A Proton Magnetic Resonance Spectroscopic Study in Autism Spectrum Disorder Using a 3-Tesla Clinical Magnetic Resonance Imaging (MRI) System: The Anterior Cingulate Cortex and the Left Cerebellum

Abstract

The pathophysiology of autism spectrum disorder (ASD) is not fully understood. We used proton magnetic resonance spectroscopy to investigate metabolite concentration ratios in the anterior cingulate cortex and left cerebellum in ASD. In the ACC and left cerebellum studies, the ASD group and intelligence quotient- and age-matched control group consisted of 112 and 114 subjects and 65 and 45 subjects, respectively. In the ASD group, γ-aminobutyric acid (GABA)+/ creatine/phosphocreatine (Cr) was significantly decreased in the anterior cingulate cortex, and glutamate (Glu)/Cr was significantly increased and GABA+/Cr was significantly decreased in the left cerebellum compared to those in the control group. In addition, both groups showed negative correlations between Glu/Cr and GABA+/Cr in the left cerebellum, and positive correlations between GABA+/Cr in the anterior cingulate cortex and left cerebellum. ASD subjects have hypoGABAergic alterations in the anterior cingulate cortex and hyperglutamatergic/hypoGABAergic alterations in the left cerebellum.

Keywords

proton magnetic resonance spectroscopy magnetic resonance imaging (MRI)autism spectrum disorder γ-aminobutyric acid glutamate

Autism spectrum disorder (ASD) is a neurobiological disorder, the main cause and specific biological markers of which are not yet fully understood. It is characterized by impaired social interaction, communication, and stereotyped interests and behaviors. ASD can show neuroanatomic abnormalities over a wide region, including the limbic system, the anterior cingulate cortex (decreased cell size and cell packing density), and the cerebellum, and especially a loss of Purkinje cells.^1
-3

The anterior cingulate cortex plays important roles in “theory of mind” and executive function, and both of these functions have been shown to be impaired in ASD.^4
-6 Magnetic resonance imaging (MRI) studies have shown a decreased volume of the anterior cingulate cortex, and various functional MRI studies have found hypoactivation of the anterior cingulate cortex in ASD.^7

-10

The cerebellum is associated with not only coordination and balance but also higher brain functions, such as language, learning, and memory,¹¹ and these functions have been shown to be impaired in ASD. There has been much research on the cerebellum in ASD. Most volumetric findings have shown an increased size of the cerebellum and a decreased size of the cerebellar vermal lobules.¹² However, there have been contradictory reports, and the results are still controversial.

Glutamate (Glu), glutamine (Gln), and γ-aminobutyric acid (GABA) are closely correlated with one another in neurons and glial cells, and for the following reasons, these metabolites may be associated with the pathophysiology in ASD.

ASD is often complicated by epilepsy or spikes in the electroencephalogram (EEG), and epileptic seizures are propagated by Glu and inhibited by GABA.

Increased plasma and serum Glu levels have been reported in ASD.^13,14

Patients with ASD show minicolumnar abnormalities in the frontal and temporal lobes of the brain. Specifically, cell columns in brains of ASD patients were more numerous, smaller, and less compact in their cellular configuration, with a reduced neuropil space in the periphery.¹⁵ Patients with ASD are suspected to exhibit GABAergic dysfunction, because the neuropil space in the periphery is occupied by GABAergic interneurons.

Moreover, uPAR knockout mice, which showed decreased GABAergic interneurons in frontal and parietal regions, showed impaired social interaction, anxiety, and seizures, which resembled ASD.¹⁶

Patients with ASD have been shown to have a reduction of the glutamic acid decarboxylase (GAD) 65- and 67-kDa proteins in the parietal and cerebellar cortices.¹⁷

In addition, abnormalities in the Glu and GABA receptor subunit genes were detected in ASD.^18,19

The balanced interaction between Glu and GABA transmission is essential for higher brain functions, such as memory, learning, cognition, and emotion. An imbalance between Glu and GABA, leading to hyperexcitation, has been linked to ASD.^20
-22

