schizophrenia is a severe psychiatric disorder that affects 1% of the population worldwide ; it is characterized by a complex phenotype , including positive , negative , affective symptoms and cognitive impairments [ 1 , 2 ] .
schizophrenia psychosis usually occurs in late adolescence and early adulthood between ages of 18 and 25 , a key period for the maturation of the prefrontal cortex .
a recent neurodevelopmental model of schizophrenia has proposed that an imbalance of excitation and inhibition in the prefrontal cortex is involved in the pathophysiology of schizophrenia .
data from human and nonhuman primate brains have provided evidence of n - methyl - d - aspartate ( nmda ) receptor hypofunction in schizophrenia , contributing to the excitation of pyramidal neurons indirectly by reducing the activity of inhibitory -aminobutyric acid ( gaba)ergic interneurons .
the activity of gaba interneurons is mediated by nmda receptors , while glutamate ( glu ) serves as a precursor for the synthesis of gaba and glutathione [ 58 ] .
gaba interneurons , in turn , project to pyramidal neurons and influence their excitation [ 911 ] .
postmodern studies have further shown reduced synapse density in glutamatergic pyramid cells in schizophrenia , linking changes of synaptic plasticity to excitation - inhibition imbalance .
excessive excitatory synaptic pruning in the prefrontal cortex may cause progressive brain tissue loss and lead to the development of psychosis .
gaba activity may be decreased in certain brain regions in schizophrenia , which may lead to reduce cortical plasticity and abnormal pruning . using advanced proton magnetic resonance spectroscopy ( h mrs )
, neurochemical concentrations such as glutamate and -aminobutyric acid can be measured in vivo [ 12 , 1416 ] .
there is increasing evidence from mrs studies linking glutamatergic or gabaergic disturbances to cognitive deficits and the pathophysiology of schizophrenia [ 15 , 1721 ] .
thus , studies on the imbalance between gabaergic and glutamatergic systems should shed light into uncovering the mechanisms of schizophrenia [ 11 , 22 ] .
aberrant gabaergic or glutamatergic levels in schizophrenia have been separately reported in previous studies [ 18 , 21 , 2331 ] .
meta - analyses focusing on h mrs studies have shown glutamate alterations across several brain regions in schizophrenia [ 12 , 16 ] .
marsman et al . reported lower glu and higher glutamine ( gln ) levels in the medial frontal cortex in schizophrenia .
merritt et al . further suggested higher glutmate+glutamine ( glx ) levels of the medial frontal cortex in subjects at high risk for schizophrenia rather than first - episode or chronic schizophrenia .
higher glu and glx levels in the basal ganglia , gln in the thalamus , and glx levels in the medial temporal lobe were also found in schizophrenia .
reduced glx or glu levels in the medial prefrontal cortex ( mpfc ) and anterior cingulate cortex ( acc ) were observed in several studies on chronic schizophrenia but could not be repeated by other studies [ 30 , 32 , 33 ] .
the mixed findings on glutamatergic alterations could be due to the illness progress , severity , duration , or pharmacological treatment .
further studies on the ultrahigh risk for psychosis ( uhr ) and first - episode schizophrenia ( fes ) , in particular , including drug - nave subjects , are necessary to exclude the effects of medications . in vivo gaba measurements have recently become available , aided by newly developed mrs sequences , meshcher - garwood point resolved spectroscopy ( mega - press ) , and j - point resolved spectroscopy ( jpress ) [ 34 , 35 ] .
gaba alterations in schizophrenia are also controversial [ 15 , 21 , 27 , 28 , 3639 ] .
several studies have reported lower gaba levels in the acc , bilateral calcarine sulci , and mpfc in chronic schizophrenia [ 15 , 38 , 39 ] . however ,
unchanged or elevated gaba levels in the acc and mpfc have also been observed [ 15 , 28 , 37 , 39 ] .
