differential projections of ciliated and microvillous olfactory receptor cells in the...

Differential Projections of Ciliatedand Microvillous Olfactory Receptor

Cells in the Catfish, Ictalurus punctatus

YASUHIRO MORITA AND THOMAS E. FINGER*

Department of Cellular and Structural Biology, Rocky Mountain Taste and Smell Center,University of Colorado School of Medicine, Denver, Colorado 80262

ABSTRACTThe primary olfactory projections of channel catfish Ictalurus punctatus have been

examined with postmortem tracing by using either 1,18-dioctadecyl-3,3,38,38-tetramethylindo-carbocyanine perchlorate or 1,1-dilinoleyl-3,3,38,38-tetramethylindocarbocyanine perchlorate(DiI). Following DiI deposition into small areas in different parts of the posterior olfactorybulb, olfactory sensory neurons always were labeled throughout the olfactory epithelium. Noobvious topographical mapping exists between the epithelium and olfactory bulb. Thedifferent dye placements, however, did result in labeling of different morphologies of receptorcells, depending on the site of injection. Retrogradely labeled neurons in the olfactoryepithelium were classified into three types on the basis of their height: tall, intermediate, andshort. Tall olfactory sensory neurons had perikarya at the bottom one-fourth of theepithelium, extended slender dendrites to the epithelial surface, and possessed numerous ciliaon the apical dendritic tips. These tall olfactory sensory neurons were labeled predominantlyfollowing DiI applications to the ventral part of the posterior olfactory bulb. In contrast, theshort olfactory sensory neurons had perikarya situated within the superficial half of theepithelium and with short apical dendrites bearing microvilli. These short olfactory sensoryneurons projected predominantly to the dorsal, posterior olfactory bulb. Thus, short microvil-lous receptor cells and tall ciliated receptor cells connect to different parts of the olfactorybulb, although the receptor cells are intermingled within the olfactory epithelium. Becausedifferent parts of the olfactory bulb are thought to respond preferentially to different classes ofodorants, these results suggest that receptor cell morphology may be related to odorantquality detection. In addition, to compare this study with previous in vivo studies, Fluoro-Goldwas injected in vivo into either the olfactory bulb or intraperotineally. These in vivo studiesshow that so-called ‘‘type II ciliar receptor cells’’ of the nonsensory epithelium are labelednonselectively by blood-borne substances, but they are not labeled by postmortem injections ofDiI anywhere in the olfactory bulb. J. Comp. Neurol. 398:539–550, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: olfactory bulb; vomeronasal organ; glomerulus; smell

Throughout the vertebrate lineage, the receptor cells ofthe olfactory epithelia, including the vomeronasal organand the septal organ of Masera, are bipolar neurons withdendritic processes that radiate upward to the surface ofthe epithelium. These apical dendritic processes beareither microvilli or cilia, which extend into the mucus layeroverlying the epithelium and which house the molecularreceptors mediating chemosensory transduction. Collec-tively, such bipolar receptor cells of the first cranial nervesystem can be considered to be varieties of olfactoryreceptor neurons (ORNs). In many terrestrial vertebrates,the microvillous and ciliated ORNs are largely segregatedin separate receptor organs. Microvillous cells constitutethe large majority of ORNs in the vomeronasal organ,

although ciliated sensory cells have been reported in somespecies (Saint Girons and Zylberberg, 1992). Conversely,the main olfactory sensory epithelium consists mostly ofciliated receptor cells, although microvillous ORNs arepresent in many species, including man (Moran et al.,1982; Rowley et al., 1989; Morrison and Costanzo, 1990,

Grant sponsor: National Institutes of Health; Grant number: PO1DC00244.

*Correspondence to: Thomas E. Finger, Department of. Cellular andStructural Biology, UCHSC B-111, 4200 East Ninth Avenue, Denver, CO80262. E-mail: [email protected]

Received 26 November 1997; Revised 25 March 1998; Accepted 10 May1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 398:539–550 (1998)

r 1998 WILEY-LISS, INC.

1992; Miller et al, 1995; however, see Carr et al., 1991).Because of this segregation, many investigators havepostulated that vomeronasal, microvillous ORNs respondto nonvolatile, pheromonal substances, whereas the cili-ated ORNs respond to volatile components (for review, seeBargmann, 1997). Microvillous ORNs, however, are notalways tuned to pheromones. In some species, e.g., gartersnakes, microvillous ORNs of the vomeronasal organrespond to odorant cues that are used in tracking prey(Kubie and Halpern, 1979).

