Brain Research, 538 (1991) 147-151 Elsevier
147
BRES 24441
Monoclonal antibody to carnosine synthetase identifies a subpopulation of frog olfactory receptor neurons Maria J. Crowe and Sarah K. Pixley Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Cincinnati, OH (U.S.A.) (Accepted 11 September 1990)
Key words: Carnosine synthetase; Neuronal subpopulation; Olfactory receptor neuron; Immunocytochemistry; Frog; Monoclonal antibody
A monoclonal antibody directed against the synthetic enzyme for the dipeptide carnosine was used on cryostat sections of olfactory epithelium from the grass frog Rana pipiens. A subpopulation of what morphologically resembled olfactory receptor neurons were immunolabelled by this antibody. Labelled cells were completely stained, including the cell body, axonai and dendritic processes, dendritic knobs and cilia-like projections from the knobs.
The dipeptide carnosine (fl-alanyl-L-histidine) has been found in muscle and/or nervous tissue of various mammalian, avian and amphibian species and is particularly enriched in the olfactory tissues of most of the species studied (for review, see ref. 17). The function of carnosine in these tissues is unknown, although it has been postulated to serve as a neurotransmitter or neuromodulator in primary olfactory neurons based on the characterization of its binding site 13, its effect on secondary olfactory mitral cells 7'28, and on its disappearance subsequent to denervation of the primary olfactory pathway 5'16. Additional support for this hypothesis comes from autoradiographic 3, biochemical 3, and immunocytochemical 2'27 localization of carnosine in the axons and/or cell bodies of primary olfactory neurons. In an attempt to further examine the role of carnosine in the olfactory pathway, several studies have focussed on the properties 14,26 and localization 11,12,24 of its biosynthetic enzyme, carnosine synthetase (CS). This enzyme, whose activity is 98% cytosolic 12, has been difficult to purify to homogeneity due to instability of the native enzyme protein and limited availability of tissue containing high specific activity 14,26. Carnosine can be synthetized from radiolabelled precursors in mammalian olfactory epithelium, suggesting that the synthetic enzyme is present there ~2,2°. Denervation experiments have also suggested that CS is localized within primary olfactory neurons H. The exact cell-type localization of CS within the olfactory epithelium has not yet been demonstrated definitively using immunocytochemistry in mammals, and
it has not been previously tested in amphibians. To approach cell-type localization and to allow further study and purification of the enzyme, a panel of monoclonal antibodies was generated against rabbit muscle CS 19. Culture supernatants from the mouse monoclonal antibody-producing cells were capable of binding enzymatically active CS in a solid-phase second antibody protocol. When the supernatants were incubated with tissue extracts (olfactory epithelium, olfactory bulb, muscle, liver and brain) from various mammalian species (mouse, rabbit, cow, rat, monkey and dog), the antibodies formed immune complexes that exhibited CS activity 19. These antibodies have been either weakly 1s'21 or not 2 effective in immunocytochemically localizing CS in mammalian tissues. The weak reactions seen suggest a neuronal localization 1s,2~. Attempts to these antibodies for immunoblotting have been unsuccessful (EL. Margolis, personal communication) regardless of the fact that these antibodies are capable of reacting with the native
CS19,21.
We report here the use of 3 of these antibodies in immunocytochemical studies of olfactory epithelial sections of the grass frog (Rana pipiens). We were able to demonstrate the localization of CS-like immunoreactivity in a subset of primary olfactory neurons with one of the 3 antibodies. Although the biochemical synthesis of carnosine in the frog brain has been demonstrated 29 and a nervous system distribution of CS in frogs similar to that seen in mammals has been shown 23, no attempts to immunocytochemically localize CS or carnosine in the
Correspondence: M.J. Crowe, Dept. of Anatomy and Cell Biology, Univ. of Cincinnati, College of Medicine, 231 Bethesda Ave. ML 521, Cincinnati, OH 45267-0521, U.S.A. 0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
148 frog have been done prior to this study. Mouse ascites fluid containing monoclonal antibodies designated as anti-CS A, D and E, generated against rabbit muscle CS, were a generous gift of Dr. Frank L. Margolis. The preparation of these antibodies has been described previously ~9. Frogs were purchased from Lemberger Co., Inc. (Oshkosh, WI). All reagents were obtained from Fisher, Inc. (Fair Lawn, N J) unless otherwise noted. Frogs were anesthetized by placing them in a 1:150 (w/v) solution of tricaine methanesulfonate (Sigma Co., St. Louis, MO). The animals were then perfused through the heart with a 6% saline solution for 5 min, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 30 min and finally 20% sucrose in PBS for 30 min. Tissues were removed and placed in 0.32 M sucrose in PBS at 4 °C until they sunk to the bottom of the container. Tissues were then blocked and frozen in M-1 Embedding Matrix (Lipshaw, Inc., Detroit, MI). Sections (12-15 /~m) were cut on a Leitz Cryostat Model 1720 and mounted onto glass slides. Sections were pre-incubated in 4% fetal calf serum (Sigma) in an isotris buffer containing 2% sodium azide, 0.5 M magnesium sulfate, 0.15 M calcium chloride and 0.5% bovine serum albumin for 20 min followed by incubation with either anti-CS A, D or E at various dilutions for 90 min. A 1:125 dilution of anti-CS E (x-CSE) in 0.2% Triton/0.1 M PBS gave the best results. Next the sections were washed 3 times in PBS, incubated in fluorescein-conjugated goat anti-mouse secondary antibody (Sigma) for 90 min, and rinsed again 3 times in PBS. The sections were cover-slipped in Bacto FA Mounting Fluid (Difco Labs, Detroit, MI), viewed on a Nikon Microphot equipped with fluorescence optics, and photographed using Kodak TMax 400 black-and-white film. Monoclonal antibodies CS A, D and E were each tested on sections of grass frog olfactory epithelium. Only the last of these (x-CSE) demonstrated any reactivity. The immunolabelled cells in the olfactory epithelium morphologically resembled primary olfactory neurons (Fig. la,b). Dendrite-like processes extended from the cell body to the luminal surface and ended in brightly labelled knob structures (Fig. la,b,c). In one case (Fig.
