Quantitative [3H]thymidine autoradiography of neurogenesis in the olfactory epithelium of developing normal, hypothyroid and hypothyroid-rehabilitated rats

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 83 (1994) 151-162

Research report

Quantitative [3H]thymidine autoradiography of neurogenesis in the olfactory epithelium of developing normal, hypothyroid and hypothyroid-rehabilitated rats Mark A. Paternostro 1, Esmail Meisami * Department of Physiology and Biophysics, Unit,ersity of Illinois, 524 Burrill Hall, 407 S. Goodwin At'e., Urbana, IL 61801, USA Accepted 28 June 1994

Abstract

We recently reported that postnatal hypothyroidism results in marked reduction in surface area and cell number in the rat olfactory epithelium (OE) and recovery from this condition is accompanied by compensatory growth and restitution of these parameters. To explore the correlative changes in olfactory neurogenesis, i.e. mitotic activity of basal cells (BCs) and migration and survival of developing olfactory receptor neurons (ORNs), hypothyroid rats at postnatal (P) days of Pll), P25 and P75 were injected with [3H]thymidine and OE was examined by quantitative autoradiography to determine the density of labeled nuclei at the BC and ORN zones at days 1, 5 and 15 post-injection. These data were compared with those of age-matched controls as well as young adult rats allowed to recover from hypothyroidism at the end of the suckling period (P25). Hypothyroidism was induced by administration of propylthiouracil (PTU) from birth in the drinking water (1 g / l ) for 10, 25 and 90 days; recovery was induced by withdrawal of PTU at P25. The results indicated that the densities of labeled nuclei in the BC and ORN zones were not significantly altered in the suckling hypothyroid rats. In the P75 hypothyroid rats density of labeled BC nuclei was unaffected l day after injection but was significantly (36%) more than controls 5 days after injection; the density of neuronal nuclei in the ORN zone of P75 injected rats was markedly and significantly reduced (56% and 37% at 5- and 15-days post-injection). Data indicate that mitotic activity of BCs and their migration into the ORN zone is not affected in the hypothyroid infant rats but migration a n d / o r survival of developing ORNs are markedly reduced in the postweaning growing rats made hypothyroid from birth. In rats allowed to recover from hypothyroidism at P25 and injected with labeled thymidine at P75, the density of labeled BC nuclei were significantly increased (48% and 43% at 1- and 5-days post-injection) compared to normal rats suggesting elevated levels of neurogenesis; density of ORN nuclei, however, were the same as controls. The results indicate critical regulatory influences of thyroid hormones on olfactory neurogenesis in the rat olfactory receptor sheet, in particular during the postweaning period. Keywords: Olfactory receptor neuron; Basal cell; Olfactory neural development; Thyroid hormone; Thyroid and neural

development; Developmental plasticity; Recovery from neural retardation; Compensatory neural growth; Neuronal proliferation and migration

1. Introduction

R e c e n t q u a n t i t a t i v e studies f r o m o u r l a b o r a t o r y have shown t h a t t h y r o i d h o r m o n e s ( T H s ) a r e essential for

* Corresponding author. Fax: (1) (217) 333 1133; E-mail: meisami(q uiuc.edu. 1 Present address: Division of Integrated Studies, Pennsylvania College of Technology, Williamsport, PA 17701, USA. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 01 6 5 - 3 8 0 6 ( 9 4 ) 0 0 8 1 1 - 6

p o s t n a t a l growth a n d d e v e l o p m e n t o f the rat olfactory e p i t h e l i u m ( O E ) [36-39]. In the rat, O E is a highly f o l d e d n e u r o e p i t h e l i a l s h e e t o f o l f a c t o r y r e c e p t o r neurons ( O R N s ) , basal cells (BCs) a n d s u p p o r t i n g cells a r r a n g e d in a p s e u d o s t r a t i f i e d m a n n e r . O u r studies have shown t h a t in the rat O E surface a r e a a n d n e u r o n n u m b e r i n c r e a s e by 8- a n d 12-fold, respectively, d u r i n g the suckling p e r i o d a n d by 2.5-fold d u r i n g the postsuckling p e r i o d [29,36,38,39]. S i m i l a r i n c r e a s e s have b e e n n o t e d in t h e m o u s e septal O E [47]. T h e s e devel-

