0361-9230/92 $5.00 + .OO
Brain Research Bulletin,Vol. 28, pp. 785-788, 1992 Printed in the USA. All rights reserved.
Copyright0 1992Pergamon Press Ltd.
BRIEF COMMUNICATION
Species Differences in Amphibian Olfactory Neuron Reactivity to a Monoclonal Antibody MARIA J. CROWE’ AND SARAH K. PIXLEY Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267 Received 8 October 199 1 CROWE, M. J. AND S. K. PIXLEY. Species differences in amphibian olfactory neuron reactivity to a monoclonal antibody. BRAIN RES BULL zS(5) 785-788, 1992.-A monoclonal antibody immunostained a subpopulation of olfactory sensory neurons in cryostat sections of the olfactory mucosa of the grass frog, Rana pipiens, and the bullfrog, Rana catesbeiana. However, in the olfactory tissues of the African clawed frog, Xenopus laevis, only mucus and mucus-secreting components were stained, and no cell-specific immunoreactivity was seen in the tiger salamander, Ambystoma tigrinum. This antibody is a useful marker of olfactory neuronal subpopulations in some amphibians and illustrates the difficulties in cross-species immunocytochemistry. Neuronal subpopulations Salamander Xenopus
Camosine synthetase
Olfactory sensory neurons
NUMEROUS research groups have searched for molecules, such
Immunocytochemistry
Frog
dent olfactory neurons [reviewed in (20)], did not stain amphibian tissues (data not shown). Our attempts to use antibodies to neuron-specific molecules such as N-CAM, neurofilaments, neuron-specific enolase, and the 2B8 antigen (1) were also unsuccessful on amphibian olfactory tissues (unpublished observations). One of the next candidates for an olfactory neuronspecific molecule we pursued was the dipeptide carnosine. Carnosine is enriched in the olfactory tissues of every species examined (19), although it is also widely distributed throughout muscle and nervous system tissues [reviewed in (6,19)]. The function of camosine remains obscure although it has been hypothesized to serve as a neurotransmitter or neuromodulator in olfactory neurons (4,8,9,13,18). Rather than use antibodies to carnosine itself, we looked at antibodies to its biosynthetic enzyme, camosine synthetase (CS). The properties and localization of CS have been determined at the regional and subcellular level in frog (nonolfactory tissues), rat, mouse, and chick (11,12,14,22,23,25,26). CS has been found in all tissues known to contain camosine, although some tissues have demonstrated lower CS concentrations than expected relative to the amount of camosine in the tissue (6,ll). Our previous studies (5) showed that a monoclonal antibody (x-CSE), generated against rabbit muscle camosine synthetase (2 l), identified olfactory sensory neurons in the grass frog Rana pipiens. Of particular interest was the result that staining was of a distinct subpopulation of neurons. This was unexpected because camosine-like immunoreactivity has been shown to be
as antibodies or lectins, that will bind specifically to subpopulations of vertebrate olfactory sensory neurons. Such reagents are needed to characterize olfactory transduction mechanisms, particularly those involved in odor discrimination. The vertebrate olfactory system responds to an enormous number of distinct odorants [reviewed in (17)], suggesting the presence of multiple receptor proteins. Supporting this is the recent descrip tion of a multigene family of proteins that may act as odorant receptors (3). What is still unknown is whether any one sensory neuron expresses some or all of these putative receptor molecules. Electrophysiological data have shown that individual sensory neurons are differentially responsive to distinct odorous stimuli (7). Therefore, it is quite likely that subpopulations of sensory neurons exist, each expressing one or a small group of receptor molecules. An additional need for identifying such neuronal subgroups is in the study of neurogenesis, which occurs in the vertebrate olfactory epithelium through adulthood (10). Most other neuronal populations are incapable of neurogenesis beyond the early postnatal stage. Identification and manipulation of neurons in different stages of maturation would allow further study of this phenomenon. There are far fewer antibody and lectin markers for amphibian neurons ( 15,16) than rodent neurons. In a broad search for neuronal markers of any specificity, we screened several antibodies that recognize mammalian olfactory neurons. Antibodies to olfactory marker protein (OMP), a specific marker of mature ro_
’ Requests for reprints should be. addressed to Maria J. Crowe, Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, ML 52 1, Cincinnati, OH 45267-0521.
