Steven L. Youngentob

Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells

We have infused replication‐incompetent retroviral vectors into the nasal cavity of adult rats 1 day after exposure to the olfactotoxic gas methyl bromide (MeBr) to assess the lineage relationships of cells in the regenerating olfactory epithelium. The vast majority of the retrovirus‐labeled clones fall into three broad categories: clones that invariably contain globose basal cells (GBCs) and/or neurons, clones that always include cells in the ducts of Bowmans glands, and clones that are composed of sustentacular cells only. Many of the GBC‐related clones contain sustentacular cells and horizontal basal cells as well. Most of the duct‐related clones contain gland cells, and some also include sustentacular cells. Thus, the destruction of both neurons and non‐neuronal cells that is caused by MeBr activates two distinct types of multipotent cells. The multipotent progenitor that gives rise to neurons and non‐neuronal cells is a basal cell, whereas the progenitor that gives rise to duct, gland, and sustentacular cells resides within the ducts, based on the pattern of sparing after lesion and the analysis of early regeneration by using cell type‐specific markers. We conclude that the balance between multipotency and selective neuropotency, which is characteristic of globose basal cells in the normal olfactory epithelium, is determined by which cell types have been depleted and need to be replenished rapidly. J. Comp. Neurol. 400:469–486, 1998.

A quantitative analysis of sniffing strategies in rats performing odor detection tasks

The sniffing strategies of rats performing two learned odor detection tasks were monitored with a pneumotachograph and quantitatively analyzed with respect to fifty-two characteristics. The results of this study demonstrated that the rats sniffing varied for different odorants, different concentrations of the same odorant, and between air and odor trials. The variations resulted from changes in such descriptors as volume, duration, average flow rate, peak flow rate and sniff number. In general, a sniffing pattern began with one or two inspirations followed by alternating inspirations and expirations. Comparison of earlier and later sniffs in a bout demonstrated a growth towards both a maximum inspiratory and expiratory sniff which had the largest duration, volume, average flow rate and peak flow rate. These maximum sniffs occurred at or near the end of a bout. Although analysis of the fifty-two characteristics was quantitatively useful in determining the physiologic values and airflow patterns generated by sniffing, a single univariate response measure incorporating twelve characteristics was the best descriptor of how sniffing patterns varied with odorant stimuli.

The aging olfactory epithelium: Neurogenesis, response to damage, and odorant-induced activity

Olfactory epithelium retains the capacity to recover anatomically after damage well into adult life and perhaps throughout its duration. None the less, olfactory dysfunctions have been reported widely for elderly humans. The present study investigates the effects of aging on the neurophysiological and anatomical status of the olfactory epithelium in barrier‐raised Fischer 344X Brown Norway F 1 hybrid rats at 7, 10, 25 and 32/35 months old. The posterior part of the olfactory epithelium in 32/35‐month‐old rats is well preserved. Globose basal cells are dividing, and new neurons are being born even at this advanced age. None the less, the numbers of proliferating basal cells and immature, GAP‐43 (+) neurons are significantly decreased. Neurophysiological status was evaluated using voltage‐sensitive dye techniques to assess inherent patterns of odorant‐induced activity in the epithelium lining the septum and the medial surface of the turbinates. In middle and posterior zones of the epithelium, there were neither age‐related changes in overall responsivity of this part of the olfactory epithelium to any of five odorants, nor shifts in the location of the odorant‐induced hotspots. The inherent activity patterns elicited by the different odorants do become more distinct as a function of age, which probably reflects the decline in immature neurons and a slight, but not statistically significant, increase in mature neurons as a function of age. In contrast with the excellent preservation of posterior epithelium, the epithelium lining the anterodorsal septum and the corresponding face of the turbinates is damaged in the 32/35‐month‐old animals: in this part, horizontal basal cells are reactive, more basal cells and sustentacular cells are proliferating than in younger animals or in posterior epithelium of the same animals, and the neuronal population is less mature on average. Our findings indicate that degeneration of the olfactory epithelium is not an inevitable or pre‐programmed consequence of the aging process, since the posterior zone of the epithelium is very well preserved in these barrier‐protected animals. However, the deterioration in the anterior epithelium suggests that environmental insults can accumulate or become more severe with age and overwhelm the regenerative capacity of the epithelium. Alternatively, the regenerative capacity of the epithelium may wane somewhat with age. Either of these mechanisms or some combination of them can account for the functional and anatomical deterioration of the sense of smell associated with senescence in humans.

