2 CUZNER, M. L., AND DAV~SON, A. N., The role of lipids in myelinogenesis of the central nervous system, Abstr. int. neurochem. Conf., (1965) 21. 3 CUZNER ...
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Influence of estradiol and cortisol on lipids and cerebrosides in the developing brain and spinal cord of the rat Functional development of the brain and spinal cord in the rat is hastened after neonatal administration of estradiol 7,15 and cortisoll6,1L Functional development has been assessed by responses elicited by brain electroshock stimulation and by direct stimulation of the spinal cord 7,15-17. Histological investigations have shown that myelin appears earlier in the rat brain after estradiol treatment during early postnatal periods of brain developmentL In the present study the effects of estradiol and cortisol on lipid and cerebroside concentrations were investigated. It has been shown that the increase of cerebrosides parallels the process of myelination in the CNS 2,14. Cerebrosides are components of the myelin sheath a,9, and, therefore, were chosen for study. Twenty litters of Long-Evans rats were kept with their lactating mothers until sacrificed. Each litter consisted of 4 males and 4 females. Estradiol and cortisol were administered to rats from the 6th until the 10th day after birth. In each litter 2 animals received, subcutaneously, estradiol dipropionate, 1 #g/g body weight per day, dissolved in 100 #g/ml sesame oil; 2 animals received, subcutaneously, cortisol, l0/zg/g body weight per day, suspended with Tween 80-0.9 ~ NaC1; the remaining animals served as controls and received the vehicles. The estradiol and cortisol doses and the duration and time periods of treatment chosen were those used previously to test effects on brain and spinal cord excitability in developing rats 7,15-17. Body weights were recorded daily throughout the experimental period. Differences were not observed between animals injected with sesame oil and those injected with 0.9 ~ NaCl, or between males and females. The data, therefore, were combined in order to obtain one control value for estradiol- and cortisol-treated rats. At 12 days after birth, animals were sacrificed by decapitation. This age was chosen because the period between l0 and 15 days is characterized by active brain myelination 4. The cerebrum, cerebellum and spinal cord were rapidly dissected out, freed from meninges and larger blood vessels, and weighed. Water content in samples of cerebrum, cerebellum and spinal cord was determined by drying in vacuo at 104°C for 4 days. Lipids were extracted from samples of cerebrum, cerebellum and spinal cord with 2 : 1 chloroform/methanol, according to the method of Folch et al. 6. Lipids were determined gravimetrically and excluded gangliosides, which were removed during washing of the extract. Values for lipids were expressed as mg/100 mg of wet tissue. The method described by Rouser et al. 1~ for the fractionation of brain lipids on Florisil columns was modified for small amounts of lipids. Two components of the cerebroside molecule, galactose and sphingosine, were analyzed. For the isolation of galactose and sphingosine the column eluants, which contained the cerebrosides, were subjected to acid hydrolysis1°,11. Sphingosine was determined by the method of Lauters and galactose was determined by the orcinol-sulphuric acid reaction 13. The galactose content of the sample was read directly from a standard curve and was multiplied by 4.66 to give the actual value of the cerebrosides. A mean molecular weight of 846 was used for the calculation of cerebrosides. Values for cerebrosides were expressed as rag/100 mg of lipid. Brain Research, 5 (1967) 524-526
525
SHORT COMMUNICATIONS TABLE I EFFECTS OF ESTRADIOL AND CORTISOL* ON DEVELOPING RATS
CNS structures Body weight (g)
Control
Estradiol
Cortisol
27.8
29.2
26.4
4- 0.5 +
1.086 + 0.008
Brain weight (g)
± 0.8
4- 0.7
1.099 4- 0.01
1.052 4- 0.03
Spinal cord Cerebrum Cerebellum
81.73 4- 0.18 85.72 4- 0.11 84.08 4- 0.12
80.92 4- 0.32 85.66 4- 0.13 83.80 4- 0.17
81.78 4- 0.24 85.80 4- 0.18 83.49 4- 0.23
Spinal cord Lipids (mg/100 mgwet tissue)** Cerebrum
5.33 4- 0.16 3.91 4- 0.04
5.51 4- 0.06 3.85 4- 0.09
Cerebellum
2.76 4- 0.09
5.35 4- 0.23 4.04 4- 0.06 ( < 0.05) ++ 2.80 4- 0.12
Cerebrosides Spinal cord (mg/100 mg lipids as determined by galactose Cerebrum content) Cerebellum
9.87 4- 0.20
11.48 4- 0.30 ( < 0.001) 2.24 4- 0.08 ( < 0.001) 2.06 4- 0.03
10.71 4- 0.28 ( < 0.01) 2.03 4- 0.08
Cerebrosides (mg/100 mg lipids as determined by sphingosine content)
Spinal cord
9.69 4- 0.21
Cerebrum
1.84 4- 0.07
10.71 4- 0.27 ( < 0.01) 1.98 4- 0.07
Cerebellum
2.01
11.28 4- 0.31 ( < 0.001) 2.22 4- 0.08 ( < 0.001) 2.00 4- 0.08
70 Water
1.84 4- 0.06 2.08 4- 0.06
4- 0.07
2.81
4- 0.16
2.03 4- 0.03
2.03 4- 0.13
* Rats received daily subcutaneously estradiol dipropionate (1/~g/g body weight) or cortisol (10 pg/g body weight) from the 6th to the 10th day after birth. ** Lipids include total lipids minus gangliosides. + Mean 4- S.E. ++ Numbers in parentheses are P values for comparison to control.
