Simultaneous monitoring of FluoZin-3 and Fura-2FF fluorescence
Figure 1 illustrates the data generation and processing method used in this report. At the beginning of each experiment, the neuronal morphology of the cells was verified using the Hoffman modulation contrast (Fig. 1a, upper panel). At the end of each experiment, the maximal Fura-2FF F340/F380 ratio (Rmax) was measured in each cell after saturating Fura-2FF with Ca2+ (Ca saturation), which was followed by measuring the maximal FluoZin-3 F488 signal (Fmax) after saturating FluoZin-3 with Zn2+ (Zn saturation). The lower panel of Fig. 1(a) shows a F488 image of the cells captured during Zn saturation. The cells were exposed for 4 min to 100 μM glutamate and 10 μM glycine (Glu/Gly) under Na+-and Zn2+-free conditions (Na+ substituted with NMDG+ and extracellular zinc chelated by CaEDTA). Upon Glu/Gly application, both F340/F380 ratio and F488 began to increase. When 10 μM TPEN (a plasma membrane-permeable chelator) was added to chelate the intracellular zinc, F488 promptly dropped in all cells but the response in F340/F380 ratio varied greatly among the cells. In 50 neurons tested (three experiments), the pattern of F340/F380 ratio response could be divided into three categories: category 1 (17 neurons, 34%) – the F340/F380 ratio failed to drop or kept increasing; category 2 (14 neurons, 28%) – the F340/F380 ratio initially dropped but then started to increase; category 3 (19 neurons, 38%) – the F340/F380 ratio dropped and remained low. Four exemplar cells representing the three categories are indicated in the lower panel of Fig. 1(a), and data from these cells are presented in Fig. 1(c). Cells A (red) and B (green) represent category 1, cell C (blue) represents category 2, and cell D (orange) category 3.
Figure 1. Simultaneous monitoring of FluoZin-3 and Fura-2FF fluorescence in single hippocampal neurons. (a) Upper panel, Hoffman modulation contrast image of hippocampal neurons cultured for 14 days. Bar = 100 μm. Lower panel, F488 fluorescence during Zn saturation in the same neurons. Indicated A (red), B (green), C (blue), D (orange) are four exemplar neurons that are discussed in the text. (b) Fura-2FF F340/F380 ratio and Fluo-Zin-3 fluorescence (F488) data from all 14 neurons shown in (a). The neurons were exposed to 100 μM glutamate and 10 μM glycine (Glu/Gly) under Na+-free conditions with Na+ substituted with N-methyl-d-glucamine (NMDG+) and in the presence of 1 mM CaEDTA to chelate extracellular Zn2+. Where indicated, 10 μM TPEN was added to chelate intracellular Zn2+. At the end of the experiment, Fura-2FF was saturated with Ca2+ to measure the maximal F340/F380 ratio (Rmax) followed by saturation of FluoZin-3 with Zn2+ to measure the maximal F488 fluorescence (Fmax). (c) Fluorescence data from the four exemplar cells indicated in (a). (d) Normalized Fura-2 FF and FluoZin-3 data expressed as a percentage of Rmax and Fmax, respectively.