785
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present in all rodent olfactory neurons (2,24). Presumably, all amphibian olfactory neurons might similarly employ the same putative neurotransmitter. Because of the potential usefulness of subgroup-specific markers of olfactory sensory neurons, as outlined above, we present here extensions of this study to amphibians other than the grass frog. We include tissues from the bullfrog, Rana catesbeiana, the African clawed frog, Xenopus laevis, and the tiger salamander, Ambystoma tigrinum. We also present here some further characterization of the specificity of the antibody and speculation on the molecular identity of the binding site. METHOD
R. catesbeiana and A. tigrinum were purchased from Charles D. Sullivan Co. (Nashville, TN). X. laevis were obtained from Nasco Co. (Fort Atkinson, WI). The procedures used to process and collect tissue sections from animals were the same as reported earlier (5). x-CSE was a generous gift of Dr. Frank L. Margolis (Roche Institute of Molecular Biology, Nutley, NJ). The preparation of this antibody has been described previously (2 1). Tissue sections were preincubated in 4% fetal calf serum (Sigma Chemical Co., St. Louis, MO) in an isotris buffer (0.14 sodium chloride, 0.0 1 M Tris, and 0.5% bovine serum albumin) for 20 min followed by incubation with either x-CSE culture supematant at a 1: 125 dilution in 0.2% t&on/O. 1 M PBS for 90 min or x-CSE ascites fluid at a 15000 dilution in 0.2% triton/ 0.1 M PBS for 4 h. For fluorescence microscopy, sections were treated as described previously (5).
utilizing normal mouse serum in place of the primary antibody demonstrated a complete lack of immunoreactivity to either the mucociliary layer or the Bowman’s glands (data not shown). In A. tigrinum, we saw no immunoreactivity with x-CSE in any portion of the olfactory mucosa in comparison to background controls that used normal mouse serum or buffer alone in place of the primary antibody (Fig. 1f). No staining was seen in any olfactory neurons, respiratory epithelial cells, or mucussecreting cells. Staining was seen in Bowman’s glands, but the specificity could not be evaluated because these glands were autofluorescent, that is, they were similarly labeled in all control sections (Fig. 1f). x-CSE was generated against mammalian CS and has been used to extract material with CS activity from tissue homogenates of mammalian olfactory epithelia and bulb (21). However, xCSE has shown no immunocytochemical staining of rodent olfactory neurons in other laboratories (2). In our hands, x-CSE immunoreactivity in rat olfactory tissue sections was not above background (data not shown). Rat muscle, heart, liver, and brain tissues also contain CS (6,14,19,2 1). We stained these rat tissues with x-CSE and obtained the same result as seen in the olfactory tissues. No study has examined CS activity in amphibian olfactory tissues, but CS has been detected in heart, muscle, liver, and brain tissues of R. pipiens (22). To determine x-CSE reactivity in tissues that should contain CS, we immunostained heart, muscle (sartorius), liver, and brain tissues of R. pipiens in parallel with olfactory tissues. The reactivity in the olfactory epithelium was as shown in Fig. Id, but no reactivity was observed in any of the nonolfactory tissues tested (not shown).