Based on these results, we were interested in the glutamatergic and GABAergic systems in ASD. Because the main findings in ASD are impaired social interaction and communication, we have been investigating metabolite concentrations in various “social brain” regions using proton magnetic resonance spectroscopy (¹H MRS).²³ Recently, we reported ¹H MRS findings in the anterior cingulate cortex and left cerebellum in Asperger syndrome (AS) according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR) criteria,²⁴ and found decreased levels of N-acetylaspartate (NAA), creatine/phosphocreatine (Cr), choline-containing compounds (Cho), and myo-inositol (Ins) in the anterior cingulate cortex but not the left cerebellum. Furthermore, the levels of both Glu · Gln complex (Glx) and GABA were normal in both the anterior cingulate cortex and left cerebellum in AS. In this report, we describe for the first time ¹H MRS findings in the anterior cingulate cortex and left cerebellum in ASD, with a particular focus on Glu, Gln, and GABA+ (where + indicates slight contamination by macromolecules), which have not yet been well studied by ¹H MRS. We hypothesize that ASD subjects will show hyperglutamatergic/hypoGABAergic changes in the anterior cingulate cortex or left cerebellum. To our knowledge, there has been no previous report on the application of ¹H MRS (3-Tesla) for determining GABA+/Cr in the left cerebellum in ASD. This study provides considerable new data about ¹H MRS in ASD from the largest ASD and control groups to date.

Methods

Subjects

Anterior Cingulate Cortex Study

The ASD group consisted of 112 subjects (4-14 years old; mean age [standard deviation (SD)] = 6.4 [2.3]; 92 boys and 20 girls), and the control group consisted of 65 subjects (2-15 years old; mean age [SD] = 6.7 [3.1]; 40 boys and 25 girls). The control group included non-autistic below-normal Intelligence Quotient (IQ) and typically developed children. Typically developed children were investigated by brain MRI and ¹H MRS due to mild headache, and no abnormalities were found. Past history of asphyxia, severe head injury, abnormal neurologic findings, abnormal MRI findings, or some specific syndromes were excluded from both the ASD and control groups (ie, we investigated “idiopathic” ASD). ASD subjects were recruited from among outpatients at the Department of Pediatrics of Tokushima University. Diagnosis was conducted by 2 experienced pediatric neurologists according to the DSM-IV-TR criteria. The IQ was determined by the Tanaka-Binet intelligence scale; the mean IQ (SD) was 71 (30) in the ASD group and 70 (18) in the control group. The characteristics of the ASD and control groups are shown in Table 1.

Table 1.

Characteristics of the ASD and Control Groups in the ACC Study.^a

	IQ, M (SD)	Age, y, M (SD)	Range	Male:female
ASD (n = 112)	71 (30)	6.4 (2.3)	4-14	92:20**
Control (n = 65)	70 (18)	6.7 (3.1)	2-15	40:25

Abbreviations: ACC, anterior cingulate cortex; ASD, autism spectrum disorder; IQ, Intelligence Quotient; M, mean; SD, standard deviation.

^aThere were no between-group differences in IQ or age. A between-group difference in gender was observed using a χ² test for independence.

**P < .01.

Left Cerebellum Study

The ASD group consisted of 114 subjects (3-14 years old; mean age [SD] = 5.6 [2.3]; 98 boys and 16 girls), and the control group consisted of 45 subjects (2-15 years old; mean age [SD] = 6.5 [3.0]; 29 boys and 16 girls). Recruitment criteria were the same as those in the anterior cingulate cortex study. Mean IQ (SD) was 67 (25) in the ASD group and 73 (20) in the control group. The characteristics of the ASD and control groups are shown in Table 2. Eighty subjects in the ASD group and 40 subjects in the control group were subjected to studies of both the anterior cingulate cortex and left cerebellum. Only the anterior cingulate cortex was studied in 32 subjects in the ASD group and 25 subjects in the control group, and only the left cerebellum was studied in the remaining 34 subjects in the ASD group and 5 subjects in the control group.

Table 2.

Characteristics of the ASD and Control Groups in the Left Cerebellum Study.^a

	IQ, M (SD)	Age, y, M (SD)	Age Range	Male:female
ASD (n = 114)	67 (25)	5.6 (2.3)	3-14	98:16**
Control (n = 45)	73 (20)	6.5 (3.0)	2-15	29:16

Abbreviations: ASD, autism spectrum disorder; IQ, Intelligence Quotient; M, mean; SD, standard deviation.

^aThere were no between-group differences in IQ or age. A between-group difference in gender was observed using a χ² test for independence.

**P < .01.

A routine MRI examination was conducted for all subjects to confirm the absence of organic disease, and the ¹H MRS measurement was conducted immediately afterward. All subjects were sedated with triclofos sodium (Tricloryl, 0.5 mL/kg body weight) 1 hour before the examination, following the guidelines for monitoring and the management of pediatric patients during and after sedation published by the American Academy of Pediatrics,²⁵ because of restlessness due to their young age or developmental delay.