three studies on drug - nave or medicine - free fes patients showed reduced gaba levels in bilateral calcarine sulci and the left basal ganglia [ 27 , 36 ] .
in addition , a recent uhr study showed higher gaba in the dorsal caudate and mpfc . therefore , more studies on gaba alterations in both drug - nave uhr and fes are needed .
one possible reason for the controversial findings of either gaba or glu alterations in schizophrenia could be that these two neurotransmitter systems interact with each other [ 22 , 4043 ] .
recently , several researchers have begun to examine the correlations between gaba and glutamatergic metabolites [ 17 , 19 ] .
kegeles et al . found a strong positive correlation between medial prefrontal gaba and glx levels across chronic schizophrenia and healthy controls ( hc ) .
similarly , de la fuente - sandoval et al . also suggested significant correlations between these two neurotransmitters in both the mpfc and dorsal caudate across uhr and hc .
in addition , these correlations could differ among various brain regions in the two groups .
for example , uhr subjects had a positive correlation between gaba and glx levels in the mpfc rather than the caudate , while hc had a positive correlation between gaba and glx in the caudate rather than the mpfc .
these results suggested that correlations between gaba and glx could be an important feature for dysfunctional gabaergic and glutamatergic neurotransmitters .
thus , more attention should be paid to studies combining the gaba and glu alterations in both drug - nave fes patients and uhr subjects .
this holds great promise for uncovering the impact of gaba and glu alterations on the onset and course of psychosis . in the present study
, we recruited drug - nave uhr subjects , drug - nave fes patients , and hc subjects and measured both gaba and glx levels in the mpfc .
the mpfc was selected as the region of interest because it has been widely implicated in schizophrenia , including by the postmortem   and mrs studies mentioned [ 17 , 19 , 46 ] .
we hypothesized that ( 1 ) medial prefrontal gaba or glx levels should change in gradient with illness progression , and these changes would be more prominent in fes patients than in uhr subjects ; ( 2 ) gaba levels should be correlated with glx levels in fes and uhr subjects , and the correlations may differ across the three groups .
twenty - one uhr individuals were recruited from the outpatients in shanghai mental health center ( smhc ) .
all uhr subjects met the criteria for at - risk mental state , as defined by the structured interview for prodromal syndromes ( sips ) and scale of prodromal syndromes ( sops ) [ 47 , 48 ] .
the method for identifying uhr subjects with prodromal symptoms was introduced in detail in our previous study .
all uhr subjects were drug - nave and completed their assessments on the day of their first visit to smhc .
other inclusion criteria were being between the ages of 16 and 40 years and having at least nine years of education .
exclusion criteria included current pregnancy , major medical or neurological illness , or a history of suicide risk or alcohol or drug abuse .
the mean duration of prodromal symptoms in the uhr group was 10.93  16.40 months .
was also obtained from his / her legal guardian if the uhr subject was younger than 18 years old .
sixteen fes patients were recruited from smhc and met the diagnostic criteria for schizophrenia or schizophreniform psychosis based on the structured clinical interview for dsm - iv ( patient edition ) .
these fes patients visited our hospital seeking help for the first time upon being diagnosed with schizophrenia and recruited , and they completed their assessments on the day of their first visit to smhc .
inclusion criteria for fes patients were having a first - episode illness , no history of exposure to antipsychotics , being between the ages of 16 and 40 years , and having at least nine years of education .
exclusion criteria included a current pregnancy , major medical or neurological illness , or a history of suicide risk or alcohol or drug abuse .
clinical symptoms of each fes patient were assessed using the 24-item brief psychiatric rating scale ( bprs , expanded version ) [ 50 , 51 ] .
written informed consent was also obtained from his / her legal guardian if the fes subject was younger than 18 years old .
other inclusion criteria were being between the ages of 16 and 40 years and having at least nine years of education .
exclusion criteria included a current pregnancy , major medical or neurological illness , and alcohol or drug abuse .
