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Scanning electron microscopy of the channel catfish olfactory lamellae
TISSUE
&CELL
1978 IO (I) l-9
hhlishd byLongmonGKW~ Ltd.PrintedinCwot Bdain
JOHN
SCANNING CHANNEL
CAPRIO”
and RANDIE
RADERMAN-LITTLE
ELECTRON MICROSCOPY OF THE CATFISH OLFACTORY LAMELLAE ABSTRACT. The olfactory lamellae of the channel catfish (Icfalurrrs punctatm) are composed of sensory and indifferent (non-sensory) epithelia organized into two distinct regions on both surfaces of each lamella. The smaller sensory region located adjacent to the midline raphe has fewer cilia per unit surface area than the indifferent epithelium and contains the olfactory neurons whose ciliated dendritic terminals occur at the epithelial surface. The indifferent epithelium, comprising the greater surface area of the olfactory lamella, is covered with a dense mat of non-sensory cilia. Fractured carbon dioxide critical point dried lamellar tissue revealed the underlying cellular structure. The lamellae are composed of two layers of epithelium enclosing a thin stromal layer. Olfactory receptors were observed in the fractured tissue only within the sensory epithelium.
Introduction MORPHOLOGICAL studies on the olfactory organ of a variety of teleosts (Holl, 1965; Bertmar, 1972; Schulte, 1972; Theisen, 1972, 1973; Breipohl et al., 1973, 1974; Andres, 1975; Lowe and MacLeod, 197.5; Zeiske and Melinkat, 1976) have shown considerable variation in the distribution patterns of sensory versus indifferent areas of the olfactory epithelium. A description of the sensory regions of the olfactory organ of catfish is lacking with the exception of the light microscopical work of Ho11 (1965) on the olfactory organ of 18 species of teleosts including the brown bullhead {Ictalurus nebulosus). This paucity of information is especially surprising since catfish have long been known for their acute sense of smell (Parker, 1910; Olmsted, I91 8) and are continuing to be used as experimental animals in olfactory research (Suzuki and Tucker, 1971; Tucker and Suzuki, 1972; Caprio, 1977). During electrophysiological studies of the Department of Biological Science, Flofida State University, Tallahassee, Florida, U.S.A. * Current address: Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, U.S.A. Received
I7 October
1977.
olfactory receptor responses of the white catfish Ictalurus cam (Tucker and Suzuki, 1972) and the channel catfish (Caprio, 1977) it was noticed that the maximal amount of olfactory neural activity was recorded when the surface microelectrode was positioned against a discrete region of an olfactory lamella near the midline raphe. As the microelectrode was advanced laterally across the face of the lamella neural activity quickly declined; if the electrode was positioned along the dorsal edge of the lamella, neural activity was also absent. It was with this information that a SEM study was begun to determine whether the sensory and indifferent epithelial regions of the channel catfish olfactory lamellae were distinguishable by surface features alone and whether these regions correlated with the microelectrode positions. Materials and Methods
Channel catfish, approximately 75-l 50 g, were anesthetized with 0.02’% MS 222. The olfactory mucosa was dissected out, rinsed in Ringer’s and cut in small pieces. Specimens were fixed for 3 hr in 3 y0 glutaraldehyde in 0.1 M cacodylate or phosphate buffer (pH 7.4). For fixation in situ, fixative was dripped on the intact mucosa for I5 min and
2
CAPRIO
dissected under glutaraldehyde in After glutaraldehyde fixation the specimens were left in buffered 7.5 ‘? sucrose for at least 3 hr or overnight and then treated for 15-24 hr with 8 % glycerin in buffer. They were then post-fixed in 2% osmium tetroxide for 30 min, rinsed several times in buffer, and dehydrated in a graded acetone series. These procedures were carried out at 4°C. The specimens were dried by the critical point method with COZ. Using slight pressure some of the dried specimens were then cracked with fine forceps in order to expose inner cell surfaces. Each fragment was then mounted on stubs using double-stick tape or conductive silver paint, and coated with a 50-100 A layer of gold/palladium. Observations were made with a Cambridge scanning electron microscope model S4-10 at voltages of 5-20 kV. Photomicrographs were taken on positive-negative Polaroid film type 105. pieces vitro.
