induction of cell division in olfactory basal epithelium following intranasal irrigation with wheat...

Induction of Cell Division in OlfactoryBasal Epithelium Following Intranasal

Irrigation With Wheat Germ Agglutinin-Horseradish Peroxidase

YOUNG WHA MOON1,2 AND HARRIET BAKER1*1Cornell University Medical College at The Burke Medical Research Institute, White Plains,

New York 106052Catholic University Medical College, Seoul, Korea 137-701

ABSTRACTThe lectin, wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP),

previously was shown to be transported into the central nervous system following applicationby intranasal irrigation. The current study investigated the hypothesis that uptake ofmolecules, such as the lectin-conjugate, by olfactory receptor cells would mimic internaliza-tion of other substances including odorants. This process would result in both prematuredeath of receptor cells and increased turnover of their precursors, globose basal cells.Tetramethylbenzidine histochemical analysis showed the presence of significant amounts ofthe lectin-conjugate in both the receptor epithelium and olfactory bulb until at least 2 weekspostintranasal application. Neither supporting nor globose basal cells contained WGA-HRP,suggesting that uptake was primarily into olfactory receptor cells. Cell turnover, assessed bytritiated-thymidine (thymidine) autoradiography, increased both 1 and 2 weeks, but not 3 and4 weeks, following intranasal irrigation with WGA-HRP. Most of the cells containingthymidine labelling appeared to be globose basal cells, although supporting cells alsooccasionally exhibited labelling. Survival of either mature or immature receptor cells in theepithelium, indicated by epithelial thickness and cell density of the septal epithelium, alsodeclined following treatment. These data suggest that uptake of substances may result in cellloss from the olfactory epithelium and increased mitotic activity of basal cells. J. Comp.Neurol. 393:472–481, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: cell death; WGA-HRP; receptor cell; sensory; odor

The adult olfactory epithelium exhibits characteristicsthat are unique in the mammalian nervous system. Theseproperties may reflect its localization in the nasal cavityand consequent exposure to environmental insult. Amongthese attributes is the ability to respond to injury byreplacement of olfactory receptor cells from populations ofstem cells located subadjacent to the basal lamina of theolfactory epithelium (Monti Graziadei and Graziadei, 1979;Costanzo and Graziadei, 1983; Calof and Chikaraishi,1989; Schwartz Levey et al., 1991; Suzuki and Takeda,1991; Carr and Farbman, 1992; Schwob et al., 1992; Carrand Farbman, 1993; Crews and Hunter, 1994, Weiler andFarbman, 1997). Following a number of natural andexperimental perturbations, which destroy mature recep-tor cells, these stem cells divide, migrate within theepithelium, produce new chemoreceptive dendrites, andsend axons to reinnervate the central target organ, themain olfactory bulb (Graziadei and Monti Graziadei, 1979;

Monti Graziadei and Graziadei, 1979; Graziadei et al.,1980; Costanzo, 1991). Although initially postulated thatcontinuous turnover of receptor cells occurs, more recentdata suggest that the rate of cell replacement may bedependent, at least partially, on environmental influence(Hinds et al., 1984; Mackay-Sim and Kittel, 1991a).

Mature receptor cells exhibit increased turnover follow-ing a number of perturbations including viral, mechanical,and chemical (see Baker, 1995, for review). Section of thefilia olfactoria by either axotomy (Harding et al., 1977;

Grant sponsor: NIH; Grant numbers: DC01710 and AG09686.*Correspondence to: Harriet Baker, Cornell University Medical College

at The Burke Medical Research Institute, 785 Mamaroneck Avenue, WhitePlains, NY 10605. E-mail: [email protected]

Received 13 March 1997; Revised 21 November 1997; Accepted 26November 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 393:472–481 (1998)

r 1998 WILEY-LISS, INC.

Graziadei et al., 1980; Camara and Harding, 1984; Sa-manen and Forbes, 1984; Morrison and Costanzo, 1989;Suzuki and Takeda, 1991) or bulbectomy (Costanzo, 1984;Carr and Farbman, 1992; Schwob et al., 1992) leads todegeneration of mature receptor neurons and increasedneurogenesis in an attempt to reconstitute the epithelium.A number of volatile substances such as methyl bromide,dibasic esters, and chloroform also produce degenerationof the olfactory receptor epithelium followed in some casesby reconstitution (Hurtt et al., 1987; Keenan et al., 1990;Hastings et al., 1991; Mery et al., 1994; Schwob et al.,1995). Intranasal irrigation with Triton X-100 also resultsin a reversible lesion of the olfactory receptor epitheliumwith reinnervation of the olfactory bulb occurring within4–8 weeks (Nadi et al., 1981; Baker et al., 1983). Incontrast, turnover appears to be reduced when the epithe-lium is protected by either housing of mice in a laminarflow hood (Hinds et al., 1984) or by using unilateral narisclosure to prevent odorant access and thus entry of othermolecules into the nasal cavity (Maruniak et al., 1989;Cummings and Brunjes, 1994).

Not yet delineated are the signals which induce receptorcell degeneration and replacement. Trophic support fromthe olfactory bulb is one factor as demonstrated by thedramatic loss in receptor cells following bulbectomy (Farb-man, 1990) as well as the ability of transforming growthfactor-a and other growth factors to stimulate olfactoryreceptor cell division in vitro (Farbman and Buchholz,1996). The importance of this support is indicated also bythe dynamics of cell death following bulbectomy. In bulbec-tomized animals, a peak in the number of degenerating,newly produced cells occurs 5–7 days after mitosis. This is,perhaps, at a time when receptor cell axons, as theymature, would normally make contact with the olfactorybulb (Schwob et al., 1992; Carr and Farbman, 1993). Inthis same time-frame (6–7 days after mitosis), olfactorymarker protein, an indicator of sensory neuron matura-tion, appears in the newly generated receptor cells (Mira-gall and Monti Graziadei, 1982).

