Chem. Senses 26: 645-652,
2001
© Oxford University Press 2001
Histochemical Identification of Carbohydrate Moieties in the Accessory Olfactory Bulb of the Mouse Using a Panel of Lectins
Department of Anatomy and Embryology, Faculty of Veterinary Medicine, E-27002 Lugo, Spain
Correspondence to be sent to: Ignacio Salazar, Department of Anatomy and Embryology, Faculty of Veterinary Medicine, E-27002 Lugo, Spain. e-mail: anigsabe{at}lugo.usc.es
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Abstract |
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Lectin binding patterns in the olfactory bulb of the mouse were investigated using 12 biotinylated lectins. Three, with specificities for galactose, N-acetylgalactosamine and L-fucose, stained only the nervous and glomerular layers of the accessory olfactory bulb; four, with specificities for galactose or N-acetylglucosamine, stained these layers in both the accessory and the main olfactory bulbs; three, with specificities for N-acetylgalactosamine or L-fucose, effected general staining with little contrast between the background and the accessory olfactory bulb or other structures; the remaining two, both of them specific for mannose, stained no part of the tissue studied. In the nervous and glomerular layers of the accessory olfactory bulb six lectins stained the anterior and posterior halves with different intensities and two of these six similarly differentiated between rostral and caudal regions of the posterior half. We conclude that: (i) three lectins binding to different monosaccharides are specific stains for the vomeronasal system when used in this area of the mouse brain; (ii) it may be appropriate to distinguish three parts in the mouse accessory olfactory bulb, instead of the hitherto generally accepted two.
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Introduction |
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Cell surface carbohydrates mediate a variety of cell interactions and functionally significant sets of sensory neurons can be identified on the basis of the carbohydrate-containing molecules they bear. Since lectins are specific for particular carbohydrate moieties they can be used to survey the distribution of glycoconjugates in tissues (Cook, 1986
; Jessell et al., 1990) and in recent years they have not infrequently been employed for this purpose in neurobiology [see articles in Acta Anat., 161, Special Issue Glycosciences, especially Sharon (Sharon, 1998)]. Their use in this field has helped to clarify such topics as the subdivision of the accessory olfactory bulb (AOB) (Ichikawa et al., 1992; Taniguchi et al., 1993; Shapiro et al., 1995) and has also shown that there is considerable between-species variation in the cell surface carbohydrate moieties expressed by homologous structures, the AOB being a case in point (Halpern, 1987; Halpern et al., 1998a). Lectins have also begun to be used in an exciting new methodology with immense potential: the introduction of a plant lectin gene into the genome of an animal species, followed by its controlled expression in tissues of interest, allows the use of the lectin as a trans-neuronal tracer, a procedure that has already begun to afford valuable information on the development of connectivity (Horowitz et al., 1999; Yoshihara et al., 1999).
As far as we know, the only previous studies of lectin binding by the mouse olfactory system have been those of Key and Giorgi and Plendl and Schmahl (Key and Giorgi, 1986; Plendl and Schmahl, 1988). Here we report the findings of a descriptive study of the distribution of carbohydrate moieties in the olfactory bulb of the mouse as determined using a battery of 12 different lectins. We found that three lectins with different monosaccharide specificities bind only to the AOB and that it may be appropriate to distinguish three parts in the mouse AOB, instead of the hitherto generally accepted two.
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Materials and methods |
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Adult, singly housed female BALB/c mice bred in the Animal House of the University of Santiago de Compostela (Registry no. 15003AE) were placed under deep anaesthesia and trans-cardially perfused with phosphate-buffered saline (PBS) followed by Bouin’s fixative (both pH 7.3). Series of 10 µm sagittal and transverse paraffin sections of the whole olfactory bulbs and frontal cortex were cut and, after elimination of endogenous peroxidase activity with H2O2, were incubated overnight at room temperature with one of the following biotinylated lectins, the monosaccharide specificities of which are listed, together with suppliers and concentrations, in Table 1: BSI-B4 (from Bandeiraea simplicifolia), DBA (from Dolichos biflorus), ECA (from Erythrina cristagalli), SBA (from Glycine max), LCA (from Lens cunicularis), LTA (from Lotus tetragonolobus), LEA (from Lycopersicum esculentum), PSA (from Pisum sativum), WGA (from Triticum vulgaris), UEA-I (from Ulex europaeus), VVA (from Vicia villosa) and WGAs (succinylated WGA). The lectin-labeled sections were then incubated with avidin–biotin–horseradish peroxidase complexes, after which fixed peroxidase was visualized by incubation with 3,3'-diaminobenzidine (0.05%) and H2O2 (0.03%) in 0.2 M Tris–HCl buffer. Two types of control were used: (i) sections processed without any lectin; (ii) sections processed with lectins pre-saturated with the corresponding monosaccharide.
