Specific metabolites in the medial prefrontal cortex are associated with the neurocognitive deficits in schizophrenia: A preliminary study

NeuroImage 49 (2010) 2783–2790

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Specific metabolites in the medial prefrontal cortex are associated with the neurocognitive deficits in schizophrenia: A preliminary study Yukihiko Shirayama a,⁎, Takayuki Obata b, Daisuke Matsuzawa a, Hiroi Nonaka b, Yoko Kanazawa b, Eiji Yoshitome b, Hiroo Ikehira b, Kenji Hashimoto c, Masaomi Iyo a a b c

Department of Psychiatry, Chiba University Graduate School of Medicine, Chiba, Japan Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Science, Chiba, Japan Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan

a r t i c l e

i n f o

Article history: Received 17 May 2009 Revised 21 August 2009 Accepted 12 October 2009 Available online 19 October 2009 Keywords: Cognitive control Medial prefrontal cortex MRS Schizophrenia Amino acids

a b s t r a c t We measured brain metabolites in the medial prefrontal cortex of 19 schizophrenic patients and 18 healthy controls by 3 T proton magnetic resonance spectroscopy (1H MRS), and examined the relationship between prefrontal cortex-related neurocognitive functions and brain metabolites in the medial prefrontal cortex. The patients with schizophrenia exhibited deficits on the verbal fluency, Wisconsin card sorting test (WCST), trail making test, Stroop test and digit span distraction test (DSDT), but not on the Iowa gambling test. The patients showed statistical significant changes in the ratio of glutamine/glutamate, the ratio of N-acetyl-Laspartate (NAA)/glycerophosphorylcholine plus phosphorylcholine (GPC + PC) and the levels of taurine in the medial prefrontal cortex compared with normal controls. Furthermore, we found significant correlations of the ratio of glutamine/glutamate with WCST and DSDT scores, the ratio of NAA/(GPC + PC) with verbal fluency and WCST scores, and the levels of taurine with scores on the Stroop test and Trail making test A among the participants. The ratios of NAA/(GPC + PC) and (GPC + PC)/(Cr + PCr) had significant relationships with the duration of untreated psychosis of the schizophrenic patients. The glutamine/glutamate ratio and levels of taurine were significantly related to the duration of illness of the patients. These data suggest that specific metabolites of the medial prefrontal cortex are associated with the neurocognitive deficits in schizophrenia. © 2009 Elsevier Inc. All rights reserved.

Introduction It seems that the cognitive deficits in schizophrenia are the core of the disorder and that working memory and attention are characteristically impaired in patients with schizophrenia (reviewed by Elvevåg and Goldberg, 2000). During clinical care, evaluation of a patients' ability to handle his or her mental problems by subjective methods is important for treatment. From the clinical point of view, frontal cortical function-related tests such as Wisconsin card sorting test (WCST), trail making test and verbal fluency test among various neuropsychological tests were found to be related to community outcome, social problem solving and skill acquisition (Green, 1996). Recent studies have shown that attention and working memory was directly related to work skills, that executive functions had a direct effect on interpersonal behaviors (Bowie et al., 2008), and that frontal

⁎ Corresponding author. Present address: Department of Psychiatry, Teikyo University Chiba Medical Center, 3426-3 Anesaki, Ichihara 299-0111, Japan. Fax: +81 436 62 1511. E-mail address: [email protected] (Y. Shirayama). 1053-8119/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2009.10.031

cortical function reflected by WCST was related to insight deficits (Shad et al., 2006). Thus, cognitive deficits in schizophrenic patients could predict the functional capacity and outcome. Previous studies have indicated that the prefrontal cortex is involved in negative symptoms and cognitive impairments in schizophrenia (Ingvar and Franzen, 1976; Weinberger, 1988). However, the negative symptoms do not have a strong relationship with cognitive functions (Kibel et al., 1993). These studies suggest the specificity of cognitive impairment in schizophrenia (reviewed by Elvevåg and Goldberg, 2000), and the brain mechanisms underlying cognitive functions have been gradually disclosed, even in the schizophrenia prodrome (Simon et al., 2007). The prefrontal cortex is divided into three parts, medial, dorsolateral and orbital (Ramnani and Owen, 2004). The medial prefrontal cortex including the anterior cingulate handles monitoring, evaluation, motivation, attention (vigilance), and switching (Ridderinkhof et al., 2004). The dorsolateral prefrontal cortex plays a role in central executive and working memory (Goldman-Rakic, 1996). The orbitofrontal cortex is implicated in executive function and decision-making and exerts an inhibitory action on the activity of the anterior cingulate (Schoenbaum et al., 2006). The functions of these regions are different

