Neurodevelopmental damage after prenatal infection: Role of oxidative stress in the fetal brain

Free Radical Biology & Medicine 42 (2007) 1231 – 1245 www.elsevier.com/locate/freeradbiomed

Original Contribution

Neurodevelopmental damage after prenatal infection: Role of oxidative stress in the fetal brain Fabien Lanté, Johann Meunier 1 , Janique Guiramand, Tangui Maurice 1 , Mélanie Cavalier, Marie-Céleste de Jesus Ferreira, Rose Aimar, Catherine Cohen-Solal, Michel Vignes, Gérard Barbanel ⁎ Oxidative Stress and Neuroprotection, IBMM, CNRS UMR-5247, University of Montpellier II, Place E. Bataillon, 34095 Montpellier Cedex 5, France Received 19 September 2006; revised 19 December 2006; accepted 15 January 2007 Available online 19 January 2007

Abstract Prenatal infection is a major risk responsible for the occurrence of psychiatric conditions in infants. Mimicking maternal infection by exposing pregnant rodents to bacterial endotoxin lipopolysaccharide (LPS) also leads to major brain disorders in the offspring. The mechanisms of LPS action remain, however, unknown. Here, we show that LPS injection during pregnancy in rats, 2 days before delivery, triggered an oxidative stress in the hippocampus of male fetuses, evidenced by a rapid rise in protein carbonylation and by decreases in α-tocopherol levels and in the ratio of reduced/oxidized forms of glutathione (GSH/GSSG). Neither protein carbonylation increase nor decreases in α-tocopherol levels and GSH/GSSG ratio were observed in female fetuses. NMDA synaptic currents and long-term potentiation in CA1, as well as spatial recognition in the water maze, were also impaired in male but not in female 28-day-old offspring. Pretreatment with the antioxidant N-acetylcysteine prevented the LPSinduced changes in the biochemical markers of oxidative stress in male fetuses, and the delayed detrimental effects in male 28-day-old offspring, completely restoring both long-term potentiation in the hippocampus and spatial recognition performance. Oxidative stress in the hippocampus of male fetuses may thus participate in the neurodevelopmental damage induced by a prenatal LPS challenge. © 2007 Elsevier Inc. All rights reserved. Keywords: LPS; Prenatal stress; Protein carbonylation; α-Tocopherol; N-Acetylcysteine; Long-term potentiation; Spatial memory; NMDA receptor; Hippocampus; Rat; Free radicals

Early life experience is critical for the development of the central nervous system. Several recent studies point to in utero and/or perinatal insults as determining factors in a number of neurodevelopmental diseases. Maternal infection during pregnancy, which produces a strong stress on both mother and fetus, is an important adverse experience in utero that has been repetitively linked to the occurrence of mental or behavioral disorders [1–8]. Of particular interest, lipopolysaccharide (LPS) prenatal injection induced the disappearance of prepulse inhibition behavior in rats [2] and in increased anxiety and decreased social interactions in mice [4]. In a previous report, we evidenced that the prenatal LPS challenge induced a clear hypofunctional state of the NMDA ⁎ Corresponding author. Fax: +33 4 67 14 42 51. E-mail address: [email protected] (G. Barbanel). 1 Present address: INSERM U. 710, EPHE, University of Montpellier II, Place E. Bataillon, 34095 Montpellier Cedex 5, France. 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.027

receptors during postnatal development in the offspring [9], without impairing the interaction of these receptors with other glutamate receptor-associated pathways [10,11]. Neonatal hypofunction of the NMDA receptor is a very well known model of schizophrenia [12], and there is also convincing evidences that later, more permanently reduced activity of NMDA receptors is implicated in control of several mood, cognition, and motor dysfunctions [13,14]. However, even though the NMDA receptors have long been considered as the most promising targets for the development of novel antipsychotic agents [15], no progress toward the validation of a specific drug has been obtained so far. The question thus arises as to other molecular signals linking the LPS challenge with the pathological processes. Because LPS itself does not cross the placental barrier in pregnant rats [16,17], involvement of proinflammatory cytokines of maternal or fetal origins has been frequently considered. In LPS-

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challenged male rats, proinflammatory cytokines increased in the peripheral circulation [18] and the time courses of their release were similar in pregnant dams [17,19] and in the amniotic fluid, the placenta, and the choroamnion [19–21]. Circulating cytokines of the pregnant female may thus reach the fetus. However, TNFα or IL-1β occurred within the fetal rat brain only when LPS was injected into the dams either directly into the cervical muscle [22] or ip at very large doses [23]. In recent studies using 10-fold lower LPS doses, these cytokines do not appear in the fetal brain [17,19], and a significant increase in IL-1β occurred only postnatally [24]. Thus, although there is no doubt that proinflammatory cytokines participate in long-term neurodevelopmental damage, they probably do so in response to another signal(s). Oxidative stress is a possible candidate in this respect. Oxidative stress may participate in most of the hypoxic-related brain damage occurring in neonates [25], in the postnatal consequences of prenatal cocaine intoxication [26–28], and in fetal alcohol syndrome [29–31]. Oxidative stress is obviously long known to participate in the host response to LPS (see review in [32]). In adult rats, oxygen free radicals are released into the central nervous system after peripheral LPS injection [33–36]. Although oxidative stress is most often considered to result from a brain cytokine induction in response to LPS, the oxygen radical release could also precede the LPS-induced cytokine increases in the brain [37]. Whether prenatal LPS induces the release of oxygen radicals and some oxidative stress in the fetal and/or neonatal rat brain has not been reported. Furthermore, whether this putative oxidative stress would induce delayed memory process dysfunctions and NMDA receptor-associated processes has also not been documented. The present study provides evidence that a prenatal LPS challenge to the dams induces a clear oxidative stress in the brain of male fetuses. The long-term potentiation (LTP) of glutamatergic synapses in the hippocampus and spatial memory performance (for review [38]) are completely blocked in male offspring of LPS-challenged dams. Supplementation with the antioxidant N-acetylcysteine (NAC) before LPS challenge prevents the oxidative stress and completely inhibits the LPSinduced electrophysiological and behavioral impairments. Oxidative stress, through a delayed impairment of NMDA receptors, is therefore a likely candidate accounting for the lateoccurring deleterious effects of prenatal LPS insults. Materials and methods Animals Pregnant Sprague–Dawley rats (Centre d'Elevage Depré) were used throughout this study. Experimental groups included: (i) a group of control animals born from saline-injected dams; (ii) a group of animals born from LPS-treated dams (500 μg·kg− 1 ip at the 19th day of gestation); (iii) a group of rats born from saline-injected dams given NAC in their drinking water (corresponding to a daily oral intake of 500 mg·kg− 1· day− 1), from E17 up to the birth of the offspring; and (iv) a

