Prenatal stress and increased susceptibility to anxiety-like behaviors: role of neuroinflammation and balance between GABAergic and glutamatergic transmission

Abstract

Neuroplasticity during the prenatal period allows neurons to regenerate anatomically and functionally for re-programming the brain development. During this critical period of fetal programming, the fetus phenotype can change in accordance with environmental stimuli such as stress exposure. Prenatal stress (PS) can exert important effects on brain development and result in permanent alterations with long-lasting consequences on the physiology and behavior of the offspring later in life. Neuroinflammation, as well as GABAergic and glutamatergic dysfunctions, has been implicated as potential mediators of behavioral consequences of PS. Hyperexcitation, due to enhanced excitatory transmission or reduced inhibitory transmission, can promote anxiety. Alterations of the GABAergic and/or glutamatergic signaling during fetal development lead to a severe excitatory/inhibitory imbalance in neuronal circuits, a condition that may account for PS-precipitated anxiety-like behaviors. This review summarizes experimental evidence linking PS to an elevated risk to anxiety-like behaviors and interprets the role of the neuroinflammation and alterations of the brain GABAergic and glutamatergic transmission in this phenomenon. We hypothesize this is an imbalance in GABAergic and glutamatergic circuits (as a direct or indirect consequence of neuroinflammation), which at least partially contributes to PS-precipitated anxiety-like behaviors and primes the brain to be vulnerable to anxiety disorders. Therefore, pharmacological interventions with anti-inflammatory activities and with regulatory effects on the excitatory/inhibitory balance can be attributed to the novel therapeutic target for anxiety disorders.

Keywords:

• Prenatal stress
• anxiety-like behaviors
• neuroinflammation
• GABA
• glutamate

1. Introduction

The stress experienced by a mother before giving birth is called prenatal stress (PS). PS can exert early and long-lasting effects on neurobehavioral development in both human and animal offspring (Ahmadzadeh et al., 2011; Edwards et al., 2002; Gholipoor et al., 2017; Kofman, 2002). It decreases learning and memory ability and increases attention deficiency, seizure susceptibility, behavioral abnormalities, and anxiety (Coe et al., 2003; Fride & Weinstock, 1988; Glover, 2011; Hashemi et al., 2013, 2016; Heshmatian et al., 2010; Mahmoodkhani et al., 2018; Saboory et al., 2015, 2019; Takahashi, 1992; Tollenaar et al., 2011; van den Bergh et al., 2018). These effects have been attributed to various mechanisms, most of which are related to the concept of fetal programming (Entringer et al., 2012). The fetal programming model suggests that the fetus phenotype can change during the prenatal period in accordance with changes in the fetal environment, such as stress exposure (Ensminger et al., 2018). However, not all infants show developmental problems due to such exposure (Dipietro, 2004). Factors such as the timing, intensity, duration, and type of exposure may partly explain some of these individual differences in early childhood behavior (Boersma & Tamashiro, 2015;; Del Giudice, 2016).

Accumulating human research shows that prenatal exposure to various types of prenatal maternal psychological distress [emotional stress (i.e. fears, depression), cognitive stress (i.e. anxiety, panic attacks), life events (loss of employment, loss of loved ones, bankruptcy, divorce), etc.] increases the risk for anxiety disorders in the offspring later in life (McLean et al., 2018; Monk et al., 2019; Van den Bergh et al., 2017). Consistently, many animal studies have indicated excessive anxiety-like behaviors in prenatally stressed rats or mice, tested in the elevated plus maze (EPM), open field test (OFT), and light/dark transition test (LDT) (Azizi et al., 2019; Cartwright-Hatton, 2006; Nakhjiri et al., 2017; Vallée et al., 1997; Zagron & Weinstock, 2006). Among the most accepted alternative hypotheses of anxiety is the dysfunction of the HPA axis, a system that is central in regulating the stress response. A considerable number of epidemiological surveys and experimental studies have established that increased anxiety-like behaviors and dysregulation of the hypothalamic-pituitary-adrenal (HPA) stress axis are commonly observed in the phenotypes of prenatally stressed humans and rodents (Akatsu et al., 2015; Hou et al., 2018; Weinstock, 2015). HPA hyperactivity has been reported in infant, young, adult, and aged animals, thus suggesting the permanent effect of PS (Darnaudéry & Maccari, 2008). HPA axis hyperactivity and subsequent increase in corticosteroid levels may induce systemic pro-inflammatory and later neuroinflammatory conditions and cause alterations in the structure or function of anxiety-related brain circuits, mainly limbic and pre-frontal structures, priming the brain to be vulnerable to anxiety disorders (French et al., 2004; Won & Kim, 2020).

An excitation–inhibition imbalance has also been suggested to cause psychiatric and paroxysmal brain disorders (Saaltink & Vreugdenhil, 2014); however, the underlying mechanisms are not entirely clear. Two emerging theories on the development of psychiatric diseases involve excessive inflammatory cytokine production and changes in GABA/glutamate neurotransmission (Haroon et al., 2017; Shim et al., 2019). Recent studies have provided evidence that these pathways converge at the level of the glial cells, major mediators of neuroinflammation, to cause behavioral changes in mood disorders (Haroon et al., 2017; Sanacora & Banasr, 2013; Shim et al., 2019). This suggests that manipulations of glial cell activity and neuroinflammatory pathways may act as effective therapeutic strategies for treating mood disorders. Neuroinflammation, as well as GABAergic and glutamatergic dysfunctions, has been implicated as potential mediators of behavioral consequences of PS and a relationship between PS, inflammation, and offspring neuropsychiatric outcomes has been demonstrated in both humans and rodents (Götz & Stefanski, 2007; Hantsoo et al., 2019).

Although the mechanisms, by which stress during pregnancy increases anxiety are unclear, a propensity for increased defensive and anxiety-like behaviors in rodent models of PS suggests that work in rodents may clarify important mechanistic details about this association. Therefore, in the present paper, we review experimental evidence linking PS to an elevated risk to anxiety-like behaviors, mostly in rodents, and interpret the role of the neuroinflammation and alterations of the brain GABAergic and glutamatergic transmission in this phenomenon. We present relevant experimental studies supporting the premise that neuroinflammation in association with structural and functional changes of GABA-ergic and glutamatergic systems contribute, at least in part, to prenatal programming of anxiety risk.