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All experiments were performed in accordance with the norms of the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW, NIH 86-23). All efforts were made to minimize animal suffering and limit the number of animals used.
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Results |
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The arrangement and general morphological characteristics of the mouse AOB are shown in selected sagittal and transverse sections in Figure 1. In neither plane is it possible to distinguish mitral from tufted cells. The internal plexiform layer of the AOB is enclosed in the lateral olfactory tract (LOT) (Figure 1A, B).
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The patterns of reactivity for each lectin are summarized below.
DBA (Figure 2A–C), BSI-B4 (Figure 2D) and UEA-I (Figure 2E–H) stained only the AOB. Of the three, UEA-I stained most densely and DBA least densely. More importantly, whereas BSI-B4 stained the whole AOB uniformly DBA stained the posterior part more intensely than the anterior, while UEA-I stained the anterior part more intensely than the posterior. UEA-I furthermore stained a particular rostral region of the posterior part much less intensely than the rest of this part of the AOB (Figure 2F, arrowhead).
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ECA (Figure 3A), WGA (Figure 3B), WGAs (Figure 3C) and LEA (Figure 3D–F) stained both the AOB and the main olfactory bulb (MOB), with intensities increasing in the order ECA < WGA < WGAs < LEA. All these lectins also effected diffuse background staining. Like UEA-I and DBA, ECA and LEA differentiated between the anterior and posterior parts of the AOB; the difference was particularly marked with LEA, which also, like UEA-I, differentiated a poorly stained rostral region of the posterior AOB from the rest of this region (Figure 3E, arrowhead).
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SBA (Figure 4A), VVA (Figure 4B) and LTA (Figure 4C) effected general staining of the whole section, with little contrast between the background and the AOB or other structures (e.g. the LOT, arrowhead in Figure 4B, C), although SBA and VVA nevertheless differentiated the anterior AOB from the posterior. These results were less reproducible than those obtained with the other lectins.
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LCA (Figure 5A, C) and PSA (Figure 5B, D) failed to stain any part of the tissue studied.
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In all cases in which the AOB or MOB bound lectins the only strata affected were the nervous layer (NL) and the glomerular layer (GL).
All lectin-less control sections were unstained and no specific binding was observed in controls with pre-saturated lectins.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The above results show that the 12 lectins used in this study are classifiable into three groups on the basis of their binding to the NL and GL of the mouse olfactory bulb (OB): Group I, binders (BSI-B4, DBA, UEA-I, ECA, WGA, WGAs and LEA, the first three of which bound only to the AOB and the others to both the AOB and MOB); Group II, non-binders (PSA and LCA); Group III, unconfirmed or dubious binders that significantly stained the background as well as the OB and/or other structures (SBA, VVA and LTA).
The finding that DBA binds to the AOB agrees with the results of the only other study in which lectins of Group I have been used to study the olfactory system of the adult mouse (Plendl and Schmahl, 1988). For the lectins of Group II no data have previously been published on their binding behaviour in the mouse. The Group III lectin SBA was considered by Key and Giorgi to bind specifically to the AOB and, hence, to the vomeronasal system, in this area of the mouse brain (Key and Giorgi, 1986).
The discrepancy between Key and Giorgi’s findings and our own with respect to the behaviour of SBA may be at least partially due to Key and Giorgi having used coronal sections and frozen tissue, but also raises the question of what objective criteria there may be for evaluation of background staining (Key and Giorgi, 1986). Background stain can of course be largely avoided by diluting the lectin, but if this procedure also significantly reduces stain density in the putatively positively stained structure then it is difficult to distinguish among the following three possibilities: (i) the target structure has only a low density of molecules bearing the carbohydrate for which the lectin is specific; (ii) the affinity of these molecules for the lectin is low; (iii) staining of both the background and the target structure is non-specific, even though the target structure may have greater non-specific affinity than the background. In this study attempts to remove background staining by SBA, VVA and LTA caused a significant reduction in OB staining; these lectins not only stained the background, but also non-OB structures such as the LOT. The results obtained with these lectins were less reproducible than those obtained with the others. We therefore regard their binding specifically to the OB as unconfirmed or dubious and group them accordingly.