Another postulate suggests that turnover rate maydepend on uptake of molecules, including odorants, fromthe environment (Graziadei and Monti Graziadei, 1979;Getchell et al., 1984; Maruniak et al., 1990). Currenthypotheses suggest that removal of odorants from theolfactory mucosa involves several mechanisms. One meansis the direct clearance of molecules into the blood (Getchellet al., 1984). Another is internalization into either receptoror sustentacular cells, where the degradation of somemolecules is catalyzed by the many metabolic enzymesfound in both cell types (Dahl and Hadley, 1991). Lastly, arole in this process also has been suggested for odorantbinding proteins (Baldaccini et al., 1986; Pevsner et al.,1986) which either carry the molecules to receptor cell ciliaor transport them away to sites either of degradation orremoval through the blood.

The consequences of ligand internalization for receptorcell turnover is the subject of the current study. Thestudies employ a lectin-conjugate, wheat germ agglutininconjugated to horseradish peroxidase (WGA-HRP), previ-ously shown to be taken up readily by receptor cells andtransported transneuronally to the olfactory bulb andother brain regions (Broadwell and Balin, 1985; Shipley,1985; Baker and Spencer, 1986; Balin et al., 1986). Thelectin-conjugate, which has been assumed to be nontoxic inother neuronal systems, remains in the olfactory epithe-

lium at relatively high concentrations for up to 2 weeksafter application and is still demonstrable at 4 weeks.Thus, the presence of the lectin may mimic longtermexposure to molecules entering the nasal cavity, includingodorants. In contrast to the effect produced by narisclosure, which prevents access of molecules to the nasalmucosa, continuous exposure to the lectin might be ex-pected to increase cell death precipitating changes in cellturnover. The current studies test the hypothesis thatinternalization of WGA-HRP by receptor cells results inaltered turnover as indicated by changes in division ofstem cells. Also examined is possible cell loss, measured byepithelial thickness, as an indication of the dynamicrelationship between cell death and replacement.

MATERIALS AND METHODS

Animals

Male CD-1 mice, either 3 weeks or 8 weeks of age, werepurchased from Charles River Breeding Laboratories(Kingston, NY), housed in hooded cages at 22°C, andprovided with food and water ad libitum. Mice wereacclimated for at least 1 week before lectin application, sothat the youngest mice used were at least 4 weeks old.Under nembutal anesthesia (30 mg/kg), wheat germ agglu-tinin conjugated to horseradish peroxidase (WGA-HRP;Sigma, St. Louis, MO) was administered as a 1% solutionin sterile saline by intranasal irrigation in a manner thatwould insure continuous contact with the nasal epithe-lium. Control mice received a similar volume of saline. Thesolutions (30 µl in 20 minutes) were administered by dropsfrom a Hamilton microsyringe (Hamilton Co., Reno, NV)without introducing the needle into the naris. This mode ofadministration mimics the natural contact of substanceswith the olfactory epithelium. All procedures were carriedout under protocols approved by the Cornell UniversityInstitutional Animal Care and Use Committee and con-form to NIH guidelines.

Tritiated-thymidine autoradiography

At 1, 2, 3, and 4 weeks postlectin treatment mice (atleast three control and three treated mice for each time-point) received three intraperitoneal injections of tritiated-thymidine (thymidine, 2 µCi/g body weight, 20 Ci/mmol,New England Nuclear, Dupont, Boston, MA) over 16hours. Two hours after the last tracer injection, mice wereperfused intracardially with Bouin’s solution (75 ml/animal), the heads were postfixed for 6 hours, decalcified(2 days) in a formic acid-sodium citrate solution, andembedded in paraffin. Horizontal, 6 µm serial sectionswere deparaffinized and dipped in NTB-2 autoradio-graphic emulsion (Kodak, Rochester, NY) by using apublished procedure (Rogers, 1979). The slides were ex-posed at 4°C for either 2 or 3 weeks before developing inKodak D-19 developer. Sections were counterstained witheither hematoxylin or Schiff reagent.

Quantification of tritiatedthymidine-labelled cells

Thymidine-labelled cells were counted in a representa-tive sample from serial, horizontal sections of the septalepithelium ipsilateral to lectin application. The data areexpressed per unit length in millimeters. For each animal,at least five sections, separated from each other dorsoven-

WGA-HRP-INDUCED OLFACTORY RECEPTOR CELL TURNOVER 473

trally by 150 µm, were selected for counting. Three posi-tions within each section were sampled, rostral, middle,and caudal. The sections counted were chosen from sets ofserial sections assuring that the same dorsoventral androstral/caudal regions of all nasal septa were sampled.Profiles counted at 4003 magnification were consideredlabelled if at least five silver grains occurred over thenucleus, a degree of labelling greater than 53 background.Only labelled profiles in the region subadjacent to thelamina propria were counted even though labelled cellswere occasionally encountered in other areas of the septalepithelium.