group of rats born from LPS-treated dams given NAC in their drinking water from E17 up to the birth of the offspring. At the concentration used, the antioxidant NAC is known to cross all barriers to reach and act on the fetus [39,40]. After birth, the size of the litters was restricted to 10 pups and offspring were weaned on postnatal day 25. LPS (from Escherichia coli, serotype 055:B5) was obtained from Sigma. To avoid possible litter-specific effects, groups of at least three independent litters were used for each parameter determination (ELISA, Oxyblots, α-tocopherol content, glutathione (GSH) oxidation). In addition, an individual litter was involved in the determination of only a single parameter. The Directory Committee of the University's Center agreed on the experimental design for laboratory animals. Protein carbonylation Fetal rats were obtained from euthanized pregnant dams. Brains were quickly removed after decapitation, and the hippocampi were dissected out, weighed, snap frozen in liquid nitrogen, and stored at−80°C until analyzed. Tissues were sonicated in 2% SDS containing PMSF as protease inhibitor, boiled for 5 min, and centrifuged. Protein concentrations were determined in the supernatant using the Bio-Rad DC protein assay kit, with BSA as standard. Protein carbonylation was first evaluated using an ELISA detection of the stable dinitrophenylhydrazone products obtained after reaction of the carbonyl groups with 2,4dinitrophenylhydrazine (DNPH), modified as recently proposed [41]. Briefly summarized, the protein samples were adjusted to 5 μg·ml− 1 and incubated overnight to adsorb on the multiwell plates (Maxisorp; Nunc). After a short reaction with DNPH at room temperature, removal of the excess of reagent, and blocking of the unreacted sites on the multiwell surface, the adsorbed hydrazone products were revealed by successively reacting with a rabbit anti-DNPH antibody (Dako) and an HRPcoupled donkey anti-rabbit antibody (Amersham). Samples were then incubated with the substrate of the peroxidase (TMB), and the absorbance was measured at 450 nm. The standard curve was prepared by mixing stock solutions of oxidized and reduced BSA (obtained as described [41]) while maintaining the same protein concentration as for experimental samples. Experimental groups were compared using one-way repeatedmeasures analysis of variance (ANOVA). Protein carbonylation was further characterized by Western blot using the Oxyblot system (Chemicon) as described by the manufacturer. Chemiluminescence signals were collected by a CCD camera (Lumi-Imager; Roche Applied System) and the intensity of the bands was analyzed by the Lumi-Quant software (Roche Applied System). Each gel contained individuals (20 μg protein per lane) from several groups of animals, using an XLarge electrophoresis system allowing 31 individual samples to be evaluated in a single run. No specific increase in a particular protein band was identified in the LPS-treated animals; thus luminescence of each lane (single animal) was expressed relative to the mean obtained for appropriate controls (stimulation index in percentage) run on the same gel, yielding a relative increase in

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protein carbonylation for each animal. Due to the semiquantitative nature of the procedure used, no comparison was performed between samples run on different gels. Experimental groups were analyzed on each single gel using one-way ANOVA. HPLC measurements of hippocampal contents of α-tocopherol α-Tocopherol was extracted from fetal tissues by sonication in 300 μl of methanol containing 0.33 mg/ml of the antioxidant butylated hydroxytoluene. Extracts were centrifuged (15 min, 20,000 g, 4°C), and the supernatants were collected and stored at − 80°C until HPLC analysis. α-Tocopherol was determined using electrochemical detection as previously described [42]. By nature, the electrochemical detection does not allow one to evaluate the oxidized α-tocopherol. One-way repeated-measures ANOVA was performed to compare each group. HPLC measurements of hippocampal contents of GSH Extracts similar to those obtained for the assay of protein carbonylation (SDS 2%), but not subjected to the boiling step of the procedure, were analyzed for GSH by a reverse-phase HPLC using fluorescence detection and precolumn derivatization with the specific reagent monobromobimane (mBBr). mBBr was added to homogenates at a final concentration of 12.5 μM and the homogenates were allowed to incubate at room temperature for 30 min. Twenty-five microliters of the reaction product was injected into the HPLC system. Chromatography was accomplished on a Lichrospher C18 (250 × 4.6 mm, 5 μm) using a curvilinear gradient elution of 3 to 20% acetonitrile in 25 mM KH2PO4 (pH 4.71) at a rate of 1 ml·min− 1. Emission of mBBr derivatives was measured at 490 nm after excitation at 394 nm and the actual concentration of GSH in each sample was quantified against a standard curve of authentic GSH. GSSG, the oxidized form of GSH, was evaluated after reduction of the samples with a large amount of NaBH4 before derivatization of the sample with mBBr. The reduction step did not interact with the derivatization procedure. Experimental groups were compared using one-way repeated-measures ANOVA. Hippocampal slice preparation Hippocampal slices (350 μm) were obtained from 28-dayold animals. After decapitation, brains were quickly dissected and placed in ice-cold buffer comprising 124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM CaCl2, 2 mM MgSO4, 10 mM glucose bubbled with O2/CO2 (95%/5%). Slices were prepared with a Vibratome (VT1000S Leica) and maintained at room temperature for at least 1 h in the same buffer containing 2 mM CaCl2. This medium was used for further recordings. Extracellular recordings with microelectrode arrays Slices were transferred to a multielectrode array (MEA-60; Multichannelsystems) comprising 60 extracellular electrodes.