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from each other, but these regions seem to have a reciprocal relationship (MacDonald et al., 2000; Fuster, 2001). It is noteworthy that during the cognitively demanding neutral portions of the Stroop counting task, the orbitofrontal cortex was suppressed, whereas the medial prefrontal cortex (including the cognitive division of the anterior cingulate) was activated (Bush et al., 2000). Cognitive control requires the dorsolateral prefrontal cortex for implementation of control and the anterior cingulate for monitoring of performance, but the two are disassociated (MacDonald et al., 2000). Schizophrenic patients showed various changes in the medial prefrontal cortex and anterior cingulate. The anterior cingulate cortex was less activated during attentional control on the Stroop task and cognitive interference in schizophrenia (Carter et al., 1997; Heckers et al., 2004). Recent studies demonstrated that decreased gray matter volume of the middle frontal gyrus was negatively correlated with performance on the WCST in patients with schizophrenia (Bonilha et al., 2008) and that reduced error-related activation in the anterior cingulate was related to impaired performance in schizophrenia (Polli et al., 2008). Recruitment of the anterior cingulate was decreased when relational memory is needed to discriminate novel pairs derived from a sequence of stimuli in schizophrenia (Ongur et al., 2006). The medial prefrontal and anterior cingulate cortices are involved in the social brain, which evaluates other people's thinking, predicts other people's behavior, and understands others (reviewed by Blakemore, 2008). This social cognitive process develops during adolescence. Interestingly, it is well known that schizophrenia is diagnosed after adolescence, indicating that system malfunction could occur during adolescence. Furthermore, adolescents showed activation of the anterior cingulate while viewing frightening images, whereas adults did not, indicating that adolescents modulate the activity of the anterior cingulate based on the emotional factor, whereas adults modulate the activity of the anterior cingulate based on attention demand (Monk et al., 2003). The medial prefrontal cortex is known to correspond to the Theory of Mind network (Dollfus et al., 2008). Proton magnetic resonance spectroscopy (1H MRS) studies have demonstrated abnormalities of levels of glutamine, glutamate and Nacetyl-L-aspartate (NAA) in the medial prefrontal cortex of schizophrenic patients (Bartha et al., 1997; Théberge et al., 2002, 2003; Yamasue et al., 2002; Wood et al., 2007). However, it remains unknown about the relationships of metabolites abnormality in the medial prefrontal cortex and prefrontal cortex-related neurocognitive deficits in schizophrenia. Recently, 1H spectra can be acquired from smaller volume of interest (VOI) with less scan numbers than before due to an increase in the magnetic field strength. It should be mentioned that weak magnetic field strength and small voxel volume result in the notcomparable big standard deviation (SD) of values, which can be decreased by increases in numbers of participants and scans. Previous studies could detect glutamine signals in the prefrontal or occipital cortex of normal controls and schizophrenic patients, and localized in vivo 1H spectra were acquired from 4.5 cm3 volume at 1.5 T (SD 40– 50%, Bartha et al., 1997), 13.5 cm3 at 2.1 T (SD 40–43%, Behar et al., 1999), 4.3 cm3 at 3 T (SD 36–41%, Tayoshi et al., 2009), 18 cm3 at 3 T (SD N30%, Shulman et al., 2006), and 1.5 cm3 at 4 T (SD not shown, Théberge et al., 2002). Under the condition with 3 T in the present study, acceptable measures of glutamine were obtained from all the participants using a large volume of 18.5 cm3 (SD 21–23%). This study was performed to aim to examine the relationships between brain metabolites and neurocognitive functions in the medial prefrontal cortex in schizophrenia. To this end, we performed in vivo 3 T 1H MRS on schizophrenic patients and normal controls and determine levels of glutamine, glutamate, myo-inositol, glycerophosphorylcholine plus phosphorylcholine (GPC + PC), creatine plus phosphocreatine (Cr + PCr), NAA and taurine in the medial prefrontal

cortex. To assess the cognitive functioning of the prefrontal cortex, six neuropsychological tests, verbal fluency, WCST, Stroop test, digit span distraction test (DSDT), trail making test, and Iowa gambling task were administered. The rationale for choosing these tests stems from the hypothesis that each test works on a region-dominant part (medial, dorsolateral or orbital portions) of the brain and could examine the region-related functions. Then we examined the relationship between these brain metabolites in the medial prefrontal cortex and neurocognitive functions. Materials and methods Subjects The subjects consisted of 19 schizophrenic patients and 18 sexand age-matched control subjects. All patients were recruited from the outpatient clinics of Chiba University Hospital. Control subjects with no past history of psychiatric disorders or drug dependence were recruited. Characteristics of the subjects are shown in Table 1. The research was approved by the ethics committee of Chiba University Graduate School of Medicine and National Institute of Radiological Science. Written informed consent was obtained after the procedure had been fully explained. All patients met the DSM-IV criteria for schizophrenia (American Psychiatric Association, 1994) and had no other psychiatric disorders. Of the patients, 12 were diagnosed as the residual type and 7 were the paranoid type. They had been clinically stable for at least 3 months. All patients except one were receiving second-generation neuroleptics; risperidone (n = 6), olanzapine (n = 6), quetiapine (n = 1), perospirone (n = 1), and aripiprazole (n = 4). The chlorpromazine-equivalent dose was 267 ± 179 (means ± SD) mg/day (Woods, 2003). Four patients were being treated with the anticholinergic drug biperiden (mean dose 2.4 mg/day), although anticholinergic drugs can lead to impairments in learning and memory (Silver and Geraisy, 1995). Eight patients were treated with benzodiazepine drugs for the anxiolytic effects and improved sleep quality. The diazepam-equivalent dose was 11.8 ± 5.4 (means ± SD) mg/day (Nelson and Chouinard, 1999; Inada et al., 2003). MRS methods Proton magnetic resonance spectroscopy was acquired from all subjects using a Signa Excite MRS system (GE, Milwaukee, WI, USA) operated at 3 T. 1H NMR used a standard quadrature coil (30 cm

Table 1 Characteristics and clinical severity of study participants.

