localization of metabotropic glutamate receptor 7 mrna and mglur7a protein in the rat basal ganglia

Localization of Metabotropic GlutamateReceptor 7 mRNA and mGluR7a Protein

in the Rat Basal Ganglia

CHRISTOPH M. KOSINSKI,1 STEFANIA RISSO BRADLEY,2 P. JEFFREY CONN,2

ALLAN I. LEVEY,3 G. BERNHARD LANDWEHRMEYER,4 JOHN B. PENNEY JR.,1

ANNE B. YOUNG,1 AND DAVID G. STANDAERT1*1Neurology Service, Massachusetts General Hospital, and Harvard Medical School,

Boston, Massachusetts 021142Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322

3Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 303224Department of Neurology, Albert-Ludwigs-University, D-79106 Freiburg, Germany

ABSTRACTMetabotropic glutamate receptors (mGluRs) coupled to G-proteins have important roles

in the regulation of basal ganglia function. We have examined the localization of the mGluR7mRNA and mGluR7a protein in the basal ganglia of the rat. Strong mGluR7 hybridizationsignals are found in cerebral cortex and striatum, but much less intense signals are present inother components of the basal ganglia. Abundant mGluR7a immunoreactivity was found instriatum, globus pallidus (GP), and substantia nigra pars reticulata (SNr). Examination usingconfocal microscopy together with dendritic and presynaptic markers as well as studies inlesion models provided evidence for the presence of mGluR7a on presynaptic terminals in allthree structures. Electron microscopic studies confirmed the presence of mGluR7a in axonterminals in both the striatum and the GP and also revealed the presence of mGluR7a atpostsynaptic sites in both of these regions. Our data demonstrate that mGluR7a is located notonly on presynaptic glutamatergic terminals of the corticostriatal pathway, where it mayserve as an autoreceptor, but also on terminals of striatopallidal and striatonigral projections,where it may modulate the release of g-aminobutyric acid (GABA). The presence of mGluR7 atthese multiple sites in the basal ganglia suggests that this receptor has a particularly crucialrole in modulating neurotransmitter release in major basal ganglia pathways. J. Comp.Neurol. 415:266–284, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: globus pallidus; corticostriatal pathway; presynaptic receptors; confocal

microscopy; electron microscopy

Glutamate is the principal excitatory transmitter in themammalian brain and has a central role in regulatingbasal ganglia function (Albin et al., 1989). Metabotropicglutamate receptors (mGluRs) couple the actions of gluta-mate to intracellular second messengers. There are threewell-established groups of mGluRs, which differ in se-quence, pharmacology, and the second messengers towhich they are linked (Nakanishi, 1992; Conn and Pin,1997). MGluR7 is a member of the Group III mGluRs,which in vitro are coupled to inhibition of cyclic AMPsynthesis and can be specifically activated by L-2-amino-4-phosphonobutyrate (L-AP4; Tanabe et al., 1992; Okamotoet al., 1994; Saugstad et al., 1994). Electrophysiologicalstudies have shown that agonists of the Group III mGluRscan inhibit the release of glutamate from the terminals ofcortical neurons projecting to the striatum, the primary

source of afferent input to the basal ganglia (Albin et al.,1989; Pisani et al., 1997). Similar presynaptic inhibition ofglutamate release by group III mGluRs has been observedin other glutamatergic projection systems (Gereau andConn, 1995; Bushell et al., 1996). Modulation of glutamaterelease by group III mGluRs has been postulated to play a

Grant sponsor: USPHS; Grant numbers: NS31579, NS34361, NS31373,NS34876, NS98011; Grant sponsor: Wyeth Ayerst Research; Grant sponsor:NARSAD; Grant sponsor: the US Army; Grant sponsor: DFG; Grantnumbers: La 701–2, SFB 505, Ko 1696/1–2; Grant sponsor: CotziasFellowship from the American Parkinson’s Disease Association.

*Correspondence to: David G. Standaert, MD, PhD, MassachusettsGeneral Hospital, Warren 408, Fruit St., Boston, MA 02114.E-mail: [email protected]

Received 29 June 1999; Revised 23 August 1999; Accepted 2 September1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 415:266–284 (1999)

r 1999 WILEY-LISS, INC.

role in excitotoxic neurodegeneration (for review see Nico-letti et al., 1996).

Expression of mGluR7 mRNA in rat neocortex as well asstriatum has been demonstrated using in situ hybridiza-tion (Kinzie et al., 1995; Ohishi et al., 1995a). The mRNAsof other members of the group III mGluRs (mGluR4, -6, -8)are expressed only at low or undetectable levels in stria-tum and neocortex (Nakajima et al., 1993; Testa et al.,1994; Duvoisin et al., 1995; Ohishi et al., 1995a). Theseobservations suggest that the inhibitory effects of GroupIII receptors on striatal glutamatergic afferents are likelyto be mediated primarily by mGluR7. In the hippocampusand other regions of the rat brain, mGluR7 protein hasbeen found to be localized predominantly to presynapticterminals, presumably serving as an autoreceptor at gluta-matergic synapses (Ohishi et al., 1995b; Bradley et al.,1996; Brandstatter et al., 1996; Li et al., 1996, 1997;Shigemoto et al., 1996, 1997; Kinoshita et al., 1998). Apostsynaptic localization has been described, however, inthe retina on amacrine cells and occasionally on hippocam-pal pyramidal cell dendrites (Bradley et al., 1996; Brand-statter et al., 1996). We have used in situ hybridization tocompare the abundance of mGluR7 mRNA expression indifferent structures of the rat basal ganglia and have usedconfocal and electron microscopy to examine the localiza-tion of the mGluR7a protein in the rat basal ganglia. Ourdata reveal that mGluR7 is predominantly a presynapticreceptor in the basal ganglia and that this receptor islikely to have an important effect on both glutamatergicand g-aminobutyric acid (GABA)ergic transmission in thissystem.

MATERIALS AND METHODS

In situ hybridization

Synthesis of DNA templates. DNA templates for syn-thesizing RNA probes were constructed using a polymer-ase chain reaction (PCR) strategy as described previously(Standaert et al., 1996). Briefly, two sets of primers wereemployed to amplify the rat mGluR7 template. The outerprimer pair used was 58-GCT CGC GCA GTG ATT ATGTT-38 and 58-TGC GGT TGA TGT TTG GTC TC-38. Thisouter primer set amplified a DNA fragment correspondingto bases 888–1765 of the published sequence (Okamoto etal., 1994). The inner upper primer used was 58-GGG ATTTAG GTG ACA CTA TAG AAG GGT TTG ACC GAT ACTTTA G-38, beginning with the promoter for SP6 polymer-ase, and the inner lower primer was 58-CTG TAA TACGAC TCA CTA TAG GGG CAT GTC TTC CAC TTT TAGG-38, ending with the promoter for T7 polymerase. Theinner set of primers was then used to amplify the finaltemplate corresponding to bases 1084–1573 of the mGluR7mRNA sequence (Okamoto et al., 1994).

In vitro transcription. Radioactively labeled RNAprobes were synthesized by in vitro transcription of 0.75µg of the PCR-generated templates using SP6 or T7 RNApolymerase (Promega, Madison, WI) with [35S]CTP (.1,000Ci/mmol; Dupont-NEN) as the radioisotope as describedpreviously (Standaert et al., 1996). The RNA product wasthen isolated by precipitation in ethanol and resuspendedin 75 µl of Tris-EDTA (TE) buffer with 100 mM dithiothrei-tol (DTT) and 80 units RNasin. Incorporation of theradioactive label was assessed by liquid scintillation count-ing. Radiolabeled probes were stored at 270°C and usedwithin 1 week.

In situ hybridization. All procedures involving ani-mals were approved by the Massachusetts General Hospi-tal Subcommittee on Research Animal Care and con-formed with NIH guidelines for use of vertebrate animals.Male Sprague-Dawley rats (200–250 g; Charles River,Wilmington, MA) were killed by rapid decapitation, andthe brains removed immediately, frozen in chilled isopen-tane, and stored at 270°C until processed further. Sections(12-µm) were cut using a cryostat (Shandon Lipshaw,Pittsburgh, PA) and also stored at 270°C. The slides weretreated as described previously (Standaert et al., 1996).Briefly, they were fixed in 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4, rinsed with 0.1 M phosphatebuffer containing 0.9% sodium chloride, pH 7.4, acetylatedin 0.1 M triethanolamine with 0.25% acetic anhydride,dehydrated in graded ethanol solutions, and delipidated inchloroform. The isotopic probe was added to achieve150,000 cpm/µl hybridization buffer. After a 4-hour hybrid-ization at 50°C, the slides were washed at 70°C in decreas-ing concentrations of saline sodium citrate (SSC), rinsedbriefly in water and 70% ethanol, and air dried. Filmautoradiograms were prepared by opposing the slides toHyperfilm b-Max (Amersham, Arlington Heights, IL) for3–7 days. Slides were then dipped in Ilford K5 emulsiondiluted 1:1 with distilled water, stored at 4°C, and devel-oped after 2 months.

