THE JOURNAL OF COMPARATIVE NEUROLOGY 32155-82 (1992)
A Quantitative Light and Electron Microscopic Analysis of Taurine-Like Immunoreactivity in the Dorsal Horn of the Rat Spinal Cord INSE S. LEE, WALEED M. RENNO, AND ALVIN J. BEITZ Department of Anatomy College of Veterinary Medicine, Seoul National University, Seoul, South Korea (I.S.L.); and Department of Veterinary Pathobiology College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 (W.R., A.J.B.)
ABSTRACT Taurine has been proposed as an inhibitory neurotransmitter or neuromodulator in the vertebrate central nervous system. Within the spinal cord, taurine has been shown to have a direct inhibitory effect on spinal neurons and to have a selective antinociceptive effect on chemically induced nociception. Although sufficient data exists to suggest that taurine plays a neurotransmitter or neuromodulatory role in the spinal cord, it is not known whether this amino acid is present in axon terminals nor if this amino acid has a unique pattern of distribution within spinal tissue. To address these questions a monoclonal antibody against taurine was employed to localize taurine-like immunoreactivity in the dorsal horn of the rat spinal cord by using both light and electron microscopic techniques. Taurine-like immunoreactivity was most dense and most prominent in laminae I and I1 of the dorsal horn. A moderate amount of immunoreactivity was also present in laminae VIII and IX and X while the remaining laminae were only lightly stained. In laminae I and I1 taurine-like immunostaining was evident within neuronal cell bodies, dendrites, myelinated and unmyelinated axons, axon terminals, and astrocytes and their processes. Cell counts of these two laminae indicated that approximately 30%of neuronal perikarya at the C2 level, 52%of neuronal perikarya at the T6 level, and 18% of neuronal perikarya at the L2 level of the cord exhibited taurine-like immunoreactivity. With preembedding diaminobenzidine staining, approximately 20% of the axons examined in laminae I and 11 were found to be immunoreactive for taurine. Using postembedding immunogold staining in combination with quantitative procedures, the highest densities of gold particles were found in axon terminals containing pleomorphic vesicles and forming symmetrical synapses (36.8 particles/ km2),in a subpopulation of myelinated axons (34.2 particles/km2), in a subpopulation of neuronal dendrites (32.6 particles/ km2),and in capillary endothelial cells (39.8 particles/pm2). Moderate labeling occurred in astrocytes (20.9 particles/pm2) and neuronal perikarya (18.7 particles/ km2).The localization of taurine to presumptive inhibitory axon terminals provides anatomical support for the hypothesis that taurine may serve an inhibitory neurotransmitter role in the superficial dorsal horn of the spinal cord. On the other hand, its localization to astrocytes and endothelial cells within both the dorsal ventral horns implies that it serves other nonneuronal functions as well. o 1992 Wiley-Liss, Inc. Key words: immunocytochemistry,lamina I, lamina 11, glia, amino acids
It has been suggested that taurine (2-aminoethanesul- McBride and Frederickson, '80; Chesney, '85; Lin et al., fonic acid) plays a wide variety of functional roles both '85) in the CNS. This proposed inhibitory transmitter role within and outside the central nervous system (CNS) is consistent with its reported antiepileptic actions (Durelli (Huxtable, '89). Several lines of evidence suggest that and Mutani, '83; Toth et al., '83). In addition to its possible taurine acts as a neuromodulator (Davison and Kaczmarek, roles as a neurotransmitter and/or a neuromodulator, '71; Kuriyama, '80; Kontro and Oja, '83; Sakai et al., '85; Taber et al., '86; Magnusson et al., '88, '89) and as an inhibitory neurotransmitter (Oja and Lahdesmaki, '74; Accepted March 2,1992. O
1992 WILEY-LISS, INC.
66 taurine has been considered to be a stabilizer of excitable membranes (Schaffer et al., '80; Hastings et al., '851, to function as an osmoregulator (Thurston et al., '80; Van Gelder, '89), to act as an antagonist of intracellular calcium ionization (vanGelder, '90; Huxtable, 'go), and to play an important role in postnatal brain development (Chesney, '85). Although the role of taurine in the spinal cord remains controversial, a number of studies suggest that this amino acid may participate as an inhibitory neurotransmitter in this region (Sonnhoff et al., '75; Nicoll et al., '76; Lane et al., '78; Kurachi and Aihara, '85; Kudo et al., '88; Yasunami et al., '88). Consistent with this postulate, Kurachi and Aihara ('85) have demonstrated that taurine possesses antiepileptic activity in the spinal cord. In addition Serrano et al. ('90) have shown that taurine has an antinociceptive effect in the spinal cord and that this effect can be inhibited by naloxone. Smullin and coworkers ('90b) have further shown that intrathecally injected taurine selectively inhibits substance P-induced biting and scratching behavior and that it also inhibits the nociceptive related writhing behavior produced by an intraperitoneal injection of acetic acid in mice. At a more cellular level taurine has been shown to hyperpolarize cell membranes and inhibit firing when iontophoretically applied to neurons in the cat spinal cord (Curtis and Johnston, '74). Kudo and coworkers ('88) have reported that low concentrations of taurine cause a marked hyperpolarization of the primary afferent fibers, while higher concentrations of taurine cause a fast-onset hyperpolarization, followed by a slow-onset depolarization. Interestingly this depolarizing effect was selectively antagonized by bicuculline while the hyperpolarizing effect of taurine was selectively reduced by strychnine, suggesting the existence of two taurine receptor subtypes in the spinal cord. Despite the fact that initial biochemical studies demonstrated a homogeneous distribution of taurine in the spinal cord (Yoneda and Kuriyama, '78; Kuriyama et al., '781, cysteine sulphinate decarboxylase (CSD), the major enzyme involved in taurine synthesis in the CNS, was found to have its highest activity in the dorsal part of the dorsal horn (Yonedaand Kuriyama, '78). This increased activity of CSD in the superficial dorsal horn would suggest an increased synthesis of taurine in this region. This is consistent with recent biochemical data demonstrating that taurine in the spinal cord is found in highest concentrations in the dorsal horn and lowest concentrations in the ventral horn (Palkovits et al., '86, '90). This differential distribution implies that taurine may serve a special functional role within the dorsal horn of the spinal cord. Since the superficial dorsal horn plays an important role in spinal mechanisms of nociception (Besson and Chaouch, '87), one possibility is that taurine plays some role in nociception or antinociception. Consistent with this hypothesis are pharmacological and behavioral data that suggest a role for taurine in analgesia via an effect in the dorsal horn of the spinal cord (Beyer et al., '88; Kuriyama and Yoneda, "78; Smullin et al., '90a, '90b). Although these data clearly support a role for taurine in the dorsal horn, it is not known whether taurine is found in axon terminals in the spinal cord nor whether its distribution in this region is in fact homogeneous as early biochemical studies would suggest. During the past 6 years, antibodies against haptenic taurine have been produced and applied to the CNS for cellular localization of this amino acid (Wu et al., '85; Yoshida et al., '86; Ida et al., '87; Ottersen et al., '85, '88;
IS. LEE ET AL. Schafer et al., '88; Magnusson et al., '88, '89). With these antibodies, taurine-like immunoreactivity (taurine-LI) has been visualized in several CNS regions, but the localization of this amino acid has not been analyzed in the spinal cord. One of the criteria that must be fulfilled in order to consider taurine as a neurotransmitterineuromodulator in the spinal cord is to demonstrate its localization in axon terminals. In the present study, we have utilized monoclonal antibodies raised against taurine conjugated to keyhole limpet hemocyanine (KLH) in order to analyze the specific distribution of taurine in the spinal cord dorsal horn and to determine if taurine-LI is present in axon terminals and/or other structures in the dorsal horn. The localization of taurine-LI was examined at cervical, thoracic, lumbar, and sacral levels by both light and electron microscopy.
