Expression of the ionotropic glutamate receptor subunits and NMDA receptor-associated intracellular proteins in the substantia nigra in schizophrenia

Molecular Brain Research 121 (2004) 60 – 69 www.elsevier.com/locate/molbrainres

Research report

Expression of the ionotropic glutamate receptor subunits and NMDA receptor-associated intracellular proteins in the substantia nigra in schizophrenia Helena T. Mueller a,*, Vahram Haroutunian b, Kenneth L. Davis b, James H. Meador-Woodruff a a

Department of Psychiatry and Mental Health Research Institute, University of Michigan, Medical School, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720, USA b Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA Accepted 14 November 2003

Abstract Multiple neurotransmitter systems have been implicated in the pathophysiology of schizophrenia. Dopamine hyperactivity has often been implicated in this illness. More recently, the glutamate hypothesis of schizophrenia suggests that NMDA receptor (NMDAR) hypofunction may also play a role in this illness. This is based primarily on studies showing that phencyclidine, an NMDAR antagonist, can induce a schizophreniform psychosis. While NMDAR dysfunction is most often implicated in schizophrenia, other components of the glutamate system, such as the AMPA and kainate receptors, as well as NMDAR-associated intracellular proteins, may also play a role in regulating NMDA receptor activity and glutamate neurotransmission. There is growing interest in the hypothesis that the pathophysiology of schizophrenia involves alterations in dopamine – glutamate interactions. The glutamate system is anatomically and functionally linked to the dopamine system, and glutamate can modulate dopaminergic activity and release by stimulating various glutamate receptor subtypes expressed by dopaminergic neurons in the substantia nigra/ventral tegmental area. In this study, we investigated dopamine – glutamate interactions by measuring the expression of transcripts encoding the subunits for the ionotropic glutamate receptors (NMDA, AMPA and kainate) and five NMDAR-associated intracellular proteins, PSD-93, PSD-95, SAP102, NF-L and yotiao in the dopaminergic neurons in the substantia nigra pars compacta (SNc) of subjects with schizophrenia and a comparison group. Tyrosine hydroxylase (TH, a marker of dopamine-synthesizing cells), NR1 (an NMDA receptor subunit) and GluR5 (a kainate subunit) transcript levels were significantly increased in the SNc in schizophrenia. These data support the hypothesis that schizophrenia may involve alterations in dopamine – glutamate interactions. D 2003 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuropsychiatric disorders Keywords: AMPA; Kainate; Dopamine; Neurotransmitter interactions; Neuropsychiatric disease

1. Introduction Multiple neurotransmitter systems have been implicated in the pathophysiology of schizophrenia. A longstanding hypothesis has been that dopaminergic hyperactivity plays a role in this illness [4,26,46,65]. Dopaminergic hyperactivity alone, however, is insufficient to account for all aspects of schizophrenia. Some patients with schizophrenia are resis* Corresponding author. Tel.: +1-734-936-2061; fax: +1-734-6474130. E-mail address: [email protected] (H.T. Mueller). 0169-328X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.11.004

tant to D2 antagonist treatment, and the onset of therapeutic effects of antipsychotics often takes weeks. Furthermore, D2 antagonists only partially alleviate the negative symptoms and cognitive deficits observed in schizophrenia [26]. While amphetamines can cause positive psychotic symptoms in normal persons, they can also improve some of the negative symptoms observed in schizophrenia [5]. Given these findings, it is likely that neural circuits and neurotransmitter systems in addition to dopamine are involved in the pathophysiology of this illness. There is growing evidence that glutamatergic dysfunction may also be involved in the pathophysiology of

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

schizophrenia; in particular, it has been hypothesized that activity of a specific class of glutamate receptor, the NMDA receptor (NMDAR), is decreased in this illness [55]. NMDAR antagonists such as phencyclidine (PCP) and ketamine can induce both positive and negative symptoms in normal individuals, and can exacerbate these symptoms in schizophrenia [40,41]. Furthermore, agonists of the glycine/D-serine co-agonist site of the NMDAR can improve some of the negative symptoms of schizophrenia [31]. The NMDAR is a heteromeric complex comprised of four or five subunits derived from seven different genes (NR1, NR2A-2D and NR3A and 3B)[3,36]. Several recent studies have found changes in the expression of NMDAR subunits in several brain regions in schizophrenia [27,30,38,47,51], consistent with the hypothesis of altered NMDAR function in the illness. While NMDAR dysfunction is most often implicated in schizophrenia, other components of the glutamate system, such as the AMPA and kainate receptors, interact with NMDARs, and thus may indirectly contribute to NMDAR dysfunction. Activation of AMPA receptors, for example, facilitates NMDAR activation by depolarizing NMDARs and releasing a Mg2 + blockade [36]. Consequently, alterations in AMPA receptor function could potentially have an effect on NMDAR activity. Like NMDARs, AMPA and kainate receptors are also comprised of multiple subunits (GluR1– GluR4 for the AMPA receptors and GluR5– GluR7 and KA1 – KA2 for the kainate receptors). There is evidence that AMPA and kainate receptors may be altered in schizophrenia [30,38,47,50]. NMDAR function may also be regulated at the intracellular level by protein – protein interactions. A number of NMDAR-associated intracellular proteins, including PSD93, PSD-95, SAP102, NF-L and yotiao bind NMDAR subunits and functionally link the receptor to specific intracellular signaling pathways. There is growing evidence that these molecules play a role in regulating glutamate receptor activity [62]. PSD-93, PSD-95 and SAP102 are all members of the membrane-associated guanylate kinase (MAGUK) superfamily characterized by 3 N-terminal PDZ domains, an SH3 domain, and a C-terminal guanylate kinase (GK) domain. PSD-95 interacts with the NR2 subunits of the NMDAR and modulates NMDAR clustering, targeting, synaptic localization, anchoring and sensitivity to glutamate [59,62]. Yotiao and NF-L interact with the NR1 subunit of the NMDAR and are also thought to play a role in modulating NMDAR function [62]. While these molecules do not appear to directly affect NMDAR activity, they may change the functional efficiency of the NMDAR and thus exert an effect on glutamate neurotransmission. Supporting the idea that these NMDAR-associated intracellular proteins may play a role in glutamatergic dysfunction in schizophrenia, expression of PSD-95 has been reported to be altered in the cortex in this illness [27,54]. Additionally, recent work in our laboratory has found alterations in the expression of a