lc), it appeared as though 5-6 labelled cilia could be seen protruding from the dendritic knob. Processes resembling axons exited the cell body basally, penetrated the basal layer of the epithelium and formed labelled nerve bundles within the lamina propria (Fig. la). Non-neuronal cell types in the olfactory epithelium were not immunolabelled by x-CSE. Sustentacular cells, whose nuclei are located immediately adjacent to the luminal surface of the epithelial layer, were clearly unmarked by x-CSE (Fig. ld). Basal cells, which sit immediately above the epithelial basal lamina, were not labelled (Fig. ld). In the lamina propria underneath the epithelium, only axons (arrow, Fig. la) and nerve bundles were immunolabelled with x-CSE, while Bowman's glands, blood vessels and connective tissue were unstained. Some bright luminal staining appeared occasionally, but this was also observed in control slides using normal mouse serum alone as the primary antibody solution. Only a minority of olfactory receptor neurons showed intense, distinct labelling. The subset pattern of immunolabelling was not due to loss of neurons during tissue processing, because adjacent tissue sections, stained with hematoxylin, showed numerous neuronal nuclei in corresponding areas (Fig. ld). If this subpopulation staining was not due to a loss of neurons during tissue processing, a second possible technical artifact could have been incomplete penetration of the antibody into the tissue, resulting in low density staining of those cells not cut during sectioning. This seems unlikely, since the permeabilizing agent Triton was used in the antibody-diluting solution to maximize penetration of the antibody. The position of labelled neuronal cell bodies in the epithelium indicated that expression of the epitope recognized by x-CSE is not developmentally regulated. Previous studies using tritiated thymidine labelling in the whole animal have shown that the primary olfactory neurons arise from division and differentiation of the basal cells, which sit adjacent and close to the basal lamina 8-1°. As the neurons mature, their cell bodies move apically in the epithelium, with immature neurons more basal and older neurons located closer to the luminal surface. Our results show that CS-like immunoreactivity was found not only in mature primary olfactory
Fig. 1. Cryostat sections of R. pipiens olfactory epithelium immunostained with anti-CSE, a: individual cell bodies are clearly immunostained. Arrowheads indicate intensely stained dendritic knobs. Arrow below the basement membrane points out axonal processes within the lamina propria. Note that most of the labelled cell bodies are located within the same region of the epithelium (Scale bar = 5/am for a,b and c). b: in this immunolabelled section, the positive cell bodies are located at different levels throughout the epithelium. Basally (arrowheads) as well as more apically (arrow) located cells have been immunostained, c: this cell demonstrates a long, labelled dendrite-like process ending in an intensely stained knob. Five or 6 projections resembling cilia (arrowhead) can be seen coming off the dendritic knob. d: a simple hematoxylin stain of a R. pipiens olfactory epithelium reveals many tightly packed cells. A few nuclei of sustentacular (filled arrowhead) and basal (unfilled arrowhead) cells are marked. Nuclei in between the layers of these two cell populations should belong predominantly to neurons. (Scale bar = 10/am).