Analysis of in situ hybridization histochemistry.

Quantitative image analysis was performed using a com-puter-assisted image analysis system (M1; Imaging Re-search Inc., St. Catharine’s, Ontario, Canada) as describedelsewhere (Testa et al., 1994). For analysis of regions onfilm autoradiograms, absolute optical density for eachregion was defined as the average absolute optical densityover the entire region minus the background absoluteoptical density of the film outside of the sections. Absoluteoptical densities from four sections per animal were ana-lyzed. Means for each region on each section were includedin a one-way repeated-measures analysis of variance byregion with post hoc pairwise comparisons (Scheffe’s) witha significance level of 1% using a statistic software pack-age (SuperANOVA; Abacus Concepts Inc., Berkeley, CA).

Immunohistochemistry

Antisera. The polyclonal antibody to mGluR7a hasbeen characterized previously (Bradley et al., 1996). It wasraised in rabbits against the synthetic peptide DKNSPAAK-KKYVSYNNLVI corresponding to a carboxy terminal re-gion of the mGluR7a protein (amino acids 896–915, withsubstitution of a lysine as position 2 to facilitate coupling),affinity purified, and used at 1.2 µg/ml. Specificity of theantibody was verified by immunoblot studies of mem-branes from rat brain, mouse brain, and Spodopterafrugiperda (Sf9) insect cells transfected with mGluR7a ormGluR7b, as previously described (Bradley et al., 1999).When the antibody was used for immunohistochemistry,immunoreactivity was abolished by preabsorbtion withthe peptide antigen (Bradley et al., 1996).

In dual-label studies, incubations were performed incombination with one of the following: mouse monoclonalantibodies to choline acetyltransferase (ChAT; ChemiconInc., Temecula, CA; 1:100), parvalbumin (Sigma, St. Louis,MO; 1:1,000), tyrosine hydroxylase (TH; Sigma; 1:1,000),microtubule-associated protein 2 (MAP2; Sigma; 1:1,000),enkephalin (ENK; Chemicon Inc.; 1:100), substance P (SP;Pharmingen, San Diego, CA; 1:400), synaptophysin (SYNP;

mGluR7 IN RAT BASAL GANGLIA 267

Sigma), or calbindin-D28K (Sigma; 1:200); goat polyclonalantibody to calretinin (Chemicon Inc.; 1:2,000); antisynap-tic vesicle protein 2 (SV2; 1:10) obtained from Dr. K.Buckley, Harvard Medical School (Buckley and Kelly,1985); a guinea pig polyclonal antibody to neuronal nitricoxide (nNOS) obtained from T. Dawson, Johns HopkinsUniversity (Huang et al., 1993; 1:500). We also used apolyclonal antibody to mGluR4a (0.5 µg/ml) as a positivecontrol in experiments with tissue from mGluR7 knockoutmice (Bradley et al., 1999).

Tissue preparation for light microscopy. Three maleSprague-Dawley rats (250–300 g, Charles River) weredeeply anesthetized with pentobarbital (100 mg/kg i.p.)and perfused with normal saline, followed by 4% parafor-maldehyde in 0.1 M sodium phosphate buffer, pH 7.4,containing 0.9% NaCl, at room temperature. The brainswere removed immediately, postfixed for 1 hour in thesame fixative at room temperature, and then cryopro-tected overnight in 30% sucrose at 4°C. Mice homozygousfor a deletion of mGluR7 (Masugi et al., 1999) wereprovided by Novartis Pharma AG (Basel, Switzerland).The mouse brains were fixed by immersion in 3% parafor-maldehyde for 6 hours and then cryoprotected as describedabove. The brains were frozen in isopentane cooled withdry ice, and 50 µm sections were cut using a freezingmicrotome. The sections were then either processed imme-diately for immunohistochemistry or stored in 50% glyc-erol in 100 mM Tris, pH 7.5, at 220°C.

Horseradish peroxidase immunohistochemistry.

Single-label horseradish peroxidase-based immunohisto-chemistry was conducted as described previously (Stan-daert et al., 1986). Briefly, sections were washed in 0.1 Msodium phosphate buffer/saline, pH 7.4, incubated in 3%hydrogen peroxide in methanol for 20 minutes followed bywashing in 0.1 M sodium phosphate buffer/saline, pH 7.4,and incubated in blocking solution (3% normal goat serumwith 0.3% Triton X-100 in 0.1 M sodium phosphate buffer/saline, pH 7.4, and 0.1% thimerosal) for 1 hour. Sectionswere then incubated in polyclonal rabbit mGluR7a antise-rum overnight at 4°C. After washing in 0.1 M sodiumphosphate buffer/saline, pH 7.4, sections were sequentiallyincubated for 1 hour in a biotinilated goat anti-rabbitantibody (Jackson Inc., West Grove, PA; 1:400), followed byavidin-biotin complex (ABC Elite, Vector Laboratories,Burlingame, CA). Immunolabeling was detected by incuba-tion in 3,38-diaminobenzidine tetrahydrochloride (0.05%;Sigma) as chromagen for 8–15 minutes.

Fluorescence double label immunohistochemistry.

Dual-label immunohistochemistry was conducted as de-scribed previously (Kosinski et al., 1998; Testa et al.,1998). Sections were washed in 0.1 M sodium phosphatebuffer/saline, pH 7.4, containing 0.9% NaCl, incubated in3% normal goat serum with 0.3% Triton X-100 in sodiumphosphate buffer/saline, pH 7.4, and 0.1% sodium azide for1 hour, and then incubated 48 hours at 4°C in a solutioncontaining the primary antibodies. For experiments thatincluded the calretinin antibody, normal donkey serumwas substituted for normal goat serum. The mGluR7aantibody was visualized using a goat anti-rabbit antise-rum coupled to Cy3 (Jackson; 1:400), and the monoclonalantibodies and the polyclonal antibodies to neuronal nitricoxide (nNOS) and calretinin were visualized with second-ary antisera labeled with Cy2 (1:800) or fluorescein isothio-cyanate (Jackson; 1:50). The sections were then mountedon gelatin-coated slides, dried, and coverslipped using

glycerol containing 100 mM Tris, pH 8.0, and 0.2% p-phenylenediamine (Sigma) to retard fading. Each experi-ment included control tissue processed with omission ofone or both primary antibodies. Preparations were exam-ined using a BioRad Laser Confocal system (MRC 1000)equipped with a Leica DMR microscope and an argon/krypton laser. High-magnification images were obtainedusing an oil-immersion lens with a numerical aperture of1.4. Images were obtained by illuminating the section witha single laser line and collecting the image using anappropriate emission filter: for Cy3, excitation at 568 nmand a 605-nm longpass filter; for Cy2 and fluoresceinisothiocyanate (FITC), excitation at 488 nm and a 522-nmbandpass filter. For each wavelength, four sequentialimages 1,024 3 1,024 pixels in size with an eight-bit pixeldepth were obtained and averaged, using a Kalman filter-ing method to reduce noise. Dual-label images were ob-tained by collecting the separate images sequentially andreconstructing the images in color using Adobe Photoshopsoftware (Adobe, Mountain View, CA). No further process-ing of the images was performed, except for the very-high-magnification images of the striatum, illustrated in Figure7A,B. These were enhanced using an unsharp mask (200%,radius 39 pixels) and a 50% threshold filter to increasecontrast; both images were processed identically.

Electron microscopy. For electron microscopy, rats(n 5 10) were perfused with 200 ml of 3% paraformalde-hyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer,pH 7.6. Brains were postfixed for 1 hour at 4°C and thensectioned at 40 µm using a vibratome (Technical ProductsInternational). Sections were collected in 0.1 M phosphatebuffer, then rinsed several times before being processed asdescribed for light microscopy. Following the treatmentwith DAB, the sections were rinsed several times and thenincubated overnight in 2% glutaraldehyde in 0.1 M phos-phate buffer. After rinsing twice for 10 minutes in phos-phate buffer and then in cacodylate buffer (0.1 M), thesections were postfixed in 1% osmium tetroxide in 0.1 Mcacodylate buffer for 30 minutes. Slices were then rinsedtwice for 10 minutes in 0.1 M cacodylate buffer, followed bya rinse in 0.05 M acetate buffer. Slices were block stainedovernight in aqueous 2% uranyl acetate, followed by arinse in 0.05 M acetate buffer. The tissue was thendehydrated in graded ethanols and finally in propyleneoxide for 5 minutes before infiltration overnight in Epon1:1 propylene oxide. The sections were finally embedded inEpon resin between glass slides and left at 60°C for 2 days.Blocks were dissected, mounted on stubs, and sectionedusing an ultramicrotome (RMC MT5000 or Reichert Ultra-cut). Ultrathin sections were collected on uncoated coppermesh grids for analysis with an electron microscope (H-7500; Hitachi).

