quantitative localization of nmdar1 receptor subunit immunoreactivity in inferotemporal and...

Ž .Brain Research 749 1997 245–262

Research report

Quantitative localization of NMDAR1 receptor subunit immunoreactivity ininferotemporal and prefrontal association cortices of monkey and human

G.W. Huntley a,), J.C. Vickers c, J.H. Morrison a,b

a Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai School of Medicine, One GustaÕe L. LeÕy Place, New York, NY 10029-6574, USAb Department of Geriatrics and Adult DeÕelopment, Box 1065, Mount Sinai School of Medicine, One GustaÕe L. LeÕy Place, New York, NY 10029-6574,

USAc Neurobiology Laboratory, DiÕision of Pathology, UniÕersity of Tasmania, Hobart, Tasmania, Australia

Accepted 2 July 1996

Abstract

Ž .The cellular and synaptic localization of immunoreactivity for the N-methyl-D-aspartate NMDA receptor subunit, NMDAR1, wasinvestigated in inferotemporal and prefrontal association neocortices of monkeys and humans. In all monkey association areas examined,the laminar distribution patterns of NMDAR1 immunoreactivity were similar, and characterized by predominant pyramidal-like neuronallabeling in layers II, III, V and VI and a dense neuropil labeling consisting of intensely stained puncta and fine-caliber processes presentthroughout layers I–III, and V–VI. Layer IV, in contrast, contained only very lightly immunostained neurons which mostly lackedextensive dendritic staining. The laminar distribution of NMDAR1 immunolabeling in human association cortex was similar to thatobserved in monkeys. Electron microscopy of monkey areas 46 and TE1 confirmed that intensely immunoreactive asymmetricalpostsynaptic densities were present throughout all cell-dense layers of prefrontal and inferotemporal association cortex. Quantitativeanalyses of the laminar proportions of immunoreactive synapses demonstrated that in both areas examined, the percentages ofimmunolabeled synapses were mostly similar across superficial layers, layer IV and infragranular layers. Finally, quantitative double-labeling immunofluorescence for non-NMDA receptor subunits or calcium-binding proteins demonstrated that virtually all GluR2r3 orGluR5r6r7-immunoreactive neurons were also labeled for NMDAR1, while regionally-specific subsets of parvalbumin-, calbindin- andcalretinin-immunoreactive neurons were co-labeled. These data indicate that in primate association cortex, NMDA receptors areheterogeneously distributed to subsets of functionally distinct types of neurons and subsets of excitatory synapses, suggesting a criticaland highly specific role in mediating the activity of excitatory connectivity which converges on cortical association areas.

Keywords: N-Methyl-D-aspartate receptor; AMPA; Calcium-binding protein; Immunocytochemistry; Primate

1. Introduction

Ž .The role of N-methyl-D-aspartate NMDA receptors inneocortical circuitry has been studied most extensively inthe visual, somatic sensory and motor cortex. In theseareas, combined pharmacological and physiological studiesin vivo indicate that NMDA receptors contribute substan-tially to the generation of normal, excitatory synapticactivity, particularly in supragranular layersw x3,30,40,71,86,90,99 , and also contribute to the kinds oflong-term synaptic modifications which may underlie

w xlearning and memory 4,8,42,57–59,97 . Such NMDA re-ceptor-mediated activity most likely arises from local,

) Ž .Corresponding author. Fax: q1 212 996-9785.

intracortical sources, since NMDA receptors do not appearto mediate monosynaptic thalamocortical activity in adultsw x3,86 . These data suggest that NMDA receptors may belocalized to particular functional circuits, which is consis-tent with anatomical studies of the cellular and synapticlocalization of immunocytochemically identified NM-

w xDAR1 subunits 2,49,50,91 . In monkey sensory-motor andvisual cortex, for example, NMDAR1 immunoreactivity is

Žfound within the majority of spiny neurons pyramidal.cells and spiny stellate cells , but is heterogeneously dis-

tributed across subpopulations of GABAergic interneuronsidentified by calcium-binding protein immunoreactivityw x49 . In addition, NMDAR1-immunoreactive asymmetricpostsynaptic densities form only a proportion of the totalexcitatory synapses in monkey primary sensory and motorcortex, and such proportions vary across layers and across

w xareas 49 .

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 96 00847-5

( )G.W. Huntley et al.rBrain Research 749 1997 245–262246

NMDA receptors are also abundant in higher-orderassociation neocortical areas of the primate frontal andtemporal lobes, as suggested by ligand-binding autoradiog-

w xraphy 32,53 and by more recent localization studies ofw xNMDA receptor subunit transcripts 1 . Administration of

ketamine and phencyclidine, two types of NMDA receptorchannel antagonists, impairs aspects of normal cognitivefunctions which are thought to depend on prefrontal cortexw x12,101 , and, in humans, can lead to disturbances similar

w xto those observed in schizophrenics 54,60 . A recent studyhas demonstrated significant changes in the expression ofNMDA receptor subunits in the prefrontal cortices of

w xschizophrenics 1 , consistent with their possible involve-ment in the kinds of cognitive disturbances which havebeen linked to dysfunction of cortical association areasw x109 . Association areas of the prefrontal and temporalcortex are also particularly vulnerable to the pathophysiol-ogy of several other major types of disorders, including,for example, Alzheimer’s disease and epileptogenesis,which may involve, in part, chronic excitotoxic mecha-nisms through excessive activation of NMDA receptorsw x37,69,70 . Overall, these data strongly support a promi-nent role for NMDA receptors in the functional activity ofneocortical association areas. However, the cellular andsynaptic distribution of NMDA receptor subunit proteinshave not been investigated in primate association cortex,details which are important for understanding their role inmediating the activity of specific cells and pathways. Theaim of the current study, therefore, is to determine thenormal cellular and synaptic distribution of immuno-reactivity for the obligatory NMDA receptor subunit R1 inmonkey and human association neocortex using an anti-body which recognizes all known alternatively spliced

w xvariants of NMDAR1 91 .

2. Materials and methods

2.1. Animals

ŽTissue from eight, male cynomolgus monkeys Macaca.fascicularis; aged 2–4 years was used in this and previous

studies. All procedures relating to the care and treatment ofthe animals were in strict accordance with institutional andNIH guidelines. Monkeys were deeply anesthetized with a

Ž .mixture of ketamine hydrochloride 25 mgrkg, i.p. andŽ .sodium pentobarbital 30 mgrkg, i.v. , and were perfused

transcardially through the left ventricle first with cold 1%paraformaldehyde in 0.1 M phosphate-buffered salineŽ .PBS for one min, followed by cold 4% paraformaldehydein PBS for 8 to 10 min. Brains were rapidly removed,blocked and postfixed at 48C in 4% paraformaldehyde foran additional 6 h. Some of the blocks containing thefrontal and temporal lobes were then washed repeatedly inPBS and sectioned on a vibratome at a setting of 50 mm.Other blocks were cryoprotected in 30% sucroserPBS,

Žfrozen and sectioned on a cryostat at 40 mm for im-. Žmunoperoxidase material or 15 mm for immunofluo-

.rescent material .

2.2. Human material

Human material was obtained both as post mortemspecimens and as biopsy specimens. For the post mortemmaterial, 40 mm cryostat sections containing the hip-pocampal formation and adjacent parahippocampus andtemporal neocortex were obtained from the Institute of

ŽBiogerontology Research Tissue Donation Program Sun.City, AZ . The post mortem intervals of all autopsy mate-

rial ranged from 3.5 to 5 h. All post mortem material wasimmersion-fixed in 4% paraformaldehyde in 0.1 M PBSfor 24 to 48 h followed by washes either in a series of

Ž .graded sucrose solutions 12%, 16% and 18% or a seriesŽof graded glycerol solutions 10% and 20% glycerol in 2%

.dimethyl sulfoxide .Samples of biopsy material of human association neo-

cortex from three individuals who underwent neurosurgicalprocedures to remove brain tumors were obtained from theDivision of Neuropathology, Mount Sinai Medical Center,where the tissues were sent for routine diagnostic evalua-tion. The samples corresponded to dorsolateral prefrontal

Ž .neocortex from one case area 9 and samples of homotyp-Žical temporal association cortex one from mid temporal

.gyrus, the other from an unspecified temporal region fromtwo cases. Biopsy material was fixed immediately in 4%paraformaldehyde in 0.1 M PBS for 6 to 24 h followed bywashes in PBS and sectioning on a vibratome at a settingof 50 mm. A parallel series of sections from each blockstained with thionin revealed a normal cellular organiza-tion.

2.3. Immunocytochemistry

The production and characterization of the anti-w xNMDAR1 antibody has been described previously 91 .

