Imaging free zinc in synaptic terminals in live hippocampal slices.

Some glutamatergic synapses in the mammalian central nervous system exhibit high levels of free ionic zinc in their synaptic vesicles. The precise role of this vesicular zinc remains obscure, despite suggestive evidence for zinc as a neuromodulator. As a step towards elucidating the role of free zinc in the brain we have developed a method for imaging zinc release in live brain slices. A newly synthesized zinc-sensitive fluorescent probe, N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide (TFLZn), was used to monitor intracellular zinc in live rat hippocampal slices. The dye loaded into the zinc-rich synaptic vesicles of the mossy fibre terminals in the hippocampal formation. Direct electrical stimulation of the mossy fibre pathway diminished the fluorescence in the mossy fibre terminals, consistent with a stimulus-dependent zinc release. The synaptic release of zinc was followed by the rapid replenishment of the zinc levels in vesicles from an as yet unidentified intracellular zinc source. Furthermore, we present evidence that zinc may play a role in a form of long-term potentiation exhibited by the mossy fibre pathway.

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol.

Distribution of a fluorescent ivermectin probe, bodipy ivermectin, in tissues of the nematode parasite Ascaris suum.

A fluorescent derivative of the anthelmintic ivermectin (4''-5,7-dimethyl bodipy proprionylivermectin, referred to hereafter as bodipy ivermectin) was synthesized for an investigation of the distribution of avermectins. Injected into adult Ascaris suum at doses greater than 0.1 micron per worm, it produced a dose-dependent immobilization. Fluorescent microscopy of frozen sections revealed the distribution of the probe in the whole nematode. Staining of collagenase-isolated muscle cells was studied using bath application of bodipy ivermectin. The trypan-blue quenching technique showed that the ivermectin probe was located in the outer monolayer of the muscle membrane. The cytoplasm was not stained. The interpretation of these observations is discussed in view of the known lipophilic nature of avermectins. Staining of the muscle membrane and nerve cord is consistent with the view that avermectins act at these sites. The significance of the hypodermal and lateral line staining is also discussed.

Fluorescent indicators for cytosolic sodium.

Fluorescent indicators sensitive to cytosolic concentrations of free Na+ have been synthesized and characterized. They consist of a crown ether, 1,7-diaza-4,10,13-trioxacyclopentadecane, linked via its nitrogens to fluorophores bearing additional liganding centers. In the currently preferred dye, SBFI (short for sodium-binding benzofuran isophthalate), the fluorophores are benzofurans linked to isophthalate groups. Selectivities for Na+ over K+ of about 20 are observed, resulting in effective dissociation constants for Na+ of about 20 mM against a background of 120 mM K+. Increasing [Na+] increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging can be performed with the same wavelengths as used with the well known Ca2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K+ over Na+.

Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.

A new group of fluorescent indicators with visible excitation and emission wavelengths has been synthesized for measurements of cytosolic free Ca2+. The five compounds, "rhod-1," "rhod-2," "fluo-1," "fluo-2," and "fluo-3" (Figs. 2 and 3), combine the 8-coordinate tetracarboxylate chelating site of 1,2-bis(2-amino-phenoxyethane-N,N,N',N'-tetraacetic acid with a xanthene chromophore to give a rhodamine-like or fluorescein-like fluorophore. Binding of Ca2+ increases the fluorescence by up to 40-fold. The Ca2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators should give better resolution of high [Ca2+] levels than previously obtainable with quin-2 or fura-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescence microscopy and flow cytometry than the UV required by previous indicators. However, the new dyes' increase in fluorescence upon binding calcium is not accompanied by a wavelength shift, so they are unsuitable for measurements using ratios at two wavelengths. The most promising dye of this series is fluo-3, whose initial biological testing in fibroblasts is described in the following paper (Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178).