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As the Glu/Gly-induced FluoZin-3 signal increase took place in the absence of extracellular Zn2+ (in the presence of 1 mM CaEDTA), the [Zn2+]i elevations represent Zn2+ release from intracellular stores (Fig. 1c, lower panel). Dineley et al. (2008) recently reported that such an intracellular Zn2+ release depends on the Glu/Gly-induced Ca2+ influx. Therefore, one would expect that cells showing a faster rate and/or onset of intracellular Ca2+ concentration ([Ca2+]i) (F340/F380 ratio) elevation would show a faster rate of [Zn2+]i (F488) increase. However, looking at the raw FluoZin-3 F488 data, this was not the case. Note that in cell A (red) and cell B (green), there is an F340/F380 ratio data overlap, which suggests that [Ca2+]i increases in both cells at the same rate (Fig. 1c, upper panel). However, the rate of F488 increase is much faster in cell B than in cell A (Fig. 1c, bottom panel), suggesting that more Zn2+ is released in cell B. On the other hand, the F488 traces in cells A, C, and D overlap (Fig. 1c, bottom panel) despite the very different patterns of F340/F380 ratio increases in these cells (Fig. 1c, upper panel), suggesting that the rates of [Zn2+]i and [Ca2+]i elevations in these cells are independent from each other. Interestingly, Fmax values measured during Zn saturation greatly differed among the cells. For example, Fmax was much higher in cell B than in cell A (Fig. 1c, bottom panel). As FluoZin-3 fluorescence intensity during Fmax measurement is a function of FluoZin-3 concentration, the data indicate that cell B loaded more FluoZin-3 than cell A. Such uneven FluoZin-3 loading among the cells creates an artifact. The cells that loaded more of the indicator elevated F488 at a faster rate, which could be misinterpreted to indicate that [Zn2+]i elevates faster in these cells. This artifact could be normalized for by expressing F488 data as a percentage of Fmax. After such normalization, the rates of FluoZin-3 fluorescence increase could be related to the onset and/or rate of [Ca2+]i increase, the fastest in cells A and B and the slowest in cell D (Fig. 1d).
In neurons in which Glu/Gly-induced elevations of Fura-2FF signal were small (did not exceed 30% of Rmax), TPEN applications promptly decreased the signal, indicating that Zn2+ rather than Ca2+ contributed to the signal (for example cells C and D in Fig. 1c, upper panel). As in this case the Fura-2FF signal increase during Glu/Gly application represented primarily [Zn2+]i elevations, no attempt was made to calibrate the Fura-2FF data in terms of [Ca2+]i. Instead, to relate the Fura-2FF signal to the maximal signal, the F340/F380 data were expressed as a percentage of Rmax (Fig. 1d, upper panel).When Zn saturation was performed after Ca saturation, the Fura-2FF F340/F380 signal dropped to about 30% of Rmax (Fig. 1b–d, upper panel). This drop was expected because Devinney et al. (2005) already demonstrated that Zn2+ and Ca2+ affect the Fura-2FF excitation spectrum differently, which is confirmed by the data shown in Figure S1a. The F340/F380 ratio of Fura-2FF saturated with Zn2+in vitro (Figure S1b), similarly as the one measured in vivo (Fig. 1d, upper panel), can reach only about 30% of the ratio measured when the indicator is saturated with Ca2+. These data suggest that when the Fura-2FF signal exceeds 30% of Rmax, the signal results primarily from Ca2+ binding to the indicator. This can explain why Zn2+ chelation with TPEN fails to affect the signal when the latter exceeds 30% of Rmax (Fig. 1c, cells A and B).
The expression of FluoZin-3 data as a percentage of Fmax does not take into account additional artifacts associated with the use of a non-ratiometric [ion] indicator. Namely, fluorescence intensity is also affected by cell swelling/shrinking or dye bleaching or leakage from the cells during experimentation. Such artifacts can be accounted for by measuring fluorescence at the isosbestic point excitation of a ratiometric indicator, which provides a measure of indicator concentration independent of intracellular [ion]. Using the approach described in the Methods, the Ca2+ isosbestic point of Fura-2FF excitation was determined to be 356 nm (Figure S1a). To account for the artifacts associated with cell swelling, dye leakage, or bleaching, we considered expressing FluoZin-3 fluorescence data as a FluoZin-3 F488/Fura-2FF F356 ratio. This method would generate valid results only if both indicators are loaded, metabolized, and/or leaked at the same rates. If this were the case, there would be a very good correlation between FluoZin-3 Fmax and Fura-2FF F356 measured during Zn saturation. Figure 2 shows that although on average the cells that loaded more FluoZin-3 also loaded more Fura-2FF, the correlation between Fura-2FF F356 and FluoZin-3 Fmax was not perfect, r2 = 0.483. Therefore, we abandoned the idea of using FluoZin-3 F488/Fura-2FF F356 ratio as an index of [Zn2+]i.
