DEVELOPMENTAL
BIOLOGY
lmmunochemical
49,
294-299
(1976)
Measurement
of Myelin Basic Protein
in Developing
Rat Brain: An Index of Myelin Synthesis STEVEN R. COHEN AND MICHAEL GUARNIERI Neurochemistry
Laboratories, Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205 Accepted October 10, 1975
The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue. INTRODUCTION
The developmental accumulation of basic protein in optic nerve (a tissue which is more than 60% myelin), the morphological appearance of myelin, the activity of an enzyme that synthesizes a myelin lipid, and the accumulation of the lipid in myelin occurred at virtually identical rates (30). Immunochemical measurements of basic protein thus furnish a precise and sensitive technique to measure myelin synthesis. Because oligodendroglial cells synthesize central nervous system myelin (reviewed in Ref. 71, the basic protein measurements also provide an index of oligodendrocyte activity.
Myelin contains a protein, the myelin basic protein, which has been monitored in several types of studies to gain information about the metabolism of the membrane (12-14, 22, 32). The effects of proteolytic enzymes on basic protein have been analyzed to gain information about the destruction of myelin during degenerative diseases (1, 3, 18, 25, 26, 33). Because quantitative measurements of basic protein required isolation of the membrane, such studies technically have been restricted to animals having appreciable amounts of myelin. For example, the yield of myelin from the brain of rats younger than 10 days is less than 400 pg/brain (24). Moreover, the composition of the membrane changes with age (2, 11, 21, 22, 24, 29, 34). Different fractions of myelin may be isolated during development or degeneration as the density of the membrane changes. Immunochemical measurements, because of their specificity, can be applied directly to proteins in untreated tissue or crude fractions (4, 8). We recently described a radioimmunoassay for basic protein and the conditions for its quantitative application to unfractionated tissue (10).
MATERIALS AND METHODS
Tissues. Pregnant SpragueDawley rats were obtained from Charles River (Boston, Mass.). Birth dates were recorded and the animals were sacrificed by decapitation at the appropriate age. The brain and spinal cord were exposed and dissected as follows: The olfactory bulb, cerebral hemisphere, and the area under the cerebral hemisphere were removed separately. The latter is designated as the midbrain. The cerebellum was removed and the tissue under it and to the first vertebrae was taken as the brain stem. The optic and 294
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
trigeminal nerves were removed from the base of the skull. The cervical spinal cord was removed from vertebrae 3-7, thoracic from B-20, and lumbar from 21-26. All tissues except optic and trigeminal nerve were weighed and 25% (w/v) homogenates were made in 0.2 M Tris-acetate buffer, pH 7.3. Optic and trigeminal nerves from single animals were homogenized in 0.5 ml of the Tris-acetate buffer. Radioimmunoassay. Samples from 0.5 pg (heavily myelinated areas) to 250 pg (younger brains) were analyzed by radioimmunoassay for myelin basic protein, as previously described (10). Protein was determined by the method of Lowry et al. cm. RESULTS
The amounts of basic protein in different areas of adult (120 day) rat brain and nerve are described in Table 1. Areas TABLE
1
CONCENTRATION OF MYELIN BASIC PROTEIN IN REGIONS OF ADULT RAT NERVOUS SYSTEM” Micrograms of myelin basic protein per milligram of tissue (wet weight) Central nervous system Cerebrum Cerebellum Olfactory bulb Midbrain Brain stem Cervical spinal cord Thoracic spinal cord Lumbar spinal cord Optic nerve Peripheral nervous system Trigeminal nerve Sciatic nerve
4.6 2.1 0.92 13.1 18.0 12.9 13.4 11.2 21.0 5.1 0.36
a Brains from 120-day-old rats were dissected and assayed for myelin basic protein as described (see Methods). The values represent the average of four tests, two determinations on two samples. Variations from the average were less than 20%. The values for wet weight of the optic, trigeminal, and sciatic nerves were estimated from protein determinations using a value of 10 mg of protein/100 mg wet weight.
