Amphibian olfactory receptor neurons express olfactory marker protein

Brain Research, 593 ( 19921 295-298 © 1992 Elsevier Science Publishers B.V. All rights reserved (X}[)6-8993/92/$05.00

295

BRES 25367

Amphibian olfactory receptor neurons express olfactory marker protein N.S. R a m a K r i s h n a a, T h o m a s V. G e t c h e l l

a,b,c, F r a n k

L. Margolis d a n d Marilyn L. G e t c h e l l b.~

'~ Departmalt of Physiology and Biophysics, h Division of Otolaryngology - Head and Neck Surgery, Departma~t of Surgel),, " Sanders-Brown Center on Aging, University of Kentucky College of Med&ine, Lexington, KY 40536 (USA) and d Department of Neuroscience, Roche Institute of Molecular Biology. Nutley, NJ 071 !0 (USA) (Accepted 23 July 1992)

Key words: Olfactory marker protein; Olfactory receptor neuron; lmmunohistochcmistry; Salamander; Frog

Expression of olfactory marker protein (OMP) in olfactory receptor neurol~ (ORNs) in two amphibians was investigated by immunohistochemical methods. The OMP immunoreactivity was observed in the cilia, apical dendritic knobs, dendrites and somas of ORNs; the axons of ORNs also showed intense immunoreactivity for OMP throughout their course from the olfactory epithelium to the glomerular layer of the olfactory bulb. Seven days after olfactory nerve transection in salamander, the number of OMP-positive ORNs was markedly reduced in the ipsilateral epithelium. The results demonstrate that amphibian ORNs express OMP and confirm its phylogenetic conservation ac.mss 0iverse species.

Olfactory marker protein (OMP) is a 19 kDa cytoplasmic protein that was isolated from the olfactory bulbs of rodents ~3. The protein has been purified and sequenced Ila~'2~'.OMP mRNA and the gene regulating its expression have been cloned3'2°':k The cellular expression of OMP mRNA and protein in olfactory receptor neurons located in the olfactory epithelium and their axons that project to the olfactory bulb has been studied in several mammalian species (e.g. refs. 1, 4, 8, 12, 18, 28), including human 7't')'24'27. The occurrence of OMP mRNA in the axons of the olfactory nerve and their terminals located in the glomeruli of the olfactory bulb has been demonstrated by in situ hybridization 2'). Although the neuroehemical and physiological significance of OMP remains enigmatic, it has proven to be an excellent reagent to identify the presence of mature olfactory receptor neurons in biochemical, molecular biological and developmental studies. Although earlier biochemical studies 1~'14 indicated the presence of OMP in lower vertebrate species such as the olfactory nerve of the garfish and the olfactory bulb of the frog, there have been no immunohistochemical studies reporting the identification and localization of OMP in the olfactory mucosa of poikilotherms. Since the function of OMP remains unknown,

study of its expression throughout vertebrate classes could lead to fundamental insights regarding its role. The olfactory epithelium of amphibians, particularly the salamander, is extensively utilized as a model system for innovative studies of sensory transduction in isolated olfactory receptor cell preparations for patch clamp experiments 5, voltage dye experiments for investigations of spatial components in the coding of odor quality z'), and identification of olfactory-specific gone products z~ associated with perireeeptor events. Further, from an evolutionary perspective, it is relevant to study the conservation of gene products and ultimately the mechanisms regulating their phylogenetic expression. In tiffs study we demonstrate for the first time the immunohistoehemical identification and localization of OMP in olfactory receptor neurons in poikilothermic species that are used extensively for neurobiological studies of the primary olfactory system. Adult tiger salamanders, Ambystoma tigrinunt, and leopard frogs, Rana pipiens, were obtained from Amphibians of North America, Nashville, TN. Unilateral olfactory nerve transections were performed on 2 salamanders anesthetized with 0.5% tricaine methanesulfonate (MS-222) as previously describedt'. For localization of OMP, 2 control salamanders, 2 nerve-sectioned

Correspondence to: T.V. Getchell, Department of Physiology and Biophysics, University of Kentucky, College of Medicine. Lexington. KY 40536-0084, USA. Fax: ( I ) (606) 258-2{139.

