Reinnervation of the rat olfactory bulb after methyl bromide-induced lesion: Timing and extent of...

Reinnervation of the Rat Olfactory BulbAfter Methyl Bromide-Induced Lesion:

Timing and Extent of Reinnervation

JAMES E. SCHWOB,1,2* STEVEN L. YOUNGENTOB,2,3 GEORGE RING,1

CARRIE L. IWEMA,1,2 AND RENEE C. MEZZA1,2

1Department of Anatomy and Cell Biology, SUNY Health Science Center,Syracuse, New York 13210

2Clinical Olfactory Research Center, SUNY Health Science Center,Syracuse, New York 13210

3Department of Physiology, SUNY Health Science Center, Syracuse, New York 13210

ABSTRACTWe used the inhalation of methyl bromide gas to produce a near-complete destruction of the rat

olfactory epithelium and analyzed the reinnervation of the bulb during reconstitution of theepithelium. The degeneration of olfactory axons elicits a transient up-regulation of glial cellproliferation and glial fibrillary acidic protein expression in the olfactory nerve and olfactory nervelayer of the bulb. Anterograde transport after intranasal infusion of wheat germ agglutininconjugated horseradish peroxidase demonstrates that the first nascent axons reach the bulb withinthe first week after lesion. Subsequently, a massive wave of fibers arrives at the bulb between 1 and2 weeks postlesion, and enters the glomeruli between 2 and 3 weeks postlesion. However, theolfactory projection does not stabilize until 8 weeks after lesion judging from the return in growthassociated protein-43 expression to control levels. The extent of reinnervation after lesion iscorrelated with the completeness with which the epithelium reconstitutes itself. In rats that arelesioned while fed ad libitum, there is near-complete reconstitution of the neuronal population, andthe projection onto the bulb fills the glomerular layer in its entirety. However, in rats that arelesioned while food restricted, a significant fraction of olfactory epithelium becomes respiratoryduring its reconstitution, and the population of reinnervating fibers is less. As a consequence, theposterior half of the bulb remains hypoinnervated overall and denervated at its caudal margin. Thepreferential reinnervation of the anterior bulb in the food-restricted, methyl bromide gas–lesionedanimals indicates that the mechanisms that guide the growth of the olfactory axons and restorereceptotopy do not operate with the same precision in this setting as they do during development orduring the lower level of turnover associated with the ‘‘normal’’ laboratory existence.Accordingly, wehypothesize that the persistence of a significant population of pre-existing neurons is needed topreserve receptotopy during reinnervation. In addition, the results suggest that in the face ofmassive turnover and a reduced afferent population, there is a tendency for reinnervating axons tofill available synaptic space. J. Comp. Neurol. 412:439–457, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: regeneration; gliosis; axonal guidance; glomerulotopy; GAP-43; olfactory marker

protein

The olfactory epithelium has the capacity, unique withinthe nervous system, to reconstitute its population ofneurons and non-neuronal cells to near-normal after beingdamaged by direct exposure to toxins or after the retro-grade neuronal degeneration caused by transection of theolfactory nerve (reviewed by Graziadei and Monti Grazia-dei, 1985; Costanzo, 1991). In particular, inhalation ofmethyl bromide gas (MeBr) at a concentration of 330 ppmfor 6 hours by rats that are maintained on food restrictionat the time of exposure reproducibly destroys all the

neurons, all the sustentacular cells, and some of the basalcells in over 95% of the main olfactory epithelium; theremaining 5% of the epithelium is damaged to a lesser

Grant sponsor: National Institutes of Health; Grant number: R01DC00467; Grant number: K04 DC00080; Grant number: P01 DC00220.

*Correspondence to: James E. Schwob, M.D., Ph.D., Department ofAnatomy and Cell Biology, SUNY Health Science Center, 750 East AdamsStreet, Syracuse, NY 13210. E-mail: [email protected]

Received 9 November 1998; Revised 21 April 1999; Accepted 14 May 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 412:439–457 (1999)

r 1999 WILEY-LISS, INC.

degree (Hurtt et al., 1987, 1988; Hastings et al., 1991;Schwob et al., 1995). Despite the massive initial damage,most areas of the epithelium recover to become indistin-guishable from normal. By 6 weeks after lesion, theseareas regenerate a normal or near-normal number ofneurons; the proportion of these neurons that are imma-ture falls to near control levels by then (Schwob et al.,1995). In parallel with, and presumably as a reflection of,neuronal recovery, basal cell proliferation falls from a peakthat is four- to fivefold increased relative to normal shortlyafter lesion to a level equivalent to controls by this 6-weektime-point (Hurtt et al., 1988; Schwob et al., 1995). Like-wise, markers of sustentacular cell function also recover tonear normal levels by this time. Some areas of what wasoriginally olfactory epithelium take on the characteristicsof respiratory epithelium during its reconstitution andremain permanently non-neuronal (Schwob et al., 1995).Nonetheless, the degree of anatomic recovery in the olfac-tory epithelium overall after MeBr exposure is striking(Hurtt et al., 1988; Hastings et al., 1991; Schwob et al.,1995). Other models of epithelial damage, such as ZnSO4or nonionic detergent irrigation, give results that aresimilar in broad outline and in many of the details to theobservations obtained by using MeBr (Mulvaney andHeist, 1971; Matulionis, 1975, 1976; Harding et al., 1978;Nadi et al., 1981; Kream and Margolis, 1984; Burd, 1993).

The kind of epithelial damage that occurs with directapplication or airborne exposure to toxins, as describedabove, differs in several respects from the damage that iscaused by lesion of the olfactory nerve. Nerve transectionis usually thought to cause only retrograde neuronaldegeneration and to leave other populations unaffected,other than the indirect response of the non-neuronal cellsto neuronal degeneration (Monti Graziadei and Graziadei,1979; Monti Graziadei et al., 1980; Walters et al., 1992). Asa consequence, studies of the reinnervation of the bulbafter peripheral olfactory lesion have tended to focus onthe nerve transection model, in part, because the lack ofdirect damage to basal cells and sustentacular cells isthought to more closely mimic the events in the epitheliumsubsequent to the death of neurons that occurs at the endof ‘‘normal’’ neuronal lifespan (Graziadei and Monti Grazia-dei, 1979; Monti Graziadei and Graziadei, 1979; MontiGraziadei et al., 1980; Morrison and Costanzo, 1995; Yeeand Costanzo, 1995; Koster and Costanzo, 1996). (It isimportant to note that the concept of an intrinsic limit tolifespan remains controversial; Farbman, 1990; Mackay-Sim and Kittel, 1991; Schwob et al., 1992).

On the other hand, the MeBr model of olfactory damagehas several important advantages for the study of thecapacity of the peripheral olfactory system to reinnervatethe bulb after damage. First, damage to the epithelium bymeans of MeBr exposure more closely mimics the directdamage to which the epithelium is subject during normallife than does nerve transection (albeit the wholesale,massive replacement of one set of mature neurons byanother represents one extreme of what is likely to be acontinuum of damage in the unprotected, nonlaboratoryenvironment where animals are exposed to toxins, infec-tious agents, etc.) (Smith, 1938; Doty, 1979; Jafek et al.,1990). Second, the olfactory nerve itself is not directlydamaged by a peripheral type of manipulation (Schwob etal., 1995). As a consequence, the analysis of reinnervationafter epithelial damage is not confounded by mechanicaldisruption of the pathway, which may interfere with the

growth of axons toward the bulb after transection (Schwobet al., 1994). In other words, the extent to which reinnerva-tion occurs and is anatomically and functionally ‘‘correct’’after reversible damage to the epithelium provides anindication of the ability of the growing olfactory axons tonavigate across a mechanically intact terrain. Further-more, this experimental setting more closely mimics theevents in the nerve that accompany the growth of axons bynewly generated neurons during neuronal turnover thandoes the transection model, whether such turnover isoccasioned by an innate limit to olfactory neuronal lifespan,by damage due to environmental causes, or both.

Thus, the availability of the MeBr lesion model raisesseveral important, unanswered questions that pertain tothe reinnervation of the olfactory bulb during the reconsti-tution of the epithelium. How do the glial cells of theolfactory nerve and bulb respond acutely to the epithelialdamage and consequent axonal degeneration? Does rein-nervation occur in this setting as it has been shown tooccur after other forms of damage to the epithelium orolfactory nerve? What is the timing of reinnervation of thebulb with relation to neuronal reconstitution? What is theextent of the reinnervation by the end of the process ofepithelial recovery? Does the process of reinnervationrecapitulate the spatial organization that was characteris-tic of the projection of the epithelium onto the bulb at theend of the recovery process? Does the process of reinnerva-tion permit functional as well as anatomic recovery? Theanalysis presented here addresses all but the last two ofthese questions, which will be the subject of future manu-scripts. An abbreviated version of a portion of the work waspresented in abstract form previously (Schwob and Young-entob, 1991, 1992).

