Differential localization of phosphoinositide-linked metabotropic glutamate receptor (mGluR1) and the inositol 1,4,5-trisphosphate receptor in rat brain

The type 1 metabotropic glutamate receptor (mGluR1) is through to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP3) and Ca2+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP3 receptor (IP3R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine. We observe a striking contrast in localizations of mGluR1 and IP3R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP3R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP3R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP3, reflecting instead influences on protein kinase C or other second messengers.

The type 1 metabotropic glutamate receptor (mGluR1) is thought to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP,) and Ca*+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP, receptor (IP,R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine.
We observe a striking contrast in localizations of mGluR1 and IP,R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP,R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP,R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP,, reflecting instead influences on protein kinase C or other second messengers.
[Key words: excitatory amino acid receptors, protein kinase C, phospholipase C, in situ hybridization, immunohistochemistry] Glutamate, the major excitatory neurotransmitter in the brain, acts through two major classes of receptors (Mayer and Westbrook, 1987;Collingridge and Lester, 1989;Monaghan et al., 1989;Miller, 199 la,b). At ionotropic receptors, glutamate directly opens ion channels. More recently, glutamate has been shown to act through a metabotropic glutamate receptor (mGluR) whereby phosphoinositide (PI) turnover is enhanced (for review, see Schoepp et al., 1990, Miller, 199 la;Baskys, 1992) via a G-protein mechanism (Nicoletti et al., 1988). Localizing neurotransmitter receptors provides valuable clues to their function. The ionotropic glutamate receptors have been localized by autoradiography with various ligands (for review, see Monaghan et al., 1989;Young and Fagg, 1990). Such localization has not been feasible for mGluR because of the lack of suitable ligands. Recently, we developed a technique to visualize PI turnover in brain slices utilizing 3H-cytidine as a precursor to the PI cycle and could demonstrate selective enhancement in discrete brain structures by glutamate derivatives (Hwang et al., 1990a). However, the limited resolution of this technique has precluded detailed analysis of mGluR localizations. mGluR has been molecularly cloned (Houamed et al.,199 1;Masu et al.,199 1) and four subtypes identified (Tanabe et al., 1992). mGluR1 is PI linked, and alternative splicing yields a long and short form designated mGluR 1 (Y and mGluR lp, respectively. In addition, mGluR1 receptor stimulation leads to increased CAMP formation and release of arachidonic acid (Aramori and Nakinishi, 1992), mGluR2 activation inhibits forskolin-induced CAMP formations, while mGluR3 and mGluR4 have no known function (Tanabe et al., 1992). Recently, another mGluR coupled to PI hydrolysis, designated mGluR5, has been cloned (Abe et al., 1992).
Utilizing an antiserum generated against peptides from the mGluR1 amino acid sequence and four oligonucleotides derived from the mGluR1 cDNA that specifically recognize mGluR1cu and mGluRl& we have conducted immunohistochemical and in situ hybridization localization of mGluR1 protein and mRNA, 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.
Materials and Methods Materials. The Vectastain immunohistochemistry kit was purchased from Vector. )H-cytidine (27.8 Ci/mmol) was obtained from New England Nuclear/Du Font. Ail other materials were purchased from Sigma, unless otherwise specified.
Preparation of anti-mGluR1 antiserum. A synthetic peptide based on amino acids 141-154 of mGluR1 protein (Masu et al.,199 1) was made and conjugated to bovine serum albumin (BSA). To ensure selective coupling through one of the carboxyl terminal lysine residues, the amino terminus of the peptide was first blocked by reaction of the peptide at pH 7.4 with a twofold molar excess of citraconic anhydride for 3 hr at room temperature. BSA was then added to the peptide, to a ratio of approximately 1 BSA molecule per 10 peptide molecules, followed by addition of glutaraldehyde (final concentration, 0.1%) for 1 hr at room temperature. The conjugation reaction was stopped by incubation with excess glycine for 1 hr at room temperature. The conjugate was dialyzed first against 100 mM sodium acetate, pH 4.2, for 5 hr and then against phosphate-buffered saline (PBS) overnight. Antiserum was raised in rabbits injected with the above BSA-conjugated peptide (Cocalico Biologicals, Inc., Reamstown, PA). Antiserum solution was purified at three steps. First, it was adsorbed overnight, at 4"C, with an affinity matrix consisting ofproteins extracted from crude brain membrane using high pH (NaOH extract) and immobilized on cyanogen bromide (CNBr)-activated Sepharose. Preliminary results showed that the mGluR1 protein adheres to a heparinagarose column (A. H. Sharp, T. M. Dawson, and S. H. Snyder, unpublished observations). Thus, the antiserum solution was further absorbed with an affinity matrix consisting of Triton X-lOO-solubilized cerebellar membranes that had been passed through a heparin-agarose column before immobilization on CNBr-activated Sepharose. Finally, it was affinity purified using a column consisting of an ovalbumin-mGluR 1 peptide conjugate immobilized on CNBr-activated Sepharose, batchwise, overnight at 4°C. The antiserum was eluted from the column with 4 M MgCl,, dialyzed first against PBS and then against PBS containing 20% sucrose, and stored in small aliquots at -70°C until use. IP,R goat affinity-purified antiserum was produced as described previously (Peng et al., 199 1;Sharp et al., 1993).
