Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry

Synapse-Specific Localization of NMDAand GABAA Receptor Subunits Revealed

by Antigen-RetrievalImmunohistochemistry

JEAN-MARC FRITSCHY,* OLIVER WEINMANN, ANDREAS WENZEL,

AND DIETMAR BENKE

Institute of Pharmacology, University of Zurich, CH-8057 Zurich, Switzerland

ABSTRACTConventional immunohistochemistry provides little evidence for the synaptic localization of

ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible insitu. Here, we have adapted antigen retrieval procedures based on microwave irradiation toenhance the immunohistochemical staining of g-aminobutyric acid type A (GABAA) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissueproduced a marked reduction of nonspecific staining, allowing an improved detection of GABAAreceptor subunits. However, staining of NMDAreceptor subunits remained suboptimal. In contrast,microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhance-ment of both NMDA and GABAA receptor subunit staining. The diffuse, partially intracellularsignals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronalsomata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, theNR2Aand NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriensand radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were un-stained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected onpyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites ofmossy fiber input. For the GABAA receptor subunits, the most striking change induced by thisprotocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidalcells. The a2 subunit immunoreactivity was particularly prominent in these synapses. In controlexperiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) wasnot influenced by prior microwave irradiation. The enhancement of cell-surface–associated stainingis therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment.These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABAAreceptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons canexpress multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210,1998. r 1998 Wiley-Liss, Inc.

Indexing terms: synapse; ionotropic neurotransmitter receptor; g-aminobutyric acid; glutamate;

subunit-specific antibody

Ionotropic neurotransmitter receptors are heterooligo-meric membrane proteins mediating fast synaptic trans-mission in the central nervous system (CNS). The immuno-histochemical detection of these receptors often results ina prominent intracellular staining of neurons, providinglittle evidence for a selective localization in the plasmamembrane or for aggregation at presumed synaptic sites.In particular, this is the case for the N-methyl-D-aspartate(NMDA) receptor subunit family (Brose et al., 1993; Hunt-ley et al., 1994; Petralia et al., 1994a,b; Farb et al., 1995;

Hof et al., 1996; Johnson et al., 1996; Rema and Ebner,1996), but has also been reported for other ionotropicreceptors including nicotinic acetylcholine receptor (Bravoand Karten, 1992; Schroder, 1992; Van der Zee et al., 1992;

Grant sponsor: Swiss National Science Foundation; Grant number:31-36327.92.

*Correspondence to: Dr. Jean-Marc Fritschy, Institute of Pharmacology,University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzer-land. E-mail: [email protected]

Received 27 May 1997; Revised 6 August 1997; Accepted 7 August 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 390:194–210 (1998)

r 1998 WILEY-LISS, INC.

Del Toro et al., 1994; Nakayama et al., 1995) andg-aminobutyric acid type A (GABAA receptor) subunits(Zimprich et al., 1991; Gutierrez et al., 1994; Hendry et al.,1994; Moreno et al., 1994; Fritschy and Mohler, 1995).Although the significance of intracellular staining of recep-tor subunits is unclear, synaptic receptors in situ appear tobe not readily accessible to antibodies unless the synapticcleft is exposed, as shown by postembedding electronmicroscopy (Nusser et al., 1994, 1995a,b, 1996; Baude etal., 1995; Kharazia et al., 1996). Thus, conventional immu-nohistochemical methods (immunoperoxidase staining,preembedding electron microscopy) appear to be of limitedvalue in visualizing synaptic neurotransmitter receptorswhile overrepresenting extrasynaptic receptors and intra-cellular subunit proteins.

The aim of the present study was to adapt antigen-retrieval procedures based on microwave irradiation fordetecting synaptic receptor epitopes at the light micro-scopic level. The use of microwave irradiation has beenshown to effectively ‘unmask’ antigens in a variety ofprotocols designed primarily for histopathological analysisof human tissue following prolonged fixation and paraffinembedding (Shi et al., 1991, 1995; Ainley and Ironside,1994; Evers and Uylings, 1994a; Login and Dvorak, 1994;Hazelbag et al., 1995; Werner et al., 1996). These antigen-retrieval procedures have been applied to a great variety ofantigens, although no single protocol is optimal becausepH and temperature appear to influence differentially theretrieval of various antigens (Evers and Uylings, 1994b;Shi et al., 1995; Werner et al., 1996). The effects ofmicrowave irradiation on histological specimens are poorlyunderstood. It is generally assumed that antigen retrievalis due to the breaking of aldehyde-based crosslinks and toprotein denaturation resulting in epitope exposure. It isunclear, however, whether these effects are merely heatinduced or dependent on microwave energy (see Werner etal., 1996, for review).

In the first series of experiments, the influence ofmicrowave treatment for unmasking GABAA and NMDAreceptor subunit immunoreactivity (IR) was tested ontissue blocks from rat brain fixed by perfusion withparaformaldehyde. This procedure resulted in an excellentpreservation of the sections and produced a marked im-provement of the GABAA receptor immunohistochemicalstaining, but not of the NMDA receptor subunit staining,which remained weak and localized intracellularly. Todetermine whether the poor antigenicity of NMDA recep-tor subunits was related to the prolonged fixation of thetissue, an alternative approach was developed. It consistedof microwave irradiation during a brief exposure of freshtissue sections to a low concentration of paraformaldehyde(Boon et al., 1988). This procedure allowed a dramaticenhancement of both the GABAA receptor and the NMDAreceptor subunit IR, which appeared frequently clusteredat sites of presumed synaptic input. Thus, microwaveirradiation of fresh tissue represents a valuable method forunmasking the epitopes of synaptic neurotransmitter re-ceptor subunits to analyze their subcellular distribution.

MATERIALS AND METHODS

Microwave irradiation of fixed tissue

Adult male Sprague-Dawley rats (Institut of LaboratoryAnimal Science, University of Zurich) were deeply anesthe-

tized with pentobarbital (Nembutal, Abbott, Chicago, IL,40 mg/kg) and perfused through the ascending aorta witha mixture of 4% paraformaldehyde and 15% saturatedpicric acid (Somogyi and Takagi, 1982). After postfixationfor 4–8 hours in the same fixative, the brain was cutparasagittally in 8- to 12-mm thick blocks to be irradiatedwith microwaves under various conditions, as detailed inTable 1. The tissue was first incubated overnight in buffer(see Table 1 for the composition of the various bufferstested) at room temperature. It was then transferred into80 ml fresh buffer and irradiated in a kitchen microwaveoven at 650 W for 30–300 seconds. Under these conditions,the buffer started to boil after approximately 60 seconds.The tissue was then cooled to approximately 40°C andtransferred into phosphate-buffered saline (PBS), pH 7.4,containing 10% dimethylsulfoxide for cryoprotection (3hours at 4° C). Thereafter, the blocks were frozen and cutat 40 µm with a sliding microtome. Sections were collectedin PBS and processed for immunohistochemistry using astandard protocol (see below).

Microwave irradiation during fixation

Adult rats were decapitated, and the brains were rapidlyexcised and frozen with powdered dry ice. Parasagittalsections were cut at 12–14 µm with a cryostat, mountedonto gelatin-coated slides, and dried at room temperaturefor exactly 30 seconds. They were then stored at 220°Cuntil use (up to 4 weeks). For the experiment, sectionswere thawed for 30 seconds at room temperature, im-mersed horizontally in a Petri dish containing 50 ml 0.5%paraformaldehyde and 5% Kryofix (Merck, Darmstadt,Germany; Marani and Horobin, 1994) in 0.1 M phosphatebuffer (pH 7.4), and immediately irradiated in a micro-wave oven (45 seconds at 650 W). They were then trans-ferred into PBS and processed for immunofluorescencestaining.

