Enzyme Histochemistry of the Olfactory Mucosa and Vomeronasal Organ in the Mouse

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J. Anat. (1974), 118, 3, pp. 477-489 477 With 16 figures Printed in Great Britain Enzyme histochemistry of the olfactory mucosa and vomeronasal organ in the mouse

ALFRED CUSCHIERI* Department ofAnatomy, Guy's Hospital Medical School, London SEl 9RT (Accepted 4 June 1974)

INTRODUCTION The enzyme histochemistry of the olfactory mucosa has been previously studied in various species of vertebrates (Baradi & Bourne, 1951, 1953; De Amicis & Zorzoli, 1957; Negri, 1957; Barbera, 1959; Bronshtein, 1960, 1965; Shantha & Nakajima, 1970). These studies have shown that the pattern of distribution of enzymes in the olfactory mucosa varies considerably among the different species. The histochemistry of the vomeronasal organ has so far received little attention and only one brief report has yet been published (Bannister & Cuschieri, 1972). The purpose of the present study was to compare the histochemical distribution of a number of enzymes in the olfactory and vomeronasal epithelia of the mouse in view of the morphological and electrophysiological differences between these epithelia which have recently been demonstrated (Tucker, 1963; Altner & Muller, 1968; Bannister, 1968; Altner, Miiller & Brachner, 1970; Graziadei & Tucker, 1970; Kratzling, 1971; Kolnberger, 1971; Muller, 1971).

MATERIALS AND METHODS Adult white mice of strain SAS ICI were used in this investigation. They were killed by cervical dislocation. Small portions of olfactory mucosa attached to underlying bone (ethmoturbinal) and intact vomeronasal organs associated with their bony capsules were excised, placed in self-sealing polythene envelopes and rapidly frozen in a dry ice-acetone mixture at about -70 'C. Sections, 8 ,um thick, were cut on a Bright's cryostat and mounted on clean slides. Sections from at least four different animals were used for each enzyme studied. The following histochemical procedures were employed: Acid phosphatase. Cryostat sections were post-fixed in acetone at 0-4 'C for 15 minutes. Alternatively, cryostat sections of tissues previously fixed in 10% formol calcium for 18 hours at 0-4 'C and washed in gum sucrose for 24 hours at 0-4 'C (Holt & Hicks, 1961) were sometimes used. The incubation procedures employed were: (a) Modified Gomori lead nitrate method for 1 hour at 37 'C (Barka & Anderson, 1962). (b) Naphthol AS-BI and hexazotized pararosanilin method (Lojda, 1962) for 20 minutes at 37 'C. Control sections were incubated in similar media containing 10-4 M sodium fluoride as inhibitor. * Present address: Department of Anatomy, The Royal University of Malta, Msida, Malta. 478 A. C USC HIERI Alkaline phosphatase. Sections were post-fixed in acetone at 0-4 °C for 15 minutes (Burstone, 1962) and incubated according to one of the following methods: (a) Calcium cobalt method (Gomori, 1952) for 75 minutes at 37 'C. (b) Naphthol AS-MX phosphate method (Burstone, 1962) for 15 minutes at room temperature. Control sections were incubated in similar media containing 0-05 M L-phenylalanine as inhibitor (Watanabe & Fishman, 1964). Non-specific esterase. Cryostat sections were post-fixed in acetone at 0-4 'C for 15 minutes and incubated in a medium containing a-naphthyl acetate and hexazotized pararosanilin for 10 minutes at room temperature (Davis & Ornstein, 1959). Control sections were incubated in a substrate-free medium. Succinic dehydrogenase (SDH). Unfixed cryostat sections were incubated according to the nitro-blue tetrazolium method (Nachlas et al. 1957) for 1 hour at 37 'C. Control sections were incubated in a substrate-free medium. Adenosine triphosphatase (ATPase): Ca++ activated. Unfixed cryostat sections were incubated according to the method of Padykula & Hermann (1955) for 1 hour at 37 'C. Control sections were incubated in a similar medium containing 2 5 x 10-3 M p-mercuribenzoate as inhibitor for 15 minutes, followed by further incubation for 45 minutes in inhibitor-free medium. Cytochrome oxidase. Unfixed cryostat sections were incubated according to the p-aminodiphenylamine method (Burstone, 1962) for 90 minutes at room temperature. Control sections were exposed to 10-3 M sodium azide for 15 minutes before incubation. fi-glucuronidase. Cryostat sections were post-fixed in acetone at 0-4 'C for 15 minutes and incubated in a medium containing naphthol AS-BI glucuronic acid and hexazotized pararosanilin (Hayashi, 1965) for 15 minutes at 37 'C. Control sections were incubated in a substrate-free medium.