61

number of NMDAR-associated intracellular proteins, including PSD-95, SAP102 and NF-L, in the thalamus in schizophrenia [21]. The glutamate system is anatomically and functionally linked to the dopamine system. Glutamatergic efferents project to both the cell bodies and axons of nigrostriatal and mesolimbic dopaminergic neurons. Glutamate can modulate dopamine release in the striatum, as well as alter dopamine release and/or activity in the substantia nigra/ ventral tegmental area (SN/VTA) [53]. For example, electrical stimulation of glutamatergic neurons in the cortex has been shown to alter dopaminergic activity in the striatum. Further, direct application of glutamate can also alter synaptic dopamine levels at the level of either the SN/VTA or the striatum [53]. The NMDA, AMPA and kainate classes of glutamate receptors have all been reported to be expressed by these midbrain dopaminergic neurons [1,2,16,23,56,67], and several studies using receptor-specific agonists and/or antagonists provide evidence that each of these receptor subtypes may mediate glutamate regulation of dopaminergic neurotransmission [53]. There is growing interest in the hypothesis that the pathophysiology of schizophrenia involves alterations of dopamine – glutamate interactions. In this model, both glutamate hypofunction and dopamine hyperactivity are thought to exist in schizophrenia due to a cortical – subcortical imbalance [15,32]. It has been proposed that a primary cortical deficit involving decreased glutamatergic function could lead to enhanced subcortical dopaminergic activity. In support of this, PCP-treated rats show enhanced amphetamine-induced striatal dopamine release, along with enhanced locomotor activity [7]. Similarly, in humans, NMDAR antagonism significantly decreases [11C]raclopride binding, which is believed to reflect enhanced striatal dopamine levels; these changes are related to the induction of positive and negative symptoms [12]. Finally, a correlation between reduced prefrontal cortical activity and increased striatal dopamine uptake has been reported [52]. These studies support the idea that alterations in dopaminergic neurotransmission thought to occur in schizophrenia may be mediated, at least in part, by changes in glutamatergic activity. In this study, we investigated the possibility of alterations in dopamine – glutamate interactions in schizophrenia. Given that glutamate regulates dopamine release, we hypothesized that changes in subcortical dopaminergic activity may be due in part to abnormalities in glutamate neurotransmission at the receptor level, as well as at the level of intracellular molecules associated with glutamate receptor signaling. Glutamate can modulate dopaminergic activity by stimulating various glutamate receptor subtypes located on axon terminals in the striatum and/or on cell bodies in the SN/VTA. Since the transcripts encoding these receptors are synthesized in the cell bodies in the SN/VTA, we measured the expression of transcripts encoding the subunits for the ionotropic glutamate receptors (NMDA, AMPA and kai-

62

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

Table 1 Subject characteristics

proteins, PSD-93, PSD-95, SAP102, NF-L and yotiao, in these same subjects.

Subject Diagnosis

Sex Age PMI Cause of death (years) (min)

82 93 97 192 230 231 232 106 123 172 190 193 195 199 212 257 309 331 337 338 356 361 363 426

F M M F F F F F M F M M M F F M F M F M M F F F

Control Control Control Control Control Control Control Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia

68 70 55 79 96 90 74 86 54 86 61 84 69 76 75 72 65 63 69 87 68 98 64 79

280 482 600 181 195 250 180 415 490 330 212 372 270 510 334 1235 350 372 820 670 335 85 392 1125

Unknown Pulmonary hypertension Cancer Cardiac failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Respiratory insufficiency Cancer Cardiac failure, pneumonia Cardiopulmonary failure Cardiopulmonary failure Myocardial infraction Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiac failure Cardiopulmonary failure Cardiopulmonary failure

nate) in the dopaminergic neurons in the substantia nigra pars compacta (SNc) of subjects with schizophrenia and a comparison group. We also examined the expression of transcripts encoding the NMDAR-associated intracellular

2. Materials and methods 2.1. Subjects and tissue preparation Sixteen subjects with schizophrenia and seven individuals with no history of psychiatric illness were studied. Subjects were from the Mount Sinai Medical Center and Bronx Veterans Affairs Medical Center, and have previously been used and described in our studies examining dopamine and glutamate receptor expression in schizophrenia [21,35,38, 49,63,64]. Characteristics of these subjects are summarized in Table 1. Age, sex, postmortem interval (PMI), tissue pH and side of brain studied were not significantly different between groups. All of the patients with schizophrenia had received antipsychotic medications for their illness, although five subjects had been medication-free for 6 or more weeks prior to death. All subjects in the comparison group were free of antipsychotic medication and had no evidence of any neurodegenerative disorder on neuropathological examination. Brains were obtained at autopsy and one hemisphere from each brain was dissected into 1 cm coronal slabs that were rapidly frozen and stored at 80 jC. Blocks containing the midbrain were cryostat sectioned (20 Am) and mounted onto poly-L-lysine subbed microscope slides. Slides for this study were matched by selecting sections at the level where the rostral third of the red nucleus ends and the middle third

Fig. 1. Dopaminergic (DA) neurons in the substantia nigra, pars compacta (SNc). Expression of tyrosine hydroxylase (TH) mRNA was used as a marker to identify DA neurons in the SNc (A and B), confirmed by visualizing these large pigmented neurons by Nissl-staining (C). Higher power views in B and C are from area shown in the box in panel A. (D) summarizes the quantification of TH mRNA in SNc in schizophrenia and a comparison group. TH mRNA levels were significantly increased in schizophrenia ( p < 0.05). Abbreviations: RN = red nucleus, A8 = retrorubral field, A9 = substantia nigra (SN), A10 = ventral tegmental area (VTA). Scale bar = 1 mm in both panels A and B.

