new structure, the ?olfactory pit,? in human olfactory mucosa

New Structure, the ‘‘Olfactory Pit,’’ inHuman Olfactory Mucosa

WEN-HUI FENG,1 JOHN S. KAUER,1 LESTER ADELMAN,2

AND BARBARA R. TALAMO1*1Department of Neuroscience, Tufts University School of Medicine,

Boston, Massachusetts 021112Department of Pathology, New England Medical Center, Boston, Massachusetts 02111

ABSTRACTAwhole-mount immunocytochemical method was devised to study the olfactory receptor

neurons on the surface of the human olfactory mucosal sheet. Antibodies to neuron-specifictubulin and/or microtubule-associated protein 5 and phosphorylated neurofilament proteinwere used. Specimens taken at autopsy from 56 patients ranging in age from 2 days to 92years revealed a structure not previously described, an olfactory pit. Round or oval openingswith a diameter of 50 to 500 µmwere observed on the surface of the olfactory epithelium in thewhole-mount specimen. The morphology, number, and distribution of these openings variedamong the different individuals. A detailed analysis of these structures was carried out byrehydrating and sectioning the whole-mount specimens. The olfactory pit (OP) is a blindpouch lined with olfactory epithelium (OE), which appears as an invagination of OE into theconnective tissue, with a depth varying between 150 and 200 µm. In some sections through anOP, a thick axon bundle emerging from the bottom of the pouch was visible. The extension andtermination of this axon bundle in the central nervous system has not been explored. We havefound OPs in monkey olfactory mucosa, but none in rodents. The function of the pitspecialization is unclear, but it appears to be a feature of normal, young epithelium. Theconfiguration of the blind pouch may prolong odorant association with the olfactory receptorneurons, or the OP may contain specialized neurons that have not yet been recognized bymorphological, biochemical, or functional techniques. J. Comp. Neurol. 378:443–453, 1997.r 1997 Wiley-Liss, Inc.

Indexing terms: immunocytochemistry; olfactory sensory neuron; neurofilament; tubulin;

Alzheimer’s disease

The organization of the human nasal cavity resemblesthat of other mammals in many features. Most of thesurface is covered by highly vascularized respiratory mu-cosa that warms and moistens the inhaled air. The olfac-tory mucosa is confined to a small area in the posteriordorsal part of the nasal cavity, where receptors for thesense of smell reside. General features are common to allspecies. The olfactory receptor neuron is a bipolar sensoryneuron which has a small, ovoid cell body and a slenderapical dendrite that extends to the mucosal surface whereit terminates as a vesicular olfactory knob. A thin, longaxon extends from the basal end of the olfactory receptorneuron through the basal lamina to join the olfactoryfascicles and projects to the olfactory bulb (e.g., Greer,1991). Non-neuronal sustentacular cells, basal cells, aswell as microvillar cells make up the rest of the epithelium(Moran et al., 1982b; Carr et al., 1991). The laminarstructure of the olfactory epithelium (OE) appears ratherhomogeneous, although classes of olfactory receptor neu-

rons can be distinguished biochemically by differences inlectin composition, monoclonal antibody staining (e.g.,Schwob, 1992), and expression of mRNA for particularolfactory specific G-protein–coupled receptors (Ressler etal., 1993; Vassar et al., 1993).Other specialized chemosensory structures reported in

the nose include the septal organ (Rodolfo-Masera, 1943),for which no special function is known, and the vomerona-sal organ (VNO), an invaginated sensory structure of the

Contract grant sponsor: Gustavus and Louise Pfeiffer Research Founda-tion; Contract grant sponsor: Alzheimer’s Association; Contract grantnumber: IIRG-89-041; Contract grant sponsor: NIH; Contract grant num-bers: R01-AG09200, R01-DC00228; Contract grant sponsor: Pew Chari-table Trusts.*Correspondence to: Barbara R. Talamo, Ph.D., Department of Neurosci-

ence, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA02111. E-mail: [email protected] 31 May 1996; Revised 9 October 1996; Accepted 9 October 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 378:443–453 (1997)

r 1997 WILEY-LISS, INC.

palato-nasal region. In humans, the VNO appears lesshighly developed than in many other terrestrial verte-brates (Halpern, 1987). In animals, each VNO is anasymmetric structure: The epithelium of one side is thick-ened and contains sensory cells; the other side is made ofnon-sensory epithelium and lies adjacent to a vascularstructure believed to contribute to pumping fluid into theorgan. The specialized sensory cells of the tubules of theVNO have microvilli rather than cilia on the surface andhave been considered as candidate chemoreceptors forpheromone detection in certain animals, although there isas yet little direct evidence for this. In the human,description of an organ analogous to the VNO in lowervertebrates is incomplete, but there appears to be a similarstructure consisting of a pair of unbranched tubules, linedby pseudostratified columnar epithelium, as described byStensaas et al. (1991) in their electron microscopy (EM)studies.A new whole-mount immunocytochemical technique has

been developed for the study of the surface topography ofthe human olfactory mucosa (Feng et al., 1991) by usingneuron-specific tubulin antibody Tu-J1 or antibody tomicrotubule-associated protein 5 (MAP5, also known asMAP1b) and antibody Ta51 to phosphorylated neurofila-ment protein heavy subunit. During the observation of thesurface structure of the olfactory mucosa stained by thismethod, a structure that has not been reported previouslywas discovered and given the name olfactory pit (OP). OPstructures were found in 45 of 48 specimens examined bythe whole-mount method. The high frequency with whichthey are observed suggests that OPs are universal featuresof human olfactory mucosa. Possible functions of thisspecialized structure are discussed. Preliminary results ofthis study have been published previously (Feng et al.,1992).

MATERIALS AND METHODS

Materials

Specimens used in this study were taken with permis-sion at autopsy from patients who died of various diseases;tissue was obtained through the Neuropathology Serviceat New England Medical Center or from the Massachu-setts General Hospital or the University of MassachusettsMedical Center. The total number of specimens investi-gated was 56, including 48 cases stained by the whole-mount technique and eight cases of randomly sectionedspecimens. Of the total, eight cases were from immaturepatients ranging in age from 2 days to 11 years; 48 caseswere from mature patients of 39–92 years. Among the 56cases, four patients had congenital malformations, six hadcentral nervous system disorders, 12 had cancer, 16 hadAlzheimer’s disease (AD) or AD along with cancer or othercentral nervous system disorders, and 18 suffered fromcardiovascular, inflammatory, or other diseases.