Lesion studies

6-Hydroxydopamine lesion of the nigrostriatal path-

way. Unilateral lesions of the nigrostriatal pathwaywere produced in rats (Sprague-Dawley, 250–300 g; CharlesRiver) by injection of 6-hydroxydopamine (6-OHDA; 8 µgin 2.5 µl of 0.9% NaCl containing 0.1% ascorbic acid) intothe left medial forebrain bundle using standard stereo-taxic methods with animals under pentobarbital anesthe-sia (50 mg/kg i.p.) as described previously (Wullner et al.,1994).Animals were pretreated with imipramine, 25 mg/kgi.p., 30 minutes prior to injection of the toxin. Two weeksafter lesioning, the animals were screened for turning

268 C.M. KOSINSKI ET AL.

behavior in response to apomorphine (0.1 mg/kg s.c.); threeanimals exhibiting more than 100 net rotations per hour inthe direction contralateral to the lesion were employed forthe anatomical studies. Transcardiac perfusion was per-formed the following day, as described above.

Decortication. Male Sprague-Dawley rats (250–300 g;Charles River) were anesthetized with sodium pentobarbi-tal (50 mg/kg i.p.) and secured in a small animal stereo-taxic apparatus. A skull flap over the left hemisphere waslifted away and cortical tissue in parietal and frontal areas(2 mm anterior and posterior to bregma, from the midlineto the temporal ridge) was removed by suction as describedelsewhere (Beal et al., 1994). A curved needle was alsopassed lateral to the aspirated area and under the remain-ing cortical gray matter to cut any remaining fibers ofpassage. Transcardiac perfusion was performed 2 weeksafter decortication as described above.

Striatal quinolinic acid lesions. Quinolinic acid (100nmol in 2 µl 0.1 M phosphate buffer, pH 7.4 (Sigma), wasinjected into the left anterior striatum of adult maleSprague-Dawley rats (200–250 g; Charles River, Wilming-ton, MA) as described previously (Orlando et al., 1995).Animals were anesthetized with sodium pentobarbital (50mg/kg i.p.), and surgery was performed in a stereotaxicapparatus using coordinates 2.6 mm lateral, 0.0 mmanterior, and 4.5 mm ventral to the dural surface (Paxinosand Watson, 1986). Injections were made over 3 minuteswith a 10-µl Hamilton syringe fitted with a 26-gaugeblunt-tipped needle. Transcardiac perfusion was per-formed 2 weeks after lesioning as described above. Theextent of the lesions was evaluated on thionin-stainedcoronal sections through the striatum. Three brains inwhich the lesion was restricted to the anterior STR and nodamage was detected in the globus pallidus (GP) werechosen for the study.

Statistical analysis of fluorescence immunohisto-

chemistry on sections from lesioned rat brains.

Double-label fluorescence immunohistochemistry formGluR7a in combination with suitable presynaptic termi-nal markers for the region of interest was performed asdescribed above on a total of six coronal sections throughthe regions of interest, two from each lesioned animal.Pairs of images were obtained from the lesioned side andthe contralateral side of the same section using identicalimaging parameters. Estimates of fluorescence intensitywere performed by measuring mean fluorescent intensityon a 256-step scale in 10 nonoverlapping fields (50 3 50pixels each) on one section of each brain using AdobePhotoshop software. Fluorescence intensity of normal sidevs. lesion side was compared using a repeated-measuresanalysis of variance with a significance level of 1% on acommercial statistics software package (SuperANOVA,Abacus Concepts Inc.).

RESULTS

In situ hybridization

In situ hybridization with the mGluR7 cRNA probeproduced a pattern of hybridization signal identical to thedistribution found in previous studies (Fig. 1A; Kinzie etal., 1995; Ohishi et al., 1995a). In cerebral cortex, thelabeling was most intense in the occipital cortex andslightly less intense in frontal cortical areas. In striatum,the signal was also intense and evenly distributed through-out the region. No dorsoventral gradient or patch/matrix

compartmentalization within the striatum was seen (Fig.1A). There was a moderately intense signal in the STN;other basal ganglia regions appeared to be only weaklylabeled if at all on the film autoradiograms.

Quantitative analysis of optical densities from autoradio-graphic films revealed that the signal was most intense inhippocampal dentate gyrus and CA3 (Fig. 2). Moderatelyintense labeling was detected in the hippocampal CA1region, cerebral cortex, striatum, and nucleus accumbens.Slightly less intense signal was found in the cerebellarPurkinje cell layer, thalamic reticular nucleus, and tha-lamic ventrolateral nucleus. In subthalamic nucleus andcerebellar granular layer, a low-intensity signal was de-tected. The signals in globus pallidus, ventral pallidum,entopeduncular nucleus, substantia nigra pars reticulata,substantia nigra pars compacta, cerebellar molecular layer,and corpus callosum were not significantly different frombackground.

Examination of the cellular distribution of hybridizationsignal on emulsion autoradiograms revealed several addi-tional features. The neuronal labeling in the striatum wasintense and was found in the vast majority of the neuronspresent in this structure (Fig. 1B). Although no signal wasdetected in the GP or SNr on film autoradiograms, micro-scopic examination showed that some cells in each of thesestructures did have moderately strong labeling for mGluR7mRNA. In the GP, the labeled cells were dispersed through-out the structure (Fig. 1C). In the SN, labeled cells werefound scattered in the SNr (Fig. 1D), but no labeling waspresent in the SNc. In the STN, there was moderatelyintense labeling of all the cells present (not illustrated).

Antibody specificity

In western blots using proteins from rat or mouse brain,the mGluR7a antibody recognized a protein with a molecu-lar weight of approximately 100 kd, as well as a largespecies of 200 kd, most likely representing a dimer ofmGluR7a as observed with other receptors in this family(Fig. 3; Testa et al., 1998). No evidence for cross-reactivitywith the alternative carboxy terminus of mGluR7b wasobserved, and no signal was detected in tissues from micehomozygous for a deletion of mGluR7 (Masugi et al., 1999).

To assess the specificity of the immunohistochemicalstaining, we also examined staining in brains from normalmice and mice homozygous for a deletion of mGluR7(Masumi et al., 1999). In normal mice, the distribution ofmGluR7a staining was similar to that observed in ratbrain. In the mGluR7 knockout mice, only faint residualcytoplasmic staining was observed; in adjacent sectionsfrom the same animal, robust staining for the closelyrelated protein mGluR4a was present (Fig. 4; Bradley etal., 1999).

Immunohistochemistry: Light microscopy

Using the mGluR7a antibody for conventional single-label immunohistochemistry, we observed a distinct stain-ing pattern throughout the rat brain (Fig. 1E). At lowpower, moderate to intense staining was observed in somebasal ganglia regions, including STR, GP, and SNr (Fig.1E). In STR no compartmentalization of mGluR7a immu-noreactivity (-ir) in patch/matrix and no dorsoventralgradient in staining intensity were detected. Little stain-ing was found in the STN or SNc (Fig. 1E). The stainingpattern in the more intensely stained basal ganglia re-


Figure 1

gions (STR, GP, and SNr) was studied in detail employingfluorescence confocal laser microscopy.

Localization of mGluR7a in the striatum:Confocal microscopy

Using confocal microscopy, we found in the striatum afine granular staining of the entire neuropil of moderateintensity (Fig. 5A). Most cell bodies contained mGluR7a-ir,and the borders of these cells were difficult to delineatefrom the neuropil staining, differing only in that thecellular staining was slightly less intense and more diffusein character (Fig. 5A). Occasionally, we observed voids inthe staining pattern, suggesting a small number of cellslacking mGluR7a-ir (Fig. 5A).

Double-label experiments with markers for subpopula-tions of striatal neurons were performed. Neurons stainingfor calbindin, a marker for projection neurons in thematrix compartment of STR (Gerfen et al., 1985), demon-strated diffuse cytoplasmic mGluR7a-ir, which was onlyslightly less intense than the surrounding neuropil (Fig.6A,D). Large cholinergic interneurons labeled for ChAT-ir

showed almost no mGluR7a-ir (Fig. 6B,E). Medium-sizedinterneurons, which were characterized either by parval-bumin-ir or calretinin-ir, demonstrated only very low or nomGluR7a-ir (Fig. 6C,F and I,L). Interneurons containingnNOS-ir were a less homogeneous population with regardto their mGluR7a-ir. Whereas about one-third of nNOS-irneurons were not mGluR7a immunoreactive (Fig. 6G,J),the other two-thirds showed diffuse cytoplasmic mGluR7-ir(Fig. 6H,K) similar in intensity to that of the calbindin-irprojection neurons (Fig. 6A,D).

