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Effects of early anosmia on two classes of granule cells in developing mouse olfactory bulbs
Neuroscience Letters, 54 (1985) 301-306 Elsevier Scientific Publishers Ireland Ltd.
301
NSL 03177
EFFECTS OF EARLY ANOSMIA ON TWO CLASSES OF GRANULE CELLS IN DEVELOPING MOUSE OLFACTORY BULBS
LESLIE C. SKEEN, BRYAN R. DUE and FELICIA E. DOUGLAS
Institute for Neuroscience and Department of Psychology, University of Delaware, Newark, DE 19716 (U.S.A.) (Received October 25th, 1984; Accepted December 12th, 1984)
Key words: anosmia - olfactory bulbs - mouse
Quantitative morphometric methods were used to examine the effects of early unilateral anosmia on two classes of granule cells in developing mouse olfactory bulbs. Volumetric results show that the internal granule cell layer in the deprived olfactory bulb is significantly smaller than the same layer in the experienced olfactory bulb. The major factor contributing to this retarded development is a selective loss of one class of interneurons; dark granule cell number is substantially reduced, while light granule cell number is not. This selective effect appears to be related to the time course of cell proliferation and differentiation and provides clues to the way early experience regulates neural development.
Early olfactory experience is known to significantly modify behavior [5]. In seeking to understand the structural basis of such modifications and the laws that govern their expression, we are studying the development of the olfactory bulbs in unilaterally anosmic mice [21]. This approach is convenient not only because the receptor input and projections of the two olfactory bulbs are entirely segregated [6, 19], but also because cytoarchitectonically distinct layers in the olfactory bulb (e.g. Fig. 1a) partition different types of neurons [20], zones of centrifugal termination [13], receptor and biochemical phenotypes [7] and modes of synaptic organization [20] .
..The most abundant neurons in the olfactory bulb form the internal granule cell layer (Fig. la). The inhibitory interneurons that give this layer its name are the principal arbiters of bulbar output [20] and undergo most of their proliferation and differentiation in the early postnatal weeks [3, 10] when olfactory cues dominate the sensory mediation of behavior [5]. Consequently, granule cells should be particularly labile to the influences of the early environment. Consistent with this notion are recent results showing that Xsirradiation of the olfactory bulbs in neonate rats selectively affects the neurons in the granule cell layer; the number of dark granule cells is significantly reduced while the light granule cells remain unaffected [24]. The present report shows that early anosmia has a similar effect on these two classes of granule cells. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
302
Three ICR strain albino mice were made unilaterally anosmic by nasal cautery [14] one day after birth (P 1). On P40, their brains were fixed via transcardial perfusion with saline followed by phosphate-buffered 10070 formalin, pH 7.2. After the brains were embedded in glycomethacrylate plastic (1B-4; Polysciences), serial sections (4 iat: thick, 120 /Lm apart) were taken with a D-profile rotary microtome knife (Hacker Instruments). The sections were floated onto chrome-alum-gelatin-subbed slides, stained with toluidine blue-O (pH 4.8), cleared with Gothard's differentiator, and enclosed in coverglass with Permount. The volumes of the deprived and experienced granule cell layers (GeLs) were then computed from X 75 drawings with a graphics tablet using the algorithm of Stephan et al. [23], Cellular measurements were obtained by sampling with rectangular grids (20 X 200 p.m): for each section, a grid was placed over each quadrant of the deprived GCL and compared with a homotopically matched grid from the experienced GeL. The nuclei of all light and dark granule cells [24] (e.g. Fig. Ib) in these grids were drawn onto graph paper at X 1000 with camera lucida. Nuclear areas were then computed with a graphics tablet, and corrected nuclear diameters (De) were
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Fig. I, A: low-power photomicrograph of mouse olfactory bulb showing its layers: ONL, olfactory nerve layer; GML, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, granule cell layer; PVL, peri ventricular layer. Black bar in the GCL shows location (lateral quadrant) and size (20 x 200 ).tm) of one grid from which cell measurements were taken. Arrow indicates approximate location from which photomicrograph in B was taken. B: high-power photomicrograph showing dark granule cell (large arrow) and light granule cell (small arrow), These two cell types were distinguished from glial cells according to the criteria of Ling et al. [12]. Scale bar = 10 ).tm.