¹H MRS Measurement

All ¹H MRS studies were performed with a 3-Tesla clinical MRI system (Signa 3 T HD; GE, Milwaukee, WI) with a standard head coil for both the MRI and ¹H MRS measurements. Conventional proton MR spectra (ie, NAA, Cr, Cho, Ins, Glu, Gln, and Glu · Gln complex (Glx) [sum of Glu and Gln]) were obtained using the Stimulated Echo Acquisition Mode (STEAM) sequence with parameters of time to response (TR) = 5 seconds, time to echo (TE) = 15 milliseconds, and sum of signals = 48 to diminish the influence of longitudinal and transverse relaxation. The scan time was 5 minutes 6 seconds. GABA+ spectra were obtained using MEshcher-GArwood Point Resolved Spectroscopy (MEGA-PRESS) with parameters of TR = 2.5 seconds, TE = 68 milliseconds, and sum of signals = 256. The scan time was 6 minutes 54 seconds. In both the anterior cingulate cortex and left cerebellum studies, we used both the STEAM (for measurements of all metabolites without GABA) and MEGA-PRESS (for measurements with GABA) sequences. We analyzed these concentrations using the external standard calibration method in LCModel (Ver. 6.2). ¹H MRS studies often use Cr as an internal reference for other peaks, on the assumption that its concentration is relatively constant. In this study, we used metabolite concentration ratios (adjusted by Cr). We acquired T1 and T2 MRI images in axial and coronal views before the ¹H MRS examination, and placed single 6.0-mL (1.5 × 2.0 × 2.0 cm) (excluding GABA+) and 27.0-mL (3.0 × 3.0 × 3.0 cm) (GABA+) volumes of interest in the anterior cingulate cortex and left cerebellum (Figure 1). The large volume of interest in GABA measurements was due to the low concentration of GABA, which required an ample signal-to-noise ratio. We made minor adjustments to the position of the volume of interest to avoid including the bone and medullary cavity. The criteria for maintaining reliable metabolite concentrations were based on the %SD of the fit for each metabolite reflecting the Cramer-Rao lower bounds; only results with %SD below 15% (excluding GABA+) and 20% (GABA+) were included in the analysis.

Statistical Analysis

We compared the ASD and control groups with respect to the metabolite concentration ratios of NAA/Cr, Cho/Cr, Ins/Cr, Glu/Cr, Gln/Cr, Glx/Cr, and GABA+/Cr. We used Student t test to determine the statistical significance of differences. Furthermore, we performed various correlation analyses using the Pearson correlation coefficient. A value of P less than .05 was considered statistically significant. These statistical analyses were 2-tailed.

Results

Characteristics of the ASD and Control Groups in Both the Anterior Cingulate Cortex and Left Cerebellum Studies

These details were mentioned in the Subjects section and are shown in Tables 1 and 2. In both studies, there were no differences between the groups with regard to IQ or age. The only between-group difference was gender, using a χ² test for independence (P < .01) (Tables 1 and 2).

Comparison of the Metabolite Concentration Ratios in the ASD and Control Groups

The metabolite concentration ratios in the anterior cingulate cortex and left cerebellum were shown in Tables 3 and 4. In the anterior cingulate cortex, GABA+/Cr was significantly decreased in the ASD group (P < .05). In the left cerebellum, Glu/Cr was significantly increased (P < .01) and GABA+/Cr was significantly decreased (P < .01) in the ASD group.

Table 3.

Metabolite Concentration Ratios in the Anterior Cingulate Cortex.^a

	NAA/Cr	Cho/Cr	Ins/Cr	Glu/Cr	Gln/Cr	Glx/Cr	GABA+/Cr
ASD (n = 112)	1.20 (0.13)	0.26 (0.03)	0.77 (0.14)	1.38 (0.20)	0.55 (0.17)	1.91 (0.30)	0.15 (0.06)*
Control (n = 65)	1.21 (0.19)	0.26 (0.03)	0.80 (0.16)	1.39 (0.22)	0.57 (0.17)	1.96 (0.27)	0.18 (0.07)
P value	.51	.84	.24	.64	.43	.29	.02

Abbreviations: ASD, autism spectrum disorder; Cho, choline-containing compounds; Cr, creatine/phosphocreatine; Glx, Glu · Gln complex; GABA+, γ-aminobutyric acid (where + indicates slight contamination by macromolecules); Ins, myo-inositol; NAA, N-acetylaspartate.

