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Inhibitory zinc-enriched terminals in mouse spinal cord
Neuroscience Vol. 105, No. 4, pp. 941^947, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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INHIBITORY ZINC-ENRICHED TERMINALS IN MOUSE SPINAL CORD G. DANSCHER,a * S. M. JO,a;b E. VAREA,a Z. WANG,a T. B. COLEc and H. D. SCHRÒDERd a b
Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
Department of Anatomy, University of Hallym College of Medicine, 1-Okchon-Dong, Chunchon 200-702, South Korea c d
Department of Pathology, Odense University Hospital, Odense, Denmark
Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195, USA
AbstractöThe ultrastructural localization of zinc transporter-3, glutamate decarboxylase and zinc ions in zinc-enriched terminals in the mouse spinal cord was studied by zinc transporter-3 and glutamate decarboxylase immunohistochemistry and zinc selenium autometallography, respectively. The distribution of zinc selenium autometallographic silver grains, and zinc transporter-3 and glutamate decarboxylase immunohistochemical puncta in both ventral and dorsal horns as seen in the light microscope corresponded to their presence in the synaptic vesicles of zinc-enriched terminals at ultrastructural levels. The densest populations of zincenriched terminals were seen in dorsal horn laminae I, III and IV, whereas the deeper laminae V and VI contained fewer terminals. At ultrastructural levels, zinc-enriched terminals primarily formed symmetrical synapses on perikarya and dendrites. Only relatively few asymmetrical synapses were observed on zinc-enriched terminals. In general, the biggest zinc-enriched terminals contacted neuronal somata and large dendritic elements, while medium-sized and small terminals made contacts on small dendrites. The ventral horn was primarily populated by big and medium-sized zinc-enriched terminals, whereas the dorsal horn was dominated by medium-sized and small zinc-enriched terminals. The presence of boutons with £at synaptic vesicles with zinc ions and symmetric synaptic contacts suggests the presence of inhibitory zinc-enriched terminals in the mammalian spinal cord, and this was con¢rmed by the ¢nding that zinc ions and glutamate decarboxylase are co-localized in these terminals. The pattern of zinc-enriched boutons in both dorsal and ventral horns is compatible with evidence suggesting that zinc may be involved in both sensory transmission and motor control. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: zinc-enriched, autometallography, zinc transporter-3 immunohistochemistry, spinal cord, mouse, glutamate decarboxylase immunohistochemistry.
the motor nuclei of the ventral horn suggests that ZEN terminals may be involved in motor control as well (SchrÖder, 1979). The systematic co-localization of zinc ions and glutamate in ZEN terminals in the brain suggests that a co-release of the two could modify the character of the postsynaptic response (Crawford and Connor, 1973; Pe¨rez-Clausell and Danscher, 1985). Until now, the majority of evidence has indicated that ZEN neurons in the CNS were a subgroup of glutamatergic neurons.
Zinc-enriched (ZEN) terminals in the spinal cord are dispersed throughout the gray matter. The super¢cial dorsal horn (laminae I, III, IV) and lamina X, involved in sensory transmission, contain relatively high concentrations of ZEN terminals (SchrÖder, 1977; SchrÖder et al., 1978; Jo et al., 2000). These results have led to a functional consideration of zinc ions being a possible neurotransmitter or neuromodulator of sensory transmission. A good deal of information has evolved from biochemical and physiological studies, suggesting that zinc ions could be involved in sensory transmission at the spinal cord level (Larson and Kitto, 1997, 1999). On the other hand, the ultrastructural localization of ZEN terminals presynaptic to dendrites and somata in
EXPERIMENTAL PROCEDURES
Male BALB/c mice (8^10 weeks; weight about 30 g) from MÖllegaard Breeding Center, Denmark, were used in the experiments of this study. The study was undertaken in accordance with the Danish and University of Aarhus guidelines for animal welfare. All e¡orts were made to minimize the number of animals used and their su¡ering.
*Corresponding author. Tel.: +45-894-230-41; fax: +45-894-230-60. E-mail address: [email protected] (G. Danscher). Abbreviations : ABC, avidin^biotin^peroxidase complex ; AMG, autometallography; BSA, bovine serum albumin; DAB, 3,3Pdiaminobenzidine; DMSO, dimethyl sulfoxide ; EM, electron microscopy ; GAD, glutamate decarboxylase ; IHC, immunohistochemistry; PB, phosphate bu¡er; RT, room temperature; TB, Tris bu¡er ; TBS, Tris-bu¡ered saline ; TSQ, N-6-metoxy-ptoluenesulfonamide quinoline ; ZEN, zinc-enriched; ZnT3, zinc transporter-3.
