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Olfactory experience modulates apoptosis in the developing olfactory bulb
BRAIN RESEARCH Brain Research 674 (1995) 245-251
ELSEVIER
Research report
Olfactory experience modulates apoptosis in the developing olfactory bulb Joseph Najbauer, Michael Leon
*
Department of Psychobiology, University of California at Irvine, Irvine, CA 92717-4550, USA
Accepted 6 December 1994
Abstract Early sensory stimulation plays a key role in shaping the structure and function of the developing olfactory system. Here, we provide the first direct evidence for apoptotic cell death in the olfactory bulbs of rat pups during normal development and we also demonstrate that olfactory deprivation by unilateral naris occlusion causes a dramatic increase in apoptotic cell death in the glomerular and granule cell layers of the deprived bulb. The accessory olfactory bulbs displayed a remarkably high basal level of apoptosis but the occluded accessory bulb did not differ in that regard from the control accessory bulb. These results suggest that apoptosis may be an important mechanism by which the olfactory system can adjust its cell numbers in response to sensory stimuli experienced in early life, thereby underlying one form of plasticity in the developing olfactory system. Keywords: Apoptosis; Cell death; Development; Olfactory bulb; Olfaction; Plasticity; Sensory deprivation
1. Introduction Physiological cell d e a t h is o n e o f t h e f u n d a m e n t a l p r o c e s s e s by w h i c h t h e d e v e l o p i n g n e r v o u s system sculpts itself into a f u n c t i o n a l n e t w o r k o f i n t e r a c t i n g cells [24,25,37]. P e r i n a t a l o l f a c t o r y b u l b s d i s p l a y a rem a r k a b l e level o f g r o w t h a n d m a t u r a t i o n [7,32] w h i c h is c h a r a c t e r i z e d by a massive influx o f cells t h a t b e c o m e i n c o r p o r a t e d into t h e b u l b circuitry [2,20]. T h e normal development of the bulbs requires sensory s t i m u l a t i o n , as o l f a c t o r y d e p r i v a t i o n i n d u c e s d r a m a t i c a n a t o m i c a l a n d n e u r o c h e m i c a l c h a n g e s in t h e b u l b s [3,6,11-13,18,19,28,31,34,49]. W h e n o l f a c t o r y cues a r e r e s t r i c t e d by s e a l i n g t h e e x t e r n a l naris on p o s t n a t a l d a y 1 ( P N D 1), t h e b u l b on t h e o c c l u d e d side is ~ 25% s m a l l e r t h a n t h e c o n t r o l b u l b o n t h e n o n - o c c l u d e d side by P N D 30 [6,8,12,31,34]. S t u d i e s e m p l o y i n g [3H]t h y m i d i n e l a b e l i n g o f t h e cells b o r n a f t e r naris occlusion i n f e r r e d f r o m t h e d e c r e a s e d n u m b e r o f l a b e l e d cells t h a t cell d e a t h m a y c o n t r i b u t e to t h e r e d u c e d size o f t h e d e p r i v e d b u l b [13]. D i r e c t o b s e r v a t i o n o f dying cells, however, was n o t p o s s i b l e with such a n analysis.
* Corresponding author. Fax: (1) (714) 824-2447. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01448-5
A p r i m a r y c a n d i d a t e m e c h a n i s m for cell d e a t h in t h e olfactory system is a p o p t o s i s , which is c h a r a c t e r ized by s h r i n k a g e o f t h e cell, c o n d e n s a t i o n of chromatin, generation of internucleosomal DNA fragments a n d ultimately, p h a g o c y t o s i s by n e i g h b o r i n g cells, witho u t t h e a c c o m p a n y i n g i n f l a m m a t i o n s e e n with n e c r o t i c cell d e a t h [27,39,40,45,47,48]. W e first d e t e r m i n e d w h e t h e r a p o p t o s i s occurs d u r i n g n o r m a l olfactory b u l b development and then we determined whether the i n c i d e n c e o f a p o p t o t i c cell d e a t h in the bulbs c o u l d b e i n c r e a s e d by early o l f a c t o r y restriction.
2. Materials and methods 2.1. Subjects and preparation of tissue
Male Wistar rats born in our colony and raised in litters of eight pups were anesthetized by hypothermia on PND 2 (day of birth is defined as PND 0). Four pups from each litter had either the left or the right naris occluded by cauterization [31]. Xylocaine then was applied topically to the cauterized region and the pups were allowed to recover on a heating pad before returning them to the dams. Littermates with non-occluded nares were used as controls, At PND 15, the brains of were removed, frozen in isopentane at -40°C and stored at -70°C until use. 20-p,m coronal sections from the bulbs were prepared, thaw-mounted onto Vectabond-coated slides (Vector Labs) and fixed in 4% paraformaldehyde in phosphate-buffered
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saline (PBS) for 10 min at room temperature. The fixed sections were air-dried and stored at - 2 0 ° C in the presence of desiccant.