149
150
Fig. 2. a: in this section, a single brightly labelled cell body is located close to the basement membrane. A dendritic process (arrow) extends the entire width of the epithelial layer, ending in a knob. A small axonal process (arrowhead) remains basally located. The position of the cell body would suggest that this is a newly differentiated neuron, b: a more apically located cell shows a thick dendritic process (arrowhead) which ends in a dendritic knob at the epithelial surface. The position of this cell body suggests that it is a relatively old neuron (Scale bar = 5 ~m for a and b). neurons in the middle of the epithelium (Fig. la,b) but also in young (Fig. 2a) and aged (Fig. 2b) neurons. Consequently, the presence of this epitope in primary olfactory neurons does not appear to be dependent on the developmental age of the cells. If the CS-like immunoreactivity seen in the frog is not due to technical artifacts or developmental age, then the subset labelling pattern observed would be surprising in light of data on the localization of carnosine and its proposed function in mammals. Carnosine has been postulated to be a general neurotransmitter or neuromodulator in olfactory receptor neurons (see beginning of this article) and no other candidate has been reported to date for either mammals or amphibians. Reports of immunostaining for carnosine in mammalian olfactory epithelium suggest staining of all mature neurons 2'27. Therefore, all mammalian neurons might be expected to contain the synthetic enzyme, and there are no data to suggest that this might be different in amphibians. The immunolabeiling of only a subset of frog olfactory receptor neurons may also be due to the possibility that x-CSE may not be recognizing the amphibian form of the
enzyme and/or it is recognizing another molecule containing a similar antigenic site. Anti-CS monoclonal antibodies we used were reported to have no binding to avian CS using immunoprecipitation 19. This suggests that there are significant species differences in the enzyme. Analysis of the epitope being recognized by x-CSE will require a biochemical/immunochemical study of the epitope, comparing mammalian and amphibian tissues. These studies may be hampered by the unavailability of purified CS to serve as a control and the reported lack of success in using the monoclonal antibodies for immunoblotting ( E L . Margolis, personal communication). The subcellular immunostaining pattern suggests that x-CSE may be binding to a molecule that is not CS. Distribution and biochemical studies of CS in mammalian, avian and frog tissues indicate that it is cytoplasmic and soluble 12"23-26"29. The immunostaining pattern appears in some cells to be associated with the plasma membrane, and in others it appears to be cytoplasmic (Figs. 1 and 2). Unless this staining pattern reflects the loss of some, but not all cytoplasmic contents, membrane-associated binding would argue against the idea
151 that true binding to CS by x-CSE is occurring. A n electron microscopic analysis of the immunoreactivity would be necessary to d e t e r m i n e the true localization of the binding observed. Finally, the subset staining pattern m a y be a result of the seasonal variation of synthetase levels that have been r e p o r t e d in o t h e r studies of frog tissues 23"29. CS may not be present in all frog olfactory neurons at all times. This is the first r e p o r t of specific cellular immunostaining with the monoclonal antibody CSE i n cryostat sections of grass frog epithelium. Only a subpopulation of neurons were positively labelled in this amphibian species. M o n o c l o n a l antibodies have been used to identify subpopulations of olfactory r e c e p t o r neurons in the rat 1 and the rabbit 6'22. A few antibodies have been generated that react with specific glycoproteins located on the ciliary m e m b r a n e s of frog olfactory r e c e p t o r neurons 4,
but they p r o d u c e a general staining p a t t e r n over the luminal surface of the epithelium and are not cell specific. To date, no o t h e r specific a m p h i b i a n olfactory neuronal m a r k e r has been d e v e l o p e d . A n t i b o d i e s to olfactory m a r k e r protein, a definitive, specific m a r k e r of m a t u r e olfactory neurons in m a n y o t h e r species 15, were negative in grass frog olfactory epithelia sections (authors, unpublished results). Regardless of the true nature of the antigen recognized by x-CSE, this is the first study demonstrating the identification of a specific subset of amphibian olfactory r e c e p t o r neurons using a monoclonal antibody marker.