Figure 2. Correlation between FluoZin-3 Fmax and Fura-2FF fluorescence excited at the Ca2+ isosbestic point (F356) and measured during Zn saturation in 47 hippocampal neurons (three coverslips).
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Glu/Gly-induced [Zn2+]i elevations depend on Ca2+ and Na+
Free Zn2+ available to the high affinity Zn2+ indicator FluoZin-3 in Locke’s buffer was estimated to be 38 nM (for details see Methods). However, the extracellular Zn2+ did not affect the Glu/Gly-induced [Zn2+]i elevations because chelation with 1 mM CaEDTA had no effect on Glu/Gly-induced elevations of the FluoZin-3 signal (Fig. 3a,c,f). Glu/Gly-induced [Zn2+]i and [Ca2+]i elevations were eliminated by preventing Ca2+ influx (Fig. 3b and e), which confirms the recent data of Dineley et al. (2008), indicating that exposure of neurons to Glu/Gly causes Zn2+ release from intracellular stores and that the Zn2+ release is triggered by Ca2+ influx. The Glu/Gly-induced [Ca2+]i elevations were greatly enhanced when extracellular Na+ was substituted with NMDG+ (Fig. 3a and e). This outcome was expected, as under such conditions the Glu/Gly-induced destabilization of Ca2+ homeostasis is exacerbated by a number of factors. These factors include a lack of Ca2+ extrusion by plasmalemmal Na+/Ca2+ exchangers, a lack of competition between Na+ and Ca2+ for NMDA and other Na+- and Ca2+-permeable channels, and an increased electrochemical driving force for Ca2+ influx because of a lack of Na+-dependent depolarization of the plasma membrane (Mattson et al. 1989; Kiedrowski 1999). Interestingly, when Na+ was present in the medium, the Glu/Gly-induced elevation of FluoZin-3 F488 signal was also greatly decreased (Fig. 3d–f), and further experiments were designed to address the mechanisms responsible for this outcome.
Figure 3. Glu/Gly-induced [Zn2+]i elevations are Ca2+- and Na+-dependent. The effects of Glu/Gly on Fura-2FF (red) and FluoZin-3 (blue) fluorescence in hippocampal neurons were monitored as explained in Fig. 1, under Na+-free conditions with Na+ substituted with NMDG+ (a–c) or in the presence of 158 mM Na+ (d). In (a) and (d), 1.3 mM CaCl2 was present in the medium. In (b), Ca2+ was omitted from the medium and 0.1 mM EGTA was added. In (c), the medium contained 1.3 mM CaCl2 and 1 mM CaEDTA. The y-axes and the 5-min time bar apply to all panels. The data are means ± SE from the indicated number of neurons monitored in a single experiment. All experiments were repeated at least three times with similar results. Panels (e) and (f) show Fura-2FF and FluoZin-3 signals, respectively, measured at the end of the 5th minute of Glu/Gly application. The data are means ± SEs from 47 to 99 individual neurons (three to five separate experiments). **p < 0.01 vs. Na+-free conditions; Kruskal–Wallis one-way anova on Ranks followed by Dunn’s test.
