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The N-methyl-d -aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications
Pharmacology & Therapeutics 97 (2003) 55 – 85
Associate editor: K.A. Neve
The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications Jennifer M. Loftisa,b,*, Aaron Janowskya,b,c,d a
Research Service, Department of Veterans Affairs Medical Center, Mental Health (P3MHDC), 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239, USA b Department of Psychiatry, Oregon Health and Science University, Portland, OR 97239, USA c Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USA d Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA
Abstract The N-methyl-D-aspartate (NMDA) receptor is an example of a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors are composed of seven known subunits (NR1, NR2A – D, NR3A – B). The present review focuses on the NR2B subunit of the receptor. Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in a variety of synaptic signaling events and protein-protein interactions. The NR2B subunit has been implicated in modulating functions such as learning, memory processing, pain perception, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structural features, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Postsynaptic density; Glutamate; Kinase; Cytoskeleton; Synaptic plasticity; Excitotoxicity Abbreviations: ADNF, activity-dependent neurotrophic factor; AP-5, DL-2-amino-5-phosphonovaleric acid; BDNF, brain-derived neurotrophic factor; CaM, calmodulin; CaMKII, Ca2+-, calmodulin-dependent protein kinase II; CAPON, carboxy-terminal postsynaptic density-95, discs-large, ZO-1 ligand of neuronal nitric oxide; CASK, a novel postsynaptic density-95 homolog with an N-terminal Ca2+-, calmodulin-dependent protein kinase domain; chapsin, channelassociated protein of synapses; CIPP, channel-interacting postsynaptic density-95, discs-large, ZO-1 domain protein; cNOS, constitutive nitric oxide synthase; Con, conantokin; CPP, 3-(R)-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; CRIPT, a novel protein that selectively binds to the third postsynaptic density-95, discs-large, ZO-1 domain of postsynaptic density-95 via its C-terminal; CTA, conditioned taste aversion; EPSC, excitatory postsynaptic current; GABA, g-aminobutyric acid; GFP, green fluorescent protein; GK, guanylate kinase; GKAP, guanylate kinase-associated protein; GST, glutathione Stransferase; HEK, human embryonic kidney; KO, knockout; LTD, long-term depression; LTP, long-term potentiation; MAGUK, membrane-associated guanylate kinase; neurabin, neural tissue-specific F-actin binding protein; NMDA, N-methyl-D-aspartate; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; PDZ, postsynaptic density-95, discs-large, ZO-1; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PP1, Type 1 protein phosphatase; PREGS, pregnenolone sulfate; PSD, postsynaptic density; PTP, protein tyrosine phosphatase; RasGAP, Ras-GTPase-activating protein; RT-PCR, reverse transcriptase-polymerase chain reaction; SAP, synapse-associated protein; SAPAP, synapse-associated protein-90/postsynaptic density-95-associated protein; SH, Src homology; S-SCAM, synaptic scaffolding molecule; SynGAP, synaptic GTPase-activating protein.
Contents 1.
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Introduction . . . . . . . . . . . . . . . . . 1.1. Structural features . . . . . . . . . . 1.2. Receptor composition/stoichiometry . Localization . . . . . . . . . . . . . . . . . 2.1. During development . . . . . . . . . 2.1.1. NR2B gene expression . . . 2.1.2. NR2B protein expression . . 2.1.3. Associated proteins . . . . .
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* Corresponding author. Research Service, Department of Veterans Affairs Medical Center, Mental Health (P3MHDC), 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239, USA. Tel.: 503-220-8262 ext. 57155; fax: 503-220-3499. E-mail address: [email protected] (J.M. Loftis). 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 2 ) 0 0 3 0 2 - 9
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2.2.
In adult tissue . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. NR2B gene expression . . . . . . . . . . . . . . . . 2.2.2. NR2B protein expression . . . . . . . . . . . . . . . 2.2.3. Cellular and subcellular distribution . . . . . . . . . 3. Functional properties/roles . . . . . . . . . . . . . . . . . . . . . . 3.1. Pharmacological . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Electrophysiological . . . . . . . . . . . . . . . . . . . . . . 3.3. Behavioral . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Endogenous regulators. . . . . . . . . . . . . . . . . . . . . 4.1.1. Cytoskeletal proteins . . . . . . . . . . . . . . . . . 4.1.2. Enzymes (e.g., phosphatases, kinases, and proteases) 4.1.3. Neuromodulators (e.g., growth factors, hormones) . . 4.2. Exogenous regulators . . . . . . . . . . . . . . . . . . . . . 4.2.1. Trauma or lesions. . . . . . . . . . . . . . . . . . . 4.2.2. Pharmacological agents (e.g., antagonists) . . . . . . 5. NR2B role in human physiology and pathophysiology . . . . . . . . 5.1. Synaptic plasticity/learning . . . . . . . . . . . . . . . . . . 5.2. Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . 5.4. Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . 5.5. Excitotoxicity/hypoxia/ischemia . . . . . . . . . . . . . . . . 5.6. Seizure disorder . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . 5.9. Drugs of abuse. . . . . . . . . . . . . . . . . . . . . . . . . 5.10. Pain perception . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The N-methyl-D-aspartate (NMDA) receptor is a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits (McBain & Mayer, 1994). NMDA receptors are concentrated at postsynaptic sites, although some appear to be presynaptic (Liu et al., 1994). Neurotransmission involving NMDA receptors has been implicated in a variety of unique roles: (1) NMDA receptor activation associated with long-lasting changes in synaptic strength (Ali & Salter, 2001), (2) organization of afferent fibers with respect to target neurons during development (Collingridge & Singer, 1990), and (3) participation in glutamate neurotoxicity (Choi & Rothman, 1990). NMDA receptors are derived from at least seven known subunit genes. One of these genes codes for the NR1 subunit, a ubiquitous and necessary component of functional NMDA receptor channels. The diversity of the NMDA receptor NR1 subunit is created by alternative splicing of the NMDAR1 subunit gene to yield eight functional splice forms, which arise via the insertion or deletion of three short exon cassettes in the N-terminal (N1) and C-terminal (C1, C2) domains of the subunit protein (Hollmann et al., 1993). Conversely, the heterogeneity of the NR2 subunits occurs due to the existence of multiple, related genes, NMDAR2A – 2D, whose specific expression profiles in the brain are developmentally and
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regionally regulated (Ishii et al., 1993; Laurie et al., 1997). Recently, NR3A and NR3B subunits have also been reported (Das et al., 1998; Eriksson et al., 2002; Nishi et al., 2001). The present review focuses on the NR2B subunit, first described as a 180-kDa glycoprotein substrate for tyrosine kinase and Ca2+-, calmodulin-dependent protein kinase II (CaMKII) in the postsynaptic density (PSD) (Gurd, 1985). Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in determining the pharmacological and functional properties of the NMDA receptor. Consequently, NR2B has been implicated in modulating functions such as learning, memory processing, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structure, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases and syndromes. 1.1. Structural features The primary structures of the NMDA NR2 subunits were revealed for both mouse (Kutsuwada et al., 1992) and rat (Monyer et al., 1992) using independent cloning strategies. The predicted amino acid sequences for the e2 (mouse
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terminology) and NR2B subunits show a sequence of 45 amino acids, specifically residues 1362– 1406, which differ between the mouse and rat homologs (McBain & Mayer, 1994). The four NR2 subunits share considerable homology. For example, the predicted proteins are 55% (NR2A and NR2C) and 70% (NR2A and NR2B) identical, but are only 20% homologous to the NR1 subunit (Monyer et al., 1992). The NR2B subunit is composed of 1456 amino acids with an approximate molecular mass of 170 –180 kDa. Hydropathy analysis of amino acid sequences predicted from the cDNA for the NR2B subunit suggests the presence of an extracellular NH2-terminal signal peptide and four putative transmembrane domains (M1-M4) (Kutsuwada et al., 1992). M2 forms a reentrant loop that lines the channel. Although the NR2 subunits have the same basic structure as NR1 and other glutamate-gated ion channels (for a review, see Hollmann & Heinemann, 1994), they differ in that they possess large intracellular C-terminal domains, in excess of 600 amino acids and containing scattered regions of conserved sequences. The size of these C-termini is larger than the extracellular NH2-terminal segment preceding the first transmembrane region (Monyer et al., 1992). However, for all NMDA receptor subunits, the NH2-terminal extracellular domain is greater than twice the size of that for other receptors ( 50 kDa) (McBain & Mayer, 1994). Finally, the NR1 and NR2 subunits possess an asparagine residue (amino acid residue 589 for NR2B) in the second transmembrane region. This domain is the putative pore-forming region for the NMDA receptor subunits. Thus, the asparagine residue may play a role in the high Ca2+ permeability of the channel. 1.2. Receptor composition/stoichiometry As will be reviewed in Section 2, although relatively comprehensive, the localization data available for NR2B are not always able to differentiate between subunit expression and the presence of functional NMDA receptors. Questions about the distribution of NR2B subunit-containing ion channels are further complicated by conflicting reports regarding NMDA receptor subunit composition. Premkumar and Auerbach (1997) inferred the stoichiometry of recombinant NMDA receptor channels from single-channel current patterns recorded from mouse NR1NR2B receptors. The authors concluded that NMDA receptors are pentamers composed of three NR1 and two NR2 subunits. In contrast, Laube et al. (1998) presented evidence for a tetrameric structure of recombinant NMDA receptors. Using solubilized mouse forebrain, the following subunit distribution of NMDA receptors was observed: NR1, 17%; NR1/NR2A, 37%; NR1/NR2B, 40%; and NR1/NR2A/ NR2B, 6% (Chazot & Stephenson, 1997). Further, in rat tissue, anti-NR2B antibodies immunoprecipitate NR2A, NR2B, and NR1 subunits from the cortex and thalamus.
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NR2D is assembled with NR1, NR2A, and NR2B subunits in the rat thalamus (Dunah et al., 1998). The NR2A and NR2B subunits also appear to co-localize in other brain regions and in vitro. For example, immunohistological methods reveal that in hippocampal subfields (CA1 –CA3), the NR2A- and NR2B-immunopositive cells exhibit extensive co-localization in the stratum radiatum and oriens (Fritschy et al., 1998). In vitro studies using human embryonic kidney (HEK)-293 cells transfected with NR1 and NR2A NMDA receptor subunits in combination with FLAG- and c-Myc epitope-tagged NR2B subunits support and extend these findings by providing data regarding the number of NR2 subunits present per receptor complex. Specifically, results show co-assembly of three NR2 subunits, NR2A/NR2B FLAG /NR2B c-myc , within the same NMDA receptor (Hawkins et al., 1999). Although characterization of NMDA receptor composition continues, existing data indicate that specific subunit assembly and receptor stoichiometry is regionally and developmentally regulated.
2. Localization 2.1. During development 2.1.1. NR2B gene expression The expression of the various NMDA receptor genes is under developmental control. Expression is thus a dynamic process, and, as will be described in the following sections, often responds to changes in the internal and external environments. Studies have recently begun to characterize the promoter regions for the glutamate receptor genes to identify regulatory elements and factors that control expression in neurons (Okamoto et al., 1999). However, the transcription factors involved in the regulation of NR2B subunits are still unknown. Although the control of expression during development remains elusive, the regional distribution of the NR2B subunit is better understood. Recent evidence suggests that NMDA receptors are critical for corticogenesis, neuronal migration, and synaptogenesis during brain development. A number of investigators have examined expression densities and localization of the NMDAR2B gene at various stages of development within different brain areas, such as the cortex, hippocampus, cerebellum, and hypothalamus. A distinct pattern of mRNAs encoding NMDA receptor subunits in the developing rat brain was revealed by in situ hybridization experiments (Monyer et al., 1994). At embryonic day 14, low levels of NR2B mRNA are evident in the spinal cord and hypothalamus. However, by embryonic day 17, there is a marked increase in NR2B transcript expression, with high levels in the cerebral cortex (especially layer 1), thalamus, and spinal cord, and lower levels in the hippocampus, colliculi, and hypothalamus. At birth, further increases in NR2B mRNA expression are apparent in the cerebral
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cortex, hippocampus, septum, neostriatum, and thalamic nuclei, but low levels are in the cerebellum. By postnatal days 7 and 12, NR2B mRNA shows robust expression, with high levels in the cerebral cortex and hippocampus, and moderate levels in the neostriatum, septum, thalamic nuclei, and cerebellum. Similar transcript expression patterns are revealed in vitro. NR2B mRNA levels are relatively low in cortical cultured neurons from embryonic day 17 rats. Between days 1 and 7 in vitro, expression increases with little further change after day 7. The level of NR2B mRNA is 4-fold higher than that of NR2A mRNA in 21-day cultures (Zhong et al., 1994). Similarly, Ritter et al. (2001) found that in human fetal brains, NR2B mRNA expression is apparent and predominant in all cerebral cortical layers as early as gestational week 8. Looking at older developmental ages, Magnusson (2000) attempted to determine whether some of the age-related changes in binding to the NMDA receptor complex could be accounted for by changes in subunit expression during the aging process. In situ hybridization of the NMDA subunits NR1, NR2A, and NR2B and receptor autoradiography using the agonist glutamate and the competitive antagonist 3-(R)-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) were performed on sections from C57Bl/6 mice at three different ages (3, 10, and 30 months). mRNA for the NR2B subunit is significantly decreased between the ages of 3 and 30 months in a majority of cerebral cortical regions and in dentate gyrus granule cells. In the cerebellum, reverse transcriptase-polymerase chain reaction (RT-PCR) assays were used to measure levels of NMDA receptor subunit mRNA levels in dissociated granule cell cultures from 8-day-old neonatal rats. Primary cultures of granule cells were maintained for 4– 14 days in vitro. In situ hybridization revealed that by 4 days in vitro, NR2B mRNA is barely detectable and that after 7 days, NR2B mRNA is below detection limits (Vallano et al., 1996). In agreement with these findings, an in situ experiment of rat NMDA receptor subunit mRNA levels shows that postnatally (days 7 and 12), the NR2B and NR2C transcript expression changes in opposite directions in the cerebellar granule cell layer, with NR2B mRNA levels decreasing in abundance while NR2C levels increase (Monyer et al., 1994). Thus, granule cell maturation is associated with down-regulation of NR2B gene expression. NR2B mRNA increases significantly between birth and postnatal day 5 in both male and female rats in the preoptic area of the anterior hypothalamus (Adams et al., 1999). Recently, in addition to localization within the brain, some NMDA receptor subunits have also been described in adult non-neuronal tissue and keratinocytes. However, their developmental expression patterns are not as well characterized. With the use of RT-PCR, the expression of the NR2B subunit was investigated in the developing rat heart. NR2B mRNA is present in the heart tissue of rats from embryonic day 14 until postnatal day 21, but disappears 10 weeks after birth. In contrast, no NR1, GluR2, or PSD-95
could be detected in the rat heart at any developmental stage (Seeber et al., 2000). Finally, mammalian osteoclasts and osteoblasts express several NMDA receptor subunit mRNAs, including NR2B, suggesting that NMDA receptors may play a role in bone cell differentiation and function (Itzstein et al., 2001). 2.1.2. NR2B protein expression NR2B protein expression, like gene expression, is developmentally regulated. Further, the regional distribution of protein expression is generally in close agreement with studies investigating gene expression. Laurie et al. (1997) used immunoblotting of whole, neonatal rat brains and found that NR2B is strongly expressed at birth (unlike the NR2A subunit) and further increases until postnatal day 6 to day 20 before declining slightly (especially in the cerebellum) to the adult level. Consistent with these findings, Wang et al. (1995) examined the ontogenic profiles of the NR2A and NR2B subunits in the rat cerebellum. At postnatal day 2, the NR2A levels are undetectable, whereas the NR2B subunit is abundant. By postnatal day 12, the expression of NR2A increases and reaches adult levels by postnatal day 22, while NR2B expression begins to decline at postnatal day 12 and is no longer apparent 22 days after birth. In contrast, Misra et al. (2000) investigated functional properties of NMDA receptor cerebellar Golgi cells using patchclamp recordings in slices from postnatal day 14 rats. Electrophysiological results reveal that ifenprodil (NR2Bselective antagonist; see Table 1) reduces whole cell NMDA-evoked currents by 80%. In addition, a population of high-conductance NMDA receptor-containing cells was detected and found to be inhibited by ifenprodil. Thus, it was concluded that in cerebellar Golgi cells, the highconductance NMDA receptor channels arise from NR2Bcontaining receptors. Interestingly, the authors found no evidence of NR2A-containing receptors in these cells. Fractionation of cortical homogenates from rat embryos reveals that the NR2B subunit is highly enriched in axonal growth cones (Wang et al., 1995). In the hippocampus, a similar distribution was found by microscopy of immature hippocampal neurons, showing preferential accumulation of NR2B in axonal growth cones and varicosities. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role for NMDA receptors in the regulation of neuronal growth and migration (Herkert et al., 1998). Sheng et al. (1994) used co-immunoprecipitation with NMDA receptor subunit-specific antibodies, and observed the composition of heteromeric receptors during development of the rat cerebral cortex. NR1 splice variantspecific antibodies for the C1- and N1-regions of the NR1 subunit co-immunoprecipitated NR2B subunits from cortical membranes throughout the period tested (postnatal days 1 – 53), although the amount of co-immunoprecipitated NR2B was consistently smaller for NR1-N1- than for C1specific antibodies. Differential co-immunoprecipitation of
J.M. Loftis, A. Janowsky / Pharmacology & Therapeutics 97 (2003) 55–85 Table 1 Pharmacological profile Drug
Action
Reference
Ifenprodil
NMDA receptor NR2Bselective antagonist; inhibits channel opening; increases NMDA receptor affinity for glutamate-site agonists NR2B-selective antagonist; binds to ifenprodil site NR2B-selective antagonist; binds to ifenprodil site NR2B-selective antagonist; binds to ifenprodil site Glycine site antagonist NR2B-selective antagonist; binds to ifenprodil site
Chenard & Menniti, 1999; Grimwood et al., 2000; Coughenour & Barr, 2001; Zhang & Shi, 2001
Eliprodil CP-101,606 CP-283,097 CGP 61594 Ro 25-6981
Con-R
Con-G
N-(phenylalkyl) cinnamides Felbamate
Grimwood et al., 2000; Pabel et al., 2000 Grimwood et al., 2000; Chazot, 2000 Grimwood et al., 2000 Honer et al., 1998 Grimwood et al., 2000; Mutel et al., 1998; Fischer et al., 1997; Lynch et al., 2001 White et al., 2000
NMDA receptor peptide antagonist; NR2B selective NMDA receptor peptide Donevan & McCabe, 2000 antagonist; NR2B selective NR2B-selective antagonist Tamiz et al., 1999
NR2B-selective antagonist Kleckner et al., 1999; Harty & Rogawski, 2000 Homoquinolinate NR2B-selective ligand Brown et al., 1998 Ro G3-1908 NR2B-selective antagonist Wyss et al., 2000 CGP 39653 NR2B-selective Christie et al., 2000 competitive antagonist MK-801 Channel-site ligand; Laurie & Seeburg, 1994 non-competitive NMDA receptor antagonist Drugs listed act at NR2B-containing NMDA receptors with varying degrees of selectivity and potency. Refer to referenced articles for specific information regarding binding affinities and mechanisms of action.
NR2A and NR2B subunits by N1- and C1-containing splice variants suggests that there must also be some segregation of NR2A and NR2B. These postnatal NR2 subunit- and NR1 splice variant-specific interactions could contribute to NMDA receptor variation and changing synaptic plasticity during cortical development. In support of their work investigating NR2B gene expression, Seeber et al. (2000) used western blot analysis to investigate the expression of the NR2B subunit in the developing rat heart. The NR2B protein is detected in heart tissue of rats from embryonic day 14 until postnatal day 21, but disappears 10 weeks after birth. As with gene expression, the NR1 and GluR2 subunits and PSD-95 protein could not be detected in the rat heart at any developmental stage. Further, no functional NMDA receptors could be detected on cardiac myocytes by whole cell recordings. In conclusion, the NR2B subunit is abundant in the developing rat heart, but its function remains unknown.
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2.1.3. Associated proteins In Section 4.1.1 we review the effects of associated PSD proteins on the regulation of the NR2B subunit. A brief discussion of the ontogeny of such proteins is included below, as it contributes to understanding the developmental and functional significance of NR2B subunits. The developing mouse brain was examined for the expression of PSD-95 and synapse-associated protein (SAP)102, which are two PDZ (PSD-95, Discs-large, ZO1) domain-containing proteins that associate with NMDA receptors (see Section 4.1; Fig. 1). Using in situ hybridization with antisense oligonucleotide probes, PSD-95 and SAP102 mRNA were shown to be expressed at embryonic day 13 in the mantle zone of various brain regions, where NR2B mRNA is also expressed at high levels. In the early postnatal period, the mRNA for both PSD-95 and SAP102 is elevated and concentrated in the telencephalon and cerebellar granular layer, where NR2A and NR2B mRNA are abundantly expressed (Fukaya et al., 1999). Thus, the regional and temporal expression profile in the brain suggests that members of the PSD-95/SAP90 protein family can interact with NMDA receptor subunits to facilitate the formation of ion channel complexes, during and after synaptogenesis. To more specifically characterize the relationship of NR2B and cytoskeletal proteins during development, Sans et al. (2000) investigated the developmental changes in expression of PSD-93, PSD-95, and SAP102 in the rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2, SAP102 is highly expressed, whereas the abundance of PSD-93 and PSD-95 is low. Interestingly, SAP102 expression increases during the first week after birth when active synaptogenesis putatively takes place in the hippocampus. SAP102 protein expression remains stable through postnatal day 35, but shows reduced expression by 6 months of age. From immunoblots of the hippocampus and immunogold analysis of CA1 synapses, the high level of NR2B expression at postnatal day 2 appears to coincide with the high level of synaptic SAP102 expression. To determine whether the developmental changes in PSD-93/PSD-95 and SAP102 reflect a preferred association with NR2A and NR2B subunits, respectively, Sans et al. (2000) measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2B/ SAP102 and NR2A/PSD-93/-95. Thus, individual cytoskeletal proteins form specific interactions with the NMDA receptor subunits and participate in functions that are critical to synapse development. 2.2. In adult tissue 2.2.1. NR2B gene expression Similar to observations made during animal development, the expression in adults of individual mRNAs for the NMDA receptor subunits overlap in some brain regions, but are specialized in many others. In particular, NR2B
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Fig. 1. Schematic representation of NR2B-containing NMDA receptor complex and associated neuronal elements. Blue proteins represent PSD-95 and related family members, other structural proteins are stippled, and proteins with less well-understood function are shown in white. Enzymes such as kinases and phosphatases are depicted in yellow. Please refer to Section 4.1 for information regarding specific protein-protein interactions and putative functions.
subunits display a unique regional and cell-specific expression profile. Early reports from Ishii et al. (1993) used northern blotting and in situ hybridization analyses to show that NR2A and NR2B are abundant in the cortex and hippocampus, NR2C is in the cerebellum, and NR2D is located in subcortical areas. More recently, select regions from the rat CNS were examined for the presence of mRNA coding for NR2B using a nuclease protection assay. The rank order of NR2B expression is as follows: cortex > olfactory bulbs > hippocampus > striatum > superior colliculus > inferior colliculus > brain stem > retina cerebellum > hypothalamus (Goebel & Poosch, 1999). Sun et al.
(2000) expanded on these findings and provided a more detailed characterization of NR2B subunit cortical expression. Quantitative RT-PCR was used to analyze the relative expressions of the NR1, NR2A, NR2B, NR2C, NR2D, and NR3 subunits of the NMDA receptor in the piriform, entorhinal, visual, and motor cortices, as well as in the olfactory bulb of adult rat. The analysis detected clear differences in the relative proportions of the NMDA receptor subunits among the brain areas examined. These regional differences are particularly prominent when the piriform and motor cortices are contrasted. Specifically, the NR2B subunit transcript is prevalent and highly expressed in the motor
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cortical region ( 135% of NR1 message), while NR2B is expressed at lower levels ( 30% of NR1) in the piriform cortex. 2.2.2. NR2B protein expression As with the studies examining NR2B localization during development, experiments investigating receptor subunit expression in adult animals demonstrate good agreement between gene and protein expression. Laurie et al. (1997) used immunoblotting techniques with rat, mouse, frog, rabbit, and human brains. Using an antibody that recognizes a portion of the C-terminal, NR2B subunit protein was observed in all species tested, except frog. In the rat and human, the NR2B subunit is primarily expressed in forebrain structures, such as the cortex, hippocampus, striatum, thalamus, and olfactory bulb. Moderate levels of NR2B subunit expression are evident in the midbrain, such as the hypothalamus, colliculi, and also the brain stem (rat only), and low expression occurs in the cerebellum and spinal cord. In agreement, Wang et al. (1995) report that in the adult rat, the NR2B subunit is expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Interestingly, regional subunit expression differences have been reported between rats and mice, since prominent NR2B immunoreactivity is found in Purkinje cell bodies and dendrites in the mouse cerebellum, but NR2B is low in the rat cerebellum (Thompson et al., 2000; Laurie et al., 1997). Although Laurie et al. (1997) were able to detect only moderate amounts of the NR2B protein in the hypothalamus, the NR2B subunits in the diencephalon participate in various physiological functions (see Section 3.3). Thus, to broaden their understanding of the role of the NR2B subunit in homeostatic processes, Khan et al. (2000) characterized the expression of the NR2B subunits within the diencephalon of adult male rats. In the hypothalamus, the highest levels of NR2B immunoreactivity were in the paraventricular and supraoptic nuclei. Further, intense NR2B immunoreactivity was present in the nucleus circularis, anterior fornical nucleus, and scattered clusters of lateral hypothalamic cells. NR2B immunoreactivity was also apparent within the arcuate nucleus, the median eminence, and the tuberal nucleus, and light immunostaining was visible in all other hypothalamic nuclei examined. In the thalamus, the highest amount of NR2B immunoreactivity was in the medial habenula and the anterodorsal, paraventricular, rhomboid, reticular, and dorsal lateral geniculate nuclei. Thus, the NR2B subunit appears to be widely distributed throughout the hypothalamus and thalamus, supporting its participation in a variety of regulatory functions. 2.2.3. Cellular and subcellular distribution In addition to investigations characterizing the localization of the NR2B subunits, a number of laboratories have been interested in discovering the cellular or subcellular specificity of NR2B abundance within different brain
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regions. Using in situ hybridization with tissue from rhesus and Japanese monkeys, Munoz et al. (1999) investigated the expression of NR2B in the sensory and motor cortices. NR2B mRNA expression is heterogeneous across the primary sensory cortex and area 5. Specifically, a region of low expression is present in layer I, higher expression is in layer II, continuous moderate expression is in layers II –IV, and alternating areas of low and high expression are in deep layers V and VI. In area 4, layer II displays the highest level of NR2B mRNA expression, whereas transcript expression appears low in layer I and moderate in the other layers. Although specific roles for NR2B-containing receptors in sensory and motor functioning are yet to be fully understood, these findings suggest that the NR2B subunits will likely be found to play a critical part in processing sensory and motor information. In addition to the cellular distribution of NR2B mRNA in the cerebral cortex, Jones et al. (1998) demonstrated with great detail cell-specific gene expression in the monkey thalamus. Similar NR2B mRNA expression has also been identified in brain structures potentially associated with the limbic system. In rats, large neurons close to the midline of the medial septal region express mRNA for glutamic acid decarboxylase and are almost exclusively g-aminobutyric acid (GABA)ergic, and all but one neuron express mRNA for the NR2B subunit (Plant et al., 1997). Further, Kuppenbender et al. (2000) used dual-label in situ hybridization techniques to assess the levels of NR1 and NR2A-D mRNA expressed in projection neurons and interneurons of the human striatum. The neuronal populations were identified with digoxigenin-tagged cRNA probes for preproenkephalin and substance P targeted to striatal projection neurons, and somatostatin, glutamic acid decarboxylase, and choline acetyltransferase targeted to striatal interneurons. Unlike other NMDA receptor subunits, intense NR2B mRNA hybridization signals were observed across all neurons in the striatum. These results suggest that, as with the cortex, thalamus, and septum, the striatum contains different populations of neurons that have unique NMDA receptor subunit compositions. Interestingly, NR2B mRNA was detected in the dendrites and soma of cultured hippocampal neurons, suggesting that protein synthesis may occur in neuronal processes (as opposed to nuclei), especially at or near postsynaptic sites (Miyashiro et al., 1994). With regard to NR2B subunit protein expression in hippocampal subfields, the NR2Bspecific antibodies labeled neuronal cell bodies and dendrites in all fields of Ammon’s horn, CA1, CA3, and in the dentate gyrus (Charton et al., 1999). Luo et al. (2002) generated functional green fluorescent protein (GFP)-tagged NR2A and NR2B subunits and studied the localization of NMDA receptors in transfected hippocampal neurons. GFPtagged NR2A- and NR2B-containing receptor clusters were most evident on dendritic shafts and spines. In the rat cerebral cortex, numerous neurons (all layers), in particular pyramidal-like cells of layers II/III and V, were
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immunolabeled for NR2B. Staining of cortical neurons in layers I and IV was relatively weak. Immunoreactivity extended into dendritic processes, especially the apical dendrites of pyramidal neurons (Charton et al., 1999). Similar results were found in studies investigating specific cortical regions in both non-human primates and rats. Employing immunocytochemistry with rhesus and Japanese monkeys, Munoz et al. (1999) also investigated the expression of the NR2B protein in the sensory and motor cortices. All cortical layers had positive immunoreactivity for the NR2B subunits. Specifically, layers II and III displayed a high density of small, round NR2B-immunopositive cells that also co-localized GABA, calbindin, or parvalbumin. In addition, a population of NR2B-containing medium-sized pyramidal neurons that also contain CaMKII-a was observed in layers II and III. Also apparent in layers II and III were NR2B-labeled apical dendrites of pyramidal neurons from layers III and IV. One group of investigators localized cell-specific NR2B expression in the spinal cord of rats. Ma and Hargreaves (2000) used immunohistochemical staining and retrograde tracing techniques to investigate the relationship between the NR2B subunits and small-diameter primary afferent dorsal root ganglion neurons that give rise to the sciatic nerve fibers. Results indicated that the NR2B subunits are predominantly expressed on small-diameter primary afferents. Consequently, these NR2B-containing NMDA receptors are hypothesized to modulate neurotransmitter release from primary afferent terminals. Although prominent NR2B subunit immunolabeling is present on neuronal processes, a recent report from Sinor et al. (2000) provides evidence for a slightly different subcellular distribution. The authors used haloperidol, an antagonist with high selectivity for the NR1/NR2B receptor configuration, to confirm that the NR1/NR2B receptors migrate to cell bodies in cultured rat cortical neurons. In mature cultures (more than 22 days old in vitro), NMDA receptor-mediated responses obtained from excised nucleated macropatches are significantly antagonized by haloperidol. NR1/NR2B receptors thus appear to be preferentially expressed in cell bodies of mature cortical neurons. In terms of cell surface expression, almost all NR2B subunits are reportedly found in the plasma membrane, as compared with less than one-half of the total NR1 subunits. These data suggest that neurons possess a large intracellular pool of NR1 subunits that await assembly with the NR2 subunits prior to expression at the plasma membrane (Hall & Soderling, 1997). Al Hallaq et al. (2001) used immunoblotting of rat cortical and cerebellar tissue to measure the prevalence of NMDA receptor subunits and PDZ domain-containing proteins. The NR2A and NR2B subunits, despite previous electrophysiological evidence (Rumbaugh & Vicini, 1999), are equally enriched in the PSD fraction. In addition, PSD-95 and channel-associated protein of synapses (chapsyn)-110 are both strongly expressed, but SAP102 is expressed to a lesser
extent, in the PSD. Consistent with these findings, there is evidence that reveals that while PSD-95 and chapsyn-110 are present only at postsynaptic sites, SAP102 is located at axonal sites, as well as postsynaptically (El Husseini et al., 2000). Collectively, these results illustrate the degree of not only regional, but also cellular and subcellular, expression specificity that is exhibited by the NR2B subunits.