To evaluate the localization of mGluR7a-ir in the striatalneuropil, sections were double labeled with either MAP2(Fig. 7A), a marker for dendrites (Huber and Matus, 1984),or SV2 (Fig. 7B), a marker of presynaptic terminals(Buckley and Kelly, 1985). Both antibodies, MAP2 andSV2, labeled the striatal neuropil very densely. Doublelabeling with mGluR7a antiserum revealed extensive over-lap between mGluR7a-ir and SV2-ir, with numerous ex-amples of puncta stained by both antibodies (Fig. 7B). Incontrast, only a minority of the striatal puncta immunore-active for mGluR7a colocalized with dendrites labeled byMAP2 (Fig. 7A).

Localization of mGluR7a in the globuspallidus: Confocal microscopy

The staining pattern observed with the mGluR7a anti-body in GP was different from that in STR. In the GP, therewere intensely labeled granules present, which in generalwere larger than the ones seen in the striatal neuropil.Most of these granules were in linear arrays and often

Fig. 2. Regional expression pattern of mGluR7 mRNA. Barsindicate mean absolute optical density (6SD) measured on filmautoradiograms in each area. ACC, nucleus accumbens; CA1, hippo-campal CA1 region; CA3, hippocampal CA3 region; CC, corpus callo-sum; CTX, cerebral cortex; DGY, hippocampal dentate gyrus; EPN,entopeduncular nucleus; GP, globus pallidus; GRA, cerebellar cortexgranular layer; MOL, cerebellar cortex molecular layer; PKC, cerebel-lar cortex Purkinje cell layer; SNC, substantia nigra pars compacta;SNR, substantia nigra pars reticulata; STN, subthalamic nucleus;STR, striatum; TRN, thalamic reticular nucleus; TVL, thalamicventrolateral nucleus. Bars shown in white were not significantlydifferent from background (P , 0.01).

Fig. 3. Western blot illustrating specificity of the mGluR7a anti-body. The first two lanes were loaded with proteins from transfectedsf9 cells expressing mGluR7a (m7a) or mGluR7b (m7b). The next fourlanes contain proteins from piriform cortex (Pir Ctx) or hippocampus(Hip) of wild-type (1/1) or mGluR7 knockout mice (2/2). In the cellstransfected with mGluR7a and both regions of the wild-type mousebrain, there is a band at about 100 kd corresponding to the size ofmGluR7a as well as a larger band most likely representing dimers ofmGluR7a. No immunoreactivity is observed in mGluR7b-expressingcells or in mGluR7 knockout mice.

Fig. 1. In situ hybridization for mGluR7 mRNA and immunohisto-chemistry with an mGluR7a specific antibody in rat brain. A: Sagittalsection of rat brain labeled with an RNA probe to a region of themGluR7 message common to all of the described isoforms. There isstrong signal in the striatum, only a modest level of signal in the STN,and no discernible labeling in the GP or SNr. B–D: Darkfield photomi-crographs showing cellular labeling for mGluR7 mRNA in striatum(B), GP (C), and SNr (D). Note that there is intense label in striatalneurons; in the GP and SNr, there is much less label overall, althougha modest number of labeled cells are present. Scale bar 5 300 µm inB–D. E: Immunohistochemical staining for mGluR7a. Note the rela-tively intense staining in striatum, GP, and SNr. ACC, nucleusaccumbens; CTX, cerebral cortex; GP, globus pallidus; SNC, substan-tia nigra pars compacta; SNr, substantia nigra pars reticulata; STN,subthalamic nucleus; STR, striatum.


appeared to demarcate fibers on two sides (Fig. 5B). Onlyrarely was mGluR7a-ir associated with cell somata in GP,and in these cases mGluR7a-ir was not found in thecytoplasm but rather outlined the outer surface of neuronsand their dendrites (Fig. 7D). In the GP, double-labelexperiments with the dendritic marker MAP2 showed thatmGluR7a-ir was associated with dendrites (Fig. 7C). Thestaining for mGluR7a was not present on all dendrites but,instead, densely invested a small subset of the fiberspresent (Fig. 7C). At very high magnification, most of theintense mGluR7a-ir staining appeared to be in closecontact with the dendritic surfaces, but not within themargins of the dendrites (Fig. 7E).

The antibody to the presynaptic terminal marker synap-tophysin produced a somewhat similar granular and ‘‘bi-laminar’’ labeling pattern along dendrites in GP (Fig.7D,F). Under very high magnification, much of themGluR7a-ir appeared to colocalized with synaptophysin-iralong the surface of underlying dendrites (Fig. 7F). Partialoverlap with synaptophysin-ir was also seen along cellsomata that were outlined by mGluR7a-ir (Fig. 7D).

Localization of mGluR7a in the substantianigra: Confocal microscopy

The staining pattern observed with the mGluR7a anti-body in SN was less homogeneous than that in STR and

Fig. 4. Immunohistochemical staining for mGluR7a (A) andmGluR4a (B) in adjacent sections from a mouse homozygous for adeletion of mGluR7. In the knockout mouse, only faint residualstaining for mGluR7a is observed in the globus pallidus (GP) and no

detectable staining is found in the striatum (STR). In contrast, thenormal, robust staining of fibers within the globus pallidus (GP) formGluR4a is preserved. Scale bar 5 50 µm.

Fig. 5. Confocal laser microscopy of mGluR7a-ir in striatum (STR;A), globus pallidus (GP; B), and substantia nigra pars reticulata (SNr;C). Arrows in A point to cells showing some diffuse mGluR7a-ir, lessintense than the surrounding neuropil staining (arrowheads); a fewcells in STR show no mGluR7a-ir (asterisk). Arrowheads in B and in C

indicate mGluR7a-ir that demarcates fibers in GP (B) and SNr (C);cells in SNr (C) show no mGluR7a-ir (asterisks). Identical experimen-tal procedure with omission of primary antibody abolished stainingalmost completely. Scale bar 5 20 µm.


Fig. 6. mGluR7a-ir in different subpopulations of striatal neurons.Paired images are shown from preparations double labeled formGluR7a (D–F,J–L) in combination with antisera to calbindin (A),choline acetyltransferase (ChAT; B), calretinin (C), nitric oxide syn-thase (nNOS; G,H), or parvalbumin (I). Arrows indicate the borders of

the neurons in corresponding images. Calbindin-ir and some of thenNOS-ir neurons showed cytoplasmic mGluR7a-ir (D,K), whereasneurons stained by ChAT, calretinin, or parvalbumin and others of thenNOS-positive neurons showed almost no mGluR7a-ir (E,F,J,L).Scale bar 5 5 µm.

GP. In SNc only rare mGluR7a-ir granules could be seenin the neuropil (Fig. 8). Dopaminergic neurons and den-drites in SNc, characterized by tyrosine hydroxylase-ir, didnot show any mGluR7a-ir. In contrast, mGluR7a-ir in SNrwas moderately intense (Fig. 5C). Cell somata in SNr,however, showed no mGluR7a-ir (Fig. 5C). Fine puncta ofstaining could be seen in the neuropil, similar to that seenin the STR. Additionally, intense staining could be de-tected along some fibers through SNr (Fig. 5C). This fiberstaining resembled the mGluR7a-ir seen in GP. Doublelabeling with the tyrosine hydroxylase (TH) antiserumdemonstrated no association between mGluR7a-ir and thefibers from dopaminergic neurons that traversed the SNr(Fig. 7G). Double labeling with the presynaptic terminalmarker synaptophysin demonstrated colocalization withmGluR7a-ir, presumably reflecting localization along thesurface of underlying dendrites (Fig. 7H).

Effect of lesions of the nigrostriatal pathwayon mGluR7a

To evaluate the extent of loss of dopaminergic projec-tions to the striatum, sections were stained with antiserato TH. Lesion of the nigrostriatal pathway yielded almostcomplete loss of TH-ir fibers and terminals in STR on thelesioned side, whereas TH-ir on the side contralateral tothe lesion was intense (Fig. 9B,D). The mean intensity ofthe TH immunofluoresence was reduced to 23.4% (P ,0.001) of that found on the contralateral side (see Fig. 11).However, the intensity of the mGluR7a-ir on the intact andlesioned sides did not differ (Figs. 9A,C, 11).