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

begins. Dopaminergic neurons of the SNc were identified by both Nissl-staining and in situ hybridization for tyrosine hydroxylase (TH) mRNA for each subject. The VTA was not consistently identified in all subjects and therefore was not included in this study. Two slides from each subject were assayed for each probe. 2.2. In situ hybridization Riboprobes were synthesized from linearized plasmids containing subclones for each of the ionotropic glutamate receptor subunits (NR1, NR2A-2D, GluR1 – GluR7 and KA1 –KA2), the NMDAR-associated intracellular proteins (PSD-93, PSD-95, NF-L, SAP102 and yotiao) and TH, as we have previously described [20,38,48]. An NR3A subclone (NCBI GeneBank accession number AF073379, nucleotide coding regions 1139 –1676) was amplified by PCR from a full-length NR3A clone [68], inserted into pCR4Blunt-TOPO vector (Zero Blunt TOPO PCR cloning kit, Invitrogen, Carlsbad, CA) and the final product confirmed by sequencing. Riboprobes were labeled by mixing 100 ACi of [35S] UTP, 2.0 Al 5  transcription buffer, 1.0 Al 0.1 M dithiothreitol (DTT), 1.0 Al each of 10 mM ATP, CTP, GTP, 2.0 Al linearized plasmid, 0.5 Al RNase inhibitor and 1.5Al Sp6, T7 or T3 RNA polymerase, and incubating the reaction mixture for 2 h at 37 jC. A total of 1 Al DNase (RNase-free) was then added and incubated for an additional 15 min at room temperature. The reaction mixture was then sieved through a 1-cm3 syringe containing G-50 Sephadex equilibrated in Tris buffer (100 mM Tris –HCl pH 7.5, 12.5 mM EDTA pH 8.0, 150 mM NaCl and 0.2% SDS) and 100 Al fractions were collected. Finally, 1 Al DTT was added to each fraction to a final concentration of 0.01 M. Slides were removed from 80 jC storage and fixed in 4% formaldehyde for 1 h at room temperature, followed by three rinses in 2  SSC (300 mM NaCl, 30 mM sodium citrate pH 7.2). Next, they were acetylated (0.1 M triethanolamine pH 8.0: acetic anhydride (400:1 vol/vol) for 10 min, washed in 2  SSC for 10 min and dehydrated in graded alcohols. 2 –8  106 cpm of radiolabeled probe in 500 Al of 50% hybridization buffer (50% formamide, 10% dextran sulfate, 3  SSC, 50 mM Na2HPO4, 1  Denhardt’s solution, 100 Ag/ml yeast tRNA, 10 mM DTT) was placed on each slide, covered and incubated overnight at 55 jC. The following day, cover slips were removed and slides were washed in 2  SSC for 5 min at room temperature followed by RNase treatment (200 Ag/ml in 10 mM Tris – HCl pH 8.0, 0.5 M NaCl) at 37 jC for 30 min. Slides were then washed twice in 2  SSC for 15 min, once in 1  SSC for 15 min at room temperature, followed by two consecutive washes in 0.5  SSC for 1 h at 55 jC, and a final wash in 0.5  SSC at room temperature for 15 min. The slides were then dehydrated in graded alcohols and apposed to film with [14C] radioactive standards for various lengths of times depending on the probe (1 week – 2 months).

63

2.3. Imaging and statistical analysis Images were acquired by digitizing in situ hybridization films with a Macintosh-based CCD imaging system, using NIH Image 1.56. For this study, the region of interest was the substantia nigra pars compacta. As a first step in imaging, the entire extent of the SNc was identified based on TH mRNA expression and Nissl-staining patterns as noted above. The pattern of labeling for each transcript was obviously punctate and not homogenous. Accordingly, final imaging and quantification was of labeling of only these discrete cellular areas within the full extent of the boundaries of the SNc. To quantify gene expression in these cells, a threshold was set at three times background expression for each probe and only transcript expression reaching that

Fig. 2. Expression of NMDA receptor subunits. In situ hybridization was used to assess the expression of NMDA receptor subunit transcripts in the SNc (top panel). NR1, NR2A-2D and NR3A mRNA levels were measured in schizophrenia and a comparison group (bottom panel). NR1 levels were significantly increased in schizophrenia ( p < 0.02).

64

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

threshold was measured. Gray scale values (GSV) of these areas above the threshold were obtained for each probe, background GSV subtracted, and converted to optical density (OD). These values were linear with respect to concentration over OD values found in this study. For each subject, values from two sections were averaged, and these mean OD values were converted to concentration [20,38]. Statistical analysis was performed using SPSS for Windows. Comparison of the mean values for each probe between the two groups was performed using Student’s t-test.

3. Results 3.1. TH mRNA expression TH is the rate-limiting enzyme for catecholamine biosynthesis and a marker of dopamine-synthesizing cells in the midbrain. Using in situ hybridization, TH mRNA expression was used to identify dopaminergic neurons in the SNc, in conjunction with examining adjacent slides that were Nissl-stained (Fig. 1). TH mRNA levels were significantly higher (t = 2.9, df = 22, p = 0.007) in schizophrenia relative to a comparison group. TH mRNA expression was not correlated with age or PMI.

Fig. 4. Expression of kainate receptor subunits. In situ hybridization was used to determine kainate receptor subunit transcript expression in the SNc (top panel). GluR5 – GluR7 and KA1 – KA2 mRNA levels were measured in schizophrenia and a comparison group (bottom panel). GluR5 levels were significantly increased in schizophrenia ( p < 0.005).

3.2. Expression of the ionotropic glutamate receptor subunits

Fig. 3. Expression of AMPA receptor subunits. In situ hybridization was used to measure AMPA receptor subunit transcript expression in the SNc (top panel). GluR1 – GluR4 mRNA levels were determined in schizophrenia and a comparison group (bottom panel). No significant differences were detected between the groups.

3.2.1. NMDA receptor subunits Expression of the NMDAR subunits, NR1, NR2A-2D and NR3A were measured in the SNc (Fig. 2). Transcripts for each of the NMDAR subunits were detected, but the levels of expression differed between the subunits. The obligatory NR1 subunit was the most abundant of the NMDAR subunit transcript in the SNc, with expression levels more than twice that of any other subunit. Both the NR2A and NR2B subunits were rare in the SNc, while the NR2C subunit was expressed at moderate levels. NR2D and NR3A were expressed at relatively low levels, yet their expression levels were greater than either NR2A or NR2B. NR1 transcript levels were significantly increased (t = 2.7, df = 21, p = 0.012) in schizophrenia. Expression levels for

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

65

the other subunits did not significantly differ between groups, although there was a trend (t = 1.8, df = 21, p = 0.08) for NR3A to be increased in schizophrenia.

significantly increased in schizophrenia (t = 3.2, df = 21, p = 0.004). None of the other subunits were changed in schizophrenia.

3.2.2. AMPA receptor subunits Transcripts for the GluR1– GluR4 subunits were detected in the SNc (Fig. 3). GluR4 was the most abundant subunit, with levels almost twice that of any other subunit. Both GluR1 and GluR3 were expressed at moderate levels, while GluR2 was present at very low levels. No significant differences were observed for any of the AMPA subunits between schizophrenia and the comparison group.