MATERIALS AND METHODS Experimental animals Twenty-one male Sprague-Dawley rats (250-340 g) were used for this study. Animals were anesthetized with chloral hydrate (0.5 mgig) and perfused through the ascending aorta with 100 ml of calcium-free Tyrode's solution, followed by either 500 ml of 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M Sorenson's phosphate buffer (pH 7.4,4"C)or 500 ml of 0.2%paraformaldehyde, 2.5% glutaraldehyde, and 0.2% picric acid in 0.1 M Sorenson's buffer. The spinal cords were removed from the animals and stored overnight in the same fixative. The following day, spinal cords were sectioned transversely (40 pm) through the cervical, thoracic, lumbar and sacral levels with the aid of a Lancer vibratome. The sections were then stored in phosphate buffered saline (PBS, pH 7.4).
Immunohistochemical procedure Free-floating sections were processed by the avidin-biotinperoxidase technique as previously described (Clements, et al., '89; Magnusson et al., '88; '89), with 3-5 rinses in PBS following each incubation step. Briefly, tissue sections were incubated overnight in primary monoclonal antibodies at room temperature. All primary antibodies used in the present study were raised against taurine conjugated to KLH with glutaraldehyde-borohydride.The antibodies have been previously characterized and found to have high specificity for taurine (Magnusson et al., '88). Under fixation conditions with 4%paraformaldehyde-0.3%glutaraldehyde the antibody has strong immunoreactivity for taurine and hypotaurine but not for any other amino acid, including glycine, beta-alanine and GABA. When high glutaraldehyde is used as a fixative, the antibody also shows some crossreactivity for cysteic acid. All light microscopic analysis and preembedding staining was performed on tissue fixed with 4% paraformaldehyde-0.3% glutaraldehyde, while all postembedding staining was performed on tissue fixed with 2.5% glutaraldehyde-0.2% paraformaldehyde and 0.2% picric acid. Sections were incubated for one hour in secondary biotinylated anti-mouse IgG and one hour in avidinbiotin-peroxidase complex (Pel-Freez). Sections were then treated for 5 minutes with DAB (3,3'-diaminobenzidine tetrahydrochloride, Sigma) in Tris buffer (pH 7.4) containing 0.009% hydrogen peroxide. Controls were performed by: 1) incubating the tissue in primary antibody preabsorbed with 2 mg/ml of original antigen (taurine conjugation to KLH) or 2) by deleting the primary or secondary antibody step.
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TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
Procedure for light microscopy Sections for light microscopy were mounted on gelatincoated slides, dehydrated in ethanol, cleared in xylene and coverslipped with Permount. The localization of taurinelike immunoreactive neurons within the dorsal horn was plotted at each spinal level with the aid of a camera lucida at a magnification of 2 5 0 ~ Two . sets of 2 km thick plastic sections, obtained from two rats, were stained with toluidine blue and used for calculating the percentage of immunoreactive neurons in laminae I and 11. Spinal cord laminae were identified according to the atlas of Paxinos and Watson ('86) and the cytoarchitectonic description of Molander et al. ('84, '89) and Gobel (Gobel and Falls, '79; Gobel et al., '80).
Preembedding procedure for electron microscopy Following immunohistochemical processing, tissue sections for electron microscopy were thoroughly rinsed in PBS and postfixed in 2% osmium tetroxide for 30 minutes. Sections were then dehydrated in an ascending series of ethanols, embedded in Polybed resin (Polysciences, Inc., Warrington, PA), and polymerized between dimethyldichlorosilane-coated slides as previously described (Clements et al., '85). After polymerization for 1day at 40°C and 2 days at 60°C, the slides were separated and tissue sections were examined by light microscopy. Areas of the dorsal horn with dense immunoreactivity were circumscribed with a diamond scribe, photographed and excised. These portions were mounted onto Polybed blocks, trimmed, sectioned (thickness, 90-110 nm), and examined with a Zeiss 10 electron microscope.
Postembedding procedure for electron microscopy Prior to immunohistochemical processing, spinal cord sections were osmicated and dehydrated as described above. The tissue sections were then embedded in Epon/Spurr's resin between dimethyldichlorosilane-treated glass slides. The slides were separated and the tissues examined by light microscopy. Areas representative of laminae I and I1 were excised, glued to epon blocks, trimmed and sectioned. Areas representative of laminae VIII and IX were also excised from the ventral horn of two rats, glued to epon blocks, trimmed and sectioned. The sections were mounted on formvar coated nickel grids for immunogold staining. The immunocytochemical postembedding protocol used was a slight modification of the procedure described by Ottersen ('88). Briefly, the sections were etched in 10% HzOzfor 15 min and rinsed with double-distilled H 2 0for a few seconds. This was followed by sequential incubations in 1) 2% normal goat serum (20 min); 2) anti-taurine monoclonal antibody (diluted 1:40) for 18 hours at room temperature; 3) polyethyleneglycol (50 mg/100 mlO.05 M Tris buffer, pH 7.2) for 5 minutes; 4) goat anti-mouse IgG conjugated to colloidal gold particles with a mean diameter of 20 nm (E-Y Laboratories, Inc., San Mateo, CA) diluted 1:20 in the solution used in step 3 for 2 hours; 5) 1%uranyl acetate (20 min) followed by lead citrate (1-2 min). The primary monoclonal antibody was diluted in 0.05 M Tris-phosphate buffered saline, pH 7.2, 0.1% BSA and 0.01% sodium azide. The sections were thoroughly rinsed with double distilled water between steps 4 and 5 and were dried prior to staining with uranyl acetate and lead citrate.
Quantitation of postembedding immunostaining Ultrathin sections were examined under a JEOL 1200EX I1 transmission microscope at 60 kV. Sections were photographed randomly at a plate magnification of x 15,000,and printed at a final magnification of x 18,000. For quantitative analysis, the number of gold particles per square micrometer for each structure was calculated according to the morphometric technique described by Bendayan et al. ('80). Briefly the surface area (Sa) of individual histological structures was first measured using a digitizing tablet (Jandel Scientific, Sigma Scan V3.90) and associated microcomputer (WYSE pc+). The number of gold particles (Nil present over individual perikarya, dendrites, axons, axon terminals, astrocytes and endothelial cells was then counted and the density of labeling (Ns) calculated as Ns = NiiSa. A background gold particle density per km2was calculated for each animal from immunohistochemically processed sections in which the taurine primary antibody was replaced with normal mouse IgG. This background density of gold particles was then subtracted from the calculated taurine gold particle densities to obtain a final density per pm2. Neuronal structures were identified according to the criteria set forth by Peters et al. ('91). A one way analysis of variance (ANOVA) was applied to assess significant differences between the mean values of gold particles among the different neuronal structures quantified. The Scheffe F-test was used to determine probability values.