TABLE 1. Influence of Buffer and Irradiation Time on Antigen Retrievalin Fixed Tissue Blocks1

Antigen

GABAAreceptor

a5 subunit

NMDAreceptor

NR2B subunit Calretinin

BufferAlCl3 (pH 2.75)2 1/2 1/2 1/2Sodium citrate (pH 2.2) 2 2 2Sodium citrate (pH 4.5) 111 1 111Glycine (pH 9.5) 1 2 1KSCN (pH 6.5)3 2 2 2Distilled water 1/2 1/2 1/2

Irradiation time (sec; Na-citrate,pH 4.5)

30 1/2 260 1 1/2

120 11 1150 111 11180 11 111240 1 11300 2 1

1Blocks of fixed tissue were preincubated overnight in the respective buffer used duringmicrowave treatment (irradiation at 650 W for 150 seconds unless indicated otherwise;see Materials and Methods for details). The buffers tested had the following concentra-tions and compositions: AlCl3: 4% AlCl3; sodium citrate: 0.1 M citric acid 1 0.2 MNa2HPO4; Glycine: 0.1 M Glycine 1 0.1 M NaCl; KSCN: 0.1–1 M KSCN. The symbolsrange from 2 (no specific staining) to 111 (marked improvement of the signal-to-noiseratio).2A pronounced shrinkage of the tissue was observed in this buffer.3Potassium thiocyanate.

SYNAPTIC GABAA AND NMDA RECEPTORS 195

Immunohistochemistry

The antibodies tested included guinea pig antisera recogniz-ing the GABAA receptor subunits a2, a3, a5, or g2 (Fritschyand Mohler, 1995) and affinity-purified antisera against theNMDA receptor subunits NR2A and NR2B raised in rabbitand guinea pig, respectively (as described in Benke et al.,1995; Wenzel et al., 1995). All GABAA and NMDA receptorsubunit antibodies were raised against extracellular epitopeslocated on the N-terminal segment of the proteins (see Fritschyand Mohler, 1995 and Wenzel et al., 1995, for details). Inaddition, several antibodies against various proteins wereused to test the general usefulness of these antigen-retrievalprocedures. They included a sheep antiserum against glu-tamic acid decarboxylase (GAD, kindly provided by Dr. W.Oertel, University of Munich, Germany) and commerciallyavailable antibodies against calbindin D28k (no. 300; Swant,Bellinzona, Switzerland), calretinin (no. 7696; Swant), heavy-chain neurofilaments (no. SMI-32; Sternberger Monoclonals,Baltimore, MD), glial fibrillary acid protein (GFAP; no. Z130;Dako Diagnostics, Zug, Switzerland), GABA(no.A2052; Sigma,St. Louis, MO), serotonin (no. 20080; Incstar, Stillwater, MN),and calcitonin-gene–related peptide (no. Ab1971; Chemicon,Temecula, CA).

Sections of postfixed tissue pretreated with microwaveirradiation were processed free-floating for immunoperoxi-dase staining by using the avidin-biotin peroxidase method ofHsu et al. (1981). The sections were incubated overnight at4°C in primary antibodies diluted in 50 mM Tris-saline, pH7.4, containing 2% normal goat serum and 0.05% Triton X-100.They were then rinsed with buffer, incubated for 30 minutes atroom temperature in biotinylated secondary antibodies raisedin goat (1:300 in Tris-saline containing 2% normal goat serum;Jackson Immunoresearch, West Grove, PA), and processed foravidin-peroxidase staining as described by the manufacturer(Vectastain Elite Kits, Vector Laboratories, Burlingame, CA)with diaminobenzidine hydrochloride (Sigma) as chromogen.The sections were then mounted on gelatin-coated slides,dehydrated, and coverslipped out of xylene. Sections fixedduring microwave irradiation were processed either for immu-noperoxidase staining as describe above or for immunofluores-cence staining by using secondary antibodies coupled to indo-carbocyanine (Cy3) or to dichlorotriazinylaminofluorescein(DTAF; Jackson Immunoresearch). These sections were cover-slipped with buffered glycerol (1:1 in sodium bicarbonatebuffer, pH 9.5).

Data analysis and photography

Sections were analyzed with a Zeiss Axiophot micro-scope (Jena, Germany) equipped for light and epifluores-cence microscopy. Photomicrographs were taken with Ko-dak T-max 100 film (Rochester, NY). Sections processed forimmunofluorescence staining were also analyzed by laserscanning confocal microscopy (TCS 4D; Leica, Heidelberg,Germany) by using Imaris software (Bitplane, Zurich,Switzerland) for image restoration and three-dimensionalreconstruction. Digital images were printed with a FujiPictrography 3000 printer.

For semiquantitative image analysis, the sections weredigitized with a high-resolution video camera and ana-lyzed with MCID M2 software (Imaging Research, St.Catherines, Ontario). For display, the images were con-

trast enhanced on a 256-gray level scale and printed on theFuji Pictrography printer.

RESULTS

Microwave irradiation of fixed tissue

The influence of microwave irradiation on retrieval ofreceptor subunit antigens was tested in fixed tissue withan antiserum against the GABAA receptor a5 subunit andnonimmune serum as control. The a5 subunit antiserumwas selected because this subunit has a characteristicregional distribution that is well known from in situhybridization histochemistry studies (Persohn et al., 1992;Wisden et al., 1992). Thus, we expected to be able todifferentiate between the effects on specific and nonspecificstaining in different brain areas. The influence of the typeof buffer, pH, and irradiation time was established bycomparison with sections from the contralateral hemi-sphere processed in parallel with standard immunoperoxi-dase staining but without microwave irradiation (Table 1).The tissue blocks were 8- to 12-mm thick slabs of rat braincut parasagittally. Sodium citrate buffer (0.1 M, pH 4.5)was consistently found to yield the best improvement of a5subunit IR following microwave irradiation for 150 sec-onds (Fig. 1; Table 1). Chaotropic ions (KSCN; Table 1) andthe other buffers tested, which have been reported tocontribute to antigen retrieval (Cattoretti et al., 1993;Evers and Uylings, 1994b; Shi et al., 1995), were noteffective. Extending the irradiation time beyond 150 sec-onds in sodium citrate buffer resulted in a gradual de-crease of staining intensity (Table 1) accompanied by arapid degradation of tissue.

The main effect of microwave irradiation in sodiumcitrate buffer on a5 subunit IR was a profound reduction ofbackground staining, particularly obvious in those regionswhere the a5 subunit is not expressed (e.g., cerebellum;Fig. 1). A semiquantitative analysis of the immunostainingintensity was performed in digitized images from sectionstreated with microwaves and from untreated sections. An86% reduction of the nonspecific staining produced by thea5 subunit antiserum was seen in the cerebellum followingmicrowave irradiation. A comparable reduction in back-ground was also seen in sections incubated with nonim-mune serum at the same concentration (1:3,000). Thenonspecific staining produced by the secondary antibodiesalone (i.e., in the absence of primary antibody) was re-duced to 0.6% of the maximal intensity seen for the a5subunit IR in the CA1 region of the hippocampus (Table 2).Most importantly, this analysis revealed that the intensityof the specific a5 subunit IR was not affected by themicrowave treatment, resulting in a marked increase ofthe signal-to-noise ratio. These results suggest that micro-wave irradiation neutralizes sites of nonspecific binding ofimmunoglobulins in fixed brain sections and reduces endog-enous peroxidase activity. Heat by itself was not sufficientto improve the a5 subunit IR or to reduce the backgroundstaining as tested by incubating the sections in a waterbath (180 seconds at either 80°C or 95°C).

The effects of microwave irradiation on GABAA receptorstaining were further evaluated on the cellular level usingantibodies against the a3, a5, and g2 subunits. With thestandard immunoperoxidase protocol, these antisera pro-

196 J.-M. FRITSCHY ET AL.

duced a rather strong intracellular staining, as shown forthe a3 subunit in the cingulate cortex (Fig. 2A). Microwaveirradiation led to a pronounced intensification of cell-surface staining and to a decrease of background staining(Fig. 2B). Under these conditions, a3 subunit IR was foundto label pyramidal neurons located in the deeper layers,revealing their dendritic tree extending toward the corti-cal surface and arborizing in layers I–II (Fig. 2C). Similarimprovements of staining quality were also seen for a5 andg2 subunit IR. At higher magnification, hot spots ofstaining suggestive of synaptic clusters became apparentalong dendrites and neuronal somata, as depicted for theg2 subunit in spinal cord neurons (Fig. 2D,E). Such hot

spots were not detectable in non-pretreated tissue (Fig.2D). These changes in the subcellular distribution ofGABAA receptor subunits were highly suggestive of anunmasking effect of microwave irradiation.