Effect of decortication on mGluR7a

The effect of removal of the cerebral cortex on corticostria-tal terminals and striatal mGluR7a were evaluated bydouble labeling the sections with mGluR7a and the presyn-aptic terminal marker SV2 (Buckley and Kelly, 1985).Decortication reduced the mean intensity of the SV2staining to 50.2% of that found on the contralateral side(P , 0.001; Figs. 9F,H, 11). The mGluR7a-ir staining in thestriatal neuropil was also reduced in intensity after decor-tication, to 66.2% of that present on the contralateral side(P , 0.001; Figs. 9E,G, 11).

Effect of quinolinic acid lesions on mGluR7a

After striatal injection of quinolinic acid, the loss ofpresynaptic terminals in GP and SNr was evaluated bystaining with antisera to enkephalin or substance P. Thesepeptides are synthesized by essentially separate popula-tions of striatal projection neurons and found withinaxonal terminals in GP and SNr, respectively (Gerfen andYoung, 1988; Reiner and Anderson, 1990). The striatalprojections are organized topographically (Gerfen andYoung, 1988). Because our quinolinic acid lesions wererestricted to the anterior part of the STR, we expected thatthe loss of projections to the GP and SNr would besegmental rather than complete. We used the staining forenkephalin-ir in GP and substance P-ir in SNr to identifyregions of these structures affected by our lesions andobtained images of mGluR7a-ir from these affected areas.

In these affected areas of the GP, enkephalin-ir wasreduced to 20.5% of the immunofluorescent intensity pres-ent on the contralateral side (P , 0.001; Figs. 10B,D, 11).Measurement of mGluR7a-ir in the same areas also re-vealed a significant reduction of the fluorescence intensity,to 54.8% of that present on the contralateral side (P ,

0.001; Figs. 10A,C, 11). In SNr a decrease of substance P-irto 27.8% that of the contralateral side was observed,similar in magnitude to the change in GP enkephalinstaining (Figs. 10F,H, 11). In these areas of SNr,mGluR7a-ir was also significantly reduced, to 61.5% ofthat present on the contralateral side (Figs. 10E,G, 11).

Localization of mGluR7a: Electronmicroscopy

Immuno-EM analysis of mGluR7a immunoreactivity instriatum revealed that labeling was found in both pre- andpostsynaptic elements. Labeled mGluR7a axons and axonterminals were quite frequent in the striatum (Fig. 12A,B).Reaction product was diffusely located, surrounding synap-tic vesicles and mitochondria. In some axon terminals,reaction product was especially dense near the synapticactive zone. In the striatum, most of the mGluR7a immuno-reactive terminals formed asymmetrical synapses on den-dritic spines (Fig. 12A) or dendritic shafts (Fig. 12B). Instriatum, evidence of postsynaptic localization of mGluR7awas also observed, in the form of labeled spines contactedby unlabeled axon terminals (Fig. 12C) and dendriticshafts showing diffuse localization of the reaction product(Fig. 12D).

In the GP, labeling was found predominantly in associa-tion with axon terminals, forming synapses on dendriticshafts (Fig. 13A,B) or, less frequently, dendritic spines(Fig. 13C). Evidence of postsynaptic labeling in the GP wasalso observed, in the form of occasional dendrites exhibit-ing diffuse reaction product (Fig. 13D).

DISCUSSION

The localization of mGluR7 mRNA, the distribution ofmGluR7a immunoreactivity, and the effects of lesions leadus to believe that mGluR7a has important functions asboth a pre- and a postsynaptic modulator of neural trans-mission in the basal ganglia. In particular, our datasuggest that much of the mGluR7a protein found in thestriatum is localized to the terminals of glutamatergiccorticostriatal afferents. MGluR7 mRNA is also expressedby the projection neurons within the striatum. Some ofthis protein is targeted to the dendritic spines of striatalcells, but a large portion is localized to the GP and SNr, inthe distal terminals of the GABAergic striatopallidal and

Fig. 7. High-magnification confocal laser microscopy images show-ing mGluR7a-ir (in red) in striatum (STR; A,B), globus pallidus (GP;C–F), and substantia nigra pars reticulata (SNr; G,H) in preparationsdouble labeled with different dendritic (A,C,E,G) and presynapticterminal markers (B,D,F,H; in green). Overlap of the two colors isillustrated in yellow. Most of the mGluR7a-ir in STR does notcolocalize with the dendritic marker MAP2 (arrows in A), whereasextensive overlap (in yellow) of mGluR7a-ir with the presynapticterminal marker SV2 is seen (arrows in B); asterisks indicate cellbodies without SV2-ir in B. In GP intense mGluR7a-ir is found alongsome but not all dendrites (arrows in C). Intense mGluR7a stainingwas also found along the surface of cell bodies in GP, often colocalizing(arrows in D) with the presynaptic terminal marker synaptophysin(SYNP). Higher magnification demonstrates that most mGluR7a-ir isnot present within the dendrites (arrows in E) but does colocalize withSYNP along dendritic surfaces (arrows in F). In SNr, mGluR7a stainedsome fibers intensely (arrows in G), but these were distinct from thetyrosine hydroxylase-ir fibers traversing the region. At higher magnifi-cation mGluR7a staining colocalized with SYNP along dendriticsurfaces (arrows in H). Scale bars 5 2 µm.


Figure 7


striatonigral pathways. In contrast, we found no evidencefor the presence of mGluR7a in the perikarya or terminalsof the dopaminergic nigrostriatal projection.

Localization of mGluR7 mRNA andmGluR7a protein

Two isoforms of mGluR7, produced by alternative splic-ing, are known to be present in human, mouse, and ratbrain (Flor et al., 1997; Shigemoto et al., 1997). The in situhybridization probe we employed was targeted to theregion of the mRNA encoding the amino terminal region ofmGluR7 and would be expected to recognize both isoforms.The distribution we observed is quite similar to thatobtained by others using different probes to conservedregions of mGluR7 (Kinzie et al., 1995; Ohishi et al.,1995a). The antibody we used was raised to a peptidecorresponding to the carboxy terminal 19 amino acids ofmGluR7a, a sequence not present in mGluR7b (Bradley etal., 1996; Flor et al., 1997), and we found that this antibodyrecognized mGluR7a, but not mGluR7b, expressed intransfected cell lines. Kinoshita et al. (1998) have recentlyreported a detailed comparison of the localizations ofmGluR7a and mGluR7b in rat brain, noting that theexpression of mGluR7b is more restricted, but in the GPand other regions the two forms are colocalized.

mGluR7 in the neostriatum

The intensity of the striatal in situ hybridization signalfor mGluR7 is comparatively strong, similar to that ob-served in the neocortex. We found that most of the striatalneurons present expressed mGluR7 mRNA. These resultsare consistent with data obtained recently using a dual-label in situ hybridization method, which revealed selec-tive concentration of the mGluR7 mRNA in striatal projec-tion neurons, and relatively low mRNA abundance in

interneurons (Kerner et al., 1997b). Examination withfluorescence confocal microscopy revealed that in STRmost mGluR7a-ir is found in the neuropil and consists ofinnumerable fine labeled puncta. In general the labeling ofneuronal perikarya was sparse. Most striatal neuronsexhibited a low-intensity, diffuse staining pattern, whereasstriatal neurons identified as cholinergic and calretinin- orparvalbumin-containing, and the majority of the nNOS-containing neurons, had no detectable perikaryal stainingfor mGluR7. These findings suggest that striatal projec-tion neurons synthesize mGluR7, but they do not accumu-late the protein within their perikarya. This is in contrastto the subunits of the AMPA and NMDA glutamate recep-tors, which are abundant in the cytoplasm of striatalneurons (Tallaksen-Greene and Albin, 1994; Chen andReiner, 1996; Chen et al., 1996; Weiss et al., 1998).

Several of our observations suggest that the origin of asubstantial portion of the mGluR7a-ir found in the striatalneuropil is the presynaptic terminals of the corticostriatalafferents. By confocal microscopy, most of the mGluR7a-irpresent did not colocalize with the dendritic marker MAP2(Fig. 7A). In contrast, there was extensive overlap betweenmGluR7a-ir and the presynaptic marker SV2 (Fig 7B).After decortication, there was a substantial loss of striatalmGluR7a-ir; in contrast, postsynaptic receptors such asmGluR1a are not reduced in intensity by such a lesion(Testa et al., 1998). Electron microscopic examinationconfirmed that a large fraction of the striatal staining wasassociated with axon terminals forming asymmetric, pre-sumably excitatory, synapses (Fig. 12; Kinoshita et al.,1998), although mGlur7 was also found at postsynapticsites. A preferential presynaptic localization of mGluR7a-irhas been demonstrated previously in the hippocampus(Bradley et al., 1996; Shigemoto et al., 1997) and in dorsalhorn afferents in the spinal cord (Li et al., 1997) using

Fig. 8. mGluR7a-ir in substantia nigra pars compacta (SNc).Corresponding images are shown from preparations that were doublelabeled for mGluR7a (A) in combination with antiserum to tyrosinehydroxylase (B) to label dopaminergic neurons and fibers. Arrows

indicate borders of the dopaminergic neurons in the correspondingimages. Tyrosine hydroxylase-positive neurons in SNc were not associ-ated with mGluR7a-ir. Scale bar 5 20 µm.