ŽBriefly, a mouse monoclonal antibody IgG type, clone.54.1 was raised against a fusion protein spanning amino

acids 660 to 811, a region which is now thought tocorrespond to a putative extracellular portion of the sub-

w xunit protein 13,46 . Specificity of the antibody was veri-fied previously by immunocytochemistry and Westernblotting of human embryonic kidney 293 cells transientlytransfected with NMDAR1 or NMDAR2A, as well as by

w xWestern blotting of monkey hippocampal tissue 91 .The effects of permeabilization on NMDAR1 immuno-

reactivity was tested in both vibratome- and cryostat-cutmonkey sections by diluting the primary antibody 1:500 ineither PBS alone or in PBS containing 0.3% Triton X-100.Monkey sections were incubated in one or the other pri-mary antibody solution for 12–15 h at 48C. The humansections were incubated only in primary antibody diluted1:500 in PBS alone. All monkey and human sections were

( )G.W. Huntley et al.rBrain Research 749 1997 245–262 247

then processed using the Vectastain ABC immunoperoxi-Ž .dase kit Vector Labs . Immunoreactivity was visualized

X Ž .with 3,3 -diaminobenzidine DAB and hydrogen peroxide.Sections were mounted and coverslipped in DPX mountingmedia. Control sections were processed immunocytochem-ically as described following the preabsorption of antibody54.1 with the fusion protein. No specific immunolabelingwas detected under these conditions. A parallel series ofsections from each block was mounted and stained withthionin. Such sections were used to establish areal andlaminar boundaries according to the nomenclature of

w xWalker 104 for delineation of prefrontal neocortex andw xSeltzer and Pandya 89 for delineation of inferotemporal

neocortex.For double labeling immunofluorescence, both vi-

bratome- and cryostat-cut monkey sections were incubatedwith 54.1 diluted 1:500 in PBS in combination with one ofthe following monoclonal antibodies or polyclonal antis-

Žera: mouse anti-GluR5r6r7 antibody 1:1000; IgM type,w x. Žclone 4F5 48 ; rabbit anti-GluR2r3 antisera 1:100;w x.Chemicon, 107 ; rabbit polyclonal antisera to the calcium

binding proteins parvalbumin, calbindin and calretininŽ .1:2000; Swant, Switzerland ; and mouse anti-MAP2 anti-

Ž .body Sigma Immunocytochemicals, USA . For mouseIgG and rabbit IgG primary antibody combinations, themouse IgG was visualized using a secondary antibody

Ž .directly conjugated to FITC Vector Labs, 1:200 , whilethe rabbit IgG was visualized using a biotinylated sec-ondary antibody followed by streptavidin-Texas RedŽ .Amersham, 1:200 . For mouse IgG and mouse IgM com-binations, the mouse IgG was visualized with a gammachain-specific, horse anti-mouse IgG conjugated to FITCŽ .Vector Labs, 1:200 , whereas the mouse IgM was visual-ized with a mu chain-specific, biotinylated goat anti-mouse

Ž .IgM antibody Vector Labs, 1:200 followed by strepta-Ž .vidin-Texas Red Amersham; 1:200 . Control sections were

used to test all secondary antibodies for non-specific bind-ing to sections lacking a primary antibody incubation, or totest for cross-reactivity to the inappropriate primary anti-body in the double labeling combinations. At the antibodyconcentrations used, non-specific binding and cross-reac-tivity of the secondary antibodies was not observed. Sec-tions were viewed under epifluorescence using a 40=Neofluor objective and filter blocks selective for visualiz-ing FITC or Texas Red.

2.4. Electron microscopy

Sections from blocks of monkey temporal cortical areaTE1 and prefrontal cortical area 46 were cut on a vi-bratome at a setting of 100 mm, incubated in primaryantibody 54.1 diluted 1:500 in PBS alone, and processedimmunocytochemically as described. Following the DABreaction, sections were treated with 1% osmium tetroxidefor 1 h, dehydrated and infiltrated with Araldite resin.After polymerization, a scalpel was used while viewing the

plastic-embedded sections under a dissecting microscopeto cut out smaller strips of tissue containing only superfi-

Ž . Ž .cial layers II and III ; layer IV; or deep layers V and VI .After flat embedding the small strips in Araldite resin,sections were cut at 50–70 nm and collected on 100-meshgrids. The sections were not counter-stained with leadcitrate or uranyl acetate. However, to increase contrast, theaperture was set to the smallest opening while viewing andphotographing the thin sections on a Zeiss CH10 electronmicroscope.

2.5. QuantitatiÕe analyses

Areas TE1 and 46 were examined quantitatively fordetermination of NMDAR1 subunit colocalization withnon-NMDA receptor subunits or calcium-binding proteins,as well as for determination of relative percentages ofimmunolabeled synapses.

For each double-labeling combination, the percentageof double-labeled cells was determined by counting the

Ž .numbers of single NMDAR1 - and double-labeled cells in15 separate, pia-to-white matter traverses from each of twoanimals. No attempt was made to correlate precisely thelocation of double-labeled cells with laminar boundaries.

For electron microscopy, the percentages of NMDAR1immunoreactive synapses were determined from thin sec-tions taken from two monkeys. Sections which had beenexcised to include only layers IIrIII, layer IV or layers

Ž .VrVI see above were viewed and photographed at amagnification of 10 000= . Unambiguously identified im-munolabeled and unlabeled asymmetrical postsynapticdensities were counted from photographs printed at a finalmagnification of approximately 31 000= by two investiga-

Ž .tors blind to the area and layer s . In most cases, theidentification of a labeled synapse was always associatedwith the presence of DAB immunoprecipitate in the adja-cent cytoplasm. Structures not clearly identified were notcounted. The regions counted were carefully chosen basedon the presence of immunoreactivity throughout the thinsection, thereby insuring adequate antibody penetration. Atotal of 2388 synapses were counted from the two areasexamined, and the percentage of the total which wereNMDAR1 immunopositive determined for each laminarsample from each region.

3. Results

3.1. Determination of optimal immunocytochemical condi-tions

Vibratome sections of monkey inferotemporal and pre-frontal association cortex incubated in primary antibody54.1 diluted in PBS alone revealed robust NMDAR1 im-

Ž .munolabeling of cell somata and dendrites Figs. 1–3 . Inaddition, a dense meshwork of mostly small, obliquely


oriented labeled processes and fine punctate labeling werealso observed throughout the neuropil of all cortical layersŽ .Fig. 2 . This general labeling pattern was identical to thatfound previously using the same monoclonal antibody insimilarly prepared material from monkey sensory-motor

w x w xand visual cortex 49 and hippocampus 91 . In contrast,vibratome sections incubated in diluent containing 0.3%Triton X-100, as well as frozen cryostat sections incubatedin diluent that either contained or lacked 0.3% TritonX-100, resulted in NMDAR1 labeling which appearedmostly restricted to cell somata and short lengths of theirmost proximal dendrites, while the dense meshwork ofneuropil labeling described above was greatly diminishedŽ .data not shown .

In the human material taken from post mortem speci-mens, NMDAR1 labeling appeared very weak to absent inboth vibratome and cryostat sections. This material wasnot examined further. However, vibratome sections takenfrom the biopsy specimens revealed a cellular labelingpattern largely similar to that observed in monkey vi-

Ž .bratome sections Fig. 8; described in more detail below .

3.2. Distribution patterns of NMDAR1 immunoreactiÕity inmonkey inferotemporal and prefrontal association cortex

Based on the optimal immunolabeling conditions de-scribed above, the following observations were taken frommonkey neocortical sections cut on a vibratome and incu-bated in primary antibody 54.1 diluted in PBS alone.

( )3.2.1. Inferotemporal cortex area TENMDAR1 immunoreactivity was present throughout all

Ž .layers of area TE Fig. 1 . Overall, the laminar distributionpatterns of NMDAR1 immunolabeling throughout area TEwere uniform, with no overt or distinguishing differences

Ž .observed between subdivisions areas TE1, TE2 and TE3 .In layer I, a dense network of fine-caliber immunolabeledprocesses and occasional immunolabeled somata were pre-

Ž .sent Fig. 1, Fig. 2 . Layers II and III were characterizedby the presence of numerous immunolabeled pyramidal-like

Ž .neurons solid, curved arrows, Fig. 2A,B whose labelingintensity and density increased with depth, reaching a

Ž .maximum in the lower half of layer III Fig. 1, Fig. 2B .Arising from such neurons were short segments of labeled,presumptive apical dendrites, and thinner, less intenselylabeled basilar dendrites. Similarly, in supragranular lay-ers, there were numerous, thicker, labeled processes and adense neuropil labeling characterized by thinner, lightlylabeled processes obliquely oriented and an intense punc-

Ž .tate labeling straight open arrows, Fig. 2A,B . In layer IV,very lightly labeled somata mostly lacking labeled pro-

Fig. 1. Photomicrographs of pair of adjacent sections taken throughmonkey temporal area TE1 showing thionin-stained lamination patternŽ .A and the corresponding laminar distribution pattern of NMDAR1immunolabeling. Bars50 mm.


Ž .cesses were present curved open arrows, Fig. 2B , as werefine-caliber, obliquely-oriented labeled processes and light,punctate labeling. Layer V was characterized by morelightly labeled neurons in comparison with supragranular

Ž .layers Fig. 1, Fig. 2C , although many of the larger

pyramidal neurons immediately subjacent to layer IV wereŽ .often more intensely labeled Fig. 1 . From labeled layer V

neurons, in general, labeled dendrites could only be fol-lowed for very short distances. The relative paucity ofextensive NMDAR1 immunolabeling of the apical den-

Ž . Ž . Ž .Fig. 2. Photomicrographs showing cellular detail of NMDAR1 immunolabeling in layers I and II A , III and IV B and V C of monkey area TE1. BothŽ . Ž .pyramidal-like cell bodies curved arrows , processes open arrows and puncta show immunoreactivity. Some small neurons in layer IV show a faint to

Ž .moderate degree of NMDAR1 immunoreactivity curved open arrows in B . Note that there are few, labeled processes which arise from infragranularŽ .neurons and extend through layer IV. In contrast, MAP2 immunoreactivity D from an adjacent section labels the apical dendrites of infragranular

Ž .pyramidal cells very intensely and extensively open arrows, D . Scale bars50 mm.


drites of infragranular pyramidal neurons is exemplifiedwhen comparing the labeling of these structures with

Ž .MAP2 Fig. 2D , in which it can be seen that bundles ofMAP2-immunolabeled apical dendrites arising from layerV neurons traverse layer IV. There was, in addition, aneuropil labeling present in layer V characterized by thin,

Žobliquely-oriented processes and labeled puncta straight.open arrows, Fig. 2C . In passing to layer VI, the density

and intensity of NMDAR1 immunolabeled somata, pro-Ž .cesses and punctate labeling increased Fig. 1 .