To determine whether the immunoreactivity of NMDAreceptor subunits could be similarly improved, the sameprocedure was tested with an antiserum against the NR2Bsubunit. This antiserum produced a rather weak, specificstaining localized intracellularly and a uniform back-ground in sections unexposed to microwave irradiation(Fig. 3A). Microwave treatment in sodium citrate bufferresulted in a decrease of background staining (Fig. 3B),revealing the characteristic regional distribution of the

Fig. 1. Effect of microwave irradiation of fixed tissue on theg-aminobutyric acid type A (GABAA) receptor a5 subunit immunoreac-tivity (IR) as seen in parasagittal sections of adult rat brain processedfor immunoperoxidase staining. The overall decrease in stainingbetween the section of control tissue, unexposed to microwaves (A),and of microwave-irradiated tissue (B) is mainly due to the loss ofnonspecific staining, whereas the specific staining (e.g., in the CA1region of the hippocampus) remains almost unchanged. Therefore,microwave treatment results in a more contrasted regional distribu-

tion of a5 subunit IR. For example, clear differences become apparentin the laminar distribution of a5 subunit IR in distinct areas of thecerebral cortex. Note in particular the lack of a5 subunit IR in layer IVof the primary somatosensory (S1) and visual cortex (V1; arrowheads).A marked difference in staining intensity between the hippocampusand dentate gyrus also becomes apparent after microwave treatment,in agreement with the corresponding difference in a5 subunit mRNAexpression levels between these two regions. Scale bar 5 1 mm.

197SYNAPTIC GABAA AND NMDA RECEPTORS

NR2B subunit reported by in situ hybridization studies(Ishii et al., 1993; Watanabe et al., 1993; Monyer et al.,1994; Wensel et al., 1997). However, the intensity of NR2Bsubunit IR remained rather weak, and most of the stainingwas confined intracellularly to neuronal somata and proxi-mal dendrites, as seen in the striatum, hippocampalformation, and cerebral cortex. The other buffers testedwere ineffective (Table 1). Therefore, microwave irradia-tion of fixed tissue did not produce the same unmaskingeffect for the NR2B subunit as for the GABAA receptorsubunits.

Several additional antibodies against various antigensexpressed in the CNS (GAD, serotonin, CGRP, calretinin,parvalbumin, GFAP, and neurofilaments) were tested af-ter microwave irradiation in sodium citrate buffer toassess the general usefulness of this method for immuno-histochemistry. The loss of background was systematicallyobserved, contributing in most cases to a marked enhance-ment of the signal-to-noise ratio and enabling a betterresolution of fine details in weakly stained structures.Cytoskeletal markers such as neurofilaments or GFAPwere otherwise not influenced by the prior microwavetreatment. Variable effects were observed, however, forcytoplasmic proteins (such as GAD or calcium-bindingproteins) and neurotransmitters. Staining was clearlyenhanced for some (e.g., calretinin; Table 1) whereas it waspartially reduced for others (e.g., GAD, GABA, serotonin,CGRP), indicating that each antibody has to be testedindividually. The reduction of staining intensity for neuro-transmitters was particularly evident in terminal fields,where some leakage outside the axonal terminals wasapparent. As tested for calretinin IR, only the sodiumcitrate buffer proved suitable to improve the immunohisto-chemical staining (Table 1), suggesting that this buffermay be of general use for antigen retrieval in brain tissuefixed by perfusion with paraformaldehyde.

Unmasking NMDA receptor subunit IR bymicrowave irradiation during fixation

The lack of improvement of NR2B subunit IR followingmicrowave irradiation of fixed tissue (Table 1) suggestedthat the prolonged fixation might prevent the access of

antibodies to synaptic receptor epitopes or alter the confor-mation of these proteins. Therefore, a procedure combin-ing microwave irradiation with a brief fixation was devel-oped based on the protocol of Boon et al. (1988). Cryostatsections prepared from fresh-frozen tissue and mountedonto gelatinized glass slides were immersed in 50 mlphosphate buffer containing 0.5% paraformaldehyde andimmediately irradiated in a microwave oven (45–90 sec-onds; 650 W). The sections were then transferred into PBSand processed for immunohistochemistry with an affinity-purified antiserum against the NR2B subunit. This proce-dure resulted in a marked increase of staining intensity inthe regions expected to contain the NR2B subunit (CA1region of the hippocampus, dentate gyrus, cerebral cortex,striatum, olfactory bulb, and thalamus) and a correspond-ing decrease of background in the rest of the brain (Fig.3C). At higher magnification, NR2B subunit IR appearedas intensely stained, discrete puncta in the neuropil,outlining the soma and proximal dendrites of neurons,which themselves were not stained. These puncta wereabsent in regions devoid of NR2B subunit expression (e.g.,inferior colliculus, cerebellum) and from the white matter.As for microwave treatment of fixed tissue, the relativestaining intensity decreased after prolonged irradiation ofthe tissue (.60 seconds; 650 W). Furthermore, tissuepreservation was enhanced by addition of 5% Kryofix(Marani and Horobin, 1994) to the fixative.

This ‘microwave fixation’ procedure was also tested withan affinity-purified rabbit antiserum against the NR2Asubunit, with very similar results. Immunofluorescencestaining revealed the presence of numerous, intenselystained puncta in the neuropil of the cerebral cortex (Fig.4A), striatum (Fig. 4B), hippocampal formation (Fig. 4C,D),thalamus, tectum, and cerebellum. The lack of intracellu-lar staining was most striking in the hippocampus CA1field, where the intense immunoreactivity in the stratumoriens and radiatum contrasted with the unstained pyra-midal layer (Fig. 4C). In the dentate gyrus, the granule cellsomata were surrounded by a moderate number of largepuncta, whereas the molecular layer appeared very in-tensely stained (Fig. 4D). The distribution of immunoreac-tive puncta thus correlated with the known distribution ofexcitatory synapses in the hippocampal formation.

Double-immunofluorescence staining for the NR2A andNR2B subunits revealed largely overlapping distributionsbut also distinct differences, including the following. In the

TABLE 2. Reduction of Nonspecific Immunoglobulin BindingAfter Microwave Treatment of Fixed Tissue Blocks1

Relative optical density value (%)

Standardprotocol

Microwave irradiationin citrate buffer

GABAA receptor a5 subunit (1:3,000)Hippocampus CA1 100 (0.21)2 100 (0.19)2

Cerebellum 38.1 5.2Nonimmune guinea pig serum

1:10 95.3 37.81:100 94.1 34.91:1,000 43.8 9.61:3,000 36.1 5.5No primary antiserum 35.2 0.6

1Parasagittal sections of rat brain were processed for immunoperoxidase staining witheither a guinea pig antiserum against the GABAA receptor a5 subunit or withnonimmune guinea pig serum and digitized with a high-resolution video camera. Therelative optical density (ROD) values of the staining are expressed in percent of thehighest value found in the CA1 region of the hippocampus for a5 subunit immunoreac-tivity. The ROD values for nonimmune serum were measured over the entire section.The data show that microwave irradiation produces a seven-fold reduction of back-ground, nonspecific staining of the a5 subunit in cerebellum without affecting thespecific staining in CA1. The background staining produced by the nonimmune serum orby the secondary antibody alone is likewise reduced by this treatment.2Actual ROD values for comparison.

Fig. 2. Effect of microwave irradiation on the regional and cellulardistribution of GABAA receptor a3 and g2 subunit IR. The a3 subunitIR in rat cingulate cortex in control (A) and microwave-treated (B)sections; a combined decrease of background staining and increase ofcell-surface staining results in a striking enhancement of the image,revealing the staining of apical dendrites arising from layer VIpyramidal cells and arborizing near the pial surface. C: High-magnification photomicrograph of the staining of individual pyrami-dal cells with the a3 subunit antiserum; note the punctate appearanceof the staining. D,E: The g2 subunit IR in neurons of Rexed lamina VIIin the spinal cord in control (D) and microwave-treated (E) sections;microwave treatment strongly reduces the intracellular staining ofthese neurons and reveals hot spots of g2 subunit IR on their soma anddendrites (arrowheads). Scale bars 5 200 µm in A,B, 50 µm in C, 30µm in D,E.


