Advance Publication
The Journal of Veterinary Medical Science
Accepted Date: 7 Dec 2012 J-STAGE Advance Published Date: 21 Dec 2012 Kondoh 1
1 Anatomy FULL PAPER
2 Histological and Lectin Histochemical Studies on the Main and Accessory
3 Olfactory Bulbs in the Japanese Striped Snake, Elaphe quadrivirgata
4
5 Daisuke KONDOH1,2), Akimi WADA1), Daisuke ENDO1,2), Nobuaki NAKAMUTA1,2)
6 and Kazuyuki TANIGUCHI1,2)*
7
8 1)Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Morioka,
9 Iwate 020-8550, Japan
10 2)Department of Basic Veterinary Science, The United Graduate School of Veterinary
11 Science, Gifu University, Gifu, Gifu 501-1193, Japan
12
13 CORRESPONDENCE TO: TANIGUCHI K., Laboratory of Veterinary Anatomy,
14 Department of Veterinary Science, Faculty of Agriculture, Iwate University, 3-18-8
15 Ueda, Morioka-shi, Iwate 020-8550, Japan
16 Tel: +81-19-621-6207
17 FAX: +81-19-621-6209
18 e-mail: [email protected]
19
20 running head: LECTIN BINDING IN SNAKE OLFACTORY BULB Kondoh 2
21
22 ABSTRACT. The main and accessory olfactory bulbs were examined by histological
23 methods and lectin histochemistry in the Japanese striped snake. As the results, the
24 histological properties are similar between the main olfactory bulb and the accessory
25 olfactory bulb. In lectin histochemistry, 21 lectins used in this study showed similar
26 binding patterns in the main olfactory bulb and the accessory olfactory bulb. In detail,
27 15 lectins stained these olfactory bulbs with similar manner, and 6 lectins did not stain
28 them at all. Two lectins, Lycopersicon esculentum lectin (LEL) and Solanum
29 tuberosum lectin (STL), stained the nerve and glomerular layers and did not stain any
30 other layers in both olfactory bulbs. Four lectins, Soybean agglutinin (SBA), Vicia
31 villosa agglutinin (VVA), Peanut agglutinin (PNA), and Phaseolus vulgaris agglutinin-L
32 (PHA-L) stained the nerve and glomerular layers more intensely than other layers in
33 both olfactory bulbs. In addition, VVA showed the dot-like stainings in the glomeruli
34 of both olfactory bulbs. These findings suggest that the degree of development and the
35 properties of glycoconjugates are similar between the main olfactory bulb and the
36 accessory olfactory bulb in the Japanese striped snake.
37
38 KEY WORDS: histology, nervous system, reptiles, Squamates, vomeronasal system Kondoh 3
39 INTRODUCTION
40 The olfactory system receives and detects chemical substances in the external
41 environment. This system is divided into two independent systems: the main olfactory
42 system and the vomeronasal system. In the main olfactory system, the receptor cells in
43 the olfactory epithelium project their axons to the glomeruli in the main olfactory bulb
44 to form synapse with output neurons and intermediate neurons. On the other hand, in
45 the vomeronasal system, the receptor cells in the vomeronasal epithelium project their
46 axons to the glomeruli in the accessory olfactory bulb [14]. Although the main
47 olfactory system exists in all vertebrate species, the vomeronasal system first appears in
48 amphibians, is lost in several species such as crocodiles, birds, whales, and humans, and
49 has various morphological and histological features among animal species [4]. The
50 localization, size and laminar structure of the main and accessory olfactory bulbs vary
51 among species [25, 29] and appear to relate with behavioral patterns and living
52 environment of each species.
53 Among all tetrapods, snakes and some lizards have the most developed
54 vomeronasal system [13], i.e. the vomeronasal system of snakes and lizards mediates
55 not only species-specific communications by pheromones such as courtship and
56 aggregative behaviors [10, 21], but also non-species-specific behaviors by odoriferous Kondoh 4
57 molecules such as predatory and defensive behaviors [22, 27, 36]. Snakes sample
58 environmental substances by the tongue-flicking and deliver concentrated chemicals to
59 the vomeronasal epithelium, and the information acquired with the tongue-flicking is
60 mediated by both the main olfactory system and the vomeronasal system [13, 36].
61 Topographically, the size of the accessory olfactory bulb is as large as that of the main
62 olfactory bulb in snakes [15, 16], although the size of the accessory olfactory bulb is
63 much smaller than that of the main olfactory bulb in many other vertebrate species.
64 Histologically, both the main and accessory olfactory bulbs in snakes are divided into 6
65 layers (the nerve, glomerular, mitral cell, internal plexiform, granule cell and ependymal
66 cell layers), and the histological properties of the constituent cells are similar between
67 these olfactory bulbs [15, 16]. However, there are few detailed reports on the
68 sublamination and cell distribution in these layers of the main and accessory olfactory
69 bulbs in snakes.