3. Functional properties/roles None of the four NR2 subunits, NR2A-NR2D, assemble by themselves into functional channels. However, when coexpressed with the NR1 subunits, receptor activation can occur. Heteromeric combinations of the NR1 and NR2B subunits are highly permeable to Ca2+, are blocked by Zn2+ and MK-801 (dizocilpine maleate; open channel blocker) and by Mg2+ in a voltage-dependent manner, and are co-activated by glutamate and glycine (for a review, see McBain & Mayer, 1994). Although the NR2A-NR2D subunits have the same basic structure as the NR1 subunit (Hollmann & Heinemann, 1994), they differ in possessing strikingly large intracellular C-terminal domains of 627, 644, 404, and 461 amino acids, respectively (see Section 1.1). As will be discussed in subsequent sections, the C-terminal has been suggested to play a role as the target domain for modulatory or accessory proteins in promoting receptor assembly, sorting, or targeting. Similarly, this region of the subunit may contribute to different channel conformations and subsequently modulate receptor function. The structural properties and functional roles attributed to the NR2B subunit of the NMDA receptor are organized into pharmacological, electrophysiological, and behavioral sections. 3.1. Pharmacological The NR2B subunit appears to be critical for a number of the basic structural and functional attributes associated with the NMDA receptor. For example, the glutamate-binding pocket is formed by the NR2B subunit (Laube et al., 1997). The high-affinity sites for CGP61594 (glycine site antagonist) were exclusively displayed by the NR1/NR2B receptors as compared with the NR1/NR2A, NR1/NR2D, or NR1/ NR2C receptors (Honer et al., 1998). The tryptophan residue in the M2 region of the NR2 subunits controls the Mg2+ block. Specifically, an NR2B leucine (W607L) mutation abolishes the block and significantly increases access to extracellular Mg2+. In addition, NR2B subunit mutations that changed tryptophan (W607) to asparagine (W607N) or alanine (W607A) also greatly attenuated the Mg2+ block. Williams et al. (1998) thus proposed a model in which the M2 loop of NR2B (W607) is positioned at the narrow constriction, at a level similar to asparagine residues in NR2B (N616) and NR1 (N616), with these three residues forming a binding site for Mg2+.
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In addition to providing information about the structural properties associated with the NR2B subunits, pharmacological experiments have facilitated the understanding of functional attributes by developing and/or characterizing a number of useful and relatively subunit-selective compounds. For instance, Grimwood et al. (2000) performed [3H]ifenprodil-binding experiments under NMDA receptorspecific assay conditions to provide the first detailed characterization of the pharmacology of the ifenprodil site on the NMDA receptors, using recombinant human NR1/NR2B receptors stably expressed in L(tk) cells, in comparison with rat cortex/hippocampus membranes. [3H]Ifenprodil binds to a single, saturable site on both human recombinant receptors and native rat receptors. In addition, trifluoroperazine was used to isolate high-affinity [3H]ifenprodil binding to NMDA receptors in a rat membrane preparation. [3H]Ifenprodil bound to a single high-affinity site, with the pharmacology of NMDA receptors containing the NR2B subunit (Coughenour & Barr, 2001). The affinity of various ifenprodil site ligands—eliprodil, CP-101,606, CP-283,097, and Ro 25-6981—is very similar for inhibition of [3H]ifenprodil binding to recombinant human NR1/NR2B and native rat receptors. These findings are in agreement with Mutel et al. (1998), who previously characterized the in vitro binding of a new subtype-selective NMDA receptor antagonist, [3H]Ro 25-6981, to rat brain membranes and sections. The compound binds a single site on the membranes with a Kd of 3 nM and a Bmax of 1.6 pmol/mg of protein. Overall, the distribution of [3H]Ro 25-6981-binding sites correlates well with that of NR2B (but not NR2A) transcripts, as revealed by in situ hybridization. Similarly, Lynch et al. (2001) used ligand binding to characterize Ro 25-6981, and found that the compound shares pharmacological actions with ifenprodil, as well as with other modulators of NR2B-containing NMDA receptors. Binding assays performed in HEK-293 cells expressing NR1/ NR2B or NR1/NR2A/NR2B receptors suggest two types of NR2B-specific NMDA receptor antagonists: (1) Ro25,6981, which binds to NR2B-containing receptors with high affinity regardless of the NR2 subunit composition, and (2) CP-101,606, whose binding is altered by the presence of other NR2 subunits (Chazot et al., 2002). In addition to the characterization of ifenprodil and the other ligands that reportedly bind to the same site on the NR2B subunit, the purification and synthesis of conantokin (Con)-R, an NMDA receptor peptide antagonist from the venom of Conus rediatus, was performed. With the use of well-defined animal seizure models, Con-R was found to possess an anticonvulsant profile superior to that of ifenprodil and MK-801. With voltage-clamp recordings of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs, Con-R exhibited the following order of preference for the NR2 subunits: NR2B = NR2A > NR2C >> NR2D (White et al., 2000). Con-G, a 17 amino acid peptide antagonist of NMDA receptors, was isolated from the venom of the marine cone
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snail, C. geographus. Using whole cell voltage-clamp recordings from cultured neurons and two electrode voltage-clamp recordings from Xenopus oocytes expressing recombinant NMDA receptors, Con-G selectively blocked NMDA receptors containing the NR2B subunits (Donevan & McCabe, 2000). The characterization of NR2B-specific compounds also supports and contributes to information regarding the neuroanatomical distribution of receptor subunits. Pharmacological properties of NMDA receptors were determined in rat brain sections with quantitative autoradiography of [3H]CGP39653 binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed. The two populations correspond anatomically to the NR2A subunits located in the cerebellar granule cell layer and the NR2B subunits found in the medial striatum (Christie et al., 2000). Another antagonist, [3H]CPP, weakly labels NMDA receptors in regions that contain predominantly NR2B mRNA (striatum and lateral septum), and agonist-preferring NMDA receptors (defined by L-[3H]glutamate binding) are located predominantly in a subset of brain regions that contain both NR2B and NR1 mRNA. The anatomical distribution of subunit mRNAs suggests that the NR2B subunits are associated with agonist-preferring NMDA receptors. Thus, the anatomical and pharmacological evidence is consistent with the hypothesis that agonist-preferring and antagonist-preferring NMDA receptors are largely determined by the NR2B and NR2A subunits, respectively (Buller et al., 1994). In addition to their usefulness as tools to elucidate regional distribution and functional properties associated with NR2B-containing receptors, a number of subunitspecific antagonists are currently being used and/or investigated as possible therapeutic agents (reviewed in Nikam & Meltzer, 2002; see Section 5). CP-101,606, an NR2B subunit-selective antagonist, is currently under development by Pfizer (New York, NY, USA) for its potential as a neuroprotectant in head injury and neurodegenerative diseases (Chazot, 2000). The analgesic activity of CP-101,606 was examined in carrageenan-induced hyperalgesia and in capsaicin- and phorbol 12-myristate 13-acetate-induced nociceptive tests in the rat. Results suggest that inhibition of the NR2B subunit of the NMDA receptor is effective in vivo at modulating nociception and hyperalgesia responses without causing the behavioral side effects often observed with currently available NMDA receptor antagonists (Taniguchi et al., 1997). CP-101,606 has also been used to investigate the role of NR2B-containing receptors in response to NMDA receptor activation in vivo. In the mouse, CP-101,606 completely inhibits increases in Foslike immunoreactivity in the dentate gyrus caused by a subconvulsant dose of NMDA. In the rat, the compound completely blocks cortical c-fos mRNA induction following focal injury in the parietal cortex and the initiation and propagation of electrically induced cortical-spreading depression. Inhibition of these responses by CP-101,606
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indicates that c-fos induction and cortical-spreading depression are dependent on activation of NMDA receptors containing the NR2B subunits (Menniti et al., 2000). Activation of NR2B-containing receptors may thus contribute to the ability of the neuron to alter its synapses via regulation of immediate early gene expression. A novel series of N-(phenylalkyl)cinnamides was synthesized recently and tested for antagonism at NMDA receptor subtypes. Electrical recordings in Xenopus oocytes expressing three different configurations of cloned rat NMDA receptor subunits (NR1 expressed in combination with either NR2A, NR2B, or NR2C) were used to assay the potency and subunit selectivity of the compounds. Results indicated that the N-(phenylalkyl)cinnamides are selective antagonists of the NR1/NR2B receptors (Tamiz et al., 1999). Thus, CP-101,606 and the N-(phenylalkyl)cinnamide series described provide new and structurally diverse models for designing NR2B-selective NMDA antagonists as potential pharmacotherapeutics. Felbamate has been used in the treatment of seizure disorders. Recombinant receptors expressed in Xenopus oocytes were used to investigate the subtype specificity and mechanisms of action of this drug. Felbamate reduces NMDA- and glycine-induced currents most effectively at NMDA receptors composed of NR1 and NR2B subunits. Further, felbamate enhances the affinity of the NR1/NR2B receptors for the agonist NMDA by 3.5-fold, suggesting a similarity in mechanism to other antagonists (Kleckner et al., 1999). Although the potential mechanism of action may be similar to that of other noncompetitive antagonists such as ifenprodil, the toxicity profile appears to be somewhat improved. Specifically, felbamate blocks NMDA receptors, but fails to exhibit the toxicity associated with other NMDA receptor antagonists. To investigate the possibility that the favorable toxicity profile of felbamate could be related to NMDA receptor subtype selectivity, Harty and Rogawski (2000) examined the specificity of felbamate block of recombinant NMDA receptors composed of the NR1 subunit and various NR2 subunits. Findings suggest that more rapid association and slower dissociation accounts for the higher affinity of felbamate block of NMDA receptors containing the NR2B subunit (data indicate more than one blocking site on the NMDA receptor channel complex). These authors concluded that felbamate exhibits modest selectivity for NMDA receptors composed of NR1/NR2B subunits. This selectivity could, in part, account for the improved clinical profile of felbamate in comparison with NMDA receptor antagonists that do not show subunit selectivity. 3.2. Electrophysiological Studies of NMDA receptor kinetics and related electrophysiological properties reveal that the mechanisms of ion channel activation and inactivation are extremely complex (for a review, see McBain & Mayer, 1994). Thus, this
section focuses on research that illuminates a unique role for NR2B-containing receptors in excitatory synaptic transmission. NR2B subunits dominate over other NR2 subunits in determining the functional properties of NMDA receptors in certain cell types [GABAergic medial septal neurons (Plant et al., 1997)]. These authors studied functional and molecular properties of septal neurons of the rat forebrain using patch clamp, fluorometric Ca2+ measurements, and single-cell RT-PCR. However, using transfected HEK-293 cells and whole cell patch-clamp recordings, NR1/NR2Amediated peak current densities are 4 times larger than NR1/NR2B. Further, peak channel open probability is significantly higher for NR1/NR2A than for NR1/NR2B, using two different open channel antagonists, MK801 and 9-aminoacridine. Results suggest that expression levels of NR2A and NR2B can regulate peak amplitude of NMDA receptor-mediated excitatory postsynaptic potentials and, therefore, play a role in mechanisms underlying synaptic plasticity (Chen et al., 1999a). Accompanying changes in channel activation, subunitspecific differences are similarly observed for the transition from the open to the closed state (i.e., deactivation kinetics). Heteromeric HEK cells expressing NMDA receptors were investigated using whole cell recordings. NR1/NR2B showed no significant inactivation with application of extracellular Ca2+. Ca2+- and glycine-independent desensitization was less pronounced in NR1/NR2B receptors as compared with NR1/NR2A receptors (Krupp et al., 1996). Further, NMDA receptors transiently transfected into HEK293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Recovery from desensitization was slower for NR1/NR2B than for NR1/ NR2A channels (Vicini et al., 1998). Taken together, these results suggest that NR2B-containing NMDA receptors desensitize less and take longer to recover than NR2Acontaining receptors. Besides cell expression systems, the use of genetically altered mouse models has also provided important information about the role of the NR2B subunit in modulating electrophysiological properties of NMDA receptors. Overexpression of NR2B in the forebrains of transgenic mice leads to enhanced activation of NMDA receptors, facilitating synaptic potentiation in response to stimulation at 10– 100 Hz (Tang et al., 1999). In contrast to overexpression, Tovar et al. (2000) studied fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the NR2B subunit. Neurons from NR2B knockout (KO) mice expressed an NMDA receptor-mediated excitatory postsynaptic current (EPSC) that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg2+ or the reversible antagonist DL-2amino-5-phosphonovaleric acid (AP-5). Whole cell currents from mouse KO neurons were also more sensitive to block by low concentrations of Zn2+ and much less sensitive to the
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NR2B-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile are consistent with the expression of NR1/NR2A heteromeric receptors in excitatory hippocampal neurons from mice lacking the NR2B subunit. Thus, NR2A can substitute and potentially provide some compensation for the absence of the NR2B subunit at synapses. Electrophysiological tests, in combination with the NR2B-selective antagonist ifenprodil and other antagonists, were used to demonstrate functional properties of the receptor. In a rat model of inflammatory pain, the effect of in vivo NMDA receptor antagonism on responses of spinal dorsal horn neurons to iontophoretic NMDA in anesthetized rats was investigated. Similar assessments were also performed using the C-fiber-evoked wind-up model. The windup phenomenon occurs under conditions of severe and persistent injury, such that C-fibers fire repetitively and, consequently, the response of dorsal horn neurons increases. Importantly, wind-up is dependent on the release of glutamate from C-fibers and the subsequent activation of postsynaptic NMDA receptor-gated ion channels. It was found that all three antagonists dose-dependently increase nociceptive thresholds in the Randall-Selitto model. Further, antinociceptive doses of the channel blockers (MK-801 and memantine) selectively antagonize NMDA responses of dorsal horn neurons, as well as inhibit C-fiber-evoked wind-up. In contrast, antinociceptive doses of ifenprodil do not exhibit NMDA antagonism in electrophysiological tests. Although ifenprodil does not inhibit single motor unit responses to noxious stimuli in rats with transected spinal cords, it markedly and dose-dependently inhibits nociceptive single motor unit responses in sham-operated rats. It was concluded that these results suggest that the spinal cord is not the principal site of antinociceptive action of ifenprodil, but that supraspinal structures are likely to mediate this effect (Chizh et al., 2001b). 3.3. Behavioral Behavioral assays, often in combination with pharmacological or genetic manipulations, have demonstrated a role for the NR2B subunit in mediating feeding and related physiological functions, learning, memory, pain, and synaptic plasticity. Kutsuwada et al. (1996) investigated the physiological significance of the NMDA receptor diversity and created NR2B subunit-defective mice. These mice show no suckling response and die shortly after birth. Similarly, mice expressing the NR2B subunit in a C-terminally truncated form die perinatally. This is the most severe phenotype compared with other KO mice (NR2A and NR2C). The lethal phenotype of NR2B-altered mice is speculated to result from an impairment in intracellular signaling due to the missing intracellular receptor domain (Sprengel et al., 1998). In Sprague-Dawley rats, the NR2A/B-selective antagonist ifenprodil attenuates NMDA-elicited feeding. Lat-
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eral hypothalamic NMDA receptors, some of which contribute to feeding control, are composed of NR2A and/ or NR2B subunits, suggesting that NR2A- and/or NR2Blinked signal transduction is involved in the feeding behavior (Khan et al., 1999; see also Stanley et al., 1996, 1997). Other reports also document hypothalamic NR2B-containing receptor participation in the control of circadian rhythms and the activity of magnocellular neuroendocrine cells (for reviews, see Brann, 1995; Ebling, 1996; Rea, 1998). Mice overexpressing the NR2B subunits exhibit superior ability in various tests of learning and memory (novel object recognition task, fear conditioning, fear extinction, and water maze). Tang et al. (1999) suggested that NR2B is critical in gating the age-dependent threshold for neuronal plasticity and memory formation. In contrast to mice overexpressing the NR2B subunit, a different, yet consistent, phenotype is observed in mice with deficient levels of NR2B. The lack of the NR2B subunit impairs the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brain stem. Further, in the hippocampi of these null-mutant mice, synaptic NMDA receptor responses and long-term depression (LTD) are absent (Kutsuwada et al., 1996). Taken together, these results suggest that NR2B plays an essential role in learning, memory, and neuronal pattern formation. Interestingly, Rosenblum et al. (1997) used a conditioned taste aversion (CTA) paradigm to investigate the role of NR2B in both feeding and learning. Blockade of NMDA receptors with the reversible antagonist AP-5 during training impaired CTA memory. When rats taste an unfamiliar flavor and, hence, learn about it either incidentally or in the context of CTA training, the tyrosine phosphorylation of NR2B in the insular cortex is increased. The level of tyrosine phosphorylation of NR2B appears to be a function of the novelty and quantity of the substance consumed. Thus, NR2Bcontaining receptors are involved in taste learning in the insular cortex, and tyrosine phosphorylation of NR2B subserves encoding the saliency shortly after an unfamiliar flavor is tasted. The NR2B subunit has also been implicated in pain perception (see Section 5.10). Wei et al. (2001) studied the effects of forebrain-targeted overexpression of NR2B on the response of mice to peripheral tissue injury. Transgenic mice overexpressing NR2B in the anterior cingulate and insular cortices exhibit enhanced responsiveness to hind paw injection of inflammatory stimuli. Thus, these results provide further support for the development and use of NR2B-selective compounds for the management of pain.
4. Regulation 4.1. Endogenous regulators The NMDA receptor is modulated by a variety of ions, amino acid neurotransmitters, and second messengers [e.g.,
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Mg2+, Ca2+, glycine, Zn2+, H+, and nitric oxide (NO)] (McBain & Mayer, 1994). NMDA receptors are also regulated (often in a subunit- and splice variant-dependent manner) by components such as PSD-anchoring proteins, enzymes, and growth factors. The potential molecular mechanisms for the regulation of NR2B expression, distribution, and function involve, but are not limited to, (1) changes in the assembly of specific NR1 splice variants and/ or other NR2 subunits with NR2B; (2) post-translational modifications such as phosphorylation; and (3) alterations in the interaction of the NR2B subunit with associated proteins, such as PSD-95 or SAP102. The following section reviews the regulation of NR2B by endogenous proteins. Given the breadth of the topic, the discussion focuses on the role of neuronal elements associated with the PSD, including cytoskeletal proteins, enzymes, and neuromodulators. 4.1.1. Cytoskeletal proteins As described in Section 3, the NR2B subunit plays an important role in synaptic plasticity, neural development, and other physiological functions. Although much has been learned in recent years about the neurochemical and biological properties of glutamate receptors, understanding the molecular mechanisms of receptor targeting, clustering, and anchoring at the synapse is only now beginning to develop. Recent findings concerning the discovery of novel proteins potentially involved in the regulation of NR2B via its specific synaptic protein interactions are reviewed below. Kornau et al. (1995) used a yeast 2-hybrid system to show that the cytoplasmic tails of NMDA receptor subunits interact with the 95-kDa PSD protein PSD-95. PSD-95 is a component of the PSD at excitatory synapses. It contains three PDZ domains, a Src homology (SH)3 domain, and a guanylate kinase (GK) domain, and has been characterized as a putative structural or scaffolding protein. The PDZ domain appears to be important for the localization of select NMDA receptor subunits. The second PDZ domain in PSD95 binds to the C-terminal containing the tSXV (t, terminal; S, serine; X, any amino acid; V, valine) motif common to the NR2 subunits, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. Recent reports indicate that each of the last five residues of the NR2B subunit is critical for binding to the PDZ domains of PSD95 and its related protein family member SAP102 (see below) (Lim et al., 2002). In support of its role as a potential scaffolding protein for NR2B, PSD-95 also binds to the cytoplasmic C-terminal of neuroligins in the mouse forebrain (neuroligins are neuronal cell adhesion molecules that interact with ß-neurexins and form intercellular junctions). In contrast to NR2B, neuroligins interact with the third PDZ domain of PSD-95 (Irie et al., 1997). Thus, PSD-95 not only localizes NR2B at synaptic sites, but also helps to form the architecture of a multi-protein signaling complex (Fig. 1). To further characterize the interactions of NMDA receptor subunits with PSD proteins, Niethammer et al. (1996) used a yeast 2-hybrid system to screen a rat brain cDNA
library. In vitro binding assays identified an interaction between NR2A and NR2B and three distinct members of the PSD-95/SAP90 family [SAP97/hdlg, PSD-95, and Clone 5 (Kim et al., 1995)]. In partial agreement with the findings of Kornau et al. (1995), the interactions appear to be mediated via the binding of C-terminal NMDA receptor subunits to the first two PDZ domains of PSD-95. A related cytoskeletal protein, chapsyn-110, was also found using a yeast 2-hybrid system. It was discovered to be a novel member of the PSD-95 subfamily of membrane-associated GKs (MAGUKs) that binds to the C-terminal of Shaker K+ channel subunits (Kim et al., 1995), as well as to NR2 subunits (Niethammer et al., 1996). Chapsyn-110 is 70– 80% identical to PSD-95 and SAP97, and shows identical domain organization with three PDZ domains in the Nterminal, a GK homology region in the C-terminal, and an intervening SH3 domain. When expressed in Cos-7 cells, Chapsyn-110 can form heteromultimers with PSD-95, but not with SAP97, and clusters NR2A and NR2B, but not NR1 (Kim et al., 1996). Using similar methodology, CASK, a novel PSD-95 homolog with an N-terminal CaM-dependent protein kinase domain, was identified by interaction with neurexins using a yeast 2-hybrid screen. The C-terminal region is similar to the intracellular junction proteins dlg-A, PSD-95/SAP90, SAP97, ZO1, and ZO2 that contain Drosophila hormone receptor, SH3, and GK domains. Given its subcellular location and binding capacities, it is hypothesized to function as a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins (Hata et al., 1996). More recently, a different function for CASK was revealed. NR2B-containing vesicles are transported by interactions within a large protein complex, including microtubule elements, CASK, and NR2B (Setou et al., 2000). Finally, Muller et al. (1996) described a novel 102kDa SAP, SAP102, detected in dendritic shafts and spines of asymmetric synapses. Like PSD-95 and related proteins, it has three PDZ domains and an SH3 and GK domain. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. Masuko et al. (1999) provided further characterization of SAP102. These authors identified the CaM-binding site on SAP102 (located near the SH3 domain of SAP102) and demonstrated that binding of CaM to SAP102 does not alter the interaction between SAP102 and NR2B. Further, SAP102 interacts with PSD-95 only in the presence of Ca2+ and CaM, thus revealing the potential for differential regulation of MAGUKs at excitatory synapses. Some evidence suggests that although PSD-95 and related scaffolding proteins appear to co-localize with NR2B subunits and contribute to the stability of NMDA receptors at the PSD, NR2B may not be required for targeting the receptor to the membrane. Tovar et al. (2000) examined whether NMDA receptor recruitment to the postsynaptic membrane was dependent on the NR2B subunit by isolating hippocampal neurons from NR2B-deficient mice
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and studying them in culture. Results indicated that NR2B is not required for targeting of NMDA receptors to the postsynaptic membrane. Nevertheless, in addition to the putative clustering of NR2B with the PSD-95 family of proteins, the synaptic association with such cytoskeletal elements may also regulate the expression and function of NR2B-containing receptors. For example, co-expression of PSD-95 with NR1/NR2A results in a decreased sensitivity to L-glutamate and an enhanced expression of NR2A and NR2B subunits. Importantly, deletion studies show that this effect is mediated via interaction of the C-terminal ESDV (E, glutamic acid; S, serine; D, aspartic acid; V, valine) motif [previously described as the tSXV motif (Kornau et al., 1995)] of the NR2 subunit with PSD-95 (Rutter & Stephenson, 2000). Further, Roche et al. (2001) demonstrated that PSD-95 inhibits NR2B-mediated internalization and that deletion of the PDZ-binding domain of NR2B increases internalization in cultured hippocampal neurons. Along with the PSD-95 family of cytoskeletal proteins, a number of associated neuronal elements recently have been identified and shown to play a role in synaptic regulation. A brief review of the proteins potentially involved in the distribution or function of NR2B containing receptors is provided below. The list of proteins is not intended to be comprehensive, as new proteins and interactions are continually being reported (Husi et al., 2000). Brain spectrin, a protein that links neuronal elements to the cytoskeleton, was investigated and found to interact with the C-terminal of NR2B subunits (Wechsler & Teichberg, 1998). Further characterization by these authors revealed that spectrin binds NR2B at sites that are different from those of PSD95. Interestingly, spectrin-NR2B interactions are antagonized by Ca2+ and Fyn-mediated phosphorylation, but not by CaM or CaMKII-mediated phosphorylation, thus demonstrating the regulatory specificity associated with the PSD. In agreement with these results illustrating the connection of NR2B with structural proteins, van Rossum et al. (1999) used affinity chromatography with glutathione Stransferase (GST)-NR2B-C-terminal fusion proteins to identify novel binding partners for this subunit. It was found that the NR2B C-terminus binds tubulin. In tubulin polymerization assays, the NR1 and NR2B C-termini significantly decrease the rate of microtubule formation without destabilizing preformed microtubules. Thus, via its interaction with such scaffolding proteins, NR2B may not only be regulated by, but also help control, the formation of cytoskeletal components. Using a yeast 2-hybrid screen, Takeuchi et al. (1997) isolated a novel protein family consisting of at least four members that specifically interact with PSD-95/SAP90 and its related proteins through the GK domain. These proteins were named SAPAPs (SAP90/PSD-95-associated proteins). SAPAPs are specifically expressed in neuronal cells and are concentrated in the PSD fraction. SAPAPs induce the enrichment of PSD-95 in the plasma membrane in transfected cells. Co-expression of PSD-95/SAP90 causes the