Sex (male/female) Age (year) Education (year) Estimated IQa Age at onset of illness (year) Duration of illness (year) Duration of untreated psychosis (year) GAFb Antipsychotic drugsc BPRS score BPRS positive scores BPRS negative scores SANS score DIEPSS score

Control

Schizophrenia

p

18 (14/4) 31.4 ± 8.4 15.4 ± 3.2 106.8 ± 11.6

19 (12/7) 30.5 ± 5.6 13.7 ± 1.8 101.7 ± 12.0 23.6 ± 5.5 7.3 ± 5.2 2.6 ± 2.4 52.9 ± 10.8 267 ± 179 23.2 ± 4.9 12.1 ± 5.3 5.8 ± 3.0 74.0 ± 12.0 0.38 ± 0.17

ns ns ns ns

Values are means (SD). ns, not significant. a Short form version of Wechsler Adult Intelligence Scale Revised (WAIS-R). b GAF: Global assessment of functioning scale. c Chlorpromazine equivalent (mg).

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Fig. 1. Voxels of interest in medial prefrontal cortex in sagittal, coronal and transverse views.

diameter). The location of the volume of interest (VOI) was chosen under the guidance of T2-weighed images pulses (repetition time 3.5 s, echo time 102 ms, slice thickness 5 mm) for the cubic voxel (28 × 30 × 22 mm) to include the medial prefrontal and anterior cingulate cortices (see Fig. 1). VOI was placed on the corpus callosum and centered on the intrahemispheric fissure, not containing the orbitofrontal cortex. A high-order shim, followed by an automated regional shim was performed until the half linewidth was accomplished to less than 8 Hz. 1H spectra were acquired using a pointresolved spectroscopy sequence (PRESS) with water suppression by CHESS pulses (repetition time 5 s, echo time 30 ms, number of scans 256). The total acquisition time was almost 60 min. Spectra were analyzed using the linear combination model (LCModel) (Provencher, 1993, 2001). Metabolite concentration for glutamine, glutamate, creatine plus phosphocreatine (Cr + PCr), myoinositol, glycerophosphorylcholine plus phosphorylcholine (GPC + PC), N-acetyl-L-aspartate (NAA), taurine, and N-acetylaspartylglutamate (NAAG) were acquired using LCModel software (Fig. 2). The standard GE libraries of model metabolite spectra, which were

provided with LCModel, were used as a basis reference for time domain fitting. Water signal was used as an internal reference. We obtained acceptable measures of various metabolites. The reliability of metabolite quantification is judged by standard deviation of the fits, which is expressed as the percent standard deviation (%SD) of the estimated concentration by Cramer-Rao lower bounds (CRLB) in LCModel. Our criterion for the reliability of the spectral fit was b25 % SD. The mean CRLB were 2.1 for Cr + PCr, 17.9 for glutamine, 5.5 for glutamate, 4.3 for myo-inositol, 3.0 for GPC + PC, 2.1 for NAA, 18.5 for taurine and 15.9 for NAAG. For glutamine, all resonances were estimated with b25 %SD. For taurine and NAAG, spectra with N25 %SD were excluded from analysis. In taurine study, three healthy controls and four schizophrenic patients were excluded. In NAAG study, four controls and six schizophrenic patients were excluded from the present data. Segmentation was performed on the VOI on T2-weighted images using the equipped software (Spectral Analysis/General Electric (SA/ GE)) and the Image J 1.41 software, tracing and calculating the region of interest. As for voxel tissue composition, the distribution of gray

Fig. 2. 1H MRS data. Spectra of the unfiltered data superimposed with the LCModel fit (A). Residual noise (B).