2.2. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling) assay T U N E L assay was performed essentially as described by Gavrieli et al. [15] with some modifications. Sections were dehydrated in ethanol (50, 70, 95 and 100% for 1 min each) and defatted in chloroform (5 min), followed by ethanol (100, 95% for 1 min each). The sections were allowed to dry at room temperature and incubated in 2% hydrogen peroxide, rinsed in distilled water and then overlaid with 300 pA/slide of Tris-HC1, pH 7.2, containing 140 m M sodium cacodylate, 1 m M cobalt chloride, 0.06 U / p , l terminal deoxynucleotidyl transferase (United States Biochemical), 4 ~tM biotinylated d U T P ( B o e h r i n g e r / M a n n h e i m ) and 0.2 m g / m l bovine serum albumin (BSA). Reactions without terminal transferase were included as controls. After incubating the slides for 3 h at 37°C in a humidified atmosphere, the reactions were terminated by immersing the slides into a solution containing 300 m M sodium chloride and 30 m M sodium citrate, p H 8.1, and were rinsed in deionized water. The slides were incubated with 2% BSA for 10 min, rinsed and overlaid with 600 # l / s l i d e of Vectastain AB Elite (Vector Labs) prepared in
PBS containing 1.6 m g / m l BSA according to the manufacturer's instructions. Reactions were developed by diaminobenzidine (0.4 m g / m l ) and hydrogen peroxide (0.003%) in PBS for 12 rain at room temperature. The sections were rinsed in PBS, dehydrated in ethanol and Histoclear and then covered with Permount.
2.3. Counting of apoptotic cells Apoptotic cells were counted in the glomerular layer, the superficial and deep granule layers as well as in the subependymal zone of the main bulb. 10 coronal sections were evaluated per bulb. The sections had been sampled at equal intervals from the region beginning at ~ 7 0 0 / z m through the anterior/posterior extent of the bulb and the beginning of the accessory bulb. The counts of labeled cells were averaged to derive the mean value for each layer for an individual animal. These averages were then used to derive the m e a n value for the group of animals (occluded and non-occluded, Fig. 3). Cells were also counted in 10 sections sampled between the beginning and the end of granule cell layer of accessory bulb and mean values were obtained similarly as for the main bulb. The counting was carried out on coded sections in a Nikon Optiphot-2 microscope at 125 x magnification.
Fig. 1. Apoptosis in glomerular (A), superficial granule (B) and deep granule cell layers (C) and subependymal zone (D) of main bulb and in granule cell layer of accessory bulb (E) detected by T U N E L assay [15]. Examples of a single apoptotic cell in superficial granule cell layer of main bulb (F) and of a group of apoptotic cells in granule cell layer of accessory bulb (G). Control T U N E L reaction lacking terminal transferase, deep granule cell layer of main bulb (H). All examples are from occluded bulbs, except (B) and (F) which are from a bulb contralateral to naris closure. Bar, 50 p.m in A - E and H; 1 0 / x m in F and G.
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
[15]. Most apoptotic cells were seen as single cells (Fig. 1F) but there were also occasional doublets, triplets and larger-size groups (Fig. 1G). The superficial granule cells located in the mitral cell layer displayed the highest density of apoptotic ceils induced by olfactory deprivation where a 297% increase relative to the contralateral bulb was detected (Fig. 2A). In the deep granule cell and glomerular layers, there was a 144 and 112% increase in apoptosis, respectively. In the subependymal zone, there was a smaller but significant induction of apoptosis with a 62% increase. The basal densities of apoptotic cells were similar in all layers of the non-occluded main bulb (Fig. 2A), however, the granule cell layer of the accessory olfactory bulb displayed > 4 × the level of basal apoptosis
2.4. Area measurements and density of apoptotic cells T h e areas of bulb layers of coded sections were m e a s u r e d using the microscope equipped with a video camera module (Sony, XC-77) using N I H Image 1.45 software. T h e areal density of apoptotic cells for each layer was derived by dividing the n u m b e r of apoptotic cells by the area of the corresponding layer. T h e density was expressed as n u m b e r of apoptotic c e l l s / m m 2. Five sections (approximately equally spaced in the r o s t r a l / c a u d a l direction) were analysed which were then averaged to obtain the m e a n density of apoptotic cells for each bulb. These m e a n s were then averaged to obtain the m e a n value for a given group of animals (Fig. 2).
3. Results
Fig. 1A-E shows examples of apoptotic cells in the different layers of the bulb revealed by this method
Unilateral
A
247
naris • []
occlusion
Occluded bulb Non-occluded bulb
30-
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o
No
B
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occlusion
40Left bulb Right bulb
30m
20-
10-
,
i
Glomerular layer cells
I
Superficial granule cells
I
Deep granule cells
i
,
, y / / / /
I
Subependymal zone cells
Accessory olfactory bulb granule cells
Fig. 2. Effect of unilateral naris occlusion on density of apoptotic cells in different layers of olfactory bulb. Apoptotic cells were labeled by T U N E L assay [15] in olfactory bulbs of rat pups with naris occlusion (A) and control animals with no occlusion (B). Data shown are m e a n + SEM values for naris-occluded animals (n = 6, one pup from each of six litters) and control animals (n = 6, littermat¢s of six occluded pups) ( * P < 0.0256; * * P < 0.0008; * * * P < 0.0002; . . . . P < 0.0001, Student's t test).