295
which are predominantly gray, the olfactory bulb, cerebellum, and cerebrum, contain approximately three- to fourfold less basic protein than white matter areas, such as optic nerve, midbrain, brain stem, and spinal cord. The trigeminal nerve, which is myelinated by both oligodendroglial and Schwann cells, contained significantly more antibody-detectable basic protein than did the sciatic nerve, which contains only peripheral myelin. It is possible to calculate the amount of myelin in the different brain areas from the dry weight of neural tissue, approximately 18% for gray and 32% for white matter (9), and the amount of basic protein in myelin. The latter value has been estimated as 30% of the total protein of myelin (13, 221,and since myelin is approximately 30% protein (241, basic protein represents 10% of the dry weight of myelin. For example, cerebrum contains 4.6 pg of basic protein per milligram (wet weight) of tissue (Table 1). The cerebrum thus contains 46 pg of myelin/l80~g (dry wt) of tissue, or 25pgllOO pg of desiccated tissue. Myelin concentrations, as a percentage of total dry weight, range from 5% (olfactory bulb), 25% (cerebrum), to 70% (optic nerve). The values will vary, depending on the value for protein concentration of the neural tissue, a difficult figure to determine (51, and the proportion of basic protein in myelin, a value which increases with age, as shown by Morel1 et al. (22) in the mouse. In preliminary studies, basic protein was never detected in rat brain before birth; therefore, the present measurements were done with postnatal tissue. The antigenicity of the basic protein was nearly constant during development. The antigen-antibody displacement curves for basic protein in lo- and 120-day-old rats differed in slope by 12%, a difference caused by changes in the relative amounts of small and large basic proteins with age, or the number of methylated arginine residues (15). The rate of basic protein accumulation in various ratios of brain and
296
DEVELOPMENTAL BIOLIXY
spinal cord are shown in Figs. l-3. The weight of brain, its water content, and protein concentration change with development. The choice of a denominator against which the accumulation of a specific protein can be measured is diffkult. The data were arbitrarily expressed as the percentage of the adult (120 day) value to show the changes in brain from Day 10 to 20 (Fig. l), the period of rapid myelination, and from Day 20 to 40 (Fig. 21, a period at which myelin accumulates at a steady rate. As shown in Fig. 1, at Day 5 the greatest amount of basic protein is in the trigeminal nerve, approximately 0.25 j.kg/mg of nerve. The spinal cord contained less than 0.15 pg of basic protein; cerebrum, cerebel-
VOLUME 49, 1976
lum, and olfactory bulb contained less than 0.05 pglmg of tissue. There was no detectable basic protein in the midbrain, brain stem, or optic nerve. The trigeminal nerve is the first site for rapid deposition of the basic proteins between the fifth and ninth day. Optic nerves follow at Day 911, and finally, the cerebrum, cerebellum, olfactory bulbs, midbrain, and brain stem rapidly deposit myelin basic protein beginning on Day 12. The synthesis of basic proteins appeared to be biphasic in the trigeminal nerve with rapid deposition occurring at Days 59 and again between 25 and 42. The contribution of CNS and PNS myelin to trigeminal nerve basic protein remains to be determined. The sciatic nerve contains a pro-
Optic I’’ Nerve I’
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80
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60-
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15
20 21
FIG. 1. Appearance of myelin basic protein in the developing rat nervous system. Ages 52.1 days. Rat brains were dissected as described and assayed for myelin basic protein by radioimmunoassay. Each point represents the average of four tests, two determinations on two animals. Variations from the average were less than 20%. The results are expressed as a percentage of the 120-day value for the particular neural area.
297
BRIEF NOTES
spinal cord (Day 1520). By the forty-second postnatal day, the content of myelin basic proteins in the spinal cord, optic, and trigeminal nerve is nearly identical to that of the 120-day-old rat. The remaining brain areas contain from 90% (cerebellum) to 75% (brain stem) of the 120-day value for myelin basic protein. DISCUSSION
40
30 Rot
50
Aqe i Days)
2. Appearance of myelin basic protein in the FIG. developing rat nervous system, Days 21-42.
Rat Age ( Days 1 FIG. 3. Appearance of myelin basic protein in the developing rat spinal cord, Days 5-42.