RESULTS
x-CSE in R. catesbeiana immunostained a subpopulation of what morphologically appeared to be olfactory sensory neurons (Fig. la). The labeled cells demonstrated the dendrite- and axonlike processes expected of olfactory sensory neurons and were similar to those described in the previous study utilizing R. pipiens (5). Perikarya of stained cells in R. catesbeiana were positioned throughout the layer of epithelium that contains the neuronal cell bodies, although the majority were found in the middle third of this area. Nerve fibers in the lamina propria were stained. A lower percentage of cells was labeled in R. catesbeiana olfactory epithelium than was seen previously in R. pipiens (compare Figs. lc and Id), but no quantitative analysis on this observation has been done. Consistent with what was seen in R. pipiens (5), no x-CSE staining was observed in other cell types in R. catesbeiana olfactory mucosa, including sustentacular, basal, and lamina propria cells. However, a subset of cells in the respiratory mucosa of R. catesbeiana were positively stained with x-CSE (Fig. 1b). These appeared to be goblet cells, based on morphology. The glands located beneath the respiratory epithelium were unstained. No staining was observed within R. pipiens respiratory epithelium. x-CSE immunostaining in olfactory mucosa from X. laevis presented a distinctly different pattern. The “mucociliary complex” at the luminal surface of the epithelium consists of the mucus layer and the mucus-embedded cilia of the olfactory sensory neurons and microvilli of the supporting ceils. This complex was intensely immunolabeled by x-CSE (Fig. le). Also positive were the mucus-secreting Bowman’s glands and their ducts within the olfactory epithelium (Fig. le). No labeling was seen in sensory neurons or sustentacular cells. The absence of staining in these cells was unambiguous in “exposed” areas of the epithelium, where the mucociliary layer had been washed off. No labeling was seen in the respiratory mucosa. Control sections
DISCUSSION
These data show distinct immunostaining differences in the tissues lining the nasal cavities of three species of amphibians using the monoclonal antibody x-CSE. Olfactory neuronal staining with x-CSE was only seen in R. pipiens and R. catesbeiana, and this staining was restricted to a subset of neurons (Figs. 1a, c, and d). As in R. pipiens (5), staining in R. catesbeiana did not appear to be due to loss of neurons during tissue processing or incomplete penetration of the antibody. Also, because neurons were stained throughout the thickness of the neuronal layer of the epithelium, the subset pattern does not appear to be due to differential developmental expression of the antigen. Previous studies have shown that position of the olfactory neuron cell body within the epithelial layer reflects the relative age of the neuron (10). The neuronal staining seen with x-CSE is unique and useful because, as stated previously, there are few other markers for amphibian olfactory sensory neurons ( 15,16) and none that label neuronal subpopulations within the olfactory epithelium. x-CSE immunoreactivity was not present in the rodent. It is possible that the fixative conditions were not optimal. Another researcher, using the same antibody in rodents, previously tested several fixatives, with very inconclusive results (Dr. A. Farbman, personal communication). x-CSE immunoreactivity was not observed in any of the R. pipiens nonolfactory tissues tested. The difficulty in interpreting these results is that the biochemical studies (22,26) did not include direct comparisons between the CS activities of frog olfactory and nonolfactory tissues. In mammals (2 l), the olfactory tissues have routinely shown about tenfold higher concentrations of CS than nonolfactory tissues. Our results with x-CSE could be explained by either 1) binding to a non-CS antigen or 2) lower amounts of CS in the nonolfactory tissues that were un-
SPECIES
DIFFERENCES
IN AMPHIBIANS
FlG. 1. Coronal cryostat sections through the amphibian olfactory sac. (a) R. cufe.sbeiana olfactory epithelium demonstrating two brightly immuno~a~ve cell bodies and processes (arrowhead). Only one dendritic. knob is labeled in this section. (b) R. c~es~e~~ff respiratory epi~elium labeled with xCSE. Note the concentration of label in goblet-shaped cells located close to the luminal surface of the epithelium (arrowheads). (c, d) Comparison between R. cutesbeiana and R. p&ens olfactory epithelium to show that the relative number of sensory neurons and dendritic knobs that demonstrate x-CSE immunoreactivity differ between these frog species. Scale bar in d = 5 pm for a, b, c, and d. (e) X. luevis olfactory epithelium immunolabeled with x-CSE. Arrowheads point to strongly labeled Bowman’s glands located underneath the epithelium. Labeled ducts pass through the epithelium. Note the highly immunoreactive luminal surface ofthe epitbelium. Scale bar = IO fim. (f) A. tigrinurn olfactory epi~elium immunola&l~ with x-CSE. No obvious immuno~~ti~ty was seen in this amphibian. Lamina propria mucosal gland staining (arrowheads) was nonspecific since it was seen in control sections containing no primary antibody. Scale bar = 10 pm.
