Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion

https://doi.org/10.1016/j.expneurol.2005.08.030Get rights and content

Abstract

“Free Zn2+” (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or “tonic” level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5–25 nM). This concentration is 15,000-fold lower than the “300 μM” concentration which is often used as the “physiological” concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.

Introduction

In the mammalian forebrain, protein-bound zinc is relatively uniformly distributed through all gray matter (Frederickson et al., 1983, Frederickson et al., 1992, Klitenick et al., 1983), whereas the “free” Zn2+ (i.e., “rapidly exchangeable” or “histochemically reactive”) is selectively sequestered in the presynaptic vesicles of specific associational fiber systems of the cerebral cortex and limbic nuclei (Casanovas-Aguilar et al., 1995, Casanovas-Aguilar et al., 2002, Dyck and Cynader, 1993, Dyck et al., 1993, Frederickson and Moncrieff, 1994, Haug, 1967, Howell et al., 1991, Perez-Clausell and Danscher, 1985, Perez-Clausell et al., 1989, Sindreu et al., 2003, Slomianka, 1992, Slomianka et al., 1990). In these zinc-containing neurons, the Zn2+ is co-localized with glutamate in the presynaptic boutons (Beaulieu et al., 1992, Sindreu et al., 2003). Therefore, the term “gluzinergic” has been proposed (Frederickson et al., 2004).

Zn2+ has been shown to be a potent modulator of glutamatergic synaptic transmission (Frederickson, 1989, Smart et al., 1994, Frederickson et al., 2005, Molnar and Nadler, 2001) and a critical component of the excitotoxic cascade occurring after ischemia, seizures and head trauma (rev Choi and Koh, 1998, Frederickson et al., 2005, Weiss et al., 2000).

Because Zn2+ and glutamate are found in the same axonal boutons (Beaulieu et al., 1992, Cole et al., 1999, Sindreu et al., 2003, Wenzel et al., 1997), it has been asserted that the two are released together (Minami et al., 2002). However, there are no published reports of free Zn2+ and glutamate co-release, nor has the release of free Zn2+ ions ever been demonstrated in the live intact brain. The release of total elemental zinc (bound + free) has been demonstrated both in vivo (Charton et al., 1985, Itoh et al., 1993, Takeda et al., 1999) and in vitro (Assaf and Chung, 1984, Howell et al., 1984), and the co-release of glutamate and elemental zinc (bound + free) has also been reported (Minami et al., 2002, Takeda et al., 2004). However, except for in vitro imaging studies (Li et al., 2001a, Quinta-Ferreira and Matias, 2004, Thompson et al., 2000, Ueno et al., 2002, Wei et al., 2004), the release of free zinc has not been demonstrated nor has the concentration of free zinc in the brain been previously measured.

The present work was undertaken to remedy these omissions. Specifically, the goals were to determine: (i) the resting baseline level of the free zinc ion (i.e., pZn) in the extracellular milieu of the brain, (ii) whether the release of Zn2+ could be observed and (iii) whether the release of Zn2+ is synchronous with the release of glutamate.

Ischemia was selected as the stimulus for releasing zinc and/or glutamate for both tactical and strategic reasons. Tactically, ischemia is known to be a potent glutamate releaser, and the protocols for detection by microdialysis have been well established (Martinez-Tica and Zornow, 2000, Koinig et al., 2001a, Koinig et al., 2001b). From a strategic perspective, ischemia is one of the excitotoxic insults in which the role of Zn2+ has been well established (Choi and Koh, 1998, Koh et al., 1996, Weiss et al., 2000) and a condition in which understanding and perhaps controlling the release of Zn2+ could be clinically important (Frederickson and Bush, 2001, Frederickson et al., 2005).

Section snippets

Rabbits

With approval from the Institutional Animal Care Committee, healthy New Zealand rabbits weighing 3.5 to 4.2 kg were anesthetized with 4% halothane in a Plexiglas box. Upon loss of the righting reflex, the animals were orally intubated using a 3.5 mm endotracheal tube, and the lungs were ventilated with 0.8–1.5% isoflurane in oxygen to produce an endtidal carbon dioxide concentration of 35–40 mm Hg. A marginal ear vein was cannulated with a 22-gauge catheter and 0.9% saline infused at the rate

Measurement issues

In our experiments, dansylamide (DNSA) fluorescence increased about 5-fold in intensity and shifted 80 nm in wavelength of peak intensity when complexed with the holoCA, providing a robust ratiometric measurement system (Fig. 1). This yielded a useful working calibration that can measure zinc from a minimum of 0.5 nM to a maximum of 500 nM (when the 500 nM DNSA is fully saturated, i.e., a 1:1 ratio of Zn:DNSA) (Fig. 2). With dialysis fluids collected from the brain, DNSA caused no appreciable

The method

The use of a ligand trap in the dialysate (apoCA) to “capture” Zn2+ ions is a procedure with two potential drawbacks. First, one might suppose that enough Zn2+ could be removed from the tissue to substantially alter the physiology, thereby compromising the results. Had convulsive EEG activity arisen (the common symptom of depletion of extracellular Zn2+; Mitchell and Barnes, 1993, Domnguez et al., 2003), or had the rate of Zn2+ release fallen progressively with time, such a depletion of Zn2+

Acknowledgments

We thank David Brantley for his superb technical work. Supported in part by NS-42882, NS-41682, NS-42894 to CJF; NS11255 and NS39161 to DJM; NS042849 to DSP, NS38585 to RBT; GM40602 to CAF; FAER (Foundation of Anesthesia education and research) grant to RNM; and NS029403 to MHZ.

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