soybean agglutinin binding to the olfactory systems of the rat and mouse
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Neuroscience Let ters, 69 (1986) 131-136 131 Elsevier Scientific Publishers Ireland Ltd.
SOYBEAN AGGLUTININ BINDING TO THE OLFACTORY SYSTEMS OF THE RAT AND MOUSE
BRIAN KEY* and PIERO P. G1ORGI
Neuroemho,ology Laboratory, School of Anatomy, University o/Queensland, St. Lucia, 4067 Brisbane, Qhl. ( A us t ralia )
(Received April 8th, 1986; Revised version received May 21st, 1986: Accepted May 26th, 1986)
Key words. olfitctory system - lectin b inding- neuronal subclass rodent
The binding of the lectin soybean agglutinin (SBA) to the olfactory system of both the rat and mouse was investigated histochemically. SBA bound to fibers in the accessory olfactory nerve and to glomeruli in the accessory olfactory bulb. In addition, SBA binding sites were present in some, but not all, glomeruli in the ventrolatcral and ventromedial portions of the main olfactory bulb of only the rat. Under standard experimental conditions, SBA did not bind to neurons in other regions of the olfactory system nor to any other neurons in the brain. This selective binding of SBA to only some glomeruli in the olfactory bulb provides additional support for the presence of, at least, two subclasses of olfactory receptor cells in the nasal cavity. Whether these neuronal subclasses are the same as those previously characterized by mono- clonal antibodies in rabbit remains to be determined.
The mammalian olfactory system consists of 3 morphologically distinct olfactory mucosae: the olfactory mucosa proper (OP), the vomeronasal organ (VNO) and the septal organ of Masera [2, 8]. Recently two subclasses of receptor cells have been identified in the OP and VNO of the rabbit on the basis of the molecular heteroge- neity observed using monoclonal antibodies [5, 9, 16]. We also have described a sub- class of olfactory neurons that innervates only the ventral portion of the main olfac- tory bulb (MOB) in Xenopus [18, 19]. These neurons were identified by cell surface glycoconjugates which were selectively labelled by the lectin soybean agglutinin (SBA). In addition, this lectin also labelled the VNO neurons. The ventral/dorsal dif- ference in the MOB was, however, particularly interesting considering a similar dis- tribution of subtypes is observed in the rabbit using monoclonal antibody technol- ogy.
The aim of the present study was to determine whether olfactory neurons, or sub- classes of these cells carry specific SBA binding sites in the rat and mouse. We wish to address the important question of whether SBA binding could identify homolo- gous neurons in the olfactory system of vertebrates.
Four adult rats of the Wistar strain and 4 adult mice of the A 6 strain were anesthe-
*Author lk~r Correspondence.
0304 3040 ~65 03.50 1986 Elsevier Scientilic Publishers Ireland Ltd.
tized with i.p. injections of Nembutal and sacrificed by cardiac perfusion with 4% para- formaldehyde in 0.2 M potassium phosphate buffer, pH 7.2. The brain was removed and serial 40-gm coronal frozen section were cut and allowed to dry on gelatin- coated microscope slides. SBA was purchased from Sigma as a conjugate to horse- radish peroxidase (SBA-HRP). Sections were incubated in SBA-HRP (13 gg/ml in 0.1 M potassium phosphate buffer, pH 7.2, containing 1 mM CaCI2, MnC12, MgCI2) for 40 min. This incubation was lbllowed by several washes in the buffered salts so- lution to remove unbound lectin before sections were reacted for the histochemical demonstration of HRP . Control sections incubated in the presence of 0.2 M N- acetyI-D-galactosamine and SBA-HRP or just HRP alone revealed that SBA--HRP labelling in the tissue was specific for this sugar.
In the mouse, SBA-HRP labelling (dark reaction product) was present in only the accessory olfactory nerve (AON) which courses along the medial surface of the MOB, and was absent from the olfactory nerve fiber layer and glomeruli of the MOB (Fig. la). In the dorso caudal MOB, SBA HRP labelling was present in the accessory olfactory bulb (AOB), the terminal field tbr accessory olfactory axons from the VNO (Fig. lb). Serial sections revealed that this labelling was present in all glomeruli m the AOB. All other neurons in the brain, at least under the present histochemical conditions, failed to bind this lectin. The specific binding of SBA-HRP to olfactory neurons in mice was observed in all animals analysed.
In the rat, in addition to the dense labelling of all glomeruli in the AOB (Fig. IC). SBA HRP lightly labelled a limited number of olfactory glomeruli in the MOB (Fig. I D). The differences in the density of reaction product between glomeruli in the AOB and MOB suggests that there are far less SBA binding molecules or sites on olfactory axons than on accessory olfactory axons. The labelled olfactory glomeruli in the MOB were located in the vemromedial and ventrolateral regions of the rostral bulb. In the caudal MOB, however, only the more ventral glomeruli were labelled. It is important to note that not all glomeruli in these regions were labelled and that unla- belled glomeruli often separated adjacently labelled glomeruli. As for mice, results observed in rats were consistent in all animals analysed.
The SBA HRP labelling in the olfactory systems of Xenopus [18, 19] as well as in mammals (rat and mouse) suggests that the specific binding molecules have impor- tant cellular functions in these neurons. This is particularly so fbr accessory olfactory neurons, since all glomeruli in the AOB of the 3 species, are labelled by SBA-HRP. In the MOB, however, there appears to be some inter-species differences. We have previously shown that all glomeruli in the ventral half of the MOB in Xenopus bind SBA [18, 19] whereas in the rat only some glomeruli in these regions were labelled. Furthermore, in the mouse no MOB glomeruli were labelled. Although the differ- ences in the labelling of the MOB between rat and mouse are most likely species- related differences, we have not excluded the possibility that higher concentrations of SBA-HRP may detect binding sites in some glomeruli of the mouse. Preliminary investigations in Xemq~us and rat [I 8] revealed that the present concentration reduced non-speeilic background labelling and gave optimal results. We are, however, cur- rently investigating the effect of different concentrations of SBA-HRP on labelling patterns in the mouse brain.
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Despite having previously shown SBA to bind to the cell surface of a subclass of receptor cells in Xenopus it is not possible from the present results to identify the cel- lular location of binding sites in the rat and mouse. Also, because SBA binds specifi- cally to the sugar N-acetyl-D-galactosamine  very little can be predicted about thc molecular nature of the binding glycoconjugate. Therefore, whether SBA binds to the same molecule or family of molecules in different species or even to the same molecules within the AOB and MOB of the rat can only be clarified by extracting and characterizing these molecules.
The results of the present study suggest that, in the rat, there are at least two mole- cularly distinct subclasses of olfactory neurons innervating the MOB. Similar results have previously been described in the rat using immunohistochemical procedures [1 ]. A subset of olfactory receptor cells were identified by the monoclonal antibody 2B8. Although the antigen recognized by this antibody was found on all glomeruli in the olfactory bulb some glomeruli c