RESULTS Acidphosphatase In the olfactory epithelium intense acid phosphatase activity was present in the supranuclear cytoplasm of the receptor cell bodies (Figs. 1, 3). The staining reaction was distinctly granular, especially when formalin-fixed tissues were used (Fig. 3). The

Fig. 1. Distribution of acid phosphatase in the olfactory epithelium. Naphthol AS-BI phosphate method. x 130. Fig. 2. Distribution of acid phosphatase in the vomeronasal organ. Naphthol AS-Bi phosphate method. x 130. Fig. 3. Acid phosphatase preparation of the olfactory mucosa showing strong granular reaction in the receptor cell bodies. The receptors in the upper layers show stronger activity than those in the deeper layers. Note moderate activity in the basal cells (b) and in Bowman's glands (g). The olfactory nerve bundles (n) show weak, diffuse activity. Naphthol AS-BI phosphate method. x 280. Fig. 4. Acid phosphatase activity in the vomeronasal sensory epithelium. Note strong granular reaction in the receptor cell bodies. Naphthol AS-BI phosphate method. x 280. The preparations in Figs. 1-4 were photographed in monochromatic light at a wavelength of about 5500 nm. Histochemistry ofolfactory and vomeronasal epithelia 479

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pi. W: _. 4l ,* W4010..'. ,.m .1 f .) V 'L -i &.. 01". 480 A. CUSCHIERI receptor cells in the deeper one-third of the olfactory epithelium contained a much lower enzyme activity than the more superficial receptors. No activity was observed in the supporting cells. A moderate reaction was obtained in the basal cells and in the secretory cells of Bowman's glands close to the luminal surface. The olfactory nerve bundles deep to the epithelium showed mild diffuse activity. The vomeronasal receptor cells showed intense activity similar to that in the olfactory receptors (Figs. 2, 4). However, there was no difference in the degree of enzyme activity between the deeper and the more superficially situated receptors. The vomeronasal nerve bundles showed mild diffuse activity. Minimal activity was present in the non-sensory columnar epithelium of the vomeronasal organ. Similar results were obtained with the two methods used. Control sections showed no reaction product. Alkaline phosphatase In the olfactory epithelium intense alkaline phosphatase activity was present in the basal cells and in the ducts of Bowman's glands (Fig. 5). In some preparations a moderate to strong reaction was also observed in the olfactory surface secretion, but in most areas the surface of the olfactory epithelium showed no activity. Mild activity was also present in the olfactory nerve bundles. No activity was observed in the supporting or receptor cell layers of the olfactory epithelium, nor was there any in the secretory cells of Bowman's glands. In the vomeronasal sensory epithelium (Fig. 6) the distribution of the enzyme was entirely different. Intense activity was present in the uppermost layer of the olfactory epithelium which is occupied by the supporting cell bodies and by the distal parts of the receptor dendrites. Mild activity was also present throughout the deeper layers of the epithelium but it was not possible tc determine whether this was associated with receptor cells or supporting cells or both. No increased activity was observable in the basal layer. The vomeronasal nerves gave a mild reaction. The non-sensory ciliated columnar epithelium of the vomeronasal organ was completely negative. Similar results were obtained with all the methods used. Control sections did not show any reaction product. Non-specific esterase A very high level of esterase activity was present in the superficial zone of the olfactory epithelium. Mild to moderate activity was also present in the deeper layers Fig. 5. Alkaline phosphatase preparation of the olfactory epithelium showing enzyme activity sharply localized to the basal cells (b) and to the ducts (d) of Bowman's glands. The surface of the epithelium is marked by arrows. Gomori's calcium cobalt method. x 130. Fig. 6. Alkaline phosphatase preparation of the vomeronasal organ showing high activity in the superficial layer (s) and mild to moderate activity in the deeper layers of the sensory epithelium. No activity is present in the columnar epithelium (c) of the vomeronasal organ. Gomori's calcium cobalt method. x 100. Fig. 7. Non-specific esterase activity in the olfactory mucosa. The enzyme is localized mainly in the superficial zone (s) of the epithelium, in Bowman's glands (g) and in the cells lining their ducts (d). a-naphthyl acetate method. x 130. Fig. 8. Non-specific esterase activity in the vomerorasal organ showing strong activity in the superficial zone (s) of the sensory epithelium and mild activity in the columnar epithelium. a.-naphthyl acetate method. x 100. Histochemistry of olfactory and vomeronasal epithelia 481 I __