70 Lectins are proteins binding nonimmunologically with glycoconjugates [3], and
71 are extensively used for the differentiation of cell types on histological sections based
72 on the staining regions and the staining intensities [23]. In lizards with well-developed
73 vomeronasal system, the lectin binding patterns are similar between the main olfactory
74 bulb and the accessory olfactory bulb [6], although the lectin binding patterns are Kondoh 5
75 different between these olfactory bulbs in many other species, such as amphibians
76 [32-34] and mammals [26, 28, 31]. According to these reports on the lizards and many
77 other species, the glycoconjugate moieties appear to be similar between the main
78 olfactory bulb and the accessory bulb in the species with well-developed vomeronasal
79 system, such as some lizards. Although snakes are equipped with the most developed
80 vomeronasal system [13, 14] and belong to Squamata as well as lizards, there is no
81 report on the lectin histochemistry on the main olfactory bulb and accessory olfactory
82 bulb in snakes. Squamata and mammals have evolved separately from primitive
83 reptiles, and it is possible that the histochemical features of olfactory system are
84 different between these two groups. In this study, we examined the main olfactory
85 bulb and the accessory olfactory bulb of the Japanese striped snake, Elaphe
86 quadrivirgata, by histological methods and 21 lectins extensively-used for screening the
87 differentiation of the glycoconjugate moieties between the main olfactory system and
88 the vomeronasal system in many species to detect possible similarities between these
89 olfactory bulbs in snakes.
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92 Kondoh 6
93 MATERIALS AND METHODS
94 Animals: Six snakes in the reproductive season (June) were studied (Table 1). They
95 were kept in near-natural conditions in the Japan Snake Institute (Ota, Japan) and
96 purchased. Based on an age estimated by the body length correlation [9], all snakes
97 were sexually mature. This study was approved and conducted according to the
98 Guideline for Animal Experiment of Iwate University. All procedures were approved
99 by the local animal ethical committee of Iwate University.
100 Histology: The animals were anesthetized by intraperitoneal injection of pentobarbital
101 (0.13-0.20 mg/g body weight) and were sacrificed by cardiac perfusion with Ringer’s
102 solution followed by Zamboni’s fixative. After decapitation, brains were removed
103 from heads, fixed in the same fixative for 3-4 hr, routinely embedded in paraffin and cut
104 frontally or horizontally at 5 m thickness. Some of these sections were stained with
105 luxol fast blue/cresyl violet (staining of Klüver-Barrera), and other sections were
106 processed for lectin histochemistry.
107 Lectin Histochemistry: Several sections were processed for histochemistry with ABC
108 method using 21 biotinylated lectins (Table 2) in the lectin screening kit I-III (Vector
109 Laboratories, Burlingame, CA, U. S. A.). After deparaffinization and rehydration,
110 sections were incubated with 0.3% H2O2 in methanol to eliminate endogenous Kondoh 7
111 peroxidase. Sections were rinsed in phosphate-buffered saline (PBS, 0.01 M, pH 7.4)
112 and incubated with 1% bovine serum albumin to block nonspecific reactions. After
113 rinsing, sections were incubated with biotinylated lectins at 4˚C overnight, reacted with
114 ABC reagent (Vector) at room temperature for 30 min, and colorized with 0.05 M
115 Tris-HCl (pH 7.4) containing 0.006% H2O2 and 0.02% 3-3’-diaminobenzidine
116 tetrahydrochloride. Staining intensities were judged as 5 grades: intense, moderate,
117 weak, faint and negative staining. Control stainings were performed (a) by the
118 preabsorption of lectins with each specific sugar residue (Table 2) or (b) by the use of
119 PBS to replace ABC reagent.
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128 Kondoh 8
129 RESULTS
130 Topographical and Histological Features of the Main and Accessory Olfactory Bulbs:
131 The olfactory bulb was located on the rostral surface of the telencephalon as a pair of
132 elongated structures, and was divided into two structures, rostrally located main
133 olfactory bulb and caudally located accessory olfactory bulb (Fig. 1A). The size of the
134 accessory olfactory bulb was as large as that of the main olfactory bulb (Fig. 1A). The
135 main olfactory bulb was a round structure and had centrally situated olfactory ventricle
136 (Fig. 1B). The glomeruli of the main olfactory bulb were laid at rostral to lateral
137 region uniformly to receive many thin olfactory nerves (Fig. 1B). On the other hand,
138 the accessory olfactory bulb was a semicircular structure and had laterally situated
139 olfactory ventricle (Fig. 1C). The glomeruli of the accessory olfactory bulb were
140 restricted to the medial region to receive a single thick vomeronasal nerve in the medial
141 region (Fig. 1C).