MATERIALS AND METHODS

Animals

Male Long-Evans hooded rats were obtained from acommercial supplier (Blue Spruce/Harlan Sprague-Daw-ley) at 200–250 g. Some rats were maintained on adlibitum rat chow and exposed to MeBr at a body weight of225–275 g; others were food-restricted and maintained at75% of ad libitum body weight (ranging from 240 to 280 g).All were maintained on ad libitum water in a heat andhumidity controlled vivarium. All animal use protocolswere approved by the Committee for Humane Use ofAnimals at the SUNY Health Science Center at Syracuse.

Methyl bromide lesion

Awake rats were placed in a wire enclosure measuring15 3 15 3 15 cm that was centered in a 30 3 30 3 30 cmplexiglas box and exposed to methyl bromide (MeBr) gas(Matheson Gas Products, East Rutherford, NJ) at 330 ppmin purified air at a flow rate of 10 L/min as described(Schwob et al., 1995). The duration of the exposure was 6hours. After lesioning, the animals were kept on the samefeeding schedule as before, and perfused at 1, 2, 3, 5, 7, or10 days, or 2, 3, 4, 6, or 8 weeks after lesion.

Bromodeoxyuridine labeling

The same three sets of animals that were used for thedetermination of proliferation rate in the epithelium in aprevious report (Schwob et al., 1995) were used to deter-mine the rate of proliferation of glial cells in the primaryolfactory projection. They were injected intravenously

440 J.E. SCHWOB ET AL.

with bromodeoxyuridine (BrdU) (Sigma Chemical, St.Louis, MO) at a dose of 90 mg/kg as described previously(Schwob et al., 1992) and killed by perfusion 1 hour later.The first set was perfused with periodate-lysine-2% para-formaldehyde (PLP), and the olfactory tissue was cryopro-tected and sectioned on a cryostat at 5 and 8 µm; a secondset was perfused with Carnoy’s solution, then further fixedby immersion, and the tissue was paraffin-embedded andsectioned at 2 and 4 µm; and the third set was perfusedwith OmniFix II (An-Con Genetics, Melville, NY), and theolfactory tissue was sectioned on a cryostat at 5 and 8 µm.

Antibody reagents

The following primary antibodies were used in theanalyses: goat antiserum against olfactory marker protein(OMP; the generous gift of Dr. Frank Margolis; MontiGraziadei et al., 1977), a rabbit antiserum that recognizesrat neural cell adhesion molecule (NCAM; the generousgift of Dr. Jonathan Covault; Covault and Sanes, 1986), arabbit antiserum that recognizes rat S-100 protein (DakoCorporation, Carpinteria, CA), the mouse monoclonal anti-body designated 2G12 that binds to the phosphorylatedform of growth associated protein-43 (GAP-43; Meiri et al.,1991), the mouse monoclonal antibody designated GA-5that is directed against porcine glial fibrillary acidicprotein (GFAP; BioGenex Laboratories, CA), and the mousemonoclonal antibody designated B44 that recognizes BrdU(Becton Dickinson, San Jose, CA). Secondary and tertiaryreagents were obtained from Vector Laboratories (Burlin-game, CA) or Jackson ImmunoResearch (West Grove, PA).

Immunohistochemistry

Bouin’s- and organic solvent-fixed paraffin sections andPLP-fixed frozen sections were immunostained with asingle antibody and visualized with peroxidase as previ-ously described (Schwob et al., 1992, 1994; Huard et al.,1998).

Anterograde transport of wheat germagglutinin-conjugated horseradish

peroxidase

At various times after MeBr exposure (1, 3, 5, 7 days, or2, 3, 4, 6 weeks), rats were anesthetized by means ofintramuscular injection of a cocktail of ketamine andacepromazine (10 mg and 0.05 mg per 100 g body weight,respectively), tracheotomized and infused with 250 µl of a1% solution of wheat germ agglutinin-conjugated horserad-ish peroxidase (WGA-HRP) in PBS (KH2PO4, 1.5 mM;NaH2PO4, 9.5 mM; KCl, 2.7 mM; NaCl; 137 mM; pH 7.2).After 2 hours, the nose was drained, the tracheotomy wasclosed, and the animal was allowed to recover for 24 hoursbefore intracardiac perfusion with 1% paraformaldehyde/1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.1 (ifcut on a sliding microtome at 30 µm and reacted asfree-floating sections, n 5 1 at 1 and 7 days and 2 and 3weeks after lesion; n 5 9 at 3 days) or 1% paraformalde-hyde in phosphate buffer (if cut on a cryostat at 8 µm andreacted on the slide; n 5 2 at each time point). For thefree-floating series, sections were harvested at 180-µmintervals through the entirety of the olfactory bulb andreacted with tetramethylbenzidine in 0.1 M acetate buffer,pH 3.3, according to published protocol (Mesulam et al.,1980).

Image analysis of the glial responseto neuronal destruction

Rats were exposed to MeBr 24 hours after one naris wasclosed by occluding with cyano-acrylate glue and oversew-ing. The naris was then reopened the day after lesion, andthe animals were allowed to survive either 3 days or 12weeks before being killed. Multiple sections through theolfactory bulbs were stained with anti-GFAP antibody andadjacent sections stained with anti-S-100 antibody. Im-ages of the peroxidase-stained sections were obtainedunder brightfield illumination at 403 total magnificationby using a CCD camera (Hamamatsu USA, Bridgewater,NJ) and Pixel Buffer frame grabber (Perceptics Corp.,Knoxville, TN) and assembled into a mosaic. The compos-ite images were segmented, and the size and stainingdensity of the segmented objects were measured by usingIPLab Spectrum image analysis software (Scanalytics,Inc., Fairfax, VA).

Image analysis of the extent of bulbardenervation

A total of three control rats, three rats lesioned while fedad libitum, and three rats lesioned while food-intake wasrestricted were killed 3 days after lesion, and images of theTMB-stained sections were obtained under brightfieldillumination with a red Wratten no. 29 filter at 163 totalmagnification as described above. A flattened map repre-senting the density of transported label across the surfaceof the bulb was constructed as follows. On the image ofeach section, a line was drawn by visual inspection throughthe center of the glomerular layer. In the anterior bulb, theline began directly opposite the point where the glomeru-lar layer is thinnest at the dorsomedial edge of the bulb,proceeds along the medial, dorsal, and lateral sides andends at the starting point. In the posterior bulb, theglomerular layer recedes from the ventrolateral margin ofthe olfactory peduncle, then successively from the lateraland dorsal sides (where it is replaced by the accessoryolfactory bulb). From the level where the glomerular layeris interrupted to the posterior limit of the bulb, the linetraversing the glomeruli began at the ventromedial mar-gin of the glomerular layer and ended at the oppositemargin of the glomerular layer. The density of reactionproduct was extracted along that line and represented asvariations in shading along a straight line whose thicknessrepresented the spacing between sections. Beginning atthe anterior pole, successive sections were aligned andstacked to assemble the flattened map of the entireglomerular layer of the bulb. The resulting map wassubjected to Gaussian blur at a six-pixel radius to providea more averaged value for the glomeruli, and pixelsequivalent to section background were arbitrarily as-signed a low value to emphasize the distribution of trans-ported label. The percentage of the glomerular layer thatlacks label was determined by segmenting the compositeimage at a value between background and the lightestlevel of transported label.

Image analysis of the extent of bulbarreinnervation

Images of OMP-stained sections of the olfactory bulbwere captured as described above. Between 16 and 24levels, spaced equally along the anteroposterior axis of thebulb, were analyzed. The lesioned cases were killed 8

REINNERVATION OF OLFACTORY BULB 441

weeks after lesion if fed ad libitum (n 5 2) or at 11–21weeks after lesion if food-restricted (n 5 6). Age-matched,unlesioned controls were analyzed in similar manner (n 53). For each section, we determined the area of theglomerular layer that is occupied by OMP-positive fibersby outlining the glomerular layer by visual inspection andsegmenting the image at a point halfway between the levelof background staining and the maximum of OMP-positivestaining in that case. The area occupied by the segmentedpixels was calculated for each section and plotted as afunction of relative distance of the imaged section alongthe axis from the anterior to posterior poles of the olfactorybulb. The anteroposterior (AP) length of the bulb maydiffer slightly between cases depending on the preciseangle of sectioning, which is the reason for using a relativemeasure. Total glomerular volume was determined byintegrating under the distance vs. glomerular area curve.The distance that marks the point dividing total glomeru-lar volume into equal halves was also determined for eachof the cases, relative to the length of the bulb.