tein with properties similar to CNS basic protein (6). The content of basic protein in other areas continues at a steady rate once the period of rapid synthesis begins. This rate is greatest in the optic nerve, which is almost fully myelinated by Day 21. The spinal cord also myelinates rapidly (Fig. 3). During development, the rate of basic protein accumulation is greatest in lumbar cord (Day 9-15) and in the cervical
The appearance of myelin basic protein in the rat central nervous system correlates with the morphological detection of myelin (17), the synthesis of myelin lipids (291, and the amount of myelin which can be isolated (24). This correlation is especially striking in the optic nerve. Our studies have shown parallel increases in basic protein and cerebroside sulfotransferase which synthesizes sulfatide, a characteristic lipid of myelin. The in vivo incorporation of radioactive sulfate into optic nerve sulfatide was also investigated by electron microscopic autoradiography (30). Myelin accumulated radioactive sulfatide at a rate which was virtually identical to the increased activity of cerebroside sulfotransferase and basic protein accumulation. These results demonstrate the close correlation between the appearance of basic protein and the synthesis of its membrane, myelin, the latter monitored both through the activity of an enzyme which synthesizes a myelin lipid and the incorporation of that lipid specifically into the membrane. It is possible to quantitatively relate basic protein and myelin synthesis, but the relations between myelin synthesis and oligodendroglial cell activities are less clear (23, 28). A single oligodendrocyte may myelinate more than one axon. Oligodendrocytes appear to synthesize myelin during all stages of their development (23). The chemical differences that have been described between immature and mature myelin (11, 22) may relate to developmental changes in the oligodendrocyte. Neurons may contribute to the synthesis of myelin. Indeed, it has not been definitely established that the basic protein is
298
DEVELOPMENTALBIOLOGY
synthesized by oligodendroglial cells. However, chemical measurements of specific cell fractions isolated from brains have shown that the specific concentration of basic protein is enriched 150-fold in oligodendrocytes compared to neuronal cell bodies (Cohen, S. R., unpublished results). Oligodendroglial cells synthesize three times their weight in myelin per day (24). Precursor pools of membrane components may be utilized for myelin synthesis. However, basic protein and myelin appear at similar rates (30). Brains may contain basic protein-like molecules of precursor molecules, analogous to proinsulin or fibrinogin, that are not detected by the antibody to the basic protein. It is difficult to imagine what advantage such a precursor molecule would confer. Our immunochemical studies provided little information about the possibility of precursor molecules. Although the antigen displacement curves changed during development, the change probably relates to differences in the proportions of the small and large basic proteins or the age-dependent methylation of arginine residues (15). Morphological studies have shown clearly that myelination occurs at specific times in various parts of the nervous system (16, 17). The morphological appearance of myelin correlates with the appearance of antibody-detectable basic protein described in the present report. In the rat spinal cord, Kornguth and Anderson (19) found essentially no change in the fluorescent antibody labeling of basic protein after Day 29 postnatally, indicating that by this age all tracts are at least partially myelinated. This agress with the finding reported there that 75-95% of the basic protein is present in the spinal cord by the twenty-fifth day. Thus, immunochemical measurements such as those for the basic protein can be coupled with morphological methods. In studies to be reported elsewhere, we have shown that the antigenic portion of the basic protein is phylogenetically conservative. Therefore,
VOLUME 49, 1976
the radioimmunoassay may be applied to a variety of species (15). More refined anatomical dissections and immunochemicalelectron microscopic measurements could provide powerful tools to correlate the physiological expression of a nerve tract with myelination. It may be possible to analyze pathological demyelination, artificially induced or naturally occurring, or genetic lesions such as the ‘ljimpy” and “quaking” mutations (21, 27, 31) by utilizing these techniques. This work was supported by U.S. Public Health Service Grants No. NS 10920 and NS 10465. The authors are grateful to R. Fingerhut for her technical assistance. S.R.C. is the recipient of a National Multiple Sclerosis Foundation Fellowship Award. REFERENCES 1. ADAMS, C. W. M., HALLPIKE, J. F., and BAYLISS, 0. B. (1971). J. Neurochem. 18, 14791483. 2. ADAMS, D. H., and OSBORNE, J. (1973). Neurobiology 3, 91-112. 3. BANIK, N. L., and DAVISON, A. H. (1974). Biothem. J. 143, 39-45. 4. BIGNAMI, A., and DAHL, D. (1975).Develop. Biol. 44, 204-209.