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o p m e n t a l c h a n g e s have b e e n c o r r e l a t e d to f u n c t i o n a l i m p r o v e m e n t s in olfactory sensitivity [1,29]. If deprived of T H s from birth, cell p r o l i f e r a t i o n a n d surface area expansion in the m a i n O E are r e d u c e d by a b o u t 45% d u r i n g the suckling period [36,39]; if T H s deficiency is c o n t i n u e d in the p o s t w e a n i n g period, these growth events cease nearly entirely [39]. R e m a r k a b l y , if T H s are restored at the e n d of the suckling period (P25), O E growth a n d d e v e l o p m e n t r e s u m e s in a c o m p e n s a t o r y m a n n e r , so that by P90 the growth deficiency in O E surface area a n d cell n u m b e r is c o m p l e t e l y restored [39]. T h e s e findings d e m o n s t r a t e that T H s have a m a j o r regulatory i n f l u e n c e on p o s t n a tal f o r m a t i o n of new O R N s a n d indicate that OE, in contrast to c e n t r a l n e u r a l structures, has the u n i q u e ability to fully recover from early p o s t n a t a l hypothyr o i d - i n d u c e d growth r e t a r d a t i o n . A u t o r a d i o g r a p h y for [3H]thymidine e n a b l e s determ i n a t i o n of b i r t h d a t e s of dividing n e u r o n s a n d their postmitotic m i g r a t i o n a n d t r a n s f o r m a t i o n . U s i n g this approach, it has b e e n shown that olfactory n e u r o g e n e sis a n d n e u r o n a l t u r n o v e r occur c o n t i n u o u s l y in the O E of y o u n g a n d adult rodents, c o n s t i t u t i n g a 30-day cycle of n e u r o n a l g e n e r a t i o n a n d d e g e n e r a t i o n . Acc o r d i n g to this scheme, new O R N s form by mitosis of p r o g e n i t o r BCs, located at or n e a r the basal lamina. This is followed by m i g r a t i o n of d a u g h t e r cells into the O R N zone a n d their s u b s e q u e n t d i f f e r e n t i a t i o n into a m a t u r e O R N [2,13-15,19,26,42,43]. In the p r e s e n t study we use q u a n t i t a t i v e [3H]thymid i n e a u t o r a d i o g r a p h y to investigate the role of T H s in olfactory n e u r o g e n e s i s in suckling a n d post-suckling rats a n d to d e t e r m i n e the status a n d i n v o l v e m e n t of n e u r o g e n e s i s in O E ' s r e m a r k a b l e plasticity a n d comp e n s a t o r y growth [39]. T h e e x p e r i m e n t s were perf o r m e d in rats kept hypothyroid from birth to P10, P25 a n d P90 by a d m i n i s t r a t i o n of the reversible goitrogen P T U (propylthiouracil). T h e s e rats were c o m p a r e d with n o r m a l controls a n d those allowed to recover from hypothyroidism ('recovery' group) by withdrawal of P T U at P25 (weaning). T h e p a r a m e t e r s investigated were the densities of l a b e l e d cells in the BC a n d O R N zones of the O E at different days following injection of [3H]thymidine. T h e results d e m o n s t r a t e the role of T H s in olfactory n e u r o g e n e s i s as well as in m i g r a t i o n a n d survival ability of the newly f o r m e d O R N s in the d e v e l o p i n g n o r m a l , hypothyroid a n d r e h a b i l i t a t e d rats.