Western blot analysis. Particulate fractions from different brain regions were prepared in 50 mM Tris HCI buffer containing 1 mM EDTA, 0.5 nM phenylmethysulfonyl fluoride, and 1 mM benzamidine. Proteins (150 pg per lane) were separated on a 7.5% SDS polyacrylamide gel, transferred to Immobilon-P membranes (Millipore), and probed with affinity-purified antibody (1:lOO) overnight. Blots were then washed and incubated with peroxidase-linked goat anti-rabbit secondary antibody (1: 1500; Boehtinger Mannheim) for 2 hr at room temperature. Bands were visualized using the chromogen 4-chloro-1 naphthol (Immuno-SELECT). For preadsorption experiments performed on fractions of different brain regions, antibody was preincubated with 20-fold excess of peptide antigen for 24 hr at 4°C.
Electron microscopy. Adult male Long-Evans Hooded and Sprague-Dawley rats (150-300 gm) were used for immunoelectron microscopic analysis. The animals were given a lethal dose of anesthetic and perfused transcardially with cold, oxygenated (95% 0,, 5% CO,) artificial cerebrospinal fluid (126 mM NaC1, 2.5 mM KCI, 1.25 mM NaH,PO,, 2 mM MgSO,, 26 mM NaHCO,) and immediately followed by 500 ml of cold fixative [4% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB) uH 7.41. The brains were removed and Dlaced into the fixative solution overnight before being cut into blocks and sectioned to 50 pm on a vibrating microtome. The sections containing the striatum were placed into a cryoprotecting solution consisting of 25% sucrose and 5% glycerol in 50 mM PB (pH 7.4). Once the sections had sunk in the cryoprotectant, they were freeze thawed in isopentane that had been chilled in liquid nitrogen.
The immunohistochemical procedure was carried out as above. Sections were then rinsed in phosphate buffer (pH 7.4) and postfixed in 1% osmium tetroxide for 30 min. They were then briefly rinsed in TBS before being incubated in a 2% uranyl acetate solution (aq) for 45-60 min. Following this, they were dehydrated through a graded series of alcohol, followed by propylene oxide prior to embedding in resin (Durcupan ACM, Fluka). Sections were placed in Durcopan overnight before being flat embedded between two silicon-coated (Sigmacote, Sigma) glass slides. The resin was then polymerized at 60°C for 48 hr. After light microscopic analysis, areas of the striatal matrix regions were selected and cut out from the slides and reembedded in blocks for further sectioning. These blocks were then sectioned for electron microscopy on a ultramicrotome and collected on either copper mesh grids or Pioloform-coated copper slot grids. The ultrathin sections were then examined with a JEOL 100s electron microscope, with some grids being stained with lead citrate.
Phosphoinositide (PI) turnover and imaging. Regions of rat brain corresponding to those areas in the regional Western blot analysis were rapidly dissected and cross-chopped into 400 pm pieces of tissue. The cross-chopped tissue was first allowed to recover for 30 min in Krebsbicarbonate buffer followed by incubating (50 ~1 ofgravitv-picked tissue) in 250 ~1 of Krebs-bicarbonate buffer containing 0.i pCi?ml 3H-cytidine (Du Porn/New Enaland Nuclear: 27.8 Ci/mmoB in an interface chamber (95% O,, 5% COZj at 37°C. LiCl (final concentration, 5 mM) was subsequently added followed 10 min later by 300 PM truns-1 -aminocyclopentane-1,3-dicarboxylate (t-ACPD). After 1 hr of incubation at 37°C the reaction was stopped on ice, membranes were lipid extracted, and the amount of radioactivity, that is, 'H-cytidine diphosphate diacylglycerol ('H-CDP-DAG) accumulation, was determined using a scintillation counter (Godfrey, 1989).
