NeuroscienceVol. 50, No. Printedin Great Britain

I, pp. I l-22,

1992

0306-4522/92 $5.00 + 0.00 PergamonPressLtd 0 1992 IBRO

LOCALIZATION OF AMPA RECEPTORS IN THE HIPPOCAMPUS AND CEREBELLUM OF THE RAT USING AN ANTI-RECEPTOR MONOCLONAL ANTIBODY D.

R. HAMPSON,*~ X. P. HUANG,~

M. D. OBERDORFER,~ J. W. GoH,$ A.

and R. J.

AuvEuNct

WENTHOLD~

iFaculty SLaboratory

of Pharmacy, University of Toronto, 19 Russell St, Toronto, Ontario, Canada M5S 2S2 of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, U.S.A. §Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Abstract-The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional a-amino-3-hydroxyl-5-methylisoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the a-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain a-amino-3-hydroxyl-S-methyl-isoxazole-4propionate receptor cDNAs (GluRl, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the a-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluRl, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.

Kainate

and

4-propionate the excitatory

capable of forming functional homomeric receptors,‘.’ while other members of the kainate group do not appear to be capable of forming functional receptors when expressed by themselves.34.37 Among the latter group are the kainate binding proteins (KBPs) that have been isolated from amphibian and avian brain.‘0,‘2,2’ The anatomical distributions of these receptors have been studied by in vitro receptor autoradiography using tritiated kainate and AMPA (see Refs 24 and 38 for reviews), and by in situ hybridization using subunit-specific probes. In the mammalian brain, high affinity [3H]kainate binding sites are concentrated in the inner one-third of the molecular layer of the dentate gyrus and CA3 stratum lucidum of the hippocampus and in the granule cell layer of the cerebellum.2s High levels of [3H]AMPA binding sites are present in stratum pyramidale and radiatum of the hippocampus and in the molecular layer of the cerebellum.27,29 In situ hybridization studies have

cc-amino-3-hydroxy-5-methyl-isoxazole(AMPA) amino

receptors acid

receptor

are two class.

subtypes

of

It is becom-

ing increasingly clear from molecular cloning and expression studies that these two subtypes of glutamate receptors are a family of receptor isotypes that are both structurally and functionally related. It has been demonstrated that at least some of the subunits that form AMPA receptors bind [3H]AMPA with high affinity and form functional ionotropic channels.2” Kainate receptors have higher affinity for kainate than AMPA; some members of this class are *To whom correspondence

should be addressed.

Abbreviarions: AMPA, cc-amino-3-hydroxy-5-methyl-isoxazole-Cpropionate; DEAE, diethylaminoethyl; Ig, immunoglobulin; KBP, kainate binding protein; Mab, monoclonal antibody; mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 11

12

11.

R.

HAMPSON ?I ul.

demonstrated that the distribution of messenger ribonucleic acid (mRNA) coding for the AMPA receptor subunits is actually greater than the distribution of [3H]AMPA binding sites,*’ suggesting that not all members of the AMPA receptor family possess binding sites for AMPA. Despite the important contributions of these receptor autoradiographic and in situ hybridization studies, these techniques possess several disadvantages that often preclude a more detailed anatomical characterization of the receptor proteins. In the case of receptor autoradiography, these disadvantages include the requirement of intact ligand binding in tissue sections, the ability to analyse only binding sites of relatively high affinity, and the low degree of resolution at the cellular and subcellular levels. Localization of mRNA via in situ hybridization techniques does not reveal the precise location of the translated products. These problems can be circumvented to a large degree by the use of immunocytochemical techniques. Immunocytochemical techniques have been used to study the distributions of the KBPs in the frog and chicken CNS. A monoclonal antibody (Mab) raised against frog KBP13 was used to analyse the distribution of this protein at the light and ultrastructural levels. In the frog CNS, the KBP is widely distributed throughout the brain; high levels are present in the cerebellum, infundibulum, the laminar nucleus of the torus semicircularis, and the entire telencephalon.6 Electron microscopy conducted on tissue sections from the frog optic tectum and telencephalon has shown that the frog KBP is present along the plasmalemma of nerve cells. Although a small proportion of the protein is located at synapses (postsynaptically), most of the antigenic sites are localized to areas of the neuronal cell surface outside of synaptic contacts. Immunocytochemical analysis conducted in the chicken CNS has indicated that a very different distribution of a highly homologous KBP exists in this species. Using a Mab that recognizes a 49,000 mol. wt KBP” in the chicken cerebellum, Somogyi et al.” demonstrated that this KBP was localized to Bergmann glial cells. No evidence for the presence of this protein on neurons was found in the chicken cerebellum. In the chicken CNS, moreover, the distribution of the KBP appears to be restricted to the cerebellum in that the protein could not be detected on tissue sections from forebrain areas. The latter observation is consistent with the reported distribution of [3H]kainic acid binding sites.14 Thus the distributions of the two homologous KBPs present in the frog and chicken CNS appear to be quite different. In light of the somewhat unexpected distributions reported for the KBPs in the frog and chick brain, it was of interest to examine the cellular and subcellular distributions of the AMPA receptors present in the mammalian CNS.

