ARTICLE |
Correspondence to: Gorm Danscher, Dept. of Neurobiology, Institute of Anatomy, U. of Aarhus, DK-8000 Aarhus C, Denmark.
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Summary |
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We present a new technique that allows zinc ions in synaptic and secretory vesicles of biopsy and early autopsy material (<2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices for subsequent autometallographic (AMG) development. Human brain biopsies, or other tissue samples containing zinc-enriched (ZEN) cells, are frozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass slides. The slides are transferred to an H2S exposure chamber placed in a -15C freezer. After 1-24 hr of gas exposure the sections are removed from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are frozen instantaneously after removal, because loosely bound or free zinc ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tissue caused by freezing, also be used to trace zinc ions at ultrastructural levels, and it is demonstrated that zinc ions in the human neocortex are located in synaptic vesicles. In the few human biopsies analyzed thus far, the light microscopic pattern created by the silver-enhanced ZEN terminals resembles that seen in the neocortex of rat brain. The technique has been applied to cryostat sections from neocortex biopsies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains removed and frozen immediately after decapitation constituted the material on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexposed sections can be stored for at least 5 months at -80C without ensuing significant loss of AMG staining intensity. (J Histochem Cytochem 45:1503-1510, 1997)
Key Words: human brain biopsy, zinc ions, autometallography, zinc-enriched (ZEN) neurons, hydrogen sulfide, silver amplification, Alzheimer, secretory vesicles
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Introduction |
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The GRANULE CELLS of fascia dentata are believed to be the zinc-enriched (ZEN) neurons in the brain that harbor the most synaptic vesicles with chelatable zinc per terminal (
Since Timm's original "sulphide silver staining" for visualization of the "Maske zinc pool" was introduced in 1958, it has been a goal for many researchers to develop a Timm technique that could be performed on tissue sections, i.e., where the chemical binding of zinc ions to sulfide could take place on tissue sections placed on glass slides.
The original Timm method used fixation of the tissue block with alcohol bubbled with H2S gas. This technique was, however, not specific for zinc, most probably because the alcoholic denaturation of the tissue proteins caused release of copper and possibly other metals. A breakthrough in the literature was Haug's introduction of transcardial perfusion with a 1% sodium sulfide solution. By using cryostat sections of brains from the perfused rats, Haug was able to obtain a satisfactory staining (for details see
Several attempts have been made to apply Timm's method to tissue sections using either H2S gas exposure or exposure to sulfide ions from a solution of sodium sulfide (
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Materials and Methods |
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Cryostat sections of biopsies from the neocortex of five individuals undergoing brain surgery and brains from 21 rats removed and frozen immediately after decapitation were used to work out the present technique.
Construction of the "Sulfidation Device"
Hydrogen sulfide is highly toxic and very reactive, and the gas must therefore be handled with a totally closed system made of inert materials. When expelled from the exposure system, e.g., by nitrogen gas, the H2S gas must bubble through a hypochlorite solution where it will be chemically destroyed (sulfide is oxidized to free sulfur).
The H2S exposure system was constructed de novo using three stainless steel (quality type 316) cylinders (100 mm in diameter and 500 mm in length) closed at both ends with lids of aluminium placed on o-rings of butyl rubber greased with silicon vacuum grease and kept in place by wing screws. The cylinders are bound together and placed in a standard freezing cabinet (Gram FS type FS100) with a thermostat ranging from -10 to -20C.
Outside the freezing cabinet is placed a low-pressure gas cylinder containing hydrogen sulfide (AGA, purity 2.8) and a high-pressure cylinder containing nitrogen (AGA, "Plus" quality). The regulator on the hydrogen sulfide gas cylinder is made of stainless steel with movable parts of teflon (AGA type TDR715S).