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. ,:. p i . ,. :, . .:...}.sZ~~~~A...'? 482 A. CUSCHIERI of the olfactory epithelium. Bowman's glands and the cells lining their ducts gave a strong reaction but the olfactory nerves were negative (Fig. 7). A similar distribution of the enzyme was observed in the vomeronasal epithelium (Fig. 8). The superficial zone contained high activity which was, however, less intense than in the corresponding part of the olfactory epithelium; the deeper layers showed mild to moderate activity which was localized mainly within the receptor cell bodies. The non-sensory columnar epithelium of the vomeronasal organ demonstrated a moderate reaction in the supranuclear parts of the cells. Control sections incubated in a substrate free medium did not show any staining. Succinic dehydrogenase (SDH) Intense SDH activity was present in the superficial zone of the olfactory epithelium (Fig. 9). The enzyme was located both in the supporting cell bodies and in the distal parts of the receptor dendrites. The olfactory surface secretion also gave a moderate reaction. The deeper layers of the epithelium showed moderate activity, discreetly localized within the receptor cell bodies (Fig. 11). A slightly stronger reaction was given by the basal cells, appearing as a distinct layer at the base of the epithelium. Bowman's glands and the cells lining their ducts at the base of the epithelium showed high activity. The olfactory nerves showed minimal activity. The vomeronasal sensory epithelium contained a high concentration of the enzyme in the superficial zone (Fig. 10). The enzyme was localized mainly in the distal parts of the dendrites (Fig. 12); the supporting cells of the vomeronasal epithelium showed a much lower activity than the supporting cells of the olfactory epithelium. The deeper layers of the vomeronasal sensory epithelium showed moderate activity localized mainly within the receptor cell bodies, but a distinct reaction was not present in the basal layer. The vomeronasal nerves showed weak activity. The non-sensory columnar epithelium of the vomeronasal organ showed intense activity. Control sections incubated in a nitro-blue tetrazolium medium without substrate did not show any staining. Adenosine triphosphatase (ATPase): Ca++ activated In the olfactory epithelium high ATPase activity was present in the supranuclear zones of the supporting cells and in the distal parts of the receptor dendrites (Fig. 13). Moderate activity was present in the deeper layers and appeared to be located within

Fig. 9. Succinic dehydrogenase preparation of the olfactory epithelium showing intense activity in the superficial zone (s) and in Bowman's glands (g). The olfactory nerves (n) show weak activity. Nitro-blue tetrazolium method. x 110. Fig. 10. Succinic dehydrogenase preparation of the vomeronasal organ showing intense activity in the superficial zone of the sensory epithelium and in the non-sensory columnar epithelium (c). Nitro-blue tetrazolium method. x 100. Fig. 11. Succinic dehydrogenase preparation of the olfactory epithelium. Intense activity is present in the superficial zone (s) of the epithelium and in Bowman's glands (g). Activity is also present in the receptor cell bodies (r) and in the basal cells (b). Note mild activity in the olfactory nerves (n) and in the surface fluid (f). Nitro-blue tetrazolium method. x 300. Fig. 12. Succinic dehydrogenase activity in the columnar epithelium (c) and in the sensory epithelium of the vomeronasal organ. Note activity in the receptor cell bodies (r) and in the peripheral processes (p) of the receptor cells. Nitro-blue tetrazolium method. x 250. Histochemistry of olfactory and vomeronasal epithelia 483