Figure 2

stratum lucidum of the CA3 field, for example, only NR2Asubunit IR was detected on the dendrites of pyramidalcells, whereas the NR2A and NR2B subunits were colocal-ized in the stratum oriens and radiatum (Fig. 5A,B); theseneurons thus appear to express two distinct NMDA recep-tor subtypes. In the cerebral cortex, both subunits weredetected in the neuropil throughout layers I–VI, with thestaining intensity of the NR2B subunit being strongest in

layers I–III and NR2A subunit IR uniformly distributedacross the six layers (not shown). In the cerebellum, NR2Asubunit IR was intense in the granule cell layer (beingselectively localized in glomeruli), whereas granule cellbodies were unstained; in contrast, NR2B subunit IR wasrestricted to a few scattered cells with a size and morphol-ogy suggestive of astrocytes (Fig. 5C,D).

The intense staining of the puncta in the neuropil madeit possible to study their subcellular distribution withhigh-resolution confocal scanning laser microscopy to pro-vide further evidence for their synaptic localization. Thedensity of puncta immunoreactive for the NR2B subunitwas found to be higher in the striatum than in layer III ofcerebral cortex, although they appeared uniformly distrib-uted in the neuropil in both areas (Fig. 6A,B). In thedentate gyrus, puncta of NR2B subunit IR were found tosurround the somata of certain granule cells (Fig. 6C,D). Aparticularly high density of such puncta was observedaround the most superficially located cells.

The specificity of the staining was verified with eithernonimmune serum or antibodies preabsorbed with theirpeptide antigen. No puncta were observed in either case,and the background was very low (not shown). Further, theprocedure was tested with an antibody against GFAP todetermine whether cytoskeletal structures are affected bythe microwave irradiation or the weak fixation. A normalstaining pattern was observed in these sections, withastrocytic processes extending toward and terminatingonto blood vessels (Fig. 7D). The number and appearanceof GFAP-positive astrocytes were indistinguishable fromthose seen with conventional immunofluorescence stain-ing, indicating that immunostaining of this cytoskeletalprotein was not influenced by the tissue treatment.

Unmasking GABAA receptor subunit IR bymicrowave irradiation during fixation

To determine whether the dramatic improvement inNMDA receptor subunit staining could be generalized toother ionotropic receptor proteins, the procedure of micro-wave irradiation during the fixation of fresh tissue sec-tions was tested for several GABAA receptor subunitantibodies (a2, a3, g2). With each of these antisera, thisprotocol resulted in the appearance of numerous intenselystained puncta, outlining cell bodies and proximal den-drites. The density of these puncta was generally lowerthan for the NR2A or NR2B subunits, but they were largerand frequently seen around neuronal somata and proximaldendrites. For all three GABAA receptor subunits, theregional distribution of these puncta corresponded to thatexpected from studies with conventional immunohisto-chemical procedures (Fritschy and Mohler, 1995).

Fig. 3. Influence of tissue processing and microwave treatment onthe regional distribution of the N-methyl-D-aspartate (NMDA) recep-tor NR2B subunit in adult rat brain. A: Conventional immunoperoxi-dase staining without microwave treatment; only small differences instaining intensity are evident across the section, due to the weak‘‘specific’’ signals in the forebrain and a uniform background. Thestrongest signals are seen in the pyramidal and granule cell layers ofthe hippocampal formation (arrowheads), reflecting the apparentintracellular localization of NR2B subunit IR. B: Microwave irradia-tion of fixed tissue. A decrease of background staining is evident in thebrainstem and cerebellum, whereas an increase of NR2B subunit IRcan be seen in the thalamus and in the dendritic layers of thehippocampal formation. However, the intracellular staining noted insections unexposed to microwave irradiation is largely retained. C:Microwave irradiation of a cryostat section during fixation; a robustincrease of staining intensity is evident in the forebrain, accompaniedby a loss of intracellular staining in the pyramidal cell layer of thehippocampus (arrowhead). A further decrease of background stainingis also evident, notably in the cerebellum. Scale bar 5 1 mm.

Fig. 4. Photomicrographs depicting the cellular distribution ofNR2A subunit IR in rat brain cryostat sections fixed during microwaveirradiation as seen with immunofluorescence. In this and the followingfigures, intense staining appears white. (A) Cerebral cortex, (B)striatum, (C) CA1 region of the hippocampus, and (D) dentate gyrus.Note the almost complete lack of intracellular staining in neuronalsomata and the punctate appearance of the staining in the neuropil.The largest puncta are seen in the molecular layer of the dentate gyrus(D), with some of them surrounding the granule cell bodies. Incontrast, no such puncta are seen around the somata of pyramidalcells in CA1 (C). Scale bars 5 30 µm in A–C, 20 µm in D.


Figure 4

The a2 subunit IR was most prominent in the hippocam-pal formation, striatum, olfactory bulb, and outer layers ofcerebral cortex, where numerous, intensely stained punctawere distributed throughout the neuropil. In subcorticalcenters and brainstem, high-magnification visualizationby confocal scanning laser microscopy revealed individualneurons outlined by numerous puncta over their soma andproximal dendrites (Fig. 7A,B); alternatively, the punctawere sometimes so numerous that individual neuronscould not be identified unambiguously. This was seen, forexample, in the thalamic reticular nucleus with a3 subunitIR (Fig. 7C). Such features were never observed withconventional immunohistochemical procedures. Thesechanges in the subcellular distribution of the immunoreac-tivity suggest that the puncta represent synaptic receptorsthat are unmasked by microwave irradiation during fixa-tion.

The most striking observation, however, was the stain-ing of presumed axon initial segment (AIS) of pyramidalneurons in the cerebral cortex (Fig. 8A), hippocampus (Fig.8B), and dentate gyrus granule cells. Although GABAergicsynapses on AIS are quite prominent (Kosaka, 1980;Somogyi et al., 1983a), GABAA receptors in these synapseshave never been reported in studies using conventionalimmunohistochemical procedures. In microwave-irradi-ated cryostat sections, however, a2 subunit IR revealed theAIS as forming 20- to 70-µm–long, tail-like structuresoutlined by numerous intensely stained puncta, as shownfor the AIS of CA3 pyramidal neurons (Figs. 8B, 9A,B).Typically, the staining started at some distance (10–30µm) from the base of the soma, and it was often difficult todetermine to which neuron a given AIS belonged. Toconfirm the preferential localization of a2 subunit IR onthe axon of pyramidal cells, a double-staining with anantibody against nonphosphorylated heavy-chain neurofila-ments (SMI-32) was performed. In both the neocortex andhippocampus, the SMI-32 staining revealed the cytoskel-eton of a subpopulation of pyramidal cells, including theirmajor dendrites and the beginning of the axon. An exampleof the localization of a2 subunit IR on the AIS of anSMI-32–positive pyramidal cell is shown in Figure 9C.Such experiments revealed that the number of a2 subunitIR puncta on the AIS outnumbered those found on thesoma and proximal dendrites of the same cell, suggesting apreferential targeting of a2–GABAA receptors to the AIS.Furthermore, the intense staining of the puncta in the AISis suggestive of a particularly high density of receptors inthese synapses.

Although the staining of AIS of hippocampal pyramidalcells was particularly prominent with the a2 subunitantiserum, it was also detected with the b2,3 and g2subunit antibodies (Fig. 9D), in line with previous evi-dence from postembedding electron microscopy (Somogyiet al., 1996). In the neocortex, a laminar segregation ofGABAA receptor subunits was evident. The a2, and to alesser extent the a1, subunits were mostly detected in theAIS of layers II–III pyramidal cells, whereas the a3subunit predominated in the AIS of layers V and VIpyramidal cells. For each a subunit variant, only a minor-ity of pyramidal cells were labeled on their AIS. Thesepreliminary observations suggest that GABAA receptors inAIS differ in subunit composition across cortical layers.

