J. Neurosci. -- eLetters for Besser et al., 29 (9) 2890-2901

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Cellular:
Limor Besser, Ehud Chorin, Israel Sekler, William F. Silverman, Stan Atkin, James T. Russell, and Michal Hershfinkel
Synaptically Released Zinc Triggers Metabotropic Signaling via a Zinc-Sensing Receptor in the Hippocampus
J. Neurosci. 2009; 29: 2890-2901 [Abstract] [Full text] [PDF]

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Electronic letters published:

The Origin of Zinc
Katalin Toth (16 March 2009)

Re: The Origin of Zinc
Michal Hershfinkel (29 March 2009)

The Origin of Zinc 16 March 2009

Katalin Toth,
Associate Professor
Centre de recherche Universite Laval Robert Giffard, Quebec City, G1J 2G3, Canada
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Re: The Origin of Zinc

toth.katalin{at}crulrg.ulaval.ca Katalin Toth

In Besser et al. (2009) the authors claim that synaptically released zinc triggers metabotropic responses in the hippocampus. However, whether zinc is in fact released from synaptic vesicles remains equivocal (Kay and Tóth, 2008; Paoletti et al., 2009). Due to this contentious issue it is crucial to design and execute experiments that provide definitive proof of the origin of zinc responsible for the effects observed by the authors. There are several important issues that should be addressed to validate the authors’ interpretation that their results relate to zinc released from synaptic vesicles.
1. Ca2+ signals measured in CA3 pyramidal cells do not return to baseline values after the stimulus within the time frame examined. This puzzling observation stands in contrast to previously published data (Miller et al., 1996; Yeckel et al., 1999; Kapur et al., 2001) and may relate to the use of a cell-permeable Ca2+ dye. Previous studies used cell-impermeable dye delivered through a patch pipette to monitor Ca2+ signals in individual CA3 pyramidal cells. Cell-permeable Ca2+ indicators permeate membranes of intracellular organelles where they tend to accumulate (Connor, 1993; Gerasimenko and Tepikin, 2005), therefore fluorescent measurements might reflect signals originating from these cellular elements obscuring signals related to Ca2+ release.
2. Despite strikingly similar Ca2+ signals in wild-type and ZnT3 KO animals the authors claim that responses in KO tissue are significantly decreased. However, when ZnT3 KO traces from Fig. 7E are scaled to Fig 7C, there is no obvious difference between Ca2+ responses from KO and WT.
3. Mossy fibre inputs have not been identified pharmacologically or by any other criteria, generally used for experiments aiming to selectively activate mossy fibre inputs (Yeckel et al., 1999; Huang et al., 2008; Castillo et al., 1997; Toth et al., 2000). In a single slice the authors see responses in 23-26 CA3 pyramidal cells after bulk stimulation in the dentate gyrus. Such stimulation undoubtedly activates multiple fibres which can lead to polysynaptic recruitment of the CA3 auto-associative network. Such polysynaptic contamination combined with the long response delay makes it is impossible to conclude with any certainty that the Ca2+ signals observed result directly from mossy fibre activation.
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Re: The Origin of Zinc 29 March 2009

Michal Hershfinkel,
Researcher
Ben Gurion University
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Re: Re: The Origin of Zinc

hmichal{at}bgu.ac.il Michal Hershfinkel

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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