Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices

Abstract

Mossy fiber termini in the hippocampus accumulate Zn(2+), which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn(2+) at the synaptic level is challenging owing to the difficulty of visualizing Zn(2+) at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn(2+) dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices. The membrane-impermeant fluorescent Zn(2+) probe, 6-CO2H-ZAP4, was loaded into presynaptic vesicles in hippocampal mossy fiber termini upon KCl-induced depolarization, which triggers subsequent endocytosis and vesicular restoration. Local tetanic stimulation decreased the Zn(2+) signal observed at individual presynaptic sites, indicating release of the Zn(2+) from vesicles in synaptic potentiation. This synapse-level two-photon Zn(2+) imaging method enables monitoring of presynaptic Zn(2+) dynamics for improving the understanding of physiological roles of mobile Zn(2+) in regular and aberrant neurologic function.

Keywords: metalloneurochemistry; zinc ion.

Fig. 1.

Properties of Zn²⁺ probes ZAP4 and 6-CO₂H-ZAP4. (A) Schematic of the structure of ZAP4 and 6-CO₂H-ZAP4. (B and C) Fluorescence enhancement of ZAP4 (B) and 6-CO₂H-ZAP4 (C) probes upon the addition of ZnCl₂ in 100 mM KCl, 50 mM Pipes, pH 7.0. (Insets) Integrated emission as a function of total Zn²⁺ present. (B) The starting concentration of ZAP4 is 1.0 µM, and the concentration of total Zn²⁺ increases to 0, 10, 20, 30, 40, 50, 60, 70, 231, 388, 686, and 2,350 µM. (C) The starting concentration of 6-CO₂H-ZAP4 is 1.0 µM, and the concentrations of total Zn²⁺ increase to 0, 10, 20, 30, 40, 50, 60, 378, and 677 µM. (D) Normalized integrated emission versus pH for ZAP4 and 6-CO₂H-ZAP4. The fit to the model is shown for each compound. (E) Metal ion sensitivity assay for ZAP4. The first bar (gray) is the fluorescent response of 0.5 µM ZAP4 to 100 µM of the listed metal ions at pH 7.0 in 100 mM KCl and 50 mM Pipes. The second bar (red) shows the emission after the addition of 100 µM ZnCl₂. All solutions were scanned at 1 min after metal addition. The samples containing Fe²⁺ and Co²⁺ displayed no further emission enhancement, as assessed at 1 h after the addition of zinc (ZnCl₂). The responses are normalized to the fluorescence of the free probe (F₀). (F) Comparison of HeLa cell fluorescence exposed to ZAP4 (Upper) or 6-CO₂H-ZAP4 (Lower). (Left) Transparent light images. (Right) Fluorescence images. Cells were treated with 5 µM of the probe for either 1 h (ZAP4) or 4 h (6-CO₂H-ZAP4). (Scale bar: 100 µm.)

Fig. 2.

Visualization of presynaptic Zn²⁺ in hippocampal mossy fiber termini with Zn²⁺ probes. (A and B) In vitro two-photon excitation profiles of Zn²⁺ probes in the absence (blue) and presence (magenta) of 100 µM ZnCl₂. The dots represent average fluorescence intensity (n = 3). The Zn²⁺ probe solutions, ZP1 (A; 25 µM) and 6-CO₂H-ZAP4 (B; 5 µM), were prepared in 50 mM Pipes and 100 mM KCl (pH 7). The emission wavelength for fluorescence detection was 495–540 nm. (C, Upper) Images from acutely isolated mouse hippocampal slices showing background levels of Zn²⁺ probe ZP1 (25 µM) and 6-CO₂H-ZAP4 (5 µM) fluorescence using various two-photon excitation wavelengths. (Scale bar: 5 µm.) (Lower) Slices were stained for 10 min, and the fluorescence intensity in the region surrounding the mossy fibers in the slices was measured and plotted. (D) Representative images of Zn²⁺ stained mossy fiber regions in hippocampal slices. Acute adult mouse hippocampal slices were stained with ZP1 (25 µM) or 6-CO₂H-ZAP4 (5 µM). (Scale bars: 200 µm.) (E) Zn²⁺ probe fluorescence images in mossy fiber region of adult wild type (WT; ^+/+), ZnT3 heterozygous (^+/−), and ZnT3 null (^−/−) mice. The white squares on images on the left represent the enlarged regions shown in the corresponding image on the right. (Scale bars: Left, 10 µm; Right, 5 µm.) (F, Upper) Fluorescent images of the mossy fiber region sparsely expressing red fluorescence (tdimer2) in adult hippocampal slices (Thy1-Brainbow 1.0 line H transgenic mouse). (Lower) Images of red fluorescent mossy fibers including boutons in the hippocampal slice. (Scale bars: Upper, 100 µm; Lower, 10 µm.) (G, Upper) Colocalization of 6-CO₂H-ZAP4 (green) and tdimer2 (red) in mossy fiber boutons of a hippocampal slice. (Lower) Enlarged, 3D view of the mossy fiber boutons indicated by a white arrow. (Scale bars: Upper, 5 µm; Lower, 2 µm.). (H, Upper) Colocalization of 6-CO₂H-ZAP4 (green) and a presynaptic active zone marker, bassoon (red), in a hippocampal slice. (Lower) Enlarged, 3D view of the boutons indicated by a white arrow. Presynaptic structures were immunolabeled by bassoon antibody after staining with 6-CO₂H-ZAP4. (Scale bars: Upper, 2 µm; Lower, 2 µm.)