Results The two olfactory capsules of the channel catfish contain bilaterally symmetric olfactory rosettes composed of numerous lamellae radiating outward from the central axis (midline raphe) (Fig. 1). The anterior end of each capsule is composed of small lamellae which become larger towards the posterior end of the capsule. Two openings occur for
AND
RADERMAN-LITTLE
water circulation in each olfactory capsule, one anteriorally (incurrent naris) and one posteriorally (excurrent naris). From dye studies water circulation occurs across the dorsal surface of the olfactory lamellae and downward across the face of each lamella. The flow then rises along the lateral walls of the capsule and exits through the excurrent naris. The sensory region of an olfactory lamella is located adjacent to the midline raphe and projects a short distance laterally (Fig. 2). The sensory region is characterized as having fewer cilia per unit surface area than the indifferent epithelium (Figs. 2, 3). Also, the tips of the olfactory neurons with cilia radiating from their periphery can be observed at the surface of the sensory epithelium (Figs. 3, 4). A sharp interface exists between the sensory and indifferent epithelia of an olfactory lamella (Figs. 2, 3). The indifferent epithelium which covers the majority of the lamellar surface contains a thick mat of non-sensory cilia. During the initial preparation of this study a fortuitous fracture occurred in the transverse plane of a carbon dioxide critical point dried specimen of olfactory tissues as we were attempting to separate adjacent lamellae with dissecting needles. This fracture which occurred within the sensory region of the lamella revealed the underlying olfactory neurons.
Fig. 1. Low power scanning electron micrograph of one olfactory end (A), Posterior end (P), Midline raphe (m). x 53.
capsule.
Fig. 2. A portion of olfactory mucosa with olfactory lamellae projecting from the midline raphe (m). Arrows indicate the interface between sensory indifferent epithelium (ie). x 230. Fig. 3. The surface of an olfactory lamella at the interface thelium (se) and the indifferent epithelium (ie). x 4830.
Anterior laterally (se) and
between the sensory epi-
Fig. 4. Portion of the sensory region of an olfactory lamella. The tips of the olfactory receptors (arrow) appear as small buttons with olfactory cilia radiating from their periphery. x 14,000. Fig. 5. Scanning electron region of a lamella showing x 1470.
micrograph of a transverse fracture face with the sensory two cell layers of olfactory receptors and supporting cells.
Fig. 6. Higher magnification of a single olfactory cell layer shown in Fig. 5. Three prominent olfactory neurons are seen. One axon (arrow) is shown leaving the receptor cell proper. x 4625.
SEM
OF
CATFISH
OLFACTORY
Subsequent studies using the technique of carefully cracking the dried specimens with fine forceps or dissecting needles have allowed us to correlate surface features with the underlying cellular organization of the olfactory lamellae. Each lamella is two-cell layers thick applied to each side of a thin stroma (Fig. 5). The sensory region containing numerous olfactory neurons and supporting cells, is approximately 50 pm wide. The center of a lamella (Fig. 5) is a pathway for olfactory axons on their way to the pedunculated olfactory bulb located immediately adjacent to the olfactory capsule. The axon of a typical olfactory receptor can be seen to course ventrally then take a sharp turn laterally to join with other olfactory axons (Fig. 6). Similar types of fractures but occurring farther laterally along the lamella and outside the sensory area (Figs. 7, 8), lack olfactory neurons. At the lateral epithelial surface a thick mat of cilia is seen which is characteristic of non-sensory regions of the channel catfish olfactory lamellae. Discussion
The general composition of the olfactory epithelium of the channel catfish is similar to that found in other fishes (Kleerekoper, 1969) and distinct from that of the higher vertebrates (Graziadei, 1971) in its lack of Bowman’s glands. In fishes, mucus is released at the epithelial surface by supporting and goblet cells which may occur only within indifferent epithelium (Schulte and Hall, 1971) or be distributed in both indifferent and sensory epithelia (Schulte, 1972; Bertmar, 1973). In the present study, goblet cells were observed only in the indifferent epithelium. In the channel catfish the olfactory lamellar surface is a flat sheet of epithelium without
Fig. 7. Scanning electron micrograph region of a lamella. x 1360.
