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Subdivisions of the accessory olfactory bulb, as demonstrated by lectin-histochemistry in the golden hamster
Neuroscience Letters, 158 (1993) 185-188 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
185
NSL 09721
Subdivisions of the accessory olfactory bulb, as demonstrated by lectin-histochemistry in the golden hamster Kazuyuki Taniguchi, Yuhsuke Nii and Kazushige O g a w a Department of Veterinary Anatomy, Faculty of Agriculture, lwate University, lwate (Japan) (Received 2 April 1993; Revised version received 6 May 1993; Accepted 7 May 1993)
Key words: Lectin; Histochemistry; Sugar residue; Olfaction; Vomeronasal organ; Accessory olfactory bulb; Golden hamster Lectin-binding patterns in the accessory olfactory bulb (AOB) of the golden hamster were investigated histochemically with 21 biotinylated lectins. The AOB was divided into rostral and caudal halves according to binding patterns of 16 lectins, WGA, s-WGA, LEL, STL, DSL, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA, ECL, UEA-I, Con A and PSA. The caudal half of the AOB was further subdivided into anterior 2/3 and posterior 1/3 by 10 lectins, WGA, s-WGA, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA and ECL. In addition, the rostral half of the AOB was subdivided into anterior 1/4 and posterior 3/4 by one lectin, PNA. Thus, the AOB of the golden hamster was divided into 4 divisions on the basis of lectin-binding patterns.
Olfactory receptor neurons of the vomeronasal organ project to the accessory olfactory bulb (AOB), constituting the vomeronasal chemosensory system [1]. In the AOB, axons of olfactory receptor neurons form the vomeronasal nerve layer, and further contribute to the formation of glomeruli in the glomerular layer [3]. Although individual receptor neurons show no distinct difference in their fine structures and are hardly dividable into subsets with conventional morphological methods [10, 11], the presence of subsets of receptor neurons to reveal subdivisions in the AOB has been suggested in the rabbit and rat by immunohistochemistry with monoclonal antibodies [2, 4-8], or by lectin-histochemistry [9]. Some of these monoclonal antibodies are reported to recognize carbohydrate antigens such as glycoconjugates [4, 5, 8]. Lectins are also known to recognize specific sugar residues of membranous structures of the cell [5, 9]. In the present study, we re-examined the AOB histochemically using 21 biotinylated lectins to demonstrate the presence of subdivisions in the AOB of the golden hamster. Adult golden hamsters (140-200 g body weight) of either sex were anesthetized with ether, and perfused with physiological saline, followed by Bouin's solution without acetic acid. The olfactory bulb was removed, immersed in the same fixative for 24 h, and embedded in
Correspondence: K. Taniguchi, Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Morioka, Iwate 020, Japan.
paraffin. Paraffin sections were cut sagittaUy at 5/tm, deparaffinized with xylene, and processed for lectin staining by the avidin-biotin complex (ABC) method with 21 biotinylated lectins (Table 1) in the commercial lectin screening kits (Vector). The sections were incubated for 48 h at 4°C with biotinylated lectins. Lectinbindings were visualized by the Vectastain ABC kit (Vector). Control lectin stainings were performed by the preabsorption of lectins with excess amount of respective specific sugar residues or by the use of phosphate-buffered saline to replace the biotinylated lectins or ABC. No specific lectin-bindings were observed after the control staining. The vomeronasal nerve and the glomerular layers of the AOB were divided into rostral and caudal halves by binding patterns of 16 lectins (Table 2) Among them, 15 lectins, WGA, s-WGA, LEL, STL, DSL, BSL-II, DBA, SBA, BSL-I, VVA, SJA, ECL, UEA-I, Con A and PSA stained the rostral half more strongly than the caudal half (Fig. 1A-D, F), while PNA stained the caudal half more strongly than the rostral half (Fig. 1E). The caudal half of the AOB was further divided into 2 parts; WGA, s-WGA, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA and ECL stained the posterior 1/3 more strongly than the anterior 2/3 (Fig. 1A-F). The rostral half of the AOB was also divided into 2 parts; PNA stained the anterior 1/4 more strongly than the posterior 3/4 (Fig. 1E). Thus, the AOB of the golden hamster was divided into 4 subdivi-
186
B
•
r~,
•
41:~,
C
Fig. 1. Binding pattern of lectins, LEL (A), SBA (B), BSL-1 (C), VVA (D), PNA (E) and ECL (F), in the accessory olfactory bulb. Rostral is left and caudal is right, x80.