142 Histologically, in both the main and accessory olfactory bulbs, round or oval
143 glomeruli were formed at the terminals of the olfactory or vomeronasal nerves and were
144 surrounded by many small periglomerular cells with round nuclei (Fig. 1D, E).
145 According to the staining of Klüver-Barrera, both the main (Fig. 1D) and accessory
146 olfactory bulbs (Fig. 1E) were divided into six layers: the nerve layer, glomerular layer, Kondoh 9
147 mitral cell layer, internal plexiform layer, granule cell layer and ependymal cell layer.
148 The internal plexiform layer of the main and accessory olfactory bulbs was divided into
149 3 sublaminae: the outer, middle, and inner sublaminae (Fig. 1D, E, a-c). These
150 sublaminae were identifiable according to the middle sublamina containing thick
151 bundles of myelin sheaths. In both the main and accessory olfactory bulbs, the
152 neurons in the mitral cell layer were classified into two types, i.e. the cells with large
153 cell bodies (Fig. 1D, E, arrowheads) and the cells with small cell bodies (Fig. 1D, E,
154 arrows), and these cells were scattered in the whole mitral cell layer.
155 Lectin Histochemistry in the Main and Accessory Olfactory Bulbs: No specific
156 staining was observed in the control staining (Figs. 2D, H, L, 4C, F), and no difference
157 based on sex or estimated age was observed. In the following sentences, we used
158 abbreviations of lectins as shown in Table 2.
159 All the lectins used in this study showed similar binding patterns between the
160 main olfactory bulb and the accessory olfactory bulb (Fig. 2). Although 5 lectins
161 among the 21 lectins, LEL, STL, RCA-120, PHA-E and PHA-L, stained the endothelial
162 cells in the whole brain intensely, these stainings were excluded from results described
163 below because the endothelial cells were not the neural components of the olfactory
164 bulb. Kondoh 10
165 Two lectins, LEL and STL, stained the nerve and glomerular layers intensely and
166 did not stain other layers at all in the main olfactory bulb (Figs. 2A, 3A). These 2
167 lectins stained the nerve and glomerular layers moderately and did not stain other layers
168 at all in the accessory olfactory bulb (Figs. 2E, I, 3D). In both the main and accessory
169 olfactory bulbs, 3 lectins, SBA, PNA and PHA-L, stained the nerve and glomerular
170 layers moderately and stained other layers weakly (Figs. 2B, F, J, 3B, E). VVA stained
171 the glomerular layer moderately, the internal plexiform layer weakly, and other layers
172 faintly (Figs. 2C, G, K, 3C, F). Four lectins, DSL, Jacalin, ConA and PHA-E, stained
173 all layers moderately. Two lectins, WGA and RCA-120, stained all layers weakly, and
174 3 lectins, ECL, PSA and LCA, stained all layers faintly. The remaining 6 lectins did
175 not stain the layers at all.
176 In detail, 6 lectins, LEL, STL, SBA, VVA, PNA and PHA-L, stained some
177 glomeruli intensely and others weakly, and the glomeruli stained intensely and those
178 stained weakly were distributed as tessellation with no reference to the region of the
179 main and accessory olfactory bulbs (Fig. 2). Among the 15 lectins staining the
180 glomeruli, VVA showed the dot-like staining in the glomeruli of both the main and
181 accessory olfactory bulbs (Fig. 4B, E), although the remaining 14 lectins stained the
182 whole region of the glomeruli equally (Fig. 4A, D). Kondoh 11
183 These findings are summarized in Table 3.
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200 Kondoh 12
201 DISCUSSION
202 In the Japanese striped snake, both the main and accessory olfactory bulbs were
203 divided into 6 layers, and these laminar structures are similar between the main
204 olfactory bulb and the accessory olfactory bulb. These histological properties
205 conformed to previous reports by a Golgi study [15, 16]. In the glomerular layer,
206 many periglomerular cells were observed in both the main and accessory olfactory bulbs.
207 In mammals, the number of the periglomerular cells in the accessory olfactory bulb is
208 much smaller than that in the main olfactory bulb [25]. In addition, the internal
209 plexiform layer was divided into 3 sublaminae in both the main and accessory olfactory
210 bulbs according to the staining of Klüver-Barrera, although it has been reported that this
211 layer is divided into 3 sublaminae only in the accessory olfactory bulb according to
212 toluidine blue staining and GABA-immunostaining [19]. The staining of
213 Klüver-Barrera enabled us to show clearly the middle sublamina containing thick
214 bundles of myelin sheaths in both the main and accessory olfactory bulbs (Fig. 1D, E).
