152
Brain Research, 99 (1975) 152~ 156 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Comparison of postnatal CNS development between male and female rats
ESTELLE G R E G O R Y
Department of Psychology, California State University at Los Angeles, Los Angeles, Cali_/i :90032
(u.s.A.) (Accepted July 30th, 1975)
A survey of the literature on the nervous system reveals that many studies either ignore or fail to recognize the possibility that sex differences might exist with regard to the parameters being investigated. This failure to consider the influence of sex appears to represent an almost tacit assumption on the part of most researchers that, apart from some neural mechanisms controlling pituitary function, the nervous system of the male and female are identical. While this may be true in some cases, there is an increasing amount of evidence 5,7,1~ that during the development of the nervous system the rate of maturation may not be equal for both sexes. Very few studies have been undertaken to compare the morphological development of the male and female nervous system. This study was undertaken to investigate whether the rate of cell growth of two areas of the nervous system is indeed equivalent between the sexes. The subjects were albino Wistar rats. Litters were limited to 6 (3 males, 3 females) within 2 days of birth. Rats 2, 7, 16, 25 and 35 days of age were examined since this age range encompasses the period o f m o s t rapid postnatal brain growth. At each age, 8 animals of each sex were studied (total N -- 80). The rats were anesthetized, sacrificed and the brains were then removed and fixed in neutral buffered formalin according to the procedure of Cammermeyer 4. Tissues were embedded in paraffin and frontal sections of 6 #m thickness were obtained. The sections were then mounted and stained with thionin according to standard histological procedures. Pyramidal cells in layer 5 of the somatosensory cortex at the level of the crossing of the anterior commissure were selected for measurement. All sections were examined under oil (magnification, x 1164). Cells were selected for measurement if they had a nucleolus and distinct nuclear and cytoplasmic outlines and were distinctly pyramidal in shape. Each cell was measured with an ocular micrometer according to the procedure of Schad6 and Baxter 1°. Base and height measurements of the cell soma were determined through the nucleolus (see Fig. 1). The diameter of the nucleus was also determined. Two sections were measured from each animal, with 25 measurements ( 12 from one hemisphere, 13 from the other) per section. The volume of each cell soma and nucleus was then calculated. The volumes of 4000 nuclei were obtained. The volume of the pyramidal cell soma was calculated with the formula derived by Schad6 and van Groenigen 11 for motoneurons, 1.04 1/6 zc AB CAB. The volume
153
Fig. l. Illustration of cell measurement. of the pyramidal cell nuclei was calculated using the formula for the volume of a sphere. The diameter of nuclei of pyramidal cells in the hippocampus at the tip of A m m o n ' s horn in the approximate areas CA2 and CA3 of Lorente de N68 were also measured. Twenty-four measurements (each diameter - 1 measurement)were taken, 12 from a section (6 from one hemisphere, 6 from the other). The volume of the 1840 nuclei was then calculated using the formula for the volume of a sphere. 22C0
?:000
I;00
i~00
/I t o~
/
1400
II/ >
/i/I
1200
i000
8o0
6oo Male ....
Female
400
200
I 7
I 16 Days of Age
25
35
Fig. 2. Pyramidal cell volume (cu./~m) from the somatosensory cortex of the male and female rat (N , 8 animals/point).
154 I00
7O0 6OO
5OO
40O
.>>~
3OO 2OO
-Male .... Female
lOa
7
16
25 Days
35
of Age
Fig. 3. Pyramidal cell nuclear volume (cu. btm)from the somatosensory cortex of the male and female rat (N = 8 animals/point).