DISCUSSION

In conventional immunohistochemistry procedures, amajor fraction of receptor subunit epitope does not appearto be accessible for the antibodies. A dramatic improve-ment of the immunohistochemical staining of NMDA andGABAA receptor subunits is achieved after brief micro-wave irradiation of adult rat brain tissue. In particular,the application of microwave irradiation during the fixa-tion of cryostat sections reveals the subcellular localiza-tion of both NMDA and GABAA receptor subunits atpresumed sites of synaptic inputs. With regard to NMDAreceptors, the differential localization of the NR2A andNR2B subunits on the dendrites of CA3 hippocampalpyramidal neurons suggests a synapse-specific segrega-tion of receptor subtypes in these neurons, correlatingwith the termination of mossy fibers in the stratumlucidum. Likewise, a synapse-specific distribution ofGABAA receptor subunits is apparent in the AIS of corticaland hippocampal pyramidal cells, notably for the a2subunit. The latter findings confirm and extend the resultsof postembedding electron microscopic studies demonstrat-ing a selective localization of the a2 and g2 subunits in AISof CA1 pyramidal neurons (Nusser et al., 1996; Somogyi etal., 1996). The remarkable specificity in the synaptictargeting of NMDA and GABAA receptor subunits inhippocampal and neocortical neurons suggests that indi-vidual neurons can express multiple receptor subtypes infunctionally distinct synapses.

Effects of microwave irradiation

The immunohistochemical staining of NMDAand GABAAreceptor subunits, which are ubiquitously expressed in thenervous tissue, is strongly dependent on the treatment ofthe tissue during or after the fixation. Microwave irradia-tion of fresh tissue sections produces a marked enhance-ment of the signal-to-noise ratio and changes the apparentcellular and subcellular distribution of GABAA and NMDAreceptor subunits. The diffuse, partially intracellular im-munoreactivity seen with conventional procedures islargely replaced by intensely immunoreactive puncta out-lining neuronal somata and dendrites. The selective en-hancement of cell-surface–associated staining suggests anunmasking of subunit epitopes, corresponding presumablyto synaptic receptors. Conventional immunohistochemicalprocedures therefore fail to label a significant fraction ofreceptors while overrepresenting extrasynaptic receptorsand intracellular subunit proteins. The reduction of intra-cellular staining on microwave irradiation is probably due

Fig. 5. Double-immunofluorescence staining depicting the differen-tial distribution of NR2A and NR2B subunit IR in cryostat sectionsfixed during microwave irradiation. A,B: CA3 region of the hippocam-pus. NR2A subunit IR (A) is very strong in the stratum oriens andradiatum and surrounds the dendrites of pyramidal cells with numer-ous puncta in the stratum lucidum (SL; arrowheads). In contrast,NR2B subunit IR (B) is absent in the stratum lucidum and has adistribution very similar to the NR2A subunit in the stratum oriens(SO) and stratum radiatum (SR). C,D: Granule cell layer of thecerebellum. NR2A subunit IR (C) is restricted to synaptic glomerulithat appear intensely stained. NR2B subunit IR (D) is absent fromthis layer, except for a few isolated cells (arrow) with an apparentmorphology of astrocytes. Note the lack of staining for either subunitin the granule neurons and in the Purkinje cells (arrowheads). Scalebars 5 30 µm.


Figure 5

to a shift in the distribution of antibodies toward thehighly concentrated synaptic epitopes exposed by thetreatment.

Although the prominent intracellular staining observedwith conventional procedures (see introductory section ofthis article) can largely be abolished following preabsorp-tion of the antiserum with its peptide antigen, the resultsof such control experiments have to be interpreted withcaution. They are necessary to determine that the epitopesrecognized by the antibodies are present in the peptideused for preabsorption, but they are insufficient to demon-strate fully the specificity of an immunohistochemicalsignal in situ. The regional distribution of the subunitsanalyzed here was not changed by the microwave treat-ment, indicating that the staining pattern achieved withconventional methods is specific. However, the microwavetreatment enables us, for the first time, to visualize thesubcellular distribution of these major types of receptorsat the sites where they are expected to be found based onprevious nonimmunocytochemical means of localization.

The marked improvement of GABAA and NMDA recep-tor immunostaining indicates that microwave irradiationenhances the access of antibodies to their epitope, presum-ably in synaptic clefts. All subunit-specific antibodiestested here were raised against extracellular epitopes,located on the N-terminal segment of the respective sub-units. In synaptic receptors, these epitopes are expected tobe exposed in the synaptic cleft. The effect of microwavesmight therefore be due primarily to the widening ofsynaptic clefts. Protein denaturation, which has beenproposed to underlie the effects of microwave irradiation(see Werner et al., 1996, for review), is an unlikelycontributing factor to the enhancement of GABAA andNMDA receptor immunoreactivity, because heat alone wasnot sufficient to cause this effect.

Microwave irradiation of tissue blocks fixed with parafor-maldehyde produced a marked reduction of backgroundstaining for all combinations of primary and secondaryantibodies tested. This indicates that sites of nonspecificbinding of immunoglobulins, as well as the endogenousperoxidase activity, are neutralized by this treatment.Such a decrease in background staining was not men-tioned in previous studies on antigen retrieval (Evers andUylings, 1994b; Shi et al., 1995; Werner et al., 1996).However, these studies were conducted mostly on paraffin-embedded sections of human tissue that had been fixed forseveral months, and much longer irradiation times wereused than were required in the present study. Although theeffects underlying background reduction remain unex-plained, our results suggest that microwave irradiation athigh temperatures (.95°C) in sodium citrate buffer, pH4.5, may be advantageous for a large number of differentantibodies in tissue fixed by vascular perfusion. However,small soluble antigens, such as neurotransmitter mol-ecules, that appear to ‘‘leak’’ out of the terminals onmicrowave irradiation were not improved in our screening.Optimal conditions remain to be established for thesemolecules.

Synaptic targeting of GABAA and NMDAreceptor subunits

Several lines of evidence indicate that the puncta ofGABAA and NMDA receptor subunit IR seen in cryostat

sections fixed during microwave irradiation representpostsynaptic receptor clusters.

First, the relative density of puncta for a particularsubunit in a given brain area correlates well with therelative staining intensity seen with conventional immuno-histochemical procedures. In addition, the puncta arecompletely absent in regions not expressing this subunitand in the white matter; they are also abolished onpreabsorption of the antibodies with their respective anti-gens.

Second, the subcellular distribution of the NR2A andNR2B subunits in the hippocampus and dentate gyruscorresponds to the known distribution of excitatory syn-apses (type I; Gray, 1969) in these brain regions. Inparticular, CA1 and CA3 pyramidal cell somata, which arenearly devoid of excitatory synapses (Blackstad, 1963;Eccles, 1964; Gray, 1969), are not labeled with eithersubunit, whereas the dendritic layers exhibit the highestdensity of puncta of the entire brain. The NR2A subunitstaining in the stratum lucidum is likely to reflect the sitesof termination of mossy fibers on CA3 pyramidal cell apicaldendrites. Likewise, the prominent puncta outlining gran-ule cells in the dentate gyrus may be localized at sites ofmossy cell input.

Third, in the cerebellum, NR2A subunit IR appears to belargely restricted to synaptic glomeruli in the granule celllayer, which represent the only site of excitatory input ontogranule cell dendrites. No labeling was detected on gran-ule cell somata that receive no synaptic input.

Fourth, the prominent punctate staining of the GABAAreceptor a2, b2,3, and g2 subunits in AIS of hippocampuspyramidal cells (Kosaka, 1980; Somogyi et al., 1983a,b)correlates with the results of postembedding electronmicroscopic studies (Nusser et al., 1996; Somogyi et al.,1996) and indicates that these synapses contain a largenumber of GABAA receptors. The moderate number ofpuncta immunoreactive for the a2 subunit around thepyramidal cell bodies (Fig. 7B) corresponds to the selectivetargeting of the a2–GABAA receptors to AIS demonstratedin these electron microscopic studies.