2. Materials and m e t h o d s 2.1. Animals

Pregnant albino rats of the Sprague-Dawley strain (Holtzman, WI) were kept singlyin plastic cages until birth. At birth, litter size was culled to 8 pups/litter and the litters were divided into two groups of normal and hypothyroid animals. After weaning (P25), males were separated and kept 4/cage and weighed twice weekly. At the same time, hypothyroid rats were divided into two separate groups, one remained hypothyroid and another was permitted to recover. The rearing and colony conditions were standard and as described before [39]. 2.2. Early hypothyroidism and recot,ery

Reversible chemical thyroidectomy was induced from birth by addition of 6-n-propyl-2-thiouracil, (PTU, Sigma) to the drinking water of the hypothyroid litters (0.1% w/v, i.e. 1 g/l), as described previously [28,36,39,47,48]. PTU passes through the mother's milk and causes depletion of THs in the pups and the mother by inhibiting synthesis and peripheral deiodination of the THs [18,49]. Hypothyroid groups received PTU until P10, P25 or P90. Recovery from hypothyroidism was induced by discontinuing PTU administration at weaning (P25). During the recovery period, plasma levels of THs return to normal and somatic growth resumes [28,36,39,47,48]. 2.3. [ 3H]Thymidine injections and histology

Normal and hypothyroid male rats were injected intraperitoneally with [3H]thymidine (specific activity 40-60 Ci/mmol, ICN Biochemicals) on P10, P25 and P75. Animals in the recovery group were injected on P75. All injections (5 mCi/g b.wt.) were given at 11:00 h. Animals in each group were sacrificed 1-, 5- or 15-days post-injection (PI). At the time of sacrifice, animals were anesthetized by ether overdose and perfused intracardially with isotonic saline followed by Bouin's fixative or 4% paraformaldehyde. The head region containing the OE was dissected, extraneous muscle and connective tissue removed and the tissue was placed in decalcifyingsolution overnight (Calex, VWR). After decalcification, the specimen were placed in 70% ethanol for storage. In all cases, the posterior third of the nasal septum, containing the region of OE, was removed and used for autoradiographic analysis. This region was defined as that portion of OE 1-2 mm anterior to the rostral edge of the OB, continuing caudally to the end of OE. This dissection was performed to insure that homologous regions of OE were selected in all animals. The tissues were dehydrated to 95% ethanol and then processed via the JB-4 Embedding System (Polysciences).After embedding, a series of 2 p.m thick frontal sections were cut on an ultramicrotome, consisting of 6-7 sections, each 500 /zm apart. We have used similar procedures in a previously published study [38]. 2.4. Autoradiography

All work with emulsion was done in complete darkness. Kodak NTB-2 emulsion was diluted 1 : 1 with distilled water and melted in a

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Fig. 1. Autoradiographs from septal olfactory epithelium (OE) of normal (N) and hypothyroid (T-) animals injected with [3H]thymidine on P25. The numbers + 1, + 5, + 15 in upper left corners indicate days post-injection. Hypothyroidism was induced by administration of propylthiouracil (PTU) from birth to day of sacrifice. 2/zm sections stained with tri-stain solution (Azure B, Methylene blue and Basic fusehin). Note that in both groups one day after injection label is found mainly in nuclei of BC layer while 15-days after injection most labeled nuclei are in ORN zone, indicating migration of young ORNs into the neuronal zone. Note also absence of marked difference between control and hypothyroid groups in these age and injection groups.

M.A. Paternostro, E. Meisami / Developmental Brain Research 83 (1994) 151-162

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Fig. 2. Quantification of autoradiographic data from samples obtained from normal and hypothyroid rats injected with [3Hlthymidine on P10 and P25 and analyzed on days 1, 5 and 15 post-injection. A: data of animals injected on PI0. B: data of animals injected on P25. Data are m e a n s +_ S.E.M. of 4 - 6 rats per age and experimental groups. Hypothyroidism was induced by administration of P T U from birth to day of sacrifice. Note gradual decrease in the density of labeled BC nuclei with days post-injections and the opposite trend for labeled nuclei in the O R N zone. Also note lack of any significant difference between m e a n s of control and hypothyroid animals in both age groups and that in 25-day injected rats (B) a lesser n u m b e r of nuclei seem to migrate to the O R N zone compared to 10-day injected rats (A).

water bath (37-45°C). Slides were dipped in the emulsion, allowed to dry to touch and placed in light tight boxes. All boxes were sealed with electrical tape, wrapped in aluminum foil and stored in the dark at 4°C for 2 - 3 weeks. Slides were developed for 2 min in D-19 (Kodak) developer, rinsed in distilled water, fixed in Rapid Fixer for 3 min, stained in JB-4 tri-stain solution (Azure B, Methylene blue and Basic fuschin) and coverslipped.

