Restraint stress during pregnancy rapidly raises kynurenic acid levels in mouse placenta and fetal brain

Abstract

Stressful events during pregnancy adversely affect brain development and may increase the risk of psychiatric disorders later in life. Early changes in the kynurenine pathway (KP) of tryptophan (TRP) degradation, which contains several neuroactive metabolites, including kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), may constitute a molecular link between prenatal stress and delayed pathological consequences. To begin testing this hypothesis experimentally, we examined the effect of a 2-h restraint stress on KP metabolism in pregnant FVB/N mice on gestational day 17. TRP, kynurenine (KYN), KYNA, 3-HK and QUIN levels were measured in maternal and fetal plasma and brain, as well as in the placenta, immediately after stress termination and 2 hours later. In the same animals, we determined the activity of tryptophan-2,3-dioxygenase (TDO) in maternal liver and in the placenta. Compared to unstressed controls, mostly transient changes in KP metabolism were observed in all tissues examined. Specifically, stress caused significant elevations of KYNA levels in maternal plasma, placenta and fetal brain, and also resulted in increased levels of TRP and KYN in placenta, fetal plasma and fetal brain. In contrast, 3-HK and QUIN levels remained unchanged from control values in all tissues at any timepoint. In the maternal liver, TDO activity was increased 2 hours after stress cessation. Taken together, these findings indicate that an acute stress during the late gestational period preferentially affects the KYNA branch of KP metabolism in the fetal brain. Possible long-term consequences for postnatal brain development and pathology remain to be examined.

Introduction

Mounting evidence has established gestation as a period of vulnerability to environmental insults. Specifically, maternal exposure to stressful conditions adversely affects brain development in the fetus and can result in long-lasting changes in adulthood [1,2]. In humans, prenatal stress is considered a risk factor for the development of a series of behavioral alterations and has been associated with aggression, hyperactivity, anxiety and cognitive problems later in life [3,4]. Moreover, exposure to adverse influences during the prenatal period has been linked to an increased risk of developing psychiatric disorders, including schizophrenia [5,6]. In rodents, prenatal stress has profound effects on the offspring, causing permanent neurobiological and behavioral alterations [7–9]. In particular, the progeny of stressed dams have an altered hypothalamic–pituitary–adrenal (HPA) axis and exhibit increased anxiety and impaired working memory [1]. Moreover, prenatal stress leads to long-lasting modifications in immune functions, exemplified by elevated cytokines in brain and periphery, alteration in microglial morphology and an enhanced immune response to inflammatory stimuli [10–12].

Stress is also known to cause changes in the kynurenine pathway (KP) of tryptophan (TRP) degradation (Fig. 1). The KP contains several neuroactive metabolites, including kynurenic acid (KYNA), which can function as an antagonist of the α7 nicotinic acetylcholine (α7nACh) and the N-methyl-D-aspartate (NMDA) receptors and also affects other biologically relevant receptors [13,14], and, in a competing branch of the pathway, the free radical generator 3-hydroxykynurenine (3-HK) and the NMDA receptor agonist quinolinic acid (QUIN) [15]. The link between stress and the KP is believed to be initiated by corticosterone (CORT), which directly activates tryptophan 2,3-dioxygenase (TDO), an enzyme that triggers the oxidative opening of TRP’s indole ring and thus catalyzes the conversion of TRP to formylkynurenine (Fig. 1) [16]. This, in turn, results in increased levels of kynurenine (KYN) and of downstream KP metabolites in both brain and periphery [17–20]. Alone or together, these effects could play a role in a number of stress-related phenomena, ranging from normal physiological responses to acute and chronic pathological events [21,22].

Fig. 1.

The kynurenine pathway of tryptophan degradation.

So far, the relationship between stress and KP metabolism has only been studied in adult animals. As maternal stress has adverse long-term consequences in the offspring and may play a role in the emergence of psychiatric diseases in adolescence and early adulthood, stress-induced prenatal KP impairments may also be functionally significant. Remarkably, in mammals, the brain levels of several key KP metabolites, including KYN, KYNA, 3-HK and QUIN, are several-fold higher in the fetus than in the postnatal period, including adulthood [23], and direct manipulation of the KP during the last week of gestation results in several, pathophysiologically interesting neurochemical and behavioral abnormalities as the offspring matures [24]. It follows that fluctuations of KP metabolites in the prenatal brain may be causally involved in the cascade linking stressful events during gestation to the emergence of a variety of clinically relevant abnormalities later in life [25–27]. In a first effort to directly investigate these putative relationships, the present study was designed to examine the effects of acute stress on KP metabolism in the fetus. To this end, we used mouse dams on gestational day (GD) 17, i.e. in a final stage of pregnancy.