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f -^J. - i -e~~~~~~~~~~~~~~~~~~~~~~~k 484 A. CUSCHIERI the receptor cell bodies. The basal cells also gave a moderate reaction, forming a discrete, moderately staining band close to the basement membrane. The olfactory nerve bundles and the blood vessels deep to the olfactory epithelium showed intense activity; Bowman's glands gave only a weak reaction. In the vomeronasal epithelium high activity was present in the supporting cell bodies and in the distal parts of the receptor dendrites (Fig. 14). However, the reaction in the supporting cells was distinctly less than that in the corresponding cells of the olfactory epithelium. Moderate activity was present throughout the rest of the epithelium. Unlike the olfactory epithelium, a distinct reaction was not present in the basal layer. The vomeronasal sensory epithelium contained loops of intra-epithelial capillary blood vessels and these showed intense activity (Fig. 14). The vomeronasal nerve bundles also demonstrated intense activity. The columnar cells of the non-sensory epithelium of the vomeronasal organ showed high activity close to their free surfaces, and weak activity in the basal parts of the cells. The cavernous vascular tissue underlying this epithelium also indicated intense activity. Control sections failed to show any reaction product. Cytochrome oxidase The olfactory epithelium showed moderate cytochrome oxidase activity in all layers (Fig. 15). Slightly higher activity was, however, present in the superficial and basal layers of the epithelium than in the intermediate receptor cell layer. Strong activity was present in the olfactory nerve bundles. Bowman's glands showed weak activity. In the vomeronasal epithelium (Fig. 16) moderate to high activity was present at the surface, and weak activity in the deeper layers. The non-sensory columnar epithelium of the vomeronasal organ showed strong enzyme activity in the supranuclear parts of the cells. f/-glucuronidase This enzyme was investigated to compare'its localization with that of acid phosphatase since both enzymes are said to be chiefly associated with lysosomes (Tappel,

Fig. 13. Ca++ activated ATPase preparation of olfactory mucosa showing strong activity in the superficial zone of the epithelium and moderate activity in the basal cells (b). Intense activity is present in the olfactory nerves (n) and mucosal blood vessels (v). Method of Padykula and Hermann. x 130. Fig. 14. Ca++ activated ATPase preparation of the vomeronasal organ showing activity in the receptor epithelium, particularly in the superficial zone. Intense activity is present in the intra- epithelial capillary vessels (v) and in the vomeronasal nerves (n). Note activity in the non-sensory columnar epithelium (c) and in the underlying cavernous vascular tissue (cv). Method of Padykula and Hermann. x 100. Fig. 15. Cytochrome oxidase preparation of olfactory mucosa showing activity of the enzyme in the superficial layer (s), in the basal cells (b) and in the olfactory nerves (n). p-phenylenediamine method. x 100. Fig. 16. Cytochrome oxidase preparation of the vomeronasal organ showing activity of the enzyme in the superficial zone of the receptor epithelium and in the columnar epithelium. p-phenylenediamine method. x 100. Histochemistry of olfactory and vomeron,,asal epithelia 485

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3I ANA II8 486 A. CUSCHIERI 1969; Barrett, 1969). However, its distribution in the olfactory and vomeronasal epithelia was very different from that of acid phosphatase. In the olfactory epithelium weak diffuse /8-glucuronidase activity was present in the superficial and basal layers and minimal activity was present in the intermediate receptor cell layer. Mild diffuse activity was present throughout the thickness of the vomeronasal epithelium, but the surface showed a slightly higher activity than the deeper layers.

