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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2019 Sep 15;11(9):5919–5931.

Elevated glutamate, glutamine and GABA levels and reduced taurine level in a schizophrenia model using an in vitro proton nuclear magnetic resonance method

Jingyu Yang 1,2,3, Huiling Guo 1,2,3, Dandan Sun 1,2, Jia Duan 1,2, Xiaoping Rao 3,4, Fuqiang Xu 3,4,5,6, Anne Manyande 7, Yanqing Tang 1,2,8, Jie Wang 3,4,9, Fei Wang 1,2
PMCID: PMC6789232  PMID: 31632560

Abstract

Accumulating evidence suggests that brain metabolic changes may be associated with the pathophysiology of schizophrenia. Both in vivo and in vitro studies have found glutamatergic and GABAergic abnormalities in different brain regions of individuals with schizophrenia. We report a longitudinal behavioral study in a methylazoxymethanol acetate (MAM) rat model of schizophrenia at three different age periods: prepuberty, late-puberty and early-adulthood. MAM-treated rats showed stable hypolocomotive activity, anxiety and cognitive deficits from late-puberty to early-adulthood. Therefore we detected the metabolites changes of adult MAM-treated rats using an in vitro proton nuclear magnetic resonance (1H-NMR) method. In the MAM-treated rats, glutamate was increased in the thalamus and hypothalamus, glutamine was increased in the hippocampus and GABA was increased in the hippocampus and prefrontal cortex, while taurine showed a decrease in the striatum, temporal cortex and parietal cortex. These abnormalities may be helped further understanding the pathophysiology of schizophrenia.

Keywords: Schizophrenia, 1H-NMR, glutamate, GABA, taurine

Introduction

Schizophrenia is regarded as a neurodevelopmental disorder characterized by positive symptoms, negative symptoms and cognitive deficits, which has clinical onset in late-puberty to early adulthood [1]. Because of ethical and practical limitations of human experimental biology, animal models are an important tool for investigating the pathophysiology and treatment of neuropsychiatric diseases [2]. Methylazoxymethanol acetate (MAM) is a mitotoxin which can interfere with neurogenesis in cortical and subcortical areas when it is administrated at embryonic day 17. As a neurodevelopmental model, there are a lot of evidences that the structural, functional and behavioral alterations in the MAM model are consistent with those observed in schizophrenia [3,4]. However, there is still no study that has explored how prenatal interference alters brain metabolic status after disease onset.

Accumulating evidences suggest that brain metabolic changes may be associate with the pathophysiology of schizophrenia [5]. Glutamatergic and GABAergic metabolites are of particular interest which could stand for the majority of excitory and inhibitory neurotransmissions among brain metabolites. Evidence for the glutamate hypothesis originated from the fact that administration of N-methyl-D-aspartate (NMDA) receptor antagonists, such as phencyclindine (PCP) and ketamine, could induce the psychotic symptoms that resemble schizophrenia in healthy volunteers [6,7]. Kim et al. first found decreased glutamate (Glu) concentration in the cerebrospinal fluid of schizophrenia patients using the fluorometric method [8]. Postmortem analysis found abnormalities on density and subunit constitution of glutamatergic receptor in the hippocampus and thalamus [9-11], and also reduced GABA-synthesizing enzyme, mRNA and protein in schizophrenia [12-14]. Proton magnetic resonance spectroscopy (1H-MRS) studies have reported elevated GABA and Glx (glutamate + glutamine) in the medial prefrontal cortex [15]. There is also evidence which showed that hypofunction of NMDA receptors on GABAergic neurons and abnormal metabolism of glutamatergic and GABAergic neurotransmission are associated with the pathophysiology of schizophrenia [16].

Additionally, taurine is a neurotransmitter and an inhibitory neuromodulator in the central nervous system which could protect neurons from glutamate-induced neuronal excitotoxicity [17]. Through different types of calcium channels, taurine can antagonize the glutamate induced elevation of intracellular free calcium ion concentrations [17]. Studies using high-performance liquid chromatography and gas chromatography-mass spectrometry showed that the schizophrenia patients had significantly lower concentrations of taurine in cerebrospinal and plasma compared with the control subjects [18,19]. However, another study using 1H-MRS found taurine to be elevated in the medial prefrontal cortex of schizophrenia patients [20].