3.2.2. Prefrontal cortexDense NMDAR1 immunoreactivity was present

throughout all areas of the monkey prefrontal cortex, withno overt differences in the laminar distribution patternsevident across areas. In addition, the laminar distributionpattern of labeling was very similar to that described forthe inferotemporal cortex. In general, immunolabeled cellsomata and dendrites were distributed throughout all lay-ers, but were mostly absent in the underlying white matterŽ .Fig. 3 . Layer I possessed a very dense network of mostlysmall, fine-caliber immunolabeled processes which was

Ž .more abundant in the inner half of the layer Fig. 3 . Inmany instances, the origin of such processes could befollowed back to the apical dendrites arising from thesomata of pyramidal neurons in superficial layers. Occa-sional small, ovoid-shaped immunolabeled cell somatawere also found in layer I. Layer II was characterized bysmall, lightly labeled pyramidal-like neurons, while inlayer III, both lightly and more intensely immunolabeledneurons were present, with both the size and staining

Žintensity of the labeled neurons increasing with depth Fig..3 . Additionally, a dense neuropil labeling was also evi-

dent in layers II and III, characterized by the presence ofnumerous thinner, lightly labeled processes obliquely ori-ented as well as an intense punctate labeling. Layer IVcontained the least densely distributed and intensely la-beled somata in comparison with other layers. Arisingfrom some, mostly lightly-labeled somata, segments oflabeled dendrites could be followed only for short dis-tances. However, from most labeled somata, no dendritic

Ž .processes could be identified Fig. 3 . There was, inaddition, a modest neuropil labeling in layer IV similar tothat described above but much less intensely immunola-beled. In layer V, the density and intensity of labeledsomata appeared lower in comparison with supragranularlayers, but higher than that in layer IV. From many labeledsomata, proximal apical dendritic labeling was observedbut such dendrites could only be followed for short dis-

Ž .tances Fig. 3 . In the neuropil, small segments of NM-DAR1-labeled processes, as well as intense punctate label-ing, was also observed. In layer VI, the density andintensity of labeled somata increased in comparison with

Ž .layer V Fig. 3 . Many labeled cells in layer VI werepolymorphic.

3.3. Co-localization of NMDAR1 with non-NMDA receptorsubunits in monkey areas TE1 and 46

The distribution of labeling for Glu5r6r7 and GluR2r3on vibratome material was very similar in the prefrontaland inferotemporal areas examined to that previously de-

Fig. 3. Photomicrographs of pair of adjacent sections taken through theŽ .fundus of the principal sulcus area 46 of a monkey showing thionin-

Ž .stained lamination pattern A and the corresponding laminar distributionpattern of NMDAR1 immunolabeling. Bars50 mm.


w xscribed in frozen sections 48,102–104 . Quantitative anal-ysis of double-labeling immunofluorescence demonstratedŽ .Table 1 that in inferotemporal area TE1 and prefrontal

Ž .area 46, all ;100% of the GluR5r6r7-immunoreactivecell somata also contained faint to moderate NMDAR1

Ž .immunofluorescence curved arrows, Fig. 4A,B . In addi-tion, many GluR5r6r7 immunoreactive puncta or seg-

ments of presumptive dendritic processes were also im-Ž .munoreactive for NMDAR1 arrowheads, Fig. 4A,B ,

while, in contrast, the majority of the GluR5r6r7-immunofluorescent apical dendrites located in granular andinfragranular layers in each cortical area examined con-tained either a low degree of, or no, NMDAR1 immuno-

Ž .reactivity open arrows, Fig. 4A,B . Similarly, the majority

Ž . Ž .Fig. 4. Pairs of fluorescence photomicrographs taken through layer III of monkey area TE1 A,B or area 46 C,D showing colocalization of non-NMDAŽ . Ž . Ž . Ž .receptor subunits GluR5r6r7 A or GluR2r3 D and NMDAR1 B,C . Many neuronal cell bodies curved arrows which are immunoreactive for

GluR5r6r7 or GluR2r3 also contain low to moderate levels of NMDAR1 immunofluorescence, while some contain only very faint NMDAR1Ž . Ž .immunofluorescence open arrows, C,D . There are also processes and puncta arrowheads, A,B that show co-localization, while some GluR5r6r7

Ž .immunoreactive processes contain comparatively low or no immunoreactivity for NMDAR1 open arrows, A,B . Bars50 mm.


Ž . Ž .Fig. 6. Pairs of fluorescence photomicrographs taken through layer III of monkey area 46 A,B or monkey area TE1 C,D showing colocalization ofŽ . Ž . Ž .NMDAR1 A,C and calbindin B,D . Many calbindin neurons lack colocalization with NMDAR1 open arrows , while in area TE1, there are greater

Ž .numbers which are double-immunofluorescent curved arrows, C,D . Bars35 mm.

Fig. 5. Pairs of fluorescence photomicrographs taken from monkey area TE1 showing colocalization of NMDAR1 and calcium-binding proteins. A,B:Ž . Ž .example of parvalbumin-immunoreactive neuron in layer IV A which completely lacks any co-labeling for NMDAR1 in either the soma solid arrows or

Ž . Ž . Ž . Ž .processes open arrows . C–F: calretinin-immunofluorescent neurons in layer II C or layer III E show limited colocalization with NMDAR1 D, F .Ž .Most of the calretinin immunoreactive neurons shown in C and E contain no NMDAR1 immunolabeling in their cell body curved arrows or processes

Ž . Ž .small, straight arrows . However, some calretinin immunoreactive cells show faint NMDAR1 immunoreactivity large, straight arrows, E and F .Barss50 mm.


Table 1Co-localization of NMDAR1 subunits with non-NMDA receptor subunitsand calcium-binding proteins in prefrontal and inferotemporal associationareas

Area 46 Area TE1

GluR2r3 95.3"2.4 94.6"3.1GluR5r6r7 100 100PV 45.2"3.7 58.9"3.4CB 24.4"2.8 81.6"4.1CR 5.1"1.7 6.2"1.4

Data are mean percentages"S.E.M.

Ž .;95% of the GluR2r3-immunoreactive cell somataŽthroughout both areas examined also contained faint open

. Ž .arrows, Fig. 4C,D to moderate curved arrows, Fig. 4C,Dlevels of NMDAR1 immunofluorescence.

3.4. Colocalization of NMDAR1 with calcium-binding pro-teins in monkey areas TE1 and 46

The distributions of calcium-binding protein immunore-active cells and processes in neocortex have been the

Ž w x.subject of many previous studies for review, see 22 , and

Ž . Ž .Fig. 7. Electron micrographs showing ultrastructural localization of NMDAR1 immunoreactivity in monkey area 46 A,B or area TE1 C,D . In superficialŽ . Ž . Ž . Ž .A,C , granular B or deep D layers of association cortex, numerous, intensely labeled asymmetrical postsynaptic densities solid arrows can be

Ž .identified on both small dendritic shafts and spines, while many asymmetrical synapses are unlabeled open arrows . Within dendritic shafts,Ž . Ž .immunoprecipitate is also associated with microtubules and the outer membranes of mitochondria curved arrow, B . Unlabeled axon terminals t and fine

Ž .astrocytic processes curved arrow, A are indicated. Bars1 mm.


will not therefore be described in detail here. In both areasŽ .examined areas TE1 and 46 , about half of the parvalbu-

min-immunoreactive cells lacked any NMDAR1 immuno-Ž . Žlabeling Table 1 in either the soma closed arrows, Fig.

. Ž .5A,B or dendrites open arrows, Fig. 5A,B . Of thosewhich were co-labeled, the somata contained relativelyintense NMDAR1 immunolabeling, but the dendritic pro-cesses of such cells were very faintly, or not all, NMDAR1immunofluorescent.

Calbindin immunoreactivity throughout each of the ar-eas examined labeled both non-pyramidal cells as well as apopulation of pyramidal neurons, as described previouslyw x22 . In general, the two populations were easily distin-guishable from each other both by obvious morphologicaldifferences, as well as by overt differences in immunofluo-

rescent intensity, with the non-pyramidal cell populationŽ .more intensely immunoreactive Fig. 6 . Only the non-

pyramidal calbindin immunoreactive cell population wasexamined quantitatively for double-immunolabeling withNMDAR1. The percentage of calbindinrNMDAR1 dou-ble-labeled cells showed the greatest disparity across the

Ž .two areas examined Table 1 . In prefrontal area 46,;25% of the calbindin-labeled cells also possessed NM-

Ž .DAR1 immunolabeling Fig. 6A,B , while the percentageof such double-labeled cells rose to about ;80% in

Ž .inferotemporal area TE1 Fig. 6C,D . In general, the NM-DAR1 immunofluorescence of such double-labeled cells

Žwas relatively faint, and restricted mostly to somata Fig..6 .

In comparison with the other two calcium-binding pro-

Ž . Ž . Ž . Ž .Fig. 8. NMDAR1 immunoreactivity in human prefrontal A,B,C and temporal association cortex D . Coronal sections through layers II A and V B,D ,Ž . Ž .and an oblique section through layer III C reveal extensive somatic immunolabeling curved arrows , as well as intensely labeled dendritic segments in all

Ž .layers open arrows . Scale bars50 mm.


Table 2Percentages of NMDAR1-immunoreactive synapses in prefrontal andinferotemporal neocortex

Area 46 Area TE1

Ž . Ž .IIrIII 20.7 84r404 19.5 90r461Ž . Ž .IV 17.4 63r362 16.4 83r506Ž . Ž .VrVI 17.8 51r286 13.8 51r369

Values represent percentages of total asymmetrical synapses which wereŽ .NMDAR1-immunoreactive raw numbers in parentheses counted from

randomly chosen fields through the indicated layers and areas.

teins examined, relatively few calretinin-immunolabeledcells also showed NMDAR1 immunolabeling in either area

Ž .examined Fig. 5C–F; Table 1 . Of those that weredouble-immunofluorescent, the intensity of NMDAR1 im-

Žmunoreactivity was relatively faint larger, straight arrows,.Fig. 5E,F .