296

Pig. [. LOcalization of olfactory marker protein (OMP) in salamander olfactory mucosa, a: OMP fluorescence is localized in the somas, dendrites, and dendritic knobs (arrows) of olfactory receptor neurons (ORN). BI3, Bowman's gland, Bar = 50 p.m. b: horizontal section of olfactory nerve (ON) showing the OMP-positive axonal bundles of ORN, Bar = 50 p.m, c: horizontal section of olfactory bulb (OB) showing OMP fluorescence in the olfactory nerve layer (ONL) and glumerular !~yer (GL), Note the absence of fluorescence in the external plexi'~orm layer (EPL). Bar = 50 pro. d: olfactory epithelium of 7-day olfactory nerve transeeted salamander showing a patch of OMP-positive receptor neurons. Transection re~ulted in reduction of epithelial thickness and number of olfactory receptor neurons (ORN) as compared to control epithelium (a). Magnification same as a, e: transverse section of olfactory nerve of nerve-transected animal showing the majority of axon fascicles are unstained for OMP. Short arrows indicate a ,~mall number of OMP-positive axon fascicles, Bar = 20 p.m, Arrowheads in a and d indicate the approximate position of basement membrane,

297 salamanders and 2 frogs were anestl~,ztized with 1% MS-222 and perfused transcardially with ice-cold physiological saline followed by Zamboni's fixative. The nasal mucosae were extirpated, post-fixed for 1-2 h, rinsed overnight at 4°C in phosphate-buffered graded (10%-20%-30%) sucrose solutions and embedded in Tissue Tek O.C.T. compound for cryostat sectioning. Sections were cut at 15 /~m thickness, thaw-mounted on chrom-alum-gelatin coated slides, air-dried and frozen at -20°C until use. Standard immunofluerescence technique was utilized with goat anti-rat OMP ~o at the optimum dilution of 1 : 200 and FITC conjugated donkey anti-goat IgG (Jackson Immuno Research, West Grove, PA) as secondary antibody. In addition, some sections were also stained with the ABC technique using a Vectastain ABC kit (Vector Labs, Burlingame, CA); Chromo-red was the chromogen. To verify the specificity of the antiserum used, negative controls were performed by omitting the primary antibody and staining with appropriate secondary antibody alone, and preabsorption of anti-OMP with rat OMP for 16 h prior to the incubation. Positive controls consisted of immunostaining sections of rat olfactory and vomeronasal mucosae, where OMP has been previously localized in several studies 4's. The olfactory mucosa consists of the sensory neuroepithclium and the subjacent lamina propria. The epithelium consists of olfactory receptor neurons (ORNs), sustentacular and basal cells. The ORN consists of a single apical dendrite, which projects distally from the soma, that terminates in an apical knob bearing several cilia located in the surface mucus. Proximally, a single axon projects through the basement membrane, where several axons in proximity to one another form fascicles enwrapped by Schwann cells; groups of these fascicles constitute the olfactory nerves. The olfactory nerves form the superficial nerve layer of the olfactory bulb; individual axons synapse in the glomerular layer with second-order neurol~s. The lamina p:opria of the olfactory mucosa contains acini of Bowman's glands (BG), melanocytes that partially envelop the acini, blood vessels, olfactory nerve and myelinated nerve bundles, and loose connective tissue. The ORNs showed bright fluorescence for OMP in the epithelia of both the salamander (Fig. la) and frog (not shown). The staining was clearly observed in the cilia, apical dendritic knobs, dendrites, and somas of the ORNs (Fig. la). The dendritic knobs typically exhibited more intense immunoreactivity than did the neuronal cell bodies (Fig. la). The axons showed bright OMP fluorescence throughout their course from the epithelium through nerve fascicles in the lamina propria and olfactory nerve (Fig. lb). In the olfactory bulb,

the immunoreactivity was restricted to the axonal bundles of the olfactory nerve layer and their terminal arborizations, which form the peripheral component of the glomerular unit (Fig. lc). Other laminae of the bulb, including the external plexiform layer, did not exhibit immunoreactivity. Seven days after transection of the salamander's olfactory nerve, the olfactory epithelial thickness and number of immunoreactive ORNs were markedly reduced when compared to normal and contralateral control epithelia (Fig. ld). In some ORNs, although the dendrites and apical dendritic knobs showed OMP staining, no fluorescence was observed in the cilia. In certain areas of the mucosa, small patches of intensely positive ORNs were evident (Fig. ld). The majority of the nerve bundles in the lamina propria and olfactory nerve of the lesioned animals did not exhibit immunoreactivity (Fig. le), although a small number of OMP-immunoreaetive axon fascicles were embedded within the unstained olfactory nerve bundle (Fig. le). Substitution of primary antibody with normal goat serum or preabsorption of the primary antibody with purified rodent OMP resulted in loss of immunoreactivity. In positive controls of rat olfactory and vomeronasal mucosae, the soma, dendrites and axons of ORNs and vomeronasal receptor neurons exhibited intense OMP immunoreactivity as described 4'~, The relative intensity of OMP fluorescence was higher in rat olfactory epithelium than amphibian. This study provides the first immunohistochcmical evidence for the expression of OMP by ORNs in two poikilotherms, the salamar~der and fl'og, it also confirms and extends the earlier biochemical studies in the frog 1°'14. Our control experiments indicate the specificity of OMP immunoreactivity for ORNs. Characteristic staining was observed in ~he cilia, apical dendritic knobs, dendrites, and somas of the ORNs in the olfactory epithelium, in the axons of ORNs in the lamina propria, and in the olfactory nerve layer in the olfactory bulb. The distribution of OMP in ORNs of amphibians is characteristic of that reported for several mammalian specieo ~4a5, including the human olfactory mucosa7'1'~. The OMP-immunoreactive axons of ORNs could be traced through the olfactory nerve and into the synaptic glomeruli formed at the periphery of the amphibian bulb as reported for mammals t~. These observations indicate that OMP antiserum crossreacts with amphibian OMP and serves as a specific reagent to identify mature ORNs in amphibians as well as mammals ~4. However, the relative intensity of OMP fluorescence was greater in rat than amphibians, suggesting either the presence of less antigen or fewer common epitopes in amphibian ORNs.