3.3. Expression of NMDAR-associated intracellular proteins

3.2.3. Kainate receptor subunits The transcripts encoding all five kainate receptor subunits were found in the SNc (Fig. 4). The GluR6 subunit was the most abundant kainate receptor subunit present, although levels of expression for GluR5 and KA2 subunits were also both high. Expression for the GluR5 subunit was

We investigated the expression of five intracellular molecules (PSD-93, PSD-95, SAP102, NF-L and yotiao) known to interact with the NMDAR subunits and modulate NMDAR function. While transcripts for each molecule studied were detected in the SNc, expression levels varied greatly (Fig. 5). Consistent with reports in other brain regions [20,70], both NF-L and PSD-95 were expressed at very high levels, while SAP102, PSD-93 and yotiao were expressed at much lower levels in the SNc. No significant differences between the two diagnostic groups were found for any of these molecules.

4. Discussion 4.1. TH expression

0.7

fmol / g tissue

0.6

Comparison group Schizophrenia

0.5 0.4 0.3 0.2 0.1 0.0

PSD-93

PSD-95

NFL

SAP-102

Yotiao

NMDA-associated proteins Fig. 5. Expressions of NMDAR-associated proteins. In situ hybridization was used to determine the expression of transcripts encoding the NMDAR-associated intracellular proteins, PSD-93, PSD-95, SAP102, NF-L and yotiao in the SNc. No significant differences were detected in schizophrenia.

The dopamine hypothesis postulates that increased dopamine release or enhanced dopamine response may be associated with schizophrenia and that the positive symptoms of schizophrenia reflect enhanced subcortical dopaminergic activity [26]. In this study, we measured TH mRNA levels in dopaminergic neurons in the SNc. Since TH is the ratelimiting enzyme in catecholamine biosynthesis, changes in TH expression may reflect alterations in mesostriatal dopaminergic activity in schizophrenia. We found significant increases in TH mRNA expression in the SNc in schizophrenia, suggesting increased dopamine synthesis. An earlier study found no differences in TH mRNA in SN in schizophrenia relative to a comparison group [39]. This discrepancy may be due to technical differences between these two studies. This earlier study used RT-PCR to measure TH mRNA from midbrain homogenates, while in situ hybridization was employed in our study. Previous findings on TH activity in schizophrenia have been varied. While one study found no differences in the levels of TH enzyme activity in the striatum (caudate, putamen and accumbens) [24], Toru et al. [71] reported increased TH enzyme activity in both the caudate and putamen, and in the SN in schizophrenia. They also found concomitant increases in the concentration of the dopamine metabolite, homovanillic acid (HVA), suggesting that dopamine turnover may be increased in schizophrenia. Bird et al. [9] also reported high HVA levels in the caudate, yet other groups have found no differences in HVA levels in the basal ganglia [6,24,29,73]. While these data are difficult to interpret, newer imaging studies, which measure synaptic dopamine levels more directly, have found elevated dopamine levels in the striatum in schizophrenia

66

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

[13,42]. Our results are consistent with these imaging studies and further support the hypothesis that subcortical dopaminergic hyperactivity plays a role in the pathophysiology of schizophrenia. A confounding variable in postmortem studies is the possible effects of antipsychotic drugs. Although we cannot exclude the possibility that increased TH expression is due to chronic antipsychotic use, most studies have found TH expression in the SN does not change, or even decreases, following antipsychotic treatment [22,44,57,72]. In our study, TH mRNA levels for the five schizophrenic subjects that were medication-free for 6 weeks or more prior to death were not significantly different from the levels of the medicated subjects. A caveat is that this type of post-hoc analysis is relatively underpowered. Nonetheless, these observations suggest that the increased TH mRNA expression found in this study is associated with the pathophysiology of schizophrenia and probably not with antipsychotic treatment. 4.2. Ionotropic glutamate receptor subunit expression Studies in multiple species have demonstrated that dopaminergic neurons in the SNc express functional NMDARs [53] and, while the expression of transcripts for the NR1 and NR2A-2D subunits have been described in human midbrain [23], there are few quantitative data on the expression of these subunits in the human SNc. In this study, we quantify the expression of the NMDAR subunit transcripts, NR1, NR2A-D and NR3A in the human SNc. The obligatory NR1 subunit, expressed at very high levels throughout much of the human brain [36], is also expressed at high levels in the SNc. The modulatory NR2A-2D subunits, which are differentially distributed throughout the brain, are all expressed in the SNc. NR2A and NR2B are both expressed at low levels, consistent with an earlier report [23]. In contrast with this same earlier study, which detected only minimal labeling of NR2C and intense labeling of NR2D, we observed moderate levels of transcript expression for both NR2C and NR2D in the SNc. The NR3A subunit, like the NR2 subunits, is thought to be modulatory. Co-assembly of the NR3A subunit with NR1/NR2 containing receptors decreases NMDAR activity [17,25,60,68,69]. In rodents, NR3A is primarily expressed during development, decreasing in the early postnatal period and reaching low levels in most regions of the adult brain. The NR3A subunit is expressed in the SN in the developing rat brain, although the level of expression in the adult SN is unknown [74]. In the adult human, the NR3A subunit is expressed in the SNc at levels greater than either NR2A or NR2B suggesting that in the SNc, NR3A expression persists at meaningful levels into adulthood. More extensive elucidation of the distribution of the NR3A subunit in human brain remains to be determined. Our results indicate that after NR1, the predominant subunit in the SNc is NR2C, followed by NR2D and