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matter (GM), white matter (WM) and cerebrospinal fluid (CSF) across groups was determined. GM ratios, WM ratios and the portions of CSF in the controls and schizophrenic patients were shown in Table 2. No statistical significances were found between the two groups. Clinical severity The Brief Psychiatry Rating Scale (BPRS) was used to evaluate the general severity (Overall and Gorham, 1961). The Scale for the Assessment of Negative Symptoms (SANS) was used to evaluate negative symptoms (Andreasen, 1982). Because many cognitive functions are influenced by extrapyramidal motor side effects, the Drug Induced Extrapyramidal Symptoms Scale (DIEPSS) was used to evaluate the effects of drug-induced extrapyramidal symptom, which could affect the clinical severity (Inada et al., 2002). Intelligence quotient (IQ) scores were estimated from the Information, Digit span, and Picture completion subscales using the short version of the Wechsler Adult Intelligence Scale Revised (WAIS-R) (Wechsler, 1997; Nakamura et al., 2000). Age at onset, duration of illness, and duration of untreated psychosis were evaluated. Neuropsychological tests The verbal fluency test accesses semantic memory functions, requiring quick and spontaneous word production (Sumiyoshi et al., 2005). This test consists of two parts. In the letter fluency test, subjects are given an initial letter as a cue, and are asked to generate as many words that begin with the cue initial letter as possible in 60 s. In the category fluency task, subjects were instructed to produce as many words of certain category as possible in 60 s. Verbal fluency requires not only retrieval from memory storage but also verbal working memory, which holds the already-generated words and inhibits repeated response. This test is thought to reflect mainly the dorsolateral prefrontal cortex function, but the anterior cingulate cortex is also involved in this task (Dolan et al., 1995). The Wisconsin card sorting test (WCST) examines executive functioning including a measure of set shifting and problem solving (Heaton et al., 1993). Subjects are instructed to sort cards according to a rule (color, shape, or number). After the subjects select six correct cards consecutively, the rule is switched without telling the subject. The number of categories achieved and perseverative errors of Nelson are measured. We used the short version of WCST (Keio version; 48 cards) to shorten the procedural time (Igarashi et al., 2002; Hori et al., 2006). This test activates the dorsolateral and medial prefrontal cortex (Mentzel et al., 1998; Riehemann et al., 2001). The trail making test examines set-shifting abilities. The trail making test consist of two parts (Reitan and Wolfson, 1985). In part A, lines are drawn to connect 25 consecutively numbered circles on one worksheet are requested. Part A examines visuomotor search skills, simple attention and processing speed. In part B, lines are drawn to connect 25 consecutively numbered and lettered circles by alternating between the two sequences (e.g. 1-A-2-B-3-). Part B examines set alternation and divided attention (mental flexibility). The trail making test is thought to use the dorsolateral and medial prefrontal cortices (Zakzanis et al., 2005). The digit span distraction test (DSDT, Oltmanns and Neale, 1975; Green et al., 1997; Moser et al., 2001) examines selective attention

and short time retention. The test consists of two conditions, with and without a distracter. Each condition has seven trials. In the control condition, subjects were requested to remember six digits that are read by a female voice. In the distracter condition, subjects were requested to remember five digits that are read by a female voice, interspersed with four digits that are read by a male voice. Subjects were requested to ignore the male voices. The percentage of digits correctly recalled was assessed. The DSDT also examines resistance to distractibility, thus verbal working memory (Moser et al., 2001). The region responsible for DSDT performance could be extrapolated from the result of a similar interference task in which distractors activated the medial prefrontal and anterior cingulate cortices (Heckers et al., 2004). The Stroop test (MacLeod, 1991; Chan et al., 2004) requires suppression of the automatic word-reading response, and examines selective attention, response inhibition, and executive function. This test consists of three parts. First, subjects are requested to names the color of dots (red, blue and green) as a baseline test (D). Next, subjects are instructed to name the color of the ink in where color-irreverent Chinese characters are printed (e.g., tree, mountain, water), establishing the tendency to respond to color. Finally, subjects are presented with a list of the color-reverent Chinese characters (e.g., the character “red” printed in green ink) and were instructed to name the color of the ink (C). This test measures the person's ability to inhibit the natural behavior of reading words and name the color of the ink instead. The difference between the reaction time (C–D) was calculated for the assessment. The Stroop test uses the functions in the medial prefrontal cortex and anterior cingulate (Carter et al., 1995; Yücel et al., 2002). The Iowa gambling task assesses the capacity to acquire a preference through reward and punishment (Bechera et al., 1994). This task requires subjects to overcome an initial attraction to highpayoff decks as losses begin to occur. Subjects sat facing four decks of cards and were instructed to pick a card from one of the decks (A, B, C, D). Subjects were informed that each time they would receive a monetary reward or penalty and the goal of the game was to maximize profits. The subjects were free to pick from any deck and to switch decks at any time. Decks A and B were disadvantageous as selections are accompanied by large monetary penalties. Decks C and D provide smaller monetary rewards and smaller penalties. Performance was measured by the total numbers of cards from advantageous decks minus those from disadvantageous decks ([C + D] − [A + B]). This test reflects the function of the orbitofrontal cortex (Fellows and Farah, 2005; Nakamura et al., 2008). Statistics The mean values of the two groups were compared using an unpaired two-tailed Student's t-test. Coefficients of brain metabolites with SANS scores, BPRS scores, and neuropsychological tests scores were estimated by Pearson coefficient. Coefficients of brain metabolites with age at onset of illness, duration of illness, and duration of untreated psychosis were estimated by Spearman rank correlation. Differences were set to be significant when p-value were b0.05. Results Cognitive impairments in schizophrenia

Table 2 Voxel tissue composition.

Gray matter ratio White matter ratio Cerebrospinal fluid ns, not significant.

Control (n = 18)

Schizophrenia (n = 19)

p

77.0 ± 7.5 14.1 ± 6.0 8.9 ± 3.4

76.1 ± 4.5 13.6 ± 4.9 10.2 ± 3.0

ns ns ns

As shown in Table 3, schizophrenic patients showed significant cognitive deficits in the prefrontal cortex-related neuropsychological tests, verbal fluency WCST, Stroop test, DSDT and Trail making test, but not in the Iowa gambling task. These results are in good agreements with past studies (Green 1996; Woodward et al., 2005; Fioravanti et al., 2005).