Among the methods that detect metabolic alterations, 1H-MRS has been developed as a powerful tool to measure metabolites of the regional brain in schizophrenia with the majority focusing on patients in vivo. Due to limited resolution and time constraint, most studies commonly use the magnetic field strength of 1.5 or 3 T [5,21], in vivo 1H-MRS can only detect a few molecules and locate predetermined voxels. However, based on previous findings of brain metabolic changes in schizophrenia, the abnormalities were found in multiple brain regions. Therefore, a specific technique could be adopted to simultaneously detect the metabolites in different brain regions, and the brain regions most associated with the metabolic abnormalities in schizophrenia could be selected. With much higher spectral resolution, the in vitro proton nuclear magnetic resonance (1H-NMR) can provide the specific metabolic information of different brain regions in the whole brain simultaneously, which can improve our understanding of the findings in vivo [22].

In this study, we selected three different age periods of MAM vs. saline treated rats to test behavioral alterations. At adult age, the 1H-NMR method was used to carry out the metabolomics analysis of extracts from nine different brain regions. To our knowledge, this is the first time the neurochemical changes in multiple brain areas of MAM model with in vitro 1H-NMR have been explored. The findings in the current study may contribute to further investigations of the pathophysiology of this disease.

Materials and methods

Animals

All procedures in these experiments were in accordance with the guidelines of the Animal Care and Use Committees at the Wuhan Institute of Physics and Mathematics, Chinese Academy of Science. The pregnant Sprague Dawley dams were obtained from VITAL RIVER (Beijing, China). The dams were randomly treated with either MAM 22 mg/kg or vehicle (0.9% saline solution) intraperitoneally (i.p.) on gestational day 17 (day 0 was defined as the day of plug observed) [23,24]. Pups were weaned 21 days after birth, and the males were kept in groups of three individuals with unlimited access to a diet of pellets and water. Animals were housed in a room under controlled temperature (21 ± 2°C) with a 12-h light/dark (lights at 8:00 am-8:00 pm) cycle.

Animal behavioral studies

In order to investigate the alterations of behaviors during the animal’s growth, a list of animal behavioral experiments (Open field test, OFT; Morris water maze, MWM) of the MAM or saline treated male rats were studied in three different age periods (n = 11 in each group), which are considered as prepuberty (PD32-39), late-puberty (PD50-57) and early-adulthood (PD80-87).

Open field test (OFT)

The apparatus used was a black box (100 × 100 × 30 cm3) with a video camera above the arena. The bottom of the box was divided into 25 (5 × 5) squares, and the central four squares were considered as the central zone. On the experimental day, a rat was placed in the center of the bottom, video recorded and timed at the same time. After 5 min, the rat was returned to its home cage. The chamber was cleaned with 70% ethanol to avoid any information left before the next test. The total distance traveled, mean speed, entries to the center area and time spent in the center area were analyzed.

Morris water maze (MWM)

The water maze apparatus was a circular black pool (diameter = 180 cm, height = 50 cm) filled with water (approximately 24 ± 1.0°C). A platform (diameter = 10 cm, height = 26 cm) was hidden 2 cm below the surface of the water in a fixed position (the center of the southwest quadrant). The room contained several visual cues on the room walls. A computerized video system was utilized to record and analyze the behaviors of the rat during the experiment.

The day before behavioral testing, the rats were exposed to the pool without the platform for 60 s to adapt to the experiment. In acquisition trial, the rats were placed in the water facing the wall and free to swim until they found the platform and stayed on it for 3 s. If an animal failed to find the platform within 60 s, it was guided to the platform and remained for 10 s. Each rat was subjected to 4 trials per day for 4 days, and the starting position was changed in each trial (starting on the N side, followed by E, S, W sides). In probe trial, the 5th day of the experiment, the platform was removed and the rats were allowed to swim freely for 90 s. Time to the platform (escape latency) in each acquisition trial and probe trial was calculated. Percentage time in the target quadrant (quadrant time) and the number of times the subject crossed the platform location (platform crossing) were calculated during the probe trial.

