Molecular Psychiatry (2014) 19, 20–29 & 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp

REVIEW Imaging glutamate in : review of findings and implications for drug discovery

EMP Poels1,2, LS Kegeles1,2, JT Kantrowitz1,2, M Slifstein1,2, DC Javitt1,2, JA Lieberman1,2, A Abi-Dargham1,2,3 and RR Girgis1,2

Currently, all treatments for schizophrenia (SCZ) function primarily by blocking D2-type dopamine receptors. Given the limitations of these medications, substantial efforts have been made to identify alternative neurochemical targets for treatment development in SCZ. One such target is brain glutamate. The objective of this article is to review and synthesize the proton magnetic resonance spectroscopy (1H MRS) and positron emission tomography (PET)/single-photon emission computed tomography (SPECT) investigations that have examined indices in SCZ, including those of modulatory compounds such as (GSH) and glycine, as well as data from ketamine challenge studies. The reviewed 1H MRS and PET/SPECT studies support the theory of hypofunction of the N-methyl-D-aspartate receptor (NMDAR) in SCZ, as well as the convergence between the dopamine and glutamate models of SCZ. We also review several advances in MRS and PET technologies that have opened the door for new opportunities to investigate the glutamate system in SCZ and discuss some ways in which these imaging tools can be used to facilitate a greater understanding of the glutamate system in SCZ and the successful and efficient development of new glutamate-based treatments for SCZ.

Molecular Psychiatry (2014) 19, 20–29; doi:10.1038/mp.2013.136; published online 29 October 2013 Keywords: glutamate; glutathione; magnetic resonance spectroscopy (MRS); NMDA; positron emission tomography (PET); schizophrenia

INTRODUCTION emission tomography (PET)/single-photon emission computed At present, the (typical or atypical) are the only tomography (SPECT) investigations that have examined glutama- approved treatments for schizophrenia (SCZ). In many patients, tergic indices in SCZ, including those of modulatory compounds 1 psychotic symptoms are only partially responsive or completely such as glutathione (GSH) and glycine. We will also review H MRS/ unresponsive to drugs,1 and there are currently no PET/SPECT work that has been performed using the ketamine agents with proven efficacy for negative symptoms or persistent challenge model in healthy subjects and discuss their patho- neurocognitive dysfunction. Even for subjects who respond well physiological implications. Preclinical and clinical evidence sup- to treatments, existing medications are associated with significant porting a role for each particular component of the glutamatergic cognitive, motoric or metabolic side effects that limit compliance. system in SCZ will be reviewed, followed by summaries of the 1 Thus, newer treatments for SCZ are desperately required. relevant H MRS and/or PET/SPECT literature. We will conclude All current treatments for SCZ function primarily by blocking D2- with a discussion of how these neurochemical findings inform the type dopamine receptors.2 Alternative neurochemical theories glutamate hypothesis of SCZ and can enhance drug development focus on disturbances in brain glutamatergic pathways, including of glutamatergic agents in SCZ. impairments in signaling at synaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs; Figure 1).3,4 Approaches for normalization of glutamatergic function are GLUTAMATE AND THE NMDA RECEPTOR currently under development. To date, promising results have Background: evidence for NMDA receptor dysfunction in SCZ been obtained with compounds such as glycine, D-serine, In initial studies with phencyclidine and ketamine in the early sarcosine5 and glycine transport inhibitors6 that target the 1960s, it was noted that both agents produced positive, negative glycine site of the NMDAR, and with N- (NAC), a and cognitive symptoms of SCZ.8,9 These compounds induce precursor compound for the amino acid cysteine in brain.5 symptoms by blocking neurotransmission at the NMDAR, sug- However, the slow rate of therapeutic development has led a gesting an alternative model for pathogenesis in SCZ. Sympto- group of leading experts from academia, government and matic effects of NMDAR blockade were better classified starting in PHARMA7 to conclude that effectively translating findings from the early 1990s in a series of ketamine challenge studies genetics and basic science into therapies will require the conducted in both normal volunteers and SCZ patients.10–13 In development of robust biomarkers and assessment of target normal volunteers, positive, negative and cognitive symptoms engagement of experimental agents.7 were observed in similar proportions as in SCZ. NMDAR blockade The objective of this article is to review and synthesize the also reproduces both the severity and type of thought disorder proton magnetic resonance spectroscopy (1H MRS) and positron seen in SCZ with both, for example, being associated with high

1Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY, USA; 2New York State Psychiatric Institute, New York, NY, USA and 3Department of Radiology, Columbia University College of Physicians and Surgeons, New York, NY, USA. Correspondence: Dr RR Girgis, Department of Psychiatry, New York State Psychiatric Institute, 1051 Riverside Drive, Unit 31, New York, NY 10032, USA. E-mail: [email protected] Received 23 March 2013; revised 25 August 2013; accepted 9 September 2013; published online 29 October 2013 Glutamate imaging in schizophrenia EMP Poels et al 21 levels of poverty of speech, circumstantiality and loss of goal, and relatively low levels of distractive or stilted speech or parapha- sias.11 These data and others suggest that reduction in NMDA functioning within the brain could serve as a single unifying feature to account for the otherwise complex pattern of deficits observed in SCZ (see review in Kantrowitz and Javitt4). The development of the glutamate hypothesis of SCZ, initially based in large part on the effects of phencyclidine, closely resembles the initial development of the dopamine hypothesis of SCZ, which was based in large part on observations of the psychotogenic effects of stimulant medications.14 PET and SPECT later supported this hypothesis by indicating increased dopamine synthesis,15–17 greater striatal amphetamine-stimulated dopamine release that is related to positive symptoms18–20 and increased baseline occupancy of striatal dopamine D2 receptors by dopa- mine that also predicts the response of positive symptoms to treatment with antipsychotic agents.21 The negative symptoms and cognitive deficits of SCZ are thought to be related, at least in part, to a cortical dopamine deficit, as originally proposed Figure 1. Schematic diagram of N-methyl-D-aspartate (NMDA) by Weinberger22 and Davis et al.23 based on preclinical and receptor showing D-serine and glutathione/redox-sensitive modula- tory sites (reprinted from Javitt Javitt DC, Int Rev Neurobiol, volume other indirect data. PET studies of cortical dopamine D1 24–27 78, Glutamate and schizophrenia: phencyclidine, N-methyl-D-aspar- receptors reported inconsistent results, and more direct tate receptors, and dopamine-glutamate interactions, pages 69–108, examination of dopaminergic transmission in the cortex is Copyright 2007 with permission from Elsevier). currently underway. A point of convergence of the dopamine and NMDA models is In summary, PET and SPECT studies testing the effects of NMDA at the level of local regulation of presynaptic dopamine release. blockade on dopaminergic indices in healthy subjects using Within striatum and frontal cortex, presynaptic dopamine release ketamine alone found mixed results in striatum,29–33,35 but is under control of intrinsic inhibitory GABAergic neurons that, in significant effects in cortex, consistent with prior rodent data.37 turn, are activated by NMDAR. In striatum, regulation of dopamine However, strikingly similar results for amphetamine-induced release appears to be modulated by GABAB receptors localized to dopamine release in individuals with SCZ and healthy subjects presynaptic dopamine terminals. GABA release, in turn, is given acute ketamine35 provide initial support for the glutamate/ modulated by NMDAR located on GABA interneurons, with 28 NMDA hypothesis of SCZ. Nevertheless, direct, in vivo measure- stimulation leading to increased GABA release. Therefore, ments of glutamatergic indices are necessary to translate some of the initial imaging studies of the glutamatergic system preclinical and clinical findings into effective therapies. Although sought to further validate the glutamate/NMDA model of SCZ by the development of PET and SPECT imaging of the glutamate examining the effects of ketamine on dopamine transmission and system has lagged behind that of the dopamine system, MRS receptors in healthy control subjects. technologies have effectively been utilized to measure gluta- matergic indices in vivo. Glutamate and dopamine PET/SPECT 1 Several studies used the D2/D3 receptor PET/SPECT ligands Glutamatergic H MRS [11C]raclopride and [123]iodobenzamide ([123]IBZM) to measure Several studies have compared glutamatergic indices—namely the effects of ketamine infusion in healthy control subjects on glutamate (Glu), (Gln) or Glu þ Gln (Glx)—in the brains 1 D2/D3 receptor availability in striatum, as a model of SCZ. Half of of individuals with SCZ with healthy control subjects using H these reported increased dopamine transmission in the stria- MRS. These findings are summarized below by brain region. tum,29,30 ventral striatum, left caudate and right putamen,31 Overall, there is a suggestion that glutamatergic levels are indicating an effect of NMDA blockade on striatal dopamine somewhat elevated in early-stage, drug-free patients, and release that is indirectly measured with this imaging technique. decrease after treatment. Future 1H MRS studies should focus on However, the remaining studies did not observe effects of the longitudinal assessment of patients in the prodromal and/or ketamine on dopamine release32,33 in the striatum. In cortex, drug-naive state and after conversion or treatment with medica- ketamine effects were reported in posterior cingulate cortex using tions in order to provide valuable information on the neurochem- 11 [ C]FLB457, a PET ligand capable of measuring cortical D2/D3 ical signatures of these groups and potentially allow researchers to receptors.34 Clinical correlations were mixed.30,33,34 One study identify biomarkers of SCZ risk. 123 used the D2/D3 SPECT ligand [ ]IBZM to study the effect of Notably, the synthesis of these data is complicated by a number ketamine on amphetamine-induced dopamine release in striatum of factors related to study heterogeneity, including different field 35 as measured by a change in D2/D3 receptor availability and strengths, description (for example, unmedicated, medicated, high reported a decrease in binding of [123]IBZM after amphetamine risk) and number of subjects in each study, neurochemicals infusion and a significantly lower binding when the subjects measured (that is, Glu, Glx and/or Gln, and whether they include received amphetamine under conditions of ketamine infusion. GABA), spectral editing/fitting techniques, use of segmentation (that This finding lends further validity to the glutamate/NMDA model is, controlling for tissue composition) and matching (that is, of SCZ, as the enhanced response to amphetamine resulting from confounds related to SCZ/healthy controls). This information for ketamine in healthy subjects is similar to the exaggerated each 1H MRS study cited herein that examined Glu, Glx or Gln is response to amphetamine seen in SCZ.18–20 summarized in Table 1. Several summary statements can be made. 36 In addition, Narendran et al. compared binding of the D1 Namely, every study examined appeared to use a method of receptor PET ligand [11C]NNC112 in chronic ketamine users spectral fitting, which is important for quantification of neurochem- and healthy control subjects, and reported higher D1 receptor ical peaks. Segmentation provides more robust measurements when availability in the dorsolateral prefrontal cortex (DLPFC) of chronic comparing across groups, but is not necessary for within-subject ketamine users similar to what has been observed in SCZ.24,25 designs. Third, although Glu, Glx or Gln are all standard measures of