7
LAMELLAE
of a transverse
secondary folding seen in salmonids (Bertmar, 1972), lungfish (Theisen, 1972) and cod and haddock (Lowe and MacLeod, 1975). However, the sensory and indifferent epithelia are easily distinguished by surface features alone. The sensory epithelium located adjacent to the midline raphe is characterized as having fewer cilia than the indifferent epithelium which is in direct contrast to that reported for similar areas of the sea trout (,Bertmar, 1972) and the cod and haddock (Lowe and MacLeod, 1975). Also, a distinct interface occurs between sensory and indifferent epithelium, as was found for similar regions in the goldfish (Breipohl et al., 1974), brook trout (Andres, 1975) and for two cyprinodonts (Zeiske and Melinkat, 1976). In contrast a transition zone containing both indifferent epithelium and olfactory receptor cells has been observed in the eel (Schulte, 1972). The location and boundaries of the sensory epithelium of the channel catfish observed with the SEM correlates well with microelectrode placement and recordings from the lamellar surface of the same species (Caprio, 1977). Numerous olfactory receptor terminals bearing cilia occur along the surface of the sensory epithelium. Although terminals of olfactory neurons may show a great polymorphism, only a single receptor type was observed in the catfish, which was similar to type la receptors reported in the goldfish (Breipohl et al., 1973). Microvillous receptors seen in elasmobranchs (Reese and Brightman, 1970), lungfish and hagfish (Theisen, 1972, 1973) and in two cyprinodonts (Zeiske and Melinkat, 1976) were not observed in the catfish. The larger surface area of the indifferent epithelium is covered with a dense mat of cilia which propel streams of incoming solutions of water and dissolved chemicals between the lamellae and over the mucous
fracture
outside
Fig. 8. Higher magnification of a fracture through indifferent epithelium. goblet cell (gc), and the thick mat of cilia at the surface. x 1330.
the sensory Note the
8
CAPRIO
layer containing the cilia of the olfactory neurons. Whether the olfactory cilia contain the receptor sites for olfactory stimuli or whether they serve as guides for chemicals to reach the olfactory terminals containing the receptor sites is not known. With the techniques used in this study not only surface features were observed, but relationships between the internal cellular structures and their surface features could be obtained. A study utilizing similar methods on the surface morphology and internal structures of catfish taste buds which included accidental fractures of the COZ dried specimens was reported recently (Ovalle and Shinn, 1977). The present data on the location of the olfactory sensory epithelium of the channel catfish is similar to that reported in the light microscopical work on the olfactory mucosa of the brown bullhead, Ictalurus nebulosus (Holl, 1965) and to the electrode placement in the electrophysiological study of the olfactory receptors of the white catfish (Tucker and Suzuki, 1972). Thus, the arrangement of sensory versus indifferent epithelium may be
AND
RADERMAN-LITTLE
similar among the different species of Ictalurids. The catfish, which has been utilized for numerous behavioral and electrophysiological experiments and has recently been used to study stimulus-taste receptor binding (Krueger and Cagan, 1976), may prove to be a model system for biochemical studies on stimulus-olfactory receptor binding (Cancalon and Beidler, in press). The olfactory lamellae are easily removed from the nasal sac and may be sectioned further to remove the larger mass of indifferent epithelium leaving a high concentration of olfactory receptors which are highly sensitive to amino acid stimuli (Suzuki and Tucker, 1971; Caprio, 1977). Acknowledgements Supported by grants from the N.I.H., PHS NS-08814 and PHS NS-05288. We thank Drs Don Tucker and Lloyd M. Beidler for their interest and support. We also thank Mr William Miller for operation of the scanning electron microscope.
