Neuronal and Glial Localization of NR1 and NR2A/B Subunits of the NMDA Receptor in the Human Cerebral Cortex

Fiorenzo Conti, Paolo Barbaresi, Marcello Melone and Alessandro Ducati1

Institute of Human Physiology and , 1 Neurosurgical Clinic, University of Ancona, Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
N-Methyl-D-aspartate (NMDA) receptors play a critical role in many cortical functions and are implicated in several neuropsychiatric diseases. In this study, the cellular expression of the NMDAR1 (NR1) and NMDAR2A and B (NR2A and B) subunits was investigated in the human cerebral cortex by immunocytochemistry with antibodies that recognize the NR1 or the NR2A and B subunits of the NMDA receptor. In frontal (areas 10 and 46) and temporal (area 21) association cortices and the cingulofrontal transition cortex (area 32), NR1 and NR2A/B immunoreactivity (ir) were similar and were localized to numerous neurons in all cortical layers. NR1- and NR2A/B-positive neurons were mostly pyramidal cells, but some nonpyramidal neurons were also labeled. Electron-microscopic observations showed that NR1 and NR2A/B ir were similar. In all cases, labeling of dendrites and dendritic spines was intense. In addition, both NR1 and NR2A/B were consistently found in the axoplasm of some axon terminals and in distal astrocytic processes. This investigation revealed that numerous NMDA receptors are localized to dendritic spines, and that they are also localized to axon terminals and astrocytic processes. These findings suggest that the effects of cortical NMDA activation in the human cortex do not depend exclusively on the opening of NMDA channels located at postsynaptic sites, and that the localization of NMDA receptors is similar in a variety of mammalian species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The N-methyl-D-aspartate (NMDA) receptor, one of the ionotropic glutamate receptor subtypes, has been the focus of much experimental work for its unique mode of operation at central nervous system synapses, for its reported role in sensory transmission, synaptic plasticity, learning and memory, and circuit development, and for its alleged involvement in numerous neuropsychiatric disorders (Ascher et al., 1991Go; Nelson and Sur, 1992Go; Bliss and Collingridge, 1993Go; Daw et al., 1993Go; Collingridge and Watkins, 1994; Scheetz and Constantine-Paton, 1994Go; Mori and Mishina, 1995Go; Conti and Hicks, 1996Go; Tsien et al., 1996Go).

Molecular cloning has shown that NMDA receptors are formed by subunits belonging to the NMDAR1 (NR1) and NMDAR2 (NR2) classes in both rodents (Monyer et al., 1992Go; Nakanishi, 1992Go; Cik et al., 1993Go; Sheng et al., 1994Go; Sucher et al., 1996Go) and humans (Karp et al., 1993Go; Planells-Cases et al., 1993Go; Foldes et al., 1994Go; Le Bourdèlles et al., 1994Go; Adams et al., 1995; Zimmer et al., 1995Go; Hess et al., 1996Go), with high interspecific homology (>90–95% respectively). NR1 has the basic features of the NMDA receptor (Moriyoshi et al., 1991Go), it can exist in several isoforms generated by alternative splicing (Zukin and Bennett, 1995Go), and is the essential subunit of the NMDA receptor, as shown by the abolishment of classical NMDA neuronal responses following targeted disruption of the NR1 gene (Forrest et al., 1994Go; Li et al., 1994Go). The NR2 class includes four different subunits, NR2A-D, encoded by separate genes (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go; Ishii et al., 1993Go). In situ hybridization has shown that these subunits are expressed differently in the brain (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go; Ishii et al., 1993Go; Watanabe et al., 1993Go) and during development (Monyer et al., 1992Go; Farrant et al., 1994Go; Sheng et al., 1994Go). Electrophysiological studies have shown that NR2 subunits produce detectable currents only when they are coexpressed with NR1 (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go; Ishii et al., 1993Go), and that when NR1 is combined with different NR2 subunits, both electrophysiological and pharmacological responses are modified (Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992, 1994; Nakanishi, 1992; Stern et al., 1992; Ishii et al., 1993; Williams et al., 1993, 1994; Hollmann and Heinemann, 1994; McBain and Mayer, 1994; Flint et al., 1997; Anson et al., 1998; see also Buller et al., 1994).

Recent insights into the molecular biology of NMDA receptors have contributed to the generation of potent and specific tools for studying the localization of these receptors at the cellular level. In several laboratories, including ours, these tools have been employed to study the cellular localization and distribution of NMDA receptors in rat and monkey neocortex, one of the brain regions where the role of NMDA receptors appears particularly well documented (Conti and Hicks, 1996Go). These studies have shown that: (i) NMDA receptors are present in many but not all cortical neurons (Aoki et al., 1994Go; Conti et al., 1994Go; Huntley et al., 1994Go; Conti and Minelli, 1996Go) and that neurons expressing NMDA receptors appear to be less numerous in layer IV than in layers II–III and V–VI (Conti and Minelli, 1996Go); (ii) in cortical neurons NMDA receptors are mostly formed by NR1 and NR2A and/or B subunits (Moriyoshi et al., 1991Go; Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go, 1994Go; Nakanishi, 1992Go; Ishii et al., 1993Go; Wenzel et al., 1995Go; Conti et al., 1997Go; Scherzer et al., 1998Go); and (iii) the vast majority of NMDA receptors are located postsynaptically on dendrites and dendritic spines (Aoki et al., 1994Go; Huntley et al., 1994Go, 1997Go; Petralia et al., 1994aGo,bGo; Conti and Minelli, 1996Go; Conti et al., 1996Go, 1997Go; De Biasi et al., 1996Go). Based on this evidence, the bulk of the effects of NMDA receptor activation appear to be generated at distal dendrites and dendritic spines.

The results of some recent investigations, however, suggest that NMDA-mediated processes may also be due to the activation of NMDA receptors located at non-conventional sites. Indeed, it has been shown that: (i) some NMDA receptors are presynaptic hetero- and autoreceptors, as NR1- and NR2A/B-immunoreactivity (ir) can be observed at axon terminals forming both asymmetric and symmetric synapses (De Biasi et al., 1996; see also Aoki et al., 1994; Huntley et al., 1994; Johnson et al., 1996); and (ii) NMDA receptors are also expressed by astrocytes (Conti et al., 1996Go).

Whether these data also apply to the human cerebral cortex is at present a matter of mere conjecture, as the only available information on the expression of NMDA receptor subunits comes from the light microscopic examination of NR1-ir (Huntley et al., 1994Go, 1997Go). We therefore investigated the cellular localization of NR1 and NR2A/B subunits of the NMDA receptor in the human cerebral cortex using immunocytochemical techniques in bioptic samples. To address this issue, we used antibodies that recognize many NR1 splice variants or the NR2A and B subunits (Petralia et al., 1994aGo,bGo; see Discussion).


