Neuron.

Vol. 9, 259-270,

August,

1992, Copyright

0 1992 by Cell Press

Cellular Localization of a Metabotropic Receptor in Rat Brain lee J. Martin,*+ Craig Richard 1. Huganir,*§

D. Blackstone,*§ and Donald 1. Price*+*11

*Department of Pathology +The Neuropathology Laboratory *Department of Neuroscience IlDepartment of Neurology §The Howard Hughes Medical Institute The Johns Hopkins University School of Medicine Baltimore, Maryland 21205-2196

Summary In rat brain, the cellular localization of a phosphoinositide-linked metabotropic glutamate receptor (mCluR1 a) was demonstrated using antibodies that recognize the C-terminus of the receptor. mCluRla, a 142 kd protein, is enriched within the olfactory bulb, stratum oriens of CA1 and polymorph layer of dentate gyrus in hippocampus, globus pallidus, thalamus, substantia nigra, superior colliculus, and cerebellum. Lower levels of mCluRla are present within neocortex, striatum, amygdala, hypothalamus, and medulla. Dendrites, spines, and neuronal cell bodies contain mCluRla. mGluRla is not detectable in presynaptic terminals. mGluRla and ionotropic a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits show differential distributions, but in Purkinje cells, mGluRla and specific AMPA receptor subunits colocalize. The postsynaptic distribution of mGluRla is consistent with postulated physiological roles of this subtype of glutamate receptor. Introduction

L-Glutamate is an excitatory neurotransmitter utilized by many neurons in thecentral nervous system. Glutamatergic neurotransmission is thought to be fundamental for learning, memory, motor control, sensory processing, development, and synaptogenesis (Collingridge and Bliss, 1987; Cotman et al., 1987; Monaghan et al., 1989) and is mediated by ionotropic receptors and metabotropic receptors (mGluR). These receptors are classified on the basis of pharmacological and electrophysiological criteria into five main subtypes: N-methyl-o-aspartate receptors, a-amino3-hydroxy-5-methyl4isoxazole propionate (AMPA) receptors, kainate receptors, 2-amino4phosphonobutyrate receptors, and trans-l-amino-cyclopentyl1,3-dicarboxylate (ACPD) receptors (Monaghan et al., 1989; Gasic and Heinemann, 1991). N-methyl-Daspartate, AMPA, and kainate receptors are ligandgated, cation-conducting channels that mediate fast excitatory postsynaptic potentials (Watkins et al., 1990; Gasic and Heinemann, 1991). Activation of the 2-amino4phosphonobutyrate receptor is associated with increased hydrolysis of cyclic guanosine mono-

Glutamate

phosphate (mediated by a G protein), which closes ion channels (Nawy and Jahr, 1990; Shiells and Falk, 1990). The Vans-ACPD receptor is a G protein-coupled receptor, which, when stimulated, initiates acascade of events, including the activation of phospholipase C, the generation of inositol triphosphate and diacylglycerol, and the subsequent mobilization of Ca*+ from intracellular stores (Sugiyama et al., 1987; Furuya et al., 1989; Schoepp et al., 1990). Stimulation of mGluRalso inhibits K+conductance, produces slow depolarization, and blocks slow afterhyperpolarization (i.e., spike accommodation) (Stratton et al., 1989, 1990; Desai and Conn, 1991). lonotropic and metabotropic glutamate receptors differ pharmacologically (selective sensitivity to agonists) and functionally (distinct signal transduction mechanisms). Moreover, recent molecular cloning studies show that non-Nmethyl-o-aspartate ionotropic glutamate receptors and mGluRexhibit different structural characteristics, supporting their classification into different receptor subfamilies (Gasic and Hollmann, 1992). The pharmacological and functional differences between ionotropic and metabotropic glutamatergic receptors may also be reflected by differences in their expression patterns and cellular distributions within the brain. An mGluR cDNA encoding a phosphoinositide-coupled mGluR (mGluRla) was cloned by the functional expression in Xenopus oocytes (Houamed et al., 1991; Masu et al., 1991). Several related cDNAs have been identified recently by cross-hybridization, including an alternatively spliced variant of mGluRla (mGluRlfl) as well as mGluR2, mGluR3, and mGluR4 (Tanabe et al., 1992). To date, only the mGluRla receptor has been shown to be coupled to the phosphoinositide second messenger system (Tanabe et al., 1992). Using the deduced amino acid sequence of the mGluRla protein, we generated antipeptide antibodies that recognize the C-terminus of this receptor in vivo. In the present study, using immunocytochemical-ultrastructural approaches, we demonstrate that mGluRla is selectively enriched within certain populations of neurons in rat brain and is localized within dendritic spines and shafts of specific subsets of neurons. These findings are consistent with many aspects of theanatomical, pharmacological, and physiological organization of glutamatergic systems. Results lmmunoblotting

lmmunoblottingwithanti-mGluRlaantibodydemonstrates the presence of immunoreactive proteins in rat brain (Figure 1). By SDS-polyacrylamide gel electrophoresis, mGluRla-immunoreactive proteins have a molecular mass of 142 kd. Although the mGluRla cDNA clone predicts a protein with a molecular mass of approximately133 kd, it has several consensus sites