The steps in PI imaging are similar and were carried out exactly as described (Hwang et al., 1990a). Briefly, rat brain slices (400 pm) of olfactory bulb, hippocampus, or cerebellum were allowed to recover for 1 hr at 20°C in an interface chamber (95% O,, 5% CO,). They were prelabeled on Whatman filter paper circles (2.1 cm in diameter) that were immersed in 0.1 ml of Krebs-bicarbonate buffer (on upside-down covers of 24-well tissue culture plates) containing 4.0 &i/ml 3H-cytidine (Du Pant/New England Nuclear; 27.8 Ci/mmol), 1 &ml actinomycin (Boehringer Mannheim), and 50 PM hydroxyurea (Sigma) for 1 hr at 30°C. LiCl(5 mM final concentration) was added beneath the Whatman filter paper'with tissue on top 10 min prior to addition of 100-300 PM t-ACPD in order to allow even diffusion of LiCI. Following 50-60 min of incubation at 37°C sections were transferred to plastic molds and embedded in O.C.T. medium (Tissue-Tek). Frozen sections were cut (16 pm) on gelatin-coated glass slides, treated in wash buffer (50 mM Tris HCl DH 4.2.2 mM EDTA. 10 mM LiCl. 1 mM cvtidine. 3% uolvethvlene glycol, 0.005% saponin, 20 &ml each of RN&e A and DNase I) for 2-5 min at 37°C quickly dried, and apposed to film (Hyperfilm-'H, Amersham) or Kodak NTB emulsion-coated coverslips for 24 weeks.
Results mGluR1 protein levels and ACPD-stimulated PI turnover direr in brain regions. A rabbit polyclonal antiserum corresponding to the peptides 14 l-l 54 of the N-terminal region of mGluR 1 (Masu et al., 1991) was developed. It recognizes mGluR1 by Western blot analysis. Interestingly, two immunoreactive bands are identified at about 140 kDa and 100 kDa, in all brain regions ( Fig. 1). Preadsorption of the antiserum with excess peptide antigen completely eliminated staining, in cerebellum and other brain regions (data not shown) (Fig. 1). These bands correspond to the predicted molecular weights of the cloned mGluR 1 (Y and mGluR 10, respectively (Tanabe et al., 1992). The relative abundance of mGluR1a and mGluRlp differ in brain regions, with mGluR 1 (Y predominating in the cerebellum and olfactory bulb and mGluRlp as the major form in other areas. A To assess the relationship between the amount of mGluR1 immunoreactivity and glutamate-mediated PI turnover, we measured t-ACPD (a selective ligand for the mGluRl)-stimulated PI turnover in various brain regions and conducted Westem blot analysis of mGluR1 protein in the same brain regions (Fig. 1). We detect t-ACPD-elicited PI turnover in all regions containing mGluR 1 protein. However, the relative intensity of mGluR1 staining is dissociated from the relative amount of t-ACPD-stimulated PI turnover. For instance, highest levels of mGluR1 protein are found in the cerebellum, which contains only moderate levels of t-ACPD-stimulated PI turnover (Fig.  1). * OB CX CP HC TH BS CB Pb mGluR1 localizations dlj$er from those of IP,R mRNA and protein. The PI cycle involves receptor-mediated stimulation of phospholipase C (PLC) activity generating diacylglycerol (DAG), which stimulates protein kinase C (PKC) activity, and IP,, which binds to the IP,R to evoke calcium release (Bet-ridge and Irvine, 1989;Ferris and Snyder, 1992). Localizations for protein and mRNA of PLC (Get-fen et al., 1988;Ross et al., 1989) PKC (Worley et al., 1986a(Worley et al., ,b, 1987Huang et al., 1988;Saito ct al., 1988;Yoshihara et al., 1991), and IP,R (Worley et al., 1989;Nakagawa et al., 199 1 a,b;Nakanishi et al., 199 1) are closely similar, though not identical (Worley et al., 1987). There are also some differences in the disposition of subtypes of PLC (Gerfen et al., 1988;Ross et al., 1989) and PKC (Yoshihara et al.,199 1). Only one major form of IP,R has been identified by protein purification and molecular cloning, though alternatively spliced forms of this IP,R exist (Danoff et al.,199 1;Nakagawa et al.,199 la,b) and recently quantitatively minor forms of distinct subtypes of IP,R derived from different genes have been identified (Siidhof et al., 199 1;Ross et al., 1992). Because the distribution of the quantitatively major, first isolated form of the IP,R resembles other markers of the PI cycle, we have compared its localization to that of mGluR1. If mGluR1 is coupled to PI turnover, one would anticipate close similarities between the distribution of mGluR1 and IP,R. In numerous areas, the localizations of mGluR1 and IP,R protein and mRNA differ strikingly (Figs. 2-5, Table 1; see also Fig. 8). For instance, in the cerebellum IP,R mRNA and protein are exclusively localized to Purkinje cells and their processes, while mGluR1 occurs both in Purkinje cells and granule cells. mGluR 1 mRNA is highly concentrated in deep cerebellar nuclei that are devoid of IP,R mRNA (Fig. 2). IP,R protein occurs in Purkinje cell terminals synapsing upon deep cerebellar nuclei, whereas mGluR 1 protein is apparent within perikarya of these nuclei (data not shown). Table 1; see also Fig. 9). The molecular layer of the dentate gyrus displays very low levels of IP,R but high levels of mGluR 1. In contrast, the granule cell layer of dentate gyrus exhibits high IP,R and low mGluR1 levels.