Fig. 9. Effect of 6-OHDA lesion (A–D) and decortication (E–H) onmGluR7a-ir in striatum. Coronal sections from 6-OHDA-treated ani-mals were double labeled with tyrosine hydroxylase (TH), and imagesof identical fields for mGluR7a-ir (A,C) and TH-ir (B,D) were obtainedfrom the lesioned side (C,D) and the contralateral side (A,B). Sectionsfrom decorticated animals were double labeled with the presynaptic

terminal marker SV2 (F,H) and mGluR7a (E,G) antiserum and imagesobtained from the decorticated (G,H) and intact sides (E,F). 6-OHDAlesion abolished TH-ir almost completely but did not affect mGluR7a-ir. Decortication decreased SV2-ir and also resulted in loss ofmGluR7a-ir in STR. Scale bar 5 20 µm.

Fig. 10. Effect of striatal quinolinic acid lesion on mGluR7a-ir inglobus pallidus (GP) and substantia nigra pars reticulata (SNr).Coronal sections through GP and SNr from lesioned animals weredouble labeled for enkephalin (B,D) or substance P (F,H), respectively,in combination with the mGluR7a antiserum (A,C,E,G). Images were

taken from the lesioned side (C,D,G,H) and the contralateral side(A,B,E,F) of the same section. Images in each row were obtained fromidentical fields. Striatal quinolinic acid lesion resulted in both a loss ofenkephalin-ir and also mGluR7a-ir in GP, whereas in the SNr bothsubstance P-ir and mGluR7a-ir were reduced. Scale bar 5 20 µm.

immunogold methods. Interestingly, in the hippocampus,mGluR7a-ir is found on terminals at the site of thesynaptic grid, whereas mGluR2/3-ir is found in pretermi-nal axons but not at the site of presynaptic membranespecializations (Shigemoto et al., 1997). Because our re-sults using immunoperoxidase do not allow us to distin-guish between these two potential localizations, furtherstudy using higher resolution methods will be important.

If mGluR7a is indeed present on corticostriatal affer-ents, it may function as a glutamatergic autoreceptor.Calabresi et al. (1993, 1996) and more recently Pisani et al.(1997) have shown that group III mGluR agonists exert apresynaptic inhibitory action on excitatory glutamatergictransmission at corticostriatal synapses in slice prepara-tions, although some earlier studies did not describe suchan effect (Lovinger and McCool, 1995). Pisiani et al. (1997)also found a strong additive inhibition when both Group IIand Group III agonists were present. In earlier studies, weobtained evidence that supports the presence of Group IIreceptors (mGluR2 and mGluR3) on striatal afferents fromthe cortex (Testa et al., 1994, 1998). Taking these findings

together, it seems likely that glutamate release fromcorticostriatal terminals is under the inhibitory control ofat least three different receptor groups: presynaptic dopa-mine D2 receptors (for review see Calabresi et al., 1996),glutamatergic mGluR2/3 autoreceptors (Calabresi et al.,1993, 1996; Lovinger and McCool, 1995; Testa et al., 1998),and glutamatergic mGluR7 autoreceptors. We also identi-fied mGluR7a-ir at postsynaptic sites in the striatum,associated with the spines of projection neurons. Most ofthese spines formed asymmetric, presumably excitatory,synapses with unlabeled terminals. It is possible theseaxon terminals are also of cortical origin but lack mGluR7a;alternatively, they may represent a component of theexcitatory input to striatum that arises from the thalamus(Parent and Hazrati, 1995).

mGluR7a on striatopallidal and striatonigralterminals

The GP and the SNr of the rat both exhibited only lowlevels of mGluR7 mRNA expression. Analyzed on a re-

Fig. 11. Quantitative analysis of mGluR7a-ir in striatum, globuspallidus, and substantia nigra pars reticulata after 6-OHDA lesions ofthe nigrostriatal pathway, decortication, or striatal quinolinic acidlesions. For each lesion type, sections from three different animalswere studied. Fluorescence intensity for mGluR7a and tyrosine hy-droxylase, enkephalin, substance P, or SV2 was measured in identical

fields. Graphs show mean fluorescence intensity (6SD) normalized tointensity on images taken from the contralateral side with identicalparameters. Asterisks indicate significant differences (P , 0.01) fromthe control sides using a one-way ANOVA with repeated-measuredesign.


gional basis by film autoradiograms, the level of signal wasnot different from background (Fig. 2), although cellularstudies revealed a modest level of mGluR7 mRNA withinsome neurons in each structure (Fig. 1C,D). Despite thepaucity of mRNA, both of these structures contained fairlystrong mGluR7a-ir. The explanation for this mismatch isthat much of the mGluR7a present in the GP and SNprarises from neurons outside these structures and is local-ized to axon terminals. Of interest, in both GP and SNr themGluR7a-ir was not uniformly distributed but rather wasfound densely investing a small number of the dendritespresent. The basis for this selective innervation and the

nature of the dendrites targeted is at present unknown.For both regions we observed colocalization of mGluR7a-irwith presynaptic markers by confocal microscopy and forthe GP found that the protein was present in axonterminals synapsing with pallidal dendrites. Intrastriatalinjection of quinolinic acid into STR, which destroysstriatal projection neurons preferentially (Beal et al.,1991), markedly reduced mGluR7a-ir in GP and SNr (Figs.10, 11). Collectively, these data support a prominentlocalization of mGluR7a on striatonigral and striatopalli-dal afferents. Using an antibody to the closely relatedGroup III receptor mGluR4a, Bradley et al. (1999) ob-

Fig. 12. Electron micrographs illustrating pre- and postsynapticlocalizations of mGluR7a-ir in the striatum. A: Example of a denselylabeled axon terminal (a*) forming an asymmetric synapse with anunlabeled dendritic spine (s). B: Immunoreactive axon terminal with a

synapse on the shaft of an unlabeled dendrite (d). C: Strongly labeleddendritic spine (s*) receiving an asymmetrical synapse from anunlabelled axon terminal (a). D: Dendrite (d*) with diffuse cytoplasmiclabeling. Scale bars 5 200 nm.


served a very similar localization of this protein on striato-pallidal, but not striatonigral, projections. At present it isnot known whether those striatopallidal afferents thatcontain mGluR7a immunoreactivity also contain mGluR4aor whether these are distinct populations.

If mGluR7a serves as a presynaptic modulator of stria-tal efferents, its function must be somewhat different fromthe autoreceptor role it appears to play on corticostriatalterminals. Striatal projection neurons use GABA in combi-nation with either enkephalin or substance P as neuro-transmitters but appear not to have a significant glutama-tergic component (Graybiel, 1990). Evidence for an

inhibitory role of mGluRs, especially presynaptic group IIand group III mGluRs, in the control of GABA release hasbeen obtained previously from electrophysiological studiesin the hippocampus (Miller, 1991; Desai et al., 1994;Gereau and Conn, 1995), the nucleus of the tractussolitarius (Glaum and Miller, 1992), and the striatum(Calabresi et al., 1992). The major glutamatergic input toGP and SNr comes from the subthalamic nucleus (Ricardo,1980; Kita and Kitai, 1987; Albin et al., 1989; Hazrati andParent, 1992). Terminals formed by these projectionsclosely interdigitate with the terminals of striatal originalong the surfaces of pallidal dendrites (DiFiglia et al.,

Fig. 13. Electron micrographs illustrating localization of mGluR7a-irin the globus pallidus. A: Labeled axon terminal (a*) forming asymmetric synapse with a pallidal dendrite (d). Additional unlabeledaxon terminals are seen making synapses with the dendrite atadjacent sites. B: Dendrite cut in cross section (d). At least four axon

terminals (a) are seen forming symmetric synapses with the dendrite,but only one terminal is strongly positive for mGluR7a (a*). C: Denselylabeled axon terminal contacting a dendritic spine (s). D: A dendritewith diffuse cytoplasmic staining for mGluR7a forms synapses withseveral unlabeled axon terminals. Scale bars 5 500 nm.


