Research ReportDistribution of metabotropic glutamate 2 and 3 receptors in the rat forebrain: Implication in emotional responses and central disinhibition
Introduction
Excessive activation of limbic brain regions, such as the basolateral nucleus of the amygdala and the hippocampus, is thought to be responsible for many symptoms of chronic stress and anxiety disorders (McEwen, 2005). Glutamate is believed to play an important role in this activation. Thus, the Gi/o-coupled group II metabotropic glutamate receptors (mGlus) have been targeted as a novel approach for the treatment of anxiety disorders. Since this approach bears a different mechanism of action than the classical benzodiazepines (Tizzano et al., 2002), it is expected that the new therapy will avoid the undesirable side effects associated with benzodiazepine treatment, which include sedation and drug dependence. In the rat and mouse, non-selective mGlu2/3 agonists are found to be efficacious in a number of anxiety models, such as blocking the expression of fear-potentiated startle, increasing the open-arm time in elevated plus maze, and reducing stress-induced hyperthermia (Monn et al., 1997, Helton et al., 1998, Ferris et al., 2001, Spooren et al., 2002, Swanson et al., 2005). The mGlu2/3 agonist LY354740 can also block the expression of fear-potentiated startle and CO2-induced anxiety in humans (Grillon et al., 2003, Schoepp et al., 2003), and successfully alleviate symptoms in patients with general anxiety disorder (Schoepp, 2004, Dunayevich et al., 2007).
The exact site of action of the mGlu2/3 agonists is not clear. Based on c-Fos activation studies, it has been hypothesized that these agonists elicit their therapeutic effect by decreasing excitation and enhancing inhibition in pathways associated with anxiety (Linden et al., 2004, Swanson et al., 2005). However, due to high homology of the mGlu2 and the mGlu3 receptors, the existing agonists bind to both receptor subtypes. Thus, it is unclear whether the effects of mGlu2/3 agonists on anxiety are mediated through mGlu2 or mGlu3, or if activation of both receptors is necessary to achieve efficacy. While development of mGlu2 and mGlu3 selective compounds is needed to clarify roles of these receptors in anxiety, detailed mapping of their expression in the brain will be helpful in exploring which receptor is involved in the therapeutic effects of non-selective agonists.
The expression of mGlu2 and mGlu3 mRNA and/or proteins in the CNS has been previously reported (Testa et al., 1994, Ohishi et al., 1993a, Ohishi et al., 1993b, Ohishi et al., 1998, Petralia et al., 1996, Tamaru et al., 2001, Crook et al., 2002). In these studies, the receptor subtype distribution is largely based on in situ hybridization experiments due to the limited availability of convincing subtype-selective antibodies. In the present study, we have re-examined and compared the detailed mGlu2 and mGlu3 mRNA distribution in the rat forebrain, with an emphasis on regions that are associated with anxiety responses. Moreover, we have further characterized and confirmed the selectivity of one antibody against mGlu2 that was previously reported (Neki et al., 1996, Ohishi et al., 1998). Using this mGlu2-selective antibody, we have mapped the mGlu2 protein expression in the pre- and postsynaptic components in the rat forebrain. To explore the possible function of mGlu3 in the thalamus, we have also investigated the effects of the mixed mGlu2/3 agonist LY379268 on GABA release from the GABAergic reticular nucleus of the thalamus, a region that expresses a high level of mGlu3 but not mGlu2.
Section snippets
Distribution of mGlu2 and mGlu3 mRNA in the forebrain
Although mGlu2 and mGlu3 belong to the same molecular family and have the highest homology within the family, the distribution of mGlu2 mRNA differed remarkably from that observed with the mGlu3 probes. These results, together with the observation that both mGlu2 and mGlu3 sense probes did not produce any specific detectable signals, suggest that the mGlu2 signals seen with the antisense probes are specific.
mGlu2 mRNA was distinctively expressed in the rat forebrain, especially in the limbic
Discussion
It has been demonstrated that among all metabotropic glutamate receptors, only mGlu2, mGlu3, mGlu5 and mGlu7 receptors are highly expressed in the rodent forebrain (Shigemoto and Mizuno, 2000). Our results confirm previous reports on the cellular localization of mGlu2 and mGlu3 receptors in the rat forebrain (Ohishi et al., 1993a, Ohishi et al., 1993b, Ohishi et al., 1998). Moreover, using confocal microcopy, our data provide direct evidence that mGlu2 receptors are extensively expressed in
Experimental procedures
All protocols regarding animal use were approved by the Merck Research Laboratory San Diego Institutional Committee for the Care and Use of Animals in Research and Education, in accordance with the guidelines of the National Institute of Health (NIH Publications No. 80-23) revised in 1996 and United States Department of Agriculture. All efforts were made to minimize the number of animals used and their suffering.
Acknowledgments
We thank Dr. Shigemoto for generously providing the monoclonal mGlu2 antibody.
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