Recovery following peripheral destruction of olfactory neurons in ...

European Journal of Neuroscience, Vol. 15, pp. 1907±1917, 2002

ã Federation of European Neuroscience Societies

Recovery following peripheral destruction of olfactory neurons in young and adult mice AngeÂlique Ducray, Jean-Robert Bondier, Germaine Michel, Karine Bon, Alain Propper and Anne Kastner Laboratoire de Neurosciences, EA 481, UniversiteÂ de Franche-ComteÂ, Place Leclerc, 25030 BesancËon Keywords: degeneration, olfactory epithelium, regeneration, ZnSO4

Abstract Olfactory neurons (ON) which are located in the olfactory epithelium are responsible of odorous molecule detection. A unique feature of these cells is their continuous replacement throughout life due to the proliferation and differentiation of local neural precursors, the basal cells. Thus, experimental destruction of all ON induces a stimulation of basal cell division followed by tissue regeneration. The fact that ON precursors display such proliferative and neurogenic activity in adults makes these cells particularly attractive as a potential tool for nervous system repair. However, basal cell proliferation and, thus, ON production, decrease in relation to age; mostly during the ®rst months of life. Therefore, we aimed to seek whether the ability of ON precursors to yield new functional ON in regenerative conditions was consequently impaired in adult. ZnSO4 intranasal perfusion administered to young (1 month) and adult (6 months) mice leads in a few days to total ON destruction and to hyposmia. Tissue and function restoration occurred in the following weeks in both mice groups and was preceded by a transient peak of cell division. In adults, although neurogenesis in the impaired olfactory epithelium was less ef®cient than in young mice, neural precursors retain their ability to provide new functional ON as indicated by the butanol detection recovery. This was achieved more rapidly than total ON regeneration, suggesting that a reduced number of reconnected ON may be suf®cient for odor discrimination.

Introduction The olfactory epithelium (OE), located in turbinates within the nasal cavity, is a useful model in which to study neural cell division and differentiation in an adult organism. This tissue contains a population of neural precursors ± present in the OE basal layer ± the globose basal cells, which are able to yield olfactory neurons (ON) throughout life (MacKay-Sim & Kittel, 1991; Farbman, 1992; Caggiano et al., 1994). This neurogenic process is essential for the renewal of mature ON that degenerate in response to noxious environmental compounds entering the nasal cavity. Moreover, experimental damage destroying the whole population of mature ON results in an almost complete regeneration and functional restoration of the tissue a few weeks after the lesion (Schultz, 1960; Graziadei & Monti Graziadei, 1983, 1984; Astic & Saucier, 2001). Such a neuronal regeneration, unique in adult mammalian nervous system, is supported by the induction of basal cells proliferation (Schwob et al., 1995; Schwartz-Levey et al., 1991; Kastner et al., 2000), which is far lower in normal conditions. The fact that olfactory neural precursors display, even occasionally, such proliferative and neurogenic activities in adults ± in contrast to most neural precursors that divide only during development ± makes these cells particularly attractive as a potential tool for nervous system repair. Other neural precursors exist in discrete regions of the adult central nervous system like hippocampus and the subventricular area (McKay, 1997; Gross, 2000), but due to their location, their putative use for autologous transplantation would be far more dif®cult. It appeared, therefore, worthwhile to know to what extent the ability of

olfactory neural cell precursors to divide and regenerate ON remains ef®cient in adults and aged animals. Different studies have reported that olfactory cell division rate decreases in relation to age, especially during the ®rst months of life (Fung et al., 1997; Weiler & Farbman, 1997; Nakamura et al., 1998; Ohta & Ichimura, 2000). This appears to be linked to the reduced expression level of numerous cell cycle regulators in relation to age (Legrier et al., 2001a). The same evolution of proliferative activity was also observed for neural precursors in the brain (Kuhn et al., 1996; Tropepe et al., 1997). Moreover, when these cells were removed from human post mortem brain and cultured in vitro, they showed a decreased number of cell cycles prior to senescence in relation with the donor age (Palmer et al., 2001). The progressive decline of neural precursor cell proliferation during ageing raises the question of whether these cells are still fully responsive to environmental cues. To answer this, we compared, in the present paper, the neurogenic activity induced by peripheral ON destruction in the OE of young and adult mice. We observed that in adult animals, olfactory neural precursors maintained after stimulation their ability to divide and to yield functional ON. Moreover, we compared, in this paper, the kinetics of tissue and function recovery of the olfactory system following ON destruction. We found that the ability to discriminate odors was fully restored, while ON regeneration was still incomplete.