In teleost fishes, both ciliated and microvillous receptorcells are present but are intermingled within the epithe-lium; no vomeronasal organ exists (e.g. Yamamoto, 1982;Cancalon, 1983; Erickson and Caprio, 1984; Moran et al.,1992). Several investigators have suggested that the differ-ent morphological types of ORNs in fishes respond todifferent classes of odorants (Thommesen, 1982, 1983), butthere is no general agreement on which cell type serveswhich function. In electrophysiological studies on salmo-nids, Thommesen describes differences in responsivenessbetween areas of epithelium that are rich in microvillousORNs compared with areas that are rich in ciliated ORNs.These studies conclude that sensitivity to bile salts corre-lates with abundant ciliated receptor cells, whereas sensi-tivity to amino acids correlates with a high abundance ofmicrovillous cells. In contrast, degeneration-regenerationstudies in goldfish suggest that sensitivity to amino acidsis related to the presence of ciliated ORNs, whereassensitivity to steroid pheromones correlates with the pres-ence of microvillous ORNs (Zippel et al., 1997a,b). Correla-tive electrophysiological and anatomical studies on chan-nel catfish failed to reveal functional differences betweenmicrovillous-rich and ciliated, ORN-rich areas (Ericksonand Caprio, 1984). Thus, the relative functional contribu-tion of each type of ORN is controversial.

In fishes as well as in all other vertebrates, the differ-ences in sensitivity of the various ORNs are sorted out intheir projection onto the olfactory bulb (OB). Thus, the OBappears to contain a mapping of odorant sensitivity, sothat different parts or glomeruli of the OB respond opti-mally to a limited group of odorants. The principle ofodotopic mapping is supported by electrophysiological(Kauer et al., 1987; Mori et al., 1992), anatomical (Skeen,1977; Stewart et al., 1979), and molecular (Ressler et al.,1993; Vassar et al., 1993; Mombaerts et al., 1996) evidencein a variety of species, including zebrafish (Friedrich andKorsching, 1997a,b) and goldfish (Hanson et al., 1997).

Thus, because the OB is organized into different func-tional domains according to receptor specificity, to testindirectly whether specific types of ORNs have differentodorant specificities, we used either 1,18-dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI-C18-[3]) or 1,1-dilinoleyl-3,3,38,38-tetramethylindocarbo-cyanine perchlorate (DiI-2; both referred herein to as DiI)as a retrograde marker to visualize the ORNs that projectto different parts of the OB. Because DiI labels fiberspassing through the region of the injection site as well asthose terminating within the injection site, and becauseolfactory axons enter from the anterior end of the OB, ourstudies were limited to application of the dye to differentquadrants in the posterior part of the OB. Thus, we werelabeling only those axons that terminated within theposterior portions of the olfactory bulb. These studies havebeen reported in abstract form at the 18th meeting of the

Association for Chemoreception Sciences (Morita et al.,1996).

MATERIALS AND METHODS

Juvenile channel catfish, Ictalurus punctatus, rangingfrom 11 cm to 15 cm in standard length, were used in thesestudies. The animals are not sexually mature at this time.Animals were obtained commercially from the Hopper-Stephens Hatcheries (Lenoke, AR) and kept for at least 1week in aquaria of the Animal Resources Center, Univer-sity of Colorado Health Sciences Center. All procedureswere carried out with the approval of the InstitutionalAnimal Care and Use Committee of this institution.

In vivo studies

In four cases, aqueous Fluoro-Gold (2%) was injected invivo either directly into the OB or, for controls, intraperito-neally to test for nonspecific uptake from circulation. Aftera 4–7 day postinjection survival, these animals wereanesthetized in 3-aminobenzoic acid ethyl ester methane-sulfonate salt (MS222; 1:5,000) and perfused with 4%buffered paraformaldehyde (0.1 M phosphate buffer, pH7.2). All of these Fluoro-Gold-injected animals, as de-scribed below, exhibited wide-spread labeling of cells in thenonsensory epithelium. The results of these cases arepresented only for comparison with the postmortem DiIresults and with a previously published study (Muller andMarc, 1984) that showed unusual presumed receptor cells(‘‘type II ciliar cells’’) within the nonsensory epithelium.

Postmortem studies

Light microscopy. Two different forms of DiI, eitherDiI-C18-[3] or DiI-2 (Molecular Probes, Eugene, OR), wereused as postmortem retrograde tracers. No appreciabledifferences in the quality or type of staining was observedfor these two dyes.

For these postmortem studies, animals were anesthe-tized with a 1:10,000 dilution of MS222 and perfusedtranscardially with buffered paraformaldehyde, as de-scribed above. The nasal rosettes and OB were dissectedout complete with the surrounding bone and connectivetissue to provide physical support. This tissue block waspermitted to remain in fixative for at least an additional 2days. To prevent inadvertent spread of the dye, the olfac-tory rosette was covered with liquid agar prior to dyeapplication.