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The interelectrode distance was 150 μm. An individual electrode from the array could either record or evoke a synaptic potential [43]. A nylon mesh was positioned above the slice to obtain a satisfactory electrical contact between the surface of the slice and the electrode array. Slices were continually superfused with the extracellular medium bubbled with O2/CO2 (95%/5%) described above (flow rate 2 ml·min− 1) and maintained at 37°C. Picrotoxin (25 μM) was included in the perfusate to eliminate GABAA-mediated synaptic transmission. Stimulation was achieved with an external stimulator by applying biphasic current pulses to one electrode of the array. Stimulation intensity (20 to 200 μA) and duration (70 to 100 μs) were adapted to avoid multiphasic responses that could appear upon a too-strong stimulation. Field excitatory postsynaptic potentials (fEPSPs) could be recorded at the same time by the different electrodes of the array. Test frequency to evoke fEPSP was 0.066 Hz. Slices that displayed epileptic-like activity were discarded. Applying a single train of stimulation at 100 Hz for 1 s triggered LTP. An individual experiment (“n”) was performed on an individual rat. One-way ANOVA or ANCOVA was performed to compare groups or regressions, respectively. Patch-clamp electrophysiological recordings Whole-cell patch-clamp recordings were undertaken to evaluate the relative contributions of AMPA and NMDA receptors to excitatory postsynaptic currents (EPSCs) in both LPS and control rats. To this aim, slices were transferred to the recording chamber of an upright microscope (DMLFS Leica) and were continually superfused with extracellular medium bubbled with O2/CO2 (95%/5%) as described above at room temperature. Whole-cell recordings were obtained from CA1 hippocampal neurons using a “blind” approach. For this, glass microelectrodes (4–5 MΩ; resistance) filled with a solution comprising 120 mM CsMeSO3, 1 mM MgCl2, 1 mM EGTA, 5 mM N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), 5 mM Hepes (pH 7.3), and 4 mM Mgadenosine 5′-triphosphate (Mg-ATP) were used. A bipolar stimulating electrode was positioned in the Schaffer collaterals/ commissural fibers to evoke EPSCs in CA1 neurons. EPSCs were isolated by eliminating the contribution of GABA receptor-mediated synaptic events with picrotoxin (25 μM) to block GABAA receptors applied in the perfusate, GABAB receptor-mediated synaptic events being eliminated by Cs+ ions in the intraelectrode medium. Under these conditions, EPSCs recorded at a holding voltage of − 50 mV were mainly due to the AMPA component. In order to isolate the NMDA component of the EPSCs, AMPA receptors were blocked by NBQX (10 μM) and Mg2+ ions removed from the extracellular medium. The ratio AMPA/NMDA was then calculated by dividing the amplitude of the AMPA component by the amplitude of the NMDA component. EPSCs were measured with a patch-clamp amplifier (Axopatch 200 B; Axon Instruments, USA) and digitized (Digidata 1200 Interface; Axon Instruments). Signals were filtered at 1 kHz and sampled at 10 kHz. Storage and data analysis were performed with Dr. William W. Anderson's software WinLTP [44].

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Place learning in the water maze The circular pool (∅ 160 cm, h 40 cm) was arbitrarily divided by the tracking system (Videotrack; Viewpoint, Champagne-au-Mont-d'Or, France) into four quadrants. A transparent Plexiglas platform (∅ 10 cm) was immersed at the center of one quadrant during training sessions. This quadrant was termed the Training quadrant and the others Opposite, Adjacent right, and Adjacent left quadrants, during the retention session. The water temperature (24 ± 2°C), light intensity, external cues in the room, and water opacity, obtained by suspension of lime carbonate, were rigorously reproduced. Swimming was recorded using a CCD camera connected to a computer and trajectories were analyzed in terms of latencies and distances. Animals were trained to learn a fixed location of the invisible platform for 5 days. Training consisted of three swims per day with a 20-min intertrial time interval. Start positions, set at each limit between quadrants, were randomly selected for each animal. Animals were allowed to swim for a maximum of 90 s to find the platform and left on it for 30 s. On day 5, 2 h after the last swim, animals were subjected to a probe test. The platform was removed from the pool and each animal was allowed 60 s of swimming. The percentage of time spent in each quadrant was determined. The median swim duration was calculated per day and expressed as mean ± SEM for each experimental group. Latencies for male and female offspring were analyzed separately over trials using the nonparametric Friedman repeated-measures test (Fr values), comparisons between groups being made using Dunn's test. Probe test data used the Dunnett multiple comparisons test after a one-way ANOVA (F values). The level of statistical significance was p < 0.05. Results Prenatal LPS induced oxidative stress in male fetuses Protein carbonylation To demonstrate the involvement of oxidative stress in the brains of fetuses after the prenatal LPS challenge, we first evaluated the concentration of carbonylated proteins through an ELISA system and tried to characterize with an Oxyblot assay whether a specific protein carbonylation occurred. A clear timedependent profile of protein carbonylation occurred in the hippocampus of male rats after LPS injection into the dams. As soon as 1 h after the LPS injection, protein carbonyl levels very significantly increased (170 ± 9% compared to controls; n = 6, p < 0.01). Carbonylation thereafter decreased, remaining significantly enhanced compared to controls, 127 ± 7% (n = 7) and 135 ± 7% (n = 6), 2 and 4 h after the LPS injection, respectively (Fig. 1A). By contrast, LPS injection into the dams did not induce any identifiable modification of the extent of protein carbonylation in the hippocampus of female fetuses (Fig. 1C). Oxyblot analyses were performed to evaluate whether a specific increase in carbonylation could be observed on some protein target after LPS. No particular increase could be

Fig. 1. Protein carbonylation. Protein samples were obtained from the hippocampus of rats after the injection of saline or LPS into the dams. Protein carbonylation was evaluated using the immunoassay described under Materials and methods. (A) Time-course plot showing the early increase in the extent of carbonylation after LPS injection in male fetuses. (B) Example of Oxyblot detection of carbonylated proteins in samples from control or LPS male fetuses 1 h after the stimulus. (C) Time-course plot of the extent of carbonylation after LPS injection in female fetuses. Basal values amounted to 2.3 ± 0.2 nmol/mg protein for both male and female fetuses. Values are means ± SEM of six independent determinations. **p < 0.01 compared to saline-injected controls at the same time.