Materials and methods Correspondence: Dr Anne Kastner, as above. E-mail: [email protected] Received 24 October 2001, revised 5 April 2002, accepted 20 April 2002 doi:10.1046/j.1460-9568.2002.02044.x

Animals The study was performed on OF1 mice, which were 1, 3 and 6 months-old. It was carried out in accordance with the `Guide for the

1908 A. Ducray et al. Care and Use of Laboratory Animals' (National Institutes of Health, USA, 1985). Olfactory neuron destruction was accomplished by bilateral ZnSO4 intranasal (i.n.) perfusion administered to lightly anaesthetised mice (5 mg/g of 0.3M chloralhydrate solution). Our aim was to destroy the ON cells without damaging, irreversibly, the supporting tissue. Preliminary experiments showed us that, whereas low doses (5 or 10 mL of 10% ZnSO4) were not suf®cient, 16 or 20 mL of 10% ZnSO4 perfused intranasally lead to signi®cant ON lesion. Both control (nontreated by ZnSO4) and treated mice were killed by decapitation under deep anaesthesia. The bulk of olfactory turbinates was immediately removed and incubated either in NP40 lysis buffer (for biochemical studies, see below) or in 4% paraformaldehyde in PBS buffer (for autoradiography and immunohistochemistry, see below). Behavioural study Test procedure establishment Preliminary experiments were performed to check ON destruction in vivo by a sensitive olfactory-mediated behavioural test. The olfactory function was evaluated by testing each mouse's ability to discriminate between two compounds within a T-maze test. The apparatus was a T-shaped maze with two dark arms containing each a watch-glass with a ®lter paper soaked with either distilled water (nonodorant) or a butanol solution (known to be a repulsive odorant for mice; Deiss & Baudoin, 1997). A random testing order was used for each mouse, which was placed in a start-box at the end of the stem and then allowed to move freely for 5 min. This task was repeated four times with 30 min intervals and the repulsive solution was put alternatively in one or the other arm at each test. The total number of entrances into each arm was counted. Discrimination ability was evaluated as the percentage of entrances in the water-containing arm. Odor discrimination in control mice was more sensitive when we used high concentrations (200 mL) of 15% butanol (almost 80% entrances in water containing-arm) rather than lower ones (40 mL of 10%; only 60% entrances in water containing-arm). Thus, for the following study of ON recovery after injury, 200 mL of 15% butanol were used to examine olfactory-mediated behavioural performance. Detection of ON lesion with this procedure test The olfactory function was tested before and after ZnSO4 i.n. perfusion. After 16±20 mL ZnSO4 10% i.n. perfusion (4±7 days), most mice (» 60% per treatment series) were unable to show a clear preference between the nonrepulsive and the repulsive odor solutions (less than 60% entrances in the water-containing arm), while the others exhibited no signi®cant behavioural changes. Only hyposmic mice (de®ned as a decrease in odor sensitivity) displayed total ON loss on the basis of histological and biochemical data (see Results), while nonhyposmic mice developed only a partial ON degeneration (not shown). Thus, hyposmia (in these experimental conditions) has been considered to be a good criterion to determine and choose which mice had to be examined to establish the time course of olfactory recovery. In a preliminary study, we analysed the ability to discriminate butanol vs. water both in six control mice and in six mice having received ZnSO4 i.n. perfusion, 0, 7 and 14 days after treatment. The treated mice displayed impaired olfactory-mediated behaviour, as compared to the control group, 7 days after the treatment (P < 0.01) but not 2 weeks after. Thus, the T-maze test may be suitable for evaluation of olfactory recovery. In this test, control mice achieved similar performances at each date. Thus, for the following tests, the performance obtained at day 0 (before ZnSO4