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
248
Table 1 Area measurements of olfactory bulb layers in animals with naris occlusion a
Occluded bulb Non-occluded bulb
Glomerular
Superficial granule
Deep granule
Subependymal zone
AOB granule layer
0.943 ___0.062 b, * ( P < 0.0045) c 1.100 ± 0.070
0.292 + 0.023 * ( P < 0.0031) 0.323 ± 0.027
1.762 ± 0.163 ( P < 0.2056) 1.852 ± 0.149
0.327 + 0.046 (P < 0.9957) 0.327 ± 0.091
0.264 + 0.045 ( P < 0.6191) 0.254 ± 0.037
Areas are expressed in mm 2. b Mean + SD. c Student's t test (paired, two-tailed), df = 5 for each layer. * Significantly different from non-occluded bulb. a
detected in the main bulb (Fig. 2A). Furthermore, t h e r e w a s n o d i f f e r e n c e in t h e d e n s i t i e s o f a p o p t o t i c cells between the accessory bulbs ipsilateral and cont r a l a t e r a l t o t h e n a r i s c l o s u r e (Fig. 2 A ) . A s e x p e c t e d , there was no difference in the density of apoptotic cells
b e t w e e n t h e l e f t a n d t h e r i g h t b u l b s in t h e n o n - o c c l u d e d g r o u p ( F i g . 2B). The areas of the glomerular and superficial granule cell l a y e r s w e r e s m a l l e r o n t h e o c c l u d e d s i d e w h e n compared with the corresponding layers on the non-oc-
Unilateral
30-
naris occlusion
A
[] []
Occluded bulb Non-occluded bulb
20-
r.
._o
10-
4) m
(J ¢J ,B
0-
o
3.
O Q. (u
No naris occlusion
30 J~ E -.z Z
B
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Left bulb Right bulb
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, J ~ ' , S , S ~ t , " / ~" / '
10.
liiiiiiilN Glomerular layer cells
Superficial granule cells
iiii:iii I
Deep granule cells
!
Subependymal Accessory zone cells olfactory bulb granule cells
Fig. 3. Effect of unilateral naris occlusion on apoptosis in different layers of olfactory bulb. Apoptotic cells were labeled by TUNEL assay [15] in olfactory bulbs of rat pups with naris occlusion (A) and control animals with no occlusion (B). Data shown are mean ± SEM values for naris-occluded animals (n = 11, one pup from each of 11 litters) and control animals (n = 12) (* P < 0.0005; * * P < 0.0001, Student's t test).
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
cluded side (Table 1). No significant differences were found in the size of the granule cell layers, the subependymal zones or the accessory bulbs of the occluded group. In addition, no differences were found in the size of any layers between the left and the right bulbs in the non-occluded group. Since a decrease in size of bulb lamina can result in higher density of apoptotic cells without an increase in apoptosis, we also determined the total numbers of apoptotic cells in different layers of the occluded and non-occluded olfactory bulbs. Cell counts of labeled cells revealed that naris closure increased apoptosis in the glomerular layer of the main olfactory bulb by 52% over the basal level of apoptosis in the non-occluded bulb (Fig. 3A). Among the superficial granule cells, there was a 204% increase in the number of apoptotic cells in the deprived bulb. In the deep granule cell layer, there was a 100% increase in apoptosis in the deprived bulb whereas the absolute numbers of affected cells in both the subependymal zone of the main bulb and the granule cell layer of the accessory bulb were unchanged by naris occlusion (Fig. 3A). Apoptosis in the subependymal zone, when expressed as number of apoptotic cells/section, had greater variance (relative SD = 47% in the occluded bulb and 46% in the non-occluded bulb) then when it was expressed as number of apoptotic cells/unit area (relative SD = 28% for the occluded and 37% for the non-occluded bulb). Variance is likely to be introduced by differences in the sizes of the bulbs among animals as well as by differences in the sizes of sections sampled from the frontal vs. the caudal part of the bulb. This may explain the statistically significant difference in the density of apoptotic cells in the subependymal zone (Fig. 2A) and the lack of significant difference for the absolute numbers of apoptotic cells in this zone (Fig. 3A). The non-occluded bulbs displayed a significant basal level of apoptosis in all layers of the bulb (Fig. 3B) and there was no difference in apoptosis between the left and right bulbs in the non-occluded group.