Fig. 3.

Synaptic activity-dependent release of Zn²⁺ at mossy fiber termini. (A and B, Left) Representative Zn²⁺ probe fluorescence images in slices stained with ZP1 (A) or 6-CO₂H-ZAP4 (B). Images were obtained before (Before) and 10 min after (After) TPA treatment. (Scale bar: 5 µm.) (Right) Quantification of Zn²⁺ chelator-dependent fluorescence intensity changes in mossy fiber boutons stained with ZP1 (A) and 6-CO₂H-ZAP4 (B). The fluorescent intensity changes were measured with or without TPA treatment of the hippocampal slices. ZP1: control, n = 14; TPA, n = 10; 6-CO₂H-ZAP4: control, n = 9; TPA, n = 15. The stained slices were perfused for 10 min with ACSF (control) or 200 µM TPA (TPA). *P < 0.01, paired t test. (C and D, Left) Representative Zn²⁺ fluorescent images in slices stained with ZP1 (C) or 6-CO₂H-ZAP4 (D). Images were collected before (Before) and immediately after (After) a K⁺-evoked depolarizing stimulus (ACSF containing 90 mM KCl for 90 s). (Scale bar: 5 µm.) (Right) Time course of the Zn²⁺ fluorescence intensity change in mossy fiber termini, normalized to the average intensity before the K⁺-evoked depolarizing stimulus (magenta bar). DCG-IV (5 µM) was added in ACSF after 6-CO₂H-ZAP4 staining (Stim. + DCG-IV). ZP1 (n = 18), 6-CO₂H-ZAP4 (Stim.; n = 8), 6-CO₂H-ZAP4 + DCG-IV (Stim. + DCG-IV; n = 14). (E) Schematic drawing of the configuration for local tetanic electrical stimulation using a glass electrode in the slice. (F) Fluorescence image from a mossy fiber region of a living hippocampal slice stained with 6-CO₂H-ZAP4. The arrow represents the position of the inserted electrode tip. The mossy fiber regions in white squares near the electrode (Stim.) and control regions at a distance from the electrode (Contr.) are enlarged in G. (Scale bar: 5 µm.) (G) 6-CO₂H-ZAP4 fluorescence at two mossy fiber regions obtained before and immediately after tetanic stimulation. (Scale bar: 5 µm.) (H) Time course of the fluorescence intensity averaged from boutons and normalized to prestimulation levels. Control (Contr.; black circles; n = 8): mossy fiber region >30 µm away from stimulation points (glass electrode). Stimulation (Stim.; magenta circles; n = 8): mossy fiber region 5–15 µm (stimulation range) away from the electrode.

Fig. 4.

Models for presynaptic Zn²⁺ imaging method at mossy fiber termini using Zn²⁺ probes. (A, Left) Schematic model of presynaptic vesicle Zn²⁺ staining using a membrane-impermeant Zn²⁺ probe. Weak depolarization stimulus induces fusion of some synaptic vesicles that allows membrane-impermeant 6-CO₂H-ZAP4 to enter into the fused vesicles. The fluorescence probe then binds to Zn²⁺ concentrated within vesicles by Zn²⁺ transporters localized to the vesicle membrane. (Right) Schematic model of Zn²⁺ release in mossy fiber termini during electric stimulation. Tetanic stimulation induces full-collapse fusion events of synaptic vesicles into the bouton membrane which is sufficient to release a large proportion of Zn²⁺ (along with the fluorescent probe) from synaptic vesicles. (B) Model for Zn²⁺ staining with the cell membrane-permeant fluorescence probe in mossy fiber termini. (Left) The Zn²⁺ probe (ZP1) stains Zn²⁺ within vesicles without synaptic activation owing to its membrane permeability. (Right) The membrane-permeant probe ZP1 stains Zn²⁺-containing vesicles throughout the mossy fiber bouton. The mossy fiber bouton contains multiple active zones (cyan region).