i.n. perfusion) was considered as the control value, and all mice received the ZnSO4 treatment. Western-blotting For biochemical studies, olfactory mucosa were homogenized in NP40 lysis buffer, (i.e., 0.5% Nonidet P40 in 50 mM Tris HCl pH, 8,) containing 120 mM NaCl, 0.1 mM Na3VO4, 1 mM EDTA and a protease inhibitor cocktail (Sigma). After 1 hour incubation at 4 °C while stirring, lysates were cleared by centrifugation at 10000 g for 2 min. Lysates from mice killed at the same time after intranasal ZhSO4 perfusion (two mice per point) were then pooled, and after protein titration by Bradford assay on each sample, they were diluted in NP40 buffer to a ®nal concentration of 8 mg/mL. For Westernblotting experiments, lysates were mixed to equal volumes of 2 3 protein sample buffer and kept frozen until used. Electrophoresis was performed on SDS polyacrylamide mini-gels (Biorad) by loading 20± 40 mg proteins in each lane, according to the standard technique of Laemmli (1970). After electrotransfer onto nitro-cellulose membrane (Millipore, ImmobilonÔ-P) and blocking in PBS±5% milk±0.1% tween solution, blots were incubated overnight at 4 °C (or 3 h at room temperature) with the primary antibody in PBS±milk±tween solution. These antibodies were anti-PCNA (proliferating cell nuclear antigen; mouse, Sigma, 1 : 1000), anti-OMP (olfactory marker protein; goat, gift from F. Margolis, 1 : 2000), anti b-3 tubulin (mouse, Sigma, 1 : 2000), anti b-actin (mouse, Sigma, 1 : 6000) and anti-Gap43 (growth associated protein 43) (rabbit, Chemicon, 1 : 1000). After incubation for 1 hour with the appropriate peroxidase-linked secondary antibody (Dako, 1 : 5000 to 1 : 1000), immunoreactivity was detected by chemiluminescence using a commercial kit (Pierce). For quanti®cation analysis, the intensity of the bands were determined by densitometry and normalized using the `Molecular Analyst' software (Biorad, CA, USA). The curves of optical density (OD) 3 mm2 (arbitrary units; a.u.) as a function of days after i.n. perfusion (PP, postperfusion) were established by expressing densitometric values of the studied protein against a housekeeping protein (where the levels don't change) such as b-actin or b-3 tubulin. Histology and immunohistochemistry Olfactory mucosa (OM) were ®xed in 4% paraformaldehyde in PBS for 8 h at 4 °C. The ®xed tissues were washed in PBS, then decalci®ed (one night), dehydrated in rising concentrations of ethanol and butanol baths and embedded in paraf®n. After paraf®n removal and rehydration, cross sections (7.5 mm) were used either for histological or immunohistochemical studies. For histological study, sections were stained with haematoxylin (Biolyon) for 2 min. Semi-quantitative analysis of OE thickness was performed `blind' by two experimenters on several section levels (n = 5±10) per mice (n = 2±3 for each days PP). Arbitrary values between 0 and 2 were attributed, 2 corresponding to control OE thickness (among 10 cell layers) and 0 to an OE reduced to one or two cell layers. Moreover, the number of cell nuclei was analysed `blind' on these stained sections with an imaging system (Olympus BX51 microscope with a DP50 camera and the Analysis program). The number of cell nuclei were counted on a piece of 200 mm OE length along the nasal septum for two adjacent sections on three different levels (for each mouse). For immunohistochemical study, sections were incubated for 5 min in PBS±0.3% Triton X-100 and then for 30 min in PBS containing 10% goat serum or fetal calf serum. Sections were next incubated with diluted primary antibodies (anti-PCNA, anti-Gap43: 1 : 50 in PBS, 10% serum) for 3 h at room temperature or one night

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 1907±1917

Recovery of olfactory neurons after lesion

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and 21 days), the nasal mucosa were removed and prepared for light microscopy autoradiography as detailed above. The slides were coated with EM-1 emulsion (Amersham), stored in light-tight boxes at 4 °C for several weeks and developed in 17±19 °C D-19 solution (Kodak) for 7 min. After dipping in the stop solution (0.5% acetic acid) for 30 s, the slides were ®xed in 30% sodium thiosulphate solution, rinsed and counterstained with haematoxylin (Biolyon). Photography and image processing Sections were observed on a Nikon microscope (Eclipse E600) and photographed with a Nikon digital camera (Coolpix 990). Adobe PhotoShop 4.0 LE on a Macintosh computer was used for contrast and light improvement, and to generate hard copy printouts.

Results Olfactory-mediated behaviour recovery after ON destruction in young and adult mice

FIG. 1. Time course of olfactory function recovery after ZnSO4 i.n. perfusion. The discrimination ability between water vs butanol was tested in young (1 month, n = 8) and adult (6 month, n = 8) mice before and after peripheral destruction of ON by intranasal ZnSO4 perfusion. The mean kinetic curves were determined by using (for each point) individual percentage scores for water vs butanol obtained with the T-maze test procedure. Notice that olfactory-mediated behaviour was impaired 4±7 days PP but recovered thereafter, similarly in young and adult mice.**(P < 0.05) between days 0, 18, 25 and 35 values for young and adult mice. The F-test of variance equality was performed followed by the Student's t-test. Percentage values + SE are indicated.