3.5. Electron microscopy

At the ultrastructural level, the NMDAR1 immunopre-cipitate was localized throughout the cytoplasm of neu-ronal somata and dendrites, but was never observed in

Ž .axons or presynaptic axon terminals t, Fig. 7 , and rarelyin fine astrocytic processes. Within somata, the immuno-precipitate was clumped, and while often too dense todiscriminate the underlying organelles with which it wasassociated, could occasionally be associated with stacks ofrough endoplasmic reticulum and the outer membranes ofmitochondria. In addition, segments of the outer somaticplasmalemma were often immunoreactive. Within den-drites, the immunoprecipitate was much less dense, whichmade it possible to readily associate the immunoprecipitate

Ž .with microtubules curved arrow, Fig. 7B , outer mem-branes of mitochondria, and occasional segments of the

Ž .plasmalemma Fig. 7 . There was, in addition, synapticlabeling, which was identified by the intense immunolabel-

Ž .ing of postsynaptic thickenings closed arrows, Fig. 7 , allof which were asymmetrical and apposed to presynapticterminals containing round vesicles, as well as by sparseDAB reaction product present within the surrounding cyto-plasm. Labeled postsynaptic densities were present alongboth fine-caliber dendritic shafts as well as within den-

Ž .dritic spines Fig. 7 , although many shaft and spineŽ .synapses were unlabeled open arrows, Fig. 7 . A more

detailed analysis of NMDAR1-immunolabeled synapseswas made of inferotemporal area TE1 and prefrontal area46 by estimating the percentages of asymmetrical post-synaptic densities which were immunolabeled in supra-

Ž .granular layers layers II and III , layer IV and infragranu-Ž .lar layers layers V and VI . As shown in Table 2, the

percentages of NMDAR1-immunoreactive synapses weresimilar across the two areas examined, and mostly similaracross the layers examined, with superficial layers contain-ing only slightly higher percentages of immunolabeled

synapses in comparison with those in layer IV and infra-granular layers. No attempt was made to determine theproportion of labeled postsynaptic elements which werelocated along shafts or within spines.

3.6. Distribution of NMDAR1 immunoreactiÕity in humanassociation cortex

Ž .The samples of human prefrontal Fig. 8A–C andŽ .temporal Fig. 8D association cortex examined contained

a cellular distribution pattern of NMDAR1 immunoreactiv-ity which closely resembled that described for monkeyassociation cortex. The majority of labeled cells in thehuman material appeared to be pyramidal-like neurons

Žlocated throughout layers II, III, V and VI curved arrows,.Fig. 8 . In addition, intensely immunoreactive, obliquely

oriented processes and puncta were densely distributedŽ .throughout all cell-dense layers open arrows, Fig. 8 .

Layer IV contained a population of more lightly stainedneurons which mostly lacked extensive dendritic labeling.

4. Discussion

4.1. Optimal tissue processing conditions for ÕisualizingNMDAR1 immunoreactiÕity

A comparison of the cellular labeling patterns followingvarious tissue processing conditions suggests that NM-DAR1 epitopes located in fine-caliber dendritic processesmay be particularly vulnerable to damage or masking dueto either freezing or permeabilization by detergents. Incontrast, such epitopes located in the intracellular somaticand proximal dendritic domains showed relatively greaterresistance to these procedures. These data may indicatethat the epitope recognized by mAb 54.1 associates withdifferent cytoplasmic elements of varying stability from itsproduction, to incorporation into the synaptic membrane,with that associated with the latter more susceptible todamage by freezing or permeabilization. Additionally, thecurrent observations indicate some potential methodologi-cal constraints on the immunocytochemical localization ofGluR subunits in human brain. In our material, the bestresults with the anti-NMDAR1 antibody were obtained inrapidly-fixed biopsy specimens, whereas NMDAR1 im-munoreactivity was greatly diminished in autopsy materialthat had a longer post-mortem interval prior to fixation.This may be true as well for the immunocytochemicallocalization of AMPA and kainate subunits in human brainw x23,101 .

4.2. Laminar and regional patterns of NMDAR1 immuno-reactiÕity in cortical association areas

The laminar distribution patterns of NMDAR1 immuno-reactivity were largely similar throughout the monkey


prefrontal and temporal association areas examined, andwere characterized by dense, immunoreactive processesand labeled somata in layers I–III, Va and VI, and, incontrast, much lighter and sparser immunostaining in layerIV. These data are generally consistent with the distribu-tion of NMDA receptor binding sites revealed autoradio-

w xgraphically 32,53 , and the laminar distribution of NM-DAR1 transcripts in human prefrontal and parietotemporal

w xcortex revealed by in situ hybridization histochemistry 1 .Ž .At the synaptic level, electron microscopy Table 2 con-

firmed that NMDAR1 immunoreactivity labeled a minorityŽ .of the asymmetric excitatory postsynaptic densities

throughout supragranular, granular and infragranular lay-ers. Taken together, these distribution patterns suggest thatNMDA receptors are operative throughout all layers ofprefrontal and temporal association cortex, not unlike thatobserved for NMDAR1 receptor subunit localizationthroughout primary somatosensory, motor, and visual cor-

w x w xtices of monkeys 49 and rats 2,20,81 .Each of the areas examined in the current study are

characterized by extensive, converging corticocortical in-puts principally to supragranular and granular layersw x6,19,88,89 ; dense plexuses of local axon collaterals mostly

w xin superficial layers 61 ; and thalamocortical inputs tow xlayer IV and the deeper half of layer III 10,33,55 . Al-

though speculative, it is unlikely that NMDA receptors aremediating thalamocortical inputs to cortical associationareas. Physiological evidence in primary sensory and mo-tor cortex of adult rats and cats indicates that the earliestmonosynaptic thalamocortical activity is mediated exclu-

w xsively by non-NMDA receptors 3,30,86 . The observationin the current study that the apical dendrites arising frominfragranular pyramidal neurons, which traverse layer IV

w xin their ascent towards the pial surface 28 , mostly lackedovert immunostaining within layer IV may be an indica-tion that NMDA receptors are selectively parceled todendritic sites outside of the major thalamocortical termi-nations. Although the mechanisms by which receptors canbe differentially targeted to specific synaptic sites within aneuron is largely unknown, anatomical evidence frommonkey hippocampus suggests that, in the case of CA3pyramidal cells, NMDAR1 immunostaining is absent fromthe postsynaptic densities of more proximal dendrites asthey traverse stratum lucidum, but are found at synapticsites in more superficial portions of the dendrites as they

w xtraverse stratum radiatum 91 . These data suggest thatNMDA receptors can be parceled within dendrites tospecific synaptic sites in conformity with particular affer-ent circuits. It is unlikely that the lack of overt apicaldendritic staining in layer IV was due to artifactual sever-ing of such processes by the plane of section, sinceadjacent sections stained with MAP2 reveal preserved

Ž .bundles of apical dendrites within layer IV e.g. Fig. 2D .The relatively more dense and intense immunostaining insupragranular neurons is consistent with a more prominentrole for NMDA receptor function in the activities of

w xcorticocortical and intrinsic connectivity 3 . Within super-ficial layers, however, there was no overt indication thatNMDAR1 immunoreactivity was clustered or concentratedwithin superficial layers in any way to suggest a preferen-tial parcellation to the interdigitating columns of cortico-cortical and callosal afferent terminations which are char-

w xacteristic of some of the regions examined 36 , suggestingthat NMDA receptors can mediate the activities of a widevariety of converging corticocortical inputs to these layers.This is consistent with the observation that the administra-tion of the reversible NMDA receptor antagonist ketamineimpairs performance on spatial working memory tasksw x12,60,101 which depend on intact prefrontal cortexw x34,79,85 and, presumably, the convergent, higher-ordersensory information conveyed by corticocortical afferents

w xwhich target these regions 35 . Given the complexity ofconvergent afferents in the primate neocortex, a definitiveconclusion regarding the anatomical correlation betweenNMDA receptors and specific afferent systems awaitsdetailed anterograde labeling and immuno-electronmicro-scopic analyses.

At the electron-microscopic level, immunoreactivity waspresent within the cytoplasm of somata and dendrites, andoften outlined non-synaptic somatic and dendritic plas-malemma and subsets of asymmetric synaptic thickenings.It is often proposed that such intracellular immunoreactiv-

w xity represents, in general, a synthetic or reserve pool 96 ,and has been observed with immunocytochemical localiza-

w x w xtion of nicotinic 41 , GABA 84,95 and other ionotropicAw xglutamate receptor subunits 7,48,52,78,82,94 . The poten-

tially dynamic nature of intracellular pools of NMDAR1immunoreactivity is suggested by a recent study in whichit was shown that the ratio of the intensity of NMDAR1immunofluorescence between the outer and the innermolecular layers of the dentate gyrus decreases in aged

w xmonkeys in comparison with young adults 31 , suggestingthat such intracellular protein levels can be modulated,possibly in the context of compensatory adaptations tochanges in afferent input. The regulation of intracellularpools of receptor subunits is likely an activity-mediatedprocess, since recent studies in vitro show changes inlevels of NMDAR1 peptide following chronic exposure to

w xNMDA receptor antagonists 29 . The significance of thesomatic plasmalemma labeling is less clear, since in neo-cortex somatic synapses are predominately of the symmet-

w xric, and therefore inhibitory, type 22 . However, electro-physiological studies of cultured rat hippocampal pyrami-dal neurons indicate that functional glutamate receptor-lin-

w xked channels are present on somatic membranes 11 ,which may correspond in part to the immunoreactivityobserved.