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue Preparation

Human cortical tissue came from the surgical specimens of five patients with brain tumors whose clinical data are summarized in Table 1Go. Three of the five tissue samples have been used also for another study (Conti et al., 1998Go). Informed consent to the surgical procedure was obtained in all cases.


View this table:
[in this window]
[in a new window]
 
Table 1 Summary of clinical data
 
The cortical tissue used in this study was not located in the vicinity of the tumor: it was macroscopically normal cortical tissue included in ‘tactical lobectomies' or that had to be resected in order to reach deep-seated tumors, and showed no signs of edema. None of the patients suffered from pre- or post-operative seizures. The approximate localization of the resected cortical samples is shown in Figure 1Go.



View larger version (58K):
[in this window]
[in a new window]
 
Figure 1.  Diagram based on the surgeon's estimate of the cortical resection and on magnetic resonance imaging findings, showing the approximate localization of the cortical samples used in the present study superimposed on Brodmann's cyto- architectonic map of the human cerebral cortex. Note that whereas in Brodmann's maps the lateral view of the brain refers to the left hemisphere, all our samples were from the right hemisphere.

 
Tissue samples, all from the right hemisphere, were immediately immersed in a cold solution of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 2–3 h, and then transferred to a fresh solution of 4% PFA for 24–48 h at 4°C. Samples were cut into small blocks that were further postfixed for 24–72 h at 4°C in the same solution. They were cut on a Vibratome in either coronal or parasagittal planes into 30–35 µm thick sections, collected in groups of five, and stored at 4°C in phosphate-buffered saline (PBS) until processing. Of the five sections, one was stained for Nissl substance with 0.1% thionine and the remaining four were used for immunocytochemistry.

Immunocytochemistry

Antibodies

The polyclonal antibodies against NR1 (AB1516/31697190) and NR2A/B (AB1548/90597149) used in the present study were purchased from Chemicon (Temecula, CA). Antibodies to NR1 were raised in rabbits against a 30 amino acid peptide corresponding to the C terminus of the rat NR1 subunit (Moriyoshi et al., 1991Go), while antibodies to NR2A/B were raised against a 20 amino acid synthetic peptide conjugated to bovine serum albumin (BSA) with glutaraldehyde which corresponds to the C-terminal sequence of NR2A (Monyer et al., 1992Go). Immunoblot analysis of human embryonic kidney cells (HEK-293) transfected with NMDA receptor subunits showed that the NR1 antibody is specific for this subunit and that it recognizes four NR1 splice variants (Petralia et al., 1994aGo), whereas the NR2A/B antibody recognizes NR2A and NR2B and, to a lesser extent, NR2C and NR2D (Petralia et al., 1994bGo). Further details on characterization and immunocytochemical application of these antibodies in the rat brain are reported in the original papers (Petralia et al., 1994aGo,bGo). In the present material, method specificity was controlled by substituting the primary antibodies with PBS or normal sheep serum (NSS) and by preadsorbing the NR1 synthetic peptide conjugated to BSA with the working dilution of both NR1 and NR2A/B (50 µl of the conjugate in 1 ml of antibodies concentrated at 1.5 µl/ml) before immunocytochemical processing.

Monoclonal antibodies to glial fibrillary acidic protein (GFAP; Eng, 1971Go; Bignami and Dahl, 1973Go), an astrocytic marker, were purchased from Sigma (St Louis, MO; clone G-A-5, G-3893/1014–4800).

Procedure

Sections from human cortical blocks were pretreated with a solution of ethanol and hydrogen peroxidase in PB to remove endogenous peroxidase activity, washed free-floating in PBS and incubated in the following solutions: 1% normal goat serum (NGS; 1 h) containing 0.3% Triton X-100 (GFAP); 10% NSS only (NR1 and NR2A/B). Sections were then washed in PBS and incubated overnight in primary antiserum at dilutions of 1.5–2.0 µg/ml (NR1 and NR2A/B) and 1:1000 (GFAP) in PBS. Sections were rinsed, incubated in biotinylated anti-rabbit (NR1 and NR2A/B) or anti-mouse (GFAP) secondary antibodies diluted 1:100 (NR1 and NR2A/B) or 1:200 (GFAP) in PBS for 1–2 h at room temperature, rinsed in PBS, then incubated in avidin–biotin peroxidase complex (ABC; Vector, Burlingame, CA) (Hsu et al., 1981Go) for 0.5 h, and rinsed in PBS. They were then incubated in 30–40 mg/100 ml 3–3'-diaminobenzidine (DAB; Sigma) with 0.02% H202 in 0.05 M Tris buffer, pH 7.6, washed in PBS, mounted on gelatin-coated slides, air-dried, washed, dehydrated and finally coverslipped.

For electron microscopy, a mild ethanol pretreatment (10%, 25%, 10%; 5 min each) was applied before the immunocytochemical procedure. NR1 and NR2A/B antibodies were used at a concentration of 1.5–2.0 µg/ml and Triton X-100 was not used. After completion of the immunocytochemical procedure, sections were washed in PB, incubated in 2.5% glutaraldehyde (30 min), washed in PB and postfixed for 1 h in 1% OsO4. After dehydration in ethanol and infiltration in Epon-Spurr resin, sections were flat-embedded between two Aclar (Sigma, 8F119)-coated coverslips. Small blocks, selected by light-microscopic inspection, were cut out, glued to blank-cured epoxy and sectioned with an ultramicrotome. Thin sections were lightly stained with lead citrate or left unstained and examined with a Philips CM10 electron microscope. Identification of neuronal and non-neuronal elements was performed using the criteria of Peters et al. (1991).

Cytoarchitectonic areas and boundaries were identified on thioninestained adjacent sections according to Brodmann (1909), von Economo (1928), Ong and Garey (1990), Rajkowska and Goldman-Rakic (1995) and Vogt et al. (1995).


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the five cases studied in the present investigation, the sections adjacent to those used for the immunocytochemical detection of NR1 and NR2A/B were processed for the visualization of Nissl substance and GFAP. Examination of Nissl-stained sections showed that they were consistent with previous descriptions and devoid of any appreciable abnormality (type="bib">Ong and Garey, 1990Go; Rajkowska and Goldman-Rakic, 1995Go; Vogt et al., 1995Go; Marco et al., 1996Go). The analysis of sections processed for GFAP evidenced abundant GFAP-positive cells and processes in layer I and in the white matter, but they were rare or absent in layers II–VI, in line with previous descriptions in normal tissue (Hansen et al., 1987Go; Marco et al., 1996Go; Perzel'ova et al., 1997Go).