215 This finding appears to allow distinction of the middle sublamina from the outer and
216 inner sublaminae. In the mitral cell layer of both the main and accessory olfactory
217 bulbs, two types of neurons, the cells with large cell bodies and the cells with small cell
218 bodies, were scattered. The cells with large cell bodies appear to correspond to the Kondoh 13
219 mitral cells as output neurons, and the cells with small cell bodies may correspond to
220 the tufted cells, which are well-known as other cell type of output neurons in the main
221 olfactory bulb of mammals [12], or the interneurons in the mitral cell layer, although we
222 could not determine these correspondences. On these present findings, the histological
223 properties are similar between the main olfactory bulb and the accessory olfactory bulb
224 in the snake. The complexity of layer organization of the olfactory bulb is
225 proportional to information-processing capability in the olfactory bulb and reflects the
226 degree of development of the olfactory bulb [29]. Therefore, these present results
227 indicate that the degree of development is similar between the main olfactory bulb and
228 the accessory olfactory bulb in the snake and that the snake depends on both the main
229 olfactory system and the vomeronasal system equally to detect information on external
230 environments.
231 Fifteen lectins stained the nerve and glomerular layers in both the main and
232 accessory olfactory bulbs. On the other hand, in the olfactory and vomeronasal
233 epithelia of the Japanese striped snake, 4 lectins among these 15 lectins, SBA, PNA,
234 ECL and PSA, stain the cell processes of the receptor cells only after sialic acid removal
235 [18] and do not stain them before this treatment [17]. These reports suggest that
236 several glycoconjugate moieties in receptor cells are capped by sialic acid residues in Kondoh 14
237 the olfactory and vomeronasal epithelia and are not capped by sialic acid residues in the
238 nerve and glomerular layers of the main and accessory olfactory bulbs. In general,
239 sialoglycoproteins have many important roles in cell migration, axonal guidance and
240 functional plasticity in the nervous system [1, 30]. It is considered that the olfactory
241 and vomeronasal receptor cells have a high plasticity in the epithelia to relocate
242 involved with turnover throughout life and that their axons have a low plasticity in the
243 olfactory or vomeronasal nerves and the glomeruli to integrate and hold the axon
244 terminals in a single glomerulus. Therefore, the glycoconjugates labeled by SBA,
245 PNA, ECL and PSA appear to mediate functional plasticity in the receptor cells by sialic
246 acid capping.
247 Six lectins, LEL, STL, SBA, VVA, PNA and PHA-L, stained individual glomeruli
248 with various intensities in both the main and accessory olfactory bulbs. These results
249 indicate that the amounts of several glycoconjugate moieties are different among
250 individual glomeruli. In the olfactory system, glycoconjugates play several important
251 roles in continuous regeneration of the olfactory neurons, i.e. neurite outgrowth and
252 synapse formation [30]. Therefore, the different amounts of glycoconjugate moieties
253 may reflect the processes of turnover stages of the receptor cells projecting their axons
254 to individual glomeruli. In addition, VVA showed the dot-like staining in the Kondoh 15
255 glomeruli in both the main and accessory olfactory bulbs. As several neurotransmitter
256 receptors, such as several muscarinic receptors, dopamine receptors and GABA
257 receptors, in the glomeruli show the dot-like staining by immunohistochemistry [2, 11,
258 20], VVA may stain some of the neurotransmitter receptors in the glomeruli of the main
259 and accessory olfactory bulbs in the snake.
260 All the lectins used in this study showed similar binding patterns between the
261 main olfactory bulb and the accessory olfactory bulb (Table 3). These results indicate
262 that glycoconjugate moieties are similar between the main olfactory bulb and the
263 accessory olfactory bulb in the snake. The binding patterns of several lectins are
264 different between the main olfactory bulb and the accessory olfactory bulb in many
265 other vertebrate species [5, 7, 8, 24, 26, 28, 32-35], although the binding patterns of
266 lectins are similar between the main and accessory olfactory bulbs in several lizards [6].
267 In particular, it is well-known that there are glycoconjugate moieties labeled by VVA in
268 only the accessory olfactory bulb in several mammals [35]. Therefore, the findings
269 from the present lectin histochemistry in the snake appear to be well accorded with the
270 reports on the lectin histochemistry in several lizards [6]. As both snakes and lizards
271 possess well-developed vomeronasal system, the present results support that
272 glycoconjugate moieties are similar between the main olfactory bulb and the accessory Kondoh 16
273 olfactory bulb in the species with well-developed vomeronasal system. In addition,
274 the lectin binding patterns in the receptor cells are reported to be similar between the
275 main olfactory system and the vomeronasal system in the Japanese striped snake [17].