Odorant Receptor Expression Patterns Are Restored in Lesion-Recovered Rat Olfactory Epithelium

Lesions of the olfactory periphery provide a means for examining the reconstitution of a diverse and highly regulated population of sensory neurons and the growth, en masse, of nascent axons to the bulb. The olfactory epithelium and its projection onto the bulb are reconstituted after ablation by methyl bromide gas, and some measure of olfactory function is restored. The extent to which the system regenerates the full repertoire of odorant receptor-expressing neurons, particularly their spatially restricted distribution across the epithelial sheet, is unknown, however, and altered odorant receptor expression might contribute to the persistent distortion of odorant quality that is observed in the lesioned-recovered animals. To address the question of receptor expression in the recovered epithelium, we performed in situ hybridization with digoxigenin-labeled riboprobes for eight odorant receptors on the olfactory epithelium from unilaterally methyl bromide-lesioned and control rats. The data demonstrate that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, is restored to normal or nearly so by 3 months after lesion. Likewise, the numbers of probe-labeled neurons in the lesioned-recovered epithelium are nearly equivalent to the unlesioned side at this time. Finally, our evidence suggests that odorant receptors are distributed in multiple overlapping bands in the normal, unlesioned, and lesioned-recovered epithelium rather than in the conventionally accepted three or four zones. Thus, the primary sensory elements required for functional recovery of the olfactory system after damage are restored, and altered function implies the persistence of a more central failure in regeneration.

Globose basal cells are required for reconstitution of olfactory epithelium after methyl bromide lesion.

Despite a remarkable regenerative capacity, recovery of the mammalian olfactory epithelium can fail in severely injured areas, which subsequently reconstitute as aneuronal respiratory epithelium (metaplasia). We contrasted the cellular response of areas of the rat epithelium that recover as olfactory after methyl bromide lesion with those undergoing respiratory metaplasia in order to identify stem cells that restore lesioned epithelium as olfactory. Ventral olfactory epithelium is at particular risk for metaplasia after lesion and patches of it are rendered acellular by methyl bromide exposure. In contrast, globose basal cells (GBCs, marked by staining with GBC‐2) are preserved in surrounding ventral areas and uniformly throughout dorsal epithelium, which consistently and completely recovers as olfactory after lesion. Over the next few days, neurons reappear, but only in those areas in which GBCs are preserved and multiply. In contrast, parts of the epithelium in which GBCs are destroyed are repopulated in part by Bowmans gland cells, which pile up above the basal lamina. Electron microscopy confirms the reciprocity between gland cells and globose basal cells. By 14 days after lesion, the areas that are undergoing metaplasia are repopulated by typical respiratory epithelial cells. As horizontal basal cells are eliminated from all parts of the ventral epithelium, the data suggest that GBC‐2(+) cells are ultimately responsible for regenerating olfactory neuroepithelium. In contrast, GLA‐13(+) cells may give rise to respiratory metaplastic epithelium where GBCs are eliminated. Thus, we support the idea that a subpopulation of GBCs is the neural stem cell of the olfactory epithelium. J. Comp. Neurol. 460:123–140, 2003.

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

We 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 the epithelium. The degeneration of olfactory axons elicits a transient up‐regulation of glial cell proliferation and glial fibrillary acidic protein expression in the olfactory nerve and olfactory nerve layer of the bulb. Anterograde transport after intranasal infusion of wheat germ agglutinin conjugated horseradish peroxidase demonstrates that the first nascent axons reach the bulb within the first week after lesion. Subsequently, a massive wave of fibers arrives at the bulb between 1 and 2 weeks postlesion, and enters the glomeruli between 2 and 3 weeks postlesion. However, the olfactory projection does not stabilize until 8 weeks after lesion judging from the return in growth associated protein‐43 expression to control levels. The extent of reinnervation after lesion is correlated with the completeness with which the epithelium reconstitutes itself. In rats that are lesioned while fed ad libitum, there is near‐complete reconstitution of the neuronal population, and the projection onto the bulb fills the glomerular layer in its entirety. However, in rats that are lesioned while food restricted, a significant fraction of olfactory epithelium becomes respiratory during its reconstitution, and the population of reinnervating fibers is less. As a consequence, the posterior half of the bulb remains hypoinnervated overall and denervated at its caudal margin. The preferential reinnervation of the anterior bulb in the food‐restricted, methyl bromide gas–lesioned animals indicates that the mechanisms that guide the growth of the olfactory axons and restore receptotopy do not operate with the same precision in this setting as they do during development or during the lower level of turnover associated with the “normal” laboratory existence. Accordingly, we hypothesize that the persistence of a significant population of pre‐existing neurons is needed to preserve receptotopy during reinnervation. In addition, the results suggest that in the face of massive turnover and a reduced afferent population, there is a tendency for reinnervating axons to fill available synaptic space. J. Comp. Neurol. 412:439–457, 1999.