References ADRIAN, E. D. and LUDWIG, C. 1938. Nervous discharges from the olfactory organs of fish. J. Physiof., 94, 441460. ANDRES, K. H. 1975. Neue morphologische Grundlagen zur Physiologie des Riechens und Schmeckens. Arch. Oto-Rhino-Laryng., 20, 1-41 (KongreObericht). BERTMAR, G. 1972. Scanning electron microscopy of olfactory rosette in sea trout. Z. Zel[forsch. mikrosk. Anat., 128, 336-346. BERTMAR,G. 1973. Ultrastructure of the olfactory mucosa in the homing baltic sea trout Salmo trutta trutta. Mar. Biol., 19, 74-88. BREIPHOL, W., BIJVANK, G. J. and PFEFFERKORN,G. E. 1974. Scanning electron microscopy of various sensory receptor cells in different vertebrates. Scanning Electron Microscopy/l974 (Part III) Proceedings of the Workshop on Advances in Biomedical Applications of the SEM. IIT Research Institute, Chicago, Illinois, U.S.A., April. CANCALON, P. and BEIDLER, L. M. Isolation of olfactory cells and characterization of olfactory receptors. In Olfaction and Taste VI (ed. J. Le Magnen). Information Retrieval Ltd, London (in press). CAPRIO, J. 1977. Electrophysiological distinctions between the taste and smell of amino acids in catfish. Nature, Lond., 266, 850-851. GRAZIADEI, P. P. C. 1971. The olfactory mucosa of vertebrates. In Handbook of Sensory physiology, Vol. IV, pp. 27-58, by L. M. Beidler. Springer, Berlin-Heidelberg-New York. HOLL, A. 1965. Vergleichende morphologische und histologische Untersuchungen am Geruchsorgan der Knochenfische. Z. Morph. okol. Tiere, 54, 707-782. KLEEREKOPER,H. 1969. O&action in Fishes. Indiana University Press, Bloomington. KRUEGER, J. and CAGAN, R. H. 1976. Biochemical studies of taste sensation. Binding of L-3H alanine to a sedimentable fraction from catfish barbel epithelium. J. biol. Chem., 251, 88-97. LOWE, G. A. and MACLEOD, N. K. 1975. The ultrastructural organization of olfactory epithelium of two species of gadoid fish. J. Fish. Biol., 7, 529-532.
SEM
OF
CATFISH
OLFACTORY
LAMELLAE
0
OLMSTED,
J. M. D. 1918. Experiments on the nature of the sense of smell in the common cattish, ,~l,,~,w,r,s nrhulosrrs (Lesueur). Am. .J. Physiol., 46, 443458. OVALLY, W. K. and SHINN, S. L. 1977. Surface morphology of taste buds in cattish barbels. C’<,/l7‘i.s.~.Kr2.s.. 178, 375-384. PARKER, G. H. 1910. Olfactory reactions in fishes. J. Exp. Zoo/.. 8, 535-542. SCHC:LTE,E. 1972. Untersuchungen an der Regio olfactoria des Aals, Anguilla anguilla L. I. Feinstruktur des Riechepithels. Z. Zdforsch. mikrosk. Anat., 125, 210-228. Scti~ LTE, E. and HOLL, A. 1971. Feinstruktur des Riechepithels van Cdamoichthys ca/ahuric~ns J. A. Smtth (Pisces, Brachiopterygii). Z. Zellforsch. mikrosk. Anat., 120, 261-279. SUZLIKI, N. and TUCKER, D. 1971. Amino acids as olfactory stimuli in freshwater catfish. I~~ta/rwrc.\wt,o (Linn.). Camp. Biochrm. Physiol., 4OA, 399404. THFISEN, B. 1972. Ultrastructure of the olfactory epithelium in the Australian lungfish Neoc~vnro&s /or.~cri. Acm THEISEN,
zool.,
Stockh.,
53, 205-218.