2.5. Quantitative analysis of autoradiographs O n each section, the total n u m b e r of labeled nuclei in both the BC and the O R N zones were counted over the entire length of the septal OE. Counts were made on one side of the nasal septum in each animal. Nuclei located within 10 tzm (approximately 2 cell layers) of the basal lamina were considered as those of the BCs. Thus our counts include the classic horizontal BCs and most (if not all) of the 'globose' BCs of Graziadei. Nuclei located between this layer and the layer of the supporting cell nuclei were considered as those of ORNs. Only nuclei showing five or more grains per nucleus were included in the counts. The total length of O E per section was estimated using camera lucida and SigmaScan Digitizing System (Jandel Scientific). For each animal and cell type, the density of labeled cells per m m was calculated and averaged for each age and experimental group.

2.6. Statistical analysis Statistical analysis was performed using the Student's t-test for unpaired sample m e a n s and the test of Analysis of Variance. Differences between the m e a n s showing a P value of less than 5% were considered significant.

3. Results

3.1. Body growth and thyroid status Compared to controls, the hypothyroid rats showed all the classic signs and developmental landmarks of hypothyroid somatic and behavioral retardation described by us and others, i.e. reduced body growth, delayed ear and eye-opening, infantile fur, facies and appearance [11,12,28,48,36-39,50]. Body growth pattern in the control, hypothyroid and recovery groups were similar to our previous studies [28,36,39,48]. At P10, P25 and P90, body weights in the hypothyroid rats were 19, 61 and 76% below controls, respectively (P <

0.01). In the recovery group, compensatory body growth began one week after PTU withdrawal, resulting in mean differences of 60, 28 and 20% in body weight (P < 0.01) at P50, P75 and P90, compared to controls. The rats of the recovery group, even though remained significantly below the weight of their age-matched controls, showed signs of markedly improved somatic and behavioral rehabilitation as previously described [28,49]. Although not measured in animals of this study, in a previous study using very similar experimental design, we have shown that hypothyroid animals had

Fig. 3. Autoradiographs from septal OE of normal (N) and hypothyroid ( T ) animals injected with [3H]thymidine on P75. The n u m b e r s + 1,+ 5 , + 15 in upper left corner indicate days post-injection. Hypothyroidism was induced by administration of P T U from birth to day of sacrifice. Other details same as in Fig. 1 legend. Note both control and hypothyroid groups show similar density of labeled BCs 1 day post,injection; by 5 and 15 days later, many labeled cells in the hypothyroid animal still remain in BC layer, while control rat BCs have migrated to O R N zone, indicating marked retardation of migration and possibly loss of young O R N s in hypothyroid rats.

M.A. Paternostro, E. Meisami / Developmental Brain Research 83 (1994) 151-162

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Fig. 5. Quantification of autoradiographic data from samples obtained from normal, hypothyroid and recovery rats injected with [3H]thymidine on P75 and analyzed 1, 5 and 15 days post-injection. Data are means _+ S.E.M. of 4-6 rats per age and experimental groups. Hypothyroidism was induced by administration of PTU from birth to day of sacrifice; recovery was induced by withdrawal of PTU on P25. A: basal cells (BCs) data. B: olfactory receptor neurons (ORNs) data. Note the gradual decrease in the density of labeled BCs with days post-injection and the opposite trend for labeled nuclei in O R N zone for all experimental groups (same as P10 and P25 data shown in Fig. 2).

completely depressed levels of plasma thyroxine from P10 to P90 and these levels returned to normal within 3 weeks of recovery onset [39,47,48].

