The Journal of Neuroscience, April 1993, 13(4): 1372-l 378

Signal Transduction, Pharmacological Properties, and Expression Patterns of Two Rat Metabotropic Glutamate Receptors, mGluR3 and mGluR4

Yasuto Tanabe,’ Akinori Nomura,’ Masayuki Maw,’ Ryuichi Shigemoto,* Noboru Mizuno,* and Shigetada Nakanishil lInstitute for Immunology and the 2Department of Morphological Brain Science, Kyoto University Faculty of Medicine, Kyoto 606, Japan

The metabotropic glutamate receptors are coupled to intra- [Key words: metabotropic glutamate receptor, cDNA clone, cellular signal transduction via G-proteins and consist of a DNA transfection, signal transduction, agonist selectivity, in family of at least five different subtypes, termed mGluRl- situ hybridization] mGluR5. We studied the signal transduction mechanism and pharmacological characteristics of the rat mGluR3 and L-Glutamate acts as a major excitatory neurotransmitter and mGluR4 subtypes in Chinese hamster ovary cells perma- plays an important role in neuronal plasticity and neurotoxicity nently expressing the cloned receptors. Both mGluR3 and in the CNS (Monaghan et al., 1989; Watkins et al., 1990).Glu- mGluR4 inhibit the forskolin-stimulated accumulation of in- tamate neurotransmissionis thus thought to be involved in tracellular CAMP formation in response to agonist interac- many integrative brain functions including memory acquisition tion. Consistent with the high degree of sequence similarity and learning, and also in someneurodegenerative disorders such to mGluR2, mGluR3 closely resembles mGluR2 in its agonist as stroke and epilepsy (Collingridgeand Singer, 1990;Meldrum selectivity; the potency rank order of agonists is L-glutamate and Garthwaite, 1990). The disparate functions of glutamate > frans-1 -aminocyclopentane- 1,3-dicarboxylate > iboten- neurotransmissionare reflected in the presenceof multiple re- ate > quisqualate. mGluR4 is totally different in its agonist ceptors that can be categorizedinto two distinct groupstermed specificity from any other member of the metabotropic re- ionotropic and metabotropic receptors(mGluRs) (Monaghan et ceptors. This receptor potently reacts with L-2-amino-4- al., 1989). The ionotropic receptors, consistingof the NMDA phosphonobutyrate (L-AP4) in a stereo-selective manner and receptors and AMPA/kainate receptors, contain cation-specific moderately responds to L-serine-O-phosphate. mGluR4 thus ion channelsand sharefundamental featureswith other ligand- corresponds well to the putative L-AP4 receptor character- gated ion channels (Hollmann et al., 1989; Monaghan et al., ized from brain preparations. Blot and in situ hybridization 1989;Moriyoshi et al., 1991). The metabotropic receptorsmod- analyses indicated that both mRNAs are widely distributed ulate the production of intracellular secondmessengers and be- in the rat brain. mGluR3 mRNA is highly expressed in neu- long to the family of G-proteinxoupled receptors(Schoepp et ronal cells of the cerebral cortex and the caudate-putamen, al., 1990; Recasenset al., 1991). An additional and novel re- and in granule cells of the hippocampal dentate gyrus. The ceptor termed L-AP4 (L-2-amino-4-phosphonobutyrate)recep- expression pattern of mGluR4 mRNA is more restricted, and tor has been identified through the actions of this compound this expression is prominent in the cerebellum, olfactory bulb, on certain glutamate-using synapses(Monaghan et al., 1989). and thalamus. Furthermore, the mGluR3 mRNA, unlike the The L-AP4 receptor has been shown to suppresssynaptic glu- other mRNAs for the metabotropic receptors, is highly ex- tamate neurotransmissionand appearsto representan autore- pressed in glial cells throughout the brain regions. The me- ceptor (Cotman et al., 1986;Forsythe and Clements,1990; Bas- tabotropic glutamate receptor subtypes can thus be clas- kys and Malenka, 1991). However, the molecular nature of the sified into three subgroups according to the similarity in their L-AP4 receptor remained to be elucidated. amino acid sequences, signal transduction, and agonist se- Recently, we isolatedcDNA clonesfor five different subtypes lectivity: mGluR1 /mGluRS, mGluRPlmGluR3, and mGluR4. The of the rat mGluR family (mGluRl-mGluR5) by molecular mRNAs for the individual receptor subtypes, however, show screeningof a rat brain cDNA library (Masu et al., 1991; Abe overlapping but distinct patterns of expression in the rat et al., 1992; Tanabe et al., 1992). The mGluR family possesses CNS. a large extracellular domain precedingthe sevenputative trans- membranesegments and sharesa high sequencesimilarity among the five different subtypes (Houamed et al., 1991; Masu et al., Received July 30, 1992; revised Oct. 2, 1992; accepted Oct. 8, 1992. 1991; Abe et al., 1992; Tanabe et al., 1992).These subtypes can We are grateful to Akira Uesugi for photographic assistance. This work was be divided further into three subgroupsaccording to their se- supported in part by research grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare of Japan, the Uehara Me- quencesimilarities: mGluRl/mGluRS, mGluR2/mGluR3, and morial Foundation, and the Semi Life Science Foundation. mGluR4 (Abe et al., 1992;Tanabe et al., 1992).Consistent with Correspondence should be addressed to Shigetada Nakanishi, Institute for Im- the sequencesimilarity, both mGluR 1 and mGluR5 are coupled munology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan. to the stimulation of phosphatidylinositol (PI) hydrolysis/Ca*+ Copyright 0 1993 Society for Neuroscience 0270-6474/93/131372-07$05.00/O signaltransduction and showstrong resemblance in their agonist The Journal of Neuroscience, April 1993, 73(4) 1373

Figure 1. Dose-response curves of ag- onists in inhibition of forskolin-stim- ulated CAMP accumulation in cells sta- bly exoressina mGluR3 (a) and mGluR4 (bj. Agonistsadded to forskolin-treated cells are as follows: in a, 0, r&rtamate; 0, tACPD, A, ibotenate; 0, quisqualate; in b, A, L-AP4; 0, L-glutamate; 0, L-ser- ine-O-phosphate; A, D-APL Intracel- lular CAMP levels in cells treated and untreated with 10 PM forskolin were 233 + 8.9 and 7.2 + 0.4 pmol/well in a, respectively, and 239 + 20 and 8.4 + 1.0 pmol/well in b, respectively. The CAMP levels in forskolin-treated cells are taken as 100%. Each point repre- Agonist (M) sents the mean ? SEM of at least two separate experiments done in triplicate. selectivity (Abe et al., 1992; Aramori and Nakanishi, 1992). by measuring the glutamate-induced inhibition of the forskolin-stim- mGluR2, on the other hand, is linked to the inhibitory CAMP mated CAMP accumulation. For the measurement of CAMP levels, a clonal cell line expressing mGluR3 or mGluR4 was seeded individually cascade and exhibits an agonist selectivity different from that in 12-well plates at a density of 1.5 x 1OS cells/well and grown for 3 d. of mGluRl/mGluRS (Tanabe et al., 1992). However, neither After 20 min preincubation in phosphate-buffered saline (PBS) con- the precise signal transduction nor the agonist selectivity of taining 1 mM 3-isobutyl-1-methylxanthine (IBMX) at 37”C, the cells mGluR3 or mGluR4 has yet been characterized. In this study, were incubated with fresh PBS containing 10 PM forskolin, 1 mM IBMX, we stably expressed mGluR3 and mGluR4 by DNA transfection and test agents for 10 min. The medium was aspirated, and the reaction was stopped by the addition of 5% trichloroacetic acid. CAMP levels into Chinese hamster ovary (CHO) cells and investigated the were measured by the RIA kit (Amersham). For pertussis toxin (PTX) intracellular signal transduction mechanisms and pharmaco- treatment, cells were preincubated with various concentrations of PTX logical characteristics of these receptor subtypes. We also ana- for 11 hr at 37°C. Each experiment was carried out at least twice in lyzed expression patterns of the mRNAs for mGluR3 and triplicate. RNA blot and in situ hybridization analyses. RNA blot analysis was mGluR4 by RNA blot and in situ hybridization. The results carried out by using 10 pg of total RNAs isolated from various regions demonstrated that both subtypes are linked to the inhibitory of the brain as described previously (Masu et al., 1991). The cDNA CAMP cascade. However, mGluR4 is totally different from other probe used was the 877 bp PstI fragment of pmGR3 and the 1263 bp members of mGluRs in agonist selectivity and reacts with L-AP4 Smal fragment of pmGR4. In situ hybridization was performed as pre- viously described (Masu et al., 199 1). Briefly, ‘S-labeled antisense ri- with a high potency that is one order more effective than L-glu- boprobe corresponding to the 1464 bp Hincll-Xbal or 648 bp EcoT141 tamate. Furthermore, the mGluR3 mRNA, unlike other mRNAs fragment of pmGR3 or the 1230 bp Xhol-Pstl or 568 bp Pstl fragment of the mGluR subtypes, has been found to be highly expressed of pmGR4 was transcribed and hybridized as described previously (Tan- in both neuronal and glial cells. abe et al., 1992). Sections were exposed to @max-film (Amersham) for 2 weeks or dinned in NTB2 emulsion (Kodak) diluted 1: 1 with distilled Materials and Methods water, developed after a 4 week exposure, and counterstained with cresyl violet. Control hybridization experiments were carried out in adjacent Materials. L-AH, D-AP4, L-serine-O-phosphate, quisqualate, and tACPD sections by using the same riboprobe in the presence of an excess of (trans- 1-aminocyclopentane- 1,3-dicarboxylate) were purchased from unlabeled probe. Tocris Neuramin. D-AP~ was more than 98% enantiomericahy pure but may have contained a trace amount of L-AP4 (less than 2%). All Results other compounds were reagent grade and were obtained as described previously (Tanabe et al., 1992). Signal transduction and agonist profiles of mGluR3 and Receptor expression in CHO cells and CAMP measurements. The cDNA mGluR4 clones for mGluR3 (pmGR3) and for mGluR4 (pmGR4) were described To investigate the pharmacological profiles and signal trans- previously (Tanabe et al., 1992). The AT-rich sequence in the 3’ non- coding region of pmGR3 was removed by digestion with PIIMI, whose duction of mGluR3 and mGluR4, we stably expressed individ- site was 65 base pairs (bp) downstream of the stop codon. The resulting ual cDNA clones encoding these receptor subtypes in mam- 2.9 kilobase pair (kbp) Clal-PflMI fragment of pmGR3 and the 3.9 kbp malian CHO cells. A mouse dihydrofolate reductase gene was EcoRI fragment of pmGR4 were inserted individually into a eukaryotic used as a selective marker gene that allowed receptor-expressing expression vector (pdKCR-dhfr) containing the mouse dihydrofolate cells to grow in a medium lacking ribonucleosides and deoxy- reductase gene as a selective marker (Tanabe et al., 1992). These plas- mids were transfected into CHO cells deficient in dihydrofolate reduc- ribonucleosides. We also removed L-glutamate and reduced the tase activity (CHO-dhfr?) (Urlaub and Chasin, 1980) by the calcium concentration of L-glutamine in the culture medium to avoid phosphate method (Graham and van der Eb, 1973). Cell populations the possible constitutive activation of these receptors by L-glu- expressing mGluR3 or mGluR4 together with dihydrofolate reductase tamate. Through this procedure, we succeeded in obtaining sev- were selected in Dulbecco’s modified Eagle’s medium lacking L-gluta- mate, ribonucleosides, and deoxyribonucleosides, and containing a re- eral cell lines stably expressing mGluR3 and mGluR4. Because duced concentration (2 mM) of L-glutamine (Tanabe et al., 1992). Clonal mGluR3 shows a high degree (- 70%) of sequence similarity to cell lines expressing high levels of mGluR3 or mGluR4 were identified mGluR2 that is coupled to the inhibitory CAMP cascade (Tan- 1374 Tanabe et al. l Properties and Expression of mGluR3 and mGluR4 = e 100 12 3 4 5 6 7 8 910 E -10 8 ;:g z ‘i; 4.4 -; s c 4.0 - 50 2.4 - 3 s! i 4 0L. I b 12 3 4 5 6 7 8 910 3 /dj * ’ _,.* / ; ., a+-”‘. 0 0 a01 0.1 1 10 100 . * pertussis toxin (rig/ml) Figure 2. Effects of PTX on agonist-mediated inhibition of forskolin- 4.4 - a* 5.0 stimulated CAMP accumulation. mGluR3-expressing cells (0) and mGluR4-expressing cells (0) were pretreated with various concentra- 2.4 - tions of PTX for 11 hr. Forskolin-stimulated CAMP levels were deter- mined with or without addition of 100 PM L-glutamaie for mGluR3 and with or without addition of 100 PM L-AP4 for mGluR4. The levels obtained without addition of L-glutamate or L-AP4 are taken as 100% Figure 3. RNA blot hybridization analysis of mGluR3 (a) and mGluR4 at each concentration of PTX indicated. Each point represents the mean (b). Total RNAs analyzed are as follows: I, whole brain; 2, olfactory ? SEM of at least two separate experiments done in triplicate. bulb; 3, medulla/pans; 4, midbrain; 5, thalamus; 6, hypothalamus; 7, hippocampus; 8, striatum; 9, cerebral cortex; 10, cerebellum. The size marker (kilonucleotides) used was the RNA ladder (Bethesda Research abe et al., 1992), we examined inhibitory effects of L-glutamate Labs). on the forskolin-stimulated CAMP accumulation in mGluR3- expressingcells. As expected, mGluR3 showedefficient L-glu- tamate-mediated inhibition of the forskolin-stimulated CAMP by measuringCAMP levels after application of various com- formation (Fig. la). Similarly, L-glutamate added to mGluR4- pounds to forskolin-treated mGluR4-expressingcells (Fig. 1b). expressingcells resultedin significant inhibition of the forskolin- The CAMP accumulation wasreduced by L-glutamatein a dose- induced CAMP accumulation (Fig. lb). This inhibition, how- dependentmanner with an EC,, value of 5 PM. Notably, L-AP~ ever, did not extend beyond about 50% of the maximally stim- more effectively inhibited the forskolin-stimulated CAMP ac- ulated levels even by the addition of higher concentrations of cumulation, and this potency with an EC,, value of 0.5 PM was L-glutamate. Becausethis partial inhibition wasconsistently ob- one order higher than that of L-glutamate. r.-Serine-O-phosphate servedin severalindependent mGluR4-expressing cell lines,this wasalso an effective agonistwith an EC,, value of 4 PM, although inhibition pattern probably representsa feature characteristic the extent of this inhibition was less than those observed for of the signal transduction mediated by mGluR4. No L-gluta- L-AP4 and L-glutamate.D-AP4, an enantiomerof L-AP4, evoked mate-mediated inhibition of the forskolin-stimulated CAMP inhibitory responsesat higher concentrations. However, the formation was observed in untransfected control cells or those D-AP4 available for our experiments was not entirely pure and transfected with the vector DNA alone (data not shown). Fur- may have contained a trace amount of L-AP4. BecauseL-AP4 thermore, no appreciable stimulation of PI hydrolysis, CAMP and D-AP4 showedabout 2 orders of magnitude difference in formation, or arachidonic acid releasewas induced by the ad- the effective concentrations,it is feasiblethat the effect of D-A&l dition of L-glutamatein either mGluR3- or mGluR4-expressing arisesfrom a small amount of L-AP4 in our D-AP4 preparation cells (data not shown). Thus, it can be concluded that both rather than reflecting its intrinsic activity. tACPD, quisqualate, mGluR3 and mGluR4 subtypesare coupled to the inhibitory and D,L-2-amino-ZLphosphonopropionate evoked slight inhi- CAMP cascade. bition (1O-20%) of the CAMP formation at the concentration We determined dose-responsecurves of various agonistsfor of 100 PM. However, this inhibition was variable among the the inhibition of the forskolin-stimulated CAMP accumulation different experiments, and was also observed in untransfected in mGluR3-expressingcells (Fig. la). L-glutamate, tACPD, ibo- control cells. Thus, the agonist activity of these compounds tenate, and quisqualatewere effective in inhibiting the CAMP could not be evaluated as being statistically significant. NMDA, accumulation in this order of agonists.Thus, the rank order of kainate, ibotenate, and AMPA did not display any inhibitory thesecompounds was in complete agreementwith that deter- activity at 100 PM each. Thus, the results presentedhere dem- mined for mGluR2 (Tanabe et al., 1992). The effective concen- onstrated that mGluR4 is clearly distinguishable from other trations of half-maximal response(EC,,) of the above com- membersof the mGluR family by its characteristic agonist se- pounds were calculated to be 3, 8, 10, and 40 PM, respectively. lectivity. Thesevalues were also very similar to thosereported for mGluR2 PTX catalyzes the ADP-ribosylation of someG-proteins, thus (Tanabe et al., 1992). NMDA, kainate, AMPA, and L-AP4 had uncoupling them from their linked receptors(Oilman, 1984;Ui, virtually no effects on the signal transduction of mGluR3 in 1984). The PTX-sensitive G-proteins include G, linked to the receptor-expressing cells. Thus, mGluR3 closely resembles inhibitory CAMP cascade,G,, and a certain type of G,, that may mGluR2 in the signal transduction and agonist selectivity. be coupled to PI hydrolysis (Gilman, 1984; Ui, 1984). To ad- BecausemGluR4 was found to be linked to the inhibitory dressthe coupling of mGluR3 and mGluR4 to G-proteins, we CAMP cascade,we determined an agonist profile of mGluR4 investigated the effectsof PTX on mGluR3- and mGluR4-me- The Journal of Neuroscience, April 1993, 73(4) 1375

Figure 4. Localization of mGluR3 mRNA (a) andmGluR4 mRNA (b) in the adultrat brainby in situ hybridiza- tion. Negativefilm images of sagittal sections are shown. IC, inferior collic- ulus; SC, superior colliculus; DC, den- tate gyrus; St, striatum; cc, corpus cal- losum; Cx, cerebral cortex; Rt, thalamic reticular nucleus; SO, supraoptic nu- cleus; UC,anterior commissure; Tu, ol- factory tubercle; Cb, cerebellar cortex; Pn, pontine nucleus; Th, thalamus; LS, lateral septum; OB, main olfactory bulb. Scale bar, 5 mm.

diated inhibition of the CAMP accumulation (Fig. 2). When Only a trace amount ofthe mRNA wasseen in the hippocampus. mGluR3- and mGluR4-expressing cells were pretreated with In situ hybridization analysis indicated that mGluR3 mRNA PTX prior to addition of forskolin and an agonist (L-glutamate was expressednot only in neuronal cells but also in glial cells for mGluR3 and L-AP4 for mGluR4), the agonist-mediated throughout the brain regions(Figs. 4a, 5a-c). This mRNA was inhibition of CAMP accumulation was reduced in a dose-de- predominantly expressedin neuronsof the cerebralcortex, tha- pendent manner and was completely abolished at 1 ng of PTX lamic reticular nucleus (Fig. 5b), and supraoptic nucleus, and per milliliter of medium in both cases. Thus, the agonist re- granule cells in the dentate gyrus, and also in glial cells in white sponses in both receptors are mediated by a PTX-sensitive G, matter such as the corpus callosum(Fig. 5a) and anterior com- protein. missure. In the cerebellum, moderate expressionwas seenin Golgi cells (Fig. 5~). mGluR4 mRNA was more restrictedly Expression patterns of mGluR3 and mGluR4 mRNAs distributed in neuronal cells (Figs. 4b, 5d-f. Prominent ex- The distribution of mGluR3 and mGluR4 transcripts in the pressionof mGluR4 mRNA was observed in neurons of the adult rat brain was examined by RNA blot and in situ hybrid- internal granular layer of the main olfactory bulb (Fig. 5d), those ization analyses. Blot hybridization analysis of mGluR3 mRNA of the thalamus (Fig. 5e), lateral septum, and pontine nucleus revealed a wide distribution of an mRNA of -4.0 kb throughout (Fig. 4b), and granule cells of the cerebellum (Fig. 5f). Only the brain regions (Fig. 3a). Additionally, a minor band corre- weak expressionof mGluR4 mRNA wasobserved in the dentate sponding to an mRNA size of - 10 kb was seen in all regions gyrus and CA3 region of the hippocampus. No significant hy- examined. Blot hybridization analysis of mGluR4 mRNA yield- bridization was observed in parallel control experiments using ed a band with an estimated mRNA size of -5.0 kb (Fig. 3b). the samemGluR3 or mGluR4 probe in the presenceof an excess A minor band with an mRNA size of - 10 kb was also found of unlabeled probe (data not shown). Furthermore, in both in the cerebellum. The expression pattern of mGluR4 mRNA mRNA analyses,a similar hybridization pattern was obtained was rather specific. The high levels of the mRNA expression with a different nonoverlapping probe derived from the mGluR3 were observed in the cerebellum, thalamus, and olfactory bulb. or mGluR4 cDNA (data not shown). 1376 Tanabe et al. l Properties and Expression of mGluR3 and mGluR4