Small crystals of DiI were inserted under visual controlinto one of the posterior quadrants of the OB: dorsal,ventral, medial, or lateral (Fig. 1). In catfish, the fibers ofthe olfactory nerve enter the OB from its anterior face andspread across the surface of the bulb to terminate inglomeruli situated around nearly the entire anteroposte-rior extent of the spherical OB. Attempts were made torestrict the dye crystal to the outer few hundred microns ofthe bulb, i.e. within the zone of termination of the primaryolfactory axons. Toward this end, the dye crystals wereapplied on the end of a broken glass micropipette and byusing a posterior approach. In a few cases, to visualize allolfactory receptor cells, DiI dissolved in methyl formamidewas applied to the distal end of the cut olfactory nerve as itentered the OB.

After the dye crystal was put in place, the tissue wascovered with agar to prevent migration of the crystal awayfrom the site of application. The tissue block then was

540 Y. MORITA AND T.E. FINGER

placed into buffered paraformaldehyde at room tempera-ture for 7–21 days to permit diffusion of the dye. After theappropriate time, as determined empirically, the olfactoryepithelium and bulb were dissected free of the bone andconnective tissue and were embedded in egg yolk. Theblock was fixed in 4% buffered paraformaldehyde over-night and then cut at 50 µm on a Vibratome. Representa-tive sections were photographed. In five cases, to assessthe distribution of labeled cells following each dye applica-tion, the lamellae were cut free from the median raphe andflat mounted. Both the anterior and posterior surfaces ofthe lamellae were photographed at low power to documentthe receptor cell distribution. All specimens were ob-served with epifluorescence on a Zeiss standard micro-scope.

The extent of the injection was assessed either bysectioning of the olfactory bulb in the horizontal plane orby visual inspection of the injection site 7–10 days afterdye application. Figure 1B shows that fascicles of labeledfibers usually could be seen radiating rostralward from thedye injection site. No labeled fascicles ever extendedaround the bulb in the transverse plane. Sections throughthe injection sites showed that all injections examined inthis manner were restricted to the outer one-third of the

bulb, i.e., no injections extended deeply enough into theOB to label olfactory nerve fibers on the side of the bulbopposite from the injection site.

Electron microscopy. To investigate the ultrastruc-ture of the retrogradely labeled olfactory receptor neurons,

Fig. 1. A: Drawing of a parasagittal section through the olfactoryorgan and the olfactory bulb (OB). To avoid labeling fibers of passage,either 1,18-dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlor-ate or 1,1-dilinoleyl-3,3,38,38-tetramethylindocarbocyanine perchlor-ate (DiI) was applied only to the posterior part of the OB, because theolfactory nerve (on) enters the anterior end of the OB. B: Photomicro-graph of a ventral view of the dissected preparation of the olfactoryorgan and OB 3 days after dye application to the posterior ventralquadrant. Small fascicles of labeled fibers (arrows) can be seenemerging from the dye application site. ot, Olfactory tract.

Fig. 2. A: Semischematic drawing of the olfactory rosette from acatfish. The rosette has been transected to reveal the lamellarorganization. The sensory epithelium (se) occupies the dorsal centralpart of the epithelium of each lamellar pair, i.e., the region surround-ing the central raphe. Line B shows the approximate plane of sectionof B. B: Horizontal section through the olfactory organ of a catfish. Thelamellae, which bear sensory epithelium (se) and nonsensory epithe-lium (nse), are supported by a central raphe. The channels betweenthe lamellae end laterally as blind pouches but are interconnectedventrally. Toluidine blue-stained preparation. C: Enlargement fromthe area indicated by the box in B showing the transition fromnonsensory epithelium (distal to the raphe) and sensory epithelium(proximal). Prominent, long cilia are evident above nonsensory epithe-lium but not sensory epithelium. on, Olfactory nerve.

OLFACTORY RECEPTOR CELL PROJECTIONS 541

olfactory epithelia from selected postmortem tracing caseswere photoconverted with diaminobenzidine. For thesecases, the nasal cavity was gently flushed with 5 ml ofbuffered 0.3% glutaraldehyde-4% paraformaldehyde, pH7.2, after anesthetization but immediately prior to trans-cardial perfusion with 4% buffered paraformaldehyde.This treatment was found to stabilize the epithelial sur-face, producing superior ultrastructural results withoutgreatly enhancing the background tissue fluorescence. Dyeapplication and handling procedures otherwise were simi-lar to the light microscopy cases. For the photoconversionprotocol, following the procedure of Sandell and Masland(1988), 1.25 mg/ml of diaminobenzidine were dissolved inTris buffer, pH 8.5. The sectioned epithelium was placedinto this solution and exposed to epifluorescence illumina-tion for 5–15 minutes until a brown precipitate appeared.Following photoconversion, the tissues were postfixed inbuffered 2% glutaraldehyde, osmicated, and embeddedfollowing the usual electron microscopy protocols (Moranet al., 1992). Thin sections were examined under a PhilipsCM 10 electron microscope. Six short ORNs and six tallORNs were photoconverted and examined under the elec-tron microscope.