RESULTS Taurine-like immunoreactivity at the light microscopic level Within the spinal cord gray matter taurine-like immunoreactivity (taurine-LI) was most dense in the dorsal portion of the dorsal horn at all spinal cord levels (Figs. 1, 21. This intense taurine-LI was localized specifically to Rexed's laminae I and I1 of the dorsal horn. In these two laminae, taurine-LI was localized to neuronal cell bodies and to many dense immunoreactive puncta throughout the neuropil (Fig. lA,C). With electron microscopy, many of these puncta appeared to be dendrites although axons, axonal terminals and glial processes also contributed to this punctal staining. Taurine-like immunostaining was moderately dense in laminae X and was also present in laminae VIII, and IX. Laminae 111, IV, V, VI, and VII of the ventral and dorsal horns were only lightly stained. Although some immunoreactive neuronal perikarya were evident in laminae 111, IV, and X, very few, if any, cell bodies were immunostained in the remaining spinal cord laminae. Taurine-like immunoreactive neurons in laminae I and I1 were elliptical or pyramidal in shape with their long axes oriented radially to the dorsal surface. The distribution of taurine-LI neurons within the dorsal horn of the spinal cord is illustrated in Figure 2. As indicated in this figure taurine-LI neurons displayed a similar distribution pattern in laminae I and I1 at all spinal levels. A few taurine-LI neurons were also found in laminae I11 and IV throughout the spinal cord with the exception of the first cervical segment. Immunoreactive neurons observed in these laminae were larger in size and weaker in immunoreactivity than those in laminae I and 11. Within the second cervical segment, a small number of immunoreactive profiles appeared in lamina I11 but none were evident in laminae IV.
Figure I
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TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
Fig. 2. Camera lucida drawings depicting the distribution of taurine-like immunoreactive perikarya in laminae I-IV of the left dorsal horn from seven representative spinal cord segments. Each immunostained neuron is represented by a single black dot.
The number of taurine-like immunoreactive profiles in lamina I11 increased from the mid-cervical to the lower cervical level of the cord. A few lightly stained immunoreactive neuronal profiles were also evident in lamina IV from mid-cervical through sacral cord levels. The average percentage of taurine-like immunoreactive neurons in laminae I and I1 per section is indicated in Table 1. Examination of 2 km thick immunoreacted plastic sections that were counterstained with toluidine blue indicated that approximately 29.6%of the neurons in laminae I and I1 in the upper cervical level (C2), 36.3% in the mid-cervical level (C4),51.8%in the thoracic level (T6), and 18.4%in the lumbar level (L2) were immunoreactive for taurine.
Taurine-like immunoreactivity at the electron microscopic level Cellular staining. Taurine-LI was observed in neuronal perikarya, astrocytes and endothelial cells within laminae I and I1 of the dorsal horn (Figs. 3, 4). With
Fig. 1. Photomicrographs of taurine-like immunoreactivity in the dorsal horn of the rat spinal cord at spinal level C1. Tissue sections were postfixed with osmium tetroxide for 30 minutes after the immunohistochemical reaction. A Transverse 50-pm section illustrating the prevalence of taurine-like immunostaining in laminae I and I1 of the dorsal horn. Bar = 36 km. B: An adjacent spinal cord section that was incubated with primary antiserum preabsorbed with a taurine-KLH conjugate. Note that laminae I and I1 appear as clear areas due to the lack of immunoreactivity. The difference between gray and white matter is evident because of the tissue staining due to treatment with osmium. Bar = 38 wm. C : High magnification view of the area indicated by the rectangle in lA, illustrating immunoreactive neuronal perikarya (arrows)and immunoreactive puncta (arrowheads).Bar = 5 pm.
TABLE 1. Percentage (2SEM) of Neurons in Laminae I and I1 in the Dorsal Horn of the Rat Spinal Cord That Contained Taurine-Like Immunoreactivity
Spinal cord segment Second cervical segment Fourth cervical segment Sixth thoracic segment Second lumbar segment
Neurons (70) Lamina I
Lamina I1
Laminae I & I1
18.0
35.4 i 2.1 46.1 ? 2.2 60.4 ? 3.0 22.4 ? 1.9
29.6 3.4 36.3 z 5.2 51.8 i_ 4.7 18.4 3.7
_f
1.1 1.3
16.8 2 34.6 i_ 2.2 10.4 ? 1.8
_f
_f
preembedding staining immunoreactive perikarya were found to contain a relatively dense DAB reaction product within the cytoplasm (Figs. 3A, 4A) and somewhat variable immunostaining in the nucleus. In some instances the nucleus was only lightly immunostained (Fig. 3A), while in many cases it appeared as dark or darker than the cytoplasm. This is consistent with the somewhat variable staining observed in the cell nucleus under the light microscope (Fig. 1C).When the density of gold particles was quantitated in postembedded tissue sections, a mean of 18.7 gold particles per pm2 was associated with the cytoplasm (Table 2) and a mean of 18.9 particles/km2 was associated with the neuronal nucleus in laminae I and 11. In general gold particles appeared randomly distributed throughout the nucleus (Fig. 3B) and cytoplasm (Figs. 3B, 4A) of taurine-LI neurons. However, there was a tendency for more gold particles to be associated with mitochondria than with other intracellular organelles. This was also noted by Ottersen ('88) in his studies of taurine-LI in the rat cerebellum. Very few gold particles were found in association with the Golgi apparatus. It is worth noting that. examination of neuronal cell bodies in laminae VIII and IX revealed little or no gold particle labeling in the
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I.S. LEE ET AL.