at 4 °C. After washing, tissue sections were incubated for 1.5 h at room temperature in 1 : 100 diluted solution of appropriate peroxidase-linked secondary antibodies. The peroxidase activity was revealed in a solution containing diaminobenzidine tetrahydrochloride (0.5 M) and 0.01% hydrogen peroxide. The number of PCNA-positive cells was counted on a piece of 600 mm length OE along the nasal septum for two adjacent sections on three different levels (for each mouse) with the same imaging system as used for cell nuclei counts. For lectin detection, sections were incubated 2 h at room temperature, in PBS±0.3% Triton X-100±20 mg/mL Dolichos bi¯orus agglutinin (DBA) (Sigma) solution and then washed in PBS. Sections were next incubated for 1.5 h at room temperature in 1 : 100 diluted solution of extra-avidin (Sigma). Peroxidase activity was revealed as for the immunohistochemistry. Autoradiography [3H] thymidine (5 mCi/g, Sigma) was injected intraperitonally in 1 (n = 2) and 6-month-old-mice (n = 2). After isotope application (10

The kinetics of ON regeneration in young (1 month, n = 8) and adult (6 months, n = 8) mice were assessed by measuring their ability to perform a simple odor-discrimination task (Fig. 1). Control mice preferred the water as opposed to the butanol-containing arm (day 0: 79.0%). After ZnSO4 i.n. perfusion (4±7 days PP) mice became hyposmic (less than 55% entrances in water-containing arm). From 10 days PP onwards, mice recovered gradually their discrimination ability, so that 18 days PP the discrimination between the two odors was similar to that observed before the treatment (» 80% entrances in water-containing arm). The same time course of olfactory sensitivity recovery after ZnSO4 i.n. perfusion was obtained with both mice populations, although the olfactory performance 10 days PP seemed better for young mice. The individual results of the olfactorymediated behavioural test showed that all hyposmic mice recovered the ability to discriminate the repulsive odor of butanol during the weeks following the lesion, although high interindividual variations were observed at some stages (see Figs 2B and 3D). ON degeneration and regeneration in young and adult mice after ZnSO4 intranasal perfusion Western-blot analysis of OMP levels showed that this protein speci®c of mature ON was highly present in OM and olfactory bulb (OB) extracts of control mice (Fig. 2A). In OM and OB, the amount of this protein dropped dramatically between 4 and 7 days PP, a fact demonstrating the destruction of mature ON by ZnSO4. This was in keeping with the loss of odor discrimination in these mice with the Tmaze test (not shown). OMP was still detected at much lower levels in the OM 25 and 35 days PP than in controls (and even 80 days PP, not shown), and remained poorly detectable in the OB whereas the amounts of b-actin in these same extracts were unchanged. These results showed that at 35 days PP, very few differentiated ON were present in the OM and reconnected to their target, the OB. Similar data were obtained in young and adult mice. The T-maze test showed, however, a good restoration of the ability to discriminate the repulsive odor of butanol in these same mice (Fig. 2B). The OE is essentially composed of only three different cell types arranged in successive layers which are, from the nasal cavity to the basal lamina: supporting cells (SC), olfactory neurons (ON) and basal cells (BC). Four days PP, OE was reduced to one or two cell layers, putatively the BC, both in young and adult mice. At 35 days PP, the OE was regenerated partially in both mice populations (Fig. 3A). At this stage, the regenerated zones were located principally in the dorsal fossa and along the nasal septum. On the left or right turbinates, the


1910 A. Ducray et al.

FIG. 2. (A) Western-blot assessment of mature ON presence after ZnSO4 i.n. perfusion. OMP was used as a marker of mature ON. Protein extracts were obtained from OM and OB of young (1 month) and adult (6 month) mice. The densitometric pro®les of OMP amounts reported on b-actin amounts are given for each age as the mean from four experiments for OM and two experiments for OB. Notice that ZnSO4 i.n. perfusion results in a long-term decline of OMP levels. (B) Individual performance of odor-mediated behavioural test until 35 days PP for young and adult mice used for Western-blotting Fig. 2A. These data show the high interindividual variations with the T-maze test used here, but all mice present an hyposmic pro®le a few days after ZnSO4 i.n. perfusion and an olfactory discrimination sensitivity recovery during the weeks following ON destruction.