Statistical analysis

To test whether the site of dye application was related tothe type of ORN labeled, a multiple analysis of variance(MANOVA; super-ANOVA) was performed by using thesite as the between factor and by using the size of thelabeled ORNs as the dependent variable. MANOVA wasutilized to take into account the likelihood that the num-bers of different sized ORNs were not independent of oneanother in each case. Wilk’s Lambda was 0.0001.

Electronic figure composition

All photomicrographs were scanned from film imagesand processed in Photoshop (Adobe Systems, MountainView, CA) on Macintosh computers (Apple Computer,Cupertino, CA). Routine adjustments to contrast and

brightness were made electronically. Occasional dirt speckswere removed electronically from areas of the figures thatdid not contain images of tissue.

RESULTS

In the 11–15 cm catfish used in this study, each nasalrosette contains 25–32 symmetric pairs of lamellae thatare separated by the nasal raphe (Fig. 2A). Several smallerlamellae at the rostral end of the rosette often are asymmet-ric. The number of lamellae on the two sides of the raphe isnot always equal. Each lamella has an anterior face and aposterior face, each of which contains both sensory andnonsensory epithelia. The sensory portion of the epithe-lium, as reported by others (Cancalon, 1983), lies close tothe nasal raphe; the distal two-thirds of each lamella arecovered by nonsensory epithelium. Histologically, the sen-sory and nonsensory epithelia are easily discernible (Fig.2B). The olfactory epithelium has a broad, upper nuclearlayer that is four or five nuclei high, whereas the nonsen-sory epithelium has only a single layer of suprabasalnuclei. Furthermore, the nonsensory epithelium is coveredby a dense mat of cilia that is visible in toluidine blue-stained sections, whereas the surface of the olfactoryepithelium appears relatively bare in such preparations(Fig. 2B). Rare, isolated ORNs may be found within thenominal confines of the nonsensory epithelium, as de-scribed below, but these are always close to the boundarybetween these tissues.

Fluoro-Gold injections

A previous study (Muller and Marc, 1984) described atype II ‘‘ciliar receptor cell’’ in the nonsensory epithelium ofcatfish following injections of horseradish peroxidase (HRP)into the OB. These putative receptor cells are described ashaving a broad distal process that measure 3.5–5.0 µm inwidth compared with the 1.5–2.0 µm wide apex of theclassical ciliated (type I) ORNs in catfish. We observelabeled cells with such broad apical processes not only

Fig. 3. A,B: Photomicrograph of a horizontal section through thenasal epithelium (cf. Fig. 2B) following in vivo injection of Fluoro-Gold.Numerous bipolar cells are labeled within the nonsensory epithelium(nse); often, a process can be seen extending to the basal lamina.

B: Higher magnification of a pair of labeled cells. These labeled cellsappear similar to the type II ciliar cells described by Muller and Marc(1984) in terms of having a broad (3–5 µm) apical surface. se, Sensoryepithelium.


following in vivo Fluoro-Gold injections into the OB butalso after injections into the peritoneal cavity. Followingany of these injections, labeled cells were of various

heights, but they included a type of elongate cell thatextended an apical process to the epithelial surface (Fig.3). This apical process was often 1–2 µm wide in its course

Fig. 4. A: Photomicrograph showing a wholemount preparation ofan entire lamella, i.e., one side of a lamellar pair. The raphe is to theleft; the plane of focus is beneath the epithelial surface, showing thefascicles of DiI-labeled fibers coursing from the sensory epithelium (se)toward the ventrally-situated olfactory nerve (on) bundles. The la-beled olfactory receptor neurons (ORNs) appear as out-of-focus blurswithin the sensory epithelium. Dorsal is up. B: Semischematic draw-ing of a pair of lamellae flanking the median raphe. The sensoryepithelium (white area) is situated dorsomedial to the nonsensoryepithelium (nse; gray area). The box indicates the area shown in A.

C: Photomicrograph similar to that shown in A but at a highermagnification and focused on the epithelial surface. Numerous punc-tate, labeled ORN apices are visible within the sensory area of theepithelium. Fascicles of labeled axons can be seen coursing (out-of-focus) beneath the surface of the nonsensory region of the epitheliumen route to the olfactory nerve, which lies ventrally. D: Semischematicdrawing of a pair of lamellae flanking the median raphe. The sensoryepithelium (white area) is situated dorsomedial to the nonsensoryepithelium (gray area). The box indicates the area of shown in C. Scalebars 5 100 µm.


through the upper half of the epithelium, but it broadenedto span approximately 3–5 µm at the epithelial surface(see Fig. 3B, inset). Occasionally, a thin basal process couldbe seen extending toward, but not past, the basal lamina.These various morphological characteristics led us toequate these labeled cells with the type II ciliar cell ofMuller and Marc.