written informed consent was also obtained from his / her legal guardian , if the hc subject was younger than 18 years old .
the study was approved by the research ethics committee of smhc and in accordance with the declaration of helsinki .
handedness was determined by self - report from each participant , and all subjects were right - handed ( see table 1 ) .
mri data were obtained using a 3-tesla siemens verio mr scanner with a 32-channel head coil ( siemens ag , erlangen , germany ) .
anatomical t1-weighted images were acquired using a three - dimensional magnetic preparation fast gradient echo ( 3d - mrpage ) sequence with echo time ( te ) = 2.96  ms , repetition time ( tr ) = 2300  ms , field of view ( fov ) = 240  240  mm , 256  256 matrix , a slice thickness of 1.0  mm , and 192 continuous sagittal slices .
the t1-weighted images were used to localize the volume - of - interest ( voi ) for the following mrs acquisition .
all scans were reviewed by a senior radiologist who evaluated whether there were obvious artifacts , signal losses , or gross pathology .
the mrs data were acquired using a mega - press sequence with tr = 1500  ms , te = 68  ms , and 128 averages with water suppression .
the voi ( 30  30  30  mm ) of the mpfc was localized in the midsagittal and coronal slices , as shown in figure 1 .
the voi included brodmann areas 24 and 32 ( containing part of the anterior cingulate cortex ) .
automated shimming followed by manual shimming was conducted to reduce the water signal full - width at half maximum ( fwhm ) below 25  hz .
mrs raw data were processed using the lcmodel software ( version 6.3 - 0i ) .
absolute gaba and glx concentrations were quantified using scaling correction , the correction for relaxation , and partial volume effects based on the lcmodel package and lcmgui , respectively .
the edited spectra were fit using lcm - basis functions that were generated from phantom measurements using the mega - press sequence with the appropriate acquisition parameters .
the gaba peak arose at 3.01  ppm and the glx peak at 3.74  ppm .
the criteria for selecting reliable metabolite concentrations were based on the % sd of the fit for each metabolite , reflecting the cramer 
rao lower bounds ( crlb ) for the lcmodel analysis [ 28 , 55 , 56 ] . only the results
we obtained high spectral quality , as the % sd of all spectra from the three groups was lower than 15% . to calculate the proportion of grey matter ( gm ) , white matter ( wm ) , and cerebrospinal ( csf ) contained in the voi
, the volumetric 3d mprage mri data were segmented using the spm8 software . in - house software developed in matlab ( mathworks natick , ma ) was then implemented to create a segmentation mask for each voxel , from which the proportions of gm , wm , and csf were calculated . to obtain the tissue - composition - corrected metabolite intensities ,
each metabolite value was corrected for the csf content of the voi using the following formula : corrected metabolite level = uncorrected metabolite level/(1  c ) , where c is the fractional csf content of the voi .
the normality for the distribution of all independent variables , including age , years of education , and metabolite concentrations , was examined using kolmogorov - smirnov tests .
demographic variables , gm , wm , and csf volumes within the voi were compared among the three groups using analysis of variance ( anova ) .
chi - squared tests were performed to assess the group effect on gender and handedness .
one - way anova with the factor of group ( uhr , fes , and hc ) was conducted to compare group differences on gaba and glx levels .
post hoc tests were performed between each two groups with bonferroni corrections for multiple comparisons ( p = 0.017 was the significant threshold level ) .
age , education , gender , and handedness were all matched among the three groups .
these three groups did not differ in their signal - to - noise ratio ( snr ) ( p = 0.65 ) and fwhm ( p = 0.97 ) from the lcmodel .
no between - group differences were found in the proportion of gm ( p = 0.83 ) , wm ( p = 0.85 ) , or csf ( p = 0.81 ) in the voi of mpfc ( shown in table 2 ) . there were significant main group effects on both gaba ( p = 0.002 ) and glx ( p < 0.001 ) levels .