Methods

The nasal mucosa was taken by isolating the bony areaof the cribriform plate by cranial access and under-cuttingthe tissue below the cribriform plate to permit removal ofthe septum and lateral bony walls of the nasal cavity as aunit. After fixation of the tissue block in 2% paraformalde-hyde plus 10% saturated picric acid in 0.1 M sodiumphosphate buffer (pH 7.4) for 41⁄2 hours, the mucosal sheetwas carefully dissected away from the septum and bony

walls. The block was pinned through tissue and cartilageflaps to a wax tray, and the epithelial sheet was pried awayfrom the underlying bone with the blunt edge of a scalpel,using the sharp edge of the blade to cut the fila olfactoriaas they passed through the bony plate. In some cases,tissue needed to be carefully cut away from calcifiedspicules projecting up into the lamina propria. The tissuewas then rinsed in cold Tris buffer (pH 7.4), opened out,pinned onto dental wax, and processed for double-labelimmunocytochemistry (Feng et al., 1991). The tissue waspresoaked in blocking serum (5% normal goat serum and0.5% Triton X-100 in 0.1 M Tris buffer) overnight, followedby incubation at 4°C in primary antibodies for 2–3 daysand secondary antibodies for 1–2 days. Primary antibodieswere mouse monoclonal antibody to either microtubule-associated protein MAP5 (1:5,000 dilution, Sigma, St.Louis, MO) or to the neuron-specific b-tubulin (class III)isoform [Tu-J1, 1:1,000 dilution, from A. Frankfurter(Moody et al., 1989)] and rat monoclonal antibody tophosphorylated heavy subunit of neurofilament protein,Ta51 (1:10 dilution, from V.M.-Y. Lee; Lee et al., 1987). Thesecondary antibody used for MAP5 or Tu-J1 visualizationwas goat anti-mouse IgG conjugated with Texas Red (1:50,Jackson Immunoresearch Laboratories, Inc., West Grove,PA) and that for Ta51 was biotinylated goat anti-rat IgG(1:100, Tago Inc., Burlingame, CA) followed by treatmentwith Streptavidin-BODIPY (1:100, Molecular Probes, Eu-gene, OR). The tissue was then dehydrated in a series ofethanol dilutions and cleared in methyl salicylate forobservation under the fluorescence microscope. For fur-ther detailed analysis of the structure of the OP, thespecimen was rehydrated and the part of the tissue thatcontained the OP was isolated, equilibrated in sucrose,and frozen for cryosectioning. Sections were restainedwith antibody Tu-J1, using the peroxidase-antiperoxidase(PAP) method with a diaminobenzidine substrate, andcounterstained byAlcian blue (Clark, 1981) and hematoxy-lin to reveal the structure of the OP and of secretory cellsand glands using an ordinary light microscope. In somecases, goat antibody to olfactory marker protein (OMP;1:500 dilution; gift of Frank Margolis) was used withsecondary biotinylated rabbit anti-goat IgG (1:100, VectorLaboratories Inc., Burlingame, CA). Normal swine serumwas substituted for goat serum in the blocking step in thiscase.

RESULTS

Specimens that were obtained during autopsy by under-cutting the cribriform plate through the floor of theanterior cranial fossa included a large area of the olfactorymucosa and a relatively small amount of adjacent respira-torymucosa. Both of themucosae are lined by pseudostrati-fied columnar epithelium. The respiratory epithelium (RE)is ciliated and contains many mucus-secreting goblet cells.Olfactory epithelium (OE) is composed of abundant olfac-tory receptor neurons, sustentacular cells, basal cells, andmicrovillar cells (Allison, 1953; Nakashima et al., 1984;Schwartz Levey et al., 1991; Greer, 1991).In the whole-mount–stained specimens of this study, the

olfactory receptor neurons were specifically labeled by theantibody Tu-J1 or MAP5 and visualized by fluorescent dyeTexas Red. When viewed from the surface of the specimen,the olfactory knobs appear as distinct red fluorescentsmall dots which are evenly distributed across the surface

444 W.-H. FENG ET AL.

of the OE (Fig. 1A). When the focus is readjusted to deeperlevels, olfactory cell bodies are seen (not shown). Withinthe olfactory epithelial sheet small, circular, nonfluores-cent areas are scattered at regular intervals. Orifices of theBowman’s gland ducts emerge at the center of these areas,and the epithelium immediately surrounding the ductopening is free of olfactory receptor neuron dendrites,giving the appearance of a dark circle (Fig. 1A–C). The

respiratory epithelium was not stained in this protocol.OPs were first discovered in OE of two infant specimens(case 1, 2 days old; case 3, 2 months old, Table 1), wherethey were clustered in two groups of eight to 12 round oroval-shaped openings on the anterior lateral and septalwalls (Fig. 1B,D). At its widest dimension, the diameter ofthe OP opening was about 50–100 µm, which is larger thanthe opening of a gland duct (15–20 µm) viewed on the

Fig. 1. Olfactory epithelial sheet in whole-mount specimens fromolfactory mucosa obtained from infants at autopsy. A: Olfactorymucosal surface of 2-month-old infant (case 3) stained with antibodiesto neuron-specific b-tubulin, class III (Tu-J1) and phosphorylatedneurofilament protein, heavy subunit (Ta51). The evenly distributedfluorescent dots (small arrows) are the dendritic knobs of olfactoryreceptor neurons, and the dark circular areas (large arrows) corre-spond to Bowman’s gland openings. B: Openings of the olfactory pits(OPs; large arrows, diameters 5 50–100 µm) and the glandular ductopenings (small arrows) in whole-mount preparation showing anterior

aspect of left olfactory mucosa of 2-day-old infant (case 1, Table 1).Tissue was stained with antibodies to Tu-J1 and Ta51. C: Highermagnification of two OPs from case 1 showing the rim of dendrites ofthe olfactory receptors around the OP openings. The large arrowpoints to gland openings and small arrows to dendritic knobs. D: Agroup of OPs in the anterior lateral wall of left olfactory mucosa of a2-month-old infant (case 3). The OP openings face in different direc-tions, as indicated by the arrows. Scale bar 5 100 µm in A,B,D;50 µm in C.

