Introduction

Schizophrenia is a severe and complex psychiatric disease affecting ca. 1% of the general population which commonly presents in late adolescence and early adulthood. It is a heterogeneous disease with a wide spectrum of different symptoms, including positive (e.g., delusions, hallucinations) and negative (e.g., social withdrawal, anhedonia) symptoms and cognitive deficits (e.g., dysfunction of attention, working memory, and executive functions) [1]. The risk of schizophrenia development depends on genetic traits and environmental factors [2]. Although the disease exhibits high heritability [3, 4], the pattern of inheritance is non-Mendelian. The etiology of schizophrenia remains unclear, but it is commonly believed that abnormalities in the operation of genetic and environmental factors during early postnatal development initiate significant structural and functional changes in the brain [2, 5] that result in the manifestation of the typical schizophrenia symptoms in the late adolescence and/or early adulthood [6]. However, in spite of decades of intensive studies, the pathomechanism of schizophrenia still remains a mystery. On the other hand, the increasing clinical and experimental evidence suggests that oxidative stress, impaired redox regulation, N-methyl-D-aspartate (NMDA) receptor hypofunction and neuroinflammation may be key factors in the development of this disease [7,8,9,10,11,12,13,14,15]. Interestingly, it is strongly suggested that redox imbalance and NMDA receptor hypofunction are interlinked with each other [8, 12, 13, 16]. Some experimental data indicate that dysregulation of glutathione (GSH) biosynthesis may lie at the basis of this interconnection.

The reduced GSH is the most abundant non-protein thiol that plays a crucial role in keeping the cellular redox balance [17, 18], both through non-enzymatic reactions with various reactive oxygen species (ROS), and by acting as a cofactor of glutathione peroxidase (GPx) which catalyzes the reduction of peroxides [19, 20]. It was found that a decrease in GSH content observed in the brain of patients with schizophrenia [21,22,23,24] correlates well with the severity of negative symptoms [25]. Furthermore, in some patients with schizophrenia, a decrease in the brain GSH concentration has been shown to be associated with polymorphisms in genes encoding two subunits of γ-glutamate-cysteine ligase (GCL) [26,27,28], a key enzyme catalyzing the first step of GSH biosynthesis in which the dipeptide γ-glutamyl-L-cysteine (γ-GluCys) is formed from glutamate (Glu) and cysteine (Cys). The second step of GSH biosynthesis is catalyzed by glutathione synthase (GS) which ligates glycine to γ-GluCys, thus forming GSH [29, 30].

The occurrence of schizophrenia-like symptoms has also been described in rodents in which deficits in GSH content in the brain have been induced by such compounds like diethyl maleate (DEM) that causes GSH depletion [31, 32] or L-buthionine-(S, R)-sulfoximine (BSO), that blocks the enzymatic activity of GCL [14, 33,34,35] and using genetic manipulations in genes encoding catalytic or modifier subunits of this enzyme [36,37,38,39]. Only catalytic subunit exhibits the entire enzymatic activity and is regulated by feedback inhibition by the end product GSH [40, 41]. In addition to the activity of the catalytic subunit, the GSH level in cells depends of the availability of cysteine (Cys), an amino acid that limits the rate of GSH biosynthesis.

Regarding the relationship between the cellular GSH content and the NMDA receptor activity, recent studies have shown that blockade of these receptors by model compounds, such as phencyclidine (PCP) or MK-801, commonly used to induce schizophrenia-like symptoms in rodents, leads to inhibition of GCL enzymatic activity [16, 42]. Consistently, Baxter et al. [16] reported that the decrease in the activity of this enzyme after administration of a single dose of MK-801 to 7-day-old newborn rats was associated with a very strong reduction in the the expression of mRNA for catalytic subunit of GCL, which resulted in a distinct decline in the concentration of the synthesized GSH. In turn, Radonjič et al. [42] demonstrated that PCP administered on the postnatal days p2, p6, p9, and p12 induced long-term decreases in both mRNA expression for catalytic subunit of GCL and GSH concentration in several brain regions of 70-day-old rats. The above effects indicate that the hypofunction of NMDA receptors promotes GSH depletion in the developing brain, and that the activity of NMDA receptors is closely related to the transcriptional control of the GCL catalytic subunit. Moreover, these data suggest that GSH deficiency in the developing rat brain resulting from hypoactivity of NMDA receptors may be an important factor responsible for schizophrenia-like behavior that is manifested in adulthood. In fact, repeated administration of PCP during embryonic and early postnatal period has been shown to impair neuronal development and produce schizophrenia-like symptoms [42,43,44,45,46].