EXPERIMENTAL

PROCEDURES

Animuls and materials

Adult male Wistar rats were from Charles R&r. Inc.. Montreal, Quebec. Domoic acid was obtained from Diagnostic Chemicals Inc., Prince Edward Island, Canada. Electrophoresis standards (high molecular weight unstained) were from Bethesda Research Laboratories. The enhanced chemiluminescence detection system was from Amersham. Production of’ antibodies

The Mab used in this study (denoted KAR-Bl) was raised in Balb/C mice by immunizing the animals with highly purified KBP from frog brain. The kainate protein was purified by ion exchange chromatography and domoic acid affinity chromatography.‘* Further details of the production and characterization of this Mab are provided in Hampson et ~1.‘~In the present study, either raw hybridoma culture supematant or Mab purified from culture supernatants (Bio Rad MAPS II kit) was used. Immunoa@nitypurification ofa-amino-3-hydroxy-Smethylisoxazole-4propionate receptors

An anti-GluRl antiserum’” was raised in rabbits by immunizing them with a synthetic peptide based on the C-terminal sequence of the rat GluRl. The antiserum was coupled to Protein A-agarose using dimethyl pimelimidate” and the solubilized receptors were immunoafhnity purified as described previously.36 Immunocytochemistry

Adult male rats (25&35Og) were anesthetized with sodium pentobarbital and perfused transcardially with 400ml of cold (4°C) 0.12 M phosphate buffer, pH 7.2, followed by the same volume of 4% paraformaldehyde in phosphate buffer or with 2% paraformaldehyde, 1.4% lysine, and 0.34% periodic acid in 0.1 M phosphate buffer, pH 7.4. After perfusing, the brains were placed in the fixative for 40 min and then left in phosphate-buffered saline (PBS) overnight at 4°C. Sections (3&5Opm) were cut on a Vibratome and rinsed in PBS. The tissue sections were incubated overnight at 4°C with Mab diluted 1: 1 to 1: 3 in blocking buffer (0.1% bovine serum albumin in PBS) followed by a 2 h incubation at 25°C. In some experiments, the sections were pretreated with 0.1% Triton X-100 to partially permeabilize the tissue. After incubation with the primary antibody, the sections were washed three times in PBS, incubated with biotinylated anti-mouse secondary antibody diluted in blocking buffer for 2 h at room temperature, washed three times in PBS, and then incubated with the Vectastain ABC reagent (Vector Laboratories) for 1 h. After washing three times with PBS, the sections were incubated with diaminobenzidine. For electron microscopy, free-floating Vibratome sections were processed as described above except that the fixative was 4% paraformaldehyde and 0.1% glutaraldehyde in 0.12 M phosphate buffer, pH 7.2. After development of the substrate, sections were postfixed in 1% osmium tetraoxide in PBS for 1 h, dehydrated in ethanol, and flat-embedded in Epon 812. Thin sections were examined using a Jeol 1OOCXII electron microscope with or without counterstaining. Electrophoresis and immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as described previously by Hampson ef al.” Culture supernatants containing Mab were diluted 1: 1 to 1: 4 in blocking buffer (50 mM Tr&itrate, pH 7.2, containing 0.1% Triton X-100,2% instant oowdered milk) for immunoblottine. The primary antibody was detected by using either an alkaline phosphatase-conjugated goat anti-mouse immunaglobulin (IgG; Promega) or a horseradish peroxidase-conjugated

Distribution sheep anti-mouse IgG and the enhanced detection system.

of AMPA

chemiluminescence

Subcellular pur(jications All subcellular fractions were prepared from adult rat brains. Purified synaptic membranes, synaptic junctions, mitochondria, microsomes, and myelin fractions were isolated as described by Cotman and Taylor.5

Brain lesions Hippocampal lesions were produced by microinjecting domoic acid into the amygdala of adult Wistar rats weighing 250@3OOg. One hundred and seventy five nanograms of domoic acid dissolved in 1 ~1 of PBS were injected into a single site in the right amygdala using a stereotaxic injection apparatus. Control animals were injected with 1 pl of PBS. The coordinates of the injections were: posterior to bregma, 3 mm; lateral, 4 mm; ventral from dura, 8 mm. Preliminary studies indicated that this dose of domoic acid was the maximal dose that could be administered to the amygdala without causing death. Seizures were controlled by injecting diazepam (20 mg/kg i.p.) 45-150 min after application of domoic acid. The animals were killed 30 days after stereotaxic injections and the hippocampi were dissected and prepared for immunoblot analysis. For the latter, hippocampi were cut into l-mm-thick sections along the rostra]-saudal axis and each section was then cut into two pieces so as to isolate the part of the section containing the CA3 pyramidal cells from the CA1 and dentate. The microdissected tissue sections were homogenized in SDS sample buffer and prepared for electrophorests.

