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Differential Localization of Phosphoinositide-Linked Metabotropic Glutamate Receptor (Mglur1) and the Lnositol 1,4,5=Trisphosphate Receptor in Rat Brain

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Differential Localization of Phosphoinositide-Linked Metabotropic Glutamate Receptor (Mglur1) and the Lnositol 1,4,5=Trisphosphate Receptor in Rat Brain The Journal of Neuroscience, May 1993, 13(5): 2001-2012 Differential Localization of Phosphoinositide-linked Metabotropic Glutamate Receptor (mGluR1) and the lnositol 1,4,5=Trisphosphate Receptor in Rat Brain Majid Fotuhi,’ Alan H. Sharp,’ Charles E. Glatt,’ Paul M. Hwang,’ Marcus von Krosigk,2 Solomon H. Snyder,’ and Ted M. Dawsonl ‘Departments of Neuroscience, Neurology Pharmacology and Molecular Sciences, and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and 3ection of Neurobiology, Yale University Medical School, New Haven, Connecticut 06511 The type 1 metabotropic glutamate receptor (mGluR1) is shownto act through a metabotropicglutamate receptor (mGluR) thought to act via the phosphoinositide (PI) system with the whereby phosphoinositide(PI) turnover is enhanced(for review, associated formation of inositol 1,4,5-trisphosphate (IP,) and see Schoepp et al., 1990, Miller, 199la; Baskys, 1992) via a Ca*+ release. Utilizing immunohistochemistry and in situ hy- G-protein mechanism(Nicoletti et al., 1988). Localizing neu- bridization, we have localized protein and mRNA, respec- rotransmitter receptorsprovides valuable cluesto their function. tively, for the mGluR1 and the IP, receptor (IP,R). We have The ionotropic glutamate receptorshave been localized by au- also localized glutamate-linked PI turnover by autoradiog- toradiography with various ligands(for review, seeMonaghan raphy with 3H-cytidine. We observe a striking contrast in et al., 1989; Young and Fagg, 1990). Such localization has not localizations of mGluR1 and IP,R both for protein and mRNA. been feasiblefor mGluR becauseof the lack of suitable ligands. For instance, mGluR1 occurs in the apparent absence of Recently, we developed a technique to visualize PI turnover in IP,R in neurons of the stratum oriens of the CA1 hippocam- brain slicesutilizing 3H-cytidine as a precursor to the PI cycle pus, islands of Calleja, anterodorsal nucleus of thalamus, and could demonstrateselective enhancementin discretebrain lateral nucleus of hypothalamus, and the granular cell layer structuresby glutamatederivatives (Hwang et al., 1990a).How- and the deep nuclei of cerebellum. mGluR1 actions in these ever, the limited resolution of this technique has precluded de- brain regions may primarily be mediated through the protein tailed analysis of mGluR localizations. mGluR has been mo- kinase C limb of the PI system, as they contain moderate lecularly cloned (Houamed et al., 1991; Masu et al., 1991) and amounts of 3H-phorbol ester binding. The subthalamic nu- four subtypes identified (Tanabe et al., 1992). mGluR1 is PI cleus, red nucleus, and Darkshevich’s nucleus, which pos- linked, and alternative splicing yields a long and short form sess high levels of mGluR1, are devoid of both IP,R immu- designatedmGluR 1(Y and mGluR lp, respectively. In addition, noreactivity and 3H-phorbol ester binding. These reciprocal mGluR1 receptor stimulation leads to increased CAMP for- localizations suggest that mGluR1 actions in many brain mation and releaseof arachidonic acid (Aramori and Nakinishi, areas may not primarily involve IP,, reflecting instead influ- 1992), mGluR2 activation inhibits forskolin-induced CAMP ences on protein kinase C or other second messengers. formations, while mGluR3 and mGluR4 have no known func- [Key words: excitatory amino acid receptors, protein ki- tion (Tanabe et al., 1992). Recently, another mGluR coupled nase C, phospholipase C, in situ hybridization, immunohis- to PI hydrolysis, designatedmGluR5, has beencloned (Abe et tochemistry] al., 1992). Utilizing an antiserum generated against peptides from the Glutamate, the major excitatory neurotransmitter in the brain, mGluR1 amino acid sequenceand four oligonucleotidesderived acts through two major classesof receptors(Mayer and West- from the mGluR1 cDNA that specifically recognize mGluR1cu brook, 1987; Collingridge and Lester, 1989; Monaghan et al., and mGluRl& we have conducted immunohistochemicaland 1989; Miller, 199la,b). At ionotropic receptors, glutamate di- in situ hybridization localization of mGluR1 protein and mRNA, rectly opens ion channels. More recently, glutamate has been respectively. We now report a striking contrast in the brain localizations of mGluR1 compared to PI turnover and inositol 1,4,Strisphosphate receptor (IP,R) protein and mRNA. Received July 6, 1992; revised Oct. 26, 1992; accepted Nov. 13, 1992. This work was supported by U.S. Public Health Service Grant MH-18501, Research Scientist Award DA-00074 to S.H.S., a grant from the International Life Materials and Methods Sciences Institute, and a gift of the Bristol-Myers-Squibb Company, Postdoctoral Materials. The Vectastainimmunohistochemistry kit waspurchased Fellowship MH-09953 to A.H.S., Predoetoral Fellowship MH-1001702 to C.E.G., from Vector. )H-cytidine(27.8 Ci/mmol) was obtained from NewEn- and Training Grant GM-07309 to P.M.H. M.F. is