Cortico-Striatal GABAergic and Glutamatergic Dysregulations in Subjects at Ultra-High Risk for Psychosis Investigated with Proton Magnetic Resonance Spectroscopy.

BACKGROUND: Dysregulations of the major inhibitory and excitatory amino neurotransmitter systems of γ-aminobutyric acid and glutamate, respectively, have been described in patients with schizophrenia. However, it is unclear whether these abnormalities are present in subjects at ultra-high risk for psychosis.
METHODS: Twenty-three antipsychotic naïve subjects at ultra-high risk and 24 healthy control subjects, matched for age, sex, handedness, cigarette smoking, and parental education, underwent proton magnetic resonance spectroscopy scans in the dorsal caudate bilaterally and the medial prefrontal cortex at 3T. Levels of γ-aminobutyric acid and of the combined resonance of glutamate and glutamine (Glx) were obtained using the standard J-editing technique and expressed as peak area ratios relative to the synchronously acquired unsuppressed voxel water signal.
RESULTS: Higher levels of γ-aminobutyric acid (P<.001) and Glx (P=.007) were found in the dorsal caudate of the subjects at ultra-high risk than in the healthy controls. In the medial prefrontal cortex, likewise, both γ-aminobutyric acid (P=.03) and Glx (P=.006) levels were higher in the ultra-high risk group than in the healthy controls. No group differences were found for any of the other metabolites (N-acetylaspartate, total choline, or total creatine) in the 2 regions of interest.
CONCLUSIONS: This study presents the first evidence of abnormal elevations, in subjects at ultra-high risk, of γ-aminobutyric acid and Glx in 2 brain regions that have been implicated in the pathophysiology of psychosis, warranting longitudinal studies to assess whether these neurotransmitter abnormalities can serve as noninvasive biomarkers of conversion risk to psychosis as well as of illness progression and treatment response.

Introduction

Schizophrenia is a highly debilitating neuropsychiatric disorder of young adulthood onset and a leading cause of disability worldwide (Whiteford et al., 2013). Therefore, there is great interest in investigating subjects at ultra-high risk (UHR) for psychosis to evaluate the dynamic phenotypic and genotypic changes that occur with illness progression to gain a better understanding of the mechanisms of schizophrenia pathogenesis necessary for assessing the risk of conversion to full psychosis or for identifying new treatment targets (Yung et al., 1998; Singh et al., 2012).

Recent research in schizophrenia has focused on dysregulations of the major inhibitory and excitatory amino neurotransmitter systems of γ-aminobutyric acid (GABA) and glutamate, respectively (Benes, 1995; Lewis et al., 2005). Using proton magnetic resonance spectroscopy (1H MRS), a number of studies have sought to document these glutamatergic and GABAergic dysregulations in vivo in patients with schizophrenia. Most, but not all, of these studies have found elevations of glutamatergic compounds (ie, glutamate, glutamine, glutamate+glutamine (Glx) or glutamine/glutamate ratios) (Bartha et al., 1997; Theberge et al., 2002; Bustillo et al., 2010; Goto et al., 2012; Kegeles et al., 2012; Ota et al., 2012; Kraguljac et al., 2013) and of GABA (Kegeles et al., 2012) in unmedicated subjects in different phases of psychosis, while decreased or unchanged levels of the 2 neurotransmitters have been reported in chronic disease or in subjects treated with antipsychotic medication (Theberge et al., 2003; Goto et al., 2009; Reid et al., 2010; Yoon et al., 2010; Bustillo et al., 2011; Kegeles et al., 2012; Kraguljac et al., 2012; de la Fuente-Sandoval et al., 2013b; Rowland et al., 2013).