Our recently published studies showed that direct inhibition of GSH biosynthesis using BSO on postnatal days p5-p16 induced a small but significant decrease in GSH content only in the prefrontal cortex (PFC) and marked changes in the concentrations of the sulfur amino acids (Cys; methionine, Met; homocysteine, Hcy), especially large increases in Cys concentration, in the PFC and hippocampus (HIP) of 16-day-old rats [14]. In these brain structures, significant changes were also found in the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), GPx, and glutathione-disulfide reductase (GR) [47]. The consequences of all biochemical alterations in the postnatal period due to the inhibition of GSH synthesis are involved the appearance of social deficits and cognitive impairment in adulthood [14, 35]. The aim of the current study was to check the extent to which the blockade of NMDA receptors induced by repeated administration of PCP during postnatal days (p2, p6, p9, p12) affects the concentration of GSH and sulfur amino acids (Cys, Met, Hcy), as well as the activity of antioxidant enzymes (SOD, GPx, GR) in the frontal cortex (FC), HIP, and striatum (STR) of 12-day-old pups, and on the manifestation of schizophrenia-like symptoms in adulthood. The results of these studies may shed new light on disturbances in the homeostasis of thiol compounds as an important factor in the action of NMDA receptor antagonists.

Materials and methods

All experimental procedures were carried out in accordance with the Act on the Protection of Animals Used for Scientific or Educational Purposes of January 21, 2005 reapproved on January 15, 2015 (published in Journal of Laws no. 23/2015 item 266, Poland), and in compliance with the Directive of the European Parliament and of the Council of Europe 2010/63/EU of 22 September 2010 on the protection of laboratory animals. The experimental protocols were approved by the Ethics Committee at the Maj Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland (permission no. 3/2018 of January 11, 2018). During the experiments, efforts were made to minimize the suffering of animals, and to reduce their numbers to the minimum required to obtain statistically reliable results (3R policy).

Animals and treatment

Pregnant Sprague-Dawley females (n = 10) were kept in individual cages under standard housing conditions; at a room temperature (21 ± 1 °C) with 40–50% humidity and under an artificial light/dark cycle (12/12; lights on from 7 am, lights off from 7 pm). Females had unrestricted access to standard breeding feed and drinking water. On the day the Sprague-Dawley pups were born (n = 124), their sex was determined (58 males, 66 females), and only males were left with their dams. To maintain an appropriate number of male pups for biochemical and behavioral studies, only 56 of the 58 born were left with their mothers. In order to standardize the mother’s influence on the development of male offspring, to females with fewer males in the litter, males from other mothers were randomly added, so that the final number of males for each female was comparable (5–6 pups/mother). The phencyclidine regimen and dose were the same as in the study by Radonjić et al. [42, 48]. On postnatal day p2, p6, p9, and p12, male pups were administered subcutaneously with phencyclidine hydrochloride (PCP dissolved in saline; 10 mg/kg, n = 28) or saline alone (NaCl 0.9%, n = 28). Before the injection of each dose of PCP, newborns were weighed and the amount of PCP was adjusted to the actual body weight. No animals died during the administration of the tested substances and during adolescence.

Biochemical experiments were carried out on 4 groups of 12-day-old pups receiving PCP (n = 16) or saline (n = 16). The first set of experiments measured the concentrations of GSH and sulfur amino acids (Cys, Met, Hcy) in brain structures, such as the FC, HIP, and STR in groups of pups treated with PCP (n = 8) or saline (n = 8). The second set of experiments determined the enzymatic activities of SOD, GPx, and GR in the same brain structures of 12-day-old pups injected with PCP (n = 8) or saline (n = 8). In both experimental sets, pups were sacrificed 4 h after the last treatment.

Behavioral tests, namely social interaction test (SIT), novel object recognition (NOR) test, and open field test (OFT) assessing the expression of schizophrenia-like symptoms were performed on 2 groups of adult rats. Male pups were treated with PCP (n = 12) or saline (n = 12) in the early days after birth, weaned on postnatal day 23, and then housed in groups of four to five per cage until adulthood (p70-p74).

Chemicals and reagents

Assay kits for the determination of enzymatic activity of SOD (Cat. No 706,002), GPx (Cat. No 703,102), and GR (Cat. No 703,202) were supplied by Cayman Chemical Company (Ann Arbor, MI, USA).