2. Factors that may contribute to differences in the effects of prenatal stress on anxiety-like behaviors

Irrespective of the mechanisms underlying PS-induced anxiety-like behaviors, the nature and arrival time of these effects depend on the timing of stress exposure; strength and duration of stress; as well as sex and age of the offspring. The type of stressor experienced also appears to have an essential role in determining the impact of PS on the offspring’s brain and behavior (Charil et al., 2010; Jafari et al., 2017; Weinstock, 2017). There are diverse types of animal models of physical and psychological stressors during pregnancy including restraint (with or without bright light), cage tilting, intermittent electric shocks, food deprivation, predatory stress, elevated platform, exposure to cold, prevention of sleep during the light cycle, forced swim, and exposure to a randomized sequence of varied stressors. Restraint stress is the most common stressor used in experimental animals and primarily is a psychological stressor, i.e. the physical discomfort induced by restraint is intentionally secondary to the cognitive appraisal of the organism's inability to gain free movement (Servatius et al., 2007). In one of the animal models of psychological stress, a rodent is allowed to observe, or witness, the PS procedure of another rodent (Finnell et al., 2017). Social isolation, crowding, and resident-intruder confrontation are commonly used animal models of psychosocial stressors.

The nature and severity of the stressor (psychological versus psychosocial) strongly influence the behavioral outcome. Götz and Stefanski (2007) used a social conflict model of stress in pregnant Long-Evans rats and reported some data which did not correspond to those of previous studies using conventional stressors. Accordingly, maternal stress was induced by using a resident–intruder confrontation (2 h each day) procedure adjusted for females. This social conflict model was based on establishing a “territory” by a resident female and its subsequent defense against unfamiliar females. Except during the confrontations, resident females were housed in pairs with males. Four- to six-month-old male offspring were subsequently assessed for anxiety-like behaviors. Prenatally stressed male rats were more active in the EPM test, showing less anxiety-like behaviors, as indicated by significantly more frequent entries into the open arms compared to the control males. In addition, prenatally stressed males had significantly lower serum corticosterone concentrations and lymphocyte counts. This was in contrast with the findings from other conventional stress studies but showed the nature of the stressor plays an important role in pregnancy outcome and anxiety-like behaviors of the offspring in later life (Götz & Stefanski, 2007). Moreover, the effects of PS vary for different gestational ages possibly, depending on the developmental stage of specific brain areas and circuits and the developmental stage of the immune system and stress system (Van den Bergh et al., 2017). The hippocampus, amygdala, and frontal cortex (FC) are among the most affected brain regions in PS (Davis & Sandman, 2012; Weinstock, 2017).

The sex-dependent impact of PS on anxiety-like behaviors is more controversial. Yet, the existing data regarding this dependence are highly mixed since some authors have reported no sex-dependent change in the anxiety-like behaviors of prenatally stressed rats, whereas our group and others have documented that PS affects anxiety-like behaviors of the offspring in a sex-dependent manner (Azizi et al., 2019; Brunton & Russell, 2010; Iturra-Mena et al., 2018; Sutherland & Brunwasser, 2018; Verstraeten et al., 2019; Weinstock, 2011). These discrepancies could be explained by the difference in the type, timing, and intensity of the stressor as well as the period of behavioral assessment. Although both male and female prenatally stressed rats usually show increased anxiety-like behaviors during adolescence and adulthood, some studies suggest that females show more variable and inconsistent effects (Darnaudéry & Maccari, 2008; Verstraeten et al., 2019), usually presenting less marked effects on anxiety-like behaviors (Iturra-Mena et al., 2018; Said et al., 2015; Van den Hove et al., 2013; Verstraeten et al., 2019; Zuena et al., 2008). For example, psychological PS mostly induces anxiety-like behaviors in males. Accordingly, in the study reported by Verstraeten et al. (2019), pregnant rats were exposed to psychological stressors and the anxiety-like behaviors of offspring adults were tested. They reported that the effect of PS on female anxiety-like behaviors was more variable and differed depending on the task performed. On the OFT, females showed no significant differences as the result of PS, contrary to those in males which showed high anxiety-like behaviors (Verstraeten et al., 2019). Consistent with this study, Iturra-Mena et al. (2018) found that anxiety-like behaviors were only evident in prepubertal male PS rats and female PS rats were less influenced by PS, indicating that only prenatally stressed males exhibited anxiety-like behaviors (Iturra-Mena et al., 2018). However, conflicting results have been reported by other researchers (Richardson et al., 2006; Schulz et al., 2011; Zagron & Weinstock, 2006). According to the study by Zagron and Weinstock (2006), male PS rats showed less marked effects on anxiety-like behaviors, as their increase in anxiety-like behaviors in the EPM was less than that in PS female rats (Zagron & Weinstock, 2006). Sex-dependent effects of PS on anxiety-like behaviors in different animal models are summarized in Table 1. As indicated in this table, differences in the anxiety-like behaviors between male and female rats exposed to PS seem to depend strongly on the type, duration, and intensity of stressor used, gestational stage targeted by PS, and age at assessment. It is not possible to state with any certainty which characteristic(s) may be implicated in the susceptibility to anxiety between female and male offspring. However, it seems that prenatal conventional stressors (psychological and psychosocial stressors) mostly induce anxiety-like behaviors in male rodents and unpredictable variable stressors mostly induce anxiety-like behaviors in females. Previously, sexual dimorphisms in brain development have been fully described and are suggested to underlie variations in expression of HPA axis-related genes and HPA stress responses, as well as the sex-dependent effects of PS on anxiety behaviors. The sex effect on anxiety-like behaviors has been reported to be related to the reduced neuroplasticity in the male and enhanced neuroplasticity in the female following PS in rats, as indicated by a significant change in two distinct phases of the neurogenesis: the number of putative neuroprogenitor cells as well as the percentage of cells differentiating into astrocytes, which has been shown to be significantly increased in the hippocampus of female PS rats compared to age-matched controls (Darnaudéry & Maccari, 2008). The influence of PS on neuronal plasticity has also been studied in the hippocampus of male rats, where it reduces neurogenesis (Lemaire et al., 2006) and increases the levels of the brain-derived neurotrophic factor (BDNF) and pro-BDNF (Zuena et al., 2008), probably as a compensatory effect of decreased neurogenesis. Moreover, in male PS rats, the protein expression and activity of type-5 metabotropic glutamate receptors (mGluR5) in the hippocampus significantly decreased, whereas in female PS rats, mGlu5 receptor protein levels showed no change and mGlu5 receptor activity significantly increased (Darnaudéry & Maccari, 2008; Zuena et al., 2008). This is relevant because mGluR5 is implicated in regulating both synaptic plasticity and neurogenesis as well as in anxiety (Di Giorgi Gerevini et al., 2004, 2005). However, in another study, female rat offspring exposed to PS showed a decrease in the mRNA expression of mGluR5 and ionotropic N-methyl d-aspartate (NMDA) receptors 1 and 2A in the hippocampus and FC (Van den Hove et al., 2013). Although mRNA expression is markedly different from protein expression, the authors suggested that the discrepancy between their study and others may be further explained by that the rats used in their study were older. They also suggested that the observed decrease in the mRNA expression of the mGlu5, together with the observed decrease in the expression of the NMDA1 and 2A receptors of female offspring exposed to PS, may be related to the resilience toward PS in this sex (Van den Hove et al., 2013). On the other hand, the decrease in mGluR function observed in PS male rats represents “an unsuccessful homeostatic mechanism aimed to restore the physiological levels of the anxiety response” (Zuena et al., 2008). Indeed, a greater number of glial cells and inflammatory mediators in the male prenatal brain induced by PS has also been suggested to enhance male susceptibility to developmental neuropsychiatric disorders versus females (McCarthy, 2016). In contrast, females appear to be less vulnerable to the effects of PS, but more vulnerable to environmental experiences that can increase the risk for anxiety later in life. Although underlying mechanisms are not yet clearly identified, sex differences in dendritic characteristics of locus coeruleus (LC), a major component of the stress response, have been suggested to underlie greater susceptibility of females to the effects of environmental insults than males. Bangasser et al. (2011) indicated a significant increase in LC dendritic density and in the length of LC dendrites of female compared to male rats. This could increase the number of synaptic contacts on LC neurons in female rats and induce a hyperarousal state in the animals, a state that has been proposed to be the primary symptom of some types of anxiety disorders such as post-traumatic stress disorder (Bangasser et al., 2011).