4. Discussion
These results indicate that apoptosis is part of normal developmental processes of the olfactory system and that early olfactory experience has a profound effect on the number of cells that undergo apoptosis in that system. Synchronized dying of cells in a group (Fig. 1G) may suggest an interdependence of these cells. Such an interdependence may arise from local generation of survival signals by the group itself, from signals generated by neighboring ceils or cells that send processes onto the group. While the granule cells appeared to be the affected
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cell population in the superficial granule layer, we cannot exclude the possibility that a small number of mitral cells also died because apoptosis is accompanied by changes in cell size and morphology. Therefore, the effect of olfactory deprivation on the survival of mitral cells remains unresolved [4,33,35]. Because mitral ceils have high expression of the bcl-2 gene [9,36] that suppresses apoptosis in other neural systems [1,14,21, 26,51], the susceptibility of mitral cells to apoptotic death might be lower than in other cells of the bulb. The finding that the subependymal zone in occluded bulbs had significant increase in apoptosis relative to the non-occluded bulbs suggests that the survival of less mature cells of this layer also can be affected by olfactory stimuli. The high basal level of apoptosis in the accessory olfactory bulb could be due to a course of developmental maturation that is different from that in the main bulb [38]. The accessory bulb receives its input from the vomeronasal organ, an area distinct from the olfactory epithelium [5] which may explain the differential effect of olfactory deprivation on the apoptosis in the main vs. the accessory bulbs. Furthermore, the developing vomeronasal organ in rats has partial access to the oral cavity [44] and, therefore, may be exposed to odorants in spite of naris occlusion. We found a significant reduction in the size of the glomerular and superficial granule layers (Table 1), while others also have reported a significant reduction in the size of the deep granule layer in occluded bulbs at PND 12 [6,11]. A possible explanation for this discrepancy is that we used PND 15 animals, an age very close to the age (PND 12) when the effect on granule cell layer laminar size just begins to be detectable. In addition, we performed the naris occlusion on PND 2 and not on PND 1 as in several other studies [6,11]. Increased cell death has been found in the olfactory epithelium upon bulbectomy [17,22,30], suggesting that the olfactory bulbs play a major role in determining the survival of olfactory receptor neurons. Cell loss and structural and functional changes have also been reported in the auditory [23,43], tactile [46] and visual [16,29] systems upon sensory deprivation brought about by deafferentation or stimulus blockade [37,41]. In systems other than the olfactory system, however, it remains to be seen whether apoptosis plays a role in these processes. In any case, these findings point toward the importance of sensory stimulation in normal postnatal neural development and emphasize the importance of proper e f f e r e n t / a f f e r e n t connections in ensuring and preserving proper physiological functioning of these systems. The total number of cells undergoing apoptosis in the bulb is likely to be much larger than what is detected at a given time. Since the clearance time for apoptotic cells (the time from which a cell dies to the
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J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
time of its phagocytosis) is rapid in other systems [10], we probably could detect only a fraction of the total number of cells that underwent apoptosis. If short clearance times were to apply in the mammalian olfactory system, apoptosis would be a dynamic process that could underlie, at least in part, the plasticity of the olfactory system. Repeated exposure of rat pups to a single odor paired with tactile stimulation increased the number of periglomerular cells in focal areas of the bulb that respond with an increase in metabolic activity as measured by 2-deoxy-D-glucose uptake in response to the trained odor [50]. Similarly, chronic exposure of rat pups to a mixture of odors results in increased numbers of cells in the superficial and deep granule cell layers [42]. These findings suggest that there might be a lower incidence of apoptosis in focal regions of the olfactory bulbs of animals raised with significant olfactory experiences. There is increasing evidence that in the developing nervous system, trophic factors and other survival signals may suppress an intrinsic suicide program of cells and, thus, may regulate cell numbers in response to sensory stimuli [39]. It is possible that apoptosis induced by olfactory deprivation is a manifestation of a suppression of local trophic factors in the bulb. Our results suggest that apoptosis may be a critical mechanism by which cells in the developing olfactory system are eliminated and that early olfactory stimulation has a profound effect on the number of cells deleted by this process.
[5] [6] [7] [8] [9]
[10] [11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements Part of this work was carried out while one of the authors (J. Najbauer) was on leave of absence from the Biophysical Research Group of the Hungarian Academy of Sciences, Medical University of P6cs, P6cs, Hungary. Supported by National Institute of Mental Health Grant MH48950 (to M. Leon).
[19]
[20]
[21]
[22]
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[39] Raft, M.C., Barres, B.A., Burne, J.F., Coles, H.S., Ishizaki, Y. and Jacobson, M.D., Programmed cell death and the control of cell survival: lessons from the nervous system, Science, 262 (1993) 695-700. [40] Raft, M.C., Cell death genes: Drosophila enters the field, Science, 264 (1994) 668-669. [41] Riesen, A.H. (Ed.), The Developmental Neuropsychology of Sensory Deprivation, Academic Press, New York, NY, 1975. [42] Rosselli-Austin, L. and Williams, J., Enriched neonatal odor exposure leads to increased numbers of olfactory bulb mitral and granule cells, Dev. Brain Res., 51 (1990) 135-137. [43] Rubel, E.W., Hyson, R.L. and Durham, D., Afferent regulation of neurons in the brain stem auditory system, J. Neurobiol., 21 (1990) 169-196. [44] Szab6, K. and Mendoza, A.S., Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. I. Light microscopy. Dev. Brain Res., 39 (1988) 253258. [45] Tomei, L.D. and Cope, F.O. (Eds.), Apoptosis: The Molecular Basis of Cell Death, Cold Spring Harbor University Press, New York, NY, 1991. [46] Van der Loos, H. and Woolsey, T.A., Somatosensory cortex: structural alterations following early injury to sense organs, Science, 179 (395-398) 1973. [47] Vaux, D.L., Toward an understanding of the molecular mechanisms of physiological cell death, Proc. Natl. Acad. Sci. USA, 90 (1993) 786-789. [48] Williams, G.T. and Smith, C.A., Molecular regulation of apoptosis: genetic controls on cell death, Cell, 74 (1993) 777-779. [49] Wilson, D.A. and Wood, J.G., Functional consequences of unilateral olfactory deprivation: time-course and age sensitivity, Neuroscience, 49 (1992) 183-192. [50] Woo, C.C. and Leon, M., Increase in a focal population of juxtaglomerular cells in the olfactory bulb associated with early learning, J. Comp. Neurol., 305 (1991) 49-56. [51] Zhong, L.-T., Sarafian, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H. and Bredesen, D.E., bcl-2 inhibits death of central neural cells induced by multiple agents, Proc. Natl. Acad. Sci. USA, 90 (1993) 4533-4537.