A NEW STRUCTURE: THE OLFACTORY PIT 445

Fig. 2. Variation of pit morphologies in whole-mount specimen of9-year-old subject (case 7). OPs are distributed over the whole area ofolfactory epithelium (OE).A: Seven adjacent OPs of different sizes andshapes (diameters range from 100 to 500 µm). B,C: Round OPillustrating openings of two subsidiary branches arising from the floorof the pit; the focus is at the level of the opening in B and at the bottomof the OP in C. Dendrites of olfactory receptor neurons rim both theopenings of the OP and its subsidiary branches in a radiating pattern.D: OP 6 of 2A is shown at high magnification, revealing three to four

openings of branches on the floor of the pit. Widest diameter5 500 µm.E: OP 3 of 2A shown at high magnification. An additional one to threebranch openings are visible within the pit. F: A spindle-shaped OP,with two to three internal branches.G:Alarge OP, greatest diameter57,300 µm, with subsidiary branch openings at either extreme. Smallarrows indicate the gland openings. H: Rectangular-shaped OP (inthis photograph, a wedge of dendrites is visible dipping down into thepit, somewhat out of the plane of focus). Long side 5 140 µm, shortside 5 70 µm. Scale bars 5 200 µm inA; 100 µm in B–H.


mucosal surface. In contrast to the duct openings, eachopening of the OP was rimmed by the dendrites of theolfactory receptor neurons in a characteristic radiatingpattern (Fig. 1C). The openings were not always flushagainst the mucosal surface and could be oriented indifferent directions, as demonstrated in Figure 1D. In twoadditional whole-mount–stained specimens from children(case 6, 7 years old; case 7, 9 years old, Table 1), OPs werenot confined to the anterior region but were observed overthe entire area of olfactory mucosa. Sixty OPs wereidentified in the left olfactory mucosa of specimen 6, and100 were observed in the right olfactory mucosa of speci-men 7. Additionally, the surface view of these two whole-mount specimens showed an OP structure that was morecomplicated than that in the two infant specimens, asshown in Figure 2A–H. In addition to the previouslyobserved small round and oval openings, irregular spindle-shaped and rectangular configurations of the openingswere observed (Fig. 2F,H). The largest diameter of theopening usually ranged between 100 and 500 µm, and wastwo to seven times larger than that observed in the twoinfant specimens. The internal structure also was morecomplex, with additional small openings at the base of theprimary OP (Fig. 2B–G). Figure 2G is an example of alarge OP in specimen 7. This shallow, oval depression is7,300 µm wide at its largest dimension and has two smallopenings within, at each of the two ends. These smallopenings are the outlets of deeper branches of the OP, asclearly demonstrated by cross-sectional analysis of thewhole-mount specimens (see below).More detailed analysis of the structure of the OP was

carried out by rehydration of the whole-mount specimensfollowed by isolation of the tissue that contained the OP.Frozen sections were prepared of the selected region.Sections were restained immunocytochemically with Tu-J1, using the PAP method. Cell bodies and dendrites ofolfactory receptor neurons were clearly labeled by the darkbrown deposits of the PAP substrate as shown in Figures3–10. Sustentacular cells and basal cells were not immuno-reactive, but their cell nuclei were stained blue withhematoxylin (e.g., Fig. 6). The small, round nuclei of thehorizontal and globose basal cells were situated just abovethe basal lamina. Nuclei of the sustentacular cells wereoval shaped and were distributed near the surface of theOE. Although microvillar cells located in the upper regionof the OE have been identified by EM (Moran et al., 1982b),none were recognized here. The OPwas a blind pouch linedby a layer of epithelium that appeared to be an invagina-tion of the surface OE into the lamina propria. In theinfant specimens, the profile of the pouch took either acircular, saccular, or tubular form. Figure 3 is an exampleof a saccular OP found in a specimen from a 7-day-oldinfant (case 2, Table 1). An enlarged ampulla-shapedstructure was observed in the specimen from a 9-year-oldchild (case 7, Table 1) (Fig. 6).Branched chambers of primary OPs are usually visible

at its base. The branches in Figures 4 and 5 are thin tubes,about 30 µm in diameter, which extend obliquely into thelamina propria of the olfactory mucosa. Some OPs haveseveral secondary branches as well (Fig. 2B–G), which areseen as openings within the principal cavity of the OP inthe whole mount, but these secondary chambers are hardto recognize in sections unless serial reconstruction iscarried out. In a few sections, a thick bundle of axons

TABLE 1. Summary of HumanAutopsy Subjects1

OEspecimen Gender Age PMI Disease OP

A. Whole-mountspecimens1 (R, L) F 2 days 24 hours Renal malformation,

respiratory failure1

2 (R, L) M 7 days 4 hours Congenital heart dis-ease, renal failure

1

3 (R, L) M 2 months 26 hours Congenital heart dis-ease

1

4 (R) M 9 months 15 hours Hydrocephalus 15 (L) M 5 years 8 hours Leukemia, renal

failure1

6 (L) F 7 years 16 hours Spinal muscularatrophy

1

7 (R, L) M 9 years 6 hours Hemolytic anemia 18 (R, L) M 11 years 6 hours Sudden death, no

diagnosis1

9 (L) F 39 years 21 hours Kidney transplantfailure

1

10 (R, L) F 40 years 18 hours Lymphoma, myocar-dial infarction

2

11 (R, L) M 44 years 15 hours Melanoma 112 (L) M 44 years 20 hours Liver transplant

failure1

13 (R) M 50 years 19 hours Cardiac transplantfailure

1

14 (R) M 52 years 20 hours Interventricularhemorrhage

1

15 (R) F 53 years 12 hours Subarachnoid hemor-rhage

1

16 (L) F 54 years 24 hours Portal hypertension 117 (R) M 54 years 10 hours Metastatic renal car-

cinoma1

18 (R, L) F 55 years 28 hours Brain ischemia 119 (L) M 56 years 26 hours Brain ischemia 220 (R) M 58 years 14 hours Pontine hemorrhage 121 (R) M 61 years 24 hours Leukemia 122 (L) M 62 years 10 hours Pancreatic cancer 123 (R) M 62 years 15 hours Down’s syndrome 1