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Na+-dependent [Zn2+]i clearance
Substitution of NMDG+ with Na+ during Glu/Gly applications caused a prompt decrease of the FluoZin-3 F488 signal (Fig. 4a, blue trace), whereas the response in Fura-2FF F340/F480 ratio greatly varied among the cells (Fig. 4a, red trace). The Na+-induced F488 decrease represented a [Zn2+]i decrease rather than a signal decrease due to cell swelling or dye leakage because a simultaneously monitored Fura-2FF F356 signal, the Ca2+ isosbestic point, was not affected (Fig. 4a, green trace). Whereas the Fura-2FF F356 signal was not affected by Na+, there was a marked, 30–40%, drop in this signal during the first 4 min of Glu/Gly application. As [Zn2+]i was increasing during that time (Fig. 4a, blue trace), one might be concerned that the drop in F356 was caused by Zn2+ binding to Fura-2FF. Such a decrease in F356 signal because of Zn2+ binding to Fura-2FF is unlikely, however, because the Zn2+ isosbestic point of Fura-2FF was found to be 364 nm (Figure S1a). Consequently, Zn2+ binding to Fura-2FF increases the fluorescence excited at the wavelengths lower than 364 nm, namely the F356. As the latter was decreasing, the decrease could not be caused by Zn2+ binding to the indicator. To further verify this line of reasoning, it was tested whether Zn2+ chelation with TPEN affects the F356 signal in neurons exposed to Glu/Gly. As shown in Fig. 4(b), TPEN failed to affect Fura-2FF F356 signal (green trace) but as expected, caused a prompt drop in the FluoZin-3 F488 signal (blue trace). The data confirmed that the drop in the F356 signal during Glu/Gly exposure could not be caused by Zn2+ binding to Fura-2FF.
Figure 4. Effects of Na+ and TPEN on FluoZin-3 F488 (blue), Fura-2FF F340/F380 ratio (red), and the Fura-2FF Ca2+ isosbestic point, F356 (green). (a) Hippocampal neurons co-loaded with FluoZin-3 and Fura-2FF were exposed to Glu/Gly under Na+-free conditions (Na+ substituted with NMDG+) followed by an application of 158 mM Na+ and removal of NMDG+. The data are means ± SE from 17 neurons monitored in a single experiment. The experiment was repeated five times with similar results. (b) The cells were treated as in (a) except that 10 μM TPEN was applied instead of Na+. The data are means ± SE from 22 neurons monitored in a single experiment. The experiment was repeated five times with similar results.
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It was then considered that the F356 decrease could be caused by the major pHi drop taking place under such conditions (Kiedrowski 1999) and the impact of pH on the Fura-2FF F356 signal was examined. To this end, excitation spectra of Fura-2FF at pH 7.4 and 5.5 in vitro were obtained. The data show that the Fura-2FF fluorescence intensity excited at 356 nm was slightly higher at pH 5.5 than at pH 7.4 (Fig. 5), although the pH drop greatly compromised the ability of Fura-2FF to report [Ca2+] increase (Fig. 5, inset). As the F356 signal was not decreased by the pH drop, the data argue against the idea that the decrease of the F356 signal during the first 4 min of Glu/Gly application (Fig. 4a and b) could be caused by a pHi decrease.
Figure 5. Excitation spectra of Fura-2FF solution under nominally Ca2+-free conditions (-Ca, blue), in the presence of 30 μM CaCl2 (+Ca, red), at pH 7.4 (solid lines), and pH 5.5 (dashed lines). Note that acidification does not decrease the isosbestic point fluorescence (F356). Inset, acidification compromises the ability of Fura-2FF to report [Ca2+] increase. To calculate the F340/F380 ratio the data shown in the main figure were used. Note that the F340/F380 ratio is greatly increased by 30 μM Ca2+ (Ca+) at pH 7.4 but not at pH 5.5. The assay was repeated three times with similar results.
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Most likely, the F356 signal decrease represents a Fura-2FF leakage. Similar leakage was observed by others (Vander Jagt et al. 2008; Medvedeva et al. 2009). It appears that this leakage occurs through pannexin-1 hemichannels which open following NMDA receptor activation and permeate a number of large molecules, including calcein (Thompson et al. 2008). One may envision that FluoZin-3 also leaks through these channels. Although such FluoZin-3 leakage is not apparent from the data (Fig. 4a and b), one has to consider that the basal FluoZin-3 F488 was so low in these experiments that any additional decrease of the signal could not be detected. Moreover, when [Zn2+]i starts to elevate, the detection of any F488 signal drop because of FluoZin-3 leakage is complicated by the simultaneous F488 signal increase because of Zn2+ binding to the indicator.