sions. These subdivisions were more obvious in the glomerular layer than in the vomeronasal nerve layer. The other lectins, RCA-I, Jacalin, LCA, PHA-E and PHA-L, showed no specific binding to the AOB. There were no sex differences in the lectin-binding patterns of the AOB. Imamura et al. [2] divided the AOB of the rabbit into the rostrolateral and caudomedial parts with monoclonal antibodies against membrane proteins. Schwarting and Crandall [8] also divided the AOB of the rat into the rostral and caudal halves with monoclonal antibodies to carbohydrate antigens. Takami et al. [9], however, divided the AOB of the rat into 2 subdivisions, anterior 1/3 and posterior 2/3, by lectin-histochemistry using BSL-! and VVA. In the present study, the AOB was divided
into 4 subdivisions on the basis of the binding patterns of 16 lectins. According to Takami et al. [9], VVA stains the posterior 2/3 of the AOB of the rat. In the present study, however, VVA strongly stained the rostral half of the AOB of the golden hamster. This discrepancy may be attributable to difference of the staining methods employed, or to species differences. The lectins used in the present study display various binding patterns. There seems to be no general relation between specific sugar residues and binding patterns.
1 Halpern, M., The organization and function of the vomeronasal system, Annu. Rev. Neurosci.. 10 (1987) 325 362.
187 TABLE 1 BINDING SPEC1FICITIES OF LECTINS USED Fuc, fucose; Gal, o-galactose; GalNAc, N-acetylgalactosamine; GIcNAc, N-acetylglucosamine; Man, mannose; NeuAc, N-acetylneuraminic acid. Lectin
Concentration (mg/ml)
Specific sugar
Wheat germ agglutinin (WGA) Succinylated wheat germ agglutinin (s-WGA) Lycopersicon esculentum lectin (LEL) Soranum tuberosum lectin (STL) Datura stramonium lectin (DSL) Bandeiraea simplicifolia lectin-II (BSL-II) Dolichos biflorus agglutinin (DBA) Soybean agglutinin (SBA) Bandeiraea simplicifolia lectin-I (BSL-I) Vicia villosa agglutinin (VVA) Sophorajaponica agglutinin (SJA) Ricinus communis agglutinin-I (RCA-I) Jacalin Peanut agglutinin (PNA) Erythrina cristagalli lectin (ECL) Ulex europaeus agglutinin-I (UEA-I) Concanavalin A (Con A) Pisum sativum agglutinin (PSA) Lens culinaris agglutinin (LCA) Phaseolus vulgaris agglutinin-E (PHA-E) Phaseolus vulgaris agglutinin-L (PHA-L)
0.002 0.0025 0.001 0.0033 0.0033 0.02 0.5 0.05 0.04 0.02 0.5 0.0006 0.0007 0.005 0.005 0.02 0.0013 0.002 0.0008 0.002 0.0083
fl-GlcNAc>0~-NeuAc fl-GlcNAc fl-GlcNAc fl-GlcNAc fl-GlcNAc ~,fl-GlcNAc ~-GalNAc 0~,fl-GalNAc, Gal ct-GalNAc, ~-Gal ~,fl-GalNAc fl-GalNAc, fl-Gal fl-GalNAc, fl-Gal galactosyl-fl-GalNAc galactosyl-fl-GalNAc galactosyl-fl-GlcNAc ~-Fuc c~-Man c~-Man s-Man oligosaccharide oligosaccharide
TABLE 2 BINDING PATTERN OF LECTINS IN THE AOB a, anterior 1/4; b, posterior 3/4; c, anterior 2/3; d, posterior 1/3; - , negative staining; +, faint staining; +, weak staining; ++, moderate staining; +++, intense staining; ++++, extremely intense staining. Vomeronasal nerve layer
Glomerular layer
Lectin
WGA s-WGA LEL STL DSL BSL-II DBA SBA BSL-I VVA SJA PNA ECL UEA-I Con A PSA
Rostral half
Caudal half
Rostral half
Caudal half
+ +++ +++ ++ -/+ +/++ + ++++ +++ ++/+++ _+ + +++ ++++ -/+ -/_+
+ ++ ++ +++ +_ + + ++(c),++++(d) + +(c),+/++(d) + +/++ _+(c),+(d) +++ + +
+ +++ +++ ++/+++ + ++ ++ ++++ +++ +++ + ++(a),+(b) +++ ++++ + +
+(c),+(d) ++(c),+++(d) ++ +++ +/++ +(c),+/++(d) +(c),+(d) ++(c),++++(d) +(c),++(d) ++(c),+++(d) +(c),+(d) ++(c),+++(d) +/+(c),+++(d) +++ +/++ +/++
188 2 Imamura, K., Mori, K., Fujita, S.C. and Obata, K., Immunochernical identification of subgroups of vorneronasal nerve fibers and their segregated terminations in the accessory olfactory bulb, Brain Res., 328 (1985) 362-366. 3 Macrides, F. and Davis, B.J., The olfactory bulb. In EC. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 391 426. 4 Mori, K., Monoclonal antibodies (2C5 and 4C9) against lactoseries carbohydrates identify subsets of olfactory and vomeronasal receptor cells and their axons in the rabbit, Brain Res., 408 (1987) 215 221. 5 Mori, K., Molecular and cellular properties of rnammalian primary olfactory axons, Microsc. Res. Technique, 24 (1993) 131-141. 6 Mori, K., Fujita, S.C., Imamura, K. and Obata, K., Imrnunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit, J. Comp. Neurol., 242 (1985) 214-229.