Expression patterns of basic helix-loop-helix transcription factors define subsets of olfactory progenitor cells

Direct damage to the olfactory epithelium by inhalation of the olfactotoxin methyl bromide activates a population of multipotent globose basal cells, which reconstitute all depleted cell populations. Because members of the basic helix‐loop‐helix family of transcription factors are known to regulate neurogenesis and cell production, we performed in situ hybridization to examine the expression of several members of that family during the recovery of the rat olfactory epithelium after methyl bromide lesion. The numbers of basal cells expressing the proneural transcriptional activators Mash1, Neurogenin1, and NeuroD all fall precipitously 1 day after lesion. Mash1 levels begin to recover by 2 days, Neurogenin1 and NeuroD by 3 days, and substantial numbers of neurons reappear by 4 days. The antineurogenic factor Hes1 is limited to the sustentacular cells of the unlesioned olfactory epithelium and to the adjoining respiratory epithelium. Immediately after methyl bromide lesion, but not at any time after bulbectomy, a large fraction of residual, marker‐confirmed globose basal cells initiate expression of Hes1. Subsequently, the Hes1‐positive cells lose their association with the basal lamina, shift apically, and differentiate into sustentacular cells. In contrast, Hes5 is expressed by a small subset of globose basal cells and by olfactory ensheathing glia in the normal mucosa; Hes5 label disappears from both transiently after lesion. In sum, the recovery of the neuronal population after peripheral lesion recapitulates the sequence of transcription factor expression observed during embryonic development of the epithelium. Moreover, expression of Hes1 marks that population of globose basal cells committed to making sustentacular cells after methyl bromide lesion. J. Comp. Neurol. 479:216–233, 2004.

OMP gene deletion causes an elevation in behavioral threshold sensitivity

To characterize the behavioral consequences of OMP gene deletion on odor processing we assessed the ability of OMP-null animals to acquire an air vs odor discrimination for five odorants, and determined whether OMP-null animals differed from controls in their threshold sensitivity to the odorant propanol. On average, control and OMP-null animals did not differ in the number of testing sessions needed to achieve criterion performance on each discrimination problem (2.04 vs 1.68, respectively; t=0.83, p=0.41). However, null animals were significantly less sensitive to the odorant propanol (3.01 x 10(-8) vs 1.06 x 10(-5), respectively; t=4.09, p=0.015). These in vivo behavioral results provide support for the hypothesis that OMP plays a modulatory role in the odor detection/signal transduction process.

OMP gene deletion results in an alteration in odorant quality perception.

To test the hypothesis that odorant quality perception is altered in olfactory marker protein (OMP)-null mice, we trained and tested adult OMP-null and control mice, using a 5-odorant identification confusion matrix task (animal odorant confusion matrix [AOCM]). On average, control and null mice performed the task at equivalent levels. The composite 5 x 5 response matrix from 40 testing sessions for each subject (both OMP-null and control) was compared with that of every other subject, yielding a dissimilarity matrix of AOCM responses. A multidimensional scaling (MDS) analysis of the dissimilarity data yielded a 4-dimensional solution, with each mouse occupying a point in MDS animal space. Statistical analysis demonstrated significant effects of genotype in determining the location of a mouse in the MDS space. These data suggest, therefore, that compared with that of controls, odorant quality perception is altered in the OMP-null mouse.

Transplantation of multipotent progenitors from the adult olfactory epithelium

MAMMALIAN olfactory epithelium produces new neurons rapidly throughout adulthood. Here, we demonstrate that precursor cells harvested from the adult olfactory epithelium, when transplanted into the nasal mucosa of host rats exposed previously to an olfactotoxic gas, engraft and participate in neuroepithelial reconstitution. In contrast to their normal neuronal fate in situ, grafted precursors harvested from bulbectomized donors produced non-neuronal cells as well as neurons. These results demonstrate that epithelial precursors activated following olfactory bulbectomy are not irreversibly committed to making neurons. Thus, olfactory progenitors are subject to a form of feedback control in vivo that regulates the types of cells that they produce within a broader-than-neuronal repertoire.