Molecular Psychiatry (2002) 7, 157–164 2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Comparative analysis of group II metabotropic glutamate receptor immunoreactivity in Brodmann’s area 46 of the dorsolateral prefrontal cortex from patients with schizophrenia and normal subjects JM Crook, M Akil, BCW Law, TM Hyde and JE Kleinman Section on Neuropathology, Clinical Brain Disorders Branch, National Institute of Mental Health, Bethesda, MD 20892, USA Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous sys- tem, and a key neurotransmitter in prefrontal cortical function. Converging lines of evidence implicate prefrontal cortical dysfunction in the neurobiology of schizophrenia. Thus, aberrant glutamate neurotransmission may underlie schizophrenia and other complex disorders of behavior. Group II metabotropic receptors (mGluRs) are important modulators of glutama- tergic and non-glutamatergic neurotransmission. Moreover, in an animal model, an agonist for group II mGluRs has been shown to reverse the behavioral, locomotor, and cognitive effects of the psychotomimetic drug phencyclidine. Accordingly, group II mGluRs constitute attractive targets for the pharmacotherapeutics and study of schizophrenia. Using immunocytochemistry and Western immunoblotting, we compared the localization and levels of group II mGluRs in Brodmann’s area 46 of the dorsolateral prefrontal cortex from patients with schizophrenia and normal subjects. Consistent with previous reports, we found that immunolabeling of group II mGluRs is prominent in Brodmann’s area 46. The majority of labe- ling was present on axon terminals distributed in a lamina-specific fashion. No apparent differ- ence in the cellular localization or laminar distribution of immunoreactive group II mGluRs was noted between the two diagnostic groups. Similarly, the levels of receptor immunoreactivity determined by quantitative Western immunoblotting were comparable between schizophrenic patients and normal subjects. We conclude that while the function of group II mGluRs in Brodmann’s area 46 of dorsolateral prefrontal cortex may be altered in patients with schizo- phrenia, this is not evident at the level of protein expression using an antibody against mGluR2 and mGluR3. Molecular Psychiatry (2002) 7, 157–164. DOI: 10.1038/sj/mp/4000966 Keywords: mGluR2/3; Western immunoblotting; immunocytochemistry; glutamate neurotransmis- sion; Brodmann’s area 46 Introduction brain.1 Taken together with the putative involvement of BA 46 in the pathophysiology of schizophrenia,5–11 Glutamate is a primary excitatory neurotransmitter in the general role of glutamate in CNS function and the the mammalian central nervous system (CNS), and a anatomy of glutamatergic neurons in BA 46 support a putative transmitter of many clinically important path- key role for glutamate dysfunction in DLPFC of schizo- ways.1 Quantitatively, glutamate is the most abundant phrenia. amino acid transmitter in the CNS,1–4 and glutamate Hypothetical models of glutamate dysfunction in receptors are located on virtually all neurons of the schizophrenia include hypofunction12,13 and hyper- CNS.4 In Brodmann’s area (BA) 46 of the dorsolateral function14,15 of glutamate neurotransmission. The prefrontal cortex (DLPFC), pyramidal neurons appear model of glutamate hypofunction is based, in part, on to be glutamatergic, forming efferent corticofugal pro- the psychotomimetic properties of phencyclidine jections to other cortical and subcortical regions of the (PCP) and ketamine. These N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor antagonists can induce positive and negative symptoms in healthy Correspondence: Dr TM Hyde, Section on Neuropathology, Clini- individuals,16–18 and the exacerbation of psychosis in cal Brain Disorders Branch, National Institute of Mental Health, patients with schizophrenia.19,20 In addition, PCP and Bldg 10, Room 4N 306 MSC 1385, Bethesda, MD 20892, USA. E-mail: hydetȰintra.nimh.nih.gov ketamine impair performance on prefrontal cortex- 21–23 Received 26 February 2001; revised 16 May 2001; accepted 27 dependent cognitive tasks, by interacting with June 2001 dopamine neurotransmission,14,22,24 mirroring cogni- Metabotropic glutamate receptors in schizophrenia JM Crook et al 158 tive impairment in schizophrenia. Assuming that PCP Materials and methods and ketamine act postsynaptically, and the resulting Tissue collection behavioral dysfunction is a valid model of schizo- After gaining the consent of a donor’s legal next of kin, phrenia, the behavioral abnormalities produced by human DLPFC was obtained at autopsy through the these drugs suggest reduced glutamate tone in the Medical Examiners’ Office of the District of Columbia DLPFC of patients