Y. Shirayama et al. / NeuroImage 49 (2010) 2783–2790 Table 3 Performance on cognitive function tests.

Verbal fluency (letter) Verbal fluency (category) WCST (category) WCST (perseverative error) Stroop (C–D) Trail making test A Trail making test B DSDT (without distractor) DSDT (with distractor) Iowa gambling task

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Table 5 Concentration of taurine and NAAG in medial prefrontal cortex.

Control (n = 18)

Schizophrenia (n = 19)

p

40.6 ± 9.3 48.3 ± 7.5 5.22 ± 1.77 1.61 ± 2.33 6.42 ± 3.48 23.5 ± 7.0 54.2 ± 20.7 87.3 ± 15.4 91.8 ± 9.7 1.67 ± 15.5

29.6 ± 8.1 41.2 ± 8.4 3.05 ± 1.75 9.37 ± 6.28 11.45 ± 6.16 31.5 ± 11.6 75.2 ± 34.3 82.1 ± 18.4 77.7 ± 20.8 − 4.74 ± 13.5

b0.001⁎⁎⁎ 0.010⁎ 0.001⁎⁎ b0.001⁎⁎⁎ 0.005⁎⁎ 0.017⁎ 0.032⁎ ns 0.014⁎ ns

⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 when compared with normal controls. ns, not significant. WCST, Wisconsin card sorting test; DSDT, digit span distraction test.

Altered glutamine/glutamate ratios, NAA/(GPC + PC) ratios, and taurine levels in medial prefrontal cortex of schizophrenia No alterations were found for glutamate, myo-inositol, GPC + PC, NAA and Cr + PCr between control and schizophrenia, although glutamine content had a tendency to increase without significance in the schizophrenic patients (Table 4). Schizophrenic patients had a significant increase in the ratios of glutamine/glutamate compared with normal controls (Table 4). The ratio of glutamine/glutamate was related to glutamine and NAA, but not glutamate among all subjects examined (data not shown), indicating that the alteration of ratio of glutamine/glutamate could have contributed to the changes in glutamine levels, but not in glutamate levels. The ratios of NAA/(GPC + PC) in the schizophrenic patients were significantly higher than those in the normal controls (Table 4). The ratio of NAA/(GPC + PC) was highly related to GPC + PC and the ratio of (GPC + PC) / (Cr + PCr), but not to NAA or Cr + PCr, among all the subjects examined (data not shown), indicating that the alteration of the ratios of NAA/(GPC + PC) could have contributed to the changes in levels of GPC + PC. Levels of taurine were significantly higher in the schizophrenic patients than in the normal controls (Table 5). However, levels of NAAG were not significantly different between the two groups (Table 5). The levels of taurine were highly related to glutamate (r = 0.55,

Taurine NAAG

Control

Schizophrenia

p

2.23 ± 0.49 (n = 16) 1.19 ± 0.36 (n = 15)

2.64 ± 0.49 (n = 14) 1.09 ± 0.29 (n = 12)

0.030⁎ ns

Mean values and SD. ⁎ p b 0.05 compared with normal controls.

p = 0.001) and myo-inositol (r = 0.50, p = 0.004) among all the subjects. Correlation of brain metabolites and their ratios with clinical measures among patients with schizophrenia The duration of untreated psychosis was found to be significantly related to NAA/(GPC + PC) ratio (r = − 0.76, p b 0.001), (GPC + PC)/ (Cr + PCr) ratio (r = 0.62, p = 0.005), but not to NAA/(Cr + PCr) ratio. The duration of illness was significantly related to glutamine/ glutamate ratio (r = 0.51, p = 0.027) and taurine (r = − 0.59, p = 0.026). Other clinical measures such as BPRS, SANS, DIEPSS and age on set failed to have any significant relationship with brain metabolites and their ratios in the medial prefrontal cortex in schizophrenia (data not shown). Correlation of glutamine/glutamate ratio, NAA/(GPC + PC) ratio, and taurine with neuropsychological tests in all the participants The glutamine/glutamate ratio was related to the results of WCST (category, perseverative error) and DSDT (without distractor, with distractor), but not to the results of the trail making test, verbal fluency, and Iowa gambling task (Table 6). The ratio of NAA/(GPC + PC) was significantly related to the results of verbal fluency (letter) and WCST (only category) and showed a tendency (although not statistically significant) to have a relationship with verbal fluency (category), the Stroop test, and duration of untreated psychosis, but did not show any relationship with the DSDT, trail making test, or Iowa gambling test (Table 6). The levels of taurine were significantly related to the Stroop test and trail making test A, but not to verbal fluency, WCST, Iowa gambling test, and trail making test B (Table 6). Discussion

Table 4 Concentration of amino acids in medial prefrontal cortex.

