Neuropsychopharmacology (2005) 30, 1557–1567 & 2005 Nature Publishing Group All rights reserved 0893-133X/05 $30.00 www.neuropsychopharmacology.org Elevated Levels of the NR2C Subunit of the NMDA Receptor in the Locus Coeruleus in Depression 1 1,3 ,1,2 Beata Karolewicz , Craig A Stockmeier and Gregory A Ordway* 1Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, MS, USA; 2Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA; 3Department of Psychiatry, Case Western Reserve University, Cleveland, OH, USA Low levels of the intracellular mediator of glutamate receptor activation, neuronal nitric oxide synthase (nNOS) were previously observed in locus coeruleus (LC) from subjects diagnosed with major depression. This finding implicates abnormalities in glutamate signaling in depression. Receptors responding to glutamate in the LC include ionotropic N-methyl-D-aspartate receptors (NMDARs). The functional NMDAR is a hetero-oligomeric structure composed of NR1 and NR2 (A–D) subunits. Tissue containing the LC and a nonlimbic LC projection area (cerebellum) was obtained from 13 and 9 matched pairs, respectively, of depressed subjects and control subjects lacking major psychiatric diagnoses. NMDAR subunit composition in the LC was evaluated in a psychiatrically normal subject. NR1 and NR2C subunit immunoreactivities in LC homogenates showed prominent bands at 120 and 135 kDa, respectively. In contrast to NR1 and NR2C, very weak immunoreactivity of NR2A and NR2B subunits was observed in the LC. Possible changes in concentrations of NR1 and NR2C that might occur in depression were assessed in the LC and cerebellum. The overall amount of NR1 immunoreactivity was normal in the LC and cerebellum in depressed subjects. Amounts of NR2C protein were significantly higher ( þ 61%, p ¼ 0.003) in the LC and modestly, but not significantly, elevated in the cerebellum ( þ 35%) of depressives as compared to matched controls. Higher levels of NR2C subunit implicate altered glutamatergic input to the LC in depressive disorders. Neuropsychopharmacology (2005) 30, 1557–1567, advance online publication, 25 May 2005; doi:10.1038/sj.npp.1300781 Keywords: depression; locus coeruleus; cerebellum; glutamate; NMDA receptor INTRODUCTION 1995; Lee et al, 1983; Melia et al, 1992; Torda et al, 1985; U’Prichard et al, 1979; Wang et al, 1998; Zafar et al, 1997). The locus coeruleus (LC) is the largest noradrenergic Together, these findings are highly suggestive of dysfunc- nucleus in the brain and projects to several cortical and tional noradrenergic neurotransmission in depression, subcortical areas. Previous observations reveal that depres- possibly through stress-induced activation and ultimate sion is associated with altered concentrations of several depletion of norepinephrine. noradrenergic proteins in the LC. For example, elevated Stress-sensitive inputs to the LC include glutamate. levels of tyrosine hydroxylase (TH) (Ordway et al, 1994a; Glutamate input to the LC originates predominately in the Zhu et al, 1999), increased agonist binding to a -adrenergic 2 glutamatergic paragigantocellularis nucleus (Aston-Jones receptors (Ordway et al, 1994b, 2003), and reduced levels of et al, 1986, 1991), and also from the cerebral cortex (Jodo norepinephrine transporters (Klimek et al, 1997) were and Aston-Jones, 1997; Jodo et al, 1998). Glutamate previously reported in the LC from major depression and in receptors that modulate LC activity include the N-methyl- suicide victims. Interestingly, depletion of norepinephrine D-aspartate receptor (NMDAR). The NMDAR is a hetero- or repeated stress in rats can increase TH expression, oligomeric structure composed of the NR1 subunit, NR2 increase binding to a -adrenergic receptors, and/or decrease 2 (A–D) subunits, and a less common NR3 subunit. The NR1 binding to the norepinephrine transporter (Cubells et al, subunit is expressed abundantly throughout the brain, while the NR2 subunits vary in their distribution in the central *Correspondence: Dr GA Ordway, Department of Psychiatry and nervous system (for a review see Loftis and Janowsky, Human Behavior, University of Mississippi Medical Center, 2500 2003). Stimulation of NMDARs results in, at least in part, North State Street, Jackson, MS 39216, USA, Tel: þ 1 601 984 5893, Fax: þ 1 601 984 5899, E-mail: [email protected] the activation of nNOS. The LC region contains NMDAR Received 19 January 2005; revised 17 March 2005; accepted 13 April (Allgaier et al, 2001; Shaw et al, 1992; Van Bockstaele and 2005 Colago, 1996), neuronal nitric oxide synthase (nNOS)- Online publication: 21 April 2005 at http://www.acnp.org/citations/ positive neurons (Cuellar et al, 2000; Karolewicz et al, 2004; Npp042105050041/default.pdf