^aGABA+/Cr was significantly decreased in the ASD group.

*P < .05.

Table 4.

Metabolite Concentration Ratios in the Left Cerebellum.^a

	NAA/Cr	Cho/Cr	Ins/Cr	Glu/Cr	Gln/Cr	Glx/Cr	GABA+/Cr
ASD (n = 114)	1.06 (0.32)	0.24 (0.20)	0.76 (0.25)	0.99 (0.32)**	0.51 (0.45)	1.54 (0.96)	0.23 (0.10)**
Control (n = 45)	0.98 (0.27)	0.25 (0.04)	0.80 (0.37)	0.84 (0.26)	0.50 (0.15)	1.35 (0.32)	0.29 (0.11)
P value	.13	.66	.42	.007	.82	.19	.001

^aGlu/Cr was significantly increased and GABA+/Cr was significantly decreased in the ASD group.

**P < .01.

Correlations Between Glu/Cr and GABA+/Cr in the Left Cerebellum in the ASD and Control Groups

There were negative correlations between Glu/Cr and GABA+/Cr in the left cerebellum in both groups (ASD group: correlation coefficient –0.62, P < .0001, n = 114; control group: correlation coefficient –0.44, P < .01, n = 45) (Figure 2). Because the ASD group showed heteroscedasticity, we also determined Spearman rank correlation coefficient in this group, and the results again suggested a significant negative correlation (P < .0001).

Correlations between Glu/Cr and GABA+/Cr in the Anterior Cingulate Cortex in the ASD and Control Groups

There were no correlations between Glu/Cr and GABA+/Cr in the anterior cingulate cortex in either group (Figure 3).

Correlations Between GABA+/Cr in the Anterior Cingulate Cortex and Left Cerebellum in the ASD and Control Groups

There were positive correlations between GABA+/Cr in the anterior cingulate cortex and left cerebellum in both groups (ASD group: correlation coefficient 0.43, P < .0001, n = 80; control group: correlation coefficient 0.54, P < .001, n = 40) (Figure 4).

Correlations Between Glu/Cr in the Anterior Cingulate Cortex and Left Cerebellum in the ASD and Control Groups

There were no correlations between Glu/Cr in the anterior cingulate cortex and left cerebellum in either group (Figure 5).

Correlations Between Age and Metabolite Concentration Ratios in the ASD and Control Groups

There were positive correlations between age and GABA+/Cr in the anterior cingulate cortex in both groups (ASD group: correlation coefficient 0.19, P < .05, n = 112; control group: correlation coefficient 0.41, P < .001, n = 65) (Figure 6). There were also positive correlations between age and GABA+/Cr in the left cerebellum in both groups (ASD group: correlation coefficient 0.25, P < .01, n = 114; control group: correlation coefficient 0.39, P < .01, n = 45) (Figure 7). No correlation was found between age and any other metabolite concentration ratio in either the anterior cingulate cortex or left cerebellum.

Gender-Associated Differences in Metabolite Concentration Ratios in the ASD and Control Groups

There was no significant difference in the metabolite concentration ratios in any region between genders in both groups.

Discussion

Glu, Gln, and GABA are closely correlated with one another in neurons and glial cells. Approximately 20% to 30% of neurons are GABAergic. Glu is converted to GABA by the rate-limiting enzyme GAD. Glu and GABA are excitatory and inhibitory neurotransmitters, respectively. There have been several reports concerning Glu and GABA+ concentrations in ASD using ¹H MRS, and the results showed decreased levels of GABA+, GABA+/NAA, and GABA+/Glu in the frontal lobe and normal levels of GABA+ in the basal ganglia,²⁶ increased levels of Glx in the pregenual anterior cingulate cortex,²⁷ increased Gln and decreased levels of GABA+/Cr in the anterior cingulate cortex,²⁸ decreased levels of Glx in the right anterior cingulate cortex,²⁹ decreased levels of Glx in the basal ganglia and normal levels of Glx in the dorsolateral prefrontal cortex in adults,³⁰ decreased levels of Glx/Cr and GABA+/Cr in the frontal lobe,³¹ decreased levels of GABA+/Cr in left auditory areas,³² decreased levels of GABA+/Cr in motor and auditory areas, and normal levels of GABA+/Cr in visual areas,³³ reduced GABAergic action in visual areas,³⁴ and increased levels of Glu in auditory areas.³⁵ To our knowledge, this is the first report on the application of ¹H MRS (3 Tesla) for determining GABA+/Cr in the left cerebellum in ASD.