Figure 3
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
71
perikarya. This finding is consistent with our observations preembedding staining many of these taurine-like immunoreactive dendrites were found to receive a predominance of at the light microscopic level. Astrocytes and endothelial cells contained high amounts asymmetrical synapses from nonimmunoreactive axon terof taurine-LI when visualized with postembeddingimmuno- minals. Occasionally immunoreactive dendrites received cytochemical procedures. Immunoreactive astrocytic cell synaptic contacts from immunoreactive axon terminals. bodies were also encountered in tissue processed with Similarly in postembedded material heavily labeled denpreembedding immunocytochemistry, but the relatively drites were seen to receive asymmetric synapses from poor structural resolution associated with our preembed- lightly labeled or unlabeled terminals (Figs. 5B, 7 C ) .Heavily ding immunohistochemical procedure made it difficult to labeled dendrites (n = 32) contained a mean of 32.5 gold identify these cells and with certainty. Analysis of postem- particles/pm2, while the mean of all dendrites sampled in bedded material revealed that the cytoplasm of astrocytes laminae I and I1 (n = 219) was 18.3 gold particles per pm2. in the dorsal horn contained a mean of 20.9 gold particles/ It is worth noting that this latter value is not significantly pm2, while the nucleus contained a mean of 22.9 particles/ different from the mean number of gold particles per pmZ pm2 (Table 2). The gold particles were found randomly calculated for all neuronal perikarya sampled in these two distributed throughout the astrocytic cytoplasm and nu- spinal cord laminae (Table 2). The number of gold particles cleus with higher numbers of particles associated with per pm2 was also calculated for dendrites of different size. mitochondria and very few particles associated with the The mean number of gold particles for dendrites measuring Golgi apparatus (Figs. 3C, 5C). In addition very few gold 0.1-0.9 pm2 was 16.4/pm2; for dendrites measuring 1.0particles were found in relationship to astrocytic filaments 9.9 pm2 the mean density was 18.6/pm2; for dendrites (Fig. 3C). The highest density of gold particles in the dorsal measuring 10-19.9 pm2 the mean density was 16.7/km2; horn of the spinal cord was found associated with endothe- and for dendrites measuring greater than 20 km2 the mean lial cells (Fig. 3D). The endothelial cytoplasm contained a density was 17.6/pm2. No significant difference in immumean density of 39.7 gold particles/pm2,while the endothe- nogold labeling was found among dendrites of different lial nucleus contained 46.2 particles/pm2. Although we did sizes indicating that taurine is distributed equally along the not perform quantitation of gold particle density in the dendritic tree. Examples of gold labeling among different spinal cord ventral horn, examination of astrocytes and size dendrites can be found in Figures 5B,D, 6D, and 7A-C. endothelial cells in laminae VIII and IX indicates that these It is interesting to note that although perikarya in laminae cells contain a relatively high density of gold particles as VIII and IX contained little if any gold particle labeling, well (Figs. 4C,D). occasional dendrites in this ventral horn region were found contain a moderate amount of gold particles (Fig. 4 0 . Immunostaining of dendrites, axons and glial to Both myelinated and unmyelinated taurine-like immunoprocesses reactive axons were identified in laminae I and I1 (Figs. 3D, Taurine-LI was observed within dendrites, axons, axon 4A,B, 5B,D, 7A). With preembedding techniques (Figs. terminals and glial processes within laminae I and 11. 4A,B) approximately 20% of myelinated axons (411210) and Ultrastructural examination of the immunoreactive punc- 21% of unmyelinated axons (32/ 155)were immunoreactive tae observed in the dorsal horn at the light microscopic level for taurine. With postembedding techniques myelinated indicated that these punctae consist primarily of immuno- axons were found to have a wide range of gold particle reactive dendrites and partially of immunoreactive axons density (Table 2). Approximately 20% (491248) of myelinand axon terminals. In laminae I and 11,where the majority ated axons had a high gold particle density (34.2 particles/ of immunoreactive neuronal perikarya was observed, a pm2),while 55%displayed a medium density (17.9 particles/ large number of the dendrites were immunoreactive for pm2) and 25% displayed a low density (7.5 particles/pm2). taurine in both pre- and postembedded immunoreacted The mean value of gold particles found in heavily labeled spinal cord sections (Figs. 4B, 6D, 7C). These stained myelinated axons (indicated as axonsidense in Table 2) was dendrites were often seen to arise from or to be in close found to be significantly different from axons containing apposition to immunoreactive neuronal cell bodies. With either a medium density (indicated as axons/moderate in Table 2) or a low density (axonsilight in Table 2) of gold particles. Examples of gold labeled taurine-LI myelinated axons are seen in Figures 5B-D, 6C, and 7D. Since unmyelinFig. 3. Electron micrographs of taurine-like immunoreactive cells ated axons were generally less than 1 pm in diameter, no in the superficial dorsal horn of the rat spinal cord. A Electron attempt was made to quantitate the number of gold partimicrograph depicting taurine-like immunoreactivity within a neuronal cles per pm2. However, unmyelinated axons were found to perikaryon (p) located in laminae I1 as visualized with the preembedding DAB procedure described in the text. Note the dense DAB reaction be either unlabeled (Fig. 7A) or to contain 2-5 gold particles product within the cytoplasm (arrow). The nucleus (n) of this cell is per profile (Fig. 3D). Approximately 75% of unmyelinated only lightly immunostained. Bar = 625 nm. B: Taurine-like immunore- axons contained no gold particles following postembedding activity within a neuronal perikaryon (p) in laminae I1 as visualized staining, which is consistent with the number of unlabeled with postembedding colloidal gold labeling. The gold particles (small unmyelinated axons observed in preembedded material. black dots indicated by the arrows) are present both throughout the Presumed astrocytic processes were found to contain cytoplasm and the nucleus. Bar = 600 nm. C: Electron micrograph taurine-LI with preembedding staining (Fig. 4A). However depicting taurine-like immunoreactivity within an astrocyte in lamina I. Note the presence of astrocytic filaments within the cytoplasm because of the poor structural resolution obtained with our (arrow). Gold particles representing taurine immunolabeling are indi- preembedding technique, the identity of these profiles as cated by arrowheads. Bar = 371 nm. D: Micrograph depicting an astrocytic processes could not be made with certainty. With endothelial cell (e) surrounding the lumen (I) of a capillary in the postembedding staining astrocytic processes, as well as, superficial dorsal horn. Both the cytoplasm and nucleus of the endothelial cell are heavily labeled with gold particles. Bundles of unmyelinated astrocytic cell bodies were found to contain gold particles axons are present near the ablumenal surface of the endothelial cell. (Fig. 7A). Because of the small size and irregular shape of astrocytic processes no attempt was made to quantitate the The majority of these fibers are unlabeled (arrows).Bar = 347 nm.
I.S. LEE ET AL.
72
Figure 4
73
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD TABLE 2. Taurine-LikeImmunoreactivityin Different Cellular Profiles in the Lamina I and I1 of Rat Spinal Cord' Statistical comparison
Cell profiles
Mean gold particle densitv -t SEM (n)
Neuron Cytoplasm
Axons
Nucleus
Dendrite
Light
Terminal
Moderate
Dense
Light
Moderate
Dense
Astrocyte cytoplasm
~~
~~~~
Neuronlcytoplasm Neuronlnucleus Dendrites Axonsilight Axonslmoderate Axonsldense Terminalilight Termindimoderate Terminalldense Astrocytelcytoplasm Astronitelnucleus
18.70 2 0.85 (36) 18.90 5 1.17 (20) 18.3 k 0 57 (219) 7.47 t 0.29 (62) 17.88 c 0.34 (138) 34.21 2 1.14 (49) 7.09 i 0.29 (91) 17.26 2 0.27 (224) 36.76 t 1.23 (71) 20.92 r 0.74 (45) 22.91 t 1.57 (19)
n.s.* n.s.
< o 01 n.s.