Materials and Methods

Chemicals

L-Tryptophan (TRP), 3-hydroxy-DL-kynurenine (3-HK), kynurenic acid (KYNA), [²H₆]L-kynurenine, quinolinic acid (QUIN), pentafluoropropionic anhydride, 2,2,3,3,3-pentafluoro-1-propanol and the TDO inhibitor 680C91 were obtained from Sigma-Aldrich (St. Louis, MO, USA). L-Kynurenine sulfate (KYN; purity: 99.4%) was obtained from Sai Advantium (Hyderabad, India). [²H₃]Quinolinic acid was purchased from Synfine Research (Richmond Hill, Ontario, Canada) and [²H₅]L-tryptophan was obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada). All other chemicals were obtained from various commercial suppliers and were of the highest available purity.

Animals

Male and female FVB/N mice (2–4 months; Jackson laboratory, Bar Harbor, ME, USA) were bred and housed in the animal facility of the University of Maryland School of Medicine. Detection of a copulation plug denoted gestational day (GD) 1. Animals were maintained on a 12 h light/dark cycle in a temperature-controlled room with food and water ad libitum. To avoid litter effects, 2 embryos per litter were used in all studies, and the data were expressed as the average of litters. All studies were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.

Restraint stress

Pregnant (GD17) mice (n=5–6 per group) were placed in a well-ventilated cylindrical restrainer (Braintree Scientific, Braintree, MA, USA) for 2 h (stress group) or left undisturbed (control group). The animals were then euthanized immediately (“0 h”) or 2 h after the cessation of stress. Maternal brain, liver and blood (“plasma”; collected in EDTA-containing vials to avoid clotting), as well as placenta, fetal brain and fetal plasma, were rapidly collected on ice and stored at −80°C until analysis.

Corticosterone determination

Levels of corticosterone (CORT) were determined by radioimmunoassay in 10 μL of thawed plasma according to protocols provided by the commercial manufacturer (MP Biochemicals, Orangeburg, NY, USA).

Tryptophan 2,3-dioxygenase assay

Maternal liver and placenta were thawed out and homogenized (1:10, w/v) in 100 mM HEPES buffer (pH 7.4), and liver homogenate was diluted further (1:10, v/v) in the same buffer. Fifty μL of the tissue homogenates were incubated at 37°C (2 h for liver, 4 h for placenta) in a solution containing 10 μM hematin, 25 μM ascorbic acid and 200 μM TRP in a total volume of 100 μL. Blanks were obtained by adding the specific TDO inhibitor 680C91 (50 μM final concentration) to the incubation solution. The reaction was stopped by the addition of 25 μL of 6% perchloric acid, and the samples were then incubated for 20 min at 60°C. After centrifugation (16,000 × g, 10 min), 20 μL of the supernatant were applied to high-performance liquid chromatography (HPLC), and KYN was detected fluorimetrically (see below).

Measurement of tryptophan and kynurenine by HPLC

For the determination of TRP and KYN in maternal and fetal plasma, the samples were thawed and diluted in ultrapure water (1:1,000, v/v for TRP; 1:2, v/v for KYN), and 25 μL of 6% perchloric acid were added to 100 μL of the preparations. Precipitated proteins were removed by centrifugation (16,000 × g, 10 min), and 20 μL of the resulting supernatant were applied to a 3 μm C18 reverse phase HPLC column (80 mm × 4.6 mm; ESA, Chelmsford, MA, USA), using a mobile phase containing 250 mM zinc acetate, 50 mM sodium acetate, and 5% (for TRP) or 3% (for KYN) acetonitrile (pH adjusted to 6.2 with glacial acetic acid). Eluted at a flow rate of 1.0 ml/min, the metabolites were detected fluorimetrically (TRP: excitation: 285 nm, emission: 365 nm; KYN: excitation: 365 nm, emission: 480 nm; 200a fluorescence detector; Perkin-Elmer, Waltham, MA, USA).