Additional experiments were performed to better understand the relationships between [Ca2+]i and [Zn2+]i changes during Glu/Gly applications. In these experiments, the neurons were exposed to Glu/Gly for 20 min. During the first 8 min, the medium was Na+-free, during the next 4 min, Na+ (158 mM) was present in the medium, and during the last 8 min, the medium was Na+-free again. Figure 6(a) shows data from a representative experiment. As in the experiment shown in Fig. 1, there was a great variability in Fura-2FF signal changes (Fig. 6a, upper panel). The pattern of signal changes could be divided into four categories. Of 131 cells tested in seven experiments, in 53 cells (40%) the Fura-2FF signal increased all the way to Rmax during the first Na+-free period and remained at this level for the rest of the experiment (Fig. 6a, upper, red). In 20 cells (15%), the Fura-2FF signal increased (often to Rmax) during the first Na+-free period, but then decreased when Na+ was added and remained low during the second Na+-free period (Fig. 6a, upper, green). In 25 cells (19%), the Fura-2FF signal increased to variable levels during the first Na+-free period, then decreased when Na+ was added and increased again when Na+ was removed (Fig. 6a, upper, blue). In 33 cells (25%), the Fura-2FF signal showed a moderate (about 50% of Rmax) increase during the first Na+-free period, decreased when Na+ was added and remained low when Na+ was removed (Fig. 6a, upper, orange). To simplify the picture, average Fura-2FF and FluoZin-3 data from the representative experiment are shown in the right panel of Fig. 6(a). In over 53% of neurons, the Fura-2FF signal remained low (below 30% of Rmax) during the second Na+-free period (green and orange traces). The persistence of low [Ca2+]i in these cells could be explained in terms of a decreased Ca2+ influx, increased Ca2+ efflux, or both. As an increased Ca2+ efflux under Na+-free conditions (when plasmalemmal Na+/Ca2+ exchangers do not remove Ca2+ from the cells) is unlikely, the persistence of low [Ca2+]i can be best explained in terms of a decreased Ca2+ influx as the NMDA currents are known to run down (Rosenmund and Westbrook 1993; Li et al. 2002).
Figure 6. Effects of Na+ and DTDP on FluoZin-3 and Fura-2FF fluorescence, and on pHi. (a) The neurons were exposed to Glu/Gly and 1 mM CaEDTA under Na+-free conditions (Na+ substituted with NMDG+). Where indicated, 158 mM Na+ (Na) was transiently applied while NMDG+ was removed. The patterns of Fura-2FF fluorescence change could be divided into four color-coded categories that are discussed in the text. The left panel shows Fura-2FF and FluoZin-3 fluorescence changes in individual neurons and the right panel shows average fluorescence changes in neurons representing each category. The experiment was repeated seven times with similar results. (b) Experiment analogous to the one shown in (a) except that 100 μM DTDP was added during the second Na+-free period, where indicated. The data are means ± SE from 16 neurons. The experiment was repeated four times with similar results. (c) Experiment analogous to the one shown in (b) except that pHi (BCECF F488/F440 ratio) was monitored. The data are means ± SE from 23 neurons. The experiment was repeated three times with similar results.