regeneration was generally not at the same stage (not shown). An electron microscopy study showed us that the regenerated tissue displayed the basic structure of normal OE (work in progress in our laboratory). A semiquantitative evaluation (Fig. 3B) showed that OE thickness decreased drastically 4 days PP in keeping with the lack of odor discrimination in these mice (not shown). Restoration occurred progressively between 18 and 35 days PP. The OE thickness values were, however, less than 60% of the control values for both mice populations 35 days PP, while olfactory performance was fully restored (Fig. 3D). Quantitative measurement (Fig. 3C) showed that the number of cell nuclei per distance along the nasal septum was low

at 4 days PP and recovered thereafter. Statistical analysis indicated that the number of nuclei at 35 days PP in adult mice was 39% less abundant than in young ones (P < 0.05). As OMP levels detected by Western-blots remained very low at 18 and 35 days PP, we analysed the differentiation state of the ON, using GAP43 as a marker for differentiating ON (Fig. 4). In control OE, GAP 43 protein was restricted to patches of cells lining the basal layer of the OE. At 18 and 35 days PP numerous ON were still differentiating GAP43-positive cells in young and adult mice, and were, therefore, not fully mature at this regenerative state. We also analysed the reappearance of a differentiated ON subpopulation


FIG. 3. Olfactory epithelium (OE) degeneration and regeneration after ZnSO4 i.n. perfusion in young and adult mice. (A) Mice OE sections of control and 4 and 35 days PP stained with haematoxylin. nc, nasal cavity; sc, supporting cells; on, olfactory neurons; bc, basal cells; ch, chorion. Arrow heads, basal lamina. Scale bar, 50 mm. (B) OE thickness (semiquantitative evaluation) of young and adult mice before and after ZnSO4 i.n. perfusion (2 or 3 mice per point). Arbitrary values (between 0 and 2) were given for OE thickness from haematoxylin-staining sections: `2' corresponding to normal control thickness, and `0' to OE reduced to one or two cell layers. Notice that OE thickness was reduced to putative globose basal cells layer at 4 days PP, and was partially recovered (60% of control OE thickness) 35 days PP. **(P < 0.05) between values compared to day 0-values. The same statistical test was the same as that used for the behavioural study. (C) The histogram represents the mean number of cell nuclei per distance expressed in percent vs control mean value. The ®lled diamonds (r) indicate individual percentage values. (D) Individual performance of odor-mediated behavioural test until 35 days PP for young and adult mice used for histological analysis (Figs 3B and C). These data show the hyposmic pro®le of these mice a few days after ZnSO4 i.n. perfusion and their olfactory discrimination sensitivity recovery during the weeks following ON destruction.



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FIG. 4. Olfactory neurogenesis after ZnSO4 i.n. perfusion. GAP43 was used as a marker of neuronal differentiation. Notice that, in both mice populations, immature ON (GAP43 positive) limited to one cell layer up to the basal cell layer in control mice, became the major ON subpopulation 18 and 35 days PP. Arrows indicate some labelled cells. Conventions are the same as in Fig. 3. Scale bars, 50 mm.

(Fig. 5) which express carbohydrates recognized selectively by the plant lectin, Dolichos bi¯orus agglutinin (DBA) and which project speci®cally to a subset of glomeruli in the OB (Key & Akeson, 1993). The number of DBA-labelled ON in the OE was very low at 18 days PP when compared to that of controls, although the mice were able to discriminate butanol. At 35 days PP a substantial number of cells were labelled with DBA indicating that differentiated ON were present again at this regenerative state. ON precursors: mitotic stimulation in young and adult mice after ZnSO4 intranasal perfusion Previous studies have shown that basal cell proliferation decreased during the ®rst months of life, suggesting that ON production may decline in relation to age (Fung et al., 1997; Weiler & Farbman, 1997; Nakamura et al., 1998; Ohta & Ichimura, 2000). Here, using an autoradiographic method, we examined directly the neurogenic activity in the control OE from young and adult mice, 10 and 21 days after [3H]thymidine incorporation. The cells that incorporated [3H]thymidine correspond to the ones that were in S-phase just after the injections. Thus, autoradiography permits us to label only the cells generated by division of these solely S-phase progenitors. Figure 6 shows that numerous basal cells and some ON were radiolabelled 10 days after [3H]thymidine incorporation in the 1 month-old mouse. In the 6 month-old mouse, only some distinct groups of basal cells exhibited silver grains. At 21 days after [3H]thymidine injection, the labelled ON were much more numerous in the young mouse than in the older one. These observations showed that less ON were produced in adult control mice when compared to younger ones. In the two mice groups, silver grains were still often present in clusters of basal cells or in the basal part of the OE, 3 weeks after [3H]thymidine incorporation in the dividing cells. This indicated that for most olfactory cells, ON differentiation was not achieved 3 weeks after ON precursor division. Olfactory neuron regeneration is supported by the induction of basal cell proliferation and differentiation. Using Western-blotting

