Neurotransmission in the hippocampus is modulated variously through presynaptic metabotropic glutamate receptors (mGluRs). To establish the precise localization of presynaptic mGluRs in the rat hippocampus, we used subtype-specific antibodies for eight mGluRs (mGluR1-mGluR8) for immunohistochemistry combined with lesioning of the three major hippocampal pathways: the perforant path, mossy fiber, and Schaffer collateral. Immunoreactivity for group II (mGluR2) and group III (mGluR4a, mGluR7a, mGluR7b, and mGluR8) mGluRs was predominantly localized to presynaptic elements, whereas that for group I mGluRs (mGluR1 and mGluR5) was localized to postsynaptic elements. The medial perforant path was strongly immunoreactive for mGluR2 and mGluR7a throughout the hippocampus, and the lateral perforant path was prominently immunoreactive for mGluR8 in the dentate gyrus and CA3 area. The mossy fiber was labeled for mGluR2, mGluR7a, and mGluR7b, whereas the Schaffer collateral was labeled only for mGluR7a. Electron microscopy further revealed the spatial segregation of group II and group III mGluRs within presynaptic elements. Immunolabeling for the group III receptors was predominantly observed in presynaptic active zones of asymmetrical and symmetrical synapses, whereas that for the group II receptor (mGluR2) was found in preterminal rather than terminal portions of axons. Target cell-specific segregation of receptors, first reported for mGluR7a , was also apparent for the other group III mGluRs, suggesting that transmitter release is differentially regulated by 2-amino-4-phosphonobutyrate-sensitive mGluRs in individual synapses on single axons according to the identity of postsynaptic neurons. Key words: metabotropic glutamate receptor; hippocampus; perforant path; mossy fiber; Schaffer collateral; axon terminal; preterminal; immunohistochemistry; lesionMetabotropic glutamate receptors (mGluRs) have various modulatory f unctions on neuronal excitability, transmitter release, and synaptic plasticity in the C NS (Pin and Duvoisin, 1995). These f unctions have been studied most extensively in the hippocampus because of its roles in learning and memory and of its architecture, which is compartmentalized well with the three major excitatory pathways: the perforant path, mossy fiber, and Schaffer collateral. The mGluRs consist of at least eight subtypes that are classified into three groups (Nakanishi and Masu, 1994;Pin and Duvoisin, 1995). Group I mGluRs (mGluR1/mGluR5) are selectively activated by 3,5-dihydroxyphenylglycine (DHPG) (Schoepp et al., 1994) and coupled to inositol phospholipid hydrolysis. On the other hand, group II mGluRs (mGluR2/ mGluR3) and group III mGluRs (mGluR4/mGluR6/mGluR7/ mGluR8), which are linked to inhibition of the cAM P cascade in receptor-transfected cell lines, are selectively activated by 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (Hayashi et al., 1993) and 2-amino-4-phosphonobutyrate (L-AP4), respectively.Excitability of hippocampal neurons is modulated directly through group I mGluRs (Davies et...

GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.

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We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis‐acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non‐permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [ Metabotropic glutamate receptors are G protein-coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. At present, eight mGluR 1 subtypes (mGluR1 through mGluR8) have been cloned and functionally expressed (1, 2). Based on their amino acid sequence homologies, pharmacology, and functional profiles, these subtypes are classified further into three groups. Members of group I (mGluR1 and -5) stimulate the activity of phospholipase C and mobilize intracellular Ca 2ϩ . Members of group II (mGluR2 and -3) and group III (mGluR4, -6, -7, -8) inhibit adenylyl cyclase. Despite the differences in primary structures and functional roles, all mGluRs feature a large conserved N-terminal extracellular domain, which is involved in the recognition of agonists and competitive antagonists (3-8).Most ligands for mGluRs were derived from amino acids and act at the conserved glutamate binding site (9). Recently, novel subtype-selective group I mGluR antagonists emerged that are structurally unrelated to amino acids and to each other. The first non-amino acid-like antagonist described was CPCCOEt (Fig. 1), a selective mGluR1 antagonist (10, 11). CPCCOEt inhibits receptor activity by a non-competitive mechanism which does not affect the binding affinity of glutamate (12, 13). Molecular characterization of the site of inhibition in mGluR1 revealed that CPCCOEt interacts with two non-conserved residues at the top of transmembrane (TM) helix VII (13). The first described selective mGluR5 antagonists, SIB-1757 and SIB-1893 (Fig. 1), are also unrelated to amino acids and were shown to act via a non-competitive mechanism (14).To address the question whether these structurally unrelated mGluR1 and mGluR5 antagonists interact with different sites of the mGluR subtypes or share a common binding site in the 7TM domain, we have studied the binding site and mode of action of the selective mGluR5 antagonist MPEP (15). MPEP is a novel derivative of SIB-1893 with nanomolar potency (Fig. 1); it is an effective antihyperalgesic in animal models of chronic inflammatory pain (16), a neuroprotectant in excitotoxin-induced striatal lesions (17) and an anticonvulsant in several epilepsy models (18). We generated a number of chimeric rec...