evidenced in LPS-treated male fetuses after densitometric analysis of several gels (Fig. 1B). Due to the semiquantitative nature of the Western blots, no attempts were made to quantify the time course of the carbonylation increase after LPS using Oxyblots. However, running on the same gel several samples from all groups allowed us to confirm that the increase in carbonylation occurred only in proteins extracted from the hippocampus of male and not female fetuses. α-Tocopherol content Under normal conditions, the toxicity of oxygen free radicals is prevented by a number of antiradical molecules, among them α-tocopherol, the major antioxidant in the brain [45]. To confirm the oxidative stress in the fetal hippocampus, we next evaluated the amount of unoxidized α-tocopherol remaining after the LPS challenge. In the hippocampus of control fetuses (E19), α-tocopherol was easily detected (Fig. 2A), and its concentration was similar in males (3.39 ± 0.46 pmol/mg protein; n = 9) and females (2.78 ± 0.24 pmol/mg protein;

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1 nmol/mg protein, n = 8; p < 0.05) fetuses 16 h after saline injection, GSSG levels remaining constant (Fig. 3A). This resulted in a large twofold increase in the GSH/GSSG ratio at this time point (94 ± 14 nmol/mg protein, n = 6, vs 44 ± 4 nmol/ mg protein, n = 8; p < 0.05 for males and 80 ± 4 nmol/mg protein, n = 10, vs 50 ± 1 nmol/mg protein, n = 8; p < 0.05 for females). LPS injections into the dams did not affect the time course of glutathione parameters up to 8 h postinjection (not shown), but clearly modulated both the GSH and the GSSG levels in male fetuses when analyzed 16 h postinjection, resulting in an highly significant almost 40% reduction in the GSH/GSSG ratio (56 ± 4%, n = 8, vs 94 ± 14%, n = 8; p < 0.01) (Fig. 3B).

Fig. 2. α-Tocopherol. (A) HPLC chromatograms of biological samples extracted from the hippocampus of control or LPS male fetuses, showing the correspondence with standard α-tocopherol. (B) Modification of the αtocopherol content 4 h after LPS infusion into the dams. Whereas α-tocopherol decreased in male fetuses, no modification occurred in females. Values are means ± SEM of 9 to 16 independent determinations. **p < 0.01 compared to saline-injected controls.

n = 11). No modification of this content occurred up to 4 h after saline infusion, but a small and significant increase occurred 24 h later, in both male (3.86 ± 0.18 pmol/mg protein; n = 8; p < 0.05) and female fetuses (3.57 ± 0.27 pmol/mg protein; n = 8; p < 0.05). α-Tocopherol levels remained constant up to 2 h after the LPS (not shown) but dropped significantly more than 40%, 4 h afterward, in male fetuses (2.31 ± 0.35 pmol/mg protein; n = 16; p < 0.05) (Fig. 2B). LPS injection into the dams did not induce any significant modification of the α-tocopherol content in the hippocampus of female fetuses (Fig. 2B). GSH content Glutathione oxidation is another mechanism protecting the cell from the deleterious effects of oxygen free radical release, and the ratio between the reduced (GSH) form and its oxidized disulfide adduct is often considered as an index of oxidative stress. We thus evaluated both GSH and GSSG levels and the GSH/GSSG ratio in the hippocampal tissues obtained from fetuses after LPS challenge of the dams. Whereas no obvious major change could be evidenced either in male or in female control fetuses up to 8 h after saline, GSH levels significantly rose in the hippocampi of both male (20 ± 2 nmol/mg protein, n = 6, vs 12 ± 1 nmol/mg protein, n = 8; p < 0.05) and female (17 ± 1 nmol/mg protein, n = 10, vs 12 ±

Fig. 3. GSH. The hippocampi were dissected out from fetuses and processed as described under Materials and methods to evaluate the levels of GSH and GSSG and the GSH/GSSG ratio. (A) Comparison of the GSH status a few minutes after saline injection into the dams and 16 h thereafter in male and female control fetuses. The GSH levels like the GSH/GSSG ratio increased after 16 h in control animals of both genders. *p < 0.05 compared to the initial (0 h) value observed in fetuses of the same gender; **p < 0.01 compared to the initial (0 h) value observed in fetuses of the same gender. (B) Modification of the GSH/GSSG ratio 16 h after LPS infusion into the dams in males or in females. Whereas the GSH/ GSSG ratio decreased in male fetuses, no modification occurred in females. **p < 0.01 compared to saline-injected controls. Values are means ± SEM of 6 to 10 independent determinations.

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By contrast, LPS injection into the dams did not induce any significant modification of the glutathione parameters in the hippocampus of female fetuses at the same time point (Fig. 3B). Prenatal LPS induced inhibition of NMDA receptor-dependent processes in male offspring The glutamate receptor system is known to be a major signaling pathway for neuronal migration during brain development [46]. As the animal matures, the activation of the NMDA receptors results in excitatory synaptic transmission and in the activity-dependent synaptic plasticity underlying learning and memory. The overall result of these mechanisms is best illustrated in the complex phenomenon of LTP in which longlasting changes in the excitability of several associated neurons occurred as a result of repeated release of L-glutamate and activation of the NMDA receptors. Because the prenatal LPS

induced a hypofunction of the NMDA receptors in the striatum of postnatal rats [9], we hypothesized that the same prenatal challenge would be able to affect the NMDA receptorassociated synaptic transmission and plasticity in mature animals. Electrophysiology We first investigated whether prenatal LPS treatment could alter the input/output (I/O) relationship in control and LPS male rats. fEPSP amplitude obtained for different stimulus intensities was plotted against the corresponding presynaptic fiber volley amplitude. A linear regression was obtained for both control and LPS rats. The slopes of the I/O curves were 2.07 and 1.95, with coefficients of determination (R2) of 0.99 and 0.98, respectively (Fig. 4A). Both correlation curves were not significantly different (ANCOVA, F1,28 = 0.08, p > 0.05), indicating that basal synaptic transmission was unaltered in LPS rats. We next