In areas 10, 46, 21 and 32, NR1 and NR2A/B ir were localized to neuronal cell bodies, to proximal portions of dendrites, to numerous small punctate structures, and to puncta and fragments of thin fibers within the neuropil (Figs 2–5GoGoGoGo). Similar patterns of immunoreactivity were observed in sections processed for the visualization of NR1 and NR2A/B (Figs 2–4GoGoGo). Light-microscopic analysis of NR1 and NR2A/B immunocytochemically stained sections revealed that in the cerebral cortex astrocytes, oligodendrocytes, microglial cells and other nonneuronal cells appeared always unlabeled.



View larger version (88K):
[in this window]
[in a new window]
 
Figure 2.  Distribution of NR1 (A) and NR2A/B (B) immunoreactivity in frontal cortex (area 46). (C) Adjacent section stained for Nissl substance. Roman numerals indicate cortical layers. Case no. HBC980510. Scale bar: 100 µm.

 


View larger version (79K):
[in this window]
[in a new window]
 
Figure 3.  Distribution of NR1 (A) and NR2A/B (B) immunoreactivity in temporal cortex (area 21). (C) Adjacent section stained for Nissl substance. Roman numerals indicate cortical layers. Case no. HBC970508. Scale bar: 100 µm.

 


View larger version (83K):
[in this window]
[in a new window]
 
Figure 4.  Distribution of NR1 (A) and NR2A/B (B) immunoreactivity in cingulofrontal transition cortex (area 32). (C) Adjacent section stained for Nissl substance. Roman numerals indicate cortical layers. Case no. HBC970507. Scale bar: 100 µm.

 


View larger version (64K):
[in this window]
[in a new window]
 
Figure 5.  Morphology of NR1- and NR2A/B-positive neurons (AD) and appearance of neuropilar staining (E) in area 21 of human cerebral cortex. (A) and (B) show some heavily stained pyramidal neurons in layers V (A) and II–III (B), that are NR1- (A) and NR2A/B (B)-positive, whereas (C) and (D) provide examples of NR1- (C) and NR2A/B (D)-positive nonpyramidal neurons. (E) shows NR1-positive puncta in the neuropil. Scale bar: 10 µm.

 
In areas 10, 46, 21 and 32 NR1- and NR2A/B-positive neurons were in all cortical layers, though they appeared more numerous in layers II and III, while in infragranular layers they were more numerous in area 21 (Figs 2–4GoGoGo). The intense perikaryal labeling, continuous with an equally intense dendritic labeling, made it possible in most cases to distinguish pyramidal from nonpyramidal neurons (Conti et al., 1987Go, 1992Go for criteria). In all sections examined, NR1- and NR2A/B-positive neurons were both pyramidal and nonpyramidal (Fig. 5Go), though labeled pyramidal cells appeared more numerous than nonpyramidal ones.

Electron-microscopic studies showed that, with both antisera, the ultrastructural pattern of labeling was similar in all layers of the cortex. The electrondense reaction product of diaminobenzidine indicating NR1 and NR2A/B ir was present in neuronal cell bodies and processes, in axon terminals and in astrocytic profiles (Figs 6 and 7GoGo).



View larger version (146K):
[in this window]
[in a new window]
 
Figure 6.  Ultrastructural localization of NR1 in human cortex. (A) Two neuronal perikarya with different degrees of ir. Arrow in (A) indicates a small clump of immunoreaction product. (B) Clumps of reaction product (arrows) scattered in the cytoplasm of the neuron framed in (A). (C) NR1-ir in the preterminal portion of an axon whose terminal forms an asymmetric synapse with an unlabeled dendritic spine. (D) an unlabeled axon terminal forming an asymmetric synapse (arrowheads) with a dendritic spine (note the spine apparatus). (E) A labeled axon terminal (asterisk) forms a symmetric synapse (open arrowheads) with an unlabeled dendritic spine that forms also an asymmetric synapse (arrowheads) with an unlabeled axon terminal. (F) A labeled (asterisk) and an unlabeled axon terminal forming asymmetric synapses (arrowheads) with a labeled dendrite. (G) A labeled distal astrocytic profile (arrow) is adjacent to a labeled (asterisk) and an unlabeled axon terminal. Abbreviations: At, axon terminal; Ax, axon; Den, dendrite; sp, spine; sa, spine apparatus; AsP, astrocytic process; Nuc, nucleus. (AG) Lead citrate counterstaining. Case no. HBC980510. Scale bars: (A) 5 µm; (B,DF) 1 µm; (C,G) 0.5 µm.

 


View larger version (160K):
[in this window]
[in a new window]
 
Figure 7.  Ultrastructural localization of NR2A/B in human cortex. (A,B) Clumps of reaction product (arrows) in a neuronal cell body. (C) A labeled perivascular astrocytic profile (arrow) beneath the endothelium. (D) A labeled axon terminal forms an asymmetric synapse (arrowhead) with a spine stemming from a labeled dendrite. Note the spine apparatus (arrow). (E) Three axon terminals, two of which are labeled (asterisk), form asymmetric synaptic contacts (arrowheads) with an unlabeled dendrite. (F) A labeled distal astrocytic profile (arrow) adjacent to an unlabeled dendrite. Abbreviations: B, basal lamina; End, endothelial cell; for other abbreviations see legend to Figure 5Go. (AF) Lead citrate counterstaining. Case no. HBC980510. Scale bars: (A) 5 µm; (BE) 1 µm; (F) 0.5 µm.

 
Neuronal labeling was found throughout the somatic and dendritic cytoplasm, but not in the nucleus (Figs 6A,B, 7A,BGoGoGo). Labeled perikarya showed a patchy distribution of reaction product, often associated with several cytoplasmic organelles (Figs 5B, 6BGoGo). In the neuropil, numerous dendrites of all sizes (Figs 6F, 7D, 8BGoGoGo) and dendritic spines of variable shape were intensely immunostained. Labeled dendritic spines received asymmetric synapses from unlabeled boutons with round, clear vesicles (Figs 6D,F, 7EGoGo). In addition, in all the samples examined, immunoreactivity was consistently found in the preterminal and terminal portions of axons (Figs 6C,E-G, 7D,E, 8BGoGoGo) and in a few myelinated axons (not shown). Labeled terminals showed mostly an asymmetric synaptic specialization (Figs 6FGo, 7D,E, 8BGoGoGo) and less frequently a symmetric one (asterisk in Fig. 6EGo) and they contacted either unlabeled dendritic spines or distal dendritic shafts.



View larger version (109K):
[in this window]
[in a new window]
 
Figure 8.  Preabsorption tests in human cerebral cortex. (A) A sample incubated in the anti-NR1 serum preasorbed with NR1 peptide does not show immunolabeling. (B,C) Samples incubated in the anti-NR2A/B serum preadsorbed with NR1 peptide show immunoreactivity in neuronal and glial processes. Case no. HBC980611. Scale bar: (AC) 0.5 µm.