3.2. Autoradiographic results from OE of normal control rats In normal control animals of various ages, essentially all of labeled nuclei at 1 day post-injection (PI) were in the BC zone (Figs. 1, 3 and 4). During the 15 days PI, density of labeled nuclei declined in the BC zone while increasing in the ORN zone (Figs. 1-5), indicating migration of labeled cells from the BC layer into the ORN zone. Comparison of normal rats injected at different ages revealed that the rate of olfactory neurogenesis as indicated by density of labeled BCs 1-day PI, declined significantly with age, decreasing from a high value of 32 BCs/mm at P10 to 75% and 40% of that value on P25 and P75 respectively (compare Figs. 2 and 5). Furthermore comparison of density of labeled ORNs at 15 days PI between normal animals of different ages revealed a marked decline in this value from a high of 25 O R N / m m at P10 to nearly 56% and 35% of that value at P25 and P75, respectively (compare Figs. 2 and 5).

3.3. Effect of hypothyroidism during the suckling period Early thyroid deficiency did not alter the rate of BC mitosis or the migration and accretion of new ORNs, compared to controls. This was true for both the P10 and P25 injected rats (Figs. 1 and 2).

3.3.1. Basal Cells The average density of labeled BC nuclei in the control group injected at P10 was 31.7 nuclei/mm one day PI. This value decreased by 55% and 91% at 5 and 15 days PI respectively (Fig. 2A). In the P25 injection group, the average density of labeled BC nuclei was 24.5 nuclei/mm one day PI and fell by 35% and 92% during the 5 and 15 days PI, respectively (Fig. 2B). Hypothyroid animals showed no significant differences compared to controls in these values at both P10 and P25 (Fig. 2A,B).

3.3.2. Olfactory receptor neurons The density of labeled ORN nuclei in control animals injected on P10 was 4 nuclei/mm at 1 day PI. This value increased 6.5-fold by 5 days PI and did not change significantly thereafter (Fig. 2A). In the P25 injection group, the average density of labeled ORN

Fig. 4. Autoradiographs from septal O E of normal (N) and recovery (R) animals injected with [3H]thymidine on P75. The numbers + 1, + 5, + 15 in upper left corner indicate days post-injection. Note greater number of labeled BCs in recovery rats at 1 day post-injection. Hypothyroidism was induced by administration of PTU from birth until day of sacrifice; recovery was induced by withdrawal of PTU on P25. Other details as in Fig. 1 legend.

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M.A. Paternostro, E. Meisami / Derelopmental Brain Research 83 (1994) 151-162

nuclei was also low (less than 2 nuclei/mm) at 1 day PI and increased 3.5- and 7-fold during the 5 and 15 days PI, respectively (Fig. 2A). No significant difference was found between the control and hypothyroid groups at both P10 and P25 in regard to density of ORN labeled nuclei (Fig. 2A,B).

tion by withdrawal of PTU at P25, showed markedly increased rates of olfactory ncurogenesis (Fig. 4). The density of labeled BCs were significantly increased, leading to markedly increased density of newly formed ORNs during the two weeks after injection (Figs. 5A and B).

3.4. Effects of extended hypothyroidism

3.5.1. Basal cells In the recovery group, like the control group, average density of labeled BCs steadily decreased during the two weeks PI (Fig. 5A). The density of labeled BCs at 1 and 5 days PI was significantly higher in the recovery group (48% and 43%, P < 0.01) compared to controls. By 15 days PI, this density fell to control levels.

As shown in autoradiographs of Figs. 3 and 4 and of the quantified data of Fig. 5A,B, unlike hypothyroidism during the suckling period, prolonged thyroid deficiency resulted in severe deficits in olfactory neuronal migration a n d / o r survival while mitotic rates of BCs remained unaltered.

3.4.1. Basal cells The appearance of BCs in the autoradiographs from rats injected on P75 and analyzed 1, 5 and 15 days PI is compared for the normal and hypothyroid rats in Fig. 3 and for the normal and recovery rats in Fig. 4. The corresponding quantitative data for these animals and ages are shown in Fig. 5A (BCs) and Fig. 5B (ORNs). In the control P75 rats average density of labeled BCs one day PI was 13.7 nuclei/ram (Fig. 5A) with no significant difference compared to the hypothyroid group. Similar to younger control animals, the density of labeled BCs declined steadily during the PI period, falling by 43% and 93% by 5 and 15 days PI, respectively (Fig. 5A). In contrast, hypothyroid animals showed no significant change in the density of labeled BCs during the 5 days PI (Figs. 3 and 5A), suggesting a marked reduction in migration of young ORNs. By 15 days PI, however, the density of labeled BCs had sharply declined in the hypothyroid group, nearing corresponding values in the control rats (Figs. 3 and 5A).