In this study, we observed hypoGABAergic changes in the anterior cingulate cortex and hyperglutamatergic/hypoGABAergic changes in the left cerebellum. Impairment of the glutamatergic/GABAergic system induced by genetic or environmental factors may inhibit normal brain development, such as neurogenesis, migration, synaptic formation, and apoptosis, and this could result in anatomical or functional neuronal association derangement and the development of ASD. Bejjani et al reviewed possible mechanisms of hyperglutamatergic alterations.²⁷ They suggested that one possible mechanism might be greater presynaptic vesicular release of Glu and colocalized N-acetylaspartylglutamate (NAAG),³⁶ faster breakdown of NAAG into NAA and Glu,³⁷ net production rather than consumption of Glu and Gln by the Krebs cycle in neurons and astrocytes,³⁸ and slower conversion of Gln to GABA.³⁹ Excess Glu in the cerebellum may cause excitotoxic cell damage. Decreased GABA may be associated with insomnia, anxiety, stress, and epileptic seizures in ASD.⁴⁰

It has been reported that patients with ASD show a reduction of the GAD 65- and 67-kDa proteins in the parietal and cerebellar cortices.¹⁷ Thus, we were interested in the percentage of GAD activity out of the total glutamatergic/GABAergic metabolic enzyme activity (“relative GAD activity”) in the anterior cingulate cortex and left cerebellum. In this study, we investigated various relationships involving Glu/Cr and GABA+/Cr in the anterior cingulate cortex and left cerebellum in both ASD and control groups using ¹H MRS. These associations have not been previously reported. In both groups, we found (1) negative correlations between Glu/Cr and GABA+/Cr in the left cerebellum (Figure 2) and no correlations between Glu/Cr and GABA+/Cr in the anterior cingulate cortex (Figure 3), and (2) positive correlations between GABA+/Cr in the anterior cingulate cortex and left cerebellum (Figure 4), and no correlations between Glu/Cr in the anterior cingulate cortex and left cerebellum (Figure 5). There is little difference in these correlation pattern between the ASD and control groups. Result (1) may be due to a difference in a physiological metabolic mechanism in the glutamatergic/GABAergic system between the anterior cingulate cortex and left cerebellum in humans. If relative GAD activity was increased, it is more likely that a negative correlation between Glu and GABA would be more apparent. Thus, result (1) may indicate that relative GAD activity in the left cerebellum is much greater than that in the anterior cingulate cortex in humans. Result (2) may indicate that, although there is some correlation between the overall GABAergic metabolic mechanism in the anterior cingulate cortex and left cerebellum, there is little association between the overall glutamatergic metabolic mechanism in the anterior cingulate cortex and left cerebellum. These results suggest that the glutamatergic/GABAergic systems in each region differ with respect to their detailed mechanisms.

Figure 2 shows substantial heteroscedasticity, with less Glu/Cr and GABA+/Cr variance at higher values of Glu/Cr and lower values of GABA+/Cr (lower-right portion in Figure 2) in the left cerebellum in the ASD group. If we compare the Glu/Cr and GABA+/Cr variance in the lower-right of Figure 2 in the ASD group with that in the upper-left of Figure 2 or the control group, we see that the variance in the lower-right of Figure 2 in the ASD group is abnormally small. ASD is a pathophysiologically heterogeneous disorder. The ASD group in the lower-right of Figure 2 can be considered to be a subgroup, which showed abnormally weak GAD activity in the left cerebellum. A dysfunction in GAD activity may contribute to hyperglutamatergic/hypoGABAergic alteration with a consistent abnormally small variance in the left cerebellum. Thus, the heteroscedasticity in Figure 2 in the ASD group may indicate pathophysiological polymorphism due to GAD activity in the left cerebellum in ASD. On the other hand, the control group showed negative correlations between Glu/Cr and GABA+/Cr with no heteroscedasticity in the left cerebellum. This lack of heteroscedasticity may indicate a lack of pathological change in GAD activity in the left cerebellum in the control group. However, the lack of heteroscedasticity might be due to the low sample size.