Alzheimer’sdisease

1

24 (R, L) M 63 years 14 hours Alcoholic cirrhosis 125 (R, L) M 64 years 9 hours Myocardial infarction 126 (R) F 64 years 19 hours Pneumonia; progres-

sive supranuclearpalsy

1

27 (R, L) F 64 years 9 hours Cardiac failure, coro-nary stenosis

1

28 (L) F 66 years 15 hours Cardiovascular dis-ease

1

29 (R) M 67 years 10 hours Cirrhosis of liver 130 (L) F 67 years 14 hours Pneumonia 131 (R, L) F 69 years 33 hours Alzheimer’s disease 132 (L) M 71 years 37 hours Alzheimer’s disease 133 (R) M 72 years ,24 hours Alzheimer’s disease 134 (R) F 73 years 16 hours Lung cancer 135 (R) M 75 years 5 hours Alzheimer’s 1 Lewy

body disease1

36 (L) F 75 years 4 hours Alzheimer’s 1 Paget’sdisease

1

37 (R) F 78 years 10 hours Alzheimer’s disease 138 (L) F 79 years 16 hours Alzheimer’s disease 139 (L) M 79 years 38 hours Prostate cancer 240 (R) M 82 years 2 hours Alzheimer’s disease 141 (R) M 83 years 8 hours Alzheimer’s disease 142 (R) F 84 years 12 hours Hypertension,

dementia1

43 (L) M 84 years 14 hours Alzheimer’s disease 144 (L) F 88 years 14 hours Alzheimer’s disease 145 (L) F 88 years 6 hours Alzheimer’s disease,

colon carcinoma1

46 (R) M 88 years 21 hours Alzheimer’s disease 147 (L) F 90 years 20 hours Urinary tract infec-

tion1

48 (L) M 92 years 30 hours Alzheimer’s disease 1B. Randomly sec-

tioned cases49 (R) F 50 years 5 hours Cervical cancer 150 (OE2) M 67 years 6 hours Internal hemorrhage 151 (OE4) F 69 years 4 hours Granulomatosis 152 (R) F 69 years 26 hours Brain edema 153 (R) M 70 years 22 hours Metastatic cancer 154 (R) M 73 years 23 hours Lung cancer 155 (L) M 75 years 5 hours Colon cancer 156 (L) F 81 years 4 hours Alzheimer’s disease 1

1Autopsy patients are identified by age, gender, and disease. The postmortem interval(PMI) also is noted. The specimen(s) of nasal olfactory epithelium (OE) examined foreach patient was from the right (R) and/or left (L) side, or was one of several olfactoryblocks that were divided and coded for storage as OE 1 . . . 4.


leading out from the bottom of the OP was observed(Fig. 7).Secretory glands and goblet cells are identified byAlcian

blue treatment of sectioned specimens. Alcian blue stainsmucus glycoproteins in the cytoplasm of the secretory cells(Figs. 3–5). Ductal openings of glands at the OE surfacehave been reported previously, but it can be appreciatedhere that the OP pouch and its branches also are wellsupplied by ducts of secretory glands.In the two specimens from infants, olfactory receptor

neurons are dispersed evenly across the surface of theolfactory mucosa. In these cases, the OPs were fully linedwith olfactory receptor neuron–containing OE. At laterages, as seen in the specimens of the 7-year-old (notshown) and 9-year-old subjects (Figs. 5, 6), metaplasticchanges are seen in some small areas of the olfactorymucosa. Within these areas, OE has degenerated and beenreplaced by nonsensory respiratory epithelium, due tounknown causes. OPs were also found to containmetaplas-tic areas, as seen in the specimen from the 9-year-oldsubject (case 7), shown in Figures 5 and 6. Within theseareas olfactory receptor neurons are less densely distrib-uted, and the number of nuclei is diminished. Metaplasiaof the OE was found at many ages, but was more frequentin specimens from aged patients. In a specimen from an84-year-old patient (case 42, Table 1), olfactory receptorneurons were sparse and did not fully encircle the openingof the OP (Fig. 11). Only a few remaining olfactory receptorneurons were associated with the OP opening in a speci-men from a 62-year-old AD patient (case 23, Table 1), andextensive degeneration of the OE was apparent across thesurface (Fig. 13A), where few olfactory receptor neuronsremained. Further analysis of sections cut through the OPof Figure 13A showed that there were metaplastic changesin the OE lining the pouch of both OPs (Fig. 13B,C). Thediversity of the OE structure is seen in adjacent OPs. Aphotomicrograph of a section from a different region of thenasal cavity in the same specimen (Fig. 8), cut through thearea near the roof of the nasal cavity, shows two OPs. OEwithin the pit and on the mucosal surface surrounding oneof these OPs (labeled ‘‘a’’) appears to have been convertedto nonsensory epithelium, rich in gland cells. However, inthe adjacent OP (labeled ‘‘b’’), olfactory receptor neuronsremain in the deepest part of the cavity and along one side