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translocation PSD-95 from the cytosol to the plasma membrane where SAPAPs are located. Using yeast 2-hybrid screening for SAPAP-interacting molecules, Hirao et al. (1998) identified a novel protein that has an inverse structure of MAGUKs with an N-terminal GK-like domain followed by five PDZ domains. It binds SAPAP through the GK-like domain and NMDA receptors and neuroligins through PDZ domains. The protein is named S-SCAM (synaptic scaffolding molecule) because S-SCAM is hypothesized to assemble receptors and cell adhesion proteins at synaptic junctions. CRIPT, a novel protein that selectively binds to the third PDZ domain of PSD-95 via its C-terminal, was similarly characterized. In heterologously expressing cells, CRIPT causes a redistribution of PSD-95 to microtubules. In the brain, CRIPT co-localizes with PSD-95 and can be coimmunoprecipitated with PSD-95 and tubulin. CRIPT may regulate the PSD-95 interaction with the tubulin-based cytoskeleton in excitatory synapses (Niethammer et al., 1998). Another putative regulator protein, cypin, a cytosolic regulator of PSD-95 postsynaptic targeting, was identified. Affinity chromatography revealed cypin to be a major PSD95 binding protein in brain extracts. Cypin is expressed in neurons, and overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD95 and SAP102 (Firestein et al., 1999), and as a consequence, may be involved in regulating the localization of NR2B to synaptic sites. Naisbitt et al. (1999) reported a novel family of PSD proteins termed Shank that are specifically enriched in the PSD of excitatory synapses. Shank contains multiple putative protein interaction domains, including an SH3 domain, a PDZ domain, the proline-rich domain, and an SAM domain. The PDZ domain of Shank mediates binding to the C-terminal of the GK-associated protein (GKAP) (see Section 4.1.2.), and this interaction is important in neurons for the synaptic localization of Shank. GKAP binds Shank and recruits Shank to PSD-95 clusters in heterologous cells. All three proteins also associate in vivo, as demonstrated using GST pull-down methods. Opportunities for such linkages appear to be numerous and are continually being discovered. Kurschner et al. (1998) identified CIPP (channel-interacting PDZ domain protein) that is expressed only in the brain and kidneys. CIPP contains four PDZ domains that could potentially interact with different PSD proteins, providing a mechanism for the simultaneous association of multiple proteins with CIPP. NR2B associates with the second and third PDZ domains of CIPP, but the functional consequences for NR2B regulation are unknown. Further increasing the list of cytoskeletal proteins, and thus adding to the complexity of NR2B regulation, is the protein neurabin (neural tissue-specific F-actin binding protein). Neurabin was cloned from rat brain cDNA. It has one F-actin binding domain at the N-terminal region and one PDZ-like domain in the middle region. Neurabin is believed to be involved in neurite formation and it is highly
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concentrated in the synapses of developed neurons. It was also enriched in the lamellipodia of growth cones during neuronal development (Nakanishi et al., 1997). The last structural protein to be discussed that is potentially involved in the regulation of NR2B subunits is densin-180. This protein was purified and found to be highly concentrated at synapses along dendrites. Densin-180 message is abundant in the forebrain. It contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. It was proposed as an adhesion molecule between pre- and postsynaptic membranes at glutamatergic synapses (Apperson et al., 1996). Further characterization of densin-180 indicates that the protein binds CaMKII, and suggests that densin-180 plays a role in the association of CaMKII with PSDs (Strack et al., 2000b). Thus, NR2B may serve to anchor and distribute NMDA receptors at synaptic sites through its interaction with the PSD (Fig. 1). Specific interactions of NR2B with PDZ domain-containing proteins is important not only for determining the localization of the receptor to the PSD, but also for regulating synaptic signaling events. 4.1.2. Enzymes (e.g., phosphatases, kinases, and proteases) Many enzymes critical for signal transduction at excitatory synapses are linked to NR2B subunits either directly or via their interactions with the cytoskeletal proteins previously described. Outlined below is information that illuminates how a number of kinases, phosphatases, and other enzymes are involved in second messenger signaling and receptor regulation. Smart (1997) reviewed experiments that demonstrate how neurotransmitter-induced activation of serine/threonine, tyrosine, and other kinases can result in the modulation of glutamate receptors. However, studies described in this section specifically emphasize the role of such enzymes in the regulation of NR2B subunits. NR2B is the most prominently tyrosine phosphorylated protein in the PSD fraction, based upon recognition by an anti-tyrosine antibody. NR2B-containing glutamate receptors may be regulated by tyrosine phosphorylation or may participate in signaling through tyrosine phosphorylation (Moon et al., 1994). In agreement with these findings, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrated that high levels of phosphotyrosine are co-localized with glutamate receptors at excitatory synapses on cultured hippocampal neurons. Using subunitspecific antibodies and rat synaptic plasma membranes, Lau and Huganir (1995) showed that NR2A and NR2B, but not NR1, are tyrosine phosphorylated. It is estimated that 3.6 ± 2.4% of NR2B and 2.1 ± 1.3% of NR2A are phosphorylated in vivo. In another review, Sala and Sheng (1999) discussed which protein tyrosine kinases phosphorylate the NR2 subunits. They explored questions regarding how this kinase-substrate specificity is determined, what the physiological significance of tyrosine phosphorylation of the NR2
subunits is, and how it is regulated. Given that Fyn, a protein tyrosine kinase, binds to the third PDZ domain of PSD-95 via its SH2 domain, they provided a model for Fyn association with the NMDA receptor/PSD-95 complex. Hisatsune et al. (1999) examined the role of Fyn and the possibility that phosphotyrosines on NR2B subunits contribute to intracellular signaling events, and demonstrated that Fyn phosphorylates NR2B at a site in NR2B that interacts with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Both the level of tyrosine phosphorylation of NR2B and the amount of the p85 subunit bound to NR2B are decreased in Fyn-deficient mice. Tyrosine phosphorylation of NR2B is important not only for the regulation of its channel activity, but also for intracellular signaling mediated through the interaction of the NMDA receptors with SH2 domain-containing molecules. Similarly, using a different KO mouse model, Manabe et al. (2000) showed that in the H-ras (one of three mammalian proto-oncogenes) null mutant hippocampus, tyrosine phosphorylation of NR2B is increased and NMDA receptor responses are subsequently enhanced. Phosphopeptide mapping of the C-terminal tail of the NR2B subunit in vitro revealed that 7 out of 25 tyrosine residues in the C-terminal region are phosphorylated. When co-expressed with Fyn in HEK-293 cells, of the 7 residues, Tyr1252, -1336, and -1472 are phosphorylated and Tyr1472 appears to be the major phosphorylation site (Nakazawa et al., 2001). It has thus been established that the NR2B subunit is phosphorylated on tyrosine residues and that this event can contribute to the potentiation of receptor responses and the transduction of specific intracellular signals. To further assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, Gurd and Bissoon (1997) investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C (PLC)-g. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, an important early event in many cell signaling pathways. PLC-g contains two SH2 domains that bind to specific phosphotyrosine residues of receptor tyrosine kinases, leading to phosphorylation by the catalytic domain of the receptor. Phosphorylation of PLC-g contributes to its activation. The authors found that NR2A and NR2B can bind to the SH2 domains of PLC-g, and they confirmed that isolated synaptic junctions contain endogenous protein tyrosine kinases that can phosphorylate both the NR2A and NR2B receptor subunits. Thus, interaction of the tyrosine-phosphorylated NR2B subunits with proteins that contain SH2 domains serves to link NR2B to signaling pathways in the postsynaptic cell (Gurd & Bissoon, 1997). Perturbations of the system further demonstrate how tyrosine phosphorylation may alter NR2B and subsequent receptor function. For example, using rat hippocampal slices and immunoprecipitation, insulin was shown to cause a
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transient phosphorylation of the NR2A and NR2B subunits on tyrosine residues. The functional consequence of insulinstimulated tyrosine phosphorylation is currently unknown (see Section 5.7), although Christie et al. (1999) speculate that it could help to potentiate the activity of the receptor. In addition to tyrosine phosphorylation, phosphorylation of serine residues by protein kinase A (PKA), protein kinase C (PKC), and CaMKII also modulates receptor function. For example, in primary hippocampal cultures, phosphorylation of NR2A/NR2B at serine residues, but not at threonine or tyrosine residues, was detected. Further, metabolic labeling with 32P reveals that NR2 subunits are highly phosphorylated under basal conditions and exhibit (more) modest increases in response to stimulation (as compared with NR1) (Hall & Soderling, 1997). The regulation by specific kinases can occur, not only directly by phosphorylation of the NR2B subunit, but also via its association with other NMDA receptor subunits or cytoskeletal proteins (see Section 4.1.1). PKA and various isoforms of PKC phosphorylate the NMDA receptor in vitro. Immunoprecipitation experiments show that all three NMDA receptor subunits (NR1, NR2A, NR2B) are substrates for PKA, as well as PKC (Leonard & Hell, 1997). PKC can affect NR1/NR2B receptor currents by direct phosphorylation of the NR2B C-terminal at residues Ser1303 and -1323 (Liao et al., 2001). Grosshans and Browning (2001) extended these findings and demonstrated that PKC activation can also induce tyrosine phosphorylation of the NR2B subunit. CamKII binds directly to the NR1 and NR2B subunits (Leonard et al., 1999). Strack et al. (2000a) showed that residues 1290 – 1309 in the cytosolic tail of NR2B are critical for CaMKII binding, and identified by site-directed mutagenesis several key residues (Lys1292, Leu1298, Arg1299, Arg1300, Glu1301, Ser1303). Phosphorylation of NR2B at Ser1303 by CaMKII inhibits binding of and promotes dissociation of CaMKII/NR2B complexes. Residues 1260 –1316 of NR2B are sufficient to recruit CaMKII to the NMDA receptor in intact cells. Leonard et al. (1999) demonstrated that activation of CamKII-a by stimulation of NMDA receptors in forebrain slices increases the association of CamKII to NR2B. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B modulates co-localization with CaMKII after NMDA receptor activation, suggesting a model for the translocation of CaMKII to postsynaptic targets (Strack et al., 2000a). Importantly, these same investigators have shown previously that the NR2B subunit, but not the NR2A or NR1 subunits, is responsible for autophosphorylation-dependent targeting of CaMKII in intact cells. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B C-terminal (Strack & Colbran, 1998). Further, glutamate-induced translocation of GFP-a-CaMKII to the PSD was blocked by AP-5, suggesting that NR2B-containing receptors are necessary for CaMKII translocation (Shen & Meyer, 1999). These
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findings illustrate that specific co-localization of CaMKII with NR2B-containing NMDA receptors depends on receptor activation, Ca2+ influx, and CaMKII autophosphorylation (on residue Thr286). Thus, translocation of CaMKII because of the interaction with the NMDA receptor ion channel may potentiate kinase activity and provide spatial and temporal control of postsynaptic substrate phosphorylation. Another kinase that has been shown to modulate receptor function is PKA. Westphal et al. (1999) isolated cDNAs encoding fragments of yotiao [a protein that binds the NMDA receptor subunit NR1 and interacts with the Cterminal C1 exon cassette (Lin, J. W. et al., 1998)] by an interaction cloning strategy to identify A-kinase anchoring proteins, and confirmed that the protein binds the NR1 subunit. Yotiao also binds to the Type 1 protein phosphatase (PP1) and to PKA. Anchored PP1 is constitutively active, limiting channel activity, whereas PKA activation overcomes PP1 activity and enables rapid enhancement of NMDA currents. Yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity. Taken together, these studies strongly support the importance of second messenger signaling via kinase and phosphatase activity for the regulation and function of NR2B-containing receptors. Another example of a second messenger molecule that is critically involved with NMDA receptor function is nitric oxide (NO) (Garthwaite, 1991), and its synthetic enzyme, neuronal NO synthase (nNOS) (NOS: EC 1.14.13.39) (Hevel & Marletta, 1994). nNOS is a Ca2+/CaM-dependent enzyme that is functionally coupled to Ca2+ influx through NMDA-type glutamate receptors (Garthwaite & Boulton, 1995), and, thus, is regulated by the gradients of Ca2+ that occur in the vicinity of open Ca2+ channels (Brenman & Bredt, 1997). Stimulation of NMDA receptors results in the activation of nNOS, catalyzing the formation of NO from Larginine (Garthwaite et al., 1989). The mechanism for selectively coupling nNOS to Ca2+ influx through NMDA receptors appears to involve the targeting of nNOS to the PSD (Brenman & Bredt, 1997). The N-terminal PDZ domain of nNOS links the enzyme to the PDZ domains of PSD-95 and PSD-93 (Brenman et al., 1996). Review of the literature indicates numerous roles for nNOS-dependent NO, including, but not limited to, stimulation of cyclic guanosine 50-monophosphate synthesis via activation of guanylate cyclase (Garthwaite, 1991), enhancement of CaM-dependent phosphorylation of PSD proteins (Wu et al., 1996), and modulation of synaptic vesicle exocytosis (Meffert et al., 1996). The regulation of NO formation appears to be mediated by the compartmentalization of nNOS with NMDA receptors at specific synaptic sites (Aoki et al., 1998). nNOS is concentrated at synaptic junctions in the brain. As discussed in Section 4.1.1, NR2B is associated with PSD-95 and related protein family members at synapses and with nNOS (Fig. 1). PSD-95 is co-expressed in numerous neuronal
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populations. nNOS isoforms lacking a PDZ domain do not associate with PSD-95 in the brain. Interaction of PDZ domains, therefore, mediates synaptic association of nNOS, and may play a more general role in formation of macromolecular signaling complexes (Brenman et al., 1996). The linking of NMDA receptors to nNOS by PSD-95 may explain why Ca2+ influx following NMDA receptor activation is tightly coupled to nNOS activation. Contributing to this synaptic regulation, Jaffrey et al. (1998) reported the identification of an nNOS-associated protein, carboxy-terminal PDZ ligand of nNOS (CAPON), which is highly enriched in the brain and has numerous regions of co-localization with nNOS. CAPON interacts with the nNOS PDZ domain through its C-terminal. CAPON competes with PSD-95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD-95/nNOS complexes in transfected cells. CAPON, therefore, may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes. In addition to nNOS and CAPON, a synaptic RasGTPase-activating protein (RasGAP) that associates with the PSD-95 protein family via its PDZ domain was identified. Named SynGAP (synaptic GTPase-activating protein), this protein interacts with all three PDZ domains of PSD95 via its C-terminal amino acids (Kim, J. H. et al., 1998). SynGAP is a brain-specific protein (130 kDa) and demonstrates RasGAP activity. It is enriched at excitatory synapses and co-immunoprecipitates with NR1 and PSD95. SynGAP plays a role in the modulation of Ras signaling at excitatory synapses. Given that Ras proteins are small Gproteins involved in the control of many signal transduction processes that affect cell growth and differentiation, SynGAP, therefore, may play a critical role in the regulation of signaling events associated with the NMDA receptor. Accompanying the neuronal elements that interact via PDZ domains are putative regulatory proteins that are linked to NR2B via the GK domains of the PSD-95 family of cytoskeletal proteins and that, therefore, may function to regulate NR2B-linked intracellular signaling. Kim et al. (1997) isolated a novel synaptic protein, GKAP, that binds directly to the GK domain of at least four known members of the PSD-95 family. GKAP co-localizes and co-immunoprecipitates with PSD-95 in vivo and co-clusters with PSD95 and K+ channels and NMDA receptors in heterologously expressing cells (Naisbitt et al., 1997). Although expressed in neurons and localized specifically in the PSD of glutamatergic synapses, little is known about the function of GKAP. Enzymatic regulation of NMDA receptors is further demonstrated by the Ca2+-dependent protease calpain. Treating synaptic membranes with calpain I results in truncation of the C-terminal domains of the NR1 and NR2A/B subunits. Changes in the channel binding of thienylphencyclidine and MK801 are observed only when the NR2A/B subunits are selectively truncated. Further, preincubation of synaptic membranes with phosphatase inhib-
itors significantly reduces the extent of calpain-mediated truncation of the C-terminal NR2A/B subunits (Bi et al., 1998). Calpain-mediated regulation of NMDA receptor structure and function could represent a feedback mechanism for the receptors that could serve to alter receptor activation. 4.1.3. Neuromodulators (e.g., growth factors, hormones) Growth factors, including members of the neurotrophin gene family, play a central role in the regulation of neuronal survival and differentiation during development. In addition to these relatively long-term actions of neurotrophins, recent studies have shown that these factors rapidly modulate transmission at glutamatergic synapses. Levine and Kolb (2000) used excised membrane patches from cultured hippocampal neurons to determine whether brain-derived neurotrophic factor (BDNF) directly modulates postsynaptic NMDA receptor activity. Acute exposure to BDNF increases NMDA single channel open probability via postsynaptic trkB receptors, an effect that is dependent on the presence of the NR2B subunit of the NMDA receptor. Incubation of BDNF with cortical or hippocampal PSDs rapidly increases tyrosine phosphorylation of NR2B in a dose-dependent manner. The actions of BDNF are specific and selective for NR2B (since nerve growth factor and NR2A do not yield the same results). NR2B phosphorylation may contribute to neurotrophin modulation of postsynaptic responsiveness (Lin, S. Y. et al., 1998). Crozier et al. (1999) also examined the mechanisms of BDNF action in dissociated embryonic hippocampal neurons. Whole cell patch-clamp recording during iontophoretic application of glutamate revealed that BDNF doubles the amplitude of induced inward current. Co-exposure to BDNF and the NMDA receptor antagonist AP-5 markedly reduces the increase in current. Co-exposure to BDNF and ifenprodil, an NR2B subunit antagonist, reproduces the response observed with AP-5, suggesting that BDNF primarily enhances activity of NR2B-containing receptors. Protein kinase involvement was confirmed with the tyrosine kinase inhibitor staurosporine, which prevents the response to BDNF. These results demonstrate the central role of NR2B-containing receptors in BDNF modulation of hippocampal synaptic transmission. To explore the molecular mechanisms underlying synaptic NR2B signaling and growth factor regulation, Lin et al. (1999) examined the effects of BDNF on the interaction and expression of protein tyrosine phosphatase (PTP)1D and NR2B. BDNF increases the association of PTP1D with tyrosine-phosphorylated proteins, including NR2B, in cortical neurons and PC12 cells. The BDNF action appears to be specific, since nerve growth factor, another member of the neurotrophin gene family, does not alter the association. These investigators reported that PTP1D is an intrinsic component of rat PSD, based on immunoblot analyses using specific anti-PTP1D antibodies. In addition, NR2B coimmunoprecipitates with PTP1D, indicating a physical
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association of the subunit with PTP1D. Taken together, these results suggest that tyrosine phosphorylation and dephosphorylation can participate in BDNF-mediated NR2B signaling cascades at distinct postsynaptic sites. Activity-dependent neurotrophic factor (ADNF), which is a protein secreted by vasoactive intestinal polypeptidestimulated astroglia, may also contribute to the regulation of NR2B. Blondel et al. (2000) used hippocampal neurons in culture to demonstrate that vasoactive intestinal polypeptide promotes neuronal differentiation through ADNF. ADNF is produced by glial cells and acts directly on neurons to promote glutamate responses and morphological development. ADNF causes secretion of neurotrophin-3, and both proteins regulate developmental changes associated with the NMDA receptor subunits NR2A and NR2B. In addition to identified growth factors, hormone modulation of the NR2B subunit has also been demonstrated. Systemic administration (10 days) of recombinant human growth hormone to young rats (11 weeks old) causes an increase in the expression of hippocampal NR2B mRNA that is not observed in elderly rats (57 –67 weeks old) (Le Greve`s et al., 2002). Estrogen treatment significantly increases the levels of hypothalamic NR2B mRNA during the late prepubertal period in female rats (Kanamaru et al., 2001). Importantly, the effects of estrogen are time- and brain region-specific, thus providing an example of how the expression of NR2B is developmentally and regionally regulated. Finally, Mukai et al. (2000) examined the subunit specificity of pregnenolone sulfate (PREGS) potentiation of NMDA receptor-mediated Ca2+ signals. Using NR1/NR2Btype NMDA receptors expressed in Chinese hamster ovary cells, PREGS enhances the Ca2+ influx through NR2Bcontaining receptors in a dose-dependent manner. Ifenprodil abolishes the NMDA-induced Ca2+ influence even in the presence of PREGS. These results imply that PREGS positively modulates the Ca2+ influx through NR2B-containing receptors that are expressed since the embryonic period. 4.2. Exogenous regulators Alterations in the expression, distribution, and function of the NR2B subunit occur via changes in brain environment. As described above, these changes can result from endogenous factors, such as kinases, phosphatases, and other regulatory enzymes. However, external variables can also influence the prevalence of NR2B subunits and consequently the function of NMDA receptors. These exogenous elements generally are identified as those resulting in trauma or lesions and those pertaining to pharmacological manipulation. 4.2.1. Trauma or lesions Watkins et al. (1998) studied the effects of single and repeated electroconvulsive shock treatment on the mRNA levels of several glutamate receptor subunits in the dentate
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gyrus and CA1 hippocampal regions of the rat. In the dentate gyrus, shock treatment elevates the levels of mRNA for NR2B. The change in NR2B transcript expression persists the longest (as compared with NR2A and metabotropic glutamate receptor subtype 5). Expression is still elevated after 24 hr, but returns to baseline by 48 hr after shock treatment. Chronic and acute treatments yield the same results. However, the functional consequences of a transient increase in NR2B mRNA expression remain unclear. In addition to providing information about the regulation of subunit expression, brain insults can also help to answer questions about local circuitry and region-specific changes in receptor function. In an attempt to elucidate the effects of decreased dopamine on glutamate receptor expression, Kayadjanian et al. (1996) performed unilateral and bilateral lesions of the medial forebrain bundle and the sensorimotor cortex, respectively. Unilateral lesions of the medial forebrain bundle resulted in no change in NR2B mRNA levels in the dopamine-depleted striatum, as compared with the normal rat striatum. Bilateral lesions of the sensorimotor cortex resulted in a significant increase in the levels of NR2B mRNA, specifically in the striatal projection area of the sensorimotor cortex. These findings suggest that NR2B expression is regulated by cortico-striatal fibers in a topographical manner. Levels of NR1 and NR2B subunits within the magnocellular nuclei of the hypothalamus are up-regulated and down-regulated, respectively, during dehydration (Decavel & Curras, 1997). Similarly, osmotic activation of the hypothalamo-neurohypophysial system reversibly down-regulates NR2B subunits in the supraoptic nucleus of the hypothalamus. These results indicate that NR2B-containing receptors on the supraoptic nucleus and paraventricular nucleus magnocellular neuroendocrine cells may contribute to neuroendocrinological functions associated with body fluid homeostasis (Curras-Collazo & Dao, 1999). Finally, the influence of heat stress on the constitutive isoform of NOS (cNOS) and NMDA receptor subunit gene expression in the hippocampus was examined in a rat model. Exposure of animals to 4 hr of heat stress at 38C results in a significant up-regulation of cNOS, but mRNA encoding NR1, N2A, and NR2B shows a marked downregulation in the hippocampus of heat-stressed animals as compared with controls (Le Greves et al., 1997). Thus, increased NO production associated with heat stress may contribute to the regulation of NMDA receptor subunit expression. 4.2.2. Pharmacological agents (e.g., antagonists) Although artificial manipulations by exogenous agents, the pharmacological studies reviewed in this section provide important information about the regulation of NR2B (Table 1). For example, treatment with certain NMDA receptor antagonists illustrates how neuronal activation controls the ability of the cell to modify its synapses. Exposure of mouse
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cortical neurons to the NMDA receptor antagonist AP-5 causes an increase in the levels of the NR2B mRNA in a concentration- and time-dependent manner (Follesa & Ticku, 1996). Treatment with AP-5 also increases the number of NR2A and NR2B clusters (Rao & Craig, 1997). Taken together, these results suggest that expression and localization of NMDA receptors are regulated by synaptic activity. Treatment with MK-801 provides concurring results. The density of NMDA receptors on membranes prepared from cultured cerebral cortical neurons was determined using binding assays with [125I]MK-801 following exposure of cultures to various antagonists of the NMDA receptor complex. The density of binding sites for MK-801 is increased by 40 – 80% after exposure to AP-5, with no change in the number or viability of neurons. Up-regulation of the NMDA receptor is observed after 2 –7 days, but not after 1 day of exposure to AP-5. The density of NMDA receptors is also increased after exposure of cells to CGS 19755 and to MK-801. Thus, the density of NMDA receptors on cultured neurons can be selectively up-regulated by exposure to specific NMDA receptor antagonists (Williams et al., 1992). Chronic treatment of cortical neurons with MK-801 enhances membrane-associated tyrosine kinase activity and up-regulates NR2B subunits, while treatment with a tyrosine kinase inhibitor results in down-regulation of NR2B subunits in cortical neurons. Thus, MK-801-induced receptor up-regulation may be mediated by NR2B phosphorylation via tyrosine kinases (Kalluri & Ticku, 1999b). In contrast, however, in vivo studies yield somewhat conflicting results. For example, when rats are injected every 12 hr for 14 days and sacrificed on day 15, chronic MK-801 administration does not alter NR1, NR2A, or NR2B subunit expression in either the hippocampus or cortex (Matthews et al., 2000). Linden et al. (2001) also investigated the effects of acute administration of MK-801 on mRNA levels of NMDA receptor subunits and on molecules known to cluster or phosphorylate the receptor. In situ hybridization of rat brain sections demonstrated that acute treatment with MK-801 (5 mg/kg, 4 hr) decreases levels of NR2B mRNA in the parietal cortex. MK-801 also increases mRNA levels of SAP90/PSD-95 and PKC-g in cortical regions. Although not in complete agreement, taken together, these results suggest that antagonist-induced changes in the density of NMDA receptors and associated neuronal elements may play a role in adaptation or toxic responses and, therefore, could complicate therapeutic approaches to the treatment of neurological disorders (see Section 5). Ifenprodil is an antagonist selective for NMDA receptors containing the NR2B subunits, and recently was used to demonstrate that antagonists not only modify the expression of subunits, but can also alter the binding properties of the receptor. Using rat brain slices, Zhang et al. (2000) found that ifenprodil enhances NMDA-induced currents in both cortical and subcortical areas. The results, combined with
previous studies, suggest that the augmenting effect is due to an increase in NMDA receptor affinity for agonists and is specific for responses induced by low NMDA concentrations. As NMDA concentrations increase, the affinityenhancing effect of ifenprodil becomes less evident and the channel-blocking effect becomes more prominent. Zhang and Shi (2001) provided further evidence for the mechanism of action of ifenprodil on NR2B-containing receptors. Specifically, based on data from whole cell recordings of rat prefrontal cortical slices, ifenprodil is hypothesized to inhibit the rising phase of fast NMDA responses by suppressing both channel opening and the association of NMDA with NMDA receptors. Thus, given the complexity of the mechanisms associated with the action of ifenprodil on NMDA receptors, interpretation of data from NMDA receptor antagonist studies should also consider variables such as agonist and antagonist concentrations, pharmacological selectivity, and the specific subunit enrichment profile (Table 1). In addition to data from NMDA receptor antagonist studies, information regarding the regulation of NR2B expression can also be gleaned via the use of other pharmacological agents. For example, cultures of rat cerebellar granule cells were treated with the glutamate transport blocker L-trans-pyrrolidine-2,4-dicarboxylate and the expression of NMDA receptor subunits was evaluated (Cebers et al., 2001). Expression of the NR2B subunit mRNA and protein is significantly lower in L-trans-pyrrolidine-2,4-dicarboxylate-treated cells as compared with controls. These findings imply that increased synaptic glutamate concentration could result in the down-regulation of NR2B-containing NMDA receptors.
