Synaptic release of zinc from brain slices: Factors governing release, imaging, and accurate calculation of concentration
Introduction
Ever since Haug suggested that certain synaptic vesicles are rich in “free” (rapidly-exchangeable) zinc (Haug, 1967), it has been proposed that the zinc would be released along with other contents of the synaptic vesicles. Such release was first shown in two reports 1984, which established that zinc release is impulse- and calcium-dependent (Howell et al., 1984, Assaf and Chung, 1984).
A variety of indirect demonstrations of zinc release have been adduced since, including the finding that vesicular zinc is present in axons of control animals, but absent in animals that have been subjected to intense and/or prolonged activation of the axons just prior to staining for zinc (Haug et al., 1971, Sloviter, 1985, Frederickson et al., 1988, Suh et al., 2000a). Another indirect replication is found in the results of experiments in which a zinc-containing pathway is activated, and some of the observed effects of activation are then blocked by blocking (typically chelating) the released zinc (Vogt et al., 2000, Molnar and Nadler, 2001, Smart et al., 2004).
Direct demonstrations of zinc release have included the release of total elemental zinc as well as the release of the free zinc ion. The total zinc release studies have been done using microdialysis in the intact brain (Charton et al., 1985, Aniksztejn et al., 1987, Itoh et al., 1993, Takeda et al., 1999, Minami et al., 2002) as well as fraction collection from brain slices in vitro (Howell et al., 1984, Assaf and Chung, 1984).
Another approach has shown that zinc labeled in situ in the vesicles is released (along with the label) over time (Perez-Clausell and Danscher, 1986) and more rapidly over time during stimulation (Kay et al., 1995, Budde et al., 1997, Varea et al., 2001, Quinta-Ferreira and Matias, 2004). Release of free zinc has been studied by microdialysis in the intact brain (Frederickson et al., 2005a) and by fluorimetric imaging of zinc release from brain or retinal slices (Thompson et al., 2000, Li et al., 2001, Ueno et al., 2002, Takeda et al., 2005, Qian and Noebels, 2005, Komatsu et al., 2005, Redenti and Chappell, 2005).
The amount of zinc that can be released from presynaptic terminals by electrical stimulation is an issue not yet fully resolved. One clear fact is that some cytoarchitectonic regions of the brain, such as the neuropil of the lateral amygdala, subiculum, and the mossy fiber neuropil of the hippocampus, contain from 200 to 600 μM of rapidly-exchangeable zinc. This has been shown by instrumental analyses indicating, for example, that the mossy fiber neuropil has from 70 to 200 ppm (dry weight) of “extra” zinc that is above and beyond what adjacent gray matter contains, which averages 70 ppm (Frederickson et al., 1983). This “extra” 70–200 ppm zinc, which corresponds to about 200–600 μM of zinc in the wet tissue, is definitely concentrated in the zinc-rich axons, as is shown by two observations. First, it is anatomically co-extensive with the axons (Frederickson et al., 1983, Wensink et al., 1987), and, second, in genetic knock-out mice that lack vesicular zinc influxers (ZnT-3 knockouts), the enrichment is not present (Linkous et al., 2006).
Most investigators who have measured the synaptic release of “free” Zn2+ from the densely innervated mossy-fiber neuropil have found that around 1–10% of the free zinc in the vesicles can be released, resulting in 1–10 μM “puffs” of free zinc being detected by fluorescent imaging techniques (Thompson et al., 2000, Li et al., 2001, Ueno et al., 2002, Komatsu et al., 2005, Takeda et al., 2005, Redenti and Chappell, 2005; cf. Kay, 2003). The present work was undertaken in search of greater understanding of the factors that govern the final estimates of the concentration of zinc released.
Section snippets
In the laboratory of NeuroBioTex Inc.
Timed pregnant rats were obtained from Sprague Dawley, and allowed to deliver in the animal colony. The pregnancies were timed to provide 1 or more litters of pups reaching the ages of 16, 23, 30, 37 and 90 days of age all on the same day so that they could be killed and brain processed all on the same day. In all the total number of pups that were processed was 16 day 5, 23 day 6, 30 day 4, 37 day 6. In addition, a group of adult rats (200–350 g) was killed.
Rats were killed with an overdose of
Development of zinc-containing mossy fibers
Sixteen-day-old rat pups have very small hippocampi, and those small hippocampi have only a very low concentration of zinc. This can be seen in fresh-frozen sections stained with TSQ (Fig. 1), or with Zinquin (Fig. 2), and in live vibratome slices in which the zinc was stained in situ in the vesicles with TSQ (Fig. 3). When quantitative microfluorimetry was used to measure the brightest spot in the dentate hilus in pups of various ages from 16 days of age to 90 (or older), the resulting
Discussion
In the following discussion, the six salient methodological issues raised in Section 1 are discussed, and the apparent impact of each upon the final calculation of zinc released is considered.
Conclusion
Six aspects of the methods one might use to study synaptic zinc release have been examined. Each can decrease the apparent concentration of released zinc by anywhere from 2- to 66-fold, and all are independent; therefore, when occurring together, the effects of these errors will be multiplicative. For example, if one were to make the methodological choice of using 16-day-old pups, release would be depressed by about 90%, incubating in CaEDTA would depress release another 50%, running
Acknowledgements
Supported in part by NS 40215, NS41682, NS 42882, to C.J.F. and NS38585 to R.B.T. Preliminary zinc release results from Hershfinkel were published in review chapters that appeared before this publication appeared (Thompson et al., 2005, Frederickson et al., 2005a, Frederickson et al., 2005b).
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