Unveiling the functions of presynaptic metabotropic glutamate receptors in the central nervous system

ABSTRACT Metabotropic glutamate (mGlu) receptors, which include mGlu1-8 receptors, are a heterogeneous family of G-proteincoupled receptors which function to modulate brain excitability via presynaptic, postsynaptic and glial mechanisms. Certain members of this receptor family have been shown to function as presynaptic regulatory mechanisms to control release of neurotransmitters. In general, Gi-coupled mGlu receptor subtypes appear to negatively modulate excitatory (and possibly also inhibitory) neurotransmitter output when activated. Localization studies have shown that mGlu7 is restricted to the presynaptic grid at the site of vesicle fusion. These studies along with other evidence suggest that mGlu7 is the nerve terminal autoreceptor that regulates physiological release of glutamate. Other mGlu subtypes, in particular mGlu2, mGlu8, and possibly mGlu4, are also localized presynaptically, but at perisynaptic sites outside the active zone of neurotransmitter release. Gi-coupled mGlu receptors also may exist on presynaptic elements of neighboring ␥-aminobutyric acid (GABA) neurons where they play a role in heterosynaptic suppressions of GABA release. This suggests that these receptors may have evolved to monitor glutamate that has "spilled" out of the synapse. Thus, they may serve as the brain's evolutionary mechanism to prevent pathological changes in neuronal excitability and thus maintain homeostasis. Recent progress on the molecular and pharmacological aspects of these presynaptic mGlu receptors is unveiling their functions and the therapeutic directions of agents designed for these novel glutamate receptor targets. In the past decade there has been considerable progress in the field of metabotropic or G-protein-coupled glutamate (mGlu) receptors. For the most part, the cloning and identification of a novel heterogeneous family of mGlu receptors has driven this progress. There are currently eight known subtypes of mGlu receptors, which have been classified into three groups (se

Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae

Microalgae have presented themselves as a strong candidate to replace diminishing oil reserves as a source of lipids for biofuels. Here we describe successful modifications of terrestrial plant lipid content which increase overall lipid production or shift the balance of lipid production towards lipid varieties more useful for biofuel production. Our discussion ranges from the biosynthetic pathways and rate limiting steps of triacylglycerol formation to enzymes required for the formation of triacylglycerol containing exotic lipids. Secondarily, we discuss techniques for genetic engineering and modification of various microalgae which can be combined with insights gained from research in higher plants to aid in the creation of production strains of microalgae

Surfactant-enhanced DNA accessibility to nuclease accelerates phenotypic β-lactam antibiotic susceptibility testing of Neisseria gonorrhoeae

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for β-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen’s slow doubling time and the lack of methods to quickly quantify the pathogen’s response to β-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the β-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to β-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15–30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats

Resource-Bound Quantification for Graph Transformation

Graph transformation has been used to model concurrent systems in software engineering, as well as in biochemistry and life sciences. The application of a transformation rule can be characterised algebraically as construction of a double-pushout (DPO) diagram in the category of graphs. We show how intuitionistic linear logic can be extended with resource-bound quantification, allowing for an implicit handling of the DPO conditions, and how resource logic can be used to reason about graph transformation systems

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [ 3 H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with ( R,S )-[ 3 H]Α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non- N -methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [ 3 H]6-cyano-7-nitro-quinoxaline-2,3-dione ([ 3 H]-CNQX) in rat brain sections. [ 3 H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [ 3 H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a K D = 67 ± 9.0 n M and B max = 3.56 ± 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, ( R,S )- Α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [ 3 H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [ 3 H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with K D1 = 9.0 ± 3.5 n M , B max = 0.15 ± 0.05 pmol/mg protein, K D2 = 278 ± 50 n M , and B max = 1.54 ± 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [ 3 H]CNQX binding sites correlated to the binding of L-[ 3 H]glutamate to quisqualate receptors and to sites labeled with [ 3 H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [ 3 H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [ 3 H]AMPA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65634/1/j.1471-4159.1990.tb01925.x.pd

Measurement of Receptor-Activated Phosphoinositide Turnover in Rat Brain: Nonequivalence of Inositol Phosphate and CDP-Diacylglycerol Formation