Fig. 4. Basal synaptic transmission and short-term and long-term synaptic plasticity in CA1 area of male LPS rats. (A) Input/output curves in slices from LPS (n = 5) and control (n = 4) rats, showing a normal basal synaptic transmission in LPS-treated rats. Each point represents a single measurement of fiber volley and fEPSP amplitude. (B) Paired-pulse facilitation ratio (PPF) is not altered in LPS rats. PPF was obtained by delivering two stimuli with different interpulse intervals. PPF was calculated by dividing the second peak amplitude by the first. Data are means (±SEM) of four individual experiments. (C) CA1 LTP is greatly compromised in LPS rats (n = 6) relative to controls (n = 6). (D) fEPSP amplitude measured 50 min after the high-frequency stimulation in both male and female offspring. Data are means ± SEM and are expressed as percentages of control fEPSP amplitude. **p < 0.01 compared to respective saline-injected controls.

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examined whether short-term synaptic plasticity was modified by prenatal LPS injection. To this aim, paired-pulse facilitation ratio was measured by applying two consecutive stimuli with different interpulse intervals ranging from 25 to 400 ms. No significant difference between control and LPS rats could be evidenced at any interpulse interval (F1,28 = 0.8, p > 0.05; Fig. 4B). High-frequency stimulation (100 Hz, 1 s)-induced LTP was studied in the CA1 area of control and LPS rats (Fig. 4C). In control male rats, the fEPSP amplitude measured 50 min after the high-frequency stimulation was 200 ± 10% of basal fEPSP amplitude. In male LPS rats, the fEPSP amplitude measured at the same time was strongly decreased (123 ± 6% of basal fEPSP amplitude) (Fig. 4D). We next examined the gender dependency of synaptic transmission and plasticity. Basal synaptic transmission was similar both in males (299 ± 11 μV, n = 5) and in females (287 ± 32 μV; n = 3). The LTP was also similar in both control and LPS female rats. In contrast to the males, fEPSP amplitude measured 50 min after the high-frequency stimulation was similar in control females (158 ± 14%; n = 3) and in LPS females (156 ± 20%; n = 3) (Fig. 4D). Impairment of the LTP in male LPS offspring may result from a direct alteration of synaptic NMDA receptors. We thus explored whether there was a decrease in the number of NMDA receptors or whether oxidation, known to be a strong inhibitor of these receptors [47], was induced in LPS male rats. To this aim, we first examined the contributions of AMPA and NMDA receptors to basal excitatory synaptic transmission, using whole-cell patch-clamp recording. EPSCs in CA1 neurons, evoked by afferent stimulation of the Schaffer collateral pathway, from controls and LPS animals are shown in Fig. 5A. On average, the AMPA component of the synaptic current (representative of the contribution of AMPA receptors to the synaptic transmission) was similar in both control and LPS rats (106 ± 30 pA, n = 3, in control males vs 168 ± 35 pA, n = 5, in LPS males). However, the NMDA component was largely reduced in LPS animals as shown by the very significant increase in the ratio AMPA/ NMDA (0.96 ± 0.09, n = 3, vs 3.55 ± 1.16, n = 4; p < 0.01) (Fig. 5A). We next evaluated whether this decrease in the contribution of NMDA receptors to basal synaptic transmission could be associated with an enhanced oxidization of these receptors. To test this hypothesis, we measured the action of DTNB, a reagent currently used to oxidize NMDA receptors [47–49]. In fact, this compound induced a comparable decrease in basal NMDA EPSC in both control and LPS rats (22 ± 2.6% of inhibition, n = 4, vs 29 ± 3.9% of inhibition, n = 5) (Fig. 5B). This tends to indicate that the oxidizing status of NMDA receptors is similar in both control and LPS rats. Therefore, an enhanced oxidization of NMDA receptors does not explain the deficit in LTP observed in LPS animals. In order to corroborate this, we investigated whether DTT, a reducing agent also used to manipulate NMDA receptor oxidizing status [47–49], could reverse LTP impairment in LPS rats. We observed that DTT (1 mM) application failed to reverse LTP deficit in LPS rats (Fig. 5C).

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Behavioral experiments As mentioned earlier, the excitatory synaptic transmission and the activity-dependent synaptic plasticity in the hippocampus underlie the processes of learning and memory. Having demonstrated a delayed effect of prenatal LPS challenge upon synaptic plasticity in the hippocampus of maturing male rats, we next investigated whether the prenatal LPS also impaired some memory processes. We chose to evaluate spatial learning in the water maze [50], as this process is frequently associated with CA1 hippocampal LTP [38]. Offspring rats were trained to locate a fixed platform position in the water maze. Control and LPS male rats showed acquisition profile differences. Latencies spent to find the platform decreased over the course of acquisition training in both saline (Fr(4,29) = 11.8, p < 0.05) and LPS groups (Fr(4,29) = 10.5, p < 0.05) (Fig. 6A). However, latencies measured for each training day were significantly higher in LPS rats compared to saline control rats (Fig. 6A). A probe-test analysis confirmed that saline animals learned the platform location, because they spent significantly more time in the Training (T) quadrant compared to the other quadrants (F(3,20) = 67.4, p < 0.0001). However, LPS male animals failed to preferentially explore the T quadrant and no statistically significant difference in time spent over quadrants was measured (F(3,20) = 2.94, p > 0.05) (Fig. 6B). Under the same experimental conditions, control and LPS female rats failed to show any difference in spatial learning. Latencies decreased over the course of acquisition training in both saline (Fr(4,29) = 16.7, p < 0.01) and LPS groups (Fr(4,29) = 13.1, p < 0.05). No difference in latencies was measured during training (Fig. 6C). The probe-test analysis confirmed that both groups learned the platform location and showed a preferential exploration of the T quadrant (F(3,20) = 44.0, p < 0.0001 in the saline group; F(3,20) = 78.9, p < 0.0001 in the LPS group) (Fig. 6D). The LPS-induced inhibition of NMDA receptor-dependent processes in male offspring is linked to the LPS-induced oxidative stress in the brain of male fetuses We next evaluated the ability of NAC to reduce the oxidative stress in the hippocampus of male fetuses. NAC is a free radical scavenger, able to directly react with several oxygen-derived radicals [51] and to increase the cellular pool of glutathione, a major cellular antioxidant that decreases during hypoxic [52] or endotoxic [53] shock. NAC was chosen as it was reported to cross both enteric and placental barriers [39,40]. In the present study NAC was given to the dams in the drinking water and its presence did not significantly modify the daily liquid intake. As expected, this procedure resulted in a very significant 3fold increase in basal GSH content in the hippocampus of male fetuses at the time of the saline or LPS injections. GSSG levels also increased 1.5-fold, resulting in an overall 2-fold increase in GSH/GSSG levels, irrespective of the gender of the animal (Fig. 7). Using our present conditions, prenatal NAC treatment did not have any effect on the extent of protein carbonylation in