〈Metabolites (mM)〉 Glutamine Glutamate Myo-inositol GPC + PC NAA Cr + PCr 〈Metabolite ratios〉 Glutamine/Glutamate Glutamate/(GPC + PC) Glutamate/(Cr + PCr) Glutamine/(GPC + PC) Glutamine/(Cr + PCr) Myo-inositol/(CR + PCr) Myo-inositol/(GPC + PC) NAA/(GPC + PC) NAA/(Cr + PCr) (GPC + PC)/(Cr + PCr)

Control (n = 18)

Schizophrenia (n = 19)

p

2.994 ± 0.704 9.568 ± 0.981 5.548 ± 0.719 1.751 ± 0.260 9.554 ± 1.016 7.527 ± 0.830

3.395 ± 0.720 9.402 ± 1.523 5.704 ± 0.677 1.804 ± 0.242 9.268 ± 1.304 7.485 ± 0.804

0.095 ns ns ns ns ns

0.312 ± 0.073 5.519 ± 0.519 1.275 ± 0.088 1.787 ± 0.682 0.417 ± 0.147 3.203 ± 0.443 0.738 ± 0.069 5.514 ± 0.584 1.273 ± 0.098 0.232 ± 0.020

0.363 ± 0.067 5.300 ± 0.886 1.256 ± 0.150 1.930 ± 0.383 0.475 ± 0.109 3.201 ± 0.515 0.762 ± 0.011 5.075 ± 0.473 1.240 ± 0.082 0.246 ± 0.022

0.034⁎ ns ns ns ns ns ns 0.017⁎ ns 0.054a

ns, not significant. Mean values with SD. ⁎ p b 0.05 compared with normal controls. a p b 0.07, a trend for changes without significance.

The first finding of the present study is that the glutamine/glutamate ratio was elevated in the medial prefrontal cortex of schizophrenic patients (Table 4) and that the level of glutamine had a tendency (although not statistically significant) to increase (Table 4). In support of this, recent studies demonstrated that the glutamine/ Table 6 Association of glutamine/glutamate ratio, NAA/(GPC + PC) ratio, and taurine levels with neuropsychological functions in all participants.

Verbal fluency (letter) Verbal fluency (category) WCST (category) WCST (perseverative error) Stroop Trail making test A Trail making test B DSDT (without distractor) DSDT (with distractor) Iowa gambling test

Glutamine/Glutamate ratio, N = 37

NAA/(GPC + PC) ratio, N = 37

Taurine, N = 30

− 0.01 − 0.02 − 0.36⁎ 0.44⁎⁎ 0.26 0.11 0.05 − 0.34⁎ − 0.41⁎ 0.07

0.34⁎ 0.30a 0.36⁎ − 0.24 − 0.32a − 0.23 − 0.13 0.04 0.05 0.07

− 0.08 − 0.29 − 0.31 0.11 0.48⁎⁎ 0.47⁎⁎ 0.29 0.09 0.02 − 0.10

⁎p b 0.05, ⁎⁎p b 0.01, coefficient in all subjects. a p b 0.07, a trend for changes without significance.

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glutamate ratio was elevated in the cerebrospinal fluid of schizophrenic patients (Hashimoto et al., 2005a) and that the level of glutamine was significantly higher in the medial prefrontal cortex of schizophrenic patients compared with controls (Bartha et al., 1997; Théberge et al., 2002). These results suggest impairment of glia– neuron interaction because the glutamine/glutamate ratio is thought to monitor the adaptation of the glutamine/glutamate cycle for high rates of synaptic glutamate-induced cell death (Ramonet et al., 2004). Dysfunction of glia–neuron communication might be involved in the pathophysiology of schizophrenia (reviewed by Hashimoto et al., 2005b). Furthermore, glutamine may protect neurons against factors that facilitate neuronal death (Watanabe et al., 1998). Glutamine is transported from astrocytes into neurons where glutaminase deaminates glutamine to produce glutamate. Therefore, levels of glutamine are relatively higher in the glial cells while levels of glutamate are relatively higher in the neurons. Thus, the increase in the ratio of glutamine/glutamate could be due to decreased glial function. In support of this, glial cell loss was found in the anterior cingulate cortex in subjects with schizophrenia (Stark et al., 2004). The second finding is that the ratio of NAA/(GPC + PC) was significantly decreased in the medial prefrontal cortex of schizophrenic patients compared with normal controls (Table 4). A previous study also showed that schizophrenic patients had reduced NAA/Cho (choline containing compounds) ratios and elevated Cho/creatine ratios in the anterior cingulate cortex compared to controls (Yamasue et al., 2002), indicating increased levels of Cho. Another study demonstrated that GPC levels significantly increased and PC levels had a trend to increase in the prefrontal cortex of schizophrenic patients, assuming that the increase in GPC levels could be due to increased phospholipase A2 or lysophospholipase activities (Shirayama et al., 2004). Furthermore, a recent study reported a significant correlation between NAA/(GPC + PC) levels and phospholipase A2 in the hippocampus of rats (Sartorius et al., 2006). It has been shown that phospholipase A2 and lysophospholipase activities are increased in schizophrenia (Ross et al., 1997; Smesny et al., 2005). Many studies showed that choline-containing compounds detected by 1H MRS are related to phospholipase A2 (reviewed by Boulanger et al., 2000). However, the present result did not show changes in levels of GPC + PC. The discrepancy might arise from differences in the VOI examined because our previous study included not only the medial prefrontal cortex but also the lateral prefrontal cortex and white matter. Further study will be needed to elucidate the possibility that choline-related compounds are involved in the pathophysiology of the prefrontal cortex in schizophrenia. Previous studies did not show any change in the NAA levels of medial prefrontal cortex in schizophrenia (reviewed by Steen et al., 2005; Stanley et al., 2007), in accordance with the present result. However, one study showed that NAA was significantly lower in the anterior cingulate of schizophrenic patients (Wood et al., 2007). We, therefore, cannot exclude the possibility of the involvement of NAA abnormality in the medial prefrontal cortex of patients with schizophrenia. The third finding is a significant increase in taurine levels of schizophrenic patients compared with normal controls (Table 5). It is likely that taurine plays an inhibitory role in neural transmission in the medial prefrontal cortex because taurine is an inhibitory neurotransmitter that activates GABAA receptors or strychninesensitive glycine receptors (del Olmo et al., 2000). Thus, increased levels of taurine might have a compensatory function against stressinduced increases in the excitatory amino acid glutamate or schizophrenia-induced decreases in the inhibitory amino acid GABA. In support, the present study showed that the levels of taurine had a significant relationship with the levels of glutamate. Although the present study failed to demonstrate an increase in glutamate levels, a previous study demonstrated that glutamate concentrations were increased in the dorsolateral prefrontal cortex in schizophrenia