1 Allen, W.K. and Akeson, R., Identification of an olfactory receptor neuron subclass: cellular and molecular analysis during development, Dev. Biol., 109 (1985) 393-401. 2 Biffo, S., Grillo, M. and Margolis, EL., Cellular localization of carnosine-like and anserine-like immunoreactivities in rodent and avian central nervous system, Neuroscience, 35 (1990) 637-651. 3 Burd, G.D., Davis, B.J., Macrides, E, Grillo, M. and Margolis, F.L., Carnosine in primary afferents of the olfactory system: an autoradiographic and biochemical study, J. Neurosci., 2 (1982) 244-255. 4 Chen, Z., Ophir, D. and Lancet, D., Monoclonal antibodies to ciliary glycoproteins of frog olfactory neurons, Brain Research, 368 (1986) 329-338. 5 Ferriero, D. and Margolis, EL., Denervation in the primary olfactory pathway of mice. II: effects on carnosine and other amine compounds, Brain Research, 94 (1975) 75-86. 6 Fujita, S.C., Mori, K., Imamura, K. and Obata, K., Subclasses of olfactory receptor cells and their segregated central projections demonstrated by a monoclonal antibody, Brain Research, 326 (1985) 192-196. 7 Gonzalez-Estrada, M.T. and Freeman, W.J., Effects of carnosine on olfactory bulb EEG, evoked potentials and DC potentials, Brain Research, 202 (1980) 373-386. 8 Graziadei, P.P.C., Cell dynamics in the olfactory mucosa, Tissue Cell, 5 (1973) 113-131. 9 Graziadei, P.P.C. and Delian, R.S., Neuronal regeneration in frog olfactory system, J. Cell Biol., 59 (1973) 525-530. 10 Graziadei, P.P.C. and Metcalf, J.F., Autoradiographic and ultrastructurai observations on the frog's olfactory mucosa, Z. Zellforsch., 116 (1971) 305-318. 11 Harding, J. and Margolis, EL., Denervation in the primary olfactory pathway of mice. III: effect on enzymes of carnosine metabolism, Brain Research, 110 (1976) 351-360. 12 Harding, J. and O'Fallon, J.V., The subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory bulbs, Brain Research, 173 (1979) 99-109. 13 Hirsch, J.D., Grillo, M. and Margolis, EL., Ligand binding studies in the mouse olfactory bulb: identification and characterization of an L-[3H]carnosine binding site, Brain Research, 158 (1978) 407-422. 14 Horinishi, H., Grillo, M. and Margolis, EL., Purification and characterization of carnosine synthetase from mouse olfactory bulbs, J. Neurochem., 31 (1978) 909-919. 15 Keller, A. and Margolis, EL., Immunological studies of the rat olfactory marker protein, J. Neurochem., 24 (1975) 1101-1106.
16 Margolis, EL., Carnosine in the primary olfactory pathway, Science, 184 (1974) 909-911. 17 Margolis, EL., Carnosine: an olfactory neuropeptide. In J.L. Barker and T.G. Smith Jr. (Eds.), The Role of Peptides in Neuronal Function, Marcel Dekker, Inc., New York, 1980, pp. 545-572. 18 Margolis, F.L., Molecular cloning of olfactory-specific gene products. In F.L. Margolis and T.V. Getchell (Eds.), Molecular Neurobiology of the Olfactory System, Plenum Press, New York, London, 1988, pp. 237-265. 19 Margolis, F.L., Grillo, M., Hempstead, J. and Morgan, J.I., Monocional antibodies to mammalian carnosine synthetase, J. Neurochem., 48 (1987) 593-600. 20 Margolis, EL., Grillo, M., Kawano, T. and Farbman, A.I., Carnosine synthesis in olfactory tissue during ontogeny: effect of exogenous beta-alanine, J. Neurochem., 44 (1985) 1459-1464. 21 Morgan, J.I., Monoclonal antibody mapping of the rat olfactory tract. In EL. Margolis and T.V. GetcheU (Eds.), Molecular Neurobiology of the Olfactory System, Plenum Press, New York, London, 1988, pp. 269-296. 22 Mori, K., Fujita, S.C., Imamura, K. and Obata, K., Immunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit, J. Comp. Neurol., 242 (1985) 214-229. 23 Ng, R.H. and Marshall, ED., Distribution of homocarnosinecarnosine synthetase in tissues of rat, mouse, chick and frog, Comp. Biochem. Physiol., 54 (1976) 519-521. 24 Ng, R.H. and Marshall, ED., Regional and subcellular distribution of homocarnosine-carnosine synthetase in the central nervous system of rats, J. Neurochem., 30 (1978) 187-190. 25 Ng, R.H., Marshall, ED., Henn, F.A. and Sellstrom, A., Metabolism of carnosine and homocarnosine in subcellular fractions and neuronal and glial cell-enriched fractions of rabbit brain, J. Neurochem., 28 (1977) 449-452. 26 Skaper, S.D., Das, S. and Marshall, ED., Some properties of a homocarnosine-carnosine synthetase isolated from rat brain, J. Neurochem., 21 (1973) 1429-1445. 27 Sakai, M., Yoshida, M., Karasawa, N., Teramura, M., Ueda, H. and Nagatsu, I., Carnosine-like immunoreactivity in the primary olfactory neuron of the rat, Experientia, 43 (1987) 298-300. 28 Tonosaki, K. and Shibuya, T., Action of some drugs on gecko olfactory bulb mitral cell responses to odor stimulation, Brain Research, 167 (1979) 180-184. 29 Yockey, W.C. and Marshall, ED., Incorporation of [14C]Histidine into homocarnosine and carnosine of frog brain in vivo and in vitro, Biochem. J., 114 (1969) 585-588.
The authors gratefully acknowledge the generous support of Dr. Robert C. Gesteland and wish to thank Dr. Steven J. Kleene for critical reading of the manuscript. This study was supported by National Institute of Health Grants PO1-NS23523 and RO1DC00347 (to R.C.G.).