Tetramethylbenzidine histochemistry

Eight mice were analyzed by tetramethylbenzidine(TMB) histochemistry to determine the time course oflectin disappearance from the epithelium. From 1 to 4weeks following lectin application, mice were perfusedwith 1% glutaraldehyde and 1% formaldehyde in 0.1Mphosphate buffer and the heads were decalcified withEDTA. Frozen sections, 30 µm, were processed for thehistochemical localization of WGA-HRP by a modificationof the method of Mesulam (1978) with TMB as thechromogen. Sections were counterstained with neutral redto provide cytoarchitectonic definition.

Determination of epithelial thicknessand cell density

The same sections and sampling procedures used fordetermination of the number of thymidine-labelled cellswere also employed for measuring the thickness of theseptal epithelium by light microscopy. Thickness wasestimated in caudal, middle, and rostral aspects of at leastsix sections spanning the dorsal ventral axis by using aneyepiece micrometer at 4003. Thickness was estimatedacross the entire epithelium including both cellular andmucus layers (the latter includes the receptor cell cilia andmicrovilli of supporting cells), as well as from only thecellular layer. Data for the entire epithelium are pre-sented, but the pattern of change was similar for bothmeasures.

The density of cells in the sensory epithelium wasdetermined in 1 week saline- and WGA-HRP-treated mice.The number of Nissl-stained nuclei was counted at 7503along a 110 µm length of the septal epithelium. To assessthe size of the Nissl-stained nuclei, an eyepiece microme-ter was used at 8003. The longest diameter was measuredin at least 90 cells in each animal. The measurements weremade in the area corresponding to the middle-region of theseptal epithelium.

Statistics

Data were analyzed by either analysis of variance withappropriate post-hoc tests or Student’s t-test. Statisticalsignificance was set at P , 0.05 level.

Photography and image processing

Either color negatives or slide positives, taken on aNikon microscope, were scanned with a Nikon scanner(Nixon Instrument Co., Melville, NY) and compositedusing Adobe Photoshop (Adobe System, Inc. MountainView, CA) on a MacIntosh computer (Apple, Carpinteria,CA). The scans were only altered to enhance contrast. InFigure 3, micrographs C and D were digitally enlarged toproduce E and F.

RESULTS

Distribution of WGA-HRP afterintranasal irrigation

One week following intranasal irrigation with WGA-HRP, the olfactory epithelium exhibited heavy labellingdemonstrated with HRP histochemistry. Label occurred inboth the cell bodies and axons of the olfactory receptor cells(Fig. 1A–C). Although the lectin-conjugate was appliedunilaterally, label occurred in both nasal cavities (Fig. 1A).This consistent finding did not permit the use of thecontralateral side as a control and required that analysisof cell turnover in lectin treated animals be compared tosaline irrigated control mice. The labelling appeared to berestricted to olfactory receptor cells as neither supporting,basal nor gland cells showed any staining (Fig. 1B). Thefasciculated axons (Fig. 1B,B’) traversed the cribriformplate and lectin labelling occurred both in the nerve fiberlayer and glomeruli of the main olfactory bulb (Fig. 1C). Atthis time, transneuronal labelling also was observed in theexternal plexiform and mitral cell layers (Fig. 1C). At 2weeks, the distribution of WGA-HRP was similar to thatobserved at 1 week in the olfactory epithelium but some-what reduced in the olfactory bulb (Fig. 1D–F). Axonallabelling in the lamina propria was reduced and transneu-ronal labelling was no longer obvious (Fig. 1F). Surpris-ingly, significant labelling, especially in the olfactory epi-thelium, was still present at 3 and 4 weeks after intranasalirrigation (not shown).

Cell turnover in the olfactory epitheliumof 8-week-old mice

Significant increases in the number of thymidine-labelled profiles, an indication of cellular labelling, wasobserved at 1 and 2 weeks following intranasal irrigationwith WGA-HRP. This is demonstrated graphically in Fig-ure 2 for both 8-week-old mice (Fig. 2A) and 4-week-oldanimals (Fig. 2B). Although an increase in the number ofthymidine-labelled profiles was apparent by visual inspec-tion (Figs. 3 and 4) in both the turbinates and septalregions of the olfactory epithelium, the analysis wasperformed on the septum which was sampled at threeregions (rostral, middle, and caudal) along its length. Thedata are expressed as the number of labelled cells per unitlength. The distribution of the labelling in lectin- vs.saline-treated mice 1 week following irrigation with WGA-HRP can be appreciated in the darkfield micrographs ofthe septal epithelium (Fig. 3A,B, respectively). Brightfieldmicrographs (Fig. 3C,D) demonstrate the large number ofsilver grains that occurred in labelled profiles as comparedto background level of grains. The higher magnificationmicrographs (Fig. 3E,F) show that most of the label wasfound in what appear to be globose basal cells that arelocalized subadjacent to the basal lamina. Labelled pro-files, presumably receptor cells, were infrequently encoun-tered in more superficial layers of the receptor epithelium(Fig. 3E,F). Labelled profiles, probably blood vessel endo-thelial cells and ependymal cells, often were observed inthe lamina propria.