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 20 – 29 Glutamate imaging in schizophrenia EMP Poels et al 22 Table 1. Scan and study parameters for Glu, Glx and Gln 1H MRS studies

Field Subjects (DN/DF/ Neurochemical (that is, Spectral Subject Source strength M/HR/HC) Sequence Glx, Glu, Gln) fitting Segmentation matchinga

Aoyama et al.57 4T 17b/0/0/0/17 STEAM Glu, Gln Y Y S, A, ED, PA ED Bartha et al.55 1.5T 10/0/0/0/10 STEAM Glu, Gln Y N S, A, ED, PA ED Bartha et al.71 1.5T 11/0/0/0/11 STEAM Glu, Gln Y N S, A, ED, PA ED Block et al.44 1.5T 0/0/25/0/19 PRESS Glx Y N c Bustillo et al.62 4T 0/0/14/0/10 STEAM Glu, Gln Y Y S, A, ET, SES Bustillo et al.58 4T 0/0/30/0/28 PEPSI Glx Y Y S, A, ET, PA ED/O Chang et al.68 4T 0/2/21/0/22 Double spin echo Glx Y N A PRESS Choe et al.64 1.5T 37/18/0/0/20 STEAM Glu (w/GABA)d YN c da Silva et al.41 3T 0/0/12e/0/23 PRESS Glx, Glu, Gln Y N S, A Egerton et al.66 3T 0/5/27/0/0 PRESS Glx, Glu Y N NA de la Fuente- 3T 22b/0/0/0/18 PRESS Glu Y Y S, A Sandoval et al.79 de la Fuente- 3T 18/0/0/18/40 PRESS Glu, Glx Y Y S, A Sandoval et al.77 de la Fuente- 3T 0/0/0/19/26 PRESS Glu, Gln Y f S, A, T Sandoval et al.80 Fusar-Poli et al.72 3T 0/0/0/24/17 PRESS Glu Y Y S, A, PR, IQ Galinska et al.38 1.5T 1/0/29/0/19 PRESS Glx Y N S, A, ED Goto et al.39 3T 0/0/18c/0/18 MEGA-PRESSg Glx Y N S, A Hutcheson et al.70 3T 0/0/28/0/28 PRESS Glx Y N S, A, PA, O Kegeles et al.47 3T 9/7/16/0/22 MEGA-PRESS Glx Y Y S, A, ET, T, PA, SES Keshavan et al.75 1.5T 0/0/0/40/46 PRESS CSI Glx Y Y S, A Kraguljac et al.61 3T 0/0/48/0/46 PRESS Glx Y Y S, A, PA, O, T Ohrmann et al.52 1.5T 18/0/21/0/21 STEAM Glx Y Y S, A, ED Ohrmann et al.53 1.5T 15/0/20/0/20 STEAM Glx Y Y S, A, ED Ohrmann et al.40 1.5T 0/0/43/0/37 PRESS Glx Y N S, A, ED Olbrich et al.48 2T 0/0/9/0/32 PRESS Glu, Gln Y N S, A, ED Ongur et al.59 4T 0/0/17/0/21 MEGA-PRESS and 4 Gln/Glu Y Y S, A pulse WET Ota et al.69 1.5T 0/0/46/0/27 PRESS Glx Y N S, A, ED Reid et al.63 3T 0/0/26/0/23 PRESS Glx Y N S, A, ET, PA, O Rowland et al.43 3T 0/0/20/0/11 PRESS Glx Y N S, A, ET Rusch et al.50 2T 0/0/29/0/31 PRESS Glu, Gln Y N S, A, ED Seese et al.45 1.5T 0/5h,i/23/0/34 PRESS Glx Y Y None Stanley et al.51 1.5T 13b/0/24/0/24 STEAM Glu, Gln Y N A, ED, PA, ED Stone et al.76 3T 0/0/0/27/27 PRESS Glu, Gln, Glx Y Y S, A, ET, SES, IQ Szulc et al.65 1.5T 0/14b/0/0/0 PRESS Glx (w/GABA) Y N NA Szulc et al.42 1.5T 0/42b/0/0/26 PRESS Glx (w/GABA) Y N S, A Tayoshi et al.78 3T 0/0/30/0/25 STEAM Glu, Gln Y Y S, A Theberge et al.54 4T 21/0/0/0/21 STEAM Glu, Gln Y Y S, A Theberge et al.74 4T 0/0/9/0/8 STEAM Glu, Gln Y Y S Theberge et al.56 4T 16b/0/0/0/16 STEAM Glu, Gln Y Y S, A, ED, PA, ED Thomas et al.67 1.5T 3i/0/10/0/12 STEAM Glx Y N S, A Valli et al.73 3T 0/0/0/22/14 PRESS Glu Y Y S, A, IQ van Elst et al.49 2T 0/0/21/0/32 PRESS Glu, Gln, Glx Y N S, A, ED Wood et al.60 3T 0/2/13/0/14 PRESS Glx Y N S, A, ED, PR, IQ Yoo et al.46 1.5T 0/0/0/22/22 PRESS Glx Y Y S, A Stone et al.81 3T 0/0/0/0/13 PRESS Glu Y Y NA Rowland et al.82 4T 0/0/0/0/10 STEAM Glu, Gln Y N NA Taylor et al.83 3T 0/0/0/0/17j PRESS and PRESS-Jk Glu, Glx Y Y NA