Fifth, a similar punctate staining, reflecting postsynap-tic receptor aggregates, has been reported for severalGABAA receptor subunits and for the NMDA receptorNR2A subunit in cryostat sections of the retina (Hartveitet al., 1994; Greferath et al., 1995; Sassoe-Pognetto et al.,1995; Koulen et al., 1996). Unlike those in the brain,synapses in the retina are not surrounded by glial end-feetand might thus be more accessible to antibodies followingfixation. Furthermore, the punctate staining in retinacould be largely abolished by prolonged fixation (Greferathet al., 1995; Sassoe-Pognetto et al., 1995), corroboratingthe hypothesis that NMDA and GABAA receptor subunitepitopes are highly sensitive to fixation.

Sixth, a prominent staining of synaptic glutamate andGABAA receptor subunits has also been obtained in pri-mary cultures of hippocampus neurons (Craig et al., 1993,1994), confirming that the inaccessibility of synaptic sub-unit epitopes is a major obstacle in situ.

Functional significance

The structural heterogeneity of ionotropic receptors isbelieved to reflect their functional and pharmacologicalheterogeneity. Whereas previous evidence suggested anallocation of receptor subtypes to defined population of


neurons (Gao and Fritschy, 1994; Gao et al., 1995; Stan-daert et al., 1996), our results indicate that receptorsubtypes may be targeted to defined synapses. This is mostclearly demonstrated for CA3 pyramidal cells, which ap-pear to express NMDA receptors containing the NR2A (but

not the NR2B) subunit at mossy fibers synapses and bothsubunits at synapses in the stratum oriens and stratumradiatum. Although the functional significance of thisdifferential receptor expression is unknown, the NR2subunit variants are major determinants of ligand affinity

Fig. 6. Video images depicting the distribution of NR2B subunit IRin the cerebral cortex (A), striatum (B), and dentate gyrus (C,D), asseen at high magnification with confocal laser scanning microscopy. Inthe cerebral cortex, immunoreactive puncta appear uniformly distrib-uted in the neuropil, surrounding the soma and proximal dendrites ofcortical neurons. A similar picture is seen in the striatum but with ahigher density of puncta. These images represent the superposition ofthree optical images spaced by 250 nm. (C) NR2B subunit IR puncta

around individual granule cell somata in the dentate gyrus as seen ina single optical image; a three-dimensional projection of the samefield, based on 36 consecutive optical sections spaced by 250 nm, isshown in D. These images demonstrate that the puncta of NR2Bsubunit IR are discrete entities entirely surrounding the soma ofcertain granule cells, notably those located at the junction with themolecular layer. Scale bars 5 10 µm.


Fig. 7. Video images from confocal scanning laser microscopydepicting the staining for the GABAA receptor a2 and a3 subunits aswell as for glial fibrillary acid protein (GFAP) in cryostat sections fixedduring microwave irradiation. A: Isolated neuron stained for the a2subunit in the globus pallidus. The soma and a thick dendrite appearcompletely surrounded by puncta of a2 subunit IR. B: Isolated neuronstained for the a3 subunit in the medullary reticular formation. Theseimages were generated from a stack of 36 confocal images spaced by250 nm with a ‘‘simulated fluorescence processing’’ algorithm, wherebythe shadows projected from a virtual light source reveal the three-dimensional features of the objects in the image. C: Punctate a3

subunit IR in the reticular nucleus of the thalamus (superposition ofeight confocal images spaced by 0.5 µm). In contrast to conventionalimmunohistochemistry that reveals numerous cell bodies in thisnucleus, microwave fixation reveals only presumptive synaptic puncta.The outline of individual neurons cannot be seen unambiguously. D:GFAP IR in the cerebral cortex (superposition of 20 confocal imagesspaced by 0.5 µm). Several astrocytes with their processes andend-feet surrounding a blood vessel are depicted, indicating thatmicrowave fixation did not influence the apparent distribution andstaining intensity of this cytoskeletal protein. Scale bars 5 10 µm.


and channel properties of NMDA receptors (Laurie andSeeburg, 1994; Monyer et al., 1994; Priestley et al., 1995;Wyllie et al., 1996). In particular, NMDA receptors contain-ing the NR2A subunit exhibit a faster decay of excitatorypostsynaptic currents (Kirson and Yaari, 1996; Flint et al.,1997). Furthermore, our finding raises the issue of thetargeting mechanisms of NMDA receptor subtypes. Theimplication of presynaptic mechanisms is conceivable inview of recent in vitro data suggesting an influence ofpresynaptic input on the subunit composition of NMDAreceptors in cultured hippocampal neurons (Gottmann etal., 1997).

With regard to GABAA receptors, a novel finding is thelaminar difference in the subunit composition of GABAAreceptors on the AIS of neocortical pyramidal cells. Thislaminar segregation, with a2–GABAA receptors presentmostly in the AIS of layer II–III pyramidal cells anda3–GABAA receptors in layers V–VI, was evident through-out the neocortex, reflecting the regional distribution of

these subunits. Because AIS are innervated selectively bychandelier cells, presynaptic mechanisms are unlikely tobe major determinants of the subunit composition of theseGABAA receptors.

CONCLUSIONS

Antigen-retrieval procedures based on microwave irra-diation represent an essential step for the immunohisto-chemical detection of synaptic GABAA and NMDA recep-tors in situ. Microwave irradiation during fixation ofcryostat sections represents a simple and powerful methodfor visualizing the subcellular distribution of NMDA andGABAA receptor subunits and for demonstrating theirselective targeting to particular synapses. The strongstaining intensity and discrete labeling achieved with thismethod are ideal for high-resolution studies by confocallaser scanning microscopy. This method is expected to

Fig. 8. Photomicrographs depicting the cellular distribution of theGABAA receptor a2 subunit in cryostat sections fixed during micro-wave irradiation. A: In layers II–III of the cerebral cortex, a moderate,partially punctate staining is seen in the neuropil surroundingunstained neuronal somata. In addition, several axon initial segments(AIS) are revealed by their intense staining for a2 subunit (arrow-

heads). B: In the CA3 region of the hippocampus, a2 subunit IR formsdiscrete puncta around the somata of pyramidal neurons (arrowheads)and on their AIS (arrow). This image illustrates typical features of theAIS, notably the gap of variable length between the pyramidal cellsoma and the dense packing of a2 subunit IR puncta on the AIS. Scalebars 5 10 µm.


permit quantitative studies on the number of synapsesimmunoreactive for either GABAA or NMDA receptors inresponse to pharmacological challenges or in pathophysi-ological situations.

ACKNOWLEDGMENTS

We are grateful to Dr. Hanns Mohler for his continuoussupport and insight and for critically reading the manu-

Fig. 9. False-color video images depicting the selective localizationand fine structure of GABAA receptor a2 and g2 subunit IR on the AISof pyramidal neurons as visualized at high magnification with confocalscanning laser microscopy. A,B: Three-dimensional visualization ofAIS IR for the a2 subunit. These images illustrate the variability inthe size and number of puncta within individual AIS in CA3 pyramidalcells. C: Targeting of a2 subunit IR puncta on the AIS of a neocorticalpyramidal cell (green) visualized by double-immunofluorescence stain-ing of neurofilaments (red) with the monoclonal antibody SMI-32.

Note the few a2 subunit IR puncta around the soma of the pyramidalcell. This image represents the superposition of 18 confocal imagesspaced by 500 nm. D: g2 subunit IR in the hippocampus CA3 field;numerous puncta can be seen distributed around the soma of severalpyramidal cells and on the AIS of a neuron not present in the field ofview. Images in A, B, and D were generated by ‘simulated fluorescenceprocessing’ following contrast enhancement by baseline subtraction.Scale bars 5 10 µm in A,B, 20 µm in C,D.


script. We thank Dr. Thomas Bachi for his friendly helpwith confocal laser microscopy and Louis Scheurer forpreparing the polypeptides used for the production ofNMDA receptor subunit antisera.

LITERATURE CITED

Ainley, C.D., and J.W. Ironside (1994) Microwave technology in diagnosticneuropathology. J. Neurosci. Methods 55:183–190.