Zinc transporter-3 (ZnT3) and glutamate decarboxylase (GAD) immunohistochemistry (IHC) Seven mice were anaesthetized with pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 50 ml isotonic saline, followed by 200 ml 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate bu¡er (PB) (pH 7.4). The entire spinal cords 941
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were removed and post¢xed using the above ¢xative for 4 h (4³C). Segmental pieces of the spinal cord were placed in PB, and 100-Wm thick transverse sections were cut on a cryostat. An a¤nity-puri¢ed rabbit antibody speci¢c for ZnT3 (provided by Dr. Richard Palmiter, Department of Biochemistry, University of Washington, USA) was used for the immunohistochemical localization. For GAD immunodetection we used an a¤nitypuri¢ed antibody speci¢c for GAD-67 (Chemicon AB-108, Chemicon International, Inc., 28835 Single Oak Drive, Temecula, CA 92590, USA). The immunolabelling procedures were performed in accordance with the routine avidin^biotin^peroxidase complex (ABC) method. Sections were rinsed in 0.1 M Tris-bu¡ered saline (TBS: 0.05 M, Tris bu¡er (TB) in/0.15 M NaCl, pH 7.4), and endogenous peroxidases were then inactivated by treatment with 1% hydrogen peroxide (H2 O2 ) in pure methanol for 15 min. The sections were rinsed three times in TBS and treated with 1% bovine serum albumin (BSA) and 3% goat serum in TBS containing 0.25% dimethyl sulfoxide (DMSO) for 2 h to reduce nonspeci¢c staining. The sections were rinsed in TBS for 30 min and incubated for 2 days at 4³C in ZnT3 antiserum, diluted 1:100 in TBS containing 3% goat serum, 1% BSA and 0.25% DMSO, or 1 day at 4³C with GAD antiserum 1:1000 in TBS containing 3% goat serum, 1% BSA and 0.25% DMSO. Following rinses for 45 min in TBS, the sections were incubated in biotinylated goat anti-rabbit IgG (diluted 1:500) for 1 day at 4³C and rinsed for 30 min in TBS. The ABC kit (DAKO, Produktionsvej 42, DK-2600 Glostrup, Denmark) was applied to visualize the reaction sites for 1 h at room temperature (RT). The ABC solution was diluted 1:100 in BSA 1% in TBS. After the sections had been rinsed in 0.1 TB (pH 7.6) and left in 0.025% 3,3P-diaminobenzidine (DAB) with 0.0033% H2 O2 for 15 min at RT, the brown color appeared in the sections. Staining was stopped by rinsing with TB, followed by mounting on 0.5% gelatin-coated slides and air-drying at RT. Sections were further processed for electron microscopy (EM) by post¢xation in 1% osmium tetroxide (OsO4 ) in 0.1 M PB (pH 7.4) for 1 h at RT. Surplus OsO4 was washed out with PB, followed by alcohol dehydration, propylene substitution, and £at-embedding in Epon 812. For light microscopy, 3-Wm thick sections were cut on an ultramicrotome and stained with 0.1% toluidine blue. Ultrathin sections were cut perpendicular to the plane of the vibratome slices, stained with uranyl acetate for 10 min, and examined at 80 kV with a Philips 208 electron microscope (Eindhoven, The Netherlands). To assess nonspeci¢c staining, a few sections in every experiment were incubated in a bu¡er without primary antibody. This procedure always resulted in a complete lack of immunoreactivity. ZnSe autometallography (ZnSeAMG ) procedure Six mice were injected i.p. under halothane anesthesia with sodium selenide (10 mg/kg) dissolved in PB. After 1.5 h the animals were killed by transcardial perfusion with a 3% glutaraldehyde solution in PB. The spinal cord segments were removed and placed in the same ¢xatives for 3 h in the refrigerator (4³C). Sections, 100 Wm thick, were cut on a vibratome and collected in the vial for AMG development. The AMG silver enhancement was performed with the original silver lactate developer (Danscher, 1982; Danscher et al., 1985). In short, sections were placed in jars £oating in the AMG developer for 60 min. AMG was stopped by replacing the developer with a 5% sodium thiosulfate solution. Ten min later the sections were rinsed with distilled water. Areas to be analyzed by EM were isolated, placed in small jars and ¢xed in 1% OsO4 in PB for 30 min. The small tissue blocks were then embedded in Epon 812, and 3-Wm thick survey sections were cut and counterstained with 0.1% toluidine blue. Sections selected for EM were reembedded on top of a blank Epon block, from which ultrathin sections (90^120 nm) were cut. The ultrathin sections were double stained with uranyl acetate (30 min) and lead citrate (5 min) and examined with a Philips 208 electron microscope (Eindhoven, The Netherlands).
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Double staining using ZnSeAMG and GADIHC Three mice were injected i.p. under halothane anesthesia with sodium selenide (10 mg/kg) dissolved in PB. After 1.5 h the animals were killed by transcardial perfusion with a 4% paraformaldehyde and 0.1% glutaraldehyde solution in PB. The spinal cord segments were removed and placed in the same ¢xatives for 3 h in the refrigerator (4³C). Sections, 100 Wm thick, were cut on a vibratome and collected in the vial for AMG development. The AMG silver enhancement was performed with the original silver lactate developer, see the ZnSeAMG procedure. After development the sections were carefully rinsed in distilled water and TBS for 10 min in order to remove the developer. The sections were then stained following the GADIHC procedure described above. A critical step in the double staining is the blocking of endogenous peroxidase. Usually it is done by methanol and hydrogen peroxidase. However, this solution oxidizes the AMG silver grains and dissolves them, and this step must, therefore, be avoided. After GADIHC the sections were post¢xed with 1% OsO4 in PB, dehydrated, embedded in Epon 812, and processed in the same way as the ZnSeAMG or ZnT3IHC sections prepared for EM analysis.