L-cysteine hydrochloride (L-Cys*HCl), glutathione (GSH), methionine (Met), homocysteine (Hcy), N-acetyl-cysteine (NAC), o-phthaldialdehyde (OPA), tris-(2-carboxyethyl)phosphine (TCEP), trichloroacetic acid (TCA), phencyclidine hydrochloride (PCP, Cat. No.: P3029) were from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA).

2-Chloro-1-methylquinolinium tetrafluoroborate (CMQT) was prepared in the Department of Environmental Chemistry, University of Łódź according to the procedure described by Bald and Głowacki [49].

Biochemical methods

Preparation of the tissue homogenates

The frozen tissue samples of the selected brain structures were weighed, and after thawing homogenized in 0.2 M phosphate buffer, pH 8.2 at the ratio 1: 10 (w/v, g/mL) for 0.5 min using an IKA-ULTRA-TURRAX T10 homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany) at an operating speed of 6000 rpm. Then, the homogenates were used for biochemical assays.

Determination of GSH and Cys content in the brain structures

Total glutathione (GSH + GSSG) and Cys concentrations in homogenates of brain structures (FC, HIP, STR) were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection based on the method described by Bald et al. [50] which was later modified by Kamińska et al. [51].

Briefly, 100 µl of a specific brain structure homogenate (FC, HIP, STR) was incubated with 7.5 µl of 0.25 M TCEP solution at room temperature for 15 min in order to reduce disulfide bonds. In the next step, 10 µl of 0.1 M CMQT was added to the homogenate, which was then vortexed and kept at a room temperature for 5 min. The mixture was acidified with 15 µl of 3 M PCA. Then, in order to remove the precipitated proteins, the mixture was centrifuged at 12,000 rpm for 10 min at 10oC. Finally, 10 µl of each collected supernatant sample was injected (using an autosampler) into the HPLC system (1220 Infinity LC system from Agilent, Waldbronn, Germany) equipped with Zorbax SB-C18 column (Agilent Technologies), a diode-array detector and controlled by OpenLAB CDS ChemStation Edition software(Rev.C.01.05, Agilent Technologies, Waldbronn, Germany). Each sample was analyzed twice. The mobile phase consisted of 0.1 M TCA adjusted with 1 M NaOH to pH = 1.6 (A) and acetonitrile (B). The flow rate was set to 1 mL/min, temperature to 25oC and the detector wavelength to 355 nm. The following gradient elution was used to separate the 2-S-quinoline derivatives of thiols from each other and from excess reagent: 0–3.5 min, 11–25% (B); 3.5–5.5 min, 25–40% (B); 5.5–9 min 40 − 11% (B).

Identification of GSH and Cys peaks was based on the comparison of retention times and spectra from a diode array detector with the corresponding set of data obtained for standard compounds in the range of 0-300 nmol/ml for GSH and 0–30 nmol/ml for Cys. Final concentrations were expressed in nmol GSH or Cys per g of tissue.

Determination of Met and Hcy levels

The HPLC method with fluorescence (FL) detection described previously by Borowczyk et al. [52] with some modifications was used to determine the content of Met and Hcy in brain structures. The method is based on an on-column derivatization with OPA.

Briefly, in order to reduce disulfide bonds, 14 µl of 0.25 M TCEP solution was added to 100 µl of homogenate at room temperature over 10 min. Then, 30 µl of 0.5 M NAC solution and 10 µl of 3 M PCA were added and the mixture was vortexed and centrifuged at 12,000 rpm at 10oC for 10 min. The collected supernatant was transferred into the HPLC system. HPLC analysis was performed on a Hewlett-Packard 1100 series system (Waldbronn, Germany) equipped with 1260 Series FL detector and controlled by HP ChemStation software (Rev. A.10.02, Hewlett-Packard Waldbronn, Germany). To perform this analysis, 5 µl of each supernatant sample was injected into the Hamilton PRP-1 column (150 nm x 4.6 nm x 5 μm). Two replicates of this analysis were performed for each supernatant sample. The mobile phase consisted of 0.01 M OPA in 0.1 M NaOH and acetonitrile in the gradient mode described earlier [52] and was applied at the flow rate 1 mL/min.

Amounts of Met and Hcy were quantified by using an FL detector at two different excitation and emission wavelengths (over 0–8 min: excitation at 348 nm and emission at 438 nm and over 8–14 min: excitation at 370 nm and emission at 480 nm). The identification of peaks was based on comparison of the retention time with data obtained for standard compounds in the range of 0–20 nmol/mL for Met and 0–7 nmol/mL for Hcy. Finally, Met and Hcy concentrations were expressed in nmoles per g tissue.