Abbreviations: A, age; DN, drug-naive schizophrenia; DF, drug-free schizophrenia; ED, education; ET, ethnicity; Gln, glutamine; Glu, glutamate; Glx, Glu þ Gln; HC, healthy control; 1H MRS, proton magnetic resonance spectroscopy; HR, high risk for schizophrenia; M, medicated schizophrenia; MRS, magnetic resonance spectroscopy; N, no; NA, not analyzed; O, occupation; PA, parental; PR, premorbid; PRESS, point-resolved spectroscopy; S, sex; SES, socioeconomic status; STEAM: stimulated echo acquisition mode; T, tobacco; Y, yes. aGroups were considered not matched if groups were statistically different. Otherwise, groups were considered matched whether or not formal statistical comparisons were performed. bMany to all of these subjects were also studied after treatment with antipsychotic medications. cNot readily apparent from the manuscript. dAppears that Glu includes Gln and Glu. eThese subjects had the 22q11 Deletion Syndrome. There were also 10 subjects with the 22q11 Deletion Syndrome who did not have SCZ. fSame sample as in de la Fuente-Sandoval et al. (2011). gMEGA- PRESS may also be referred to as PRESS with J-editing. hNot readily apparent from the manuscript whether these were drug-naive or drug-free subjects. iSubjects were children or adolescents. jEight subjects received ketamine, and nine received placebo. kPRESS-J may also be referred to as TE-averaged PRESS.

glutamatergic neurochemistry, Glu itself is the most pure measure, most studies that compared across groups controlled for gender and not all studies clearly indicated whether or not GABA was and age, several also controlled for additional characteristics, included in the Glx measure (that is, as opposed to only Glu and including education, IQ and/or parental education, although Gln), complicating the interpretation of findings. Finally, although statistical comparisons were not always reported.