Preparation of figures

Photomicrographs of histologic sections were taken on35 mm Ilford XP-2 film by using a Nikon Eclipse 800photomicroscope (Nikon Instruments, Melville, NY). Indi-vidual images were digitized using a Nikon LS-100 SuperCoolscan film scanner attached to an Apple Macintosh G3computer. The images were assembled into panels usingAdobe Photoshop 5.0 (Adobe Systems, Inc., San Jose, CA).Unless otherwise noted in the figure legends, the imageswere not enhanced except for minor adjustments to bright-ness and contrast.

RESULTS

Reaction of glial cells in the olfactory nerveand bulb to MeBr-induced damage to the

olfactory epithelium

The death of 90–98% of the neurons as a consequence ofMeBr exposure inevitably produces anterograde degenera-tion of an equally large percentage of olfactory axons in theolfactory nerve and bulb. The reaction of the glial cells inthe nerve and in the bulb was assessed by means ofchanges in the number of actively proliferating glia and inthe distribution and intensity of staining with antibodiesagainst GFAP and S-100.

By 24 hours after exposure to MeBr, the number ofproliferating cells in the fascicles of the olfactory nerve andin the olfactory nerve layer (ONL) and the glomerularlayer (GL) of the bulb increases substantially. The numbercontinues to increase over the next 2 days and remainsgreater than normal 1 week after lesion (Fig. 1). Countsindicate that the rate of proliferation at 3 days is increasedeightfold over control (Table 1). The proliferating cellshave the characteristic shape and position of glial cells inthe primary olfactory projection; the nuclei are small andhighly elongate and sit within the fascicles of the olfactorynerve and in the septa between glomeruli where glialsomata are found (Fig. 2; Barber and Lindsay, 1982;Doucette, 1984, 1993; Valverde and Lopez-Mascaraque,1991; Bailey and Shipley, 1993; Chiu and Greer, 1996).

In addition to the proliferative response, the expressionof GFAP is markedly increased in the olfactory nerve, theONL, and the GL after lesion; the increased expression ismost strikingly evident when comparing lesioned and

unlesioned sides of rats in which closure of one naris beforeMeBr restricts the damage to the contralateral side of thenose (Fig. 2). Likewise, there is an increase, although moremodest, in the level of S-100 expression in these samelayers of the bulb and in the nerve (not shown). We usedcomputer-based densitometric analysis to compare thenumber and size of the GFAP-stained profiles between thetwo sides. The intensity of the reaction product is in-

Fig. 1. Glial cell proliferation in the olfactory nerve and glomerularlayers increases acutely after methyl bromide gas lesion. A: Sectionfrom mid-anterior bulb of animal lesioned 3 days before injection ofbromodeoxyuridine (BrdU) and then perfused. Arrowheads point toexamples of nuclei labeled by the incorporation of BrdU. The nucleiare elongate and situated preferentially either between axon fasciclesin the olfactory nerve, or in the septa between glomeruli, indicatingthat they are glia. B: Comparable level from age-matched controlinjected with BrdU. There are no labeled nuclei in this field. onl,olfactory nerve layer; gl, glomerular layer; epl, external plexiformlayer. Scale bar 5 100 µm in B (applies to A,B).

TABLE 1. Bromodeoxyuridine Labeling of Glial Cells in Olfactory Nerveand Glomerular Layers After Methyl Bromide Gas Lesion1

1 Daypostlesion

(n 5 3)

3 Dayspostlesion

(n 5 2)

7 Dayspostlesion

(n 5 3)

Increase in labeling relative tomatched control 2.25 6 0.40 7.92 6 1.58 3.33 6 1.01

1Labeled cells in the olfactory nerve and glomerular layers were counted on three ormore sections per case, expressed per unit area of the two layers and compared withcontrol animals prepared in an equivalent manner at the same levels of the bulb.Analysis of variance demonstrates a significant effect of length of postlesion survival onthe increase in labeling (F 5 8.56, P , 0.05, 2 degrees of freedom).


creased between three- and fourfold in the ONL and theGL on the lesioned side. In these layers, the density ofstaining is such that individual profiles are difficult todistinguish and the glomerular neuropil is significantlydarker than in the deeper layers after lesion; no suchdistinction between the GL and the deeper layers, i.e., theexternal plexiform layer, is evident on the undamagedside.

The aforementioned results are expected from the mas-sive degeneration of axons in the ON and outer layers ofthe bulb. However, GFAP expression is also increased inthe deeper layers of the bulb in response to the lesion. Inthe internal granular layer (IGL) by itself and the extraglo-merular layers as a whole, the number of stained profileson the lesioned side is 1.83-fold higher than on theunlesioned side, which is a statistically significant in-crease (t 5 4.59, P , 0.025, one-tailed test, 2 degrees offreedom). Likewise, the number of stained somata in theIGL, determined by segmenting images by size as well asintensity of staining, are increased on the lesioned side bytwofold (t 5 8.41, P , 0.01, one-tailed test, 2 degrees offreedom). Thus, the number of GFAP (1) glia as well as thenumber of stained processes, is increased in the extraglo-merular layers of the bulb. GFAP expression graduallydiminishes with increasing time after lesion. By 8 weeksafter MeBr, the immunostaining for GFAP and S-100 havereturned to levels that are indistinguishable from control.

Extent of denervation of the olfactory bulbdue to MeBr exposure

Anterograde transport of WGA-HRP after its infusioninto the nose of rats 3 days after lesion was used to assess

which areas of the bulb are reliably and completelydenervated by exposure to MeBr. Transport of marker wasassessed in three sets of animals: age-matched, unlesionedcontrol rats (n 5 3); rats that were food-restricted for aperiod of time before MeBr exposure (n 5 3); and rats thatwere not food-restricted (n 5 6). As we have shown earlier(Schwob et al., 1995), food-restriction accentuates theeffect of the MeBr on the epithelium (for reasons that areincompletely understood at present). Thus, exposure toMeBr destroys well over 95% of the epithelium in food-restricted rats compared with 90–95% of the epithelium inanimals fed ad libitum. In addition to being less severeoverall, the extent of the lesion is also more variable acrossthe group of animals fed ad libitum (Schwob et al., 1995).Furthermore, the increased severity of initial lesion due tofood-restriction causes a greater degree of replacement ofolfactory by respiratory epithelium during the reconstitu-tion of the lesioned areas. The two different groups of ratswere used to evaluate the effect of lesion severity on theparameters of reinnervation of the bulb.

In keeping with the extent of the destruction in theolfactory epithelium, exposure of either group of rats toMeBr markedly and reproducibly diminishes the trans-port of label into the glomeruli and the olfactory nervelayer of the olfactory bulb by comparison with control (Fig.3). The olfactory nerve layer is markedly thinner and morefriable to handling than in normal and is largely devoid oflabel. Likewise, the glomeruli are shrunken by comparisonwith control (Fig. 3C,D,G,H vs. 3K,L). In the food-restricted rats, a small amount of transported label isfound in isolated glomeruli in the ventral and posterior

Fig. 2. Expression of glial fibrillary acidic protein (GFAP) inolfactory nerve and olfactory bulb is up-regulated after methyl bro-mide gas (MeBr) lesion. A: Exposure to MeBr, when limited to one sideof the nose by unilateral naris closure, destroys the epithelium on theopen side (right) and produces little or no damage to the closed side(left), as shown by staining with anti-olfactory marker protein (OMP)at 3 days after lesion. B: An adjacent section demonstrates thatstaining with anti-GFAP is increased in the fascicles of the olfactorynerve on the lesioned side (right) compared with the unexposed side

(left). Representative fascicles are indicated by arrowheads. C: Exami-nation of the olfactory bulb reveals a several-fold increase in GFAP-levels in the olfactory nerve (onl) and glomerular layers (gl) whencomparing lesioned (right) to unexposed (left) sides. The slightlydenser staining in the external plexiform (epl) and internal granular(igl) layers is confirmed by densitometric comparisons (see text).Boxed areas are shown in higher power in D and E as indicated. Scalebars 5 250 µm in B (applies to A,B); 500 µm in C; 100 µm in E (appliesto D,E).