10.001
A Quantitative Light and Electron Microscopic Analysis of Taurine-Like Immunoreactivity in the Dorsal Horn of the Rat Spinal Cord INSE S. LEE, WALEED M. RENNO, AND ALVIN J. BEITZ Department of Anatomy College of Veterinary Medicine, Seoul National University, Seoul, South Korea (I.S.L.); and Department of Veterinary Pathobiology College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 (W.R., A.J.B.)
ABSTRACT Taurine has been proposed as an inhibitory neurotransmitter or neuromodulator in the vertebrate central nervous system. Within the spinal cord, taurine has been shown to have a direct inhibitory effect on spinal neurons and to have a selective antinociceptive effect on chemically induced nociception. Although sufficient data exists to suggest that taurine plays a neurotransmitter or neuromodulatory role in the spinal cord, it is not known whether this amino acid is present in axon terminals nor if this amino acid has a unique pattern of distribution within spinal tissue. To address these questions a monoclonal antibody against taurine was employed to localize taurine-like immunoreactivity in the dorsal horn of the rat spinal cord by using both light and electron microscopic techniques. Taurine-like immunoreactivity was most dense and most prominent in laminae I and I1 of the dorsal horn. A moderate amount of immunoreactivity was also present in laminae VIII and IX and X while the remaining laminae were only lightly stained. In laminae I and I1 taurine-like immunostaining was evident within neuronal cell bodies, dendrites, myelinated and unmyelinated axons, axon terminals, and astrocytes and their processes. Cell counts of these two laminae indicated that approximately 30%of neuronal perikarya at the C2 level, 52%of neuronal perikarya at the T6 level, and 18% of neuronal perikarya at the L2 level of the cord exhibited taurine-like immunoreactivity. With preembedding diaminobenzidine staining, approximately 20% of the axons examined in laminae I and 11 were found to be immunoreactive for taurine. Using postembedding immunogold staining in combination with quantitative procedures, the highest densities of gold particles were found in axon terminals containing pleomorphic vesicles and forming symmetrical synapses (36.8 particles/ km2),in a subpopulation of myelinated axons (34.2 particles/km2), in a subpopulation of neuronal dendrites (32.6 particles/ km2),and in capillary endothelial cells (39.8 particles/pm2). Moderate labeling occurred in astrocytes (20.9 particles/pm2) and neuronal perikarya (18.7 particles/ km2).The localization of taurine to presumptive inhibitory axon terminals provides anatomical support for the hypothesis that taurine may serve an inhibitory neurotransmitter role in the superficial dorsal horn of the spinal cord. On the other hand, its localization to astrocytes and endothelial cells within both the dorsal ventral horns implies that it serves other nonneuronal functions as well. o 1992 Wiley-Liss, Inc. Key words: immunocytochemistry,lamina I, lamina 11, glia, amino acids
It has been suggested that taurine (2-aminoethanesul- McBride and Frederickson, '80; Chesney, '85; Lin et al., fonic acid) plays a wide variety of functional roles both '85) in the CNS. This proposed inhibitory transmitter role within and outside the central nervous system (CNS) is consistent with its reported antiepileptic actions (Durelli (Huxtable, '89). Several lines of evidence suggest that and Mutani, '83; Toth et al., '83). In addition to its possible taurine acts as a neuromodulator (Davison and Kaczmarek, roles as a neurotransmitter and/or a neuromodulator, '71; Kuriyama, '80; Kontro and Oja, '83; Sakai et al., '85; Taber et al., '86; Magnusson et al., '88, '89) and as an inhibitory neurotransmitter (Oja and Lahdesmaki, '74; Accepted March 2,1992. O
1992 WILEY-LISS, INC.
66 taurine has been considered to be a stabilizer of excitable membranes (Schaffer et al., '80; Hastings et al., '851, to function as an osmoregulator (Thurston et al., '80; Van Gelder, '89), to act as an antagonist of intracellular calcium ionization (vanGelder, '90; Huxtable, 'go), and to play an important role in postnatal brain development (Chesney, '85). Although the role of taurine in the spinal cord remains controversial, a number of studies suggest that this amino acid may participate as an inhibitory neurotransmitter in this region (Sonnhoff et al., '75; Nicoll et al., '76; Lane et al., '78; Kurachi and Aihara, '85; Kudo et al., '88; Yasunami et al., '88). Consistent with this postulate, Kurachi and Aihara ('85) have demonstrated that taurine possesses antiepileptic activity in the spinal cord. In addition Serrano et al. ('90) have shown that taurine has an antinociceptive effect in the spinal cord and that this effect can be inhibited by naloxone. Smullin and coworkers ('90b) have further shown that intrathecally injected taurine selectively inhibits substance P-induced biting and scratching behavior and that it also inhibits the nociceptive related writhing behavior produced by an intraperitoneal injection of acetic acid in mice. At a more cellular level taurine has been shown to hyperpolarize cell membranes and inhibit firing when iontophoretically applied to neurons in the cat spinal cord (Curtis and Johnston, '74). Kudo and coworkers ('88) have reported that low concentrations of taurine cause a marked hyperpolarization of the primary afferent fibers, while higher concentrations of taurine cause a fast-onset hyperpolarization, followed by a slow-onset depolarization. Interestingly this depolarizing effect was selectively antagonized by bicuculline while the hyperpolarizing effect of taurine was selectively reduced by strychnine, suggesting the existence of two taurine receptor subtypes in the spinal cord. Despite the fact that initial biochemical studies demonstrated a homogeneous distribution of taurine in the spinal cord (Yoneda and Kuriyama, '78; Kuriyama et al., '781, cysteine sulphinate decarboxylase (CSD), the major enzyme involved in taurine synthesis in the CNS, was found to have its highest activity in the dorsal part of the dorsal horn (Yonedaand Kuriyama, '78). This increased activity of CSD in the superficial dorsal horn would suggest an increased synthesis of taurine in this region. This is consistent with recent biochemical data demonstrating that taurine in the spinal cord is found in highest concentrations in the dorsal horn and lowest concentrations in the ventral horn (Palkovits et al., '86, '90). This differential distribution implies that taurine may serve a special functional role within the dorsal horn of the spinal cord. Since the superficial dorsal horn plays an important role in spinal mechanisms of nociception (Besson and Chaouch, '87), one possibility is that taurine plays some role in nociception or antinociception. Consistent with this hypothesis are pharmacological and behavioral data that suggest a role for taurine in analgesia via an effect in the dorsal horn of the spinal cord (Beyer et al., '88; Kuriyama and Yoneda, "78; Smullin et al., '90a, '90b). Although these data clearly support a role for taurine in the dorsal horn, it is not known whether taurine is found in axon terminals in the spinal cord nor whether its distribution in this region is in fact homogeneous as early biochemical studies would suggest. During the past 6 years, antibodies against haptenic taurine have been produced and applied to the CNS for cellular localization of this amino acid (Wu et al., '85; Yoshida et al., '86; Ida et al., '87; Ottersen et al., '85, '88;
IS. LEE ET AL. Schafer et al., '88; Magnusson et al., '88, '89). With these antibodies, taurine-like immunoreactivity (taurine-LI) has been visualized in several CNS regions, but the localization of this amino acid has not been analyzed in the spinal cord. One of the criteria that must be fulfilled in order to consider taurine as a neurotransmitterineuromodulator in the spinal cord is to demonstrate its localization in axon terminals. In the present study, we have utilized monoclonal antibodies raised against taurine conjugated to keyhole limpet hemocyanine (KLH) in order to analyze the specific distribution of taurine in the spinal cord dorsal horn and to determine if taurine-LI is present in axon terminals and/or other structures in the dorsal horn. The localization of taurine-LI was examined at cervical, thoracic, lumbar, and sacral levels by both light and electron microscopy.