1H-NMR studies

Brain samples collection

After the behavioral studies in early-adulthood periods, the animals were anesthetized (sevoflurane) and microwaved (1.0 kw, 19 s) using a commercial head focused microwave machine (Tangshan Nanosource Microwave Thermal Instrument Manufacturing Co. Ltd.) to prevent postmortem chemical changes [25]. The cooked brain tissue was quickly removed from the skull and manually divided into nine different brain regions, such as midbrain (MID), thalamus (THA), hypothalamus (HYP), hippocampus (HIP), striatum (STR), temporal cortex (TC), prefrontal cortex (PFC), parietal cortex (PC) and occipital cortex (OC) according to the anatomical landmarks [26,27]. The tissues were placed into a 2 ml pre-weighed EP tube, reweighed and immediately frozen in liquid nitrogen. At the end, all samples were stored in -80°C refrigerator for further analysis.

Samples extraction

HCl/methanol (300 μL, 0.1 M HCl) and ethanol (600 μL, 60%) were added separately to the samples and homogenized twice (20 Hz, 90 s, Tissuelyser II, QIAGEN, Germany) [28]. The mixture was centrifuged (4°C, 11,000 g, 10 min) and the supernatant was placed into a 5.0 ml EP tube. The extraction process was repeated twice with 700 μl ethanol each time. All supernatants were collected together and desiccated with the centrifugal drying apparatus (Thermo Scientific 2010, Germany) for 3 h. Samples were further dried with a freeze dryer for 24 h.

Phosphate buffer (pH = 7.2, 540 μL) and double distilled water (60 μl, 120 mg/L 3-(Trimethylsilyl) propionic-2, 2, 3, 3, d4 acid sodium salt (TSP, 269913-1G, Sigma Aldrich) in D2O) were added to dissolve the dried product and the mixture was vortexed for 30 s. TSP was set as the internal standard. The mixed solution was centrifuged (4°C, 14,000 g, 10 min) with high-speed centrifuge, and the supernatant (500 μl) was transferred to a 5 mm NMR tube for the 1H-NMR study.

1H-NMR study

One dimensional (1D) NMR spectra were acquired on a BrukerAvance III 600 MHz NMR spectrometer equipped with an inverse cryogenic probe (BrukerBiospin, Germany). All data were collected at 298 K with a standard WATERGATE pulse sequence. The 90° pulse length of each individual sample was adjusted to 10.1 μs. The scan was performed with the following parameters: Dummy scans (DS), 8; Acquisition scans (NS), 128; Acquisition time (AQ), 1.8 s; Relaxation delay (D1), 3.0 s; Spectral width, 20 ppm; Data points, 32 k. Finally, it took about 10 minutes for a sample to be loaded and scanned. For a random sample, a series of two-dimensional (2D) NMR spectra were also collected to assign the metabolites in the 1H-NMR spectra (online spectral databases). The 2D spectra include the following: 1H-1H correlation spectroscopy (COSY), J-resolved spectroscopy (JRES), 1H-1H total correlation spectroscopy (TOCSY), 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-13C heteronuclear multiple bond correlation (HMBC) 2D-NMR spectra data (online spectral databases). All the NMR data collections were completed using the TOPSPIN software (V3.0, BrukerBiospin, Germany).

Statistical analysis

Behavioral experiments

For analysis of OFT, all data were analyzed using two-factor mixed ANOVA, with group (MAM vs. Control) as between-subject factor and age period (prepuberty, late-puberty, early-adulthood) as within-subject factors. Post hoc analysis was performed using the Bonferroni test. For analysis of the acquisition trial of MWM, all data of the four trials were used as an average on each training day. The effect of the prenatal MAM treatment on escape latency (s), was analyzed using two-way repeated measures (RM) ANOVA with days as RM. The Student’s t-test was used to further test the difference between the two groups on each training day. For analysis of the probe trial of MWM, all data were analyzed by two-factor mixed ANOVA, with group (MAM vs. Control) as between-subject factor and age period (prepuberty, late-puberty, early-adulthood) as within-subject factors. The criterion for statistical significance was a probability value of 0.05. All results are presented as means ± SEM.

NMR data

For Fourier transformation, all spectra were firstly manually phased, baseline corrected, and referenced to the internal standard TMSP (δ = 0) using Topspin software 3.2 (Bruker Biospin, Germany). The raw data were automatically loaded into NMRSpec software [29], a tool using MATLAB code. Then, the peak alignment and integration were completed. Most amino acids were located in the region of 1.1-4.2 ppm, thus the other regions were removed before spectral alignment. Furthermore, ethanol was difficult to completely remove during the sample lyophilizing, that could affect the spectral analysis of the relevant spectral regions (δ: 1.15-1.22 and 3.597-3.687), thus the relevant ethanol signals were also discarded. All spectral data were divided into 0.04 ppm spectral buckets and automatically integrated in NMRSpec. The bucketed spectral data and integrated areas were normalized with the tissue weight and related peak area of TSP.