Fig. 3. Methyl bromide gas lesion of the olfactory epitheliumcauses near-complete denervation of the olfactory bulb. A–D: Inanimals lesioned while food-restricted (fd-restr), intranasal infusion ofwheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) at 3 day after exposure produces little or no transported label 24hours later at any level in the bulb in contrast with the uniform, denseglomerular labeling observed in control animals (cf. I–L). The arrow-heads in D indicate small foci of staining in the glomeruli. In each ofthe low-magnification photomicrographs, dorsal is toward the top andmedial is toward the left. Boxed areas are shown at higher magnifica-tion in the corresponding panels. E–H: In animals that were adlibitum fed (ad lib) at the time of lesion, the amount of transported

label is profoundly decreased in all parts of the bulb, although there ismore WGA-HRP in the olfactory nerve layer of the ventral posteriorbulb (open arrow in F) than in animals lesioned while food restricted(cf. B vs. F); that result is expected given the greater degree ofepithelial sparing in animals fed ad libitum. The arrowheads in Hindicate small foci of staining in glomeruli. Other conventions are thesame as in A–D. I–L: Age-matched control animal killed 24 hours afterWGA-HRP infusion. A,E,I: Sections of anterior bulb. B,F,J: Sections ofmid-posterior bulb at the level where the glomerular layer begins toretract from the ventrolateral quadrant of the bulb. Abbreviations areas in Figure 1. Scale bars 5 500 µm in J (applies to A,B,E,F,I,J); 200µm in L (applies to C,D,G,H,K,L).


parts of the bulb as shown in the illustrated case, which istypical of the extent of transport acutely after lesion (Figs.3D, 4). Nonetheless, most of the bulb, other than theventral and posterior margins, completely lacks label in allmembers of the group. For example, 98% of the glomerularlayer lacks label in the illustrated case, and in that partwhich retains some innervation, the label is markedlylighter than control (Fig. 4). Likewise, exposure of thegroup of rats fed ad libitum to MeBr markedly andreproducibly diminishes the transport of label to theolfactory bulb. Overall, there is a slightly greater degree oflabel in the glomerular layer compared with food-re-stricted animals (cf. Fig. 3A–D vs. 3E–H and Fig. 4B vs.4C); for example, 94% of the glomerular layer lackstransported WGA-HRP in the illustrated case (Fig. 4). Inboth groups of animals, the distribution and extent of thelabel parallels the pattern of sparing in the olfactoryepithelium, which is limited to isolated patches of rela-tively intact epithelium lining the lateral, posterior, andventral cul de sacs of the nasal cavity, the nasal septalorgan, and a scattering of neurons at the anterior marginof the olfactory epithelium (Schwob et al., 1995; Youngen-tob et al., 1997). It is important to note that WGA-HRP isdispersed throughout the nasal cavity of the lesionedanimals after infusion to the same, if not greater, extent asin controls (data not shown); indeed, the posterior nasalcavity is more patent after lesion than in controls due tothe reduction in the thickness of its epithelial lining.

The comparison of the six cases fed ad libitum allowedus to define an area of the bulb that lacks transportedWGA-HRP. The aggregate area that lacked transport in allsix cases was the dorsal third of the lateral surface of thebulb and the dorsal two-thirds of the medial surface of thebulb and is indicated by asterisks on the flattened compos-ite map (Fig. 4). Of course, the label-less zone extendedwell beyond the confines of that region in any individuallesioned rat. Given the number of cases examined, it is notunreasonable to assume that the designated area consis-tently lacks transported label and is suitable for ananalysis of the timing of reinnervation (see below).

Timing of reinnervation of the olfactory bulbafter MeBr lesion

Two measures were used to assess the time course ofreinnervation of the olfactory bulb: the anterograde trans-port of WGA-HRP from the nose to the glomerular layer ofthe bulb and phosphorylated–GAP-43 (phospho–GAP-43)immunoreactivity within the glomeruli of the bulb. Eachmeasure of reinnervation was used in rats that were fed adlibitum at the time of lesion, and the results with each willbe described in turn.

Anterograde transport of WGA-HRP. As describedabove, the pattern of anterograde transport acutely afterlesion indicates that the dorsomedial half of the bulb iseither completely denervated or retains innervation byfibers that are so few in number that they are insufficientto generate recognizable transported label. Accordingly,the appearance of transported WGA-HRP in the corre-sponding region of the bulb reflects new axons and pro-vides an indication of the length of time it takes theepithelium to reinnervate the bulb.

By 1 week after lesion the level of transported WGA-HRP in the olfactory nerve layer has increased by compari-

Fig. 4. Methyl bromide gas (MeBr) exposure denervates the vastmajority of the bulb. Flattened maps of the glomerular layer of thebulb (constructed as described in the Materials and Methods section)illustrating the extent of transported wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) label in a representa-tive animal that was unlesioned (Control), another that was food-restricted before MeBr-lesion, infused intranasally with WGA-HRP 3days after lesion and perfused 24 hours later (Food-restricted), and athird that was fed ad libitum before MeBr lesion and infusion. Parts ofthe glomerular layer that contain label at or below background arerendered as uniform light-gray to highlight the transported label,which is indicated as shades of darker gray. In animals fed ad libitum,the projection to the ventromedial bulb is partially spared (openarrow). However, the dorsomedial bulb lacked transported label (thearea marked by asterisks) in all eight animals fed ad libitum. In theillustrated food-restricted case, 98.5% of the glomerular layer is at orbelow background. In the illustrated case, 94.3% of the glomerularlayer is at or below background. The extent of denervation is consis-tent with the small percentage of the epithelium that is spared theeffects of the MeBr (Schwob et al., 1995). VM, ventromedial edge of thebulb; VL, ventrolateral edge of the bulb. Arrow indicates dorsomedialbulb. For all flattened maps, anterior is up and posterior is down.


son with the amount observed immediately after lesion;the label is concentrated ventrally in the mid-posteriorbulb (Fig. 5A–D). The density of the transported label hereexceeds the amount observed in any of the 11 casesexamined at 3 days after lesion and must, in part, repre-sent new fibers (cf. Fig. 5A–D vs. Fig. 3). Indeed, some ofthe label observed in the glomerular layer in the posteriorbulb most likely represents reinnervation of the glomerulifor the same reason. The appearance of new fibers herefirst is not surprising, because this is the part of the bulbthat is closest to the epithelium. In the dorsomedial bulb,where any label reflects transport in newly arrived axons,there are few labeled fibers in the olfactory nerve layer andthey are limited to the anterior bulb. In this area, a rareglomerulus may contain WGA-HRP and even then thelabel in the glomerulus is spotty and weak.

Between 1 and 2 weeks after lesion (cf. Fig. 5A–D vs.5E–H) and again between 2 and 3 weeks after lesion (cf.Fig. 5E–H vs. 5I–L), the amount of transported WGA-HRPand the thickness of the olfactory nerve layer both increasesharply. Indeed, substantial label extends to the posteriormargins of the bulb by 2 weeks. Some WGA-HRP–labeledfibers extend into the underlying glomeruli in all parts ofthe bulb at 2 weeks, although glomerular reinnervation isheavier in the ventrolateral bulb compared with thedorsomedial bulb. Nonetheless, the thickness of the nervelayer and the amount of transported material in it remainless at this time compared with control rats or animalsinfused at 3 weeks after lesion. Similarly, there is less labelin the glomerular layer, and the glomeruli appear some-what smaller at 2 weeks compared with either control ratsor to rats that survive 3 weeks after lesion.

By 3 weeks after lesion, the olfactory nerve layer isnormal or near normal in its thickness and in the amountof transported WGA-HRP that it contains. Moreover, theglomeruli in all parts of the bulb are plump, filled withlabel, and indistinguishable from control.

Staining with anti-phospho-GAP-43 and anti-OMP.