Baude, A., Z. Nusser, E. Molnar, R.A.J. McIlhinney, and P. Somogyi (1995)High-resolution immunogold localization of AMPA type glutamatereceptor subunits at synaptic and non-synaptic sites in rat hippocam-pus. Neurosci. 69:1031–1055.

Benke, D., A. Wenzel, L. Scheurer, J.M. Fritschy, and H. Mohler (1995)Immunobiochemical characterization of the NMDA-receptor subunitNR1 in the developing and adult rat brain. J. Recept. Res. 15:393–411.

Blackstad, T.W. (1963) Ultrastructural studies on the hippocampal region.Prog. Brain Res. 3:122–148.

Boon, M.E., P.O. Gerrits, H.E. Moorlag, P. Nieuwenhuis, and L.P. Kok(1988) Formaldehyde fixation and microwave irradiation. Histochem. J.20:313–322.

Bravo, H., and H.J. Karten (1992) Pyramidal neurons of the rat cerebralcortex, immunoreactive to nicotinic acetylcholine receptors, projectmainly to subcortical targets. J. Comp. Neurol. 320:62–68.

Brose, N., G.P. Gasic, D.E. Vetter, J.M. Sullivan, and S.F. Heinemann(1993) Protein chemical characterization and immunocytochemicallocalization of the NMDA receptor subunit NMDA R1. J. Biol. Chem.268:22663–22671.

Cattoretti, G., S. Pileri, C. Parravicini, M.H.G. Becker, S. Poggi, C. Bifulco,G. Key, L. D’Amato, E. Sabattini, E. Feudale, F. Reynolds, J. Gerdes,and F. Rilke (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J. Pathol. 171:83–98.

Craig, A.M., C.D. Blackstone, R.L. Huganir, and G. Banker (1993) Thedistribution of glutamate receptors in cultured rat hippocampal neu-rons: Postsynaptic clustering of AMPA-selective subunits. Neuron10:1055–1068.

Craig, A.M., C.D. Blackstone, R.L. Huganir, and G. Banker (1994) Selectiveclustering of glutamate and g-aminobutyric acid receptors oppositeterminals releasing the corresponding neurotransmitters. Proc. Natl.Acad. Sci. USA 91:12373–12377.

Del Toro, E.D., J.M. Juiz, X. Peng, J. Lindstrom, and M. Criado (1994)Immunocytochemical localization of the a7 subunit of the nicotinicacetylcholine receptor in the rat central nervous system. J. Comp.Neurol. 349:325–342.

Eccles, J.C. (ed) (1964) The Physiology of Synapses. Berlin: SpringerVerlag.

Evers, P., and H.B.M. Uylings (1994a) Effects of microwave pretreatmenton immunocytochemical staining of vibratome sections and tissueblocks of human cerebral cortex stored in formaldehyde fixative for longperiods. J. Neurosci. Methods 55:163–172.

Evers, P., and H.B.M. Uylings (1994b) Microwave-stimulated antigenretrieval is pH and temperature dependent. J. Histochem. Cytochem.42:1555–1563.

Farb, C.R., C. Aoki, and J.E. Ledoux (1995) Differential localization ofNMDA and AMPA receptor subunits in the lateral and basal nuclei ofthe amygdala: A light and electron microscopic study. J. Comp. Neurol.362:86–108.

Flint, A.C., U.S. Maisch J.H. Weishaupt, A.R. Kriegstein, and H. Monyer(1997) NR2A subunit expression shortens NMDA receptor synapticcurrents in developing neocortex. J. Neurosci. 17:2469–2476.

Fritschy, J.M., and H. Mohler (1995) GABAA-receptor heterogeneity in theadult rat brain: Differential regional and cellular distribution of sevenmajor subunits. J. Comp. Neurol. 359:154–194.

Gao, B., and J.M. Fritschy (1994) Selective allocation of GABAA-receptorscontaining the a1-subunit to neurochemically distinct subpopulationsof hippocampal interneurons. Eur. J. Neurosci. 6:837–853.

Gao, B., J.P. Hornung, and J.M. Fritschy (1995) Identification of distinctGABAA-receptor subtypes in cholinergic and parvalbumin-positive neu-rons of the rat and marmoset medial-septum-diagonal band complex.Neurosci. 65:101–117.

Gottmann K., A. Mehrle, G. Gisselmann, and H. Hatt (1997) Presynapticcontrol of subunit composition of NMDA receptors mediating synapticplasticity. J. Neurosci. 17:2766–2774.

Gray, E.G. (1969) Electron microscopy of excitatory and inhibitory syn-apses: A brief review. Prog. Brain Res. 31:141–155.

Greferath, U., U. Grunert, J.M. Fritschy, A. Stephenson, H. Mohler, and H.Wassle (1995) GABAA-receptor subunits have differential distributionsin the rat retina: In situ hybridization and immunohistochemistry. J.Comp. Neurol. 353:553–571.

Gutierrez, A., Z.U. Khan, and A.L. de Blas (1994) Immunocytochemicallocalization of g2 short and g2 long subunits of the GABAA receptor inthe rat brain. J. Neurosci. 14:7168–7179.

Hartveit, E., J.H. Brandstatter, M. Sassoe-Pognetto, D.J. Laurie, P.H.Seeburg, and H. Wassle (1994) Localization and developmental expres-sion of the NMDA receptor subunit NR2A in the mammalian retina. J.Comp. Neurol. 348:570–582.

Hazelbag, H.M., L.J.C.M. van den Broek, E.B.L. van Dorst, G.J.A. Offer-haus, G.J. Fleuren, and P.C.W. Hogendoorn (1995) Immunostaining ofchain-specific keratins on formalin-fixed, paraffin-embedded tissues: Acomparison of various antigen retrieval systems using microwaveheating and proteolytic pre-treatments. J. Histochem. Cytochem. 43:429–437.

Hendry, S.H.C., M.M. Huntsman, A. Vinuela, H. Mohler, A.L. de Blas, andE.G. Jones (1994) GABAA receptor subunit immunoreactivity in pri-mate visual cortex: Distribution in macaques and humans and regula-tion by visual input in adulthood. J. Neurosci. 14:2383–2401.

Hof, P.R., P. Vissavajjhala, R.E. Rosenthal, G. Fiskum, and J.H. Morrison(1996) Distribution of glutamate receptor subunit proteins GluR2(4),GluR5/6/7, and NMDAR1 in the canine and primate cerebral cortex—Acomparative immunohistochemical analysis. Brain Res. 723:77–89.

Hsu, S.M., L. Raine, and H. Fanger (1981) Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase techniques: A comparison be-tween ABC and unlabeled antibody (PAP) procedures. J. Histochem.Cytochem. 29:577–580.

Huntley, G.W., J.C. Vickers, W. Janssen, N. Brose, S.F. Heinemann, andJ.H. Morrison (1994) Distribution and synaptic localization of immuno-cytochemically identified NMDA receptor subunit proteins in sensory-motor and visual cortices of monkey and man. J. Neurosci. 14:3603–3619.

Ishii, T., K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C.Akazawa, R. Shigemoto, N. Mizuno, M. Masu, and S. Nakanishi (1993)Molecular characterization of the family of the N-methyl-D-aspartatereceptor subunits. J. Biol. Chem. 268:2836–2843.

Johnson, R.R., X.P. Jiang, and A. Burkhalter (1996) Regional and laminardifferences in synaptic localization of NMDA receptor subunit NR1splice variants in rat visual cortex and hippocampus. J. Comp. Neurol.368:335–355.

Kharazia, V.N., K.D. Phend, A. Rustioni, and R.J. Weinberg (1996) EMcolocalization of AMPA and NMDA receptor subunits at synapses in ratcerebral cortex. Neurosci. Lett. 210:37–40.

Kirson, E.D., and Y. Yaari (1996) Synaptic NMDA receptors in developingmouse hippocampal neurones—Functional properties and sensitivity toifenprodil. J. Physiol. (Lond.) 497:437–455.

Kosaka, T. (1980) The axon initial segment as a synaptic site: Ultrastruc-ture and synaptology of the initial segment of the pyramidal cell in therat hippocampus (CA3 region). J. Neurocytol. 9:861–882.