3. 5. 2. Olfactory receptor neurons At 1 day PI, a low average of 1.5 nuclei/mm were labeled in the recovery group (Figs. 4 and 5B). By days 5 and 15 PI this density value increased by about 3 and 8-fold respectively. The density values in the recovery rats, however, were not significantly higher than the age-matched control values (Fig. 5B). 3.6. Survival of developing ORNs during migration and maturation Comparing the density of labeled BCs 1-day PI with those in the ORN zone 15-day later revealed a reduction of 40-60% for animals of different age groups, indicating that not all of the BCs migrate into the ORN-zone. Dividing the density of labeled ORNs at 15 100" i.~ ::3

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3.4.2. Olfactory neurons The rate of formation of new ORNs in the P75 control animals was similar to that observed for the younger groups (Fig. 5B). At 1 day PI the number of labeled nuclei in the ORN zone was low (about 1 nuclei/mm), rising steadily during the post-injection period to reach values of about 5- and 9-fold higher on days 5 and 15 PI, respectively (Fig. 5B). In the hypothyroid group, the accretion of labeled nuclei in the ORN zone occurred at significantly slower rates compared to controls, as indicated by 56% and 38% (P<0.01) reduction in the density of labeled ORN nuclei by 5 and 15 days PI (Figs. 3 and 5B). 3.5. Recovery from early hypothyroidism Young adult rats (P75), kept hypothyroid during the suckling period, and allowed to recover thyroid func-

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Fig. 6. Ability of labeled BC cells to survive during the two weeks after injection of labeled thymidine in neonatal (P10-P25), weanling (P25-P40) and young adult rats (P75-P90). To obtain these percentages, the average number of labeled cells in the BC and O R N layers at day 15 post-injection were divided by the corresponding value at day 1 post-injection. Note that the relative ability of labeled cells to survive during migration and maturation declines with development in all groups and that the survival of the prospective ORNs is significantly diminished in the young adult hypothyroid rat compared to normal and rehabilitated animals, although the younger hypothyroid rats seem to be spared.

M.A. Paternostro, E. Meisami / Developmental Brain Research 83 (1994) 151-162

days PI by that of labeled BCs at 1 day PI yields the percent of labeled ORNs forming and surviving during the two weeks PI. As shown in Fig. 6, this percentage decreases with age in all groups, the decrease being moderate (25% between P10 and P95) in controls compared to twice as much in hypothyroid animals. Recovery from hypothyroidism enhanced the survival of developing ORNs, since the percentage of forming and surviving ORNs in the P90 recovery rats was 56% compared to 42% in the age-matched hypothyroid rats (Fig. 6).

4. Discussion Unique to the olfactory system is that neurogenesis occurs continuously in the O E of both developing and adult animals. In the developing animal this process serves to form the population of ORNs required for adult olfactory function. In the adult animal neurogenesis serves to maintain a constant population of olfactory sensory neuron as they die due to normal cycle of degeneration and regeneration or exposure to toxic enviornmental agents. The turn-over cycle of ORNs is estimated to be about a month in the rat and the duration of ORN development, from the time of division of BC to the time of formation of fully mature ORN with connection to the OB is estimated to be 7 days [2,13-15,19,26,42,43]. Our results in the normal young adult rats confirm this general pattern. One day after [3H]thymidine injection the label was found predominantly in the nuclei of BCs. Five days later labeled cells were found in both the BC and O R N zones of OE, indicating that some of the BCs have migrated apically into the O R N zone while others remained in the BC zone. Two weeks after labeled thymidine injection, BC zone was largely devoid of labeled cells, the latter found mainly in the ORN zone. The labeled cells in the younger neonates and weanling normal rats exhibited similar trends except for a higher number of labeled BCs, compared to the young adult, indicating a relative decline in the rate of neurogenesis in the O E with age. These observations are consistent with the current notion that division of progenitor cells in the BC layer gives rise to cells which subsequently migrate apically into the O R N zone, forming new ORNs [2,15,19,42]. We also noted that only a portion of the labeled BCs find their way into the O R N zone and that the relative amount of this portion declines with the animal's growth and development. Presumably some of the labeled BCs disperse within the BC layer to increase the surface area of O E during growth while others do not survive the process of migration, differentiation and maturation. While several experimental manipulations have been shown to alter the rate of olfactory neurogenesis