A similar distribution of dividing cells occurred 2 weeksfollowing application of the lectin-conjugate (Fig. 4A–F).The low power micrographs illustrate the large number oflabelled neurons in lectin-treated as compared to saline-treated mice. This group of saline-treated mice displayed a

474 Y.W. MOON AND H. BAKER

Fig. 1. Histochemical demonstration of wheat germ agglutininconjugated to horseradish peroxidase (WGA-HRP) in the olfactoryepithelium (OE) and bulb (OB) 1 (A-C) and 2 weeks (D-F) afterintranasal irrigation with lectin conjugate. The low magnification,darkfield micrographs (A,D) indicate the presence of label in the OE,olfactory nerve layer (on) and OB. Brightfield micrographs (B,E) showthe presence of stained receptor (rc) but not sustentacular (sc) andbasal (bc) cells. Insets (B’,E’) are higher power micrographs of the

regions indicated by the arrows in B and E. In B, granular label also isobserved in the nerve layer (on). In the olfactory bulb (C,F), thedarkfield micrographs demonstrate the presence of label in the nervelayer and glomeruli (gl) at both posttreatment time points, but inmitral cells (m) only at 1 week after irrigation. ep, External plexiformlayer; g, glomerulus; gr, granule cell layer; NC, nasal cavity. Scalebar 5 800 µm in A and D, 50 µm in B and E, 100 µm in B’ and E’, and 80µm in C and F.


relatively low number of thymidine-labelled cells, perhapsexaggerating the difference between the lectin-irrigatedand saline-treated mice.

At 3 weeks postirrigation, the number of labelled pro-files, in the lectin-treated mice, although slightly higherthan in saline-treated animals, is no longer significantlydifferent (Fig. 2A). By 4 weeks, the number of labelledprofiles is identical in the two treatment groups (Fig. 2A).

Cell turnover in 4-week-old mice

The effects of intranasal application of WGA-HRP alsowere assessed in 4-week-old mice. A significant increase inthe number of thymidine-labelled profiles was observedboth 1 and 2 weeks postlectin application (Fig. 2B). Thenumber of cells labelled was lower than observed in the 8week-old mice since the exposure to emulsion was shorter.The pattern of labelling, however, was similar indicatingthat both 4- and 8-week-old animals show the sameresponse to the application of the lectin-conjugate.

Olfactory epithelial thickness and celldensity following WGA-HRP application

The thickness of the cellular and mucus layers of theolfactory receptor epithelium also was determined. Thisanalysis assessed if the increase in cell division of thebasal cells produced by WGA-HRP, as indicated by thymi-dine labelling, also was associated with an increase in celldeath within the olfactory epithelium. The sample in-cluded three regions of the septal epithelium (rostral,middle, and caudal). The width of the epithelium wasreduced by 40% in the WGA-HRP-treated mice on allaspects measured 1 week following intransal irrigationwith the lectin-conjugate (Mean thickness [µm] 6 SEM,saline, 54.0 6 8.91 µm vs. WGA-HRP, 31.6 6 3.36 µm;P , 0.05). At 2 weeks posttreatment, no significant differ-ences in the thickness occurred in the receptor epithelium,suggesting that in these areas if cell death occurred it wasexactly matched by addition of new cells (Fig. 5). The

Fig. 2. Histograms demonstrating the number of thymidine-labelled cells per millimeter observed 1 to 4 weeks after intranasalirrigation with either wheat germ agglutinin conjugated to horserad-ish peroxidase (WGA-HRP; hatched bar) or saline (open bar) in 8- (A)and 4- (B) week-old mice. In A the tissues were exposed to emulsion for

3 weeks and in B for 2 weeks. Analysis of variance indicated significantinteraction with time and treatment. Post hoc least significant differ-ence (LSD) tests showed that irrigation with WGA-HRP resulted in anincrease in the number of labelled cells 1 and 2, but not at 3 and 4,weeks posttreatment. Asterisk indicates P , 0.05.


decreased thickness can also be appreciated by comparingthe marked areas in Figure 3C and D.

The number of cells was estimated in the middle regionof the septal epithelium at 1 week post-WGA-HRP applica-tion when there was a large difference in epithelial thick-ness. The percent decrease in the WGA-HRP treated mice,about 40%, in the number of nuclear profiles in a knownlength of epithelium was about the same as the reductionin the epithelial thickness (see above) suggesting thatsignificant cell loss occurred (Mean number of cells per 110µm 6 SEM, saline, 88.8 6 2.21 vs. WGA-HRP, 56.7 6 3.61;P , 0.05). In addition, the nuclear diameter did not differbetween groups (Mean nuclear size in µm 6 SEM, saline,

4.48 6 0.18 µm vs. WGA-HRP, 4.42 6 0.28 µm; P . 0.05)demonstrating that there was no systematic error inestimating the number of nuclear profiles.

DISCUSSION

These studies demonstrate that intranasal irrigationwith the lectin conjugate, WGA-HRP, produces an increasein turnover of olfactory stem cells 1 and 2 weeks posttreat-ment as monitored by changes in the number of thymidine-labelled cells in the septal olfactory epithelium. Turnover,indicated by these methods, returned to control values by 4weeks after treatment. The alteration in the number of

Fig. 3. Darkfield (A,B) and brightfield (C–F) micrographs show theincrease in the number of thymidine-labelled cells 1 week followingintranasal irrigation with wheat germ agglutinin conjugated to horse-radish peroxidase (WGA-HRP; A,C,E) as compared to saline (B,D,F).The darkfield micrographs show the larger number of thymidine-labelled cells in the septal epithelium of lectin-versus saline-treatedmice. The brightfield micrographs suggest that most of the labelled

cells in the receptor cell (rc) layer were globose basal cells (arrows).The sections were selected to show the occasional thymidine-labelledsupporting cells (sc) observed (arrowheads). Note also that the recep-tor epithelium is thinner in WGA-HRP- than saline-treated mice(double arrows). lp, lamina propria; nc, nasal cavity; s, septum. Scalebar 5 100 µm in A and B, 20 µm in C and D, 10 µm in E and F.


labelled cells at 1 week was associated with a modestchange in the thickness of the septal epithelium.