Recently, we and others reported the results of 1H MRS studies that found elevated glutamate levels in the dorsal caudate of subjects at UHR (de la Fuente-Sandoval et al., 2011) and of those at familial high risk for schizophrenia (Tandon et al., 2013), suggesting the involvement of this excitatory amino acid neurotransmitter in the early phases of schizophrenia. However, confirmation studies are needed, since not all prior 1H MRS studies had found such elevations in UHR subjects (Stone et al., 2009, 2010; Wood et al., 2010; Natsubori et al., 2014). Moreover, no prior 1H MRS study has, to our knowledge, measured in vivo brain GABA levels in subjects at UHR. The primary objectives of the present study were, therefore, to use 1H MRS to test, for the first time, the hypothesis that, as in schizophrenia (Kegeles et al., 2012), prefrontal GABA levels are abnormally elevated in the early stages of schizophrenia and to replicate our previous observation of abnormal elevations of glutamate levels in antipsychotic-naïve UHR subjects (de la Fuente-Sandoval et al., 2011).

METHODS

Participants

The present study, conducted between May 2012 and March 2013, was approved by the Ethics and Scientific Committees of the National Institute of Neurology and Neurosurgery (INNN) of Mexico. Written informed consent of all subjects was obtained, as were written consent of both parents and assent of subjects younger than 18 years of age, prior to participation.

Together, 47 subjects participated in the study and of these 23 met the criteria for UHR, as assessed with the Structured Interview for Prodromal Syndromes (SIPS) (Miller et al., 2003). The UHR participants were recruited through the Emergency Service and the Adolescent Program of Neuropsychiatric and Imaging Study of the INNN. All UHR participants included in this study were treatment seeking and were excluded if they (1) had a concomitant medical or neurological illness, current substance abuse, or history of substance dependence (excluding nicotine), or comorbidity of any other axis I disorders; (2) were considered to be at high risk for suicide; or (3) presented with psychomotor agitation. All UHR participants, furthermore, were required to be antipsychotic medication-naïve, and use of benzodiazepines, mood stabilizers, or antidepressants within 12 weeks of the scans was exclusionary. Information about previous use of psychoactive medication, including antipsychotics, was collected as part of the diagnostic interviews of the participants and their family members.

Twenty-four age- and sex-matched healthy subjects, as established using the Structured Clinical Interview for DSM-IV (First et al., 1997), were recruited to serve as the normal comparison group. Participants in the control group with a history of psychiatric illness or family history of schizophrenia were excluded.

All enrolled subjects were screened for drugs of abuse (cannabis, cocaine, heroin, opioids, and benzodiazepines) at screen and at 1 hour prior to the 1H MRS scans. Working memory performance was assessed in all subjects with the Letter-Number Span test (Wechsler, 1997).

Magnetic Resonance Neuroimaging Procedures

All the neuroimaging studies were conducted at the INNN on a 3T GE Signa EXCITE MRI system (GE Healthcare, Milwaukee, WI) equipped with an 8-channel head coil (Invivo, Orlando, FL). To ensure reproducible scan prescription and minimize head motion, each participant’s head was positioned along the cantho-meatal line and then immobilized with a forehead strap.

Structural MRI

A 3-plane localizer imaging series was obtained, followed by a volumetric T1 weighed spoiled gradient-recalled (SPGR) echo acquisition (TE=5ms, TR=12ms, TI=450ms, flip angle=20°, FOV=25.6cm, 256×256 matrix, 186 slices, and a slice thickness=1mm) oriented above and parallel to the anterior-posterior commissures line. The resulting 3D SPGR images were reformatted to sagittal and coronal views and used for optimal 1H MRS voxel placement and brain tissue segmentation.

1H MRS

In vivo brain GABA and Glx spectra were obtained from a 3.0×2.5×2.5-cm3 medial prefrontal cortex (MPFC) voxel (including portions of Brodmann areas 24, 32 and 10, and the pregenual anterior cingulate cortex) (Figure 1A) and from a 4.5×2.5×2.0-cm3 voxel that was prescribed to include primarily the dorsal caudate bilaterally (the lower end of voxel was located 3mm dorsal to the anterior commissure, including the maximum amount of gray matter and with a dorsal extension (thickness) of 2cm (Figure 1C). Levels of GABA and Glx in each voxel were recorded with the standard J-edited spin echo difference method (Rothman et al., 1993; Sailasuta et al., 2001), as fully described recently (Geramita et al., 2011) and illustrated in Figure 1B. Briefly, a pair of a frequency-selective inversion pulses was inserted into the standard point-resolved spectroscopy method and then applied on the GABA C-3 resonance at 1.9 ppm on alternate scans using TE/TR 68/1500ms. This resulted in 2 subspectra (Figure 1B, traces a-b) in which the GABA C-4 resonance at 3.03 ppm and Glx C-2 at 3.71 ppm were alternatively inverted or not inverted.