The gas cylinders are connected with the three gas chambers inside the freezing cabinet through a system of 1/8" stainless steel (316) high-pressure tubes and fittings known from high-pressure chromatography. This system is made with gas valves to keep the hydrogen sulfide and nitrogen gas cylinders isolated from each other. Otherwise the high-pressure nitrogen may flow into the low-pressure hydrogen sulfide gas cylinder by accident. In the freezing cabinet, 3 m of the tube is wound up as a spiral and placed in a plastic container filled with water ice. This part of the arrangement ensures that the gas temperature is reduced to a temperature close to the freezing cabinet.
The outlet from the gas chambers is likewise made with 1/8" tubes connected to a common output into an empty washing bottle, followed by two washing bottles each containing 200 ml of a 25% sodium hypochlorite solution. The bottles are closed tightly with silicon vacuum grease and plastic rings and placed in a fume cupboard. When the nitrogen-expelled H2S gas passes through the hypochlorite solution, the sulfide is reduced to free sulfur.
Handling of Tissue Sections
Biopsies from human patients were frozen in liquid nitrogen immediately after removal in connection with surgical intervention. Likewise, brain and other organs removed from deeply anesthetized or decapitated Wistar rats were frozen on removal. These biopsies and tissue blocks were kept at -80C.
The frozen tissue blocks were glued to a cryostat stage by Tissue-Tek OCT 4583 Compound (Miles; Elkhart, IN), placed in a cryostat, and allowed to increase in temperature to -17C. Then 30-µm-thick sections were cut and mounted on Farmer cleaned (see below for explanation) glass slides. The sections were thawed for a few seconds to adhere to the slide and then stored at -80C until moved to the sulfidation chamber. The sections are placed in the chamber for 30 min before being flushed with H2S gas for 2 min, followed by 5 min of intermission and another 2 min of H2S flushing. Gas flow is adjusted to a level at which vivid bubbling is seen in the washing bottles.
The exposure time in the now H2S-filled chamber varied from 1 to 24 hr. In most cases an exposure period of 6-8 hr was found to be optimal, but in order not to miss any traces of zinc ions in the sections it is advisable to expose initially for 24 hr.
After H2S exposure, the chamber was flushed with nitrogen gas for 10 min. Then the chamber was opened and the cradles removed and placed in 70% alcohol for 30 min. Later, the sections were rehydrated and finally dipped in a 0.5% gelatin solution.
Because the AMG process is very sensitive, just 1 gold atom, 3-6 silver atoms or zinc sulfide molecules can catalyze silver precipitation on their surfaces (
Preparation of the AMG Solution
AMG Development for Light Microscopic Analysis
In daylight the AMG developer was poured into Farmer-rinsed jars and placed at 26C in a waterbath equipped with a shaking device and placed in a light-tight box. Fifteen minutes later the slides were placed in the jars and the lid was replaced (
After 60 min of development the AMG process was stopped by replacing the developer in the jars with a 5% thiosulfate solution. After 10 min the jars were placed under running 40C tapwater for 20 min to remove the gelatin coat and then dipped twice in distilled water. Every second slide was immersed in a 2% Farmer solution for 30 sec and carefully rinsed in distilled water. All sections were finally counterstained with toluidine blue. After rinsing and dehydration the sections were mounted in DePex mounting medium (BDH Laboratory Supplies; Poole, UK).
A test slide with cryostat sections from a rat brain taken from an animal transcardially perfused with 0.1% sodium sulfide for 7 min (the NeoTimm method;
AMG Development for Ultrastructural Analysis
Cryostat sections to be analyzed in the electron microscope were cut 50-70 µm thick and placed in a small (volume 8 ml) teflon bowl kept at -17C. The bowl was transferred to the gas chamber. After H2S exposure it was filled with cold 3% glutaraldehyde in a 0.1 M phosphate solution. The sections were allowed to thaw for 30 min in this solution at room temperature, then rinsed in distilled water three times, submerged in AMG, and developed at 26C for 60 min in the AMG device described above. After 10 min in the stopbath the sections were analyzed in a stereo microscope, and selected parts of the sections to be analyzed in the EM were cut out, fixed in a 1% osmium tetroxide solution in 0.1 M phosphate buffer (pH 7.4), and embedded in Epon. Two-µm-thick semithin sections were cut and, after light microscopic evaluation, re-embedded on top of a blank Epon block. From this preparation ultrathin sections were cut and stained with uranyl acetate and lead citrate (
Effect of Long-term Low-temperature Storage of Tissue Sections
Six rats were sacrificed by decapitation, the brains removed quickly, frozen in CO2, and stored at -17C. The next day one of the hemispheres of each brain was placed in a cryostat and 30-µm sections were cut as described previously. The slides and remaining hemisphere from each animal were wrapped with plastic freezer film and stored at -80C.