5. NR2B role in human physiology and pathophysiology Both endogenous and exogenous factors interact with NMDA receptors and play a role in a number of human disorders and physiological processes. 5.1. Synaptic plasticity/learning Although changes in synaptic function have not been directly linked to learning in humans, long-term potentiation (LTP) and LTD have been used to model and to generate inferences regarding learning-related phenomena. Both LTP and LTD can be triggered by NMDA receptor activation. Hrabetova et al. (2000) sought to test the hypothesis that the specific patterns of afferent stimulation producing LTP and LTD activate separate receptor subpopulations composed of different NMDA receptor subunits. The authors examined the inhibition of LTP and LTD by a series of competitive NMDA receptor antagonists that varied in their affinities for NR2A/B and NR2C/D. Antagonists with higher affinity for the NR2A/B subunits relative to NR2C/D show more potent inhibition of LTP than LTD. Thus, distinct subpopulations of
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NMDA receptors characterized by different NR2 subunits contribute to the induction mechanisms of potentiation and depression. Earlier studies also support the involvement of NR2 subunits in synaptic plasticity and specifically implicate NR2B in LTP-related events. Quantitative in situ hybridization revealed that following the induction of hippocampal LTP in the dentate gyrus of freely moving rats, specific increases in the expression of the NR2B subunit are evident in postsynaptic dentate granule cells. However, there are no changes in the expression of the NR2A, NR2C, or NR2D subunits. The elevation in NR2B subunit expression is delayed, occurring days after LTP induction. NR2B subunit expression is enhanced significantly by 48 hr after LTP, but starts to decrease toward basal levels by 96 hr. The transient increase in the expression of the NR2B subunits is temporarily related to an increase in the expression of the NR1 subunits (Thomas et al., 1994). Changes in the expression of individual glutamate receptor subunits that occur days after the induction of LTP may reflect activation of so-called lateonset genes that are needed for the maintenance of LTP (Thomas et al., 1996). In an NR2B knock-down model for aged rats, NR2B antisense treatment abolished LTP and impaired spatial learning, as measured by the Morris water maze (Clayton et al., 2002). Collectively, these results support a role for NR2B-containing receptors in LTP, and suggest a relationship among the NR2B subunits, LTP, and specific cognitive functions. In addition to alterations in subunit expression, changes in the phosphorylation state of NR2B are also associated with LTP. CaMKII-dependent phosphorylation of NR2A and NR2B is decreased in rats with hippocampal damage and impaired LTP caused by methylazoxymethanol-induced prenatal ablation of CA1 neurons (Caputi et al., 1999). LTP in the dentate gyrus is also correlated with tyrosine phosphorylation of the NR2B subunit, persisting in vivo for several hours. Consequently, NR2B phosphorylation is hypothesized to play a role in short- and intermediate-term mechanisms of experience-dependent plasticity (Rosenblum et al., 1996). Nakazawa et al. (2001) examined alterations in the level of tyrosine residue 1472 phosphorylation on the NR2B subunits after the induction of LTP. Using extracellular field potential recording techniques, excitatory postsynaptic potentials were recorded in the hippocampal CA1 region of mice. Sixty minutes after the induction of LTP, enhanced phosphorylation of NR2B Tyr1472 was observed. The data suggest that residue-specific tyrosine phosphorylation of NR2B is involved in the expression of LTP. Further evidence for the role of tyrosine phosphorylation of NR2B subunits in synaptic plasticity is provided by Manabe et al. (2000). KO-mice lacking the H-ras gene were generated and then used to study the roles of NMDA receptors, phosphorylation, and Ras in synaptic transmission and LTP. In the H-ras null mutant hippocampus, the tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor is increased, and correspondingly,
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NMDA synaptic responses are selectively enhanced. LTP is markedly enhanced in mutant mice, most likely because of a selective enhancement of NMDA synaptic responses. It was concluded, therefore, that the regulation of activitydependent synaptic plasticity in the adult animals by downregulation of the phosphorylation of specific NMDA receptor subunits might be another major role for the H-ras protein. In humans (children), chronic lead exposure produces deficits in learning and memory. Recent studies assessed the effects of this potential toxin on NMDA receptor subunits. Exposure to lead during development has no effect on NR1 or NR2B subunit protein expression in the hippocampus or the cortex. However, the expression of the NR2A subunits is reduced in the rat hippocampus (Nihei & Guilarte, 1999). In a subsequent study, Nihei et al. (2000) demonstrated that impairments of spatial learning and hippocampal LTP in rats chronically exposed to lead are associated with changes in gene and protein expression of the NMDA receptor subunits NR1 and NR2A, but not NR2B. In vitro studies investigating the effects of lead on the expression of the NR1 and NR2B subunits provide somewhat different results. Primary cortical and hippocampal cultures were treated with lead. In cortical neurons, dose-dependent increases in the expression of NR2B mRNA and subunit protein were observed, but lead-induced changes in the expression of NR1 mRNA or subunit protein were not present. However, in hippocampal neurons, both NR1 and NR2B mRNA and subunit protein levels were decreased (Lau et al., 2002). Thus, the role of NR2B-containing receptors in mediating the toxic effects of lead and its subsequent impact on learning and memory remains unclear. 5.2. Schizophrenia Accompanying the long-standing theories regarding dopamine and schizophrenia are more recent reports implicating glutamate and NMDA receptors. The NMDA receptor hypothesis of schizophrenia is based on the observation that hypofunction of the NMDA receptors induced by various NMDA receptor antagonists precipitates a transient psychotic state in healthy individuals. Accordingly, researchers are beginning to look for evidence that the NMDA receptor system may be dysfunctional in schizophrenia. Clinical trials of glycinergic agents are currently being conducted (for a review, see Farber et al., 1999). The specific rationale for these studies is that activation of the glycine site within the NMDA receptor complex is necessary for the normal function of this receptor system. Early results from clinical trials of glycinergic agents indicate that these agents either have no effect or are only modestly beneficial effects on the negative symptoms accompanying schizophrenia (Farber et al., 1999). Goff and Coyle (2001) reviewed evidence from more recent clinical trials, as well as from basic science reports, and suggest that pharmacotherapeutic modulation of the glycine site on the NMDA
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receptors can be helpful for the treatment of negative symptoms associated with schizophrenia. Several investigators have specifically been looking at the role of the NR2B subunit in schizophrenia. Expression patterns of mRNA for individual receptor subunits were assessed using in situ hybridization in prefrontal, parietotemporal, and cerebellar cortices of postmortem brains from people with schizophrenia and from neuroleptic-treated and non-medicated control subjects (Akbarian et al., 1996). Although a shift in the relative proportions of NR2A, NR2B, NR2C, and NR2D mRNA expression is suggested, the data revealed no significant changes in the expression of NR2A, NR2B, NR2C, or NR2D mRNA in schizophrenic patients as compared with control subjects. However, using different methodology, Grimwood et al. (1999) found somewhat conflicting results. As measured by radioligand binding with [3H]ifenprodil, NR2B-containing receptors are selectively up-regulated in the superior temporal cortex in schizophrenia, but not in the pre-motor cortex (Grimwood et al., 1999). Although changes in transcript levels are not always indicative of corresponding alterations in protein expression (and visa versa), these findings suggest that further investigation of the role of NR2B in schizophrenia is needed. Gao et al. (2000) analyzed postmortem hippocampal tissue from people with schizophrenia and from control subjects. Glutamate receptor autoradiography of various subfields in the hippocampus provided no evidence of ligand-binding differences between the groups. Interestingly, in situ hybridization experiments were also performed and the level of mRNA for the NR2B subunit in CA2 of the hippocampus was found to be 40% higher in schizophrenia tissue than in control tissue. Finally, Nishiguchi et al. (2000) screened for genetic variations in the region of the NR2B subunit gene encoding the C-terminal intracellular domain in subjects with schizophrenia and studied the association between schizophrenia and a novel polymorphism of the NR2B subunit gene, the silent mutation 2664C/T. No significant differences in the frequencies of 2664C/T genotypes exist between patients with schizophrenia and control subjects. Thus, although the NR2B subunit may be involved in certain aspects of the illness, these findings provide no evidence of an association between schizophrenia and the 2664C/T polymorphism of the NR2B subunit gene. 5.3. Parkinson’s disease Another disorder traditionally associated with disturbances in the dopaminergic system is Parkinson’s disease. Overactivity of the striatolateral pallidal pathway, the ‘‘indirect’’ striatal output pathway to the external pallidal segment, is thought to be responsible for the generation of some Parkinson’s symptoms. However, as with schizophrenia, the glutamatergic system and NMDA receptors recently have been shown to interact with dopaminergic circuitry and to contribute to neurochemical alterations accompanying the disorder. Oh et al. (1998) examined the effects of unilateral
nigrostriatal dopamine system ablation with 6-hydroxydopamine, followed by twice daily treatment with levodopa (LDOPA), on the phosphorylation state of the rat striatal NR2A and NR2B subunits. After 3 weeks of L-DOPA treatment, tyrosine phosphorylation of the NR2A subunit, and especially the NR2B subunit, is increased ipsilateral to the lesion (20 ± 5% and 46 ± 7% of the intact striatum, respectively) without accompanying changes in subunit protein levels. Augmented tyrosine phosphorylation of the NR2B subunits alone or in combination with the smaller rise in NR2A phosphorylation contributes to the apparent enhancement in striatal NMDA receptor sensitivity (see Section 4.1.2) and, thus, to the changes in dopaminergic responses in L-DOPA-treated Parkinsonian rats. Using a similar animal model, Oh et al. (1999) looked at CaMKII phosphorylation of serine. L-DOPA treatment elevates serine phosphorylation of striatal NR2A subunits, but not NR2B subunits. Further, chronic treatment with SKF-38393, a D1agonist, increases NR2A and decreases NR2B serine phosphorylation, while chronic exposure to quinpirole, a D2agonist, has no effect on NR2A, but increases NR2B, phosphorylation. Kinase-specific changes in the phosphorylation of the NR2B subunits may thus contribute to the compensatory neurochemical adaptations associated with LDOPA treatment. Dunah et al. (2000) also studied the expression and phosphorylation of NMDA receptor subunits in the rat 6hydroxydopamine lesion model of Parkinson’s disease. In the lesioned striatum, the levels of NR1 and NR2B subunit expression are decreased to 68 ± 3.2 and 62 ± 4.4% of control, respectively. Co-immunoprecipitation studies indicate that there is a selective reduction of the NR1/NR2B, but not NR1/NR2A, receptors. However, using tissue fractionation techniques, the authors found that the decrease in NR2B abundance could be attributed to an intracellular redistribution of subunit proteins rather than to a reduction in subunit protein expression. Further, in partial agreement with previous studies, tyrosine phosphorylation of NR2B (but not NR2A) is decreased in the lesioned striatum. LDOPA treatment normalizes the lesion-induced alterations in receptor subunit enrichment and causes hyperphosphorylation of the NR1 serine and NR2A and NR2B tyrosine residues. NMDA receptor antagonist studies also support a role for the glutamatergic system, specifically mediated by NR2Bcontaining receptors, in modulating features of Parkinson’s disease. Selective blockade of NR2B-containing NMDA receptors with ifenprodil and eliprodil causes a significant increase in locomotor activity in the reserpine-treated rat model of Parkinson’s disease (Nash et al., 1999). Additionally, Steece-Collier et al. (2000) examined the antiparkinsonian effects of CP-101,606 in rodents and non-human primates. In rats, this selective antagonist of the NR2Bcontaining receptors decreases haloperidol-induced catalepsy. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinetreated monkeys, CP-101,606 reduces parkinsonian motor
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symptoms by 20%. CP-101,606 also significantly potentiates the effect of L-DOPA as indicated by a 50% reduction in motor symptoms when compared with vehicle and a 30% reduction when compared with L-DOPA alone. Drug-related side effects were not observed at any dose of CP-101,606 administered. The authors concluded that CP-101,606 has direct antiparkinsonian actions in both rodents and monkeys and that it synergistically potentiates the motoric improvements accompanying L-DOPA treatment. Consequently, NR2B-selective antagonism may prove useful in the treatment of Parkinson’s disease. 5.4. Huntington’s disease Huntington’s disease, a hyperkinetic movement disorder, may also be associated with changes in the function of the NR2B-containing NMDA receptors. The condition is generated by an expanded CAG repeat in the first exon of the Huntington’s disease gene. The mutant huntingtin protein, therefore, has an expanded polyglutamine (Q) tract in its Nterminal region, causing a toxic gain of function, as well as possibly a loss of function of normal huntingtin. Receptors composed of NR1 and NR2B subunits exhibit significantly larger currents when co-expressed with mutant huntingtin. Further, mutant huntingtin may increase the number of functional NR1/NR2B-type receptors at the cell surface (Chen et al., 1999b). Follow-up experiments provide further evidence for the involvement of NMDA receptors in Huntington’s disease. Transfected HEK-293 cells co-expressing full-length mutant huntingtin-138Q and either NR1/NR2A or NR1/NR2B heteromeric receptors exposed to NMDA exhibit a significant increase in excitotoxic cell death as compared with cells expressing huntingtin-15Q or GFP (Zeron et al., 2001). Interestingly, the difference in cell death is larger for cells containing NR1/NR2B receptors. Zeron and colleagues thus concluded that subunit-specific interactions between NR2B-containing receptors and mutant huntingtin potentiate NMDA receptor-initiated apoptotic cell death and, consequently, may contribute to the development of neuropathology associated with Huntington’s disease. 5.5. Excitotoxicity/hypoxia/ischemia Excitotoxicity is a critical mechanism contributing to neurodegeneration during ischemia or hypoxia. Major events in the cascade triggered by hypoxia or ischemia include overstimulation of NMDA-type glutamate receptors, Ca2+ entry into cells, activation of Ca2+-sensitive enzymes such as nNOS, production of oxygen free radicals, mitochondrial impairment, and subsequent necrosis or apoptosis (for reviews, see Sattler & Tymianski, 2000; Johnston et al., 2000). In support of the specific role of the NMDA receptor complex in excitotoxic damage, Sattler et al. (1999) used cultured cerebral cortical neurons to demonstrate that suppressing the expression of PSD-95 selectively attenuates the
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excitotoxicity triggered via NMDA receptors, but not by other glutamate or Ca2+ channels. NMDA receptor function is not affected because receptor expression, NMDA currents, and [45Ca2+] loading are unchanged. Moreover, suppressing PSD-95 expression blocks Ca2+-activated NO production by NMDA receptors selectively, without affecting nNOS expression or function. Thus, PSD-95 is required for efficient coupling of NMDA receptor activity to NO toxicity and imparts specificity to excitotoxic Ca2+ signaling (Sattler et al., 1999). Importantly, specific changes in NR2B-containing NMDA receptors may accompany aspects of excitotoxic damage associated with hypoxia and ischemia. Cheng et al. (1999) examined several factors related to the increase in susceptibility to excitotoxicity that occurs in cultured embryonic forebrain neurons over time. For instance, RNase protection assays were used to measure NR1, NR2A, and NR2B mRNA levels, demonstrating that NR2B mRNA levels increase dramatically during the first 10 days and subsequently remain stable. It was concluded that increased glutamate-stimulated [Ca2+]i responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons. To more comprehensively address the question of NR2B subunit involvement in neurotoxicity associated with ischemia and hypoxia, Zhang et al. (1997) assessed rat receptor gene expression, protein levels, and function after severe global ischemia using a 4-vessel occlusion ischemia model. At 12 and 24 hr after ischemic challenge, decreased expression of NR2A and NR2B mRNA in CA1 and other hippocampal subfields was observed. A significant decrease in NR2A/B protein (total hippocampus) was observed at both 6 and 24 hr after ischemia, as compared with controls. Further, electrophysiological assessment of single-channel NMDA receptor activity in the CA1 indicates that the main conductance state of NMDA receptor channels is maintained 6 hr after the ischemic challenge, but that by 18– 24 hr, this main conductance state is rarely observed. Thus, the ischemia-induced decrease in the expression of NR2A and/ or NR2B alters the function of the NMDA receptors. Kim, W. T. et al. (1998) created hypoxic conditions in newborn piglets. Animals were ventilated until paO2 was < 20 mm Hg. Unlike the previous study that used 6– 24 hr of ischemic exposure, 1 hr after a hypoxic challenge there were no changes in the expression of NR2B or other NMDA receptor subunits (NR1, NR2A) in regions of the frontal cortex, hippocampus, thalamus, basal ganglia, or cerebellum, as assessed by western analysis. In addition to potentially altering NR2B subunit expression, ischemia has also been shown to alter protein interactions. NR2A and NR2B bind to each of the SH2 domains of the tyrosine kinases Src and Fyn (expressed as GST-SH2 fusion proteins), and binding is increased 2-fold after ischemia and reperfusion. Transient ischemia induces a marked increase in the tyrosine phosphorylation of NR2A
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and NR2B and, consistent with previous findings (Kim, W. T. et al., 1998), has no effect on the amount of individual NMDA receptor subunits (Takagi et al., 1999). Similarly, regional levels of tyrosine phosphorylation of the NR2B subunit are enhanced after ischemia in the rat brain in both the synaptosomal fraction and the whole-tissue homogenate of rat neocortex and hippocampus, but return to control levels only in the preconditioned brain (ischemic preconditioning was 3 min). Using the 2-vessel occlusion model of global cerebral ischemia, preconditioning provides maximum neuroprotection when induced 2 days prior to a potentially lethal ischemic insult of 9-min duration. Interestingly, ischemic preconditioning induces a selective decrease in the levels of the NR2A and NR2B subunits and a modest decrease in the levels of NR1 in the synaptosomal fraction of the neocortex, but not hippocampus (Shamloo & Wieloch, 1999). To further examine such findings, which demonstrate that transient cerebral ischemia results in an enhancement of tyrosine phosphorylation of proteins associated with the PSD, Cheung et al. (2000) investigated possible mechanisms behind this increase. Isolated PSDs were analyzed for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Tyrosine phosphorylation of several PSD proteins, including the NR2A and NR2B subunits, is enhanced relative to sham-treated animals after 20 min and 6 hr of reperfusion. The ability of intrinsic tyrosine kinases to phosphorylate PSD proteins, including the NMDA receptor subunits, increases 3-fold after ischemia. While ischemia can increase interactions among some proteins, it also can decrease the binding of other NMDA receptor-associated proteins. Takagi et al. (2000) examined the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. The association between PSD-95 and NR2A and NR2B, as indicated by co-immunoprecipitation, is less in post-ischemic samples than in sham-operated controls. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge. Recently, lithium was proposed as a drug that might be protective against glutamate-induced excitotoxicity. Lithium pretreatment of cultured cerebral cortical neurons reduced the level of tyrosine phosphorylation (Tyr1472) without altering the expression of the NR2B subunits. Further, the decrease in residue-specific phosphorylation was associated with its neuroprotective effects (Hashimoto et al., 2002). 5.6. Seizure disorder Previous research has consistently demonstrated a connection between glutamatergic neurotransmission and certain types of seizure activity; however, the degree and nature of NMDA receptor involvement, the subunit specificity, and the effects of seizure activity on subsequent receptor expres-
sion and function have yet to be fully understood. In an attempt to characterize the potential involvement of individual NMDA receptor subunits, Narita et al. (2000a) pretreated mice intracerebroventricularly with antibodies specific to the C-terminal region of the NR2B (as well as NR1 and NR2A) subunit 1 and 3 days before treatment with NMDA. The pretreatment regimes for all three NMDA receptor subunits produced a significant increase in the seizure threshold for NMDA, suggesting that the NR1, NR2A, and NR2B subunits may be involved in seizure sensitivity to glutamatergic agonists and consequently have an important role in regulating seizure activity. Kindling has been used as an animal model of epilepsy, such that application of initially subconvulsive electrical stimulation eventually results in the development of limbic and clonic motor seizures. To investigate the possible effects of seizures on NMDA receptor gene expression, transcript levels of NR1, NR2A, NR2B, NR2C, and NR2D were measured using the rat hippocampus (Kraus et al., 1994). Kindling in hippocampal regions does not alter NMDA receptor gene expression 24 hr or 28 days after the last seizure. Similarly, in a follow-up study that assessed NMDA receptor subunit levels at 28 days post-seizure, Kraus and McNamara (1998) reported that kindling does not produce long-lasting changes in the levels of the NR1, NR2A, or NR2B proteins in the hippocampus. Taken together, these data suggest that seizure activity does not appear to transiently or permanently alter the expression of NMDA receptor subunits. In contrast, Mathern et al. (1998) investigated hippocampal NMDA receptor subunit immunoreactivity in tissue taken at autopsy from temporal lobe epilepsy patients compared with receptor subunits in tissue from non-epileptic control patients. Using an antibody that labeled both the NR2A and NR2B subunits, the authors observed increased NR2A/B expression throughout all hippocampal subfields. It was concluded that subunit up-regulation might contribute to seizure generation or propagation by altering NMDA receptor-mediated neurotransmission. Given these disparate findings, more research appears to be needed to understand the relationship between NMDA receptor subunit expression and seizure activity. Toward this end, Kojima et al. (1998) generated transgenic mice that expressed either a constitutively active form of Fyn or native Fyn in neurons of the forebrain. The threshold for pentylenetetrazole-induced seizures is reduced and electrical stimulation of the amygdala elicits higher seizure activity in transgenic mice expressing the mutant Fyn as compared with wild-type mice. In forebrains of both native and mutant Fyn transgenic mice, tyrosine phosphorylation of NR2B is enhanced, confirming that this subunit is a substrate of Fyn. In addition, MK-801 administration attenuates kindling in mice overexpressing native Fyn, as well as in wild-type mice. Consequently, tyrosine phosphorylation of NR2B by Fyn might be involved in the susceptibility to NMDA receptor-mediated kindling.
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5.7. Diabetes Quantitative autoradiography suggests that the antagonist affinity and density of the NMDA receptor are greater in diabetic mice than in control animals (Li et al., 1999). However, to date, there is little information in the literature regarding diabetes and potential NMDA receptor subunit involvement. There exists an animal model, the streptozotocin-diabetic rat, which manifests impairments in spatial memory and LTP expression—two areas of functioning previously shown to involve subunit-specific NMDA receptor activation (see Section 3). Using the streptozotocindiabetic rat, Di Luca et al. (1999) studied the effects of experimental diabetes on the expression of NMDA receptors and associated proteins regulating synaptic transmission at the PSD. In situ hybridization and immunoblot analyses were used to assess the expression of NMDA receptor subunits and CaMKII. In the hippocampus, the mRNA and immunoreactivity for NR2B is reduced by 40% in diabetic rats, while NR1, NR2A, GluR1, GluR2/3, PSD-95, and CaMKII transcript and protein levels are unchanged. In addition, NMDA receptor subunit phosphorylation by CaMKII was studied in the hippocampus and cortex of control, streptozotocin-diabetic, and insulin-treated rats. Although the expression of CaMKII is unaltered, the ability of CaMKII to phosphorylate NR2A/B subunits is reduced in hippocampal PSD fractions from streptozotocin-diabetic rats as compared with the PSD from control rats. Insulin intervention for 3 months after streptozotocin treatment partially restores both CaMKII activity and NR2B levels. Thus, these findings suggest that the metabolic changes associated with diabetes alter NMDA receptors in a kinaseand subunit-specific manner. In a follow-up study, Gardoni et al. (2002) provide additional evidence to support this hypothesis. Using the same rat model, 4 months (but not 1 month) after the onset of diabetes, NR2B expression and phosphorylation is reduced in the hippocampus. Insulin treatment also prevents the reduction in CaMII and tyrosine kinase activity and NR2B subunit expression levels. 5.8. Alzheimer’s disease Alzheimer’s disease is often associated with a loss of NMDA receptors in selected brain regions. For example, Maragos et al. (1987) demonstrated a marked loss of NMDA receptors in the hippocampus of patients with Alzheimer-type dementia, as measured by [3H]thienylphencyclidine binding. Recently, investigators have begun to examine the effects of Alzheimer’s disease on individual NMDA receptor subunits. Wang et al. (2000) estimated the levels of all a-amino-3-hydroxy-5-methyl-isoxazole-4-propionate and NMDA receptor subunits in selected (i.e., hippocampus, frontal, and entorhinal cortex) brain tissue samples obtained from control subjects and patients who died with Alzheimer’s disease. Modest decreases in the NMDA receptor subunits NR1, NR2A, and NR2B were
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found in the hippocampus and frontal cortex, while little or no change in any of these subunits was observed in the entorhinal cortex. Sze et al. (2001) assessed levels of both non-phosphorylated and phosphorylated receptor subunit proteins in various areas of post-mortem brains from patients who died with Alzheimer’s disease and control subjects. Significant reductions in NR2B expression are present in the hippocampus (40%) and entorhinal cortex (31%). In addition, tyrosine phosphorylated NR2B is decreased (56%) in the entorhinal cortex. The authors found no correlation between phosphorylated and non-phosphorylated NR2B levels and, thus, suggest that the Alzheimer’s disease-related reduction in phosphorylation is subunitindependent. Collectively, these results suggest that NMDA receptor subunits are selectively and differentially reduced in areas of Alzheimer’s-diseased brains. However, questions remain regarding the magnitude and location of protein loss, as well as the nature of the relationship among decreased subunit expression, phosphorylation, and the cognitive impairments and cell loss observed in Alzheimer’s disease. In a study of 132 patients with Alzheimer’s disease and 114 control subjects, Tsai et al. (2002) investigated the prevalence of the allelic variant (C2664T) of the NR2B. The distribution of the NR2B genotypes and alleles did not differ between the groups, thus suggesting that C2664T does not significantly contribute to the development of Alzheimer’s disease. 5.9. Drugs of abuse Many abused drugs alter the expression and function of NMDA receptors. It is further hypothesized that NMDA receptors mediate the adaptive mechanisms that are involved in the development, maintenance, and expression of drug addiction. In this section, evidence for the role of NR2B-containing NMDA receptors in alcohol, cocaine, and opioid abuse is discussed. NMDA receptors are an important site of action for ethanol (Lovinger et al., 1989; Carter et al., 1995), and importantly, dependence, tolerance, and withdrawal symptoms seem to be mediated in part by NMDA receptors. Narita et al. (2000b) treated mice with ethanol for 5 days. Withdrawal signs (piloerection, jerking, tremor, and handling-elicited convulsions) are present at 3, 6, 9, 12, and 48 hr after the discontinuation of the liquid-ethanol diet. Treatment during withdrawal with a selective NR2B-containing NMDA receptor antagonist, ifenprodil, significantly reduces the expression of ethanol withdrawal signs. In addition, during chronic ethanol treatment, protein abundance for the NR2B subunit in the mouse limbic forebrain, but not the cerebral cortex, is significantly increased with respect to control levels, as measured by immunoblotting. The significant up-regulation of NR2B lasts for at least 9 hr after the discontinuation of ethanol, and returns to basal levels by 48 hr. Similarly, Kalluri et al. (1998) investigated the effects of chronic ethanol administration and withdrawal on NR2B
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expression in the rat cerebral cortex and hippocampus. Chronic ethanol treatment up-regulates NR2B expression in both the cortex ( 37%) and hippocampus ( 35%). At 48 hr of withdrawal, the expression of NR2B returns to almost control levels. When ethanol is repeatedly administered to rats for 8 days by gastric intubation and withdrawn for 24 hr, changes in NR2B subunit expression are also evident. Specifically, NR2B mRNA is not substantially altered in cholinergic and GABAergic neurons, but shows a decrease in specific brain areas (the CA region and neostriatum) (Darstein et al., 2000). Taken together, these studies thus suggest that ethanol-induced alterations in the abundance of the NR2B subunit may contribute to the initial development of physical dependence on ethanol. However, there is not complete agreement in the literature regarding the effects of ethanol exposure on NR2B expression. Using primary cultures of rat cerebellar granule cells, Cebere et al. (1999) found that chronic ethanol exposure (50 – 100 mM for 3 days) has no effect on the relative levels of mRNA for NR2B or on the expression of the NR2B protein. Although methodologically very disparate, collectively these studies illustrate the need for further investigation of the effects of ethanol on NR2B expression. Ethanol may also modulate the phosphorylation of NR2B-containing receptors. Mice lacking Fyn are hypersensitive to the hypnotic effects of ethanol. The administration of ethanol enhances tyrosine phosphorylation of the NR2B, but not of the NR1, subunit in the hippocampus of control mice, but not in Fyn-deficient mice. Interestingly, an acute tolerance to ethanol inhibition of NMDA receptormediated excitatory postsynaptic potentials in hippocampal slices develops in control mice, but not in Fyn-deficient mice (Miyakawa et al., 1997). Anders et al. (1999) also investigated the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK-293 cells. Unlike previous findings, using whole cell patch clamp and ratiometric Ca2+ imaging, Anders and colleagues found that the degree of ethanol inhibition of NR2B-containing receptors is unaffected by Fyn tyrosine kinase. Kalluri and Ticku (1999a) determined that in mouse fetal cortical neurons, acute or chronic ethanol treatments do not affect the total phosphorylation of the NR2B subunit. The effects of acute ethanol treatment on the phosphorylation of the NR2B subunit in the adult cortex and hippocampus were also examined. In adult mice, acute ethanol treatment increases the tyrosine phosphorylation of the NR2B in the hippocampus, but not in the cortex. Thus, although the functional significance of ethanol-induced changes in the phosphorylation of NR2B remains in question, it does appear that ethanol exposure contributes to alterations in the expression and phosphorylation of NR2Bcontaining receptors. Cocaine is a potent psychostimulant drug that acts by blocking dopamine uptake, thus increasing dopamine neurotransmission. The actions of cocaine, however, appear to be contingent not only upon the dopaminergic system, but
also upon the glutamatergic system, since glutamate antagonists inhibit many effects of cocaine. Cocaine administration also alters the expression of NMDA receptor subunits. Fitzgerald et al. (1996) showed that chronic cocaine administration up-regulates the NR1 subunit in the ventral tegmental area of the rat brain. Cocaine-induced changes in NR2A/B expression were not observed; however, the antibodies used were not able to differentiate between NR2A and NR2B subunits (Fitzgerald et al., 1996). Using a different method (immunohistochemistry) and more selective antibodies, Loftis and Janowsky (2000) observed that cocaine treatment caused brain region-specific and withdrawal time-dependent alterations in NR2B expression. After 24 hr of withdrawal, cocaine-induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine causes an 50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. Collectively, these results suggest that regionally specific changes in NMDA receptor protein expression may underlie neuronal adaptations following repeated cocaine administration and, therefore, may contribute to withdrawal-related symptomology. As with ethanol exposure, receptor subunit- and kinasespecific phosphorylation is likely an important regulator of the cocaine-induced changes in neurotransmission. Although there is little information on the effects of cocaine treatment on NR2B phosphorylation, the influence of protein kinases on the expression of behavioral sensitization to cocaine has been assessed. Prior to a challenge dose of cocaine, inhibitors of CaMKII (KN93), PKA (H-89), or PKC (bisindolymaleimide-I) were microinjected into the medial nucleus accumbens of rats repeatedly administered either cocaine or saline. None of the kinase inhibitors influenced the behavioral response induced by cocaine in saline-pretreated rats. Among cocaine-sensitized animals, KN93 or bisindolymaleimide-I blocks the expression of behavioral sensitization to cocaine, whereas H-89 has no effect (Pierce et al., 1998). Thus, specific kinases are hypothesized to promote the expression of behavioral sensitization to cocaine, as well as to influence the neurochemical adaptations accompanying repeated cocaine exposure (J. M. Loftis & A. Janowsky, in press). The NMDA receptor has also been implicated in opioid tolerance and withdrawal. The effects of continuous intracerebroventricular infusion of butorphanol, a mixed m/k opioid agonist, on the modulation of NR1, NR2A, NR2B, and NR2C gene expression were investigated using in situ techniques. Drug treatment results in significant modulations of NR1, NR2A, and NR2B mRNA levels. Specifically, NR2B mRNA is decreased in the cerebral cortex, caudate putamen, thalamus, and CA3 region of the hippocampus in withdrawn rats (7 hr after stopping the infusion) (Oh et al., 2000). In addition to assessing drug-induced changes in subunit expression, behavioral measures have also been
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used to evaluate the functional significance of individual receptor subunits on drug-related behaviors. Narita et al. (2000a) conducted a study to determine whether the NMDA receptor (NR1, NR2A, or NR2B) subunits influence the development of morphine-induced place preference using specific antibodies against the respective subunits in mice. Morphine produces a significant dose-dependent preference for the drug-associated place that is completely antagonized by intracerebroventricular pretreatment with an antibody specific to the C-terminal region of the NR2B subunits (but not the NR1 or NR2A subunits). A significant increase in NR2B subunit protein levels is apparent 24 hr after the last conditioning trial in the limbic forebrain obtained from morphine-conditioned mice, as compared with those in the saline-conditioned mice. The protein levels of NR1 or NR2A do not change after morphine conditioning, thus showing the morphine-induced changes to be specific for the NR2B subunit. 5.10. Pain perception The NR2B subunit is implicated in pain perception and, consequently, is being targeted for the development of novel antinociception pharmacotherapeutics (reviewed in Chizh et al., 2001a; Gurwitz & Weizman, 2002). In forebrain structures and regions of the spinal cord, NMDA receptors have an important role in persistent inflammatory pain by reinforcing glutamate sensory transmission (reviewed in Zhuo, 2002). Mice overexpressing the NR2B subunit in the anterior cingulate and insular cortices demonstrate a selective enhancement of persistent pain and allodynia. Specifically, transgenic and wild-type mice display no differences in their tail-flick response to pain or in their latencies to react to hot and cold noxious stimuli. Forebrain-targeted NR2B overexpression, however, does significantly enhance pawlicking behavior associated with prolonged noxious stimulation (peripheral formalin injection) and mechanical allodynia (Wei et al., 2001). NMDA receptor gene expression and neuronal activity in the rostral ventromedial medulla also plays a role in mediating pain perception. After complete Freund’s adjuvant-induced hindpaw inflammation, RTPCR analyses indicate that there is an up-regulation of the NR1, NR2A, and NR2B mRNA expression levels that is apparent 5 hr after inflammation, persists for 7 days, and is returned to baseline by 2 weeks following the inflammation (Miki et al., 2002). Thus, drugs targeting NR2B-containing NMDA receptors in pain-relevant structures, such as the dorsal horn of the spinal cord (Boyce et al., 1999) or anterior cingulate cortex (Wei et al. 2001), could serve as a new class of medicine for managing chronic pain in humans.
6. Conclusions The NR2B subunit of the NMDA-type glutamate receptor is involved in a diverse and extensive array of functions
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and pathologies. Given its region- and cell-specific and developmentally regulated pattern of expression in the CNS, there exist numerous and unique opportunities for modulatory interactions via PSD proteins, enzymes, and other neuronal elements. Future investigation, in combination with new technologies, will permit further characterization of the NR2B subunit, such that the functional consequences of receptor subunit alterations might be more completely understood. References Adams, M. M., Flagg, R. A., & Gore, A. C. (1999). Perinatal changes in hypothalamic N-methyl-D-aspartate receptors and their relationship to gonadotropin-releasing hormone neurons. Endocrinology 140, 2288 – 2296. Akbarian, S., Sucher, N. J., Bradley, D., Tafazzoli, A., Trinh, D., Hetrick, W. P., Potkin, S. G., Sandman, C. A., Bunney, W. E., Jr., & Jones, E. G. (1996). Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics. J Neurosci 16, 19 – 30. Al Hallaq, R. A., Yasuda, R. P., & Wolfe, B. B. (2001). Enrichment of Nmethyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities. J Neurochem 77, 110 – 119. Ali, D. W., & Salter, M. W. (2001). NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity. Curr Opin Neurobiol 11, 336 – 342. Anders, D. L., Blevins, T., Sutton, G., Swope, S., Chandler, L. J., & Woodward, J. J. (1999). Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells. J Neurochem 72, 1389 – 1393. Aoki, C., Bredt, D. S., Fenstemaker, S., & Lubin, M. (1998). The subcellular distribution of nitric oxide synthase relative to the NR1 subunit of NMDA receptors in the cerebral cortex. Prog Brain Res 118, 83 – 97. Apperson, M. L., Moon, I. S., & Kennedy, M. B. (1996). Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family. J Neurosci 16, 6839 – 6852. Bi, R., Bi, X., & Baudry, M. (1998). Phosphorylation regulates calpainmediated truncation of glutamate ionotropic receptors. Brain Res 797, 154 – 158. Blondel, O., Collin, C., McCarran, W. J., Zhu, S., Zamostiano, R., Gozes, I., Brenneman, D. E., & McKay, R. D. (2000). A glia-derived signal regulating neuronal differentiation. J Neurosci 20, 8012 – 8020. Boyce, S., Wyatt, A., Webb, J. K., O’Donnell, R., Mason, G., Rigby, M., Sirinathsianghi, D., Hill, R. G., & Rupniak, N. M. (1999). Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology 38, 611 – 623. Brann, D. W. (1995). Glutamate: a major excitatory transmitter in neuroendocrine regulation. Neuroendocrinology 61, 213 – 225. Brenman, J. E., & Bredt, D. S. (1997). Synaptic signaling by nitric oxide. Curr Opin Neurobiol 7, 374 – 378. Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., Froehner, S. C., & Bredt, D. S. (1996). Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and a1-syntrophin mediated by PDZ domains. Cell 84, 757 – 767. Brown, J. C., III, Tse, H. W., Skifter, D. A., Christie, J. M., Andaloro, V. J., Kemp, M. C., Watkins, J. C., Jane, D. E., & Monaghan, D. T. (1998). [3H]homoquinolinate binds to a subpopulation of NMDA receptors and to a novel binding site. J Neurochem 71, 1464 – 1470. Buller, A. L., Larson, H. C., Schneider, B. E., Beaton, J. A., Morrisett, R. A., & Monaghan, D. T. (1994). The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition. J Neurosci 14, 5471 – 5484.