Figure 1.

(A) Sagittal (top) and axial localizer images showing the size and location of the medial prefrontal cortex voxel of interest. (B) Demonstration of in vivo human brain γ-aminobutyric acid (GABA) and glutamate+glutamine (Glx) detection by proton magnetic resonance spectroscopy (1H MRS) in the dorsal caudate: (a-b), single-voxel subspectra acquired in 13.4 minutes with the editing pulse on and off and 256 (512 total) interleaved averages; spectrum (c), difference between spectra (a) and (b) showing the edited brain GABA and Glx resonances; spectrum (d), model-fitting of the experimental spectrum (c) to obtain the GABA and Glx peak areas; spectrum (e), individual components of the fits; spectrum (f), residual of the difference between spectra (c) and (d). (C) Coronal (top) and axial localizer images showing dorsal caudate voxel size and location. NAA, N-acetyl-aspartate; tCho, total choline; tCr, total creatine. Comparable spectral quality and signal-to-noise ratios were also obtained for the MPFC voxel.

Subtracting these 2 subspectra yielded a spectrum consisting only of the edited GABA C-4 and Glx C-2 resonances, with all overlapping resonances eliminated (Figure 1B). For each voxel, the data were acquired in 13.4 minutes using 256 interleaved excitations (512 total), with the editing pulse on or off. While, in principle, the J-editing method cannot separate the individual components of the Glx peak, our recent data suggest that this resonance is predominantly glutamate due to the relatively low concentration of glutamine and the poor editing efficiency of the method for both compounds (Shungu, 2013; Shungu et al., 2013). The resulting raw 8-channel phased-array coil data were combined into a single regular 1D signal using the relative coil sensitivities derived from the unsuppressed voxel tissue water signal acquired with each receiver coil. The magnetic field homogeneity for the acquisitions was typically ≤12Hz, as assessed from the full-width at half maximum of the unsuppressed water resonance.

1H MRS Data Processing and Quantification

Details of the MRS data quality assessment criteria and procedures used in this study to retain or reject spectra for inclusion in group analyses are provided in the supplementary Material. The areas under the individual spectral peaks, which are proportional to the concentrations of the associated metabolites, were obtained as illustrated in Figure 1B (traces a-f) by frequency-domain fitting of each resonance to a Gauss-Lorentz (ie, pseudo-Voigt) function using a highly optimized public-domain Levenberg-Marquardt nonlinear least-squares minimization routine (Markwardt, 2009). The levels of GABA and Glx in the edited spectra were then expressed as ratios of peak areas relative to the synchronously acquired and similarly fitted unsuppressed voxel water signal (W), a method shown to have a high test-retest reliability for both GABA and Glx (Kegeles et al., 2006). Levels of N-acetylaspartate, total choline, and total creatine were likewise derived by fitting the subspectra recorded with the editing pulses turned off (Figure 1B, trace a).

Voxel Tissue Heterogeneity

To estimate the proportions of gray matter, white matter, and cerebrospinal fluid contained in each voxel of interest, the volumetric SPGR MRI data were segmented using the SPM8 software (Friston et al., 1995). In-house software developed in MATLAB (MathWorks, Natick, MA) was then implemented to generate a segmentation mask for each voxel, from which the proportions of gray matter, white matter, and cerebrospinal fluid were determined. These were then compared between the groups and, in case of significant differences, included in the statistical model as covariates.

Statistical Analysis

To identify potential confounding factors, unequal variance t tests compared the groups for age, gender, and voxel tissue heterogeneity. Factors that differed (P < .05) or trended toward a significant difference (.05