 
Glial labeling was confined to distal astrocytic processes (Figs 6F,G, 7C,F, 8CGoGoGo), whereas astroglial cell bodies were occasionally weakly labeled. No reaction product was found in oligodendrocytes (not shown). Astrocytic processes were characterized by extremely irregular contours (Figs 6F,G, 7FGoGo) or by their perivascular location beneath the endothelial basal lamina of capillaries (Fig. 7CGo). Labeled astrocytic processes in the neuropil generally surrounded labeled (Fig. 6FGo) or unlabeled (Figs 6F, 7E, 8CGoGoGo) axonal terminals with asymmetric specialization, but were also present in areas not related with synapses.

In sections incubated in the antiNR1 serum preadsorbed with the NR1 peptide, no labeling was observed either at light- (not shown) or electron-microscopic (Fig. 8AGo) level, whereas in sections incubated in the antiNR2A/B serum preadsorbed with the NR1 peptide the pattern of immunolabeling was analogous to that observed in sections processed with the NR2A/B antibody at both the light- (not shown) and electron-microscopic level (Fig. 8B,CGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present investigation of the distribution and cellular localization of the NR1 and NR2A/B subunits of the NMDA receptors in the human cerebral cortex confirmed the robust expression of this subtype of glutamate (Glu) receptors suggested by previous binding studies (Jansen et al., 1989Go; Albin et al., 1991Go) and revealed that: (i) the distribution of the NR2A/B subunits of the NMDA receptor is in all respects similar to that of NR1; (ii) dendritic spines express high levels of NR1 and NR2A/B subunits; (iii) both NR1 and NR2A/B subunits are localized also to excitatory and inhibitory axon terminals; and (iv) both NR1 and NR2A/B subunits are expressed by astrocytes. Thus, the effects of cortical NMDA activation in human cortex do not seem to depend exclusively on the opening of NMDA channels located at postsynaptic sites, and the localization of NMDA receptors appears to be similar in a variety of mammalian species.

Methodological Considerations

This study was designed to investigate the distribution of NR1 and NR2A/B ir in normal samples of human cerebral cortex. Accordingly, we selected cortical tissue that was macroscopically normal in appearance and that was included in tactical lobectomies or resected to reach deep-seated tumors in patients who had never suffered from pre- or postoperative seizures. We also verified histologically that the sections adjacent to those used for the immunocytochemical visualization of NR1 and NR2A/B exhibited no obvious abnormalities in their architectonics or GFAP ir. These strict criteria of sample selection and the histological controls allow us to assume that the cortical tissue samples used in the present study were normal. Since receptor immunoreactivity in rapidly fixed biopsy specimens is much stronger than in autoptic samples (e.g. Huntley et al., 1997Go), our study seems to have been performed under optimal experimental conditions.

To investigate the distribution of NR1 and NR2A/B in the cerebral cortex, affinity-purified polyclonal antibodies raised against synthetic peptides corresponding to the predicted C termini of rat NR1 (Moriyoshi et al., 1991Go) and NR2A (Monyer et al., 1992Go; Nakanishi, 1992Go) were used. As reported in Materials and Methods, immunoblot studies showed that the NR1 antibody is specific for this subunit and that it recognizes four NR1 splice variants (Petralia et al., 1994aGo), whereas the NR2A antibody recognizes NR2A and NR2B and, to a lesser extent, NR2C and NR2D (Petralia et al., 1994bGo). Preadsorption tests performed on the tissue used in these studies showed that NR1 ir is totally abolished in sections processed with antibodies that had been incubated overnight with the conjugate peptide, and that NR2A/B ir is unaffected by preadsorbtion of the NR2A/B antibodies with the NR1 conjugate peptide, thus indicating that NR2A/B antibodies does not cross-react with NR1. Although the possibility of a cross-reaction of NR1 or NR2A/B antibodies with unidentified proteins cannot be excluded, these data suggest that the antigens we have localized in these tissue samples are the NR1 and NR2A/B subunits of the NMDA receptors.

The rationale for the choice of the NR1 antibody used here lies in the fact that it recognizes many of the splice variants demonstrated to date (Zukin and Bennett, 1995Go). Given that NR1 splice variants are expressed differentially both at the cellular and the subcellular level (Laurie and Seeburg, 1994Go; Sheng et al., 1994Go; Standaert et al., 1994Go; Johnson et al., 1996Go), the recognition of several NR1 splice variants allows to achieve the labeling of most NR1 subunits and, therefore, of most, if not all, NMDA receptors. The NR2A antibody selected for this study recognizes both NR2A and NR2B subunits: this property is particularly useful for the analysis of NR2 expression in the cerebral cortex, where NR2A and NR2B are the only NR2 subunits expressed prominently (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go; Ishii et al., 1993Go; Watanabe et al., 1993Go; Wenzel et al., 1995Go).

Three methods can be used for the ultrastructural immunocytochemical localization of receptors, i.e. pre-embedding immunoperoxidase and pre- and postembedding immunogold (see Lujan et al., 1996; Ottersen and Landsend, 1997, for recent reviews). Although the postembedding immunogold method is the procedure of choice to detect the synaptic localization of receptors and for the quantitative analysis of receptor density, its major drawbacks are its very low sensitivity and the need to use nonstandard fixation and embedding procedures (Nusser et al., 1994Go; Baude et al., 1995Go; Phend et al., 1995Go; Propatiloff et al., 1996Go). Conversely, pre-embedding immunoperoxidase is the most sensitive procedure (Ottersen and Landsend, 1997Go) and allows correlations to be made between light- and electron-microscopical findings. It is therefore the method of choice to obtain information on the regional distribution of labeling and to visualize receptors at nonsynaptic sites (e.g. in distal glial processes). For the purpose of the present study, therefore, pre-embedding immunoperoxidase is clearly the method of choice.

Distribution of NR1 and NR2A/B Subunits of the NMDA Receptor in the Human Cerebral Cortex

Whereas the distribution of the NR1 subunit of the NMDA receptor has been analyzed at the light microscope in area 17 (Huntley et al., 1994Go) and in the prefrontal and temporal association cortices (Huntley et al., 1997Go), the distribution of the NR2A/B subunits has never been investigated in the human cerebral cortex. The results of the present light-microscopic analysis show that in areas 10, 46, 21 and 32 the distribution pattern of neurons expressing the NR2A and/or the NR2B subunit(s) is similar to that of neurons expressing NR1, and that the morphological features of NR1- and NR2A/B-positive neurons are also similar. These similarities in distribution are also evident at the electron-microscopic level, as the cellular localization of NR2A/B is indistinguishable from that of NR1.