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[2,9,14,16,19,21,43] little is known about possible hormonal control of this process in the adult and during development. As THs profoundly affect the differentiation of central neural tissue [11,12,34,50], they may be a factor in regulation of olfactory neurogenesis. Adult hypothyroid mice show reduced survival of newly formed ORNs [25]. Studies by Burd and associates have shown that in the frog tadpoles, deficiency in THs decreases the number of developing olfactory axons while excess thyroxine increases this number [5,6]. Our recent quantitative and morphometric studies have shown that in growing hypothyroid rats, the normal postnatal increase in surface area and cell number in the O E is reduced by nearly one half and that this deficiency is essentially completely restored by compensatory growth after restoration of THs status [36,39]. Autoradiographic results from the P10 and P25 hypothyroid animals observed during days 1, 5 and 15 following [3H]thymidine injection indicate that the absence of THs during the suckling period of development does not alter the density of BCs, i.e. number of BCs undergoing division per unit length of OE; nor does hypothyroidism in the young rat alter the density of ORNs formed from the division of the labeled BCs. However, since the surface area of O E and total number of BCs and ORNs are reduced by 40-45% in the P25 rat kept hypothyroid from birth [36,39], it follows that hypothyroidism reduces the total number of BCs dividing, without altering their density. Similarly, the daughter cells thus formed in the hypothyroid animals can migrate apically and form new ORNs at a rate similar to that seen in normal control rat, but the total number of the ORNs in the hypothyroid animal is also reduced due to reduction in O E surface area. How can OE surface area and total cell number decrease without a change in density of dividing BCs or newly formed ORNs? We have suggested previously [30,36,39] that postnatal O E growth may be considered as a combination of two developmental processes: one, a vertical, differentiative proliferation of BCs, leading to formation of progenitor BCs or new ORNs; this process leads to increased thickness of OE; a second process consisting of horizontal proliferation of BCs and ORN progenitors leads to expansion of O E surface area [36,39]. The mechanisms regulating these two growth phenomena may involve different types of BCs. In the suckling animal, both the horizontal and vertical growth processes occur actively while in the adult animal where O E expansion has ceased, the vertical proliferation of BCs and O R N progenitor cells continues as a consequence of turn-over of ORNs. In support of these differential mechanisms, recent immunocytochemical evidence suggests that the BCs of O E are of two types: a keratin-positive type, akin to the classic BC, which may be responsible for horizontal and expansive growth, and a second keratin-negative type of

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BC which may act as the stem cell and progenitor of ORNs [24,33,45,46]. The relationship between these two types of BCs is poorly understood. One possible explanation for our results in the developing animals may be that neonatal hypothyroidism selectively interferes with the ability of the horizontal keratin-positive BCs to divide, leading to retarded growth of OE surface area while allowing the division of keratin-negative stem cells to continue. This explanation agrees with our previous findings that in the P25 hypothyroid rat OE thickness was unchanged while OE area was markedly reduced [36,39]. The reason(s) for this selective vulnerability of the horizontal BCs to hypothyroidism is not known but may involve differential concentration of receptors for THs or growth factors dependent on THs. The reduced ability of OE to expand in the hypothyroid rat may in part be secondary to the markedly reduced growth of the submucosal connective tissue, which requires THs for its growth and maturation [4,22]. The results from older hypothyroid animals indicate more pronounced and direct effects of THs on the development of OE cells. As in the younger rats, hypothyroidism had no noticeable effect on the ability of BCs to incorporate [3H]thymidine into DNA. However, prolonged deficiecny in THs was detrimental to migration and survival of newly formed ORNs, as both processes were severely retarded in the older (P75) hypothyroid groups. These results are similar to those found by Mackay-Sim and Beard [25] who observed that hypothyroidism in adult mice did not affect the incorporation of [3H]thymidine into dividing BCs, but led to reduced survival of developing or newly formed ORNs. Thus, in the absence of THs newly formed ORNs may fail to mature fully and consequently degenerate. During the course of their differentiation and maturation, ORNs undergo a specific pattern of development including dendritic growth, knob formation and ciliogenesis, axonal growth and innervation of OB followed by synaptogenesis with central target neurons [13,15,43]. Thyroid hormones, known to be critical for normal differentiation of central nervous tissue [ 11,12,34,41,50], may also be critical for maturation of ORNs. We have shown that maturation of ORNs as indicated by the number of ORNs containing Olfactory Marker Protein or intact knobs is markedly diminished in the developing hypothyroid rats [37,38]. Thus it is possible that those ORNs which do not mature properly in the absence of thyroid hormones fail to survive and consequently degenerate and die. Another possibility for the marked retardation of neurogenesis in the hypothyroid rats might be associated with developmental deficits in the olfactory bulb. Several studies indicate that the olfactory bulb, as the target for ORNs, is critical for maturation and survival