Cell and nuclear volume calculations were completed with the aid of an IBM 360/44 computer. The resultant volumes were then tested for differences between the sexes at each age using a two-way analysis of variance 14. The cellular lamination of the cortex underwent extensive changes during the age period examined. The cortex of the 2-day-old rat (and to a lesser extent the 7-dayold rat) was not well differentiated; the cortical cells were closely packed and very similar in appearance. Pyramidal cell nuclei were very large relative to soma size. As the cortex matured the layers became clearer, the cells differentiated, and the volume of the nuclei of the cells decreased relative to total soma volume. Pyramidal cell volume increased quite rapidly from 2 to 16 days of age. The cell volume increased at a slightly slower rate from 16 to 25 days of age. F r o m 25 to 35 days of age the cell volume of b o t h sexes decreased markedly. At 25 days of age the volume of pyramidal cells in the female was slightly (t0 ~ ) less than that of the males. At 35 days the volume of the soma in the males was larger by 3 0 ~ (P < 0.001) than the volume of the soma in the female (Fig. 2). The volume of the nuclei of the pyramidal cells followed a similar growth pattern (Fig. 3); nuclear volume increased to 25 days and then decreased by 35 days. The volume of the nuclei of hippocampal cells also increased rapidly from 2 to 25 days of age. F r o m 25 to 35 days of age the volume changed very little (Fig. 4). In this study the development of the size of the hippocampal cells did not differ between the sexes. Since the hippocampus matures at an earlier age than the neocortex it is possible that a pattern similar to that observed in the neocortex also occurs in the hippocampus but at an earlier age. The present study only observed postnatal development and it is plausible that differential development in the hippocampus might occur prenatally. Differences in pyramidal cell soma volume were observed between the male and female rats at 35 days o f age. The volume o f the males' cells was significantly larger
155
7oo
.2. >o
400
30C -....
2LK
Mal,~ 5emal,,
IOC
I 7
2
I 16
I 25 Days
I 35
of Age
Fig. 4. Hippocampal cell nuclear volume (cu./~m) in areas CA2 and CA3 of Lorente de N6. than that of the females. There are at least two possible explanations for these results. The first explanation could be related to body size, that is, at 35 days of age the males' body weight was significantly greater than the females (P < 0.001) for the first time (Fig. 5). The difference in cell size of neurons involved in somatosensory function could then simply be a reflection of a longer axon in the male. The present study, however, also noted that the nuclei of the pyramidal cells were also larger in the males than in the females by about 25 o//o.If this argument were valid, then one would expect to see this same nuclear size differential in adult rats. Pfaff 9, however, does not report a difference in nuclear volume of pyramidal cells between normal adult male and female rats. Further, since human neurons are not notably larger than those of the rat despite larger axons it seems unlikely that length of axon, per se, necessarily influences soma size (Ford, unpublished data).
F2G
g,
ioc
E
8C
t/
6c
20
2
7
16
25
35
Days of Age
Fig. 5. Average body weight (-k S.E.M.) of male and female rats at various ages. Hatched bar represents females, unhatched bar represents males.
156 An alternative interpretation would involve the following argument. The developing neuron closely resembles the neuron that is regenerating after axonal section. Brattgard e t al. 1,2 have noted that during regeneration the soma accumulates water, thereby greatly increasing the cellular volume; once synaptic contact is reestablished the somatic swelling recedes. One sees this same pattern in the developing pyramidal cell. The cell volume increases rapidly until 25 days of age when most synaptic contacts have been established. The cell volume is smaller when measured at 35 days of age (this same type of decrease in pyramidal cell volume has been noted previously in the cat by Brizzee and Jacobs 3, and in the rat by Sugita13). It is suggested that while the cell is developing, neuronal metabolism is directed toward both growth and maintenance (as in the regenerating neuron). The soma size may reflect this degree of metabolic and synthetic activity. Following this period of extremely rapid growth during which the neuron established the majority of its synaptic contacts 6, the cellular metabolism reverts primarily to a maintenance role; this in turn is reflected in the soma by a reduction of volume. This suggests that the larger pyramidal cell volume of the male at 35 days of age may reflect a less mature cell, and possibly one with fewer synaptic connections. The advice and guidance of Dr. Marion Diamond and Dr. Donald Ford is gratefully acknowledged.