303
calculated with the method of Hendry [9]. Corrected nuclear volumes (Vc) for each sample were obtained by multiplying summed nuclear areas by section thickness and applying the correction factor of Bayer [2]. Cell densities were then calculated by dividing Vc by the average nuclear volume (4/3 where r= 1/2 De), Population estimates were then obtained by multiplying cell density by GCL volume. Paired ttests were used to compare measures from the deprived and experienced GCLs. Results from the volumetric measurements show that early anosmia dramatically retards the development of the GCL. As shown in Fig. 2a, the deprived GCL is onethird smaller in volume than the experienced GCL (P< 0.01). However, microscopic examination of the deprived and experienced GCLs reveals no obvious differences in neuronal density. This observation suggests that the deprived GCL has substantially fewer neurons. Subsequent cellular measurements confirm that inference. Neither the diameter (Fig. 2b) nor the density (Fig. 2c) of light or dark granule cells is significantly altered by early olfactory deprivation. Instead, anosmia has a profound effect on the number of granule cells, and this effect is limited almost exclusively to one subtype. Population estimates, shown in Fig. 2d, reveal that the deprived GCL has 32070 fewer dark granule cells than the experienced GCL (P
7fr
304
granule cells in the deprived GCL is due to an acceleration of a phenomenon which occurs throughout the nervous system during normal development: natural cell death [8]. An alternative which must also be considered is that the loss of dark granule cells in the deprived GCL is a consequence of retarded neural proliferation. The synthesis of dopamine in a subpopulation of tufted cells is now known to depend on the integrity [1, 11], and possibly the normal activity [22], of the olfactory receptor axons. Since monoamines have been implicated in the regulation of neural proliferation [17, 18], early experience could also modulate the rate of dark granule cell prolifera-
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Fig. 2. A: mean volumes of experienced and deprived (shaded) GCLs in unilaterally anosmic mice. Note significant difference in deprived vs experienced GCL volumes (*P< 0.001). B: mean nuclear diameters of dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. C: mean nuclear densities of dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. D: population estimates for dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. Note significant difference in the number of DGs in deprived vs experienced GCLs (*P
305
tion via the trophic influence of compounds synthesized in tufted cells. Greater insight into the relative contributions of accelerated cell death versus retarded cell proliferation to the loss of these neurons must await the results of studies now in progress. This research was supported by NIH Grant NS-19604. We thank Bob Rafetto for his many hours spent refining the computer algorithms so necessary for these analyses.
2 3
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Baker, 1., Kawano, T., Margolis, F.L. and loh, T.H., Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat, 1. Neurosci., 3 (1983) 69-78. Bayer, S.A., Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study, Exp. Brain Res., 46 (1982) 315-323. Bayer, S.A., 3H-Thymidine-radiographic studies of neurogenesis in the rat olfactory bulb, Exp. Brain Res., 50 (1983) 329-340. Benson, T.E., Ryugo, D.K. and Hinds, 1.W., Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis, 1. Neurosci., 4 (1984) 638-653. Cheal, M., Social olfaction: a review of the ontogeny of olfactory influences on vertebrate behavior, Behav. BioI., 15 (1975) 1-25. Costanzo, R.M. and O'Connell, R.l., Spatially organized projections of hamster olfactory nerves, Brain Res., 139 (1978) 327-332. Halasz, N. and Shepherd, G.M., Neurochemistry of the vertebrate olfactory bulb, Neuroscience, 10 (1983) 579-619. Hamburger, V. and Oppenheim, R.W., Naturally occurring neuronal death in vertebrates, Neurosci. Comment., 1 (1982) 39-55. Hendry, LA., A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment, 1. Neurocytol., 5 (1976) 337-349. Hinds, J. W., Autoradiographic study of histogenesis in the mouse olfactory bulbs. r. Time of origin of neurons and neuroglia, 1. Compo Neurol., 134 (1968) 287-304. Kream, R.M., Davis, B.J., Kawano, T., Margolis, F.L. and Macrides, F., Substance P and catecholaminergic expression in neurons of the hamster olfactory bulb, 1. Compo Neurol., 222 (1984) 140-154. Ling, E.A., Paterson, 1.A., Privat , A., Mori, S. and Leblond, C.P., Investigation of glial cells in semithin sections. L Identification of glial cells in the brain of young rats, 1. Compo Neurol., 149 (1973) 43-72. Luskin, M.B. and Price, 1.L., The topographic organization of association fibers of the olfactory system in the rat, including centrifugal fibers to the olfactory bulb, J. Compo Neurol. 216 (1983) 264-291. Meisami, E. and Safari, L., A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb, Brain Res., 221 (1981) 81-107. Orona, E., Rainer, E.C. and Scott, J.W., Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb, 1. Compo Neurol., 226 (1984) 346-356. Orona, E., Scott, l.W. and Rainer, E.C., Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb, J. Compo Neurol., 217 (1983) 227-237. Patel, A.1., Bendek, G., Balazs, R. and Lewis, P.D., Effect of reserpine on cell proliferation in the developing rat brain: a biochemical study, Brain Res., 129 (1977) 283-297.
306 18 Patel, A.J., Vertes, Zs., Lewis, P.O. and Lai, M., Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological study, Brain Res., 202 (1980) 415-428. 19 Price, J .L., An autoradiographic study of complementary laminar patterns of termination of afferent fibers to the olfactory cortex, J. Compo Neurol., 150 (1973) 87-108. 20 Shepherd, G.M., The Synaptic Organization of the Brain, 2nd edn., Oxford University Press, New York, 1979, 436 pp. 21 Skeen, L.C., Due, B.R. and Douglas, F.E., Effects of olfactory deprivation on layers and neural populations in the mouse olfactory bulb, Abstr. 5th Amer, Chern. Soc. Meeting, Sarasota, FL, U.S.A. 1983, No. 130. 22 Smith, L.K., Brunjes, P.C. and McCarty, R., Unilateral odor deprivation: time-course of neurochemical changes, Abstr. 5th Arner. Chern. Soc. Meeting, Sarasota, FL, U.S.A., 1983, No. 131. 23 Stephan, H., Frahm, H. and Baron, G., New and revised data on volumes of brain structures in insectivores and primates, Folia Prirnatol., 35 (1981) 1-29. 24 Struble, R.G. and Walters, C.P., Light microscopic differentiation of two populations of rat olfactory bulb granule cells, Brain Res., 236 (J 982) 237-251.
301
NSL 03177
EFFECTS OF EARLY ANOSMIA ON TWO CLASSES OF GRANULE CELLS IN DEVELOPING MOUSE OLFACTORY BULBS
LESLIE C. SKEEN, BRYAN R. DUE and FELICIA E. DOUGLAS
Institute for Neuroscience and Department of Psychology, University of Delaware, Newark, DE 19716 (U.S.A.) (Received October 25th, 1984; Accepted December 12th, 1984)
Key words: anosmia - olfactory bulbs - mouse
Quantitative morphometric methods were used to examine the effects of early unilateral anosmia on two classes of granule cells in developing mouse olfactory bulbs. Volumetric results show that the internal granule cell layer in the deprived olfactory bulb is significantly smaller than the same layer in the experienced olfactory bulb. The major factor contributing to this retarded development is a selective loss of one class of interneurons; dark granule cell number is substantially reduced, while light granule cell number is not. This selective effect appears to be related to the time course of cell proliferation and differentiation and provides clues to the way early experience regulates neural development.
Early olfactory experience is known to significantly modify behavior [5]. In seeking to understand the structural basis of such modifications and the laws that govern their expression, we are studying the development of the olfactory bulbs in unilaterally anosmic mice [21]. This approach is convenient not only because the receptor input and projections of the two olfactory bulbs are entirely segregated [6, 19], but also because cytoarchitectonically distinct layers in the olfactory bulb (e.g. Fig. 1a) partition different types of neurons [20], zones of centrifugal termination [13], receptor and biochemical phenotypes [7] and modes of synaptic organization [20] .
..The most abundant neurons in the olfactory bulb form the internal granule cell layer (Fig. la). The inhibitory interneurons that give this layer its name are the principal arbiters of bulbar output [20] and undergo most of their proliferation and differentiation in the early postnatal weeks [3, 10] when olfactory cues dominate the sensory mediation of behavior [5]. Consequently, granule cells should be particularly labile to the influences of the early environment. Consistent with this notion are recent results showing that Xsirradiation of the olfactory bulbs in neonate rats selectively affects the neurons in the granule cell layer; the number of dark granule cells is significantly reduced while the light granule cells remain unaffected [24]. The present report shows that early anosmia has a similar effect on these two classes of granule cells. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
302
Three ICR strain albino mice were made unilaterally anosmic by nasal cautery [14] one day after birth (P 1). On P40, their brains were fixed via transcardial perfusion with saline followed by phosphate-buffered 10070 formalin, pH 7.2. After the brains were embedded in glycomethacrylate plastic (1B-4; Polysciences), serial sections (4 iat: thick, 120 /Lm apart) were taken with a D-profile rotary microtome knife (Hacker Instruments). The sections were floated onto chrome-alum-gelatin-subbed slides, stained with toluidine blue-O (pH 4.8), cleared with Gothard's differentiator, and enclosed in coverglass with Permount. The volumes of the deprived and experienced granule cell layers (GeLs) were then computed from X 75 drawings with a graphics tablet using the algorithm of Stephan et al. [23], Cellular measurements were obtained by sampling with rectangular grids (20 X 200 p.m): for each section, a grid was placed over each quadrant of the deprived GCL and compared with a homotopically matched grid from the experienced GeL. The nuclei of all light and dark granule cells [24] (e.g. Fig. Ib) in these grids were drawn onto graph paper at X 1000 with camera lucida. Nuclear areas were then computed with a graphics tablet, and corrected nuclear diameters (De) were
0•
•
•
. _
•
,~
. .l \
A
" -,
"
Fig. I, A: low-power photomicrograph of mouse olfactory bulb showing its layers: ONL, olfactory nerve layer; GML, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, granule cell layer; PVL, peri ventricular layer. Black bar in the GCL shows location (lateral quadrant) and size (20 x 200 ).tm) of one grid from which cell measurements were taken. Arrow indicates approximate location from which photomicrograph in B was taken. B: high-power photomicrograph showing dark granule cell (large arrow) and light granule cell (small arrow), These two cell types were distinguished from glial cells according to the criteria of Ling et al. [12]. Scale bar = 10 ).tm.
303
calculated with the method of Hendry [9]. Corrected nuclear volumes (Vc) for each sample were obtained by multiplying summed nuclear areas by section thickness and applying the correction factor of Bayer [2]. Cell densities were then calculated by dividing Vc by the average nuclear volume (4/3 where r= 1/2 De), Population estimates were then obtained by multiplying cell density by GCL volume. Paired ttests were used to compare measures from the deprived and experienced GCLs. Results from the volumetric measurements show that early anosmia dramatically retards the development of the GCL. As shown in Fig. 2a, the deprived GCL is onethird smaller in volume than the experienced GCL (P< 0.01). However, microscopic examination of the deprived and experienced GCLs reveals no obvious differences in neuronal density. This observation suggests that the deprived GCL has substantially fewer neurons. Subsequent cellular measurements confirm that inference. Neither the diameter (Fig. 2b) nor the density (Fig. 2c) of light or dark granule cells is significantly altered by early olfactory deprivation. Instead, anosmia has a profound effect on the number of granule cells, and this effect is limited almost exclusively to one subtype. Population estimates, shown in Fig. 2d, reveal that the deprived GCL has 32070 fewer dark granule cells than the experienced GCL (P
7fr
304
granule cells in the deprived GCL is due to an acceleration of a phenomenon which occurs throughout the nervous system during normal development: natural cell death [8]. An alternative which must also be considered is that the loss of dark granule cells in the deprived GCL is a consequence of retarded neural proliferation. The synthesis of dopamine in a subpopulation of tufted cells is now known to depend on the integrity [1, 11], and possibly the normal activity [22], of the olfactory receptor axons. Since monoamines have been implicated in the regulation of neural proliferation [17, 18], early experience could also modulate the rate of dark granule cell prolifera-
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Fig. 2. A: mean volumes of experienced and deprived (shaded) GCLs in unilaterally anosmic mice. Note significant difference in deprived vs experienced GCL volumes (*P< 0.001). B: mean nuclear diameters of dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. C: mean nuclear densities of dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. D: population estimates for dark (DG) and light (LG) granule cells in the experienced and deprived (shaded) GCLs. Note significant difference in the number of DGs in deprived vs experienced GCLs (*P
305
tion via the trophic influence of compounds synthesized in tufted cells. Greater insight into the relative contributions of accelerated cell death versus retarded cell proliferation to the loss of these neurons must await the results of studies now in progress. This research was supported by NIH Grant NS-19604. We thank Bob Rafetto for his many hours spent refining the computer algorithms so necessary for these analyses.
2 3
4
5 6 7 8 9 10 11
12
13
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Baker, 1., Kawano, T., Margolis, F.L. and loh, T.H., Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat, 1. Neurosci., 3 (1983) 69-78. Bayer, S.A., Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study, Exp. Brain Res., 46 (1982) 315-323. Bayer, S.A., 3H-Thymidine-radiographic studies of neurogenesis in the rat olfactory bulb, Exp. Brain Res., 50 (1983) 329-340. Benson, T.E., Ryugo, D.K. and Hinds, 1.W., Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis, 1. Neurosci., 4 (1984) 638-653. Cheal, M., Social olfaction: a review of the ontogeny of olfactory influences on vertebrate behavior, Behav. BioI., 15 (1975) 1-25. Costanzo, R.M. and O'Connell, R.l., Spatially organized projections of hamster olfactory nerves, Brain Res., 139 (1978) 327-332. Halasz, N. and Shepherd, G.M., Neurochemistry of the vertebrate olfactory bulb, Neuroscience, 10 (1983) 579-619. Hamburger, V. and Oppenheim, R.W., Naturally occurring neuronal death in vertebrates, Neurosci. Comment., 1 (1982) 39-55. Hendry, LA., A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment, 1. Neurocytol., 5 (1976) 337-349. Hinds, J. W., Autoradiographic study of histogenesis in the mouse olfactory bulbs. r. Time of origin of neurons and neuroglia, 1. Compo Neurol., 134 (1968) 287-304. Kream, R.M., Davis, B.J., Kawano, T., Margolis, F.L. and Macrides, F., Substance P and catecholaminergic expression in neurons of the hamster olfactory bulb, 1. Compo Neurol., 222 (1984) 140-154. Ling, E.A., Paterson, 1.A., Privat , A., Mori, S. and Leblond, C.P., Investigation of glial cells in semithin sections. L Identification of glial cells in the brain of young rats, 1. Compo Neurol., 149 (1973) 43-72. Luskin, M.B. and Price, 1.L., The topographic organization of association fibers of the olfactory system in the rat, including centrifugal fibers to the olfactory bulb, J. Compo Neurol. 216 (1983) 264-291. Meisami, E. and Safari, L., A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb, Brain Res., 221 (1981) 81-107. Orona, E., Rainer, E.C. and Scott, J.W., Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb, 1. Compo Neurol., 226 (1984) 346-356. Orona, E., Scott, l.W. and Rainer, E.C., Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb, J. Compo Neurol., 217 (1983) 227-237. Patel, A.1., Bendek, G., Balazs, R. and Lewis, P.D., Effect of reserpine on cell proliferation in the developing rat brain: a biochemical study, Brain Res., 129 (1977) 283-297.
306 18 Patel, A.J., Vertes, Zs., Lewis, P.O. and Lai, M., Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological study, Brain Res., 202 (1980) 415-428. 19 Price, J .L., An autoradiographic study of complementary laminar patterns of termination of afferent fibers to the olfactory cortex, J. Compo Neurol., 150 (1973) 87-108. 20 Shepherd, G.M., The Synaptic Organization of the Brain, 2nd edn., Oxford University Press, New York, 1979, 436 pp. 21 Skeen, L.C., Due, B.R. and Douglas, F.E., Effects of olfactory deprivation on layers and neural populations in the mouse olfactory bulb, Abstr. 5th Amer, Chern. Soc. Meeting, Sarasota, FL, U.S.A. 1983, No. 130. 22 Smith, L.K., Brunjes, P.C. and McCarty, R., Unilateral odor deprivation: time-course of neurochemical changes, Abstr. 5th Arner. Chern. Soc. Meeting, Sarasota, FL, U.S.A., 1983, No. 131. 23 Stephan, H., Frahm, H. and Baron, G., New and revised data on volumes of brain structures in insectivores and primates, Folia Prirnatol., 35 (1981) 1-29. 24 Struble, R.G. and Walters, C.P., Light microscopic differentiation of two populations of rat olfactory bulb granule cells, Brain Res., 236 (J 982) 237-251.