A

B

C

D

receptors

13

in rat brain

Cell transfections

COS- 1 cells transiently expressing the rat GluR 1,GluR2, or GluR3 cDNAs were provided by T. Livelli of the Institute for Cancer Research, Columbia University. Glutamate receptor subunit cDNAs were subcloned into the pCOS expression vector, the cells were transfected using the Transient Expression Transfection Kit (Specialty Media Inc.) which uses the diethylaminoethyl (DEAE)dextran method combined with chloroquine shock, and a total membrane fraction was prepared from the cells for immunoblot analysis 48 h later. Protein measurements Protein

was quantified

the BCA kit from Pierce.

using RESULTS

Reactivity

of Mab KAR-BI

on immunoblots

A single major immunoreactive band with a relative molecular weight of 107,000 was observed in the rat hippocampus (Fig. 1, lane B); this band corresponds to the 99,000 mol. wt band previously reported by Hampson et al.13 The mol. wt values were determined

in the

acrylamide

gel

listed

in

present

and

Experimental

calculated

for this band

the

was

range

99,000-l

the

study

using

a 10%

electrophoresis Procedures.

varied 12,000

slightly and

poly-

standards

The

M,

from the

values

gel to gel;

average

EFGH

Fig. 1. Immunoblot of rat cerebellum (lane A), rat hippocampus (lane B), immunoaffinity purified GluRl (lane H), and transfected COS cell membranes (lanes C-G): lane C, GluRI; lane D, GluR2: lane E, GluR3; lane F, GluRl + GluR2; lane G, mock transfection with no GluR cDNA. The blot was probed with culture supernatant containing Mab KAR-Bl diluted 1: 1 with blocking buffer. Forty-five to 65 pg of total membrane protein was applied to lanes AG; 1 pg of protein was applied to lane H. Enhanced chemiluminescence detection system was used in lanes A-G; an alkaline phosphatase system was used in lane H. Arrow on the right indicates 107,000 mol. wt AMPA receptor subunits. Arrows on the left show electrophoresis standards: top to bottom 200,000, 97,400, 68,000. 43,000, and 29,000 mol. wt.

was

14

D. R. HAMPSONef al.

107,000. The migration rate of the immunoreactive bands present in the rat hippocampus (and cerebellum) were not affected by the presence or absence of reducing reagents. In addition to the 107,000 mol. wt band, an additional major immunoreactive band with mol. wt of 128,000 was seen in the cerebellum (Fig. 1, lane A). The 128,000 mol. wt immunoreactive protein was not detected in any of the other brain areas examined (hippocampus, cerebral cortex, striatum, thalamus, globus palhdus, brainstem). This protein was not labeled by an antiserum raised against GluRl (see below). The identity of this band is not known at the present time (see Discussion). To confirm that the 107,000 mol. wt immunoreactive protein(s) in rat brain were AMPA receptor subunit(s), COS cells were transfected with GluRl, GluR2, GIuR3, and GluRl + GluR2 cDNAs. Immunoblots of membranes from these cells showed an immunoreactive band migrating to the same position as the bands seen in the hippocampus and cerebellum (Fig. I). These bands were absent in COS cells that were subjected to a mock transfection with no GluR cDNA. The slight differences in the mol. wt of the AMPA receptor subunits seen on the blots correspond to the differences in the primary amino acid sequences as determined from the cloned cDNAs. In addition to the major immunoreactive band of mol. wt 107,000, other immunoreactive bands were seen at lower relative molecular weights. These proteins are unlikely to be. proteolytic products of AMPA receptors since they were also present in the mocktransfected COS cells, and in addition, most of the immunoreactive bands seen at the lower mol. wt were absent from rat brain tissue. As a further confirmation that the KAR-Bl Mab recognizes the AMPA receptor subunits, an antipeptide antiserum specific for the GluRl receptor was employed. This antiserum was produced by immunizing rabbits with a peptide corresponding to a portion of the rat GluRl sequence. The antiserum was then used to immunoaffinity purify the GluRl receptor complex from solubilized brain tissue. Immunoblots of the immunoaffinity purified GluRl showed a band labeled by KAR-Bl at the same relative molecular weight as the bands seen in brain and in transfected COS cells (Fig. 1, lane H). The appearance of only a single major immunoreactive band of mol. wt 107,~ (except in cerebellum as noted above) on immunoblots from various areas of rat brain known to contain multiple forms of the AMPA receptors can be explained by the inability of the gel system to resolve subunits with very similar molecular weights. The higher mol. wt values reported here from calculations based on SDS-PAGE gels and immunoblots compared to the values calculated from the cDNA sequences may be explained by the presence of oligosaccharide side chains present on AMPA receptors in rat brain.” The epitope recognized by Mab KAR-Bl is apparently highly conserved since immunoblots from various species have

demonstrated that the 107,000 mol. wt band is also present in mouse, guinea-pig, bovine, and human brain tissue (unpublished observation). ~mm~o~ytochem~ca~ analyses in the hip~ocampus and cerebellum-light microscopy