Measurement of tryptophan, kynurenine and quinolinic acid by GC/MS

To measure TRP, KYN and QUIN levels in tissue, the stored samples were thawed and sonicated (1:5, w/v for maternal brain; 1:10, w/v for fetal brain and placenta,) in ultrapure water. The homogenates were further diluted in 0.1% ascorbic acid (1:20 final for maternal brain, 1:50 final for fetal brain, 1:100 final for placenta). For the determination of QUIN in plasma, thawed maternal and fetal samples were diluted in 0.1% ascorbic acid (1:10, v/v).

Fifty μl of an internal standard mix ([²H₃]quinolinic acid, [²H₆]L-kynurenine and [²H₅]L-tryptophan) were added to 50 μL of each sample, and proteins were precipitated with 50 μL of acetone. After centrifugation (13,700 × g, 5 min), 50 μL of methanol:chloroform (20:50) were added to the supernatant, and the samples were centrifuged (13,700 × g, 10 min). The upper layer was added to a glass tube and dried down for 90 min. The samples were then derivatized with 120 μL of 2,2,3,3,3-pentafluoro-1-propanol and 130 μL of pentafluoropropionic anhydride at 75°C for 30 min, dried down and reconstituted in 50 μL of ethyl acetate. One μL was then used for analysis by gas chromatography/mass spectrometry (Notarangelo et al., 2012).

Measurement of 3-hydroxykynurenine and kynurenic acid

For measurement of 3-HK and KYNA, thawed fetal plasma (1:4, v/v for 3-HK, 1:50, v/v for KYNA) and fetal brain homogenate (1:20 final for 3-HK, 1:50 final for KYNA) were diluted in ultrapure water. Placental homogenate (1:20 final for 3-HK, 1:100 final for KYNA) and maternal plasma (1:2, v/v for 3-HK and 1:10, v/v for KYNA) were diluted in ultrapure water. A dilution of 1:5 (w/v) was used for the determination of both KYNA and 3-HK in maternal brain homogenate.

Twenty-five μL of 6% perchloric acid were added to 100 μL of the samples, and precipitated proteins were removed by centrifugation (16,000 × g, 10 min). For KYNA determination, 20 μL of the resulting supernatant were injected to a 3 μm C18 reverse phase HPLC column (HR-80?; 80 mm × 4.6 mm; ESA), using a mobile phase containing 250 mM zinc acetate, 50 mM sodium acetate, and 3% acetonitrile (pH adjusted to 6.2 with glacial acetic acid) at a flow rate of 1.0 ml/min. In the eluate, KYNA was detected fluorimetrically (excitation: 344 nm, emission: 398 nm; S200a fluorescence detector; Perkin Elmer). For 3-HK determination, 20 μL of the supernatant were applied to a 3 μm HPLC column (80 mm × 4.6 mm; ESA), using a mobile phase consisting of 1.5 % acetonitrile, 0.9 % triethylamine, 0.59 % phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid, and a flow rate of 0.5 mL/min. In the eluate, 3-HK was detected electrochemically using a HTEC 500 detector (Eicom Corp., San Diego, CA; oxidation potential: +0.5 V).

Statistical analysis

Results are expressed as the mean ± SEM. One-way and two-way Anova followed by Bonferroni’s post-hoc test or Student’s t-test were used to determine statistical significance. To measure the relationship between metabolites in different tissues, Pearson correlation analysis was performed. A p value of <0.05 was considered significant.

Results

Effect of acute stress on plasma CORT and TDO activity

To evaluate the response of pregnant mice to acute restraint stress, we measured CORT in maternal and fetal plasma. Compared to basal values, CORT levels were significantly elevated (p<0.001) in both samples immediately after the termination of stress and returned to basal levels after an additional 2 h (Fig. 2). The increase in CORT was slightly higher in maternal than in fetal plasma (256% and 192%, respectively; p<0.05, two-way Anova followed by Bonferroni’s post-hoc test). Interestingly, CORT levels in maternal plasma correlated positively with CORT levels in fetal plasma (p<0.0001).

Fig. 2.

Stress-induced elevation of CORT levels in maternal and fetal plasma. CORT was determined in control mice (ctr) or immediately and 2 h after the cessation of stress. Data are the mean ± SEM (n=5–6 per group) and are expressed as a percentage of endogenous values. *** p<0.001 vs. ctr (one-way Anova, followed by Bonferroni’s post-hoc test).