7 Mori, K., Imamura, K., Fujita, S.C. and Obata, K., Projections of two subclasses of vomeronasal nerve fibers to the accessory olfactory bulb in the rabbit, Neuroscience, 20 (1987) 259 278. 8 Schwarting, G.A. and Crandall, J.E., Subsets of olfactory and vomeronasal sensory epithelial cells and axons revealed by monoclonal antibodies to carbohydrate antigens, Brain Res., 547 (1991) 239-248. 9 Takami, S., Graziadei, P.RC. and Ichikawa, M., The differential staining patterns of two lectins in the accessory olfactory bulb of the rat, Brain Res., 598 (1992) 337-342. 10 Taniguchi, K., Matusaki, Y., Ogawa, K. and Saito, T.R., Fine structure of the vorneronasal organ in the common marmoset (Callithrixjacchus), Folia Primatol., 59 (1992) 169-176. 11 Taniguchi, K. and Mikami, S., Fine structure of the vomeronasal organ of horse and cattle. A comparative study, Cell Tissue Res., 240 (1985) 41~,8.
185
NSL 09721
Subdivisions of the accessory olfactory bulb, as demonstrated by lectin-histochemistry in the golden hamster Kazuyuki Taniguchi, Yuhsuke Nii and Kazushige O g a w a Department of Veterinary Anatomy, Faculty of Agriculture, lwate University, lwate (Japan) (Received 2 April 1993; Revised version received 6 May 1993; Accepted 7 May 1993)
Key words: Lectin; Histochemistry; Sugar residue; Olfaction; Vomeronasal organ; Accessory olfactory bulb; Golden hamster Lectin-binding patterns in the accessory olfactory bulb (AOB) of the golden hamster were investigated histochemically with 21 biotinylated lectins. The AOB was divided into rostral and caudal halves according to binding patterns of 16 lectins, WGA, s-WGA, LEL, STL, DSL, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA, ECL, UEA-I, Con A and PSA. The caudal half of the AOB was further subdivided into anterior 2/3 and posterior 1/3 by 10 lectins, WGA, s-WGA, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA and ECL. In addition, the rostral half of the AOB was subdivided into anterior 1/4 and posterior 3/4 by one lectin, PNA. Thus, the AOB of the golden hamster was divided into 4 divisions on the basis of lectin-binding patterns.
Olfactory receptor neurons of the vomeronasal organ project to the accessory olfactory bulb (AOB), constituting the vomeronasal chemosensory system [1]. In the AOB, axons of olfactory receptor neurons form the vomeronasal nerve layer, and further contribute to the formation of glomeruli in the glomerular layer [3]. Although individual receptor neurons show no distinct difference in their fine structures and are hardly dividable into subsets with conventional morphological methods [10, 11], the presence of subsets of receptor neurons to reveal subdivisions in the AOB has been suggested in the rabbit and rat by immunohistochemistry with monoclonal antibodies [2, 4-8], or by lectin-histochemistry [9]. Some of these monoclonal antibodies are reported to recognize carbohydrate antigens such as glycoconjugates [4, 5, 8]. Lectins are also known to recognize specific sugar residues of membranous structures of the cell [5, 9]. In the present study, we re-examined the AOB histochemically using 21 biotinylated lectins to demonstrate the presence of subdivisions in the AOB of the golden hamster. Adult golden hamsters (140-200 g body weight) of either sex were anesthetized with ether, and perfused with physiological saline, followed by Bouin's solution without acetic acid. The olfactory bulb was removed, immersed in the same fixative for 24 h, and embedded in
Correspondence: K. Taniguchi, Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Morioka, Iwate 020, Japan.