276 These similarities of lectin binding patterns between the main olfactory system and the
277 vomeronasal system may reflect that these systems mediate similar behaviors elicited by
278 odoriferous molecules such as predatory and defensive behaviors in snakes [21, 27, 36].
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290 Kondoh 17
291 ACKNOWLEDGMENTS. The authors are very grateful to late Dr. Michihisa Toriba,
292 the Japan Snake Institute, Japan, for providing animals used in this study and Mr.
293 Daisuke Nagase, Iwate University, Japan, for helpful discussions on several points in
294 this paper.
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308 Kondoh 18
309 REFERENCES
310 1. Brusés, J. L. and Rutishauser, U. 2001. Roles, regulation, and mechanism of
311 polysialic function during neural development. Biochimie 83: 635–643.
312 2. Crespo, C., Blasco-Ibáñez, J. M., Briñón, J. G., Alonso, J. R., Domínguez, M. I.
313 and Martínez-Guijarro, F. J. 2000. Subcellular localization of m2 muscarinic
314 receptors in GABAergic interneurons of the olfactory bulb. Eur. J. Neurosci. 12:
315 3963–3974.
316 3. Damjanov, I. 1987. Lectin cytochemistry and histochemistry. Lab. Invest. 57: 5–20.
317 4. Eisthen, H. L. 1992. Phylogeny of the vomeronasal system and of receptor cell
318 types in the olfactory and vomeronasal epithelia of vertebrates. Microsc. Res. Tech.
319 23: 1–21.
320 5. Franceschini, V., Lazzari, M. and Ciani, F. 1996. Identification of surface
321 glycoconjugates in the olfactory system of turtle. Brain Res. 725: 81–87.
322 6. Franceschini, V., Lazzari, M. and Ciani, F. 2000. Lectin cytochemical localisation
323 of glycoconjugates in the olfactory system of the lizards Lacerta viridis and
324 Podarcis sicula. Anat. Embryol. 202: 49–54.
325 7. Franceschini, V., Lazzari, M. and Ciani, F. 2001. Lectin-binding patterns in the
326 olfactory system of the lizard, Physignathus lesueurii. J. Morphol. 247: 34–38. Kondoh 19
327 8. Franceschini, V., Lazzari, M. and Ciani, F. 2003. Surface glycoconjugates in the
328 olfactory system of Ambystoma mexicanum. J. Morphol. 256: 301–305.
329 9. Fukada, H. 1960. Biological studies on the snakes. VII. Growth and maturity of
330 Elaphe quadrivirgata (Boie). Bull. Kyoto Gakugei Univ. B 16: 6–21.
331 10. Graves, B. M., Halpern, M. and Friesen, J. L. 1991. Snake aggregation
332 pheromones: source and chemosensory mediation in western ribbon snakes
333 (Thamnophis proximus). J. Comp. Psychol. 105: 140–144.
334 11. Gutièrrez-Mecinas, M., Crespo, C., Blasco-Ibáñez, J. M., Gracia-Llanes, F. J.,
335 Marqués-Marí, A. I., Nácher, J., Varea, E. and Martínez-Guijarro, F. J. 2005.
336 Distribution of D2 dopamine receptor in the olfactory glomeruli of the rat olfactory
337 bulb. Eur. J. Neurosci. 22: 1357–1367.
338 12. Haberly, L. B. and Price, J. L. 1977. The axonal projection patterns of the mitral
339 and tufted cells of the olfactory bulb in the rat. Brain Res. 129: 152–157.
340 13. Halpern, M. 1987. The organization and function of the vomeronasal system. Annu.
341 Rev. Neurosci. 10: 325–362.
342 14. Halpern, M. and Martínez-Marcos, A. 2003. Structure and function of the
343 vomeronasal system: an update. Prog. Neurobiol. 70: 245–318.
344 15. Iwahori, N., Nakamura, K. and Mamiya, C. 1989. A Golgi study on the main Kondoh 20
345 olfactory bulb in the snake, Elaphe quadrivirgata. Neurosci. Res. 6: 411–425.
346 16. Iwahori, N., Nakamura, K. and Mamiya, C. 1989. A Golgi study on the accessory
347 olfactory bulb in the snake, Elaphe quadrivirgata. Neurosci. Res. 7: 55–70.
348 17. Kondoh, D., Yamamoto, Y., Nakamuta, N., Taniguchi, K. and Taniguchi, K. 2010.
349 Lectin histochemical studies on the olfactory epithelium and vomeronasal organ in
350 the Japanese striped snake, Elaphe quadrivirgata. J. Morphol. 271: 1197–1203.
351 18. Kondoh, D., Yamamoto, Y., Nakamuta, N., Taniguchi, K. and Taniguchi, K. 2012.
352 Seasonal changes in the histochemical properties of the olfactory epithelium and
353 vomeronasal organ in the Japanese striped snake, Elaphe quadrivirgata. Anat.