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Associate editor: K.A. Neve
The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications Jennifer M. Loftisa,b,*, Aaron Janowskya,b,c,d a
Research Service, Department of Veterans Affairs Medical Center, Mental Health (P3MHDC), 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239, USA b Department of Psychiatry, Oregon Health and Science University, Portland, OR 97239, USA c Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USA d Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA
Abstract The N-methyl-D-aspartate (NMDA) receptor is an example of a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors are composed of seven known subunits (NR1, NR2A – D, NR3A – B). The present review focuses on the NR2B subunit of the receptor. Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in a variety of synaptic signaling events and protein-protein interactions. The NR2B subunit has been implicated in modulating functions such as learning, memory processing, pain perception, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structural features, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Postsynaptic density; Glutamate; Kinase; Cytoskeleton; Synaptic plasticity; Excitotoxicity Abbreviations: ADNF, activity-dependent neurotrophic factor; AP-5, DL-2-amino-5-phosphonovaleric acid; BDNF, brain-derived neurotrophic factor; CaM, calmodulin; CaMKII, Ca2+-, calmodulin-dependent protein kinase II; CAPON, carboxy-terminal postsynaptic density-95, discs-large, ZO-1 ligand of neuronal nitric oxide; CASK, a novel postsynaptic density-95 homolog with an N-terminal Ca2+-, calmodulin-dependent protein kinase domain; chapsin, channelassociated protein of synapses; CIPP, channel-interacting postsynaptic density-95, discs-large, ZO-1 domain protein; cNOS, constitutive nitric oxide synthase; Con, conantokin; CPP, 3-(R)-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; CRIPT, a novel protein that selectively binds to the third postsynaptic density-95, discs-large, ZO-1 domain of postsynaptic density-95 via its C-terminal; CTA, conditioned taste aversion; EPSC, excitatory postsynaptic current; GABA, g-aminobutyric acid; GFP, green fluorescent protein; GK, guanylate kinase; GKAP, guanylate kinase-associated protein; GST, glutathione Stransferase; HEK, human embryonic kidney; KO, knockout; LTD, long-term depression; LTP, long-term potentiation; MAGUK, membrane-associated guanylate kinase; neurabin, neural tissue-specific F-actin binding protein; NMDA, N-methyl-D-aspartate; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; PDZ, postsynaptic density-95, discs-large, ZO-1; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PP1, Type 1 protein phosphatase; PREGS, pregnenolone sulfate; PSD, postsynaptic density; PTP, protein tyrosine phosphatase; RasGAP, Ras-GTPase-activating protein; RT-PCR, reverse transcriptase-polymerase chain reaction; SAP, synapse-associated protein; SAPAP, synapse-associated protein-90/postsynaptic density-95-associated protein; SH, Src homology; S-SCAM, synaptic scaffolding molecule; SynGAP, synaptic GTPase-activating protein.
Contents 1.
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Introduction . . . . . . . . . . . . . . . . . 1.1. Structural features . . . . . . . . . . 1.2. Receptor composition/stoichiometry . Localization . . . . . . . . . . . . . . . . . 2.1. During development . . . . . . . . . 2.1.1. NR2B gene expression . . . 2.1.2. NR2B protein expression . . 2.1.3. Associated proteins . . . . .
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* Corresponding author. Research Service, Department of Veterans Affairs Medical Center, Mental Health (P3MHDC), 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239, USA. Tel.: 503-220-8262 ext. 57155; fax: 503-220-3499. E-mail address: [email protected] (J.M. Loftis). 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 2 ) 0 0 3 0 2 - 9
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2.2.
In adult tissue . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. NR2B gene expression . . . . . . . . . . . . . . . . 2.2.2. NR2B protein expression . . . . . . . . . . . . . . . 2.2.3. Cellular and subcellular distribution . . . . . . . . . 3. Functional properties/roles . . . . . . . . . . . . . . . . . . . . . . 3.1. Pharmacological . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Electrophysiological . . . . . . . . . . . . . . . . . . . . . . 3.3. Behavioral . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Endogenous regulators. . . . . . . . . . . . . . . . . . . . . 4.1.1. Cytoskeletal proteins . . . . . . . . . . . . . . . . . 4.1.2. Enzymes (e.g., phosphatases, kinases, and proteases) 4.1.3. Neuromodulators (e.g., growth factors, hormones) . . 4.2. Exogenous regulators . . . . . . . . . . . . . . . . . . . . . 4.2.1. Trauma or lesions. . . . . . . . . . . . . . . . . . . 4.2.2. Pharmacological agents (e.g., antagonists) . . . . . . 5. NR2B role in human physiology and pathophysiology . . . . . . . . 5.1. Synaptic plasticity/learning . . . . . . . . . . . . . . . . . . 5.2. Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . 5.4. Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . 5.5. Excitotoxicity/hypoxia/ischemia . . . . . . . . . . . . . . . . 5.6. Seizure disorder . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . 5.9. Drugs of abuse. . . . . . . . . . . . . . . . . . . . . . . . . 5.10. Pain perception . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The N-methyl-D-aspartate (NMDA) receptor is a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits (McBain & Mayer, 1994). NMDA receptors are concentrated at postsynaptic sites, although some appear to be presynaptic (Liu et al., 1994). Neurotransmission involving NMDA receptors has been implicated in a variety of unique roles: (1) NMDA receptor activation associated with long-lasting changes in synaptic strength (Ali & Salter, 2001), (2) organization of afferent fibers with respect to target neurons during development (Collingridge & Singer, 1990), and (3) participation in glutamate neurotoxicity (Choi & Rothman, 1990). NMDA receptors are derived from at least seven known subunit genes. One of these genes codes for the NR1 subunit, a ubiquitous and necessary component of functional NMDA receptor channels. The diversity of the NMDA receptor NR1 subunit is created by alternative splicing of the NMDAR1 subunit gene to yield eight functional splice forms, which arise via the insertion or deletion of three short exon cassettes in the N-terminal (N1) and C-terminal (C1, C2) domains of the subunit protein (Hollmann et al., 1993). Conversely, the heterogeneity of the NR2 subunits occurs due to the existence of multiple, related genes, NMDAR2A – 2D, whose specific expression profiles in the brain are developmentally and
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regionally regulated (Ishii et al., 1993; Laurie et al., 1997). Recently, NR3A and NR3B subunits have also been reported (Das et al., 1998; Eriksson et al., 2002; Nishi et al., 2001). The present review focuses on the NR2B subunit, first described as a 180-kDa glycoprotein substrate for tyrosine kinase and Ca2+-, calmodulin-dependent protein kinase II (CaMKII) in the postsynaptic density (PSD) (Gurd, 1985). Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in determining the pharmacological and functional properties of the NMDA receptor. Consequently, NR2B has been implicated in modulating functions such as learning, memory processing, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structure, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases and syndromes. 1.1. Structural features The primary structures of the NMDA NR2 subunits were revealed for both mouse (Kutsuwada et al., 1992) and rat (Monyer et al., 1992) using independent cloning strategies. The predicted amino acid sequences for the e2 (mouse
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terminology) and NR2B subunits show a sequence of 45 amino acids, specifically residues 1362– 1406, which differ between the mouse and rat homologs (McBain & Mayer, 1994). The four NR2 subunits share considerable homology. For example, the predicted proteins are 55% (NR2A and NR2C) and 70% (NR2A and NR2B) identical, but are only 20% homologous to the NR1 subunit (Monyer et al., 1992). The NR2B subunit is composed of 1456 amino acids with an approximate molecular mass of 170 –180 kDa. Hydropathy analysis of amino acid sequences predicted from the cDNA for the NR2B subunit suggests the presence of an extracellular NH2-terminal signal peptide and four putative transmembrane domains (M1-M4) (Kutsuwada et al., 1992). M2 forms a reentrant loop that lines the channel. Although the NR2 subunits have the same basic structure as NR1 and other glutamate-gated ion channels (for a review, see Hollmann & Heinemann, 1994), they differ in that they possess large intracellular C-terminal domains, in excess of 600 amino acids and containing scattered regions of conserved sequences. The size of these C-termini is larger than the extracellular NH2-terminal segment preceding the first transmembrane region (Monyer et al., 1992). However, for all NMDA receptor subunits, the NH2-terminal extracellular domain is greater than twice the size of that for other receptors ( 50 kDa) (McBain & Mayer, 1994). Finally, the NR1 and NR2 subunits possess an asparagine residue (amino acid residue 589 for NR2B) in the second transmembrane region. This domain is the putative pore-forming region for the NMDA receptor subunits. Thus, the asparagine residue may play a role in the high Ca2+ permeability of the channel. 1.2. Receptor composition/stoichiometry As will be reviewed in Section 2, although relatively comprehensive, the localization data available for NR2B are not always able to differentiate between subunit expression and the presence of functional NMDA receptors. Questions about the distribution of NR2B subunit-containing ion channels are further complicated by conflicting reports regarding NMDA receptor subunit composition. Premkumar and Auerbach (1997) inferred the stoichiometry of recombinant NMDA receptor channels from single-channel current patterns recorded from mouse NR1NR2B receptors. The authors concluded that NMDA receptors are pentamers composed of three NR1 and two NR2 subunits. In contrast, Laube et al. (1998) presented evidence for a tetrameric structure of recombinant NMDA receptors. Using solubilized mouse forebrain, the following subunit distribution of NMDA receptors was observed: NR1, 17%; NR1/NR2A, 37%; NR1/NR2B, 40%; and NR1/NR2A/ NR2B, 6% (Chazot & Stephenson, 1997). Further, in rat tissue, anti-NR2B antibodies immunoprecipitate NR2A, NR2B, and NR1 subunits from the cortex and thalamus.
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NR2D is assembled with NR1, NR2A, and NR2B subunits in the rat thalamus (Dunah et al., 1998). The NR2A and NR2B subunits also appear to co-localize in other brain regions and in vitro. For example, immunohistological methods reveal that in hippocampal subfields (CA1 –CA3), the NR2A- and NR2B-immunopositive cells exhibit extensive co-localization in the stratum radiatum and oriens (Fritschy et al., 1998). In vitro studies using human embryonic kidney (HEK)-293 cells transfected with NR1 and NR2A NMDA receptor subunits in combination with FLAG- and c-Myc epitope-tagged NR2B subunits support and extend these findings by providing data regarding the number of NR2 subunits present per receptor complex. Specifically, results show co-assembly of three NR2 subunits, NR2A/NR2B FLAG /NR2B c-myc , within the same NMDA receptor (Hawkins et al., 1999). Although characterization of NMDA receptor composition continues, existing data indicate that specific subunit assembly and receptor stoichiometry is regionally and developmentally regulated.
2. Localization 2.1. During development 2.1.1. NR2B gene expression The expression of the various NMDA receptor genes is under developmental control. Expression is thus a dynamic process, and, as will be described in the following sections, often responds to changes in the internal and external environments. Studies have recently begun to characterize the promoter regions for the glutamate receptor genes to identify regulatory elements and factors that control expression in neurons (Okamoto et al., 1999). However, the transcription factors involved in the regulation of NR2B subunits are still unknown. Although the control of expression during development remains elusive, the regional distribution of the NR2B subunit is better understood. Recent evidence suggests that NMDA receptors are critical for corticogenesis, neuronal migration, and synaptogenesis during brain development. A number of investigators have examined expression densities and localization of the NMDAR2B gene at various stages of development within different brain areas, such as the cortex, hippocampus, cerebellum, and hypothalamus. A distinct pattern of mRNAs encoding NMDA receptor subunits in the developing rat brain was revealed by in situ hybridization experiments (Monyer et al., 1994). At embryonic day 14, low levels of NR2B mRNA are evident in the spinal cord and hypothalamus. However, by embryonic day 17, there is a marked increase in NR2B transcript expression, with high levels in the cerebral cortex (especially layer 1), thalamus, and spinal cord, and lower levels in the hippocampus, colliculi, and hypothalamus. At birth, further increases in NR2B mRNA expression are apparent in the cerebral
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cortex, hippocampus, septum, neostriatum, and thalamic nuclei, but low levels are in the cerebellum. By postnatal days 7 and 12, NR2B mRNA shows robust expression, with high levels in the cerebral cortex and hippocampus, and moderate levels in the neostriatum, septum, thalamic nuclei, and cerebellum. Similar transcript expression patterns are revealed in vitro. NR2B mRNA levels are relatively low in cortical cultured neurons from embryonic day 17 rats. Between days 1 and 7 in vitro, expression increases with little further change after day 7. The level of NR2B mRNA is 4-fold higher than that of NR2A mRNA in 21-day cultures (Zhong et al., 1994). Similarly, Ritter et al. (2001) found that in human fetal brains, NR2B mRNA expression is apparent and predominant in all cerebral cortical layers as early as gestational week 8. Looking at older developmental ages, Magnusson (2000) attempted to determine whether some of the age-related changes in binding to the NMDA receptor complex could be accounted for by changes in subunit expression during the aging process. In situ hybridization of the NMDA subunits NR1, NR2A, and NR2B and receptor autoradiography using the agonist glutamate and the competitive antagonist 3-(R)-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) were performed on sections from C57Bl/6 mice at three different ages (3, 10, and 30 months). mRNA for the NR2B subunit is significantly decreased between the ages of 3 and 30 months in a majority of cerebral cortical regions and in dentate gyrus granule cells. In the cerebellum, reverse transcriptase-polymerase chain reaction (RT-PCR) assays were used to measure levels of NMDA receptor subunit mRNA levels in dissociated granule cell cultures from 8-day-old neonatal rats. Primary cultures of granule cells were maintained for 4– 14 days in vitro. In situ hybridization revealed that by 4 days in vitro, NR2B mRNA is barely detectable and that after 7 days, NR2B mRNA is below detection limits (Vallano et al., 1996). In agreement with these findings, an in situ experiment of rat NMDA receptor subunit mRNA levels shows that postnatally (days 7 and 12), the NR2B and NR2C transcript expression changes in opposite directions in the cerebellar granule cell layer, with NR2B mRNA levels decreasing in abundance while NR2C levels increase (Monyer et al., 1994). Thus, granule cell maturation is associated with down-regulation of NR2B gene expression. NR2B mRNA increases significantly between birth and postnatal day 5 in both male and female rats in the preoptic area of the anterior hypothalamus (Adams et al., 1999). Recently, in addition to localization within the brain, some NMDA receptor subunits have also been described in adult non-neuronal tissue and keratinocytes. However, their developmental expression patterns are not as well characterized. With the use of RT-PCR, the expression of the NR2B subunit was investigated in the developing rat heart. NR2B mRNA is present in the heart tissue of rats from embryonic day 14 until postnatal day 21, but disappears 10 weeks after birth. In contrast, no NR1, GluR2, or PSD-95
could be detected in the rat heart at any developmental stage (Seeber et al., 2000). Finally, mammalian osteoclasts and osteoblasts express several NMDA receptor subunit mRNAs, including NR2B, suggesting that NMDA receptors may play a role in bone cell differentiation and function (Itzstein et al., 2001). 2.1.2. NR2B protein expression NR2B protein expression, like gene expression, is developmentally regulated. Further, the regional distribution of protein expression is generally in close agreement with studies investigating gene expression. Laurie et al. (1997) used immunoblotting of whole, neonatal rat brains and found that NR2B is strongly expressed at birth (unlike the NR2A subunit) and further increases until postnatal day 6 to day 20 before declining slightly (especially in the cerebellum) to the adult level. Consistent with these findings, Wang et al. (1995) examined the ontogenic profiles of the NR2A and NR2B subunits in the rat cerebellum. At postnatal day 2, the NR2A levels are undetectable, whereas the NR2B subunit is abundant. By postnatal day 12, the expression of NR2A increases and reaches adult levels by postnatal day 22, while NR2B expression begins to decline at postnatal day 12 and is no longer apparent 22 days after birth. In contrast, Misra et al. (2000) investigated functional properties of NMDA receptor cerebellar Golgi cells using patchclamp recordings in slices from postnatal day 14 rats. Electrophysiological results reveal that ifenprodil (NR2Bselective antagonist; see Table 1) reduces whole cell NMDA-evoked currents by 80%. In addition, a population of high-conductance NMDA receptor-containing cells was detected and found to be inhibited by ifenprodil. Thus, it was concluded that in cerebellar Golgi cells, the highconductance NMDA receptor channels arise from NR2Bcontaining receptors. Interestingly, the authors found no evidence of NR2A-containing receptors in these cells. Fractionation of cortical homogenates from rat embryos reveals that the NR2B subunit is highly enriched in axonal growth cones (Wang et al., 1995). In the hippocampus, a similar distribution was found by microscopy of immature hippocampal neurons, showing preferential accumulation of NR2B in axonal growth cones and varicosities. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role for NMDA receptors in the regulation of neuronal growth and migration (Herkert et al., 1998). Sheng et al. (1994) used co-immunoprecipitation with NMDA receptor subunit-specific antibodies, and observed the composition of heteromeric receptors during development of the rat cerebral cortex. NR1 splice variantspecific antibodies for the C1- and N1-regions of the NR1 subunit co-immunoprecipitated NR2B subunits from cortical membranes throughout the period tested (postnatal days 1 – 53), although the amount of co-immunoprecipitated NR2B was consistently smaller for NR1-N1- than for C1specific antibodies. Differential co-immunoprecipitation of
J.M. Loftis, A. Janowsky / Pharmacology & Therapeutics 97 (2003) 55–85 Table 1 Pharmacological profile Drug
Action
Reference
Ifenprodil
NMDA receptor NR2Bselective antagonist; inhibits channel opening; increases NMDA receptor affinity for glutamate-site agonists NR2B-selective antagonist; binds to ifenprodil site NR2B-selective antagonist; binds to ifenprodil site NR2B-selective antagonist; binds to ifenprodil site Glycine site antagonist NR2B-selective antagonist; binds to ifenprodil site
Chenard & Menniti, 1999; Grimwood et al., 2000; Coughenour & Barr, 2001; Zhang & Shi, 2001
Eliprodil CP-101,606 CP-283,097 CGP 61594 Ro 25-6981
Con-R
Con-G
N-(phenylalkyl) cinnamides Felbamate
Grimwood et al., 2000; Pabel et al., 2000 Grimwood et al., 2000; Chazot, 2000 Grimwood et al., 2000 Honer et al., 1998 Grimwood et al., 2000; Mutel et al., 1998; Fischer et al., 1997; Lynch et al., 2001 White et al., 2000
NMDA receptor peptide antagonist; NR2B selective NMDA receptor peptide Donevan & McCabe, 2000 antagonist; NR2B selective NR2B-selective antagonist Tamiz et al., 1999
NR2B-selective antagonist Kleckner et al., 1999; Harty & Rogawski, 2000 Homoquinolinate NR2B-selective ligand Brown et al., 1998 Ro G3-1908 NR2B-selective antagonist Wyss et al., 2000 CGP 39653 NR2B-selective Christie et al., 2000 competitive antagonist MK-801 Channel-site ligand; Laurie & Seeburg, 1994 non-competitive NMDA receptor antagonist Drugs listed act at NR2B-containing NMDA receptors with varying degrees of selectivity and potency. Refer to referenced articles for specific information regarding binding affinities and mechanisms of action.
NR2A and NR2B subunits by N1- and C1-containing splice variants suggests that there must also be some segregation of NR2A and NR2B. These postnatal NR2 subunit- and NR1 splice variant-specific interactions could contribute to NMDA receptor variation and changing synaptic plasticity during cortical development. In support of their work investigating NR2B gene expression, Seeber et al. (2000) used western blot analysis to investigate the expression of the NR2B subunit in the developing rat heart. The NR2B protein is detected in heart tissue of rats from embryonic day 14 until postnatal day 21, but disappears 10 weeks after birth. As with gene expression, the NR1 and GluR2 subunits and PSD-95 protein could not be detected in the rat heart at any developmental stage. Further, no functional NMDA receptors could be detected on cardiac myocytes by whole cell recordings. In conclusion, the NR2B subunit is abundant in the developing rat heart, but its function remains unknown.
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2.1.3. Associated proteins In Section 4.1.1 we review the effects of associated PSD proteins on the regulation of the NR2B subunit. A brief discussion of the ontogeny of such proteins is included below, as it contributes to understanding the developmental and functional significance of NR2B subunits. The developing mouse brain was examined for the expression of PSD-95 and synapse-associated protein (SAP)102, which are two PDZ (PSD-95, Discs-large, ZO1) domain-containing proteins that associate with NMDA receptors (see Section 4.1; Fig. 1). Using in situ hybridization with antisense oligonucleotide probes, PSD-95 and SAP102 mRNA were shown to be expressed at embryonic day 13 in the mantle zone of various brain regions, where NR2B mRNA is also expressed at high levels. In the early postnatal period, the mRNA for both PSD-95 and SAP102 is elevated and concentrated in the telencephalon and cerebellar granular layer, where NR2A and NR2B mRNA are abundantly expressed (Fukaya et al., 1999). Thus, the regional and temporal expression profile in the brain suggests that members of the PSD-95/SAP90 protein family can interact with NMDA receptor subunits to facilitate the formation of ion channel complexes, during and after synaptogenesis. To more specifically characterize the relationship of NR2B and cytoskeletal proteins during development, Sans et al. (2000) investigated the developmental changes in expression of PSD-93, PSD-95, and SAP102 in the rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2, SAP102 is highly expressed, whereas the abundance of PSD-93 and PSD-95 is low. Interestingly, SAP102 expression increases during the first week after birth when active synaptogenesis putatively takes place in the hippocampus. SAP102 protein expression remains stable through postnatal day 35, but shows reduced expression by 6 months of age. From immunoblots of the hippocampus and immunogold analysis of CA1 synapses, the high level of NR2B expression at postnatal day 2 appears to coincide with the high level of synaptic SAP102 expression. To determine whether the developmental changes in PSD-93/PSD-95 and SAP102 reflect a preferred association with NR2A and NR2B subunits, respectively, Sans et al. (2000) measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2B/ SAP102 and NR2A/PSD-93/-95. Thus, individual cytoskeletal proteins form specific interactions with the NMDA receptor subunits and participate in functions that are critical to synapse development. 2.2. In adult tissue 2.2.1. NR2B gene expression Similar to observations made during animal development, the expression in adults of individual mRNAs for the NMDA receptor subunits overlap in some brain regions, but are specialized in many others. In particular, NR2B
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Fig. 1. Schematic representation of NR2B-containing NMDA receptor complex and associated neuronal elements. Blue proteins represent PSD-95 and related family members, other structural proteins are stippled, and proteins with less well-understood function are shown in white. Enzymes such as kinases and phosphatases are depicted in yellow. Please refer to Section 4.1 for information regarding specific protein-protein interactions and putative functions.
subunits display a unique regional and cell-specific expression profile. Early reports from Ishii et al. (1993) used northern blotting and in situ hybridization analyses to show that NR2A and NR2B are abundant in the cortex and hippocampus, NR2C is in the cerebellum, and NR2D is located in subcortical areas. More recently, select regions from the rat CNS were examined for the presence of mRNA coding for NR2B using a nuclease protection assay. The rank order of NR2B expression is as follows: cortex > olfactory bulbs > hippocampus > striatum > superior colliculus > inferior colliculus > brain stem > retina cerebellum > hypothalamus (Goebel & Poosch, 1999). Sun et al.
(2000) expanded on these findings and provided a more detailed characterization of NR2B subunit cortical expression. Quantitative RT-PCR was used to analyze the relative expressions of the NR1, NR2A, NR2B, NR2C, NR2D, and NR3 subunits of the NMDA receptor in the piriform, entorhinal, visual, and motor cortices, as well as in the olfactory bulb of adult rat. The analysis detected clear differences in the relative proportions of the NMDA receptor subunits among the brain areas examined. These regional differences are particularly prominent when the piriform and motor cortices are contrasted. Specifically, the NR2B subunit transcript is prevalent and highly expressed in the motor
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cortical region ( 135% of NR1 message), while NR2B is expressed at lower levels ( 30% of NR1) in the piriform cortex. 2.2.2. NR2B protein expression As with the studies examining NR2B localization during development, experiments investigating receptor subunit expression in adult animals demonstrate good agreement between gene and protein expression. Laurie et al. (1997) used immunoblotting techniques with rat, mouse, frog, rabbit, and human brains. Using an antibody that recognizes a portion of the C-terminal, NR2B subunit protein was observed in all species tested, except frog. In the rat and human, the NR2B subunit is primarily expressed in forebrain structures, such as the cortex, hippocampus, striatum, thalamus, and olfactory bulb. Moderate levels of NR2B subunit expression are evident in the midbrain, such as the hypothalamus, colliculi, and also the brain stem (rat only), and low expression occurs in the cerebellum and spinal cord. In agreement, Wang et al. (1995) report that in the adult rat, the NR2B subunit is expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Interestingly, regional subunit expression differences have been reported between rats and mice, since prominent NR2B immunoreactivity is found in Purkinje cell bodies and dendrites in the mouse cerebellum, but NR2B is low in the rat cerebellum (Thompson et al., 2000; Laurie et al., 1997). Although Laurie et al. (1997) were able to detect only moderate amounts of the NR2B protein in the hypothalamus, the NR2B subunits in the diencephalon participate in various physiological functions (see Section 3.3). Thus, to broaden their understanding of the role of the NR2B subunit in homeostatic processes, Khan et al. (2000) characterized the expression of the NR2B subunits within the diencephalon of adult male rats. In the hypothalamus, the highest levels of NR2B immunoreactivity were in the paraventricular and supraoptic nuclei. Further, intense NR2B immunoreactivity was present in the nucleus circularis, anterior fornical nucleus, and scattered clusters of lateral hypothalamic cells. NR2B immunoreactivity was also apparent within the arcuate nucleus, the median eminence, and the tuberal nucleus, and light immunostaining was visible in all other hypothalamic nuclei examined. In the thalamus, the highest amount of NR2B immunoreactivity was in the medial habenula and the anterodorsal, paraventricular, rhomboid, reticular, and dorsal lateral geniculate nuclei. Thus, the NR2B subunit appears to be widely distributed throughout the hypothalamus and thalamus, supporting its participation in a variety of regulatory functions. 2.2.3. Cellular and subcellular distribution In addition to investigations characterizing the localization of the NR2B subunits, a number of laboratories have been interested in discovering the cellular or subcellular specificity of NR2B abundance within different brain
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regions. Using in situ hybridization with tissue from rhesus and Japanese monkeys, Munoz et al. (1999) investigated the expression of NR2B in the sensory and motor cortices. NR2B mRNA expression is heterogeneous across the primary sensory cortex and area 5. Specifically, a region of low expression is present in layer I, higher expression is in layer II, continuous moderate expression is in layers II –IV, and alternating areas of low and high expression are in deep layers V and VI. In area 4, layer II displays the highest level of NR2B mRNA expression, whereas transcript expression appears low in layer I and moderate in the other layers. Although specific roles for NR2B-containing receptors in sensory and motor functioning are yet to be fully understood, these findings suggest that the NR2B subunits will likely be found to play a critical part in processing sensory and motor information. In addition to the cellular distribution of NR2B mRNA in the cerebral cortex, Jones et al. (1998) demonstrated with great detail cell-specific gene expression in the monkey thalamus. Similar NR2B mRNA expression has also been identified in brain structures potentially associated with the limbic system. In rats, large neurons close to the midline of the medial septal region express mRNA for glutamic acid decarboxylase and are almost exclusively g-aminobutyric acid (GABA)ergic, and all but one neuron express mRNA for the NR2B subunit (Plant et al., 1997). Further, Kuppenbender et al. (2000) used dual-label in situ hybridization techniques to assess the levels of NR1 and NR2A-D mRNA expressed in projection neurons and interneurons of the human striatum. The neuronal populations were identified with digoxigenin-tagged cRNA probes for preproenkephalin and substance P targeted to striatal projection neurons, and somatostatin, glutamic acid decarboxylase, and choline acetyltransferase targeted to striatal interneurons. Unlike other NMDA receptor subunits, intense NR2B mRNA hybridization signals were observed across all neurons in the striatum. These results suggest that, as with the cortex, thalamus, and septum, the striatum contains different populations of neurons that have unique NMDA receptor subunit compositions. Interestingly, NR2B mRNA was detected in the dendrites and soma of cultured hippocampal neurons, suggesting that protein synthesis may occur in neuronal processes (as opposed to nuclei), especially at or near postsynaptic sites (Miyashiro et al., 1994). With regard to NR2B subunit protein expression in hippocampal subfields, the NR2Bspecific antibodies labeled neuronal cell bodies and dendrites in all fields of Ammon’s horn, CA1, CA3, and in the dentate gyrus (Charton et al., 1999). Luo et al. (2002) generated functional green fluorescent protein (GFP)-tagged NR2A and NR2B subunits and studied the localization of NMDA receptors in transfected hippocampal neurons. GFPtagged NR2A- and NR2B-containing receptor clusters were most evident on dendritic shafts and spines. In the rat cerebral cortex, numerous neurons (all layers), in particular pyramidal-like cells of layers II/III and V, were
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immunolabeled for NR2B. Staining of cortical neurons in layers I and IV was relatively weak. Immunoreactivity extended into dendritic processes, especially the apical dendrites of pyramidal neurons (Charton et al., 1999). Similar results were found in studies investigating specific cortical regions in both non-human primates and rats. Employing immunocytochemistry with rhesus and Japanese monkeys, Munoz et al. (1999) also investigated the expression of the NR2B protein in the sensory and motor cortices. All cortical layers had positive immunoreactivity for the NR2B subunits. Specifically, layers II and III displayed a high density of small, round NR2B-immunopositive cells that also co-localized GABA, calbindin, or parvalbumin. In addition, a population of NR2B-containing medium-sized pyramidal neurons that also contain CaMKII-a was observed in layers II and III. Also apparent in layers II and III were NR2B-labeled apical dendrites of pyramidal neurons from layers III and IV. One group of investigators localized cell-specific NR2B expression in the spinal cord of rats. Ma and Hargreaves (2000) used immunohistochemical staining and retrograde tracing techniques to investigate the relationship between the NR2B subunits and small-diameter primary afferent dorsal root ganglion neurons that give rise to the sciatic nerve fibers. Results indicated that the NR2B subunits are predominantly expressed on small-diameter primary afferents. Consequently, these NR2B-containing NMDA receptors are hypothesized to modulate neurotransmitter release from primary afferent terminals. Although prominent NR2B subunit immunolabeling is present on neuronal processes, a recent report from Sinor et al. (2000) provides evidence for a slightly different subcellular distribution. The authors used haloperidol, an antagonist with high selectivity for the NR1/NR2B receptor configuration, to confirm that the NR1/NR2B receptors migrate to cell bodies in cultured rat cortical neurons. In mature cultures (more than 22 days old in vitro), NMDA receptor-mediated responses obtained from excised nucleated macropatches are significantly antagonized by haloperidol. NR1/NR2B receptors thus appear to be preferentially expressed in cell bodies of mature cortical neurons. In terms of cell surface expression, almost all NR2B subunits are reportedly found in the plasma membrane, as compared with less than one-half of the total NR1 subunits. These data suggest that neurons possess a large intracellular pool of NR1 subunits that await assembly with the NR2 subunits prior to expression at the plasma membrane (Hall & Soderling, 1997). Al Hallaq et al. (2001) used immunoblotting of rat cortical and cerebellar tissue to measure the prevalence of NMDA receptor subunits and PDZ domain-containing proteins. The NR2A and NR2B subunits, despite previous electrophysiological evidence (Rumbaugh & Vicini, 1999), are equally enriched in the PSD fraction. In addition, PSD-95 and channel-associated protein of synapses (chapsyn)-110 are both strongly expressed, but SAP102 is expressed to a lesser
extent, in the PSD. Consistent with these findings, there is evidence that reveals that while PSD-95 and chapsyn-110 are present only at postsynaptic sites, SAP102 is located at axonal sites, as well as postsynaptically (El Husseini et al., 2000). Collectively, these results illustrate the degree of not only regional, but also cellular and subcellular, expression specificity that is exhibited by the NR2B subunits.