The significant increase in Glu/Cr and decrease in GABA+/Cr in the left cerebellum in ASD (Table 4) may be due to a subgroup with abnormally weak GAD activity (ASD group in the lower-right of Figure 2). This result is consistent with a report by Fatemi et al,¹⁷ which showed a reduction in GAD proteins in the cerebellum in ASD. The decreased GABA+/Cr in the anterior cingulate cortex in ASD (Table 3) may be due to a somewhat common overall glutamatergic/GABAergic metabolic mechanism between the anterior cingulate cortex and left cerebellum, based on positive correlations between GABA+/Cr in the anterior cingulate cortex and left cerebellum, as shown in Figure 4. However, the absence of a correlation between Glu/Cr and GABA+/Cr in the anterior cingulate cortex in the ASD group (Figure 3) may suggest that relative GAD activity in the anterior cingulate cortex in the ASD group is low compared to that in the left cerebellum.

With regard to the effect of aging on the GABAergic system, it has been reported that the activity of the GAD and the concentration of GABA both decrease with age in several cerebral cortical areas, whereas the number of GABA-receptor-binding sites is either increased or unchanged.⁴¹ With regard to the correlation between aging and ¹H MRS findings, previous studies have reported no correlation between Glu or GABA+ and aging, a correlation between Glu or Gln and aging, and a negative correlation between Glu and aging in any region in normal subjects.^42
-44 The subjects in these reports were older than those in this child study. In our study, there were positive correlations between age and GABA+/Cr in both the anterior cingulate cortex and left cerebellum in the ASD and control groups (Figures 6 and 7). This result suggests that there are positive physiological correlations between age and GABA+/Cr in children.

This study was limited by a gender mismatch between the ASD and control groups. ASD is seen more frequently in males than females, and gender differences have been reported regarding clinical symptoms.^45
-47 Female subjects tend to show smaller sizes of the cerebellum compared to males by brain MRI.¹² With regard to gender differences in ¹H MRS findings, significant between-group differences in Glu, Glx, and GABA+, as well as, in contrast, the lack of a significant between-group difference in Glu, have been reported in normal subjects.^42,43 In this study, there was no significant between-gender difference in any of the metabolite concentration ratios for any region in the ASD and control groups. Therefore, our results should not be greatly influenced by gender differences.

Another limitation is the use of triclofos sodium for sedation. Triclofos sodium is a nonopioid and nonbenzodiazepine oral sedative drug that is used for pediatric sedation and produces hypnosis for 6 to 8 hours. The effect of triclofos sodium on the measured metabolites of ¹H MRS was unclear, and this issue needs to be investigated further.

In summary, we found hypoGABAergic alterations in the anterior cingulate cortex and hyperglutamatergic/hypoGABAergic alterations in the left cerebellum in ASD subjects, using in vivo ¹H MRS. This finding may contribute to a better understanding of the pathogenesis of autism.

Footnotes

Acknowledgments

The work was carried out at Tokushima University Hospital, Tokushima, Japan.

Author Contributions

HI contributed to the study conception and design; acquisition, analysis, and interpretation of data; and drafting the manuscript. KM and SK contributed to study conception and design and revising the manuscript critically for important intellectual content. MH and SH contributed to the acquisition, analysis, and interpretation of data and revising the manuscript critically for important intellectual content. YT, TM, AG, YA, and MM contributed to the conception and design of data.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

This study was carried out in accordance with the latest version of the Declaration of Helsinki and was approved by the ethics committee of Tokushima University Hospital (No. 998). Written informed consent was obtained from all of the subjects’ parents. Children who could understand the content and purpose of this study also provided their consent.

References

Bauman

Kemper

. Histoanatomic observations of the brain in early infantile autism. Neurology. 1985;35:866–874.

Lotspeich

Ciaranello

. The neurobiology and genetics of infantile autism. Int Rev Neurobiol. 1993;35:87–129.

Simms

Kemper

Timbie

. The anterior cingulate cortex in autism: heterogeneity of qualitative and quantitative cytoarchitectonic features suggests possible subgroups. Acta Neuropathol. 2009;118:673–684.

Baron-Cohen

Ring

Wheelwright

. Social intelligence in the normal and autistic brain: an fMRI study. Eur J Neurosci. 1999;11:1891–1898.

Brunet

Sarfati

Hardy-Baylé

Decety

. A PET investigation of the attribution of intentions with a nonverbal task. Neuroimage. 2000;11:157–166.