147
BRES 24441
Monoclonal antibody to carnosine synthetase identifies a subpopulation of frog olfactory receptor neurons Maria J. Crowe and Sarah K. Pixley Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Cincinnati, OH (U.S.A.) (Accepted 11 September 1990)
Key words: Carnosine synthetase; Neuronal subpopulation; Olfactory receptor neuron; Immunocytochemistry; Frog; Monoclonal antibody
A monoclonal antibody directed against the synthetic enzyme for the dipeptide carnosine was used on cryostat sections of olfactory epithelium from the grass frog Rana pipiens. A subpopulation of what morphologically resembled olfactory receptor neurons were immunolabelled by this antibody. Labelled cells were completely stained, including the cell body, axonai and dendritic processes, dendritic knobs and cilia-like projections from the knobs.
The dipeptide carnosine (fl-alanyl-L-histidine) has been found in muscle and/or nervous tissue of various mammalian, avian and amphibian species and is particularly enriched in the olfactory tissues of most of the species studied (for review, see ref. 17). The function of carnosine in these tissues is unknown, although it has been postulated to serve as a neurotransmitter or neuromodulator in primary olfactory neurons based on the characterization of its binding site 13, its effect on secondary olfactory mitral cells 7'28, and on its disappearance subsequent to denervation of the primary olfactory pathway 5'16. Additional support for this hypothesis comes from autoradiographic 3, biochemical 3, and immunocytochemical 2'27 localization of carnosine in the axons and/or cell bodies of primary olfactory neurons. In an attempt to further examine the role of carnosine in the olfactory pathway, several studies have focussed on the properties 14,26 and localization 11,12,24 of its biosynthetic enzyme, carnosine synthetase (CS). This enzyme, whose activity is 98% cytosolic 12, has been difficult to purify to homogeneity due to instability of the native enzyme protein and limited availability of tissue containing high specific activity 14,26. Carnosine can be synthetized from radiolabelled precursors in mammalian olfactory epithelium, suggesting that the synthetic enzyme is present there ~2,2°. Denervation experiments have also suggested that CS is localized within primary olfactory neurons H. The exact cell-type localization of CS within the olfactory epithelium has not yet been demonstrated definitively using immunocytochemistry in mammals, and
it has not been previously tested in amphibians. To approach cell-type localization and to allow further study and purification of the enzyme, a panel of monoclonal antibodies was generated against rabbit muscle CS 19. Culture supernatants from the mouse monoclonal antibody-producing cells were capable of binding enzymatically active CS in a solid-phase second antibody protocol. When the supernatants were incubated with tissue extracts (olfactory epithelium, olfactory bulb, muscle, liver and brain) from various mammalian species (mouse, rabbit, cow, rat, monkey and dog), the antibodies formed immune complexes that exhibited CS activity 19. These antibodies have been either weakly 1s'21 or not 2 effective in immunocytochemically localizing CS in mammalian tissues. The weak reactions seen suggest a neuronal localization 1s,2~. Attempts to these antibodies for immunoblotting have been unsuccessful (EL. Margolis, personal communication) regardless of the fact that these antibodies are capable of reacting with the native
CS19,21.
We report here the use of 3 of these antibodies in immunocytochemical studies of olfactory epithelial sections of the grass frog (Rana pipiens). We were able to demonstrate the localization of CS-like immunoreactivity in a subset of primary olfactory neurons with one of the 3 antibodies. Although the biochemical synthesis of carnosine in the frog brain has been demonstrated 29 and a nervous system distribution of CS in frogs similar to that seen in mammals has been shown 23, no attempts to immunocytochemically localize CS or carnosine in the
Correspondence: M.J. Crowe, Dept. of Anatomy and Cell Biology, Univ. of Cincinnati, College of Medicine, 231 Bethesda Ave. ML 521, Cincinnati, OH 45267-0521, U.S.A. 0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
148 frog have been done prior to this study. Mouse ascites fluid containing monoclonal antibodies designated as anti-CS A, D and E, generated against rabbit muscle CS, were a generous gift of Dr. Frank L. Margolis. The preparation of these antibodies has been described previously ~9. Frogs were purchased from Lemberger Co., Inc. (Oshkosh, WI). All reagents were obtained from Fisher, Inc. (Fair Lawn, N J) unless otherwise noted. Frogs were anesthetized by placing them in a 1:150 (w/v) solution of tricaine methanesulfonate (Sigma Co., St. Louis, MO). The animals were then perfused through the heart with a 6% saline solution for 5 min, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 30 min and finally 20% sucrose in PBS for 30 min. Tissues were removed and placed in 0.32 M sucrose in PBS at 4 °C until they sunk to the bottom of the container. Tissues were then blocked and frozen in M-1 Embedding Matrix (Lipshaw, Inc., Detroit, MI). Sections (12-15 /~m) were cut on a Leitz Cryostat Model 1720 and mounted onto glass slides. Sections were pre-incubated in 4% fetal calf serum (Sigma) in an isotris buffer containing 2% sodium azide, 0.5 M magnesium sulfate, 0.15 M calcium chloride and 0.5% bovine serum albumin for 20 min followed by incubation with either anti-CS A, D or E at various dilutions for 90 min. A 1:125 dilution of anti-CS E (x-CSE) in 0.2% Triton/0.1 M PBS gave the best results. Next the sections were washed 3 times in PBS, incubated in fluorescein-conjugated goat anti-mouse secondary antibody (Sigma) for 90 min, and rinsed again 3 times in PBS. The sections were cover-slipped in Bacto FA Mounting Fluid (Difco Labs, Detroit, MI), viewed on a Nikon Microphot equipped with fluorescence optics, and photographed using Kodak TMax 400 black-and-white film. Monoclonal antibodies CS A, D and E were each tested on sections of grass frog olfactory epithelium. Only the last of these (x-CSE) demonstrated any reactivity. The immunolabelled cells in the olfactory epithelium morphologically resembled primary olfactory neurons (Fig. la,b). Dendrite-like processes extended from the cell body to the luminal surface and ended in brightly labelled knob structures (Fig. la,b,c). In one case (Fig.