of the upper wall of the OP. This is an example of thesurvival of olfactory receptor neurons in an area wheremost of the sensory cells at the surface have been lost. Insome cases, including case 50 (Table 1), extreme metapla-sia was observed. Figure 9 is a photomicrograph of asection of this specimen cut through a highly branched OP.Most of the epithelium on the mucosal surface and in theOP lacks olfactory receptor neurons and is composed ofnonsensory epithelium containing many gland cells. Onlya few olfactory receptor neurons appear on the bottom andnear the opening of the OP (arrows). Serial section recon-struction of another case (75-year-old colon cancer patient,case 55, Table 1) revealed a tortuous tunnel-like pit. Onlypatchy regions of OE were present in this pit, interspersedwith nonsensory epithelium (data not shown). In additionto the metaplastic areas, degenerative neuropathologicalchanges characteristic of OE in aged patients includingthose withAlzheimer’s disease can also be seen in olfactorymucosa of whole-mount specimens. Swollen olfactory recep-tor neurons packed with material immunoreactive withantibody to phosphorylated neurofilament protein in thesoma were often observed, along with infiltration of ectopicnerve fibers in the area of degenerating OE, similar to thatreported previously in sections (Talamo et al., 1989, 1991a,1994). These structures also can be observed in the whole-mount specimens double-stained with antibodies to phos-phorylated neurofilament (Ta51) along with either Tu-J1or MAP 5 (data not shown). Such pathological changes areobserved both in the surface OE and in the OE lining theOP (Figs. 8, 10).OPs in adult patients were usually distributed near the

roof of the nasal cavity. The number of OPs observed inmature whole-mount specimens usually ranged from fiveto 20 in a given case. The quantitation is not definitivebecause detection can be compromised by deterioration ofthe specimen, by the possibility that the entire region ofthe olfactory mucosa was not obtained at autopsy, anddepending on the extent of the metaplasia. The number ofOPs may be underestimated, although OPs were observedeven after long postmortem intervals (Table 1). If olfactoryreceptor neurons deteriorated during the postmortem inter-val and no dendritic staining remained, a positive identifi-cation of a surface opening as an OPwould not be possible.Moreover, none of the mature specimens had as high a

Fig. 3. Staining in Figures 3–10 is carried out as described inFigure 3. Mucopolysaccharide and neuron-specific tubulin staining insection of OP derived from whole-mount specimen of a 7-day-oldsubject (case 2), re-labeled after sectioning, using the peroxidase-antiperoxidase (PAP)method andAlcian blue stain. The OP is sac-like;diameter 5 80 µm, depth 5 100 µm. Glands open to the surface of OE(large arrow) and also into the OP (small arrow). Other glandular profilescontaining blue mucopolysaccharide material are seen within the laminapropria, but do not open to the surface in this section. Scale bar 5 100 µm.

Fig. 4. Broad OP in 9-year-old-child (case 7). This section passesthrough two subsidiary branches of the OP (arrows). The left branch isconnected with a gland. OP diameter 5 375 µm; depth 5 160 µm. Scalebar5 100 µm.

Fig. 5. Section of anOP from the same patient as Figure 4, cut througha long subsidiary branch. The epithelium lining the right half of the branchis nonsensory and rich in gland cells, as shown byAlcian blue staining. Onthe left wall and upper lining of the primaryOP, the soma and dendrites ofmany olfactory receptor neurons are visible (arrows). Scale bar5 50 µm.

Fig. 6. Section from the same patient as Figures 4 and 5. Two glands(asterisks) open into the cavity of a large ampulla-shapedOP (diam-

eter 5 300µm;depth 5 375µm). Small areas containing olfactory receptorneurons are seen deep in theOP (arrows). Scale bar5 50 µm.

Fig. 7. Axon bundle (arrow) emerging from the bottom of the organ insection of an OP of the 9-year-old child (case 7). OP diameter 5 63 µm;depth5 45 µm. Scale bar5 50 µm.

Fig. 8. Olfactory mucosa of a 62-year-old patient with Alzheimer’sdisease (case 23). Metaplastic epithelium has replaced most of the OElining the mucosal surface and one of two OPs, OPa. A second OP, OPb,retains OE in its deepest portion (short arrows). Degenerating, swollenolfactory receptor neurons appear in surface epithelium and in OPb (longarrows). Scale bar5 200 µm.

Fig. 9. Complex OP in section from 67-year-old patient (case 50). Boththe epithelium on the surface and in the OP have been converted intorespiratory epithelium with many goblet cells stained blue; only a fewolfactory receptorneuronsare seen in theOP(arrows). Scale bar5200µm.

Fig. 10. Abnormal neuriticmass andmetaplasia inOP in a 82-year-oldpatient (case 40). Metaplastic epithelium is interspersed with OE on boththe surface and in the OP.An abnormal neuritic mass of the type found inAlzheimer’s disease patients is seen within the OP (arrow). Scale bar 550 µm.


Figures 3–10


Fig. 11. Degenerating OE in an 84-year-old demented patient (case42) stained with antibodies Tu-J1 and Ta51, and photographedthrough a rhodamine filter set to visualize Tu-J1 staining. Olfactoryreceptor neurons and olfactory axons are sparse. The opening of theOP (star) is incompletely lined by olfactory receptor neurons. Widestdiameter 5 250 µm. Scale bar 5 100 µm.

Fig. 12. Degenerative changes in OE from young child. Fluores-cent micrograph of whole-mount specimen from a 5-year-old leukemiapatient (case 5) stained with antibodies Tu-J1 and Ta51 and photo-graphed through filters revealing Tu-J1 staining. OE shows extensivedegenerative change. In this area, the only remaining olfactoryreceptor neurons are associated with the OP, and the surface epithe-lium is metaplastic. Scale bar 5 100 µm.

Fig. 13. Pair of OPs in whole-mount and corresponding crosssections from a 62-year-old patient (case 23) photographed afterstaining with Tu-J1 and Ta51 with filter set revealing Tu-J1 staining.A: Whole-mount specimen shows two OPs (a and b); degenerative

changes are similar to those in Figure 12, with olfactory receptorneurons remaining only in association with the OPs. B: Section cutthrough OPa of Figure 13A. Metaplastic changes have occurred alongthe surface and the OE lining of the OP. Some olfactory receptorneurons remain in the OP (arrows). C: Section cut through OPb ofFigure 13A. Similar changes appear in OPb as in OPa. The two pits donot seem to be connected at any point in serial sections. Some remnantolfactory receptor neurons are indicated by the arrow. RE, respiratoryepithelium; OE, olfactory epithelium. Scale bar 5 100 µm.