5. BOGOCH,S. (1969).In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 1, pp. 75-92. Plenum Press, New York. 6. BROSTOFF,S. W., KARKHANIS, Y. D., CARLO,D. J., REUTER, W., and EYLAR, E. H. (1975). Brain Res. 86, 44%458. 7. BUNGE, R. P. (1968).Physiol. Reu. 48,197-251. 8. CICERO,T. J., FERRENDELLIK,J. A., SUNTZEFF, U., and MOORE,B. W. (1972). J. Neurochem.
19, 21192125. 9. CLAUSEN, J. (1969). In “Handbook of Neurochemistry” (A. Lajtha, ed.) Vol. 1, pp. 273 300. Plenum Press, New York. 10. COHEN, S. R., MCKHANN, G. M., and GUARNIERI, M. (1975). J. Neurochem., 25, 371-376. 11. CUZNER,M. L., and DAVISON, A. N. (1968). Biothem. J. 106, 29-34. 12. EINSTIN, E. R., DALAL, K. B., and CSEJTEY,J. (1970). Brain Res. 18, 35-49.
13. ENG, L. F., CHAO, F. C., GERSTL,B., PRATT, D., and TAVASTJERNA,M. S. (1968). Biochemistry 7, 44554465. 14. FISCHER,C. A., and MORELL, P. (1974). Brain Res. 74, 51-65. 15. GUARNIERI,M., and COHEN, S. R. (1975). Brain Res. 100, 226-230. 16. JACOBSON,M. (1970). “Developmental Neurobiology,” pp. 174-180. Holt, Rinehart and
BRIEF NOTES Winston, New York. 17. JACOBSON, S. (1963). J. Comp. New. 121,5-29. 18. KIES, M. W., THOMPSON, E. B., and ALVORD, E. C. JR. (1965). Ann. N.Y. Acad. Sci. 122, 14b 160. 19. KORNGUTH, S. E., and ANDERSON, J. W. (1965). J. Cell. Biol. 26, 157-166. 20. LOWRY, 0. H., ROSEBROLJGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). J. Biol. Chem. 193, 265-275. 21. MATTHIEU, J.-M., QUARLES, R. H., WEBSTER, H. DEF., HOGAN, E. L., and BRADY, R. 0. (1974). J. Neurochem. 23, 517-523. 22. MORELL, P., GREENFIELD, S., CONSTANTINO-CECCARINI, E., and WISNIEWSKI, H. (1972). J. Neurochem. 19, 2545-2554. 23. MORI, S., and LEBLOND, C. P. (1970). J. Comp. Neur. 139, l-30. 24. NORTON, W. T., and PODUSLO, S. E. (1973) J. Neurochem. 21, 75%773. 25. RAGHAVAN, S. S., RHOADS, P. B., and KANFER,
26.
27. 28. 29. 30.
31. 32.
33. 34.
299
J. N. (1973). Biochem. Biophys. Acta 328,205212. ROYATTA, M., FREY, H., RIEKKINEN, P. J., LAUKSONEN, H., and RINNE, U. K. &per. Neurol. 45, 174-185. SIDMAN, R. L., DICKIE, M. M., and APPEL, S. H. (1964). Science 144, 309-311. SKOFF, R. P. (1975). J. Comp. Neur. 161,59&612. SMITH, M. E. (1973). J. Lipid Res. 14, 541-551. TENNEKOON, G., COHEN, S. R., PRICE, D. L., and MCKHANN, G. M. (1975). Trans. Amer. Sot. Neurochem. 6, 211. WATANABE, I., and BINGLE, G. J. (1972). J. Neuropath. Exp. Neurology 31, 352-369. WIGGINS, R. C., BENJAMINS, J. A., KRIGMAN, M. R., and MORELL, D. (1974). Bruin Res. 80, 345-349. WOOD, J. G., DAWSON, R. M. L., and HAUSER, H. (1974). J. Neurochem. 22, 637-643. ZGORZALEWICZ, B., NEUHOFF, U., and WAEHNELDT, T. U. (1974). Neurobiology 4, 266276.
