We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 M NaCl, and 6 M urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 M urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG

A highly potent and selective metabotropic glutamate receptor (mGluR) 1 antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2, 3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC), is described. FTIDC inhibits, with equal potency, L-glutamate-induced intracellular Ca 2ϩ mobilization in Chinese hamster ovary cells expressing human, rat, or mouse mGluR1a. The IC 50 value of FTIDC is 5.8 nM for human mGluR1a and 6200 nM for human mGluR5. The maximal response in agonist concentration-response curves was reduced in the presence of higher concentrations of FTIDC, suggesting the inhibition in a noncompetitive manner. FTIDC at 10 M showed no agonistic, antagonistic, or positive allosteric modulatory activity toward mGluR2, mGluR4, mGluR6, mGluR7, or mGluR8. FTIDC did not displace [ 3 H]L-quisqualate binding to human mGluR1a, indicating FTIDC is an allosteric antagonist. Studies using chimeric and mutant receptors of mGluR1 showed that transmembrane (TM) domains 4 to 7, especially Phe801 in TM6 and Thr815 in TM7, play pivotal roles in the antagonism of FTIDC. FTIDC inhibited the constitutive activity of mGluR1a, suggesting that FTIDC acts as an inverse agonist of mGluR1a. Intraperitoneally administered FTIDC inhibited face-washing behavior elicited by a group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine in mice at doses that did not produce motor impairment. Oral administration of FTIDC also inhibited the face-washing behavior. FTIDC is a highly potent and selective allosteric mGluR1 antagonist and a compound having oral activity without species differences in its antagonistic activity on recombinant human, mouse, and rat mGluR1. FTIDC could therefore be a valuable tool for elucidating the functions of mGluR1 not only in rodents but also in humans.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors, and they are thought to contribute to the fine-tuning of fast synaptic response, neuronal excitability, and neurotransmitter release. To date, eight mGluR subArticle, publication date, and citation information can be found at

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Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the specificity of GD3G7 toward various glycosaminoglycan preparations showed that this antibody specifically reacted with squid CS-E (rich in the GlcUA␤1-3GalNAc(4,6-O-sulfate) disaccharide unit E), hagfish CS-H (rich in the IdoUA␣1-3GalNAc(4,6-Osulfate) unit iE), and shark skin DS (rich in both E and iE units). In situ hybridization for the expression of N-acetylgalactosamine-4-sulfate 6-O-sulfotransferase in the postnatal mouse brain, which is involved in the biosynthesis of CS/DS-E, showed a widespread expression of the transcript in the developing brain except at postnatal day 7, where strong expression was observed in the external granule cell layer in the cerebellum. The expression switched from the external to internal granule cell layer with development. Immunohistochemical localization of GD3G7 in the mouse brain showed that the epitope was relatively abundant in the cerebellum, hippocampus, and olfactory bulb. GD3G7 suppressed the growth of neurites in embryonic hippocampal neurons mediated by CS-E, suggesting that the epitope is embedded in the neurite outgrowth-promoting motif of CS-E. In addition, a CS-E decasaccharide fraction was found to be the critical minimal structure needed for recognition by GD3G7. Four discrete decasaccharide epitopic sequences were identified. The antibody GD3G7 has broad applications in investigations of CS/DS chains during the central nervous system's development and under various pathological conditions.

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