdala, e.g., by impairing neurogenesis, inhibiting migration and differentiation, and/or inducing cell loss. However, the precise mechanisms by which toxin-induced MIA induces fetal brain inflammation and function remain elusive. It has been demonstrated that it is not the toxin itself that directly induces the effects but the soluble mediators (cytokines, chemokines, reactive species) produced via downstream signaling cascades [2]. Therefore, other glial cells and infiltrating macrophages and/or monocytes should not be ignored. CCL2 is expressed by other myeloid cells, such as monocytes, and acts as a chemoattractant; in particular, it is involved in recruiting cells to sites of injury. Increased expression of CCL2, as seen in our data, may induce recruitment of other myeloid cell populations to the CNS in response to neuroinflammation. Alternatively, activated astrocytes and microglial cells may release CCL2 after MIA insult. The involvement and mechanisms of recruitment of different inflammatory cells to the developing CNS should be explored in future studies. Furthermore, MIA may influence immune mediators at the protein level through various mechanisms, e.g., miRNAs can alter the expression of TAB3 downstream of IL-1β signaling after MIA induction [18] or posttranslational modifications may take place.

In mice, a disturbance in cytokine signaling via receptor blockade or genetic deletion can produce alterations in ASD-like phenotypes including memory deficits and social withdrawal behaviors [3]. Furthermore, prenatal MIA on E9-E12 enhances anxiety- and depressive-like behavior in adult offspring [10, 24]. The central nucleus is the major output station of amygdalar projections, and as our data shows, the Ce structure is compromised after MIA, which could have negative effects for neurobehavioral outcomes. For example, MIA-induced changes in the Ce could alter visceral and motor responses to anxiogenic and emotionally arousing stimuli. Because the Ce is a major regulator of the HPA axis, alterations in the cell types and function of the Ce may also affect inflammation by altering glucocorticoid hormone function [20]. Thus, despite comparable neuronal cell numbers between MIA and saline groups at P40, the function of the Ce may be permanently altered after MIA exposure, because specific subsets of peptidergic Ce projection neurons innervating certain hypothalamic and brainstem regions may be affected. While our study examined the cytoarchitectural effects of MIA challenge over development, further studies using our model may be beneficial for correlating the structural changes in the amygdala with behavioral outcomes of this insult, e.g., ASD-like phenotypes.

In conclusion, there is a complex interplay between the immune and neural signaling in the developing brain, and when an insult or disturbance such as MIA occurs, this may negatively affect the cytoarchitecture and function of the amygdala. Our model of maternal inflammation induced an elevated cytokine and TLR response in the fetal brain, which is associated with moderate astrogliosis, activated microglia, and cytoarchitectural changes in the postnatal amygdala. Our data suggests that the developing amygdala may be particularly sensitive to maternal stimuli that may influence genetically determined developmental processes.