BIOLOGY
lmmunochemical
49,
294-299
(1976)
Measurement
of Myelin Basic Protein
in Developing
Rat Brain: An Index of Myelin Synthesis STEVEN R. COHEN AND MICHAEL GUARNIERI Neurochemistry
Laboratories, Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205 Accepted October 10, 1975
The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue. INTRODUCTION
The developmental accumulation of basic protein in optic nerve (a tissue which is more than 60% myelin), the morphological appearance of myelin, the activity of an enzyme that synthesizes a myelin lipid, and the accumulation of the lipid in myelin occurred at virtually identical rates (30). Immunochemical measurements of basic protein thus furnish a precise and sensitive technique to measure myelin synthesis. Because oligodendroglial cells synthesize central nervous system myelin (reviewed in Ref. 71, the basic protein measurements also provide an index of oligodendrocyte activity.
Myelin contains a protein, the myelin basic protein, which has been monitored in several types of studies to gain information about the metabolism of the membrane (12-14, 22, 32). The effects of proteolytic enzymes on basic protein have been analyzed to gain information about the destruction of myelin during degenerative diseases (1, 3, 18, 25, 26, 33). Because quantitative measurements of basic protein required isolation of the membrane, such studies technically have been restricted to animals having appreciable amounts of myelin. For example, the yield of myelin from the brain of rats younger than 10 days is less than 400 pg/brain (24). Moreover, the composition of the membrane changes with age (2, 11, 21, 22, 24, 29, 34). Different fractions of myelin may be isolated during development or degeneration as the density of the membrane changes. Immunochemical measurements, because of their specificity, can be applied directly to proteins in untreated tissue or crude fractions (4, 8). We recently described a radioimmunoassay for basic protein and the conditions for its quantitative application to unfractionated tissue (10).
MATERIALS AND METHODS
Tissues. Pregnant SpragueDawley rats were obtained from Charles River (Boston, Mass.). Birth dates were recorded and the animals were sacrificed by decapitation at the appropriate age. The brain and spinal cord were exposed and dissected as follows: The olfactory bulb, cerebral hemisphere, and the area under the cerebral hemisphere were removed separately. The latter is designated as the midbrain. The cerebellum was removed and the tissue under it and to the first vertebrae was taken as the brain stem. The optic and 294
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
trigeminal nerves were removed from the base of the skull. The cervical spinal cord was removed from vertebrae 3-7, thoracic from B-20, and lumbar from 21-26. All tissues except optic and trigeminal nerve were weighed and 25% (w/v) homogenates were made in 0.2 M Tris-acetate buffer, pH 7.3. Optic and trigeminal nerves from single animals were homogenized in 0.5 ml of the Tris-acetate buffer. Radioimmunoassay. Samples from 0.5 pg (heavily myelinated areas) to 250 pg (younger brains) were analyzed by radioimmunoassay for myelin basic protein, as previously described (10). Protein was determined by the method of Lowry et al. cm. RESULTS
The amounts of basic protein in different areas of adult (120 day) rat brain and nerve are described in Table 1. Areas TABLE
1
CONCENTRATION OF MYELIN BASIC PROTEIN IN REGIONS OF ADULT RAT NERVOUS SYSTEM” Micrograms of myelin basic protein per milligram of tissue (wet weight) Central nervous system Cerebrum Cerebellum Olfactory bulb Midbrain Brain stem Cervical spinal cord Thoracic spinal cord Lumbar spinal cord Optic nerve Peripheral nervous system Trigeminal nerve Sciatic nerve
4.6 2.1 0.92 13.1 18.0 12.9 13.4 11.2 21.0 5.1 0.36
a Brains from 120-day-old rats were dissected and assayed for myelin basic protein as described (see Methods). The values represent the average of four tests, two determinations on two samples. Variations from the average were less than 20%. The values for wet weight of the optic, trigeminal, and sciatic nerves were estimated from protein determinations using a value of 10 mg of protein/100 mg wet weight.