Owen

Evans

Petrides

. Evidence for a two-stage model of spatial working memory processing within the lateral frontal cortex: a positron emission tomography study. Cerebral Cortex. 1996;6:31–38.

Agam

Joseph

Barton

Manoach

. Reduced cognitive control of response inhibition by the anterior cingulate cortex in autism spectrum disorders. Neuroimage. 2010;52:336–347.

Gomot

Bernard

Davis

. Change detection in children with autism: an auditory event-related fMRI study. Neuroimage. 2006;29:475–484.

Kana

Keller

Minshew

Just

. Inhibitory control in high-functioning autism: decreased activation and underconnectivity in inhibition networks. Biol Psychiatry. 2007;62:198–206.

10.

Kennedy

Courchesne

. Functional abnormalities of the default network during self- and other-reflection in autism. Soc Cogn Affect Neurosci. 2008;3:177–190.

11.

Desmond

Fiez

. Neuroimaging studies of the cerebellum: language, learning and memory. Trends Cogn Sci. 1998;2:355–362.

12.

Stanfield

McIntosh

Spencer

. Towards a neuroanatomy of autism: a systematic review and meta-analysis of structural magnetic resonance imaging studies. Eur Psychiatry. 2008;23:289–299.

13.

Aldred

Moore

Fitzgerald

Waring

. Plasma amino acid levels in children with autism and their families. J Autism Dev Disord. 2003;33:93–97.

14.

Shinohe

Hashimoto

Nakamura

. Increased serum levels of glutamate in adult patients with autism. Prog Neuropsychopharmacol Biol Psychiatry. 2006;30:1472–1477.

15.

Casanova

Buxhoeveden

Switala

Roy

. Minicolumnar pathology in autism. Neurology. 2002;58:428–432.

16.

Levitt

. Disruption of interneuron development. Epilepsia. 2005;46(suppl 7):22–28.

17.

Fatemi

Halt

Stary

. Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. Biol Psychiatry. 2002;52:805–810.

18.

Cook

Jr Courchesne

Cox

. Linkage-disequilibrium mapping of autistic disorder, with 15q11-13 markers. Am J Hum Genet. 1998;62:1077–1083.

19.

Serajee

Zhong

Nabi

Huq

. The metabotropic glutamate receptor 8 gene at 7q31: partial duplication and possible association with autism. J Med Genet. 2003;40:e42.

20.

Chao

Chen

Samaco

. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature. 2010;468:263–269.

21.

Coghlan

Horder

Inkster

. GABA system dysfunction in autism and related disorders: from synapse to symptoms. Neurosci Biobehav Rev. 2012;36:2044–2055.

22.

Gogolla

Leblanc

Quast

. Common circuit defect of excitatory-inhibitory balance in mouse models of autism. J Neurodev Disord. 2009;1:172–181.

23.

Mori

Toda

Ito

. A proton magnetic resonance spectroscopic study in autism spectrum disorders: amygdala and orbito-frontal cortex. Brain Dev. 2013;35:139–145.

24.

Goji

Ito

Mori

. Assessment of anterior cingulate cortex (ACC) and left cerebellar metabolism in Asperger’s syndrome with proton magnetic resonance spectroscopy (MRS). PLoS One. 2017;12:e0169288.

25.

Cote

Wilson

. Guidelines for monitoring and management of pediatric patients during and after sedation for diagnostic and therapeutic procedures: an update. Pediatrics. 2006;118:2587–2602.

26.

Harada

Taki

Nose

. Non-invasive evaluation of the GABAergic/glutamatergic system in autistic patients observed by MEGA-editing proton MR spectroscopy using a clinical 3 Tesla instrument. J Autism Dev Disord. 2011;41:447–454.

27.

Bejjani

O’Neill

Kim

. Elevated glutamatergic compounds in pregenual anterior cingulate in pediatric autism spectrum disorder demonstrated by ¹H MRS and ¹H MRSI. PLoS One.2012;7:e38786.

28.

Cochran

Sikoglu

Hodge

. Relationship among glutamine, γ-aminobutyric acid, and social cognition in autism spectrum disorders. J Child Adolesc Psychopharmacol. 2015;25:314–322.

29.

Bernardi

Anagnostou

Shen

. In vivo ¹H-magnetic resonance spectroscopy study of the attentional networks in autism. Brain Res. 2011;1380:198–205.

30.