These data are in line with the results of previous in situ hybridization (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go, 1994Go; Ishii et al., 1993Go), immunocyto-chemical (Petralia et al., 1994aGo,bGo; Conti et al., 1997Go) and histoblot (Wenzel et al., 1995Go) studies in rodent cerebral cortex which showed that the NR1 and NR2A and B subunits exhibit similar distribution patterns, and with the demonstration of triple subunit heteromeric NMDA receptors (NR1 + NR2A + NR2B) in cortical neurons (Sheng et al., 1994; see also Conti et al., 1997). Given that NR2C and NR2D are weakly expressed in rodent neocortex (Kutsuwada et al., 1992Go; Meguro et al., 1992Go; Monyer et al., 1992Go, 1994Go; Ishii et al., 1993Go), these results have generated the notion that in the rodent neocortex most cortical NMDA receptors are formed by NR1 and NR2A and/or B subunits. Analysis of the expression patterns of mRNAs for NMDA receptor subunits in the human cerebral cortex (Akbarian et al., 1996Go) supports this conclusion. Although the present results in human cerebral cortex do not address the stoichiometry of the human cortical NMDA receptors, they are nevertheless compatible with the notion that NMDA receptors in the cerebral cortex are formed preferentially by NR1 and NR2A and/or 2B subunits.

Neuronal Expression of NMDA Receptors in the Human Cerebral Cortex

The present investigation shows that, in areas 10, 46, 21 and 32 of the human cerebral cortex, neurons expressing NMDA receptors are located in all layers, but are more numerous in layers II–III, followed by infragranular layers, and that in these layers they are in most cases pyramidal neurons. In previous studies, Huntley et al. (1994, 1997) have shown a similar distribution pattern in human area 17 and in prefrontal and temporal association cortices. This organizational feature thus appears to be consistent across different cortical fields of the human cerebral cortex. Interestingly, the same pattern has been described in several neocortical areas of adult rats (Aoki et al., 1994Go; Conti et al., 1994Go, 1997Go) and monkeys (Huntley et al., 1994Go, 1997Go), which suggests that the distribution of NMDA receptors exhibits a striking interspecific similarity.

The electron-microscopic analysis performed in the present study has allowed us to garner information on the localization of NR1 and NR2A/B in the human cerebral cortex: both NR1 and NR2A/B exhibited a major localization to dendritic shafts and spines, and a minor localization to axon terminals. NR1 and NR2A/B immunoreactivities were concentrated in patches in the cytoplasm and dendrites, but the salient feature of NMDA receptors in human cortex is their localization to dendritic spines and shafts, which are in apposition to unstained axon terminals containing round vesicles. This localization pattern is identical to that observed in the neocortex of rats (Aoki et al., 1994Go; Conti et al., 1997Go) and monkeys (Huntley et al., 1994Go, 1997Go), and is in line with a large amount of anatomical, electro-physiological and pharmacological data pointing to a preferential relationship between NMDA receptors and dendritic spines (see Conti et al., 1997 for literature).

Ultrastructural studies showed that in the human cerebral cortex NMDA subunits R1 and R2A/B are localized to axon terminals forming both asymmetric and symmetric synapses. In previous investigations of the cellular localization of NMDA receptor subunits in the rat cerebral cortex, it has been shown that NR1 is localized to axon terminals forming asymmetric (Aoki et al., 1994Go; De Biasi et al., 1996Go) and symmetric (De Biasi et al., 1996Go) synapses, and that NR2A/B is expressed by axon terminals forming both symmetric and asymmetric synapses (Petralia et al., 1994bGo; De Biasi et al., 1996Go). In the monkey, rare NR1-positive axon terminals have been reported in primary cortices (Huntley et al., 1994Go), but not in association cortices (Huntley et al., 1997Go). Since presynaptic NMDA receptors have been reported also in the spinal cord (Liu et al., 1994Go), locus coeruleus (Van Bockstaele and Colago, 1996Go) and hippocampus (Siegel et al., 1994Go), presynaptic NMDA receptors appear to be a consistent, although minor, feature of brain organization. The identity of the synaptic transmitter released by NMDA-positive axon terminals has not been investigated directly in the present study; however, given that axon terminals forming asymmetric synapses are either Glu- or aspartate (Asp)-positive (DeFelipe et al., 1988Go; Conti et al., 1989Go), NMDA receptors in axon terminals forming asymmetric synapses are in all likelihood autoreceptors. The large majority of symmetric synapses in the neocortex are GABAergic (Kisvarday et al., 1990Go), and double-labeling studies have shown that in rat neocortex some NMDA receptors are heteroreceptors on GABAergic axon terminals (De Biasi et al., 1996Go); accordingly, the axon terminals forming symmetric synapses and expressing NMDA receptors observed in the present study are very likely to be GABAergic.

Glial Expression of NMDA Receptors in the Human Cerebral Cortex

This study shows that in the human cerebral cortex, astrocytes express the NR1 and NR2A and/or NR2B subunits of the NMDA receptor. Given the specificity of the antibodies used in the present investigation, the NR1 and NR2A/B ir observed in astrocytic processes is unlikely to reflect cross-reactivity with other Glu receptors expressed by astrocytes (Blankenfeld, 1995Go; Gallo and Russell, 1995Go), though the possibility that the antibodies might have cross-reacted with other unidentified membrane proteins cannot be ruled out. The use of techniques for achieving good preservation of nervous tissue has allowed us to define the electron-microscopic features of astrocytes and astrocytic processes (Peters et al., 1991Go), which can now be identified unambiguously (Peters et al., 1991; Privat et al., 1995; see also Minelli et al., 1995, 1996 for recent data from our laboratory). The specificity of the antibodies used here and the conservative criteria used to identify astrocytic processes support the conclusion that some astrocytic processes in the human cerebral cortex do express NMDA receptors. Under the present experimental conditions, the electron microscopic approach was deemed preferable to that based on molecular astrocytic markers such as GFAP or S-100, since GFAP does not label all astrocytes (Peters et al., 1991Go; Privat et al., 1995Go) and since S-100 also labels some neurons and oligodendrocytes (Rickmann and Wolff, 1995Go; Richterlandsberg and Heinrich, 1995Go). In addition, the localization of NR1 and NR2A/B ir to distal astrocytic processes makes their visualization in light-microscopic double-labeling experiments with GFAP or S-100 extremely difficult and therefore unreliable. The present results are in agreement with our previous observations of GFAP- positive cells expressing mRNA for NR1 and of the presence of NR1 and NR2A/B ir distal astrocytic processes in the rat neocortex (Conti et al., 1994Go, 1996Go), and emphasize the striking similarity in the localization of NMDA receptors between rodent and human cerebral cortex.