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Fig. 5. Effect of prenatal LPS on NMDA synaptic responses in male rats. (A) The contribution of NMDA receptors to basal synaptic transmission was examined in CA1 neurons of LPS male rats. For this purpose, the EPSCs due to NMDA receptor activation (EPSCNMDA) were isolated by blocking AMPA receptor-mediated responses with the AMPA receptor antagonist NBQX (10 μM) in a Mg2+-free medium containing 25 μM picrotoxin to block GABAA receptor-mediated EPSCs. The ratio AMPA/NMDA was further calculated by dividing the amplitude of the EPSCs measured in normal extracellular medium by the amplitude of the IPSCs measured in Mg2+-free medium containing NBQX. Experiments were carried out in neurons voltage-clamped at − 50 mV. On the left, representative traces obtained from experiments performed in control and LPS rats are shown. On the right, averages of pooled AMPA/NMDA ratios are displayed. Data are means ± SEM. **p < 0.01 compared to AMPA/NMDA ratio obtained in control rats. (B) The effects of the oxidizing agent DTNB on EPSCNMDA were tested in control and LPS rats. For this purpose, EPSCNMDA were isolated as described above and DTNB (100 μM) was applied for 15 min. Illustrative traces extracted from experiments performed in control and LPS rats are shown on the left. On the right, a recapitulative graph plotting the levels of inhibition induced by DTNB is shown. Data are averages of pooled levels of inhibition calculated by normalizing EPSCNMDA amplitude in the presence of DTNB to basal EPSCNMDA amplitude (control). They are expressed as means ± SEM. (C) The effects of the reducing agent dithiothreitol (DTT) on NMDA receptor-dependent LTP were tested in LPS rats. To this aim, DTT (1 mM) was applied for 10 min in slices prepared from LPS rats after a first LTP induction protocol (100 Hz for 1 s, as indicated by the arrow) was delivered. After the DTT was washed off, a second induction protocol (second arrow) was applied. On the graph, data are expressed as percentages of fEPSP measured under basal synaptic transmission before highfrequency stimulation. The graph is illustrative of at least three independent experiments.

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Fig. 6. Performance of rats exposed in utero to LPS or saline solution in the water-maze test. Acquisition profiles and probe test performances for (A, B) male offspring rats or (C, D) female offspring. Probe test: 1 h after the last swim trial, the platform was removed and animals were subjected to a 60-s swim. The presence in each quadrant was measured. Quadrants: T, training; AL, adjacent left; O, opposite; AR, adjacent right. The number of animals was n = 6 per group. *p < 0.05, **p < 0.01 vs the control group, ++p < 0.01 vs time spent in the T quadrant for the same experimental group; Dunn's test in (A, C) and Dunnett's test in (B, D).

male controls. However, NAC pretreatment completely prevented the LPS-induced increase in male fetuses (107 ± 4%, 4 h after the LPS; n = 5) (Fig. 8A). The prenatal NAC treatment also prevented the LPS-induced enhancement of carbonylation observed in males using the Oxyblot analysis (not shown). Similarly, NAC pretreatment completely prevented the LPSinduced decrease in α-tocopherol content (3.25 ± 0.21 pmol/mg protein, 4 h after the LPS, n = 6; p < 0.01) in the hippocampus of male fetuses (Fig. 8B). NAC-induced enhancements of both the GSH and the GSSG levels were still observed 16 h after saline injection in male fetuses, indistinguishable from the basal levels, resulting in a similar GSH/GSSG ratio. NAC pretreatment completely prevented all GSH or GSSG variations observed in male rats 16 h after the LPS injection into the dams and, more interestingly, prevented the LPS-induced decrease of the GSH/GSSG ratio in male fetuses (119 ± 11%, 16 h after the LPS, n = 8) (Fig. 8C).

NAC pretreatment did not induce any effect on protein carbonylation or on the α-tocopherol levels in the hippocampus of female fetuses, whether obtained from saline or LPSchallenged dams, at any time point tested. On the other hand, basal levels of GSH and GSSG in female fetuses and their ratio brought about by the NAC pretreatment were very similar to those observed in male fetuses (Fig. 7). No further changes could be detected 16 h after the injection of either saline or LPS (not shown). NAC pretreatment did not have any effect on the LTP of saline-injected controls, but completely prevented the LPSinduced inhibition of the LTP in male rats (fEPSP measured 50 min after the high-frequency stimulation was 225 ± 27% of basal fEPSP amplitude, n = 6) (Figs. 9A and 9B). Finally, as concerned the spatial memory tests, NAC treatment failed to affect the acquisition profile of control animals (Fr(4,29) = 16.8, p < 0.01 in NAC-treated saline group, vs Fr(4,29) = 19.1, p < 0.001 for H2O-treated saline group) (Fig. 9C). By contrast,

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Fig. 7. NAC-induced changes in GSH redox status in male and female fetuses. Pretreatment of the dams with NAC at 500 mg·kg− 1·day− 1 in their drinking water for 2 days promoted an increase in the GSH and GSSG levels in the hippocampus of E19 male and female fetuses and an increase in the GSH/GSSG ratio. Values are means ± SEM of six independent determinations. **p < 0.01 compared to the value observed at the same time point in fetuses of the same gender obtained from dams given only water as drink.