RESULTS
Light microscopy The neuropil throughout the spinal cord exhibited a highly di¡erentiated GADIHC , ZnT3IHC and ZnSeAMG pattern composed of puncta showing immunoreactivity and AMG silver grains demonstrating zinc selenide clusters. The white matter was unstained apart from rows of ZnSeAMG grains and ZnT3IHC and GADIHC puncta along dendritic projections radiating from the gray matter (Fig. 1a^c). At higher magni¢cation the ZnSeAMG , ZnT3IHC and GADIHC staining can be seen as patterns of stained ZEN terminals in the dorsal horn (Fig. 1d^f) and around motor neurons in the ventral horn (Fig. 1g^i). Ultrastructural localization of ZnT3 The dotted distribution of ZnT3IHC puncta in both ventral and dorsal horns corresponded ultrastructurally to ZEN terminals. The electron opaque reaction products for peroxidase-labelled ZnT3 antibodies were located in presynaptic terminals (Fig. 2). The immunostaining was found to be associated with synaptic vesicles. A majority of the ZnT3 terminals in the mouse spinal cord contained £attened synaptic vesicles and made symmetrical contacts (Fig. 2a,b), but some ZEN terminals, mostly medium-sized or small boutons, contained round vesicles and made asymmetrical synaptic contacts (Fig. 2c). Most of the ZnT3-labelled terminals found in the dorsal horn made axo-dendritic contacts, but a small number were axo-somatic. In the super¢cial laminae of the dorsal horn a few ZEN axoaxonal synaptic complexes were encountered (data not shown). In the ventral horn ZEN terminals were much bigger, less numerous and more variable in size. Most of them made axo-dendritic and axo-somatic symmetrical synaptic contacts to motor neurons and contained irregular or £attened vesicles.
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Fig. 1. Light micrographs of mouse spinal cord sections stained with ZnSeAMG (a,d,g), ZnT3IHC (b,e,h) and GADIHC (c,f,i). The three techniques show almost identical staining patterns (a^c). (d^f) Show details of the staining patterns in the dorsal horn and (g^i) show stained terminals around motor neurons in the ventral horn. a^c and d^i are same magni¢cation.
Ultrastructural localization of ZnSeAMG ZEN terminals making symmetric synapses contained ZnSeAMG silver grains in £attened synaptic vesicles (Fig. 3a). The less abundant ZEN terminals making asymmetric contacts contained homogeneous round synaptic vesicles (Fig. 3b). Both kinds of terminals had
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AMG grains in only a fraction of the synaptic vesicles, and AMG grains were found in both symmetric and asymmetric synaptic clefts (Fig. 3a,b). In the ventral horn, large ZEN terminals were found around motor neurons and made axo-dendritic and axo-somatic synaptic contacts. AMG silver grains were often concentrated close to the synaptic specializations.
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Fig. 2. Electron micrographs from the super¢cial dorsal horn, laminae I^II, of a mouse lumbo-sacral spinal cord slice stained with ZnT3IHC . The electron opaque puncta of peroxidase-labelled ZnT3 antibodies are located in presynaptic terminals contacting dendritic elements with typical symmetrical synapses (a,b). A few make asymmetrical synapses with postsynaptic membrane thickenings (c, arrowheads). a^c are same magni¢cation.
Double staining using ZnSeAMG and GADIHC The double-stained sections showed a high amount of synaptic terminals stained with both techniques, but contained terminals loaded only with GADIHC puncta or ZnSeAMG grains. In most cases the ZnSeAMG -positive
GADIHC -negative terminals could be identi¢ed as having round vesicles and asymmetric synaptic specializations. Fig. 4 shows two boutons, one double stained with ZnSeAMG and GADIHC , one containing ZnSeAMG grains only. In order to show that the ZnSeAMG and the TSQ
Fig. 3. Electron micrographs showing axo-dendritic synapses in the dorsal horn, laminae II^IV, from lower thoracic segment. (a) A majority of the ZEN terminals contain £attened synaptic vesicles and make symmetric contacts to dendritic elements (arrowheads). Note terminals making typical asymmetrical synapses (open arrows), devoid of ZnSeAMG grains. (b) Some ZEN terminals, mostly medium-sized or small boutons, contain round vesicles and make asymmetrical synaptic contacts (arrows). d, dendrite.
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Fig. 4. Electron micrograph showing a GAD-positive and a GADnegative ZEN terminal.
(N-6-metoxy-p-toluenesulfonamide quinoline) techniques reveal the same pool of zinc ions in the spinal cord we stained 20-Wm thick sections with TSQ (Frederickson et al., 1987) (Fig. 5a). The dorsal horn of a spinal cord section stained with ZnSeAMG is shown in Fig. 5b. Spinal cord sections from mice that had been treated with sodium selenide did not stain with TSQ proving that the two methods mark the same pool of zinc ions.