ELSEVIER
Research report
Olfactory experience modulates apoptosis in the developing olfactory bulb Joseph Najbauer, Michael Leon
*
Department of Psychobiology, University of California at Irvine, Irvine, CA 92717-4550, USA
Accepted 6 December 1994
Abstract Early sensory stimulation plays a key role in shaping the structure and function of the developing olfactory system. Here, we provide the first direct evidence for apoptotic cell death in the olfactory bulbs of rat pups during normal development and we also demonstrate that olfactory deprivation by unilateral naris occlusion causes a dramatic increase in apoptotic cell death in the glomerular and granule cell layers of the deprived bulb. The accessory olfactory bulbs displayed a remarkably high basal level of apoptosis but the occluded accessory bulb did not differ in that regard from the control accessory bulb. These results suggest that apoptosis may be an important mechanism by which the olfactory system can adjust its cell numbers in response to sensory stimuli experienced in early life, thereby underlying one form of plasticity in the developing olfactory system. Keywords: Apoptosis; Cell death; Development; Olfactory bulb; Olfaction; Plasticity; Sensory deprivation
1. Introduction Physiological cell d e a t h is o n e o f t h e f u n d a m e n t a l p r o c e s s e s by w h i c h t h e d e v e l o p i n g n e r v o u s system sculpts itself into a f u n c t i o n a l n e t w o r k o f i n t e r a c t i n g cells [24,25,37]. P e r i n a t a l o l f a c t o r y b u l b s d i s p l a y a rem a r k a b l e level o f g r o w t h a n d m a t u r a t i o n [7,32] w h i c h is c h a r a c t e r i z e d by a massive influx o f cells t h a t b e c o m e i n c o r p o r a t e d into t h e b u l b circuitry [2,20]. T h e normal development of the bulbs requires sensory s t i m u l a t i o n , as o l f a c t o r y d e p r i v a t i o n i n d u c e s d r a m a t i c a n a t o m i c a l a n d n e u r o c h e m i c a l c h a n g e s in t h e b u l b s [3,6,11-13,18,19,28,31,34,49]. W h e n o l f a c t o r y cues a r e r e s t r i c t e d by s e a l i n g t h e e x t e r n a l naris on p o s t n a t a l d a y 1 ( P N D 1), t h e b u l b on t h e o c c l u d e d side is ~ 25% s m a l l e r t h a n t h e c o n t r o l b u l b o n t h e n o n - o c c l u d e d side by P N D 30 [6,8,12,31,34]. S t u d i e s e m p l o y i n g [3H]t h y m i d i n e l a b e l i n g o f t h e cells b o r n a f t e r naris occlusion i n f e r r e d f r o m t h e d e c r e a s e d n u m b e r o f l a b e l e d cells t h a t cell d e a t h m a y c o n t r i b u t e to t h e r e d u c e d size o f t h e d e p r i v e d b u l b [13]. D i r e c t o b s e r v a t i o n o f dying cells, however, was n o t p o s s i b l e with such a n analysis.
* Corresponding author. Fax: (1) (714) 824-2447. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01448-5
A p r i m a r y c a n d i d a t e m e c h a n i s m for cell d e a t h in t h e olfactory system is a p o p t o s i s , which is c h a r a c t e r ized by s h r i n k a g e o f t h e cell, c o n d e n s a t i o n of chromatin, generation of internucleosomal DNA fragments a n d ultimately, p h a g o c y t o s i s by n e i g h b o r i n g cells, witho u t t h e a c c o m p a n y i n g i n f l a m m a t i o n s e e n with n e c r o t i c cell d e a t h [27,39,40,45,47,48]. W e first d e t e r m i n e d w h e t h e r a p o p t o s i s occurs d u r i n g n o r m a l olfactory b u l b development and then we determined whether the i n c i d e n c e o f a p o p t o t i c cell d e a t h in the bulbs c o u l d b e i n c r e a s e d by early o l f a c t o r y restriction.