Horder

Lavender

Mendez

. Reduced subcortical glutamate/glutamine in adults with autism spectrum disorders: a [¹H]MRS study. Transl Psychiatry. 2013;3:e279.

31.

Kubas

Kułak

Sobaniec

. Metabolite alterations in autistic children: a ¹H MR spectroscopy study. Adv Med Sci. 2012;57:152–156.

32.

Rojas

Singel

Steinmetz

. Decreased left perisylvian GABA concentration in children with autism and unaffected siblings. Neuroimage. 2014;86:28–34.

33.

Gaetz

Bloy

Wang

. GABA estimation in the brains of children on the autism spectrum: measurement precision and regional cortical variation. Neuroimage. 2014;86:1–9.

34.

Robertson

Ratai

Kanwisher

. Reduced GABAergic action in the autistic brain. Curr Biol. 2016;26:80–85.

35.

Brown

Singel

Hepburn

Rojas

. Increased glutamate concentration in the auditory cortex of persons with autism and first-degree relatives: a (1)H-MRS study. Autism Res. 2013;6:1–10.

36.

Williamson

Neale

. Calcium-dependent release of N-acetylaspartylglutamate from retinal neurons upon depolarization. Brain Res. 1988;475:151–155.

37.

Cassidy

Neale

. N-Acetylaspartylglutamate catabolism is achieved by an enzyme on the cell surface of neurons and glia. Neuropeptides. 1993;24:271–278.

38.

Petroff

Mattson

Rothman

Proton

MRS

: GABA and glutamate. Adv Neurol. 2000;83:261–271.

39.

Ross

Bluml

. Magnetic resonance spectroscopy of the human brain. Anat Rec. 2001;265:54–84.

40.

Kang

Barnes

. A common susceptibility factor of both autism and epilepsy: functional deficiency of GABA A receptors. J Autism Dev Disord. 2013;43:68–79.

41.

Rehman

Masson

. Neuroendocrinology of ageing. Age Ageing. 2001;30:279–287.

42.

Chang

Jiang

Ernst

. Effects of age and sex on brain glutamate and other metabolites. Magn Reson Imaging. 2009;27:142–145.

43.

O’Gorman

Michels

Edden

. In vivo detection of GABA and glutamate with MEGA-PRESS: reproducibility and gender effects. J Magn Reson Imaging. 2011;33:1262–1267.

44.

Kaiser

Schuff

Cashdollar

Weiner

. Age-related glutamate and glutamine concentration changes in normal human brain: ¹H MR spectroscopy study at 4 T. Neurobiol Aging. 2005;26:665–672.

45.

Baron-Cohen

Knickmeyer

Belmonte

. Sex differences in the brain: implications for explaining autism. Science. 2005;310:819–823.

46.

Giarelli

Wiggins

Rice

. Sex differences in the evaluation and diagnosis of autism spectrum disorders among children. Disabli Health J. 2010;3:107–116.

47.

Hartley

Sikora

. Sex differences in autism spectrum disorder: an examination of developmental functioning, autistic symptoms, and coexisting behavior problems in toddlers. J Autism Dev Disord. 2009;39:1715–1722.

A Proton Magnetic Resonance Spectroscopic Study in Autism Spectrum Disorder Using a 3-Tesla Clinical Magnetic Resonance Imaging (MRI) System: The Anterior Cingulate Cortex and the Left Cerebellum

Abstract

Keywords

Methods

Subjects

Anterior Cingulate Cortex Study

Left Cerebellum Study

1H MRS Measurement

Statistical Analysis

Results

Characteristics of the ASD and Control Groups in Both the Anterior Cingulate Cortex and Left Cerebellum Studies

Comparison of the Metabolite Concentration Ratios in the ASD and Control Groups

Correlations Between Glu/Cr and GABA+/Cr in the Left Cerebellum in the ASD and Control Groups

Correlations between Glu/Cr and GABA+/Cr in the Anterior Cingulate Cortex in the ASD and Control Groups

Correlations Between GABA+/Cr in the Anterior Cingulate Cortex and Left Cerebellum in the ASD and Control Groups

Correlations Between Glu/Cr in the Anterior Cingulate Cortex and Left Cerebellum in the ASD and Control Groups

Correlations Between Age and Metabolite Concentration Ratios in the ASD and Control Groups

Gender-Associated Differences in Metabolite Concentration Ratios in the ASD and Control Groups

Discussion

Footnotes

Acknowledgments

Author Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

References

¹H MRS Measurement