NAC treatment allowed LPS animals to show a correct acquisition profile (Fr(4,29) = 18.7, p < 0.001 for NAC-treated LPS group, vs Fr(4,29) = 4.5, p > 0.05 for H2O-treated LPS group) (Fig. 9C). Indeed, significant differences were observed in the latencies measured during trials 3–5 between NACdrinking and H2O-drinking LPS animals. The probe test confirmed this beneficial effect of the NAC treatment, because the time spent in the T quadrant measured in NAC-treated LPS rats returned to normal (F(3,20) = 22.2, p < 0.0001) (Fig. 9D). Discussion The four major findings of this study can be summarized as follows: (i) an LPS challenge to the dams during late gestation

generated a transient oxidative stress in the hippocampus of fetuses, (ii) this immune challenge produced a delayed impairment of synaptic plasticity and spatial memory processes in offspring, (iii) a prenatal antioxidant treatment reversed all these impairments, and (iv) all these effects specifically occurred in male, not in female, offspring. The release of oxygen radicals is first illustrated by the enhanced carbonylation of proteins in the hippocampus of male fetuses, as early as 1 h postinjection of LPS into the dams. The origin of this oxidative stress in the rat fetus is uncertain, but may involve a nonspecific response to LPS in the pregnant dam. Systemic infection in the sheep indeed results in microcirculation failures in the uterus [54] and impaired the oxygen delivery to the fetus [55]. This could result in a drop in the pH of the fetal blood [56], inducing the release of free iron from its protein stores [57]. In a recent study in newborn piglets, non-proteinbound iron was indeed released after a severe hypoxic shock [58]. Free iron entering the Fenton reaction would thus induce the oxidative stress. On the other hand, more specific routes, or at least some additional mechanisms, must occur under our conditions, as nonspecific mechanisms could not explain why the increase in carbonylation occurred only in male fetuses and not in females. The release of oxygen radicals is further substantiated by the decrease in α-tocopherol content in male fetuses after the LPS challenge. The concentration of α-tocopherol in the fetal brain depends exclusively on maternal diet. The decrease we observed results either from a disturbed transfer of maternal α-tocopherol after LPS (itself resulting from a decrease in maternal circulating levels of α-tocopherol or from a reduced trans-placental transfer) or from the burning up of fetal α-tocopherol. The fetal supply of α-tocopherol seems very robust and a 2-week vitamin E-deficient diet of the mothers is necessary to yield only a small 20% decrease in α-tocopherol levels in the fetal brain [59]. A possible decreased availability of α-tocopherol, restricted to the 4 h of experiment seems thus very unlikely under our acute conditions. Even so, if unavailability of α-tocopherol

Fig. 8. NAC-induced reversal of oxidative stress markers in male fetuses. (A) Pretreatment of the dams with NAC at 500 mg·kg− 1·day− 1 in their drinking water 2 days before LPS injection blunted the increase in carbonylation observed in males 4 h after LPS. (B) Pretreatment of the dams with NAC blunted the decrease in αtocopherol occurring in male fetuses 4 h after LPS. (C) Pretreatment of the dams with NAC blunted the decrease in the GSH/GSSG ratio in males 16 h after LPS. Values are means ± SEM of at least six independent determinations. **p < 0.01 compared to saline-injected controls at the same time point.

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Fig. 9. NAC-induced reversal of the NMDA receptor-dependent correlates in male offspring. (A) NAC pretreatment (n = 5) allowed the restoration of a normal LTP at CA1 synapses (compare with Fig. 3). (B) fEPSP amplitude measured 50 min after the high-frequency stimulation. Data are means (±SEM) and are expressed as percentages of control fEPSP amplitude. **p < 0.01 compared to respective saline-injected controls. (C) Acquisition profile in the water-maze test in LPS male offspring after NAC pretreatment was indistinguishable from that of control animals. **p < 0.01 vs the control saline-treated group, ##p < 0.01 vs the H2O-treated LPS group. (D) Probe test after NAC pretreatment. Only the Training (T) quadrant is represented. **p < 0.01 vs the control saline-treated group.

from the mother's side was involved after LPS injection, complex mechanisms would be necessary to explain why the decrease in brain α-tocopherol occurred only in male and not in female fetuses and why the supplementation with the antioxidant NAC normalized this decrease. Thus the decrease in αtocopherol content most likely reflects the oxidative stress occurring in the hippocampus of male fetuses after the prenatal LPS challenge. Like α-tocopherol, GSH is a major antioxidant in the brain. In the present study, we demonstrated a GSH/GSSG ratio decrease in male fetuses, but not in female fetuses, 16 h postLPS challenge, i.e., with a delay similar to that already reported for maternal and fetal hepatic GSH [39]. Evaluating the GSH/ GSSG ratio instead of the GSH levels proved to be more reliable in the present study, as we observed an unexpected increase in both GSH and GSSG levels at this time point. At the present time, we do not have any clear explanation for that effect. However, because it was also observed in control fetuses, this rise does not relate to the LPS challenge itself. Most likely, this

late rise in antioxidant capacity may correspond to a normal adaptive mechanism preparing the fetal (male or female) brain a few hours before delivery, the most important change in oxygen supply that the brain will normally assume. An increase in αtocopherol content was similarly observed in both male and female fetuses 24 h after saline or LPS injections (not shown). Nevertheless, whatever the mechanisms of GSH regulation involved, prior supplementation with the antioxidant and GSH precursor NAC completely blunted the decrease in the GSH/ GSSG ratio in male fetuses, again supporting the involvement of an LPS-induced oxidative stress in male fetal hippocampus. In a previous study, we reported that a prenatal LPS challenge induced a definite hypofunction of the NMDA receptors [9], a perturbation that, according to pharmacological evidences [12,60], may be responsible for the behavioral impairments already reported [2]. We thus explored whether the LPS-induced oxidative stress in the fetal hippocampus indeed contributed to the damages induced in the offspring. To this aim, we evaluated two physiological parameters, namely the long-