Table 1. Sex-dependent effects of PS on anxiety-like behaviors in different animal models with prenatal stress.

Species	Stress type and duration	Stress protocol	Sex and age at assessment	Methods of behavioral evaluation	Outcome	Reference
Sprague–Dawley rats	G14-G21 Restraint stress	3 Stress sessions/day, 45 min per session, with bright light	Male and female PND 24	OFT	PS males spend more time on the perimeter and less time in the center compered to control. No significant differences on the OFT in PS females. (↑anxiety-like behavior in PS males)	Iturra-Mena et al. (2018)
CD rats	G14–G21 Restraint and a randomized sequence of varied stressors	–Restraint stress: (3 sessions/day, 45 min/session, with bright light) –Foot shock stress: (one session/day, 30 min/session) –Injection stress: (once/day, 0.5 ml SC injection of isotonic saline)	Male and female Adulthood	EPM	PS females engaged in fewer entries to the open arms than control females. No significant difference in this parameter in PS males. (↑anxiety-like behavior in PS females)	Richardson et al. (2006)
Wistar rats	Last 10 days of pregnancy Foot shock stress	80 Electric chocks (0.5 mA, for 5 s, 1–2 min apart) during sessions that lasted 100 min, for 8 hours	Male and female PND 80	EPM	PS males spent significant shorter time in the open arms. PS females spent shorter time in central squares and open arms compared to control but no significant changes. (↑anxiety-like behavior in PS males)	Said et al. (2015)
Sprague–Dawley rats	G14–G21 Unpredictable variable stress (2–3 stress sessions/day)	–Restraint stress: (30 min/session) –Forced swimming stress: (15 min/session) –Exposure to a cold room (4 °C, 6-h) –Social stress: 5 rats/cage for 9-h –Overnight fasting	Male and female PND 180	EPM	Each stretch-attend bout was significantly longer for PS females than control females. No significant difference in this parameter in PS males. (↑anxiety-like behavior in PS females)	Schulz et al. (2011)
Long-Evans hooded rats	G1–G18 Psychological stress	–Restraint stress: 20 min/day –Forced swimming stress: (5 min/day) (administered alternatingly)	Male and female PND 90–100	OFT EPM	PS males spent more time along the walls on the OFT. No significant differences on the OFT in PS females. (↑anxiety-like behavior in PS males)	Verstraeten et al. (2019)
Wistar rats	G14–G21 Restraint stress	Mild restraint stress, 30 min/day	Male and female PND 35	EPM	PS females spent significant shorter time in the open arms compared to control. No significant difference in PS males. (↑anxiety-like behavior in PS females)	Zagron and Weinstock (2006)
Sprague–Dawley rats	G16–G20 Social defeat stress	Pregnant rats were paired with a different and unfamiliar lactating “resident” rat for five consecutive days, 10 min/day	Male and female 12–13 weeks	EPM	PS males showed a significant grater latency to enter an open arm and lower number of entries than controls. No differences between control and PS female rats. (↑anxiety-like behavior in PS males)	Brunton and Russell (2010)

EPM: elevated plus maze; EZM: elevated zero maze; G: gestational day; OFT: open field test; PND: postnatal day; PS: prenatal stress; SC: subcutaneous.

Altogether, PS has a substantial effect on structural plasticity in male rats, while female rats are protected by the effects of PS on neurogenesis, probably by an increase in the number of astrocytes and activity of mGluR (Darnaudéry & Maccari, 2008; Zuena et al., 2008), which highlights the sexual dimorphism in vulnerability to PS (Van den Hove et al., 2013; Verstraeten et al., 2019).

3. Possible mechanisms underlying PS-induced anxiety-like behaviors: role of neuroinflammation and alterations of the brain GABAergic and glutamatergic transmission