After 1, 2, 3, 4, 5, and 6 months, sections were cut from a hemisphere and together with the previously prepared sections were exposed to hydrogen sulfide in the gas chamber and AMG developed as described above.
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Results and Comments |
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The period of time necessary to convert all the zinc ions in a certain batch of sections to zinc sulfide crystal lattices in the H2S chamber was found to depend solely on the temperature in the chamber. The 30-µm-thick cryostat sections of rat brain used as a test tissue in the present study showed creation of zinc sulfide crystal lattices after 10 min at -15C, and 24-hr exposure resulted in an AMG staining that technically was of almost equally high quality to that of transcardially perfused rat brains (Figure 1a and Figure 1b). The temperature of the chamber should always be below -5C because the H2S gas exposure at higher temperatures was found to be harmful to tissue morphology and to cause creation of nonspecific AMG grains. The reason for these problems might be a high pH in the tissue created by the H2S gas.
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The time scale for leakage of zinc ions from their vesicular environment was analyzed by sacrificing rats and keeping them at room temperature for 2, 4, or 6 hr before the brains were removed and frozen. A substantial decline in staining intensity was found after 2 hr, and after 6 hr the diffusion leakage and chemical binding of zinc ions to molecules in the cytosol reduced the intensity of the AMG staining, which also became blurred and was of low diagnostic value. Only in the hippocampal mossy fiber system and the amygdaloid complex could the AMG staining still be recognized as bouton related, possibly because of the "autoTimm" effect (
The human biopsy material used in this study was frozen in liquid nitrogen immediately after removal (Figure 1c-e). The quality was satisfactory in three of the five cases, and the staining pattern created by the ZEN terminals was almost identical to that found in rat neocortex (Figure 1e, Homo; Figure 1f, rat). Ultrastructural analysis of ultrathin sections from the 50-µm-thick cryostat sections showed that the zinc ions are located in the synaptic vesicles of ZEN terminals in the human neocortex (Figure 2a). Because of freezing of the tissue the quality of the electron microscopic sections is far from satisfactory, and we are presently trying to improve the technique to obtain tissue preservation closer to that seen in perfused experimental animals (Figure 2b).
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Some of the human brain biopsies did not stain at all. This is most probably because they have been deprived of oxygen, e.g., by arteries being clamped or heated (burned) during surgery.
To ensure that the zinc pattern demonstrated by the H2S gas technique was caused by zinc ions, deeply anesthetized rats were treated IP with 1000 mg diethyldithiocarbamate (DEDTC) per kg bw. One hour later the still-anesthetized animals were sacrificed, and the brains were removed and frozen (see above). Cryostat sections processed according to the present AMG technique, i.e., frozen sections exposed to H2S, were blank.
From previous work it is known that chelation with dithizone results in the same lack of staining and that proton activation (PIXE) multielement analysis (
The long-term storage of tissue blocks and sections at -80C did not influence the staining pattern, although a minor reduction in intensity was seen after 6 months. A difference between storage as tissue blocks or sections was not noticeable, but the risk for freeze-drying effects is higher for the sections.