TDO activity in the maternal liver trended to be elevated immediately after the termination of stress, and the increase reached statistical significance 2 h later (p<0.05; Fig. 3A). We also observed a trend toward increased TDO activity in the placenta immediately after the termination of stress (p=0.07, one-way Anova, followed by Bonferroni’s post-hoc test) (Fig. 3B). However, TDO activity was undetectable in the fetal liver of both control and stressed mice (data not shown).

Fig. 3.

Effect of stress on TDO activity in maternal liver (A) and placenta (B). Enzyme activity was measured in control mice (ctr) or immediately and 2 h after the cessation of stress. Data are the mean ± SEM (n=5–6 per group). ** p<0.01 vs. ctr (one-way Anova, followed by Bonferroni’s post-hoc test).

TRP and KP metabolites in maternal brain and plasma

To examine the effect of acute stress on the dynamics of maternal KP metabolism, we determined TRP, KYN, KYNA, 3-HK and QUIN levels in brain and plasma of control and stressed dams immediately and 2 h after the termination of stress. With one exception (an increase in KYNA levels in the maternal plasma immediately after the termination of stress; p<0.05), acute stress had no significant effect on TRP or any of the KP metabolites in either the maternal brain (Table 1) or the maternal plasma (Table 2) (p>0.05 in both cases; one-way Anova, followed by Bonferroni’s post-hoc test).

Table 1.

Effect of restraint stress on TRP and KP metabolites in the maternal brain

	Controls	0 h after stress	2 h after stress
TRP (pmoles/mg protein)	308.1 ± 9.8	367.7 ± 25.8	302.3 ± 3.0
KYN (pmoles/mg protein)	12.6 ± 0.5	14.1 ± 2.0	14.2 ± 1.2
KYNA (fmoles/mg protein)	28.5 ± 2.0	36.7 ± 4.5	35.9 ± 2.8
3-HK (fmoles/mg protein)	862.6 ± 89.0	815.6 ± 157.7	1041.9 ± 58.5
QUIN (fmoles/mg protein)	473.0 ± 25.4	472.4 ± 55.0	500.0 ± 37.7

Data are the mean ± SEM (n=5–6 per group).

Table 2.

Effect of restraint stress on TRP and KP metabolites in maternal plasma

	Controls	0 h after stress	2 h after stress
TRP (pmoles/μL)	58.6 ± 4.6	70.2 ± 5.7	68.9 ± 6.5
KYN (pmoles/μL)	3.7 ± 0.5	3.3 ± 0.3	3.5 ± 0.2
KYNA (fmoles/μL)	62.4 ± 6.6	116.3 ± 19.4*	103.1 ± 12.5
3-HK (fmoles/μL)	27.3 ± 6.1	17.2 ± 2.8	35.3 ± 7.1
QUIN (fmoles/μL)	176.2 ± 13.4	215.0 ± 15.2	171.7 ± 10.9

Data are the mean ± SEM (n=5–6 per group).

^*p<0.05 vs controls (one-way Anova, followed by Bonferroni’s post-hoc test).

TRP and KP metabolites in placenta and fetal plasma

Despite a tendency towards higher TDO activity (see above), TRP levels in the placenta were transiently elevated immediately after stress (p<0.05, Fig. 4). KYN and KYNA levels, too, were increased immediately after the cessation of stress (p<0.05 and p<0.01, respectively) but returned to control levels after 2 h. In contrast, stress did not affect either 3-HK or QUIN levels in the placenta (Fig. 4).

Fig. 4.

Effect of stress on TRP, KYN and KYNA in the placenta. TRP and KP metabolites were measured in control mice (ctr) or immediately and 2 h after the cessation of stress. Data are the mean ± SEM (n=5–6 per group). * p<0.05, ** p<0.01 vs. ctr (one-way Anova, followed by Bonferroni’s post-hoc test).

Analyzed by t-test, fetal plasma contained higher basal levels of KYN (p<0.001), KYNA (p<0.05) and QUIN (p<0.001) than maternal plasma (cf. Fig. 5 and Table 2). Acute restraint stress caused significant elevations in TRP and KYN levels in fetal plasma (p<0.01 each; Fig. 5) immediately after the termination of stress, and KYN levels remained elevated 2 h after stress (p<0.05). However, we did not detect significant stress-induced changes in KYNA, 3-HK or QUIN levels in these samples (Fig. 5).