354 Histol. Embryol. 41: 41–53.
355 19. Kosaka, T., Kosaka, K. and Nagatsu, I. 1991. Tyrosine hydroxylase-like
356 immunoreactive neurons in the olfactory bulb of the snake, Elaphe quadrivirgata,
357 with special reference to the colocalization of tyrosine hydroxylase- and
358 GABA-like immunoreactivities. Exp. Brain Res. 87: 353–362.
359 20. Kratskin, I., Kenigfest, N., Rio, J. P., Djediat, C. and Repérant, J. 2006.
360 Immunocytochemical localization of the GABAB2 receptor subunit in the glomeruli
361 of the mouse main olfactory bulb. Neurosci. Lett. 402: 121–125.
362 21. Kubie, J. L., Vagvolgyi, A. and Halpern, M. 1978. Roles of the vomeronasal and Kondoh 21
363 olfactory systems in courtship behavior of male garter snakes. J. Comp. Physiol.
364 Psychol. 92: 627–641.
365 22. Kubie, J. L. and Halpern, M. 1979. Chemical senses involved in garter snake prey
366 trailing. J. Comp. Physiol. Psychol. 93: 648–667.
367 23. Leathem, A. and Atkins, N. 1983. Lectin binding to formalin-fixed paraffin sections.
368 J. Clin. Pathol. 36: 747–750.
369 24. Matsui, T., Saito, S., Kobayashi, Y. and Taniguchi, K. 2011. Lectin histochemical
370 study on the olfactory bulb of the newt, Cynopus pyrrhogaster. Anat. Histol.
371 Embryol. 40: 419–425.
372 25. Meisami, E. and Bhatnagar, K. P. 1998. Structure and diversity in mammalian
373 accessory olfactory bulb. Microsc. Res. Tech. 43: 476–499.
374 26. Mendoza, A. S. and Kühnel, W. 1991. Lectin histochemical investigations on the
375 regio olfactoria and the vomeronasal organ of rat and golden hamster. Acta
376 Histochem. 91: 173–184.
377 27. Miller, L. R. and Gutzke, W. H. 1999. The role of the vomeronasal organ of
378 crotalines (Reptilia: Serpentes: Viperidae) in predator detection. Anim. Behav. 58:
379 53–57.
380 28. Nakajima, T., Shiratori, K., Ogawa, K., Tanioka, Y. and Taniguchi, K. 1998. Kondoh 22
381 Lectin-binding patterns in the olfactory epithelium and vomeronasal organ of the
382 common marmoset. J. Vet. Med. Sci. 60: 1005–1011.
383 29. Nieuwenhuys, R. 1967. Comparative anatomy of the olfactory centers and tracts.
384 Prog. Brain Res. 23: 1–64.
385 30. Plendl, J. and Sinowatz, F. 1998. Glycobiology of the olfactory system. Acta Anat.
386 Basel 161: 234–253.
387 31. Saito, H., Ogawa, K. and Taniguchi, K. 1994. Lectin-binding patterns of the
388 olfactory receptors (olfactory epithelium, vomeronasal organ and septal olfactory
389 organ of Masera) in the rat. Exp. Amin. 43: 51–60.
390 32. Saito, S. and Taniguchi, K. 2000. Expression patterns of glycoconjugates in the
391 three distinctive olfactory pathways of the clawed frog, Xenopus laevis. J. Vet. Med.
392 Sci. 62: 153–159.
393 33. Saito, S., Matsui, T., Kobayashi, N., Wakisaka, H., Mominoki, K., Matsuda, S. and
394 Taniguchi, K. 2003. Lectin histochemical study on the olfactory organ of the newt,
395 Cynops pyrrhogaster, revealed heterogeneous mucous environments in a single
396 nasal cavity. Anat. Embryol. 206: 349–356.
397 34. Saito, S., Kobayashi, N. and Atoji, Y. 2006. Subdivision of the accessory olfactory
398 bulb in the Japanese common toad, Bufo japonicus, revealed by lectin Kondoh 23
399 histochemical analysis. Anat. Embryol. 211: 395–402.
400 35. Shapiro, L. S., Ee, P. L. and Halpern, M. 1995. Lectin histochemical identification
401 of carbohydrate moieties in opossum chemosensory systems during development,
402 with special emphasis on VVA-identified subdivisions in the accessory olfactory
403 bulb. J. Morphol. 224: 331–349.
404 36. Zuri, I. and Halpern, M. 2003. Differential effects of lesions of the vomeronasal and
405 olfactory nerves on garter snake (Thamnophis sirtalis) responses to airborne
406 chemical stimuli. Behav. Neurosci. 117: 169–183.