paraffin. Paraffin sections were cut sagittaUy at 5/tm, deparaffinized with xylene, and processed for lectin staining by the avidin-biotin complex (ABC) method with 21 biotinylated lectins (Table 1) in the commercial lectin screening kits (Vector). The sections were incubated for 48 h at 4°C with biotinylated lectins. Lectinbindings were visualized by the Vectastain ABC kit (Vector). Control lectin stainings were performed by the preabsorption of lectins with excess amount of respective specific sugar residues or by the use of phosphate-buffered saline to replace the biotinylated lectins or ABC. No specific lectin-bindings were observed after the control staining. The vomeronasal nerve and the glomerular layers of the AOB were divided into rostral and caudal halves by binding patterns of 16 lectins (Table 2) Among them, 15 lectins, WGA, s-WGA, LEL, STL, DSL, BSL-II, DBA, SBA, BSL-I, VVA, SJA, ECL, UEA-I, Con A and PSA stained the rostral half more strongly than the caudal half (Fig. 1A-D, F), while PNA stained the caudal half more strongly than the rostral half (Fig. 1E). The caudal half of the AOB was further divided into 2 parts; WGA, s-WGA, BSL-II, DBA, SBA, BSL-I, VVA, SJA, PNA and ECL stained the posterior 1/3 more strongly than the anterior 2/3 (Fig. 1A-F). The rostral half of the AOB was also divided into 2 parts; PNA stained the anterior 1/4 more strongly than the posterior 3/4 (Fig. 1E). Thus, the AOB of the golden hamster was divided into 4 subdivi-
186
B
•
r~,
•
41:~,
C
Fig. 1. Binding pattern of lectins, LEL (A), SBA (B), BSL-1 (C), VVA (D), PNA (E) and ECL (F), in the accessory olfactory bulb. Rostral is left and caudal is right, x80.
sions. These subdivisions were more obvious in the glomerular layer than in the vomeronasal nerve layer. The other lectins, RCA-I, Jacalin, LCA, PHA-E and PHA-L, showed no specific binding to the AOB. There were no sex differences in the lectin-binding patterns of the AOB. Imamura et al. [2] divided the AOB of the rabbit into the rostrolateral and caudomedial parts with monoclonal antibodies against membrane proteins. Schwarting and Crandall [8] also divided the AOB of the rat into the rostral and caudal halves with monoclonal antibodies to carbohydrate antigens. Takami et al. [9], however, divided the AOB of the rat into 2 subdivisions, anterior 1/3 and posterior 2/3, by lectin-histochemistry using BSL-! and VVA. In the present study, the AOB was divided
into 4 subdivisions on the basis of the binding patterns of 16 lectins. According to Takami et al. [9], VVA stains the posterior 2/3 of the AOB of the rat. In the present study, however, VVA strongly stained the rostral half of the AOB of the golden hamster. This discrepancy may be attributable to difference of the staining methods employed, or to species differences. The lectins used in the present study display various binding patterns. There seems to be no general relation between specific sugar residues and binding patterns.