The olfactory bulb provides further evidence for a dissociation between mGluR1 and IP,R mRNA and protein (Figs. 2-4, 7; Table 1; see also Fig. 9). Substantial levels of mGluR1 protein occur in the glomeruli, external plexiform, and mitral cells of the olfactory bulb, while IP,R is concentrated within the periglomerular and granule cells with moderate levels in tufted cells (Fig. 7A,B). IP,R protein and mRNA are concentrated within pyramidal cells of all regions and lamina of the cerebral cortex . In contrast, mGluR 1 mRNA is confined to occasional nonpyramidal neurons throughout the cortex (Fig. 5C). Substantial mGluR1 protein is evident within the cortical neuropil, apparently reflecting terminal patterns that may arise from the thalamus, where mGluR1 mRNA and protein levels are high.
In the hippocampus, IP,R and mGluR1 protein and mRNA (Figs. 2-4, 6) display notably reciprocal localizations. IP,R is concentrated in a dense band comprising the CA1 pyramidal layer with much lower levels in CA3, whereas mGluR1 is enriched in CA3 with low levels in CA 1. mGluR 1 immunoreactivity is concentrated in fine fibers in the stratum oriens of CA 1 and the subiculum, where staining for IP,R is minimal (Fig. 6, Divergent patterns in the thalamus (Figs. 24, Table 1) are evident for mGluR1 and IP,R mRNA and protein. In contrast to high mGluR1 levels, IP,R levels are much lower in most of the thalamus. The anterior dorsal thalamic nucleus contains abundant mGluR1 but no IP,R at all. The anterior ventral nucleus and the ventral lateral posterior nucleus are enriched in mGluR1 with low levels of IP,R. The medial and lateral geniculate nuclei contain high levels of mGluR 1, but only low to moderate levels of IP,R (Fig. 4). Similarly, the gelatinosum nucleus and subthalamic nucleus are enriched in mGluR1 but lack substantial staining for IP,R. In the hypothalamus, dramatic reciprocity of mGluR1 and IP,R is evident in the lateral hypothalamic nucleus with high levels of mGluR 1 but no IP,R (Figs. 4, 7E,F). By contrast, the dorsal medial hypothalamic Figure 2. Comparison of the localization of mGluR 1 (LY and p) and IP,R mRNAs. Pairs of adjacent thin (12 pm) sections of rat brain were processed for in situ hybridizations with 35S-labeled oligonucleotides specific for mGluR1 (left) and IP,R (right) cDNAs. Labeled structures appear white in these dark-field images. The relatively higher level of mGluR1 versus IP,R labeling is evident in the mitral layer (Mi) of the olfactory bulb, CA3 region of hippocampus (HC), dentate gyrus (DG), globus pallidus (GP), thalamic nuclei (7') including medial geniculate nucleus (MG), and in the mammillary bodies (M). mGluR1 labeling also occurs in apparent absence of IP,R labeling in the Darkshevich's nucleus (DK), deep cerebellar nuclei (DCN), lateral vestibular nucleus (L v), facial nucleus (7), and spinal motor nucleus of the trigeminal nerve (SW). Conversely, higher amounts of IP,R labeling appear in cortex (Cx), caudate-putamen (CPU), CA1 region of hippocampus, upper layer (pars compacta) of substantia nigra (J&V), and Purkinje layer of cerebellum (Cb). Amygdala (A) and ventral medial nucleus of hypothalamus (VMH) contains low levels of labeling for both mGluR1 and IP,R. Scale bar, 100 pm. nucleus displays abundant IP,R with negligible mGluR1 (data not shown).
The striatum possesses low levels of mGluR1 mRNA but high levels of IP,R mRNA (Fig. 2). However, both mGluR1 and IP,R proteins are abundant (Figs. 3, 4). IP,R is primarily localized to perikarya and dendrites of medium spiny neurons (Figs. 3,4,8) and is enriched within the striosomal compartment (Fotuhi et al., 1991). In contrast, mGluR1 is enriched in the presynaptic terminal fields in neuropil within the matrix compartment, although occasional postsynaptic neurons stain for mGluR1 (Figs. 3, 8, and data not shown). The nearby islands of Calleja are enriched in mGluR1 with no IP,R, whereas the surrounding olfactory tubercle contains abundant IP,R but is devoid of mGluR1 (Figs. 4, 7C,D).