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The bottom panel of Fig. 6(a) shows that the FluoZin-3 signal increased during the first Na+-free period and decreased following Na+ application, as expected. Interestingly, during the second Na+-free period, the FluoZin-3 signal remained low in all neurons despite the above discussed variability in the Fura-2FF signal among the neurons. As virtually all cells failed to increase [Zn2+]i during the second Na+-free period (Fig. 6a, bottom), it appears that either the intracellular store from which Zn2+ was released during the first Na+-free period had been depleted or that the mechanism involved in Zn2+ release is inactive during the second Na+-free period. To clarify this, it was tested whether an oxidizing agent, 2,2′dithiopyridine (DTDP) known to induce Zn2+ release from internal stores (Aizenman et al. 2000), is able to elevate [Zn2+]i during the second Na+-free period. As shown in Fig. 6(b), DTDP application promptly increased the FluoZin-3 signal, indicating that prior to DTDP addition the intracellular stores still contained Zn2+.
pHi increase plays a role in Na+-dependent [Zn2+]i clearance
A pHi drop is expected to promote Zn2+ release from thiols (Li et al. 1954) and other ligand types as well as promote Zn2+ efflux from organelles, for example from the Golgi apparatus, by activating H+/Zn2+ exchange (Ohana et al. 2009). Therefore, it was considered that the difference in [Zn2+]i elevation between the first and the second Na+-free period (Fig. 6b) could be explained in terms of a different pHi. To this end, using BCECF fluorescence, pHi was monitored under analogous experimental conditions. Glu/Gly application under Na+-free conditions profoundly decreased BCECF F488/F440 ratio, indicating a pHi decrease, and Na+ application caused the pHi to increase. However, during the second Na+-free period, the Glu/Gly-induced pHi drop was less profound than during the first Na+-free period (Fig. 6c). This result may be interpreted to indicate that the pHi decrease during the second Na+-free period was insufficient to elevate [Zn2+]i. No pHi drop was associated with DTDP application (Fig. 6c), confirming that the mechanism of [Zn2+]i elevation induced by this agent represents oxidation (Aizenman et al. 2000), not a pHi decrease.
To clarify the role of pHi in [Zn2+]i changes, the relationships between pHi and [Zn2+]i were studied in a greater detail. To this end, the effects of Na+ vs. Li+ and Cs+ on the rates of [Zn2+]i drop and pHi increase were compared. The efficacy of these cations in elevating pHi (Fig. 7a) and clearing [Zn2+]i (Fig. 7b) was similar, namely, Na+ > Li+ > Cs+. This result confirms that pHi fluctuations may play a role in the mechanism of [Zn2+]i changes. To further explore this possibility, gramicidin was used as an experimental tool. Gramicidin is an antibiotic that forms channels that permeate small monovalent but not divalent cations (Hladky and Haydon 1972) and therefore when pHi is low, gramicidin is expected to increase it by promoting H+ efflux. Indeed, as shown in Fig. 7(c), the rate of pHi elevation increased when gramicidin was added. The rate of pHi elevation increased even more when gramicidin (5 μM) was co-applied with Cs+ (NMDG+ was substituted with Cs+). Gramicidin also greatly increased the rate of pHi elevation when extracellular pH was increased from 7.4 to 9.0 (Fig. 7c). As shown in Fig. 7(d), the experimental maneuvers leading to the pHi increase also resulted in [Zn2+]i clearance and the efficacy of these maneuvers in provoking [Zn2+]i decrease and pHi increase was similar, namely, gramicidin + pH 9.0 > gramicidin + Cs+ > pH 9.0 alone > gramicidin alone.
Figure 7. The relationships between pHi and [Zn2+]i in neurons exposed to Glu/Gly. (a) Effects of Na+, Li+, and Cs+ on pHi in neurons treated with Glu/Gly. During the Na+-free period, Na+ was substituted with NMDG+. The left panel shows averages from 17 to 24 neurons monitored in a single experiment. The right panel shows average rates of pHi increase calculated from three such experiments. **p < 0.01, *p < 0.05; one-way anova followed by Student–Newman–Keuls test. (b) Analogous experiments as those shown in (a) except that [Zn2+]i was monitored. **p < 0.01, one-way anova followed by Student–Newman–Keuls test. (c) Analogous experiments as those shown in (a) except that the effects of cesium ± gramicidin (Gram; 5 μM) or pH 9.0 ± gramicidin on the rate of pHi increase were studied. *p < 0.05; Kruskal–Wallis one-way anova on Ranks followed by Dunn’s test. (d) Analogous experiments as those shown in (c) except that [Zn2+]i was monitored. **p < 0.01, one-way anova followed by Student–Newman–Keuls test.