lc), it appeared as though 5-6 labelled cilia could be seen protruding from the dendritic knob. Processes resembling axons exited the cell body basally, penetrated the basal layer of the epithelium and formed labelled nerve bundles within the lamina propria (Fig. la). Non-neuronal cell types in the olfactory epithelium were not immunolabelled by x-CSE. Sustentacular cells, whose nuclei are located immediately adjacent to the luminal surface of the epithelial layer, were clearly unmarked by x-CSE (Fig. ld). Basal cells, which sit immediately above the epithelial basal lamina, were not labelled (Fig. ld). In the lamina propria underneath the epithelium, only axons (arrow, Fig. la) and nerve bundles were immunolabelled with x-CSE, while Bowman's glands, blood vessels and connective tissue were unstained. Some bright luminal staining appeared occasionally, but this was also observed in control slides using normal mouse serum alone as the primary antibody solution. Only a minority of olfactory receptor neurons showed intense, distinct labelling. The subset pattern of immunolabelling was not due to loss of neurons during tissue processing, because adjacent tissue sections, stained with hematoxylin, showed numerous neuronal nuclei in corresponding areas (Fig. ld). If this subpopulation staining was not due to a loss of neurons during tissue processing, a second possible technical artifact could have been incomplete penetration of the antibody into the tissue, resulting in low density staining of those cells not cut during sectioning. This seems unlikely, since the permeabilizing agent Triton was used in the antibody-diluting solution to maximize penetration of the antibody. The position of labelled neuronal cell bodies in the epithelium indicated that expression of the epitope recognized by x-CSE is not developmentally regulated. Previous studies using tritiated thymidine labelling in the whole animal have shown that the primary olfactory neurons arise from division and differentiation of the basal cells, which sit adjacent and close to the basal lamina 8-1°. As the neurons mature, their cell bodies move apically in the epithelium, with immature neurons more basal and older neurons located closer to the luminal surface. Our results show that CS-like immunoreactivity was found not only in mature primary olfactory
Fig. 1. Cryostat sections of R. pipiens olfactory epithelium immunostained with anti-CSE, a: individual cell bodies are clearly immunostained. Arrowheads indicate intensely stained dendritic knobs. Arrow below the basement membrane points out axonal processes within the lamina propria. Note that most of the labelled cell bodies are located within the same region of the epithelium (Scale bar = 5/am for a,b and c). b: in this immunolabelled section, the positive cell bodies are located at different levels throughout the epithelium. Basally (arrowheads) as well as more apically (arrow) located cells have been immunostained, c: this cell demonstrates a long, labelled dendrite-like process ending in an intensely stained knob. Five or 6 projections resembling cilia (arrowhead) can be seen coming off the dendritic knob. d: a simple hematoxylin stain of a R. pipiens olfactory epithelium reveals many tightly packed cells. A few nuclei of sustentacular (filled arrowhead) and basal (unfilled arrowhead) cells are marked. Nuclei in between the layers of these two cell populations should belong predominantly to neurons. (Scale bar = 10/am).