Determination of total SOD activity

SOD activity was determined by colorimetry using SOD Cayman’s kit according to the manufacturer’s instruction. SOD activity was expressed in the conventional units U/mg of protein (1U is defined as the amount of enzyme needed for 50% dismutation of O2•− radical). A SOD solution of known activity was used as a standard (0–0.05 U/mL).

Determination of GPx activity

GPx activity was assayed using the GPx Cayman’s Assay Kit. The reaction of oxidized glutathione (GSSG) formation catalyzed by GPx is coupled with the GR reaction. In GR-catalyzed reaction, GSSG is reduced to GSH at the expense of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation. A decrease in absorbance at 340 nm, which can be easily quantified spectrophotometrically is due to oxidation of NADPH to NADP+.

The measure of GPx activity in the tested samples is defined as the difference in the rate of absorbance change (∆A340/min) measured for the sample containing the enzyme, and in the rate of decrease in absorbance (∆A340/min) for the control sample.

Determination of GR activity

Determination of GR activity was carried out using the GR Cayman’s Assay Kit. GR catalyzes the reduction of GSSG to its reduced form GSH, which is accompanied by NADPH oxidation to NADP+. The oxidation of NADPH causes a decrease in absorbance at 340 nm, and is directly proportional to the GR activity in the sample.

Both GPx and GR activities were expressed in nmol/mg protein/min (nmol of NADPH oxidized to NADP+ by the enzyme during 1 min per mg of protein).

Determination of protein content

Protein content was assayed by the method described by Lowry et al. [53].

Behavioral methods

The behavioral tests used: Social Interaction Test (SIT), Novel Object Recognition (NOR) test, and Open Field Test (OFT) have been described in our previous articles [14, 35]. They were performed on the same groups of PCP or saline treated rats by experienced experimenters on consecutive days of adulthood (p70, p72 and p73).

SIT

Each measurement of social behavior between two rats was carried out during the light phase of the light/dark cycle. For this purpose, rats selected from separate housing cages were placed in a black PVC box measuring 67 cm × 57 cm × 30 cm (length × width × height). The arena was dimly illuminated with an indirect light of 18 lx. The difference in individual body weight between rats of a given pair was within 15 g. Rats of each pair were placed diagonally in opposite corners of the box. Social interactions between two rats were measured over a 10-min period as the total time spent in social behaviors, such as sniffing, genital investigation, chasing and fighting with each other. Every rat was used only once in the SIT. The number of social interactions was counted as a separate parameter. After each measurement session, the test box was cleaned with a 10% ethyl alcohol solution. SIT was conducted on day p70, and each group was represented by 12 rats (6 pairs).

NOR

After completing the SIT, on the postnatal day 71 the habituation procedure for the NOR test began. The rat was placed in an empty PVC box with dimensions: length 67 cm x width 57 cm x height 30 cm in which the floor surface was divided into 6 symmetrical sectors for 10 min to adapt it to the environment. The next day (p72), the rat was placed again in this box containing two identical objects (white tins 5 cm wide and 14 cm high) for 5 min (T1 session). One hour after T1 session, each rat was placed in the same box for 5 min (T2 session), but the box contained two different objects, i.e., the object known from the previous session, and the other new one. In both T1 and T2 sessions, for each of the two objects, the time of its exploration by the tested rat was measured separately (sniffing, touching or climbing). The recognition index was calculated for each rat [(time spent exploring the novel object– time spent exploring the familiar object)/(total time spent exploring both objects during the recognition session), and was expressed in percentages. In the NOR test, mobility was an additional parameter measured, assessed as the number of sector line crossings.

OFT

The elevated OFT is usually used to assess a rat’s exploratory activity in the open space. Briefly, a black circular platform without walls with a diameter of 1 m was divided into six symmetrical sectors and was raised 50 cm above the floor. The experimental room was dark and only the center of the platform was illuminated by a 75 W bulb placed 75 cm above it. In the first phase of the experiment, a rat was placed in the center of the platform and allowed to explore it. The exploratory behavior ambulation, peeping, and rearing in the open field were assessed for 5 min, by measuring, respectively, the time of walking, the number of sector crossings, and the number of episodes of peeping under the edge of the arena and rearing. The OFT was performed on day p73. Each group consisted of 12 rats.

Statistics

The significance of differences in the exploration of the two objects by rats in the NOR test (session T1 and T2) was assessed using a two-way analysis of variance (ANOVA) followed, by the Tukey test for post hoc comparisons, when appropriate. Statistical analysis of biochemical and other behavioral data was performed using Student’s t-test for independent samples.