The transport of WGA-HRP serves as a highly sensitiveassay for the beginning phase of the reinnervation of thebulb, but it is unlikely to be a good measure of when theprocess of reinnervation is complete, i.e., when the fibersinnervating the bulb are as close to normal in theircharacteristics as possible. On the other hand, immuno-staining with the monoclonal antibody 2G12, which isselective for the protein kinase C–phosphorylated form ofGAP-43 (that form which is limited to distal axons in theperipheral olfactory system and elsewhere) (Meiri et al.,1991), is very useful for identifying immature olfactoryaxons and determining whether they persist at varioustimes after lesion in excess of the numbers observed inage-matched controls. Similarly, the increase in OMP-immunoreactive material in animals killed 2 weeks ormore after lesion provides a further indication of thestatus of the reinnervation process. Before that time, thepersistence of OMP- and GAP-43-immunoreactive debrisprevents their use as indicators of reinnervation. However,debris is cleared from the GL and ONL by 2 weeks asshown by assessing the immunostaining in animals le-sioned by the combination of MeBr inhalation and 3-methylindole (3-MI) injection as a function of time after lesion(Schwob et al., 1994). The co-administration of thesetoxins causes a more severe lesion in the nose than MeBr

alone and results in the permanent replacement of essen-tially the whole dorsomedial zone of the olfactory epithe-lium by respiratory epithelium or scar tissue and thepermanent denervation of dorsomedial bulb (Schwob etal., 1994). In this part of the bulb, immunostaining withGAP-43 and with OMP disappear by 2 weeks after com-bined lesion never to return (data not shown). Thus,GAP-43- or OMP-immunoreactive material that is ob-served in the ONL and GL of the bulb at or after the end ofthe second week after MeBr exposure is found in reinner-vating axons rather than debris from ones that havedegenerated as a consequence of the initial lesion.

The appearance of phospho-GAP-43 labeling in thedorsomedial bulb is congruent with the observations madeafter anterograde transport of WGA-HRP. Thus, at 2weeks after lesion, the olfactory nerve layer contains ahigh density of phospho-GAP-43 (1) fibers, but only a fewextend from the olfactory nerve layer into the underlyingglomeruli (Fig. 6I–L). By 3 weeks after lesion, the nervelayer has expanded and a substantial contingent of phos-pho-GAP-43 (1) axons project from the ONL into theglomeruli (Fig. 7A–D). The glomerular content of phospho-GAP-43-stained fibers gradually declines between 4 and 8weeks after lesion (Fig. 7E–L). Densitometric analysis of 8weeks postlesion animals and age-matched controls dem-onstrate no significant difference by this time (the densityof glomerular 2G12-labeling in animals at 8 weeks postle-sion is 1.05 6 0.05 times that of control; mean 6 SEM).There is a parallel increase in the bulbar content of OMPbetween 2 and 6 weeks after lesion (not shown). Both thedecline in phospho-GAP-43 and the increase in OMP in thebulb reflect the changes in the type of olfactory neuronsthat have emerged in the periphery; i.e., most of theneurons are immature and GAP-43 (1) at the beginning ofthat period, but mature, OMP (1) neurons predominate bythe end of that time (Schwob et al., 1995).

Both the transport of WGA-HRP and the phospho-GAP-43 data suggest that there is little discernible differ-ence between anterior and posterior bulb in the timing ofthe reinnervation. The one exception to that rule seems tobe the ventromedial quadrant of the mid-posterior bulb inwhich the glomeruli and olfactory nerve layer apparentlyare reinnervated by a noticeable component of phospho-GAP-43 (1) fibers around 1 week after lesion (Fig. 6F). Asubstantial number of axons innervating that area arespared the effects of the lesion, which may contribute to itsrapid reinnervation by serving as a substrate for thegrowth of nascent axons.

Extent of reinnervation of the olfactory bulbafter MeBr lesion

As indicated above, the density of GAP-43 labeling in theglomeruli is roughly equivalent to age-matched controls by8 weeks after lesion. Likewise, the rate of basal cellproliferation and the proportion of immature to matureneurons in the olfactory epithelium have also returned tonormal levels by that time (Schwob et al., 1995). Thus, theprocess of reinnervation that is elicited in response toMeBr lesion is essentially complete within that 8-weektime-frame. Consequently, animals killed at survivals of 8weeks or longer provide a measure of the extent to which


Fig. 5. The bulb is reinnervated rapidly in the first weeks afterlesion. A–D: Representative case infused with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) 1 week after lesion;exposed to methyl bromide gas (MeBr) while fed ad libitum. Theamount of transported label in the olfactory nerve layer is increased at1 week compared with 3 days after lesion (cf. Fig. 3E–H). In addition, anumber of glomeruli in the posterior half of the ventromedial bulb aredensely labeled by transported WGA-HRP (open arrow in Fig. 3B).Some of that label derives from new axons that have reinnervatedthose glomeruli, given that the label in this area is denser at this timecompared with the acute postlesion period (cf. Fig. 3F). E–H: Represen-tative case infused with WGA-HRP 2 weeks after lesion; MeBr-exposed while fed ad libitum. By this time, the density of label in the

nerve and glomerular layers has increased quite markedly. However,the vast majority of the glomeruli contain less label and are smaller,than in control rats or at later time-points in the reinnervationprocess, and some glomeruli lack label completely (arrowheads inG,H). I–L: Representative case infused with WGA-HRP 3 weeks afterlesion; MeBr-exposed while fed ad libitum. At this time, all of theglomeruli have been reinnervated to a substantial degree throughoutthe anteroposterior extent of the bulb. In each of the low-magnificationphotomicrographs, dorsal is toward the top and medial is toward theleft. Boxed areas are shown at higher magnification in the correspond-ing panels. Abbreviations are as in Figure 1. Scale bars 5 500 µm in J(applies to A,B,E,F,I,J); 200 µm in L (applies to C,D,G,H,K,L).


Fig. 6. Immunostaining with antibody to phospho–GAP-43 con-firms the arrival of a wave of reinnervating fibers between 1 and 2weeks after lesion. A–D: Unexposed, age-matched control rat. Someglomeruli contain a substantial number of labeled fibers (curvedarrows in C,D), but most lack immunoreactivity (examples indicatedby arrowheads in C,D). Boxed areas are shown at higher magnificationin the corresponding panels. E–H: Representative case killed 1 weekafter lesion; exposed to methyl bromide gas (MeBr) while fed adlibitum. The glomeruli are devoid of label (arrowheads in G,H), withthe exception of ones that contain a few positive fibers (curved arrow inH) and others in the ventromedial quadrant (open arrow in F). The

latter result is confirmatory of the experiments assessing transport oflabel from the nose at 1 week after lesion (cf. Fig. 5B). I–L: Represen-tative case killed 2 weeks after lesion; MeBr-exposed while fed adlibitum. The olfactory nerve layer contains more label, and phospho–GAP-43 (1) fibers have grown into a substantial percentage of theglomeruli (curved arrows in K), although many others remain devoidof label (e.g., arrowhead in L). In each of the low-magnificationphotomicrographs, dorsal is toward the top and medial is toward theright. Abbreviations are as in Figures 1, 2. Scale bars 5 500 µm in J(applies to A,B,E,F,I,J); 200 µm in L (applies to C,D,G,H,K,L).

Fig. 7. After reaching a peak, the numbers of newly arrived axonsslowly decline to the control levels as the projection stabilizes.A–D: Representative case killed 3 weeks after lesion; exposed tomethyl bromide gas (MeBr) while fed ad libitum. The density ofglomerular labeling with phospho–GAP-43 reaches a peak at thistime, when all glomeruli contain substantial numbers of axonscontaining phospho–GAP-43. Boxed areas are shown at higher magni-fication in the corresponding panels. E–H: Representative case killed4 weeks after lesion; MeBr-exposed while fed ad libitum. The declinein staining intensity in individual glomeruli, and the re-emergence ofunlabeled glomeruli (arrowhead in G) indicates that existing fibers

make the transition away from GAP-43 expression and that fewer newfibers are arriving at that time-point. I–L: Representative case killed 8weeks after lesion; MeBr-exposed while fed ad libitum. The furtheremergence of unstained and lightly stained glomeruli (arrowheads inK) indicates that the projection is no longer in flux as at earliertime-points, i.e., the projection returns to control levels of turnover. Ineach of the low-magnification photomicrographs, dorsal is toward thetop and medial is toward the right. Abbreviations are as in Figures 1,2. Scale bars 5 500 µm in J (applies to A,B,E,F,I,J); 200 µm in L(applies to C,D,G,H,K,L).

Figure 8


parts of the bulb remain uninnervated or less than fullyreinnervated after lesion.