B. 1973. The olfactory system in the hagfish Myxine ghrtinostr. I. Fine structure of the apical part of the olfactory epithelium. Acta zool., Stockh., 54, 271-284. TUCKER, D. and SUZUKI, N. 1972. Olfactory responses to schreckstoff of catfish. In Olfhction crnd Ta.\tc (ed. D. Schneider) Vol. IV, pp. 121-127. Wissenschaftliche Verlagsgesellschaft MBH, Stuttgart. Germany. ZEISKE, E. and MELINKAT, R. 1976. Ultrastructure studies on the epithelia of the olfactory organ ofcyprinodonts (Teleostei, Cyprinodontoidea). Cell 7%. Res., 172, 2455267.
&CELL
1978 IO (I) l-9
hhlishd byLongmonGKW~ Ltd.PrintedinCwot Bdain
JOHN
SCANNING CHANNEL
CAPRIO”
and RANDIE
RADERMAN-LITTLE
ELECTRON MICROSCOPY OF THE CATFISH OLFACTORY LAMELLAE ABSTRACT. The olfactory lamellae of the channel catfish (Icfalurrrs punctatm) are composed of sensory and indifferent (non-sensory) epithelia organized into two distinct regions on both surfaces of each lamella. The smaller sensory region located adjacent to the midline raphe has fewer cilia per unit surface area than the indifferent epithelium and contains the olfactory neurons whose ciliated dendritic terminals occur at the epithelial surface. The indifferent epithelium, comprising the greater surface area of the olfactory lamella, is covered with a dense mat of non-sensory cilia. Fractured carbon dioxide critical point dried lamellar tissue revealed the underlying cellular structure. The lamellae are composed of two layers of epithelium enclosing a thin stromal layer. Olfactory receptors were observed in the fractured tissue only within the sensory epithelium.
Introduction MORPHOLOGICAL studies on the olfactory organ of a variety of teleosts (Holl, 1965; Bertmar, 1972; Schulte, 1972; Theisen, 1972, 1973; Breipohl et al., 1973, 1974; Andres, 1975; Lowe and MacLeod, 197.5; Zeiske and Melinkat, 1976) have shown considerable variation in the distribution patterns of sensory versus indifferent areas of the olfactory epithelium. A description of the sensory regions of the olfactory organ of catfish is lacking with the exception of the light microscopical work of Ho11 (1965) on the olfactory organ of 18 species of teleosts including the brown bullhead {Ictalurus nebulosus). This paucity of information is especially surprising since catfish have long been known for their acute sense of smell (Parker, 1910; Olmsted, I91 8) and are continuing to be used as experimental animals in olfactory research (Suzuki and Tucker, 1971; Tucker and Suzuki, 1972; Caprio, 1977). During electrophysiological studies of the Department of Biological Science, Flofida State University, Tallahassee, Florida, U.S.A. * Current address: Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, U.S.A. Received
I7 October
1977.
olfactory receptor responses of the white catfish Ictalurus cam (Tucker and Suzuki, 1972) and the channel catfish (Caprio, 1977) it was noticed that the maximal amount of olfactory neural activity was recorded when the surface microelectrode was positioned against a discrete region of an olfactory lamella near the midline raphe. As the microelectrode was advanced laterally across the face of the lamella neural activity quickly declined; if the electrode was positioned along the dorsal edge of the lamella, neural activity was also absent. It was with this information that a SEM study was begun to determine whether the sensory and indifferent epithelial regions of the channel catfish olfactory lamellae were distinguishable by surface features alone and whether these regions correlated with the microelectrode positions. Materials and Methods
Channel catfish, approximately 75-l 50 g, were anesthetized with 0.02’% MS 222. The olfactory mucosa was dissected out, rinsed in Ringer’s and cut in small pieces. Specimens were fixed for 3 hr in 3 y0 glutaraldehyde in 0.1 M cacodylate or phosphate buffer (pH 7.4). For fixation in situ, fixative was dripped on the intact mucosa for I5 min and
2
CAPRIO
dissected under glutaraldehyde in After glutaraldehyde fixation the specimens were left in buffered 7.5 ‘? sucrose for at least 3 hr or overnight and then treated for 15-24 hr with 8 % glycerin in buffer. They were then post-fixed in 2% osmium tetroxide for 30 min, rinsed several times in buffer, and dehydrated in a graded acetone series. These procedures were carried out at 4°C. The specimens were dried by the critical point method with COZ. Using slight pressure some of the dried specimens were then cracked with fine forceps in order to expose inner cell surfaces. Each fragment was then mounted on stubs using double-stick tape or conductive silver paint, and coated with a 50-100 A layer of gold/palladium. Observations were made with a Cambridge scanning electron microscope model S4-10 at voltages of 5-20 kV. Photomicrographs were taken on positive-negative Polaroid film type 105. pieces vitro.