787
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detectable in these assays. Thus, at present, we have no positive staining control to aid identification of the antigen. Previous attempts to use this particular antibody in Western blots had been unsuccessful (F. L. Margolis, personal communication) so further analysis would most likely require immunoprecipitation. One observation that suggests that x-CSE is binding to CS was the fact that tissues from A. tigrinum were negative while tissues from frog (R. catesbeiana and R. pipiens) were positive. Carnosine was undetectable in biochemical analysis of the olfactory mucosa (F. L. Margolis, personal communication), olfactory bulb ( 19) or brain (19) of A. tigrinum, while in the same analysis camosine was detectable in frog (R. catesbeiana) olfactory mucosa and brain (F. L. Margolis, personal communication). Therefore, in these amphibians the immunochemical findings are consistent with the biochemical presence or absence of carnosine. More numerous observations suggest the x-CSE binding site was not the CS molecule in amphibians. First, as discussed earlier, x-CSE staining in the frog was in a subpopulation of olfactory sensory neurons. Second, staining of mucus and mucus-secreting cells or glands was seen in X. laevis (Fig. le) and R. catesbeiana (Fig. lb). No reports have described CS or camosine in respiratory tissues, Bowman’s glands, or other glandular tissues. Finally, the x-CSE immunoreactivity pattern in individual olfac-
AND
PIXLEY
tory neurons was suggestive of membrane-associated binding, as discussed previously (5). CS is a soluble, cytoplasmic protein ( 12,23) that should not be membrane associated. Despite the lack of progress in identification ofthe molecule(s) that x-CSE recognizes, this monoclonal antibody is a useful marker of subsets of olfactory sensory neurons in two amphibian species. The general lack of neuronal markers in amphibian species and the robust staining observed with this antibody make it a reagent of potential use in functional studies involving electrophysiological experiments on dispersed cells and in studies comparing x-CSE distribution with that of molecular probes for olfactory receptor molecules. It is hoped that such studies would lead to an understanding of the functional differences of the xCSE-positive neuronal subpopulations, in particular, the possibility that they are associated with detection of a specific odorant or group of odorants. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the generous support of Dr. Robert C. Gesteland and thank Dr. Frank L. Margolis of the Roche Institute of Molecular Biology for use of the x-CSE antibody and critical advice in the preparation of the manuscript. This study was supported by National Institutes of Health Grants DC00347 and DC00352 awarded to Dr. Gesteland.
REFERENCES 1. Allen, W. K.; Akeson, R. Identification ofa cell surface glycoprotein family on olfactory receptor neurons with a monoclonal antibody. J. Neurosci. 5:284-296; 1985. 2. Biffo, S.; GrilIo, M.; Margolis, F. L. Cellular localization of camosinelike and anserine-like immunoreactivities in rodent and avian central nervous system. Neuroscience 35:637-65 I; 1990. 3. Buck, L.; Axel, R. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65: I75- 187; 1991. 4. Burd, G. D.; Davis, B. J.; Macrides, F.; Grille, M.; Margolis, F. L. Camosine in primary afferents of the olfactory system: An autoradioaranhic and biochemical studv. J. Neurosci. 2:244-255: 1982. 5. Crowe, M. J.; Pixley, S. K. Mon&onal antibody to camosine synthetase identifies a subpopulation of frog olfactory receptor neurons. Brain Res. 538:147-151; 1991. 6. Crush, K. G. Camosine and related substances in animal tissues. Comp. B&hem. Physiol. 34:3-30; 1970. 7. Duchamp, A.; Revial, M. F.; Holley, A.; MacLeod, P. Odor discrimination by frog olfactory receptors. Chem. Senses 1:213-233; 1974. 8. Ferriero, D.; Margolis, F. L. Denervation in the primary olfactory pathway of mice. II: Effects on camosine and other amine comnounds. Brain Res. 94:75-86; 1975. 9. Gonzalez-Estrada, M. T.; Freeman, W. J. Effects of camosine on olfactorv bulb EEG. evoked notentials and DC potentials. Brain Res. 202:373-386; 1980. . IO. Graziadei, P. P. C. Cell dynamics in the olfactory mucosa. Tissue Cell 5:113-131; 1973. Il. Harding, J.; Margolis, F. L. Denervation in the primary olfactory pathway of mice. III: Effect on enzymes of camosine metabolism. Brain Res. 1l&351-360; 1976. 12. Harding, J.; O’FalIon, J. V. The subcellular distribution of camosine, carnosine synthetase, and camosinase in mouse olfactory bulbs. Brain Res. 173:99-109; 1979. 13. Hirsch, J. D.; Grille, M.; Margolis, F. L. Ligand binding studies in the mouse olfactory bulb: Identification and characterization of an L-[3H]camosine binding site. Brain Res. 158:407-422; 1978.