Fig. 5.

Stress-induced increases in the levels of TRP and KYN in the fetal plasma. TRP and KP metabolites were measured in control mice (ctr) or immediately and 2 h after the cessation of stress. Data are the mean ± SEM (n=5–6 per group). * p<0.05, ** p<0.01 vs. ctr (one-way Anova, followed by Bonferroni’s post-hoc test).

TRP and KP metabolites in the fetal brain

The tissue concentrations of all metabolites analyzed were substantially higher (all p<0.001, t-test) in the fetal brain than in the maternal brain. Specifically, TRP and KYN levels were ~3 times and ~7 times higher, respectively, KYNA levels were ~20-fold higher, and 3-HK and QUIN levels were both ~4–5 times higher in fetal compared to maternal brain (cf. Fig. 6 and Table 1).

Fig. 6.

Stress-induced increases in the levels of TRP, KYN and KYNA in the fetal brain after. TRP and KP metabolites were measured in control mice (ctr) or immediately and 2 h after the cessation of stress. Data are the mean ± SEM (n=5–6 per group). ** p<0.01, *** p<0.001 vs. ctr (one-way Anova, followed by Bonferroni’s post-hoc test).

Similar to the results obtained in placenta and fetal plasma, acute stress caused significant increases in TRP (p<0.001), KYN (p<0.01) and KYNA (p<0.01) levels in the fetal brain immediately after stress. Two hours later, KYN levels remained elevated, whereas TRP and KYNA levels had returned to control values (Fig. 6). No stress-related changes in 3-HK or QUIN content were detected in fetal brain (Fig. 6).

Interestingly, TRP levels in fetal brain correlated with TRP levels in fetal plasma (p<0.001) and placenta (p<0.0001), and KYNA levels in the fetal brain correlated positively with TRP levels in placenta (p<0.01), fetal plasma (p=0.056) and fetal brain (p<0.001), as well as with KYNA levels in fetal plasma (p<0.01).

Discussion

The present study, which was designed to examine the short-term effects of an acute physical stress on KP metabolism at a late gestational stage, revealed several interesting phenomena. First, most of the changes seen following the 2-h restraint were transient, i.e. they were more pronounced immediately after cessation of the restraint than 2 hours later. Second, with the exception of an increase in plasma KYNA levels, stress did not cause significant changes in KP metabolite levels in maternal blood or brain. Third, stress-induced KP changes in the placenta may have affected fetal KP metabolism. Finally, the relatively brief episode of restraint did not influence either 3-HK or QUIN levels in any of the tissues studied, indicating an unexpected, selective effect of stress on the KYNA branch of the metabolic cascade.

As confirmed here, acute stress raises circulating levels of CORT not only in maternal but also in fetal plasma [28,29]. In line with the fact that TDO activity is readily stimulated by CORT and therefore up-regulated in adult animals under stressful conditions [18,20,30,31], we also observed a stress-induced increase in TDO activity in the maternal liver. Although not reaching statistical significance, a similar response to stress was observed in the placenta, which was found to contain comparatively low but clearly detectable TDO activity – in agreement with reports of placental TDO expression [32,33]. In accordance with a previous study reporting the absence of TDO activity in the liver of young rats [34], we were not able to measure TDO activity in fetal liver tissue homogenate in either basal conditions or after stress. Of note in this context, the fetal liver is not fully functional during the prenatal period, as maturation is gradual and continues after birth [35].

In spite of the trend toward higher TDO activity, the stress-induced increase in placental and fetal TRP levels seen in the present study was not unexpected since rapid TRP elevations in response to immobilization have been previously observed in both plasma and brain of adult male rats [36,37]. TRP levels are also raised in the fetal brain when pregnant rats are stressed by crowding during the final week of gestation [38], though, to our knowledge, this effect has so far not been authenticated in mice. Although the underlying mechanism(s) may also include compensatory down-regulation of other TRP-degrading placental enzymes during the prenatal period [39] and/or direct effects of steroid hormones on TRP metabolism [40–42], the elevated TRP levels seen in the highly vascularized placenta [43] immediately after restraint of the dam were more likely related to increased free TRP or to the presence of maternal blood where we observed a trend toward increased TRP levels (Table 2).