DISCUSSION
The present study supports a recent report showing GABAergic ZEN terminals in lamprey spinal cord (Byrinyi et al., 2000). In the spinal cord, the light microscopic patterns revealed by AMG and ZnT3 immuno-
staining seem to be identical (Jo et al., 2000). ZnT3 knockout mice have been shown to have lost all their zinc-containing synaptic vesicles, or at least ZnSAMG staining is lacking in the brains of mice without ZnT3 transporter molecules (Cole et al., 1999). The spinal cords of ZnT3-Null mice have not yet been studied, but we expect to ¢nd the same lack of zinc ions in the ZEN terminals. The ¢nding of inhibitory GABAergic ZEN terminals in the spinal cord shows that at least this part of the CNS contains two morphologically and functionally di¡erent types of ZEN terminals. Particularly apparent are the large ZEN boutons with symmetric synapses and £at synaptic vesicles terminating on the somata and big dendrites of ventral horn motor neurons. The staining pattern of GAD is almost identical to that of ZnT3 and ZnSeAMG showing heavier staining in laminae I, III, IV and X. Most of the ZEN terminals observed in the mouse spinal cord were located presynaptic to dendrites and neuronal somata. They are presumed to be inhibitory, since they contained pleomorphic or £attened vesicles and made symmetrical synaptic contacts. With a few exceptions, symmetrical synaptic contacts are typical for GABAergic terminals presumed to have inhibitory functions throughout the CNS (Onteniente et al., 1987; Babb et al., 1988). Asymmetrical synapses, on the other hand, are characteristic for the excitatory glutamatergic neurons (Liu and Jones, 1996; Rubio and Juiz, 1998). Only a minor fraction of the spinal cord ZEN terminals contains clear round vesicles and makes asymmetrical synaptic contacts. Our ¢ndings, therefore, support the notion that at least two populations of ZEN terminals are present in the mouse spinal cord, a subset of glutamatergic boutons as found in the brain (Frederickson and Danscher, 1988, 1990) and a subset of GABAergic terminals. Several antibodies have been developed to show GABAergic terminals, one detects the neurotransmitter (GABA) and others detect the protein that produces it (GAD). They have both been used in the spinal cord (McLaughlin et al., 1975; Magoul et al., 1987). Two isoforms of the GAD protein have been characterized, one of 65 kDa and the other of 67 kDa. Usually it is accepted that both types are expressed in the GAD-pos-
Fig. 5. Micrographs of a spinal cord sections stained with TSQ (a) and ZnSeAMG (b). There appeared to be a smaller di¡erence in the intensity of staining in color photographs than in the black and white photos shown here.
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itive neurons (Bu et al., 1992). In this study we have used a polyclonal antibody against GAD-67 (Chemicon). The double staining with AMG and IHC has been described previously (De Biasi and Bendotti, 1998). We used the ZnSeAMG tracing of zinc ions together with GAD immunostaining at EM levels. The GABAergic terminals are in particular abundant in the super¢cial dorsal horn where they make axo-dendritic or axo-somatic contacts (Magoul et al., 1987; Dumoulin et al., 1999). If zinc ions are co-released with GABA into the synaptic cleft, as occurs with glutamate in excitatory terminals (Assaf and Chung, 1984; Howell et al., 1984; Budde et al., 1997), it is not unlikely that zinc ions could modulate pre- and/or postsynaptic GABA receptors (Westbrook and Mayer, 1987; Smart et al., 1991) and thus a¡ect neuronal inhibition in a manner unique to the spinal cord. Although the level of ZEN terminals seems to be lower in the spinal cord than in telencephalic regions like the neocortex, hippocampus and amygdala (Slomianka, 1992; Pe¨rez-Clausell, 1996), there is still a relatively high concentration of ZEN terminals in the gray matter, in particular in the super¢cial dorsal horn lamina (Jo et al., 2000). Thus, the modulation of inhibitory, as well as excitatory, responses by zinc could construct a signi¢cant additional control of motor neuron activity in the ventral horn or primary a¡erent terminals in the dorsal horn (SchrÖder, 1979). It has been found that GABAA receptors are distributed homogeneously in the gray matter, while GABAB receptors are mainly located in laminae I, II, III and X (Price et al., 1984). These are the main locations of GABAergic ZEN terminals in the mouse spinal cord.
GABA responses have been shown to be modulated by zinc ions (Westbrook and Mayer, 1987). The GABA receptors sensitive to zinc ions were found to be GABAB receptors, whereas GABAA receptors were almost una¡ected (Turgeon and Albin, 1992). Zinc a¡ects the a¤nity constant for GABA to its receptor: Within the range of 10^100 WM, zinc ions signi¢cantly enhance GABAB binding, while they decrease it at higher concentrations (Turgeon and Albin, 1992). Inhibitory neurons in the spinal cord have been assigned to an anti-nociceptive e¡ect, and zinc ions have been found to modulate nociception. Decreasing the level of zinc ions by intrathecal injection of chelating agents increases nociception, while increasing the level of zinc ions results in a lowering of the pain (Larson and Kitto, 1997, 1999).
CONCLUSION
The present study demonstrates that the spinal cord contains a population of GABAergic ZEN boutons in addition to ZEN terminals with excitatory morphology, comparable to the glutaminergic ZEN terminals found in the brain. AcknowledgementsöWe thank Herdis Krunderup, Dorete Jensen, Albert Meier, Thorkild Nielsen and Karin Wiedemann for excellent technical assistance. The study was supported by the Danish Medical Research Council, the Danish Research Academy, the Lundbeck Foundation, the Leo Foundation, the Gangsted Foundation, and by a Korea Science and Engineering Foundation grant to Seung Mook Jo.