The absolute concentration (μmol/g wet weight) of the metabolite was calculated based on the following equation:

Cm = (Am*CTSP*VTSP*9)/(Rm*nH)

Where Am is the related peak area of the target metabolite; CTSP and VTSP are the concentration and volume of TSP; 9 is the number of protons in TSP; nH is the number of protons of the metabolites within the peaks area of Am; Rm is the ratio between the area of partial NMR signal of the selected metabolites during actual detection and the area of the whole proton signal in the standard spectrum. During sample detection, many signals overlapped, thus it was impossible to use them to calculate the absolute concentration. It was better to select a pure signal which did not overlap with other signals or part of the related signal. For example, Rm values of the metabolites Asp, taurine, Gly and creatine were equal to 1, whereas the signals of NAA (0.5871), GABA-4 (0.5728), Glu-4 (0.9001), Gln-4 (0.3901) and myo-instol (0.4933) overlapped with the other signals.

Data analysis was performed using SPSS software (Version 22) and codes in MATLAB. Student’s t-test were employed to determine the significant differences in metabolite concentrations. Probability value of 0.05 was considered statistically significant. Metabolite concentrations were presented as means ± SEM.

Results

Behavioral experiments

Open field test

For the spontaneous locomotor activity analysis, the total travel distance was significantly reduced in MAM rats (Figure 1A, F = 19.578, P<0.001). There was no age period effect as well as group × age period interaction displayed. The group difference was present in late-puberty (P<0.001) and early-adulthood (P = 0.004) compared to control rats following post hoc analysis. However, there was no significant difference between the two groups in prepuberty (P = 0.257). Total travel distance of MAM rats was significantly reduced in late-puberty (P = 0.045) and early-adulthood (P = 0.035) compared to prepuberty, but there was no difference between late-puberty and early-adulthood. There was no difference in total travel distance between any two age periods in control rats. Thus, MAM rats showed impairment in spontaneous locomotor activity from late-puberty to early-adulthood.

Figure 1.

Figure 1

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Effect of MAM vs. saline treatment on performance in the open field test (OFT) at three age periods. Each column represents the mean (± S.E.M.) on the experiment day of OFT. *P<.05 compared with control rats at the same age period; **P<.01 compared with control rats at the same age period. ***P<.001 compared with control rats at the same age period.

For the anxiety reactivity analysis (Figure 1B and 1C), MAM rats showed less time spent in the center area (F = 26.696, P<0.001) and fewer entries to the center area (F = 31.251, P<0.001). There was no age period effect as well as group × age period interaction displayed. Post hoc analysis showed that the group difference was consistently present in the three age periods: prepuberty, time spent in the center area (P<0.001), entries of center area (P<0.001); late-puberty, time spent in the center area (P = 0.012), entries to center area (P<0.001); early-adulthood, time spent in the center area (P = 0.012), entries to center area (P = 0.031). There was no difference between any two age periods in MAM and control rats, indicating that MAM rats exhibited stability of anxiety state in all age periods.

Morris water maze

In the acquisition trial, MAM rats showed deficits in spatial learning ability from late-puberty to early-adulthood, but not in prepuberty (Figure 2A). The two-way ANOVA with day as repeated measures revealed an effect of group on escape latency in late-puberty (F = 0.323, P = 0.004) and early-adulthood (F = 7.250, P = 0.015), but not in prepuberty (F = 0.148, P = 0.704). Further analysis of each training day using Student’s t test, showed that MAM rats spent more time to find the platform on day 1 (F = 0.777, P = 0.002) in late-puberty, day 2 (F = 3.960, P = 0.016) and day 4 (F = 7.274, P = 0.002) in early-adulthood. There was no difference on any day in prepuberty. The two-way ANOVA with day as repeated measurements also showed an effect of training days in three age periods (prepuberty, F = 4.529, P = 0.006; late-puberty, F = 5.331, P = 0.003; early-adulthood, F = 12.792, P<0.001), which indicates the learning process of the test. There was no interaction between groups and training days in any age periods.

Figure 2.