NWPX 260

12 205-

I) 11697-

45-

Figure 1. Rat Cerebellar Synaptic Plasma Membranes (5 bg of Protein per Lane) Were Subjected to SDS-Polyacrylamide Gel Electrophoresis and lmmunoblotted with Antibodies (2 pg of IgC per ml) Raised against a Synthetic Peptide Corresponding to the C-Terminus of mCluRla mGluRla-immunoreactive proteins have a molecular mass of 142 kd (lanel). lmmunoreactivity is abolished completely when antibodies are preadsorbed with 50 FM synthetic peptide prior to immunoblotting (lane 2). Positions of molecular mass standards (in kd) are indicated at left.

for N-linked glycosylation (Masu et al., 1991), and, therefore, this molecular mass difference is likely caused by in vivo posttranslational glycosylation. Detection of this band of proteins is completely abolished when the antibody is preadsorbed with 50 PM synthetic peptide. mGluRla immunoreactivity is not detected by immunoblotting of tissue homogenates from the gastrocnemius muscle, testes, diaphragm, kidney, liver, spleen, bladder, and small intestine (data not shown). lmmunocytochemistry mGluRla immunoreactivity is selectively enriched within neurons in a variety of brain regions (see Figures 2-7). Levels of mGluRla are particularly high in olfactory bulb,certain nuclei of the basal ganglia, thalamus, and cerebellum. Punctate immunoreactivity within the neuropil is the predominant pattern of staining, although immunoreactive proximal dendrites and perikarya are also present. The number of mGluRla-immunoreactive neuronal cell bodies was greater in rats that received intracerebroventricular injections of colchicine, presumably because transport of this receptorwas reduced. mGluRla immunoreactivity is not associated with glial cells. In control immunocytochemical sections, preadsorption of mCluRla antibodies with synthetic mGluRla peptide abolishes all immunocytochemical staining (compare Figures 4B and 4C). The distributions of mCluRla immunoreactivity in rat brain are highly selective. Within olfactory bulb, punctate labeling is abundant within glomeruli, and many mitral cell bodies express mGluRla (Figure 2). Virtually all thalamic nuclei show immunoreactivity within the neuropil, although distinct thalamic sub-

nuclei are differentially enriched in mGluRla (Figure 3). In the cerebellum, mGluRla is abundant in the molecular layer; many Purkinje cell bodies are immunoreactive,and Golgicells(identified byglutamicacid decarboxylase immunoreactivity), but not granule cells, are mGluRla positive (Figure 4). The main dendritic shafts of Purkinje cells are immunoreactive (Figure 46). In cerebellar cortex, comparisons between the distributions of mGluRa and AMPA receptor subunits show that GluRl and GluR4 are enriched in the neuropil of molecular layer but not in Purkinje cell bodies (see Figures 8C and 8E), whereas GluR213 immunoreactivity is abundant in Purkinje cell bodies and dendritic shafts but not within the neuropil of the molecular layer(see Figure8F). In Purkinjecell bodies, mGluRla colocalizes with GluR213 immunoreactivity (Figure 5). There is no colocalization of mGluRla and GIuR4 or GluRl within these neurons. l.evels of mGluRla are lower within cerebral cortex, but fine punctate immunoreactivity decorates the contours of some neocortical perikaryaand dendrites. Within hippocampus, the superficial region of stratum oriens of CAI, at the stratum oriens-alveus border, and the polymorph layer of the dentategyrus show high levels of mGluRla(Figure6).Thecell bodiesof many nonpyramidal neurons (presumptive interneurons), predominantly within CAI, are mGluRla positive. Fewer pyramidal and granule cell bodies are mGluRla positive. lmmunoreactive dendritic profiles are found within the stratum oriens and stratum radiatum of most CA subfields but primarily in CAI. In contrast to the distribution of mGluRla in hippocampus, GluRl and GluR2/3 immunoreactivities are enriched highly within the dendritic shafts and spines in the striatum oriens and striatum radiatum of CAI-CA3 and within the dentate gyrus (see Figures 8A and 86; compare with Figure 6). mGluRla is low within the striatum (Figure 7), but faint staining is associated with profiles of perikarya and long dendrites of some large neurons. The globus pallidus, ventral pallidum, and entopeduncular nucleus are highly enriched in mGluRla-containing neuronal cell bodies and processes (Figure 7). mGluRla-immunoreactive neuronal processes are also present within the compact and reticular divisions of the substantia nigra. Punctate and perikaryal immunoreactivities are enriched within the subthalamic nucleus (Figure 7). At the light microscopic level, it is not clear whether mGluRla-positive puncta are pre-or postsynaptic elements. In 1 brn thick plastic sections of cerebellar cortex, thalamus, and globus pallidus, very fine and discrete mGluRla-immunoreactive puncta are enriched within the neuropil of thecerebellar molecular layer (Figure4D) and neuropil of thalamus and globus pallidus. In cerebellum, immunoreactive Colgi cell bodies and processes are distributed within the granulecell layer, butgranulecellsarenot labeled. Preliminary electron microscopic examination demonstrates that mGluRla is localized within dendriticspines in the cerebellar molecular layer (Figure 4E) and in small-

Metabotropic

Glutamate

Receptor

in Rat Brain

261

Figure

2. mCluRla

lmmunoreactivity

Is Enriched

in Brain

Regions

That

Receive

Primary

Sensory

Afferents

(A) In olfactory bulb (inset), glomeruli are intensely immunoreactive for mGluRla. Arrow (inset) identifies a glomerulus seen at higher magnification (asterisk). The external plexiform layer (EPL) has diffuse immunoreactivity within the neuropil. Some mitral cell bodies (arrowhead) and cell bodies of neurons located in deeper layers (arrow) are immunoreactive. Abbreviations: CCL, granule cell layer; CL, glomerular layer; MCL, mitral cell layer; ONL, olfactory nerve layer. Bar, 47 pm; inset, 286 urn. (6) The superior colliculus (SC) and medial geniculate nucleus (MC) are enriched in mCluRla immunoreactivity. Abbreviations: DC, clentate gyrus; P, polymorph layer of the dentate gyrus. Bar, 585 pm.

caliber dendritic In contrast, CluRl in the cerebellar GluR2/3 is present

shafts within is enriched molecular within main

the granule cell layer. within Bergmann glia layer (Figure 8D), and dendritic shafts of Pur-

kinje cells and is considerably less prevalent than mGluRla in dendritic spines of Purkinje cells (Figure BG). Within the thalamus, mCluRla is enriched within largeand small-caliber dendritic shafts (Figure 3). In

NelJKXl 262

Figure 3. TheThalamus mCluRla lmmunoreactivity

Has High

Levels

of

(A) Individual thalamic nuclei show differential patterns of mGluRla expression. Abbreviations: CL, centrolateral nucleus; G, gelatinosus nucleus; H, habenula, ic, internal capsule; LD, laterodorsal nucleus; MD, mediodorsal nucleus; mt, mammillothalamic tract; VM, ventromedial nucleus; VPL, ventroposterolateral nucleus; R, reticular nucleus. Bar, 481 km. (B) mGluRla immunoreactivity within the thalamus is localized within dendritir shafts of thalamic neurons. In this electron micrograph, an immunoreactive dendritic profile (dl) is postsynaptic to an unlabeled terminal (t). mGluRla immunoreactivity is enriched at postsynaptic densities (arrowheads). A nonimmunoreactive axon (ax) and dendrite (d2) are at the left and the lower right, respectively. Bar, 0.2 Wm.

our

of cerebellar immunoreactivitywas

preparations

mGluRla synaptic

cortex and thalamus, not visualized in pre-

terminals.

Discussion This report describes thecellular abotropic glutamate receptor the distribution of mGluRla, linked glutamate receptor,

localization of a metin rat brain. To visualize a phosphoinositideantipeptide antibodies

were raised against asynthetic peptidecorresponding to the C-terminus of mGluRla (Masu et al., 1991). The characterization of these antibodies by immunoblot analysis of rat brain synaptic membranes shows that this antibody reacts with a single band of protein with an M, of 142,000, consistent with the molecular weight predicted by analysis of the cloned mCluRla cDNA (Houamed et al., 1991; Masu et al., 1991; Tanabe et al., 1992). No cross-reactivity with other known mGluRs (e.g., mGluRlf3, mGluR2, mGluR3, and mGluR4) is

Metabotropic 263

Figure

4. The

Glutamate

Cerebellar

Receptor

Cortex

in Rat Brain

Has Abundant

mGluRla

lmmunoreactivity

(A) mGluRla is localized within the molecular (ML), Purkinje cell (PL), and granule cell (CL) layers; the underlying white matter fwm) is devoid of immunoreactivity. The ML has the most abundant mGluRla immunoreactivity. Bar, 260 pm. (B) In the Purkinje cell layer (PL), cell bodies of Purkinje neurons show mCluRla immunoreactivity. In the granule cell layer (GL), the somata of Colgi cells (arrowheads), but not granule cells, are immunoreactive for mGluRla. The neuropil of the molecular layer (ML) is enriched in mGluRla. Bar, 16 pm. (C) Preadsorption of antibodies with the synthetic peptide, corresponding to the C-terminus of the mCluRla receptor, abolishes all immunoreactivity within cerebellum, demonstrating the specificity of antibodies. Abbreviations: CL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar, 16 pm. (D) In ‘I pm thick plastic sections of cerebellar cortex, the molecular layer contains numerous discrete mGluRla-positive puncta (arrowheads). Bar, 10 pm. (E) Puncta within the cerebellar molecular layer correspond to mCluRla-immunoreactive spines of Purkinje cells. In this electron micrograph, a nonimmunoreactive parallel fiber terminal (t) forms an asymmetrical synapse with an mCluRla-immunoreactive spine (sp). mCluRla is enriched within the cytoplasm of the spine and at the postsynaptic density (arrowheads). Bar, 0.15 pm.

Neuron 264

Figure5. Within the Cerebellum, Rla and the CIuR2 and/or GIuR3 of the AMPA Receptor Colocalize Purkinje Cell Bodies (Arrows)

mGluSubunit in Many

GluR2&immunoreactive Purkinje cell bodies (brown reaction product) are covered with bluegreen granules, corresponding to mGluRla immunoreactivity. mGluRla was visualized with benzidine dihydrochloride (blue-green, granular reaction product); CluR2/3 was visualized with diaminobenzidine (brown reaction product). Abbreviations: CL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar, 20 pm.

likely because these receptors are predicted to be approximately 100 kd proteins and exhibit no similarity to mGluRla in thedomain that is recognized bythese antibodies (Tanabe et al., 1992). Northern blot and in situ hybridization analyses have been used to begin to map the distributions of mGluRla metabotropic receptor mRNA in rat brain (Houamed et al., 1991; Masu et al., 1991), and our immunocytochemical findings on the cellular localization of mGluRla in rat brain are consistent, for the most part, with these preliminary reports. The mRNA transcripts and mGluRla protein are both expressed differentially within a variety of regions in rat brain, and levels are high in olfactory bulb, cerebellum, thalamus, and hippocampus (Masu et al., 1991). At the cellular level, mitral cells of the olfactory bulbs, Purkinje cells of the cerebellum, pyramidal and nonpyramidal cells of the hippocampus, and thalamic neurons express high levels of mGluRla mRNA (Masu et al,, 1991). In our immunocytochemical preparations, many of these neuronal perikarya are enriched in mGluRla immunoreactivity, and we further demonstrate that this receptor protein is enriched in dendrites and spines within the neuropil. Before the mGluRs were cloned, an autoradiographic binding study showed that AMPA-insensitive, quisqualate-sensitive [3H]glutamate-binding (AiQsGB)

Figure

6. mCluRla

lmmunoreactivity

Is Distributed

Differentially

sites are present in rat brain (Cha et al., 1990). AiQsGB was highest in the lateral septum and cerebellar molecular layer and was also present in striatum, cingulate cortex, and entorhinal cortex. Although the thalamus had a low number of AiQsGB sites (Cha et al., 1990), pharmacological and electrophysiological results strongly indicate that ACPD-preferring phosphoinositide-linked receptors participate in synaptic transmission within the thalamus (Salt and Eaton, 1991). Our data, showing that mGluRla is abundant at postsynaptic sites within the thalamus, are consistent with pharmacological and electrophysiological studies (Salt and Eaton, 1991). Prominent differences between AiQsGB and mGluRla immunoreactivities in thalamus suggest the existence of more than one molecular subtype of mGluR--an idea supported by the recent cloning of a family of mGluRs (Tanabe et al., 1992). Our studies of cerebellum show that mGluRla is enriched in Purkinje cell bodies and spines and that these neurons express both mGluRla and AMPA receptors with a particular subunit composition. These observations are consistent with the coparticipation of metabotropic and ionotropic quisqualate receptors in synaptic interactions and with the induction of long-term depression in cerebellum (Kano and Kato, 1987). Parallel fibers, originating from glutamatergic

in Hippocampus

(A) The superficial region of CA1 (arrowheads) and the polymorph layer (P) of dentate gyrus (DC) are selectively enriched in mCluRla. The cingulate cortex (arrows) and lateral geniculate nucleus (LC) also show intense immunoreactivity. Bar, 293 urn. (B)Thestratum oriens layer (0r)of CAl, immediatelydeep to thealveus (alv),and somecell bodiesof nonpyramidal neurons(arrowheads) are immunoreactive for mGluRla. Bar, 47 km. (C) In the medial part of CA1 (box in [A]), a plexus of dendrites is enriched in mGluRla. Some of these dendrites have numerous immunoreactive spines. Bar, 22.5 urn. (D) Within the stratum oriens of CAI, mGluRla-immunoreactive cell bodies of nonpyramidal neurons and dendrites are intermingled extensively. Bar, 11.3 pm. (E) Dendrites in CA1 also show punctate mGluRla immunoreactivity apparently on the surface of the plasmalemma (arrowheads). Bar, 3.8 urn.

Metabotropic 265

Glutamate

Receptor

in Rat Brain

NeUrO” 266

Figure

7. mCluRla

lmmunoreactivity

Is Enriched

in Some

Regions

of the

Basal Ganglia

(A) mGluRla is abundant within the globus pallidus (CP) and entopeduncular nucleus (EP) but is sparse tions: ic, internal capsule; R, reticular thalamic nucleus; VPL, ventroposterolateral thalamic nucleus. (6) Fine cellular processes (arrowheads) within globus pallidus (GP) show mGluRla immunoreactivity. right. Bar, 62.5 urn. (C) At a higher magnification, mCluRla-positive processes within globus pallidus appear to be axons 7.0 urn. (D) The subthalamic nucleus (STh) is enriched in mCluRla-immunoreactive neuropil and neuronal cell tions: cp, cTTerebral peduncle; LH, lateral hypothalamus. Bar, 128 urn.

granule cells, form numerous synapses with Purkinje cell dendritic spines in the molecular layer (Young et al., 1974; Sandoval and Cotman, 1978). The activation of parallel fibers (via vestibular nerve stimulation) evokes excitatory postsynaptic potentials at parallel fiber-Purkinje cell sTTed for extended periods bytheconcomitant activation of climbing fiber inputs to Purkinje cells (Ito et al., 1982). This postsynaptic alteration requires the activation of

Figure

8. Distributions

of AMPA

Receptor

Subunits

in Hippocampus

within striatum Bar, 163 urn. The striatum or dendrites bodies

(CPU). Abbrevia(CPU)

IS seen

(arrowheads).

(arrowheads).

at Bar,

Abbrevia-

both metabotropic quisqualate receptors and ionotropic (AMPA) receptors as well as the presence of Ca2+ (Linden et al., 1991). Purkinjecells have abundant metabotropic AMPA-insensitive, quisqualate-sensitive, glutamate-binding sites (Cha et al., 1990), mGluRla and AMPA receptor subunit mRNA (Keinanen et al., 1990; Masu et al., 1991), inositol triphosphate receptors (Maeda et al., 1990; Ross et al., 1990), and Ca”binding proteins (Celia, 1990; Sequier et al., 1990). In

and Cerebellum

(A) Within hippocampus, GluRl is highly enriched in subfields of Ammon’s horn (CAI, CAZ, and CA3) and in dentate gyrus (DC). The stratum oriens and stratum radiatum have intense immunoreactivity within the neuropil. CA1 shows slightly higher levels of CluRl immunoreactivity than CA2 and CA3. Bar, 260 urn. (B) Main dendritic shafts (dl and d2) and spines (spl and sp2) of hippocampal neurons of CA1 are enriched in CIuRl. Bar, 0.3 pm. (C) Within cerebellar cortex, GluRl is abundant in the molecular layer (ML) and virtually devoid in the granule cell layer (CL). The Purkinje cell layer (PL) contains CluRl-positive processes and pericellular arrays, but Purkinje cell bodies are not immunoreactive for CIuRl. Bar, 68 urn. (D) Within cerebellar molecular layer, nonimmunoreactive dendritic spines (dl, d2, and d3) forming synapses with presumed parallel fiber terminals (tl, t2, and t3) are ensheathed by GluRl-positive glial processes (arrowheads). Bar, 0.25 pm. (E) CIuR4 is enriched in the cerebellar molecular layer (ML) and less abundant in the Purkinje cell layer (PL) and granule cell layer (GL). GluRQimmunoreactive profiles of granule cells are present (arrowheads) but Purkinje cell bodies are not immunoreactive. Bar, 68 pm.

Metabotropic

Glutamate

Receptor

in Rat Brain

267

(F) In cerebellar cortex, Purkinje cell bodies (arrows) and dendritic shafts (arrowheads) are GluR2/3 immunoreactive. The neuropil staining in the molecular layer (ML) is considerably less abundant than in GIuRl, GIuR4, and mGluRla preparations (compare with Figure 4; Figures 8C and BE). In the granule cell layer (CL), some CluR2/3Ammunoreactive profiles of granule cells are discernible (thin arrows). Bar, 19 urn. (C) In the molecular layer of the cerebellum, GluR2/3 is visualized within large dendritic shafts (d). In this cross-sectioned profile of dendrite (d), immunoreactivity is present on or near the plasmalemma (arrowheads). GluR2/3 is not associated with dendritic spines 1:asterisks). Bar, 0.36 urn.

Nf?UK?” 268

cerebellar slices, quisqualate (Blackstone et al., 1989) and ACPD (Hwang et al., 1990) can stimulate phosphoinositide turnover. Our results show that phosphoinositide-linked metabotropic and AMPA-preferring ionotropic glutamate receptors colocalize within Purkinje cells and that mGluRla is enriched particularly within dendritic shafts and spines of cerebellar Purkinje cells, whereas the GIuR2 and/or GIuR3 subunits of the AMPA receptor are concentrated in dendritic shafts, but not spines, of these neurons (Figure 4E; Figure 8G). Furthermore, GluRl and GluR4 subunits of the AMPA receptor are enriched in processes of Bergmann glia that ensheath synaptic complexes formed by parallel fiber terminals and Purkinje cell spines (Figure 8D). These subcellular distributions of distinct glutamate receptors are reminiscent of the differential targetingof parallel and climbing fiber inputsto Purkinje cells and, thus, provide direct anatomical evidence to support the reported physiological roles of glutamate receptors in cerebellar long-term depression. In addition, our results suggest that parallel fibers synapse predominantly with Purkinje cell spines that express mGluRla and, possibly, that climbing fibers synapse predominantly with Purkinje cell dendritic shafts that express ionotropic receptorscomposed of GIuR2 and/ or GIuR3 but not GluRl or GluR4. The structure of the ionotropic glutamate receptor that mediates the excitatory postsynaptic potentials at parallel fiberPurkinje cell spine synapses is still unclear. Thus, glutamatergic presynaptic axonal terminals arising from parallel and climbing fibers may have different functions by virtue of interactions with distinct glutamate receptor subtypes on different anatomical domains of Purkinje cells. In hippocampus, an ACPD-sensitive, phosphoinositide-linked glutamate receptor has been implicated in long-term potentiation (Palmer et al., 1989; Baskys and Malenka, 1991). Thecellular location (i.e., presynaptic, postsynaptic, or both) of mGluRs participating in long-term potentiation within CA1 of the hippocampus is unclear. Pharmacological and physiological studies suggest that mGluRs are likely to be located pre- and postsynaptically in CA1 (Stratton et al., 1989; Baskys and Malenka, 1991). lonotropic AMPA receptors are enriched postsynaptically in dendritic shafts and spines of hippocampal pyramidal cells. Although our immunocytochemical studies of hippocampus are preliminary with respect to the precise ultrastructural localization of mGluRla within this region, the present results suggest that, within CAI, mGluRla is predominantly localized postsynaptically to dendritic shafts and dendritic spines as well as to many cell bodies of nonpyramidal neurons that may be interneuronal basket cells. These mGluRla-enriched nonpyramidal neurons are located near the stratum oriens-alveus border and may correspond to the electrophysiologically identified interneurons, which receive excitatory afferents and are thought to mediate feedforward and feedback inhibition of CA1 pyrami-

dal cells that express ionotropic glutamate receptors (Lacaille et al., 1987). These nonpyramidal cells, which are generally thought to use y-aminobutyric acid as a neurotransmitter (Frotscher et al., 1984), may have a postsynaptic role in long-term potentiation. In addition, some dendrites within hippocampus may have mGluRla-enriched bouton-like puncta on their surface; thus, a presynaptic distribution originating from terminals of mGluRla-positive interneurons cannot be excluded at this time. In any event, theenrichment of mGluRlawithin dendrites in CA1 is consistentwith the postulated role of mGluR in hippocampal longterm potentiation.

Experimental Preparation

Procedures of Affinity-Purified

Antibodies to mCluRla KPNVTYASVILRDYKQSSSTL), corre20 aa (residues 1180-1199) of the pre dieted polypeptide sequence encoded by the mCluRla cDNA in rat brain (Houamed et al., 1991; Masu et al., 1991; Tanabe et al., 19921, was synthesized on an Applied Biosystems 430A Peptide Synthesizer (Applied Biosystems, Inc., Foster City, CA). An N-terminal lysine residue was added to the peptide to facilitate coupling of peptide to carrier protein. After synthesis, the peptide was purified using reverse-phase high performance liquid chromatography; then, using glutaraldehyde, the synthetic peptide was coupled to thyroglobulin. Antisera against carrier proteinconjugated peptides were raised in New Zealand White rabbits (Hazleton Research Products, Denver, PA). The mGluRla antiserum was affinity purified on a column prepared by coupling bovine serum albumin-conjugated mCluRla peptide to Affi-Gel 15 (Bio-Rad, Rockville Center, NY).

A synthetic peptide (i.e., sponding to the C-terminal

lmmunoblotting The specificity of the antibody to mGluRla was evaluated by immunoblotting of rat brain synaptic membranes. Synaptic membranes from adult male Sprague-Dawley rat cerebellum were prepared using procedures described previously (Blackstone et al., 1992). Aliquots (5 kg of protein) of synaptic membranes were subjected to SDS-polyacrylamide gel electrophoresis and were transferred by electroblotting to nitrocellulose (Blackstone et al., 1992). Blots were blocked (1 hr) in 0.5% nonfat drymilk,O.l% Tween20,50mMTri+bufferedsaline(TBS)at room temperature and then incubated (I hr) with affinity-purified mGluRla antibody diluted to 2 pg of immunoglobulin C (IgC) per ml in blocking buffer. Blots were subsequentlywashed, incubated (I hr) with horseradish peroxidase-conjugated donkey anti-rabbit IgG (I:5000 in blocking buffer), and finally washed with TBS. lmmunoreactive proteins were visualized with enhanced chemiluminescence (Amersham, Arlington Heights, IL). lmmunocytochemistry The brains of adult male and female Sprague-Dawley rats (n = 5) were prepared for both light and electron microscopic evaluation of mCluRla. Some rats (n = 3) received bilateral intracerebroventricular injections of colchicine (120 pg) and were sacrificed 48 hrafter injection. Ratswere perfused intra-aorticallywith cold 0.1 M phosphate-buffered 0.9% saline, followed by either 4% paraformaldehyde/O.l% glutaraldehyde/l5% saturated picric acid or 4% paraformaldehyde/2.5% acrolein, both prepared in phosphate-buffered saline. Brains were removed, blocked, and postfixed (1 hr, 4OC). Some brains were cryoprotected (overnight at 4°C) in 20% glycerol, phosphate-buffered saline and frozen in isopentane chilled by dry ice. Other brains were rinsed (overnight) in phosphate-buffered saline (4°C). Coronal sections (40 pm) were cut on a sliding microtome or Vibratome and were transferred to cold TBS (pH 7.2). Sections intended solely for light microscopy were permeabilized (30 min) in 0.4% Triton X-100