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The faster rate of gramicidin-induced pHi increase in the presence of Cs+ than in the presence of NMDG+ was expected. The gramicidin-formed channels do not permeate NMDG+. Therefore, K+ plus H+ efflux through the channels generates electrical potential that hampers exit of these ions. This does not occur in the presence of Cs+ (or another cation that permeates gramicidin channels) because the electrical potential generated by the K+ plus H+ efflux is dissipated by the Cs+ influx via the gramicidin channels.
The data shown in Fig. 7 indicate that pHi fluctuations play a role in [Zn2+]i changes. However, should the pHi increase be the sole mechanism responsible for the clearance of cytosolic Zn2+, there should be a perfect correlation between the rate of pHi increase and the rate of [Zn2+]i drop. As shown in Fig. 8(a), this was indeed the case for all experimental conditions except for the Na+ application. The rate of Na+-induced [Zn2+]i decrease was about twice as fast as expected from the rate of the Na+-induced pHi increase. This suggests that while the pHi increase plays a role in Na+-dependent [Zn2+]i decrease, Na+ also promotes additional pHi-independent mechanisms of cytosolic Zn2+ clearance.
Figure 8. Na+-dependent [Zn2+]i clearance involves pHi-dependent and pHi-independent mechanisms. (a) Correlation between the rate of pHi increase and the rate of [Zn2+]i decrease using the data from Fig. 7(a–d) (right panels). The linear regression with r2 = 0.968 is calculated for all the data except Na+ (the red point). Note that the rate of Na+-induced [Zn2+]i drop is about twice as fast as could be predicted based on the rate of Na+-induced pHi increase. (b) The Na+-dependent [Zn2+]i clearance (Na, blue trace) is not inhibited by 10 μM La3+ (Na + La3+, green trace) and is fully active under Ca-free conditions (Na Ca-free, red trace). The experimental details are the same as in Fig. 4(a). The data are averages from 18 to 23 neurons. The experiments were repeated four times with similar results.
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In cortical neurons, Qin et al. (2008) recently described a Na+-dependent mechanism of Zn2+ efflux, which requires the presence of extracellular Ca2+ and is inhibited by 10 μM La3+. To test whether this mechanism plays a role in Na+-induced cytosolic Zn2+ clearance, the impact of 10 μM La3+ and the Ca2+-free medium on the rate of Na+-dependent [Zn2+]i decrease was tested. However as shown in Fig. 8(b), the rate of the Na+-dependent [Zn2+]i decrease was not affected by either treatment.
Glu/Gly-induced [Zn2+]i elevations do not exceed 20 nM
In an attempt to calibrate FluoZin-3 signals in terms of [Zn2+]i, possible complications from overloading neurons with indicator (Dineley et al. 2002) were minimized by loading the neurons with very low concentrations of FluoZin-3 AM (0.1 μM) for only 5–6 min. Using the calibration approach described in Methods, [Zn2+]i elevations appeared to reach the low nanomolar range (Fig. 9). However, as mentioned earlier, FluoZin-3 most likely leaks from the Glu/Gly-treated neurons. Therefore, Fmax values measured at the end of the experiments and used to calibrate the data are likely underestimated. Nevertheless, such calibration defines the upper limit of [Zn2+]i increase in these experiments. While the exact value of [Zn2+]i elevation is not known, one can be confident that it does not exceed 20 nM (Fig. 9).
Figure 9. FluoZin-3 fluorescence data calibrated for [Zn2+]i. Experimental conditions were analogous to those described in Fig. 1. The data are means ± SE from 20 cells monitored in a single experiment. The experiment was repeated five times with similar results.
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