2. Materials and methods 2.1. Subjects and preparation of tissue
Male Wistar rats born in our colony and raised in litters of eight pups were anesthetized by hypothermia on PND 2 (day of birth is defined as PND 0). Four pups from each litter had either the left or the right naris occluded by cauterization [31]. Xylocaine then was applied topically to the cauterized region and the pups were allowed to recover on a heating pad before returning them to the dams. Littermates with non-occluded nares were used as controls, At PND 15, the brains of were removed, frozen in isopentane at -40°C and stored at -70°C until use. 20-p,m coronal sections from the bulbs were prepared, thaw-mounted onto Vectabond-coated slides (Vector Labs) and fixed in 4% paraformaldehyde in phosphate-buffered
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saline (PBS) for 10 min at room temperature. The fixed sections were air-dried and stored at - 2 0 ° C in the presence of desiccant.
2.2. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling) assay T U N E L assay was performed essentially as described by Gavrieli et al. [15] with some modifications. Sections were dehydrated in ethanol (50, 70, 95 and 100% for 1 min each) and defatted in chloroform (5 min), followed by ethanol (100, 95% for 1 min each). The sections were allowed to dry at room temperature and incubated in 2% hydrogen peroxide, rinsed in distilled water and then overlaid with 300 pA/slide of Tris-HC1, pH 7.2, containing 140 m M sodium cacodylate, 1 m M cobalt chloride, 0.06 U / p , l terminal deoxynucleotidyl transferase (United States Biochemical), 4 ~tM biotinylated d U T P ( B o e h r i n g e r / M a n n h e i m ) and 0.2 m g / m l bovine serum albumin (BSA). Reactions without terminal transferase were included as controls. After incubating the slides for 3 h at 37°C in a humidified atmosphere, the reactions were terminated by immersing the slides into a solution containing 300 m M sodium chloride and 30 m M sodium citrate, p H 8.1, and were rinsed in deionized water. The slides were incubated with 2% BSA for 10 min, rinsed and overlaid with 600 # l / s l i d e of Vectastain AB Elite (Vector Labs) prepared in
PBS containing 1.6 m g / m l BSA according to the manufacturer's instructions. Reactions were developed by diaminobenzidine (0.4 m g / m l ) and hydrogen peroxide (0.003%) in PBS for 12 rain at room temperature. The sections were rinsed in PBS, dehydrated in ethanol and Histoclear and then covered with Permount.
2.3. Counting of apoptotic cells Apoptotic cells were counted in the glomerular layer, the superficial and deep granule layers as well as in the subependymal zone of the main bulb. 10 coronal sections were evaluated per bulb. The sections had been sampled at equal intervals from the region beginning at ~ 7 0 0 / z m through the anterior/posterior extent of the bulb and the beginning of the accessory bulb. The counts of labeled cells were averaged to derive the mean value for each layer for an individual animal. These averages were then used to derive the m e a n value for the group of animals (occluded and non-occluded, Fig. 3). Cells were also counted in 10 sections sampled between the beginning and the end of granule cell layer of accessory bulb and mean values were obtained similarly as for the main bulb. The counting was carried out on coded sections in a Nikon Optiphot-2 microscope at 125 x magnification.
Fig. 1. Apoptosis in glomerular (A), superficial granule (B) and deep granule cell layers (C) and subependymal zone (D) of main bulb and in granule cell layer of accessory bulb (E) detected by T U N E L assay [15]. Examples of a single apoptotic cell in superficial granule cell layer of main bulb (F) and of a group of apoptotic cells in granule cell layer of accessory bulb (G). Control T U N E L reaction lacking terminal transferase, deep granule cell layer of main bulb (H). All examples are from occluded bulbs, except (B) and (F) which are from a bulb contralateral to naris closure. Bar, 50 p.m in A - E and H; 1 0 / x m in F and G.
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
[15]. Most apoptotic cells were seen as single cells (Fig. 1F) but there were also occasional doublets, triplets and larger-size groups (Fig. 1G). The superficial granule cells located in the mitral cell layer displayed the highest density of apoptotic ceils induced by olfactory deprivation where a 297% increase relative to the contralateral bulb was detected (Fig. 2A). In the deep granule cell and glomerular layers, there was a 144 and 112% increase in apoptosis, respectively. In the subependymal zone, there was a smaller but significant induction of apoptosis with a 62% increase. The basal densities of apoptotic cells were similar in all layers of the non-occluded main bulb (Fig. 2A), however, the granule cell layer of the accessory olfactory bulb displayed > 4 × the level of basal apoptosis
2.4. Area measurements and density of apoptotic cells T h e areas of bulb layers of coded sections were m e a s u r e d using the microscope equipped with a video camera module (Sony, XC-77) using N I H Image 1.45 software. T h e areal density of apoptotic cells for each layer was derived by dividing the n u m b e r of apoptotic cells by the area of the corresponding layer. T h e density was expressed as n u m b e r of apoptotic c e l l s / m m 2. Five sections (approximately equally spaced in the r o s t r a l / c a u d a l direction) were analysed which were then averaged to obtain the m e a n density of apoptotic cells for each bulb. These m e a n s were then averaged to obtain the m e a n value for a given group of animals (Fig. 2).