1982). It is possible that glutamate input from the STNacts not only directly to excite the GP and SNr targetneurons but also by means of mGluR7a to reduce inhibi-tory GABA release. This combined action would increasethe overall synaptic afferent transmission from STN to GPand SNr and might be of considerable importance underpathological conditions such as Parkinson’s disease, inwhich the subthalamic nucleus is overactive (Bergman etal., 1990).

SUMMARY

The present data together with previous investigationssuggest that mGluRs modulate basal ganglia function atmultiple sites (Fig. 14; Testa et al., 1994, 1995, 1998;Kerner et al., 1997a; Kosinski et al., 1998; Bradley et al.,1999). Both mGluR2/3 and mGluR7a appear to be associ-ated with presynaptic terminals of corticostriatal projec-tions. The Group 1 receptor proteins (mGluR1, mGluR5)appear to be localized largely to postsynaptic sites, includ-ing the spines and/or dendrites of striatal medium spinyneurons. MGluR4a is found on the terminals of striatopal-lidal projections, whereas mGluR7a is found on both the

pallidal and nigral projections of striatal cells. Pallidal andnigral neurons also express mGluR1 isoforms, and theseproteins are found clustered within and along their den-drites, whereas dopaminergic neurons express predomi-nantly the mGluR1d isoform.

ACKNOWLEDGMENTS

This work was supported by USPHS grants NS31579and NS34361 (D.G.S.) and NS31373, NS34876, andNS98011 (P.J.C.); by grants from Wyeth Ayerst Research(P.J.C.), NARSAD (P.J.C.), and the U.S. Army (A.I.L.); byDFG grants La 701–2 and SFB 505 (G.B.L.) and DFGgrant Ko 1696/1–2 (C.M.K.). D.G.S. is the recipient of aCotzias Fellowship from the American Parkinson’s Dis-ease Association.

LITERATURE CITED

Albin RL, Young AB, Penney JB. 1989. The functional anatomy of basalganglia disorders. Trends Neurosci 12:366–375.

Fig. 14. Summary diagram of our results with respect to mGluR7ain context with previous findings. Illustrated in schematic form are thetwo principal sources of afferent projections to the striatum (STR), thecerebral cortex (CTX), which employs glutamate (GLU) as a transmit-ter, and the dopaminergic (DA) projection from the substantia nigrapars compacta (SNc). The striatal projection neurons employ GABA asa neurotransmitter and project to the globus pallidus (GP) andsubstantia nigra pars reticulata (SNr). Subthalamic efferents provideexcitatory glutamatergic input to the GP and SNr. In this schematic,the labels 1–7 refer to the mRNAs encoding the metabotropic gluta-mate receptors (mGluRs); the plus symbols (1, 11, and 111)indicate the relative intensity of expression as determined by in situhybridization. Cortical projection neurons exhibit prominent expres-sion of mGluR2, mGluR3, mGluR5, and mGluR7 mRNA and lessintense expression of mGluR1 mRNA. Our data suggest that there is

mGluR2/3-ir and mGluR7a-ir present on the terminals or preterminalaxons of the corticostriatal fibers. These may serve as autoreceptors inthis glutamatergic pathway. Immunoreactivity for mGluR1 (mostlikely the mGluR1d isoform) is found on the dendrites of dopamineneurons in the SNc, but appears not to be present on the axonalprocesses of these cells. Intense expression of mGluR7 mRNA wasfound in striatal projection neurons, and the mGluR7a protein wasdetected in association with striatal terminals in GP and SNr. At thissite, mGluR7 may regulate GABA release, perhaps in response toglutamatergic inputs from the STN. MGluR4a is also found onstriatopallidal but not striatonigral terminals. Expression of mGluR1mRNA is found in both the STR and the GP/SNr; this protein is likelyto be a postsynaptic receptor on dendrites of neurons in each of theseregions.


Beal MF, Ferrante RJ, Swartz KJ, Kowall NW. 1991. Chronic quinolinicacid lesions in rats closely resemble Huntington’s disease. J Neurosci11:1649–1659.

Beal MF, Finn SF, Brouillet E. 1994. Evidence for the involvement ofmetabotropic receptors in striatal excitotoxin lesions in vivo. Neurode-generation 2:81–91.

Bergman H, Wichmann T, DeLong M. 1990. Reversal of experimentalparkinsonism by lesions of the subthalamic nucleus. Science 249:1436–1438.

Bradley SR, Levey AI, Hersch SM, Conn PJ. 1996. Immunocytochemicallocalization of group III metabotropic glutamate receptors in thehippocampus with subtype-specific antibodies. J Neurosci 16:2044–2056.

Bradley SR, Standaert DG, Rhodes KJ, Rees HD, Testa CM, Levey AI, ConnPJ. 1999. Immunohistochemical localization of subtype 4a metabo-tropic glutamate receptors in the rat and mouse basal ganglia. J CompNeurol 407:33–46.

Brandstatter JH, Koulen P, Kuhn R, Vanderputten H, Wassle H. 1996.Compartmental localization of a metabotropic glutamate receptor(mGluR7)—two different active sites at a retinal synapse. J Neurosci16:4749–4756.

Buckley K, Kelly RB. 1985. Identification of a transmembrane glycoproteinspecific for secretory vesicles of neural and endocrine cells. J Cell Biol100:1284–1294.

Bushell TJ, Jane DE, Tse HW, Watkins JC, Garthwaite J, Collingridge GR.1996. Pharmacological antagonism of the actions of group II and IIImGluR agonists in the lateral perforant path of rat hippocampal slices.Br J Pharmacol 117:1457–1462.

Calabresi P, Mercuri NB, Bernardi G. 1992. Activation of quisqualatemetabotropic receptors reduces glutamate and GABA-mediated synap-tic potentials in the rat striatum. Neurosci Lett 139:41–44.

Calabresi P, Pisani A, Mercuri NB, Bernardi G. 1993. Heterogeneity ofmetabotropic glutamate receptors in the striatum: electrophysiologicalevidence. Eur J Neurosci 5:1370–1377.

Calabresi P, Pisani A, Mercuri NB, Bernardi G. 1996. The corticostriatalprojection: from synaptic plasticity to dysfunctions of the basal ganglia.Trends Neurosci 19:19–24.

Chen Q, Reiner A. 1996. Cellular distribution of the NMDA receptorNR2A/2B subunits in the rat striatum. Brain Res 743:346–352.

Chen Q, Veenman CL, Reiner A. 1996. Cellular expression of ionotropicglutamate receptor subunits on specific striatal neuron types and itsimplications for striatal vulnerability in glutamate receptor-mediatedexcitotoxicity. Neuroscience 73:715–731.

Conn PJ, Pin JP. 1997. Pharmacology and functions of metabotropicglutamate receptors. Annu Rev Pharmacol Toxicol 37:205–237.

Desai MA, McBain CJ, Kauer JA, Conn PJ. 1994. Metabotropic glutamatereceptor-induced disinhibition is mediated by reduced transmission atexcitatory synapses onto interneurons and inhibitory synapses ontopyramidal cells. Neurosci Lett 181:78–82.

DiFiglia M, Pasik P, Pasik T. 1982. A golgi and ultrastructural study of themonkey globus pallidus. J Comp Neurol 212:53–73.

Duvoisin RM, Zhang C, Ramonell K. 1995. A novel metabotropic glutamatereceptor expressed in the retina and olfactory bulb. J Neurosci 15:3075–3083.

Flor PJ, Van der Putten H, Ruegg D, Lukic S, Leonhardt T, Bence M, SansigG, Knopfel T, Kuhn R. 1997. A novel splice variant of a metabotropicglutamate receptor, human mGluR7b. Neuropharmacology 36:153–159.

Gereau RW, Conn PJ. 1995. Roles of specific metabotropic glutamatereceptor subtypes in regulation of hippocampal CA1 pyramidal cellexcitability. J Neurophysiol 74:122–129.

Gerfen CR, Young WS. 1988. Distribution of striatonigral and striatopalli-dal peptidergic neurons in both patch and matrix compartments: an insitu hybridization histochemistry and fluorescent retrograde tracingstudy. Brain Res 460:161–167.

Gerfen CR, Baimbridge KG, Miller JJ. 1985. The neostriatal mosaic:compartmental distribution of calcium-binding protein and parvalbu-min in the basal ganglia of the rat and monkey. Proc Natl Acad Sci USA82:8780–8784.

Glaum SR, Miller RJ. 1992. Metabotropic glutamate receptors mediateexcitatory transmission in the nucleus of the solitary tract. J Neurosci12:2251–2258.

Graybiel AM. 1990. Neurotransmitters and neuromodulators in the basalganglia. Trends Neurosci 13:244–253.