The type of cell labeled in the nonsensory epitheliumwas similar regardless of the site of injection. Accordingly,the label in these cells was attributed to nonspecific uptakeof blood-borne substances. Because these cells were neverlabeled by postmortem injections of DiI into the OB (seebelow), we do not consider the type II ciliar cell to be anolfactory receptor neuron. Although the majority of theselabeled cells lies in the nonsensory epithelium, followingintraperitoneal injections, similar, elongate, labeled cellsalso occur rarely within the sensory epithelium (Fig. 3A).

DiI injections

Following application of DiI to any of the sites in theposterior OB, retrogradely labeled ORNs occurred through-out the olfactory epithelium of all lamellae. Retrogradelylabeled axons of ORNs could be followed in arcing trajecto-ries within each lamella (Fig. 4A). Axons arising fromORNs in the proximal parts of the lamella course ventrallythrough the lamina propria to join the olfactory nervebundles beneath each rosette. In contrast, axons originat-ing from outlying regions of the rosette arc marginally andventrally, beneath the nonsensory epithelium, to reach theunderlying nerve bundles (Fig. 4A). Only a few, scattered,labeled ORNs could be found within the nominal area ofthe nonsensory epithelium; these always were situatednear the boundary between olfactory and nonsensoryareas (Fig. 4C).

Within the olfactory epithelium, a diversity of receptorcell morphologies could be discerned according to theirheight and structure (Fig. 5). To classify the ORNs (at leastcrudely), the depth of the epithelium was divided intoquadrants, and the location of the nucleus of each receptorwas recorded. A layer of endogenous fluorescence in the

upper three quadrants of the epithelium (Fig. 6B) provideda convenient intrinsic marker of depth. Retrogradely la-beled ORNs were categorized as tall (nucleus in the lowestquadrant), intermediate (nucleus in the third lowest quad-rant), or short (nucleus in the upper half). Few ORNs had anucleus in the uppermost quadrant of the epithelium. AllORNs had dendrites apparently extending to reach theepithelial surface (Fig. 5). All of these cells met themorphological criteria for being mature ORNs, in that they1) were labeled retrogradely from the OB; 2) exhibited afine, axon-like process emerging from their base andpenetrating the basal lamina; and 3) had an apical den-drite reaching to the epithelial surface. In most cases, long,filamentous processes could be seen extending outwardfrom the apical knob of intermediate and tall ORNs (Figs.5, 6A). These labeled apical processes were assumed to becilia based on their length and the ability to resolve themat light microscopic levels. Few short ORNs exhibitedfluorescent cilia.

Differential bulbar connectivity

In cases in which the DiI was restricted to one of theposterior quadrants of the OB, different sizes of ORNswere labeled, depending on the site of the dye application.(Table 1, Fig. 6A,B). The extent and intensity of labelingvaried somewhat, depending on the diffusion time anddistance as well as the size of the DiI crystal used.However, the predominant type and size of ORN that waslabeled from a given quadrant was consistent. In cases ofdye application to the ventral quadrant, the majority oflabeled ORNs were tall, whereas, after dye application tothe dorsal OB, the majority were short. On the other hand,dye application to the medial or lateral quadrants resultedin labeling of all types of ORNs.

Statistical analysis of these data (Table 1) shows thatthe four sites of injection have different percentages ofshort (F 5 17.19; degrees of freedom [df] 5 3; P , 0.0001)and tall (F 5 20.62; df 5 3; P , 0.001) ORNs but do notdiffer with respect to percentage of labeled intermediateneurons. Post-hoc comparisons show that significantly

Fig. 5. Nine photomicrographs showing the variety of olfactoryreceptor neurons (ORNs) that were labeled following DiI injectionsinto the olfactory bulb. For purposes of statistical analysis, we dividedthese into three size groups according to the position of the nucleuswithin the height of the epithelium: short, intermediate, and tall. This

is not meant to imply that these represent unique or distinctive types.Indeed, a continuum of sizes can be found, especially between theintermediate and tall groups.(cf. far right ORN in the intermediategroup with the far left ORN in the tall group). The level of the basallamina is indicated.


fewer tall ORNs were labeled from the dorsal OB than forany other application site, and significantly more shortORNs were labeled from dorsal OB compared with eitherthe ventral or medial sites (Scheffe test; P , 0.05).Injections into the lateral OB also resulted in significantlymore labeled short ORNs than the ventral site. In sum-mary, short ORNs project differentially to the dorsalposterior OB, whereas tall ORNs generally do not projectto this region.