1 BRATTGARD,S. O., EDSTR()M,J. E., AND HYDF,N, H., The chemical changesin regenerating neurons, J. Neurochem., 1 (1957) 3t6-325. 2 BRATTGARD,S. O., EDSTROM, J. E., AND HYDEN, H., The productive capacity of the neuron in retrograde reaction, Exp. Cell Res., 5 (1958) 185-200. 3 BRIZZEE,K. R., AND JACOBS, L. A., Postnatal changes in volumetric and density relationships of neurons in cerebral cortex of cat, Acts anat. (Basel), 38 (1959) 291-303. 4 CAMMERMEYER,J., Morphologic distinctions between oligodendrocytes and microglia cells in the rabbit cerebral cortex, Amer. J. Anat., 118 (1966) 227-248. 5 DORNER,G., AND STAUDT,J., Perinatal structural sex differentiation of the hypothalamus in rats, Neuroendocrinology, 4 (1969) 20-24. 6 EAYRS, J. T., AND GOODHEAD, B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Lond.), 93 (1959) 385-402. 7 ILLINGSWORTH,R. S., The Normal Child, Little, Brown and Co., New York, 1968. 8 LORENTE DE NO, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 9 PFAFF, n . W., Morphological changes in the brains of adult male rats after neonatal castration, J. Endocrin., 36 (1966) 415~,16. 10 SCHADI~,J. P., AND BAXTER, C. F., Changes during growth in the volume and surface area of cortical neurons in the rabbit, Exp. Neurol., 12 (1960) 158-178. 11 SCHADE,J. P., AND VAN GROENIGEN,W. B., Structural organization of the human cerebral cortex, Acts anat. (Basel), 47 (1961) 74-111. 12 SORmRO,O., AND FORD, n . H., Age and sex: the effect on the composition of different regions of the neonatal rat brain. In D. H. FORD (Ed~), Influence of Hormones on the Nervous System, Proc. Int. Soc. Psychoneuroendocrinol,, Brooklyn, 1970, Karger, Basel, 1971, pp. 322-333. 13 SUGITA, N., Comparative studies on the growth of the cerebral cortex, J. comp. NeuroL, 29 (1918) 61-96 and 119-151. 14 WINER, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, pp. 228-248.
Brain Research, 99 (1975) 152~ 156 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Comparison of postnatal CNS development between male and female rats
ESTELLE G R E G O R Y
Department of Psychology, California State University at Los Angeles, Los Angeles, Cali_/i :90032
(u.s.A.) (Accepted July 30th, 1975)
A survey of the literature on the nervous system reveals that many studies either ignore or fail to recognize the possibility that sex differences might exist with regard to the parameters being investigated. This failure to consider the influence of sex appears to represent an almost tacit assumption on the part of most researchers that, apart from some neural mechanisms controlling pituitary function, the nervous system of the male and female are identical. While this may be true in some cases, there is an increasing amount of evidence 5,7,1~ that during the development of the nervous system the rate of maturation may not be equal for both sexes. Very few studies have been undertaken to compare the morphological development of the male and female nervous system. This study was undertaken to investigate whether the rate of cell growth of two areas of the nervous system is indeed equivalent between the sexes. The subjects were albino Wistar rats. Litters were limited to 6 (3 males, 3 females) within 2 days of birth. Rats 2, 7, 16, 25 and 35 days of age were examined since this age range encompasses the period o f m o s t rapid postnatal brain growth. At each age, 8 animals of each sex were studied (total N -- 80). The rats were anesthetized, sacrificed and the brains were then removed and fixed in neutral buffered formalin according to the procedure of Cammermeyer 4. Tissues were embedded in paraffin and frontal sections of 6 #m thickness were obtained. The sections were then mounted and stained with thionin according to standard histological procedures. Pyramidal cells in layer 5 of the somatosensory cortex at the level of the crossing of the anterior commissure were selected for measurement. All sections were examined under oil (magnification, x 1164). Cells were selected for measurement if they had a nucleolus and distinct nuclear and cytoplasmic outlines and were distinctly pyramidal in shape. Each cell was measured with an ocular micrometer according to the procedure of Schad6 and Baxter 1°. Base and height measurements of the cell soma were determined through the nucleolus (see Fig. 1). The diameter of the nucleus was also determined. Two sections were measured from each animal, with 25 measurements ( 12 from one hemisphere, 13 from the other) per section. The volume of each cell soma and nucleus was then calculated. The volumes of 4000 nuclei were obtained. The volume of the pyramidal cell soma was calculated with the formula derived by Schad6 and van Groenigen 11 for motoneurons, 1.04 1/6 zc AB CAB. The volume
153
Fig. l. Illustration of cell measurement. of the pyramidal cell nuclei was calculated using the formula for the volume of a sphere. The diameter of nuclei of pyramidal cells in the hippocampus at the tip of A m m o n ' s horn in the approximate areas CA2 and CA3 of Lorente de N68 were also measured. Twenty-four measurements (each diameter - 1 measurement)were taken, 12 from a section (6 from one hemisphere, 6 from the other). The volume of the 1840 nuclei was then calculated using the formula for the volume of a sphere. 22C0
?:000
I;00
i~00
/I t o~
/
1400
II/ >
/i/I
1200
i000
8o0
6oo Male ....