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Discussion |
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The pool of zinc ions that can be silver-amplified by AMG is localized in a subclass of synaptic vesicles (
The pool of zinc ions contained in synaptic vesicles of ZEN neurons is quite stable after transformation to zinc sulfide as long as the sections are not heated above 45C and the pH is kept neutral to weakly basic (
Timm introduced the "sulphide silver method" in 1958, and until now a successful technique for Timm staining of tissue sections has not been published. As mentioned earlier, the use of H2S gas has been suggested by several authors (
When the vesicular zinc ion pool is chelated by diethyldithiocarbamate (DEDTC) in vivo (in rats) the present technique, like all other zinc-specific AMG techniques, results in blank AMG sections. This is because the zinc-DEDTC bonds cannot be broken by the sulfide ions, and it proves that the AMG pattern results from zinc ions. If the pattern were caused by copper ions the hypothetical copper-DEDTC would be transformed into copper sulfide crystals that could be AMG silver-enhanced (
It can be concluded, therefore, that it is imperative that the zinc ion pool in the synaptic vesicles of ZEN neuronal terminals is chemically bound as stable crystals before it can escape from the vesicles. In animals this can be done in several ways: by transcardial perfusion (
AMG analysis of human brains 6 hr post mortem has given most unsatisfactory results because of the diffusion of zinc ions from the zinc ion-containing synaptic vesicles of ZEN boutons, a process that most likely starts as soon as the terminals are deprived of oxygen. Therefore, only biopsies from surgical or diagnostic intervention in the human brain can be expected to give an optimal AMG demonstration of the ZEN terminals. However, the possibility that human brain autopsy specimens taken shortly after death could still contain appreciable amounts of sulfide-available zinc ions in their ZEN terminals cannot be excluded. In rat, a usable AMG staining of the ZEN terminals can be obtained from animals that have died 2 hr before the brain was removed.
The "autoTimm" method shows some staining in, i.e., hippocampus and amygdala after 24 hr (
The finding of an AMG-zinc pattern in biopsies from human neocortex that appears to be almost identical to the pattern of ZEN terminals in the rat brain makes the technique a promising tool for future studies in this particular population of neurons in the human brain, particularly since new studies suggest an increase in the zinc content of hippocampus and amygdala in Alzheimer-diseased brains (
The presence of zinc ions in synaptic vesicles of human ZEN neurons suggests that ZEN neurons in humans and animals might be phylogenetically identical.
Apart from its use in brain studies and possible future use in pathological diagnostics, the present AMG technique can be applied to sections from many organs that contain zinc-enriched cells, e.g., Paneth cells in the gut, ß-cells in the Langerhans islets of the pancreas, secretory cells in prostate and epididymis, or zinc ions in semen.
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Acknowledgments |
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Supported by the Danish Medical Research Council, Direktør E. Danielsen og Hustrus Fond, Beckett-Fonden, and Karen Elise Jensens Fond.
We thank Prof H. Su for valuable comments on the manuscript and Ms H. Brandstrup, Ms D. Jensen, Mr A. Meier, Mr T.A. Nielsen, and Ms K. Wiedemann for expert technical assistance.
