Title

 

Authors

M. Summa^{*  
 
*}Deptof Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy  

 

Abstract

We have recently characterized a population of hippocampal NMDA autoreceptors able to elicit glutamate exocytosis (Luccini et al. 2007). Our study was carried out by applying NMDA plus glycine in the absence of Mg²⁺ ions, to overcome the cation-mediated occlusion of the NMDA associated ionic channel. It has to be reminded, however, that under physiological conditions, extracellular solutions do contain Mg²⁺ ions while hypomagnesia is positively correlated to some pathological conditions in CNS (Benga et al. 1985; Michaelis and Michaelis 1994). To better understand the function of NMDA autoreceptors under more physiological conditions, but also considering that hippocampal NMDA autoreceptor ionic profiles differ from presynaptic NMDA heteroreceptors in terms of Na⁺ dependence (Breukel et al. 1998) and Mg²⁺ sensitivity (Jourdain et al. 2007), we investigated the sensitivity of NMDA autoreceptors to Mg²⁺ ions. Our results indicate that rat hippocampal glutamatergic terminals possessNMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis also in the presence of physiological Mg²⁺ (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg²⁺-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg²⁺, while MK801, CGS19755 and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [³H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg²⁺ strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker NFPS, but not the GlyT2 blocker Org25534, prevented it. Accordingly, [³H]glycine was taken up during superfusion, while lowering the external concentration of Na⁺, the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western Blot analysis of sub-synaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the presynaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA presynaptic autoreceptors.
 
This work was supported by grants from “Italian Ministero dell’Istruzione, dell’Università e della Ricerca Scientifica” and from University of Genoa ‘Progetto Ricerca Ateneo’