Comparison of OMP-stained sections from food-re-stricted/lesioned animals with ones from controls demon-strates that the posterior part of the bulb is not fullyreinnervated after lesions of that severity. We have ana-lyzed a total of 13 animals that were lesioned whilefood-restricted and allowed to survive 11–21 weeks afterlesion. In all of the lesioned animals, the glomeruli in thecaudal half of the bulb contain a less than normal contin-gent of OMP-stained fibers by comparison with control(Fig. 8). Indeed, most of the glomeruli that are situatedwithin about a millimeter of the posterior margin of thebulb on both the lateral and medial sides of the peduncleare completely lacking in OMP-positive fibers (Fig.8I,L,O,R). As indicated above, OMP provides an accurateassessment of the innervation state of the glomeruli bythis time after lesion. As further proof of that point, we alsoused antibodies directed against GAP-43 and NCAM,which label and have the capacity to highlight against thesurrounding neuropil olfactory axons that lack OMP (Ringet al., 1997); staining of adjacent or nearby sections withthese antibodies confirms the hypoinnervation and persis-tent denervation of posterior glomeruli (not shown). Thus,a number of glomeruli at the posterior margin of the bulbare not reinnervated to any extent, even after very longsurvivals. It is important to note again that a substantialproportion of the olfactory epithelium does not recoverfrom the effects of the lesion in the food-restricted/lesionedanimals due to the greater initial severity of the lesion andis replaced, in patches, by respiratory epithelium in theanterior, ventral and lateral parts of the olfactory area(Schwob et al., 1995).

In contrast with the status of the bulb in food-restrictedanimals that are lesioned, glomeruli in the posterior bulbare usually re-innervated by 6–8 weeks after lesion inanimals that were fed ad libitum at the time of MeBrexposure (Fig. 8H,K,N,Q). A few glomeruli at the posteriormargin of the glomerular layer were uninnervated (by thecriterion of lacking OMP-, GAP-43-, or NCAM-stainedolfactory axons) in one of a total of six cases of this type(Fig. 8H,K,N,Q). In the other cases, all of the glomerulicontain demonstrable olfactory axons. The status of theepithelium at long survivals after lesion also differs be-tween rats that are lesioned when fed ad libitum and onesthat are food-restricted. In rats fed ad libitum at the timeof exposure, the vast majority of the lesioned epitheliumrecovers as olfactory, and the extent of patchy replacementof olfactory by respiratory epithelium is much less than infood-restricted/lesioned rats (Schwob et al., 1995).

We analyzed further the distribution of the reinnervat-ing fibers across the bulb by measuring the area of theglomerular layer that contains OMP-stained fibers in six ofthe food-restricted/lesioned cases, two of the lesioned casesfed ad libitum and three of the controls, as described in theMaterials and Methods section. The area of the GL occu-pied by OMP-stained fibers provides an indirect, butuseful, measure of the number of mature olfactory axonsthat are innervating the glomeruli at a particular level ofthe bulb. In properly prepared material, the intensity ofstaining is reproducible from glomerulus to glomerulus,section to section, and animal to animal. Indeed, thedensity of staining in an OMP-positive region of a glomeru-lus is equivalent between glomeruli that are large andwell-innervated, such as those in the anterior bulb ofanimals lesioned while fed ad libitum vs. glomeruli thatare small and hypoinnervated such as those in the poste-rior bulb of the food-restricted/lesioned animals (Fig. 6).Thus, olfactory axons, even when present in smaller-than-usual numbers, are not dispersed throughout the glomeru-lus (otherwise, the average density of the staining wouldbe less in the hypoinnervated glomeruli), but apparentlyremain tightly clustered together as in the glomeruli ofnormal rats (Pinching and Powell, 1971; Halasz and Greer,1993). Consequently, the area occupied with OMP-stainingprovides an estimate of the number of mature olfactoryaxons by itself, and we have plotted that value as afunction of distance along the anterior-posterior (A-P) axisof the bulb for each of the three groups of animals (Fig. 9).The total volume of olfactory axons in a bulb is derivedfrom these plots by integrating the curve as a function ofdistance. The determination of volume also permits thederivation of the point along the A-P axis at which half ofthe axons are anterior and half are posterior, which weterm the midway point, in each of the cases.

A comparison of the plots of glomerular area occupied byOMP-stained fibers vs. distance along the A-P axis indi-cates that the extent and distribution of innervation issimilar between control and lesioned animals fed adlibitum (Fig. 9). However, the plots from food-restricted/lesioned rats differ from either of the other two conditionsover the posterior three-fifths of the bulb (Fig. 9). In thebulb of food-restricted/lesioned animals, the plot reaches aplateau/peak below that of control at a point in theanterior half of the bulb, begins to decline at a moreanterior level, and reaches a nadir at a more anterior level

Fig. 8. After stabilization of the projection, the distribution ofolfactory marker protein (OMP)–stained axons in animals lesionedwhile fed ad libitum approximates the unlesioned control, whereas inanimals lesioned while food-restricted, the projection is weightedtoward more anterior levels, resulting in hypoinnervation of theposterior bulb and persistent denervation of along its posteriormargin. A,G,M: Age-matched control animal illustrating three levelsof the olfactory bulb, i.e., anterior, mid-posterior, and far-posterior,respectively. D,J,P: Boxed areas in A,G,M at higher magnification.The two posterior levels are situated at the caudal margin of theglomerular layer on the lateral and medial sides of the bulb, respec-tively. Curved arrows indicate ‘‘necklace’’ glomeruli situated at theposterior margin of the glomerular layer, which stain more lightlywith anti-OMP (Ring et al., 1997). B,H,N: Rat exposed to methylbromide gas (MeBr) while fed ad libitum and killed 8 weeks later;same conventions as the control rat (immediately above). E,K,Q: Boxedareas in B,H,N at higher magnification. C,I,O: Rat exposed to MeBrwhile food-restricted and killed 15 weeks later. Same conventionsapply. In each of the low-magnification photomicrographs, dorsal istoward the top and medial is toward the left. F,L,R: Boxed areas inC,I,O at higher magnification. Glomeruli at the two posterior levelsare smaller in the animal lesioned while food-restricted and contain asmaller volume of OMP-stained fibers (cf. regions indicated by openarrows in I vs. G,H and in O vs. M,N). Furthermore, many of theglomeruli at the posterior margin on the lateral (I,L) and medial (O,R)sides of the bulb are completely devoid of OMP-stained fibers. Like-wise, the low-density labeling of the necklace glomeruli with OMP isdisplaced or disappears in lesioned-recovered, food-restricted rats (cf.curved arrows indicating the necklace glomerulus on the lateral side ofthe bulb in G, H vs. its absence in I. Abbreviations are as in Figures 1,2. Scale bars 5 500 µm in O (applies to A–C, G–I, M–O); 200 µm in R(applies to D–F, J–L, P–R).


than in either the control animals or the rats lesionedwhile fed ad libitum (Fig. 9). Thus, the hypoinnervation ofthe bulb in food-restricted/lesioned rats begins well ante-rior to the glomeruli at the posterior edge of the bulb wherethe lack of reinnervation is apparent by inspection.

The midway point, because it is an internal referenceand is unaffected by variation in immunostaining betweenanimals, is a valid measure for performing statisticalcomparisons across the different treatment groups (Fig. 9).The position of the midway point correlated highly withthe area of the glomerular layer occupied by OMP-stainedfibers across all conditions (r 5 0.836). Thus, it is com-pletely unaffected by variation in immunostaining be-tween animals, and it is a highly reliable measure forassessing whether the differences in the distribution oflabel among the different groups of rats are significant

(Fig. 9). Single-factor analysis of variance demonstrates asignificant effect of treatment condition on the midwaypoint (F 5 13.26, P , 0.005, 2 degrees of freedom [d.f.]).Furthermore, Dunnett’s test for multiple comparisons tocontrol demonstrates that the difference between controland food-restricted/lesioned rats is statistically significant(q8 5 4.37, P , 0.01, 7 d.f., two-tailed test). There is nosignificant difference between the group of lesioned ratsfed ad libitum and the controls on that measure (q8 5 0.44,P . 0.05, 6 d.f., two-tailed test).