Female
400
200
I 7
I 16 Days of Age
25
35
Fig. 2. Pyramidal cell volume (cu./~m) from the somatosensory cortex of the male and female rat (N , 8 animals/point).
154 I00
7O0 6OO
5OO
40O
.>>~
3OO 2OO
-Male .... Female
lOa
7
16
25 Days
35
of Age
Fig. 3. Pyramidal cell nuclear volume (cu. btm)from the somatosensory cortex of the male and female rat (N = 8 animals/point).
Cell and nuclear volume calculations were completed with the aid of an IBM 360/44 computer. The resultant volumes were then tested for differences between the sexes at each age using a two-way analysis of variance 14. The cellular lamination of the cortex underwent extensive changes during the age period examined. The cortex of the 2-day-old rat (and to a lesser extent the 7-dayold rat) was not well differentiated; the cortical cells were closely packed and very similar in appearance. Pyramidal cell nuclei were very large relative to soma size. As the cortex matured the layers became clearer, the cells differentiated, and the volume of the nuclei of the cells decreased relative to total soma volume. Pyramidal cell volume increased quite rapidly from 2 to 16 days of age. The cell volume increased at a slightly slower rate from 16 to 25 days of age. F r o m 25 to 35 days of age the cell volume of b o t h sexes decreased markedly. At 25 days of age the volume of pyramidal cells in the female was slightly (t0 ~ ) less than that of the males. At 35 days the volume of the soma in the males was larger by 3 0 ~ (P < 0.001) than the volume of the soma in the female (Fig. 2). The volume of the nuclei of the pyramidal cells followed a similar growth pattern (Fig. 3); nuclear volume increased to 25 days and then decreased by 35 days. The volume of the nuclei of hippocampal cells also increased rapidly from 2 to 25 days of age. F r o m 25 to 35 days of age the volume changed very little (Fig. 4). In this study the development of the size of the hippocampal cells did not differ between the sexes. Since the hippocampus matures at an earlier age than the neocortex it is possible that a pattern similar to that observed in the neocortex also occurs in the hippocampus but at an earlier age. The present study only observed postnatal development and it is plausible that differential development in the hippocampus might occur prenatally. Differences in pyramidal cell soma volume were observed between the male and female rats at 35 days o f age. The volume o f the males' cells was significantly larger
155
7oo
.2. >o
400
30C -....
2LK
Mal,~ 5emal,,
IOC
I 7
2
I 16
I 25 Days
I 35
of Age
Fig. 4. Hippocampal cell nuclear volume (cu./~m) in areas CA2 and CA3 of Lorente de N6. than that of the females. There are at least two possible explanations for these results. The first explanation could be related to body size, that is, at 35 days of age the males' body weight was significantly greater than the females (P < 0.001) for the first time (Fig. 5). The difference in cell size of neurons involved in somatosensory function could then simply be a reflection of a longer axon in the male. The present study, however, also noted that the nuclei of the pyramidal cells were also larger in the males than in the females by about 25 o//o.If this argument were valid, then one would expect to see this same nuclear size differential in adult rats. Pfaff 9, however, does not report a difference in nuclear volume of pyramidal cells between normal adult male and female rats. Further, since human neurons are not notably larger than those of the rat despite larger axons it seems unlikely that length of axon, per se, necessarily influences soma size (Ford, unpublished data).