NR3A. NR2A and NR2B are expressed at much higher levels in other brain regions, while the NR2C subunit is primarily restricted to the cerebellum and thalamus [36]. Both NR2D and NR3A are thought to be important mainly during development [19,36,68]. This pattern of nigral expression suggests that the SNc contains a unique set of functional NMDARs, which are likely to be associated with glutamate regulation of dopaminergic activity. The expression of AMPA receptor subunits in the human SNc has not previously been described. In both rodents and primates, each of the AMPA subunits, GluR1 – GluR4, are expressed in dopamine-containing cells in the midbrain [1,16,18,45,56,61]. Consistent with this, all four AMPA subunits were detected in the human SNc. The GluR4 subunit was the most abundant subunit, with expression levels even greater than the NR1 subunit, unlike patterns seen in other brain regions [36]. Relatively less is known regarding the distribution of kainate receptors (GluR5 – GluR7 and KA1 – KA2) in the SN. In rodents, reports of kainate receptor subunit expression in SN have been inconsistent [10,75]. We detected transcripts for all of the kainate subunits in the human SNc. 4.3. Altered expression of NMDA, AMPA and kainate receptor subunits in schizophrenia Because glutamate is a critical modulator of subcortical dopamine function, and abnormal dopamine – glutamate interactions may be involved in the pathophysiology of schizophrenia, we examined the expression of the ionotropic glutamate receptor subunits in the SNc in schizophrenia. The NMDAR subunit, NR1, and the kainate subunit, GluR5, are abnormally expressed in the SNc in schizophrenia. NR1 mRNA expression was significantly increased in schizophrenia, consistent with previous findings of increased NR1 expression in multiple cortical regions [27,33,43]. There are also reports of decreased NR1 expression in brain regions including hippocampus, frontal cortex, thalamus and superior temporal cortex in schizophrenia [30,37,38,66]. Collectively, these findings suggest that the expression of the NR1 subunit is altered in a region-specific fashion in schizophrenia, and further supports the hypothesis that the pathophysiology of schizophrenia involves abnormalities in NMDAR expression. Elevated levels of NR1 mRNA in the SNc may increase the number of NR1 subunits available to form functional NMDARs, which in turn may lead to increased NMDAR expression and activity. While the glutamate hypothesis usually posits a general decrease in NMDAR activity in schizophrenia, it is possible that within a local circuit, NMDAR activity is enhanced. In the SNc, increasing NMDAR activity may increase dopaminergic activity and/or dopamine release, consistent with our finding of increased TH mRNA and the hypothesis of subcortical dopaminergic hyperactivity in schizophrenia. We did not detect any differences in expression of the AMPA receptor subunits. Kainate receptor expression, how-

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

ever, was altered: GluR5 mRNA was significantly elevated in schizophrenia. While this is the first report of GluR5 abnormalities in schizophrenia, previous studies indicate that like the NMDAR subunits, there are region-specific changes in kainate receptors in schizophrenia. Transcript expression for other kainate receptor subunits have been found to be altered in regions including the thalamus, prefrontal cortex and the hippocampus [28,34,50,58]. There are also changes in kainate receptor binding and immunoreactivity in this illness [8,48]. Kainate receptors have been reported to modulate dopamine release [53]. A change in subunit stoichiometry, in this case, an increase in GluR5 expression, may have an effect on kainate receptor function. GluR5 homomers and GluR5/6 containing receptors both desensitize faster and recover from desensitization slower than homomeric GluR6 receptors [11] suggesting a mechanism by which elevated GluR5 expression may influence glutamate regulation of dopaminergic activity. Past antipsychotic treatment is a potential confound when interpreting our results. In general, there is evidence that both typical and atypical drugs can regulate glutamate receptor gene expression, although these effects are complex and vary depending on drug, subunit and region studied. In most regions examined, NR1 expression appears not to change or to decrease in animals following chronic antipsychotic treatment. While there are a few reports of increased NR1 expression following antipsychotic treatment, the effects of antipsychotics on NR1 expression in the SNc are unknown. Similar to what we observed for TH mRNA, both NR1 and GluR5 mRNA expression were not different between the medication-free and medicated subjects, suggesting that antipsychotic treatment may have minimal effects on the expression of these subunits in the SNc in these subjects. 4.4. NMDAR-associated intracellular proteins in schizophrenia PSD-93, PSD-95 SAP102, NF-L and yotiao are cytoplasmic molecules, which can directly modulate NMDAR activity or activate intracellular signaling pathways linked to NMDAR activity. These molecules are co-localized with NMDARs in the post-synaptic density and have been shown to play roles in mediating NMDAR clustering, targeting, and synaptic localization, as well as anchoring the NMDAR to the cytoskeleton [62] and altering NMDAR sensitivity to glutamate [59]. They also link NMDARs to downstream signaling molecules such as protein phosphatase-1 that activate other proteins including Ca2 +/calmodulin-dependent kinase II (CaMKII) [62] and neuronal nitric oxide synthase (nNOS) [14]. The ability of these molecules to couple the NMDAR to intracellular pathways provides a secondary functional role for NMDARs, perhaps independent of its electrophysiological activity. Both PSD-95 and NF-L were found to be abundantly expressed in the SNc, while PSD-93, SAP102 and yotiao were expressed at much

67

lower levels, consistent with transcript levels previously reported in the primate and human thalamus [20,21]. While no significant differences were detected in the expression levels of any of these molecules in schizophrenia, other studies have found changes in other brain regions in schizophrenia, including the prefrontal cortex, occipital cortex and thalamus [21,27,54]. These findings suggest that these molecules, like the glutamate receptor subunits, exhibit regionspecific changes in their expression in schizophrenia and may not be critical for NMDAR function in the SNc. While speculative, perhaps glutamate regulation of dopaminergic activity in the SNc does not need this intracellular machinery or signaling, but rather requires only the ion gating and depolarizing properties associated with NMDAR activity. 4.5. Conclusions All subunits of the NMDA, AMPA and kainate classes of glutamate receptors and a set of NMDAR-associated intracellular proteins are expressed in the human SNc, and may play a role in glutamate regulation of dopaminergic activity and/or release. TH mRNA levels were significantly increased in schizophrenia, supporting the hypothesis that the pathophysiology of schizophrenia involves alterations in subcortical dopaminergic activity. Expression of both the NR1 subunit of the NMDAR and the GluR5 subunit of the kainate receptor were significantly elevated in schizophrenia, suggesting glutamatergic neurotransmission in mesencephalic dopamine neurons is abnormal in schizophrenia. Collectively, these findings further support the hypothesis that schizophrenia may involve alterations in dopamine – glutamate interactions in mesostriatal circuitry. Finally, it would be interesting to study the specificity of these findings to schizophrenia by examining the expression of these molecules in other psychiatric disorders, especially in major depressive and bipolar disorders.

Acknowledgements This work was supported by MH650101 (HTM) and MH53327 (JMW). This work was presented at the 56th annual meeting of the Society of Biological Psychiatry, New Orleans, LA, May 3 –5, 2001.