with schizophrenia. Paradoxically, (Washington DC, USA). All tissue was obtained and recent studies have shown that psychotomimetic processed in accordance with a research protocol NMDA receptor antagonists increase glutamate efflux, reviewed and approved by the Institutional Review perhaps via presynaptic autoreceptors,25–27 thereby Board of the National Institute of Mental Health, in potentiating PFC-glutamate neurotransmission at post- conformance with the standards and guidelines of the synaptic non-NMDA (alpha-amino-3-hydroxy-5- National Institutes of Health. Tissue specimens were methyl-4-isoxazolepropionic acid (AMPA), and collected from 20 patients with a diagnosis of schizo- kainate) receptors.14 Importantly, antagonism of iono- phrenia and 20 subjects with no clinical history of psy- tropic AMPA/kainate receptors in PFC attenuates mne- chiatric illness or neuroleptic exposure (normal monic and other behavioral effects of NMDA receptor subjects). Patient diagnoses were confirmed after an antagonism.14 independent review of medical records by two board While most studies of glutamate receptor mediated certified psychiatrists according to DSM-IV criteria.42 neurotransmission in schizophrenia have focused on A board certified neuropathologist conducted an exam- ionotropic glutamate receptors, recently, G-protein ination of each case in order to exclude any subject coupled mGluRs have received increased attention.28,29 with significant neuropathological abnormalities. As modulators of synaptic neurotransmission,15,30,31 Microscopic examination of brain tissue sections taken mGluRs are functionally related to NMDA, AMPA, and from multiple cerebral areas and stained with Biel- kainate receptors. Also, mGluRs are involved in neur- schowsky’s silver stain was performed to exclude the oplasticity,30,32,33 are characterized by a diversity of presence of pathology associated with Alzheimer’s dis- receptor subtypes and heterogeneous localization,30,34 ease. Toxicology screens conducted on blood or brain and constitute attractive targets for the pharmacothera- samples showed no evidence of alcohol or drug abuse peutics of psychiatric disorders associated with by any of the subjects studied. increased or decreased glutamate neurotransmis- Wherever possible, normal subjects were matched sion.34,35 for gender, age at death, postmortem interval (PMI; the time between death and freezing of brain tissue), and The mGluRs currently recognized are classified into pH of tissue to subjects who had schizophrenia (Table three groups according to sequence homology, signal 1). Where death was not witnessed (normal subjects: transduction mechanism, and agonist selectivity.34,36 ID 7, 8; schizophrenic patients: ID 4, 6; Table 1), PMI Group II mGluRs consist of mGluR2 and mGluR3, was taken as the interval half way between the last which modulate synaptic transmission through inhi- 37,38 sighting of a subject while still alive and being found bition of cyclic AMP or calcium ion channels. dead (less than 12 h), to the time of tissue freezing. For Group II mGluRs are typically presynaptic autorecep- 30,34 34 schizophrenic patients, duration of illness (DOI; the tors, but also serve as presynaptic heteroceptors. time from first hospital admission to death), and final Moreover, they provide neuroprotection against gluta- and average daily antipsychotic drug doses (calculated 39,40 mate-induced excitotoxicity, and are prevalent in as chlorpromazine equivalents) were determined from 41 frontal cortex. Finally, in an animal model, group II the medical records (Table 1). mGluRs have been shown to mediate, in part, the dis- Following autopsy, the cerebral hemispheres of each ruptive behavioral, locomotor, and cognitive effects of brain were blocked coronally and flash frozen using a PCP, and the concomitant cortical glutamate mixture of dry ice and isopentane. Tissue blocks were efflux.15,30,34 stored at −80°C until required (freezer time, FT; Table Based on current understanding of the functional 1). The selection of tissue blocks containing BA 46 of and anatomical characteristics of group II mGluRs, we DLPFC was standardized between subjects. Blocks hypothesized that the levels and/or anatomical distri- extended rostral to the genu of the corpus callosum and bution of these receptors are altered in BA 46 of the contained the middle frontal gyrus. For immunocyto- DLPFC from patients with schizophrenia compared to chemistry, 14-␮m tissue sections were cut at −20°C normal subjects. To investigate this hypothesis, we using a cryo-microtome (CM 3050, Leica Microsystems, used immunocytochemistry with light microscopy to Deerfield, IL, USA), and thaw-mounted onto chrome- identify the localization of group II