The antigens detected by Mab KAR-Bl were present in high levels in several cell types in the hippocampus and the cerebellum. Similar patterns of labeling were obtained using either the crude hybridoma culture supernatant or the purified Mab. In the hippocampus, better results were obtained with the paraformaldehyde-lysineperiodic acid fixative than with 4% paraformaldehyde alone. The Triton X-100 treatment also improved the level of specific staining whereas methanol-hydrogen peroxide pretreatment reduced the immunostaining. In the hip~ampus, the most heavily stained areas were the pyramidal and granule cell layers (Fig. 2). All the pyramidal cell subfields (CAl, CA2, CA3, CA4) and the entire granule cell layer were labeled. Antibody labeling was of approximately the same intensity throughout each of the pyramidal cell subfields. The staining in the pyramidal and granule cell layers was distributed throughout the entire cell somata in the cells that were labeled. At higher magnifications, immunostaining can be seen on the apical dendrites of pyramidal cells in the striatum radiatum. In addition to pyramidal and granule cells, scattered cells outside these cellular layers in the hilus were also labeled. These cells may be GABAergic interneurons although no attempt was made in the present study to identify them. In the cerebellum, specific immunostaining was observed in several different cell types present in this structure (Fig. 3). Purkinje cell bodies and their dendrites were intensely stained. At higher magnifications, the Purkinje cell dendrites in the molecular layer displayed a pattern of labeling suggestive of postsynaptic sites apposing afferent terminals of parallel and climbing fibers. Bergmann processes extending into the molecular layer were also clearly labeled. In the granule cell layer, the granule cells were immunostained, as were some of the Golgi cells. In the granule cell layer, cells which resembled astrocytes were also labeled.

Three different methodologies were employed to examine the subcellular distribution of the AMPA receptors: subcellular fractionation, lesion studies, and electron microscopy. To determine which subcellular compartments contained the AMPA receptors, purified subcellular fractions were prepared from rat brain and subjected to SDS-PAGE and immunoblotting. Receptor subunits were absent in the soluble and myelin fractions, present at low levels in mitochondria and microsomes, and highly concentrated in synaptic membranes and particularly in purified synaptic junctions (Fig. 4). Synaptic junctions are

Distribution

of AMPA

receptors

in rat brain

15

Fig. 2. Immunostaining of adult rat hippocampus with Mab KAR-BI. (A) Low magnification ( x 25) of hippocampus showing continuous labeling of the pyramidal and granule cell layers. (B) Higher magnification ( x 150) of the dentate gyrus showing immunostaining of granule cells (G), pyramidal cells in CA4, and interneurons outside the CA4 pyramidal cell layer (arrowhead). A higher magnification of the pyramidal cells in CA1 and their labeled dendrites in the stratum rddiatum is shown in C.

by treating synaptic membranes with Triton X-100; this detergent treatment removes much of the neuronal membrane outside the synapse leaving a structure that is composed primarily of postsynaptic components and a relatively small amount of presynaptic material. The staining observed in the cell bodies of neurons in the hippocampus and cerebellum (Figs 2 and 3) is presumably reflected in the receptor detected on immunoblots of the microsomal fraction (Fig. 4, lane E). To examine the pre- vs postsynaptic location of these receptors at the mossy fiber terminal-CA3 pyramidal cell synapse, excitotoxic lesions were produced by injecting the potent kainic acid analog, domoic acid, into the amygdala and assessing the level of AMPA receptors in the hippocampus via immunoblotting 30 days later. Although the mechanism of this phenomenon is not entirely understood, excitotoxic lesions of the amygdala are known to

prepared

cause degeneration of the CA3 pyramidal cells while sparing the presynaptic input, the mossy fibers emanating from the granule cells.” The amygdaloid lesions produced by domoic acid caused a significant reduction in the amount of hippocampal receptor detected on immunoblots compared to animals injected with saline (Fig. 5). Domoic acid (this study) was approximately six to 10 times more potent than kainic acid” in producing this type of lesion. Separate animals were injected with saline and used as controls rather than using the contralateral hippocampus in the domoic acid-treated animals because decreases, albeit smaller than those seen in the ipsilateral hippocampus, were also observed in the contralateral hippocampus in the domoic acid-treated animals. The efficacy of the lesion was confirmed by Cresyl Violet staining of tissue sections from the lesioned animals; substantial pyramidal cell loss was apparent in CA3/CA4 while the pyramidal cells in CA 1 and CA2

appeared normal, as did the granule cells (data not shown). The reduction in the immunostaining produced in CA3 suggests that the AMPA receptors present at these synapses are located postsynaptically. As a final verification of the subcellular location of these receptors, electron microscopy was performed on ultrathin sections from the dendritic fields (stratum radiatum) of the CA1 and CA3 hippocampal

pyramidal cells and from the molecular layer of the cerebellar cortex. In each of these areas. analysis at the ultrastructural level shows that the immunostaining apparent in the dendritic layers at the light microscopy level represents both cytoplasmic and postsynaptic labeling (Fig. 6). Antibody reactivity was concentrated along the cytoplasmic face of the postsynaptic membrane as well as in the dendritic

Fig. 3. Immunostaining in sections from adult rat cerebellum, Purkinje cell bodies (A, arrow) and their dendrites (B) are intensely labeled. Labeling of Bergmann glial processes in the molecular layer is also apparent (A, arrowheads), as well as occasional labeling of Golgi cells in the granule layer (A, open arrowhead). In the granule cell layer, granule cell bodies appear lightly labeled, and processes resembling astrocytes (C, arrowhead) also appear immunoreactive.