~8 Additional evidence on the cellular expression of OMP was obtained from olfactory nerve section experiments. Within 7 days following olfactory nerve transection, there was a marked reduction in the epithelial thickness and in the number of OMP-immunoreactive neurons in the ipsilateral olfactory epithelium compared to contrah~teral and control epithelia. These obscr,'ations are consistent with lesion-induced retrograde degeneration of ORNs, confirming and extending our earlier electrophysiological studies ~2:-~. However, small patches of ORNs continued to exhibit OMP immunoreactivity in the ipsilateral side of the olfactory nerve transected animal. These may represent the newly differentiated ORNs, since olfactory neurons are replaced by cell division and differentiation of progenitors from a population of basal cells -~2, or they may reflect an incomplete nerve section. These results are consistent with similar studies on mammals (see refs. 14, 28) and further confirms the expression of OMP in the primary tdfactory system of amphibians. In conclusion, our immunohistoehemical and lesion data demonstrate that amphibian ORNs express OMP. Since OMP is selectively expressed in mature ORNs, these observations could facilitate the unambiguous identification of isolated ORNs used in patch clamp studies s''~, Furthermore. the presence of OMP in the primary olfactory system of a wide variety of species that range from fish to human ns coupled with the phylogenetic conservation of cellular localization to ORNs, strongly supports the supposition that this protein plays an important role in function of ORNs,

The aufitors wish Io Ihank Dr. Nikk Kalzman for technical assislance. This work was supporled by Nlli Grant DC.(IIII59 (T,V,G,) and NSF Grant BNS-88.2ll~74 (M.L,G.), I Baker, I1., Grillo, M. and Margolis, F.L,, Biochemical and imn~un()cyh~chenticalcharacterization af olIaclory marker protein in Ihe rodent central nervous system, J, ('prop. Nt.urol., 285 (1989) 24¢~=2¢~1. 2 (,inelli, A.R. and Kauer, J.S,, Voltage-sensitive dyes and funclional activity in Ihe olfaclory palhway, Atom, Rer. Neurosci., 15 (1992) 321-351. 3 Danciger, E., Metlling. C., Vidal, M., Morris, R, and Margolis, F,L,, Olfactory marker protein gone: Its structure and olfactory neuron-specific expression in Iransgenic mice, PnR'. Nat/, Actul, Sci. USA, 86 (IC)89) 8565-8569. 4 [:arl~ma,, A.I. and Margolis, F.L,, Olfactory marker prolcin during ontogcny: immunohistochemical Iocalizalion, Or.r, Biol,, 74 (19~0) 205-215. 5 Fircslcin, S., Zuf~dl, F. and Shepherd, G,M., Single odor-sensitive channels in tdfactory receptor neurons are also gated by cyclic nuclcotidcs, J, Neurosci,, II (1991) 3555-3572, h (;~tcllell, M.L. and Getehcll, T.V,, Immunohistoehemical localization or components of the immune barrier in the olfactory mucosae of salamander and rats, Anat. Rec,, 231 (1991) 358-374. 7 Getchell, M.L. and Mellerl, T.K,, Olfactory mucus secretion. In T,V.. Gctchcll, R,L, Doty, L,M. Bartoshuk and J.B, Snow, Jr.