Results The two olfactory capsules of the channel catfish contain bilaterally symmetric olfactory rosettes composed of numerous lamellae radiating outward from the central axis (midline raphe) (Fig. 1). The anterior end of each capsule is composed of small lamellae which become larger towards the posterior end of the capsule. Two openings occur for
AND
RADERMAN-LITTLE
water circulation in each olfactory capsule, one anteriorally (incurrent naris) and one posteriorally (excurrent naris). From dye studies water circulation occurs across the dorsal surface of the olfactory lamellae and downward across the face of each lamella. The flow then rises along the lateral walls of the capsule and exits through the excurrent naris. The sensory region of an olfactory lamella is located adjacent to the midline raphe and projects a short distance laterally (Fig. 2). The sensory region is characterized as having fewer cilia per unit surface area than the indifferent epithelium (Figs. 2, 3). Also, the tips of the olfactory neurons with cilia radiating from their periphery can be observed at the surface of the sensory epithelium (Figs. 3, 4). A sharp interface exists between the sensory and indifferent epithelia of an olfactory lamella (Figs. 2, 3). The indifferent epithelium which covers the majority of the lamellar surface contains a thick mat of non-sensory cilia. During the initial preparation of this study a fortuitous fracture occurred in the transverse plane of a carbon dioxide critical point dried specimen of olfactory tissues as we were attempting to separate adjacent lamellae with dissecting needles. This fracture which occurred within the sensory region of the lamella revealed the underlying olfactory neurons.
Fig. 1. Low power scanning electron micrograph of one olfactory end (A), Posterior end (P), Midline raphe (m). x 53.
capsule.
Fig. 2. A portion of olfactory mucosa with olfactory lamellae projecting from the midline raphe (m). Arrows indicate the interface between sensory indifferent epithelium (ie). x 230. Fig. 3. The surface of an olfactory lamella at the interface thelium (se) and the indifferent epithelium (ie). x 4830.
Anterior laterally (se) and
between the sensory epi-
Fig. 4. Portion of the sensory region of an olfactory lamella. The tips of the olfactory receptors (arrow) appear as small buttons with olfactory cilia radiating from their periphery. x 14,000. Fig. 5. Scanning electron region of a lamella showing x 1470.
micrograph of a transverse fracture face with the sensory two cell layers of olfactory receptors and supporting cells.
Fig. 6. Higher magnification of a single olfactory cell layer shown in Fig. 5. Three prominent olfactory neurons are seen. One axon (arrow) is shown leaving the receptor cell proper. x 4625.
SEM
OF
CATFISH
OLFACTORY
Subsequent studies using the technique of carefully cracking the dried specimens with fine forceps or dissecting needles have allowed us to correlate surface features with the underlying cellular organization of the olfactory lamellae. Each lamella is two-cell layers thick applied to each side of a thin stroma (Fig. 5). The sensory region containing numerous olfactory neurons and supporting cells, is approximately 50 pm wide. The center of a lamella (Fig. 5) is a pathway for olfactory axons on their way to the pedunculated olfactory bulb located immediately adjacent to the olfactory capsule. The axon of a typical olfactory receptor can be seen to course ventrally then take a sharp turn laterally to join with other olfactory axons (Fig. 6). Similar types of fractures but occurring farther laterally along the lamella and outside the sensory area (Figs. 7, 8), lack olfactory neurons. At the lateral epithelial surface a thick mat of cilia is seen which is characteristic of non-sensory regions of the channel catfish olfactory lamellae. Discussion
The general composition of the olfactory epithelium of the channel catfish is similar to that found in other fishes (Kleerekoper, 1969) and distinct from that of the higher vertebrates (Graziadei, 1971) in its lack of Bowman’s glands. In fishes, mucus is released at the epithelial surface by supporting and goblet cells which may occur only within indifferent epithelium (Schulte and Hall, 1971) or be distributed in both indifferent and sensory epithelia (Schulte, 1972; Bertmar, 1973). In the present study, goblet cells were observed only in the indifferent epithelium. In the channel catfish the olfactory lamellar surface is a flat sheet of epithelium without
Fig. 7. Scanning electron micrograph region of a lamella. x 1360.