supported in part by FCAR of glandNuclear/Du Font. Ail othermaterials were purchased from Sigma, Ouebee. Canada. T.M.D. is a Pfizer Postdoctoral Fellow and is sunoorted bv the unless otherwise specified. ,&e&n Academy of Neurology and the French Foundation f& Alzhe&e& Research, the Dana Foundation, and U.S. Public Health Service CIDA NS 01578- Preparation of anti-mGluR1 antiserum. A synthetic peptide based on 01_.. amino acids 141-154 of mGluR1 protein (Masu et al., 199 1) was made Correspondence should be addressed to Solomon H. Snyder, Department of and conjugated to bovine serum albumin (BSA). To ensure selective Neuroscience, The Johns Hopkins University School of Medicine, 725 North coupling through one of the carboxyl terminal lysine residues, the amino Wolfe Street, Baltimore, MD 21205. terminus of the peptide was first blocked by reaction of the peptide at Copyright 0 1993 Society for Neuroscience 0270-6474/93/132001-12$05.00/O pH 7.4 with a twofold molarexcess of citraconicanhydride for 3 hr at 2002 Fotuhi et al. - mGluR1 Metabotropic Glutamate Receptor room temperature. BSA was then added to the peptide, to a ratio of fixative [4% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phos- approximately 1 BSA molecule per 10 peptide molecules, followed by phate buffer (PB) uH 7.41. The brains were removed and Dlaced into addition of glutaraldehyde (final concentration, 0.1%) for 1 hr at room the fixative solution overnight before being cut into blocks and sectioned temperature. The conjugation reaction was stopped by incubation with to 50 pm on a vibrating microtome. The sections containing the striatum excess glycine for 1 hr at room temperature. The conjugate was dialyzed were placed into a cryoprotecting solution consisting of 25% sucrose first against 100 mM sodium acetate, pH 4.2, for 5 hr and then against and 5% glycerol in 50 mM PB (pH 7.4). Once the sections had sunk in phosphate-buffered saline (PBS) overnight. Antiserum was raised in the cryoprotectant, they were freeze thawed in isopentane that had been rabbits injected with the above BSA-conjugated peptide (Cocalico Bi- chilled in liquid nitrogen. ologicals, Inc., Reamstown, PA). The immunohistochemical procedure was carried out as above. Sec- Antiserum solution was purified at three steps. First, it was adsorbed tions were then rinsed in phosphate buffer (pH 7.4) and postfixed in 1% overnight, at 4”C, with an affinity matrix consisting ofproteins extracted osmium tetroxide for 30 min. They were then briefly rinsed in TBS from crude brain membrane using high pH (NaOH extract) and im- before being incubated in a 2% uranyl acetate solution (aq) for 45-60 mobilized on cyanogen bromide (CNBr)-activated Sepharose. Prelim- min. Following this, they were dehydrated through a graded series of inary results showed that the mGluR1 protein adheres to a heparin- alcohol, followed by propylene oxide prior to embedding in resin (Dur- agarose column (A. H. Sharp, T. M. Dawson, and S. H. Snyder, cupan ACM, Fluka). Sections were placed in Durcopan overnight before unpublished observations). Thus, the antiserum solution was further being flat embedded between two silicon-coated (Sigmacote, Sigma) absorbed with an affinity matrix consisting of Triton X- lOO-solubilized glass slides. The resin was then polymerized at 60°C for 48 hr. After cerebellar membranes that had been passed through a heparin-agarose light microscopic analysis, areas of the striatal matrix regions were se- column before immobilization on CNBr-activated Sepharose. Finally, lected and cut out from the slides and reembedded in blocks for further it was affinity purified using a column consisting of an ovalbumin- sectioning. These blocks were then sectioned for electron microscopy mGluR 1 peptide conjugate immobilized on CNBr-activated Sepharose, on a ultramicrotome and collected on either copper mesh grids or Piolo- batchwise, overnight at 4°C. The antiserum was eluted from the column form-coated copper slot grids. The ultrathin sections were then exam- with 4 M MgCl,, dialyzed first against PBS and then against PBS con- ined with a JEOL 100s electron microscope, with some grids being taining 20% sucrose, and stored in small aliquots at -70°C until use. stained with lead citrate. IP,R goat affinity-purified antiserum was produced as described pre- Phosphoinositide (PI) turnover and imaging. Regions of rat brain cor- viously (Peng et al., 199 1; Sharp et al., 1993). responding to those areas in the regional Western blot analysis were Western blot analysis. Particulate fractions from different brain regions rapidly dissected and cross-chopped into 400 pm pieces of tissue. The were prepared in 50 mM Tris HCI buffer containing 1 mM EDTA, 0.5 cross-chopped tissue was first allowed to recover for 30 min in Krebs- nM phenylmethysulfonyl fluoride, and 1 mM benzamidine. Proteins (150 bicarbonate buffer followed by incubating (50 ~1 ofgravitv-picked tissue) pg per lane) were separated on a 7.5% SDS polyacrylamide gel, trans- in 250 ~1 of Krebs-bicarbonate buffer containing 0.i pCi?ml 3H-cytidine ferred to Immobilon-P membranes (Millipore), and probed with affin- (Du Porn/New Enaland Nuclear: 27.8 Ci/mmoB in an interface chamber ity-purified antibody (1:lOO) overnight.
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