(Tebartz van Elst et al., 2005). However, other studies showed that levels of glutamate were decreased in the anterior cingulate of medicated patients with chronic schizophrenia, but not never-treated patients with schizophrenia (Théberge et al., 2002, 2003). Meanwhile, a deficit in GABAergic neural transmission is assumed to exist in the prefrontal cortex of schizophrenia and is documented in relation to cognitive dysfunction (Volk and Lewis, 2005). A future study of taurine will be performed to elucidate its relationship with glutamate and GABA. The fourth finding is that the duration of untreated psychosis was significantly related to the ratios of NAA/(GPC + PC) and (GPC + PC)/ (Cr + PCr), and the duration of illness was significantly related to the glutamine/glutamate ratio and taurine levels among patients with schizophrenia. A previous study demonstrated a significant negative correlation between the NAA/Cho ratio in the anterior cingulate and the severity of blunted affect of schizophrenics (Yamasue et al., 2002). Here, it should be noted that the duration of untreated psychosis is associated with clinical outcome and response to antipsychotic treatment in schizophrenia (Ho et al., 2000; Marshall et al., 2005; Perkins et al., 2005). Therefore, it may that the ratios of NAA/(GPC + PC) and (GPC + PC)/(Cr + PCr) in the medial prefrontal cortex is related to the duration of untreated psychosis, being in association with clinical outcome and the response to antipsychotic treatment in schizophrenia. Meanwhile, since it is likely that the glutamine/ glutamate ratio is related with neuroprotection and taurine plays an inhibitory role in neurotransmission as discussed above, it seems true the glutamine/glutamate ratio and taurine levels are associated with the duration of illness in schizophrenia. Future study will be needed to elucidate the relationships between the biological markers and clinical measures. The final finding is that there was a significant correlation of DSDT with the glutamine/glutamate ratio, but not with the NAA/ (GPC + PC) ratio whereas there was a significant correlation of verbal fluency with the NAA/(GPC + PC) ratio, but not with the glutamine/ glutamate ratio (Table 6), suggesting that DSDT and verbal fluency are associated with the glutamine/glutamate ratio and the NAA/ (GPC + PC) ratio, respectively. Meanwhile, there were significant correlations of WCST with both glutamine/glutamate ratio and NAA/ (GPC + PC) ratio (Table 6), indicating that WCST is associated with both glutamine/glutamate ratio and NAA/(GPC + PC) ratio. In support, a recent study reported that a significant correlation was observed between NAA and glutamate/glutamine in the anterior cingulate cortex and learning potential on the WCST (Ohrmann et al., 2008). Furthermore, there were significant correlations of Stroop test and trail making test A with levels of taurine, but not with the ratios of glutamine/glutamate and NAA/(GPC + PC) (Table 6), suggesting that the Stroop test and trail making test A are associated with levels of taurine. It is likely that specific metabolites in the medial prefrontal cortex are associated with specific neurocognitive functions of the prefrontal cortex. The medial frontal cortex is thought to monitor failure (errors or negative feedback) or reduced probability (conflicts or decision uncertainty) (reviewed by Ridderinkhof et al., 2004). The cognitive tests related to the medial prefrontal cortex, Stroop test, trail making test and DSDT, involve sustained or switching attention while monitoring the failure or reduced probability. Meanwhile, the dorsolateral prefrontal cortex-dominant cognitive tests, WCST and verbal fluency, use the ability to monitor outcomes as well to evaluate them. Both the trail making and Stroop tests require performance of a visuomotor set-shifting task (Warner et al., 2006). Executive function includes cognitive flexibility and sifting cognitive sets. The WCST, trail making test B, Stroop test and verbal fluency are often used to examine executive function (Purdon et al., 2000). However, both the WCST and Stroop test require inhibitory control (Kosmidis et al., 2006), indicating that executive function also requires inhibitory control. A recent study demonstrated that reduced attentional