295
which are predominantly gray, the olfactory bulb, cerebellum, and cerebrum, contain approximately three- to fourfold less basic protein than white matter areas, such as optic nerve, midbrain, brain stem, and spinal cord. The trigeminal nerve, which is myelinated by both oligodendroglial and Schwann cells, contained significantly more antibody-detectable basic protein than did the sciatic nerve, which contains only peripheral myelin. It is possible to calculate the amount of myelin in the different brain areas from the dry weight of neural tissue, approximately 18% for gray and 32% for white matter (9), and the amount of basic protein in myelin. The latter value has been estimated as 30% of the total protein of myelin (13, 221,and since myelin is approximately 30% protein (241, basic protein represents 10% of the dry weight of myelin. For example, cerebrum contains 4.6 pg of basic protein per milligram (wet weight) of tissue (Table 1). The cerebrum thus contains 46 pg of myelin/l80~g (dry wt) of tissue, or 25pgllOO pg of desiccated tissue. Myelin concentrations, as a percentage of total dry weight, range from 5% (olfactory bulb), 25% (cerebrum), to 70% (optic nerve). The values will vary, depending on the value for protein concentration of the neural tissue, a difficult figure to determine (51, and the proportion of basic protein in myelin, a value which increases with age, as shown by Morel1 et al. (22) in the mouse. In preliminary studies, basic protein was never detected in rat brain before birth; therefore, the present measurements were done with postnatal tissue. The antigenicity of the basic protein was nearly constant during development. The antigen-antibody displacement curves for basic protein in lo- and 120-day-old rats differed in slope by 12%, a difference caused by changes in the relative amounts of small and large basic proteins with age, or the number of methylated arginine residues (15). The rate of basic protein accumulation in various ratios of brain and
296
DEVELOPMENTAL BIOLIXY
spinal cord are shown in Figs. l-3. The weight of brain, its water content, and protein concentration change with development. The choice of a denominator against which the accumulation of a specific protein can be measured is diffkult. The data were arbitrarily expressed as the percentage of the adult (120 day) value to show the changes in brain from Day 10 to 20 (Fig. l), the period of rapid myelination, and from Day 20 to 40 (Fig. 21, a period at which myelin accumulates at a steady rate. As shown in Fig. 1, at Day 5 the greatest amount of basic protein is in the trigeminal nerve, approximately 0.25 j.kg/mg of nerve. The spinal cord contained less than 0.15 pg of basic protein; cerebrum, cerebel-
VOLUME 49, 1976
lum, and olfactory bulb contained less than 0.05 pglmg of tissue. There was no detectable basic protein in the midbrain, brain stem, or optic nerve. The trigeminal nerve is the first site for rapid deposition of the basic proteins between the fifth and ninth day. Optic nerves follow at Day 911, and finally, the cerebrum, cerebellum, olfactory bulbs, midbrain, and brain stem rapidly deposit myelin basic protein beginning on Day 12. The synthesis of basic proteins appeared to be biphasic in the trigeminal nerve with rapid deposition occurring at Days 59 and again between 25 and 42. The contribution of CNS and PNS myelin to trigeminal nerve basic protein remains to be determined. The sciatic nerve contains a pro-
Optic I’’ Nerve I’
I’ I’
80
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60-
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3 z 5 2 a”
50-
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,’
,’
,'
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x Trigeminal Nerve Cerebrum Cerebellum o’k”:bory o Midbrain Brainstem
?’ 8’ I’
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.’
IO Rat Age (Days)
15
20 21
FIG. 1. Appearance of myelin basic protein in the developing rat nervous system. Ages 52.1 days. Rat brains were dissected as described and assayed for myelin basic protein by radioimmunoassay. Each point represents the average of four tests, two determinations on two animals. Variations from the average were less than 20%. The results are expressed as a percentage of the 120-day value for the particular neural area.