Molecular Psychiatry (2014), 20 – 29 & 2014 Macmillan Publishers Limited Glutamate imaging in schizophrenia EMP Poels et al 23 DLPFC. Most studies of glutamatergic indices in the DLPFC show Table 2. Summary of MRS studies with ketamine measuring no differences between patient and control groups in either glutamatergic levels in healthy subjects medicated or unmedicated subjects,38–47 whereas some have 48–53 shown either increases or decreases in medicated subjects. Field Number Results in ACC Longitudinal studies investigating the effect of treatment with Source strength KS/PS Glu/Gln/Glx antipsychotic medications on glutamatergic measures in DLPFC show either decreases or no change.39,42,51 Finally, most of these Rowland et al.82a 2.0T 10/10 —/m/NA studies report no significant relationships between glutamatergic Stone et al.81 3.0T 13/0 m/NA/— 83 measures in DLPFC and clinical/behavioral symptoms. Taylor et al. 3.0T 8/9 —/NA/— Abbreviations: ACC, anterior cingulate cortex; Gln, glutamine; Glu, MPFC including ACC. It has consistently been reported that glutamate; Glx, Glu þ Gln; KS, ketamine subjects; MRS, magnetic resonance unmedicated patients have elevated glutamatergic levels in the spectroscopy; NA, not analyzed; PS, placebo subjects. ‘—’ Indicates no m a medial prefrontal cortex (MPFC) compared with healthy control difference. ‘ ’ Indicates higher levels. Same subjects had two scans, one with ketamine and one with placebo. subjects,47,54–56 although one study reported no differences.57 The pattern in medicated patients is less clear, although the majority of studies suggest that glutamatergic levels in medicated patients 40,47,58–63 Cerebellum. One study measured Glu and Glx levels in the are similar to those in healthy control subjects. cerebellum of a group of drug-naive prodromal and FE patients77 Longitudinal studies of subjects in unmedicated and medicated and reported no difference in Glu or Glx levels between patients states are few in number and do not consistently support an effect 56,57,62,64,65 and healthy control subjects in this region. The effect of of medications on glutamatergic indices. In addition, antipsychotic medication on glutamatergic levels in this region studies investigating relationships between glutamatergic levels in has not been investigated. the MPFC and clinical/behavioral symptoms have been mostly negative. Egerton et al.66 investigated Glu levels in a group of first- Basal ganglia. There seem to be no differences in glutamatergic episode (FE) patients, most of whom were medicated. Patients in indices between chronic medicated patients and healthy control remission had lower levels of Glu in the anterior cingulate cortex 44,78 subjects, although FE subjects demonstrated elevated Glx or (ACC) than nonremitted patients. In addition, across the whole 39,77,79 Glu. The results in high-risk or prodromal subjects are sample, ACC Glu levels were associated with greater negative mixed,75,77 although a longitudinal study demonstrated that symptoms and worse global functioning. In contrast, Reid et al.63 higher levels of Glu were observed in the psychosis transition reported that Glx levels were associated with less negative group when compared with the nontransition group and healthy symptoms in their sample. 80 control subjects. Another longitudinal study of antipsychotic- naive individuals with FE psychosis demonstrated that Glu levels Parietal and occipital lobe. It is difficult to draw conclusions in associative striatum decrease after 4 weeks of treatment with about glutamatergic indices in this area because of the paucity risperidone.79 The majority of clinical correlations have not been of studies, as well as significant differences in regions of significant. interest placement and patient populations. Some studies suggest abnormalities in glutamatergic levels in parietal or 1H MRS studies with ketamine challenge occipital cortex,67–69 whereas others do not.39,43,58,59 Relation- ships between neurochemical indices and clinical measures are To further test the glutamate/NMDA hypofunction theory of SCZ, also inconsistent. several investigations have examined the effect of ketamine on glutamatergic levels in the ACC, measured by 1H MRS (Table 2). Stone et al.81 scanned healthy subjects while receiving a ketamine Temporal lobe. There are no patterns of glutamatergic abnorm- infusion. Glutamatergic levels were measured by 1H MRS before 42,45 alities in the temporal lobe of patients with SCZ. One study and after ketamine infusion. This study reported increased levels also reports that treatment with antipsychotic medications may of Glu after ketamine infusion and observed a positive correlation 42 decrease levels of Glx. between Glu levels after ketamine administration and score on the PANSS (Positive and Negative Syndrome Scale) positive subscale. 82 Hippocampus/medial temporal lobe. Studies of glutamatergic Rowland et al. scanned healthy subjects with ketamine and with levels in hippocampus are mostly negative,48,50,61,70–73 although placebo and observed an increase in Gln levels during the first some have reported increased glutamatergic levels in chronic 10 min of ketamine infusion. After 10 min, ketamine was medicated patients.41,49,68 One longitudinal study reported no administered in a lower maintenance dose and Gln levels effect of medication in this population,65 although Glu, Gln and normalized. No correlations were observed between SANS (Scale GABA were measured together and the region included for the Assessment of Negative Symptoms) score and Gln levels. 83 hippocampus and temporal lobe. In addition, some Taylor et al. compared subjects who received ketamine with investigations suggest relationships between glutamate levels subjects who received placebo and reported no difference in Glx and executive functioning50 and global clinical state,49 although or Glu levels between these subject groups. Overall, these findings these findings need to be replicated. are consistent with findings of increased glutamatergic indices in MPFC in unmedicated patients with SCZ. Thalamus. There seems to be no clear pattern of glutamatergic abnormalities in the thalamus. Patients are found to be either no NMDA and SPECT different than control subjects,38,42,46,62,73–75 have increased One main limitation of glutamate 1H MRS studies is that 1H MRS indices in some FE drug-naive patients54,56,57 or have decreased provides a total tissue measure of neurochemicals and does not indices in some high-risk populations.72,76 The effects of distinguish between intracellular or extracellular compartments, or medication treatment are also mostly negative,57,62,65 although between intra- or extrasynaptic compartments, further limiting the one study reported a decrease in Gln levels after 30 months of specificity of such measurements. treatment with antipsychotic medications.56 Similarly, there do not PET and SPECT allow for a more selective measurement of brain appear to be relationships between glutamatergic levels in the neurochemistry than does 1H MRS. Although development of thalamus and symptomatology. tracers for glutamatergic targets with suitable kinetic properties