Distribution of AMPA receptors

I7

in rat brain

ABCDEFG -200

kDa

-97 4-68

-29 4-14 Fig. 4. Distribution of AMPA receptors in purified subcellular fractions from rat brain Each lane contains 15 30 jig of protein. Lane A, hippocampal tissue; B, soluble fraction; C. my&n fraction: D, nllto~h(~ndri~~l fraction: E, microso~dl fraction; F. synaptic membranes; G. synaptic junctions.

adjacent to the postsynaptic membrane. The concentration of the immunostaining on the cytoplasmic face of the membrane indicates that the cytoplasm

c

is located intraepitope recognized by Mab KAR-BI cellularly. In addition to the numerous labeled synapses. there were also many unlabeled synapses.

E

F

Fig. 5. The effects of excitotoxic lesions of the amygdala on the level of AMPA receptors in the hippocampus. Domoic acid (175 ng) was injected into the right amygdala and the right hippocampus was removed for immunoblot analysis 30 days later. The hippocampus was dissected into a CA3 section and a dentate section as described in Experimental Procedures and the CA3 section was prepared for electrophoresis and immunoblotting. Each lane represents a separate animal: lanes A-C. domoic acid-injected rats: lanes D-F. saline-injected rats. Twenty micrograms of protein were applied to each lane.

13. R. HAMPSON 1’1 cd.

IX

Fig. 6. Electron micrographs ofimmunostained postsynaptic localization of AMPA receptors.

synapses in the rat hippocampus and cerebellum showmg

Hippocampus CA1 (A and C); hippocampus CA3 (B): molecular layer of the cerebellum (D). The postsynaptic density is intensely stained (arrowheads): intracellular staining is present in the postsynaptic profile. Numerous unstained synaptic vesicles (asterisks) are present in the presynaptic terminals. Uncounterstained sections. Scale bars = 0.25 Iin:

The lack of staining at these synapses could be attributed to both the inac~ssibility of the Mab to the antigen at some of the synapses in the tissue section, and the absence of the receptor subunits at other synapses. DISCUSSION

Antibody characterization A Mab raised against a 48,000 mol. wt KBP purified from frog brain was shown to cross-react

with several forms of the functionat AMPA receptors present in rat brain. In a previous study, we demonstrated that this Mab recognizes proteins that are present in the rat hippocampus, cerebellum, thalamus, globus pallidus, and cerebral cortex, absent or present in low quantities in the brainstem and are nervous system-specific in both the rat and frog.” Results from the present study demonstrate that Mab KAR-Bl recognizes subunits of the rat brain AMPA receptors that have been designated GluRl, GluR2, and G~uR~~,‘~*‘~or GluR-A, GluR-B, and GluR-CZO

Distribution of AMPA receptors in rat brain The finding that there is immunological cross-reactivity between the two groups of proteins is in accord with the fact that the KBPs are structurally homologous with the functional AMPA receptors identified in rat brain.35 A comparison of the primary amino acid sequences of the frog and chicken KBPs with the carboxy terminal half of the functional rat GluRl receptor reveals that 35% of the amino acids are identical among all three proteins; the level of homology is increased to 62% if conservative amino acid substitutions are considered. The demonstration that a Mab raised against the KBP from frog brain cross-reacts with the AMPA receptors present in rat brain that mediate excitatory amino acid-induced ion fluxes and current depolarizations, lends further support to the postulate that the KBPs may be part of an ion channel comSeveral of the models proposed for the plex. 3~1’.‘3.34.35 kainate binding proteins and the AMPA receptors indicate that both groups of proteins have a number of features in common, including the presence of polar or charged residues in the second and third transmembrane domains that may be part of an ion-conducting pore. In addition to the proposed models, preliminary experiments have indicated that Mab KAR-Bl recognition of the receptor subunits is eliminated after chemical or protease digestion and that the intensity of antibody labeling on immunoblots is increased after pretreatment of the immobilized receptor with non-denaturing detergents (unpublished observations). These findings suggest that the epitope recognized by Mab KAR-Bl may be conformation-dependent. A conformation-dependent epitope common to both the KBPs and the AMPA receptors suggests that, in addition to primary amino acid sequence identity, the two classes of proteins may also possess similar higher order structures. Our preliminary results indicate that Mab-Bl does not recognize the chicken KBP nor a high affinity kainate receptor present in rat brain, termed GluR6;* future studies may determine whether the KAR-Bl antibody recognizes another rat brain kainate binding protein termed KA- 1.37 Receptor localization Immunocytochemical analysis of the AMPA receptors in the hippocampus and cerebellum at the light microscopy level showed that high densities of the receptor proteins are present in both structures. In the hippocampus, high levels of immunoreactivity were observed in the granule and pyramidal cells. The intensity of antibody labeling in the pyramidal cell layer and dendritic fields corresponds closely to the reported distributions of AMPA receptor mRNAs4,” and with the density of [3H]AMPA binding sites in these layers. *’ The highest density of antibody labeling and ligand binding is in the pyramidal cell layer with lower levels of immunostaining and ligand binding in the dendritic fields. The presence of high levels