The preferential subcellular localization of NR1 and NR2A/B ir in astrocytic processes rather than in cell bodies presumably reflects the low cytoplasmic levels of a protein that is antero gradely transported, and suggests that they can monitor Glu release by neighboring axon terminals (DeFelipe et al., 1988Go; Conti et al., 1989Go) of thalamic and corticocortical origin, as well as by axon collaterals of cortical Gluergic neurons (Conti et al., 1987Go, 1988Go; Kharazia and Weinberg, 1994Go), thereby mediating part of the neuron–glia signaling mechanisms that regulate gene expression and responses to changes in Glu levels (e.g. Stewart et al., 1991Go; Herrera and Cuello, 1992Go; Uchihori and Puro, 1993Go). Astrocytic NMDA receptors may also participate in the mechanism(s) subserving activity-dependent cortical plasticity (Catalano et al., 1997Go; Rao and Craig, 1997Go).


    Notes
 
We wish to thank Dr Robert Wenthold (NIDCD/NIH, Bethesda) for providing us with the NR1 conjugate peptide, and Dr S. Modena for editorial assistance. This work was made possible by a grant from Telethon (962/1997) and was supported by funds from MURST and CNR.

Address correspondence to Fiorenzo Conti, Istituto di Fisiologia Umana, Università di Ancona, Via Tronto, 10/A Torrette di Ancona, I-60020 Ancona, Italy. E-mail: f.conti{at}popcsi.unian.it.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adams S-L, Foldes RL, Kamboy RK (1995) Human N-methyl-D-aspartate receptor modulatory subunit hNR3: cloning and sequencing of the cDNA and primary structure of the protein. Biochim Biophys Acta 1260:105–108.[ISI][Medline]

Akbarian S, Sucher NJ, Bradley D, Tafazzoli A, Trinh D, Hetrick WP, Potkin SG, Sandman CA, Bunney WE Jr, Jones EG (1996) Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics. J Neurosci 16:19–30.[Abstract]

Albin RL, Sakurai SY, Makowiec RL, Higgins DS, Young AB, Penney JB (1991) Excitatory amino acid, GABA-A, and GABA-B binding sites in human striate cortex. Cereb Cortex 1:499–509.[Abstract]

Anson LC, Chen PE, Wyllie DJA, Colquhoun D, Schoepfer R (1998) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. J Neurosci 18:581–589.[Abstract/Free Full Text]

Aoki C, Venkatesan C, Go C-G, Mong JA, Dawson TM (1994) Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats. J Neurosci 14:5202–5222.[Abstract]

Ascher P, Choi DW, Christen Y (1991) Glutamate, cell death, and memory. Berlin: Springer-Verlag.

Baude A, Nusser Z, Roberts JDB, Mulvihill E, McIllhinney RAJ, Somogyi (1995) High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus. Neuroscience 69:1031–1055.[ISI][Medline]

Bignami A, Dahl D (1973) Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibody to the glial fibrillary acidic protein. J Comp Neurol 153:27–38.[ISI]

Blankenfeld G, Enkuist K, Kettenmann H (1995) Gamma-aminobutyric acid and glutamate receptors. In: Neuroglia (Kettenmann H, Ransom B, eds), pp. 335–345. New York: Oxford University Press.

Bliss TVP, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31–39.[ISI][Medline]

Brodmann K (1909) Vergleichende Lokalisationslehre der Grosshirnrinde in ihren Prinzipien dargestellt auf Grund des Zellenbaues. Translated and edited by Garey LJ (1994) Localization in the cerebral cortex. London: Smith-Gordon.

Buller AL, Larson HC, Schneider BE, Beaton JA, Morrisett RA, Monaghan DT (1994) The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition. J Neurosci 14:5471–5484.[Abstract]

Catalano SM, Chang CK, Shatz CJ (1997) Activity-dependent regulation of NMDAR1 immunoreactivity in the developing visual cortex. J Neurosci 17:8376–8390.[Abstract/Free Full Text]

Cik M, Chazot PL, Stephenson FA (1993) Optimal expression of cloned NMDAR1/NMDAR2 heteromeric glutamate receptors: a biochemical characterization. Biochem J 296:877–883.[ISI][Medline]

Collingridge GL, Watkins JC (1994) The NMDA receptor. Oxford: Oxford University Press.

Conti F, Hicks TP (1996) Excitatory amino acids and the cerebral cortex. Cambridge, MA: MIT Press.

Conti F, Minelli A (1996) The anatomy of glutamatergic transmission. In: Excitatory amino acids and the cerebral cortex (Conti F, Hicks TP, eds), pp. 81–98. Cambridge, MA: MIT Press.

Conti F, Rustioni A, Petrusz P, Towle AC (1987) Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys. J Neurosci 7:1887–1901.[Abstract]

Conti F, Fabri M, Manzoni T (1988) Glutamate-positive cortico-cortical neurons in the somatic sensory areas I and II of cats. J Neurosci 8:2948–2960.[Abstract]

Conti F, DeFelipe J, Farinas I, Manzoni T (1989) Glutamate-positive neurons and axon terminals in cat sensory cortex: a correlative light and electron microscopic study. J Comp Neurol 290:141–153.[ISI][Medline]

Conti F, De Biasi S, Fabri M, Manzoni T (1992) Substance P-containing pyramidal neurons in the cat somatic sensory cortex. J Comp Neurol 322:136–148.[ISI][Medline]

Conti F, Minelli A, Molnar M, Brecha NC (1994) Cellular localization and laminar distribution of NMDAR1 mRNA in the rat cerebral cortex. J Comp Neurol 343:554–565.[ISI][Medline]

Conti F, De Biasi S, Minelli A, Melone M (1996) Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes. Glia 17:254–258.[ISI][Medline]

Conti F, Minelli A, De Biasi S, Melone M (1997) Neuronal and glial localization of NMDA receptors in the cerebral cortex. Mol Neurobiol 14:1–18.[ISI][Medline]

Conti F, Melone M, De Biasi S, Minelli A, Brecha NC, Ducati A (1998) Neuronal and glial localization of GAT-1, a high-affinity GABA plasma membrane transporter, in human cerebral cortex; with a note on its distribution in monkey cortex. J Comp Neurol 396:51–63.[ISI][Medline]

Daw NW, Stein PSG, Fox K (1993) The role of NMDA receptors in information processing. Annu Rev Neurosci 16: 207–222.[ISI][Medline]

De Biasi S, Minelli A, Melone M, Conti F (1996) Presynaptic NMDA receptors in the neocortex are both auto- and heteroreceptors. NeuroReport 7:2773–2776.[ISI][Medline]

DeFelipe J, Conti F, Van Eyck SL, Manzoni T (1988) Demonstration of glutamate-positive axon terminals forming asymmetric synapses in the cat neocortex. Brain Res 455:162–165.[ISI][Medline]

Eng LF (1971) An acidic protein isolated from fibrous astrocytes. Brain Res 28:351–354.[ISI][Medline]

Farrant M, Feldmeyer D, Takahashi T, Cull-Candy SG (1994) NMDA- receptor channel diversity in developing cerebellum. Nature 368: 335–339.[ISI][Medline]

Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H (1997) NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J Neurosci 17:2469–2476.[Abstract/Free Full Text]

Foldes RL, Adams S-L, Fantaske RP, Kamboj RK (1994) Human N-methyl-D-aspartate receptor modulatory subunit hNR2A: cloning and sequencing of the cDNA and primary structure of the protein. Biochim Biophys Acta 1223:155–159.[ISI][Medline]

Forrest D, Yuzaki M, Soares HD, Ng L, Luk DC, Sheng M, Stewart CL, Morgan JL, Connor JA, Curran T (1994) Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death. Neuron 13:325–338.[ISI][Medline]

Gallo V, Russell JT (1995) Excitatory amino acid receptors in glia: different subtypes for distinct functions? J Neurosci Res 42:1–8.[ISI][Medline]

Hansen LA, Armstrong DM, Terry RD (1987) An immunohistochemical quantification of fibrous astrocytes in the aging human cerebral cortex. Neurobiol Aging 8:1–6.[ISI][Medline]

Herrera DG, Cuello AC (1992) MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain. Brain Res 598:286–293.[ISI][Medline]

Hess SD, Daggett LP, Crona J, Deal C, Lu C-C, Urrutia A, Chavez-Noriega L, Ellis SB, Johnson EC, Velicelebi G (1996) Cloning and functional characterization of human heteromeric N-methyl-D-aspartate recep- tors. J Pharmacol Exp Ther 278:808–816.[Abstract]

Hollmann M, Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neurosci 17:31–108.[ISI][Medline]

Hsu SM, Raine L, Fanger H (1981) Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:557–580.

Huntley GW, Vickers JC, Janssen W, Brose N, Heinemann SF, Morrison JH (1994) Distribution and synaptic localization of immunocytochem- ically identified NMDA receptor subunit proteins in sensory-motor and visual cortices of monkey and human. J Neurosci 14:3603–3619.[Abstract]

Huntley GW, Vickers JC, Morrison JH (1997) Quantitative localization of NMDAR1 receptor subunit immunoreactivity in inferotemporal and prefrontal association cortices of monkey and human. Brain Res 749:245–262.[ISI][Medline]

Ishii T, Moriyoshi K, Sugihara H, Sakurada K, Kadotani H, Yokoi M, Akawaza C, Shigemoto R, Mizuno N, Masu M, Nakanishi S (1993) Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. J Biol Chem 268:2836–2843.[Abstract/Free Full Text]

Jansen KLR, Faull RLM, Dragunow M (1989) Excitatory amino acid receptors in the human cortex: a quantitative autoradiographic study comparing the distributions of [3H]TCP, [3H]glycine, L-[3H]glutamate, [3H]AMPA, and [3H]kainic acid binding sites. Neuroscience 32: 587–607.[ISI][Medline]

Johnson RR, Jiang X, Burkhalter A (1996) Regional and laminar differences in synaptic localization of NMDA receptor subunit NR1 splice variants in rat visual cortex and hippocampus. J Comp Neurol 368:335–355.[ISI][Medline]

Karp SJ, Masu M, Eki T, Ozawa K, Nakanishi S (1993) Molecular cloning and chromosomal localization of the key subunit of the human N-methyl-D-aspartate receptor. J Biol Chem 268:3728–3733.[Abstract/Free Full Text]

Kharazia VN, Weinberg RJ (1994) Glutamate in thalamic fibers termin- ating in layer IV of primary sensory cortex. J Neurosci 14:6021–6032.[Abstract]

Kisvarday ZF, Gulyas A, Beroukas D, North JB, Chubb IW, Somogyi P (1990) Synapses, axonal and dendritic patterns of GABA-immuno- reactive neurons in human cerebral cortex. Brain 113:793–812.[Abstract]

Kutsuwada T, Kashiwabuchi N, Mori M, Sakimura K, Kushiya E, Araki K, Meguro H, Masaki H, Kumanishi T, Arakawa M, Mishina M (1992) Molecular diversity of the NMDA receptor channel. Nature 358:36–41.[ISI][Medline]

Laurie DJ, Seeburg PH (1994) Regional and developmental heterogeneity in splicing of the rat brain NR1 mRNA. J Neurosci 14:3180–3194.[Abstract]

Le Bourdèlles B, Wafford KA, Kemp JA, Marshall G, Bain C, Wilcox AS, Sikela JM, Whiting PJ (1994) Cloning, functional coexpression, and pharmacological characterisation of human cDNAs encoding NMDA receptor NR1 and NR2A subunits. J Neurochem 62:2081–2098.

Li Y, Erzurumlu R, Chen C, Zhaveri S, Tonegawa S (1994) Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice. Cell 76:427–437.[ISI][Medline]

Liu H, Wang H, Sheng M, Yan LY, Jan YN, Basbaum AI (1994) Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn. Proc Natl Acad Sci USA 91:8383–8387.[Abstract]

Lujan R, Nusser Z, Roberts JDB, Shigemoto R, Somogyi P (1996) Peri- synaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur J Neurosci 8:1488–1500.[ISI][Medline]

Marco P, Sola RG, Pulido P, Alijarde MT, Sanchez A, Ramon y Cajal S, DeFelipe J (1996) Inhibitory neurons in the human epileptogenic temporal neocortex. An immunocytochemical study. Brain 119: 1327–1347.[Abstract]

McBain CJ, Mayer ML (1994) N-Methyl-D-aspartic receptor structure and function. Physiol Rev 74:723–760.[Free Full Text]

Meguro H, Mori H, Araki K, Kushiya E, Kutsuwada T, Yamazaki M, Kumanishi T, Arakawa M, Sakimura K, Mishina M (1992) Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs. Nature 357:70–74.[ISI][Medline]

Minelli A, Brecha NC, Karschin C, De Biasi S, Conti F (1995) GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex. J Neurosci 15: 7734–7746.[Abstract]

Minelli A, De Biasi S, Brecha NC, Vitellaro-Zuccarello L, Conti F (1996) GAT-3, a high-affinity GABA plasma membrane transporter, is local- ized to astrocytic processes, and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex. J Neurosci 16:6255–6264.[Abstract/Free Full Text]

Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH (1992) Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256:1217–1221.[ISI][Medline]

Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529–540.[ISI][Medline]

Mori H, Mishina M (1995) Structure and function of the NMDA receptor channel. Neuropharmacology 34:1219–1237.[ISI][Medline]

Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S (1991) Molecular cloning and characterization of the rat NMDA receptor. Nature 354:31–37.[ISI][Medline]

Nakanishi S (1992) Molecular diversity of glutamate receptors and implications for brain function. Science 258:597–603.[ISI][Medline]

Nelson S, Sur M (1992) NMDA receptors in sensory information processing. Curr Opin Neurobiol 2:484–488.[Medline]

Nusser Z, Mulvihill E, Streit P, Somogyi P (1994) Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization. Neuroscience 61:421–427.[ISI][Medline]

Ong WY, Garey LJ (1990) Neuronal architecture in the human temporal cortex. Anat Embryol 181:351–364.[ISI][Medline]

Ottersen OP, Landsend AS (1997) Organization of glutamate receptors at the synapse. Eur J Neurosci 9:2219–2224.[ISI][Medline]

Perzel’ova A, Macikova I, Mraz P, Bizik I, Steno J (1997) What is the role of protoplasmic astrocytes in adult human brain cortex? DFG Study Group on Functions of Glial Cells: 120.

Peters A, Palay SL, Webster H de F (1991) The fine structure of the nervous system. neurons and their supportive cells. New York: Oxford University Press.

Petralia RS, Yokotani N, Wenthold RJ (1994a) Light and electron micro- scope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody. J Neurosci 14:667–696.[Abstract]

Petralia RS, Wang Y-X, Wenthold RJ (1994b) The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. J Neurosci 14:6102–6120.[Abstract]

Phend KD, Rustioni A, Weinberg RJ (1995) An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. J Histochem Cytochem 43:283–292.[Abstract/Free Full Text]

Planells-Cases R, Sun W, Ferrer-Montiel AV, Montal M (1993) Molecular cloning, functional expression, and pharmacological characterization of an N-methyl-D-aspartate receptor subunit from human brain. Proc Natl Acad Sci USA 90:5057–5061.[Abstract]

Privat A, Gimenez-Ribotta M, Ridet J-L (1995) Morphology of astrocytes. In: Neuroglia (Kettenmann H, Ransom B, eds), pp. 3–22. New York: Oxford University Press.

Propatiloff A, Weinberg RJ, Rustioni A (1996) AMPA receptor subunits underlying terminals of fine-caliber primary afferent fibers. J Neurosci 16:3363–3372.[Abstract/Free Full Text]

Rajkowska G, Goldman-Rakic PS (1995) Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria. Cereb Cortex 5:307–322.[Abstract]

Rao A, Craig AM (1997) Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 19:801–812.[ISI][Medline]

Richterlandsberg C, Heinrich M (1995) S-100 immunoreactivity in rat brain glial cultures is associated with both astrocytes and oligo- dendrocytes. J Neurosci Res 42:657–665.[ISI][Medline]

Rickmann M, Wolff JR (1995) S100 protein expression in subpopulations of neurons of rat brain. Neuroscience 67:977–991.[ISI][Medline]

Scheetz AJ, Constantine-Paton M (1994) Modulation of NMDA receptor function: implications for vertebrate neural development. FASEB J 8:744–752.

Scherzer CR, Landwehrmeyer GB, Kerner JA, Counihan TJ, Kosinski CM, Standaert DG, Daggett LP, Velicelebi G, Penney JB Jr, Young AB (1998) Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: hippocampus and cortex. J Comp Neurol 390:75–90.[ISI][Medline]

Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY (1994) Changing subunit composition of heteromeric NMDA receptors during develop- ment of rat cortex. Nature 368:144–147.[ISI][Medline]

Siegel SJ, Brose N, Janssen WG, Gasic GP, Jahn R, Heinemann SF, Morrison JH (1994) Regional, cellular, and ultrastructural distribution of N-methyl-D-aspartate receptor subunit 1 in monkey hippocampus. Proc Natl Acad Sci USA 91:564–568.[Abstract]

Standaert DG, Testa CM, Young AB, Penney JB (1994) Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat. J Comp Neurol 343:1–16.[ISI][Medline]

Stern P, Béhè P, Schoepfer R, Colquhoun D (1992) Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors. Proc R Soc Lond B 250:271–277.[ISI][Medline]

Stewart O, Torre ER, Tomasulo R, Lothman E (1991) Neuronal activity up-regulates astroglial gene expression. Proc Natl Acad Sci USA 88:6819–6823.[Abstract]

Sucher NJ, Awobuluyi, Choi Y-B, Lipton SA (1996) NMDA receptors: from genes to channels. Trends Pharmacol Sci 17:348–355.[ISI][Medline]

Tsien JZ, Huerta PT, Tonegawa S (1996) The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity. Cell 87: 1317–1326.[ISI][Medline]

Uchihori Y, Puro DG (1993) Glutamate as a neuron-to-glia signal for mitogenesis: role of glial N-methyl-D-aspartate. Brain Res 613:212–220.[ISI][Medline]

Van Bockstaele EJ, Colago EOE (1996) Selective distribution of the NMDA-R1 glutamate receptor in astrocytes and presynaptic axon terminals in the nucleus locus coeruleus of the rat brain: an immuno- electron microscopic study. J Comp Neurol 369:483–496.[ISI][Medline]

Vogt BA, Nimchinsky EA, Vogt LJ, Hof PR (1995) Human cingulate cortex: surface features, flat maps, and cytoarchitecture. J Comp Neurol 359:490–506.[ISI][Medline]

von Economo C (1928) La citoarchitettonica della corteccia cerebrale. Bologna: Cappelli.

Watanabe M, Inoue Y, Sakimura K, Mishina M. (1993) Distinct distri- butions of five N-methyl-D-aspartate receptor channel subunit mRNAs in the forebrain. J Comp Neurol 338:377–390.[ISI][Medline]

Wenzel A, Scheurer L, Kunzi R, Fritschy JM, Mohler H, Benke D (1995) Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C, and 2D in rat brain. NeuroReport 7:45–48.[ISI][Medline]

Williams K, Russell SL, Shen YM, Molinoff PB (1993) Developmental switch in the expression of NMDA receptors occurs in vivo and in vitro. Neuron 10:267–278.[ISI][Medline]

Williams K, Zappia AM, Pritchett DB, Shen YM, Molinoff PB (1994) Sensitivity of the N-methyl-D-aspartate receptor to polyamine is controlled by NR2 subunits. Mol Pharmacol 45:803–809.[Abstract]

Zimmer M, Fink TM, Franke Y, Lichter P, Spiess J (1995) Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1). Gene 159:219–223.[ISI][Medline]

Zukin RS, Bennett MVL (1995) Alternatively spliced isoforms of the NMDAR1 receptor subunit. Trends Neurosci 18:306–313.[ISI][Medline]