The significance of differences in the body weight between the studied groups was evaluated using the repeated measures ANOVA followed by the Newman-Keuls test for post hoc comparisons, when appropriate.

A p values of 0.05 or less were considered to indicate statistical significance. The statistical analysis was done using STATISTICA 10.0 Software (Statsoft, Inc, USA).

Results

The effect of repeated PCP administration on the contents of GSH and sulfur amino acids in brain structures

Repeated PCP treatment in the early postnatal days resulted in significant decreases in the GSH concentration, measured in brain structures of 12-day-old male pups 4 h after the last dose, i.e. in the FC (t14 = 3.383, p < 0.01), HIP (t14 = 4.173, p < 0.001), and STR (t14 = 4.265, p < 0.001) compared to the control group (Fig. 1A–C).

Fig. 1
figure 1

The effects of repeated PCP administration in the early postnatal days (p2, p6, p9, p12) on the concentrations of GSH, Cys, Met, and Hcy in the frontal cortex (A), hippocampus (B), and striatum (C) of 12-day-old pups. Data in nmole/g of tissue are presented as the mean ± SEM, n = 8 for each group. Statistical analysis was carried out with the Student’s t-test for independent samples, ***p < 0.001, **p < 0.01 and *p < 0.05 vs. the control group

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Parallel to the decreases in the GSH level, the content of Cys, a rate-limiting factor in the GSH synthesis was significantly decreased in all examined brain structures (for the FC t14 = 10.248, p < 0.001; for the HIP t14 = 4.628, p < 0.001; for the STR t14 = 6.486, p < 0.001). In the PCP-treated groups, the Met concentration in the FC and HIP was at a similar level as in the control group, and only in the STR, it was significantly reduced (t14 = 5.345, p < 0.001). Hcy, which is a Met metabolite, was significantly decreased in the FC (t14 = 3.470, p < 0.01) and STR (t14 = 2.518, p < 0.05) compared to the control group, while in the HIP, only a downward trend in its content was observed (Fig. 1).

The effect of repeated PCP administration on the enzymatic activities of SOD, GPx, and GR in brain structures

The activity of antioxidant enzymes (SOD, GPx, GR) in the FC, HIP, and STR of 12-day-old Sprague–Dawley male pups, was measured in tissue homogenates, 4 h after administration of the last dose of PCP or vehicle (0.9% NaCl)(Figs. 2A–C).

Fig. 2
figure 2

The effects of repeated PCP administration in the early postnatal days (p2, p6, p9, p12) on the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-disulfide reductase (GR) in the frontal cortex (A), hippocampus (B), and striatum (C) of 12-day-old pups. SOD activity is expressed in the conventional unit U/mg of protein while GPx and GR activities are expressed in nmol/mg of protein/min. Further explanations are provided in the Material and Methods section. Data are shown as the mean ± SEM, n = 7–8 for each group. Statistical analysis was carried out with the Student’s t-test for independent samples, **p < 0.01 and *p < 0.05 vs. the control group

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Of the antioxidant enzymes studied, PCP treatment affected only SOD activity in the HIP and GR activity in the FC. In particular, SOD activity in the HIP was significantly reduced (t14 = 2.377, p < 0.05), while GR activity in the FC was significantly increased (t14 = -3.399, p < 0.01) vs. the appropriate control group.

Repeated PCP administration in the early postnatal period impairs social behavior in adulthood

Statistical analysis of social behavior in 70-day-old rats revealed that repeated administration of PCP in the early postnatal period shortened the total time spent by two rats in social interactions (t10 = 5.361, p < 0.001) and reduced the number of these interactions (t10 = 4.420, p < 0.001) in adulthood (Fig. 3A and B).

Fig. 3
figure 3

The effects of repeated PCP treatment in the early postnatal days (p2, p6, p9, p12) on social behavior deficits assessed in the SIT in 70-day-old rats as the total social interaction time expressed in seconds (A) and the number of interactions (B). Data are presented as the mean ± SEM, n = 12 (6 pairs) for each group. Statistical analysis was carried out with the Student’s t-test for independent samples, ***p < 0.001 vs. the control group

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Repeated PCP administration in the early postnatal period impairs cognitive performance in adulthood

The NOR test designed to assess cognitive impairment was performed in 72-day-old rats on the next day after SIT (Fig. 4).