Figure 2

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A. Effect of MAM vs. saline treatment on performance in the acquisition trial of the Morris water maze (MWM) at three age periods. The two-way ANOVA with day as repeated measures revealed an effect of group on escape latency in late-puberty (F = 7.250, P = 0.015) and early-adulthood (F = 8.603, P = 0.008). Each point represents mean (± S.E.M.) of the four trials on the same experiment day. *P<.05 compared with control rats on the same experiment day by Student’s t test; **P<.01 compared with control rats on the same experiment day using Student’s t test. B-D. Effect of MAM vs. saline treatment on performance in the probe trial of the Morris water maze at three age periods. Each column represents the mean (± S.E.M.) of four consecutive trials. *P<.05 compared with control rats at the same age period.

In the probe trial, MAM showed a slight differential in reference memory in early-adulthood. Overall analysis showed MAM rats spent more time to find the platform (F = 6.372, P = 0.003, Figure 2B). An age period effect was present (F = 6.372, P = 0.003), but not a group × age period interaction (F = 0.439, P = 0.647). Post hoc analysis revealed no group difference in the three age periods (prepuberty, P = 0.421; late-puberty, P = 0.184; early-adulthood, P = 0.951). Overall analysis displayed no group effect on the time of swimming in the target quadrant (F = 0.727, P = 0.397, Figure 2C). There was also no age period effect (F = 1.094, P = 0.342) and group × age period interaction (F = 2.718, P = 0.074). Post hoc analysis showed that there was group difference present in early-adulthood (P = 0.019). MAM rats spent less time in crossing the platform location (F = 4.891, P = 0.031, Figure 2D). An age period effect (F = 10.028, P<0.001) was also found, but no group × age period interaction (F = 0.462, P = 0.633). The difference was present in early-adulthood (P = 0.049) by the post hoc analysis.

NMR data

In the current study, there were nine different brain regions involved. In order to show the results clearly, only a sample from the brain region (occipital cortex) were selected for illustration. At first, the metabolites in the 1H-NMR spectrum were identified using related published works and 2D-NMR spectra. The identification of these metabolites is shown in (Figure 3). Furthermore, the average NMR spectrum of the extracts in the represented brain region (occipital cortex) in the MAM-treated and control groups were also calculated and illustrated (Figure 3). During the extraction of the brain samples, methanol and ethanol were involved, which were detected using 1H-NMR methods. During the lyophilization procedure, these two chemicals were evaporated. However, the results showed that it was very difficult to totally erase them. Thus, the NMR signal regions related to ethanol and methanol were discarded for further analysis.

Figure 3.

Figure 3

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The average normalized 1H-NMR spectra of the metabolites of occipital cortex of MAM vs. saline treatment. Red line: control; black line: MAM. Note: Lowercase, position of the hydrogen signal which is connected with the carbon position; Lac, lactate; Myo, myo-institol; Cre, creatine; Asp, aspartic acid; Ala, alanine; Glx, glutamine + glutamate; EtoH, ethanol; Gly, glycine; Tau, taurine; GABA, gamma amino acid butyric acid; NAA, N-aceytl aspartate; Gln, glutamine; Glu, glutamate.

In order to primarily estimate the difference of the metabolites in different brain regions, the differences of the average 1H-NMR spectra in the MAM-treated and control groups were calculated (Figure 4). The different metabolites in these brain regions were identified. However, there is no information of the standard deviation of these metabolites in this figure. Thus, it was very difficult to verify the major differences between brain regions in the MAM group compared with the control group.

Figure 4.

Figure 4

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Differences in 1H-NMR spectra of the metabolites of different brain regions of MAM vs. saline treatment.

Statistical analysis of the metabolites

The absolute concentrations of all detected metabolites in different brain regions were calculated. Then the metabolites in different brain regions of these two different groups were compared. For precise analysis, group differences were found in Glu, Gln, GABA and taurine. Glu was significantly increased in the thalamus (F = 0.476, P = 0.004) and hypothalamus (F<0.001, P = 0.025, Figure 5A). Gln was elevated in the hippocampus (F≤0.001, P = 0.015, Figure 5B). GABA was found raised in the hippocampus (F = 0.040, P = 0.029) and the prefrontal cortex (F = 0.201, P = 0.203) of MAM rats (Figure 5C). Taurine decreased in the striatum (F = 0.044, P = 0.026), temporal cortex (F = 5.438, P = 0.020) and parietal cortex (F = 0.067, P = 0.028, Figure 5D).