3.1. Neuroinflammation and PS-precipitated anxiety-like behaviors

Neuroinflammation may play an important role in mediating PS effects on the offspring’s anxiety-like behaviors (Calcia et al., 2016; Chen et al., 2020; Verstraeten et al., 2019). Inflammation has been considered as a mediator in the relationship between PS and offspring neuropsychiatric consequences and potentially influenced by poor HPA regulation of an inflammatory response (Götz & Stefanski, 2007; Hantsoo et al., 2019). Nevertheless, during neurodevelopment, the immune cytokines can act as neurotrophic substances protecting and promoting neurite growth; with excess and prolonged activation, these cytokines can be very destructive. Numerous studies have assessed the potential mediatory role of neuroinflammation and elevated pro-inflammatory cytokines in the association between PS and the offspring’s behavioral outcomes, finding associations between PS, morphology, and biological activity of microglia cells and the inflammatory response of the hippocampus and prefrontal cortex with increased offspring neuropsychiatric risk. Accordingly, in the studies reported by Diz-Chaves et al. (2012, 2013), pregnant C57BL/6 mice were subjected to restraint stress as well as a bright light from gestational (G) day 12 to delivery three times/day and for 45 min. Subsequently, morphology of microglia cells as well as inflammatory response of the hippocampus were investigated in their 4-month-old female and male offspring. PS increased interleukin (IL)-1β mRNA levels and the total number of ionized calcium-binding adaptor molecule 1 (Iba1)-immunoreactive microglial cells in the hippocampus. In addition, peripheral inflammation induced by systemic administration of lipopolysaccharide induced a significant increase in mRNA levels of IL-6, tumor necrosis factor α (TNF-α), interferon γ-inducible protein 10 (IP10), and proportion of Iba1-immunoreactive cells in the hippocampus. These findings indicated that the hippocampi of prenatally stressed mice display a pro-inflammatory status and an exaggerated response to an inflammatory challenge (Diz-Chaves et al., 2012, 2013), which may predispose these animals to the development of anxiety-like behaviors. Consistent with these studies, Ślusarczyk et al. (2015) subjected pregnant Sprague–Dawley rats daily to three stress sessions, during which they were exposed to restraint stress and a bright light for 45 min from G12 to delivery. For in vitro experiments, a 1–2-day-old male offspring and, for in vivo studies, a 3-month-old male offspring were investigated. PS induced extended inflammation in the fetal brain, with elevated ex vivo microglial production of pro-inflammatory cytokines IL-1β, IL-18, TNF-α, and IL-6, while insulin-like growth factor 1 production was suppressed in the cultures of microglia from PS rats. Moreover, the adult PS rats showed behavioral disturbances, which indicated prolonged immobility time as well as shortened swimming and climbing time in the forced swim test, enhanced expression of microglial activation markers, and increased number of Iba1-immunopositive cells in the hippocampus and FC. These results suggested that PS may lead to enhanced neuroinflammatory response through excessive microglia activation and contribute to increased depressive- and anxiety-like behaviors observed in prenatally stressed offspring (Ślusarczyk et al., 2015). Supporting these observations, Gur et al. (2017) subjected pregnant C57/Bl6 mice to restraint stress between G10 and G16 for a period of 2 h/day and the offspring were subsequently assessed on postnatal days 60–70 for anxiety-like behaviors using EPM. A cohort of pregnant females was sacrificed at E17.5 for tissue collection. According to their findings, IL-1β increased, whereas BDNF decreased in the placenta and fetal brain from the offspring exposed to PS. Prenatally stressed female offspring also demonstrated a significant increase in anxiety-like behaviors as reflected by a decreased amount of time and distance traveled in the open arms of the EPM (Gur et al., 2017). Other similar animal studies have also reported that PS increased the expression of immune response genes in the placenta, including IL-6 and IL-1β, resulting in male-specific locomotor hyperactivity and increased HPA axis response (Bronson & Bale, 2014; Mueller & Bale, 2008); the pretreatment with nonsteroidal anti-inflammatory drugs (NSAIDS) prevented stress-induced immune gene expression changes and ameliorated the behavior defects (Bronson & Bale, 2014), providing further support for a mechanistic link between the immune system and behavioral outcomes. Poor maternal glucocorticoid-immune coordination is a potential mechanism for the suggested relationship between PS, inflammation, and offspring anxiety-like behaviors. In view of the high level of glucocorticoid receptors expression in the hippocampus and prefrontal cortex, these regions are likely to be particularly sensitive to the PS-induced corticosterone release, leading to indirect effects on microglia in these regions. In addition, as microglia express both glucocorticoid and mineralocorticoid receptors (Sierra et al., 2008), there may be direct effects of PS-induced corticosterone surges on microglia. It may not be glucocorticoids per se, but poor regulation of cytokine-glucocorticoid negative feedback that influences the relationship between PS and offspring outcomes (Hantsoo et al., 2019). These findings support the hypothesis that PS increases the risk of a number of behavioral disturbances including anxiety, in part through activating microglia and other centrally-mediated immune responses. Indeed, previous reports have indicated increased microglial activity prevalent among patients with anxiety (Frick et al., 2013) and in animals with anxiety-like symptoms (Wohleb et al., 2011, 2012). The importance of these findings and impact of PS on subsequent later-life behaviors and health have been reviewed by Réus et al. (2015) and Calcia et al. (2016), explaining that stressful life experiences, in association with elevated pro-inflammatory cytokines, may induce microglia hyperactivation, which in turn may be associated with structural and functional changes in the brain that predisposes individuals to behavioral and mental disturbances (Calcia et al., 2016; Réus et al., 2015).

3.2. Gabaergic system and PS-precipitated anxiety-like behaviors

While in the adult brain, GABA acts as an inhibitory neurotransmitter, it depolarizes targeted cells and triggers calcium influx during the perinatal period. GABA-mediated calcium signaling regulates a number of important developmental processes which include cell proliferation, differentiation, synapse maturation, and cell death (Owens & Kriegstein, 2002). PS alters early developmental processes of the GABAergic system and, therefore, the effects of PS on numerous neuropsychiatric disorders can be related, at least in part, to abnormalities in the GABAergic system. GABAergic abnormalities have been implicated in the pathogenesis of autism (Yip et al., 2008), schizophrenia (Hashimoto et al., 2008; Matrisciano et al., 2013), seizures (Baek et al., 2016; Bagheri et al., 2020; Ben-Ari et al., 2012; Lopim et al., 2020; Saboory et al., 2014), and anxiety (Ackermann et al., 2008; Gholami & Saboory, 2013; Laloux et al., 2012; Lussier & Stevens, 2016; Zhu et al., 2018). The GABAergic inhibitory circuits in different regions of the mammalian brain including the neocortex, hippocampus, dorsomedial hypothalamus, amygdala, and cerebellum have been identified as key regulators of anxiety-like behaviors (Ackermann et al., 2008; Bennett et al., 2017; Bueno et al., 2005; Ehrlich et al., 2015; Stevens et al., 2013; Zhu et al., 2018). Animal models have implicated the involvement of the GABAergic system in PS-precipitated anxiety-like behaviors due to its importance in the processes of brain development and associations with the pathology of anxiety. Supporting this deduction, it has been reported that prenatal administration of drugs which indirectly activate GABA transmission partially ameliorates PS-precipitated anxiety-like behaviors in adult rats, as indicated by increased spent time in the open arms of the EPM (Zimmerberg & Blaskey, 1998). In both human and animal studies, negative modulators of GABAergic neurotransmission generally possess anxiogenic activity, while positive modulators exert anxiolytic effects (Kalueff & Nutt, 2007). PS is thought to negatively modulate GABAergic neurotransmission. Accordingly, PS disturbs early developmental processes of the GABAergic system in rodents, including diminishing embryonic neurogenesis of GABAergic progenitors (Uchida et al., 2014), delaying GABAergic progenitors' migration to the cerebral cortex (Gumusoglu et al., 2017; Stevens et al., 2013), and methylation in cortical interneurons (Matrisciano et al., 2013). With regard to this latter finding, increased DNA methyltransferase (DNMT) expression and decreased glutamic acid decarboxylase (GAD) 67 expression, which are two of the GABAergic neuronal markers, have been reported in the brain of adult mice prenatally exposed to stress (Matrisciano et al., 2013; Zhu et al., 2018). The subsequent maturation of GABAergic cells is also affected by PS. It has been reported that PS increased GABAergic synapses in the hypothalamus of adult rats, as indicated by a higher basal expression of a marker of GABAergic synapses, the vGAT (vesicular GABA transporter) (Viltart et al., 2006). Some of the above-mentioned studies have documented that the influences of PS on the GABAergic system during developmentally dynamic periods may be relevant to the anxiety-like behaviors that occur after PS. A recent study by Zhu et al. (2018) consistently provided evidence that DNA epigenetic modifications of GABAergic interneurons in the basolateral amygdala participated in the etiology of anxiety-like phenotype in PS mice (Zhu et al., 2018). Accordingly, PS mice developed an anxiety-like phenotype accompanied by a significant increase of DNMT1 and a reduced expression of GAD67 in the basolateral amygdala. Blockade of DNMT1 restored the increased synaptic transmission and anxiety-like behaviors in PS mice via improving the GABAergic system. Supporting this finding, Gumusoglu et al. (2017) and Stevens et al. (2013) have demonstrated that the density of GABAergic progenitors is reduced and their tangential movement from these cells’ birthplace in the ventral telencephalon to their destination in the developing cortical plate is delayed in a mice model of PS. They have suggested that increased anxiety-like behaviors in prenatally stressed offspring may be related to these GABAergic delays (Gumusoglu et al., 2017; Stevens et al., 2013). Consistently, Lussier and Stevens (2016) reported a positive correlation between the frontal cortex parvalbumin/GAD67GFP1 cell ratio and the time spent in the closed arm of the EPM, and a positive correlation between hippocampal GAD67GFP1 cell density and the time spent in the center of the OFT, in PS adult mice. They concluded that PS resulted in a delay in GABAergic cell number and maturation of the parvalbumin subtype, which may be relevant to the observed anxiety-like behaviors (Lussier & Stevens, 2016). In the study reported by Ehrlich et al. (2015) on prenatally stressed female rats, PS increased anxiety-like behaviors, as indicated by a reduced central tendency in the OFT, as well as decreased gene expression of KCC2 (K-Cl cotransporter 2) and increased gene expression of NKCC1 (Na-K-Cl cotransporter 1), two genes which regulate GABAergic function in the amygdala of the offspring (Ehrlich et al., 2015). The relative contribution of the GABAergic system alterations in anxiety-like behaviors in different animal models is summarized in Table 2.