post hoc tests demonstrated significantly lower gaba levels in fes patients ( 2.27  0.56  mmol / l ) than hc ( 3.04  0.69  mmol / l , p = 0.003 ) and uhr participants ( 2.96  0.75  mmol / l , p = 0.01 ) .
post hoc tests also revealed significantly lower glx levels in fes patients ( 12.75  2.66  mmol / l ) compared with hc ( 16.57  3.17  mmol / l , p
< 0.001 ) and uhr subjects ( 16.37  2.56  mmol / l , p = 0.001 ) . however , both gaba and glx levels in uhr subjects were comparable with hc subjects ( p = 1.00 for gaba , p = 1.00 for glx ) ( shown in figure 2 ) . removing the four smoking hc subjects
there were positive correlations between gaba and glx levels in each group ( r = 0.53 , p = 0.009 for hc ; r = 0.56 , p = 0.008 for uhr ; r = 0.79 , p < 0.001 for fes ) , as shown in figure 3 .
further direct comparison of the correlation coefficients with z - transformation between the three groups did not indicate any significant differences ( z = 1.35 , p = 0.18 for hc versus fes ; z = 0.13 , p = 0.89 for hc versus uhr ; z = 1.21 , p = 0.23 for uhr versus fes ) .
in addition , the glx / gaba ratio did not indicate a significant main group effect ( f = 0.11 , p = 0.90 ; 5.63  1.21 for hc , 5.79  1.24 for uhr ; 5.73  0.88 for fes ) .
the present study measured both gaba and glx concentrations among drug - nave uhr subjects , drug - nave fes patients , and hc subjects .
significantly lower gaba and glx levels in the mpfc were observed in drug - nave fes patients than uhr or hc subjects , while gaba and glx levels in uhr subjects were comparable with hc subjects .
our findings , which are not confounded by medications , suggested that reduced gaba and glx levels should play an important role in the early stages of psychosis .
reduced medial prefrontal gaba concentration in fes patients indicates the existence of a dysfunctional gaba neurotransmitter system in the early stages of schizophrenia . in vivo measurements of gaba concentrations
however , findings showing gaba alterations in fes patients are relatively limited and less consistent [ 27 , 36 ] .
kelemen et al . found lower gaba / cr levels in the bilateral calcarine sulci in drug - nave fes patients .
lower gaba / cr ratios were also found in the left basal ganglia in early - onset fes and remained after six months of treatment with antipsychotic medications in another study .
however , unchanged gaba / cr ratios have been revealed in both the frontal lobe and parietooccipital lobe .
all these results suggest that alterations in gaba levels are related to the onset of schizophrenia but depend on the specific brain region and medication [ 27 , 36 ] .
our findings demonstrating reduced medial prefrontal gaba levels in drug - nave fes patients provide more evidence that gaba alterations have an important impact on the early stages of psychosis .
deficits in the gaba neurotransmitter system in schizophrenia have also been proven by postmortem and electrophysiological studies [ 5759 ] .
postmortem studies have demonstrated gaba deficits in a subclass of fast - spiking interneurons expressing ca - binding protein parvalbumin in patients with schizophrenia . reduced message rna and expression of gad67 ( the synthetic enzyme for gaba ) and gat1 ( the transporter that clears synaptic gaba )   as well as an apparent compensatory upregulation in postsynaptic gabaa receptors in the dorsolateral prefrontal cortex [ 57 , 61 ] , acc , and prefrontal cortex [ 45 , 59 , 62 ]
electrophysiological studies have provided more evidence for the dysfunctional action of gabaergic transmission in schizophrenia reflected by deficits in gamma oscillation , p50 , and the cortical inhibition index [ 6367 ] .
reduced auditory p50 suppression has reflected disrupted inhibitory gating of the brain in response to repeated auditory stimuli in drug - nave fes patients [ 63 , 65 ] .