Figure 5. Localization of mGluR3 mRNA (u-c) and mGluR4 mRNA (d-f) in the adult rat brain by in situ hybridization. Bright-field photo- micrographs of emulsion-dipped sections through the corpus callosum (a), thalamic reticular nucleus (b), cerebellar cortex (c, fl, olfactory bulb (d), and ventrolateral thalamic nucleus (e) are shown. In a, intense signals are seen in small glial cells. Arrowheads in c indicate labeled Golgi cells. M, molecular layer; P, Purkinje cell layer; G, granular layer; EP, external plexiform layer; Mi, mitral cell layer; ZG, internal granular layer. Scale bar, 50 pm.

occur in the mossy fiber-CA3 synapsesof guinea pig, but not Discussion in the same synapsesof rat (Lanthom et al., 1984). The ex- This article reports the characterization of the signal transduc- pressionof mGluR4 mRNA is not necessarilyhigh in neuronal tion and agonist profiles of mGluR3 and mGluR4 and their cells projecting theseneuronal pathways. However, the putative expressionpatterns in the adult rat brain. mGluR3 resembles L-AP4 receptor has been shown to exhibit high stereospecificity mGluR2 in both signal transduction and agonist selectivity. for the L-isomerof AP4 and to interact moderately with L-serine- Similar to mGluR3, mGluR4 iscoupled to the inhibitory CAMP O-phosphate (Cotman et al., 1986).Thus, the agonist profile of cascade,but its agonist selectivity is totally different from not this receptor agreeswell with that of mGluR4. Recent electro- only mGluR2/mGluR3 but also mGluRl/mGluRS. Interest- physiological studies indicated that the L-AP4 receptor is in- ingly, mGluR4 effectively and selectively interacts with L-AP4. volved in suppressionof excitatory synaptic transmission by It has previously beensuggested by electrophysiologicalstudies reducing glutamate releaseat a presynaptic site (Cotman et al., that there is a novel receptor that potently respondsto L-AP4 1986;Forsythe and Clements,1990; Baskys and Male&a, 199I). (Koemer et al., 1981; Hori et al., 1982;Yamamoto et al., 1983; Thus, the L-AP4 receptor appears to represent a presynaptic Lanthom et al., 1984; Heam et al., 1986). These studiesindi- autoreceptor. It is thus feasiblethat mGluR4 correspondsto the cated that L-AP4 suppressedneurotransmission in various syn- L-AP4 receptor at least at some of neuronal synapses and reg- apsesof the rat CNS, including the lateral entorhinal-dentate ulates glutamate neurotransmission by the action at presynaptic gyms pathway and the lateral olfactory tract-prepyriform cortex sites. pathway. L-AP4-sensitive responseshave also beenreported to Another interesting system in which L-AP4 has been exten- The Journal of Neuroscience, April 1993, 13(4) 1377