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 20 – 29 Glutamate imaging in schizophrenia EMP Poels et al 24 for imaging has proven to be more difficult than for some other Table 3. Summary of MRS studies measuring glutathione levels in the transmitter systems, several important studies have been brains of patients with schizophrenia performed. 123 The [ I]CNS-1261 is an intrachannel NMDA receptor SPECT Field Number PN/PF/ Results ligand used to study the NMDA receptor function in vivo in Source strength PM/HC Region GSH humans. Binding of [123I]CNS-1261 has been examined in a sample of patients compared with healthy control subjects,84–86 and in a Do et al.98 1.5T 5/4/5/10 MPFC k sample of healthy subjects after ketamine infusion.87 Total Matsuzawa 3.0T 0/0/20/16 PMFC — et al.99 distribution volume (VT), a measure of both specific and Terpstra 4.0T 0/0/13/9 ACC — nonspecific binding, was lower in all brain regions of clozapine 100 medicated patients and in healthy subjects after treatment with et al. Wood et al.97 3.0T 13/0/17/18 MTL m ketamine except in putamen, temporal and pericallosal regions. 123 The [ I]CNS-1261 BI1 and BI2 (measures of specific binding, Abbreviations: ACC, anterior cingulate cortex; GSH, glutathione; HC, 88 similar to BPP and BPND, respectively, using whole cortex as a healthy control subjects; MPFC, medial prefrontal cortex; MRS, magnetic reference region) were lower in the left hippocampus of drug-free resonance spectroscopy; MTL, medial temporal lobe; PF, drug-free patients; patients and in the right frontal lobe of clozapine-treated patients PM, medicated patients; PMFC, posterior medial frontal cortex; PN, drug- m k when compared with healthy control subjects. Higher specific naive patients. ‘—’ Indicates no difference. ‘ ’ Indicates higher levels. ‘ ’ Indicates lower levels. binding was observed in the right cerebral regions (including occipital lobe) of clozapine-treated patients compared with healthy control subjects. The results of these SPECT studies are consistent with the Glutathione and 1H MRS current understanding of the glutamate/NMDA hypothesis of SCZ AlthoughGSHquantificationwithspectroscopyischallenging, that states that hypofunction of this system contributes to the evidence from 1H MRS studies also supports a role for GSH in etiology of SCZ. Although these data need to be replicated with SCZ (Table 3). Four studies have used 1HMRStomeasure additional, specific ligands, the field has recently been taking GSH levels in the brains of individuals with SCZ and healthy advantage of technological advances in brain imaging to examine control subjects. Using a PRESS (Point-RESolved Spectroscopy) additional parameters of the glutamate system, with a focus on sequence and the linear combination model for spectral neurochemicals that modulate the NMDAR, such as glycine and analysis, increased levels of GSH were measured in the temporal GSH. lobe of a group of FE patients, some of whom were medicated97 and no significant relationship was observed between GSH levels and PANSS total score.97 Do et al.98 used a double NMDA RECEPTOR MODULATION quantum coherence filter technique with PRESS and observed Background: NMDAR modulation decreased levels of GSH in the MPFC of a heterogenous group of patients with SCZ. Matsuzawa et al.99 used the MEGA-PRESS NMDARs are activated by glutamate, which is the main excitatory sequence to acquire GSH spectra and reported no difference in neurotransmitter in the brain. NMDARs are modulated at two GSH levels in the posterior medial frontal cortex in atypical independent sites—a glycine/D-serine modulatory site and a proton medicated chronic patients compared with healthy control (redox)-sensitive site (Figure 1)—that therefore may serve as subjects, although negative clinical correlations were observed independent targets for pharmacological manipulation. The redox between GSH levels and scores on total SANS, negative site is sensitive to compounds that modulate local pH and redox BPRS (Brief Psychiatric Rating Scale) and the Trail Making Test. state, including polyamines and GSH. GSH (g-L-glutamyl-L-cysteinyl- Similarly, a study that used STEAM (STimulated Echo Acquisition glycine) is synthesized in brain from the amino acids cysteine, Mode) spectroscopy revealed no difference in GSH levels in the glutamate and glycine. The availability of cysteine is rate limiting for ACC between chronic medicated patients and healthy control GSH synthesis. GSH is a nonprotein thiol that is the primary brain subjects.100 In this study, Terpstra et al.100 supported their antioxidant and thus important in protecting the brain against findings by cross-validating their STEAM and MEGA-PRESS oxidative stress.3,89 When presented extracellularly, GSH or other sequences. reducing agents increase glutamate-induced depolarization via Although the use of spectral fitting and sequences that utilize NMDAR activation, whereas extracellular oxidizing conditions J-coupling (for example, MEGA-PRESS) produce robustly quantifi- decrease this response.90 NAC is a prodrug of cysteine with high able spectra, other techniques for measuring GSH such as double oral bioavailability and has primary effects as an antioxidant by quantum coherence filter have been validated.101,102 Therefore, stimulating the formation of GSH from cysteine.3,91 the contribution of differences in sequences and spectral editing Disturbances of GSH metabolism have been linked to SCZ based techniques to the discrepancies in the GSH literature remains upon several lines of evidence. Mice with a chronic deficit in GSH unclear. In addition, other factors, such as medication status and induced by knocking out the gene for the glutamate–cysteine phase of illness, may contribute to these different findings. ligase modulatory subunit demonstrated elevations in anterior Although only a few studies have examined GSH levels in SCZ cortical glutamate and glutamine levels that were similar to what is and the results are mixed, these results suggest abnormalities of observed in unmedicated patients or early SCZ.92 Treatment with GSH in SCZ. Future research should examine the effects of NAC normalized these levels.92 Experimentally induced GSH treatment on GSH levels in drug-naive/free subjects, as well as deficits in rodents lead to impairments in long-term potentiation examine the effects of NAC challenge and ketamine challenge on and NMDAR dysfunction,93 and may also contribute to cognitive GSH levels. deficits, suggesting a role of GSH depletion in cognitive impairments in SCZ.94,95 Finally, NAC, which is converted to GSH in vivo, has been used in one long-term clinical study of SCZ to date. In this study, Berk et al.96 randomized 140 medicated patients Glutathione and PET/SPECT with SCZ to either placebo or 1 mg NAC b.i.d. for 24 weeks. Patients PET103 and SPECT104 ligands for GSH and GSH transferase are who received NAC had greater improvements on the PANSS total, being developed but remain in the preclinical stage of general and negative subscales, as well as on the CGI-S (Clinical development. Furthermore, their penetration of the blood–brain Global Impression–Severity) scale (effects sizes 0.43–0.57). barrier has not been established.