7
LAMELLAE
of a transverse
secondary folding seen in salmonids (Bertmar, 1972), lungfish (Theisen, 1972) and cod and haddock (Lowe and MacLeod, 1975). However, the sensory and indifferent epithelia are easily distinguished by surface features alone. The sensory epithelium located adjacent to the midline raphe is characterized as having fewer cilia than the indifferent epithelium which is in direct contrast to that reported for similar areas of the sea trout (,Bertmar, 1972) and the cod and haddock (Lowe and MacLeod, 1975). Also, a distinct interface occurs between sensory and indifferent epithelium, as was found for similar regions in the goldfish (Breipohl et al., 1974), brook trout (Andres, 1975) and for two cyprinodonts (Zeiske and Melinkat, 1976). In contrast a transition zone containing both indifferent epithelium and olfactory receptor cells has been observed in the eel (Schulte, 1972). The location and boundaries of the sensory epithelium of the channel catfish observed with the SEM correlates well with microelectrode placement and recordings from the lamellar surface of the same species (Caprio, 1977). Numerous olfactory receptor terminals bearing cilia occur along the surface of the sensory epithelium. Although terminals of olfactory neurons may show a great polymorphism, only a single receptor type was observed in the catfish, which was similar to type la receptors reported in the goldfish (Breipohl et al., 1973). Microvillous receptors seen in elasmobranchs (Reese and Brightman, 1970), lungfish and hagfish (Theisen, 1972, 1973) and in two cyprinodonts (Zeiske and Melinkat, 1976) were not observed in the catfish. The larger surface area of the indifferent epithelium is covered with a dense mat of cilia which propel streams of incoming solutions of water and dissolved chemicals between the lamellae and over the mucous
fracture
outside
Fig. 8. Higher magnification of a fracture through indifferent epithelium. goblet cell (gc), and the thick mat of cilia at the surface. x 1330.
the sensory Note the
8
CAPRIO
layer containing the cilia of the olfactory neurons. Whether the olfactory cilia contain the receptor sites for olfactory stimuli or whether they serve as guides for chemicals to reach the olfactory terminals containing the receptor sites is not known. With the techniques used in this study not only surface features were observed, but relationships between the internal cellular structures and their surface features could be obtained. A study utilizing similar methods on the surface morphology and internal structures of catfish taste buds which included accidental fractures of the COZ dried specimens was reported recently (Ovalle and Shinn, 1977). The present data on the location of the olfactory sensory epithelium of the channel catfish is similar to that reported in the light microscopical work on the olfactory mucosa of the brown bullhead, Ictalurus nebulosus (Holl, 1965) and to the electrode placement in the electrophysiological study of the olfactory receptors of the white catfish (Tucker and Suzuki, 1972). Thus, the arrangement of sensory versus indifferent epithelium may be
AND
RADERMAN-LITTLE
similar among the different species of Ictalurids. The catfish, which has been utilized for numerous behavioral and electrophysiological experiments and has recently been used to study stimulus-taste receptor binding (Krueger and Cagan, 1976), may prove to be a model system for biochemical studies on stimulus-olfactory receptor binding (Cancalon and Beidler, in press). The olfactory lamellae are easily removed from the nasal sac and may be sectioned further to remove the larger mass of indifferent epithelium leaving a high concentration of olfactory receptors which are highly sensitive to amino acid stimuli (Suzuki and Tucker, 1971; Caprio, 1977). Acknowledgements Supported by grants from the N.I.H., PHS NS-08814 and PHS NS-05288. We thank Drs Don Tucker and Lloyd M. Beidler for their interest and support. We also thank Mr William Miller for operation of the scanning electron microscope.