Table 1. Properties and expressions of the five subtypes of the mGluR family

Receptor Signal Agonist Subgroup (amino acids) transduction selectivity mRNA expression sitesa I mGluR 1 Purkinje cells of cerebellum, mitral and (I 199 or 906) tufted cells of olfactory bulb, granule cells of dentate gyrus, pyramidal cells of CA2-CA4, neurons of thalamus IP,/Ca2+ QA > Glu z Ibo > tACPD cascade mGluR5 Neurons of striatum, cerebral cortex, and (1171) internal granular layer of olfactory bulb, granule cells of dentate gyrus; pyramidal cells of CA l-CA4 II mGluR2 Golgi cells of cerebellum, granule cells of (872) dentate gyrus, neurons of cerebral cortex and main and accessory olfactory bulb Inhibitory Glu 2 tACPD > Ibo > QA CAMP cascade mGluR3 Neurons of cerebral cortex and thalamic (879) reticular nucleus, granule cells of dentate gyrus, glial cells throughout the CNS III mGluR4 Inhibitory L-AP4 > Glu > SOP Granule cells of cerebellum, neurons of (912) CAMP cascade thalamus and internal granular layer of olfactory bulb

IP,, inositol &phosphate; QA, quisqualate; Glu, glutamate; Ibo, ibotenate; SOP, L-serine-O-phosphate. 0 Only characteristic expression sites are described.

sively studied is within the retina. L-glutamate hyperpolarizes expected, and the role of this receptor in glial functions will be ON-bipolar cellsand depolarizesOFF-bipolar cells in the retina. interesting for further investigations. L-AP4 mimics the action of L-glutamateat ON-bipolar cells and Taking into account the results presentedhere, together with selectively hyperpolarizes these cells (Nawy and Jahr, 1990, thosereported previously (Abe et al., 1992;Aramori and Naka- 1991). The hyperpolarization has been shown to result from nishi, 1992;Tanabe et al., 1992),Table 1 summarizesthe prop- erties and expressionsof five subtypes of the mGluR family. lowering intracellular cGMP levels through the activation of The five receptors can be classifiedinto three subgroups.The G-protein

Aramori I, Nakanishi S (1992) Signal transduction and pharmacolog- the function of the central nervous system. Annu Rev Pharmacol ical characteristics of a metabotropic glutamate receptor, mGluk1, Toxic01 29:365-402. in transfected CHO cells. Neuron 8:757-765. Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S Baskys A, Malenka RC (1991) Agonists at metabotropic glutamate (199 1) Molecular cloning and characterization of the rat NMDA receptors presynaptically inhibit EPSCs in neonatal rat hippocampus. receptor. Nature 354:3 l-37. J Physiol (Land) 444:687-701. Nawy S, Jahr CE (1990) Suppression bv glutamate of cGMP-activated Collingridge’GL, Singer W (1990) Excitatory amino acid receptors and conductance in retinal bipolar cells. Na