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engagement contributes to deficits in prefrontal inhibitory control in schizophrenia, suggesting that alterations in attentional and executive control functions can synergistically disrupt voluntary behavioral responses in schizophrenia (Reilly et al., 2008). It is well known that the medial prefrontal and anterior cingulate cortices are substantially different from each other. The anterior cingulate cortex is involved in a form of attention and emotional activity (Bush et al., 2000). When the cognitive demands of task switching increase, the anterior cingulate cortex is recruited efficiently rather than the medial prefrontal cortex (Johnston et al., 2007). Further, in depression, changes in blood flow in the dorsal anterior cingulate and medial frontal cortex occur on opposite sites after deep brain stimulation (Mayberg et al., 2005). Anatomically, the anterior and posterior portions of the medial frontal cortex play different roles in action monitoring and social cognition (Amodio and Frith, 2006). Therefore, a future 1H MRS study will be performed to separate the medial prefrontal cortex from the anterior cingulate cortex using the distinct localization of interest. As for experimental condition, the present result for glutamine is that glutamine spectrum did not always have standard deviation of the fits b20 %SD. Field strength and voxel volume are important points to obtain small fitting errors. For the purpose of studying the change in glutamine levels between the medial prefrontal cortex and anterior cingulate cortex, magnetic field of 4 T will be needed to obtain a typical standard deviation of the fit b20 %SD for glutamine. The same things can be stated for the study of taurine levels in the prefrontal cortex. Under the magnetic field of 3 T, a larger volume and an increase in numbers of scan will be needed to obtain reliable data from all the participants. In conclusion, schizophrenic patients showed statistically significant differences in the ratios of glutamine/glutamate and NAA/(GPC + PC) and the level of taurine in the medial prefrontal cortex compared with normal controls. Furthermore, we found significant correlations of the glutamine/glutamate ratio with DSDT and WCST results, the NAA/(GPC + PC) ratio with verbal fluency and WCST results, and the levels of taurine with the Stroop test and trail making test A results among the participants. The ratios of NAA/(GPC + PC) and (GPC + PC)/(Cr + PCr) had significant relationships with the duration of untreated psychosis among the schizophrenic patients. The glutamine/glutamate ratio and levels of taurine were significantly related to the duration of illness of the patients. These data suggest that specific metabolites in the medial prefrontal cortex are associated with the neurocognitive deficits in schizophrenia. References American Psychiatric Association, 1994. Diagnostic and Statistical Manual of Mental Disorders, 4th ed (DSM-IV). Washington, DC. Amodio, D.M., Frith, C.D., 2006. Meeting of minds: the medial frontal cortex and social cognition. Nat. Rev. Neurosci. 7, 268–277. Andreasen, N.C., 1982. Negative symptoms in schizophrenia: definition and reliability. Arch. Gen. Psychiatry 139, 784–788. Bartha, R., Williamson, P.C., Drost, D.J., Malla, A., Carr, T.J., Cortese, L., Canaran, G., Rylett, R.J., Neufeld, R.W., 1997. Measurement of glutamate and glutamine in the medial prefrontal cortex of never-treated schizophrenic patients and healthy controls by proton magnetic resonance spectroscopy. Arch. Gen. Psychiatry 54, 959–965. Bechera, A., Damasio, A.R., Damasio, H., Anderson, S.W., 1994. Insensitivity to future consequences following damage to human prefrontal cortex. Cognition 50, 7–15. Behar, K.L., Rothman, D.L., Petersen, K.F., Hooten, M., Delaney, R, Petroff, O.A.C., Shulman, G.I., Nvarro, V., Petrakis, I.L., Charney, D.S., Krystal, J.H., 1999. Preliminary evidence of low cortical GABA levels in localized 1H-MR spectra of alcoholdependent and hepatic encephalopathy patients. Am. J. Psychiatry 156, 952–954. Blakemore, S.J., 2008. The social brain in adolescence. Nat. Rev. Neurosci. 9, 267–277. Bonilha, L., Molnar, C., Horner, M.D., Anderson, B., Forster, L., George, M.S., Nahas, Z., 2008. Neurocognitive deficits and prefrontal cortical atrophy in patients with schizophrenia. Schizophr. Res. 101, 142–151. Boulanger, Y, Labelle, M, Khiat, A., 2000. Role of phospholipase A2 on the variations of the choline signal intensity observed by 1H magnetic resonance spectroscopy in brain diseases. Brain Res. Rev. 33, 380–389. Bowie, C.R., Leung, W.W., Reichenberg, A., McClure, M.M., Patterson, T.L., Heaton, R.K., Harvey, D., 2008. Predicting schizophrenia patients' real-world behavior with specific neuropsychological and functional capacity measures. Biol. Psychiatry 63, 505–511.

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