Time-course of disappearanceof WGA-HRP-labelling

One week postirrigation, intense labelling for WGA-HRP occurred throughout the olfactory epithelium and theolfactory bulb. Even though the lectin-conjugate was ap-plied unilaterally, the distribution of label was almost asstrong on the contralateral side. The presence of the septalwindow may account for the leakage of the WGA-HRP toboth nasal cavities. Similar phenomena have previouslybeen observed (Broadwell and Balin, 1985). In mice,unilateral intranasal irrigation with zinc sulfate alsoresults in bilateral lesions of the epithelium (Harding andWright, 1979; Baker et al., 1983). The lectin-conjugate waspresent primarily in the receptor cells. Neither supporting

cells nor glandular elements contained significant label.The lack of label was unexpected in view of previous datashowing uptake of various molecules including HRP (notWGA-HRP), thorium dioxide, and ferritin into both olfac-tory receptor cells and supporting cells (Bannister andDodson, 1992). The longest interval between applicationand observation in the Bannister and Dodson (1992)studies was 24 hours, while in the current studies theshortest interval was 1 week. Either the lectin-conjugatenever entered or it was cleared from the supporting cellsbefore 1 week while it was maintained for up to 4 weeks inthe receptor cells. Several studies indicate that WGA-HRPundergoes receptor-mediated endocytosis in contrast toHRP-alone which is taken up by bulk endocytosis(Broadwell and Balin, 1985; Baker and Spencer, 1986;Balin et al., 1986). The current data support the hypoth-esis that uptake of the lectin is receptor mediated through

Fig. 4. Darkfield (A-D) and brightfield (E,F) micrographs show theincrease in the number of thymidine-labelled cells 2 weeks followingintranasal irrigation with wheat germ agglutinin conjugated to horse-radish peroxidase (WGA-HRP; A,C,E) as compared to saline (B,D,F).The low power micrographs demonstrate the large number of thymi-

dine-labelled cells in all regions of the receptor epithelium in thelectin- vs. the saline-treated mice. The higher magnification bright-field micrographs show that the label is found primarily in globosebasal cells. Scale bar 5 800 µm in A and B, 100 µm in C and D, 20 µm inE and F.


a mechanism specific to receptor cells. Studies in rat,however, do not indicate the nature of the lectin receptor,as a number of parameters including sugar specificity andmolecular weight of the lectins were not predictive of theefficacy of uptake (Baker and Franzen, 1994). In other exvivo labelling studies, WGA-HRP exhibited relatively non-specific binding, labelling many cell types (Lundh et al.,1989; Foster et al., 1991). However, these studies do notdistinguish between cell-surface molecules which wouldprovide a substrate for specific uptake of the lectins andintracellular ligands which also may bind the lectin butnot be relevant for receptor mediated internalization.

The lectin-conjugate was found in both the perikaryaand axons of receptor cells. Interestingly, by 1 weekpostirrigation receptor cell dendrites exhibited little HRP-label suggesting that the lectin-conjugate was transportedto and sequestered in the cell body. Axonal labellingoccurred in the lamina propria as well as in the nerve fiberlayer and glomeruli of the olfactory bulb. As previouslyobserved in rat and mouse olfactory bulb (Broadwell andBalin, 1985; Shipley, 1985; Baker and Spencer, 1986),transneuronal labelling also was apparent in the externalplexiform layer and in mitral cells.

The WGA-HRP-labelling pattern manifested at 2 weekswas similar to that observed at 1 week, but of lesserintensity in all compartments. At 3 and 4 weeks limitedlabelling occurred in the receptor cells of the olfactoryepithelium as well as the axonal projections in the olfac-tory bulb. Transneuronal labelling could no longer bedemonstrated. These data indicate that the lectin is cleared

relatively slowly from the receptor cells and this processmay mimic the sequestering of other molecules includingperhaps odorants which gain access to the olfactory epithe-lium under physiological conditions (Bannister and Dod-son, 1992). The consequences of this process for receptorcell turnover are discussed below.

Distribution and identity of tritiatedthymidine-labelled cells

In agreement with previous studies, the position andmorphology of the 3H-thymidine-labelled cells in the olfac-tory epithelium suggested that they are globose basal cells(Schwartz Levey et al., 1991; Suzuki and Takeda, 1991;Caggiano et al., 1994; Crews and Hunter, 1994; Holbrooket al., 1995; Huard and Schwob, 1995). Recent retrovirusdata from other laboratories shows that following perturba-tions to the epithelium, reconstitution occurs from theglobose basal cells and that horizontal basal cells are notinvolved (Caggiano et al., 1994; Schwob et al., 1994).Similarly, in the current studies, few if any horizontalbasal cells appeared to be labelled, supporting the hypoth-esis that globose basal cells divide to produce immaturecells which either mature or die (see below). The concentra-tion of labelled cells in the epithelial region adjacent to thelamina propria is consistent with the fact that the intervalbetween thymidine injection and perfusion was too shortto permit migration of the immature population of receptorcells to other epithelial layers (Mackay-Sim and Kittel,1991a; Schwob et al., 1992; Carr and Farbman, 1993;

Fig. 5. Bar graphs showing the changes in the thickness of theolfactory receptor epithelium in wheat germ agglutinin conjugated tohorseradish peroxidase (WGA-HRP) (hatched bars)- and saline (openbars)-treated mice 1 to 4 weeks post-intranasal irrigation. The thick-

ness of the epithelium, estimated in caudal, medial and rostral aspectsof the septal epithelium, differed in all aspects only at 1 weekpost-treatment. Asterisk indicates P , 0.05.