14. Horinishi, H.; Grille, M.; Margolis, F. L. Purification and characterization of camosine synthetase from mouse olfactory bulbs. J. Neurochem. 31:909-919; 1978. markers for the 15 Key, B.; Akeson, R. A. Immunochemical frog olfactory neuroepithelium. Dev. Brain Res. 57: 103- 117: 1990. . 16 Key, B.; Giorgi, P. P. Selective binding of soybean agglutinin to the olfactory system of Xenopus. Neuroscience 18:507-5 15: 1986. 17. Lance& D. Vertebrate olfactory reception. Annu. Rev. Neurosci. 9: 329-355; 1986. 18. Margolis, F. L. Camosine in the primary olfactory pathway. Science 184:909-911; 1974. 19. Margolis, F. L. Camosine: An olfactory neuropeptide. In: Barker, J. L.; Smith, T. G., Jr., eds. The role of peptides in neuronal function. New York: Marcel Dekker; 1980~545-572. 20. Margolis, F. L. Molecular cloning of olfactory-specific gene products. In: Margolis, F. L.; Getchell, T. V., eds. Molecular neurobiology of the olfactory system. New York: Plenum Press; 1988:237-26 1. 21. Margolis, F. L.; Griho, M.; Hempstead, J.; Morgan, J. I. Monoclonal antibodies to mammalian camosine svnthetase. J. Neurochem. 48: 593-600; 1987. 22. Ng, R. H.; Marshall, F. D. Distribution of homocamosine-carnosine svnthetase in tissues of rat. mouse, chick and frog. Comp. Biochem. Physiol. 54:519-521; 1976. 23. Ng, R. H.; Marshall, F. D. Regional and subcellular distribution of homocamosine-camosine synthetase in the central nervous system of rats. J. Neurochem. 30:187-190, 1978. 24. Sakai, M.; Yoshida, M.; Karasawa, N.; Teramura, M.; Ueda, H.; Nagatsu, I. Camosine-like immunoreactivity in the primary olfactory neuron of the rat. Experientia 43:298-300; 1987. 25. Skaper, S. D.; Das, S.; Marshall, F. D. Some properties of a homocamosine-camosine synthetase isolated from rat brain. J. Neurothem. 21:1429-1445; 1973. 26. Yockey, W. C.; Marshall, F. D. Incorporation of [‘4C]-histidine into homocamosine and carnosine of frog brain in vivo and in vitro. Biochem. J. 114:585-588; 1969.
Brain Research Bulletin,Vol. 28, pp. 785-788, 1992 Printed in the USA. All rights reserved.
Copyright0 1992Pergamon Press Ltd.
BRIEF COMMUNICATION
Species Differences in Amphibian Olfactory Neuron Reactivity to a Monoclonal Antibody MARIA J. CROWE’ AND SARAH K. PIXLEY Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267 Received 8 October 199 1 CROWE, M. J. AND S. K. PIXLEY. Species differences in amphibian olfactory neuron reactivity to a monoclonal antibody. BRAIN RES BULL zS(5) 785-788, 1992.-A monoclonal antibody immunostained a subpopulation of olfactory sensory neurons in cryostat sections of the olfactory mucosa of the grass frog, Rana pipiens, and the bullfrog, Rana catesbeiana. However, in the olfactory tissues of the African clawed frog, Xenopus laevis, only mucus and mucus-secreting components were stained, and no cell-specific immunoreactivity was seen in the tiger salamander, Ambystoma tigrinum. This antibody is a useful marker of olfactory neuronal subpopulations in some amphibians and illustrates the difficulties in cross-species immunocytochemistry. Neuronal subpopulations Salamander Xenopus
Camosine synthetase
Olfactory sensory neurons
NUMEROUS research groups have searched for molecules, such
Immunocytochemistry
Frog
dent olfactory neurons [reviewed in (20)], did not stain amphibian tissues (data not shown). Our attempts to use antibodies to neuron-specific molecules such as N-CAM, neurofilaments, neuron-specific enolase, and the 2B8 antigen (1) were also unsuccessful on amphibian olfactory tissues (unpublished observations). One of the next candidates for an olfactory neuronspecific molecule we pursued was the dipeptide carnosine. Carnosine is enriched in the olfactory tissues of every species examined (19), although it is also widely distributed throughout muscle and nervous system tissues [reviewed in (6,19)]. The function of camosine remains obscure although it has been hypothesized to serve as a neurotransmitter or neuromodulator in olfactory neurons (4,8,9,13,18). Rather than use antibodies to carnosine itself, we looked at antibodies to its biosynthetic enzyme, camosine synthetase (CS). The properties and localization of CS have been determined at the regional and subcellular level in frog (nonolfactory tissues), rat, mouse, and chick (11,12,14,22,23,25,26). CS has been found in all tissues known to contain camosine, although some tissues have demonstrated lower CS concentrations than expected relative to the amount of camosine in the tissue (6,ll). Our previous studies (5) showed that a monoclonal antibody (x-CSE), generated against rabbit muscle camosine synthetase (2 l), identified olfactory sensory neurons in the grass frog Rana pipiens. Of particular interest was the result that staining was of a distinct subpopulation of neurons. This was unexpected because camosine-like immunoreactivity has been shown to be
as antibodies or lectins, that will bind specifically to subpopulations of vertebrate olfactory sensory neurons. Such reagents are needed to characterize olfactory transduction mechanisms, particularly those involved in odor discrimination. The vertebrate olfactory system responds to an enormous number of distinct odorants [reviewed in (17)], suggesting the presence of multiple receptor proteins. Supporting this is the recent descrip tion of a multigene family of proteins that may act as odorant receptors (3). What is still unknown is whether any one sensory neuron expresses some or all of these putative receptor molecules. Electrophysiological data have shown that individual sensory neurons are differentially responsive to distinct odorous stimuli (7). Therefore, it is quite likely that subpopulations of sensory neurons exist, each expressing one or a small group of receptor molecules. An additional need for identifying such neuronal subgroups is in the study of neurogenesis, which occurs in the vertebrate olfactory epithelium through adulthood (10). Most other neuronal populations are incapable of neurogenesis beyond the early postnatal stage. Identification and manipulation of neurons in different stages of maturation would allow further study of this phenomenon. There are far fewer antibody and lectin markers for amphibian neurons ( 15,16) than rodent neurons. In a broad search for neuronal markers of any specificity, we screened several antibodies that recognize mammalian olfactory neurons. Antibodies to olfactory marker protein (OMP), a specific marker of mature ro_
’ Requests for reprints should be. addressed to Maria J. Crowe, Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, ML 52 1, Cincinnati, OH 45267-0521.
785
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CROWE AND PIXLEY
present in all rodent olfactory neurons (2,24). Presumably, all amphibian olfactory neurons might similarly employ the same putative neurotransmitter. Because of the potential usefulness of subgroup-specific markers of olfactory sensory neurons, as outlined above, we present here extensions of this study to amphibians other than the grass frog. We include tissues from the bullfrog, Rana catesbeiana, the African clawed frog, Xenopus laevis, and the tiger salamander, Ambystoma tigrinum. We also present here some further characterization of the specificity of the antibody and speculation on the molecular identity of the binding site. METHOD
R. catesbeiana and A. tigrinum were purchased from Charles D. Sullivan Co. (Nashville, TN). X. laevis were obtained from Nasco Co. (Fort Atkinson, WI). The procedures used to process and collect tissue sections from animals were the same as reported earlier (5). x-CSE was a generous gift of Dr. Frank L. Margolis (Roche Institute of Molecular Biology, Nutley, NJ). The preparation of this antibody has been described previously (2 1). Tissue sections were preincubated in 4% fetal calf serum (Sigma Chemical Co., St. Louis, MO) in an isotris buffer (0.14 sodium chloride, 0.0 1 M Tris, and 0.5% bovine serum albumin) for 20 min followed by incubation with either x-CSE culture supematant at a 1: 125 dilution in 0.2% t&on/O. 1 M PBS for 90 min or x-CSE ascites fluid at a 15000 dilution in 0.2% triton/ 0.1 M PBS for 4 h. For fluorescence microscopy, sections were treated as described previously (5).