The two mannose-specific lectins, LCA and PSA, both failed to stain anything and the three N-acetylglucosamine-specific lectins, LEA, WGA and WGAs, all stained both the AOB and the MOB. In contrast, the N-acetylgalactosamine-specific group (DBA, SBA and VVA), the galactose-specific group (ECA and BSI-B4) and the fucose-specific group (UEA-I and LTA) were all heterogeneous as regards the staining behaviour of their members (in particular, ECA stained both the AOB and the MOB but BSI-B4 only the former). These results suggest, subject to the limitations of the lectin battery used, that in neither the AOB nor the MOB are there cells bearing lectin-bindable mannose, show that in both structures there are cells bearing lectin-bindable N-acetylglucosamine and that BSI-B4 binds to only a subset of the galactose-bearing molecules bound by ECA. The behaviour of DBA, ECA, UEA-I and LEA in the AOB also shows that the anterior and posterior AOB differ as regards the density and/or nature of their lectin-bindable N-acetylgalactosamine, galactose, fucose and N-acetylglucosamine and that the rostroventral part of the posterior AOB differs from the rest of the posterior AOB as regards the density and/or nature of lectin-bindable fucose and N-acetylglucosamine. It was pointed out by Sharon and Lis that some lectins are specific for just one anomer while others bind both, and many other factors may also influence the affinity of a monosaccharide-bearing molecule for a lectin that in principle is specific for that monosaccharide (Sharon and Lis, 1989).
As noted above, the mouse AOB has previously been studied with lectins by Key and Giorgi and Plendl and Schmahl (Key and Giorgi, 1986; Plendl and Schmahl, 1988). Lectins have also been used to study the AOB and other areas of the vomeronasal system in the rat (Barber, 1989; Ichikawa et al., 1992; Salazar and Sánchez Quinteiro, 1998), dog (Salazar et al., 1992), hamster (Taniguchi et al., 1993), sheep (Salazar et al., 2000), pig (Salazar et al., 2000) and opossum (Shapiro et al., 1995). These studies show both similarities and differences among different species as regards the distribution of lectin-bindable carbohydrate moieties. Unfortunately, the available data are an insufficient basis for any convincing explanation of these differences. Like Shapiro et al. (Shapiro et al., 1995), we can only point out that care must be taken in comparing results obtained for different systems in different species using different lectins.
This study is the first in which the distinction between the anterior and posterior regions of the NL and GL of the AOB in the mouse has been observed using lectin histochemistry, but this distinction has previously been observed in the mouse at the molecular level (Belluscio et al., 1999; Malnic et al., 1999; Rodríguez et al., 1999) and has been seen using a variety of methods in a number of other species [for a review see Halpern et al. (Halpern et al., 1998a,b)]. Similarly, our observation that with UEA-I and LEA the rostral part of the posterior AOB stained differently from the caudal part has a precedent in Sugai et al.’s finding that in rat the Ricinus communis agglutinin RCA120 bound strongly to the anterior AOB NL/GL, weakly to the rostral two-thirds of the posterior AOB GL and not at all to the caudal third of the posterior AOB NL/GL (Sugai et al., 2000). Again, there seem to be between-species differences as regards the identity of the lectin-bindable carbohydrate, since RCA120 is specific for galactose but neither of the galactose-specific lectins used in this study, BSI-B4 and ECA, differentiated between rostral and caudal regions in the posterior AOB. Nevertheless, the binding pattern of the galactose-specific RCA120 in rat is the same as those of the fucose-specific UEA-I and poly(N-acetylglucosamine)-specific LEA in mouse and, following Sugai et al.’s reasoning for rat (Sugai et al., 2000), we suggest that in mouse, too, this pattern probably reflects a functional division of the AOB into three parts (anterior, rostral posterior and caudal posterior) that may correspond to three input/output routes.
On a more technical level, we stress that the three region organization of the AOB described above might well have gone unnoticed if we had not systematically stained and examined whole series of sections in both transverse and sagittal planes [see also Salazar and Sánchez-Quinteiro for other aspects of the use of lectins in histochemistry (Salazar and Sánchez-Quinteiro, 1998)].
In conclusion, the results of this study show that in the region of the mouse brain studied the lectins BSI-B4, DBA and UEA-I stain only the AOB, that six lectins (ECA, DBA, SBA, VVA, UEA-I and LEA) stain the anterior and posterior halves of the AOB with different intensities and that two of these six (UEA-I and LEA) stain the caudal part of the posterior AOB more intensely than the rostral part. The latter two findings suggest that the AOB comprises three distinct regions. The functional significance of this should be a fruitful subject for future research.
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Acknowledgments |
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The authors wish to thank Mr J. Castiñeiras for technical assistance and Mr I. C. Coleman for revising the English text. This work was supported in part by the Spanish Ministry of Education and Culture under project PB-0535.
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Accepted March 6, 2001
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