Research ReportInhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate
Introduction
By rapidly taking up extracellular glutamate, astroglial cells play the critical role of protecting neurons from the excitotoxic buildup of glutamate, thereby ensuring the fidelity of glutamatergic transmission at high frequency (Danbolt, 2001). Astrocytic glutamate uptake also plays an important role in the coupling between synaptic activity and glucose utilization, i.e. neurometabolic coupling (Pellerin et al., 2007). Astrocytes are equipped with efficient glutamate transporters that surround the synaptic cleft and use the electrochemical gradient of Na+ to take up glutamate against its electrochemical gradient. In these cells, the Na+-coupled glutamate transport system is so efficient that the astrocytic intracellular Na+ concentration (Na+i) undergoes rapid and large elevations when glutamate is applied extracellularly, even at low micromolar concentration (Chatton et al., 2000). The bulk of glutamate uptake in the adult brain is mediated by astroglia that express mostly the excitatory amino acid transporter (EAAT) isoforms 1 and 2, whereas EAAT3 and EAAT4 are considered to be neuronal transporters, and EAAT5 is retina-specific (Danbolt, 2001).
Inhibitors of Na+-dependent glutamate transporters are therefore invaluable tools for elucidating the physiological roles of these transporters in detail. Earlier inhibitors of glutamate transporters, such as threo-β-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC), are transported competitive inhibitors that lead to astrocytic coupled Na+ influx (Chatton et al., 2001). A more recently synthesized compound, dl-threo-β-benzyloxyaspartate (TBOA), has been introduced and since then widely used as a non-transported competitive inhibitor of glutamate transporters (Shimamoto et al., 1998). This compound is a non-selective inhibitor of all EAATs subtypes with activity in the micromolar range. While this compound was a real breakthrough for the study of glutamate transport, its relatively low potency imposes the use of fairly high concentrations, increasing the risk of unwanted effects in particular when used in situ (Bernardinelli and Chatton, 2008), or when in vivo use is envisaged for instance as a possible treatment for mood disorder pathologies (Lee et al., 2007, Sanacora et al., 2003).
Recently, a series of analogues of TBOA have been reported with both improved potency and selectivity (Shimamoto et al., 2004). Among them, the most promising one appears to be TFB-TBOA with nanomolar affinity for EAATs. In the present study, we characterized the effects of TFB-TBOA on the Na+i response to glutamate in primary mouse astrocytes and on the electrical properties of pure cortical neurons in primary culture.
Section snippets
Results
Glutamate evokes a robust elevation of Na+i in mouse astrocytes in primary culture (Chatton et al., 2000) that is primarily due to Na+/glutamate cotransport activity. The first set of experiments was aimed at determining whether the new inhibitor of the glutamate transporter TFB-TBOA applied alone influenced baseline Na+i levels and at characterizing its inhibitory properties on the Na+i responses to glutamate application.
Fig. 1A shows an original experimental trace of the Na+i response to
Discussion
The aim of this report was to characterize the effects of the new glutamate transporter inhibitor TFB-TBOA on astrocytes in primary culture. As astrocytes are equipped with a high density of Na+-coupled glutamate transporter, Na+i concentration undergoes rapid and robust increases in these cells, which can be measured by microspectrofluorimetric methods (see e.g. Chatton et al., 2000, Rose et al., 1997). This Na+i increase is almost entirely attributable to the activity of the Na+-glutamate
Cell culture and solutions
Every effort was made to minimize suffering and the number of animals used in all experiments. In addition, all the procedures used to prepare living cells have been approved by the Swiss legislation and follows their guidelines. Primary cultures of mouse astrocytes were prepared as previously described (Chatton et al., 2000). After microdissection of cortices from 1 to 4 day-old C57bl6 mice, tissue was mechanically dissociated by successive aspirations through sterile syringes. The isolated
Acknowledgments
We gratefully acknowledge Guillaume Azarias, Christophe Lamy, and Anita Luthi for their comments on the manuscript and Steeve Menetrey for his excellent technical assistance. This study was supported by grant #3100A0-119827 from the Swiss National Science Foundation to JY Chatton.
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2016, NeuroscienceCitation Excerpt :This uptake mechanism was first demonstrated in cultured astrocytes, in which application of glutamate or its transportable agonist d-aspartate readily evoke substantial increases (>10 mM) in intracellular sodium concentration (Kimelberg et al., 1989; Rose and Ransom, 1996b; Chatton et al., 2000, 2001). These are suppressed by the glutamate uptake blocker TBOA (Chatton et al., 2001; Bernardinelli and Chatton, 2008) or its higher affinity version (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) (Tsukada et al., 2005; Bozzo and Chatton, 2010). Cytosolic sodium elevations induced by glutamate are accompanied by sodium signals in mitochondria as well, indicating a link between glutamate-evoked sodium signaling and mitochondrial function as discussed below (see chapter 4.
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