Caggiano et al., 1994). These stem cell aggregates presum-ably further divide to provide a population of immaturecells which can replace damaged or aged receptor neurons.While the quantitative analysis was limited to the septum,visual inspection of the same sections showed that asimilar upregulation in basal cell turnover occurred in theturbinates. The distribution of labelled cells was patchy,that is, alternating areas of labelled and unlabelled cells.The significance of this phenomenon is not clear, but couldrepresent the active zones previously described (Graziadeiand Monti Graziadei, 1979; Schwob et al., 1992). Also, aspreviously reported, a small population of thymidine-labelled cells occurred in the region of the supporting celllayer and in various regions of the lamina propria, oftenassociated with blood vessels (Schwob et al., 1992). Label-ling in the latter two populations was not assessed quanti-tatively, but did not appear to change with the WGA-HRP-treatment.

Evidence for dynamic equilibrium of celldeath and replacement

A decrease in the thickness of the epithelium at 1 weekpostapplication suggests that neuronal loss occurred inconjunction with the increased turnover. The epithelium,however, did not exhibit the extent of neuronal degenera-tion and damage to all cell types associated with treat-ments such as exposure to methylbromide where extensivecell loss and pyknotic nuclei occur in even relatively sparedregions of the epithelium (Schwob et al., 1995). The lack ofchange in the thickness of the septal epithelium at 2 weeksindicated that the increased number of new cells, gener-ated by the dividing globose basal cells, was matchedexactly by cell loss. Several hypotheses could be postulatedto explain the lack of change in thickness at 2 weeks. First,degeneration may occur in a population of either mature orimmature receptor cells and the loss may be matchedexactly by generation of new cells. Other investigatorshave found that at 2 weeks significant regeneration canoccur even after lesions which cause massive destructionof receptor cells (see Farbman, 1990, for review). Thus,reconstitution may occur within this time-frame.

Second, mature neurons, that is those already connectedto the olfactory bulb, may not die. This hypothesis requiresthat all the newly produced cells die before reachingmaturity. The premature death of new, immature cells hadbeen documented previously (Mackay-Sim and Kittel,1991b; Carr and Farbman, 1992; Schwob et al., 1992). Thecontinuing presence of WGA-HRP-labelled receptor cellsand their axons 2 and even 3 or 4 weeks after irrigationsuggests that at least some of the cells labelled by theinitial treatment continue to survive in the epithelium. Itis possible that the WGA-HRP leaks out of receptorneurons, especially those that are degenerating, and isinternalized subsequently from the extracellular space byadjacent neurons. However, this type of nonspecific trans-fer is not thought to occur for this lectin-conjugate. In fact,in the olfactory system, transneuronal transfer appears tooccur specifically in both the anterograde and retrogradedirections only in regions displaying direct synaptic conti-guity (Broadwell and Balin, 1985; Shipley, 1985; Bakerand Spencer, 1986).

Lastly, a situation may occur that is intermediate be-tween the first two hypotheses. A population of vulnerablecells, either mature or immature receptor cells, may dieand be replaced from the newly produced cells which in

turn reconnect with the olfactory bulb. Other less suscep-tible cells may continue to be present in the epithelium.The patchy nature of the label could represent regions ofspecific vulnerability. If it is assumed that there is someregulated turnover of receptor cells in the normal epithe-lium, those neurons closer to the end of their life-spancould be more vulnerable to the otherwise nontoxic effectsof the WGA-HRP.

Mechanisms regulating cell turnover

Reciprocal regulatory relationships have been postu-lated to exist between the olfactory epithelium and bulb.The continuing presence of the olfactory bulb in theseexperiments may act to stabilize and prevent massivedegeneration of the epithelium. Even at 1 week afterirrigation with WGA-HRP no evidence of massive degenera-tion was apparent. Current studies will determine if acontinuing process of apoptotic cell death occurs in thecells containing the lectin-conjugate.

The specific signals are not known which produce theincrease in cell division of the globose basal cells. Theremay be ‘‘multiple levels of epigenetic regulation’’ (Carr andFarbman, 1993). Perhaps the presence of WGA-HRP in thecells causes them to produce a signal up-regulating celldivision. If cell death occurs in a population of cells in theepithelium, this may be the signal for production of newreplacement cells. In either case, the current data suggestthat the presence in receptor neurons of a molecule, eitherWGA-HRP or perhaps other molecules including odorants,may be a sufficient stimulus to induce cell death andreplacement. The fact that keeping animals in a laminarflow system, where presumably they would be exposed tolower levels of xenobiotics, results in maintenance ofpopulations of long lived neurons, suggests that sequestra-tion of molecules in receptor cells is one important mecha-nism regulating their turnover.

ACKNOWLEDGMENTS

The authors thank Linda Franzen for excellent technicalassistance, Dr. Elena Cigola for technical and conceptualinput, Dr. Alan Blau for assistance with statistical analy-sis, and Charlie Carver for assistance in computer compos-iting of the figures.

These studies were supported by NIH grant DC01710and AG09686 (to H.B.).

LITERATURE CITED

Baker, H. (1995) Transport phenomena within the olfactory system. In R.L.Doty (ed): Handbook of Olfaction and Gustation. New York: MarcelDekker, pp. 173–190.