19

of immunoreactive receptor dispersed throughout cell somata indicates that the antibody may be labeling receptors being transported and processed in the endoplasmic reticulum and Golgi apparatus. The observation that the receptors present in stratum pyramidale are located primarily intracellularly provides an explanation for the high levels of [3H]AMPA binding found in a cellular layer with relatively few synaptic contacts. *’ Receptor subunits produced in the cell body may be capable of binding hgand; the subunits are then transported to dendrites and nerve terminals where they are incorporated into functional synapses. The results of our electron microscopy studies (see below) corroborate the suggestion that Mab-labeled intracellular subunits are destined for synaptic contacts in the dendritic fields. Intracellular labeling of neuronal cell bodies with anti-receptor Mabs has been reported for other neurotransmitter receptor systems including GABA receptors (cerebellar stellate and basket cells)33 and nicotinic acetylcholine receptors (cihary ganglion neurons).‘* The large intracellular pool of AMPA receptor proteins seen in the pyramidal and granule cell bodies could allow for the rapid exchange of receptor subunits between the cytoplasm and the synaptic membrane. Such a rapid exchange of subunits may underlie some aspects of synaptic plasticity associated with this class of excitatory amino acid receptors. For example, the observation that enhanced current responses mediated by AMPA receptors occur after the induction of long-term potentiation in CA1 neurons” is compatible with the hypothesis that there exists a large intracellular pool of presynthesized subunits that may be rapidly exchanged with subunits in existing synapses or incorporated into the cell membrane to form new excitatory synapses. Since the analysis of the subcellular distribution of the AMPA receptors was one of the fundamental objectives of the present study, we used three different approaches to address this issue: subcellular fractionation, brain lesions, and electron microscopy. The finding that the receptor is highly concentrated in purified synaptic junctions suggests that these receptors are primarily localized to the synaptic membrane. The results of the lesioning experiments point to a postsynaptic location at the mossy fiber-CA3 pyramidal neuron synapse; excitotoxic lesions of the amygdala significantly reduced receptor protein present in the CA3 pyramidal cells of the hippocampus. Previous studies have shown that excitotoxic lesions of the amygdala cause degeneration of the CA3 that pyramidal cells. 2,30 Our results, demonstrating amygdaloid lesions cause a sharp reduction in the AMPA receptors present in the hippocampus, are in contrast to the results of a previous study investigating the pre- vs postsynaptic location of high affinity kainic acid binding sites which used a similar experimental design in terms of the lesion produced and the time interval between lesioning and analysis

20

D. K. HAMPSONCI al

(30 days).3” The principal conclusion in the study by Represa et uL,‘~ was that the majority of the high affinity [‘Hlkainic acid binding sites in the stratum lucidum of the CA3 pyramidal cell field are located presynaptically on the terminals of the mossy fibers. However, since the proteins labeled by KAR-Bl (GluRl, GluR2, GluR3) bind [3H]AMPA with high affinity but not [3H]kainic acid,*” the difference in the results between Represa et al. and the present study could be explained by the fact that two different sites were being analysed in the two studies. In addition to the subcellular fractionation and lesioning experiments, the synaptic localization of the AMPA receptors was also demonstrated by direct visualization at the ultrastructural level. In electron micrographs from the dendritic fields of CA1 and CA3 pyramidal cells, immunoreactivity was highly concentrated on the cytoplasmic side of the postsynaptic membrane. A similar profile was also observed in the dendrites of neurons in the cerebellar cortex. No evidence of presynaptic labeling was seen in either the hippocampus or the cerebellum. As noted above, the pattern of labeling that we have observed in the rat hippocampus using Mab KAR-BI corresponds to the distribution of the AMPA receptors as determined by ligand binding and in situ hybridization studies. However, the pattern of immunoreactivity differs from that of two glutamate binding proteins identified in rat brain.’ Antibodies raised against glutamate binding proteins with mol. wts of 71,000 and 63,000 label pyramidal cells in CA3 and CA1 cells near the subiculum more intensely than the other pyramidal cells, whereas the pattern of immunostaining with Mab KAR-Bl is uniformly distributed throughout the pyramidal cell layer. Although the identity of these glutamate binding proteins remains to be determined, it has been proposed that they are part of the N-methyl-D-aspartate receptor complex.” In the cerebellar cortex, several cell types in the granule cell, Purkinje cell, and molecular layers were labeled by Mab KAR-B 1. As in the hippocampus, the distribution of antibody labeling generally corresponds to the pattern of [3H]AMPA binding; higher densities of antibody labeling and ligand binding are found in the molecular layer compared to the granule cell layer. However, the pattern and intensity of the immunostaining correlates more closely with the distribution of the AMPA receptor mRNA than with [‘H]AMPA binding, particularly in the granule cell layer where both antibody labeling and mRNA are observed, but where very little [3H]AMPA binding is seen. As noted previously by Keinanen et al.,*’ it appears as though [3H]AMPA may only label the subpopulation of the total pool of AMPA receptors present in the cerebellum. Very high levels of the antigen were seen in Purkinje cell bodies and dendrites. The intense staining observed on Purkinje cell dendrites at higher magnifi-

cations probably represents mature, functional receptor complexes associated with the postsynaptic membrane. The very high levels of receptor detected in Purkinje cells are consistent with observations made in mutant mice lacking these cells where it has been shown that [‘H]AMPA binding in the molecular layer of the cerebella of these animals is reduced to 29% of control values.” Neurotoxicity studies conducted on cerebellar slices have also demonstrated that in mature animals, Purkinje cells are highly susceptible to the toxic effects of the excitotoxin. quisqualic acid.’ Antibody recognition of’ a novel cerebellar-spec$c protein with immunological homology to the AMPA receptors