References ADRIAN, E. D. and LUDWIG, C. 1938. Nervous discharges from the olfactory organs of fish. J. Physiof., 94, 441460. ANDRES, K. H. 1975. Neue morphologische Grundlagen zur Physiologie des Riechens und Schmeckens. Arch. Oto-Rhino-Laryng., 20, 1-41 (KongreObericht). BERTMAR, G. 1972. Scanning electron microscopy of olfactory rosette in sea trout. Z. Zel[forsch. mikrosk. Anat., 128, 336-346. BERTMAR,G. 1973. Ultrastructure of the olfactory mucosa in the homing baltic sea trout Salmo trutta trutta. Mar. Biol., 19, 74-88. BREIPHOL, W., BIJVANK, G. J. and PFEFFERKORN,G. E. 1974. Scanning electron microscopy of various sensory receptor cells in different vertebrates. Scanning Electron Microscopy/l974 (Part III) Proceedings of the Workshop on Advances in Biomedical Applications of the SEM. IIT Research Institute, Chicago, Illinois, U.S.A., April. CANCALON, P. and BEIDLER, L. M. Isolation of olfactory cells and characterization of olfactory receptors. In Olfaction and Taste VI (ed. J. Le Magnen). Information Retrieval Ltd, London (in press). CAPRIO, J. 1977. Electrophysiological distinctions between the taste and smell of amino acids in catfish. Nature, Lond., 266, 850-851. GRAZIADEI, P. P. C. 1971. The olfactory mucosa of vertebrates. In Handbook of Sensory physiology, Vol. IV, pp. 27-58, by L. M. Beidler. Springer, Berlin-Heidelberg-New York. HOLL, A. 1965. Vergleichende morphologische und histologische Untersuchungen am Geruchsorgan der Knochenfische. Z. Morph. okol. Tiere, 54, 707-782. KLEEREKOPER,H. 1969. O&action in Fishes. Indiana University Press, Bloomington. KRUEGER, J. and CAGAN, R. H. 1976. Biochemical studies of taste sensation. Binding of L-3H alanine to a sedimentable fraction from catfish barbel epithelium. J. biol. Chem., 251, 88-97. LOWE, G. A. and MACLEOD, N. K. 1975. The ultrastructural organization of olfactory epithelium of two species of gadoid fish. J. Fish. Biol., 7, 529-532.
SEM
OF
CATFISH
OLFACTORY
LAMELLAE
0
OLMSTED,
J. M. D. 1918. Experiments on the nature of the sense of smell in the common cattish, ,~l,,~,w,r,s nrhulosrrs (Lesueur). Am. .J. Physiol., 46, 443458. OVALLY, W. K. and SHINN, S. L. 1977. Surface morphology of taste buds in cattish barbels. C’<,/l7‘i.s.~.Kr2.s.. 178, 375-384. PARKER, G. H. 1910. Olfactory reactions in fishes. J. Exp. Zoo/.. 8, 535-542. SCHC:LTE,E. 1972. Untersuchungen an der Regio olfactoria des Aals, Anguilla anguilla L. I. Feinstruktur des Riechepithels. Z. Zdforsch. mikrosk. Anat., 125, 210-228. Scti~ LTE, E. and HOLL, A. 1971. Feinstruktur des Riechepithels van Cdamoichthys ca/ahuric~ns J. A. Smtth (Pisces, Brachiopterygii). Z. Zellforsch. mikrosk. Anat., 120, 261-279. SUZLIKI, N. and TUCKER, D. 1971. Amino acids as olfactory stimuli in freshwater catfish. I~~ta/rwrc.\wt,o (Linn.). Camp. Biochrm. Physiol., 4OA, 399404. THFISEN, B. 1972. Ultrastructure of the olfactory epithelium in the Australian lungfish Neoc~vnro&s /or.~cri. Acm THEISEN,
zool.,
Stockh.,
53, 205-218.
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