of newly formed ORNs [8,32,43]. This suggests that reduced survival of ORNs in the hypothyroid rats may be secondary to hypothyroid-induced retardation in development of olfactory bulb neurons or glia. Indeed, deficiency in THs results in a variety of bulbar deficits including reduced cell number, volume, weight, DNA and RNA content and enzyme activity [27,44]. We have recently reported that restoration of THs in weanling rats, kept hypothyroid during the suckling period, initiates a compensatory growth in the OE such that by P90, complete recovery of OE surface area and total ORN number is observed [39]. These results suggest that olfactory neurogenesis was accelerated to overcome the hypothyroid deficits. Our present results support this mechanism as the OE of rats in the recovery group showed significant increases in the density of proliferating BCs and of newly formed ORNs. Compensatory changes in the rate of BC mitosis have been observed by other investigators under differing experimental conditions. Unilateral bulbectomy leads to a two-fold increase in olfactory neurogenesis on the operated side [14,43], while chemical destruction of ORNs by ethyl bromide results in an accelerated rate of neurogenesis in OE [21]. In these types of studies, the OE is subjected to severe neuronal degeneration prior to the recovery period. Chemical signals originating from the degenerating neurons may initiate and maintain the accelerated rate of BC proliferation. The recovery animals in our study, however, possess at the onset of the recovery process, a structurally normal, albeit smaller and hypoplastic OE, with n o marked evidence of neuronal degeneration or death. Thus, the accelerated proliferation of BCs and OE growth observed in the recovery rats might be the result of a different compensatory growth phenomena involving separate molecular signals. One such a mechanism underlying the increased neurogenesis may be changes in levels of various growth factors. Receptors for THs, NGF and NT-3 have been localized in the olfactory tissue [10,40,48] and THs have been found to act synergistically with such growth factors as NGF and NT-3 in other regions of CNS [17,20,52]. Restoration of THs may enhance these synergistic actions through up-regulation of for THs or receptors for NGF and NT-3. In conclusion, THs appear to be critical in postnatal growth and development of OE, including proliferation of BCs and migration and survival of developing ORNs within the rat OE, although the relative effects on BC proliferation and the development of the young ORN are age-dependent. Thus, in the suckling rat, proliferative growth processes underlying OE expansion is dependent on THs while in the young adult animal, ORN survival is more critically dependent on these hormones. The results also indicate that compensatory growth of OE in the post-hypothyroid rehabilitated

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animals occurs as a result of increased proliferation rate of BCs and survival of developing ORNs.

Acknowledgments This work was supported by grants from the Research Board of the University of Illinois, NIH-SITG Funds and a NIH predoctoral fellowship to Mark Paternostro. The authors are grateful to Prof. Matilde Holzwarth for valuable technical advice. A preliminary report of this study was presented to the 1991 meeting of the Society for Neuroscience (Paternostro, M.A. and Meisami, E., Role of thyroid hormones on olfactory neurogenesis in the postnatal rat: a quantitative

[3H]thymidine study, Soc. Neurosci. Abstr., 17 (1991) 1320).

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