Table 2. Association of the GABAergic system alterations with PS-precipitated anxiety-like behaviors in different animal models with prenatal stress.

Species	Stress type and duration	Stress protocol	Sex and age at assessment	Brain region	Behavioral outcome	Cellular and molecular outcome	Reference
Sprague- Dawley rats	G9–G20 Chronic unpredictable mild stress	Consisted of restraint, cage tilt, damp bedding, cage changes, noise and overnight illumination	Female PND 21 and 90	Amygdala	Increased anxiety-like behaviors. (OFT: reduced central tendency)	Decreased gene expression of KCC2 and increased gene expression of NKCC1	Ehrlich et al. (2015)
CD1 mice	G12-Delivery Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3–4-h intervals	Male 8–11 week-old	Cortex	Increased anxiety-like behaviors. (EPM: decreased time spent in the open arm)	Decreased density and delaying of GABAergic progenitors migration	Gumusoglu et al. (2017)
CD1 mice	G12-Delivery Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3–4-h intervals	Male Cellular and molecular assessments: PND 24 and 150 Behavior testing: 2–3 month-old	Hippocampus, medial frontal cortex	Increased anxiety-like behaviors. (OFT: decreased time spent in the central area; EPM: increased time spent in the closed arms)	Increased parvalbumin/GAD67GFP1 cell ratio in the FC. Decreased hippocampal GAD67GFP1 cell density.	Lussier and Stevens (2016)
C57BL/6J mice	G10-Delivery Restraint stress	3 Stress sessions/day, 45 min/session, 3–4-h intervals, exposed to 24-h constant light	Female PND 60–70	Basolateral amygdala	Increased anxiety-like behaviors. (OFT: decreased time spent in the central area; EPM: decreased time spent in the open arms; LDT: decreased time spent in the light-box)	Increased binding of DNMT1 and methyl CpG binding protein 2 to GAD67 promoter region. Decrease of GAD67 transcript. Enrichment of 5-methylcytosine in GAD67 promoter regions.	Zhu et al. (2018)

BDNF: brain-derived neurotropic factor; DNMT: DNA methyltransferase; EPM: elevated plus maze; FC: frontal cortex; G: gestational day; GABA: γ-amino butyric acid; GAD: glutamic acid decarboxylase; KCC2: K-Cl cotransporter 2; LDT: light/dark transition test; NKCC1: Na-K-Cl cotransporter 1; OFT: open field test; PFC: prefrontal cortex; PND: postnatal day; PS: prenatal stress.

Briefly, there seems to be a direct correlation between anxiety state and the GABAergic neurotransmitter system in the adult offspring raised by their biological mothers during gestational development. Considering that stress during the gestational period has adverse effects on GABAergic neuron development, one can conclude that PS may increase anxiety-related behaviors through negatively modulating GABAergic neurotransmission and epigenetic GABAergic dysfunction in different parts of the developing brain, particularly in the frontal cortex and hippocampus.

3.3. Glutamatergic system and PS-precipitated anxiety-like behaviors

The glutamatergic system is believed to play a crucial role in the neurobiological mechanisms underlying stress–response and anxiety-like behaviors. The relative contribution of the glutamatergic system in the modulation of stress consequences is known to be through regulating the HPA stress axis (Evanson & Herman, 2015; Joca et al., 2007). A disturbance of glutamatergic transmission in the brain impairs the normal responses of the body to challenging situations and may contribute to the pathophysiology of anxiety (Riaza Bermudo-Soriano et al., 2012). Further confirming of this interpretation are the results showing that drugs targeting glutamatergic neurotransmission, in particular NMDA and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors’ antagonists, and several modulators of mGlu receptors (Riaza Bermudo-Soriano et al., 2012) or drugs correcting the defect in glutamate release (Marrocco et al., 2012) show anxiolytic-like effects in animal or human studies. With respect to PS, as glutamate receptors are involved in neural stem cell self-renewal, proliferation, differentiation, and survival (Duan et al., 2013; Xiao et al., 2013), it has been suggested that glutamate receptors are involved in the often-reported deleterious effects of PS on behavioral responses to stress (Wang et al., 2015). Chronic maternal distress alters the responsiveness of the glutamatergic system following a challenge in adulthood. In fact, while in control animals, acute stress enhances the phosphorylation levels of the NMDA receptor subunits, PS attenuates such activation (Fumagalli et al., 2009). Therefore, PS decreases the responsiveness of the glutamatergic system to challenging conditions in adulthood (Fumagalli et al., 2009), which may impair cellular stress response and increase susceptibility to subsequent cognitive or stress-related disorders, such as anxiety (Marrocco et al., 2012; Sun et al., 2013; Wang et al., 2015).

