http://www.paper.edu.cn Brain Research 928 (2002) 165±174 www.bres-interactive.com Interactive report Localization of zinc-enriched neurons in the mouse peripheral q sympathetic system Zhan-You Wanga,b,c,* , Jia-Yi Lia , Gorm Danscherb , Annica DahlstromÈ a aDepartment of Anatomy and Cell Biology, University of Gothenburg, Box 420, SE-405 30 Gothenburg, Sweden bDepartment of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark cDepartment of Histology and Embryology, China Medical University, Shenyang 110001, PR China Accepted 17 November 2001 Abstract Growing evidence supports the notion that zinc ions located in the synaptic vesicles of zinc-enriched neurons (ZEN) play important physiological roles and are involved in certain pathological changes in the central nervous system. Here we present data revealing the distribution of zinc ions and the co-localization of zinc transporter 3 (ZnT3) and tyrosine hydroxylase (TH) in crush-operated sciatic nerves and lumbar sympathetic ganglia of mice, using zinc selenide autometallography (ZnSeAMG ) and ZnT3 immuno¯uorescence combined with confocal scanning microscopy, respectively. Six hours after the crush operation, ZnSeAMG grains and ZnT3 immunoreactivity were predominantly present in a subpopulation of thin unmyelinated sciatic nerve axons. In order to identify the type(s) of ZEN axons involved, double labeling with ZnT3 and (1) TH, (2) vesicular acetylcholine transporter (VAChT), (3) calcitonin gene-related peptide (CGRP), and (4) neuropeptide Y (NPY) was performed. Confocal microscopic observations showed that ZnT3 was located in a subpopulation of sciatic axons in distended parts proximal and distal to the crush site. Most, if not all, ZnT3-positive axons contained TH immuno¯uorescence, a few showed co-localization of ZnT3 and VAChT with very weak immunostaining, while no congruence was observed between ZnT3 and CGRP or NPY. Studies of the lumbar sympathetic ganglia showed that not more than 5% of the neurons were ZnT3-positive and that almost all of these were TH-positive. Furthermore, approximately 5% of total lumbar sympathetic ganglionic cells were ZnSeAMG positive, 48 h after a local injection of sodium selenide into the sciatic nerve. The present data support the notion that a subgroup of mouse sympathetic postganglionic neurons are ZEN neurons. 2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Uptake and transporters Keywords: Zinc transporter 3; Tyrosine hydroxylase; Sciatic nerve; Sympathetic; Confocal laser scanning microscopy; Autometallography 1. Introduction lated by zinc transporter 3 (ZnT3) in the synaptic vesicles of zinc-enriched neurons (ZEN) [7,14,15,17,41]. Fortu- Zinc is an important trace element in the mammalian nately, this zinc pool can be traced by several different body, serving structural, catalytic and regulatory roles in techniques including the autometallographic techniques cellular biology [4,10,46]. In the brain, the vast majority of (ZnSAMG and ZnSe AMG ) and N-(6-methoxy-8-quinolyl)-p- intracellular zinc is tightly bound to proteins, where it toluene sulfonamide (TSQ) ¯uorescence staining exerts structural functions or acts as a cofactor [7]. [12,13,20]. Several ZEN neuronal pathways have been However, a particular pool of zinc in the brain (about identi®ed in the mammalian CNS 10±15%) is present as free or loosely-bound ions accumu- [8,16,25,26,28,33,34,36,39,47]. Most of these ZEN path- ways were found to be glutamatergic [18]. The co-localiza- qPublished on the World Wide Web on 14 January 2002. tion of zinc and glutamate in ZEN terminals analyzed in *Corresponding author. Tel.: 146-31-773-3354; fax: 146-31-829-690. the brain supported the idea that zinc may function as a E-mail address: [email protected] (Z.-Y. Wang). modulator of glutamatergic neurotransmission [3,19,37]. 0006-8993/02/$ ± see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993(01)03344-3 中国科技论文在线 http://www.paper.edu.cn 166 Z.-Y. Wang et al. / Brain Research 928 (2002) 165 ±174 However, zinc ions have been found to modulate the sciatic crush operations, the mice were anesthetized and function of both the excitatory and inhibitory receptors in perfused transcardially with saline followed by 3% brain slices [44,50], and recently the presence of inhibitory glutaraldehyde in 0.1 M PB. The sciatic nerves were ZEN terminals was demonstrated in the spinal cord of dissected and post®xed for 2 h in the same ®xative. After lamprey, mouse and rat [5,16,29,47]. A possible existence rinsing in PB, the samples were cryoprotected overnight in of GABAergic and glycinergic ZEN terminals also in the 30% sucrose. They were then frozen with CO2 gas and cut cerebellum and brain stem is presently under investigation. in 20-mm-thick cryostat sections. The sections were im- It appears that ZnT3, despite of its presence