06-676 Sigma-AldrichAnti-mGluR2/3 Antibody

As part of our ongoing small-molecule metabotropic glutamate (mGlu) receptor positive allosteric modulator (PAM) research, we performed structure-activity relationship (SAR) studies around a series of group II mGlu PAMs. Initial analogues exhibited weak activity as mGlu2 receptor PAMs and no activity at mGlu3. Compound optimization led to the identification of potent mGlu2/3 selective PAMs with no in vitro activity at mGlu1,4-8 or 45 other CNS receptors. In vitro pharmacological characterization of representative compound 44 indicated agonist-PAM activity toward mGlu2 and PAM activity at mGlu3. The most potent mGlu2/3 PAMs were characterized in assays predictive of ADME/T and pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu2/3 PAMs. On the basis of its overall profile, compound 74 was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in rats after intraperitoneal administration. These mGlu2/3 receptor PAMs have significant potential as small molecule tools for investigating group II mGlu pharmacology.

Glutamatergic neurotransmission plays an important role in the behavioral and molecular plasticity observed in cocaine mediated locomotor sensitization. Recent studies show that glutamatergic signaling is regulated by receptor trafficking, synaptic localization, and association with scaffolding proteins. The trafficking of the glutamate receptors was investigated in the dorsal and ventral prefrontal cortex at 1 and 21 days after repeated cocaine administration which produced robust locomotor sensitization. A subcellular fractionation technique was used to isolate the cellular synaptosomal fraction containing the postsynaptic density. At early withdrawal, the prefrontal cortex displayed a reduction in the synaptosomal content of the AMPA and NMDA receptor subunits. In contrast, after extended withdrawal, there was a significant increase in the trafficking of the receptors into the synaptosomal compartment. These changes were accompanied by corresponding trafficking of the postsynaptic glutamatergic scaffolding proteins. Thus, enhanced trafficking of glutamate receptors from cytosolic to synaptosomal compartment is associated with prolonged withdrawal from repeated exposure to cocaine and may have functional consequences for the synaptic and behavioral plasticity.

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 (Shigemoto et al,., 1996), 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.

Glutamate neurotransmission involves numerous ionotropic and metabotropic glutamate receptor types in postsynaptic, presynaptic and glial locations. Distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was studied with an affinity-purified, characterized polyclonal antibody made from a C-terminus peptide. This antibody, mGluR2/3, recognized both mGluR2 and mGluR3, but did not cross-react with any other type of metabotropic glutamate receptor except for a very slight recognition of mGluR5. Light microscope distribution of the antibody binding sites in the nervous system matched the combined distributions of messenger RNA for mGluR2 and mGluR3. For example, dense staining seen in the accessory olfactory bulb and cerebellar Golgi cells matched high levels of mGluR2 messenger RNA in these structures, while moderately dense staining in the reticular nucleus of the thalamus and light to moderate staining in glia throughout the brain matched significant levels of mGluR3 messenger RNA in these structures. In the rostral olfactory structures, the densest stained neurons belonged to presumptive "necklace olfactory glomeruli." In the hippocampus, staining was densest in the neuropil of the stratum lucidum/pyramidale, stratum lacunosum/moleculare, hilus and middle third of the molecular layer of the dentate gyrus. Ultrastructural studies of the cerebral cortex, hippocampus and caudate-putamen revealed significant staining in postsynaptic and presynaptic structures and glial wrappings of presumptive excitatory synapses; frequently, this staining was concentrated in discrete patches at or near active zones. In the hippocampus, presynaptic staining appeared to be concentrated in terminals of two populations of presumptive glutamatergic axons: mossy fibers originating from granule cells and perforant path fibers originating from the entorhinal cortex. These data suggest that populations of mGluR2 and/or mGluR3 receptors are localized differentially in synapses, i.e. those in and near the postsynaptic and presynaptic membranes and in glial wrappings of synapses, in several regions of the brain. In addition, we provide immunocytochemical evidence of mGluR2 or mGluR3 receptors in presynaptic terminals of glutamatergic synapses. Thus, mGluR2 and mGluR3 are found in various combinations of presynaptic, postsynaptic and glial localizations that may reflect differential modulation of excitatory amino acid transmission.

The dorsal cochlear nucleus (DCN) is a major brain center for integration of auditory information, and excitatory amino acid neurotransmission plays a central role in the processing of this information. In this study, the distribution of glutamate receptors was examined with preembedding immunocytochemistry, using 14 antibodies to ionotropic (GluR1, GluR2/3, GluR4, GluR5-7, GluR6/7, KA2, NR1, NR2A/B, delta 1/2) and metabotropic (mGluR1 alpha, mGluR2/3, mGluR5) glutamate receptor subtypes. Each of these antibodies produced a specific immunolabeling pattern, including a variety of postsynaptic, presynaptic, and glial localizations. Some antibodies showed widespread distribution patterns, notably the antibodies to the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits, GluR2 and GluR3, and the N-methyl-D-aspartate (NMDA) receptor subunit, NR1. In contrast, antibodies to other glutamate receptor subunits produced more restricted distribution patterns, especially that to GluR1, which stained the outer neuropil of the DCN, cartwheel cells, and a small population of presumptive interneurons associated with the dorsal acoustic stria, but produced little or no staining in fusiform cells or deep DCN neurons. Staining of the postsynaptic density and membrane of the granule cell-parallel fiber/cartwheel cell spins synapse was most prevalent with delta 1/2 and mGluR1 alpha antibodies. A unique pattern of staining was found with mGluR2/3 antibody--with staining concentrated in Golgi cells and unipolar brush cells of the middle to deep DCN. Distribution of some glutamate receptors in the DCN shows similarities to that of the cerebellum, where delta 2 and mGluR1 alpha may modulate neurotransmission at parallel fiber synapses, while mGluR2 and/or mGluR3 may modulate mossy terminal function.

Glutamic acid is the major excitatory amino acid of the central nervous system which interacts with two receptor families, the ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are coupled to G proteins and can be divided into three subgroups based on their sequence homology, signal transduction pathway and pharmacology. In this study, we describe the cloning of the cDNA encoding the human metabotropic glutamate receptor type 3 (HmGluR3). It was obtained by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to highly conserved sequences between rat mGluRs. The receptor shows 879 amino acids with 96% amino acid sequence identity with rat mGluR3. It is strongly expressed in fetal and adult whole brain, especially in caudate nucleus and corpus callosum. The gene was identified by fluorescence in situ hybridization on chromosome 7 band q22. Activation of the human mGluR3, permanently expressed in Baby Hamster Kidney (BHK) cells, by excitatory amino acid inhibits the forskolin-stimulated accumulation of intracellular cAMP. The rank order of potency is L-glutamic acid > or = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD) >> ibotenic acid > quisqualic acid. (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mM] is without effect on inhibition of forskolin-induced cAMP accumulation by L-glutamic acid.