Baker, H., and L. Franzen (1994) Variable transport of lectin-conjugates inthe rat olfactory system. Soc. Neurosci. Abst. 20:140.

Baker, H., T. Kawano, F.L. Margolis, and T.H. Joh (1983) Transneuronalregulation of tyrosine hydroxylase expression in olfactory bulb of mouseand rat. J. Neurosci. 3:69–78.

Baker, H. and R.F. Spencer (1986) Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactoryepithelium to the brain of the adult rat. Exp. Brain Res. 63:461–473.

Baldaccini, N.E., A. Gagliardo, P. Pelosi, and A. Topazzini (1986) Occur-rence of a pyrazine binding protein in the nasal mucosa of somevertebrates. Comp. Biochem. Physiol. 84B:249–253.

Balin, B.J., R.D. Broadwell, M. Salcman, and M. El-Kalliny (1986) Avenuesfor entry of peripherally administered protein to the central nervoussystem in mouse, rat and squirrel monkey. J. Comp. Neurol. 251:260–280.


Bannister, L.H. and H.C. Dodson (1992) Endocytic pathways in theolfactory and vomeronasal epithelia of the mouse: Ultrastructure anduptake of tracers. Microscopy Res. Tech. 23:128–141.

Broadwell, R.D. and B.J. Balin (1985) Endocytic and exocytic pathways ofthe neuronal secretory process and trans-synaptic transfer of wheatgerm agglutinin-horseradish peroxidase in vivo. J. Comp. Neurol.242:632–650.

Caggiano, M., J.S. Kauer, and D.D. Hunter (1994) Globose basal cells areneuronal progenitors in the olfactory epithelium: A lineage analysisusing a replication-incompetent retrovirus. Neuron 13:339–352.

Calof, A.L. and D.M. Chikaraishi (1989) Analysis of neurogenesis in amammalian neuroepithelium: Proliferation and differentiation of anolfactory neuron precursor in vitro. Neuron 3:115–127.

Camara, C.G. and J.W. Harding (1984) Thymidine incorporation in theolfactory epithelium of mice: early exponential response induced byolfactory neurectomy. Brain Res. 308:63–68.

Carr, V.M. and A.I. Farbman (1992) Ablation of the olfactory bulb up-regulates the rate of neurogenesis and induces precocious cell death inolfactory epithelium. Exp. Neurol. 115:55–59.

Carr, V.M. and A.I. Farbman (1993) The dynamics of cell death in theolfactory epithelium. Exp. Neurol. 124:308–314.

Costanzo, R.M. (1984) Comparison of neurogenesis and cell replacement inthe hamster olfactory system with and without a target (olfactory bulb).Brain Res. 307:295–301.

Costanzo, R.M. (1991) Regeneration of Olfactory Receptor Cells. CibaFoundation Symp. 160, New York: Wiley, pp. 233–248.

Costanzo, R.M., and P.P.C. Graziadei (1983) A quantitative analysis ofchanges in the olfactory epithelium following bulbectomy in hamster. J.Comp. Neurol. 215:370–381.

Crews, L. and D. Hunter (1994) Neurogenesis in the olfactory epithelium.Perspec. Dev. Neurobiol. 2:151–161.

Cummings, D.M. and P.C. Brunjes (1994) Changes in cell proliferation inthe developing olfactory epithelium following neonatal unilateral narisocclusion. Exp. Neurol. 128:124–128.

Dahl, A.R. and W.M. Hadley (1991) Nasal cavity enzymes involved inxenobiotic metabolism: Effects on the toxicity of inhalants. Toxicology21:345–372.

Farbman, A.I. (1990) Olfactory neurogenesis: genetic or environmentalcontrols? TINS 13:362–365.

Farbman, A.I. and J.A. Buchholz (1996) Transforming growth factor-a andother growth factors stimulate cell division in olfactory epithelium invitro. J. Neurobiol. 30:267–280.

Foster, J.D., M.L. Getchell, and T.V. Getchell (1991) Identification of sugarresidues in secretory glycoconjugates of olfactory mucosae using lectinhistochemistry. Anat. Rec. 229:525–544.

Getchell, T.V., F.L. Margolis, and M.L. Getchell (1984) Perireceptor andreceptor events in vertebrate olfaction. Prog. Neurobiol. 23:317–345.

Graziadei, P.P.C., M.S. Karlan, G.A. Monti Graziadei, and J.J. Bernstein(1980) Neurogenesis of sensory neurons in the primate olfactory systemafter section of the fila olfactoria. Brain Res. 186:289–300.

Graziadei, P.P.C. and G.A. Monti Graziadei (1979) Neurogenesis andneuron regeneration in the olfactory system of mammals. I. Morphologi-cal aspects of differentiation and structural organization of the olfactorysensory neurons. J. Neurocytol. 8:1–18.

Harding, J., P.P.C. Graziadei, G.A. Monti Graziadei, and F.L. Margolis(1977) Denervation in the primary olfactory pathway of mice. IV.Biochemical and morphological evidence for neuronal replacementfollowing nerve section. Brain Res. 132:11–28.

Harding, J.W. and J.W. Wright (1979) Effects of intranasal irrigation withmitotic inhibitors on olfactory behavior and biochemistry in mice. BrainRes. 168:31–41.

Hastings, L., M.L. Miller, D.J. Minnema, J. Evans and M. Radike (1991)Effects of methyl bromide on the rat olfactory system. Chem. Senses16:43–55.

Hinds, J.W., P.L. Hinds, and N.A. McNelly (1984) An autoradiographicstudy of the mouse olfactory epithelium: evidence for long-lived recep-tors. Anat. Rec. 210:375–383.