Stress during the gestational period also causes several long-lasting changes in glutamatergic neurotransmission and neuroplasticity, which results in an increased vulnerability to anxiety-like behaviors. PS-induced glutamatergic alterations may happen in various brain regions related to anxiety, such as the hippocampus, amygdala, striatum, and prefrontal cortex (Marrocco et al., 2012; Sun et al., 2013). Accordingly, NMDA receptors increase in the frontal cortex, hippocampus, medial caudate-putamen, as well as core regions of the nucleus accumbens of prenatally stressed rats (Berger et al., 2002; Tavassoli et al., 2013). A synaptic reduction in the AMPA (Biala et al., 2011; Yaka et al., 2007) and NMDA receptor subunits (Biala et al., 2011; Burt et al., 2013; Sun et al., 2013) in the hippocampus, prefrontal cortex, and striatum of prenatally stressed rats has also been reported. Similar results are reported with an impaired function of NMDA receptors and reduce long-term potentiation in the hippocampus (Markham et al., 2010; Son et al., 2006) and the FC (Sowa et al., 2015) in rodents. Sexual differences in long-term effects of PS on regional glutamate receptor expression have been reported in rat offspring. Accordingly, PS induces an opposite activity or expression of mGluRs and NMDARs in the hippocampus or prefrontal cortex between male and female rats (Biala et al., 2011; Fumagalli et al., 2009; Wang et al., 2015; Zuena et al., 2008). However, Sun et al. (2013) found no sex difference of NMDAR expression induced by PS in the hippocampus (Sun et al., 2013). Glutamate synaptic transmission and expression of glutamate transporters are also affected in prenatally stressed offspring in a brain region-specific manner. PS produces a higher level of vesicular glutamate transporter (GLT) 1 protein and mRNA, and increases the uptake capacity for glutamate in the FC and hippocampus of adult male rats (Adrover et al., 2015). Moreover, PS significantly reduces the excitatory amino acid transporters (EAAT) mRNA levels in the same brain regions (Zhang et al., 2013). The decreased EAATs level may result in the decrease of glutamate reuptake and, then, potentially induce the accumulating of glutamate in the synaptic cleft, which might be a risk factor for increasing synaptic glutamate levels. However, selective reductions of glutamate release (Mairesse et al., 2015), in association with large reductions in the levels of synaptic vesicle-related proteins (Marrocco et al., 2012), in the ventral hippocampus of prenatally stressed rats (with no changes in the dorsal hippocampus or perirhinal cortex) are also reported.

Changes in glutamate synaptic transmission, glutamate receptor subtypes levels, and activity in different forebrain regions of adult rats suggest that the development and formation of the corticostriatal and corticolimbic pathways may be permanently altered as the result of stress suffered prenatally and maldevelopment of these pathways may provide a neurobiological substrate for developing neurological disorders, such as anxiety. A study by Marrocco et al. (2012) consistently provided evidence that depolarization-evoked glutamate release was largely reduced in the ventral hippocampus of male PS rats and this reduction correlated positively with anxiety-like behaviors in these rats. They showed a positive correlation between the time spent in the open arm of the EPM as well as in the light compartment of the LDT and the extent of depolarization-evoked glutamate release in the ventral hippocampus and concluded that anxiety-like behaviors were inversely related to the evoked release of glutamate. Pharmacological enhancement of glutamate release in the ventral hippocampus using a mixture of drugs that block presynaptic mGlu2/3 and GABA_B receptors, that negatively regulate glutamate release, corrects the above-mentioned anxiety-like behavior in male PS rats (Marrocco et al., 2012). Consistently, Mairesse et al. (2015) reported that adult male PS rats exhibited an anxiety-like phenotype associated with an abnormal glucocorticoid feedback regulation of the HPA axis and with a reduced depolarization-evoked glutamate release in the ventral hippocampus. Pharmacological enhancement of glutamate release in PS rats corrected anxiety-like behaviors (Mairesse et al., 2015). These findings indicate again that an impairment of glutamate release in the ventral hippocampus is a key component of the neuroplastic program induced by PS and that strategies aimed to enhance glutamate release in the ventral hippocampus correct the “anxious phenotype” caused by PS. A recent study by Jia et al. (2015) reported that PS either during the middle or late gestation could induce anxiety-like behaviors in both male and female offspring, especially in male rats in the late gestation. They suggested that elevated mGluR1 and mGluR5 may contribute to anxiety-like behaviors of prenatally stressed offspring rats, as they found that PS-precipitated anxiety-like behaviors were associated with the increased expression of mGluR1/5 in the hippocampus and/or prefrontal cortex (Jia et al., 2015). However, Morley-Fletcher et al. (2011) reported an anxiety-like behavioral phenotype in adult male PS rats, which was correlated with reduced hippocampal levels of mGlu2/3 and mGlu5 (Morley-Fletcher et al., 2011). Wang et al. (2015) reported sex differences in PS-induced anxiety-like behavior and anxiety-related alterations in glutamate receptor expression. PS increased the baseline expression of mGluRs and NMDARs in the prefrontal cortex, only in male rat offspring. They showed that the effects of PS on glutamate receptor expression in female offspring were not apparent during the baseline expression, but rather emerged under the anxiogenic circumstance. They used the EPM test as an anxiogenic challenge which evoked anxiety-like behavior in rats to investigate the involvement of glutamate receptors during anxiety. Under these anxiogenic circumstances, anxiety-like behaviors induced by the EPM test were associated with increased levels of mGluRs and NMDARs in the hippocampus and prefrontal cortex in male PS rats and with increased levels of mGluRs in the hippocampus in female PS rats (Wang et al., 2015). Consistently, Zuena et al. (2008) suggested that the epigenetic changes in hippocampal mGluRs expression and activity as well as hippocampal neuroplasticity induced by PS may be related to anxiety-like behaviors in these rats and the behavioral outcome was critically sex-dependent and may diverge in males and females (Zuena et al., 2008). However, in the study reported by Sun et al. (2013), both male and female PS rats showed increased anxiety-like behaviors associated with decreased NMDARs expression and suggested that PS-induced glutamatergic abnormity may participate in the anxiety-like behaviors in the adult male and female offspring (Sun et al., 2013). The relative contribution of the glutamatergic system alterations in anxiety-like behaviors in different animal models is summarized in Table 3.

Table 3. Association of the glutamatergic system alterations with PS-precipitated anxiety-like behaviors in different animal models with prenatal stress.

Species	Stress type and duration	Stress protocol	Sex and age at assessment	Brain region	Behavioral outcome	Cellular and molecular outcome	Reference
Sprague–Dawley rats	Mid: G7–G13 Late:G14–G20 Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3–4-h intervals	Male and female 1-month-old	Hippocampus, PFC, striatum	Increased anxiety-like behaviors in both sex, especially in male. (OFT: reduction in the time spent in the center, in the number of center crossing, in the number of total crossing, and in the number of rearing).	Increased expression of mGluR1/5 in the hippocampus and/or PFC.	Jia et al. (2015)
Sprague–Dawley rats	G11-Delivery Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3–4-h intervals	Male 3-month-old	Hippocampus	Increased anxiety-like behaviors. (EPM: reduced number of entries into the open arm, increased latency to enter the open arm, reduced latency to enter the closed arm and time spent in the open arm; LDT: reduced time spent in the light compartment).	Decreased extent of depolarization-evoked glutamate release in the ventral hippocampus	Marrocco et al. (2012)
Sprague–Dawley rats	G11-Delivery Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3 to 4-h intervals	Male 2–3 month-old	Hippocampus	Increased anxiety-like behaviors (EPM: decreased time spent in the open arm.	Reduced hippocampal levels of mGluR2/3 and mGluR5.	Morley-Fletcher et al. (2011)
Sprague–Dawley rats	G14–G21 Restraint stress	3 Stress sessions/day, 45 min/session, with bright light, 3–4-h intervals	Male and female 3-month-old	Hippocampus, PFC, striatum	Increased anxiety-like behaviors in both sex (OFT: reduction of center crosses, total cross and rearing counts, and time spent in the center, in both sex. EPM: decreased percentage of open arm entries and time in male)	Reduced NMDAR1 and NMDAR2A expression in the hippocampus as well as NMDAR2A expression in the PFC and striatum in both sexes	Sun et al. (2013)
Sprague–Dawley rats	G7–G20 Unpredicted chronic mild stress 1–2 of the stressors randomly/day	Space restriction, cage tilt, housing in an empty cage (no sawdust), wet bedding, crowding, food and water deprivation, flashing light during the dark phase.	Male and female 12–13 week-old	Amygdala, hippocampus, PFC	Increased anxiety-like behavior in both sex. (EPM: decrease in the percentage of time spent and entries in the open arm).	Increased baseline expression of mGluR2/3, mGluR5, and NMDAR1 in the PFC in male. No change in baseline expression of any proteins in different brain regions in female rats.	Wang et al. (2015)
Sprague–Dawley rats	G11-Delivery Restraint stress	3 Stress sessions/day, 45 min per session, under bright light conditions, 3–4-h intervals	Male and female 3-month-old	Hippocampus	Male: increased anxiety-like behavior (EPM: decreased the time spent in the open arm. Female: decreased anxiety-like behavior (EPM: increased the time spent in the open arm).	Decreased expression of mGluR5 and the activity of mGluR1/5. Increased levels of BDNF and pro-BDNF in males. No change in the expression of mGluR1/5, increased the activity of mGluR1/5, and no changes in neurogenesis and BDNF level in females	Zuena et al. (2008)

AMPA: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate; BDNF: brain derived neurotropic factor; EPM: elevated plus maze; FC: frontal cortex; G: gestational day; GluRs: glutamate receptors; LDT: light/dark transition test; mGluRs: metabotropic glutamate receptors; NMDA: N-methyl-d-Aspartate; OFT: open field test; PFC: prefrontal cortex; PND: postnatal day; PS: prenatal stress; R: receptor.

Taken together, the precise impact of PS on the glutamatergic system is controversial and these discrepancies render it extremely difficult to determine the exact direction of PS-induced structural and functional changes in the glutamatergic system. These conflicting results could be explained by the differences in experimental conditions, such as the type, timing, duration, and intensity of the stressor as well as the period and method of anxiety assessment and the sex and age of the offspring. Despite these discrepancies, the observations that PS causes several long-lasting alterations in the glutamatergic system and the altered glutamatergic system correlates with the development of anxiety-like behaviors, suggest that an impairment of glutamatergic system and glutamate synaptic transmission in the hippocampus and prefrontal cortex may lie at the core of the neuroplastic program induced by PS and strongly mediate PS effects on the offspring’s anxiety-like behaviors.

3.4. Role of neuroinflammation and imbalance between GABAergic and glutamatergic transmission in PS-precipitated anxiety-like behaviors

The above-mentioned converging evidence suggests that PS-induced alterations in GABAergic and glutamatergic signaling, which lead to an imbalance in excitatory/inhibitory tone in the brain, are risk factors that at least partially contributes to appearance of anxiety-like behaviors. Since GABA derives from glutamate and glutamate derives from GABA, PS-induced alterations in both neurotransmitters can affect each other and, therefore, disrupt the excitatory/inhibitory balance. Hyperexcitation due to enhanced excitatory transmission or reduced inhibitory transmission can promote anxiety. Therefore, disrupted excitatory/inhibitory signaling balance in the brain, particularly in the frontal cortex and hippocampus, has been suggested as a major contributor to the development of neurobehavioral disorders like anxiety (Wu et al., 2008). A recent study by Heslin and Coutellier (2018) consistently provided evidence that a potential interaction between imbalanced excitatory/inhibitory signaling in the cortex and PS exposure increased anxiety-like behaviors in mice (Heslin & Coutellier, 2018). They used heterozygous knock-out (HET) mice with decreased expression of the transcription factor Npas4. The neuronal PAS domain protein 4 (Npas4) gene is instrumental in regulating the excitatory/inhibitory balance in the cortex and hippocampus in response to activation. Npas4 heterozygous and wild-type male and female mice offspring were exposed to either PS or standard gestation. The PS protocol consisted of restraint stress for 30 min twice per day, under bright light conditions, from G7 to G19. The anxiety-like behavior of offspring adults was then tested on the OFT on postnatal days 62 ± 2. Their results showed that a combination of PS and Npas4 deficiency in male mice increased anxiety-like behaviors. PS HETs spent less time in the center zone and more time in the wall zone in the OFT than the standard gestation HETs. This behavioral deficit was associated with decreased parvalbumin-positive GABAergic interneurons in the prefrontal cortex (PFC) in Npas4 heterozygous males. On the other hand, PS males showed lower mRNA expression levels of NMDAR1 in association with some other molecular changes. Thus, the reduced number of parvalbumin-expressing cells in the PFC of HET mice, when associated with other PS-induced dysregulation, could contribute to the reduced time spent in the center of the OFT observed in PS HET mice. In contrast, females displayed fewer behavioral effects and molecular changes in PFC in response to PS and decreased Npas4. These authors suggested that the pattern of increased anxiety-like behaviors observed in PS males may be the consequence of disrupted prefrontal excitatory/inhibitory balance, associated with disturbances in other brain regions and systems not tested in their work. In support of this hypothesis, in another study, Van den Hove et al. (2013) subjected Sprague–Dawley pregnant rats to a restraint stress protocol while being exposed to bright light (3 stress sessions per day, 45 min each session), from G14 until G21 and both male and female adult offspring were subsequently assessed on postnatal day 120 for anxiety-like behaviors (Van den Hove et al., 2013). In addition, genes encoding proteins known to be involved in the pathophysiology and/or treatment of affective disorders, including genes involved in glutamate and GABA neurotransmission, within the hippocampus and FC were also examined. They showed that, in addition to increased anxiety-related behaviors in male PS rats, the expression of multiple GABAergic and glutamatergic receptor subunits were affected after PS. For example, within the FC of male offspring, the expression of the alpha 4 GABA receptor subunit as well as NMDAR2A and mGluR5 expression decreased in PS as compared to control animals. Female PS rats showed a decrease in the expression of the NMDAR1 and 2A within the FC, as well as a decreased mGluR5 expression in both the hippocampus and FC. Moreover, the expression of various GABA receptor subunits and the expression of GAD decreased in both brain regions in female PS rats. These data once again showed distinct sex-dependent changes in gene expression after PS and also suggested that changes in glutamatergic and GABAergic neurotransmissions may play a prominent role in regulating the affective state and showed their possible therapeutic value in disorders like anxiety. Another research study by Laloux et al. (2012) on prenatally stressed male rats consistently demonstrated an association between GABAergic and glutamatergic epigenetic modifications and anxiety-like behaviors. Accordingly, Sprague–Dawley pregnant rats were subjected to the repeated episodes of restraint stress under bright light for 45 min three times on the daily basis from G11 until delivery and anxiety-like behaviors; GABA and glutamate receptor protein expression of male offspring were subsequently investigated on different postnatal days. On PND 10 and 14, PS rats emitted more ultrasonic vocalizations (USVs) in response to isolation from their mothers and showed later suppression of USV production when being exposed to an unfamiliar male odor, indicating the pronounced anxiety-like profile. Moreover, PS rats showed reduced expression of the γ2 subunit of GABA_A receptors in the amygdala on PND 22, the increased expression of mGlu5 receptors in the amygdala on PND 22, reduced expression of mGluR5 in the hippocampus on PND 14 and PND 22, and reduced expression of mGluRs2/3 in the hippocampus on PND 22. These PS-induced GABAergic and glutamatergic epigenetic modifications were associated with increased anxiety-like behaviors in the OFT and EPM. The percentage of visits and the time spent in the open arms of the EPM as well as the time spent in the central area of the OFT decreased in PS rats on PND 22 as compared to age-matched controls (Laloux et al., 2012).