Fig. 4
figure 4

The effects of repeated (PCP administration in the early postnatal days (p2, p6, p9, p12) on cognitive performance assessed in the NOR test in adult rats. The effects of PCP on: (A) exploration of two objects in the T1 session, (B) exploration of a novel and familiar object in the T2 session, (C) the recognition index, and on (D) motility of rats in T1 and T2 sessions. Data are presented as the mean ± SEM, n = 12 for each group. Statistical analysis for data presented in figures A and B was performed using two-way ANOVA; asterisks indicate the significance of difference according to the Tukey post hoc test, **p < 0.01 vs. familiar object (A). Statistical analysis for data presented in figures C, D and E was carried out with the Student’s t-test for independent samples, ***p < 0.001 vs. the control group

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A two-way ANOVA performed for the exploration time of two identical objects by each rat from PCP- or saline-treated groups during the acquisition trial (T1 session) showed a significant effect of the treatment (F1,44 = 7.669, p < 0.01), no effect of the object (F1,44 = 0.0027, p > 0.05), and no interaction between treatment and objects (F1,44 = 0.614, p > 0.05). In the T1 session both control and PCP-treated rats spent the same amount of time on exploration of two identical objects (Fig. 4A). The same analysis performed for the exploration time of two different objects (familiar and novel) by each rat from PCP or saline treated groups during the retention trial (session T2) showed no treatment effect (F1,44 = 0.263, p > 0.05), but a significant effect of the object (F1,44 = 4.505, p < 0.05), and an interaction between the treatment and object (F1,44 = 9.449, p < 0.01). Post hoc analysis showed that in the session T2, control rats explored the novel object significantly longer than the familiar one, in contrast to the PCP-treated rats which explored both objects with the same intensity (Fig. 4B).

As for the calculated value of the recognition index in the group of rats receiving PCP, it was significantly lower compared to the control group (t22 = 7.748, p < 0.0001) by 25.7% (Fig. 4C).

Regarding the motility of the tested rats, in the T1 session there was no significant differences between the PCP-pretreated and control rats in this respect, but in the T2 session motility of the PCP-pretreated rats was significantly lower than in the control group (t22 = 2.924, p < 0.01; Fig. 4D and E).

Repeated PCP administration in the early postnatal period reduces the time of walking in adulthood

On postnatal day 72, control rats and those exposed to PCP in the early postnatal period were tested for exploratory behavior (time of walking, ambulation, peeping and rearing) in the OFT over 5-minute sessions.

Among the parameters studied, only the time of walking was affected by PCP, namely, it was significantly reduced in the PCP-pretreated group vs. the control group (t22 = 2.442, p < 0.05; Fig. 5A).

Fig. 5
figure 5

The effects of repeated PCP administration in the early postnatal days (p2, p6, p9, p12) on exploratory behavior evaluated in the OFT in adult rats as (A) the time of walking expressed in seconds, (B) the number of sector crossings, (C) the number of peeping and rearing episodes. Data is shown as the mean ± SEM, n = 12 for each group. Statistical analysis was carried out with the Student’s t-test for independent samples, *p < 0.05 vs. the control group

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The effect of repeated PCP administration in the early postnatal period on body weight of Sprague-Dawley rats

The repeated measures ANOVA performed for body weight of rats treated with saline (control) or PCP (10 mg/kg sc) during postnatal days p2, p6, p9, and p12 revealed significant effects of treatment (F1,22 = 21.240, p < 0.001) and time (F5,110 = 3125.28, p < 0.0001), and the interaction of time x treatment (F5,110 = 8.7899, p < 0.0001). Post hoc comparison of the examined groups showed significantly reduced weight gain in rats receiving PCP compared with the controls, and this effect was particularly pronounced in the late adolescence and early adulthood (p60 and p70) (Table 1).

Table 1 The effect of repeated PCP (10 mg/kg s.c.) administration in the early postnatal days (p2, p6, p9, p12) on weight gain during development and at two time points in late adolescence (p60) and adulthood (p70)
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Discussion

The biochemical results obtained in our study showed that repeated injections of PCP at a dose of 10 mg/kg to Sprague-Dawley pups on postnatal days p2, p6, p9, p12, according to the schedule previously described by Radonjić et al. [42, 48], significantly decreased the contents of both GSH and sulfur amino acid Cys in the FC, HIP, and STR of 12-day-old rats. i.e. in the brain structures involved in the manifestation of schizophrenia-like behaviors in adulthood. Interestingly, the decreases in GSH concentration in these structures amounting to 5.4% in the FC, 10.1% in the HIP, and 11.6% in the STR were much smaller than the decreases in Cys concentrations in these structures: by 30.2% in the FC, by 12.6% in the HIP, and by 24.2% in the STR, although Cys availability is a primary factor limiting GSH synthesis [29, 54]. Compared to the PCP-mediated blockade of NMDA receptor, direct inhibition of the GSH-synthesizing enzyme (GCL) by BSO resulted in only a slight 7% decrease in GSH concentration in the PFC of 16-day-old rats, whereas Cys levels increased significantly in the PFC by 47.5% and in the HIP by 34.9% with no changes in the STR [14, 47]. This comparative analysis shows that in these two models of schizophrenia, a different way of modulating GSH synthesis in the early days of postnatal life leads to opposite changes in Cys concentrations in specific brain structures. These results seem to suggest potential differences in further pathways of metabolism of this amino acid.