DISCUSSION The present study illustrates some similarities and differences in the histochemical distribution of enzymes in the olfactory and the vomeronasal sensory epithelia. Although the fine structure of the constituent cells of the olfactory and vomeronasal epithelia has been extensively studied, the functions of the supporting and the basal cells and the mechanisms underlying olfactory perception and discrimination are still subjects of speculation. The association of certain enzymes with particular cell types may help to elucidate at least some of the metabolic activities of these cells. Likewise, variations in the histochemical distribution of enzymes within the olfactory and vomeronasal epithelia may reflect differences in the metabolic and functional activities of the two epithelia. However, interpretation of the functional significance of histochemically demonstrated enzymes must proceed with caution because of the known limitations of histochemical methods (Pearse, 1968). Furthermore, the incon- sistencies in the distribution of enzymes within the olfactory epithelium of different species introduce additional difficulties in assessing particular functions and cast grave doubts as to whether certain enzymes are as specifically important in olfactory perception and discrimination as they have been claimed to be (Baradi & Bourne, 1951, 1953; Negri, 1957). The results reported in this paper show that the olfactory and vomeronasal receptors in the mouse both contain the enzymes acid phosphatase, adenosine triphosphatase, succinic dehydrogenase and cytochrome oxidase. Of these, acid phosphatase is present in particularly high concentration, suggesting that this enzyme plays an important role in the metabolism of the receptor cells. The distinctly granular appearance of the acid phosphatase reaction within the receptors suggests furthermore that the enzyme is associated with lysosomes (Fishman, Hiroyuki & Rufo, 1969). These are known to be abundant in the supranuclear cytoplasm of the olfactory and vomeronasal receptor cell bodies (Frisch, 1967; Bannister & Cuschieri, 1972). If these results do indeed indicate a high autophagic activity in olfactory and vomeronasal receptors, then, taking into account the probability that the terminal knobs of the olfactory receptors, and particularly the receptor membrane, are con- stantly exposed to damage, while vomeronasal receptor cells are not replaceable (Moulton, Celebri & Fink, 1970), it is clear that mechanisms ought to be available for lysosomal destruction of aged or damaged organelles and subsequent cell repair. However, it is surprising that /8-glucuronidase and non-specific esterase were not found in significant amounts in the olfactory and vomeronasal receptors because, in many cells, these enzymes and acid phosphatase have a similar lysosomal distribution (Barrett, 1969; Tappel, 1969). The presence of the enzymes ATPase, SDH, cytochrome oxidase and non-specific Histochemistry of olfactory and vomeronasal epithelia 487 esterase in all the cells of the olfactory and vomeronasal epithelia in particularly high concentration in the supranuclear region of the supporting cells points to very high metabolic activity here. The fine structure of the supporting cells support this (Frisch, 1967; Cuschieri, 1972) for the supranuclear region in these cells contains a great abundance of mitochondria associated with profiles of smooth endoplasmic reticulum. High concentrations of oxidative enzymes have also been reported in the supporting cells of the rhesus monkey olfactory mucosa (Shantha & Nakajima, 1970). However, just the opposite was reported by Bronshtein (1965). In the vomeronasal epithelium the supporting cell bodies also showed a very high activity of the enzymes ATPase, SDH, cytochrome oxidase and esterase but never- theless distinctly less than in the corresponding cells of the olfactory epithelium. Mitochondria are also less numerous. In the present study the most striking histochemical difference between the olfactory and vomeronasal sensory epithelia was in the distribution of alkaline phosphatase. In the olfactory epithelium this enzyme was present in high concentration in the basal cells, but in the vomeronasal epithelium this was not so. The presence of alkaline phosphatase in the basal cells of the olfactory epithelium has been previously demonstrated in the rat and the rabbit (Baradi & Bourne, 1953), in the cat (Negri, 1957) and in the rhesus monkey (Shantha & Nakajima, 1970) but not in man (Baradi & Bourne, 1953). Possibly the enzyme is concemed with active transport of molecules across the cell membrane (Bradfield, 1950; Danielli, 1954). The absence of discrete alkaline phosphatase activity in the basal layer of the vomeronasal epithelium is not surprising since true basal cells are not present in the vomeronasal epithelium of the mouse (Cuschieri, 1972). Likewise the basal layer of the vomeronasal epithelium fails to show adenosine triphosphatase, succinic dehydrogenase and cytochrome oxidase. The vomeronasal epithelium however is well supplied with intra-epithelial capillary vessels, and so active transport of metabolites by basal cells may not be required. On the other hand, unlike the olfactory epithelium, the vomeronasal epithelium demonstrates high alkaline phosphatase activity in the surface layer and in the receptor layer. Here the enzyme appears to be associated with the supporting cells and possibly also with the distal parts of the receptor dendrites, though it is difficult to be sure of this.

SUMMARY The histochemical distributions of the enzymes acid phosphatase, alkaline phosphatase, adenosine triphosphatase, esterase, succinic dehydrogenase, cytochrome oxidase and ,8-glucuronidase in the olfactory and vomeronasal epithelia were compared in the mouse. Intense acid phosphatase activity was present in the receptor cell bodies of both epithelia, suggesting high autophagic activity. However, ,/-glucuronidase was not present in the receptor cells. Adenosine triphosphatase, succinic dehydrogenase, cytochrome oxidase and non-specific esterase all showed very high activity in the supporting cells of the olfactory epithelium and slightly lower activity in the supporting cells of the vomeronasal epithelium. These enzymes also exhibited mild activity in the receptor cell layer of both epithelia and moderate activity in the basal cells of the olfactory epithelium only. 3I-2 488 A. CUSCHIERI Alkaline phosphatase activity was present in the basal cells of the olfactory epithelium, suggesting the possibility that this enzyme might be concerned with active transport processes across the base of the epithelium. Alkaline phosphatase was also present in the ducts of Bowman's glands. In contrast, the vomeronasal epithelium showed high alkaline phosphatase activity in the supporting cells and mild activity in the receptor cell layer, but none in the basal layer. I am grateful to Dr L. H. Bannister for his help and invaluable advice during the course of this work and to Dr K. L. Richardson for reading and criticizing the manuscript.

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