Molecular Psychiatry (2014), 20 – 29 & 2014 Macmillan Publishers Limited Glutamate imaging in schizophrenia EMP Poels et al 25 Post-Synaptic Neuron

Modulation of Glu Excitation and Plasticity Glu Depolarize Glu Glu Glu Ca++, Na+ Glu Glu Glu Glu Glu Glu Glu NMDA

Glu Glu IPs, Ca++

Glu Glu Glu Glu Glu Glu Gly N-Acetyl cysteine Glu Cystine Glu Glu Glu GlyT1 Xc- mGlu5

Glu Gly Glu Glial Cell Figure 2. Schematic diagram of glutamatergic synapse showing potential interactions between type 5 metabotropic 7 (mGluR5), N-methyl-D-aspartate (NMDA) receptors and the cystine/glutamate antiporter (reprinted from Javitt et al. Reprinted and modified with permission from The American Association for the Advancement of Science).

Glycine and PET/SPECT mGluR5 is coupled to the NMDAR via a protein complex including As described above, the NMDAR is modulated at both a redox site postsynaptic density protein (PDS95), shank and homer 116–118 and a glycine/D-serine site (Figure 1). To date, promising clinical proteins. results have been obtained with compounds such as glycine, Selective modulation of mGluR5 in preclinical models of SCZ 5 6 D-serine, sarcosine and glycine transport inhibitors that target has demonstrated potential as a therapeutic strategy. Treatment the glycine site of the NMDAR. Although no MRS, PET or SPECT of rodents with selective, positive modulators of mGluR5 improves 119 120,121 studies of the glycine transporter have been performed in SCZ, models of positive symptoms and cognitive deficits, several PET tracers that target the glycine transporter type 1 (GlyT-1), whereas inhibiting or knocking out the mGluR5 leads to negative 122 123 such as [11C]GSK931145 (see refs. 105 and 106) and symptoms or cognitive deficits. mGluR5 knockout mice also 124 [11C]RO5013853,107,108 have been developed. These have proven exhibit SCZ-like deficits in prepulse inhibition that are not 125 useful for quantifying occupancy of GlyT-1 by other, unlabeled reversible with antipsychotic treatment. Furthermore, phen- compounds, but it is not yet clear whether they are suitably sensitive cyclidine-induced deficits in prepulse inhibition were potentiated enough for examining GlyT-1 availability in psychiatric populations in rats treated with MPEP (2-methyl-6-phenylethynylpyridine), an 124,126 including SCZ. [18F]MK-6577 is another promising GlyT-1 tracer but antagonist of the mGlu5 receptor. remains to be characterized in humans.109,110 An inherent problem In sum, preclinical models suggest that mGluR5 may be a for any radiotracer for this target is the lack of a reference tissue, that promising target for drug development in SCZ, as decreasing is, a brain region mostly devoid of GlyT-1 that can be used to transmission through the mGluR5 appears to mimic some of the estimate nonspecific binding. This limits the information that can be clinical phenomena of SCZ, whereas enhancing transmission obtained from a single scan with any of these tracers to regional appears to ameliorate these phenomena. Although studies in distribution volume, a parameter that is the sum of several terms, preclinical species are encouraging, few post-mortem studies not all of which are related to GlyT-1 availability.88 comparing mGluR5 levels and expression in individuals with SCZ and healthy control subjects have been performed, and the results from these studies have been equivocal.115 Therefore, clinical studies are needed to validate this target for therapeutic development. The TYPE 5 METABOTROPIC GLUTAMATE RECEPTOR (MGLUR5) recent development of the PET ligand [11C]ABP688, an mGluR5 mGluR5 and SCZ allosteric antagonist, has made it possible for in vivo imaging to Recent preclinical evidence suggests that targeting the mGluR5 make an important contribution to this process.127 may be an alternate and effective way of ameliorating NMDAR hypofunction in SCZ. mGluR5 belongs to group I of metabotropic mGluR5 PET receptors (mGluR1 and mGluR5) that signals via phospholipase C [11C]ABP688 is a glutamatergic radioligand that binds to an and potentiates NMDAR currents (Figure 2), as opposed to group II allosteric site on mGluR5, suggesting it may have potential as an and III receptors (mGlu2, 3, 4, 6, 7, 8) that tend to limit glutamate in vivo probe for glutamatergic transmission,127 as well as mGluR5 release (see review in refs. 111 and 112). mGluR5 is particularly levels. The use of [11C]ABP688 to measure mGluR5 has been implicated as a potentiator of NMDA function as agonists of either validated in humans.127 Although one study demonstrated mGluR1 or mGluR5 potentiate NMDAR currents, but only an questionable test–retest properties128 in humans, numerous mGluR5 selective antagonist blocked this potentiation.113,114 preclinical129–131 and human studies have demonstrated good Similarly, group I mGluR agonists only potentiated NMDAR effects test–retest properties as well as binding characteristics that allow in mGluR1 knockout mice, but not in mGluR5 knockout mice.113,114 for comparison between clinical populations,127,132,133 including The importance of mGluR5 receptors on NMDAR function is major depressive disorder133 and sleep deprivation.132 There are highlighted not only by their functional interconnection but also currently no studies using [11C]ABP688 to measure mGluR5 levels by their physical interconnection. The mGluR5 colocalizes with the in SCZ. The [18F]FPEB is another promising mGluR5 tracer that has NMDAR in pyramidal cells (Figure 1).115 Studies have shown that recently been characterized in humans.134,135 The development of