utilizing normal mouse serum in place of the primary antibody demonstrated a complete lack of immunoreactivity to either the mucociliary layer or the Bowman’s glands (data not shown). In A. tigrinum, we saw no immunoreactivity with x-CSE in any portion of the olfactory mucosa in comparison to background controls that used normal mouse serum or buffer alone in place of the primary antibody (Fig. 1f). No staining was seen in any olfactory neurons, respiratory epithelial cells, or mucussecreting cells. Staining was seen in Bowman’s glands, but the specificity could not be evaluated because these glands were autofluorescent, that is, they were similarly labeled in all control sections (Fig. 1f). x-CSE was generated against mammalian CS and has been used to extract material with CS activity from tissue homogenates of mammalian olfactory epithelia and bulb (21). However, xCSE has shown no immunocytochemical staining of rodent olfactory neurons in other laboratories (2). In our hands, x-CSE immunoreactivity in rat olfactory tissue sections was not above background (data not shown). Rat muscle, heart, liver, and brain tissues also contain CS (6,14,19,2 1). We stained these rat tissues with x-CSE and obtained the same result as seen in the olfactory tissues. No study has examined CS activity in amphibian olfactory tissues, but CS has been detected in heart, muscle, liver, and brain tissues of R. pipiens (22). To determine x-CSE reactivity in tissues that should contain CS, we immunostained heart, muscle (sartorius), liver, and brain tissues of R. pipiens in parallel with olfactory tissues. The reactivity in the olfactory epithelium was as shown in Fig. Id, but no reactivity was observed in any of the nonolfactory tissues tested (not shown).
RESULTS
x-CSE in R. catesbeiana immunostained a subpopulation of what morphologically appeared to be olfactory sensory neurons (Fig. la). The labeled cells demonstrated the dendrite- and axonlike processes expected of olfactory sensory neurons and were similar to those described in the previous study utilizing R. pipiens (5). Perikarya of stained cells in R. catesbeiana were positioned throughout the layer of epithelium that contains the neuronal cell bodies, although the majority were found in the middle third of this area. Nerve fibers in the lamina propria were stained. A lower percentage of cells was labeled in R. catesbeiana olfactory epithelium than was seen previously in R. pipiens (compare Figs. lc and Id), but no quantitative analysis on this observation has been done. Consistent with what was seen in R. pipiens (5), no x-CSE staining was observed in other cell types in R. catesbeiana olfactory mucosa, including sustentacular, basal, and lamina propria cells. However, a subset of cells in the respiratory mucosa of R. catesbeiana were positively stained with x-CSE (Fig. 1b). These appeared to be goblet cells, based on morphology. The glands located beneath the respiratory epithelium were unstained. No staining was observed within R. pipiens respiratory epithelium. x-CSE immunostaining in olfactory mucosa from X. laevis presented a distinctly different pattern. The “mucociliary complex” at the luminal surface of the epithelium consists of the mucus layer and the mucus-embedded cilia of the olfactory sensory neurons and microvilli of the supporting ceils. This complex was intensely immunolabeled by x-CSE (Fig. le). Also positive were the mucus-secreting Bowman’s glands and their ducts within the olfactory epithelium (Fig. le). No labeling was seen in sensory neurons or sustentacular cells. The absence of staining in these cells was unambiguous in “exposed” areas of the epithelium, where the mucociliary layer had been washed off. No labeling was seen in the respiratory mucosa. Control sections
DISCUSSION
These data show distinct immunostaining differences in the tissues lining the nasal cavities of three species of amphibians using the monoclonal antibody x-CSE. Olfactory neuronal staining with x-CSE was only seen in R. pipiens and R. catesbeiana, and this staining was restricted to a subset of neurons (Figs. 1a, c, and d). As in R. pipiens (5), staining in R. catesbeiana did not appear to be due to loss of neurons during tissue processing or incomplete penetration of the antibody. Also, because neurons were stained throughout the thickness of the neuronal layer of the epithelium, the subset pattern does not appear to be due to differential developmental expression of the antigen. Previous studies have shown that position of the olfactory neuron cell body within the epithelial layer reflects the relative age of the neuron (10). The neuronal staining seen with x-CSE is unique and useful because, as stated previously, there are few other markers for amphibian olfactory sensory neurons ( 15,16) and none that label neuronal subpopulations within the olfactory epithelium. x-CSE immunoreactivity was not present in the rodent. It is possible