Hazrati LN, Parent A. 1992. Convergence of subthalamic and striatalefferents at pallidal level in primates: an anterograde double-labelingstudy with biocytin and PHA-L. Brain Res 569:336–340.

Huang PL, Dawson TM, Bredt DS, Synder SH, Fishman MC. 1993.Targeted disruption of the neuronal nitric oxide synthase gene. Cell75:1273–1286.

Huber G, Matus A. 1984. Differences in the cellular distribution of twomicrotubule-associated proteins, MAP1 and MAP2, in rat brain. JNeurosci 4:151–160.

Kerner JA, Standaert DG, Penney JB, Young AB, Landwehrmeyer GB.1997a. Expression of group I metabotropic receptors by populations ofneurons in the rat neostriatum. Mol Brain Res 48:259–269.

Kerner JA, Standaert DG, Penney JB, Young AB, Landwehrmeyer GB.1997b. Expression of Group II and III mGluR mRNAs in identifiedstriatal and cortical neurons. Soc Neurosci Abstr 23:938.

Kinoshita A, Shigemoto R, Ohishi H, van der Putten H, Mizuno N. 1998.Immunohistochemical localization of metabotropic glutamate recep-tors, mGluR7a and mGluR7b, in the central nervous system of the adultrat and mouse: a light and electron microscopic study. J Comp Neurol393:332–352.

Kinzie JM, Saugstad JA, Westbrook GL, Segerson TP. 1995. Distribution ofmetabotropic glutamate receptor 7 messenger RNA in the developingand adult rat brain. Neuroscience 69:167–176.

Kita H, Kitai ST. 1987. Efferent projections of the subthalamic nucleus inthe rat: light and electron microscopic analysis with the PHA-L method.J Comp Neurol 260:435–452.

Kosinski CM, Standaert DG, Testa CM, Penney JB, Young AB. 1998.Expression of metabotropic glutamate receptor 1 isoforms in thesubstantia nigra pars compacta of the rat. Neuroscience 86:783–798.

Li H, Ohishi H, Kinoshita A, Shigemoto R, Nomura S, Mizuno N. 1997.Localization of a metabotropic glutamate receptor, mGluR7, in axonterminals of presumed nociceptive, primary afferent fibers in thesuperficial layers of the spinal dorsal horn: an electron microscopestudy in the rat. Neurosci Lett 223:153–156.

Li JL, Ohishi H, Kaneko T, Shigemoto R, Neki A, Nakanishi S, Mizuno N.1996. Immunohistochemical localization of a metabotropic glutamatereceptor, mGluR7, in ganglion neurons of the rat—with special refer-ence to the presence in glutamatergic ganglion neurons. Neurosci Lett204:9–212.

Lovinger DM, McCool BA. 1995. Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involvesmGluR2 or mGluR3. J Neurophysiol 73:1076–1083.

Masugi M, Yokoi M, Shigemoto R, Muguruma K, Watanabe Y, Sansig G, vander Putten H, Nakanishi S. 1999. Metabotropic glutamate receptorsubtype 7 ablation causes deficit in fear response and conditioned tasteaversion. J Neurosci 19:955–963.

Miller RJ. 1991. Metabotropic excitatory amino acid receptors reveal theirtrue colors. Trends Pharmacol Sci 12:365–67.

Nakajima Y, Iwakabe H, Akazawa C, Nawa H, Shigemoto R, Mizuno N,Nakanishi S. 1993. Molecular characterization of a novel retinalmetabotropic glutamate receptor mGluR6 with a high agonist selectiv-ity for L-2-amino-4-phosphonobutyrate. J Biol Chem 268:11868–11873.

Nakanishi S. 1992. Molecular diversity of glutamate receptors and implica-tions for brain function. Science 258:597–603.

Nicoletti F, Bruno V, Copani A, Casabona G, Knopfel T. 1996. Metabotropicglutamate receptors: a new target for the therapy of neurodegenerativedisorders? Trends Neurosci 19:267–271.

Ohishi H, Akazawa C, Shigemoto R, Nakanishi S, Mizuno N. 1995a.Distributions of the mRNAs for L-2-amino-4-phosphonobutyrate-sensitive metabotropic glutamate receptors, mGluR4 and mGluR7, inthe rat brain. J Comp Neurol 360:555–570.

Ohishi H, Nomura S, Ding YQ, Shigemoto R, Wada E, Kinoshita A, Li JL,Neki A, Nakanishi S, Mizuno N. 1995b. Presynaptic localization of ametabotropic glutamate receptor, mGluR7, in the primary afferentneurons—an immunohistochemical study in the rat. Neurosci Lett202:85–88.

Okamoto N, Hori S, Akazawa C, Hayashi H, Shigemoto R, Mizuno N,Nakanishi S. 1994. Molecular characterization of a new metabotropicglutamate receptor mGluR7 coupled to inhibitory cyclic AMP signaltransduction. J Biol Chem 269:1231–1236.

Orlando LR, Standaert DG, Penney JB, Young AB. 1995. Metabotropicreceptors in excitotoxicity: (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG) protects against rat striatal quinolinic acid lesions. NeurosciLett 202:109–112.


Parent A, Hazrati LN. 1995. Functional anatomy of the basal ganglia . 1.The cortico-basal ganglia-thalamo-cortical loop. Brain Res Rev 20:91–127.

Pisani A, Calabresi P, Centonze D, Bernardi G. 1997. Activation of group IIImetabotropic glutamate receptors depresses glutamatergic transmis-sion at corticostriatal synapse. Neuropharmacology 36:845–851.

Reiner A, Anderson KD. 1990. The patterns of neurotransmitter andneuropeptide cooccurence among striatal projection neurons: conclu-sions based on recent findings. Brain Res Rev 15:251–265.

Ricardo JA. 1980. Efferent connections of the subthalamic region in the rat.I. The subthalamic nucleus of Luys. Brain Res 202:257–271.

Saugstad JA, Kinzie JM, Mulvill ER, Segerson TP, Westbrook GL. 1994.Cloning and expression of a new member of the L-2-amino-4-phosphonic acid-sensitive class of metabotropic glutamate receptors.Mol Pharmacol 45:367–372.

Shigemoto R, Kulik A, Roberts JDB, Ohishi H, Nusser Z, Kaneko T,Somogyi P. 1996. Target-cell-specific concentration of a metabotropicglutamate receptor in the presynaptic active zone. Nature 381:523–525.

Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi S, Takada M, FlorPJ, Neki A, Abe T, Nakanishi S, Mizuno N. 1997. Differential presynap-tic localization of metabotropic glutamate receptor subtypes in the rathippocampus. J Neurosci 17:7503–7522.

Standaert DG, Needleman P, Saper CB. 1986. Organization of atriopeptin-like immunoreactive neurons in the central nervous system of the rat. JComp Neurol 253:315–341.

Standaert DG, Landwehrmeyer GB, Kerner JA, Penney JB, Young AB.1996. Expression of NMDAR2D glutamate receptor subunit mRNA inneurochemically identified interneurons in the rat neostriatum, neocor-tex, and hippocampus. Mol Brain Res 42:89–102.

Tallaksen-Greene SJ, Albin RL. 1994. Localization of AMPA-selectiveexcitatory amino acid receptor subunits in identified populations ofstriatal neurons. Neuroscience 61:509–519.

Tanabe Y, Masu M, Ishii T, Shigemoto R, Nakanishi S. 1992. A family ofmetabotropic glutamate receptors. Neuron 8:169–179.

Testa CM, Standaert DG, Young AB, Penney JB. 1994. Metabotropicglutamate receptor mRNA expression in the basal ganglia of the rat. JNeurosci 14:3005–3018.

Testa CM, Standaert DG, Landwehrmeyer GB, Penney JB, Young AB. 1995.Differential expression of the mGluR5 metabotropic glutamate receptorby rat striatal neurons. J Comp Neurol 354:241–252.

Testa CM, Friberg IK, Weiss SW, Standaert DG. 1998. Immunohistochemi-cal localization of metabotropic glutamate receptors mGluR1a andmGluR2/3 in the rat basal ganglia. J Comp Neurol 390:5–19.

Weiss SW, Albers DS, Iadarola MJ, Dawson TM, Dawson VL, StandaertDG. 1998. NMDAR1 glutamate receptor subunit isoforms in neostria-tal, neocortical and hippocampal nitric oxide synthase neurons. JNeurosci 18:1725–1734.

Wullner U, Testa CM, Catania MV, Young AB, Penney JB. 1994. Glutamatereceptors in striatum and substantia nigra—effects of medial forebrainbundle lesions. Brain Res 645:98–102.


localization of metabotropic glutamate receptor 7 mrna and mglur7a protein in the rat basal ganglia

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