149
150
Fig. 2. a: in this section, a single brightly labelled cell body is located close to the basement membrane. A dendritic process (arrow) extends the entire width of the epithelial layer, ending in a knob. A small axonal process (arrowhead) remains basally located. The position of the cell body would suggest that this is a newly differentiated neuron, b: a more apically located cell shows a thick dendritic process (arrowhead) which ends in a dendritic knob at the epithelial surface. The position of this cell body suggests that it is a relatively old neuron (Scale bar = 5 ~m for a and b). neurons in the middle of the epithelium (Fig. la,b) but also in young (Fig. 2a) and aged (Fig. 2b) neurons. Consequently, the presence of this epitope in primary olfactory neurons does not appear to be dependent on the developmental age of the cells. If the CS-like immunoreactivity seen in the frog is not due to technical artifacts or developmental age, then the subset labelling pattern observed would be surprising in light of data on the localization of carnosine and its proposed function in mammals. Carnosine has been postulated to be a general neurotransmitter or neuromodulator in olfactory receptor neurons (see beginning of this article) and no other candidate has been reported to date for either mammals or amphibians. Reports of immunostaining for carnosine in mammalian olfactory epithelium suggest staining of all mature neurons 2'27. Therefore, all mammalian neurons might be expected to contain the synthetic enzyme, and there are no data to suggest that this might be different in amphibians. The immunolabeiling of only a subset of frog olfactory receptor neurons may also be due to the possibility that x-CSE may not be recognizing the amphibian form of the
enzyme and/or it is recognizing another molecule containing a similar antigenic site. Anti-CS monoclonal antibodies we used were reported to have no binding to avian CS using immunoprecipitation 19. This suggests that there are significant species differences in the enzyme. Analysis of the epitope being recognized by x-CSE will require a biochemical/immunochemical study of the epitope, comparing mammalian and amphibian tissues. These studies may be hampered by the unavailability of purified CS to serve as a control and the reported lack of success in using the monoclonal antibodies for immunoblotting ( E L . Margolis, personal communication). The subcellular immunostaining pattern suggests that x-CSE may be binding to a molecule that is not CS. Distribution and biochemical studies of CS in mammalian, avian and frog tissues indicate that it is cytoplasmic and soluble 12"23-26"29. The immunostaining pattern appears in some cells to be associated with the plasma membrane, and in others it appears to be cytoplasmic (Figs. 1 and 2). Unless this staining pattern reflects the loss of some, but not all cytoplasmic contents, membrane-associated binding would argue against the idea
151 that true binding to CS by x-CSE is occurring. A n electron microscopic analysis of the immunoreactivity would be necessary to d e t e r m i n e the true localization of the binding observed. Finally, the subset staining pattern m a y be a result of the seasonal variation of synthetase levels that have been r e p o r t e d in o t h e r studies of frog tissues 23"29. CS may not be present in all frog olfactory neurons at all times. This is the first r e p o r t of specific cellular immunostaining with the monoclonal antibody CSE i n cryostat sections of grass frog epithelium. Only a subpopulation of neurons were positively labelled in this amphibian species. M o n o c l o n a l antibodies have been used to identify subpopulations of olfactory r e c e p t o r neurons in the rat 1 and the rabbit 6'22. A few antibodies have been generated that react with specific glycoproteins located on the ciliary m e m b r a n e s of frog olfactory r e c e p t o r neurons 4,
but they p r o d u c e a general staining p a t t e r n over the luminal surface of the epithelium and are not cell specific. To date, no o t h e r specific a m p h i b i a n olfactory neuronal m a r k e r has been d e v e l o p e d . A n t i b o d i e s to olfactory m a r k e r protein, a definitive, specific m a r k e r of m a t u r e olfactory neurons in m a n y o t h e r species 15, were negative in grass frog olfactory epithelia sections (authors, unpublished results). Regardless of the true nature of the antigen recognized by x-CSE, this is the first study demonstrating the identification of a specific subset of amphibian olfactory r e c e p t o r neurons using a monoclonal antibody marker.