The substantia nigra pars compacta contains low mGluR1 but high IP,R mRNA and protein (Figs. 24) in neurons with a pattern characteristic of dopamine-containing cell bodies. Within the substantia nigra pars reticula& both mGluR1 mRNA and protein are present. In contrast, IP,R mRNA is absent in pars reticulata, but IP,R protein is enriched, apparently reflecting terminal patterns from striatal projection neurons.
In the brainstem and midbrain regions (Figs. 2-4, Table 1) striking differences in mGluR1 and IP,R dispositions are evident in the nucleus of Darkshevich, the lateral vestibular nuclei, the red nucleus, and the cranial nerve nuclei. mGluR1 mRNA and protein are highly enriched in these structures, but IP,R mRNA and protein are virtually absent (Figs. 2E,F, 4). The superior colliculus contains high levels of mGluR1 protein, but only moderate levels of IP,R. In contrast, the pontine nuclei are enriched in IP,R but low in mGluR1 (Fig. 3).
Electron microscopy reveals dissociation of mGluR1 and IP,R at the ultrastructural level. In the striatum, mGluR 1 is primarily localized to presynaptic terminals (Fig. S), although occasional postsynaptic densities do occur (data not shown). In contrast, IP,R immunoreactivity is predominantly enriched in all postsynaptic structures. These include somata, dendritic shafts, and spines (Fig. 8).
Dissociation of mGluR1 protein from IP,R protein and t-ACPD-stimulated PI turnover. To explore further the dissociation of mGluR1 and IP,R proteins, we compared the anatomical distribution of mGluR1 and IP,R proteins with t-ACPIL stimulated PI turnover in tissue slices (Fig. 9). In the olfactory bulb, both t-ACPD-stimulated PI turnover and mGluR1 immunoreactivity are concentrated within the glomeruli. In con-The Journal of Neuroscience, May 1993, f3 (5) 2005 trast, IP,R is concentrated in periglomerular cells (Figs. 7,9). The hippocampus displays striking differences between t-ACPDelicited PI turnover, mGluR1, and IP,R protein (Fig. 9). Both t-ACPD-stimulated PI turnover and mGluR1 are enriched in CA3 with low levels of IP,R. In contrast, mGluR1 protein is abundant within the molecular layer of the dentate gyrus, where there are low amounts of IP,R and t-ACPD-stimulated PI tumover. The CA 1 region contains low levels of t-ACPD-stimulated PI turnover, which might reflect mGluR1 within the stratum oriens. In the cerebellum all three markers are enriched within the molecular layer. However, there is virtually no t-ACPDstimulated PI turnover in the deep cerebellar nuclei, where both IP,R and mGluR 1 proteins are present in high concentrations. Discussion mGluR1 is distributed distinctly throughout all regions of the brain, with highest levels in the olfactory bulb, CA3 and the dentate gyrus of the hippocampal formation, the thalamus, lateral hypothalamus, cranial nuclei of the brainstem, and the cerebellum. mGluR1 immunoreactivity in some areas (e.g., lateral hypothalamus) is limited to neuronal perikarya, while in other areas it is enriched in the neuropil (e.g., cerebral cortex).
Our antibody apparently recognizes both the long and short forms of mGluR1, mGluR 1 cu, and mGluRl& respectively. It is not known whether mGluR l/3 is a functional receptor (Tanabe et al., 1992), but the identification of a protein of appropriate molecular weight (100 kDa) by our antibody suggests that mGluR l/3 is expressed in several brain regions. The anatomical distribution of our antibody is much more extensive, although completely inclusive of the distribution of an antibody specific for mGluR ILY (Martin et al., 1992). .Presumably, the immunostaining shown here represents both mGluRla and mGluRlp as staining is completely blocked by preadsorption with excess peptide. Interestingly, Western blot analysis reveals differences between the distribution of mGluR1ol and mGluR l& mGluR lcu is present in the olfactory bulb and is present in high amounts in the thalamus, and cerebellum, while mGluRlP is enriched in the cortex, caudate-putamen, and hippocampus as well as in the olfactory bulb, thalamus, and cerebellum (Fig. 1). mGluRlp may be located primarily presynaptically, as our electron microscopy in the striatum shows a predominant presynaptic labeling pattern and the Western blot analysis demonstrates that the striatum contains very low levels of mGluR1cu. Consistent with mGluR1 immunoreactivity primarily limited to presyn- Figure 3. Immunohistochemical localization of mGluR1 and IP,R proteins in sagittal brain sections. Adjacent thick (40 pm) rat sagittal brain sections were stained with affinity-purified antibodies against mGluR1 (a and P) (A) and IP,R (B). C, Preadsorption (PA) of antibodies against mGluR 1 with its respective antigen completely abolishes immunostaining; similar results were obtained with the IP,R antibody (data not shown).
Immunoreactive structures appear white in these dark-field images. Areas high in mGluR1 immunoreactivity but low in IP,R immunoreactivity include olfactory bulb (OS), CA3 of the hippocampus, dentate gyrus (DG), thalamus (T), granular layer of cerebellum (Cb), superior colliculus (SC), and lateral hypothalamus (LH). Areas low in mGluR 1 and high in IP,R immunostaining include the anterior olfactory nucleus (AO), olfactory tubercle (Tu), pyramidal cell layer of cortex (Cx), caudate-putamen (CP), and pontine nucleus (Pn). SN, substantia nigra. Scale bar, 2.5 mm. Figure 4. Contrasting localization of mGluR1 and IP,R immunoreactivity in coronal brain sections. Pairs of adjacent thick (40 pm) coronal rat brain sections were immunohistochemically processed with affinity-purified mGluR1 (left) and IP,R (right) antibodies. Positive staining appears white in these dark-field images. The contrasting distributions of these two proteins revealed by in situ hybridization in Figure 2 is more clearly apparent in these immunostained sections. Much higher amounts of mGluR1 staining are present in the olfactory bulb (OB) and in the following thalamic nuclei: anterodorsal (AD), anteroventral (A V), ventrolateral posterior (VEX), medial geniculate (MG), and nucleus gelatinosum (G). The lateral hypothalamus (LH), islands of Calleja (1Cj), mammillary bodies (M), and nucleus of Darkshevich (OK) also exhibit more mGluR1 than IP,R immunoreactivity. Similarly, in the brainstem, many of the cranial nuclei including the motor nucleus of the trigeminal nerve (5) as well as the substantia gelatinosa and ventral horn of spinal cord (SC) contain higher amounts of mGluR1 immunoreactivity. Areas with higher IP,R immunoreactivity depicted in these sections include the internal capsule (ic), CA1 of hippocampus, pars reticulata (the lower portion) of substantia nigra (SN), and Purkinje cell layer of cerebellum (Cb). DG, dentate gyrus; CP, caudate-putamen; Cx, cortex. Scale bar, 2.5 mm.
MGIuR IP3R Figure 5. Contrasting localizations of mGluR1 and IP,R immunoreactivity in the cerebral cortex and cerebellum. Adjacent sections were immunostained with mGluR 1 (left) and IP,R (right) antibodies. mGluR 1 immunostaining is mostly in the neuropil as well as in the cell bodies of nonpyramidal cells enriched in layers II, III, V, and VI (A, C). IP,R immunostaining differs from that of mGluR1 in being more concentrated in the cell bodies of pyramidal cells (B, D). Neither antibody stains glia, as the corpus callosum (cc) appears blank. In the cerebellum (E, F), Purkinje cell bodies (P) and their dendrites in the molecular layer (MOL) both contain high levels of mGluR1 and IP,R immunoreactivity, though with somewhat different patterns. The granule cells (CR), on the other hand, are enriched in mGluR1 but lack IP,R. As in the corpus callosum, the white matter (w) of cerebellum is devoid of immunoreactivity. Axonal staining (Ax) appears in the white matter for IP,R but not for mGluR1. Scale bars, 100 pm.
aptic terminals in the caudate-putamen (Fig. 8), electrophysiological studies indicate that t-ACPD decreases synaptic excitation via presynaptic mechanisms, perhaps through inhibition ofglutamate release (Baskys and Malenka,199 1;Lovinger,199 1). The ultrastructural localization of mGluR1 in the striatum is interesting in light of the fact the PI-linked glutamate receptors were first identified in cultured striatal neurons (Sladeczek et   The distribution of mGluR1 corresponds to that of IP,R in some areas and to that of PKC in other areas. In some areas enriched in mGluR1, high amounts of both IP,R and PKC are present, yet in other areas neither IP,R nor PKC are detected. The values in mGluRI and IP,R columns are from the immunohistochemical stainings presented here and range from lowest (0) to highest (5) based on visual inspection of the average of six sets of immunostaining experiments.
The values for PKC are from Worley et aI. (1986aWorley et aI. ( ,b, 1987and M. Fotuhi, T. M. Dawson, and S. H. Snyder, unpublished observations). al., 1985). Presumably these receptors are postsynaptic. Perhaps the ultrastructural localization of the novel PI-linked glutamate receptor (mGluR5) will be postsynaptic in striatum.
