Fluorescence imaging study of extracellular zinc at the hippocampal mossy fiber synapse

Abstract

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Figure 1

Synaptically released Zn²⁺ induced by electrical stimulation at the mossy fiber pathway. (A) Images of the hilus of the hippocampal dentate gyrus perfused with 10 μM NG. The yellow arrow represents the tip of the electrode placed in the hilar region close to the granule cells. The three circles represent the three regions of interest (ROIs) studied. (B) Depiction of a hippocampal slice. Three open circles represent ROIs, the number of which corresponds with ROIs in A and with the curves plotted in C. (C) Electrical stimulation (100 Hz) evoked release of Zn²⁺ from mossy fiber terminals measured by changes in fluorescence intensity. Changes of fluorescence over time at three separate ROIs are plotted as curves ROI1, ROI2, and ROI3. Arrow indicates the beginning of stimulation. (D) Summary of NG fluorescence responses in hilus and molecular layer to the same source of electrical stimulation. Values plotted are the means ± S.E.M., N = 6; P < 0.01. (E) The Zn²⁺ chelator CaEDTA (1 mM) inhibited the NG fluorescent response to electrical stimulation.

Figure 2

Application of Zinpyr-4 in detecting synaptically released Zn²⁺ in response to electrical stimulation. (A) Increase in Newport Green, Zinpyr-4 and FluoZin-3 fluorescence evoked by electrical stimulation. Values plotted are the means ± S.E.M., N = 6 (NG), 5 (Zinpyr-4), 6 (FluoZin-3); P < 0.01. (B) A representative experiment showing the effect of exogenous Zn²⁺ added into the recording chamber without and with a hippocampal slice. For in vitro tests, Zn²⁺ was added to ACSF that already contained 10 μM Zinpyr-4. Additions of 1 μM or 10μM Zn²⁺ did not change fluorescence intensity of the hippocampal slice loaded with Zinpyr-4.

Figure 3

Stimulation-induced increase of FluoZin-3 fluorescence. A. Electrical stimulation (100 Hz for 10 seconds) in the mossy fiber pathways evoked increases in FluoZin-3 fluorescence. However, there was an increase in response in the hilar-CA3 region as well as the molecular region. The placement of the electrode was the same as in Figure 1. Insert: The average value of fluorescence increases (ΔF) in hippocampal regions with high frequency electrical stimulation in the presence of FluoZin-3. B. Representative experiments showing the fluorescence responses of FluoZin-3 to exogenous Zn²⁺. In the absence of hippocampal slices,