and immunohistochemistry (using PCNA as a marker of cell division) we investigated the ability of these cells to proliferate after ON destruction in young and adult mice. Previous studies have shown that PCNA Western-blotting was a suitable method to compare the level of olfactory cell division in the OE (Kastner et al., 2000; Legrier et al., 2001a). A peak of PCNA expression appeared a few days after ZnSO4 i.n. perfusion both in young and adult mice. Then progressively, the PCNA protein amounts dropped to control levels (Fig. 7). A densitometric analysis of another set of four experiments revealed that the PCNA/b-actin amounts 7 days PP were, however, » 50% lower in adults (n = 6) than in young mice (n = 6). Thus, olfactory cell division, which is almost absent in adult mice, may be highly stimulated following neuronal destruction, although it reached lower values than in young mice. This was also observed by PCNA immunohistochemistry in the OE (Fig. 8), which showed that PCNApositive basal cells became less numerous in the ageing OE, but at 4 days PP, a higher number of PCNA-positive cells was present whatever mice age.

Discussion Neuronal loss and regeneration after intranasal ZnSO4 lesions The present study examined the time course of functional, biochemical and anatomical recovery after olfactory lesion with ZnSO4 in mice. Intranasal perfusion of ZnSO4 has been one of the most commonly used methods to destroy ON (Schultz, 1960; Alberts & Galef, 1971). It is less dif®cult to execute and less stressful for the animals than surgical lesions of the olfactory tract. However, in some cases, this method leads to surprising results. For instance in rats, ZnSO4 has been shown to be poorly ef®cient in inducing anosmia and complete ON loss (Slotnick et al., 2000), but in general, that problem was not observed in mice. In contrast, the damage induced in mice OE can be so severe, that it precludes reconstitution happening. Also, ZnSO4 may destroy both neuronal and non-neuronal cells in contrast



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FIG. 5. ON subpopulation labelled with Dolichos bi¯orus agglutinin (DBA) after ZnSO4 i.n. perfusion. (A) DBA-positive ON in the control and regenerating OE: arrows indicate labelled neuron cell bodies. The dendrite of some DBA-positive ON are also labelled. Notice that DBA-labelled ON are almost absent in the OE 18 days PP but no more at 35 days PP. Conventions are the same than in Fig. 3. Scale bars, 50 mm. (B) Quanti®cation analysis of DBA-labelled ON subpopulation after ZnSO4 i.n. perfusion. Histogram represents the mean number of DBA-labelled ON expressed in percent vs control mean values.

to olfactory transection, which causes the retrograde lesion of solely ON. Thus, two previous studies (Harding et al., 1978; Burd, 1993) reported a poor functional or biochemical recovery after ZnSO4 perfusion in mice (100 mL, 5%) while OE reconstitution was described 30 days after olfactory nerve transection (Harding et al., 1977). Our results show that concentration and volumes of ZnSO4 perfused may be important factors contributing to the extent of neuronal loss and recovery. Thus, by using relatively low amounts of ZnSO4 (16 mL, 10%) we succeed ®rstly, in obtaining complete neuronal cell loss and hyposmia in a large proportion of mice, 4± 7 days after intranasal perfusion, and secondly, we observed a recovery a few weeks after the lesion. Indeed, OE was reduced to essentially one or two cell layers in hyposmic animals but reacquired progressively a multilayered structure during the following weeks. Our results are in agreement with those obtained by Matulionis, 1979) and Herzog & Otto (1999) who worked with ZnSO4 in mice and rats, respectively. The fact that the levels of OMP protein remained surprisingly low in the OE and the OB several weeks after the lesion may be due to incomplete ON regeneration (see below) or alterna-

tively to a failure of these ON to properly express the marker. However, our histological results are globally similar to what is observed after surgical treatment, as several studies reported the reappearance of new ON from 3 weeks on following axotomy in rodents (Graziadei & Monti Graziadei, 1984; Costanzo, 1985; Yee & Costanzo, 1995). Thus, at least in mice, we con®rmed that ZnSO4 treatment may be a convenient technique to induce ON degeneration and regeneration and to analyse its time frame in various physiological conditions. We showed in the present paper that in most hyposmic mice, the ability to discriminate butanol vs water was restored about 20 days following the chemiotoxic treatment. Restoration of odormediated behaviour following olfactory lesion by various methods has been shown previously in ®sh (Cancalon, 1982; Von Rekowski & Zippel, 1993), birds (Oley et al., 1975), amphibia (Simmons & Getchell, 1981) and rodents (Yee & Costanzo, 1995; Yee & Rawson, 2000). Yee & Costanzo (1995) have shown that following nerve transection in hamster, the ability to discriminate between odors was restored between 15 and 30 days after surgery,



FIG. 6. [3H] thymidine-labelled cells in OE of young and adult control mice. 10 days after [3H] thymidine incorporation only basal cells exhibited silver grains in adult mice, whereas also some ON were labelled in young ones. 21 days after [3H] thymidine incorporation some clusters of ON were labelled in both mice population but were more numerous in young mice. Arrows indicate some labelled cells. Conventions are the same than in Fig. 3. Scale bar, 50 mm.