3. Functional properties/roles None of the four NR2 subunits, NR2A-NR2D, assemble by themselves into functional channels. However, when coexpressed with the NR1 subunits, receptor activation can occur. Heteromeric combinations of the NR1 and NR2B subunits are highly permeable to Ca2+, are blocked by Zn2+ and MK-801 (dizocilpine maleate; open channel blocker) and by Mg2+ in a voltage-dependent manner, and are co-activated by glutamate and glycine (for a review, see McBain & Mayer, 1994). Although the NR2A-NR2D subunits have the same basic structure as the NR1 subunit (Hollmann & Heinemann, 1994), they differ in possessing strikingly large intracellular C-terminal domains of 627, 644, 404, and 461 amino acids, respectively (see Section 1.1). As will be discussed in subsequent sections, the C-terminal has been suggested to play a role as the target domain for modulatory or accessory proteins in promoting receptor assembly, sorting, or targeting. Similarly, this region of the subunit may contribute to different channel conformations and subsequently modulate receptor function. The structural properties and functional roles attributed to the NR2B subunit of the NMDA receptor are organized into pharmacological, electrophysiological, and behavioral sections. 3.1. Pharmacological The NR2B subunit appears to be critical for a number of the basic structural and functional attributes associated with the NMDA receptor. For example, the glutamate-binding pocket is formed by the NR2B subunit (Laube et al., 1997). The high-affinity sites for CGP61594 (glycine site antagonist) were exclusively displayed by the NR1/NR2B receptors as compared with the NR1/NR2A, NR1/NR2D, or NR1/ NR2C receptors (Honer et al., 1998). The tryptophan residue in the M2 region of the NR2 subunits controls the Mg2+ block. Specifically, an NR2B leucine (W607L) mutation abolishes the block and significantly increases access to extracellular Mg2+. In addition, NR2B subunit mutations that changed tryptophan (W607) to asparagine (W607N) or alanine (W607A) also greatly attenuated the Mg2+ block. Williams et al. (1998) thus proposed a model in which the M2 loop of NR2B (W607) is positioned at the narrow constriction, at a level similar to asparagine residues in NR2B (N616) and NR1 (N616), with these three residues forming a binding site for Mg2+.
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In addition to providing information about the structural properties associated with the NR2B subunits, pharmacological experiments have facilitated the understanding of functional attributes by developing and/or characterizing a number of useful and relatively subunit-selective compounds. For instance, Grimwood et al. (2000) performed [3H]ifenprodil-binding experiments under NMDA receptorspecific assay conditions to provide the first detailed characterization of the pharmacology of the ifenprodil site on the NMDA receptors, using recombinant human NR1/NR2B receptors stably expressed in L(tk) cells, in comparison with rat cortex/hippocampus membranes. [3H]Ifenprodil binds to a single, saturable site on both human recombinant receptors and native rat receptors. In addition, trifluoroperazine was used to isolate high-affinity [3H]ifenprodil binding to NMDA receptors in a rat membrane preparation. [3H]Ifenprodil bound to a single high-affinity site, with the pharmacology of NMDA receptors containing the NR2B subunit (Coughenour & Barr, 2001). The affinity of various ifenprodil site ligands—eliprodil, CP-101,606, CP-283,097, and Ro 25-6981—is very similar for inhibition of [3H]ifenprodil binding to recombinant human NR1/NR2B and native rat receptors. These findings are in agreement with Mutel et al. (1998), who previously characterized the in vitro binding of a new subtype-selective NMDA receptor antagonist, [3H]Ro 25-6981, to rat brain membranes and sections. The compound binds a single site on the membranes with a Kd of 3 nM and a Bmax of 1.6 pmol/mg of protein. Overall, the distribution of [3H]Ro 25-6981-binding sites correlates well with that of NR2B (but not NR2A) transcripts, as revealed by in situ hybridization. Similarly, Lynch et al. (2001) used ligand binding to characterize Ro 25-6981, and found that the compound shares pharmacological actions with ifenprodil, as well as with other modulators of NR2B-containing NMDA receptors. Binding assays performed in HEK-293 cells expressing NR1/ NR2B or NR1/NR2A/NR2B receptors suggest two types of NR2B-specific NMDA receptor antagonists: (1) Ro25,6981, which binds to NR2B-containing receptors with high affinity regardless of the NR2 subunit composition, and (2) CP-101,606, whose binding is altered by the presence of other NR2 subunits (Chazot et al., 2002). In addition to the characterization of ifenprodil and the other ligands that reportedly bind to the same site on the NR2B subunit, the purification and synthesis of conantokin (Con)-R, an NMDA receptor peptide antagonist from the venom of Conus rediatus, was performed. With the use of well-defined animal seizure models, Con-R was found to possess an anticonvulsant profile superior to that of ifenprodil and MK-801. With voltage-clamp recordings of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs, Con-R exhibited the following order of preference for the NR2 subunits: NR2B = NR2A > NR2C >> NR2D (White et al., 2000). Con-G, a 17 amino acid peptide antagonist of NMDA receptors, was isolated from the venom of the marine cone
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snail, C. geographus. Using whole cell voltage-clamp recordings from cultured neurons and two electrode voltage-clamp recordings from Xenopus oocytes expressing recombinant NMDA receptors, Con-G selectively blocked NMDA receptors containing the NR2B subunits (Donevan & McCabe, 2000). The characterization of NR2B-specific compounds also supports and contributes to information regarding the neuroanatomical distribution of receptor subunits. Pharmacological properties of NMDA receptors were determined in rat brain sections with quantitative autoradiography of [3H]CGP39653 binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed. The two populations correspond anatomically to the NR2A subunits located in the cerebellar granule cell layer and the NR2B subunits found in the medial striatum (Christie et al., 2000). Another antagonist, [3H]CPP, weakly labels NMDA receptors in regions that contain predominantly NR2B mRNA (striatum and lateral septum), and agonist-preferring NMDA receptors (defined by L-[3H]glutamate binding) are located predominantly in a subset of brain regions that contain both NR2B and NR1 mRNA. The anatomical distribution of subunit mRNAs suggests that the NR2B subunits are associated with agonist-preferring NMDA receptors. Thus, the anatomical and pharmacological evidence is consistent with the hypothesis that agonist-preferring and antagonist-preferring NMDA receptors are largely determined by the NR2B and NR2A subunits, respectively (Buller et al., 1994). In addition to their usefulness as tools to elucidate regional distribution and functional properties associated with NR2B-containing receptors, a number of subunitspecific antagonists are currently being used and/or investigated as possible therapeutic agents (reviewed in Nikam & Meltzer, 2002; see Section 5). CP-101,606, an NR2B subunit-selective antagonist, is currently under development by Pfizer (New York, NY, USA) for its potential as a neuroprotectant in head injury and neurodegenerative diseases (Chazot, 2000). The analgesic activity of CP-101,606 was examined in carrageenan-induced hyperalgesia and in capsaicin- and phorbol 12-myristate 13-acetate-induced nociceptive tests in the rat. Results suggest that inhibition of the NR2B subunit of the NMDA receptor is effective in vivo at modulating nociception and hyperalgesia responses without causing the behavioral side effects often observed with currently available NMDA receptor antagonists (Taniguchi et al., 1997). CP-101,606 has also been used to investigate the role of NR2B-containing receptors in response to NMDA receptor activation in vivo. In the mouse, CP-101,606 completely inhibits increases in Foslike immunoreactivity in the dentate gyrus caused by a subconvulsant dose of NMDA. In the rat, the compound completely blocks cortical c-fos mRNA induction following focal injury in the parietal cortex and the initiation and propagation of electrically induced cortical-spreading depression. Inhibition of these responses by CP-101,606
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indicates that c-fos induction and cortical-spreading depression are dependent on activation of NMDA receptors containing the NR2B subunits (Menniti et al., 2000). Activation of NR2B-containing receptors may thus contribute to the ability of the neuron to alter its synapses via regulation of immediate early gene expression. A novel series of N-(phenylalkyl)cinnamides was synthesized recently and tested for antagonism at NMDA receptor subtypes. Electrical recordings in Xenopus oocytes expressing three different configurations of cloned rat NMDA receptor subunits (NR1 expressed in combination with either NR2A, NR2B, or NR2C) were used to assay the potency and subunit selectivity of the compounds. Results indicated that the N-(phenylalkyl)cinnamides are selective antagonists of the NR1/NR2B receptors (Tamiz et al., 1999). Thus, CP-101,606 and the N-(phenylalkyl)cinnamide series described provide new and structurally diverse models for designing NR2B-selective NMDA antagonists as potential pharmacotherapeutics. Felbamate has been used in the treatment of seizure disorders. Recombinant receptors expressed in Xenopus oocytes were used to investigate the subtype specificity and mechanisms of action of this drug. Felbamate reduces NMDA- and glycine-induced currents most effectively at NMDA receptors composed of NR1 and NR2B subunits. Further, felbamate enhances the affinity of the NR1/NR2B receptors for the agonist NMDA by 3.5-fold, suggesting a similarity in mechanism to other antagonists (Kleckner et al., 1999). Although the potential mechanism of action may be similar to that of other noncompetitive antagonists such as ifenprodil, the toxicity profile appears to be somewhat improved. Specifically, felbamate blocks NMDA receptors, but fails to exhibit the toxicity associated with other NMDA receptor antagonists. To investigate the possibility that the favorable toxicity profile of felbamate could be related to NMDA receptor subtype selectivity, Harty and Rogawski (2000) examined the specificity of felbamate block of recombinant NMDA receptors composed of the NR1 subunit and various NR2 subunits. Findings suggest that more rapid association and slower dissociation accounts for the higher affinity of felbamate block of NMDA receptors containing the NR2B subunit (data indicate more than one blocking site on the NMDA receptor channel complex). These authors concluded that felbamate exhibits modest selectivity for NMDA receptors composed of NR1/NR2B subunits. This selectivity could, in part, account for the improved clinical profile of felbamate in comparison with NMDA receptor antagonists that do not show subunit selectivity. 3.2. Electrophysiological Studies of NMDA receptor kinetics and related electrophysiological properties reveal that the mechanisms of ion channel activation and inactivation are extremely complex (for a review, see McBain & Mayer, 1994). Thus, this
section focuses on research that illuminates a unique role for NR2B-containing receptors in excitatory synaptic transmission. NR2B subunits dominate over other NR2 subunits in determining the functional properties of NMDA receptors in certain cell types [GABAergic medial septal neurons (Plant et al., 1997)]. These authors studied functional and molecular properties of septal neurons of the rat forebrain using patch clamp, fluorometric Ca2+ measurements, and single-cell RT-PCR. However, using transfected HEK-293 cells and whole cell patch-clamp recordings, NR1/NR2Amediated peak current densities are 4 times larger than NR1/NR2B. Further, peak channel open probability is significantly higher for NR1/NR2A than for NR1/NR2B, using two different open channel antagonists, MK801 and 9-aminoacridine. Results suggest that expression levels of NR2A and NR2B can regulate peak amplitude of NMDA receptor-mediated excitatory postsynaptic potentials and, therefore, play a role in mechanisms underlying synaptic plasticity (Chen et al., 1999a). Accompanying changes in channel activation, subunitspecific differences are similarly observed for the transition from the open to the closed state (i.e., deactivation kinetics). Heteromeric HEK cells expressing NMDA receptors were investigated using whole cell recordings. NR1/NR2B showed no significant inactivation with application of extracellular Ca2+. Ca2+- and glycine-independent desensitization was less pronounced in NR1/NR2B receptors as compared with NR1/NR2A receptors (Krupp et al., 1996). Further, NMDA receptors transiently transfected into HEK293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Recovery from desensitization was slower for NR1/NR2B than for NR1/ NR2A channels (Vicini et al., 1998). Taken together, these results suggest that NR2B-containing NMDA receptors desensitize less and take longer to recover than NR2Acontaining receptors. Besides cell expression systems, the use of genetically altered mouse models has also provided important information about the role of the NR2B subunit in modulating electrophysiological properties of NMDA receptors. Overexpression of NR2B in the forebrains of transgenic mice leads to enhanced activation of NMDA receptors, facilitating synaptic potentiation in response to stimulation at 10– 100 Hz (Tang et al., 1999). In contrast to overexpression, Tovar et al. (2000) studied fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the NR2B subunit. Neurons from NR2B knockout (KO) mice expressed an NMDA receptor-mediated excitatory postsynaptic current (EPSC) that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg2+ or the reversible antagonist DL-2amino-5-phosphonovaleric acid (AP-5). Whole cell currents from mouse KO neurons were also more sensitive to block by low concentrations of Zn2+ and much less sensitive to the
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NR2B-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile are consistent with the expression of NR1/NR2A heteromeric receptors in excitatory hippocampal neurons from mice lacking the NR2B subunit. Thus, NR2A can substitute and potentially provide some compensation for the absence of the NR2B subunit at synapses. Electrophysiological tests, in combination with the NR2B-selective antagonist ifenprodil and other antagonists, were used to demonstrate functional properties of the receptor. In a rat model of inflammatory pain, the effect of in vivo NMDA receptor antagonism on responses of spinal dorsal horn neurons to iontophoretic NMDA in anesthetized rats was investigated. Similar assessments were also performed using the C-fiber-evoked wind-up model. The windup phenomenon occurs under conditions of severe and persistent injury, such that C-fibers fire repetitively and, consequently, the response of dorsal horn neurons increases. Importantly, wind-up is dependent on the release of glutamate from C-fibers and the subsequent activation of postsynaptic NMDA receptor-gated ion channels. It was found that all three antagonists dose-dependently increase nociceptive thresholds in the Randall-Selitto model. Further, antinociceptive doses of the channel blockers (MK-801 and memantine) selectively antagonize NMDA responses of dorsal horn neurons, as well as inhibit C-fiber-evoked wind-up. In contrast, antinociceptive doses of ifenprodil do not exhibit NMDA antagonism in electrophysiological tests. Although ifenprodil does not inhibit single motor unit responses to noxious stimuli in rats with transected spinal cords, it markedly and dose-dependently inhibits nociceptive single motor unit responses in sham-operated rats. It was concluded that these results suggest that the spinal cord is not the principal site of antinociceptive action of ifenprodil, but that supraspinal structures are likely to mediate this effect (Chizh et al., 2001b). 3.3. Behavioral Behavioral assays, often in combination with pharmacological or genetic manipulations, have demonstrated a role for the NR2B subunit in mediating feeding and related physiological functions, learning, memory, pain, and synaptic plasticity. Kutsuwada et al. (1996) investigated the physiological significance of the NMDA receptor diversity and created NR2B subunit-defective mice. These mice show no suckling response and die shortly after birth. Similarly, mice expressing the NR2B subunit in a C-terminally truncated form die perinatally. This is the most severe phenotype compared with other KO mice (NR2A and NR2C). The lethal phenotype of NR2B-altered mice is speculated to result from an impairment in intracellular signaling due to the missing intracellular receptor domain (Sprengel et al., 1998). In Sprague-Dawley rats, the NR2A/B-selective antagonist ifenprodil attenuates NMDA-elicited feeding. Lat-
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eral hypothalamic NMDA receptors, some of which contribute to feeding control, are composed of NR2A and/ or NR2B subunits, suggesting that NR2A- and/or NR2Blinked signal transduction is involved in the feeding behavior (Khan et al., 1999; see also Stanley et al., 1996, 1997). Other reports also document hypothalamic NR2B-containing receptor participation in the control of circadian rhythms and the activity of magnocellular neuroendocrine cells (for reviews, see Brann, 1995; Ebling, 1996; Rea, 1998). Mice overexpressing the NR2B subunits exhibit superior ability in various tests of learning and memory (novel object recognition task, fear conditioning, fear extinction, and water maze). Tang et al. (1999) suggested that NR2B is critical in gating the age-dependent threshold for neuronal plasticity and memory formation. In contrast to mice overexpressing the NR2B subunit, a different, yet consistent, phenotype is observed in mice with deficient levels of NR2B. The lack of the NR2B subunit impairs the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brain stem. Further, in the hippocampi of these null-mutant mice, synaptic NMDA receptor responses and long-term depression (LTD) are absent (Kutsuwada et al., 1996). Taken together, these results suggest that NR2B plays an essential role in learning, memory, and neuronal pattern formation. Interestingly, Rosenblum et al. (1997) used a conditioned taste aversion (CTA) paradigm to investigate the role of NR2B in both feeding and learning. Blockade of NMDA receptors with the reversible antagonist AP-5 during training impaired CTA memory. When rats taste an unfamiliar flavor and, hence, learn about it either incidentally or in the context of CTA training, the tyrosine phosphorylation of NR2B in the insular cortex is increased. The level of tyrosine phosphorylation of NR2B appears to be a function of the novelty and quantity of the substance consumed. Thus, NR2Bcontaining receptors are involved in taste learning in the insular cortex, and tyrosine phosphorylation of NR2B subserves encoding the saliency shortly after an unfamiliar flavor is tasted. The NR2B subunit has also been implicated in pain perception (see Section 5.10). Wei et al. (2001) studied the effects of forebrain-targeted overexpression of NR2B on the response of mice to peripheral tissue injury. Transgenic mice overexpressing NR2B in the anterior cingulate and insular cortices exhibit enhanced responsiveness to hind paw injection of inflammatory stimuli. Thus, these results provide further support for the development and use of NR2B-selective compounds for the management of pain.
4. Regulation 4.1. Endogenous regulators The NMDA receptor is modulated by a variety of ions, amino acid neurotransmitters, and second messengers [e.g.,
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Mg2+, Ca2+, glycine, Zn2+, H+, and nitric oxide (NO)] (McBain & Mayer, 1994). NMDA receptors are also regulated (often in a subunit- and splice variant-dependent manner) by components such as PSD-anchoring proteins, enzymes, and growth factors. The potential molecular mechanisms for the regulation of NR2B expression, distribution, and function involve, but are not limited to, (1) changes in the assembly of specific NR1 splice variants and/ or other NR2 subunits with NR2B; (2) post-translational modifications such as phosphorylation; and (3) alterations in the interaction of the NR2B subunit with associated proteins, such as PSD-95 or SAP102. The following section reviews the regulation of NR2B by endogenous proteins. Given the breadth of the topic, the discussion focuses on the role of neuronal elements associated with the PSD, including cytoskeletal proteins, enzymes, and neuromodulators. 4.1.1. Cytoskeletal proteins As described in Section 3, the NR2B subunit plays an important role in synaptic plasticity, neural development, and other physiological functions. Although much has been learned in recent years about the neurochemical and biological properties of glutamate receptors, understanding the molecular mechanisms of receptor targeting, clustering, and anchoring at the synapse is only now beginning to develop. Recent findings concerning the discovery of novel proteins potentially involved in the regulation of NR2B via its specific synaptic protein interactions are reviewed below. Kornau et al. (1995) used a yeast 2-hybrid system to show that the cytoplasmic tails of NMDA receptor subunits interact with the 95-kDa PSD protein PSD-95. PSD-95 is a component of the PSD at excitatory synapses. It contains three PDZ domains, a Src homology (SH)3 domain, and a guanylate kinase (GK) domain, and has been characterized as a putative structural or scaffolding protein. The PDZ domain appears to be important for the localization of select NMDA receptor subunits. The second PDZ domain in PSD95 binds to the C-terminal containing the tSXV (t, terminal; S, serine; X, any amino acid; V, valine) motif common to the NR2 subunits, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. Recent reports indicate that each of the last five residues of the NR2B subunit is critical for binding to the PDZ domains of PSD95 and its related protein family member SAP102 (see below) (Lim et al., 2002). In support of its role as a potential scaffolding protein for NR2B, PSD-95 also binds to the cytoplasmic C-terminal of neuroligins in the mouse forebrain (neuroligins are neuronal cell adhesion molecules that interact with ß-neurexins and form intercellular junctions). In contrast to NR2B, neuroligins interact with the third PDZ domain of PSD-95 (Irie et al., 1997). Thus, PSD-95 not only localizes NR2B at synaptic sites, but also helps to form the architecture of a multi-protein signaling complex (Fig. 1). To further characterize the interactions of NMDA receptor subunits with PSD proteins, Niethammer et al. (1996) used a yeast 2-hybrid system to screen a rat brain cDNA
library. In vitro binding assays identified an interaction between NR2A and NR2B and three distinct members of the PSD-95/SAP90 family [SAP97/hdlg, PSD-95, and Clone 5 (Kim et al., 1995)]. In partial agreement with the findings of Kornau et al. (1995), the interactions appear to be mediated via the binding of C-terminal NMDA receptor subunits to the first two PDZ domains of PSD-95. A related cytoskeletal protein, chapsyn-110, was also found using a yeast 2-hybrid system. It was discovered to be a novel member of the PSD-95 subfamily of membrane-associated GKs (MAGUKs) that binds to the C-terminal of Shaker K+ channel subunits (Kim et al., 1995), as well as to NR2 subunits (Niethammer et al., 1996). Chapsyn-110 is 70– 80% identical to PSD-95 and SAP97, and shows identical domain organization with three PDZ domains in the Nterminal, a GK homology region in the C-terminal, and an intervening SH3 domain. When expressed in Cos-7 cells, Chapsyn-110 can form heteromultimers with PSD-95, but not with SAP97, and clusters NR2A and NR2B, but not NR1 (Kim et al., 1996). Using similar methodology, CASK, a novel PSD-95 homolog with an N-terminal CaM-dependent protein kinase domain, was identified by interaction with neurexins using a yeast 2-hybrid screen. The C-terminal region is similar to the intracellular junction proteins dlg-A, PSD-95/SAP90, SAP97, ZO1, and ZO2 that contain Drosophila hormone receptor, SH3, and GK domains. Given its subcellular location and binding capacities, it is hypothesized to function as a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins (Hata et al., 1996). More recently, a different function for CASK was revealed. NR2B-containing vesicles are transported by interactions within a large protein complex, including microtubule elements, CASK, and NR2B (Setou et al., 2000). Finally, Muller et al. (1996) described a novel 102kDa SAP, SAP102, detected in dendritic shafts and spines of asymmetric synapses. Like PSD-95 and related proteins, it has three PDZ domains and an SH3 and GK domain. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. Masuko et al. (1999) provided further characterization of SAP102. These authors identified the CaM-binding site on SAP102 (located near the SH3 domain of SAP102) and demonstrated that binding of CaM to SAP102 does not alter the interaction between SAP102 and NR2B. Further, SAP102 interacts with PSD-95 only in the presence of Ca2+ and CaM, thus revealing the potential for differential regulation of MAGUKs at excitatory synapses. Some evidence suggests that although PSD-95 and related scaffolding proteins appear to co-localize with NR2B subunits and contribute to the stability of NMDA receptors at the PSD, NR2B may not be required for targeting the receptor to the membrane. Tovar et al. (2000) examined whether NMDA receptor recruitment to the postsynaptic membrane was dependent on the NR2B subunit by isolating hippocampal neurons from NR2B-deficient mice
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and studying them in culture. Results indicated that NR2B is not required for targeting of NMDA receptors to the postsynaptic membrane. Nevertheless, in addition to the putative clustering of NR2B with the PSD-95 family of proteins, the synaptic association with such cytoskeletal elements may also regulate the expression and function of NR2B-containing receptors. For example, co-expression of PSD-95 with NR1/NR2A results in a decreased sensitivity to L-glutamate and an enhanced expression of NR2A and NR2B subunits. Importantly, deletion studies show that this effect is mediated via interaction of the C-terminal ESDV (E, glutamic acid; S, serine; D, aspartic acid; V, valine) motif [previously described as the tSXV motif (Kornau et al., 1995)] of the NR2 subunit with PSD-95 (Rutter & Stephenson, 2000). Further, Roche et al. (2001) demonstrated that PSD-95 inhibits NR2B-mediated internalization and that deletion of the PDZ-binding domain of NR2B increases internalization in cultured hippocampal neurons. Along with the PSD-95 family of cytoskeletal proteins, a number of associated neuronal elements recently have been identified and shown to play a role in synaptic regulation. A brief review of the proteins potentially involved in the distribution or function of NR2B containing receptors is provided below. The list of proteins is not intended to be comprehensive, as new proteins and interactions are continually being reported (Husi et al., 2000). Brain spectrin, a protein that links neuronal elements to the cytoskeleton, was investigated and found to interact with the C-terminal of NR2B subunits (Wechsler & Teichberg, 1998). Further characterization by these authors revealed that spectrin binds NR2B at sites that are different from those of PSD95. Interestingly, spectrin-NR2B interactions are antagonized by Ca2+ and Fyn-mediated phosphorylation, but not by CaM or CaMKII-mediated phosphorylation, thus demonstrating the regulatory specificity associated with the PSD. In agreement with these results illustrating the connection of NR2B with structural proteins, van Rossum et al. (1999) used affinity chromatography with glutathione Stransferase (GST)-NR2B-C-terminal fusion proteins to identify novel binding partners for this subunit. It was found that the NR2B C-terminus binds tubulin. In tubulin polymerization assays, the NR1 and NR2B C-termini significantly decrease the rate of microtubule formation without destabilizing preformed microtubules. Thus, via its interaction with such scaffolding proteins, NR2B may not only be regulated by, but also help control, the formation of cytoskeletal components. Using a yeast 2-hybrid screen, Takeuchi et al. (1997) isolated a novel protein family consisting of at least four members that specifically interact with PSD-95/SAP90 and its related proteins through the GK domain. These proteins were named SAPAPs (SAP90/PSD-95-associated proteins). SAPAPs are specifically expressed in neuronal cells and are concentrated in the PSD fraction. SAPAPs induce the enrichment of PSD-95 in the plasma membrane in transfected cells. Co-expression of PSD-95/SAP90 causes the