fes patients showed reduced short - interval cortical inhibition ( sici ) and a prolonged cortical silence period ( csp ) compared with healthy control subjects , suggesting weakened gabaa - mediated inhibition [ 64 , 66 ] .
all these findings further support the hypothesis that dysfunctional gabaergic transmission exists in fes patients [ 6367 ] . in the present study , low medial prefrontal gaba levels
were only observed in fes patients not in uhr subjects . a positron emission tomography ( pet )
study demonstrated the reduced binding potential of gabaa / benzodiazepine receptors in the right caudate in uhr individuals .
a recent mrs study indicated an elevated gaba / water ratio in the mpfc in uhr subjects . in our previous study
, we found that gabab - mediated cortical inhibition was impaired in uhr , whereas gabaa - mediated cortical inhibition was not altered yet . whether gaba dysfunction has occurred before the onset of psychosis remains unclear .
there was interplay between two gaba - mediated neurotransmitter systems ( gabaa - mediated and gabab - mediated ones ) [ 70 , 71 ] .
thus , there might be a compensatory effect between two gaba subsystems , leading to gaba deficits in fes patients rather than uhr subjects [ 64 , 69 ] .
previous studies indicated that fes patients showed a reduced sici and a prolonged duration of csp , implying both gabaa and gabab dysfunction , whereas uhr subjects only had a reduced sici or prolonged duration of csp , suggesting partially impaired gaba functions [ 64 , 69 ] .
thus , medial prefrontal gaba reductions could be a candidate biomarker for the early stages of psychosis .
in addition , we found significant correlations between glx and gaba levels in fes patients , in accordance with previous studies [ 17 , 19 ] . kegeles et al . reported a positive correlation between gaba and glx levels in the mpfc in medicated and unmedicated patients with schizophrenia and hc subjects
. de la fuente - sandoval et al . found a positive correlation between gaba and glx levels across uhr and hc subjects .
glu serves as a precursor for the synthesis of gaba and glutathione [ 58 ] .
we hypothesized that the reduction of gaba levels might be secondary to a reduction in the glutamatergic system .
there was also a significant correlation between gaba and glx levels in uhr subjects , and both metabolites remained unchanged .
these findings suggest that the interplay of gaba and glx systems might act in a mutually compensatory manner .
both impaired gabaergic and glutamatergic systems may contribute to the development of psychosis [ 25 , 27 ] .
there are limitations of the present study ; thus , we should be cautious when interpreting our results .
first , mrs data were only acquired in one region ( mpfc ) , which made our results less comparable with previous studies on other regions , for example , the striatum , which will be improved in our further work .
second , we only recruited drug - nave subjects , which made our sample size for each group relatively small . considering that the heterogeneity of fes patients and uhr subjects may affect metabolic levels , further studies with a large sample size
in summary , we found both reduced medial prefrontal gaba and glx concentrations only in drug - nave fes patients but not in drug - nave uhr subjects , suggesting that gaba and glx alterations may be associated with the early stages of psychosis .
 altered -aminobutyric acid ( gaba ) , glutamate ( glu ) levels , and an imbalance between gabaergic and glutamatergic neurotransmissions have been involved in the pathophysiology of schizophrenia . however , it remains unclear how these abnormalities impact the onset and course of psychosis . in the present study , 
 21 drug - nave subjects at ultrahigh risk for psychosis ( uhr ) , 16 drug - nave patients with first - episode schizophrenia ( fes ) , and 23 healthy controls ( hc ) were enrolled . in vivo gaba and glutamate+glutamine ( glx ) levels in the medial prefrontal cortex 
 were measured using proton magnetic resonance spectroscopy . 
 medial prefrontal gaba and glx levels in fes patients were significantly lower than those in hc and uhr , respectively . 
 gaba and glx levels in uhr were comparable with those in hc . in each group , there was a positive correlation between gaba and glx levels . 
 reduced medial prefrontal gaba and glx levels thus may play an important role in the early stages of schizophrenia . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions