Synaptically Released Zinc Triggers Metabotropic Signaling via a Zinc-Sensing Receptor in the Hippocampus

The hypothesis that the free-Zn2+ contained in synaptic vesicles is co-released with glutamate has been evaluated and confirmed by numerous studies using a variety of approaches; from earlier work using 65Zn or atomic absorption spectrometry (Assaf and Chung, 1984; Howell et al., 1984; Aniksztejn et al., 1987), to more recent studies in which Zn2+- sensitive fluorescent dyes were employed (Thompson et al., 2000; Li et al., 2001; Ueno et al., 2002; Qian and Noebels, 2005; Frederickson et al., 2006).

Of particular note and significance to our work, Qian and Noebels (2005) elegantly demonstrated that Zn2+ rise is detected with the glutamate release following mossy fiber (MF) stimulation. Importantly, they showed that synaptic Zn2+ is a cotransmitter released with glutamate under physiological conditions. This directly attests to the relevance of this process in neuronal signaling in the CA3 region. Our results demonstrate, for the first time, a unique target for synaptically Zn2+, the ZnR, which is activated following MF stimulation. Additionally, we identified a molecular moiety, GPR39, that putatively mediates ZnR signaling.

1. The studies cited by Toth focus on the response of single neurons using approaches appropriate for the aims of those studies. In contrast, we employed bulk loading of the Ca2+-sensitive dye Fura-2, in order to monitor cytoplasmic Ca2+ signals in populations of neurons (Beierlein et al., 2002). As a result, we show prolonged signals representing the average of many cells, responding at slightly different times. Application of an agonist of the group I mGluR induced a Ca2+ response similar to that triggered by Zn2+.

The metabotropic nature of the ZnR response is strongly supported by the controls we used, among them, depletion of intracellular Ca2+ stores as well as the Gq and PLC inhibitors. Therefore, the most likely mechanism for the observed Zn2+-dependent signaling is a metabotropic Gq-coupled receptor.

2. The Ca2+ response is the delta of the peak from the baseline obtained prior to stimulation. In ZnT3 KO mice, Fig. 7E, this response was approximately 50% of that obtained in WT mice, Fig. 7C. The average of the responses is shown in the bar graph of Fig. 7F in which the scale differs from that of Fig. 7D.

3. Attenuation of the Ca2+ signal in the ZnT3 KO mice, or following application of the extracellular Zn2+ chelator in WT animals, indicates that the metabotropic signaling is largely mediated by synaptically- released Zn2+, demonstrated to occur within and to be released from the mossy fibers. The protocol we employed to trigger this release is well established (Qian and Noebels, 2005). Thus, the possibility that other pathways might have been activated in our system in no way alters the conclusion that synaptic Zn2+ induces the ZnR-metabotropic response in CA3 neurons.

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