FIG. 7. ON precursors mitotic stimulation in young and adult mice. PCNA serves as a marker of proliferation. Western-blot assessment of olfactory cell proliferation in young and adult mice (two mice per point) before and after ZnSO4 i.n. perfusion. b-3 tubulin was used as a calibration marker. Graphs give densitometric mean values with arbitrary units (a.u.) of PCNA/b-3 tubulin signals from n = 8 independent experiments.

in correlation with the reappearance of ON terminals in the OB. In our study conditions, it cannot be excluded that the trigeminal nerve, which is sensitive to butanol, may also be involved in the ability of mice to discriminate between this compound and water. However, we think that the functional restoration observed in the present study may be principally linked to the activity of the olfactory system for the following reasons. (i) The curves of discrimination recovery (Fig. 1) are identical to that obtained in our laboratory by measuring mitral cells (the ON target cells in the OB) activity by electrophysiological techniques (Michel, V., unpublished data). (ii) These recovery kinetics of Fig. 1 are also quite similar to those obtained after olfactory nerve transection using another functional task (see below) (Yee & Rawson, 2000). (iii) Preliminary data obtained in our laboratory with bulbectomised mice indicated that the ability to discriminate water vs butanol was de®nitively impaired in these animals in relation with the absence of proper olfactory projections. In our study, an interesting result was the observation of a complete ability to discriminate butanol odor in most mice 18 days after ZnSO4 i.n. perfusion, despite unachieved tissue regeneration as indicated by the poor OMP protein levels and by the low number of cell nuclei or DBA-labelled ON in the OE at this regenerative state. In addition, the

OE was still much thinner than in control mice several weeks after the lesion, a fact also previously described by others (Costanzo, 1985; Burd, 1993; Herzog & Otto, 1999). Moreover, numerous cells were still in an immature state as indicated by the presence of GAP43 in these cells 35 days after the lesion. Thus, a limited proportion of functional ON reconnections may be suf®cient to perform properly a simple olfactory-mediated behaviour task. Similarly, the ability of axotomised mice to ®nd a buried piece of cheese was completely restored 16 days following the lesion (Yee & Rawson, 2000), although complete ON regeneration cannot be expected at this time of regeneration. Also, in the line with our observations, Harding et al. (1978) showed that 13 or 16 months after intranasal irrigation of mice with ZnSO4, olfactory function was fully restored, but biochemical parameters such as carnosine synthesis and its transport were markedly reduced in comparison to control values in the OB and OE. However, due to the long delay before examination, the latter de®ciency may also re¯ect the age-related decrease in OE thickness (Hinds & McNelly, 1981) rather than incomplete regeneration. Our results are rather surprising in regards to recent data obtained after nerve transection. Indeed, they showed an impairment both in olfactory topographic projections to the OB (Schwob et al., 1999; Costanzo, 2000) and in sensory perception, as animals must be



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FIG. 8. PCNA immunohistochemistry on control and 4 days PP OE. (A) The number of PCNA-positive cells decrease in control mice in relation to age. However, 4 days PP a higher number of PCNA-positive cells were present in each mice group. Arrows indicate some labelled cells. Conventions are the same as in Fig. 3. Scale bar, 50 mm. (B) Quanti®cation in control and 4 days PP mice of the PCNA-positive cells on two pieces of 600 mm length OE along the nasal septum at three different levels (for each mice). Histogram gives the mean of PCNA-positive cell number in 1-month-old mice (n = 2) and 6-monthold mice (n = 2). rIndividual numbers of PCNA positive cells.

trained again to recover the ability to perform an odor discrimination task (Yee & Costanzo, 1998). However, on the other hand, olfactory function is considerably preserved even after severe damage in the OE or OB (Lu & Slotnick, 1994; Youngentob et al., 1997; Setzer & Slotnick, 1998) so that 5±10% of the ON may be suf®cient to detect a large variety of odors. In line with that assumption is also the observation that during rat postnatal development, the number of ON converging on each mitral cell in the OB increases by a factor of more than 10 during the ®rst 25 days (Meisami, 1989), although it is known that rats display a functional olfactory system at birth. Thus, in the case of total ON injury and regeneration, animals may recover function as soon as the ®rst subpopulation of ON will reconnect to the OB, so that functional recovery may precede complete tissue formation, as during the ontogenic process. Behavioural analysis may therefore be a convenient method to assess neuronal loss and recovery in the olfactory system and to determine the effect of various factors on its time course (Yee & Rawson, 2000). Age-related changes in OE neurogenic activity In the present study, we showed an increase of the neurogenic activity of ON progenitors in response to ON degeneration in both mice populations, although it was less ef®cient in adults.

