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

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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 pathways 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].

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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 ganglia, 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

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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. 2a±d). Malmo,È Sweden), dilution 1:800. Neither ZnSeAMG grains nor ZnT3 dots were found in Goat anti-vesicular acetylcholine transporter (VAChT): normal uncrushed sciatic axons (data not shown). Produced against a synthetic peptide corresponding to the carboxyl-terminus (amino acids 511±530) predicted from 3.2. Double labeling of ZnT3 and different types of the cloned rat VAChT (Chemicon, Malmo,È Sweden), neuronal markers in sciatic nerves dilution 1:4000. Sheep anti-calcitonin gene-related peptide (CGRP): A large number of TH-positive axons were detected on Raised against synthetic CGRP (Af®nity, Exeter, UK), both sides of the crushes in sciatic nerves, and TH-IR dilution 1:200. could also be seen in thin axons far away from the crush Sheep anti-neuropeptide Y (NPY): Raised against syn- sites. Axons showing intense ZnT3-IR and TH-IR were thetic neuropeptide Y (Chemicon, Malmo,È Sweden), dilu- seen close to the crush sites. It was evident that most, if tion 1:800. not all, ZnT3-positive axons contained TH-IR (Fig. 2a±a0). Double labeling with ZnT3/TH, ZnT3/CGRP, ZnT3/ However, not all TH-containing ®bers were ZnT3-positive. NPY, and ZnT3/VAChT was carried out on consecutive In sections double-incubated with anti-ZnT3 and anti- sections of pieces of crushed sciatic nerves taken from VAChT, a co-localization between ZnT3 and VAChT could formaldehyde-perfused mice. All immunostaining proce- be seen in a few axons of three crush-operated mice. dures were performed at room temperature. Brie¯y, sec- Unlike the co-localization pattern of ZnT3 and TH there tions were preincubated with normal donkey serum (1:50) was an inverse relationship in staining intensity between for 30 min, followed by primary antibodies against rabbit ZnT3 and VAChT; strongly ¯uorescent VAChT-IR axons anti-ZnT3 together with sheep anti-TH, sheep anti-CGRP, had weak ZnT3 ¯uorescence, and vice versa (Fig. 2b±b0). sheep anti-NPY, or goat anti-VAChT overnight. After Sections double-labeled with anti-ZnT3 and anti-CGRP rinsing with PBS, the sections were incubated for 2 h with or NPY showed no co-localization at all even 6 h after the donkey anti-rabbit IgG conjugated with ¯uorescein iso- sciatic nerve crush (Fig. 2c±c0,d±d0). thiocyanate (FITC) (for labeling ZnT3, Jackson Immuno- Research, dilution 1:50) and donkey anti-sheep IgG conjugated with Texas Red (for labeling TH, CGRP and NPY, 3.3. Retrograde tracing of ZEN neurons in the lumbar Jackson ImmunoResearch, dilution 1:50). Sections double- sympathetic ganglia incubated with rabbit anti-ZnT3 and goat anti-VAChT were incubated with donkey anti-rabbit IgG conjugated with Retrogradely labelled ZEN neurons were observed in the FITC (1:50) or donkey anti-goat IgG conjugated with lumbar sympathetic ganglia 48 h after sodium selenide Texas Red (Jackson ImmunoResearch, dilution 1:50) for 2 injections into the sciatic nerves. However, no AMG h. After mounting, the sections were analyzed under a positive staining could be found in the dorsal root ganglia confocal laser scanning microscope (CLSM) with a and the ventral horn of the spinal cord (Fig. 3). About 5% of the total number of sympathetic cells krypton/argon laser (BIO-RAD MRC1024). Images were AMG collected and processed using Adobe Photoshop (version displayed ZnSe positive staining (Fig. 3a, b). In 5.5). general, ZEN somatae were sparsely distributed in the Control sections were incubated with normal donkey sympathetic ganglia. At higher magni®cation, the AMG serum instead of the primary antibodies, followed by all grains were located in the perinuclear zone but also subsequent incubations as described above. distributed in the more peripheral cytoplasm (Fig. 3b). Some neurites from the labelled somatae also showed strong AMG staining (Fig. 3a). In the control sections of SCG without sodium selenide 3. Results treatment, no AMG grains could be seen in the lumbar sympathetic ganglia (data not shown). 3.1. Accumulation of ZnSeAMG grains and ZnT3 in crush-operated sciatic nerve ®bres 3.4. Double labeling with ZnT3 and TH or NPY in sections from lumbar sympathetic ganglia The `stop-¯ow' operation of the sciatic nerve showed post-operative ZnSeAMG staining 1, 3 and 6 h later. In non-operated and operated mice, the lumbar sympa- ZnSeAMG grains were observed both proximally and thetic ganglia showed ZnT3 staining in approximately 5% distally to the two crushes (Fig. 1). The accumulations of of the ganglionic neurons (Fig. 4). The ZnT3-positive ZnSeAMG grains were mainly located close to the crush neurons seemed to be randomly distributed in the ganglia sites, but were absent in the segment between two crushes. and their staining was concentrated in the perinuclear The concentration of ZnSeAMG grains increased with time Golgi region (Fig. 4). and was only present in a subpopulation of the sciatic The ZnT3/TH double staining showed that almost all 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. Dark-®eld microphotographs of the crush-operated sciatic nerve of a mouse. The sections were stained with ZnSeAMG . At 1, 3 and 6 h after the operation the accumulations of ZnSeAMG grains increase with time and appear in a subpopulation of the sciatic axons. Arrows indicate the crush sites. Scale bar is 100 mm.