1 Allen, W.K. and Akeson, R., Identification of an olfactory receptor neuron subclass: cellular and molecular analysis during development, Dev. Biol., 109 (1985) 393-401. 2 Biffo, S., Grillo, M. and Margolis, EL., Cellular localization of carnosine-like and anserine-like immunoreactivities in rodent and avian central nervous system, Neuroscience, 35 (1990) 637-651. 3 Burd, G.D., Davis, B.J., Macrides, E, Grillo, M. and Margolis, F.L., Carnosine in primary afferents of the olfactory system: an autoradiographic and biochemical study, J. Neurosci., 2 (1982) 244-255. 4 Chen, Z., Ophir, D. and Lancet, D., Monoclonal antibodies to ciliary glycoproteins of frog olfactory neurons, Brain Research, 368 (1986) 329-338. 5 Ferriero, D. and Margolis, EL., Denervation in the primary olfactory pathway of mice. II: effects on carnosine and other amine compounds, Brain Research, 94 (1975) 75-86. 6 Fujita, S.C., Mori, K., Imamura, K. and Obata, K., Subclasses of olfactory receptor cells and their segregated central projections demonstrated by a monoclonal antibody, Brain Research, 326 (1985) 192-196. 7 Gonzalez-Estrada, M.T. and Freeman, W.J., Effects of carnosine on olfactory bulb EEG, evoked potentials and DC potentials, Brain Research, 202 (1980) 373-386. 8 Graziadei, P.P.C., Cell dynamics in the olfactory mucosa, Tissue Cell, 5 (1973) 113-131. 9 Graziadei, P.P.C. and Delian, R.S., Neuronal regeneration in frog olfactory system, J. Cell Biol., 59 (1973) 525-530. 10 Graziadei, P.P.C. and Metcalf, J.F., Autoradiographic and ultrastructurai observations on the frog's olfactory mucosa, Z. Zellforsch., 116 (1971) 305-318. 11 Harding, J. and Margolis, EL., Denervation in the primary olfactory pathway of mice. III: effect on enzymes of carnosine metabolism, Brain Research, 110 (1976) 351-360. 12 Harding, J. and O'Fallon, J.V., The subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory bulbs, Brain Research, 173 (1979) 99-109. 13 Hirsch, J.D., Grillo, M. and Margolis, EL., Ligand binding studies in the mouse olfactory bulb: identification and characterization of an L-[3H]carnosine binding site, Brain Research, 158 (1978) 407-422. 14 Horinishi, H., Grillo, M. and Margolis, EL., Purification and characterization of carnosine synthetase from mouse olfactory bulbs, J. Neurochem., 31 (1978) 909-919. 15 Keller, A. and Margolis, EL., Immunological studies of the rat olfactory marker protein, J. Neurochem., 24 (1975) 1101-1106.
16 Margolis, EL., Carnosine in the primary olfactory pathway, Science, 184 (1974) 909-911. 17 Margolis, EL., Carnosine: an olfactory neuropeptide. In J.L. Barker and T.G. Smith Jr. (Eds.), The Role of Peptides in Neuronal Function, Marcel Dekker, Inc., New York, 1980, pp. 545-572. 18 Margolis, F.L., Molecular cloning of olfactory-specific gene products. In F.L. Margolis and T.V. Getchell (Eds.), Molecular Neurobiology of the Olfactory System, Plenum Press, New York, London, 1988, pp. 237-265. 19 Margolis, F.L., Grillo, M., Hempstead, J. and Morgan, J.I., Monocional antibodies to mammalian carnosine synthetase, J. Neurochem., 48 (1987) 593-600. 20 Margolis, EL., Grillo, M., Kawano, T. and Farbman, A.I., Carnosine synthesis in olfactory tissue during ontogeny: effect of exogenous beta-alanine, J. Neurochem., 44 (1985) 1459-1464. 21 Morgan, J.I., Monoclonal antibody mapping of the rat olfactory tract. In EL. Margolis and T.V. GetcheU (Eds.), Molecular Neurobiology of the Olfactory System, Plenum Press, New York, London, 1988, pp. 269-296. 22 Mori, K., Fujita, S.C., Imamura, K. and Obata, K., Immunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit, J. Comp. Neurol., 242 (1985) 214-229. 23 Ng, R.H. and Marshall, ED., Distribution of homocarnosinecarnosine synthetase in tissues of rat, mouse, chick and frog, Comp. Biochem. Physiol., 54 (1976) 519-521. 24 Ng, R.H. and Marshall, ED., Regional and subcellular distribution of homocarnosine-carnosine synthetase in the central nervous system of rats, J. Neurochem., 30 (1978) 187-190. 25 Ng, R.H., Marshall, ED., Henn, F.A. and Sellstrom, A., Metabolism of carnosine and homocarnosine in subcellular fractions and neuronal and glial cell-enriched fractions of rabbit brain, J. Neurochem., 28 (1977) 449-452. 26 Skaper, S.D., Das, S. and Marshall, ED., Some properties of a homocarnosine-carnosine synthetase isolated from rat brain, J. Neurochem., 21 (1973) 1429-1445. 27 Sakai, M., Yoshida, M., Karasawa, N., Teramura, M., Ueda, H. and Nagatsu, I., Carnosine-like immunoreactivity in the primary olfactory neuron of the rat, Experientia, 43 (1987) 298-300. 28 Tonosaki, K. and Shibuya, T., Action of some drugs on gecko olfactory bulb mitral cell responses to odor stimulation, Brain Research, 167 (1979) 180-184. 29 Yockey, W.C. and Marshall, ED., Incorporation of [14C]Histidine into homocarnosine and carnosine of frog brain in vivo and in vitro, Biochem. J., 114 (1969) 585-588.
The authors gratefully acknowledge the generous support of Dr. Robert C. Gesteland and wish to thank Dr. Steven J. Kleene for critical reading of the manuscript. This study was supported by National Institute of Health Grants PO1-NS23523 and RO1DC00347 (to R.C.G.).