Two methods for the measurement of receptor-activated phosphoinositide turnover were evaluated for their degree of correspondence in slices of rat brain; they involved the Li + -dependent accumulations of either [ 3 H]-inositol-labeled inositol phosphates or [ 3 H]cytidine-labeled CDP-diacylglycerol. In contrast to the expectation that the ratio of these two responses would remain approximately constant, varying degrees of correspondence were obtained. The two extremes are exemplified by carbachol, which elicited large increases in both inositol phosphate and CDP-diacylglycerol labeling, and endothelin, which gave a robust inositol phosphate response with little or no accumulation of 3 H-CDP-diacylglycerol. No instance of the presence of the latter response in the absence of 3 H-inositol phosphate accumulation was observed. Measurement of 3 H-CDP-diacylglycerol accumulation thus may add additional insight into the regulation of phosphoinositide turnover and the complex actions of Li + .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66135/1/j.1471-4159.1993.tb03258.x.pd

Contribution of Cystine-Glutamate Antiporters to the Psychotomimetic Effects of Phencyclidine

Altered glutamate signaling contributes to a myriad of neural disorders, including schizophrenia. While synaptic levels are intensely studied, nonvesicular release mechanisms, including cystine–glutamate exchange, maintain high steady-state glutamate levels in the extrasynaptic space. The existence of extrasynaptic receptors, including metabotropic group II glutamate receptors (mGluR), pose nonvesicular release mechanisms as unrecognized targets capable of contributing to pathological glutamate signaling. We tested the hypothesis that activation of cystine–glutamate antiporters using the cysteine prodrug N-acetylcysteine would blunt psychotomimetic effects in the rodent phencyclidine (PCP) model of schizophrenia. First, we demonstrate that PCP elevates extracellular glutamate in the prefrontal cortex, an effect that is blocked by N-acetylcysteine pretreatment. To determine the relevance of the above finding, we assessed social interaction and found that N-acetylcysteine reverses social withdrawal produced by repeated PCP. In a separate paradigm, acute PCP resulted in working memory deficits assessed using a discrete trial t-maze task, and this effect was also reversed by N-acetylcysteine pretreatment. The capacity of N-acetylcysteine to restore working memory was blocked by infusion of the cystine–glutamate antiporter inhibitor (S)-4-carboxyphenylglycine into the prefrontal cortex or systemic administration of the group II mGluR antagonist LY341495 indicating that the effects of N-acetylcysteine requires cystine–glutamate exchange and group II mGluR activation. Finally, protein levels from postmortem tissue obtained from schizophrenic patients revealed significant changes in the level of xCT, the active subunit for cystine–glutamate exchange, in the dorsolateral prefrontal cortex. These data advance cystine–glutamate antiporters as novel targets capable of reversing the psychotomimetic effects of PCP

Translating Glutamate: From Pathophysiology to Treatment

The neurotransmitter glutamate is the primary excitatory neurotransmitter in mammalian brain and is responsible for most corticocortical and corticofugal neurotransmission. Disturbances in glutamatergic function have been implicated in the pathophysiology of several neuropsychiatric disorders—including schizophrenia, drug abuse and addiction, autism, and depression—that were until recently poorly understood. Nevertheless, improvements in basic information regarding these disorders have yet to translate into Food and Drug Administration–approved treatments. Barriers to translation include the need not only for improved compounds but also for improved biomarkers sensitive to both structural and functional target engagement and for improved translational models. Overcoming these barriers will require unique collaborative arrangements between pharma, government, and academia. Here, we review a recent Institute of Medicine–sponsored meeting, highlighting advances in glutamatergic theories of neuropsychiatric illness as well as remaining barriers to treatment development.National Institute of Mental Health (U.S.) (grant R37MH49334)National Institute of Mental Health (U.S.) (Intramural Research Program)National Institute of Mental Health (U.S.) (R01DA03383)National Institute of Mental Health (U.S.) (P50MH086385)National Institutes of Health (U.S.)FRAXA Research FoundationHoward Hughes Medical InstituteSimons Foundatio