References [1] D.S. Albers, S.W. Weiss, M.J. Iadarola, D.G. Standaert, Immunohistochemical localization of N-methyl-D-aspartate and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionate receptor subunits in the substantia nigra pars compacta of the rat, Neuroscience 89 (1999) 209 – 220. [2] R.L. Albin, R.L. Makowiec, Z.R. Hollingsworth, L.S.t. Dure, J.B. Penney, A.B. Young, Excitatory amino acid binding sites in the basal ganglia of the rat: a quantitative autoradiographic study, Neuroscience 46 (1992) 35 – 48.

68

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

[3] O. Andersson, A. Stenqvist, A. Attersand, G. von Euler, Nucleotide sequence, genomic organization, and chromosomal localization of genes encoding the human NMDA receptor subunits NR3A and NR3B, Genomics 78 (2001) 178 – 184. [4] B. Angrist, S. Gershon, Dopamine and psychotic states: preliminary remarks, Adv. Biochem. Psychopharmacol. 12 (1974) 211 – 219. [5] B. Angrist, E. Peselow, M. Rubinstein, J. Corwin, J. Rotrosen, Partial improvement in negative schizophrenic symptoms after amphetamine, Psychopharmacology (Berl.) 78 (1982) 128 – 130. [6] N.C. Bacopoulos, E.G. Spokes, E.D. Bird, R.H. Roth, Antipsychotic drug action in schizophrenic patients: effect on cortical dopamine metabolism after long-term treatment, Science 205 (1979) 1405 – 1407. [7] A. Balla, R. Koneru, J. Smiley, H. Sershen, D.C. Javitt, Continuous phencyclidine treatment induces schizophrenia-like hyperreactivity of striatal dopamine release, Neuropsychopharmacology 25 (2001) 157 – 164. [8] F.M. Benes, M.S. Todtenkopf, P. Kostoulakos, GluR5,6,7 subunit immunoreactivity on apical pyramidal cell dendrites in hippocampus of schizophrenics and manic depressives, Hippocampus 11 (2001) 482 – 491. [9] E.D. Bird, E.G. Spokes, J. Barnes, A.V. MacKay, L.L. Iversen, M. Shepherd, Increased brain dopamine and reduced glutamic acid decarboxylase and choline acetyl transferase activity in schizophrenia and related psychoses, Lancet 2 (1977) 1157 – 1158. [10] S. Bischoff, J. Barhanin, B. Bettler, C. Mulle, S. Heinemann, Spatial distribution of kainate receptor subunit mRNA in the mouse basal ganglia and ventral mesencephalon, J. Comp. Neurol. 379 (1997) 541 – 562. [11] D. Bleakman, Kainate receptor pharmacology and physiology, Cell. Mol. Life Sci. 56 (1999) 558 – 566. [12] A. Breier, T.P. Su, R. Saunders, R.E. Carson, B.S. Kolachana, A. de Bartolomeis, D.R. Weinberger, N. Weisenfeld, A.K. Malhotra, W.C. Eckelman, D. Pickar, Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: evidence from a novel positron emission tomography method, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 2569 – 2574. [13] A. Breier, C.M. Adler, N. Weisenfeld, T.P. Su, I. Elman, L. Picken, A.K. Malhotra, D. Pickar, Effects of NMDA antagonism on striatal dopamine release in healthy subjects: application of a novel PET approach, Synapse 29 (1998) 142 – 147. [14] J.E. Brenman, D.S. Chao, S.H. Gee, A.W. McGee, S.E. Craven, D.R. Santillano, Z. Wu, F. Huang, H. Xia, M.F. Peters, S.C. Froehner, D.S. Bredt, Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains, Cell 84 (1996) 757 – 767. [15] M. Carlsson, A. Carlsson, Schizophrenia: a subcortical neurotransmitter imbalance syndrome? Schizophr. Bull. 16 (1990) 425 – 432. [16] B.T. Chatha, V. Bernard, P. Streit, J.P. Bolam, Synaptic localization of ionotropic glutamate receptors in the rat substantia nigra, Neuroscience 101 (2000) 1037 – 1051. [17] J.E. Chatterton, M. Awobuluyi, L.S. Premkumar, H. Takahashi, M. Talantova, Y. Shin, J. Cui, S. Tu, K.A. Sevarino, N. Nakanishi, G. Tong, S.A. Lipton, D. Zhang, Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits, Nature 415 (2002) 793 – 798. [18] L.W. Chen, L.C. Wei, B. Lang, G. Ju, Y.S. Chan, Differential expression of AMPA receptor subunits in dopamine neurons of the rat brain: a double immunocytochemical study, Neuroscience 106 (2001) 149 – 160. [19] A.M. Ciabarra, J.M. Sullivan, L.G. Gahn, G. Pecht, S. Heinemann, K.A. Sevarino, Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family, J. Neurosci. 15 (1995) 6498 – 6508. [20] S.M.a.M.-W. Clinton, H. James, Nucleus-specific expression of NMDA receptor associated postsynaptic density proteins in primate thalamus, Thalamus Relat. Syst. 1 (2002) 303 – 316.