The present study shows substantial differences between the localizations of mGluR1 and IP,R mRNA and protein. As mGluR 1 is believed to be linked to PI turnover, one might have expected its protein and mRNA to be distributed similarly to IP,R. Instead, in numerous parts of the brain, we have observed a reciprocal localization of mGluR1 and IP,R. In some areas, such as the islands of Calleja and anterodorsal nucleus of thal-MGIuR IP3R Fzgure 6. Reciprocal localizations of mGluR1 and IP,R immunoreactivity in the hiDDocamDa1 formation. In these bright-field images of an adjacent pair of sections immunostained with amus, we observe high levels of mGluR1 in the complete absence of IP,R. The PI cycle can be differentiated in terms of subtypes of PLC that have been selectively localized in the brain (Gerfen et al., 1988;Ross et al., 1989). Still, since the various forms of PLC all presumably generate IP,, one would not anticipate brain areas enriched in mGluR1 but devoid of IP,R. While other subtypes of IP,R have been identified, they appear to be of much lower abundance than the form of IP,R described here (Danoff et al., 1991;Nakagawa et al., 1991a,b;Stidhof et al., 1991;Ross et al., 1992). However, the minor subtypes of IP,R may be concentrated in certain brain regions that more closely parallel the distribution of mGluRla! and p. In such regions of dissociation of mGluR 1 from IP,R, it is conceivable that mGluR acts through the PI cycle primarily to activate PKC. Several areas that are devoid of IP,R possess high levels of both mGluR1 and PKC (Table 1). Consistent with this possibility, quisqualate, which potently activates mGluR, stimulates a rapid and transient translocation of PKC activity in striatal neurons (Manzoni et al., 1990). In addition, glutamate causes a transient phosphorylation of three PKC substrates in a time scale comparable to DAG production (Scholz and Palfrey,199 1).
Perhaps the distribution of mGluR5 may better fit with IP,R localizations in some brain regions. The existence of other subtypes of PI-linked mGluR as yet unidentified may also parallel the distribution IP,R better. For instance, in the molecular cloning studies, cross-hybridized cDNA clones were isolated from a cDNA library prepared from a certain size of mRNA (ap-\ mGluR1 (left) and IP,R (right) anti--bodies, durk areas represent positive --staining. Stratum oriens of CA1 and CA3 (Or in A and C) and CA4 contain an abundance of mGluR 1 -positive cells and processes in the absence of any appreciable IP,R immunostaining. The reverse occurs for pyramidal (Pv) cells and processes in stratum radiatum (Rad) of CAl, the lacunosum molecular (LMol), and granule cells of dentate gyrus (GrDG). LD, lateral dorsal nucleus ofthalamus; DC, dentate gyrus; Mel, molecular layer of dentate gyrus. Scale bars, 100 pm.
proximately 3-4 kilonucleotides and greater than 4 kilonucleotides), and it is possible that a different size of cDNA may encode a distinct subtype of PI-linked mGluR. Furthermore, several groups have reported the possible existence of other subtypes of the PI-linked mGluR, such as the ibotenate-preferring mGluR (see Schoepp et al., 1990, for review).
We have also localized PI turnover by autoradiographical localization of 3H-CDP-DAG following the stimulation of mGluR1 by t-ACPD in the presence of 3H-cytidine. 3H-CDP-DAG accumulation is a reliable technique for visualization of PI cycle as it is stoichiometrically linked to agonist-stimulated inositol formation (Godfrey, 1989;Hwang et al., 1990a,b;Kennedy et al., 1990). Similar to contrasting localization between mGluR1 and IP,R immunoreactivities, there are discrepancies in t-ACPD-stimulated PI turnover, mGluR 1, and IP,R immunoreactivity. For instance, 3H-CDP-DAG accumulation and mGluR1 are high in the external plexiform layer of the olfactory bulb, where minimal amounts of IP,R are detected (Fig. 9). Moreover, t-ACPD-stimulated PI turnover and IP,R are low in the molecular layer of the dentate gyrus, where there are high levels of mGluR1 protein. Both these areas contain abundant amounts of PKC, suggesting that the actions of mGluR1 may be mediated through the PKC limb of the PI cycle. We also noted a lack of correlation of mGluR1 protein levels as determined by Western blot analysis and t-ACPDstimulated PI turnover assessed by 3H-CDP-DAG. Similar results in the level of t-ACPD-stimulated PI turnover in various Figure 7. Reciprocal distribution of mGluR and IP,R immunoreactivity in other brain regions. Pairs of thick (40 pm) rat brain sections were immunostained with mGluR1 (left) and IP,R (right) antibodies. Positive staining appears as dark areus in these bright-field images. In the olfactory bulb (A, B), mGluR1 immunostaining appears dense in the glomeruli (GZ), in the external plexiform layer (EPZ), but low in the internal plexiform layer (ZPZ). In contrast, the IP,R immunostaining is enriched in periglomeruli cells (PC) and tufted cells (Z'ufl in the mitral cell layer (Mi), where mGluR1 protein level is low. In a pair of coronal sections (C, D) through rostra1 striatum, seen at high magnification, islands of Calleja (ZC') and olfactory tubercle (Tu) contain extremely high and low amounts of mGluR, respectively. The exact opposite occurs for IP,R immunoreactivity. Similarly, the arcuate nucleus (Arc), the lateral hypothalamic nucleus (HZ), subthalamic nucleus (STh), dorsal perimammillary nucleus (PMD), fomix (A, and internal capsule (ic) exhibit reciprocal immunostaining for mGluR and IP,R proteins. 3 V, third ventricle. Scale bar, 100 pm.