While such ultrastructural localization observed in thepresent study was generally consistent with that describedpreviously for other monkey cortical areas using the same

w xantibody 49,91 or for rat cortical areas using other anti-w xNMDAR1 antisera 2,81 , some notable differences were


observed. Previous studies of NMDAR1 localization in ratvisual cortex using polyclonal antisera described localiza-tion to axon terminals and astrocytes, which, while virtu-

Žally never observed in monkey neocortex present study;w x.49 , has been observed in monkey hippocampus, wherepresynaptic mossy fibers are intensely immunoreactive

w xwhen visualized with mAb 54.1 91 . The most parsimo-nious explanation for such discrepancies is relative differ-ences in the specificities and sensitivities of differentantibodies. Consistent with this, a recent study of rat andcat cerebellum in which several anti-NMDAR1 antibodies

Ž .were used and compared including mAb 54.1 , revealedslight differences in the cellular localization patterns and

w xintensities of immunoreactivity 52 . It is also possible thatŽ .the fixation used 4% paraformaldehyde , which is optimal

for light microscopy, may be less than optimal for suffi-cient membrane preservation at the ultrastructural level,leading to inadequate retention of detectable levels ofantigen in fine astrocytic or axonal processes. Finally, thepossibility also remains that species differences betweenrat and monkey account for slightly different localizationpatterns.

The laminar proportions of immunoreactive synapseswere similar between the prefrontal and temporal associa-tion areas examined, suggesting a very similar organizationof NMDA receptors and similar contributions to excitatoryactivities in different association areas. In contrast, previ-ous studies of primary motor and sensory cortex usingidentical methods revealed more heterogeneity in the lami-nar proportions of immunoreactive synapses, with higherproportions evident particularly in superficial layers in

w xcomparison with those of association areas 49 . Althoughspeculative, these data may indicate a greater involvementof NMDA receptor-mediated activity in sensory and motorcortical areas in comparison with association cortical areas.

The distribution of NMDAR1 subunit immunoreactivityin the limited samples of human cortex was similar to thedistribution patterns described for corresponding monkeycortical regions in prefrontal and temporal association

Ž .cortex present study and in the primary visual cortexw x49 . These data suggest that in human cortex, NMDAreceptors function in similar cells and circuits as they do inmonkey cortex. Functionally, studies of human neocorticalslice preparations show that NMDA receptors appear tocontribute more significantly to EPSPs recorded in supra-

w xgranular neurons than infragranular neurons 5,17,51 ,which is likely a reflection of the dense immunolabeledprocesses found in the superficial layers of the humancortex. In the infragranular layers, it is less clear whatfunctional role NMDA receptors play in normal synaptictransmission. However, epileptiform activity induced byfocal stimulation of infragranular neurons in human corti-

w xcal slices can be blocked by NMDA antagonists 5 , whichsuggests that at least under certain abnormal, or pathologi-cal conditions, NMDA receptors in infragranular neuronsmay become active.

4.3. Heterogeneity in GluR profile of neocortical neurons

The results of the co-localization experiments suggestheterogeneity in the GluR profile across different popula-tions of neocortical neurons. However, it must be kept inmind that such an interpretation is based on labelingmethods in which non-immunoreactive neurons could beones which either truly lack NMDAR1 proteins, or whichactually have NMDAR1 proteins, but at levels which arebelow the threshold for detection by immunofluorescence.With this caveat in mind, the results of the present study

Ž .indicate that the vast majority of kainate GluR5r6r7 -Ž .and AMPA GluR2r3 -containing pyramidal neurons in

monkey association areas also possess NMDAR1 subunitsand, therefore, presumably functional NMDA receptorsŽ w x.since R1 is thought to be an obligatory subunit 76 .These data are similar to the patterns of co-localizednon-NMDA and NMDA receptors subunits reported earlierfor the pyramidal neurons of monkey primary motor,

w xsomatic sensory and visual cortex 49 , suggesting thatthroughout all regions of monkey neocortex, non-NMDAreceptor-containing pyramidal cells also possess NMDAreceptors. However, studies have either shown directly orimplied that the particular combinations of individual sub-units which compose them can vary from cell to cell inb o t h n e o c o r t e x a n d h i p p o c a m p u sw x9,15,39,49,66,68,78,82,102,106 , indicating a tremendousheterogeneity in the functional properties of the variousreceptor complexes. It remains to be determined whethernon-NMDA and NMDA receptor subunits are co-localizedat the level of individual synapses in the cerebral cortex,and where, precisely, such synapses are located. A widevariety of physiological studies have shown that non-NMDA and NMDA receptors may not necessarily be

Ž w x.co-localized at all synapses e.g. 56,72,73 , suggesting adifferential parcellation of functional receptor subtypes todifferent synaptic locations. This would be consistent with

w xthe present and previous 2,49,81 electron microscopicobservations throughout rat and monkey cerebral cortexthat NMDAR1-immunoreactive synapses form a minorityof the total asymmetrical synapses in each of the layersexamined.

The co-distribution of immunoreactivities for NMDAR1and calcium-binding proteins in monkey association cortexexamined in the present study extends previous findingsindicating extensive glutamate receptor subunit hetero-geneity amongst structurally and functionally distinct sub-

Žpopulations of cortical GABAergic interneurons reviewedw x.in 50 . There are two striking observations in this regard.

First, there was wide variability in the percentage ofcolocalization across the three subpopulations of interneu-rons labeled by the calcium-binding proteins. It has beenpreviously established that calcium-binding protein im-munoreactivity in the monkey neocortex labels mostlynon-overlapping subpopulations of GABAergic interneu-

w xrons 38,74,100 . In the cortical association areas exam-


ined, the population labeled by parvalbumin, which in-w xcludes basket cells and chandelier cells 24,25,38,63,108 ,

and the population labeled by calbindin, which includesw xdouble-bouquet cells 23,26 , showed a greater percentage

of colocalization in comparison with the population la-beled by calretinin, a finding similar to one made previ-

w xously in other areas of monkey cortex 49 . These anatomi-cal data are consistent with physiological studies showingthat rat hippocampal and cortical inhibitory interneurons invitro possess a heterogeneous glutamate receptor subunit

w xprofile 15,39,68 , which, collectively, suggests that NMDAreceptors play a differential role in modulating the activityof different types of GABAergic neurons, each with dis-tinct targets and roles in inhibitory function. Second, acrossdifferent areas of monkey cerebral cortex, there was widevariability in the percentage of colocalization within agiven calcium-binding protein-labeled population. For ex-ample, for the population of interneurons labeled by par-valbumin, the percentage of colocalization with NMDAR1varied from 80–90% in primary motor and sensory areasw x49 , to 45–60% in association areas. Likewise, the per-centage of calbindin colocalization varied from 25% inprefrontal area 46, to 82% in temporal area TE1. Previousstudies have demonstrated a regionally specific distributionof morphologically distinct subtypes of GABAergic cellswhich are neurochemically heterogeneous with regard to

w x w xtachykinin 23 , somatostatin 16,27 and corticotropin-re-w xleasing factor 63 immunoreactivities. The results of the

present and previous studies indicate that the parvalbuminand calbindin subpopulations of cortical GABAergic in-terneuron also exhibit an NMDA receptor subunit hetero-geneity which is regionally specific. In contrast, the per-centage of colocalization of the calretinin-labeled popula-tion remained relatively consistent across motor, sensoryw x49 and association cortical areas at approximately 5–8%,which may be a reflection of the selective resistance ofneocortical calretinin-containing neurons maintained in

w xvitro to calcium overload and excitotoxicity 64 . Simi-larly, in vitro studies of mouse cortical neurons haveshown GABAergic interneurons in general to be particu-

w xlarly resistant to NMDA-induced cellular toxicity 98 ,which may be due to the relative paucity of NMDAreceptor subunits localized to GABAergic cells when con-sidered as a whole population.

4.4. Implications for neurodegeneratiÕe diseases

Selective vulnerability of subpopulations of corticalneurons to toxicity and death triggered by overactivationof NMDA and other types of glutamate receptors has beensuggested for a variety of disorders, including Alzheimer’s

Ž . w xdisease AD 17,18,37 , which affects particularly thecorticocortical circuits of higher-order association areasfurnished by the pyramidal cells of layers III and Vw x62,75,80 , while apparently sparing the calcium-binding

w xprotein-labeled interneuronal populations 43–45 . How-

ever, the laminar, cellular and synaptic localization pat-terns of NMDAR1 immunoreactivity do not support anyovert correspondence between the localization of NM-DAR1 subunits and the layers or cells which appear

Ž .selectively vulnerable or resistant in Alzheimer’s disease.Such an observation does not preclude an involvement ofNMDA receptor-mediated excitotoxicity in AD and otherdiseases, but more likely points to what is undoubtedly amore complex and interactive set of conditions whichrender neurons selectively vulnerable. For example, attenu-ation of intracellular energy levels has been shown torender neurons more vulnerable to NMDA receptor in-

w xduced toxicity 65,77,92 , and experimental models ofexcitotoxicity have been shown to result in alterations ofcytoskeletal proteins that are similar to those found inm a n y n e u r o d e g e n e r a t i v e c o n d i t i o n sw x14,21,47,67,87,93,105 . A recent study has shown thatglutamate exposure causes differential regulation of thephosphorylation state of microtubule-associated proteinMAP2 depending on whether glutamate activates

w xmetabotropic or NMDA receptors 83 , suggesting a com-plex interaction between glutamatergic neurotransmission,receptor specificity, and variable cascades of intracellularsignaling affecting phosphoproteins. Thus, if excitotoxicityis a component of the degeneration of specific corticalneurons, then the precise mechanism is likely mediated byadditional cell-type specific properties including, for exam-ple, the ability to buffer calcium, maintenance of energyhomeostasis, free radical scavenging or subsequent interac-tions with second-messenger systems and their target pro-teins.