In general, Cys is a semi-essential proteinogenic amino acid supplied in food and also produced endogenously via the transsulfuration pathway from homocysteine (Hcy), which is formed during the metabolism of Met [30, 55, 56]. Cys, in addition to being a substrate for GSH synthesis, is also a substrate for the endogenous production of hydrogen sulfide (H2S), which is an important regulator of the redox state and has cytoprotective and neuromodulatory properties [57, 58]. Physiological concentrations of H2S and its metabolites enhance the function of NMDA receptors and facilitate the induction of long-term potentiation (LTP) in the HIP [57, 59]. In patients with schizophrenia, both decreases [60] and increases [61, 62] in H2S concentration were found, which correlated well with the manifestation of positive and negative symptoms assessed according to appropriate diagnostic scales [60, 63]. However, the effect of H2S on the expression of pathological changes is unclear.

In the PCP-induced model of schizophrenia presented in this study, the level of H2S was not determined in the examined brain structures. Therefore, we can only speculate that the decreases in Cys content, on the one hand, may be a factor limiting production of H2S, and on the other, they may result from the use of Cys for the production of H2S. The latter assumption seems more likely. Assuming this alternative, the further action of H2S by adding sulfur to Cys residues at the redox site of the NMDA receptor [64, 65] can be considered as a mechanism attempting to overcome the lack of stimulation of this receptor by glutamate due to the inhibition of its activity by PCP. Unlike the PCP model, in the model induced by the BSO + GBR 12,909 combination, in 16-day-old rats, despite the high concentration of Cys, a significant decrease in H2S synthesis was observed in the HIP, while in adulthood, in this brain structure, an increase in the concentration of H2S metabolite, i.e. bound sulfane sulfur was found, while the level of free H2S remained the same as in the control [66]. This increase in the concentration of bound sulfane sulfur in the HIP of adult BSO + GBR 12,909 model rats coincided with an intensification of locomotor activity, while its attenuation after administration of N-acetylcysteine coincided with a decrease in the level of bound sulfane sulfur [66]. The above data indicate an important role of anaerobic Cys metabolites in the expression of some symptoms of schizophrenia and their modulation in the therapy.

On the other hand, it is also worth mentioning that Cys is an essential amino acid for the proteins synthesis, correct folding and stabilization of native, physiologically active conformations [67]. Therefore, it can be assumed that significant reduction in the Cys content in the brain structures of rats receiving PCP in the early postnatal period, impairing the synthesis of various proteins, may lead to structural and functional abnormalities. Perhaps the reduction in weight gain over time, observed in PCP-treated rats in our study, is a consequence of Cys deficiency in the early postnatal period.

In addition to lowering the Cys levels in the examined brain structures of 12-day-old pups, PCP treatment also reduced the levels of Met in the STR, and Hcy in the FC and STR. Met, like Cys, is a sulfur amino acid supplied by the diet and also endogenously synthesized via the re-methylation route from Hcy [68, 69]. The first metabolite formed during the conversion of Met to Hcy is S-adenosylmethionine (SAM) [55], which serves as the principal donor of methyl groups for different methyltransferases [70, 71], including catechol-O-methyltransferase (COMT) that metabolizes the extracellular DA to 3-methoxytyramine (3-MT). Chronic PCP treatment induces excessive release of DA in the STR [72], but significantly reduces it in the PFC [73]. Hence, in our study, the decrease in the level of Met only in the STR, 4 h after the last dose of PCP, may be due to the use of a significant pool of this amino acid for the synthesis of SAM, which may then participate in the COMT-dependent catabolism of extracellular DA to 3-MT. In our study, a significant decrease in the Hcy level in the PFC and a downward trend in the content of this amino acid in the HIP with a simultaneous decrease in the concentration of Cys and maintaining the control level of Met in both these structures suggest a shift in the balance between the processes of Hcy transsulfuration and remethylation in favor of the remethylation reaction. The intensification of the Hcy remethylation reaction in 12-day-old rats subchronically administered PCP in the early postnatal period may mean an increase in SAM synthesis in the examined brain structures, and thus may lead to abnormal epigenetic changes, some of which may be related to the etiology of schizophrenia. Further biochemical studies are required to substantiate the proposed mechanism implicating the changes in the metabolism of sulfur amino acids Cys and Met in the pathogenesis of schizophrenia.