Intralamellar distribution

Wholemounts of individual labeled lamellae were usedto plot the distribution of labeled receptor cells followingthe dorsal and ventral bulbar injections, because theseinjections preferentially labeled either tall or short ORNs(see above). Labeled ORNs were distributed across theentire extent of the olfactory epithelium of each lamella.No consistent segregation of tall or short ORNs to either

Fig. 6. Photomicrographs of the periraphe region of the olfactoryorgan after postmortem application of DiI to different quadrants of theposterior olfactory bulb (OB). A: Ventral dye application. Tall olfactory

receptor neurons (ORNs) predominate. B: Dorsal dye application.Many labeled short cells are evident, although some tall ORNs also areapparent.


paramedian or peripheral portions of the receptor epithe-lium was apparent (Fig. 7).

Dendritic morphology

In the fluorescence microscope, long (3–5 µm) cilia oftencould be observed extending from the tall and intermedi-ate ORNs (Figs. 5, 6A), whereas short receptors seldomexhibited such apical extensions. To determine whetherthe apical specialization differed on tall and short ORNs,several of each type were photoconverted for ultrastruc-tural analysis following either dorsal or ventral place-ments of DiI into the OB. The tall ORNs bore cilia at theirapex (Fig. 8A), as has been described for teleost ciliatedORNs (Yamamoto, 1982). Often, labeled cilia could befound above the epithelial surface tens of microns from thelabeled apical dendrite. In contrast, the short ORNs hadmicrovillous extensions reaching 1.5–2.0 µm from theapical dendrite (Fig. 9). For both types of labeled ORN, theapical morphology was obvious, and each type of ORNexhibited well-preserved intracellular organelles, e.g., mi-tochondria, nucleus, and, where present, ciliary basalbodies.

In summary, we have shown in a catfish that a type ofmicrovillous ORN with a short cell body projects preferen-tially to the dorsal quadrant of the posterior part of theOB. Conversely, a type of tall, ciliated ORN projectspreferentially to the ventral OB. Furthermore, the so-called type II ciliar cell, which Muller and Marc (1984)previously suggested to be a type of ORN, does not have anaxon contacting the OB; therefore, it is not an ORN.

TABLE 1. Relative Distribution of Short, Intermediate, and Tall OlfactoryReceptor Neurons After Injections of 1,18-Dioctadecyl- or

3,3,38,38-Tetramethylindocarbocyanine Perchlorate Into DifferentParts of the Olfactory Bulb1

Injection site

Percentage of labeled ORNsTotalORNsTall* Intermediate Short*

Ventral1 79.7 13.1 7.1 2,5332 89.8 6.9 3.2 1,5023 60.6 32.9 6.5 4334 74.3 21.0 4.7 1,6075 62.0 27.8 10.1 503

Mean (total n) 73.3 20.3 6.3a,b 6,578Dorsal

1 10.3 18.7 71.0 2522 0.0 12.0 88.1 4523 0.3 11.5 88.3 3754 3.4 16.2 80.5 507

Mean (total n) 3.5c 14.6 82a 1,586Medial

1 17.9 36.9 45.2 842 14.6 27.0 58.4 3,1913 72.9 20.1 7.0 4,1284 44.9 37.7 17.4 867

Mean (total n) 37.6 30.4 32a 8,270Lateral

1 6.1 51.2 42.7 822 20.9 24.5 54.5 3,4263 3.5 14.8 81.6 365

Mean (total n) 10.2 30.2 59.6b 3,873Overall mean and total 35.1 23.3 41.6 20,307

1The numbered rows under each site refer to different cases. Multiple analysis ofvariance (MANOVA) showed that the tall and short olfactory receptor neurons (ORNs)had significantly different distributions across the four injection sites. The dorsalinjections labeled significantly fewer tall ORNs than injections at any other site andsignificantly more short ORNs than the ventral or medial injections.*The four injection sites had significantly different percentages of short (P , 0.0001)and tall (P , 0.05) ORNs (MANOVA).aDorsal injections were significantly (P , 0.05) more than ventral or medial injections.bLateral injections were significantly (P , 0.05) more than ventral injections.cDorsal injections were significantly (P , 0.05) different than all other sites.

Fig. 7. Chartings of positions of labeled olfactory receptor neurons(ORNs; black dots) in wholemount lamellae from two different cases ofDiI application. The chartings include the sensory epithelium (se) andimmediately adjacent nonsensory epithelium (nse) in the same orien-tation as that shown in Figure 4C. For each case, representativelamella from the anterior (D1 and V1), middle (D2 and V2), andposterior (D3 and V3) regions of the olfactory organ are shown. Thecharts in the right column (D1, D2, and D3) are from a case injected inthe dorsal olfactory bulb (OB); charts in the left column (V1, V2, andV3) are from a case injected in the ventral OB. No systematicdifferences in distribution were apparent between anterior and poste-rior faces of each lamella. Likewise, no obvious differences in distribu-tion of labeled ORNs occur following dorsal OB injections comparedwith ventral OB injections.


