Alan R. Kay

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.

Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.

A heart-like Na+ current in the medial entorhinal cortex

Acutely dissociated neurons from the superficial layers of the medial entorhinal cortex of the rat were studied under voltage clamp using the whole-cell patch-clamp configuration. Neurons from the medial entorhinal cortex exhibit a tetrodotoxin (TTX)-resistant Na+ current (ITTX-R; IC50 approximately 146 nM), in addition to the normal TTX-sensitive Na+ current (ITTX-S; IC50 approximately 6 nM). ITTX-R was found in both putative stellate and putative pyramidal neurons from the medial entorhinal cortex. ITTX-R is kinetically indistinguishable from ITTX-S, but can be distinguished from ITTX-S based on its enhanced sensitivity to block by Cd2+, La3+, and Zn2+. ITTX-R is kinetically and pharmacologically similar to the TTX-resistant Na+ current found in cardiac muscle.

Fluorescent Detection of Zn2+-Rich Vesicles with Zinquin: Mechanism of Action in Lipid Environments

High concentrations of free Zn2+ ions are found in certain glutamatergic synaptic vesicles in the mammalian brain. These terminals can be visualized histochemically with quinoline sulfonamide compounds that form fluorescent complexes with Zn2+. The present study was undertaken to examine the interaction of the water-soluble quinoline sulfonamide probe, Zinquin (2-methyl-8-(toluene-p-sulfonamido)-6-quinolyloxyacetic acid) with the complex heterogeneous cellular environment. Experiments on rat hippocampal and neocortical slices gave indications that Zinquin in its free acid form was able to diffuse across the plasma and synaptic vesicle membranes. Further experiments were undertaken on unilamellar liposomes to study the interaction of Zinquin and its metal complexes in membranes. These experiments confirmed that Zinquin is able to diffuse across lipid bilayers. Steady-state and time-resolved fluorimetric studies showed that Zinquin in aqueous solution mainly forms a 1:2 (metal:ligand) complex with small amounts of a 1:1 complex. Formation of the 1:1 complex was favored by the presence of lipid, suggesting that it partitions into membranes. Evidence is presented that Zinquin can act as a Zn(2+)-ionophore, exchanging Zn2+ for two protons. The presence of a pH gradient across vesicles traps the Zn(2+)-probe complex within the vesicles. Zinquin is useful as a qualitative probe for detecting the presence of vesicular Zn2+; however, its tendency to partition into membranes and to serve as an ionophore should be borne in mind.

Imaging synaptic zinc: promises and perils

It is well established that some excitatory nerve terminals have high concentrations of Zn(2+) in their synaptic vesicles. For some time, it has been believed that synaptic Zn(2+) is released during neurotransmission and acts as a neuromodulator. Fluorescent Zn(2+) indicators that do not penetrate membranes offer the prospect of rendering the release of Zn(2+) visible. Here, I take a critical look at fluorimetric imaging experiments devised to determine whether Zn(2+) is released and show that they are particularly susceptible to artifacts. Moreover, I will argue that recent experiments suggest that, rather than being released, Zn(2+) is presented to the extracellular space firmly coordinated to presynaptic macromolecules.

Is Zinc a Neuromodulator

The vesicles of certain glutamatergic terminals in the mammalian forebrain are replete with ionic zinc. It is believed that during synaptic transmission zinc is released, binds to receptors on the pre- or postsynaptic membranes, and hence acts as a neuromodulator. Although exogenous zinc modulates a wide variety of channels, whether synaptic zinc transits across the synaptic cleft and alters the response of channels has been difficult to establish. We will review the evidence for zinc as a neuromodulator and propose diagnostic criteria for establishing whether it is indeed one. Moreover, we will delineate alternative ways in which zinc might act at synapses. This review, with 3 figures and 57 references, describes the evidence that the cation Zn2+ acts as a modulator of synaptic activity. Beginning with a discussion of the criteria that zinc would have to meet in order to be classified as a neuromodulator and the use of chelators for intercepting zinc in the synaptic cleft, the authors then consider different models for zinc’s action at synapses, ranging from free diffusion of zinc to bound zinc in the extracellular space and a possible action within synaptic vesicles. The article wraps up with suggested experimental approaches that may help resolve the questions surrounding the role of zinc in the central nervous system.

An intracellular medium formulary

Whole-cell patch-clamp recordings allow diffusible intracellular ions and molecules to be replaced by the contents of the recording pipette. In this review, the formulation of intracellular media is considered with a view to improving the stability of recordings and emulating the intracellular environment.

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green.

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.

The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium

There has been some dispute in the literature as to the sensitivity of the zinc indicator FluoZin-3 to calcium, with suggestions that physiological levels of calcium and magnesium effectively occlude the response of the probe to zinc. In this communication we demonstrate that calcium concentrations as high as 10 mM do not prevent FluoZin-3 from detecting zinc elevations as low as 100 pM. Moreover, the inclusion of a few microM Ca-EDTA does not prevent FluoZin-3 from responding to increases in zinc concentration but does extend the dynamic range of the probe by reducing contaminating zinc levels and allowing the probe to respond to multiple zinc additions. In addition, we have derived a mathematical model to account for the kinetics of FluoZin-3 response to zinc in the presence of an additional zinc and calcium chelator.

The interplay between inorganic phosphate and amino acids determines zinc solubility in brain slices

Inorganic phosphate (Pi) is an important polyanion needed for ATP synthesis and bone formation. As it is found at millimolar levels in plasma, it is usually incorporated as a constituent of artificial CSF formulations for maintaining brain slices. In this paper, we show that Pi limits the extracellular zinc concentration by inducing metal precipitation. We present data suggesting that amino acids like histidine may counteract the Pi‐induced zinc precipitation by the formation of soluble zinc complexes. We propose that the interplay between Pi and amino acids in the extracellular space may influence the availability of metals for cellular uptake.