The cerebellum was unique in terms of the pattern observed on immunoblots probed with Mab KARBl. In addition to GluRl, GluR2, and GluR3, this antibody also labeled a protein on immunoblots with a mol. wt of approximately 128,000. The cerebellum was the only structure in which we were able to detect this protein. Whether this immunoreactive protein represents an AMPA receptor precursor, a novel AMPA receptor subunit, or is unrelated to the glutamate receptor family is not presently known. The molecular weight estimated from immunoblots is similar to the calculated size of the metabotropic glutamate receptor based on the cDNA sequence.‘6.‘2 In addition to having some sequence homology to the AMPA receptors, the metabotropic glutamate receptor is also highly concentrated in Purkinje receptor is cells. However, the metabotropic also present in other areas of the CNS, whereas the distribution of the 128,000 mol. wt immunoreactive protein is restricted to the cerebellum. Cloning studies using the KAR-Bl Mab as a screening probe may prove useful in the further structural and functional characterization of this cerebellar-specific protein. CONCLUSIONS A Mab that was raised against a KBP from frog brain was shown to cross-react with the GluRl, GluR2, and GluR3 forms of the rat brain AMPA receptor. This Mab labeled AMPA receptors on immunoblots of membranes prepared from rat brain and from transfected COS cells. In the mammalian cerebellum, the Mab also labeled another protein with a mol. wt of 128,000; this protein was not detected in other areas of the rat CNS. The identity of this apparently cerebellar-specific protein is not known. On tissue sections, the most intense immunostaining was on Purkinje cells in the cerebellum and in pyramidal cells in the hippocampus. Immunostaining was concentrated in both the dendrites and cell bodies of Purkinje cells and pyramidal cells. The intense intracellular staining observed indicates that there is a large intracellular pool of presynthesized

Distribution of AMPA receptors subunits present in these cells in the adult rat brain. Subce]lular fractionation, domoic acid lesions, and electron micrographic studies all demonstrated that these AMPA receptors are located postsynaptically in the hippocampus and cerebellum of the rat.

in rat brain

21

Acknowledgements-The authors would like to thank Dr E. Theriault for helpful advice and comments and T. Livelli for providing COS cells. This work was supported by grants to

D.R.H. from the MRC Group on Nerve Cells and Synapses, and Health and Welfare Canada. J.W.G. is a Medical Research Council Scholar.