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translocation PSD-95 from the cytosol to the plasma membrane where SAPAPs are located. Using yeast 2-hybrid screening for SAPAP-interacting molecules, Hirao et al. (1998) identified a novel protein that has an inverse structure of MAGUKs with an N-terminal GK-like domain followed by five PDZ domains. It binds SAPAP through the GK-like domain and NMDA receptors and neuroligins through PDZ domains. The protein is named S-SCAM (synaptic scaffolding molecule) because S-SCAM is hypothesized to assemble receptors and cell adhesion proteins at synaptic junctions. CRIPT, a novel protein that selectively binds to the third PDZ domain of PSD-95 via its C-terminal, was similarly characterized. In heterologously expressing cells, CRIPT causes a redistribution of PSD-95 to microtubules. In the brain, CRIPT co-localizes with PSD-95 and can be coimmunoprecipitated with PSD-95 and tubulin. CRIPT may regulate the PSD-95 interaction with the tubulin-based cytoskeleton in excitatory synapses (Niethammer et al., 1998). Another putative regulator protein, cypin, a cytosolic regulator of PSD-95 postsynaptic targeting, was identified. Affinity chromatography revealed cypin to be a major PSD95 binding protein in brain extracts. Cypin is expressed in neurons, and overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD95 and SAP102 (Firestein et al., 1999), and as a consequence, may be involved in regulating the localization of NR2B to synaptic sites. Naisbitt et al. (1999) reported a novel family of PSD proteins termed Shank that are specifically enriched in the PSD of excitatory synapses. Shank contains multiple putative protein interaction domains, including an SH3 domain, a PDZ domain, the proline-rich domain, and an SAM domain. The PDZ domain of Shank mediates binding to the C-terminal of the GK-associated protein (GKAP) (see Section 4.1.2.), and this interaction is important in neurons for the synaptic localization of Shank. GKAP binds Shank and recruits Shank to PSD-95 clusters in heterologous cells. All three proteins also associate in vivo, as demonstrated using GST pull-down methods. Opportunities for such linkages appear to be numerous and are continually being discovered. Kurschner et al. (1998) identified CIPP (channel-interacting PDZ domain protein) that is expressed only in the brain and kidneys. CIPP contains four PDZ domains that could potentially interact with different PSD proteins, providing a mechanism for the simultaneous association of multiple proteins with CIPP. NR2B associates with the second and third PDZ domains of CIPP, but the functional consequences for NR2B regulation are unknown. Further increasing the list of cytoskeletal proteins, and thus adding to the complexity of NR2B regulation, is the protein neurabin (neural tissue-specific F-actin binding protein). Neurabin was cloned from rat brain cDNA. It has one F-actin binding domain at the N-terminal region and one PDZ-like domain in the middle region. Neurabin is believed to be involved in neurite formation and it is highly
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concentrated in the synapses of developed neurons. It was also enriched in the lamellipodia of growth cones during neuronal development (Nakanishi et al., 1997). The last structural protein to be discussed that is potentially involved in the regulation of NR2B subunits is densin-180. This protein was purified and found to be highly concentrated at synapses along dendrites. Densin-180 message is abundant in the forebrain. It contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. It was proposed as an adhesion molecule between pre- and postsynaptic membranes at glutamatergic synapses (Apperson et al., 1996). Further characterization of densin-180 indicates that the protein binds CaMKII, and suggests that densin-180 plays a role in the association of CaMKII with PSDs (Strack et al., 2000b). Thus, NR2B may serve to anchor and distribute NMDA receptors at synaptic sites through its interaction with the PSD (Fig. 1). Specific interactions of NR2B with PDZ domain-containing proteins is important not only for determining the localization of the receptor to the PSD, but also for regulating synaptic signaling events. 4.1.2. Enzymes (e.g., phosphatases, kinases, and proteases) Many enzymes critical for signal transduction at excitatory synapses are linked to NR2B subunits either directly or via their interactions with the cytoskeletal proteins previously described. Outlined below is information that illuminates how a number of kinases, phosphatases, and other enzymes are involved in second messenger signaling and receptor regulation. Smart (1997) reviewed experiments that demonstrate how neurotransmitter-induced activation of serine/threonine, tyrosine, and other kinases can result in the modulation of glutamate receptors. However, studies described in this section specifically emphasize the role of such enzymes in the regulation of NR2B subunits. NR2B is the most prominently tyrosine phosphorylated protein in the PSD fraction, based upon recognition by an anti-tyrosine antibody. NR2B-containing glutamate receptors may be regulated by tyrosine phosphorylation or may participate in signaling through tyrosine phosphorylation (Moon et al., 1994). In agreement with these findings, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrated that high levels of phosphotyrosine are co-localized with glutamate receptors at excitatory synapses on cultured hippocampal neurons. Using subunitspecific antibodies and rat synaptic plasma membranes, Lau and Huganir (1995) showed that NR2A and NR2B, but not NR1, are tyrosine phosphorylated. It is estimated that 3.6 ± 2.4% of NR2B and 2.1 ± 1.3% of NR2A are phosphorylated in vivo. In another review, Sala and Sheng (1999) discussed which protein tyrosine kinases phosphorylate the NR2 subunits. They explored questions regarding how this kinase-substrate specificity is determined, what the physiological significance of tyrosine phosphorylation of the NR2
subunits is, and how it is regulated. Given that Fyn, a protein tyrosine kinase, binds to the third PDZ domain of PSD-95 via its SH2 domain, they provided a model for Fyn association with the NMDA receptor/PSD-95 complex. Hisatsune et al. (1999) examined the role of Fyn and the possibility that phosphotyrosines on NR2B subunits contribute to intracellular signaling events, and demonstrated that Fyn phosphorylates NR2B at a site in NR2B that interacts with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Both the level of tyrosine phosphorylation of NR2B and the amount of the p85 subunit bound to NR2B are decreased in Fyn-deficient mice. Tyrosine phosphorylation of NR2B is important not only for the regulation of its channel activity, but also for intracellular signaling mediated through the interaction of the NMDA receptors with SH2 domain-containing molecules. Similarly, using a different KO mouse model, Manabe et al. (2000) showed that in the H-ras (one of three mammalian proto-oncogenes) null mutant hippocampus, tyrosine phosphorylation of NR2B is increased and NMDA receptor responses are subsequently enhanced. Phosphopeptide mapping of the C-terminal tail of the NR2B subunit in vitro revealed that 7 out of 25 tyrosine residues in the C-terminal region are phosphorylated. When co-expressed with Fyn in HEK-293 cells, of the 7 residues, Tyr1252, -1336, and -1472 are phosphorylated and Tyr1472 appears to be the major phosphorylation site (Nakazawa et al., 2001). It has thus been established that the NR2B subunit is phosphorylated on tyrosine residues and that this event can contribute to the potentiation of receptor responses and the transduction of specific intracellular signals. To further assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, Gurd and Bissoon (1997) investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C (PLC)-g. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, an important early event in many cell signaling pathways. PLC-g contains two SH2 domains that bind to specific phosphotyrosine residues of receptor tyrosine kinases, leading to phosphorylation by the catalytic domain of the receptor. Phosphorylation of PLC-g contributes to its activation. The authors found that NR2A and NR2B can bind to the SH2 domains of PLC-g, and they confirmed that isolated synaptic junctions contain endogenous protein tyrosine kinases that can phosphorylate both the NR2A and NR2B receptor subunits. Thus, interaction of the tyrosine-phosphorylated NR2B subunits with proteins that contain SH2 domains serves to link NR2B to signaling pathways in the postsynaptic cell (Gurd & Bissoon, 1997). Perturbations of the system further demonstrate how tyrosine phosphorylation may alter NR2B and subsequent receptor function. For example, using rat hippocampal slices and immunoprecipitation, insulin was shown to cause a
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transient phosphorylation of the NR2A and NR2B subunits on tyrosine residues. The functional consequence of insulinstimulated tyrosine phosphorylation is currently unknown (see Section 5.7), although Christie et al. (1999) speculate that it could help to potentiate the activity of the receptor. In addition to tyrosine phosphorylation, phosphorylation of serine residues by protein kinase A (PKA), protein kinase C (PKC), and CaMKII also modulates receptor function. For example, in primary hippocampal cultures, phosphorylation of NR2A/NR2B at serine residues, but not at threonine or tyrosine residues, was detected. Further, metabolic labeling with 32P reveals that NR2 subunits are highly phosphorylated under basal conditions and exhibit (more) modest increases in response to stimulation (as compared with NR1) (Hall & Soderling, 1997). The regulation by specific kinases can occur, not only directly by phosphorylation of the NR2B subunit, but also via its association with other NMDA receptor subunits or cytoskeletal proteins (see Section 4.1.1). PKA and various isoforms of PKC phosphorylate the NMDA receptor in vitro. Immunoprecipitation experiments show that all three NMDA receptor subunits (NR1, NR2A, NR2B) are substrates for PKA, as well as PKC (Leonard & Hell, 1997). PKC can affect NR1/NR2B receptor currents by direct phosphorylation of the NR2B C-terminal at residues Ser1303 and -1323 (Liao et al., 2001). Grosshans and Browning (2001) extended these findings and demonstrated that PKC activation can also induce tyrosine phosphorylation of the NR2B subunit. CamKII binds directly to the NR1 and NR2B subunits (Leonard et al., 1999). Strack et al. (2000a) showed that residues 1290 – 1309 in the cytosolic tail of NR2B are critical for CaMKII binding, and identified by site-directed mutagenesis several key residues (Lys1292, Leu1298, Arg1299, Arg1300, Glu1301, Ser1303). Phosphorylation of NR2B at Ser1303 by CaMKII inhibits binding of and promotes dissociation of CaMKII/NR2B complexes. Residues 1260 –1316 of NR2B are sufficient to recruit CaMKII to the NMDA receptor in intact cells. Leonard et al. (1999) demonstrated that activation of CamKII-a by stimulation of NMDA receptors in forebrain slices increases the association of CamKII to NR2B. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B modulates co-localization with CaMKII after NMDA receptor activation, suggesting a model for the translocation of CaMKII to postsynaptic targets (Strack et al., 2000a). Importantly, these same investigators have shown previously that the NR2B subunit, but not the NR2A or NR1 subunits, is responsible for autophosphorylation-dependent targeting of CaMKII in intact cells. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B C-terminal (Strack & Colbran, 1998). Further, glutamate-induced translocation of GFP-a-CaMKII to the PSD was blocked by AP-5, suggesting that NR2B-containing receptors are necessary for CaMKII translocation (Shen & Meyer, 1999). These
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findings illustrate that specific co-localization of CaMKII with NR2B-containing NMDA receptors depends on receptor activation, Ca2+ influx, and CaMKII autophosphorylation (on residue Thr286). Thus, translocation of CaMKII because of the interaction with the NMDA receptor ion channel may potentiate kinase activity and provide spatial and temporal control of postsynaptic substrate phosphorylation. Another kinase that has been shown to modulate receptor function is PKA. Westphal et al. (1999) isolated cDNAs encoding fragments of yotiao [a protein that binds the NMDA receptor subunit NR1 and interacts with the Cterminal C1 exon cassette (Lin, J. W. et al., 1998)] by an interaction cloning strategy to identify A-kinase anchoring proteins, and confirmed that the protein binds the NR1 subunit. Yotiao also binds to the Type 1 protein phosphatase (PP1) and to PKA. Anchored PP1 is constitutively active, limiting channel activity, whereas PKA activation overcomes PP1 activity and enables rapid enhancement of NMDA currents. Yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity. Taken together, these studies strongly support the importance of second messenger signaling via kinase and phosphatase activity for the regulation and function of NR2B-containing receptors. Another example of a second messenger molecule that is critically involved with NMDA receptor function is nitric oxide (NO) (Garthwaite, 1991), and its synthetic enzyme, neuronal NO synthase (nNOS) (NOS: EC 1.14.13.39) (Hevel & Marletta, 1994). nNOS is a Ca2+/CaM-dependent enzyme that is functionally coupled to Ca2+ influx through NMDA-type glutamate receptors (Garthwaite & Boulton, 1995), and, thus, is regulated by the gradients of Ca2+ that occur in the vicinity of open Ca2+ channels (Brenman & Bredt, 1997). Stimulation of NMDA receptors results in the activation of nNOS, catalyzing the formation of NO from Larginine (Garthwaite et al., 1989). The mechanism for selectively coupling nNOS to Ca2+ influx through NMDA receptors appears to involve the targeting of nNOS to the PSD (Brenman & Bredt, 1997). The N-terminal PDZ domain of nNOS links the enzyme to the PDZ domains of PSD-95 and PSD-93 (Brenman et al., 1996). Review of the literature indicates numerous roles for nNOS-dependent NO, including, but not limited to, stimulation of cyclic guanosine 50-monophosphate synthesis via activation of guanylate cyclase (Garthwaite, 1991), enhancement of CaM-dependent phosphorylation of PSD proteins (Wu et al., 1996), and modulation of synaptic vesicle exocytosis (Meffert et al., 1996). The regulation of NO formation appears to be mediated by the compartmentalization of nNOS with NMDA receptors at specific synaptic sites (Aoki et al., 1998). nNOS is concentrated at synaptic junctions in the brain. As discussed in Section 4.1.1, NR2B is associated with PSD-95 and related protein family members at synapses and with nNOS (Fig. 1). PSD-95 is co-expressed in numerous neuronal
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populations. nNOS isoforms lacking a PDZ domain do not associate with PSD-95 in the brain. Interaction of PDZ domains, therefore, mediates synaptic association of nNOS, and may play a more general role in formation of macromolecular signaling complexes (Brenman et al., 1996). The linking of NMDA receptors to nNOS by PSD-95 may explain why Ca2+ influx following NMDA receptor activation is tightly coupled to nNOS activation. Contributing to this synaptic regulation, Jaffrey et al. (1998) reported the identification of an nNOS-associated protein, carboxy-terminal PDZ ligand of nNOS (CAPON), which is highly enriched in the brain and has numerous regions of co-localization with nNOS. CAPON interacts with the nNOS PDZ domain through its C-terminal. CAPON competes with PSD-95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD-95/nNOS complexes in transfected cells. CAPON, therefore, may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes. In addition to nNOS and CAPON, a synaptic RasGTPase-activating protein (RasGAP) that associates with the PSD-95 protein family via its PDZ domain was identified. Named SynGAP (synaptic GTPase-activating protein), this protein interacts with all three PDZ domains of PSD95 via its C-terminal amino acids (Kim, J. H. et al., 1998). SynGAP is a brain-specific protein (130 kDa) and demonstrates RasGAP activity. It is enriched at excitatory synapses and co-immunoprecipitates with NR1 and PSD95. SynGAP plays a role in the modulation of Ras signaling at excitatory synapses. Given that Ras proteins are small Gproteins involved in the control of many signal transduction processes that affect cell growth and differentiation, SynGAP, therefore, may play a critical role in the regulation of signaling events associated with the NMDA receptor. Accompanying the neuronal elements that interact via PDZ domains are putative regulatory proteins that are linked to NR2B via the GK domains of the PSD-95 family of cytoskeletal proteins and that, therefore, may function to regulate NR2B-linked intracellular signaling. Kim et al. (1997) isolated a novel synaptic protein, GKAP, that binds directly to the GK domain of at least four known members of the PSD-95 family. GKAP co-localizes and co-immunoprecipitates with PSD-95 in vivo and co-clusters with PSD95 and K+ channels and NMDA receptors in heterologously expressing cells (Naisbitt et al., 1997). Although expressed in neurons and localized specifically in the PSD of glutamatergic synapses, little is known about the function of GKAP. Enzymatic regulation of NMDA receptors is further demonstrated by the Ca2+-dependent protease calpain. Treating synaptic membranes with calpain I results in truncation of the C-terminal domains of the NR1 and NR2A/B subunits. Changes in the channel binding of thienylphencyclidine and MK801 are observed only when the NR2A/B subunits are selectively truncated. Further, preincubation of synaptic membranes with phosphatase inhib-
itors significantly reduces the extent of calpain-mediated truncation of the C-terminal NR2A/B subunits (Bi et al., 1998). Calpain-mediated regulation of NMDA receptor structure and function could represent a feedback mechanism for the receptors that could serve to alter receptor activation. 4.1.3. Neuromodulators (e.g., growth factors, hormones) Growth factors, including members of the neurotrophin gene family, play a central role in the regulation of neuronal survival and differentiation during development. In addition to these relatively long-term actions of neurotrophins, recent studies have shown that these factors rapidly modulate transmission at glutamatergic synapses. Levine and Kolb (2000) used excised membrane patches from cultured hippocampal neurons to determine whether brain-derived neurotrophic factor (BDNF) directly modulates postsynaptic NMDA receptor activity. Acute exposure to BDNF increases NMDA single channel open probability via postsynaptic trkB receptors, an effect that is dependent on the presence of the NR2B subunit of the NMDA receptor. Incubation of BDNF with cortical or hippocampal PSDs rapidly increases tyrosine phosphorylation of NR2B in a dose-dependent manner. The actions of BDNF are specific and selective for NR2B (since nerve growth factor and NR2A do not yield the same results). NR2B phosphorylation may contribute to neurotrophin modulation of postsynaptic responsiveness (Lin, S. Y. et al., 1998). Crozier et al. (1999) also examined the mechanisms of BDNF action in dissociated embryonic hippocampal neurons. Whole cell patch-clamp recording during iontophoretic application of glutamate revealed that BDNF doubles the amplitude of induced inward current. Co-exposure to BDNF and the NMDA receptor antagonist AP-5 markedly reduces the increase in current. Co-exposure to BDNF and ifenprodil, an NR2B subunit antagonist, reproduces the response observed with AP-5, suggesting that BDNF primarily enhances activity of NR2B-containing receptors. Protein kinase involvement was confirmed with the tyrosine kinase inhibitor staurosporine, which prevents the response to BDNF. These results demonstrate the central role of NR2B-containing receptors in BDNF modulation of hippocampal synaptic transmission. To explore the molecular mechanisms underlying synaptic NR2B signaling and growth factor regulation, Lin et al. (1999) examined the effects of BDNF on the interaction and expression of protein tyrosine phosphatase (PTP)1D and NR2B. BDNF increases the association of PTP1D with tyrosine-phosphorylated proteins, including NR2B, in cortical neurons and PC12 cells. The BDNF action appears to be specific, since nerve growth factor, another member of the neurotrophin gene family, does not alter the association. These investigators reported that PTP1D is an intrinsic component of rat PSD, based on immunoblot analyses using specific anti-PTP1D antibodies. In addition, NR2B coimmunoprecipitates with PTP1D, indicating a physical
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association of the subunit with PTP1D. Taken together, these results suggest that tyrosine phosphorylation and dephosphorylation can participate in BDNF-mediated NR2B signaling cascades at distinct postsynaptic sites. Activity-dependent neurotrophic factor (ADNF), which is a protein secreted by vasoactive intestinal polypeptidestimulated astroglia, may also contribute to the regulation of NR2B. Blondel et al. (2000) used hippocampal neurons in culture to demonstrate that vasoactive intestinal polypeptide promotes neuronal differentiation through ADNF. ADNF is produced by glial cells and acts directly on neurons to promote glutamate responses and morphological development. ADNF causes secretion of neurotrophin-3, and both proteins regulate developmental changes associated with the NMDA receptor subunits NR2A and NR2B. In addition to identified growth factors, hormone modulation of the NR2B subunit has also been demonstrated. Systemic administration (10 days) of recombinant human growth hormone to young rats (11 weeks old) causes an increase in the expression of hippocampal NR2B mRNA that is not observed in elderly rats (57 –67 weeks old) (Le Greve`s et al., 2002). Estrogen treatment significantly increases the levels of hypothalamic NR2B mRNA during the late prepubertal period in female rats (Kanamaru et al., 2001). Importantly, the effects of estrogen are time- and brain region-specific, thus providing an example of how the expression of NR2B is developmentally and regionally regulated. Finally, Mukai et al. (2000) examined the subunit specificity of pregnenolone sulfate (PREGS) potentiation of NMDA receptor-mediated Ca2+ signals. Using NR1/NR2Btype NMDA receptors expressed in Chinese hamster ovary cells, PREGS enhances the Ca2+ influx through NR2Bcontaining receptors in a dose-dependent manner. Ifenprodil abolishes the NMDA-induced Ca2+ influence even in the presence of PREGS. These results imply that PREGS positively modulates the Ca2+ influx through NR2B-containing receptors that are expressed since the embryonic period. 4.2. Exogenous regulators Alterations in the expression, distribution, and function of the NR2B subunit occur via changes in brain environment. As described above, these changes can result from endogenous factors, such as kinases, phosphatases, and other regulatory enzymes. However, external variables can also influence the prevalence of NR2B subunits and consequently the function of NMDA receptors. These exogenous elements generally are identified as those resulting in trauma or lesions and those pertaining to pharmacological manipulation. 4.2.1. Trauma or lesions Watkins et al. (1998) studied the effects of single and repeated electroconvulsive shock treatment on the mRNA levels of several glutamate receptor subunits in the dentate
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gyrus and CA1 hippocampal regions of the rat. In the dentate gyrus, shock treatment elevates the levels of mRNA for NR2B. The change in NR2B transcript expression persists the longest (as compared with NR2A and metabotropic glutamate receptor subtype 5). Expression is still elevated after 24 hr, but returns to baseline by 48 hr after shock treatment. Chronic and acute treatments yield the same results. However, the functional consequences of a transient increase in NR2B mRNA expression remain unclear. In addition to providing information about the regulation of subunit expression, brain insults can also help to answer questions about local circuitry and region-specific changes in receptor function. In an attempt to elucidate the effects of decreased dopamine on glutamate receptor expression, Kayadjanian et al. (1996) performed unilateral and bilateral lesions of the medial forebrain bundle and the sensorimotor cortex, respectively. Unilateral lesions of the medial forebrain bundle resulted in no change in NR2B mRNA levels in the dopamine-depleted striatum, as compared with the normal rat striatum. Bilateral lesions of the sensorimotor cortex resulted in a significant increase in the levels of NR2B mRNA, specifically in the striatal projection area of the sensorimotor cortex. These findings suggest that NR2B expression is regulated by cortico-striatal fibers in a topographical manner. Levels of NR1 and NR2B subunits within the magnocellular nuclei of the hypothalamus are up-regulated and down-regulated, respectively, during dehydration (Decavel & Curras, 1997). Similarly, osmotic activation of the hypothalamo-neurohypophysial system reversibly down-regulates NR2B subunits in the supraoptic nucleus of the hypothalamus. These results indicate that NR2B-containing receptors on the supraoptic nucleus and paraventricular nucleus magnocellular neuroendocrine cells may contribute to neuroendocrinological functions associated with body fluid homeostasis (Curras-Collazo & Dao, 1999). Finally, the influence of heat stress on the constitutive isoform of NOS (cNOS) and NMDA receptor subunit gene expression in the hippocampus was examined in a rat model. Exposure of animals to 4 hr of heat stress at 38C results in a significant up-regulation of cNOS, but mRNA encoding NR1, N2A, and NR2B shows a marked downregulation in the hippocampus of heat-stressed animals as compared with controls (Le Greves et al., 1997). Thus, increased NO production associated with heat stress may contribute to the regulation of NMDA receptor subunit expression. 4.2.2. Pharmacological agents (e.g., antagonists) Although artificial manipulations by exogenous agents, the pharmacological studies reviewed in this section provide important information about the regulation of NR2B (Table 1). For example, treatment with certain NMDA receptor antagonists illustrates how neuronal activation controls the ability of the cell to modify its synapses. Exposure of mouse
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cortical neurons to the NMDA receptor antagonist AP-5 causes an increase in the levels of the NR2B mRNA in a concentration- and time-dependent manner (Follesa & Ticku, 1996). Treatment with AP-5 also increases the number of NR2A and NR2B clusters (Rao & Craig, 1997). Taken together, these results suggest that expression and localization of NMDA receptors are regulated by synaptic activity. Treatment with MK-801 provides concurring results. The density of NMDA receptors on membranes prepared from cultured cerebral cortical neurons was determined using binding assays with [125I]MK-801 following exposure of cultures to various antagonists of the NMDA receptor complex. The density of binding sites for MK-801 is increased by 40 – 80% after exposure to AP-5, with no change in the number or viability of neurons. Up-regulation of the NMDA receptor is observed after 2 –7 days, but not after 1 day of exposure to AP-5. The density of NMDA receptors is also increased after exposure of cells to CGS 19755 and to MK-801. Thus, the density of NMDA receptors on cultured neurons can be selectively up-regulated by exposure to specific NMDA receptor antagonists (Williams et al., 1992). Chronic treatment of cortical neurons with MK-801 enhances membrane-associated tyrosine kinase activity and up-regulates NR2B subunits, while treatment with a tyrosine kinase inhibitor results in down-regulation of NR2B subunits in cortical neurons. Thus, MK-801-induced receptor up-regulation may be mediated by NR2B phosphorylation via tyrosine kinases (Kalluri & Ticku, 1999b). In contrast, however, in vivo studies yield somewhat conflicting results. For example, when rats are injected every 12 hr for 14 days and sacrificed on day 15, chronic MK-801 administration does not alter NR1, NR2A, or NR2B subunit expression in either the hippocampus or cortex (Matthews et al., 2000). Linden et al. (2001) also investigated the effects of acute administration of MK-801 on mRNA levels of NMDA receptor subunits and on molecules known to cluster or phosphorylate the receptor. In situ hybridization of rat brain sections demonstrated that acute treatment with MK-801 (5 mg/kg, 4 hr) decreases levels of NR2B mRNA in the parietal cortex. MK-801 also increases mRNA levels of SAP90/PSD-95 and PKC-g in cortical regions. Although not in complete agreement, taken together, these results suggest that antagonist-induced changes in the density of NMDA receptors and associated neuronal elements may play a role in adaptation or toxic responses and, therefore, could complicate therapeutic approaches to the treatment of neurological disorders (see Section 5). Ifenprodil is an antagonist selective for NMDA receptors containing the NR2B subunits, and recently was used to demonstrate that antagonists not only modify the expression of subunits, but can also alter the binding properties of the receptor. Using rat brain slices, Zhang et al. (2000) found that ifenprodil enhances NMDA-induced currents in both cortical and subcortical areas. The results, combined with
previous studies, suggest that the augmenting effect is due to an increase in NMDA receptor affinity for agonists and is specific for responses induced by low NMDA concentrations. As NMDA concentrations increase, the affinityenhancing effect of ifenprodil becomes less evident and the channel-blocking effect becomes more prominent. Zhang and Shi (2001) provided further evidence for the mechanism of action of ifenprodil on NR2B-containing receptors. Specifically, based on data from whole cell recordings of rat prefrontal cortical slices, ifenprodil is hypothesized to inhibit the rising phase of fast NMDA responses by suppressing both channel opening and the association of NMDA with NMDA receptors. Thus, given the complexity of the mechanisms associated with the action of ifenprodil on NMDA receptors, interpretation of data from NMDA receptor antagonist studies should also consider variables such as agonist and antagonist concentrations, pharmacological selectivity, and the specific subunit enrichment profile (Table 1). In addition to data from NMDA receptor antagonist studies, information regarding the regulation of NR2B expression can also be gleaned via the use of other pharmacological agents. For example, cultures of rat cerebellar granule cells were treated with the glutamate transport blocker L-trans-pyrrolidine-2,4-dicarboxylate and the expression of NMDA receptor subunits was evaluated (Cebers et al., 2001). Expression of the NR2B subunit mRNA and protein is significantly lower in L-trans-pyrrolidine-2,4-dicarboxylate-treated cells as compared with controls. These findings imply that increased synaptic glutamate concentration could result in the down-regulation of NR2B-containing NMDA receptors.