REFERENCES
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INHIBITORY ZINC-ENRICHED TERMINALS IN MOUSE SPINAL CORD G. DANSCHER,a * S. M. JO,a;b E. VAREA,a Z. WANG,a T. B. COLEc and H. D. SCHRÒDERd a b
Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
Department of Anatomy, University of Hallym College of Medicine, 1-Okchon-Dong, Chunchon 200-702, South Korea c d
Department of Pathology, Odense University Hospital, Odense, Denmark
Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195, USA
AbstractöThe ultrastructural localization of zinc transporter-3, glutamate decarboxylase and zinc ions in zinc-enriched terminals in the mouse spinal cord was studied by zinc transporter-3 and glutamate decarboxylase immunohistochemistry and zinc selenium autometallography, respectively. The distribution of zinc selenium autometallographic silver grains, and zinc transporter-3 and glutamate decarboxylase immunohistochemical puncta in both ventral and dorsal horns as seen in the light microscope corresponded to their presence in the synaptic vesicles of zinc-enriched terminals at ultrastructural levels. The densest populations of zincenriched terminals were seen in dorsal horn laminae I, III and IV, whereas the deeper laminae V and VI contained fewer terminals. At ultrastructural levels, zinc-enriched terminals primarily formed symmetrical synapses on perikarya and dendrites. Only relatively few asymmetrical synapses were observed on zinc-enriched terminals. In general, the biggest zinc-enriched terminals contacted neuronal somata and large dendritic elements, while medium-sized and small terminals made contacts on small dendrites. The ventral horn was primarily populated by big and medium-sized zinc-enriched terminals, whereas the dorsal horn was dominated by medium-sized and small zinc-enriched terminals. The presence of boutons with £at synaptic vesicles with zinc ions and symmetric synaptic contacts suggests the presence of inhibitory zinc-enriched terminals in the mammalian spinal cord, and this was con¢rmed by the ¢nding that zinc ions and glutamate decarboxylase are co-localized in these terminals. The pattern of zinc-enriched boutons in both dorsal and ventral horns is compatible with evidence suggesting that zinc may be involved in both sensory transmission and motor control. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: zinc-enriched, autometallography, zinc transporter-3 immunohistochemistry, spinal cord, mouse, glutamate decarboxylase immunohistochemistry.
the motor nuclei of the ventral horn suggests that ZEN terminals may be involved in motor control as well (SchrÖder, 1979). The systematic co-localization of zinc ions and glutamate in ZEN terminals in the brain suggests that a co-release of the two could modify the character of the postsynaptic response (Crawford and Connor, 1973; Pe¨rez-Clausell and Danscher, 1985). Until now, the majority of evidence has indicated that ZEN neurons in the CNS were a subgroup of glutamatergic neurons.
Zinc-enriched (ZEN) terminals in the spinal cord are dispersed throughout the gray matter. The super¢cial dorsal horn (laminae I, III, IV) and lamina X, involved in sensory transmission, contain relatively high concentrations of ZEN terminals (SchrÖder, 1977; SchrÖder et al., 1978; Jo et al., 2000). These results have led to a functional consideration of zinc ions being a possible neurotransmitter or neuromodulator of sensory transmission. A good deal of information has evolved from biochemical and physiological studies, suggesting that zinc ions could be involved in sensory transmission at the spinal cord level (Larson and Kitto, 1997, 1999). On the other hand, the ultrastructural localization of ZEN terminals presynaptic to dendrites and somata in
EXPERIMENTAL PROCEDURES
Male BALB/c mice (8^10 weeks; weight about 30 g) from MÖllegaard Breeding Center, Denmark, were used in the experiments of this study. The study was undertaken in accordance with the Danish and University of Aarhus guidelines for animal welfare. All e¡orts were made to minimize the number of animals used and their su¡ering.
*Corresponding author. Tel.: +45-894-230-41; fax: +45-894-230-60. E-mail address: [email protected] (G. Danscher). Abbreviations : ABC, avidin^biotin^peroxidase complex ; AMG, autometallography; BSA, bovine serum albumin; DAB, 3,3Pdiaminobenzidine; DMSO, dimethyl sulfoxide ; EM, electron microscopy ; GAD, glutamate decarboxylase ; IHC, immunohistochemistry; PB, phosphate bu¡er; RT, room temperature; TB, Tris bu¡er ; TBS, Tris-bu¡ered saline ; TSQ, N-6-metoxy-ptoluenesulfonamide quinoline ; ZEN, zinc-enriched; ZnT3, zinc transporter-3.