MGIuR IP3R brain regions have been obtained by assessment of 3H-inositol phosphate accumulation, and there is also a marked discrepancy depending on the relative distribution of the various components. Many other brain regions such as the subthalamic nuin these levels with the levels of mGluR1 mRNA (Condorelli et al., 1992). cleus, red nucleus, and Darkshevich's nucleus are highly en-Together, these findings raise the possibility that mGluR1 riched in mGluR1 but are devoid of both IP,R and PKC, may act through the PI cycle to activate primarily IP,R or PKC suggesting the possibility that mGluR1 may also act through other second messengers. For instance, activation of phospho- Figure 8. Contrasting localization of mGluR 1 and IP,R viewed at the ultrastructural level. Electron micrographs show the pattern of mGluR limmunoreactive (A) and IP,R-immunoreactive (B) structures. A depicts mGluR1 -immunoreactive terminal (star) forming an asymmetrical synaptic contact (region between arrowheads) with two postsynaptic structures (diamonds). B illustrates a dendrite containing IP,R immunoreactivity (star) receiving a synaptic contact (region between arrowheads) from a nonlabeled presynaptic terminal (diamond). Scale bar: 0.28 pm for A; 0.5 wm for R.
Fimre 9. Comparison of the distri-b&on of t-ACPD-stimulated PI imaging with the localization of mGluR1 and IP,R immunostaining.
t-ACPDstimulated PI turnover (A, D, G) occurs in parts of olfactory bulb (OB), hippocampus (HC), and cerebellum (Cb) that contain high amounts of mGluR1 immunostaining (B, E, H) whether or not high amounts of IP,R immunostaining are also present (C, F, I). PI imaging in A, D, and G was carried out as described in the Materials and Methods. In the olfactory bulb (A-C) PI imaging is concentrated in the glomeruli (GI). which are also enriched in mGluR1 \-II but not in IP,R immunoreactivity. Similarly, in the hippocampus (D-F), PI imaging is abundant in CA3 and stratum oriens of CAl, regions that contain high and low amounts of mGluR1 and IP,R, respectively. Interestingly, t-ACPD-stimulated PI turnover is absent from the molecular layer of the dentate gyrus (DC), an area with abundant mGluR1 proteins. In the cerebellum (G-Z), however, PI imaging, mGluR1 and IP,R proteins are all enriched in the molecular layer (MOL). lipase A, (PLA,) and associated formation of arachidonic acid metabolites can involve G-protein-linked receptors (Piomelli and Greengard, 1990;Marin et al., 199 1). Moreover, stimulation by mGluR agonists together with agonists of AMPA receptors enhances the formation of arachidonic acid in cultured striatal neurons (Dumuis et al., 1990). Whether this involves direct activation of PLA, or DAG stimulation of PLA, (Burch, 1988) awaits further study. The N-type calcium channel labeled by w-conotoxin (McEnery et al., 1991) is influenced by GTP derivatives and appears linked to G-proteins (Kasai, 1991). mGluR may be associated with a voltage-dependent calcium channel, as quisqualate in the presence of glutamate receptorgated ion channel antagonists can depress voltage-dependent Ca*+ currents in hippocampal neurons in culture in a G-proteindependent fashion (Lester and Jahr, 1990). The most convincing evidence for the association of other second messengers with mGluR1 comes from Chinese hamster ovary cells expressing this receptor. In these cells, mGluR 1 agonists not only enhance PI turnover, but also stimulate the formation of CAMP and the release of arachidonic acid (Aramori and Nakanishi, 1992).