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term potentiation of synaptic transmission in the hippocampus [61] and the spatial learning in the water maze [50], which strongly rely on NMDA receptor activation. As expected, both the LTP and the spatial learning performance were almost fully blunted in male offspring from LPS-treated dams, but not in female offspring. Next, to substantiate the possible link between oxidative stress in the fetal brain and impairment of hippocampal functions, we pretreated the dams with NAC. The in vivo protective effect of NAC has been repeatedly reported [39,40,62]. In the present study, a simple tapwater treatment of the dams with NAC clearly (i) increased the GSH levels in the hippocampus of the fetuses; (ii) prevented altogether the increase in protein carbonylation, the decrease in α-tocopherol levels, and the decrease in the GSH/GSSG ratio in the hippocampus of male fetuses; and (iii) obliterated both the LPS-induced LTP inhibition and the LPS-induced memory process impairments. Together with the results of our previous study [9], the present data therefore point to a clear link between prenatal oxidative stress and delayed postnatal inactivation of the NMDA receptors, impairing both the synaptic plasticity and the spatial memory in maturing animals. A series of additional electrophysiological explorations further indicated that the inactivation of the synaptic NMDA receptors in the hippocampus of male offspring most likely arise from a reduction in the number of these receptors at the synapse, and not from their possible inactivation by oxidation. Several mechanisms may drive the link between prenatal oxidative stress and the postnatal effect on NMDA receptors. A first possibility emerges from the present study. α-Tocopherol is known to specifically interact with enzymes, structural proteins, lipids, and transcription factors, resulting in a number of cellular functions that are independent of its antioxidant or radical scavenging abilities [45]. We furthermore recently reported that α-tocopherol also protected neurons against free radical toxicity through an unexpected genomic action [42]. The amount of available α-tocopherol temporarily decreases after prenatal LPS and this unavailability during a decisive period of development could, in itself, constitute a significant detrimental mechanism. Similarly, the glutathione deficit we observed, if occurring permanently, or at least extending beyond birth up to the first week of life, could alter the NMDA synaptic plasticity in the offspring. Impairment of GSH synthesis during the early postnatal period indeed resulted in a definite NMDA receptor hypofunction in more mature CA1 neurons, also evidenced by a clear impairment of LTP [49]. However, at variance with this study demonstrating that the NMDA receptors were more extensively oxidized in the GSH impaired animals, the redox status was very similar between LPS and control male offspring in the present study. Alternatively, oxidative stress may promote a number of specific and nonexclusive deregulations. Among them, neuronal migration is a possible target of the LPS-induced oxidative stress. Migration starts around E18 and proceeds up to E20 in the rat [63]. Neuronal migration is an essential but complex process, which may be affected by a number of environmental factors, among which glutamate and the NMDA receptor play a

key role [64]. Oxidative stress after prenatal LPS might thus impair the normal migration of neurons during corticogenesis. A similar malfunction of glutamate-mediated chemotaxis has been described in an in vitro model of Down syndrome, a normal migration being furthermore reestablished by antioxidants such as NAC or dithiothreitol [65]. Future studies are required to evaluate whether the NMDA receptors themselves in the fetus are a possible target for the oxidative processes. There are finally a number of other possibilities for fetal oxidative stress to interact with the postnatal development of the brain. Reactive oxygen species mediate the induction of proinflammatory cytokines in microglia [66]. Thus, the prenatal LPS-induced oxidative stress, associated with a misregulation of proinflammatory cytokines during the first week of life, may participate in the neonatal brain inflammation resulting in an enhanced cell death compared to nonstressed animals [24]. It should next be pointed out that other prenatal stresses, possibly unrelated to oxidative stress, may also result in a delayed inhibition of NMDA receptor-associated signaling. Indeed, severe immobilization stress to pregnant rats produced deficits in spatial learning [67] and in LTP [68] in the offspring, together with a remodeling of NMDA receptors, with a relative decrease in the NR2B and increase in the NR2A subunits. The consequences of prenatal LPS on the development and on the relative abundance of NR2A and NR2B subunits in the offspring have not been evaluated. However, at variance with the consequences of immobilization stress that resulted in learning deficits more pronounced in female than in male offspring [69], the present stress generated opposite gender differences, the learning deficits being more pronounced in male than in female offspring. Finally, the gender selectivity of the responses to prenatal LPS challenge is somewhat unexpected. The LTP inhibition and deficits in spatial reference memory are indeed observed only in males and perfectly correlate with the gender selectivity of the increase in protein carbonylation and of the decrease in αtocopherol. The sex hormone environment might possibly account for these differences. Earlier studies indeed reported that circulating levels of estradiol, which is an efficient antioxidant, differ between male and female fetuses [70]. The difference seems rather small (20%), but nothing is known about possible gender differences in the free fraction of the steroid or in its occurrence in the brain of fetuses. This gender selectivity seems, however, unique among all prenatal stress paradigms. Indeed, sustained hypoxic episodes during gestation can produce white-matter damage without any gender selectivity [71]. Restraint stress applied between E17 and E19 gives rise to neuronal damage associated with oxidative stress in mature female but not in male offspring [72] and to behavioral deficits more pronounced in female than in male offspring [69]. In utero cocaine exposure, which induced an attenuation of uterine blood flow in the rat [73], also induced an oxidative stress in young male adults [28]. However, the cocaine-induced oxidative stress in the fetus [26], as well as memory deficits [27], impairment of LTP (M. Vignes, unpublished observation) in rats, and behavioral alterations in

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rabbits [74], were not all gender-specific. In human beings, a number of psychiatric issues have been linked to perturbations occurring during gestation. Among them, the risk of schizophrenia associated with preeclampsia was greater in males than in females [75]. Summing up, the present study demonstrates that LPS challenge in pregnant dams results in a transient oxidative stress in the hippocampus of fetuses and in delayed impairments of both CA1 LTP and spatial recognition in the offspring. All these effects are gender-dependent, occurring only in males, and may be reversed by a simple antiradical treatment. Together with the recent reports that prenatal oxidative stress may be involved in the etiology of psychiatric issues [49,76], our results open up new possibilities to explore novel therapeutic and/or preventive strategies.

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Acknowledgments F.L. and J.M. are recipients of fellowships from the Fondation pour la Recherche Médicale.

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