DISCUSSION

The presence of multiple morphological types of ORNs inolfactory epithelium has been recognized since microscopicstudies in the middle of this century (Le Gros Clark, 1956).Later ultrastructural studies in a variety of species showedtwo classes of ORN with different apical morphologies:ciliated and microvillous. Both ciliated and microvillousORNs are present in the olfactory epithelium of diversevertebrates, including most teleost fishes, salamanders,and some mammals (for review, see Eisthen, 1992).

The short, microvillous ORNs described in the presentstudy are distributed broadly, if not randomly, across theentire expanse of olfactory epithelium within each lamella.This contrasts with the scanning electron microscopicreport of Erikson and Caprio (1984), who found in thesame species that microvillous ORNs are more prevalentin the dorsomedial portion of each lamella and are lessprevalent in the more marginal parts of the sensoryepithelium. The lack of concurrence between their studyand ours might be due to several factors in addition to thedifferences in techniques used. First, the fish that wereused in the present study were juveniles (11–15 cm),whereas larger animals (20–30 cm) were used in theformer study. Changes may occur in the relative distribu-tion of ciliated and microvillous ORNs during maturation.Second, it is possible that the short, microvillous ORN wedescribe is only one of several types of microvillous ORNsthat may or may not look similar in the scanning electronmicroscope. Indeed, as summarized by Yamamoto (1982),at least two types of microvillous ORNs, stellate andtufted, have been described in fishes, although Eriksonand Caprio (1984) report that the stellate microvilloustype predominates in these catfish. Perhaps the labeledmicrovillous ORNs we see are not the stellate type, andperhaps the more numerous stellate or other microvillousORNs that project elsewhere in the OB are clustereddorsomedially.

The type II ciliar cells of Muller and Marc (1984) werelabeled only following in vivo injections of Fluoro-Gold andnever after postmortem injections of DiI, even into theolfactory nerve. Thus, these cells do not appear to be ORNsbut are capable of nonspecific uptake of foreign substancesfrom circulation. Our results are consistent with those ofMoran et al. (1992), who were unable to label this type ofcell retrogradely by application of small amounts of HRP tothe cut olfactory nerve in trout, although such an applica-tion robustly labels microvillar and ciliated ORNs.

The present study has shown that ORNs with differentmorphologies connect to different parts of the OB. Short,microvillous ORNs project to the dorsal posterior OB,whereas tall, ciliated ORNs project to the ventral posteriorOB. Because other morphological types of ORNs also arepresent in the epithelium (Morita and Finger, 1996; Han-sen et al., 1997; Hansen and Zeiske, 1998), the segregationof projections according to ORN morphology, as describedin this report, is likely to be indicative of a more generalplan of organization rather than an exceptional situation.Because various classes of odorants preferentially activateparticular regions of the OB of many vertebrates, includ-ing some teleosts (see, e.g., Friedrich and Korsching,1997a,b), it is tempting to speculate that a particularmorphological type of ORN is responsible for transductionof a particular type of odorant.

The concept of a correlation between ORN morphologyand the odorant detected dates back to the seminal work ofLe Gros Clark (1956). In that paper, he described differentclasses of ciliated receptor cells in rabbits based on thelength of the apical dendrite, the size of the apical knob, itsstaining intensity, and the number of cilia extending fromit. Le Gros Clark suggested that the different morpholo-gies were related to differing odorant specificities. Thistheme was extended in the later works by Thommesen(1983), who suggested that, in teleosts, ciliated ORNs andmicrovillous ORNs each detect one of the two main groupsof odorants that were then known for these fishes—either

Fig. 8. Electron photomicrographs of a photoconverted tall olfac-tory receptor neuron (ORN) following an injection into the ventralolfactory bulb (OB). A: Low-power photomicrograph showing thelabeled apical dendrite of a tall ORN. The cell body was situated nearthe basal lamina, below the bottom of this photomicrograph, and out ofthe plane of this section. This cell is from the case shown in Figure 1B.B: Higher magnification of the apical portion of the labeled dendrite.Labeled as well as unlabeled cilia are clearly evident. Unlabeledmicrovilli (mv) also can be seen in the vicinity.


amino acids or bile salts. By recording from regions ofepithelium that were rich in ciliated ORNs vs. regions thatwere rich in microvillous ORNs, Thommesen suggestedthat ciliated ORNs are responsible for detection of bilesalts and that microvillous ORNs detect amino acids. Asimilar electrophysiological study in catfish failed to find adifference in responsiveness for areas of the olfactoryepithelium that were rich in either microvillous or ciliatedORNs (Erickson and Caprio, 1984). More recent degenera-tion-regeneration studies in goldfish indicate a correlationbetween the presence of microvillous ORNs and sensitivityto steroid sex pheromones (Zippel et al., 1997b). Thus, thegeneral idea of morphological correlates for differences in

ORN chemical sensitivity has supportive physiologicalevidence, although species may differ in terms of whichmorphology correlates with which odorant sensitivity.