MATERIALS AND METHODS Experimental animals Twenty-one male Sprague-Dawley rats (250-340 g) were used for this study. Animals were anesthetized with chloral hydrate (0.5 mgig) and perfused through the ascending aorta with 100 ml of calcium-free Tyrode's solution, followed by either 500 ml of 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M Sorenson's phosphate buffer (pH 7.4,4"C)or 500 ml of 0.2%paraformaldehyde, 2.5% glutaraldehyde, and 0.2% picric acid in 0.1 M Sorenson's buffer. The spinal cords were removed from the animals and stored overnight in the same fixative. The following day, spinal cords were sectioned transversely (40 pm) through the cervical, thoracic, lumbar and sacral levels with the aid of a Lancer vibratome. The sections were then stored in phosphate buffered saline (PBS, pH 7.4).
Immunohistochemical procedure Free-floating sections were processed by the avidin-biotinperoxidase technique as previously described (Clements, et al., '89; Magnusson et al., '88; '89), with 3-5 rinses in PBS following each incubation step. Briefly, tissue sections were incubated overnight in primary monoclonal antibodies at room temperature. All primary antibodies used in the present study were raised against taurine conjugated to KLH with glutaraldehyde-borohydride.The antibodies have been previously characterized and found to have high specificity for taurine (Magnusson et al., '88). Under fixation conditions with 4%paraformaldehyde-0.3%glutaraldehyde the antibody has strong immunoreactivity for taurine and hypotaurine but not for any other amino acid, including glycine, beta-alanine and GABA. When high glutaraldehyde is used as a fixative, the antibody also shows some crossreactivity for cysteic acid. All light microscopic analysis and preembedding staining was performed on tissue fixed with 4% paraformaldehyde-0.3% glutaraldehyde, while all postembedding staining was performed on tissue fixed with 2.5% glutaraldehyde-0.2% paraformaldehyde and 0.2% picric acid. Sections were incubated for one hour in secondary biotinylated anti-mouse IgG and one hour in avidinbiotin-peroxidase complex (Pel-Freez). Sections were then treated for 5 minutes with DAB (3,3'-diaminobenzidine tetrahydrochloride, Sigma) in Tris buffer (pH 7.4) containing 0.009% hydrogen peroxide. Controls were performed by: 1) incubating the tissue in primary antibody preabsorbed with 2 mg/ml of original antigen (taurine conjugation to KLH) or 2) by deleting the primary or secondary antibody step.
67
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
Procedure for light microscopy Sections for light microscopy were mounted on gelatincoated slides, dehydrated in ethanol, cleared in xylene and coverslipped with Permount. The localization of taurinelike immunoreactive neurons within the dorsal horn was plotted at each spinal level with the aid of a camera lucida at a magnification of 2 5 0 ~ Two . sets of 2 km thick plastic sections, obtained from two rats, were stained with toluidine blue and used for calculating the percentage of immunoreactive neurons in laminae I and 11. Spinal cord laminae were identified according to the atlas of Paxinos and Watson ('86) and the cytoarchitectonic description of Molander et al. ('84, '89) and Gobel (Gobel and Falls, '79; Gobel et al., '80).
Preembedding procedure for electron microscopy Following immunohistochemical processing, tissue sections for electron microscopy were thoroughly rinsed in PBS and postfixed in 2% osmium tetroxide for 30 minutes. Sections were then dehydrated in an ascending series of ethanols, embedded in Polybed resin (Polysciences, Inc., Warrington, PA), and polymerized between dimethyldichlorosilane-coated slides as previously described (Clements et al., '85). After polymerization for 1day at 40°C and 2 days at 60°C, the slides were separated and tissue sections were examined by light microscopy. Areas of the dorsal horn with dense immunoreactivity were circumscribed with a diamond scribe, photographed and excised. These portions were mounted onto Polybed blocks, trimmed, sectioned (thickness, 90-110 nm), and examined with a Zeiss 10 electron microscope.
Postembedding procedure for electron microscopy Prior to immunohistochemical processing, spinal cord sections were osmicated and dehydrated as described above. The tissue sections were then embedded in Epon/Spurr's resin between dimethyldichlorosilane-treated glass slides. The slides were separated and the tissues examined by light microscopy. Areas representative of laminae I and I1 were excised, glued to epon blocks, trimmed and sectioned. Areas representative of laminae VIII and IX were also excised from the ventral horn of two rats, glued to epon blocks, trimmed and sectioned. The sections were mounted on formvar coated nickel grids for immunogold staining. The immunocytochemical postembedding protocol used was a slight modification of the procedure described by Ottersen ('88). Briefly, the sections were etched in 10% HzOzfor 15 min and rinsed with double-distilled H 2 0for a few seconds. This was followed by sequential incubations in 1) 2% normal goat serum (20 min); 2) anti-taurine monoclonal antibody (diluted 1:40) for 18 hours at room temperature; 3) polyethyleneglycol (50 mg/100 mlO.05 M Tris buffer, pH 7.2) for 5 minutes; 4) goat anti-mouse IgG conjugated to colloidal gold particles with a mean diameter of 20 nm (E-Y Laboratories, Inc., San Mateo, CA) diluted 1:20 in the solution used in step 3 for 2 hours; 5) 1%uranyl acetate (20 min) followed by lead citrate (1-2 min). The primary monoclonal antibody was diluted in 0.05 M Tris-phosphate buffered saline, pH 7.2, 0.1% BSA and 0.01% sodium azide. The sections were thoroughly rinsed with double distilled water between steps 4 and 5 and were dried prior to staining with uranyl acetate and lead citrate.
Quantitation of postembedding immunostaining Ultrathin sections were examined under a JEOL 1200EX I1 transmission microscope at 60 kV. Sections were photographed randomly at a plate magnification of x 15,000,and printed at a final magnification of x 18,000. For quantitative analysis, the number of gold particles per square micrometer for each structure was calculated according to the morphometric technique described by Bendayan et al. ('80). Briefly the surface area (Sa) of individual histological structures was first measured using a digitizing tablet (Jandel Scientific, Sigma Scan V3.90) and associated microcomputer (WYSE pc+). The number of gold particles (Nil present over individual perikarya, dendrites, axons, axon terminals, astrocytes and endothelial cells was then counted and the density of labeling (Ns) calculated as Ns = NiiSa. A background gold particle density per km2was calculated for each animal from immunohistochemically processed sections in which the taurine primary antibody was replaced with normal mouse IgG. This background density of gold particles was then subtracted from the calculated taurine gold particle densities to obtain a final density per pm2. Neuronal structures were identified according to the criteria set forth by Peters et al. ('91). A one way analysis of variance (ANOVA) was applied to assess significant differences between the mean values of gold particles among the different neuronal structures quantified. The Scheffe F-test was used to determine probability values.
