N-acetylaspartylglutamate (NAAG), a dipeptide derivative of N-acetylaspartate (NAA) and glutamate (Glu), is present in neurons. Upon neurostimulation, NAAG is exported to astrocytes where it activates a specific metabotropic Glu surface receptor (mGluR3), and is then hydrolyzed by an astrocyte-specific enzyme, NAAG peptidase, liberating Glu, which can then be taken up by the astrocyte. NAAG is a selective mGluR3 agonist, one of several mGluRs that, when activated, triggers Ca2+ waves that spread to astrocytic endfeet in contact with the vascular system, where a secondary release of vasoactive agents induces a focal hyperemic response providing increased oxygen and nutrient availability to the stimulated neurons. Changes in blood oxygen levels can be assessed in vivo using a blood oxygenation level-dependent (BOLD) magnetic resonance imaging technique that reflects a paramagnetic effect of deoxyhemoglobin. In this study we used the competitive NAAG peptidase inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) as a probe to interrupt the NAAG-mGluR3- Glu-astrocyte Ca2+ activation sequence. Using this probe, we investigated the relationship between release of the endogenous neuropeptide NAAG and brain blood oxygenation levels, as measured by changes in BOLD signals. In an anesthetized mouse, using an overtly nontoxic dose of 2-PMPA of 250 mg/kg i.p., there was an initial global BOLD signal increase of about 3% above control, lasting about 4 min, followed by a decrease from control of about 4%, sustained over a 32.5-min period of the drug test procedure. Similar changes, but of reduced magnitude and duration, were observed at a dose of 167 mg/kg. The 2-PMPA-induced decreases in BOLD signals appear to indicate that blood deoxyhemoglobin is elevated when endogenous NAAG cannot be hydrolyzed, thus linking the efflux of NAAG from neurons and its hydrolysis by astrocytes to hyperemic oxygenation responses in brain.

The data support a role for the endocannabinoid (EC) system in EtOH tolerance/dependence and suggest that drugs targeted against EC system could be therapeutically useful in treating alcohol-related disorders.

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