Holbrook, E.H., K.E. Mieleszko Szumowski, and J.E. Schwob (1995) Animmunochemical, ultrastructural, and developmental characterizationof the horizontal basal cells of rat olfactory epithelium. J. Comp. Neurol.363:129–146.

Huard, J.M.T. and J.E. Schwob (1995) Cell cycle of globose basal cells in ratolfactory epithelium. Dev. Dyn. 203:17–26.

Hurtt, M.E., K.T. Morgan, and P.K. Working (1987) Histopathology of acutetoxic responses in selected tissues from rats exposed by inhalation tomethyl bromide. Fund. App. Toxicol. 9:352–365.

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

Lundh, B., U. Brockstedt, and K. Kristensson (1989) Lectin-binding patternof neuroepithelial and respiratory epithelial cells in the mouse nasalcavity. Histochem. J. 21:33–43.

Mackay-Sim, A. and P. Kittel (1991a) Cell dynamics in the adult mouseolfactory epithelium: a quantitative autoradiographic study. J. Neuro-sci. 11:979–984.

Mackay-Sim, A. and P.W. Kittel (1991b) On the life span of olfactoryreceptor neurons. Europ. J. Neurosci. 3:209–215.

Maruniak, J.A., J.R. Henegar, and T.P. Sweeney (1990) Effects of long-termunilateral naris closure on the olfactory epithelia of adult mice. BrainRes. 526:65–72.

Maruniak, J.A., P.J. Lin, and J.R. Henegar (1989) Effects of unilateral narisclosure on the olfactory epithelia of adult mice. Brain Res. 490:212–218.

Mery, S., J.L. Larson, B.E. Butterworth, D.C. Wolf, R. Harden, and K.T.Morgan (1994) Nasal toxicity of chloroform in male F-344 rats andfemale B6C3F1 mice following a 1-week inhalation exposure. Toxicol.Appl. Pharmacol. 125:214–227.

Mesulam, M.M. (1978) Tetramethylbenzidine for horseradish peroxidaseneurohistochemistry: a non-carcinogenic blue reaction product withsuperior sensitivity for visualizing neural afferents and efferents. J.Histochem. Cytochem. 26:106–117.

Miragall, F. and G.A. Monti Graziadei (1982) Experimental studies on theolfactory marker protein. II. Appearance of the olfactory marker proteinduring differentiation of the olfactory sensory neurons of mouse: Animmunocytochemical and autoradiographic study. Brain Res. 239:245–250.

Monti Graziadei, G.A. and P.P.C. Graziadei (1979) Neurogenesis andneuron regeneration in the olfactory system of mammals. II. Degenera-tion and reconstitution of the olfactory sensory neurons after axotomy.J. Neurocytol. 8:197–213.

Morrison, E.E. and R.M. Costanzo (1989) Scanning electron microscopicstudy of degeneration and regeneration in the olfactory epithelium afteraxotomy. J. Neurocytol. 18:393–405.

Nadi, N.S., R. Head, M. Grillo, J. Hempstead, N. Grannot-Reisfeld, and F.L.Margolis (1981) Chemical deafferentation of the olfactory bulb: Plastic-ity of the levels of tyrosine hydroxylase, dopamine and norepinephrine.Brain Res. 213:365–377.

Pevsner, J., P.B. Sklar, and S.H. Snyder (1986) Odorant-binding protein:Localization to nasal glands and secretions. Proc. Natl. Acad. Sci. USA83:4942–4946.

Rogers, A.W. (1979) Techniques in Autoradiography., 3rd Ed., Amsterdam,Elsevier.

Samanen, D.W., and W.B. Forbes (1984) Replication and differentiation ofolfactory receptor neurons following axotomy in the adult hamster: Amorphometric analysis of postnatal neurogenesis. J. Comp. Neurol.225:201–211.

Schwartz Levey, M., D.M. Chikaraishi, and J.S. Kauer (1991) Characteriza-tion of potential precursor populations in the mouse olfactory epithe-lium using immunocytochemistry and autoradiography. J. Neurosci.11:3556–3564.

Schwob, J.E., J.M.T. Huard, M.B. Luskin, and S.L. Youngentob (1994)Retroviral lineage studies of the rat olfactory epithelium. Chem. Senses19:671–682.

Schwob, J.E., K.E. Mieleszko Szumowski, and A.A. Stasky (1992) Olfactorysensory neurons are trophically dependent on the olfactory bulb fortheir prolonged survival. J. Neurosci. 12:3896–3919.

Schwob, J.E., S.L. Youngentob, and R.C. Mezza (1995) Reconstitution of therat olfactory epithelium after methyl bromide-induced lesion. J. Comp.Neurol. 359:15–37.

Shipley, M.T. (1985) Transport of molecules from nose to brain: Transneuro-nal anterograde and retrograde labeling in the rat olfactory system bywheat germ agglutinin-horseradish peroxidase applied to the nasalepithelium. Brain Res. Bull. 15:129–142.

Suzuki, Y. and M. Takeda (1991) Basal cells in the mouse olfactoryepithelium after axotomy: immunohistochemical and electron-micro-scopic studies. Cell Tiss. Res. 266:239–245.

Weiler, E. and A.I. Farbman (1997) Proliferation in the rat olfactoryepithelium: Age-dependent changes. J. Neurosci. 17:3610–3622.


induction of cell division in olfactory basal epithelium following intranasal irrigation with wheat...

Documents