Koulen, P., M. Sassoe-Pognetto, U. Grunert, and H. Wassle (1996) Selectiveclustering of GABAA and glycine receptors in the mammalian retina. J.Neurosci. 16:2127–2140.

Laurie, D.J., and P.H. Seeburg (1994) Ligand affinities at recombinantN-methyl-D-aspartate receptors depend on subunit composition. Eur. J.Pharmacol. 268:335–345.

Login, G.R., and A.M. Dvorak (1994) Application of microwave fixationtechniques in pathology to neuroscience studies: A review. J. Neurosci.Methods 55:173–182.

Marani, E., and R.W. Horobin (1994) Overview of microwave applications inthe neurosciences. J. Neurosci. Methods 55:111–117.

Monyer, H., N. Burnashev, D.J. Laurie, B. Sakmann, and P.H. Seeburg(1994) Developmental and regional expression in the rat brain andfunctional properties of four NMDA receptors. Neuron 12:529–540.

Moreno, J.I., M.A. Piva, C.P. Miralles, and A.L. Deblas (1994) Immunocyto-chemical localization of the b2 subunit of the g-aminobutyric acidA

receptor in the rat brain. J. Comp. Neurol. 350:260–271.Nakayama, H., S. Shioda, H. Okuda, T. Nakashima, and Y. Nakai (1995)

Immunocytochemical localization of nicotinic acetylcholine receptor inrat cerebral cortex. Mol. Brain. Res. 32:321–328.


Nusser, Z., E. Mulvihill, P. Streit, and P. Somogyi (1994) Subsynapticsegregation of metabotropic and ionotropic glutamate receptors asrevealed by immunogold localization. Neurosci. 61:421–427.

Nusser, Z., J.D.B. Roberts, A. Baude, J.G. Richards, W. Sieghart, and P.Somogyi (1995a) Immunocytochemical localization of the a1 and b2/3subunits of the GABAA receptor in relation to specific GABAergicsynapses in the dentate gyrus. Eur. J. Neurosci. 7:630–646.

Nusser, Z., J.D.B. Roberts, A. Baude, J.G. Richards, and P. Somogyi (1995b)Relative densities of synaptic and extrasynaptic GABAA receptors oncerebellar granule cells as determined by a quantitative immunogoldmethod. J. Neurosci. 15:2948–2960.

Nusser, Z., W. Sieghart, D. Benke, J.M. Fritschy, and P. Somogyi (1996)Differential synaptic localization of two major g-aminobutyric acid typeA receptor alpha subunits on hippocampal pyramidal neurons. Proc.Natl. Acad. Sci. USA 93:11939–11944.

Persohn, E., P. Malherbe, and J.G. Richards (1992) Comparative molecularneuroanatomy of cloned GABAA receptor subunits in the rat CNS. J.Comp. Neurol. 326:193–216.

Petralia, R.S., Y.X. Wang, and R.J. Wenthold (1994a) The NMDA receptorsubunits NR2A and NR2B show histological and ultrastructural local-ization patterns similar to those of NR1. J. Neurosci. 14:6102–6120.

Petralia, R.S., N. Yokotani, and R.J. Wenthold (1994b) Light and electronmicroscope distribution of the NMDA-receptor subunit NMDAR1 in therat nervous system using a selective anti-peptide antibody. J. Neurosci.14:667–696.

Priestley, T., P. Laughton, J. Myers, B. Lebourdelles, J. Derby, and P.Whiting (1995) Pharmacological properties of recombinant humanN-methyl-D-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mousefibroblast cells. Mol. Pharmacol. 48:415–421.

Rema, V., and F.F. Ebner (1996) Postnatal changes in NMDAR1 subunitexpression in the rat trigeminal pathway to barrel field cortex. J. Comp.Neurol. 368:165–184.

Sassoe-Pognetto, M., J. Kirsch, U. Grunert, U. Greferath, J.M. Fritschy, H.Mohler, H. Betz, and H. Wassle (1995) Colocalization of gephyrin andGABAA-receptor subunits in the rat retina. J. Comp. Neurol. 357:1–14.

Schroder, H. (1992) Immunohistochemistry of cholinergic receptors. Anat.Embryol. 186:407–429.

Shi, S.R., M.E. Key, and K.L. Kalra (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immuno-histochemical staining based on microwave oven heating of tissuesections. J. Histochem. Cytochem. 39:741–748.

Shi, S.R., A. Imam, L. Young, R.J. Cote, and C.R. Taylor (1995) Antigenretrieval immunohistochemistry under the influence of pH using mono-clonal antibodies. J. Histochem. Cytochem. 43:193–201.

Somogyi, P., and H. Takagi (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and elec-tron microscopic immunocytochemistry. Neurosci. 7:1779–1783.

Somogyi, P., M.G. Nunzi, A. Gorio, and A.D. Smith (1983a) A new type ofspecific interneuron in the monkey hippocampus forming synapsesexclusively with the axon initial segments of pyramidal cells. Brain Res.259:137–142.

Somogyi, P., A.D. Smith, M.G. Nunzi, A. Gorio, H. Takagi, and J.Y. Wu(1983b) Glutamate decarboxylase immunoreactivity in the hippocam-pus of the cat: Distribution of immunoreactive synaptic terminals withspecial reference to the axon initial segment of pyramidal neurons. J.Neurosci. 3:1450–1468.

Somogyi, P., J.M. Fritschy, D. Benke, J.D.B. Roberts, and W. Sieghart(1996) The g2 subunit of the GABAA-receptor is concentrated insynaptic junctions containing the a1 and b2/3 subunits in hippocampus,cerebellum and globus pallidus. Neuropharmacology 35:1425–1444.

Standaert, D.G., G.B. Lanwehrmeyer, J.A. Kerner, J.B. Penney, and A.B.Young (1996) Expression of NMDAR2D glutamate receptor subunitmRNA in neurochemically identified interneurons in the rat neostria-tum, neocortex and hippocampus. Mol. Brain. Res. 42:89–102.

Van der Zee, E.A., C. Streefland, A.D. Strosberg, H. Schroder, and P.G.M.Luiten (1992) Visualization of cholinoceptive neurons in the rat neocor-tex: Colocalization of muscarinic and nicotinic acetylcholine receptors.Mol. Brain. Res. 14:326–336.

Watanabe, M., Y. Inoue, K. Sakimura, and M. Mishina (1993) Distinctdistributions of five N-methyl-D-aspartate receptor channel subunitmRNAs in the forebrain. J. Comp. Neurol. 338:377–390.

Wenzel, A., L. Scheurer, R. Kunzi, J.M. Fritschy, H. Mohler, and D. Benke(1995) Distribution of NMDA receptor subunit proteins NR2A, 2B, 2Cand 2D in rat brain. Neuroreport 7:45–48.

Wenzel, A., J.M. Fritschy, H. Mohler, and D. Benke (1997) NMDA receptorheterogeneity during postnatal development of the rat brain: Differen-tial expression of the NR2A, NR2B, and NR2C subunit proteins. J.Neurochem. 68:469–478.

Werner, M., R.von Wasielewski, and P. Komminoth (1996) Antigen re-trieval, signal amplification and intensification in immunohistochemis-try. Histochem. Cell Biol. 105:253–260.

Wisden, W., D.J. Laurie, H. Monyer, and P.H. Seeburg (1992) The distribu-tion of 13 GABAA receptor subunit mRNAs in the rat brain. I.Telencephalon, diencephalon, mesencephalon. J. Neurosci. 12:1040–1062.

Wyllie, D.A.J., P. Behe, M. Nassar, R. Schoepfer, and D. Colquhoun (1996)Single-channel currents from recombinant NMDA NR1a/NR2D recep-tors expressed in Xenopus oocytes. Proc. R. Soc. Lond. B 263:1079–1086

Zimprich, F., J. Zezula, W. Sieghart, and H. Lassmann (1991) Immunohisto-chemical localization of the a1, a2 and a3 subunit of the GABAAreceptor in the rat brain. Neurosci. Lett. 127:125–128.


Documents

Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry