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Olfactory deprivation enhances normal spine loss in the olfactory bulb of developing ferrets
Neuroscience Letters, 62 (1985) 169 173 Elsevier Scientific Publishers Ireland Ltd.
169
NSL 03651
OLFACTORY D E P R I V A T I O N E N H A N C E S N O R M A L SPINE L O S S IN THE OLFACTORY BULB OF D E V E L O P I N G FERRETS
R. A P F E L B A C H and E. W E I L E R
hzstitut li~r Biologic III. Universitiit Tiihin~en, Auf der Morgenstelle, 28, D-7400 Tiihin£,en ( F. R.G. (Received May 61h, 1985; Revised version received and accepted August 29th, 1985)
Key wor.(s.
ferret rearing environment - odor exposure technique
olfactory system
dendritic spine
Golgi
Ferrets show a sensitive phase in their postnatal development during which they can become imprintcd to food odors. At the same time the number of granule cell spines in the olfactory bulb reaches a maximum, declining significantly thereafter. In ferrets, exposed continuously to saturated levels of gcraniol odor in the cage environment, the normal decline in spine number (occurring between day 60 and 90) is significantly enhanced. No such effects were observed during earlier ontogenetic phases. This late postnatal phase is further associated with a marked and significant decrease in total brain weight. The signilicance of these events to olfactory imprinting and plasticity in the developing brain is discussed.
We have recently shown that odor imprinting in the developing ferret (Musteh, putoriusL.luro) occurs during a sensitive phase lasting throughout the third postnatal month of life [3]. We have further shown that the number of dendritic spines as well as the number of reciprocal synapses in the internal granular cells of the ferret olfactory bulb (OB) increases significantly from day 30 to day 60 but decreases significantly thereafter, reaching adult levels by 150 days of age [4, 14]. In order to study the influence of early olfactory experience on the developmental changes in the number of granule cell spines, we investigated and report here the effects of early olfactory deprivation on this parameter. However, we wished to study such effects by using a non-invasive approach. Therefore, olfactory deprivation was induced by rearing litters of ferrets from birth in an artificial environment saturated with geraniol odor. The apparatus and other conditions were similar to those described by Laing and Panhuber [10]. The continuous overexposure to a single odor masks the ability of the animals to experience other odors in the environment, bringing about a relative state of olfactory deprivation [7, 9 11]. At different ages, groups (n = 3-4 animals) of normal and geraniol exposed ferrets were sacrificed by Nembutal anesthesia followed by intracranial perfusion with saline and 3.2", glutaraldehyde-2.61~/ paraformaldehyde in 0.09 M sodium cacodylate buffer (pH 7.35) at room temperature. The OBs were removed and processed liar rapid Golgi technique. The bulbs were embedded in Agar, cut in 100-Hm slices, dehydrated and mounted on slides with DePeX. Examining the material under the light microscope, spines were counted on differ0304-394085 $ 03.'~0 © 1985 Elsevier Scientitic Publishers Ireland Ltd.
170
ent, randomly selected apical branchings of granule cells dendrites, along segments of 50/~m in length. In this context the term spine is used for all spine-like processes. as classification of these structures into spines, gemmules or other protrusions is not possible at the light microscopical level. For each age and each group up to 500 dendritic segments and 9000 spines were counted. Ontogenetic dependent changes in the number of spines per dendritic segment in normal animals are shown in Fig. 1. it is evident that during the second month of postnatal life there is a marked increase in the mean number of spines per dendritic segment. This number remains constant during the third month but significantly (227~i,; P<0.001, Kolmogoroff-Smirnoff test) decreases thereafter, reaching adul~ levels by about 150 days. The results on the number of spines in the normal and geraniol-exposed animals at the ages of 60 and 150 days are shown in Fig. 2. It is evident that exposure to geraniol from birth to day 60 has no effect on spine number, but this condition significantly enhances (by an additional 25°11; P < 0.001) the normal decline (22~..~i)in spine number observed in the normal animals. The results reveal that the relative state of odor deprivation does not impart its effects on the initial phase of growth where spine number is increasing, but rather on the later phase of growth where this parameter is either stable or decreasing. This indicates that not only this later phase of granule cell development in the OB is a plastic and vulnerable phase, but that early olfactory experience may regulate the normal decline in this parameter. It should be noted that the effect of geraniol overexposure, presumably reflecting olfactory deprivation, occurs during the sensitive phase of olfactory imprinting to food and prey odors in this carnivorous species, as shown by us previously.
°
Gr)
~
o,,
:J
•
15. Q..
.E
5
if)
3'0
60
9'0
120
' ' 400 Age (days) 150
"
Fig. 1. Age-dependent distribution of granule cell spines in the external plexiform layer. Striped range indicates the sensitive phase when olfactory food imprinting is possible.
171
-22,, %
E
$ Q" 1 0 _
-
- 3 0 , 4 °/o p
o)
.5
e~
m
5
60
150
Age
(days)
Fig. 2. Spree density of control animals (~1) and experimental animals raised under gcranio[ exposure (~{]'). l)ue to geraniol exposure (olfactory deprivation) the number of spines per 50 Itm dendritic length i'~ signiticamlv lower in 150 day old experimental animals than in normal animals of the same age.
In order to determine the developmental stage of the ferret's brain during the late age growth phase when the above effects on OB and olfactory behavior are observed, brain weights of ferrets at different age levels between birth and 4 years were measured. The results, shown Fig. 3, reveal both expected and unexpected growth patterns. Thus, as expected, between birth to day 60, brain growth shows a continuous increase (ca. 30-fold). However, after that age until adulthood, mean brain weigh! actually shows a highly significant reduction of about 20'}~, (P<0.001). The main decline occurs between day 60 and day 120, adult values being reached by day 150. Wc found identical growth patterns in both sexes, although males show higher brain weights at all ages. Our unpublished results indicate further that this particular growth-related, normal decline observed in subadults, is restricted to brain only, not including other organs. Thus, it appears that the decline in spine numbers tk~llowing olthctory deprivation coincides with the late reduction phase of brain growth, indicating that this specific phase, in contrast to earlier stages, seems to be an important plastic phase during which environmental effects (e.g. imprinting, sensory stimuli or sensory deprivation) exert their influence and shape the developing neural structures and behavior. The internal granule cells in the OB are microneurons showing most of their proliferation and maturation in the postnatal phase [2]. Airman [1] first proposed that the brain neurons which proliferate postnatally may be particularly involved in the plasticity and vulnerability of the developing brain. Several studies [5, 12. 13] indicate
172
n g-
oqp oo
6
5-
w
. .• - , ..eW, •
= 147
"" ' i : , . 4
"
O O
I
4-
ea e
zm
~3-
2-
0
8 2'5
5'0
1 01 0
2 0,0
4 0,0
8 0, 0
1600 AGE
(DAYS)
Fig. 3. Postnatal brain weight development of female ferrets. The brain of 50-120 day old subadult animals surpasses that of adult conspecifics.
that altricial mammals (rat, mouse) show a critical phase during postnatal ontogeny when growth and development of the OB depend on olfactory stimulation. Thus, unilateral olfactory deprivation by nare closure resulted in reduced growth, diminished proliferation of cells, retarded differentiation of synaptic enzymes and abnormal development of synaptic structures in the OB. Closure of the external nare in later ages will not cause similar effects [6]. The spines of the internal granule cells are the sites of synapses between these cells and the secondary relay neurons (mitral and tufted cells) in the OB. Our finding that spine number is reduced in ferrets exposed to continuous odor of geraniol (olfactory deprivation) is in agreement with studies in the visual system where reductions in spine number were observed in the cortical cells of dark-reared mice [16]. However, it is interesting that the reduction in the spine number was not observed in the phase when the spine number increases but rather in a later phase when the spine number shows a reduction, even in the normal animal. Such a normal developmental reduction is not restricted to the ferret and has been recently reported by Greer [8] for granule cells of the rat, as well. Similarly, Rausch and Scheich [15], working with the mynah bird, also found a normal overshoot (overproduction followed by a reduction) in the density of spines in trhe central neurons controling speech. Another interesting aspect of our findings is the fact that the normal overproduction and reduction changes in the spine and synaptic number [14], the enhance-
173
ment of this event by olfactory deprivation (this study) and the critical sensitivity to food odor imprinting [3, 4] all occur during a phase when the growth of the brain in the subadult ferret, as measured by weight, also shows an overshoot followed by a reduction, This indicates that the developing brain of the ferret undergoes a special phase when it is susceptible to environmental influences. Further studies are needed to investigate the underlying causes of the overshoot and decline in neural structures and any relationship that may exist between the latter and behavioral development. This study was supported by the D F G (Ap 14/8-6). 1 Allman, J., Postnatal growth and differentiation of the mammalian brain, with implications for a morphological theory of memory. In G.C. Quarton, T. Melnechuk and F.O. Schmitt (Eds.I, The Neurosciences, Rockefeller University Press, New York, 1967, pp. 723 743. 2 Altman, J., Auloradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol,, 137 (1969) 433M.58. 3 Apfelbach, R., A sensitive phase for the development of olfactory preference in ferrets (Mu.vtela putorius.liluro L.), Z. Siiugetierkde., 43 (1978) 289 295. 4 Apfclbach, R., Weiler, E. and Rehn, B., Is there a neural basis for olfactory food imprinting in ferrets?, Naturwiss., 72 (1985) 106-107. 5 Benson, T.E., Ryugo, D.K. and Hinds, J.W., Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic morphometric analysis, J. Neurosci., 4 (1984) 638653. 6 Brunjes, P.C. and Borror, M.J., Unilateral odor deprivation: differential effects due to time of treatment, Brain Res. Bull., 11 (1983)501 503. 7 Doving, K.B. and Pinching, A.J., Selective degeneration of neurons in the olfactory bulb l'ollowing prolonged odour exposure, Brain Res., 52 ( 19731 115 129. 8 Greet, C.A., A quantitative Golgi analysis of granule cell development in the neonalal rat olfactory bulb, In Proc. VII Meeting of Ass. Chemoreception Sci., April 1985, Sarasota, FL, U.S.A.. No. 80 (abstract), 9 Harvey, F.E. and Cowley, J.J., Effects of external chemical environment on the developing olfactory bulbs of the mouse (Mus rnusculus), Brain Res., Bull., 13 (1984) 541- 547. 10 Laing, D.G. and Panhuber, H., Neural and behavioral changes in rats following conlmuous exposure to an odor, J. Comp. Physiol., 124 (1978) 259-265. 11 Laing. D.G. and Panhuber, H., Olfactory sensitivity of rats reared in an odorous or deodorised environment. Physiol, Behav,, 25 (1980) 555-558. 12 Meisami, E., The developing rat olfactory bulb: prospects of a new model system in developmental neurobiology. In E. Meisami and M.A.B. Brazier (Eds.), Neural Growth and Differntialion, Raven Press, New York, 1979, pp. 183-206. 13 Meisami, E. and Moussavi, R., Lasting effects of early olfactory deprivation on the growth, DNA, RNA and protein content and Na-K-ATPase and AChE activity of the rat olfactory bulb, Dev. Brain Rcs., 2 (1982) 217 229. 14 Mendoza, A.S., Rehn, B. and Apfelbach, R., Postnatale Synapsenausbildung im Bulbus olfactorius von Frettchen. Befunde an der externen plexiformen Schicht, Verh. Anat. Ges., 79, in press. 15 Rausch, G. and Scheich, H., Dendritic spine loss and enlargement during maturation of thc speech control system in the mynah bird (Gracula reliy;iosa), Neurosci, Lett., 46 (1982) 131 136. 16 Valverde. F., Apical dendritic spines of the visual cortex and light deprivation the mouse, Exp. Brain Res.,3(1967) 337 352.
169
NSL 03651
OLFACTORY D E P R I V A T I O N E N H A N C E S N O R M A L SPINE L O S S IN THE OLFACTORY BULB OF D E V E L O P I N G FERRETS
R. A P F E L B A C H and E. W E I L E R
hzstitut li~r Biologic III. Universitiit Tiihin~en, Auf der Morgenstelle, 28, D-7400 Tiihin£,en ( F. R.G. (Received May 61h, 1985; Revised version received and accepted August 29th, 1985)
Key wor.(s.
ferret rearing environment - odor exposure technique
olfactory system
dendritic spine
Golgi
Ferrets show a sensitive phase in their postnatal development during which they can become imprintcd to food odors. At the same time the number of granule cell spines in the olfactory bulb reaches a maximum, declining significantly thereafter. In ferrets, exposed continuously to saturated levels of gcraniol odor in the cage environment, the normal decline in spine number (occurring between day 60 and 90) is significantly enhanced. No such effects were observed during earlier ontogenetic phases. This late postnatal phase is further associated with a marked and significant decrease in total brain weight. The signilicance of these events to olfactory imprinting and plasticity in the developing brain is discussed.
We have recently shown that odor imprinting in the developing ferret (Musteh, putoriusL.luro) occurs during a sensitive phase lasting throughout the third postnatal month of life [3]. We have further shown that the number of dendritic spines as well as the number of reciprocal synapses in the internal granular cells of the ferret olfactory bulb (OB) increases significantly from day 30 to day 60 but decreases significantly thereafter, reaching adult levels by 150 days of age [4, 14]. In order to study the influence of early olfactory experience on the developmental changes in the number of granule cell spines, we investigated and report here the effects of early olfactory deprivation on this parameter. However, we wished to study such effects by using a non-invasive approach. Therefore, olfactory deprivation was induced by rearing litters of ferrets from birth in an artificial environment saturated with geraniol odor. The apparatus and other conditions were similar to those described by Laing and Panhuber [10]. The continuous overexposure to a single odor masks the ability of the animals to experience other odors in the environment, bringing about a relative state of olfactory deprivation [7, 9 11]. At different ages, groups (n = 3-4 animals) of normal and geraniol exposed ferrets were sacrificed by Nembutal anesthesia followed by intracranial perfusion with saline and 3.2", glutaraldehyde-2.61~/ paraformaldehyde in 0.09 M sodium cacodylate buffer (pH 7.35) at room temperature. The OBs were removed and processed liar rapid Golgi technique. The bulbs were embedded in Agar, cut in 100-Hm slices, dehydrated and mounted on slides with DePeX. Examining the material under the light microscope, spines were counted on differ0304-394085 $ 03.'~0 © 1985 Elsevier Scientitic Publishers Ireland Ltd.
170
ent, randomly selected apical branchings of granule cells dendrites, along segments of 50/~m in length. In this context the term spine is used for all spine-like processes. as classification of these structures into spines, gemmules or other protrusions is not possible at the light microscopical level. For each age and each group up to 500 dendritic segments and 9000 spines were counted. Ontogenetic dependent changes in the number of spines per dendritic segment in normal animals are shown in Fig. 1. it is evident that during the second month of postnatal life there is a marked increase in the mean number of spines per dendritic segment. This number remains constant during the third month but significantly (227~i,; P<0.001, Kolmogoroff-Smirnoff test) decreases thereafter, reaching adul~ levels by about 150 days. The results on the number of spines in the normal and geraniol-exposed animals at the ages of 60 and 150 days are shown in Fig. 2. It is evident that exposure to geraniol from birth to day 60 has no effect on spine number, but this condition significantly enhances (by an additional 25°11; P < 0.001) the normal decline (22~..~i)in spine number observed in the normal animals. The results reveal that the relative state of odor deprivation does not impart its effects on the initial phase of growth where spine number is increasing, but rather on the later phase of growth where this parameter is either stable or decreasing. This indicates that not only this later phase of granule cell development in the OB is a plastic and vulnerable phase, but that early olfactory experience may regulate the normal decline in this parameter. It should be noted that the effect of geraniol overexposure, presumably reflecting olfactory deprivation, occurs during the sensitive phase of olfactory imprinting to food and prey odors in this carnivorous species, as shown by us previously.
°
Gr)
~
o,,
:J
•
15. Q..
.E
5
if)
3'0
60
9'0
120
' ' 400 Age (days) 150
"
Fig. 1. Age-dependent distribution of granule cell spines in the external plexiform layer. Striped range indicates the sensitive phase when olfactory food imprinting is possible.
171
-22,, %
E
$ Q" 1 0 _
-
- 3 0 , 4 °/o p
o)
.5
e~
m
5
60
150
Age
(days)
Fig. 2. Spree density of control animals (~1) and experimental animals raised under gcranio[ exposure (~{]'). l)ue to geraniol exposure (olfactory deprivation) the number of spines per 50 Itm dendritic length i'~ signiticamlv lower in 150 day old experimental animals than in normal animals of the same age.
In order to determine the developmental stage of the ferret's brain during the late age growth phase when the above effects on OB and olfactory behavior are observed, brain weights of ferrets at different age levels between birth and 4 years were measured. The results, shown Fig. 3, reveal both expected and unexpected growth patterns. Thus, as expected, between birth to day 60, brain growth shows a continuous increase (ca. 30-fold). However, after that age until adulthood, mean brain weigh! actually shows a highly significant reduction of about 20'}~, (P<0.001). The main decline occurs between day 60 and day 120, adult values being reached by day 150. Wc found identical growth patterns in both sexes, although males show higher brain weights at all ages. Our unpublished results indicate further that this particular growth-related, normal decline observed in subadults, is restricted to brain only, not including other organs. Thus, it appears that the decline in spine numbers tk~llowing olthctory deprivation coincides with the late reduction phase of brain growth, indicating that this specific phase, in contrast to earlier stages, seems to be an important plastic phase during which environmental effects (e.g. imprinting, sensory stimuli or sensory deprivation) exert their influence and shape the developing neural structures and behavior. The internal granule cells in the OB are microneurons showing most of their proliferation and maturation in the postnatal phase [2]. Airman [1] first proposed that the brain neurons which proliferate postnatally may be particularly involved in the plasticity and vulnerability of the developing brain. Several studies [5, 12. 13] indicate
172
n g-
oqp oo
6
5-
w
. .• - , ..eW, •
= 147
"" ' i : , . 4
"
O O
I
4-
ea e
zm
~3-
2-
0
8 2'5
5'0
1 01 0
2 0,0
4 0,0
8 0, 0
1600 AGE
(DAYS)
Fig. 3. Postnatal brain weight development of female ferrets. The brain of 50-120 day old subadult animals surpasses that of adult conspecifics.