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre-and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTP␥S binding (EC50-values, 64 -290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (<10 M) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the bloodbrain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.G protein ͉ mGluR G protein-coupled receptors in vertebrates constitute a superfamily of 1,000-2,000 transmembrane heptahelical proteins that can be activated by a large number of extracellular signals such as photons, hormones, neurotransmitters, and growth and development factors. These receptors transduce and amplify cellular signals by the activation of G proteins, which in turn modulates cytoplasmic second-messenger and ion levels (1). Sequence comparison among the different G protein-coupled receptors revealed the existence of at least six receptor families (2). One of them, family 3, comprises receptors for extracellular calcium, pheromones, GABA, and L-glutamate. This receptor family is characterized by a large N-terminal extracellular domain that has been demonstrated by site-directed mutagenesis and x-ray crystallography to contain the binding site for orthosteric agonists (3-5). Metabotropic glutamate receptors (mGluRs) are members of family 3 and are activated by the major excitatory neurotransmitter of the mammalian brain, L-glutamate. The mGluRs act as important pre-and postsynaptic regulators of neurotransmission in the CNS, and there are at least eight subtypes (mGluR1 to m...

Glutamatergic neurotransmission has been strongly implicated in the pathophysiology of affective disorders, such as major depression and anxiety. Of all glutamate receptors, the role of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8) in such disorders is the least investigated because of the lack of specific pharmacological tools. To this end, we examined the behavioural profiles of mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) in animal models of depression and anxiety. mGluR7-/- mice were compared with wild-type (mGluR7+/+) littermates and showed substantially less behavioural immobility in both the forced swim test and the tail suspension test. Both behavioural paradigms are widely used to predict antidepressant-like activity. Further, mGluR7-/- mice displayed anxiolytic activity in four different behavioural tests, i.e. the light-dark box, the elevated plus maze, the staircase test, and the stress-induced hyperthermia test, while their cognitive performance was normal in the passive avoidance paradigm. Analysis of locomotor activity in a novel environment demonstrated that mGluR7-/- mice were slightly more active in the initial minutes following placement in the chamber only. Together, these data suggest that mGluR7 may play a pivotal role in mechanisms that regulate behavioural responses to aversive states. Therefore, drugs acting at mGluR7 may provide novel treatments for psychiatric disorders such as depression and anxiety.

Summary: Metabotropic glutamate (mGlu) receptors have been considered as potential targets for neuroprotective drugs, but the lack of specific drugs has limited the development of neuroprotective strategies in experimental models of acute or chronic central nervous system (CNS) disorders. The advent of potent and centrally available subtype-selective ligands has overcome this limitation, leading to an extensive investigation of the role of mGlu receptor subtypes in neurodegeneration during the last 2 years. Examples of these drugs are the noncompetitive mGlu1 receptor antagonists, CPCCOEt and BAY-36-7620; the noncompetitive mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)pyridine, SIB-1893, and SIB-1757; and the potent mGlu2/3 receptor agonists, LY354740 and LY379268. Pharmacologic blockade of mGlu1 or mGlu5 receptors or pharmacologic activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of N-methyl-d-aspartate (NMDA) receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes. A series of observations suggest a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer disease. MGlu2/3 receptor agonists inhibit glutamate release, but also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of CNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders. The advantage of all these drugs with respect to NMDA or AMPA receptor agonists derives from the evidence that mGlu receptors do not "mediate," but rather "modulate" excitatory synaptic transmission. Therefore, it can be expected that mGlu receptor ligands are devoid of the undesirable effects resulting from the inhibition of excitatory synaptic transmission, such as sedation or an impairment of learning and memory.