To determine whether the parameters measured in control and hormonetreated animals differed significantly in their means, the t test for non-paired data was applied< Although brain and body weights were slightly higher in the estradiol-treated animals and slightly lower in the cortisol-treated animals, when brain : body weight ratios were calculated, there were no differences between controls and treated animals (Table I). Lipids of the cerebrum were higher in the estradiol-treated animals than in controls, whereas in the cerebellum and spinal cord the concentration of lipids was the same in both groups. No differences were found in lipids of the cortisol-treated animals and those of controls. When lipids were fractionated, there was a significantly higher amount of cerebrosides in the spinal cord and cerebrum of the estradioltreated than in controls. Cerebrosides in the spinal cord of the cortisol-treated were higher than those in controls. The increase in cerebrosides after estradiol and cortisol suggests that hormones may influence the process of myelination which is very active at this age period. Histological studies1 have also shown that myelin appears earlier in the rat brain after estradiol treatment during postnatal' periods of development. It is suggested that Brain Research, 5 (1967) 524-526
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SHORT COMMUNICATIONS
precocious f u n c t i o n a l m a t u r a t i o n of the central n e r v o u s system after n e o n a t a l estradiol a n d cortisol treatmentT, t5-t7 may be partly a result of e n h a n c e d m y e l i n a t i o n at a n early period of development. This work was supported by C o n t r a c t AT(11-1)-34, Project 82, from the U.S. A t o m i c Energy Commission. Dr. Regina Casper is a U S P H S HD-101 postdoctoral fellow. Department of Physiology, University of California, Berkeley, Calif. 94720 (U.S.A.)
REGINA CASPER ANTONIA VERNADAKIS PAOLA S. TIMIRAS
1 CURRY, J. J., AND HELM, L. M., Brain myelination after neonatal administrationof oestradiol, Nature (Lond.), 209 (1966) 915-916. 2 CUZNER,M. L., AND DAV~SON,A. N., The role of lipids in myelinogenesis of the central nervous system, Abstr. int. neurochem. Conf., (1965) 21. 3 CUZNER,M. L., DAVtSON,A. N., AND GREGSON,N. A., The chemical composition of vertebrate myelin and microsomes, J. Neurochem., 12 (1965) 469-481. 4 DAVISON,A. N., ANDDOBBING,J'., Myelination as a vulnerable period in brain development, Brit. reed. Bull., 22 (1966) 40-44. 5 FISHER,R. A., Statistical Methods for Research Workers, Hafner, New York, 1950. 6 FOLCH,J., LEES,M., ANDSLOANESTANLEY, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 226 (1957) 497-509. 7 HEIM, L. M., AND TIMIRAS,P. S., Gonad-brain relati6nship: Precocious brain maturation after estradiol in rats, Endocrinology, 72 (1963) 598-606. 8 LAUTER,C. J., AND TRAMS,E. G., A spectrophotometric determination of sphingosine, J. Lipid Res., 3 (1962) 136-138. 9 NORTON,W. T., AND AUTILIO,L. A., The lipid composition of purified bovine brain myelin, J. Neurochem., 13 (1966) 213-222. 10 O'BRmN, J. S., FILLERUP, D. L., AND MEAD, J. F., Brain lipids: I. Quantification and fatty acid composition of cerebroside sulfate in human cerebral gray and white matter, J. Lipid Res., 5 (1964) 109-116. ll RADIN,N. S., BROWN,J. R., AND LAVIN,F. B., The preparative isolation of cerebrosides, J. biol. Chem., 219 (1956) 977-983. 12 ROUSER, G., BAUMANN,A. J., KRITCHEVSKY,G., HEELER, D., AND O'BRIEN,J. S., Quantitative chromatographic fractionation of complex lipid mixtures: Brain lipids, J. Amer. Oil Chem. Soc., 38 (1961) 544-555. 13 SVENNERHOLM,L., The quantitative estimation of cerebrosides in nervous tissue, J. Neurochem., 1 (1956) 42-53. 14 TORVIK,A., AND SIDMAN,R. L., Autoradiographic studies on lipid synthesis in the mouse brain during postnatal development, J. Neurochem., 12 (1965) 555-565. 15 VERNADAKIS,A., AND TIMIRAS, P. S., Effect of estradiol on spinal cord convulsions in developing rats, Nature (Lond.), 197 (1963) 906. 16 VERNADAKIS,A., ANDWOODBURY,D. M., Effect of cortisol on the electroshock seizure thresholds in developing rats, J. Pharmacol. exp. Ther., 139 (1963)110-113. 17 VERNADAKIS,A., AND WOODBURY,D. M., Effects of cortisol and diphenylhydantoin (Dilantin) on spinal cord convulsions in developing rats, J. Pharmacol. exp. Ther., 144 (1964) 316-320. (Received May 2nd, 1967)
Brain Research, 5 (1967) 524-526