3. Results
Fig. 1A-E shows examples of apoptotic cells in the different layers of the bulb revealed by this method
Unilateral
A
247
naris • []
occlusion
Occluded bulb Non-occluded bulb
30-
l
20. Ik ~k ~k
I E _=
10.
Q O
.o
0.
o D. ¢1l N,-
o
No
B
[] []
J~
E Z
naris
occlusion
40Left bulb Right bulb
30m
20-
10-
,
i
Glomerular layer cells
I
Superficial granule cells
I
Deep granule cells
i
,
, y / / / /
I
Subependymal zone cells
Accessory olfactory bulb granule cells
Fig. 2. Effect of unilateral naris occlusion on density of apoptotic cells in different layers of olfactory bulb. Apoptotic cells were labeled by T U N E L assay [15] in olfactory bulbs of rat pups with naris occlusion (A) and control animals with no occlusion (B). Data shown are m e a n + SEM values for naris-occluded animals (n = 6, one pup from each of six litters) and control animals (n = 6, littermat¢s of six occluded pups) ( * P < 0.0256; * * P < 0.0008; * * * P < 0.0002; . . . . P < 0.0001, Student's t test).
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
248
Table 1 Area measurements of olfactory bulb layers in animals with naris occlusion a
Occluded bulb Non-occluded bulb
Glomerular
Superficial granule
Deep granule
Subependymal zone
AOB granule layer
0.943 ___0.062 b, * ( P < 0.0045) c 1.100 ± 0.070
0.292 + 0.023 * ( P < 0.0031) 0.323 ± 0.027
1.762 ± 0.163 ( P < 0.2056) 1.852 ± 0.149
0.327 + 0.046 (P < 0.9957) 0.327 ± 0.091
0.264 + 0.045 ( P < 0.6191) 0.254 ± 0.037
Areas are expressed in mm 2. b Mean + SD. c Student's t test (paired, two-tailed), df = 5 for each layer. * Significantly different from non-occluded bulb. a
detected in the main bulb (Fig. 2A). Furthermore, t h e r e w a s n o d i f f e r e n c e in t h e d e n s i t i e s o f a p o p t o t i c cells between the accessory bulbs ipsilateral and cont r a l a t e r a l t o t h e n a r i s c l o s u r e (Fig. 2 A ) . A s e x p e c t e d , there was no difference in the density of apoptotic cells
b e t w e e n t h e l e f t a n d t h e r i g h t b u l b s in t h e n o n - o c c l u d e d g r o u p ( F i g . 2B). The areas of the glomerular and superficial granule cell l a y e r s w e r e s m a l l e r o n t h e o c c l u d e d s i d e w h e n compared with the corresponding layers on the non-oc-
Unilateral
30-
naris occlusion
A
[] []
Occluded bulb Non-occluded bulb
20-
r.
._o
10-
4) m
(J ¢J ,B
0-
o
3.
O Q. (u
No naris occlusion
30 J~ E -.z Z
B
[] []
Left bulb Right bulb
20-
, J ~ ' , S , S ~ t , " / ~" / '
10.
liiiiiiilN Glomerular layer cells
Superficial granule cells
iiii:iii I
Deep granule cells
!
Subependymal Accessory zone cells olfactory bulb granule cells
Fig. 3. Effect of unilateral naris occlusion on apoptosis in different layers of olfactory bulb. Apoptotic cells were labeled by TUNEL assay [15] in olfactory bulbs of rat pups with naris occlusion (A) and control animals with no occlusion (B). Data shown are mean ± SEM values for naris-occluded animals (n = 11, one pup from each of 11 litters) and control animals (n = 12) (* P < 0.0005; * * P < 0.0001, Student's t test).
J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
cluded side (Table 1). No significant differences were found in the size of the granule cell layers, the subependymal zones or the accessory bulbs of the occluded group. In addition, no differences were found in the size of any layers between the left and the right bulbs in the non-occluded group. Since a decrease in size of bulb lamina can result in higher density of apoptotic cells without an increase in apoptosis, we also determined the total numbers of apoptotic cells in different layers of the occluded and non-occluded olfactory bulbs. Cell counts of labeled cells revealed that naris closure increased apoptosis in the glomerular layer of the main olfactory bulb by 52% over the basal level of apoptosis in the non-occluded bulb (Fig. 3A). Among the superficial granule cells, there was a 204% increase in the number of apoptotic cells in the deprived bulb. In the deep granule cell layer, there was a 100% increase in apoptosis in the deprived bulb whereas the absolute numbers of affected cells in both the subependymal zone of the main bulb and the granule cell layer of the accessory bulb were unchanged by naris occlusion (Fig. 3A). Apoptosis in the subependymal zone, when expressed as number of apoptotic cells/section, had greater variance (relative SD = 47% in the occluded bulb and 46% in the non-occluded bulb) then when it was expressed as number of apoptotic cells/unit area (relative SD = 28% for the occluded and 37% for the non-occluded bulb). Variance is likely to be introduced by differences in the sizes of the bulbs among animals as well as by differences in the sizes of sections sampled from the frontal vs. the caudal part of the bulb. This may explain the statistically significant difference in the density of apoptotic cells in the subependymal zone (Fig. 2A) and the lack of significant difference for the absolute numbers of apoptotic cells in this zone (Fig. 3A). The non-occluded bulbs displayed a significant basal level of apoptosis in all layers of the bulb (Fig. 3B) and there was no difference in apoptosis between the left and right bulbs in the non-occluded group.