Acknowledgements

This work was supported by NIH Grants AG05138 andAG06447; the Aaron Diamond Foundation and theAlexandrine and Alexander Sinsheimer Fund. J.C.V. is arecipient of a R.D. Wright Fellowship from the Australian

ŽNH and MRC. The assistance of Drs. S. Morgello Depart-.ment of Pathology, Mount Sinai Medical Center , and K.

ŽPost and J. Bederson Department of Neurosurgery, Mount.Sinai Medical Center in providing the human biopsy

material is gratefully acknowledged. We thank A.M. Ed-wards, Y.-L. Hsu and William Janssen for excellent techni-cal assistance, and R. Woolley for help in preparation ofthe figures.

References

w x1 Akbarian, S., Sucher, N.J., Bradley, D., Tafazzoli, A., Trinh, D.,Hetrick, W.P., Potkin, S.G., Sandman, C.A., Bunney Jr., W.E. andJones, E.G., Selective alterations in gene expression for NMDAreceptor subunits in prefrontal cortex of schizophrenics, J. Neu-

Ž .rosci., 16 1996 19–30.w x2 Aoki, C., Venkatesan, C., Go, C.-G., Mong, J.A. and Dawson,


T.M., Cellular and subcellular localization of NMDA-R1 subunitimmunoreactivity in the visual cortex of adult and neonatal rats, J.

Ž .Neurosci., 14 1994 5202–5222.w x3 Armstrong-James, M., Welker, E. and Callahan, C.A., The contri-

bution of NMDA and non-NMDA receptors to fast and slowtransmission of sensory information in the rat SI barrel cortex, J.

Ž .Neurosci., 13 1993 2149–2160.w x4 Artola, A. and Singer, W., Long-term potentiation and NMDA

Ž .receptors in rat visual cortex, Nature, 330 1987 649–652.w x5 Avoli, M. and Olivier, A., Electrophysiological properties and

synaptic responses in the deep layers of the human epileptogenicŽ .neocortex in vitro, J. Neurophysiol., 61 1989 589–606.

w x6 Barbas, H. and Mesulam, M.M., Cortical afferent input to theŽ .principalis region of the rhesus monkey, Neuroscience, 15 1985

619–637.w x7 Baude, A., Molnar, E., Latawiec, J., McIlinney and Somogyi, P.,

Synaptic and nonsynaptic localization of the GluR1 subunit of theAMPA-type excitatory amino acid recetor in the rat cerebellum, J.

Ž .Neurosci., 14 1994 2830–2843.w x8 Bear, M.F., Kleinschmidt, A., Gu, Q. and Singer, W., Disruption of

experience-dependent synaptic modifications in striate cortex byŽ .infusion of an NMDA receptor antagonist, J. Neurosci., 10 1990

909–925.w x9 Bekkers, J.M., Stevens, C.F., NMDA and non-NMDA receptors

are co-localized at individual excitatory synapses in cultured ratŽ .hippocampus, Nature, 341 1989 230–233.

w x10 Benevento, L.A. and Rezak, M., The cortical projections of theinferior pulvinar and adjacent lateral pulvinar in the rhesus monkeyŽ .Macaca mulatta : an autoradiographic study, Brain Res., 108Ž .1976 1–24.

w x11 Benke, T.A., Jones, O.T., Collingridge, G.L. and Angelides, K.J.,N-Methyl-D-aspartate receptors are clustered and immobilized ondendrites of living cortical neurons, Proc. Natl. Acad. Sci. USA, 90Ž .1993 7819–7823.

w x12 Bennett, A., Abi-Saab, D., Karper, L., D’Souza, D.C., Abi-Dargham, A., Charney, D. and Krystal, J., Ketamine impairslearning, but not procedural expression, on the wisconsin card

Ž .sorting test, Schizophrenia Res., 15 1995 108–109.w x13 Bennett, J.A. and Dingledine, R., Topology profile for a glutamate

receptor: three transmembrane domains and a channel-lining reen-Ž .trant loop, Neuron, 14 1995 373–384.

w x14 Bigot, D. and Hunt, S.P., Effect of excitatory amino acids onmicrotubule-associated proteins in cultured cortical and spinal neu-

Ž .rons, Neurosci. Lett., 111 1990 275–280.w x15 Bochet, P., Audinat, E., Lambolez, B., Crepel, F., Rossier, J., Iino,´

M., Tsuzuki, K. and Ozawa, S., Subunit composition at the single-cell level explains functional properties of a glutamate-gated chan-

Ž .nel, Neuron, 12 1994 383–388.w x16 Campbell, M., Lewis, D.A., Benoit, R. and Morrison, J.H., Re-

gional heterogeneity in the distribution of somatostatin-28 andŽ .somatostatin-28 1–12 -immunoreactive profiles in monkey neo-

Ž .cortex, J. Neurosci., 7 1987 1133–1144.w x17 Carlson, M.D., Penney, Jr. J.B. and Young, A.B., NMDA, AMPA,

and benzodiazepine binding site changes in Alzheimer’s diseaseŽ .visual cortex, Neurobiol. Aging, 14 1993 343–352.

w x18 Cepeda, C., Peacock, W., Levine, M.S. and Buchwald, N.A.,Iontophoretic application of NMDA produces different types ofexcitatory responses in developing human cortical and caudate

Ž .neurons, Neurosci. Lett., 126 1991 167–171.w x19 Chavis, D.A. and Pandya, D.N., Further observations on corti-

cofrontal connections in the rhesus monkey, Brain Res., 117Ž .1976 369–386.

w x20 Conti, F., Minelli, A., Molnar, M. and Brecha, N., Cellular local-ization and laminar distribution of NMDAR1 mRNA in the rat

Ž .cerebral cortex, J. Comp. Neurol., 343 1994 554–565.w x21 De Boni, U and Crapper McLachlan, D.R., Controlled induction of

paired helical filaments of the Alzheimer type in cultured human

Ž .neurons, by glutamate and aspartate, J. Neurol. Sci., 68 1985105–118.

w x22 DeFelipe, J., Neocortical neuronal diversity: chemical heterogene-ity revealed by colocalization studies of classic neurotransmitters,neuropeptides, calcium-binding proteins, and cell surface molecules,

Ž .Cereb. Cortex, 3 1993 273–289.w x23 DeFelipe, J., Hendry, S.H.C., Hashikawa, T., Molinari, M. and

Jones, E.G., A microcolumnar structure of monkey cerebral cortexrevealed by immunocytochemical studies of double bouquet cell

Ž .axons, Neuroscience, 37 1990 655–673.w x24 DeFelipe, J., Hendry, S.H.C. and Jones, E.G., Visualization of

chandelier cell axons by parvalbumin immunoreactivity in monkeyŽ .cerebral cortex, Proc. Natl. Acad. Sci. USA, 86 1989 2093–2097.

w x25 DeFelipe, J., Huntley, G.W., del Rıo, M.R., Sola, R.G. and Morri-´son, J.H., Microzonal decreases in the immunostaining for non-NMDA ionotropic excitatory amino acid receptor subunits GluR2r3and GluR 5r6r7 in the human epileptogenic neocortex, Brain

Ž .Res., 657 1995 150–158.w x26 DeFelipe, J. and Jones, E.G., High-resolution light and electron

microscopic immunocytochemistry of colocalized GABA and cal-bindin D-28k in somata and double bouquet cell axons of monkey

Ž .somatosensory cortex, Eur. J. Neurosci., 4 1992 46–60.w x27 de Lima, A.D. and Morrison, J.H., Ultrastructural analysis of

somatostatin-immunoreactive neurons and synapes in the temporaland occipital cortex of the macaque monkey, J. Comp. Neurol.,

Ž .283 1989 212–237.w x28 Feldman, M.L., Morphology of the neocortical pyramidal neuron.

Ž .In E.G. Jones and A. Peters, Eds. , The Cerebral Cortex, Vol. 5,Plenum, New York, 1984, pp. 123–200.

w x29 Follesa, P. and Ticku, M.K., NMDA receptor upregulation: molec-ular studies in cultured mouse cortical neurons after chronic antag-

Ž .onist exposure, J. Neurosci., 16 1996 2172–2178.w x30 Fox, K., Sato, H. and Daw, N., The location and function of

NMDA receptors in cat and kitten visual cortex, J. Neurosci., 9Ž .1989 2443–2454.

w x31 Gazzaley, A.H., Siegel, S.J., Kordower, J.H., Mufson, E.J. andMorrison, J.H., Circuit-specific alterations of N-methyl-D-aspartatereceptor subunit 1 in the dentate gyrus of aged monkeys, Proc.