5. NR2B role in human physiology and pathophysiology Both endogenous and exogenous factors interact with NMDA receptors and play a role in a number of human disorders and physiological processes. 5.1. Synaptic plasticity/learning Although changes in synaptic function have not been directly linked to learning in humans, long-term potentiation (LTP) and LTD have been used to model and to generate inferences regarding learning-related phenomena. Both LTP and LTD can be triggered by NMDA receptor activation. Hrabetova et al. (2000) sought to test the hypothesis that the specific patterns of afferent stimulation producing LTP and LTD activate separate receptor subpopulations composed of different NMDA receptor subunits. The authors examined the inhibition of LTP and LTD by a series of competitive NMDA receptor antagonists that varied in their affinities for NR2A/B and NR2C/D. Antagonists with higher affinity for the NR2A/B subunits relative to NR2C/D show more potent inhibition of LTP than LTD. Thus, distinct subpopulations of
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NMDA receptors characterized by different NR2 subunits contribute to the induction mechanisms of potentiation and depression. Earlier studies also support the involvement of NR2 subunits in synaptic plasticity and specifically implicate NR2B in LTP-related events. Quantitative in situ hybridization revealed that following the induction of hippocampal LTP in the dentate gyrus of freely moving rats, specific increases in the expression of the NR2B subunit are evident in postsynaptic dentate granule cells. However, there are no changes in the expression of the NR2A, NR2C, or NR2D subunits. The elevation in NR2B subunit expression is delayed, occurring days after LTP induction. NR2B subunit expression is enhanced significantly by 48 hr after LTP, but starts to decrease toward basal levels by 96 hr. The transient increase in the expression of the NR2B subunits is temporarily related to an increase in the expression of the NR1 subunits (Thomas et al., 1994). Changes in the expression of individual glutamate receptor subunits that occur days after the induction of LTP may reflect activation of so-called lateonset genes that are needed for the maintenance of LTP (Thomas et al., 1996). In an NR2B knock-down model for aged rats, NR2B antisense treatment abolished LTP and impaired spatial learning, as measured by the Morris water maze (Clayton et al., 2002). Collectively, these results support a role for NR2B-containing receptors in LTP, and suggest a relationship among the NR2B subunits, LTP, and specific cognitive functions. In addition to alterations in subunit expression, changes in the phosphorylation state of NR2B are also associated with LTP. CaMKII-dependent phosphorylation of NR2A and NR2B is decreased in rats with hippocampal damage and impaired LTP caused by methylazoxymethanol-induced prenatal ablation of CA1 neurons (Caputi et al., 1999). LTP in the dentate gyrus is also correlated with tyrosine phosphorylation of the NR2B subunit, persisting in vivo for several hours. Consequently, NR2B phosphorylation is hypothesized to play a role in short- and intermediate-term mechanisms of experience-dependent plasticity (Rosenblum et al., 1996). Nakazawa et al. (2001) examined alterations in the level of tyrosine residue 1472 phosphorylation on the NR2B subunits after the induction of LTP. Using extracellular field potential recording techniques, excitatory postsynaptic potentials were recorded in the hippocampal CA1 region of mice. Sixty minutes after the induction of LTP, enhanced phosphorylation of NR2B Tyr1472 was observed. The data suggest that residue-specific tyrosine phosphorylation of NR2B is involved in the expression of LTP. Further evidence for the role of tyrosine phosphorylation of NR2B subunits in synaptic plasticity is provided by Manabe et al. (2000). KO-mice lacking the H-ras gene were generated and then used to study the roles of NMDA receptors, phosphorylation, and Ras in synaptic transmission and LTP. In the H-ras null mutant hippocampus, the tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor is increased, and correspondingly,
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NMDA synaptic responses are selectively enhanced. LTP is markedly enhanced in mutant mice, most likely because of a selective enhancement of NMDA synaptic responses. It was concluded, therefore, that the regulation of activitydependent synaptic plasticity in the adult animals by downregulation of the phosphorylation of specific NMDA receptor subunits might be another major role for the H-ras protein. In humans (children), chronic lead exposure produces deficits in learning and memory. Recent studies assessed the effects of this potential toxin on NMDA receptor subunits. Exposure to lead during development has no effect on NR1 or NR2B subunit protein expression in the hippocampus or the cortex. However, the expression of the NR2A subunits is reduced in the rat hippocampus (Nihei & Guilarte, 1999). In a subsequent study, Nihei et al. (2000) demonstrated that impairments of spatial learning and hippocampal LTP in rats chronically exposed to lead are associated with changes in gene and protein expression of the NMDA receptor subunits NR1 and NR2A, but not NR2B. In vitro studies investigating the effects of lead on the expression of the NR1 and NR2B subunits provide somewhat different results. Primary cortical and hippocampal cultures were treated with lead. In cortical neurons, dose-dependent increases in the expression of NR2B mRNA and subunit protein were observed, but lead-induced changes in the expression of NR1 mRNA or subunit protein were not present. However, in hippocampal neurons, both NR1 and NR2B mRNA and subunit protein levels were decreased (Lau et al., 2002). Thus, the role of NR2B-containing receptors in mediating the toxic effects of lead and its subsequent impact on learning and memory remains unclear. 5.2. Schizophrenia Accompanying the long-standing theories regarding dopamine and schizophrenia are more recent reports implicating glutamate and NMDA receptors. The NMDA receptor hypothesis of schizophrenia is based on the observation that hypofunction of the NMDA receptors induced by various NMDA receptor antagonists precipitates a transient psychotic state in healthy individuals. Accordingly, researchers are beginning to look for evidence that the NMDA receptor system may be dysfunctional in schizophrenia. Clinical trials of glycinergic agents are currently being conducted (for a review, see Farber et al., 1999). The specific rationale for these studies is that activation of the glycine site within the NMDA receptor complex is necessary for the normal function of this receptor system. Early results from clinical trials of glycinergic agents indicate that these agents either have no effect or are only modestly beneficial effects on the negative symptoms accompanying schizophrenia (Farber et al., 1999). Goff and Coyle (2001) reviewed evidence from more recent clinical trials, as well as from basic science reports, and suggest that pharmacotherapeutic modulation of the glycine site on the NMDA
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receptors can be helpful for the treatment of negative symptoms associated with schizophrenia. Several investigators have specifically been looking at the role of the NR2B subunit in schizophrenia. Expression patterns of mRNA for individual receptor subunits were assessed using in situ hybridization in prefrontal, parietotemporal, and cerebellar cortices of postmortem brains from people with schizophrenia and from neuroleptic-treated and non-medicated control subjects (Akbarian et al., 1996). Although a shift in the relative proportions of NR2A, NR2B, NR2C, and NR2D mRNA expression is suggested, the data revealed no significant changes in the expression of NR2A, NR2B, NR2C, or NR2D mRNA in schizophrenic patients as compared with control subjects. However, using different methodology, Grimwood et al. (1999) found somewhat conflicting results. As measured by radioligand binding with [3H]ifenprodil, NR2B-containing receptors are selectively up-regulated in the superior temporal cortex in schizophrenia, but not in the pre-motor cortex (Grimwood et al., 1999). Although changes in transcript levels are not always indicative of corresponding alterations in protein expression (and visa versa), these findings suggest that further investigation of the role of NR2B in schizophrenia is needed. Gao et al. (2000) analyzed postmortem hippocampal tissue from people with schizophrenia and from control subjects. Glutamate receptor autoradiography of various subfields in the hippocampus provided no evidence of ligand-binding differences between the groups. Interestingly, in situ hybridization experiments were also performed and the level of mRNA for the NR2B subunit in CA2 of the hippocampus was found to be 40% higher in schizophrenia tissue than in control tissue. Finally, Nishiguchi et al. (2000) screened for genetic variations in the region of the NR2B subunit gene encoding the C-terminal intracellular domain in subjects with schizophrenia and studied the association between schizophrenia and a novel polymorphism of the NR2B subunit gene, the silent mutation 2664C/T. No significant differences in the frequencies of 2664C/T genotypes exist between patients with schizophrenia and control subjects. Thus, although the NR2B subunit may be involved in certain aspects of the illness, these findings provide no evidence of an association between schizophrenia and the 2664C/T polymorphism of the NR2B subunit gene. 5.3. Parkinson’s disease Another disorder traditionally associated with disturbances in the dopaminergic system is Parkinson’s disease. Overactivity of the striatolateral pallidal pathway, the ‘‘indirect’’ striatal output pathway to the external pallidal segment, is thought to be responsible for the generation of some Parkinson’s symptoms. However, as with schizophrenia, the glutamatergic system and NMDA receptors recently have been shown to interact with dopaminergic circuitry and to contribute to neurochemical alterations accompanying the disorder. Oh et al. (1998) examined the effects of unilateral
nigrostriatal dopamine system ablation with 6-hydroxydopamine, followed by twice daily treatment with levodopa (LDOPA), on the phosphorylation state of the rat striatal NR2A and NR2B subunits. After 3 weeks of L-DOPA treatment, tyrosine phosphorylation of the NR2A subunit, and especially the NR2B subunit, is increased ipsilateral to the lesion (20 ± 5% and 46 ± 7% of the intact striatum, respectively) without accompanying changes in subunit protein levels. Augmented tyrosine phosphorylation of the NR2B subunits alone or in combination with the smaller rise in NR2A phosphorylation contributes to the apparent enhancement in striatal NMDA receptor sensitivity (see Section 4.1.2) and, thus, to the changes in dopaminergic responses in L-DOPA-treated Parkinsonian rats. Using a similar animal model, Oh et al. (1999) looked at CaMKII phosphorylation of serine. L-DOPA treatment elevates serine phosphorylation of striatal NR2A subunits, but not NR2B subunits. Further, chronic treatment with SKF-38393, a D1agonist, increases NR2A and decreases NR2B serine phosphorylation, while chronic exposure to quinpirole, a D2agonist, has no effect on NR2A, but increases NR2B, phosphorylation. Kinase-specific changes in the phosphorylation of the NR2B subunits may thus contribute to the compensatory neurochemical adaptations associated with LDOPA treatment. Dunah et al. (2000) also studied the expression and phosphorylation of NMDA receptor subunits in the rat 6hydroxydopamine lesion model of Parkinson’s disease. In the lesioned striatum, the levels of NR1 and NR2B subunit expression are decreased to 68 ± 3.2 and 62 ± 4.4% of control, respectively. Co-immunoprecipitation studies indicate that there is a selective reduction of the NR1/NR2B, but not NR1/NR2A, receptors. However, using tissue fractionation techniques, the authors found that the decrease in NR2B abundance could be attributed to an intracellular redistribution of subunit proteins rather than to a reduction in subunit protein expression. Further, in partial agreement with previous studies, tyrosine phosphorylation of NR2B (but not NR2A) is decreased in the lesioned striatum. LDOPA treatment normalizes the lesion-induced alterations in receptor subunit enrichment and causes hyperphosphorylation of the NR1 serine and NR2A and NR2B tyrosine residues. NMDA receptor antagonist studies also support a role for the glutamatergic system, specifically mediated by NR2Bcontaining receptors, in modulating features of Parkinson’s disease. Selective blockade of NR2B-containing NMDA receptors with ifenprodil and eliprodil causes a significant increase in locomotor activity in the reserpine-treated rat model of Parkinson’s disease (Nash et al., 1999). Additionally, Steece-Collier et al. (2000) examined the antiparkinsonian effects of CP-101,606 in rodents and non-human primates. In rats, this selective antagonist of the NR2Bcontaining receptors decreases haloperidol-induced catalepsy. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinetreated monkeys, CP-101,606 reduces parkinsonian motor
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symptoms by 20%. CP-101,606 also significantly potentiates the effect of L-DOPA as indicated by a 50% reduction in motor symptoms when compared with vehicle and a 30% reduction when compared with L-DOPA alone. Drug-related side effects were not observed at any dose of CP-101,606 administered. The authors concluded that CP-101,606 has direct antiparkinsonian actions in both rodents and monkeys and that it synergistically potentiates the motoric improvements accompanying L-DOPA treatment. Consequently, NR2B-selective antagonism may prove useful in the treatment of Parkinson’s disease. 5.4. Huntington’s disease Huntington’s disease, a hyperkinetic movement disorder, may also be associated with changes in the function of the NR2B-containing NMDA receptors. The condition is generated by an expanded CAG repeat in the first exon of the Huntington’s disease gene. The mutant huntingtin protein, therefore, has an expanded polyglutamine (Q) tract in its Nterminal region, causing a toxic gain of function, as well as possibly a loss of function of normal huntingtin. Receptors composed of NR1 and NR2B subunits exhibit significantly larger currents when co-expressed with mutant huntingtin. Further, mutant huntingtin may increase the number of functional NR1/NR2B-type receptors at the cell surface (Chen et al., 1999b). Follow-up experiments provide further evidence for the involvement of NMDA receptors in Huntington’s disease. Transfected HEK-293 cells co-expressing full-length mutant huntingtin-138Q and either NR1/NR2A or NR1/NR2B heteromeric receptors exposed to NMDA exhibit a significant increase in excitotoxic cell death as compared with cells expressing huntingtin-15Q or GFP (Zeron et al., 2001). Interestingly, the difference in cell death is larger for cells containing NR1/NR2B receptors. Zeron and colleagues thus concluded that subunit-specific interactions between NR2B-containing receptors and mutant huntingtin potentiate NMDA receptor-initiated apoptotic cell death and, consequently, may contribute to the development of neuropathology associated with Huntington’s disease. 5.5. Excitotoxicity/hypoxia/ischemia Excitotoxicity is a critical mechanism contributing to neurodegeneration during ischemia or hypoxia. Major events in the cascade triggered by hypoxia or ischemia include overstimulation of NMDA-type glutamate receptors, Ca2+ entry into cells, activation of Ca2+-sensitive enzymes such as nNOS, production of oxygen free radicals, mitochondrial impairment, and subsequent necrosis or apoptosis (for reviews, see Sattler & Tymianski, 2000; Johnston et al., 2000). In support of the specific role of the NMDA receptor complex in excitotoxic damage, Sattler et al. (1999) used cultured cerebral cortical neurons to demonstrate that suppressing the expression of PSD-95 selectively attenuates the
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excitotoxicity triggered via NMDA receptors, but not by other glutamate or Ca2+ channels. NMDA receptor function is not affected because receptor expression, NMDA currents, and [45Ca2+] loading are unchanged. Moreover, suppressing PSD-95 expression blocks Ca2+-activated NO production by NMDA receptors selectively, without affecting nNOS expression or function. Thus, PSD-95 is required for efficient coupling of NMDA receptor activity to NO toxicity and imparts specificity to excitotoxic Ca2+ signaling (Sattler et al., 1999). Importantly, specific changes in NR2B-containing NMDA receptors may accompany aspects of excitotoxic damage associated with hypoxia and ischemia. Cheng et al. (1999) examined several factors related to the increase in susceptibility to excitotoxicity that occurs in cultured embryonic forebrain neurons over time. For instance, RNase protection assays were used to measure NR1, NR2A, and NR2B mRNA levels, demonstrating that NR2B mRNA levels increase dramatically during the first 10 days and subsequently remain stable. It was concluded that increased glutamate-stimulated [Ca2+]i responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons. To more comprehensively address the question of NR2B subunit involvement in neurotoxicity associated with ischemia and hypoxia, Zhang et al. (1997) assessed rat receptor gene expression, protein levels, and function after severe global ischemia using a 4-vessel occlusion ischemia model. At 12 and 24 hr after ischemic challenge, decreased expression of NR2A and NR2B mRNA in CA1 and other hippocampal subfields was observed. A significant decrease in NR2A/B protein (total hippocampus) was observed at both 6 and 24 hr after ischemia, as compared with controls. Further, electrophysiological assessment of single-channel NMDA receptor activity in the CA1 indicates that the main conductance state of NMDA receptor channels is maintained 6 hr after the ischemic challenge, but that by 18– 24 hr, this main conductance state is rarely observed. Thus, the ischemia-induced decrease in the expression of NR2A and/ or NR2B alters the function of the NMDA receptors. Kim, W. T. et al. (1998) created hypoxic conditions in newborn piglets. Animals were ventilated until paO2 was < 20 mm Hg. Unlike the previous study that used 6– 24 hr of ischemic exposure, 1 hr after a hypoxic challenge there were no changes in the expression of NR2B or other NMDA receptor subunits (NR1, NR2A) in regions of the frontal cortex, hippocampus, thalamus, basal ganglia, or cerebellum, as assessed by western analysis. In addition to potentially altering NR2B subunit expression, ischemia has also been shown to alter protein interactions. NR2A and NR2B bind to each of the SH2 domains of the tyrosine kinases Src and Fyn (expressed as GST-SH2 fusion proteins), and binding is increased 2-fold after ischemia and reperfusion. Transient ischemia induces a marked increase in the tyrosine phosphorylation of NR2A
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and NR2B and, consistent with previous findings (Kim, W. T. et al., 1998), has no effect on the amount of individual NMDA receptor subunits (Takagi et al., 1999). Similarly, regional levels of tyrosine phosphorylation of the NR2B subunit are enhanced after ischemia in the rat brain in both the synaptosomal fraction and the whole-tissue homogenate of rat neocortex and hippocampus, but return to control levels only in the preconditioned brain (ischemic preconditioning was 3 min). Using the 2-vessel occlusion model of global cerebral ischemia, preconditioning provides maximum neuroprotection when induced 2 days prior to a potentially lethal ischemic insult of 9-min duration. Interestingly, ischemic preconditioning induces a selective decrease in the levels of the NR2A and NR2B subunits and a modest decrease in the levels of NR1 in the synaptosomal fraction of the neocortex, but not hippocampus (Shamloo & Wieloch, 1999). To further examine such findings, which demonstrate that transient cerebral ischemia results in an enhancement of tyrosine phosphorylation of proteins associated with the PSD, Cheung et al. (2000) investigated possible mechanisms behind this increase. Isolated PSDs were analyzed for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Tyrosine phosphorylation of several PSD proteins, including the NR2A and NR2B subunits, is enhanced relative to sham-treated animals after 20 min and 6 hr of reperfusion. The ability of intrinsic tyrosine kinases to phosphorylate PSD proteins, including the NMDA receptor subunits, increases 3-fold after ischemia. While ischemia can increase interactions among some proteins, it also can decrease the binding of other NMDA receptor-associated proteins. Takagi et al. (2000) examined the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. The association between PSD-95 and NR2A and NR2B, as indicated by co-immunoprecipitation, is less in post-ischemic samples than in sham-operated controls. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge. Recently, lithium was proposed as a drug that might be protective against glutamate-induced excitotoxicity. Lithium pretreatment of cultured cerebral cortical neurons reduced the level of tyrosine phosphorylation (Tyr1472) without altering the expression of the NR2B subunits. Further, the decrease in residue-specific phosphorylation was associated with its neuroprotective effects (Hashimoto et al., 2002). 5.6. Seizure disorder Previous research has consistently demonstrated a connection between glutamatergic neurotransmission and certain types of seizure activity; however, the degree and nature of NMDA receptor involvement, the subunit specificity, and the effects of seizure activity on subsequent receptor expres-
sion and function have yet to be fully understood. In an attempt to characterize the potential involvement of individual NMDA receptor subunits, Narita et al. (2000a) pretreated mice intracerebroventricularly with antibodies specific to the C-terminal region of the NR2B (as well as NR1 and NR2A) subunit 1 and 3 days before treatment with NMDA. The pretreatment regimes for all three NMDA receptor subunits produced a significant increase in the seizure threshold for NMDA, suggesting that the NR1, NR2A, and NR2B subunits may be involved in seizure sensitivity to glutamatergic agonists and consequently have an important role in regulating seizure activity. Kindling has been used as an animal model of epilepsy, such that application of initially subconvulsive electrical stimulation eventually results in the development of limbic and clonic motor seizures. To investigate the possible effects of seizures on NMDA receptor gene expression, transcript levels of NR1, NR2A, NR2B, NR2C, and NR2D were measured using the rat hippocampus (Kraus et al., 1994). Kindling in hippocampal regions does not alter NMDA receptor gene expression 24 hr or 28 days after the last seizure. Similarly, in a follow-up study that assessed NMDA receptor subunit levels at 28 days post-seizure, Kraus and McNamara (1998) reported that kindling does not produce long-lasting changes in the levels of the NR1, NR2A, or NR2B proteins in the hippocampus. Taken together, these data suggest that seizure activity does not appear to transiently or permanently alter the expression of NMDA receptor subunits. In contrast, Mathern et al. (1998) investigated hippocampal NMDA receptor subunit immunoreactivity in tissue taken at autopsy from temporal lobe epilepsy patients compared with receptor subunits in tissue from non-epileptic control patients. Using an antibody that labeled both the NR2A and NR2B subunits, the authors observed increased NR2A/B expression throughout all hippocampal subfields. It was concluded that subunit up-regulation might contribute to seizure generation or propagation by altering NMDA receptor-mediated neurotransmission. Given these disparate findings, more research appears to be needed to understand the relationship between NMDA receptor subunit expression and seizure activity. Toward this end, Kojima et al. (1998) generated transgenic mice that expressed either a constitutively active form of Fyn or native Fyn in neurons of the forebrain. The threshold for pentylenetetrazole-induced seizures is reduced and electrical stimulation of the amygdala elicits higher seizure activity in transgenic mice expressing the mutant Fyn as compared with wild-type mice. In forebrains of both native and mutant Fyn transgenic mice, tyrosine phosphorylation of NR2B is enhanced, confirming that this subunit is a substrate of Fyn. In addition, MK-801 administration attenuates kindling in mice overexpressing native Fyn, as well as in wild-type mice. Consequently, tyrosine phosphorylation of NR2B by Fyn might be involved in the susceptibility to NMDA receptor-mediated kindling.
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5.7. Diabetes Quantitative autoradiography suggests that the antagonist affinity and density of the NMDA receptor are greater in diabetic mice than in control animals (Li et al., 1999). However, to date, there is little information in the literature regarding diabetes and potential NMDA receptor subunit involvement. There exists an animal model, the streptozotocin-diabetic rat, which manifests impairments in spatial memory and LTP expression—two areas of functioning previously shown to involve subunit-specific NMDA receptor activation (see Section 3). Using the streptozotocindiabetic rat, Di Luca et al. (1999) studied the effects of experimental diabetes on the expression of NMDA receptors and associated proteins regulating synaptic transmission at the PSD. In situ hybridization and immunoblot analyses were used to assess the expression of NMDA receptor subunits and CaMKII. In the hippocampus, the mRNA and immunoreactivity for NR2B is reduced by 40% in diabetic rats, while NR1, NR2A, GluR1, GluR2/3, PSD-95, and CaMKII transcript and protein levels are unchanged. In addition, NMDA receptor subunit phosphorylation by CaMKII was studied in the hippocampus and cortex of control, streptozotocin-diabetic, and insulin-treated rats. Although the expression of CaMKII is unaltered, the ability of CaMKII to phosphorylate NR2A/B subunits is reduced in hippocampal PSD fractions from streptozotocin-diabetic rats as compared with the PSD from control rats. Insulin intervention for 3 months after streptozotocin treatment partially restores both CaMKII activity and NR2B levels. Thus, these findings suggest that the metabolic changes associated with diabetes alter NMDA receptors in a kinaseand subunit-specific manner. In a follow-up study, Gardoni et al. (2002) provide additional evidence to support this hypothesis. Using the same rat model, 4 months (but not 1 month) after the onset of diabetes, NR2B expression and phosphorylation is reduced in the hippocampus. Insulin treatment also prevents the reduction in CaMII and tyrosine kinase activity and NR2B subunit expression levels. 5.8. Alzheimer’s disease Alzheimer’s disease is often associated with a loss of NMDA receptors in selected brain regions. For example, Maragos et al. (1987) demonstrated a marked loss of NMDA receptors in the hippocampus of patients with Alzheimer-type dementia, as measured by [3H]thienylphencyclidine binding. Recently, investigators have begun to examine the effects of Alzheimer’s disease on individual NMDA receptor subunits. Wang et al. (2000) estimated the levels of all a-amino-3-hydroxy-5-methyl-isoxazole-4-propionate and NMDA receptor subunits in selected (i.e., hippocampus, frontal, and entorhinal cortex) brain tissue samples obtained from control subjects and patients who died with Alzheimer’s disease. Modest decreases in the NMDA receptor subunits NR1, NR2A, and NR2B were
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found in the hippocampus and frontal cortex, while little or no change in any of these subunits was observed in the entorhinal cortex. Sze et al. (2001) assessed levels of both non-phosphorylated and phosphorylated receptor subunit proteins in various areas of post-mortem brains from patients who died with Alzheimer’s disease and control subjects. Significant reductions in NR2B expression are present in the hippocampus (40%) and entorhinal cortex (31%). In addition, tyrosine phosphorylated NR2B is decreased (56%) in the entorhinal cortex. The authors found no correlation between phosphorylated and non-phosphorylated NR2B levels and, thus, suggest that the Alzheimer’s disease-related reduction in phosphorylation is subunitindependent. Collectively, these results suggest that NMDA receptor subunits are selectively and differentially reduced in areas of Alzheimer’s-diseased brains. However, questions remain regarding the magnitude and location of protein loss, as well as the nature of the relationship among decreased subunit expression, phosphorylation, and the cognitive impairments and cell loss observed in Alzheimer’s disease. In a study of 132 patients with Alzheimer’s disease and 114 control subjects, Tsai et al. (2002) investigated the prevalence of the allelic variant (C2664T) of the NR2B. The distribution of the NR2B genotypes and alleles did not differ between the groups, thus suggesting that C2664T does not significantly contribute to the development of Alzheimer’s disease. 5.9. Drugs of abuse Many abused drugs alter the expression and function of NMDA receptors. It is further hypothesized that NMDA receptors mediate the adaptive mechanisms that are involved in the development, maintenance, and expression of drug addiction. In this section, evidence for the role of NR2B-containing NMDA receptors in alcohol, cocaine, and opioid abuse is discussed. NMDA receptors are an important site of action for ethanol (Lovinger et al., 1989; Carter et al., 1995), and importantly, dependence, tolerance, and withdrawal symptoms seem to be mediated in part by NMDA receptors. Narita et al. (2000b) treated mice with ethanol for 5 days. Withdrawal signs (piloerection, jerking, tremor, and handling-elicited convulsions) are present at 3, 6, 9, 12, and 48 hr after the discontinuation of the liquid-ethanol diet. Treatment during withdrawal with a selective NR2B-containing NMDA receptor antagonist, ifenprodil, significantly reduces the expression of ethanol withdrawal signs. In addition, during chronic ethanol treatment, protein abundance for the NR2B subunit in the mouse limbic forebrain, but not the cerebral cortex, is significantly increased with respect to control levels, as measured by immunoblotting. The significant up-regulation of NR2B lasts for at least 9 hr after the discontinuation of ethanol, and returns to basal levels by 48 hr. Similarly, Kalluri et al. (1998) investigated the effects of chronic ethanol administration and withdrawal on NR2B
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expression in the rat cerebral cortex and hippocampus. Chronic ethanol treatment up-regulates NR2B expression in both the cortex ( 37%) and hippocampus ( 35%). At 48 hr of withdrawal, the expression of NR2B returns to almost control levels. When ethanol is repeatedly administered to rats for 8 days by gastric intubation and withdrawn for 24 hr, changes in NR2B subunit expression are also evident. Specifically, NR2B mRNA is not substantially altered in cholinergic and GABAergic neurons, but shows a decrease in specific brain areas (the CA region and neostriatum) (Darstein et al., 2000). Taken together, these studies thus suggest that ethanol-induced alterations in the abundance of the NR2B subunit may contribute to the initial development of physical dependence on ethanol. However, there is not complete agreement in the literature regarding the effects of ethanol exposure on NR2B expression. Using primary cultures of rat cerebellar granule cells, Cebere et al. (1999) found that chronic ethanol exposure (50 – 100 mM for 3 days) has no effect on the relative levels of mRNA for NR2B or on the expression of the NR2B protein. Although methodologically very disparate, collectively these studies illustrate the need for further investigation of the effects of ethanol on NR2B expression. Ethanol may also modulate the phosphorylation of NR2B-containing receptors. Mice lacking Fyn are hypersensitive to the hypnotic effects of ethanol. The administration of ethanol enhances tyrosine phosphorylation of the NR2B, but not of the NR1, subunit in the hippocampus of control mice, but not in Fyn-deficient mice. Interestingly, an acute tolerance to ethanol inhibition of NMDA receptormediated excitatory postsynaptic potentials in hippocampal slices develops in control mice, but not in Fyn-deficient mice (Miyakawa et al., 1997). Anders et al. (1999) also investigated the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK-293 cells. Unlike previous findings, using whole cell patch clamp and ratiometric Ca2+ imaging, Anders and colleagues found that the degree of ethanol inhibition of NR2B-containing receptors is unaffected by Fyn tyrosine kinase. Kalluri and Ticku (1999a) determined that in mouse fetal cortical neurons, acute or chronic ethanol treatments do not affect the total phosphorylation of the NR2B subunit. The effects of acute ethanol treatment on the phosphorylation of the NR2B subunit in the adult cortex and hippocampus were also examined. In adult mice, acute ethanol treatment increases the tyrosine phosphorylation of the NR2B in the hippocampus, but not in the cortex. Thus, although the functional significance of ethanol-induced changes in the phosphorylation of NR2B remains in question, it does appear that ethanol exposure contributes to alterations in the expression and phosphorylation of NR2Bcontaining receptors. Cocaine is a potent psychostimulant drug that acts by blocking dopamine uptake, thus increasing dopamine neurotransmission. The actions of cocaine, however, appear to be contingent not only upon the dopaminergic system, but
also upon the glutamatergic system, since glutamate antagonists inhibit many effects of cocaine. Cocaine administration also alters the expression of NMDA receptor subunits. Fitzgerald et al. (1996) showed that chronic cocaine administration up-regulates the NR1 subunit in the ventral tegmental area of the rat brain. Cocaine-induced changes in NR2A/B expression were not observed; however, the antibodies used were not able to differentiate between NR2A and NR2B subunits (Fitzgerald et al., 1996). Using a different method (immunohistochemistry) and more selective antibodies, Loftis and Janowsky (2000) observed that cocaine treatment caused brain region-specific and withdrawal time-dependent alterations in NR2B expression. After 24 hr of withdrawal, cocaine-induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine causes an 50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. Collectively, these results suggest that regionally specific changes in NMDA receptor protein expression may underlie neuronal adaptations following repeated cocaine administration and, therefore, may contribute to withdrawal-related symptomology. As with ethanol exposure, receptor subunit- and kinasespecific phosphorylation is likely an important regulator of the cocaine-induced changes in neurotransmission. Although there is little information on the effects of cocaine treatment on NR2B phosphorylation, the influence of protein kinases on the expression of behavioral sensitization to cocaine has been assessed. Prior to a challenge dose of cocaine, inhibitors of CaMKII (KN93), PKA (H-89), or PKC (bisindolymaleimide-I) were microinjected into the medial nucleus accumbens of rats repeatedly administered either cocaine or saline. None of the kinase inhibitors influenced the behavioral response induced by cocaine in saline-pretreated rats. Among cocaine-sensitized animals, KN93 or bisindolymaleimide-I blocks the expression of behavioral sensitization to cocaine, whereas H-89 has no effect (Pierce et al., 1998). Thus, specific kinases are hypothesized to promote the expression of behavioral sensitization to cocaine, as well as to influence the neurochemical adaptations accompanying repeated cocaine exposure (J. M. Loftis & A. Janowsky, in press). The NMDA receptor has also been implicated in opioid tolerance and withdrawal. The effects of continuous intracerebroventricular infusion of butorphanol, a mixed m/k opioid agonist, on the modulation of NR1, NR2A, NR2B, and NR2C gene expression were investigated using in situ techniques. Drug treatment results in significant modulations of NR1, NR2A, and NR2B mRNA levels. Specifically, NR2B mRNA is decreased in the cerebral cortex, caudate putamen, thalamus, and CA3 region of the hippocampus in withdrawn rats (7 hr after stopping the infusion) (Oh et al., 2000). In addition to assessing drug-induced changes in subunit expression, behavioral measures have also been
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used to evaluate the functional significance of individual receptor subunits on drug-related behaviors. Narita et al. (2000a) conducted a study to determine whether the NMDA receptor (NR1, NR2A, or NR2B) subunits influence the development of morphine-induced place preference using specific antibodies against the respective subunits in mice. Morphine produces a significant dose-dependent preference for the drug-associated place that is completely antagonized by intracerebroventricular pretreatment with an antibody specific to the C-terminal region of the NR2B subunits (but not the NR1 or NR2A subunits). A significant increase in NR2B subunit protein levels is apparent 24 hr after the last conditioning trial in the limbic forebrain obtained from morphine-conditioned mice, as compared with those in the saline-conditioned mice. The protein levels of NR1 or NR2A do not change after morphine conditioning, thus showing the morphine-induced changes to be specific for the NR2B subunit. 5.10. Pain perception The NR2B subunit is implicated in pain perception and, consequently, is being targeted for the development of novel antinociception pharmacotherapeutics (reviewed in Chizh et al., 2001a; Gurwitz & Weizman, 2002). In forebrain structures and regions of the spinal cord, NMDA receptors have an important role in persistent inflammatory pain by reinforcing glutamate sensory transmission (reviewed in Zhuo, 2002). Mice overexpressing the NR2B subunit in the anterior cingulate and insular cortices demonstrate a selective enhancement of persistent pain and allodynia. Specifically, transgenic and wild-type mice display no differences in their tail-flick response to pain or in their latencies to react to hot and cold noxious stimuli. Forebrain-targeted NR2B overexpression, however, does significantly enhance pawlicking behavior associated with prolonged noxious stimulation (peripheral formalin injection) and mechanical allodynia (Wei et al., 2001). NMDA receptor gene expression and neuronal activity in the rostral ventromedial medulla also plays a role in mediating pain perception. After complete Freund’s adjuvant-induced hindpaw inflammation, RTPCR analyses indicate that there is an up-regulation of the NR1, NR2A, and NR2B mRNA expression levels that is apparent 5 hr after inflammation, persists for 7 days, and is returned to baseline by 2 weeks following the inflammation (Miki et al., 2002). Thus, drugs targeting NR2B-containing NMDA receptors in pain-relevant structures, such as the dorsal horn of the spinal cord (Boyce et al., 1999) or anterior cingulate cortex (Wei et al. 2001), could serve as a new class of medicine for managing chronic pain in humans.
6. Conclusions The NR2B subunit of the NMDA-type glutamate receptor is involved in a diverse and extensive array of functions
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