F2G
g,
ioc
E
8C
t/
6c
20
2
7
16
25
35
Days of Age
Fig. 5. Average body weight (-k S.E.M.) of male and female rats at various ages. Hatched bar represents females, unhatched bar represents males.
156 An alternative interpretation would involve the following argument. The developing neuron closely resembles the neuron that is regenerating after axonal section. Brattgard e t al. 1,2 have noted that during regeneration the soma accumulates water, thereby greatly increasing the cellular volume; once synaptic contact is reestablished the somatic swelling recedes. One sees this same pattern in the developing pyramidal cell. The cell volume increases rapidly until 25 days of age when most synaptic contacts have been established. The cell volume is smaller when measured at 35 days of age (this same type of decrease in pyramidal cell volume has been noted previously in the cat by Brizzee and Jacobs 3, and in the rat by Sugita13). It is suggested that while the cell is developing, neuronal metabolism is directed toward both growth and maintenance (as in the regenerating neuron). The soma size may reflect this degree of metabolic and synthetic activity. Following this period of extremely rapid growth during which the neuron established the majority of its synaptic contacts 6, the cellular metabolism reverts primarily to a maintenance role; this in turn is reflected in the soma by a reduction of volume. This suggests that the larger pyramidal cell volume of the male at 35 days of age may reflect a less mature cell, and possibly one with fewer synaptic connections. The advice and guidance of Dr. Marion Diamond and Dr. Donald Ford is gratefully acknowledged.
1 BRATTGARD,S. O., EDSTR()M,J. E., AND HYDF,N, H., The chemical changesin regenerating neurons, J. Neurochem., 1 (1957) 3t6-325. 2 BRATTGARD,S. O., EDSTROM, J. E., AND HYDEN, H., The productive capacity of the neuron in retrograde reaction, Exp. Cell Res., 5 (1958) 185-200. 3 BRIZZEE,K. R., AND JACOBS, L. A., Postnatal changes in volumetric and density relationships of neurons in cerebral cortex of cat, Acts anat. (Basel), 38 (1959) 291-303. 4 CAMMERMEYER,J., Morphologic distinctions between oligodendrocytes and microglia cells in the rabbit cerebral cortex, Amer. J. Anat., 118 (1966) 227-248. 5 DORNER,G., AND STAUDT,J., Perinatal structural sex differentiation of the hypothalamus in rats, Neuroendocrinology, 4 (1969) 20-24. 6 EAYRS, J. T., AND GOODHEAD, B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Lond.), 93 (1959) 385-402. 7 ILLINGSWORTH,R. S., The Normal Child, Little, Brown and Co., New York, 1968. 8 LORENTE DE NO, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 9 PFAFF, n . W., Morphological changes in the brains of adult male rats after neonatal castration, J. Endocrin., 36 (1966) 415~,16. 10 SCHADI~,J. P., AND BAXTER, C. F., Changes during growth in the volume and surface area of cortical neurons in the rabbit, Exp. Neurol., 12 (1960) 158-178. 11 SCHADE,J. P., AND VAN GROENIGEN,W. B., Structural organization of the human cerebral cortex, Acts anat. (Basel), 47 (1961) 74-111. 12 SORmRO,O., AND FORD, n . H., Age and sex: the effect on the composition of different regions of the neonatal rat brain. In D. H. FORD (Ed~), Influence of Hormones on the Nervous System, Proc. Int. Soc. Psychoneuroendocrinol,, Brooklyn, 1970, Karger, Basel, 1971, pp. 322-333. 13 SUGITA, N., Comparative studies on the growth of the cerebral cortex, J. comp. NeuroL, 29 (1918) 61-96 and 119-151. 14 WINER, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, pp. 228-248.