[21] S.M. Clinton, V. Haroutunian, K. Davis, J. Meador-Woodruff, Altered Expression of NMDA receptor-associated post-synaptic density proteins in the thalamus in schizophrenia, Am. J. Psychiatry (in press). [22] S.L. Cottingham, D. Pickar, T.K. Shimotake, P. Montpied, S.M. Paul, J.N. Crawley, Tyrosine hydroxylase and cholecystokinin mRNA levels in the substantia nigra, ventral tegmental area, and locus ceruleus are unaffected by acute and chronic haloperidol administration, Cell. Mol. Neurobiol. 10 (1990) 41 – 50. [23] T.J. Counihan, G.B. Landwehrmeyer, D.G. Standaert, C.M. Kosinski, C.R. Scherzer, L.P. Daggett, G. Velicelebi, A.B. Young , J.B. Penney Jr., Expression of N-methyl-D-aspartate receptor subunit mRNA in the human brain: mesencephalic dopaminergic neurons, J. Comp. Neurol. 390 (1998) 91 – 101. [24] T.J. Crow, H.F. Baker, A.J. Cross, M.H. Joseph, R. Lofthouse, A. Longden, F. Owen, G.J. Riley, V. Glover, W.S. Killpack, Monoamine mechanisms in chronic schizophrenia: post-mortem neurochemical findings, Br. J. Psychiatry 134 (1979) 249 – 256. [25] S. Das, Y.F. Sasaki, T. Rothe, L.S. Premkumar, M. Takasu, J.E. Crandall, P. Dikkes, D.A. Conner, P.V. Rayudu, W. Cheung, H.S. Chen, S.A. Lipton, N. Nakanishi, Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A, Nature 393 (1998) 377 – 381. [26] K.L. Davis, R.S. Kahn, G. Ko, M. Davidson, Dopamine in schizophrenia: a review and reconceptualization, Am. J. Psychiatry 148 (1991) 1474 – 1486. [27] S. Dracheva, S.A. Marras, S.L. Elhakem, F.R. Kramer, K.L. Davis, V. Haroutunian, N-Methyl-D-aspartic acid receptor expression in the dorsolateral prefrontal cortex of elderly patients with schizophrenia, Am. J. Psychiatry 158 (2001) 1400 – 1410. [28] S.L. Eastwood, B. McDonald, P.W. Burnet, J.P. Beckwith, R.W. Kerwin, P.J. Harrison, Decreased expression of mRNAs encoding non-NMDA glutamate receptors GluR1 and GluR2 in medial temporal lobe neurons in schizophrenia, Mol. Brain Res. 29 (1995) 211 – 223. [29] I.J. Farley, K.S. Shannak, O. Hornykiewicz, Brain monoamine changes in chronic paranoid schizophrenia and their possible relation to increased dopamine receptor sensitivity, Adv. Biochem. Psychopharmacol. 21 (1980) 427 – 433. [30] X.M. Gao, K. Sakai, R.C. Roberts, R.R. Conley, B. Dean, C.A. Tamminga, Ionotropic glutamate receptors and expression of Nmethyl-D-aspartate receptor subunits in subregions of human hippocampus: effects of schizophrenia, Am. J. Psychiatry 157 (2000) 1141 – 1149. [31] D.C. Goff, J.T. Coyle, The emerging role of glutamate in the pathophysiology and treatment of schizophrenia, Am. J. Psychiatry 158 (2001) 1367 – 1377. [32] A.A. Grace, Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia, Neuroscience 41 (1991) 1 – 24. [33] S. Grimwood, P. Slater, J.F. Deakin, P.H. Hutson, NR2B-containing NMDA receptors are up-regulated in temporal cortex in schizophrenia, NeuroReport 10 (1999) 461 – 465. [34] P.J. Harrison, D. McLaughlin, R.W. Kerwin, Decreased hippocampal expression of a glutamate receptor gene in schizophrenia, Lancet 337 (1991) 450 – 452. [35] D.J. Healy, V. Haroutunian, P. Powchik, M. Davidson, K.L. Davis, S.J. Watson, J.H. Meador-Woodruff, AMPA receptor binding and subunit mRNA expression in prefrontal cortex and striatum of elderly schizophrenics, Neuropsychopharmacology 19 (1998) 278 – 286. [36] M. Hollmann, S. Heinemann, Cloned glutamate receptors, Annu. Rev. Neurosci. 17 (1994) 31 – 108. [37] C. Humphries, A. Mortimer, S. Hirsch, J. de Belleroche, NMDA receptor mRNA correlation with antemortem cognitive impairment in schizophrenia, NeuroReport 7 (1996) 2051 – 2055. [38] H.M. Ibrahim , A.J. Hogg Jr., D.J. Healy, V. Haroutunian, K.L. Davis, J.H. Meador-Woodruff, Ionotropic glutamate receptor binding and

H.T. Mueller et al. / Molecular Brain Research 121 (2004) 60–69

[39]

[40] [41]

[42]

[43]

[44]

[45]

[46] [47] [48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

[57]

subunit mRNA expression in thalamic nuclei in schizophrenia, Am. J. Psychiatry 157 (2000) 1811 – 1823. H. Ichinose, T. Ohye, K. Fujita, F. Pantucek, K. Lange, P. Riederer, T. Nagatsu, Quantification of mRNA of tyrosine hydroxylase and aromatic L-amino acid decarboxylase in the substantia nigra in Parkinson’s disease and schizophrenia, J. Neural Transm. Parkinson’s Dis. Dement. Sect. 8 (1994) 149 – 158. D.C. Javitt, S.R. Zukin, Recent advances in the phencyclidine model of schizophrenia, Am. J. Psychiatry 148 (1991) 1301 – 1308. A.C. Lahti, B. Koffel, D. LaPorte, C.A. Tamminga, Subanesthetic doses of ketamine stimulate psychosis in schizophrenia, Neuropsychopharmacology 13 (1995) 9 – 19. M. Laruelle, A. Abi-Dargham, C.H. van Dyck, R. Gil, C.D. D’Souza, J. Erdos, E. McCance, W. Rosenblatt, C. Fingado, S.S. Zoghbi, R.M. Baldwin, J.P. Seibyl, J.H. Krystal, D.S. Charney, R.B. Innis, Single photon emission computerized tomography imaging of amphetamineinduced dopamine release in drug-free schizophrenic subjects, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 9235 – 9240. S. Le Corre, C.G. Harper, P. Lopez, P. Ward, S. Catts, Increased levels of expression of an NMDARI splice variant in the superior temporal gyrus in schizophrenia, NeuroReport 11 (2000) 983 – 986. A.J. Levinson, S. Garside, P.I. Rosebush, M.F. Mazurek, Haloperidol induces persistent down-regulation of tyrosine hydroxylase immunoreactivity in substantia nigra but not ventral tegmental area in the rat, Neuroscience 84 (1998) 201 – 211. L.J. Martin, C.D. Blackstone, A.I. Levey, R.L. Huganir, D.L. Price, AMPA glutamate receptor subunits are differentially distributed in rat brain, Neuroscience 53 (1993) 327 – 358. S. Matthysse, Antipsychotic drug actions: a clue to the neuropathology of schizophrenia? Fed. Proc. 32 (1973) 200 – 205. J.H. Meador-Woodruff, D.J. Healy, Glutamate receptor expression in schizophrenic brain, Brain Res. Rev. 31 (2000) 288 – 294. J.H. Meador-Woodruff, S.P. Damask, S.J. Watson Jr., Differential expression of autoreceptors in the ascending dopamine systems of the human brain, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 8297 – 8301. J.H. Meador-Woodruff, V. Haroutunian, P. Powchik, M. Davidson, K.L. Davis, S.J. Watson, Dopamine receptor transcript expression in striatum and prefrontal and occipital cortex. Focal abnormalities in orbitofrontal cortex in schizophrenia, Arch. Gen. Psychiatry 54 (1997) 1089 – 1095. J.H. Meador-Woodruff, K.L. Davis, V. Haroutunian, Abnormal kainate receptor expression in prefrontal cortex in schizophrenia, Neuropsychopharmacology 24 (2001) 545 – 552. J.H. Meador-Woodruff, A.J. Hogg Jr., R.E. Smith, Striatal ionotropic glutamate receptor expression in schizophrenia, bipolar disorder, and major depressive disorder, Brain Res. Bull. 55 (2001) 631 – 640. A. Meyer-Lindenberg, R.S. Miletich, P.D. Kohn, G. Esposito, R.E. Carson, M. Quarantelli, D.R. Weinberger, K.F. Berman, Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia, Nat. Neurosci. 5 (2002) 267 – 271. M. Morari, M. Marti, S. Sbrenna, K. Fuxe, C. Bianchi, L. Beani, Reciprocal dopamine – glutamate modulation of release in the basal ganglia, Neurochem. Int. 33 (1998) 383 – 397. T. Ohnuma, H. Kato, H. Arai, R.L. Faull, P.J. McKenna, P.C. Emson, Gene expression of PSD95 in prefrontal cortex and hippocampus in schizophrenia, NeuroReport 11 (2000) 3133 – 3137. J.W. Olney, N.B. Farber, Glutamate receptor dysfunction and schizophrenia, Arch. Gen. Psychiatry 52 (1995) 998 – 1007. M. Paquet, M. Tremblay, J.J. Soghomonian, Y. Smith, AMPA and NMDA glutamate receptor subunits in midbrain dopaminergic neurons in the squirrel monkey: an immunohistochemical and in situ hybridization study, J. Neurosci. 17 (1997) 1377 – 1396. G.M. Pasinetti, D.G. Morgan, S.A. Johnson, S.L. Millar, C.E. Finch, Tyrosine hydroxylase mRNA concentration in midbrain dopaminergic neurons is differentially regulated by reserpine, J. Neurochem. 55 (1990) 1793 – 1799.