Fig. 14. OP in OE of 29-year-old monkey olfactory mucosa whichhas been double-labeled by antibodies to microtubule-associated pro-tein 5 (MAP 5; shown in A, photographed with fluorescein isothiocya-nate (FITC) filters, using BODIPY, a Molecular Probes fluorophore, asa marker) and olfactory marker protein (OMP; shown in B, usinglong-wavelength filter, with Texas Red as a marker). MAP 5 and OMPare in the olfactory receptor neurons in both surface OE (large arrow)and in the OP (small arrows). Scale bar 5 100 µm.


number of OPs as found in the specimens from the 7- and9-year-old subjects.A limited number of studies also were carried out in

nonhuman primates. Figure 14A,B show sections from arhesus monkey OE which were cut through an OP anddouble-labeled with antibodies to MAP5 and OMP, respec-tively. OMP is found only in mature olfactory receptorneurons in the OE. Similar co-localization of Tu-J1 andOMP also was observed in OPs of human specimens (datanot shown). The structure of themonkey OP is very similarto that of the human OP, but no OPs have been observed inrats and mice using similar staining techniques.

DISCUSSION

This study describes a new, elaborate olfactory struc-ture, the OP, in human olfactory mucosae. Use of awhole-mount method combined with immunocytochemis-try has permitted visualization of openings of OPs on thesurface of the OE. These complicated invaginationsmay befully lined with olfactory receptor neurons, or may be linedonly partially, possibly as a consequence of pathologicalchanges or age. Subsequent sectioning and restaining ofwhole-mount sections has confirmed that OPs are neitherartifacts of fixation nor glands. Furthermore, the OP doesnot appear to be related to the VNO. In humans, as inanimals, the structure identified as the human VNO issituated ventrally in the nasal septum near the floor of thenasal cavity; in humans, it opens into the nasal cavity. TheOPs, however, are located within the olfactory epithelialsheet, mostly near the roof of the nasal cavity, and arenumerous. They are lined by epithelium that is continuouswith and appears identical with the OE, when using thestains described in this study. The major neuronal elementin the human OE is the OMP-containing bipolar olfactoryreceptor neuron (Nakashima et al., 1985b; Chuah andZheng, 1987; Talamo et al., 1989), and OMP-immunoreac-tive cells also are abundant in the human OP. Morphologi-cally, they resemble the bipolar olfactory receptor neuronsand are Tu-J1 or MAP5-immunoreactive, with long, slen-der dendrites, unlike the flask-shaped microvillar cell ofthe OE. However, the issue of whether cells in the OP havemicrovilli or cilia at the surface cannot be determined atthe resolution of these micrographs, which was not suffi-cient to detect cilia. Further, the neuron-type cells of theOP were not confined to the upper layers of the epitheliumas reported for the microvillar cells (Moran et al., 1982b).The specimens of the present study included only tissuefrom the roof, dorsal nasal wall, and dorsal septum of thenose and did not include the tissue of the ventral nasalcompartment. Clearly, the OP structures are entirelydistinct from the VNO.The absence of a description of a morphological struc-

ture similar to the OP in previous reports of humanolfactory epithelium might be due to the limitations of themethods employed. Many investigations have employedboth light and electron microscopy in examination of thestructure of both normal (Moran et al., 1982a; Nakashimaet al., 1984; Morrison and Costanzo, 1990; Chuah andZheng, 1992; Paik et al., 1992) and pathologically alteredhuman olfactory mucosa (Moran et al., 1985, 1992; Na-kashima et al., 1985a, 1991; Trojanowski et al., 1991;Schwob et al., 1993), including specimens from very youngto aged subjects. Some earlier studies on the surfacetopography of human olfactory mucosa were done before

the use of immunocytochemical techniques was wide-spread (Naessen, 1970, 1971). Apparently the structurewas not obvious by scanning EM (Morrison and Costanzo,1990). The opening of an OP in a whole-mount preparationis very difficult to distinguish from the opening of aglandular duct without prior knowledge of the size differ-ences of the openings and the use of a specific labelingprobe like Tu-J1 or MAP5 antibodies, which stain theolfactory receptor neurons and dendrites that characteris-tically rim the surface of the OPs (Fig. 1C). Although manystudies of sectioned tissue have been carried out, thesections may not have passed through the OP, especially inEM studies where the ultrathin sections cover only smalldistances. In investigations which utilized only hematoxy-lin and eosin staining, it is difficult to distinguish sensoryfrom nonsensory epithelia and to tell the difference be-tween OPs and gland openings, particularly when OE inthe OPmay have undergone metaplastic changes.Although the specimens of this study were collected

from patients who died of various diseases, the OPdoes notappear to be a pathological structure. Most of the OPs arelined with OE, especially in the infant specimens whereolfactory receptor neuron density is high and the OE liningthe OPs is intact. There is no sign of inflammation, such asexudate, engorgement of vessels, or infiltration of inflam-matory cells. Moreover, we did not find any correlationbetween the presence or number of OPs and any particulardisease state.As noted above, both the OE on the mucosal surface and

the OE in the OP are subject to metaplastic change. Theetiology of this metaplasia is unknown. It might be causedby environmental insults such as toxins (Keenan et al.,1990), bacterial and viral infections (Kolmer, 1927; Yamagi-shi and Nakano, 1992) or long exposure to the vicissitudesof heat, cold, drying or to diminished capacity for regenera-tion of olfactory receptor neurons and sustentacular cellsor to aging changes (Doty and Snow, 1988; Paik et al.,1992). If most of the lining of the OP has been convertedinto nonsensory epithelium, the OP would not be recogniz-able as a distinctive structure in our whole-mount speci-mens by our criteria and therefore might be confused withlarge glandular openings. This may be one of the reasonsfor the lack of recognition of this structure in earlierreports and for the large variability in OP numbers acrossindividuals in this study.We also found OPs in monkey specimens. The specimens

were acquired from collaborators studying the brain inaging animals (Presty et al., 1987). The histology of theolfactory mucosa from monkeys appears to be normalbased on the various immunocytochemical probes thathave been applied (Talamo et al., 1991b). No swollenolfactory receptor neurons stained for phosphorylatedneurofilament protein were observed nor was there ectopicgrowth of neurites in the OE. Thus, it is likely that the OPis a normal structure in the human as well as in highlyevolved primates such as rhesus, which have a similarnasal epithelial cavity with little turbinate structure whichcould contribute increased sensory epithelial surface area.The presence of OPs in young infants, as early as 2 days