Figure 5.

Figure 5

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Differences of metabolite concentration in nine brain regions of MAM vs. saline treatment. Each column represents the mean (± S.E.M.) of metabolite concentration of each group in certain brain region. *P<0.05 compared with control rats at the same brain region; **P<.01 compared with control rats at the same brain region. ***P<.001 compared with control rats at the same brain region.

Discussion

Behavioral patterns of MAM-treated rats

Gestational MAM exposure model has been suggested to produce similar behavioral abnormalities to those seen in schizophrenia. A previous study screened the MAM-treated rats in three different age periods: prepuberty (PD28-42), late-puberty (PD56-70) and adulthood (PD98-112). The results indicated that the MAM rats had elevated locomotor activity in late puberty and adulthood, but not in prepuberty. The anxiolytic effect had different age patterns which was present in prepuberty and adulthood, but had disappeared in late puberty [30]. Another study found that MAM rats had the same locomotor activity with control rats in late-puberty (PD58-60) [31]. Elevated anxiety-like behaviors of MAM rats were observed both in peripuberty [32] and adulthood [33] in other studies. However, there are few studies that confirmed whether the MAM model, like schizophrenia, had onset in late-puberty or adulthood. Our findings are consistent with the clinical observation that anxiety is highly prevalent in schizophrenia patients [34]. In the current study, MAM-treated rats showed hypoactivity from late-puberty to early-adulthood and heightened anxiety levels throughout the three age periods. These abnormalities might be correlated to hypersensitivity to stress [35] around puberty and resulted in the dopaminergic hyper responsivity which occurs in adulthood [32].

Cognitive deficits are believed to be core symptoms of schizophrenia. One study assessed MAM rats on an attentional set-shifting task which is a rodent analog of the Wisconsin card sort task. They found that MAM rats required more time to learn the task [36]. Furthermore, another study examined the performance on Morris water maze of juvenile (PD12-21) and adolescent (PD28-45) MAM-treated rats. Both groups presented deficits in learning the location of the platform [37]. In the present study, we found MAM-treated rats had similar performance compared with the control during prepuberty, but the spatial learning ability were impaired from late-puberty and persisted in early-adulthood.

Overall, the behavioral abnormalities indicate that the MAM rats were in a relatively constant onset state from late-puberty to early-adulthood. This provided favorable evidence for our further study of metabolic changes using the MAM treated animal model.

Metabolic changes in adult MAM-treated rats

Glutamatergic and GABAergic metabolites

The glutamate hypothesis of schizophrenia highlighted that NMDA receptor hypofunction might be a key mechanism that could help explain major clinical and pathophysiological aspects of schizophrenia [38]. The NMDA receptor hypofunction was associated with reduced parvalbumin-containing GABA interneurons [39], leading to the imbalance between neuronal excitation and inhibition [40,41], which may contribute to the disinhibition of cortical excitatory neurons resulting in excess glutamate release [41]. Mice with reduced expression of NMDA receptor manifested schizophrenia-like symptoms, and these kinds of symptoms can be alleviated by haloperidol or clozapine [42]. The long-time treatment with antipsychotic drugs could induce adaptive changes on glutamate receptor expression in different brain regions of rats [43]. Postmortem analysis found reduction of glutamate in the hippocampus and prefrontal cortex [44] and alteration of the density and subunit constitution of glutamate receptor in the thalamus and hippocampus of schizophrenia patients [9-11]. A recent meta-analysis which included 59 in vivo 1H-MRS studies revealed that there was a broad elevation of glutamatergic metabolites (glutamate, glutamine or glutamate plus glutamine) across several brain regions of schizophrenia patients and individuals at high risk, such as the thalamus, basal ganglia, medial frontal cortex and medial temporal cortex [45].

GABA is the main inhibitory neurotransmitter which is formed by the decarboxylation of glutamate catalyzed by glutamate decarboxylase (GAD). Postmortem analysis found a subclass of GABA interneurons in the prefrontal cortex with significant low expression of GAD67 mRNA [13], which is consistent with other studies [12,14]. However, in 1H-MRS studies, there have been inconsistent results of GABA levels in schizophrenia individuals, with reports showing a reduction [46], unchanged [47,48] and elevation [15,49,50], mainly located in the striatum, dorsal caudate nucleus, anterior cingulate, medial prefrontal cortex, temporal cortex, parietal cortex and occipital cortex. With the in vitro 1H-NMR method, we observed elevated GABA concentrations in the hippocampus and prefrontal cortex of MAM-treated rats. Our result is not exactly consistent with these former postmortem findings. That might be due to two main factors: the postmortem metabolite changes in the bio-system and the different living systems. However, all findings support that the GABA disorders do occur in schizophrenia.