(Eds.), Smell and Taste in Heahh and Disease, Raven, New York, 1991, pp. 83-95. 8 Hartman, B.K. and Margolis, F.L., lmmunofluorescence of the olfaclory marker protein, Brabs Res,, 96 (1975) 176-180, 9 Kauer. J.S., Contributions of topography and parallel processing to odor coding in the vertebrate olfactory pathway, Trends Neurosci., 14 (19911 79-85. ll} Keller, A. and Margolis, F.L., Immunological studies of the rat olfactory marker protein, J. Neurochem., 24 (1975) 1101-110b. I I Keller, A. and Margolis, F.L., Isolation and characterization of rat olfactory, marker protein, J. Biol. Chem., 251 (1976) 6232-6237. 12 Lin, P.JJ,, Phelix, C. and Krause, WJ., An immunohistochemical study of olfactory epithelium in the opossum before and after birth, Z. mikrosk.-anat, Forsch., 102 (1988)272-282. 13 Margolis, F.L., A brain protein unique to the olfactory bulb, Proc. Natl. Acad. SCL USA, 69 (1972) 1221-1224. 14 Margolis, F,L., A marker protein for the olfactory chemoreceptor neuron. In R.A. Bradshaw and D.M. Schneider (Eds.), Prote#)s of the Nt'n'ous System, Raven. New York, 1980, pp. 59-84, 15 Margolis, F.L., Olfactory marker protein: from PAGE band to eDNA clone, Tremls Neurosci., 8 (1985) 542-546. 16 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, New York, 1988, pp. 237-265. 17 Maue. R.A. and Dionne, V.E., Preparation of isolated mouse olfactory receptor neurons, Pfliigers Arch., 409 (1987) 244-250. 18 Monti Graziadei, G.A,. Margolis, F.L., Harding, J.W. and Graziadei, P.P.C., Immunocytochcmistry of the olfactory marker protein, J. Histochem. Cytochem., 25 (1977) 1311-1316. 19 Nakashima, T., Kimmelman, C.P. and Snow, J.B., Jr.. Immunohistopathology of human olfactory epithelium, nerve and bulb, Laryngoscope. 95 (1985) 391-396. 20 Rogers, K.E., Grillo, M., Sydor, W., Poonian, M. and Margolis, F.L., Olfilctory neuron-specific protein is translated from a large poly(A+) mRNA, Prec. Nut/. Acad. Sci. USA, 82 (1985) 52185222. 21 Rogers, K.E,, Dasgupla, P,, Guhler, O., Grillo. M., Khew-Goodall, Y.S. and Mnrgolis, F,L, Molecular cloning and sequencing of a eDNA t'or olfitetory marker protein, Proc. Natl. ,4cad. Sc,. USA, 84 (1987) 1704-1708. 22 Simmons, PA, ,nd Getchell, T.V., Physiological activity of newly differentiated olfaclory receptor neurons correlated witlt morpho. logical recovery from olfactory ~terve section in the salamander, J. Nt'dm~phy,ffol,, 45 ( 1981) 529-549. 23 Simmons, P.A., Rahfls, J.A, and Getchell, T.V,, Ultrastructural changes in olfactory receptor neurons following olfactory nerve section, J. Cotnp. Neurol,, 197 (1981) 237-257, 24 Smith, R.L., Baker, H., Kolstad, K,, Spencer, D,D. and Greer, C.A,, Localization of tyrosine hydmxylase and olfactory marker protein immunoreactivities in the human and macaque olfactory bulb, Bratty Re,~,, 548 (1991) 140-148. 25 Snyder, D,A,, Rivers, A,M,, Yokoe, H,, Menco. B,Ph,M. and Anholt, R,R,II,, OIfactomedin: Purification, characterization, and localization of a novel olfactory glycoprotein, Bit~'hemistt3,, 30 (1991) 9143-9153, 2f~ Sydor, W,, Teitelbaum, Z, Blacher, R,, Sun, S., Benz, W. and Margolis, F,L,, Amino acid sequence of a unique protein: Ral olfactory marker protein, Ar~'h, Biochem, Bioph.v,~., 249 (1986) 351-362, 27 Talamo, B.R,, Rudel, R.A,, Kosil~, K,S,, Lee, V.M.-Y., Neff, S., Adelman, L. and Kaucr, J,S,, Pathological changes in olfactory neurons in patients with Alzheimer's disease, Nature, 337 (1989) 736-739, 28 Verhaagen, J., Oestreicher, A.B., Grillo, M., Khew-Goodall, Y.-S., Gispen, W,H, and Margolis, F,L,, Neumplasticity in the olfactory system: differential effects of central and peripheral lesions of the primary olfactory pathway on the expression of B-50/GAP43 and the olfactory marker protein, J. Neurosci. Res,, 26 (1990) 31-44. 29 Wensley, C,H,, Kauer, J.S., Margolis, F,L, and Chikaraishi, D.M., OMP mRNA is transported to the axon terminals of olfactory receptor cells, Soc, NeuroscL Abstr., 17 (1991) 637.