Zinc transporter-3 (ZnT3) and glutamate decarboxylase (GAD) immunohistochemistry (IHC) Seven mice were anaesthetized with pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 50 ml isotonic saline, followed by 200 ml 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate bu¡er (PB) (pH 7.4). The entire spinal cords 941
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were removed and post¢xed using the above ¢xative for 4 h (4³C). Segmental pieces of the spinal cord were placed in PB, and 100-Wm thick transverse sections were cut on a cryostat. An a¤nity-puri¢ed rabbit antibody speci¢c for ZnT3 (provided by Dr. Richard Palmiter, Department of Biochemistry, University of Washington, USA) was used for the immunohistochemical localization. For GAD immunodetection we used an a¤nitypuri¢ed antibody speci¢c for GAD-67 (Chemicon AB-108, Chemicon International, Inc., 28835 Single Oak Drive, Temecula, CA 92590, USA). The immunolabelling procedures were performed in accordance with the routine avidin^biotin^peroxidase complex (ABC) method. Sections were rinsed in 0.1 M Tris-bu¡ered saline (TBS: 0.05 M, Tris bu¡er (TB) in/0.15 M NaCl, pH 7.4), and endogenous peroxidases were then inactivated by treatment with 1% hydrogen peroxide (H2 O2 ) in pure methanol for 15 min. The sections were rinsed three times in TBS and treated with 1% bovine serum albumin (BSA) and 3% goat serum in TBS containing 0.25% dimethyl sulfoxide (DMSO) for 2 h to reduce nonspeci¢c staining. The sections were rinsed in TBS for 30 min and incubated for 2 days at 4³C in ZnT3 antiserum, diluted 1:100 in TBS containing 3% goat serum, 1% BSA and 0.25% DMSO, or 1 day at 4³C with GAD antiserum 1:1000 in TBS containing 3% goat serum, 1% BSA and 0.25% DMSO. Following rinses for 45 min in TBS, the sections were incubated in biotinylated goat anti-rabbit IgG (diluted 1:500) for 1 day at 4³C and rinsed for 30 min in TBS. The ABC kit (DAKO, Produktionsvej 42, DK-2600 Glostrup, Denmark) was applied to visualize the reaction sites for 1 h at room temperature (RT). The ABC solution was diluted 1:100 in BSA 1% in TBS. After the sections had been rinsed in 0.1 TB (pH 7.6) and left in 0.025% 3,3P-diaminobenzidine (DAB) with 0.0033% H2 O2 for 15 min at RT, the brown color appeared in the sections. Staining was stopped by rinsing with TB, followed by mounting on 0.5% gelatin-coated slides and air-drying at RT. Sections were further processed for electron microscopy (EM) by post¢xation in 1% osmium tetroxide (OsO4 ) in 0.1 M PB (pH 7.4) for 1 h at RT. Surplus OsO4 was washed out with PB, followed by alcohol dehydration, propylene substitution, and £at-embedding in Epon 812. For light microscopy, 3-Wm thick sections were cut on an ultramicrotome and stained with 0.1% toluidine blue. Ultrathin sections were cut perpendicular to the plane of the vibratome slices, stained with uranyl acetate for 10 min, and examined at 80 kV with a Philips 208 electron microscope (Eindhoven, The Netherlands). To assess nonspeci¢c staining, a few sections in every experiment were incubated in a bu¡er without primary antibody. This procedure always resulted in a complete lack of immunoreactivity. ZnSe autometallography (ZnSeAMG ) procedure Six mice were injected i.p. under halothane anesthesia with sodium selenide (10 mg/kg) dissolved in PB. After 1.5 h the animals were killed by transcardial perfusion with a 3% glutaraldehyde solution in PB. The spinal cord segments were removed and placed in the same ¢xatives for 3 h in the refrigerator (4³C). Sections, 100 Wm thick, were cut on a vibratome and collected in the vial for AMG development. The AMG silver enhancement was performed with the original silver lactate developer (Danscher, 1982; Danscher et al., 1985). In short, sections were placed in jars £oating in the AMG developer for 60 min. AMG was stopped by replacing the developer with a 5% sodium thiosulfate solution. Ten min later the sections were rinsed with distilled water. Areas to be analyzed by EM were isolated, placed in small jars and ¢xed in 1% OsO4 in PB for 30 min. The small tissue blocks were then embedded in Epon 812, and 3-Wm thick survey sections were cut and counterstained with 0.1% toluidine blue. Sections selected for EM were reembedded on top of a blank Epon block, from which ultrathin sections (90^120 nm) were cut. The ultrathin sections were double stained with uranyl acetate (30 min) and lead citrate (5 min) and examined with a Philips 208 electron microscope (Eindhoven, The Netherlands).
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Double staining using ZnSeAMG and GADIHC Three mice were injected i.p. under halothane anesthesia with sodium selenide (10 mg/kg) dissolved in PB. After 1.5 h the animals were killed by transcardial perfusion with a 4% paraformaldehyde and 0.1% glutaraldehyde solution in PB. The spinal cord segments were removed and placed in the same ¢xatives for 3 h in the refrigerator (4³C). Sections, 100 Wm thick, were cut on a vibratome and collected in the vial for AMG development. The AMG silver enhancement was performed with the original silver lactate developer, see the ZnSeAMG procedure. After development the sections were carefully rinsed in distilled water and TBS for 10 min in order to remove the developer. The sections were then stained following the GADIHC procedure described above. A critical step in the double staining is the blocking of endogenous peroxidase. Usually it is done by methanol and hydrogen peroxidase. However, this solution oxidizes the AMG silver grains and dissolves them, and this step must, therefore, be avoided. After GADIHC the sections were post¢xed with 1% OsO4 in PB, dehydrated, embedded in Epon 812, and processed in the same way as the ZnSeAMG or ZnT3IHC sections prepared for EM analysis.