Metabotropic 269

Glutamate

Receptor

in Rat Brain

(TX), TBS, whereas sections for electron microscopy were treated (IO min) with 0.08% TX, TBS. Subsequent immunocytochemical steps were identical for both groups of sections, but sections for electron microscopy were not exposed to TX. Sections were preincubated (1 hr) with 4% normal goat serum diluted in 0.1% TXiTBS and were incubated (48 hr, 4°C) in affinity-purified rabbit polyclonal antimGluRla antibodies at a concentration of 0.5 bg of IgC per ml in 0.1% TX, 2% normal goat serum, TBS. Control sections were incubated with the following: comparable amounts of rabbit IgG, mGluRla antibody preadsorbed overnight with excess (IO pglml) synthetic mCluRla peptide, or with primary antisera omitted. To compare thedistributions of mCluRla to ionotropic glutamate receptors, selected sections through the hippocampus and cerebellum were stained with antibodies that recognize AMPA receptor subunits (CIuRl, GIuR4, and a common epitope on GIuR2 and GIuR3). These antibodies have been characterized previously (Blackstone et al., 1992; L. J. Martin, C. D. Blackstone, A. I. Levey, R. L. Huganir, and D. L. Price, submitted). Following incubation, sections were rinsed (30 min) in TBS, incubated (1 hr) with goat anti-rabbit immunoglobulin (Cappel, West Chester, PA) diluted at l:lOO, rinsed (30 min) in TBS, and incubated (1 hr) with rabbit peroxidase-antiperoxidase complex (Sternberger Monoclonals, Baltimore, MD) diluted at 1:200. For additional controls, the secondary antibody and peroxidaseantiperoxidase were omitted from the incubation solution. After the final incubation, sections were rinsed (30 min) in TBS and developed using a standard diaminobenzidine reaction. After the disclosing reaction, samples (approximately 2 mm*) were taken from Vibratome sections, treated (1 hr) with 2% osmium tetroxide, dehydrated, stained en bloc with uranyl acetate, and flat embedded in resin. Plastic-embedded sections were mounted on an Araldite block and cut into semithin (1 pm) and ultrathin (gold to silver interference color) sections for light and electron microscopy, respectively. Ultrathin sections, with and without lead citrate staining, were viewed and photographed with a Philips CM12 or JEOL 100s electron microscope. Selected sections of cerebellum were processed using double-label immunocytochemistry (Levey et al., 1986) to establish whether mCluRla colocalizes with ionotropic AMPA receptor subunits in individual Purkinje cells. Sections were first blocked in 4% normal goat serum and incubated (48 hr) with antibodies that recognize the GluR2 and GIuR3 subunits (CIuR2/ 3) or the GIuR4 or CluRl subunits of the AMPA receptor (Blackstone et al., 1992; L. 1. Martin, C. D. Blackstone, A. I. Levey, R. L. Huganir, and D. L. Price, submitted) and were developed using diaminobenzidine as chromagen. Subsequently, these sections were blocked (overnight) in 10% normal goat serum, incubated (48 hr) with mGluRla antibody, and developed using benzidine dihydrochloride as chromagen.

The authors thank Dr. Clark Riley and The Howard Hughes Medical Institute Biopolymer Laboratory for the preparation of synthetic peptide. Wealso thank Ms. JudithVan Lareand Mr. Wayne Voris for their excellent technical assistance. Thisworkwassupported bygrantsfromtheU.S.PublicHealth Service (National lnstititutes of Health NS20471 and AG05146), the Medical Scientist Training Program (GM07309), and The Howard Hughes Medical Institute and funds from the American Health Assistance Foundation and The Metropolitan Life Foundation. D. L. P. is the recipient of a Leadership and Excellence in Alzheimer’s Disease (LEAD) award (NIAAG07914) and a Javits Neuroscience Investigator award (NIH NS 10580). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘adverfisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact. March

Baskys, A., and Malenka, R. C. (1991). Trans-ACPD depresses synaptic transmission in the hippocampus. Eur. J. Pharmacol. 193, 131-132. Blackstone, C. D., Supattapone, S., and Snyder, S. H. (1989). lnositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission. Proc. Natl. Acad. Sci. USA 86, 4316-4320. Blackstone, C. D., Moss, S. J., Martin, L. J., Levey, A. I., Price, D. L., and Huganir, R. L. (1992). Biochemical characterization and localization of a non-N-methyl-o-aspartate glutamate receptor in rat brain. J. Neurochem. 58, 1118-1126. Celio, M. R. (1990). Calbindin nervous system. Neuroscience

D-28k and parvalbumin 35, 375-475.

in the

rat

Cha, J.-H. J., Makowiec, R. L., Penney, J. B., and Young, A. B. (1990). L-[3H]glutamate labels the metabotropic excitatory amino acid receptor in rodent brain. Neurosci. Lett. 773, 78-83. Collingridge, C. L., and Bliss, T. V. P. (1987). their role in long-term potentiation. Trends 293.

NMDA receptorsNeurosci. 70, 288-

Cotman, C. W., Monaghan, D. T., Ottersen, 0. P., and StormMathisen, J. (1987). Anatomical organization of excitatory amino acid receptorsand their pathways. Trends Neurosci. 70,273-280. Desai, M. A., and Conn, P. J. (1991). Excitatory effects of ACPD receptor activation in the hippocampus are mediated by direct effects on pyramidal cells and blockade of synaptic inhibition. 1. Neurophysiol. 66, 40-52. Frotscher, M., L&Bnth, C., Li.ibbers, K., and Oertel, W. H. (1984). Commissural afferents innervate glutamate decarboxylase immunoreactive non-pyramidal neurons in the guinea pig hippocampus. Neurosci. Lett. 46, 137-143. Furuya, S., Ohmori, Intracellular calcium ceptor in rat cultured

H., Shigemoto, mobilization hippocampal

Gasic, G. P., and Heinemann, ionic channels: the glutamate robiol. 7, 20-26.

T., and Sugiyama, H. (1989). triggered by a glutamate recells. J. Physiol. 474,539-548.