REFERENCES I. Ambrosini A., Barnard E. A. and Prestipino G. (1991) AMPA and kainate-operated channels reconstituted in artificial bilayers. Fedn Eur. biochem. Sacs Lett. 281, 27-29. complex of the rat: 2. Ben-Ari Y., Tremblay E. and Ottersen 0. P. (1980) Injections of kainic acid into the amygdaloid an electrographic, clinical and histological study in relation to the pathology of epilepsy. Neuroscience 5, 515m 528. 3 Betz H. (1990) Ligand-gated ion channels in the brain: the amino acid receptor superfamily. Neuron 5, 3833392. 4: Boulter J., Hollmann M., O’Shea-Greenfield A., Hartley M.. Denneris E., Maron C. and Heinemann S. (1990) Molecular cloning and functional expression of glutamate receptor subunit genes. Science 249, 103331037. 5. Cotman C. W. and Taylor D. (1972) Isolation and structural studies on synaptic complexes from rat brain. J. Cell Biol. 55, 696-7 I I. 6. Dechesne C. J., Oberdorfer M. D., Hampson D. R., Wheaton K. D., Nazarali A. J., Goping G. and Wenthold R. J. (I 990) Distribution of a putative kainic acid receptor in the frog central nervous system determined with monoclonal and polyclonal antibodies: evidence for synaptic and extrasynaptic localization. J. Neurosci. 10, 479-490. characterization of I. Eaton M. J.. Chen J.-W., Kumar K. N., Cong Y. and Michaelis E. K. (1990) Immunochemical brain synaptic membrane glutamate-binding proteins. J. biol. Chem. 265, 16,195.-16,204. 8. Egebjerg J., Bernhard B., Hermans-Borgmeyer I. and Heinemann S. (1991) Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351, 7455748. G. and Garthwaite J. (1986) In vitro neurotoxicity of excitatory acid analogues during cerebellar 9. Garthwaite development. Neuroscience 17, 755-767. IO. Gregor P., Eshar N., Ortega A. and Teichberg V. I. (1988) Isolation, immunochemicdl characterization and localization of the kainate sub-class of glutamate receptor from chick cerebellum. Eur. molec. Biol. Org. J. 7, 267332679. II. Gregor P., Mano I.. Maoz I., McKeown M. and Teichberg V. I. (1989) Molecular structure of the chick cerebellar kainate-binding subunit of a putative glutamate receptor. Nature 342, 689-692. 12. Hampson D. R. and Wenthold R. J. (1988) A kainic acid receptor purified from frog brain using domoic acid alhnity chromatography. J. biol. Chem. 263, 2500-2505. 13. Hampson D. R., Wheaton K. D., Dechesne C. J. and Wenthold R. J. (1989) Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping. J. hiol. Chem. 264, 13,329913,335. 14. Henley J. M. and Barnard E. A. (1989) Solubilization and characterization of a putative quisqualate-type glutamate receptor from chick brain. J. Neurochem. 53, 140&148. IS. Hollman M., O’Shea-Greenfield A., Rogers S. W. and Heinemann S. (1989) Cloning by functional expression of a member of the glutamate receptor family. Nature 342, 6433648. 16. Houamed K. M., Kuijper J. L., Gilbert T. L.. Haldeman B. A., O’Hara P. J.. Mulvihill E. R., Almers W. and Hagen F. S. (1991) Cloning expression, and gene structure of a G-protein coupled glutamate receptor from rat brain. %ienc,e 252, 1318~1320. 17. Hullebroeck M. F. and Hampson D. R. (1992) Characterization of the oligosaccharide side chains on high affinity kainate and AMPA receptors. Brain Res. (in press). 18. Jacob M. H.. Lindstrom J. M. and Berg D. K. (1986) Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor. J. Cell Biol.103, 205 -214. 19. Kauer J. A., Malenka R. C. and Nicoll R. A. (1988) A persistent postsynaptic modification mediates long-term potentiation in the hippocampus. Neuron 1, 91 I-917. 20. Keinanen K., Wisden W., Sommer B., Werner P., Herb A., Verndoorn T. A., Sakmann B. and Seeberg P. (1990) A family of AMPA-selective glutamate receptors. Science 249, 5566560. 21. Klein A. U., Niederoest B., Winterhalter K. H.. Cuenod M. and Streit P. (1988) A kainate binding protein in pigeon cerebellum: purification and localization by monoclonal antibody. Neurosci. Lett. 95, 359-364. 22. Masu M.. Tanabe Y., Tsuchida K.. Shigemoto R. and Nakanishi S. (1991) Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. 23. Mattson M. P., Wang H. and Michaelis E. K. (1991) Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons. Brain Res. 565, 94 -108. 24. Monaghan D. T., Bridges R. J. and Cotman C. W. (1989) The excitatory amino acid receptors: their classes. pharmacology, and distinct properties in the function of the central nervous system. A. Ret. Pharmac. Toxic. 29, 365-402. 25. Monaghan D. T. and Cotman C. W. (1982) The distribution of [‘Hlkainic acid binding sites in the rat CNS as determined using autoradiography. Brain Res. 252, 91-100. 26. Nakanishi N., Shneider N. A. and Axe1 R. (1990) A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5, 569-581. 27. Olsen R. W.. Szamraj 0. and Houser C. R. (1987) [‘HIAMPA binding to glutamate receptor subpopulations in rat brain. Brain Res. 402, 243-254. 2x. Olson J. M. M., Greenamyre J. T., Penney J. B. and Young A. B. (1987) Autoradiographic localization of cerebellar excitatory amino acid binding sites in the mouse. Neuroscience 22, 913-923. 29. Rainbow T. C., Wieczorek C. M. and Halpain S. (1984) Q uantitative autoradiography of binding sites for [‘HIAMPA. a structural analogue of glutamic acid. Brain Res. 309, 1733177. mossy fibers: localization 30. Represa A., Tremblay E. and Ben-Ari Y. (1987) Kainate binding sites in the hippocampal and plasticity. Neuroscience 20, 739-748.

22

D. R.

FIAMPS~N et al.

31. Schneider C., Newman R. A., Sutherland D. R., Asser U. and @eaves M. F. (1982) A one-step purification ot membrane proteins using high efficiency immunomatrix. J. biol. Chem. 257, 10,76610,769. 32. Somogyi P., Eshar N., Teichberg V. I. and Roberts J. D. B. (1990) Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellum. Neuroscience 35, g-30. 33. Somogyi P., Takagi H., Richards J. G. and Mohler H. (1989) Subcellular localization of benzodiazepine/GABA, receptors in the cerebellum of the rat, cat, and monkey using monoclonal antibodies. J. Neurosci. 9, 2197-2209. 34. Wada K., Dechesne C. J., Shimasaki S., King R. G., Kusano K., Buonanno A., Hampson D. R., Banner C., Wenthold R. J. and Nakatani Y. (1989) Sequence and expression of a frog brain complementary DNA encoding a kainate-binding protein. Nature 342, 684689. 35. Wenthold R. J., Hampson D. R., Wada K., Hunter C., Oberdorfer M. D. and Dechesne C. J. (1990) Isolation, localization and cloning of a kainic acid binding protein from frog brain. J. Hisrochem. Cytochem. 38, 1717-1728. 36. Wenthold R. J., Hunter C., Wada K. and Dechesne C. J. (1990) Antibodies to a C-terminal peptide of the rat brain glutamate receptor subunit, GluR-A, recognize a subpopulation of AMPA binding sites but not kainate sites. Fedn Eur. biochem. Sots L&t. 276, 147-150. 37. Werner P., Voight M., Keinanen K., Wisden W. and Seeburg P. H. (1991) Cloning of a putative high-affinity kainate receptor expressed predominantly in hippocampal CA3 cells. Nature 351, 742-748.

38. Young A. B. and Fagg G. E. (1990) Excitatory amino acid receptors in the brain: membrane binding and receptor autoradiographic approaches. Trends pharmac. Ski. 11, 126-132. (Accepted

10 March 1992)