Ž .Natl. Acad. Sci. USA, 93 1996 3121–3125.w x32 Geddes, J.W., Cooper, S.M., Cotman, C.W., Patel, S. and Mel-

drum, B.S., N-methyl-D-aspartate receptors in the cortex and hip-Ž .pocampus of baboon Papio anubis and Papio papio , Neuro-

Ž .science, 32 1989 39–47.w x33 Giguere, M. and Goldman-Rakic, P.S., Mediodorsal nucleus: areal,

laminar, and tangential distribution of afferents and efferents in theŽ .frontal lobe of rhesus monkeys, J. Comp. Neurol., 277 1988

195–213.w x34 Goldman-Rakic, P.S., Circuitry of primate prefrontal cortex and the

regulation of behaviour by representational memory. In V.B.Ž .Mountcastle, F. Plum and S.R. Geiger Eds. , Handbook of Physi-

ology –The NerÕous System, Vol. 5, Am. Physiol. Soc., Bethesda,MD, 1987, pp. 373–417.

w x35 Goldman-Rakic, P.S., Topography of cognition: parallel distributednetworks in primate association cortex, Annu. ReÕ. Neurosci., 11Ž .1988 37–156.

w x36 Goldman-Rakic, P.S. and Schwartz, M.L., Interdigitation of con-tralateral and ipsilateral columnar projections to frontal association

Ž .cortex in primates, Science, 216 1982 755–757.w x37 Greenamyre, J.T. and Young, A.B., Excitatory amino acids and

Ž .Alzheimer’s disease, Neurobiol. Aging, 10 1989 593–602.w x38 Hendry, S.H.C., Jones, E.G., Emson, P.C., Lawson,D.E.M., Heiz-

mann, C.W. and Streit, P., Two classes of cortical GABA neuronsdefined by differential calcium binding protein immunoreactivities,

Ž .Exp. Brain Res., 76 1989 467–472.w x39 Hestrin, S., Different glutamate receptor channels mediate fast

excitatory synaptic currents in inhibitory and excitatory corticalŽ .neurons, Neuron, 11 1993 1083–1091.


w x40 Hicks, T.P. and Guedes, R.C.A., Neuropharmacological propertiesof electophysiologically identified, visually responsive neurones ofthe posterior lateral suprasylvian area. A microionophoretic study,

Ž .Exp. Brain Res., 49 1983 157–173.w x41 Hill, J.A., Zoli, M., Bourgeois, J.-P. and Changeaux, J.-P., Im-

munocytochemical localization of a neuronal nicotinic receptor: theŽ .b 2-subunit, J. Neurosci., 13 1993 1551–1568.

w x42 Hirsch, J.C. and Crepel, F., Use-dependent changes in synapticŽ .efficiency in rat prefrontal neurons in vitro, J. Physiol., 427 1990

31–49.w x43 Hof, P.R., Cox, K., Young, W.G., Celio, M.R., Rogers, J. and

Morrison, J.H., Parvalbumin-immunoreactive neurons in the neo-cortex are resistant to degeneration in Alzheimer’s disease, J.

Ž .Neuropath. Exp. Neurol., 50 1991 451–462.w x44 Hof, P.R. and Morrison, J.H., Neocortical neuronal subpopulations

labeled by a monoclonal antibody to calbindin exhibit differentialŽ .vulnerability in Alzheimer’s disease, Exp. Neurol., 111 1991

293–301.w x45 Hof, P.R., Nimchinsky, E.A., Celio, M.R., Bouras, C. and Morri-

son, J.H., Calretinin-immunoreactive neocortical interneurons areŽ .unaffected in Alzheimer’s disease, Neurosci. Lett., 152 1993

145–149.w x46 Hollmann, M., Maron, C. and Heinemann, S., N-glycosylation site

tagging suggests a three transmembrane domain topology for theŽ .glutamate receptor GluR1, Neuron, 13 1994 1331–1343.

w x47 Hugon, J. and Vallat, J.M., Abnormal distribution of phosphory-lated neurofilaments in neuronal degeneration induced by kainic

Ž .acid, Neurosci. Lett., 119 1990 45–48.w x48 Huntley, G.W., Rogers, S.W., Moran, T., Janssen, W., Archin, N.,

Vickers, J.C., Cauley K., Heinemann, S.F. and Morrison, J.H.,Selective distribution of kainate receptor subunit immunoractivityin monkey neocortex revealed by a monoclonal antibody thatrecognizes glutamate receptor subunits GluR5r6r7, J. Neurosci.,

Ž .13 1993 2965–2981.w x49 Huntley, G.W., Vickers, J.C., Janssen, W., Brose, N., Heinemann,

S.F. and Morrison, J.H., Distribution and synaptic localization ofimmunocytochemically identified NMDA receptor subunit proteinsin the sensory-motor cortex and visual cortices of monkey and

Ž .human, J. Neurosci., 14 1994 3603–3619.w x50 Huntley, G.W., Vickers, J.C. and Morrison, J.H., Cellular and

synaptic localization of NMDA and non-NMDA receptor subunitsin neocortex: organizational features related to cortical circuitry,

Ž .function and disease, Trends Neurosci., 17 1994 536–543.w x51 Hwa, G.G.C. and Avoli, M., Excitatory synaptic transmission

mediated by NMDA and non-NMDA receptors in thesuperficialrmiddle layers of the epileptogenic human neocortex

Ž .maintained in vitro, Neurosci. Lett., 143 1992 83–86.w x52 Jaarsma, D., Wenthold, R.J. and Mugnaini, E., Glutamate receptor

subunits at mossy fiber-unipolar brush cell synapses: light andelectron microscopic immunocytochemical study in cerebellar cor-

Ž .tex of rat and cat, J. Comp. Neurol., 357 1995 145–160.w x53 Jansen, K.L.R., Faull, R.L.M. and Dragunow, M., Excitatory amino

acid receptors in the human cerebral cortex: a quantitative autora-w 3 xdiographic study comparing the distributions of H TCP,

w3 x w3 x w3 x w3 xH glycine, L- H glutamate, H AMPA and H kainic acidŽ .binding sites, Neuroscience, 32 1989 587–607.

w x54 Javitt, D.C. and Zukin, S.R., Recent advances in the phencyclidineŽ .model of schizophrenia, Am. J. Psych., 148 1991 1301–1308.

w x55 Jones, E.G., The Thalamus, Plenum Press, New York, 1985.w x56 Jones, K.A. and Baughman, R.W., Both NMDA and non-NMDA

subtypes of glutamate receptors are concentrated at synapses onŽ .cerebral cortical neurons in culture, Neuron, 7 1991 593–603.

w x57 Kimura, F., Nishigori, A., Shirokawa, T. and Tsumoto, T., Long-term potentiation and NMDA receptors in the visual cortex of

( ) Ž .young rats, J. Physiol. Lond. , 414 1989 125–144.w x58 Kirkwood, A. and Bear, M.F., Hebbian synapses in visual cortex,

Ž .J. Neurosci., 14 1994 1634–1645.

w x59 Kirkwood, A., Dudek, S.M., Gold, J.T., Aizenman, C.D. and Bear,M.F., Common forms of synaptic plasticity in the hippocampus and

Ž .neocortex in vitro, Science, 260 1993 1518–1521.w x60 Krystal, J.H., Karper, L.P., Seibyl, J.P., Freeman, G.K, Delaney,

R., Bremner, J.D., Heninger, G.R., Bowers, M.B. and Charney,D.S., Subanesthetic effects of the noncompetitive NMDA antago-nist, ketamine, in humans: psychotomimetic, perceptual, cognitive,

Ž .and neuroendocrine responses, Arch. Gen. Psych., 51 1994 199–214.

w x61 Levitt, J.B., Lewis, D.A., Yoshioka,T. and Lund, J.S., Topographyof pyramidal neuron intrinsic connections in macaque monkey

Ž . Ž .prefrontal cortex areas 9 and 46 , J. Comp. Neurol., 338 1993360–376.

w x62 Lewis, D.A., Campbell, M.C., Terry, R.D. and Morrison, J.H.,Laminar and regional distributions of neurofibrillary tangles andneuritic plaques in Alzheimer’s disease: a quantitative study of

Ž .visual and auditory cortices, J. Neurosci., 7 1987 1799–1808.w x63 Lewis, D.A. and Lund, J.S. Heterogeneity of chandelier neurons in

monkey neocortex: corticotropin-releasing factor- and parvalbu-Ž .min-immunoreactive populations, J. Comp. Neurol., 293 1990

599–615.w x64 Lukas, W. and Jones, K.A., Cortical neurons containing calretinin

are selectively resistant to calcium overload and excitotoxicity inŽ .vitro, Neuroscience, 61 1994 307–316.

w x65 Lysko, P.G., Cox, J.A., Vigano, A. and Henneberry, R.C., Excita-tory amino acid neurotoxicity at the N-methyl-D-aspartate receptorin cultured neurons: pharmacological characterization, Brain Res.,

Ž .499 1989 258–266.w x66 Martin, L.J., Blackstone, C.D., Levey, A.I., Huganir, R.L. and

Price, D.L., AMPA receptor subunits are differentially distributedŽ .in rat brains, Neuroscience, 53 1993 327–358.

w x67 Mattson, M.P., Antigenic changes similar to those seen in neurofib-rillary tangles are elicited by glutamate and Ca2q influx in cultured

Ž .hippocampal neurons, Neuron, 2 1990 105–117.w x68 McBain, C.J. and Dingledine, R., Heterogeneity of synaptic gluta-

mate receptors on CA3 stratum radiatum interneurones of ratŽ .hippocampus, J. Physiol., 462 1993 373–392.

w x69 McDonald, J.W. and Johnston, M.V., Physiological and pathophys-iological roles of excitatory amino acids during central nervous

Ž .system development, Brain Res. ReÕ., 15 1990 41–70.w x70 Meldrum, B. and Garthwaite, J., Excitatory amino acid neurotoxic-

Ž .ity and neurodegenerative disease, Trends. Pharm. Sci., 11 199154–61.

w x71 Miller, K.D., Chapman, B. and Stryker, M.P., Visual responses inadult cat visual cortex depend on N-methyl-D-aspartate receptors,

Ž .Proc. Natl. Acad. Sci. USA, 86 1989 5153–5187.w x72 Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M.,

Lomeli, H., Burnashev, N., Sakmann, B. and Seeburg, P.H., Het-eromeric NMDA receptors: molecular and functional distinction of

Ž .subtypes, Science, 256 1992 1217–1221.w x73 Mooney, R, Konishi, M., Two distinct inputs to an avian song

nucleus activate different glutamate receptor subtypes on individualŽ .neurons, Proc. Natl. Acad. Sci. USA, 88 1991 4075–4079.

w x74 Morino-Wannier, P., Fujita, S.C. and Jones, E.G., GABAergicneuronal populations in monkey primary auditory cortex defined byco-localized calcium binding proteins and surface antigens, Exp.