that the fixative conditions were not optimal. Another researcher, using the same antibody in rodents, previously tested several fixatives, with very inconclusive results (Dr. A. Farbman, personal communication). x-CSE immunoreactivity was not observed in any of the R. pipiens nonolfactory tissues tested. The difficulty in interpreting these results is that the biochemical studies (22,26) did not include direct comparisons between the CS activities of frog olfactory and nonolfactory tissues. In mammals (2 l), the olfactory tissues have routinely shown about tenfold higher concentrations of CS than nonolfactory tissues. Our results with x-CSE could be explained by either 1) binding to a non-CS antigen or 2) lower amounts of CS in the nonolfactory tissues that were un-
SPECIES
DIFFERENCES
IN AMPHIBIANS
FlG. 1. Coronal cryostat sections through the amphibian olfactory sac. (a) R. cufe.sbeiana olfactory epithelium demonstrating two brightly immuno~a~ve cell bodies and processes (arrowhead). Only one dendritic. knob is labeled in this section. (b) R. c~es~e~~ff respiratory epi~elium labeled with xCSE. Note the concentration of label in goblet-shaped cells located close to the luminal surface of the epithelium (arrowheads). (c, d) Comparison between R. cutesbeiana and R. p&ens olfactory epithelium to show that the relative number of sensory neurons and dendritic knobs that demonstrate x-CSE immunoreactivity differ between these frog species. Scale bar in d = 5 pm for a, b, c, and d. (e) X. luevis olfactory epithelium immunolabeled with x-CSE. Arrowheads point to strongly labeled Bowman’s glands located underneath the epithelium. Labeled ducts pass through the epithelium. Note the highly immunoreactive luminal surface ofthe epitbelium. Scale bar = IO fim. (f) A. tigrinurn olfactory epi~elium immunola&l~ with x-CSE. No obvious immuno~~ti~ty was seen in this amphibian. Lamina propria mucosal gland staining (arrowheads) was nonspecific since it was seen in control sections containing no primary antibody. Scale bar = 10 pm.
787
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CROWE
detectable in these assays. Thus, at present, we have no positive staining control to aid identification of the antigen. Previous attempts to use this particular antibody in Western blots had been unsuccessful (F. L. Margolis, personal communication) so further analysis would most likely require immunoprecipitation. One observation that suggests that x-CSE is binding to CS was the fact that tissues from A. tigrinum were negative while tissues from frog (R. catesbeiana and R. pipiens) were positive. Carnosine was undetectable in biochemical analysis of the olfactory mucosa (F. L. Margolis, personal communication), olfactory bulb ( 19) or brain (19) of A. tigrinum, while in the same analysis camosine was detectable in frog (R. catesbeiana) olfactory mucosa and brain (F. L. Margolis, personal communication). Therefore, in these amphibians the immunochemical findings are consistent with the biochemical presence or absence of carnosine. More numerous observations suggest the x-CSE binding site was not the CS molecule in amphibians. First, as discussed earlier, x-CSE staining in the frog was in a subpopulation of olfactory sensory neurons. Second, staining of mucus and mucus-secreting cells or glands was seen in X. laevis (Fig. le) and R. catesbeiana (Fig. lb). No reports have described CS or camosine in respiratory tissues, Bowman’s glands, or other glandular tissues. Finally, the x-CSE immunoreactivity pattern in individual olfac-
AND
PIXLEY
tory neurons was suggestive of membrane-associated binding, as discussed previously (5). CS is a soluble, cytoplasmic protein ( 12,23) that should not be membrane associated. Despite the lack of progress in identification ofthe molecule(s) that x-CSE recognizes, this monoclonal antibody is a useful marker of subsets of olfactory sensory neurons in two amphibian species. The general lack of neuronal markers in amphibian species and the robust staining observed with this antibody make it a reagent of potential use in functional studies involving electrophysiological experiments on dispersed cells and in studies comparing x-CSE distribution with that of molecular probes for olfactory receptor molecules. It is hoped that such studies would lead to an understanding of the functional differences of the xCSE-positive neuronal subpopulations, in particular, the possibility that they are associated with detection of a specific odorant or group of odorants. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the generous support of Dr. Robert C. Gesteland and thank Dr. Frank L. Margolis of the Roche Institute of Molecular Biology for use of the x-CSE antibody and critical advice in the preparation of the manuscript. This study was supported by National Institutes of Health Grants DC00347 and DC00352 awarded to Dr. Gesteland.
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