As for the activities of antioxidant enzymes in the brain of 12-day-old rats, it was shown that 4 h after the last dose of PCP, the activity of SOD in the HIP was significantly reduced, while the activity of GR in the FC increased significantly, but no significant changes in GPx activity were observed in any of the examined brain structures.

At the behavioral level, the results of the present study showed that repeated subcutaneous injections of PCP at a dose of 10 mg/kg to Sprague-Dawley rats during early postnatal days resulted in long-term social and cognitive deficits of schizophrenic type manifested in adulthood as assessed in the SIT and NOR test, respectively. The emergence of social and cognitive deficits in this neurodevelopmental model of schizophrenia is consistent with the effects shown in previous studies where PCP administered mainly to adult rodents, either acutely or repeatedly, induced these behavioral changes [73,74,75,76,77]. In addition to social and cognitive deficits, PCP administration to adult rats also induced hyperlocomotion [72, 78,79,80], which is considered as the equivalent of positive symptoms in humans. Moreover, PCP-pretreated adult rats exhibited exaggerated hyperlocomotion compared to control in response to amphetamine [72, 79]. However, in our study, administration of PCP to male pups in the early postnatal period did not increase locomotor activity in adulthood, as measured in the OFT as the time of walking. On the contrary, the walking time was significantly decreased suggesting a decrease in locomotor activity. On the other hand, Wang et al. [43] showed that PCP administration at a single 2 mg/kg dose to adolescent 42-day-old rats, that had been repeatedly pre-treated with a high PCP dose of 10 mg/kg on the postnatal days p7, p9, p11, increased the locomotor activity to a much greater extent than in control rats receiving saline. The effect described above suggests that perinatal administration of PCP sensitized the locomotor response to PCP challenge, similarly to chronic PCP treatment in adult rats, as previously shown [80,81,82,83]. However, our study did not test the effect of a PCP challenge dose on locomotor activity in adulthood. Therefore, it can be assumed that also in the model of schizophrenia used in our study, the administration of a challenge dose of PCP to adult rats might result in an increase in locomotor activity.

In conclusion, the results of the present study showed that blockade of NMDA receptors in the early postnatal life induced by subchronic administration of a high dose of PCP (10 mg/kg) led at the biochemical level to a decrease in the concentrations of GSH and essential sulfur amino acid Cys in the FC, HIP, and STR of 12-day-old rats and at the behavioral level to the emergence of social and cognitive deficits in adulthood.

Limitations of the study

One of the limitation factors of this work is the lack of a prepulse inhibition test. However, literature data show that PCP administered at a dose of 10 mg/kg in the same regimen as in the current work, induces sensorimotor gating deficits in rats [44]. Furthermore, PCP given in a single dose has been documented to produce transient schizophrenia-like psychoses in healthy volunteers, including positive and negative symptoms and cognitive dysfunctions [84, 85], and to exacerbate existing symptoms in schizophrenic patients [86]. The long-term use of PCP causes neuropsychological deficits that persist for several weeks. Moreover, PCP-induced psychosis is clinically difficult to distinguish from the primary psychosis of schizophrenia [85]. Therefore, PCP is commonly used as a model compound to induce schizophrenia-like behavior in basic research performed on laboratory animals. Hence, it seems reasonable to assume that the behavioral changes observed in the SIT and NOR tests in our study (despite the lack of the prepulse inhibition test) reflect changes of a schizophrenic nature, resembling the negative symptoms and cognitive deficits observed in patients with schizophrenia rather than similar changes in other psychiatric diseases (e.g. depression).

The second limiting factor is the exclusion of female rats from these studies. The rationale for this exclusion was the documented effect of estradiol on GSH synthesis. Studies by Dabrosin and Őllinger [87] indicate that 20-hour exposure of primary culture hepatocytes from Sprague-Dawley females to estradiol increased both GSH content and protein expression of GCL, the rate-limiting enzyme in GSH synthesis. Moreover, this study showed that in women, estradiol increased the concentration of GSH in subcutaneous adipose tissue in the luteal phase of the menstrual cycle. The above data suggest that in females, the presence of estradiol could interfere with the effect of NMDA receptor blockade on GSH synthesis, which we avoided by performing experiments only on males.