The qualitative and quantitative similarity between stress-related changes in the levels of TRP, KYN and KYNA in the placenta and the fetal brain shown here suggests a close functional relationship between the two tissues. The relevance of the placenta for healthy fetal development is indeed well-established [44], and its pathophysiologically significant role during maternal insults has been documented [45–47]. More specifically, the placenta is known to regulate the transport of amino acids [48] and the synthesis of several neuroactive factors that can influence fetal brain development, including the TRP metabolite serotonin [49]. KP metabolites and enzymes, too, may play a role in the placenta both under physiological conditions and when the organ is compromised [32,50–53]. Notably, as both TRP and KYN pass easily from the placenta to the fetus [42,51,54,55], the transient increases in the fetal levels of these two compounds seen here in response to maternal stress may be at least in part caused by direct transfer from the placenta. KYNA, in contrast, does not penetrate readily from the placenta into the fetus [55], i.e. the stress-induced KYNA elevation in the embryonic brain likely reflected local synthesis in the fetal tissue itself due to higher substrate availability, as KAT activity in placenta or fetal brain is not affected by stress (data not shown). KYN is indeed enzymatically transaminated to KYNA in the fetal brain [56], and we recently demonstrated the efficient synthesis of KYNA from KYN, as well as the prompt liberation of newly produced KYNA, using fetal brain tissue slices [57]. Interestingly, however, 3-HK production from KYN was essentially undetectable in these and other in vitro experiments (Notarangelo et al., manuscript in preparation). Therefore, while the source of 3-HK and QUIN in the fetal brain remains to be elaborated, the present results are consistent with a scenario in which the placenta, by efficiently providing TRP and KYN to the fetus, critically mediates the effects of maternal stress on the embryo.

Stress during pregnancy not only affects the fetal brain acutely but also increases the risk of the offspring for developing major psychiatric disorders, including depression and schizophrenia [25,58]. Elevated fetal brain levels of KYNA, an established neuromodulator in the adult mammalian brain [24], may be one of the factors that contribute to these impairments and dysfunctions later in life. Thus, KYNA is an endogenous antagonist of α7nACh and NMDA receptors, both of which play critical roles in brain development [59,60], and can also target two other receptors, the aryl hydrocarbon receptor and the G protein-coupled receptor GPR35, which have not yet been evaluated as participants in early life events [13,14]. Alone or together, these effector sites may react unconventionally to a sudden KYNA surge in the prenatal period and cause abnormal effects downstream. Of potential relevance in this context is the fact that basal brain concentrations of several KP metabolites, including KYNA, are substantially higher prenatally than postnatally [23]. Cause(s) and physiological ramifications of these elevated metabolite levels, as well as possible implications for pathological events during the perinatal period [61], await further clarification.

In rodents, pharmacological interventions that produce increases in brain KYNA levels during the last week of gestation result in chemical, structural, electrophysiological and cognitive impairments in adulthood [62–67]. First support for causality has recently been obtained by the observation that these cognitive impairments can be prevented by perinatal administration of the α7nACh receptor agonist choline [23]. Notably, prenatal stress causes α7nACh receptor abnormalities in the offspring [68,69], and prenatal dietary choline supplementation has been shown to have positive behavioral effects in a variety of informative animal models [70–72]. It is therefore tempting to speculate that the acute effect of prenatal stress on KYNA described here, especially if accompanied and potentially exacerbated by KP abnormalities caused by infections or other immune activations during pregnancy [32,53,73–75], may be directly related to behavioral impairments later in life and the etiology of major brain diseases.

In summary, the present study demonstrated a significant, transient increase of KYNA levels in the fetal brain after acute stress, raising the prospect that KYNA plays a causative role in the development of postnatal abnormalities and, possibly, of psychiatric disorders in humans. Ongoing studies in our laboratory are designed to evaluate the consequences of repeated or chronic stress and infections during pregnancy on fetal KP metabolism, to examine possibly sex-specific, long-term effects in adolescence and adulthood, and to predictably exacerbate or ameliorate long-term impairments by targeted interventions at various stages in life.

Abbreviations

TRP

Tryptophan

KYN

Kynurenine

KYNA

Kynurenic acid

3-HK

3-Hydroxykynurenine

QUIN

Quinolinic acid

KP

Kynurenine pathway

CORT

Corticosterone

TDO

Tryptophan 2,3-dioxygenase