Within the last decade, Buck and Axel (1991) andcoworkers (Ngai et al., 1993) have described several sub-families of odorant-receptor genes that are thought to codefor distinct seven-transmembrane odorant-receptor mol-ecules. In mammals, those ORNs that express the samegene product project to the same glomerulus or to smallgroup of glomeruli (Vassar et al., 1993; Ressler et al., 1994;Mombaerts et al., 1996). Because the morphology of anORN also appears to correlate with its bulbar projectionsite, one would expect that a particular ORN morphology

Fig. 9. Electron photomicrographs of a photoconverted short olfac-tory receptor neuron (ORN) following a dorsal olfactory bulb (OB)injection. A: Low-power view showing the position of the cell withinthe epithelium. The soma of this ORN is situated within 5 µm of the

epithelial surface. B: Higher magnification view showing the morphol-ogy of the labeled ORN. The section grazes the edge of the nucleus(nuc). C: Enlargement of the apical portion of the dendrite. Labeledmicrovilli (mv) as well as unlabeled, nearby cilia are visible.


also should correlate with expression of a particularodorant-receptor molecule. Some evidence for this correla-tion exists from studies on rodents, in which a givenreceptor molecule probe hybridizes with ORNs at a particu-lar height in the epithelium, i.e., ORNs of the same size(Strotmann et al., 1996). This is the pattern that would beexpected if the height of an ORN were related to its bulbarprojection and receptor molecule rather than to its relativeage in the epithelium, as is typically thought (Farbman,1992). In the vomeronasal organ of mammals, two differ-ent heights of microvillous receptor neuron exist, eachusing a different G protein, using a different family ofreceptor molecules, and projecting to different sites in theaccessory OB (Halpern, 1995; Jia and Halpern, 1996;Herrada and Dulac, 1997; Ryba and Tirindelli, 1997).Taken together, these findings imply that, for a receptorcell, the determination for expression of a particularodorant-receptor gene is not an independent selectionprocess but is related to structural features of the ORN,e.g., size, shape, and nuclear position within the epithe-lium.

The most obvious separation of connections of somemicrovillous ORNs from other ORNs is in the vomeronasalorgan of tetrapods. The vomeronasal organ occurs in mostterrestrial vertebrates but is no longer considered to be anadaptation to terrestrial life, because premetamorphicamphibia and aquatic salamanders have such an organ(Eisthen, 1992). The vomeronasal organ in rodents con-tains only microvillous ORNs and projects uniquely to theaccessory OB, which is situated along the dorsal posteriormargin of the main OB. Our findings show that, in catfish,too, a set of microvillous receptor cells projects to thedorsal posterior OB. This coincidence in both ORN typeand projection site may be indicative of a phyletic relation-ship between the short, microvillous ORNs of catfish andthe vomeronasal ORNs of tetrapods. Accordingly, it will beinteresting to see whether the microvillous ORNs of cat-fish possess receptor molecules similar to the class ofso-called pheromone receptor molecules that occur in thevomeronasal ORNs instead of those typical of the mainolfactory epithelium (Buck and Axel, 1991; Ngai et al.,1993; Dulac and Axel, 1995; Herrada and Dulac, 1997;Matsunami and Buck, 1997; Ryba and Tirindelli, 1997).Even if such a molecular continuity cannot be established,it is still possible that a phyletic continuity exists betweenthe short, microvillous ORNs in catfish and the microvil-lous vomeronasal receptors of tetrapods. If the short,microvillous ORNs of catfish are forerunners of the vomero-nasal system (Dulka, 1993), then the dorsal segregation oftheir inputs may be a preadaptation to the formation of theaccessory OB and vomeronasal organ. However, an ad-equate cladistic analysis would require examination of thissituation in numerous outgroups, e.g., lungfishes or Latime-ria, both of which are reported to have microvillous ORNs.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Kjell Døving for stimulat-ing discussion and ideas that led to the development of thisresearch. The authors thank Barbel Bottger and Pam Ellerfor histological assistance and Dr. Miriam Linschoten forperforming the statistical analysis. We also thank Drs.Kjell Døving, Anne Hansen, and Rona Delay for commentsand suggestions on drafts of this paper. This work wassupported by NIH grant PO1 DC00244 to T.E.F.

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differential projections of ciliated and microvillous olfactory receptor cells in the...

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