S. (1991). Receptors receptor family. Curr.

coupled to Opin. Neu-

Gasic, G. P., and Hollmann, M. (1992). Molecular neurobiology of glutamate receptors. Annu. Rev. Physiol. 54, 507-536. 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. Science 25.7, 1318-1321. Hwang, P. M., Bredt, D. S., and Snyder, S. H. (1990). Autoradiographic imaging of phosphoinositide turnover in the brain. Science 249, 802-804. Ito, M., Sakurai, M., and Tongroach, P. (1982). Climbing fibre induced depression of both mossy fibre responsiveness and glutamate sensitivity of cerebellar Pu rkinje cells. J. Physiol. 324,113134.

Acknowledgments

Received

References

25, 1992;

revised

June

10, 1992.

Kano, M., and Kato, M. (1987). Quisqualate receptors are specifically involved in cerebellar synaptic plasticity. Nature 325, 276279. KeinBnen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T. A., Sakmann, B., and Seeburg, P. H. (1990). A family of AMPA-selective glutamate receptors. Science 249, 556-560. Lacaille, J.-C., Mueller, A. L., Kunkel, D. D., and Schwartzkroin, P. A. (1987). Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippcampal slices: electrophysiology and morphology. J. Neurosci. 7, 1979-1993. Levey, A. I., Bolam, J. P., Rye, D. B., Hallanger, A. E., Demuth, R. M., Mesulam, M.-M., and Wainer, 8. H. (1986). A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride. J. Histochem. Cytochem. 34,1449-1457. Linden, D. I., Dickinson, M. H., Smeyne, M., and Connor, J. A. (1991). A long-term depression of AMPA currents in cultured

cerebellar

Purkinje

neurons.

Neuron

7, 81-89.

Maeda, N., Niinobe, M., and Mikoshiba, K. (1990). A cerebellar Purkinjecell marker Pa protein isan inositol1,4,5-trisphosphate (InsP,) receptor protein. Purification and characterization of InsP, receptor complex. EMBO J. 9, 61-67. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., and shi, S. (1991). Sequence and expression of a metabotropic mate receptor. Nature 349, 760-765.

Nakanigluta-

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. Annu. Rev. Pharmacol. Toxicol. 29, 365-402. Nawy, S., and cGMP-activated 269-271.

Jahr, C. E. (1990). Suppression conductance in retinal bipolar

by glutamate of cells. Nature 346,

Palmer, E., Monaghan, D. T., and Cotman, C. W. (1989). TransACPD, a selective agonist of the phosphoinositide-coupled excitatory amino acid receptor. Eur. J. Pharmacol. 766, 585-587. Ross, C. A., Bredt, D., and Snyder, S. H. (1990). Messenger cules in the cerebellum. Trends Neurosci. 13, 216-222.

mole-

Salt, T. E., and Eaton, S. A. (1991). Excitatory actions of the metabotropic excitatory amino acid receptor agonist, tram-( &)-l-aminocyclopentane-1,3-dicarboxylate (t-ACPD), on rat thalamic neurons in vivo. Eur. J. Neurosci. 3, 1104-1111. Sandoval, M. E., and Cotman, C. W. (1978). mateasaneurotransmitterofcerebellarparallelfibers. ence 3, 199-206.

Evaluation

of glutaNeurosci-

Schoepp, D., Bockaert, J., and Sladeczek, F. (1990). Pharmacologlcal and functional characteristics of metabotropic excitatory amino acid receptors. Trends Pharmacol. Sci. II, 74-81. Skquier, J. M., Hunziker, W., Andressen, (1990). Calbindin D-28k protein and mRNA brain. Eur. J. Neurosci. 2, 1118-1126.

C., and Celio, M. R. localization in the rat

Shiells, R. A., and Falk, C. (1990). Glutamate receptors of rod bipolar cells are linked to a cyclic CMP cascade via a G-protein. Proc. R. Sot. Lond. (B) 242, 91-94. Stratton, K. R., Worley, P. F., and Baraban, J. M. (1989). Excitation of hippocampal neurons by stimulation of glutamate Qp receptors. Eur. J. Pharmacol. 773, 235-237. Stratton, K. R., Worley, P. F., and Baraban, 1. M. (1990). Pharmacological characterization of phosphoinositide-linked glutamate receptor excitation of hippocampal neurons. Eur. J. Pharmacol. 186, 357-361. Sugiyama, H., Ito, I., and Hirono, mate receptor linked to inositol ture 325, 531-533.

C. (1987). A new type of glutaphospholipid metabolism. Na-

Tanabe, Y., Masu, M., Ishii, T., Shigemoto, (1992). A family of metabotropic glutamate 169-179.

R., and Nakanishi, receptors. Neuron

S. 8,

Watkins, J. C., Krogsgaard-Larsen, P., and Honor&, T. (1990). Structure-activity relationships in thedevelopment of excitatory amino acid receptor agonists and competitive antagonists. Trends Pharmacol. Sci. 17, 25-33. Young, A. B., Oster-Granite, S. H. (1974). Clutamic acid: granule cell loss in hamster

M. L., Herndon, R. M., and Snyder, selective depletion by viral induced cerebellum. Brain Res. 73, 1-13.