The assumption that the volume occupied by OMP-immunoreactive material is a direct reflection of axonalnumber underlies this analysis. Although the potentialexists for subtle shifts in size between reinnervating axonsand those in control bulb, the results from food-restricted,lesioned animals are sufficiently different from controls to

Fig. 9. Reinnervation fills the entirety of the glomerular layer inanimals lesioned while fed ad libitum, but the posterior bulb remainshypoinnervated in animals in which the severity of the lesion wasaccentuated by food-restriction. The area occupied by the olfactorymarker protein labeling in the glomerular layer was determined foreach case at a minimum of 20 equally spaced levels and plotted alongthe anteroposterior extent of the bulb. The point along the anteropos-

terior axis that divides the projection into halves was determined fromintegrating the area under the individual curves; the arrows indicatethe group average of the position of the half-way point and the errorbars at the tail of the arrow indicate the SEM. The individual curveswere averaged geometrically across each condition at fixed equivalentlevels in the bulb, and the SEM at each of these points is indicated.


render subtle effects such as axon size irrelevant for themain result. Both the demonstration that the midwaypoint correlates with total area and the statistical signifi-cance demonstrated by the analysis of midway point as afunction of treatment group are strong indications thatreinnervating fibers are concentrated anteriorly in thebulb of rats exposed to MeBr while food-restricted and donot reinnervate all areas of the bulb equally.

DISCUSSION

The successful reinnervation of the olfactory bulb afterthe olfactory epithelium is damaged, i.e., the restoration ofthe premorbid anatomic organization, is a daunting task:neurons from each of the epithelial zones need to projectonto glomeruli in the corresponding bulbar zone, restoringthe rhinotopy that is characteristic of the primary olfac-tory projection (Schwob and Gottlieb, 1986; Schoenfeld etal., 1994), and widely dispersed cells expressing a commonodorant receptor need to converge their axons onto one or afew glomeruli (Ressler et al., 1994; Vassar et al., 1994;Mombaerts et al., 1996; Wang et al., 1998), which, for wantof a better term, can be termed receptotopy. Despiteknowing of the capacity of the olfactory epithelium toreconstitute after lesion and for newly generated neuronsto reinnervate the olfactory bulb for a number of years(beginning with the work of Nagahara, 1940), the timing,extent, and accuracy of that reinnervation has not beenanalyzed to an equivalent degree. The present study hasassessed the glial response, and the time course and extentof reinnervation after peripheral lesion caused by inhala-tion of MeBr. The results suggest that there are limits onthe process of reinnervation that prevent perfect recoveryafter lesion under some circumstances.

We find that the massive destruction of the epitheliumelicited by MeBr causes a several-fold enhancement ofglial cell proliferation and in the number of GFAP-expressing glia in the olfactory nerve and in the nerve andglomerular layers of the bulb. Although not unexpected,given the gliotic reaction noted previously after nervetransection (Barber and Dahl, 1987) and ZnSO4 irrigation(Burd, 1993; Chuah et al., 1995), it is worth noting that theresponse is elicited without mechanical disruption of thetissue. Thus, the proliferation and enhanced expression ofGFAP by the glia in the ONL and GL in response to MeBrlesion is likely elicited by axonal debris, the subsequentinvasion of phagocytic cells (Burd, 1993), or both, ratherthan an effect of gross disruption of the tissue. Moresurprising was the response of glia in the IGL, given thatthe surrounding granule cells do not receive a directafferent input from the nose. Further investigation will beneeded to determine whether an indirect effect on thegranule cells causes the response. Published results indi-cate that interneurons in the bulb are sensitive to periph-eral damage. Epithelial disruption by ZnSO4 infusioncauses a profound down-regulation of tyrosine hydroxy-lase expression by dopaminergic periglomerular cells(Baker at al., 1983, 1984) and a significant reduction inGABA levels in the bulb (Margolis, 1974), which may notreflect changes in periglomerular cells (Baker at al., 1988).In addition, preliminary results suggest that there isaccelerated proliferation of granule cell precursors, periglo-merular cell precursors, or both, in the anterior neurogenic

stream (unpublished data, B. Kirschenbaum, C. Alvarez-Buylla, J.E. Schwob, and S.L. Youngentob).

With respect to the time course of reinnervation, thedata from the WGA-HRP transport experiments indicatethat a massive wave of reinnervating fibers washes overthe bulb between 1 and 2 weeks after lesion, whichparallels the timing of epithelial reconstitution after MeBrexposure. The first few olfactory neurons reappear in theepithelium at 3 days after lesion, and by 2 weeks, a greaterthan normal number of neurons has been elaborated,although the vast majority are still immature and expressGAP-43. Our findings are also congruent with the timingof reinnervation of the bulb after other forms of experimen-tal lesion, i.e., transection of the olfactory nerve (Hardinget al., 1977; Graziadei and Monti Graziadei, 1980; Yee andCostanzo, 1995; Koster and Costanzo, 1996) or Triton-X100 irrigation (Nadi et al., 1981; Kawano and Margolis,1982; Kream and Margolis, 1984). ZnSO4 irrigation usu-ally disrupts the epithelium so severely that the regenera-tion of olfactory neurons, and consequently the reinnerva-tion of the bulb, is spotty, incomplete, and significantlydelayed (Harding et al., 1978; Burd, 1993). Nonetheless,the concordance of the results with nerve transection,Triton-X irrigation, and MeBr inhalation provides assur-ance that the timing of reinnervation is not dependent onthe method of lesioning.

Furthermore, we estimate from these data that it takesindividual axons about 3–4 days to grow from the epithe-lium to the bulb. Our estimate is derived from the firstlarge scale reappearance of neurons at 4 days after lesion,and the first significant contact with the bulb at 7 daysafter lesion, when some axons are found in the ventral partof the olfactory nerve layer. However, these same datasuggest that there is a further lag between axons enteringthe ONL and when they enter the glomeruli. Even at 2weeks after lesion, the reinnervation of the glomeruli isspotty and incomplete, despite the generation of a popula-tion of sensory neurons, albeit immature and GAP-43-expressing, larger than that of age-matched controls(Schwob et al., 1995).

Beyond the 2-week postlesion time point, transportmeasures are no longer effective for assessing the furthercourse of reinnervation of the glomeruli, and particularlythe return toward normality, due to unavoidable, technique-inherent variability among animals. However, GAP-43expression, and more specifically the kinase C-phosphory-lated form, is a marker that is selective for immatureolfactory axons (Verhaagen et al., 1989, 1990; Meiri et al.,1991; Schwob et al., 1992, 1994). Thus, the degree to whichthe olfactory axons in the glomeruli express GAP-43 is ameasure of the maturation of the innervating fibers and isless subject to technical variability than transport of label.By using this measure, we demonstrate that the popula-tion of reinnervating fibers return to ‘‘normal’’, i.e., astatus equivalent to age-matched controls, between 6 and8 weeks after lesion. These data have obvious implicationsfor experiments that assess the time-course and degree ofrecovery of olfactory guided behavior after epithelial lesion(Youngentob and Schwob, 1991, 1992).

Our most striking finding relates to the extent of reinner-vation in rats that were food-restricted when lesioned withMeBr. At the end of the regeneration process, the posteriorbulb was hypoinnervated, as evidenced by the reduction in


the volume of the glomerular layer that contains OMP-(1)fibers in the posterior half of the bulb. Indeed, glomeruli atthe posterior margin remain persistently denervated afterepithelial reconstitution. Most of the denervated glomeruliare typical ones, but the specialized necklace glomeruli,which are identifiable by their position at the extreme edgeof the glomerular sheet (Ring et al., 1997), also remaindenervated. In contrast, hypoinnervation/denervation ofthe posterior bulb was not a prominent feature of theregenerated projection in animals lesioned while fed adlibitum. In these animals, innervation was restored tolevels that are indistinguishable from control throughoutthe full extent of the bulb.

The lesion caused by MeBr exposure affects a greaterpercentage of the epithelium and is demonstrably moresevere in the food-restricted animals. As a consequence,there is a substantial degree of replacement of olfactory byrespiratory epithelium after reconstitution in the food-restricted rats (Schwob et al., 1995). In contrast, mostareas of the epithelium recover completely as olfactory inanimals that are fed ad libitum when lesioned (Schwob etal., 1995). The difference in epithelial recovery is a likelyreason for the difference in extent of reinnervation be-tween the two groups of animals. Indeed, the failure toreinnervate the posterior bulb, more specifically the extentof that failure as assayed by the mid-point in the distribu-tion of the reinnervating fibers, correlates strongly withthe ‘‘mass’’ of reinnervating axons across animals, whenthat is estimated as the volume of glomerular stainingwith OMP.