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416 Kondoh 24
417 FIGURE LEGENDS
418 Fig. 1. Topographical (A) and histological features stained with the staining of
419 Klüver-Barrera (B to E) of the main and accessory olfactory bulbs in the Japanese
420 striped snake. A: Dorsal aspect of the brain. The right of the figure is rostral. Bar =
421 3 mm. B: Frontal section of the main olfactory bulb. The left sides of the figures are
422 medial, and the uppers are dorsal in (B) and (C). The box indicates the region shown
423 in (D). Bar = 300 m. C: Frontal section of the accessory olfactory bulb. The box
424 indicates the region shown in (E). Bar = 300 m. D: Histological structure of the
425 main olfactory bulb. Character (a) indicates the outer sublamina, (b) indicates the
426 middle sublamina and (c) indicates the inner sublamina of the internal plexiform layer
427 in (D) and (E). Arrow and arrowhead indicates the cells with small cell bodies and the
428 cells with large cell bodies in the mitral cell layer, respectively, in (D) and (E). Bar =
429 50 m. E: Histological structure of the accessory olfactory bulb. Bar = 50 m.
430 AOB, accessory olfactory bulb; ECL, ependymal cell layer; GCL, granule cell layer;
431 GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer; MOB, main
432 olfactory bulb; NL, nerve layer; OV, olfactory ventricle; T, telencephalon.
433
434 Fig. 2. Binding patterns of lectins in the main olfactory bulb and the accessory Kondoh 25
435 olfactory bulb in the Japanese striped snake. A to H: Frontal section of the main (A to
436 D) and accessory (E to H) olfactory bulbs labeled by LEL (A and E), SBA (B and F)
437 and VVA (C and G) stainings and control staining by the use of PBS to replace ABC
438 reagent (D and H). The left side of the figure is medial, and the upper, dorsal in (A) to
439 (H). Arrows in (A) and (E) indicate the endothelial cells. Asterisks in (C) and (G)
440 indicate the internal plexiform layer. I-L: Horizontal section of the accessory olfactory
441 bulb labeled by LEL (I), SBA (J) and VVA (K) stainings and control staining by the use
442 of PBS to replace ABC reagent (L). Arrow in (I) indicates the endothelial cells. Bars
443 = 300 m. GL, glomerular layer; NL, nerve layer; VNN, vomeronasal nerve.
444
445 Fig. 3. Higher magnifications of the main olfactory bulb (A to C) and the accessory
446 olfactory bulb (D to F) labeled by LEL (A and D), SBA (B and E) and VVA (C and F)
447 stainings in the Japanese striped snake. Arrows in (A) and (D) indicate the endothelial
448 cells. Bar = 50 m. ECL, ependymal cell layer; GCL, granule cell layer; GL,
449 glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.
450
451 Fig. 4. Higher magnifications of the glomeruli in the main olfactory bulb (A to C) and
452 the accessory olfactory bulb (D to F) labeled by LEL (A and D) and VVA (B and E) Kondoh 26
453 stainings and control staining by the use of PBS to replace ABC reagent (C and F) in the
454 Japanese striped snake. Bar = 50 m.
For Review Only
Fig. 1. Topographical (A) and histological features stained with the staining of Klüver-Barrera (B to E) of the main and accessory olfactory bulbs in the Japanese striped snake. A: Dorsal aspect of the brain. The right of the figure is rostral. Bar = 3 mm. B: Frontal section of the main olfactory bulb. The left sides of the figures are medial and the uppers are dorsal in (B) and (C). The box indicates the region shown in (D). Bar = 300 µm. C: Frontal section of the accessory olfactory bulb. The box indicates the region shown in (E). Bar = 300 µm. D: Histological structure of the main olfactory bulb on horizontal section. Character (a) indicates the outer sublamina, (b) indicates the middle sublamina, and (c) indicates the inner sublamina of the internal plexiform layer in (D) and (E). Arrow and arrowhead indicates the cells with small nuclei and the cells with large nuclei in the mitral cell layer, respectively, in (D) and (E). Bar = 50 µm. E: Histological structure of the accessory olfactory bulb. Bar = 50 µm. AOB, accessory olfactory bulb; ECL, ependymal cell layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer; MOB, main olfactory bulb; NL, nerve layer; OV, olfactory ventricle; T, telencephalon.
For Review Only
Fig. 2. Binding patterns of lectins in the main olfactory bulb and the accessory olfactory bulb in the Japanese striped snake. A to H: Frontal section of the main (A to D) and accessory (E to H) olfactory bulbs labeled by LEL (A and E), SBA (B and F), VVA (C and G), and control (D and H) stainings. The left side of the figure is medial, and the upper, dorsal in (A) to (H). Arrows in (A) and (D) indicate the endothelial cells. Asterisks in (C) and (G) indicate the internal plexiform layer. I-L: Horizontal section of the accessory olfactory bulb labeled by LEL (I), SBA (J), VVA (K), and control (L) stainings. Arrow in (I) indicates the endothelial cells. Bars = 300 µm. GL, glomerular layer; NL, nerve layer; VNN, vomeronasal nerve.