Xu et al, 1994), and guanosine 30,50 cyclic monophosphate NMDA receptor in the LC in depression B Karolewicz et al 1558 (cGMP) (Vulliemoz et al, 1999; Xu et al, 1998), suggesting next-of-kin for all subjects. Blood and urine samples from the existence of a glutamate/nitrergic transduction pathway. all subjects were examined by the coroner’s office for The exact cellular localization of these glutamate/nitrergic psychotropic medications and substances of abuse. signaling proteins within the human LC is still unknown. Retrospective, informant-based psychiatric assessments However, immunohistochemical labeling of nNOS, an were performed for all depressed and control subjects. The intracellular mediator of glutamate-NMDAR signaling, Structured Clinical Interview for DSM-IV Psychiatric revealed localization of nNOS in neurons and glial cells in Disorders (SCID-IV) was administered to next-of-kin of the region of the LC of a psychiatrically normal subject the 10 of depressed subjects (First et al, 1996). A trained (Karolewicz et al, 2004). interviewer administered the Schedule for Affective Dis- Interestingly, several lines of evidence suggest a crucial orders and Schizophrenia: lifetime version (SADS-L) to involvement of glutamate signaling in the pathophysiology knowledgeable next-of-kin of the three remaining depressed of depression and in the mechanism of action of subjects. Axis I psychopathology was assessed and con- antidepressant drugs. Compounds that decrease glutama- sensus diagnosis was reached in conference using informa- tergic transmission via blockade of ionotropic NMDA or tion from the interview and medical records. All subjects group I metabotropic glutamate receptors produce anti- with dysthymia and adjustment disorder and seven of the 10 depressant-like effect in animal screening procedures major depressive subjects died as a result of suicide. (Layer et al, 1995; Moryl et al, 1993; Papp and Moryl, Information regarding medication history came from the 1994; Skolnick, 1999; Tatarczynska et al, 2001). Moreover, coroner’s records (prescriptions issued to the deceased recent studies have revealed that agonists of group III around the time of death) as well as medical records. metabotropic glutamate receptors, known to inhibit gluta- Compliance with prescriptions at the time of death was mate release, exhibit antidepressant-like activity in animals assessed by a pill count (versus the date of prescription), (Palucha et al, 2004; Tatarczynska et al, 2002). Furthermore, post-mortem determination of blood or urine levels of the ketamine, an NMDAR antagonist, exhibits antidepressant medication, and the interview with the next-of-kin. No activity in humans (Berman et al, 2000). antidepressant drugs were detected in the post-mortem Hence, converging evidence from laboratory and clinical toxicology screening of subjects in the present study studies provide the basis for the hypothesis that glutama- (Table 1), as the presence of such drugs was a criterion of tergic input to the LC may be altered in depression, leading exclusion from the study. Information on smoking was also to compensatory changes in LC proteins. Recently, a low collected in the interview. In the present study, there were amount of nNOS was reported in subjects diagnosed with eight pairs of active cigarette smokers, one pair of subjects major depression (Karolewicz et al, 2004). The aim of the with past histories of smoking, and four pairs of present study was to (1) examine the composition of the nonsmokers (never smoked). NMDAR in the human LC, and (2) investigate potential The control subjects did not meet criteria for an Axis I abnormalities in the concentrations of NMDAR subunits in disorder at the time of their deaths, except as indicated the LC that might occur in depressed subjects. For the study above for nicotine dependence. Blocks of brain tissue were of depressive disorders, subjects were matched for age, sex, dissected, frozen in dry-ice cooled isopentane and stored cigarette smoking history, and as close as possible for post- at À801C. mortem interval (PMI). Brain tissue was collected from carefully screened subjects (post-mortem) who were diag- Dissection and Anatomical Positioning of nosed retrospectively with depressive disorders at the time Measurements of death, including major depression, dysthymia and adjustment disorder with depressed mood, and from Frozen tissue blocks were cut along the entire length of the controls who lacked major (Axis I) psychiatric disorders, LC, with histological sections taken at 1 mm intervals to except as indicated below for nicotine dependence. evaluate anatomical position along the LC axis. The LC was punched from sections and punches were