Received for publication April 8, 1997; accepted June 5, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assaf SY, Chung S-H (1984) Release of endogenous Zn2+ from brain tissue during activity. Nature 308:734-736 [Medline]
Brunk U, Brun A, Sköld G (1968) Histochemical demonstration of heavy metals with the sulphide-silver method: a methodological study. Acta Histochem 31:345-357 [Medline]
Chafetz MD (1986) Timm's method modified for human tissue and compatible with adjacent section histofluorescence in the rat. Brain Res Bull 16:19-24 [Medline]
Constantinidis J (1990) Démence d'Alzheimer et zinc. Schweiz Arch Neurol Psychiatr 141:523-566 [Medline]
Danscher G (1981) Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochemistry 71:1-16 [Medline]
Danscher G (1982) Exogenous selenium in the brain. A histochemical technique for light and electron microscopical localization of catalytic selenium bonds. Histochemistry 76:281-293 [Medline]
Danscher G (1984) Dynamic changes in the stainability of rat hippocampal mossy fiber boutons after local injection of sodium sulphide, sodium selenite, and sodium diethyldithiocarbamate. In Frederickson CJ, Howell GA, Kasarkis E, eds. The Neurobiology of Zinc, Part B: Deficiency, Toxicity, and Pathology. New York, Alan R Liss, 177-191
Danscher G (1993) A silver method for counterstaining plastic embedded tissue. Stain Technol 58:365
Danscher G (1994) Autometallographic (AMG) nerve tracing: demonstration of retrograde axonal transport of zinc selenide in zinc-enriched (ZEN) neurons. In Gu J, Hacker GW, eds. Modern Methods in Analytical Morphology. New York, Plenum Press, 327-339
Danscher G (1996) The autometallographic zinc-sulphide method. A new approach involving in vivo creation of nanometer sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles. J Histochem 28:363-373
Danscher G, Howell G, Pérez-Clausell J, Hertel N (1985) The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. Histochemistry 83:419-422 [Medline]
Danscher G, Montagnese C (1994) Autometallographic localization of synaptic vesicular zinc and lysosomal gold, silver and mercury. J Histotechnol 17:15-22
Danscher G, Zimmer J (1978) An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues. Histochemistry 55:27-40 [Medline]
Deibel MA, Ehmann WD, Markesbery WR (1996) Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress. J Neurol Sci 143:137-142 [Medline]
Farmer EH (1884) Yearbook Phot 59:65, 118, 123
Frederickson CJ (1989) Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 31:145-238 [Medline]
Frederickson CJ, Danscher G (1990) Zinc-containing neurons in hippocampus and related CNS structures. Prog Brain Res 83:71-84 [Medline]
Frederickson RE, Frederickson CJ, Danscher G (1990) In situ binding of bouton zinc reversibly disrupts performance on a spatial memory task. Behav Brain Res 38:25-33 [Medline]
Friedman B, Price JL (1984) Fiber systems in the olfactory bulb and cortex: a study in adult and developing rats, using the Timm method with light and electron microscope. J Comp Neurol 223:88-109 [Medline]
Haug F-MS (1973) Heavy metals in the brain. A light microscope study of the rat with Timm's sulphide silver method. Methodological considerations and cytological and regional staining patterns. Adv Anat Embryol Cell Biol 47:1-71
Howell GA, Welch MG, Frederickson CJ (1984) Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308:736-738 [Medline]
Jaarsma D, Korff J (1990) A novel non-perfusion Timm method for human brain tissue. J Neurosci Methods 35:125-131 [Medline]
James TH (1977) The Theory of the Photographic Process. New York, Macmillan
Kemp K, Danscher G (1979) Multi-element analysis of the rat hippocampus by proton-induced X-ray emission spectroscopy (phosphorous, sulphur, chlorine, potassium, calcium, iron, zinc, copper, lead, bromine and rubidium). Histochemistry 59:167-176 [Medline]
Maske H (1955) Über den topochemischen Nachweis von Zink im Ammonshorn verschiedener Säugetiere. Naturwissenschaften 42:424
Pérez-Clausell J, Danscher G (1985) Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study. Brain Res 337:91-98 [Medline]
Pérez-Clausell J, Danscher G (1986) Release of zinc sulphide accumulations into synaptic clefts after in vivo injection of sodium sulphide. Brain Res 362:358-361 [Medline]
Rassendren FA, Lory P, Pin JP, Nargeot J (1990) Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes. Neuron 4:733-740 [Medline]
Timm F (1958) Zur Histochemie der Schwermetalle. Das Sulfid-Silberverfahren. Dtsch Z Gesamte Gerichtl Med 46:706-711
Xie X, Smart TG (1993) Properties of GABA-mediated synaptic potentials induced by zinc in adult rat hippocampal pyramidal neurons. J Physiol (Lond) 460:503-523 [Abstract]