Ž .Brain Res., 88 1992 422–432.w x75 Morrison, J.H., Hof, P.R., Campbell, M.J., de Lima, A.D., Voigt,

T., Bouras, C., Cox, K. and Young, W.G., Cellular pathology inAlzheimer’s disease: implications for corticocortical disconnectionand differential vulnerability. In S.I. Rapoport, H. Petit, D. Leys

Ž .and Y. Christen Eds. , Imaging, Cerebral Topography andAlzheimer’s Disease, Springer–Verlag, New York, 1990, pp. 19–40.

w x76 Nakanishi, S., Molecular diversity of glutamate receptors andŽ .implications for brain function, Science, 258 1992 587–603.

w x77 Novelli, A., Reilly, J.A., Lysko, P.G. and Henneberry, R.C., Gluta-


mate becomes neurotoxic via the N-methyl-D-aspartate receptorwhen intracellular energy levels are reduced, Brain Res., 451Ž .1988 205–212.

w x78 Nusser, Z., Mulvihill, E., Streit, P. and Somogyi, P., Subsynapticsegregation of metabotropic and ionotropic glutamate receptors as

Ž .revealed by immunogold localization, Neuroscience, 61 1994421–427.

w x Ž .79 Passingham, R.E., Memory of monkeys Macaca mulatta withŽ .lesions in prefrontal cortex, BehaÕ. Neurosci., 99 1985 3–21.

w x80 Pearson, R.C.A., Esiri, M.M., Hiorns, R.W., Wilcock, G.K., Pow-ell, T.P.S., Anatomical correlates of the distribution of the patho-logical changes in the neocortex in Alzheimer’s disease, Proc.

Ž .Natl. Acad. Sci. USA, 82 1985 4531–4534.w x81 Petralia, R.S., Yokotani, N. and Wenthold, R.J., Light and electron

microscope distribitution of the NMDA receptor subunit NMDAR1in the rat nervous system using a selective anti-peptide antibody, J.

Ž .Neurosci., 14 1994 667–696.w x82 Petralia, R.S. and Wenthold, R.J., Light and electron immunocyto-

chemical localization of AMPA-selective glutamate receptors in theŽ .rat brain, J. Comp. Neurol., 318 1992 329–354.

w x83 Quinlan, E.M. and Halpain, S., Postsynaptic mechanisms for bidi-rectional control of MAP2 phosphorylation by glutamate receptors,

Ž .Neuron, 16 1996 357–368.w x84 Richards, J.G., Schoch, P., Haring, P., Takacs, B. and Mohler, H.,¨

Resolving GABA rbenzodiazepine receptors: cellular and subcel-A

lular localization in the CNS with monoclonal antibodies, J. Neu-Ž .rosci., 7 1987 1866–1886.

w x85 Rosvold, H.E., Szwarcbart, M.K., Mirsky, A.F. and Mishkin, M.,The effect of frontal-lobe damage on delayed response performance

Ž .in chimpanzees, J. Comp. Physiol. Psychol., 54 1961 368–374.w x86 Salt, T.E., Meir, C.L., Seno, N., Krucker, T. and Herrling, P.L.,

Thalamocortical and corticocortical excitatory postsynaptic poten-tials mediated by excitatory amino acid receptors in the cat motor

Ž .cortex in vivo, Neuroscience, 64 1995 433–442.w x87 Sautiere, P.-E., Sindou, P., Couratier, P., Hugon, J., Wattez, A. and´

Delacourte, A., Tau antigenic changes induced by glutamate in ratprimary culture model: a biochemical approach, Neurosci. Lett.,

Ž .140 1992 206–210.w x88 Schwartz, M.L. and Goldman-Rakic, P.S., Callosal and intrahemi-

spheric connectivity of the prefrontal association cortex in rhesusmonkey: relation between intraparietal and principal sulcal cortex,

Ž .J. Comp. Neurol., 226 1984 403–420.w x89 Seltzer, B. and Pandya, D.N., Afferent connections and architecton-

ics of the superior temporal sulcus and surrounding cortex in theŽ .rhesus monkey, Brain Res., 149 1978 1–24.

w x90 Shima, K. and Tanji, J., Involvement of NMDA and non-NMDAreceptors in motor task-related activity in the primary and sec-

Ž .ondary cortical motor areas of the monkey, Cereb. Cortex, 3 1993330–347.

w x91 Siegel, S.J., Brose, N., Janssen, W.G., Gasic, G.P., Jahn, R.,Heinemann, S.F. and Morrison, J.H., Regional, cellular and ultra-structural distribution of the glutamate receptor subunit NMDAR1

Ž .in monkey hippocampus, Proc. Natl. Acad. Sci. USA, 91 1994564–568.

w x92 Simpson, J.R. and Isacson, O., Mitochondrial impairment reducesthe threshold for in vivo NMDA-mediated neuronal death in the

Ž .striatum, Exp. Neurol., 121 1993 57–64.w x93 Sindou, P., Couratier, P., Barthe, D. and Hugon, J., A dose-depen-

dent increase of tau immunostaining is produced by glutamate

Ž .toxicity in primary neuronal cultures, Brain Res., 572 1992242–246.

w x94 Somogyi, P., Eshhar, N., Teichberg, V.I. and Roberts, J.D.B.,Subcellular localization of a putative kainate receptor in Bergmannglial cells using a monoclonal antibody in the chick and fish

Ž .cerebellar cortex, Neuroscience, 35 1990 9–30.w x95 Somogyi, P., Takagi, H., Richards, J.G. and Mohler, H., Subcellu-¨

lar localization of benzodiazepinerGABAA receptors in the cere-bellum of rat, cat, and monkey using monoclonal antibodies, J.

Ž .Neurosci., 9 1989 2197–2209.w x96 Stollberg, J. and Berg, D.K., Neuronal acetylcholine receptors: fate

Ž .of surface and internal pools in cell culture, J. Neurosci., 7 19871809–1815.

w x97 Sutor, B. and Hablitz, J.J., Long-term potentiation in frontal cortex:role of NMDA-modulated polysynaptic excitatory pathways, Neu-

Ž .rosci. Lett., 97 1989 111–117.w x98 Tecoma, E.S. and Choi, D.W., GABAergic neocortical neurons are

Ž .resistant to NMDA receptor-mediated injury, Neurology, 39 1989676–682.

w x99 Tsumoto, T., Haghihara, H., Sato, H. and Hata, Y., NMDA recep-tors in the visual cortex of young kittens are more effective than

Ž .those of adult cats, Nature, 327 1987 513–514.w x100 Van Brederode, J.F.M., Mulligan, K.A. and Hendrickson, A.E.,

Calcium-binding proteins as markers for subpopulations ofGABAergic neurons in monkey striate cortex, J. Comp. Neurol.,

Ž .298 1990 1–22.w x101 Verma, A. and Moghaddam, B., NMDA receptor antagonists im-

pair prefrontal cortex function as assessed via spatial delayedalternation performance in rats: modulation by dopamine, J. Neu-

Ž .rosci., 16 1996 373–379.w x102 Vickers, J.C., Huntley, G.W., Edwards, A.M., Moran, T., Rogers,

S.W., Heinemann, S.F. and Morrison, J.H., Quantitative localiza-tion of AMPArkainate and kainate glutamate receptor subunits inneurochemically identified subpopulations of neurons in the pre-

Ž .frontal cortex of the macaque monkey, J. Neurosci., 13 19932982–2992.

w x103 Vickers,J.C., Huntley, G.W., Hof, P.R., Bederson, J., DeFelipe, J.and Morrison, J.H., Immunocytochemical localization of non-NMDA ionotropic excitatory amino acid receptor subunits in hu-

Ž .man neocortex, Brain Res., 671 1995 175–180.w x104 Walker, A.E., A cytoarchitectural study of the prefrontal area of the

Ž .macaque monkey, J. Comp. Neurol., 73 1940 59–86.w x105 Wang, S., Lees, G.J., Rosengren, L.E., Karlsson, J.-E., Stigbrand,

T., Hamberger, A. and Haglid, K.G., The effect of an N-methyl-D-aspartate lesion in the hippocampus on glial and neuronal marker

Ž .proteins, Brain Res., 541 1991 334–341.w x106 Wenthold, R.J., Petralia, R.S., Blahos II, J. and Niedzielski, A.S.,

Evidence for multiple AMPA receptor complexes in hippocampalŽ .CA1rCA2 neurons, J. Neurosci., 16 1996 1982–1989.

w x107 Wenthold, R.J., Yokotani, N., Doi, K. and Wada, K., Immuno-chemical characterization of the non-NMDA glutamate receptor

Ž .using subunit-specific antibodies, J. Biol. Chem., 267 1992 501–507.

w x108 Williams, S.M., Goldman-Rakic, P.S. and Leranth, C., The synap-tology of parvalbumin-immunoreactive neurons in the primate pre-

Ž .frontal cortex, J. Comp. Neurol., 320 1992 353–369.w x109 Wolf, S.S., Hyde, T.M. and Weinberger, D.R., Neurobiology of

Ž .schizophrenia, Curr. Opin. Neurol. Neurosurg., 6 1993 86–92.

quantitative localization of nmdar1 receptor subunit immunoreactivity in inferotemporal and...

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