that altricial mammals (rat, mouse) show a critical phase during postnatal ontogeny when growth and development of the OB depend on olfactory stimulation. Thus, unilateral olfactory deprivation by nare closure resulted in reduced growth, diminished proliferation of cells, retarded differentiation of synaptic enzymes and abnormal development of synaptic structures in the OB. Closure of the external nare in later ages will not cause similar effects [6]. The spines of the internal granule cells are the sites of synapses between these cells and the secondary relay neurons (mitral and tufted cells) in the OB. Our finding that spine number is reduced in ferrets exposed to continuous odor of geraniol (olfactory deprivation) is in agreement with studies in the visual system where reductions in spine number were observed in the cortical cells of dark-reared mice [16]. However, it is interesting that the reduction in the spine number was not observed in the phase when the spine number increases but rather in a later phase when the spine number shows a reduction, even in the normal animal. Such a normal developmental reduction is not restricted to the ferret and has been recently reported by Greer [8] for granule cells of the rat, as well. Similarly, Rausch and Scheich [15], working with the mynah bird, also found a normal overshoot (overproduction followed by a reduction) in the density of spines in trhe central neurons controling speech. Another interesting aspect of our findings is the fact that the normal overproduction and reduction changes in the spine and synaptic number [14], the enhance-
173
ment of this event by olfactory deprivation (this study) and the critical sensitivity to food odor imprinting [3, 4] all occur during a phase when the growth of the brain in the subadult ferret, as measured by weight, also shows an overshoot followed by a reduction, This indicates that the developing brain of the ferret undergoes a special phase when it is susceptible to environmental influences. Further studies are needed to investigate the underlying causes of the overshoot and decline in neural structures and any relationship that may exist between the latter and behavioral development. This study was supported by the D F G (Ap 14/8-6). 1 Allman, J., Postnatal growth and differentiation of the mammalian brain, with implications for a morphological theory of memory. In G.C. Quarton, T. Melnechuk and F.O. Schmitt (Eds.I, The Neurosciences, Rockefeller University Press, New York, 1967, pp. 723 743. 2 Altman, J., Auloradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol,, 137 (1969) 433M.58. 3 Apfelbach, R., A sensitive phase for the development of olfactory preference in ferrets (Mu.vtela putorius.liluro L.), Z. Siiugetierkde., 43 (1978) 289 295. 4 Apfclbach, R., Weiler, E. and Rehn, B., Is there a neural basis for olfactory food imprinting in ferrets?, Naturwiss., 72 (1985) 106-107. 5 Benson, T.E., Ryugo, D.K. and Hinds, J.W., Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic morphometric analysis, J. Neurosci., 4 (1984) 638653. 6 Brunjes, P.C. and Borror, M.J., Unilateral odor deprivation: differential effects due to time of treatment, Brain Res. Bull., 11 (1983)501 503. 7 Doving, K.B. and Pinching, A.J., Selective degeneration of neurons in the olfactory bulb l'ollowing prolonged odour exposure, Brain Res., 52 ( 19731 115 129. 8 Greet, C.A., A quantitative Golgi analysis of granule cell development in the neonalal rat olfactory bulb, In Proc. VII Meeting of Ass. Chemoreception Sci., April 1985, Sarasota, FL, U.S.A.. No. 80 (abstract), 9 Harvey, F.E. and Cowley, J.J., Effects of external chemical environment on the developing olfactory bulbs of the mouse (Mus rnusculus), Brain Res., Bull., 13 (1984) 541- 547. 10 Laing, D.G. and Panhuber, H., Neural and behavioral changes in rats following conlmuous exposure to an odor, J. Comp. Physiol., 124 (1978) 259-265. 11 Laing. D.G. and Panhuber, H., Olfactory sensitivity of rats reared in an odorous or deodorised environment. Physiol, Behav,, 25 (1980) 555-558. 12 Meisami, E., The developing rat olfactory bulb: prospects of a new model system in developmental neurobiology. In E. Meisami and M.A.B. Brazier (Eds.), Neural Growth and Differntialion, Raven Press, New York, 1979, pp. 183-206. 13 Meisami, E. and Moussavi, R., Lasting effects of early olfactory deprivation on the growth, DNA, RNA and protein content and Na-K-ATPase and AChE activity of the rat olfactory bulb, Dev. Brain Rcs., 2 (1982) 217 229. 14 Mendoza, A.S., Rehn, B. and Apfelbach, R., Postnatale Synapsenausbildung im Bulbus olfactorius von Frettchen. Befunde an der externen plexiformen Schicht, Verh. Anat. Ges., 79, in press. 15 Rausch, G. and Scheich, H., Dendritic spine loss and enlargement during maturation of thc speech control system in the mynah bird (Gracula reliy;iosa), Neurosci, Lett., 46 (1982) 131 136. 16 Valverde. F., Apical dendritic spines of the visual cortex and light deprivation the mouse, Exp. Brain Res.,3(1967) 337 352.