Taurine

Increasing evidences suggest that the taurine-induced neuroprotective effect may be mediated by its antagonism to the glutamate-induced excitotoxicity [17]. In an analysis of a rodent model of maternal immune activation (MIA) which is associated with schizophrenia, taurine was significantly reduced in hippocampus of the adult offspring [51]. Moreover, in another MIA model with longitudinal observation, the reduction of taurine in the prefrontal cortex was found in adult offspring but not adolescent offspring [52]. In vitro 1H-MRS study that used a rodent model of neonatal maternal deprivation revealed increased taurine in the hippocampus and prefrontal cortex, which reflect that the increased taurine might protect the neonatal brain after more severe damage [53]. At present, fewer taurine in vivo 1H-MRS studies have been reported, which only found elevated taurine in plasma [54] and the medial prefrontal cortex [20] of schizophrenia individuals. Our results suggest that taurine is significantly reduced in the striatum, temporal cortex and parietal cortex of adult MAM-treated rats, this could probably be a decompensation state for excess glutamate.

Abnormal metabolic state in corticolimbic system

Our findings revealed that there was metabolic dysfunction in the corticolimbic system, such as the thalamus, hypothalamus, hippocampus, striatum, prefrontal cortex, temporal cortex and the parietal cortex. Structural changes in the corticolimbic system are important features of schizophrenia. In this pathway, a major function of the NMDA receptor is to drive GABA interneurons, thus maintaining the inhibition of primary neurons in the limbic system. NMDA receptor antagonists could inhibit the activation of GABA interneurons, which in turn could increase glutamate release [55] and lead to excessive excitatory activity release [56]. Further, evidence showed that the sensitivity to NMDA receptor antagonists of rats developed during adolescence and continued into early adulthood [56] and NMDA receptor hypofunction was present in adolescence of the MAM model [57], which suggests that early developmental interference with the nervous system does not begin to take effect until adolescence, which is consistent with our findings of behavioral changes. We postulate that the impairment of the NMDA receptor, which was caused by interference during the early developmental period, damaged GABAergic neurons, which led to the disinhibitory effect on downstream neurons. In the beginning, the nervous system could compensate for these damages through some mechanisms, and taurine might be one of these compensatory mechanisms. However, with the continuous maturation of the nervous system, the level of metabolites in the brain gradually become a decompensation state, as we observed with the elevation of Glu, Gln, GABA and the reduction of taurine, which eventually leads to damage of the cortical limbic system. The mechanism by which the imbalance in brain function and metabolic status and the role played in the pathophysiology of schizophrenia needs further research.

According to our experimental design, the detection of metabolites cannot be realized in prepuberty and late-puberty stage. Thus, we did not conduct dynamic monitoring of metabolic alterations during the growth process. In future experiments, we will conduct multi-point monitoring to better explain this problem.

The present study demonstrated the effect of gestational exposure to MAM on the behavioral changes in different developmental periods and the metabolic alteration in different brain regions of adult rats. MAM-treated rats showed significant hypoactivity and high levels of anxiety mainly from late-puberty to early-adulthood, and slightly cognitive impairment in early-adulthood. Metabolic analysis of adult MAM-treated rats showed Glu was increased in the hypothalamus and thalamus, GABA was increased in the prefrontal cortex and hippocampus, and Gln was increased in the hippocampus. However, taurine showed a significant decrease in the striatum, temporal cortex and parietal cortex. These findings may have some significance when studying how metabolic abnormalities in different brain regions of schizophrenia patients are involved in the pathophysiological mechanism of the disease. In conclusion, the behavioral changes of MAM rats, as a neurodevelopmental model, manifested in a time-dependent pattern and the metabolic changes in the corticolimbic system could be critical to the pathophysiology of schizophrenia.

Acknowledgements

This work was supported by the China Postdoctoral Science Foundation (2018M640265) and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Y6Y0021004).

Disclosure of conflict of interest

None.

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