RESULTS Taurine-like immunoreactivity at the light microscopic level Within the spinal cord gray matter taurine-like immunoreactivity (taurine-LI) was most dense in the dorsal portion of the dorsal horn at all spinal cord levels (Figs. 1, 21. This intense taurine-LI was localized specifically to Rexed's laminae I and I1 of the dorsal horn. In these two laminae, taurine-LI was localized to neuronal cell bodies and to many dense immunoreactive puncta throughout the neuropil (Fig. lA,C). With electron microscopy, many of these puncta appeared to be dendrites although axons, axonal terminals and glial processes also contributed to this punctal staining. Taurine-like immunostaining was moderately dense in laminae X and was also present in laminae VIII, and IX. Laminae 111, IV, V, VI, and VII of the ventral and dorsal horns were only lightly stained. Although some immunoreactive neuronal perikarya were evident in laminae 111, IV, and X, very few, if any, cell bodies were immunostained in the remaining spinal cord laminae. Taurine-like immunoreactive neurons in laminae I and I1 were elliptical or pyramidal in shape with their long axes oriented radially to the dorsal surface. The distribution of taurine-LI neurons within the dorsal horn of the spinal cord is illustrated in Figure 2. As indicated in this figure taurine-LI neurons displayed a similar distribution pattern in laminae I and I1 at all spinal levels. A few taurine-LI neurons were also found in laminae I11 and IV throughout the spinal cord with the exception of the first cervical segment. Immunoreactive neurons observed in these laminae were larger in size and weaker in immunoreactivity than those in laminae I and 11. Within the second cervical segment, a small number of immunoreactive profiles appeared in lamina I11 but none were evident in laminae IV.
Figure I
69
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
Fig. 2. Camera lucida drawings depicting the distribution of taurine-like immunoreactive perikarya in laminae I-IV of the left dorsal horn from seven representative spinal cord segments. Each immunostained neuron is represented by a single black dot.
The number of taurine-like immunoreactive profiles in lamina I11 increased from the mid-cervical to the lower cervical level of the cord. A few lightly stained immunoreactive neuronal profiles were also evident in lamina IV from mid-cervical through sacral cord levels. The average percentage of taurine-like immunoreactive neurons in laminae I and I1 per section is indicated in Table 1. Examination of 2 km thick immunoreacted plastic sections that were counterstained with toluidine blue indicated that approximately 29.6%of the neurons in laminae I and I1 in the upper cervical level (C2), 36.3% in the mid-cervical level (C4),51.8%in the thoracic level (T6), and 18.4%in the lumbar level (L2) were immunoreactive for taurine.
Taurine-like immunoreactivity at the electron microscopic level Cellular staining. Taurine-LI was observed in neuronal perikarya, astrocytes and endothelial cells within laminae I and I1 of the dorsal horn (Figs. 3, 4). With
Fig. 1. Photomicrographs of taurine-like immunoreactivity in the dorsal horn of the rat spinal cord at spinal level C1. Tissue sections were postfixed with osmium tetroxide for 30 minutes after the immunohistochemical reaction. A Transverse 50-pm section illustrating the prevalence of taurine-like immunostaining in laminae I and I1 of the dorsal horn. Bar = 36 km. B: An adjacent spinal cord section that was incubated with primary antiserum preabsorbed with a taurine-KLH conjugate. Note that laminae I and I1 appear as clear areas due to the lack of immunoreactivity. The difference between gray and white matter is evident because of the tissue staining due to treatment with osmium. Bar = 38 wm. C : High magnification view of the area indicated by the rectangle in lA, illustrating immunoreactive neuronal perikarya (arrows)and immunoreactive puncta (arrowheads).Bar = 5 pm.
TABLE 1. Percentage (2SEM) of Neurons in Laminae I and I1 in the Dorsal Horn of the Rat Spinal Cord That Contained Taurine-Like Immunoreactivity
Spinal cord segment Second cervical segment Fourth cervical segment Sixth thoracic segment Second lumbar segment
Neurons (70) Lamina I
Lamina I1
Laminae I & I1
18.0
35.4 i 2.1 46.1 ? 2.2 60.4 ? 3.0 22.4 ? 1.9
29.6 3.4 36.3 z 5.2 51.8 i_ 4.7 18.4 3.7
_f
1.1 1.3
16.8 2 34.6 i_ 2.2 10.4 ? 1.8
_f
_f
preembedding staining immunoreactive perikarya were found to contain a relatively dense DAB reaction product within the cytoplasm (Figs. 3A, 4A) and somewhat variable immunostaining in the nucleus. In some instances the nucleus was only lightly immunostained (Fig. 3A), while in many cases it appeared as dark or darker than the cytoplasm. This is consistent with the somewhat variable staining observed in the cell nucleus under the light microscope (Fig. 1C).When the density of gold particles was quantitated in postembedded tissue sections, a mean of 18.7 gold particles per pm2 was associated with the cytoplasm (Table 2) and a mean of 18.9 particles/km2 was associated with the neuronal nucleus in laminae I and 11. In general gold particles appeared randomly distributed throughout the nucleus (Fig. 3B) and cytoplasm (Figs. 3B, 4A) of taurine-LI neurons. However, there was a tendency for more gold particles to be associated with mitochondria than with other intracellular organelles. This was also noted by Ottersen ('88) in his studies of taurine-LI in the rat cerebellum. Very few gold particles were found in association with the Golgi apparatus. It is worth noting that. examination of neuronal cell bodies in laminae VIII and IX revealed little or no gold particle labeling in the
70
I.S. LEE ET AL.