For Review Only
Fig. 3. Higher magnifications of the main olfactory bulb (A to C) and the accessory olfactory bulb (D to F) labeled by LEL (A and D), SBA (B and E), and VVA (C and F) stainings in the Japanese striped snake. Arrows in (A) and (D) indicate the endothelial cells. Bar = 50 µm. GL, glomerular layer; ECL, ependymal cell layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.
Fig. 4. Higher magnifications of the glomeruli in the main olfactory bulb (A to C) and the accessory olfactory bulb (D to F) labeled by LEL (A and D), VVA (B and E), and control (C and F) stainings in the Japanese striped snake. Bar = 50 µm. Table 1. Details of animals used in the present study
Date TL (cm) W (g) Sex
No. 22 June 2009 116 202.1 Male
No. 23 June 2009 119 225.7 Female No. 24 June 2009 114 202.9 Male No. 25 June 2009 124 458.7 Male No. 36 June 2010 130 341.1 Male No. 37 June 2010 89 226.8 Female
TL, total length; W, weight Table 2. Concentrations and binding specificities of lectins used in the present study
Lectins Abbreviation Concentration Binding specificity (mg ml-1) Wheat germ agglutinin WGA 1.0×10-2 GlcNAc, NeuAc -2 Succinylated-wheat germ agglutinin s-WGA 1.0×10 (GlcNAc)n -3 Lycopersicon esculentum lectin LEL 2.0×10 (GlcNAc)2-4 -2 Solanum tuberosum lectin STL 1.0×10 (GlcNAc)2-4 -3 Datura stramonium lectin DSL 4.0×10 (GlcNAc)2-4 Bandeiraea simplicifolia lectin-II BSL-II 5.0×10-2 GlcNAc Dolichos biflorus agglutinin DBA 5.0×10-2 Gal, GalNAc Soybean agglutinin SBA 1.0×10-2 Gal, GalNAc Bandeiraea simplicifolia lectin-I BSL-I 5.0×10-3 Gal, GalNAc Vicia villosa agglutinin VVA 1.0×10-2 Gal, GalNAc Sophora japonica agglutinin SJA 5.0×10-2 Gal, GalNAc Ricinus communis agglutinin-I RCA-120 2.0×10-3 Gal, GalNAc Jacalin 5.0×10-4 Gal, GalNAc Peanut agglutinin PNA 4.0×10-3 Gal Erythrina cristagalli lectin ECL 2.0×10-2 Gal, GalNAc Ulex europaeus agglutinin-I UEA-I 5.0×10-2 Fuc Concanavalin A ConA 3.3×10-3 Man, Glc Pisum sativum agglutinin PSA 4.0×10-3 Man, Glc Lens culinaris agglutinin LCA 4.0×10-3 Man, Glc Phaseolus vulgaris agglutinin-E PHA-E 5.0×10-3 Oligosaccharide Phaseolus vulgaris agglutinin-L PHA-L 2.5×10-3 Oligosaccharide
Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuAc, N-acetylneuraminic acid. Table 3. Lectin binding patterns in the olfactory bulb of the Japanese striped snake
Lectin Main olfactory bulb Accessory olfactory bulb Nerve Glomerular Mitral cell, internal Nerve Glomerular Mitral cell, internal layer layer plexiform, granule cell layer layer plexiform, granule cell and ependymal cell and ependymal cell layers layers WGA + + + + + + s-WGA － － － － － － LEL +++ +++ － ++ ++ － STL +++ +++ － ++ ++ － DSL ++ ++ ++ ++ ++ ++ BSL-II － － － － － － DBA － － － － － － SBA ++ ++ + ++ ++ + BSL-I － － － － － － VVA ± ++ ± or + a) ± ++ ± or + a) SJA － － － － － － RCA -120 + + + + + + Jacalin ++ ++ ++ ++ ++ ++ PNA ++ ++ + ++ ++ + ECL ± ± ± ± ± ± UEA-I － － － － － － ConA ++ ++ ++ ++ ++ ++ PSA ± ± ± ± ± ± LCA ± ± ± ± ± ± PHA-E ++ ++ ++ ++ ++ ++ PHA-L ++ ++ + ++ ++ +
－, negative staining; ±, faint staining; +, weak staining; ++, moderate staining; +++, intense staining. a) The internal plexiform layer was stained weakly and the mitral cell, granule cell, and ependymal cell layer were stained faintly.