Altogether, the above-mentioned findings are relevant to neurobehavioral disorders where evidence of PS-induced disruption of excitatory/inhibitory signaling balance over the PFC and hippocampus is associated with enhanced anxiety-like behaviors and might contribute to a better understanding of the underlying mechanism for the increased risk of PS offspring to develop neurobehavioral disorders.

Multiple mechanisms may compromise excitatory/inhibitory signaling balance, one of which is neuroinflammation. Neuroinflammation highly contributes to the imbalance between GABAergic and glutamatergic transmission and, consequently, to the etiology of anxiety. El-Ansary and Al-Ayadhi (2014) in a human study on autistic patients reported an increase of brain TNF-α and the association between this cytokine and the impaired glutamate/GABA ratio, which could be explained on the basis of the relationship between neurons, glial cells, and TNF-α, leading to glutamate excitotoxicity and increasing the susceptibility to neurobehavioral disorders. They showed that elevated TNF-α concentrations could also be contributed to the decrease of the inhibitory transmission (El-Ansary & Al-Ayadhi, 2014). Consistently, an in vitro culture study in mature rat and mouse hippocampal neurons demonstrated that the acute application of TNF-α induced a rapid and persistent decrease of inhibitory synaptic strength and downregulated cell-surface levels of GABA_A receptors (Pribiag & Stellwagen, 2013). Inflammatory mediators have also been shown to induce anxiety-like behaviors via regulating GABA release in the paraventricular nucleus of the hypothalamus (PVN). For example, intracerebroventricular infusion of IL-1β was found to activate astrocytes in the PVN, which in turn resulted in the release of a considerable amount of GABA from astrocytes and induced anxiety-like behaviors in Sprague–Dawley male adult rats. Pretreatment with astrocytes toxin prevented IL-1β-induced GABA release and markedly reduced anxiety-like behaviors (Shim et al., 2019). Although it has been shown that GABA agonists can exert anti-inflammatory effects via stimulating GABA_A and GABA_B receptors on glial cells (Yoon et al., 2012), an abnormal enhancement in GABA release secondary to the astrocytes hyperactivity may contribute to the pathophysiology of anxiety. Moreover, Luo et al. (2020) reported that pharmacological inhibition of the microglia activation and suppression of both peripheral and central IL-1β, IL-6, and TNF-α levels, as well as the pharmacological regulation of the imbalance in excitatory/inhibitory receptors and neurotransmitters in the basolateral nucleus ameliorated anxiety-like behaviors in a mouse model of complete Freund’s adjuvant-induced chronic inflammation anxiety (Luo et al., 2020). These explanations could provide some evidence suggesting that the impaired GABAergic and glutamatergic neurotransmission could be related to neuroinflammation as one of the etiological hypotheses involved in developing neurobehavioral disorders, like anxiety.

4. Conclusions

While the short-term activation of the HPA axis allows adaptive responses to the challenge, the prolonged and enhanced HPA axis responses, such as those that occur following PS can be devastating for the organism. PS leads to HPA axis disruption, which in turn may induce systemic pro-inflammatory conditions. Systemic inflammation leads to neuroinflammation and enhanced levels of pro-inflammatory cytokines through excessive microglia activation in the brain exerting neurotoxic effects on specific brain regions, either directly or indirectly. This may cause alterations in the structure or function of anxiety-related brain circuits priming the brain to be vulnerable to anxiety disorders. While we hypothesize that this is an imbalance in GABAergic and glutamatergic circuits (mainly in limbic and pre-frontal circuits), that at least partially contributes to PS-induced anxiety; disturbances in other brain regions and systems cannot be excluded. Therefore, pharmacological interventions with anti-inflammatory activities and with regulatory effects on the excitatory/inhibitory balance can be attributed to the novel therapeutic target for PS-precipitated anxiety disorders.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Notes on contributors

Shiva Roshan-Milani

Shiva is a professor in physiology at Urmia University of medical sciences (MD, PhD).

Behdad Seyyedabadi

Behdad is a student of pharmacy at Urmia University of medical sciences.

Ehsan Saboory

Ehsan is a professor in physiology at Zanjan University of medical sciences.

Negin Parsamanesh

Negin is an assistant professor at Zanjan University of medical sciences.

Nasrin Mehranfard

Nasrin is an assistant professor at Urmia University of medical science.