ZnT3-immunoreactive ganglionic neurons are also TH- blocking fast axonal transport by a nerve crush technique positive (Fig. 4a±a0,b±b0). Occasionally, neurons showed [30,31]. Thus the technique of concentrating material near ZnT3 and NPY staining, but they always presented an the crush can reveal the presence of material otherwise too extremely weak NPY immuno¯uorescence (Fig. 4c±c0, dilute to allow detection in normal nerves. d±d0). Several studies have demonstrated that TH is a conveni- ent marker for sympathetic adrenergic neurons [22], whereas VAChT is speci®cally expressed in cholinergic 4. Discussion neurons [21] and has not been found in non-cholinergic neurons so far. Therefore, anti-TH and VAChT have been The short-term crush-operation of the sciatic nerves has considered excellent markers for solving the present been used for decades to study axonal transport of various questions [32]. The peptides CGRP and NPY both reside endogenous materials in the PNS. Distinct accumulations in large dense-core vesicles. CGRP has been found in of various synaptic vesicle proteins can be observed after motor and sensory axons in the rat sciatic nerve [6], while 中国科技论文在线 http://www.paper.edu.cn

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Fig. 2. Confocal microscopic images of mouse sciatic nerve sections double-labeled with anti-ZnT3 and different neuronal markers 6 h after crush operation. All images are from the proximal parts and the crush sites are at the bottom of the ®gures. (a±a0) Double immunostaining for ZnT3 (green) and TH (red). All ZnT3-positive axons are positive also for TH, indicating that these two proteins are co-localized in sciatic axons. Note that both the ZnT3-immunoreactivity (IR) and TH-IR are strong. (b±b0) Double incubation with anti-ZnT3 and anti-VAChT. Axons containing both ZnT3 (green) and VAChT (red) are always weakly stained. (c±c0) Double staining for ZnT3 (green) and CGRP (red). No co-localization can be observed. (d±d0) Double staining for ZnT3 (green) and NPY (red), no co-localization. Scale bars are 25 mm(a±a0), 150 mm(b±b0) and 100 mm(c±c0 and d±d0).

NPY has been observed in a population of rat adrenergic also TH-positive, but that not all TH-positive ®bers were axons [11], claimed to speci®cally innervate the car- ZnT3-positive, suggests that a subgroup of sympathetic diovascular system [35]. neurons are ZEN neurons. We also showed that a small Our ®nding that almost all ZnT3-positive axons were fraction (about 5%) of the total lumbar sympathetic 中国科技论文在线 http://www.paper.edu.cn

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Fig. 2. (continued)

ganglion cells were ZnT3-positive and TH-positive. The proteins are present in the ZEN terminals, but little is lack of co-localization between ZnT3 and NPY suggests known about the function of this zinc pool. At the cellular that the ZEN subgroup lacks NPY or has very low levels level, there are speci®c membrane-bound transporters, and of NPY. a growing family of mammalian zinc transporter proteins, In the CNS, signi®cant amounts of zinc ions and ZnT3 ZnT1, ZnT2, ZnT3 and ZnT4, has been identi®ed [27,40± 中国科技论文在线 http://www.paper.edu.cn

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Fig. 3. Light microphotographs from ZnSeAMG -stained sections 48 h after sodium selenide injection into the mouse sciatic nerves. The retrogradely labelled ZEN neurons are found in the lumbar sympathetic ganglia (a, b), but not in the dorsal root ganglia (c) and the ventral horn of the spinal cord (d). Arrowheads indicate the AMG-positive somatae. Arrows indicate the AMG-positive neurites in the lumbar sympathetic ganglia.