The preferential reinnervation of the anterior half of thebulb in the food-restricted group means one of two things.Either the olfactory neurons that normally innervate theposterior glomeruli are not reconstituted after exposure ofthe food-restricted animals to MeBr to the same extent asthose projecting to anterior glomeruli, or the mechanismsthat guide axons to the posterior glomeruli during develop-ment and during the limited turnover associated with‘‘normal’’ life are no longer operating in adult rats subjectto massive peripheral lesion.

We cannot fully exclude the first of these hypotheses.However, several pieces of data suggest that the findingscannot be explained by a selective dysgenesis of posterior-projecting neurons during epithelial reconstitution. First,any one type of olfactory neuron, as defined by theexpression of a specific member of the odorant receptorgene family, is dispersed throughout the anteroposteriorextent of the corresponding zone of the normal epitheliummore or less at random (Strotmann et al., 1992, 1994;Ressler et al., 1993; Vassar et al., 1993; Mombaerts et al.,1996). Although there are exceptions to that general rule,for example, the neurons expressing the odorant receptorOR37 are clustered rather than widely distributed (Strot-mann et al., 1992, 1994), they are apparently few. Thus,the patchy replacement of olfactory by respiratory epithe-lium will not, a priori, have the effect of eliminatingposterior-projecting types of neurons, and no zone in theepithelium is completely replaced by respiratory epithe-lium as a consequence of the lesion (Schwob et al., 1995).Moreover, we have used cRNA probes to eight differentodorant receptors to directly assess the distribution ofeight types of olfactory neurons by in situ hybridization inlesioned/reconstituted epithelium vs. unlesioned control in

rats subjected to unilateral MeBr exposure. For eachreceptor type, including the neurons labeled by OR37, thenumber and position of the labeled neurons on the open/lesioned side was roughly equivalent to the control side(Iwema et al., 1997; C.L. Iwema and J.E. Schwob, unpub-lished data). Second, the subsets of neurons that normallyproject to the necklace glomeruli at the posterior-mostmargin of the bulb are regenerated during the reconstitu-tion of the epithelium and reinnervate the bulb, but aremistargeted to glomeruli situated anterior and dorsal totheir usual target glomeruli (Ring et al., 1995). Thus, wecan find no evidence for a preferential depletion of posterior-expressing neurons.

Instead, we interpret the hypoinnervation and persis-tent denervation of the posterior bulb as a consequence oftwo failures: the failure of axon guidance mechanisms atsome level, which is caused by the near-total destruction ofthe pre-existing population of neurons by MeBr exposurein food-restricted animals, and the failure of epithelialreconstitution, whereby the permanent replacement of asignificant proportion of olfactory by respiratory epithe-lium has the effect of limiting the population of newlygenerated neurons to a less than normal number. Anoverall reduction in the number of neurons, i.e., a reduc-tion that more or less equally affects all types of olfactoryneurons, is not sufficient by itself to cause a selectivehypoinnervation of the posterior bulb, if receptotopic speci-ficity is maintained. Rather, guidance mechanisms mustfail under these circumstances. Had they remained opera-tive, all parts of the bulb would be reinnervated but to areduced extent, which is not what we find.

Other observations speak to the nature of the guidancefailure. We have previously presented data that the rhino-topic projection of the epithelium onto the bulb is restored,i.e., the projection of dorsal epithelium remains limited todorsal bulb and ventral to ventral bulb, when assayedeither by retrograde transport experiments or by thedistribution of axons from ventral epithelium that areselectively labeled with the monoclonal antibody RB-8(Schwob and Youngentob, 1991; Schwob et al., 1993). Thus,axons are apparently being guided from the epithelium tothe correct zone in the bulb. Likewise, the subsets ofneurons that project to different sets of necklace glomerulireinnervate roughly the same area of the bulb as in normalbut are not targeted into the correct glomeruli. In theseexamples, axons are guided to a region within 5–10glomeruli of their normal location at the extreme marginsof the glomerular sheet (Ring et al., 1995). The datasuggest that the mechanisms persist for targeting axonsinto the vicinity of the correct glomerulus, but once theaxons near their target, guidance mechanisms fail to steerthem to their usual glomerulus.

The failure to reinnervate the correct target glomerulusreveals something about the nature of the mechanismsthat function normally. Glomeruli appear to be reinner-vated correctly by newly generated neurons during the lowrate of turnover that is characteristic of ‘‘normal’’ adult life.The persistence of significant reinnervation is indicated bythe substantial number of GAP-43-stained axons in glo-meruli in the equivalent of middle-aged rats (Meiri et al.,1991). Furthermore, the innervation to the posterior bulb,and the selective innervation of necklace glomeruli by theappropriate types of neurons are preserved in older barrier-


isolated rats (G. Ring and J.E. Schwob, unpublishedobservations). Thus, the failure to innervate the posteriorbulb after MeBr is not an indication that adult animalslack mechanisms to target olfactory axons precisely underall circumstances.

Instead, other aspects of the lesion paradigm must beresponsible for the differences in the capacity for correctreinnervation between it and normal. One difference be-tween the two settings is the degree to which pre-existingaxons of like-type, i.e., axons made by neurons thatexpress the same odorant receptor, are available to guidethe newly arrived axons into the specific target glomeru-lus. We hypothesize that the absence of the pre-existingaxons deprives the newly growing axons of a scaffold alongwhich they can navigate and is an essential reason whythe posterior bulb remains uninnervated in the severelylesioned animals. A dependence on pre-existing fibers oflike-type is not inconsistent with demonstrations thatodorant receptor expression plays a role in targeting axonsof a specific type into a glomerulus during the embryonicdevelopment of the olfactory system (Singer et al., 1995;Mombaerts et al., 1996; Wang et al., 1998). Furthermore,and central to our hypothesis, the choice of receptor maysubserve the fasciculation of like-axons, because the expres-sion of a receptor-less locus (where the coding portion waseliminated by homologous recombination) causes that setof axons to distribute throughout the olfactory nerve layer(Wang et al., 1998).

Data acquired with the nerve transection model are alsoconsistent with our hypothesis. With this paradigm aswell, the axons from a specific type of neuron, namely onesmarked by the insertion of tau-LacZ in tandem with the P2odorant receptor, do not innervate the same glomerulusafter regeneration of the nerve as they would in unoper-ated mice (Costanzo, 1997). The marked axons do bundleand converge onto a few glomeruli, which suggests that thefasciculation of like axons occurs in this setting (Costanzo,1997). Thus, glomerular targeting also does not operatewith the same precision during the massive turnovercaused by nerve section. In the case of misrouting afterMeBr lesion, mechanical disruption is not playing a role inthe misguiding of the axons.

Given that turnover is nearly as profound in the ratslesioned with MeBr while fed ad libitum, why then is theposterior bulb reinnervated in those animals but not inones that were food-restricted when lesioned? One poten-tially relevant difference between these two groups is thefar greater extent of patchy replacement in the food-restricted animals, which has the effect of reducing thepopulation of re-innervating fibers, as noted above. Thesedata suggest that reinnervating fibers prefer the anteriorbulb, which may reflect some predilection for the closestavailable synaptic space. From the data presented here,we cannot be certain whether receptotopy is restored afterreinnervation in the animals fed ad libitum. However, weknow that one example of an identified subset of neurons,i.e., those marked by the antibody 2A4 (Carr et al., 1994),projects to more than its usual pair of glomeruli after thecompletion of the lesion-induced reinnervation in rats fedad libitum (Carr et al., 1998). That result provides oneindication that receptotopy is not as precise as normaleven when there is nearly complete reconstitution of theepithelium.

In summary, MeBr-mediated damage to the epitheliumprovokes a rapid reconstitution of the population of olfac-tory neurons and reinnervation of the bulb. However, theextent of the lesion does extract a cost. The accuracy withwhich the epithelium projects onto the bulb, i.e., theexquisitely precise receptotopy that is established duringdevelopment and maintained throughout normal life isapparently compromised and affects the functioning of theolfactory system (Youngentob and Schwob, 1997). In thatregard, our findings in rat are reminiscent of humans.Deterioration of glomerular innervation is noted in man(Esiri and Wilcock, 1984; Smith et al., 1991; J.E. Schwob,unpublished observations) and may contribute to thehyposmia and parosmia that accompany severe peripherallesions (Doty, 1979).

ACKNOWLEDGMENTS

We thank Dr. Frank Margolis and Dr. Karina Meiri fortheir generous gifts of antiserum against OMP and mono-clonal antibody 2G12 against the phosphorylated form ofGAP-43, respectively. J.E.S., G.R., and S.L.Y. receivedsupport from the National Institutes of Health.

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