1 Halpern, M., The organization and function of the vomeronasal system, Annu. Rev. Neurosci.. 10 (1987) 325 362.
187 TABLE 1 BINDING SPEC1FICITIES OF LECTINS USED Fuc, fucose; Gal, o-galactose; GalNAc, N-acetylgalactosamine; GIcNAc, N-acetylglucosamine; Man, mannose; NeuAc, N-acetylneuraminic acid. Lectin
Concentration (mg/ml)
Specific sugar
Wheat germ agglutinin (WGA) Succinylated wheat germ agglutinin (s-WGA) Lycopersicon esculentum lectin (LEL) Soranum tuberosum lectin (STL) Datura stramonium lectin (DSL) Bandeiraea simplicifolia lectin-II (BSL-II) Dolichos biflorus agglutinin (DBA) Soybean agglutinin (SBA) Bandeiraea simplicifolia lectin-I (BSL-I) Vicia villosa agglutinin (VVA) Sophorajaponica agglutinin (SJA) Ricinus communis agglutinin-I (RCA-I) Jacalin Peanut agglutinin (PNA) Erythrina cristagalli lectin (ECL) Ulex europaeus agglutinin-I (UEA-I) Concanavalin A (Con A) Pisum sativum agglutinin (PSA) Lens culinaris agglutinin (LCA) Phaseolus vulgaris agglutinin-E (PHA-E) Phaseolus vulgaris agglutinin-L (PHA-L)
0.002 0.0025 0.001 0.0033 0.0033 0.02 0.5 0.05 0.04 0.02 0.5 0.0006 0.0007 0.005 0.005 0.02 0.0013 0.002 0.0008 0.002 0.0083
fl-GlcNAc>0~-NeuAc fl-GlcNAc fl-GlcNAc fl-GlcNAc fl-GlcNAc ~,fl-GlcNAc ~-GalNAc 0~,fl-GalNAc, Gal ct-GalNAc, ~-Gal ~,fl-GalNAc fl-GalNAc, fl-Gal fl-GalNAc, fl-Gal galactosyl-fl-GalNAc galactosyl-fl-GalNAc galactosyl-fl-GlcNAc ~-Fuc c~-Man c~-Man s-Man oligosaccharide oligosaccharide
TABLE 2 BINDING PATTERN OF LECTINS IN THE AOB a, anterior 1/4; b, posterior 3/4; c, anterior 2/3; d, posterior 1/3; - , negative staining; +, faint staining; +, weak staining; ++, moderate staining; +++, intense staining; ++++, extremely intense staining. Vomeronasal nerve layer
Glomerular layer
Lectin
WGA s-WGA LEL STL DSL BSL-II DBA SBA BSL-I VVA SJA PNA ECL UEA-I Con A PSA
Rostral half
Caudal half
Rostral half
Caudal half
+ +++ +++ ++ -/+ +/++ + ++++ +++ ++/+++ _+ + +++ ++++ -/+ -/_+
+ ++ ++ +++ +_ + + ++(c),++++(d) + +(c),+/++(d) + +/++ _+(c),+(d) +++ + +
+ +++ +++ ++/+++ + ++ ++ ++++ +++ +++ + ++(a),+(b) +++ ++++ + +
+(c),+(d) ++(c),+++(d) ++ +++ +/++ +(c),+/++(d) +(c),+(d) ++(c),++++(d) +(c),++(d) ++(c),+++(d) +(c),+(d) ++(c),+++(d) +/+(c),+++(d) +++ +/++ +/++
188 2 Imamura, K., Mori, K., Fujita, S.C. and Obata, K., Immunochernical identification of subgroups of vorneronasal nerve fibers and their segregated terminations in the accessory olfactory bulb, Brain Res., 328 (1985) 362-366. 3 Macrides, F. and Davis, B.J., The olfactory bulb. In EC. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 391 426. 4 Mori, K., Monoclonal antibodies (2C5 and 4C9) against lactoseries carbohydrates identify subsets of olfactory and vomeronasal receptor cells and their axons in the rabbit, Brain Res., 408 (1987) 215 221. 5 Mori, K., Molecular and cellular properties of rnammalian primary olfactory axons, Microsc. Res. Technique, 24 (1993) 131-141. 6 Mori, K., Fujita, S.C., Imamura, K. and Obata, K., Imrnunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit, J. Comp. Neurol., 242 (1985) 214-229.
7 Mori, K., Imamura, K., Fujita, S.C. and Obata, K., Projections of two subclasses of vomeronasal nerve fibers to the accessory olfactory bulb in the rabbit, Neuroscience, 20 (1987) 259 278. 8 Schwarting, G.A. and Crandall, J.E., Subsets of olfactory and vomeronasal sensory epithelial cells and axons revealed by monoclonal antibodies to carbohydrate antigens, Brain Res., 547 (1991) 239-248. 9 Takami, S., Graziadei, P.RC. and Ichikawa, M., The differential staining patterns of two lectins in the accessory olfactory bulb of the rat, Brain Res., 598 (1992) 337-342. 10 Taniguchi, K., Matusaki, Y., Ogawa, K. and Saito, T.R., Fine structure of the vorneronasal organ in the common marmoset (Callithrixjacchus), Folia Primatol., 59 (1992) 169-176. 11 Taniguchi, K. and Mikami, S., Fine structure of the vomeronasal organ of horse and cattle. A comparative study, Cell Tissue Res., 240 (1985) 41~,8.