postnatal, suggests that the development of this organprobably begins before birth. From the limited samples wehad available, it appears that OPs are first established inthe anterior aspect of the dorsal nasal wall and laterextend to the entire olfactory mucosa after birth. Themeaning of this developmental pattern is not clear. In


favorable sections, axons of the olfactory receptor neuronswithin the OP are observed to converge at the bottom ofthe organ into a thick nerve bundle. Information aboutextension and synaptic termination of this axon bundlemay provide insight into whether the OP has a specializedfunction or represents a collection of neurons destined tosynapse in a particular olfactory bulb region, but the axonshave not been traced because these specimens includedonly the peripheral part of the olfactory system and not theolfactory bulb. The entire specimen would need to remainintact to carry out tracing experiments. One possibleconsequence of the physical configuration is that odorantmolecules in the inspired air may be trapped in the blindpit and flushed out only slowly, thus prolonging odorantassociation with the olfactory receptor neurons locatedwithin the pit. Local metabolism during this prolongedassociation may convert odorant molecules to active prod-ucts which are detected only locally. In two specimens(case 6, 7 years, and case 7, 9 years), the number of OPs ismuch larger (approximately 60 and 100 for the left andright side, respectively) than in specimens from other ages.A rough calculation of the area of OE contributed by theOPs in case 7 gives an estimatedmaximal increment to thesurface area. The surface OE was reconstructed in aphotomicrograph montage of the whole-mount specimenand measured by computer-aided planimetry using theSigma Scan program. This gave a total surface area of 500mm2. The average diameter of the OP was assumed to be0.3 mm, and the surface area, S, for each OP was calcu-lated as the surface area of half of a sphere, by the formulaS 5 0.5 (4pr2). Here, r is the radius of the OP and is equalto 0.15 mm. The calculated total surface area of 100 OPs isabout 14 mm2. This value is equal to approximately 3% ofthe whole area of OE on one side of the nasal cavity of thisspecimen. From these calculations, the increase in OEsurface area contributed by even a large number of pits isnot likely to be substantial, and therefore is unlikely to beamajor function of these structures. The particularly largenumber of OPs in young specimens may reflect an adapta-tion to certain physiological functions of this structure atthat developmental stage. Moreover, it seems reasonableto speculate that the OP may contain specialized neuronsthat are adapted for the detection of particular moleculesof importance to the organism. Alternatively, the anatomi-cal structure may confer some advantage for odorantprocessing or convergence of odorant information withother local hormonal or signaling molecules. Olfactoryreceptor neurons in OPs also seem to be somewhat pro-tected from damage, surviving where surface olfactoryreceptor neurons are severely depleted, as in Figure 9.However, if there is a specialized neuron in the OP, wehave not yet been able to distinguish it morphologically orimmunocytochemically from other olfactory receptor neu-rons in the surface OE.Further examination of OPs using tracing techniques to

examine their projections to the olfactory bulb andmolecu-lar and immunocytochemical studies will be required toexplore the function of this intriguing structure.

ACKNOWLEDGMENTS

We are grateful to several sources for tissue: Dr. WilliamCano, New England Medical Center; Dr. Jim Hamos,University of Massachusetts Medical Center; Drs. John

Growden at the Alzheimer’s Disease Research Center andTessa Hedley-Whyte at Massachusetts General Hospital.

LITERATURE CITED

Allison, A.C. (1953) The morphology of the olfactory system of vertebrates.Biol. Rev. 28:195–202.

Carr, V.M., A.I. Farbman, L.M. Colletti, and J.I. Morgan (1991) Identifica-tion of a new non-neuronal cell type in rat olfactory epithelium.Neuroscience 45:433–449.

Chuah, M.I., and D.R. Zheng (1987) Olfactory marker protein is present inolfactory receptor cells of human fetuses. Neuroscience 23:363–370.

Chuah,M.I., and D.R. Zheng (1992) The human primary olfactory pathway:fine structural and cytochemical aspects during development and inadults. Microsc. Res. Tech. 23:76–85.

Clark, G. (1981) Staining Procedures. Baltimore: Williams andWilkins.Doty, R.L., and J. Snow (1988)Age related alterations in olfactory structure

and function. In F.L. Margolis and T.V. Getchell (eds): MolecularNeurobiology of the Olfactory System. NewYork, NY: Plenum Press, pp.355–374.

Feng, W.H., J.S. Kauer, and B.R. Talamo (1991) Distribution of pathologicalneurites across the surface of Alzheimer’s nasal epithelium: a newwhole-mount immunocytochemical method. Soc. Neurosci. Abstr. 17:691.

Feng, W.H., J.S. Kauer, M. Stockmayer, and B.R. Talamo (1992) A newstructure, the ‘‘pit organ,’’ in human and monkey olfactory mucosa. Soc.Neurosci. Abstr. 18:1198.

Greer, C.A. (1991) Structural Organization of the Olfactory System. In T.V.Getchell, et al. (eds): Smell and Taste in Health and Disease. NewYork:Raven Press, pp. 65–79.

Halpern, M. (1987) The Organization and Function of the VomeronasalSystem.Annu. Rev. Neurosci. 10:325–362.

Keenan, C.M., D.P. Kelly, and M.S. Bogdanffy (1990) Degeneration andrecovery of rat olfactory epithelium following inhalation of dibasicesters. Fundam.Appl. Toxicol. 15:381–393.

Kolmer, W. (1927) Geruchsorgan. In W. von Mollendorff (ed): Handbuch dermikroskopischen Anatomie des Menschen. Berlin: Julius Springer, pp.192–249.

Lee, V.M.-Y., M.J. Carden, W.W. Schlaepfer, and J.Q. Trojanowski (1987)Monoclonal antibodies distinguish several differentially phosphory-lated states of the two largest rat neurofilament subunits (NF-H andNF-M) and demonstrate their existence in the normal nervous systemof adult rats. J. Neurosci. 7:3474–3488.