69

[58] R.H. Porter, S.L. Eastwood, P.J. Harrison, Distribution of kainate receptor subunit mRNAs in human hippocampus, neocortex and cerebellum, and bilateral reduction of hippocampal GluR6 and KA2 transcripts in schizophrenia, Brain Res. 751 (1997) 217 – 231. [59] A.R. Rutter, F.A. Stephenson, Coexpression of postsynaptic density95 protein with NMDA receptors results in enhanced receptor expression together with a decreased sensitivity to L-glutamate, J. Neurochem. 75 (2000) 2501 – 2510. [60] Y.F. Sasaki, T. Rothe, L.S. Premkumar, S. Das, J. Cui, M.V. Talantova, H.K. Wong, X. Gong, S.F. Chan, D. Zhang, N. Nakanishi, N.J. Sucher, S.A. Lipton, Characterization and comparison of the NR3A subunit of the NMDA receptor in recombinant systems and primary cortical neurons, J. Neurophysiol. 87 (2002) 2052 – 2063. [61] K. Sato, H. Kiyama, M. Tohyama, The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1 – 4) in the rat brain, Neuroscience 52 (1993) 515 – 539. [62] M. Sheng, D.T. Pak, Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses, Ann. N.Y. Acad. Sci. 868 (1999) 483 – 493. [63] R.E. Smith, V. Haroutunian, K.L. Davis, J.H. Meador-Woodruff, Expression of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia, Am. J. Psychiatry 158 (2001) 1393 – 1399. [64] R.E. Smith, V. Haroutunian, K.L. Davis, J.H. Meador-Woodruff, Vesicular glutamate transporter transcript expression in the thalamus in schizophrenia, NeuroReport 12 (2001) 2885 – 2887. [65] S.H. Snyder, The dopamine hypothesis of schizophrenia: focus on the dopamine receptor, Am. J. Psychiatry 133 (1976) 197 – 202. [66] B.P. Sokolov, Expression of NMDAR1, GluR1, GluR7, and KA1 glutamate receptor mRNAs is decreased in frontal cortex of ‘‘neuroleptic-free’’ schizophrenics: evidence on reversible up-regulation by typical neuroleptics, J. Neurochem. 71 (1998) 2454 – 2464. [67] D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurol. 343 (1994) 1 – 16. [68] N.J. Sucher, S. Akbarian, C.L. Chi, C.L. Leclerc, M. Awobuluyi, D.L. Deitcher, M.K. Wu, J.P. Yuan, E.G. Jones, S.A. Lipton, Developmental and regional expression pattern of a novel NMDA receptorlike subunit (NMDAR-L) in the rodent brain, J. Neurosci. 15 (1995) 6509 – 6520. [69] L. Sun, F.L. Margolis, M.T. Shipley, M.S. Lidow, Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor: its regional distribution and developmental expression in the rat brain, FEBS Lett. 441 (1998) 392 – 396. [70] M. Takeuchi, Y. Hata, K. Hirao, A. Toyoda, M. Irie, Y. Takai, SAPAPs. A family of PSD-95/SAP90-associated proteins localized at postsynaptic density, J. Biol. Chem. 272 (1997) 11943 – 11951. [71] M. Toru, T. Nishikawa, N. Mataga, M. Takashima, Dopamine metabolism increases in post-mortem schizophrenic basal ganglia, J. Neural Transm. 54 (1982) 181 – 191. [72] L.T. Weiss-Wunder, M.F. Chesselet, Acute and repeated administration of fluphenazine-N-mustard alters levels of tyrosine hydroxylase mRNA in subsets of mesencephalic dopaminergic neurons, Neuroscience 49 (1992) 297 – 305. [73] B. Winblad, G. Bucht, C.G. Gottfries, B.E. Roos, Monoamines and monoamine metabolites in brains from demented schizophrenics, Acta Psychiatr. Scand. 60 (1979) 17 – 28. [74] H.K. Wong, X.B. Liu, M.F. Matos, S.F. Chan, I. Perez-Otano, M. Boysen, J. Cui, N. Nakanishi, J.S. Trimmer, E.G. Jones, S.A. Sucher, N.J. Sucher, Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain, J. Comp. Neurol. 450 (2002) 303 – 317. [75] U. Wullner, D.G. Standaert, C.M. Testa, J.B. Penney, A.B. Young, Differential expression of kainate receptors in the basal ganglia of the developing and adult rat brain, Brain Res. 768 (1997) 215 – 223.