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 20 – 29 Glutamate imaging in schizophrenia EMP Poels et al 26 [11C]ABP688 and [18F]FPEB brings with it the possibility of directly CONFLICT OF INTEREST investigating the mGluR5 system in SCZ and assessing its potential EMP Poels declares no conflict of interest. Dr Kegeles has received research support for drug development. from Pfizer and Amgen. Dr Kantrowitz reports having received consulting payments within the last 2 years from Quadrant Health, RTI Health solutions, the Sacoor Medical Group, the Healthcare Advisory Board and AgencyRx. He has conducted clinical research supported by the NIMH, Roche-Genetech, EnVivo, Psychogenics, Sunovion, SUMMARY Novartis, Pfizer, Lilly and GlaxoSmithKline. He owns a small number of shares of The current glutamate model of SCZ suggests hypofunction of the common stock in GlaxoSmithKline. Dr Slifstein has consulted for Amgen and has NMDAR. This is supported by the 1H MRS and PET/SPECT studies received research support from Pierre Fabre. Dr Javitt holds intellectual property for reviewed above. Namely, ketamine infusion in healthy control use of glycine, D-serine and glycine transport inhibitors in the treatment of schizophrenia, and holds equity in Glytech. Dr Lieberman serves on the Advisory subjects results in increased amphetamine-induced dopamine Board of Bioline, Intracellular Therapies and PsychoGenics. He does not receive direct release in striatum. In addition, chronic ketamine use leads to financial compensation or salary support for participation in research, consulting or increased D1 receptor availability in cortex, consistent with what is advisory board activities. He receives grant support from Allon, F Hoffman-La Roche, 1 observed in SCZ, at least in some studies. The H MRS studies have GlaxoSmithKline, Eli Lilly, Merck, Novartis, Pfizer, Psychogenics, Sepracor (Sunovion) shown that ketamine infusion leads to increased cortical and Targacept; and he holds a patent from Repligen. Dr Abi-Dargham has received glutamate levels, likely compensation for acute NMDAR blockade research support from Pierre Fabre and Forest, and has been a consultant for or on and hypofunction. These findings are consistent with 1H MRS the scientific advisory board of Roche, Pfizer, Takeda, Otsuka, Amgen and Shire. studies of glutamatergic indices in SCZ in which glutamatergic Dr Girgis has received research support from Eli Lilly through APIRE. levels are elevated in MPFC and basal ganglia in early-stage, drug- naive or drug-free patients. These findings are also supported by SPECT studies in which ketamine infusion in healthy control ACKNOWLEDGMENTS subjects led to decreases in NMDAR availability, as well as by studies in individuals with SCZ in which NMDAR availability was This work was supported by the National Institute of Mental Health (P50 MH066171- decreased in the hippocampus of drug-free patients. Together, 01A1) and NCRR grant 2KL2RR024157-06. these studies provide pathophysiologic support for the glutamate hypofunction model of SCZ, are consistent with preclinical glutamatergic models of SCZ and encourage further development 6 REFERENCES of agents such as glycine, D-serine, glycine transport inhibitors, and NAC5 that increase the activity of NMDAR. 1 Miyamoto S, Miyake N, Jarskog LF, Fleischhacker WW, Lieberman JA. 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