RESULTS
Light microscopy The neuropil throughout the spinal cord exhibited a highly di¡erentiated GADIHC , ZnT3IHC and ZnSeAMG pattern composed of puncta showing immunoreactivity and AMG silver grains demonstrating zinc selenide clusters. The white matter was unstained apart from rows of ZnSeAMG grains and ZnT3IHC and GADIHC puncta along dendritic projections radiating from the gray matter (Fig. 1a^c). At higher magni¢cation the ZnSeAMG , ZnT3IHC and GADIHC staining can be seen as patterns of stained ZEN terminals in the dorsal horn (Fig. 1d^f) and around motor neurons in the ventral horn (Fig. 1g^i). Ultrastructural localization of ZnT3 The dotted distribution of ZnT3IHC puncta in both ventral and dorsal horns corresponded ultrastructurally to ZEN terminals. The electron opaque reaction products for peroxidase-labelled ZnT3 antibodies were located in presynaptic terminals (Fig. 2). The immunostaining was found to be associated with synaptic vesicles. A majority of the ZnT3 terminals in the mouse spinal cord contained £attened synaptic vesicles and made symmetrical contacts (Fig. 2a,b), but some ZEN terminals, mostly medium-sized or small boutons, contained round vesicles and made asymmetrical synaptic contacts (Fig. 2c). Most of the ZnT3-labelled terminals found in the dorsal horn made axo-dendritic contacts, but a small number were axo-somatic. In the super¢cial laminae of the dorsal horn a few ZEN axoaxonal synaptic complexes were encountered (data not shown). In the ventral horn ZEN terminals were much bigger, less numerous and more variable in size. Most of them made axo-dendritic and axo-somatic symmetrical synaptic contacts to motor neurons and contained irregular or £attened vesicles.
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Fig. 1. Light micrographs of mouse spinal cord sections stained with ZnSeAMG (a,d,g), ZnT3IHC (b,e,h) and GADIHC (c,f,i). The three techniques show almost identical staining patterns (a^c). (d^f) Show details of the staining patterns in the dorsal horn and (g^i) show stained terminals around motor neurons in the ventral horn. a^c and d^i are same magni¢cation.
Ultrastructural localization of ZnSeAMG ZEN terminals making symmetric synapses contained ZnSeAMG silver grains in £attened synaptic vesicles (Fig. 3a). The less abundant ZEN terminals making asymmetric contacts contained homogeneous round synaptic vesicles (Fig. 3b). Both kinds of terminals had
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AMG grains in only a fraction of the synaptic vesicles, and AMG grains were found in both symmetric and asymmetric synaptic clefts (Fig. 3a,b). In the ventral horn, large ZEN terminals were found around motor neurons and made axo-dendritic and axo-somatic synaptic contacts. AMG silver grains were often concentrated close to the synaptic specializations.
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Fig. 2. Electron micrographs from the super¢cial dorsal horn, laminae I^II, of a mouse lumbo-sacral spinal cord slice stained with ZnT3IHC . The electron opaque puncta of peroxidase-labelled ZnT3 antibodies are located in presynaptic terminals contacting dendritic elements with typical symmetrical synapses (a,b). A few make asymmetrical synapses with postsynaptic membrane thickenings (c, arrowheads). a^c are same magni¢cation.
Double staining using ZnSeAMG and GADIHC The double-stained sections showed a high amount of synaptic terminals stained with both techniques, but contained terminals loaded only with GADIHC puncta or ZnSeAMG grains. In most cases the ZnSeAMG -positive
GADIHC -negative terminals could be identi¢ed as having round vesicles and asymmetric synaptic specializations. Fig. 4 shows two boutons, one double stained with ZnSeAMG and GADIHC , one containing ZnSeAMG grains only. In order to show that the ZnSeAMG and the TSQ
Fig. 3. Electron micrographs showing axo-dendritic synapses in the dorsal horn, laminae II^IV, from lower thoracic segment. (a) A majority of the ZEN terminals contain £attened synaptic vesicles and make symmetric contacts to dendritic elements (arrowheads). Note terminals making typical asymmetrical synapses (open arrows), devoid of ZnSeAMG grains. (b) Some ZEN terminals, mostly medium-sized or small boutons, contain round vesicles and make asymmetrical synaptic contacts (arrows). d, dendrite.
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Fig. 4. Electron micrograph showing a GAD-positive and a GADnegative ZEN terminal.
(N-6-metoxy-p-toluenesulfonamide quinoline) techniques reveal the same pool of zinc ions in the spinal cord we stained 20-Wm thick sections with TSQ (Frederickson et al., 1987) (Fig. 5a). The dorsal horn of a spinal cord section stained with ZnSeAMG is shown in Fig. 5b. Spinal cord sections from mice that had been treated with sodium selenide did not stain with TSQ proving that the two methods mark the same pool of zinc ions.