in the testis, mersed in an AMG developer [12] and placed in a 26 8C is almost speci®c for zinc ion transport in the mammalian water bath for 1 h. The silver enhancement was stopped by CNS. ZnT3 has been found in high concentrations in the a 5% thiosulfate stop solution. The sections were rinsed hippocampus and neocortex [42] and in the spinal cord with running tap water (38 8C) for 10 min in order to [29]. The pattern of ZnT3 immunohistochemistry is identi- remove the gelatin membrane, and then rinsed in distilled cal to that of ZnSAMG and ZnSeAMG staining for zinc ions water and counterstained with 0.1% Toluidine Blue. The [28,29]. Ultrastructurally, ZnT3 immunoreactivity and sections were analyzed with a Nikon microscope equipped ZnSAMG grains are located in the synaptic vesicles of ZEN with an Easy Image Analysis System (Bergstrom Instru- terminals [28,29,49]. ments AB, Gothenburg, Sweden), and the images were Recently we found that a stop-¯ow nerve crush on the further processed with Adobe Photoshop (version 5.5). rat sciatic nerve resulted in the accumulation of zinc ions Two normal mice, with intact sciatic nerves, were in a subgroup of unmyelinated axons [48]. In the present treated as above in order to study whether ZnSeAMG grains study we extend the studies to mouse tissues. We decided could be observed in the normal nerve. therefore to con®rm the existence of ZEN neurons in the Three mice were used for retrograde tracing of ZEN PNS using ZnT3 immunohistochemistry, and to test the neurons in the lumbar sympathetic ganglia. They were hypothesis that the peripheral ZEN neurons are post- anesthetized and the sciatic nerves were exposed bilateral- ganglionic sympathetic neurons by applying double label- ly. A crush was made on each nerve and about 1 mlof ing with ZnT3 and adrenergic markers to crushed sciatic sodium selenide (0.5% in 0.1 M PB, pH 7.4) was injected nerves, and retrograde tracing the ZEN neurons in the slowly into the crush site. Forty-eight hours later, the mice lumbar sympathetic ganglia. were perfusion ®xed with 3% glutaraldehyde and the lumbar sympathetic ganglia, the lumbar dorsal root gan- glia, the lumbar spinal cords and the sciatic nerves were dissected for post®xation. The samples were further treated 2. Materials and methods and the cryostat sections were stained with AMG as described above. 2.1. Sciatic nerve crush operation 2.3. Double immuno¯uorescence labeling Male BALB/c mice, weighing approximately 20±25 g, were used as experimental animals (Mùllegaard Breeding Eight mice were used for immuno¯uorescence staining. Center, Denmark). They were housed in a 12 h light/dark They were reanesthetized 6 h after the nerve crush and cycle and with food and tap water available ad libitum. transcardially perfused with saline followed by 4% Under deep anesthesia with pentobarbital, the sciatic paraformaldehyde in 0.1 M PB (pH 7.4). The sciatic nerves were exposed bilaterally and double-crushed in the nerves were removed and post®xed with the same ®xative midthigh region by a pair of watchmaker tweezers for 5 s. for 3 h at 4 8C. The samples were rinsed overnight in 20% The distance between the two crushes was about 1 mm, in sucrose in phosphate-buffered saline (PBS), frozen with order to avoid the contamination between anterogradely CO gas, sectioned longitudinally at 10 mm in a cryostat and retrogradely accumulated material. After suturing of 2 (Leitz), and placed on gelatin-coated glass slides. the muscles and skin, the mice were kept in a warm Five non-operated mice were used to study the normal environment before sacri®ce. This procedure was carried level of ZnT3 in the sciatic nerves and lumbar sympathetic out in accordance with the regulations of the Animal ganglia. Their sciatic nerves and lumbar sympathetic Ethical Committee in Gothenburg. ganglia were dissected and treated as above. To study whether the distribution of ZnT3 co-localized 2.2. Zinc selenide autometallography with one or more of the following neuronal markers, double immunohistochemical incubations were carried out In order to observe the distribution of zinc ions by using the following primary antibodies: ZnSeAMG staining in the crushed sciatic nerve, nine mice Rabbit anti-ZnT3: An af®nity-puri®ed antibody speci®c were anesthetized and injected intraperitoneally with so- for ZnT3 (provided by Dr R. D. Palmiter, Department of dium selenite (20 mg/kg, dissolved in 0.1 M phosphate Biochemistry, University of Washington, USA), dilution buffer [PB]) 1 h before sacri®ce. One, 3 and 6 h after the 1:100. 中国科技论文在线 http://www.paper.edu.cn Z.-Y. Wang et al. / Brain Research 928 (2002) 165 ±174 167 Sheep anti-tyrosine hydroxylase (TH): Produced against axons. ZnSeAMG grains and ZnT3 were present only in a the native TH from rat pheochromocytoma (Chemicon, subgroup of axons (Fig.