Moody, S.A., M.S. Quigg, and A. Frankfurter (1989) Development of theperipheral trigeminal system in the chick revealed by an isotype-specific anti-beta-tubulin monoclonal antibody. J. Comp. Neurol. 279:567–580.

Moran, D.T., J.C. Rowley III, B.W. Jafek, and M.A. Lovell (1982a) The finestructure of the olfactory mucosa in man. J. Neurocytol. 11:721–746.

Moran, D.T., J.C. Rowley III, and B.W. Jafek (1982b) Electronmicroscopy ofhuman olfactory epithelium reveals a new cell type: the microvillar cell.Brain Res. 253:39–46.

Moran, D.T., B.W. Jafek, J.C. Rowley, III, and P.M. Eller (1985) Electronmicroscopy of olfactory epithelia in two patients with anosmia. Arch.Otolaryngol. (Stockh.) 111:122–126.

Moran, D.T., B.W. Jafek, P.M. Eller, and J.C. Rowley, III (1992) Ultrastruc-tural histopathology of human olfactory dysfunction. Microsc. Res.Tech. 23:103–110.

Morrison, E.E., and R.M. Costanzo (1990) Morphology of the humanolfactory epithelium. J. Comp. Neurol. 297:1–13.

Naessen, R. (1970) The identification and topographical location of theolfactory epithelium in man and other mammals. Acta Otolaryngol.(Stockh.) 70:51–57.

Naessen, R. (1971) An enquiry on the morphological characteristics andpossible changes with age in the olfactory region of man.Acta Otolaryn-gol. (Stockh.) 71:49–62.

Nakashima, T., C.P. Kimmelman, and J.B. Snow (1984) Structure of humanfetal and adult olfactory neuroepithelium. Arch. Otolaryngol. HeadNeck Surg. 110:641–646.

Nakashima, T., C.P. Kimmelman, and J. Snow (1985a) Immunohistopathol-ogy of human olfactory epithelium, nerve and bulb. Laryngoscope95:391–396.

Nakashima, T., C.P. Kimmelman, and J.B. Snow (1985b) Olfactory markerprotein in the human olfactory pathway. Arch. Otolaryngol. Head NeckSurg. 111:294–297.


Nakashima, T., M. Tanaka,M. Inamitsu, and T. Uemura (1991) Immunohis-topathology of variations of human olfactory mucosa. Eur.Arch. Otorhi-nolaryngol. 248:370–375.

Paik, S.I., M.N. Lehman, A.M. Seiden, H.J. Duncan, and D.V. Smith (1992)Human olfactory biopsy. The influence of age and receptor distribution.Arch. Otolaryngol. Head Neck Surg. 118:731–738.

Presty, S.K., J. Bachevalier, L.C. Walker, R.G. Struble, D.L. Price, M.Mishkin, and L.C. Cork (1987)Age differences in recognition memory ofthe rhesus monkey (Macaca mulatta). Neurobiol. Aging 8:435–440.

Ressler, K.J., S.L. Sullivan, and L.B. Buck (1993) A zonal organization ofodorant receptor gene expression in the olfactory epithelium. Cell73:597–609.

Rodolfo-Masera, T. (1943) Su l’esistenza di un particolare organo olfattivonel setto nasale della cavia e di altri roditori. Arch. Ital. Anat. Embriol.48:157–212.

Schwartz Levey, M.S., D.M. Chikaraishi, and J.S. Kauer (1991) Character-ization of potential precursor populations in the mouse olfactoryepithelium using immunocytochemistry and autoradiography. J. Neuro-sci. 11:3556–3564.

Schwob, J.E. (1992) The biochemistry of olfactory neurons: stages ofdifferentiation and neuronal subsets. In M.J. Serby and K.L. Chobor(eds): Science of Olfaction. NewYork: Springer-Verlag, pp. 80–125.

Schwob, J.E., K.E. Szumowski, D.A. Leopold, and P. Emko (1993) Histopa-thology of olfactory mucosa in Kallmann’s syndrome. Ann. Otol. Rhinol.Laryngol. 102:117–122.

Stensaas, L.J., R.M. Lavker, L. Monti-Bloch, B.I. Grosser, and D.L. Berliner

(1991) Ultrastructure of the human vomeronasal organ. J. SteroidBiochem. Mol. Biol. 39:553–560.

Talamo, B.R., R.A. Rudel, K.S. Kosik, V.M.-Y. Lee, S. Neff, L. Adelman, andJ.S. Kauer (1989) Pathological changes in olfactory neurons in patientswithAlzheimer’s disease. Nature 337:736–739.

Talamo, B.R., W.H. Feng, M. Perez-Cruet, L. Adelman, K. Kosik, V.M.-Y.Lee, L.C. Cork, and J.S. Kauer (1991a) Pathological Changes inOlfactory Neurons in Alzheimer’s disease. In J.H. Growden, S. Corkin,E. Ritter-Walker, and R.J. Wurtman (eds): Aging and Alzheimer’sDisease. NewYork: N.Y. Acad. Sci. pp. 1–7.

Talamo, B.R., W.H. Feng, M. Stockmayer, L. Cork, and J.S. Kauer (1991b)Monkey and human olfactory epithelium: a comparative study. Chem.Senses 16:589.

Talamo, B.R., W.H. Feng, and M. Stockmayer (1994) Human olfactoryepithelium: normal patterns and types of lesions found in the generalpopulation. Inhalation Toxicol. [Suppl.] 6:249–275.

Trojanowski, J.Q., P.D. Newman, W.D. Hill, and V.M.-Y. Lee (1991) Humanolfactory epithelium in normal aging, Alzheimer’s disease, and otherneurodegenerative disorders. J. Comp. Neurol. 310:365–376.

Vassar, R., J. Ngai, and R. Axel (1993) Spatial segregation of odorantreceptor expression in themammalian olfactory epithelium. Cell 74:309–318.

Yamagishi, M., and Y. Nakano (1992) A re-evaluation of the classification ofolfactory epithelia in patients with olfactory disorders. Eur. Arch.Otorhinolaryngol. 249:393–399.


new structure, the ?olfactory pit,? in human olfactory mucosa

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