Previous work has shown that normal mitogenic activity in the OE is reduced in relation to age (Weiler & Farbman, 1997) mainly during the ®rst months of life. For instance, basal cells division rhythm in 3 month-old mice is among three times lower than in 3 week-old animals and among 10 times lower than in new-born animals. The level of ON production and turnover is therefore reduced in mature mice (Fung et al., 1997; present data). This may account for the reduced number of ON and OE thickness in senescent mice (Hinds & McNelly, 1981) and contribute to the age-related decline of the olfactory sensibility observed in older animals or humans. Actually, although neural precursors are less active in adult vs developing mice in normal conditions (up to 10 times), our paper shows that they retain a substantial neurogenic potential in response to environmental cues. Even in a latent quiescent state, these cells may thus have kept the molecular machinery necessary to drive ef®ciently the neurogenic process; especially growth factor receptors. At this point, several extracellular signals as FGF 2 (®broblast growth factor 2), LIF (leukemia inhibitory factor), NPY (neuropeptide Y) and TGFa (transforming growth factor) have indeed been shown to be mitogenic for olfactory cells (Mahanthappa & Schwarting, 1993; DeHamer et al., 1994; Satoh & Yoshida, 1997; Farbman & Ezeh, 2000; Newman et al., 2000; Hansel et al., 2001; Nan et al., 2001), but the


1916 A. Ducray et al. putative age-related changes in their expression are not known. It has been shown previously that ON regeneration and reconnection within the OB following nerve transection occurs even in old hamsters (12± 24 month-old). This demonstrates that the ability to undergo neural cell division, differentiation, axonal growth and reconnection to the OB still persists in old age (Morrison & Costanzo, 1995), but direct comparison between young and older animals was not reported. In their study, the fact that the glomeruli in the reinnervated OB were always smaller than normal, even at the longest survival times, suggests, however, that the mitogenic or neurogenic activity of ON precursors may not be as ef®cient in adult as during development. In our study, it indeed appeared that these latter parameters measured in regenerative conditions are quantitatively higher in young mice than in adult mice. Thus, it cannot been excluded that the whole mitogenic and neurogenic potential of ON precursors is limited. In vitro studies will allow us to seek whether the total number of olfactory neural cell divisions before senescence may depend on the age of the donor, as shown recently for cells of the central nervous system in humans (Palmer et al., 2001). Neural cell precursors, which are able to generate neurons in adults, are also present in a few regions of the brain (hippocampus and subventricular area), and are thought to be of worthwhile interest for putative regenerative strategies (Isacson et al., 2001). It would, therefore, be important to know whether these cells and the olfactory ones share common properties in their neurogenic potential. The fact that olfactory cells, even in a quiescent state, may retain their mitogenic and neurogenic potential in adult animals suggests that adult OE may be a suitable tissue to provide a population of neural precursors for therapeutic use. Olfactory neuronal precursors already have real advantages over other precursors of the brain: they are more numerous (Legrier et al., 2001b) and easier to obtain (Roisen et al., 2001). The OE tissue is accessible via a simple biopsy through the external naris (Feron et al., 1998; Roisen et al., 2001), so that the technical dif®culties associated with the use of tissue from the brain of adult donor are avoided. Moreover, the use of ON precursors would allow autologous transplantation. For a potential use of these cells in cell replacement therapies, it is essential to know whether they may give rise to neurons other than ON. Adult neuronal stem cells taken from the brain are indeed noticeable by the fact that they are able to differentiate into different cell types from those of the tissues to which they belong (Vescovi & Gritti, 2001). Although neural precursors displaying stem cell properties have been isolated from the OE (Mumm et al., 1996; Roisen et al., 2001), it is not yet known whether they are also multipotent neural stem cells or not. Further studies including heterologous transplantation and cell culture would be necessary to answer this question.

Acknowledgements We thank Drs Jean-Louis Millot and Danielle Henquell for their technical assistance for the behavioural test procedure establishment, Dr Franck Margolis for his generous gift of OMP antibody. We are also grateful to Dr Philippe Thibert for helping with statistical analyses.

Abbreviations GAP43, growth associated protein 43; OB, olfactory bulb; OE, olfactory epithelium; OM, olfactory mucosa; OMP, olfactory marker protein; ON, olfactory neurons; PCNA, proliferating cell nuclear antigen; PP , post ZnSO4 i.n. perfusion.

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