Figure 3
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD
71
perikarya. This finding is consistent with our observations preembedding staining many of these taurine-like immunoreactive dendrites were found to receive a predominance of at the light microscopic level. Astrocytes and endothelial cells contained high amounts asymmetrical synapses from nonimmunoreactive axon terof taurine-LI when visualized with postembeddingimmuno- minals. Occasionally immunoreactive dendrites received cytochemical procedures. Immunoreactive astrocytic cell synaptic contacts from immunoreactive axon terminals. bodies were also encountered in tissue processed with Similarly in postembedded material heavily labeled denpreembedding immunocytochemistry, but the relatively drites were seen to receive asymmetric synapses from poor structural resolution associated with our preembed- lightly labeled or unlabeled terminals (Figs. 5B, 7 C ) .Heavily ding immunohistochemical procedure made it difficult to labeled dendrites (n = 32) contained a mean of 32.5 gold identify these cells and with certainty. Analysis of postem- particles/pm2, while the mean of all dendrites sampled in bedded material revealed that the cytoplasm of astrocytes laminae I and I1 (n = 219) was 18.3 gold particles per pm2. in the dorsal horn contained a mean of 20.9 gold particles/ It is worth noting that this latter value is not significantly pm2, while the nucleus contained a mean of 22.9 particles/ different from the mean number of gold particles per pmZ pm2 (Table 2). The gold particles were found randomly calculated for all neuronal perikarya sampled in these two distributed throughout the astrocytic cytoplasm and nu- spinal cord laminae (Table 2). The number of gold particles cleus with higher numbers of particles associated with per pm2 was also calculated for dendrites of different size. mitochondria and very few particles associated with the The mean number of gold particles for dendrites measuring Golgi apparatus (Figs. 3C, 5C). In addition very few gold 0.1-0.9 pm2 was 16.4/pm2; for dendrites measuring 1.0particles were found in relationship to astrocytic filaments 9.9 pm2 the mean density was 18.6/pm2; for dendrites (Fig. 3C). The highest density of gold particles in the dorsal measuring 10-19.9 pm2 the mean density was 16.7/km2; horn of the spinal cord was found associated with endothe- and for dendrites measuring greater than 20 km2 the mean lial cells (Fig. 3D). The endothelial cytoplasm contained a density was 17.6/pm2. No significant difference in immumean density of 39.7 gold particles/pm2,while the endothe- nogold labeling was found among dendrites of different lial nucleus contained 46.2 particles/pm2. Although we did sizes indicating that taurine is distributed equally along the not perform quantitation of gold particle density in the dendritic tree. Examples of gold labeling among different spinal cord ventral horn, examination of astrocytes and size dendrites can be found in Figures 5B,D, 6D, and 7A-C. endothelial cells in laminae VIII and IX indicates that these It is interesting to note that although perikarya in laminae cells contain a relatively high density of gold particles as VIII and IX contained little if any gold particle labeling, well (Figs. 4C,D). occasional dendrites in this ventral horn region were found contain a moderate amount of gold particles (Fig. 4 0 . Immunostaining of dendrites, axons and glial to Both myelinated and unmyelinated taurine-like immunoprocesses reactive axons were identified in laminae I and I1 (Figs. 3D, Taurine-LI was observed within dendrites, axons, axon 4A,B, 5B,D, 7A). With preembedding techniques (Figs. terminals and glial processes within laminae I and 11. 4A,B) approximately 20% of myelinated axons (411210) and Ultrastructural examination of the immunoreactive punc- 21% of unmyelinated axons (32/ 155)were immunoreactive tae observed in the dorsal horn at the light microscopic level for taurine. With postembedding techniques myelinated indicated that these punctae consist primarily of immuno- axons were found to have a wide range of gold particle reactive dendrites and partially of immunoreactive axons density (Table 2). Approximately 20% (491248) of myelinand axon terminals. In laminae I and 11,where the majority ated axons had a high gold particle density (34.2 particles/ of immunoreactive neuronal perikarya was observed, a pm2),while 55%displayed a medium density (17.9 particles/ large number of the dendrites were immunoreactive for pm2) and 25% displayed a low density (7.5 particles/pm2). taurine in both pre- and postembedded immunoreacted The mean value of gold particles found in heavily labeled spinal cord sections (Figs. 4B, 6D, 7C). These stained myelinated axons (indicated as axonsidense in Table 2) was dendrites were often seen to arise from or to be in close found to be significantly different from axons containing apposition to immunoreactive neuronal cell bodies. With either a medium density (indicated as axons/moderate in Table 2) or a low density (axonsilight in Table 2) of gold particles. Examples of gold labeled taurine-LI myelinated axons are seen in Figures 5B-D, 6C, and 7D. Since unmyelinFig. 3. Electron micrographs of taurine-like immunoreactive cells ated axons were generally less than 1 pm in diameter, no in the superficial dorsal horn of the rat spinal cord. A Electron attempt was made to quantitate the number of gold partimicrograph depicting taurine-like immunoreactivity within a neuronal cles per pm2. However, unmyelinated axons were found to perikaryon (p) located in laminae I1 as visualized with the preembedding DAB procedure described in the text. Note the dense DAB reaction be either unlabeled (Fig. 7A) or to contain 2-5 gold particles product within the cytoplasm (arrow). The nucleus (n) of this cell is per profile (Fig. 3D). Approximately 75% of unmyelinated only lightly immunostained. Bar = 625 nm. B: Taurine-like immunore- axons contained no gold particles following postembedding activity within a neuronal perikaryon (p) in laminae I1 as visualized staining, which is consistent with the number of unlabeled with postembedding colloidal gold labeling. The gold particles (small unmyelinated axons observed in preembedded material. black dots indicated by the arrows) are present both throughout the Presumed astrocytic processes were found to contain cytoplasm and the nucleus. Bar = 600 nm. C: Electron micrograph taurine-LI with preembedding staining (Fig. 4A). However depicting taurine-like immunoreactivity within an astrocyte in lamina I. Note the presence of astrocytic filaments within the cytoplasm because of the poor structural resolution obtained with our (arrow). Gold particles representing taurine immunolabeling are indi- preembedding technique, the identity of these profiles as cated by arrowheads. Bar = 371 nm. D: Micrograph depicting an astrocytic processes could not be made with certainty. With endothelial cell (e) surrounding the lumen (I) of a capillary in the postembedding staining astrocytic processes, as well as, superficial dorsal horn. Both the cytoplasm and nucleus of the endothelial cell are heavily labeled with gold particles. Bundles of unmyelinated astrocytic cell bodies were found to contain gold particles axons are present near the ablumenal surface of the endothelial cell. (Fig. 7A). Because of the small size and irregular shape of astrocytic processes no attempt was made to quantitate the The majority of these fibers are unlabeled (arrows).Bar = 347 nm.
I.S. LEE ET AL.
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Figure 4
73
TAURINE IMMUNOREACTIVITY IN THE SPINAL CORD TABLE 2. Taurine-LikeImmunoreactivityin Different Cellular Profiles in the Lamina I and I1 of Rat Spinal Cord' Statistical comparison
Cell profiles
Mean gold particle densitv -t SEM (n)
Neuron Cytoplasm
Axons
Nucleus
Dendrite
Light
Terminal
Moderate
Dense
Light
Moderate
Dense
Astrocyte cytoplasm
~~
~~~~
Neuronlcytoplasm Neuronlnucleus Dendrites Axonsilight Axonslmoderate Axonsldense Terminalilight Termindimoderate Terminalldense Astrocytelcytoplasm Astronitelnucleus
18.70 2 0.85 (36) 18.90 5 1.17 (20) 18.3 k 0 57 (219) 7.47 t 0.29 (62) 17.88 c 0.34 (138) 34.21 2 1.14 (49) 7.09 i 0.29 (91) 17.26 2 0.27 (224) 36.76 t 1.23 (71) 20.92 r 0.74 (45) 22.91 t 1.57 (19)
n.s.* n.s.
< o 01 n.s.
10.001