42]. Generally, zinc transporter proteins are predicted to appearance of ZnT3 and ensuing zinc ions. However, the have a common structure, consisting of six transmembrane absence of ZnT3 or zinc ions between the two crushes domains and a histidine-rich cytoplasmic rood [27]. ZnT1 makes this explanation less likely. Therefore, we conclude appears to be expressed ubiquitously on the plasma that ZnT3 and zinc ions, albeit present in low amounts in membrane in a variety of tissues, including the intestine, the intact nerve, are subjected to a fast anterograde and kidney and liver, serving as a zinc ion ef¯ux transporter of retrograde transport, resulting in the observed accumula- the cells [40]. ZnT2 resides on vesicular membranes of the tions of material. intestine, kidney and testis and transports zinc into endo- Studies of the CNS have demonstrated that the zinc ions somal/lysosomal compartments [41]. ZnT3 has been found are released from ZEN terminals into the synaptic clefts as to be expressed restrictedly in the brain and testis [42] a response to synaptic activity [1,2,24,43], and that it can implying some speci®c roles of zinc ions in these two modulate certain amino acid receptors. The relation be- tissues [38]. ZnT4 is essential for adding zinc ions to milk, tween zinc and amino acid neurotransmission has been and may also participate in the intestinal uptake of zinc shown in the zinc-rich neuronal pathways [9,44,45,51]. We [27]. Apart from the above-mentioned ZnT family, a therefore hypothesize that zinc ions may have a regulatory divalent cation transporter 1 (DCT1) involved in the function in some adrenergic neurons. transport of a number of trace elements, including zinc, copper and iron, has also been cloned [23]. The functional role(s) of zinc ions in sympathetic 5. Conclusion ganglionic neurons are completely unknown. The fact that we have not been able to show zinc ions or zinc pumps in The rapid appearance of zinc ions and ZnT3 in crushed undamaged sciatic sympathetic ®bers could be because of sciatic axons both proximally and distally to the crush extremely small quantities of ZnT3 and zinc ions in normal suggests that these axons belong to ZEN showing fast sympathetic ®bers, but also that the crush ignited an anterograde and retrograde axonal transport. The observa- intra-axonal cascade of chemical events leading to the tion of a subset of sympathetic ganglionic neurons that 中国科技论文在线 http://www.paper.edu.cn

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Fig. 4. Confocal microscopic images of sections of mouse lumbar sympathetic ganglia double-labeled with anti-ZnT3 and TH or NPY. (a±a0) Double labeling of ZnT3 (green) and TH (red); only a small fraction of sympathetic TH-positive cells are ZnT3-positive. (b±b0) ZnT3 immuno¯uorescence is predominantly present in the perinuclear zone. Note two cells that contain both ZnT3 and TH immuno¯uorescence (yellow), and one ZnT3-positive neuron lacking TH immunoreactivity. (c±c0 and d±d0) Double staining for ZnT3 and NPY, revealing a weak NPY-IR of the ZnT3-positive neurons. Scale bars are 250 mm(a±a0), 50 mm(b±b0), 100 mm(c±c0) and 50 mm(d±d0).

have co-localized zinc ions, and zinc transporter molecules Acknowledgements in their somata, and the uniformity in the appearance of ZnSeAMG and ZnT3 immunostaining in crushed mouse and Supported by The Swedish Medical Research Council rat sciatic nerves [48], further support the notion that a (2207), Loo och Hans Ostermans Foundation, The Royal subset of sympathetic adrenergic neurons in mammals are Society of Arts and Science in Gothenburg, The Swedish ZEN neurons. Medical Society, The Swedish Foundation for Parkinson 中国科技论文在线 http://www.paper.edu.cn

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