DISCUSSION
The present study supports a recent report showing GABAergic ZEN terminals in lamprey spinal cord (Byrinyi et al., 2000). In the spinal cord, the light microscopic patterns revealed by AMG and ZnT3 immuno-
staining seem to be identical (Jo et al., 2000). ZnT3 knockout mice have been shown to have lost all their zinc-containing synaptic vesicles, or at least ZnSAMG staining is lacking in the brains of mice without ZnT3 transporter molecules (Cole et al., 1999). The spinal cords of ZnT3-Null mice have not yet been studied, but we expect to ¢nd the same lack of zinc ions in the ZEN terminals. The ¢nding of inhibitory GABAergic ZEN terminals in the spinal cord shows that at least this part of the CNS contains two morphologically and functionally di¡erent types of ZEN terminals. Particularly apparent are the large ZEN boutons with symmetric synapses and £at synaptic vesicles terminating on the somata and big dendrites of ventral horn motor neurons. The staining pattern of GAD is almost identical to that of ZnT3 and ZnSeAMG showing heavier staining in laminae I, III, IV and X. Most of the ZEN terminals observed in the mouse spinal cord were located presynaptic to dendrites and neuronal somata. They are presumed to be inhibitory, since they contained pleomorphic or £attened vesicles and made symmetrical synaptic contacts. With a few exceptions, symmetrical synaptic contacts are typical for GABAergic terminals presumed to have inhibitory functions throughout the CNS (Onteniente et al., 1987; Babb et al., 1988). Asymmetrical synapses, on the other hand, are characteristic for the excitatory glutamatergic neurons (Liu and Jones, 1996; Rubio and Juiz, 1998). Only a minor fraction of the spinal cord ZEN terminals contains clear round vesicles and makes asymmetrical synaptic contacts. Our ¢ndings, therefore, support the notion that at least two populations of ZEN terminals are present in the mouse spinal cord, a subset of glutamatergic boutons as found in the brain (Frederickson and Danscher, 1988, 1990) and a subset of GABAergic terminals. Several antibodies have been developed to show GABAergic terminals, one detects the neurotransmitter (GABA) and others detect the protein that produces it (GAD). They have both been used in the spinal cord (McLaughlin et al., 1975; Magoul et al., 1987). Two isoforms of the GAD protein have been characterized, one of 65 kDa and the other of 67 kDa. Usually it is accepted that both types are expressed in the GAD-pos-
Fig. 5. Micrographs of a spinal cord sections stained with TSQ (a) and ZnSeAMG (b). There appeared to be a smaller di¡erence in the intensity of staining in color photographs than in the black and white photos shown here.
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itive neurons (Bu et al., 1992). In this study we have used a polyclonal antibody against GAD-67 (Chemicon). The double staining with AMG and IHC has been described previously (De Biasi and Bendotti, 1998). We used the ZnSeAMG tracing of zinc ions together with GAD immunostaining at EM levels. The GABAergic terminals are in particular abundant in the super¢cial dorsal horn where they make axo-dendritic or axo-somatic contacts (Magoul et al., 1987; Dumoulin et al., 1999). If zinc ions are co-released with GABA into the synaptic cleft, as occurs with glutamate in excitatory terminals (Assaf and Chung, 1984; Howell et al., 1984; Budde et al., 1997), it is not unlikely that zinc ions could modulate pre- and/or postsynaptic GABA receptors (Westbrook and Mayer, 1987; Smart et al., 1991) and thus a¡ect neuronal inhibition in a manner unique to the spinal cord. Although the level of ZEN terminals seems to be lower in the spinal cord than in telencephalic regions like the neocortex, hippocampus and amygdala (Slomianka, 1992; Pe¨rez-Clausell, 1996), there is still a relatively high concentration of ZEN terminals in the gray matter, in particular in the super¢cial dorsal horn lamina (Jo et al., 2000). Thus, the modulation of inhibitory, as well as excitatory, responses by zinc could construct a signi¢cant additional control of motor neuron activity in the ventral horn or primary a¡erent terminals in the dorsal horn (SchrÖder, 1979). It has been found that GABAA receptors are distributed homogeneously in the gray matter, while GABAB receptors are mainly located in laminae I, II, III and X (Price et al., 1984). These are the main locations of GABAergic ZEN terminals in the mouse spinal cord.
GABA responses have been shown to be modulated by zinc ions (Westbrook and Mayer, 1987). The GABA receptors sensitive to zinc ions were found to be GABAB receptors, whereas GABAA receptors were almost una¡ected (Turgeon and Albin, 1992). Zinc a¡ects the a¤nity constant for GABA to its receptor: Within the range of 10^100 WM, zinc ions signi¢cantly enhance GABAB binding, while they decrease it at higher concentrations (Turgeon and Albin, 1992). Inhibitory neurons in the spinal cord have been assigned to an anti-nociceptive e¡ect, and zinc ions have been found to modulate nociception. Decreasing the level of zinc ions by intrathecal injection of chelating agents increases nociception, while increasing the level of zinc ions results in a lowering of the pain (Larson and Kitto, 1997, 1999).
CONCLUSION
The present study demonstrates that the spinal cord contains a population of GABAergic ZEN boutons in addition to ZEN terminals with excitatory morphology, comparable to the glutaminergic ZEN terminals found in the brain. AcknowledgementsöWe thank Herdis Krunderup, Dorete Jensen, Albert Meier, Thorkild Nielsen and Karin Wiedemann for excellent technical assistance. The study was supported by the Danish Medical Research Council, the Danish Research Academy, the Lundbeck Foundation, the Leo Foundation, the Gangsted Foundation, and by a Korea Science and Engineering Foundation grant to Seung Mook Jo.
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