4. Discussion
These results indicate that apoptosis is part of normal developmental processes of the olfactory system and that early olfactory experience has a profound effect on the number of cells that undergo apoptosis in that system. Synchronized dying of cells in a group (Fig. 1G) may suggest an interdependence of these cells. Such an interdependence may arise from local generation of survival signals by the group itself, from signals generated by neighboring ceils or cells that send processes onto the group. While the granule cells appeared to be the affected
249
cell population in the superficial granule layer, we cannot exclude the possibility that a small number of mitral cells also died because apoptosis is accompanied by changes in cell size and morphology. Therefore, the effect of olfactory deprivation on the survival of mitral cells remains unresolved [4,33,35]. Because mitral ceils have high expression of the bcl-2 gene [9,36] that suppresses apoptosis in other neural systems [1,14,21, 26,51], the susceptibility of mitral cells to apoptotic death might be lower than in other cells of the bulb. The finding that the subependymal zone in occluded bulbs had significant increase in apoptosis relative to the non-occluded bulbs suggests that the survival of less mature cells of this layer also can be affected by olfactory stimuli. The high basal level of apoptosis in the accessory olfactory bulb could be due to a course of developmental maturation that is different from that in the main bulb [38]. The accessory bulb receives its input from the vomeronasal organ, an area distinct from the olfactory epithelium [5] which may explain the differential effect of olfactory deprivation on the apoptosis in the main vs. the accessory bulbs. Furthermore, the developing vomeronasal organ in rats has partial access to the oral cavity [44] and, therefore, may be exposed to odorants in spite of naris occlusion. We found a significant reduction in the size of the glomerular and superficial granule layers (Table 1), while others also have reported a significant reduction in the size of the deep granule layer in occluded bulbs at PND 12 [6,11]. A possible explanation for this discrepancy is that we used PND 15 animals, an age very close to the age (PND 12) when the effect on granule cell layer laminar size just begins to be detectable. In addition, we performed the naris occlusion on PND 2 and not on PND 1 as in several other studies [6,11]. Increased cell death has been found in the olfactory epithelium upon bulbectomy [17,22,30], suggesting that the olfactory bulbs play a major role in determining the survival of olfactory receptor neurons. Cell loss and structural and functional changes have also been reported in the auditory [23,43], tactile [46] and visual [16,29] systems upon sensory deprivation brought about by deafferentation or stimulus blockade [37,41]. In systems other than the olfactory system, however, it remains to be seen whether apoptosis plays a role in these processes. In any case, these findings point toward the importance of sensory stimulation in normal postnatal neural development and emphasize the importance of proper e f f e r e n t / a f f e r e n t connections in ensuring and preserving proper physiological functioning of these systems. The total number of cells undergoing apoptosis in the bulb is likely to be much larger than what is detected at a given time. Since the clearance time for apoptotic cells (the time from which a cell dies to the
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J. Najbauer, M. Leon/Brain Research 674 (1995) 245-251
time of its phagocytosis) is rapid in other systems [10], we probably could detect only a fraction of the total number of cells that underwent apoptosis. If short clearance times were to apply in the mammalian olfactory system, apoptosis would be a dynamic process that could underlie, at least in part, the plasticity of the olfactory system. Repeated exposure of rat pups to a single odor paired with tactile stimulation increased the number of periglomerular cells in focal areas of the bulb that respond with an increase in metabolic activity as measured by 2-deoxy-D-glucose uptake in response to the trained odor [50]. Similarly, chronic exposure of rat pups to a mixture of odors results in increased numbers of cells in the superficial and deep granule cell layers [42]. These findings suggest that there might be a lower incidence of apoptosis in focal regions of the olfactory bulbs of animals raised with significant olfactory experiences. There is increasing evidence that in the developing nervous system, trophic factors and other survival signals may suppress an intrinsic suicide program of cells and, thus, may regulate cell numbers in response to sensory stimuli [39]. It is possible that apoptosis induced by olfactory deprivation is a manifestation of a suppression of local trophic factors in the bulb. Our results suggest that apoptosis may be a critical mechanism by which cells in the developing olfactory system are eliminated and that early olfactory stimulation has a profound effect on the number of cells deleted by this process.
[5] [6] [7] [8] [9]
[10] [11]
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Acknowledgements Part of this work was carried out while one of the authors (J. Najbauer) was on leave of absence from the Biophysical Research Group of the Hungarian Academy of Sciences, Medical University of P6cs, P6cs, Hungary. Supported by National Institute of Mental Health Grant MH48950 (to M. Leon).
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