297
BRIEF NOTES
spinal cord (Day 1520). By the forty-second postnatal day, the content of myelin basic proteins in the spinal cord, optic, and trigeminal nerve is nearly identical to that of the 120-day-old rat. The remaining brain areas contain from 90% (cerebellum) to 75% (brain stem) of the 120-day value for myelin basic protein. DISCUSSION
40
30 Rot
50
Aqe i Days)
2. Appearance of myelin basic protein in the FIG. developing rat nervous system, Days 21-42.
Rat Age ( Days 1 FIG. 3. Appearance of myelin basic protein in the developing rat spinal cord, Days 5-42.
tein with properties similar to CNS basic protein (6). The content of basic protein in other areas continues at a steady rate once the period of rapid synthesis begins. This rate is greatest in the optic nerve, which is almost fully myelinated by Day 21. The spinal cord also myelinates rapidly (Fig. 3). During development, the rate of basic protein accumulation is greatest in lumbar cord (Day 9-15) and in the cervical
The appearance of myelin basic protein in the rat central nervous system correlates with the morphological detection of myelin (17), the synthesis of myelin lipids (291, and the amount of myelin which can be isolated (24). This correlation is especially striking in the optic nerve. Our studies have shown parallel increases in basic protein and cerebroside sulfotransferase which synthesizes sulfatide, a characteristic lipid of myelin. The in vivo incorporation of radioactive sulfate into optic nerve sulfatide was also investigated by electron microscopic autoradiography (30). Myelin accumulated radioactive sulfatide at a rate which was virtually identical to the increased activity of cerebroside sulfotransferase and basic protein accumulation. These results demonstrate the close correlation between the appearance of basic protein and the synthesis of its membrane, myelin, the latter monitored both through the activity of an enzyme which synthesizes a myelin lipid and the incorporation of that lipid specifically into the membrane. It is possible to quantitatively relate basic protein and myelin synthesis, but the relations between myelin synthesis and oligodendroglial cell activities are less clear (23, 28). A single oligodendrocyte may myelinate more than one axon. Oligodendrocytes appear to synthesize myelin during all stages of their development (23). The chemical differences that have been described between immature and mature myelin (11, 22) may relate to developmental changes in the oligodendrocyte. Neurons may contribute to the synthesis of myelin. Indeed, it has not been definitely established that the basic protein is
298
DEVELOPMENTALBIOLOGY
synthesized by oligodendroglial cells. However, chemical measurements of specific cell fractions isolated from brains have shown that the specific concentration of basic protein is enriched 150-fold in oligodendrocytes compared to neuronal cell bodies (Cohen, S. R., unpublished results). Oligodendroglial cells synthesize three times their weight in myelin per day (24). Precursor pools of membrane components may be utilized for myelin synthesis. However, basic protein and myelin appear at similar rates (30). Brains may contain basic protein-like molecules of precursor molecules, analogous to proinsulin or fibrinogin, that are not detected by the antibody to the basic protein. It is difficult to imagine what advantage such a precursor molecule would confer. Our immunochemical studies provided little information about the possibility of precursor molecules. Although the antigen displacement curves changed during development, the change probably relates to differences in the proportions of the small and large basic proteins or the age-dependent methylation of arginine residues (15). Morphological studies have shown clearly that myelination occurs at specific times in various parts of the nervous system (16, 17). The morphological appearance of myelin correlates with the appearance of antibody-detectable basic protein described in the present report. In the rat spinal cord, Kornguth and Anderson (19) found essentially no change in the fluorescent antibody labeling of basic protein after Day 29 postnatally, indicating that by this age all tracts are at least partially myelinated. This agress with the finding reported there that 75-95% of the basic protein is present in the spinal cord by the twenty-fifth day. Thus, immunochemical measurements such as those for the basic protein can be coupled with morphological methods. In studies to be reported elsewhere, we have shown that the antigenic portion of the basic protein is phylogenetically conservative. Therefore,
VOLUME 49, 1976
the radioimmunoassay may be applied to a variety of species (15). More refined anatomical dissections and immunochemicalelectron microscopic measurements could provide powerful tools to correlate the physiological expression of a nerve tract with myelination. It may be possible to analyze pathological demyelination, artificially induced or naturally occurring, or genetic lesions such as the ‘ljimpy” and “quaking” mutations (21, 27, 31) by utilizing these techniques. This work was supported by U.S. Public Health Service Grants No. NS 10920 and NS 10465. The authors are grateful to R. Fingerhut for her technical assistance. S.R.C. is the recipient of a National Multiple Sclerosis Foundation Fellowship Award. REFERENCES 1. ADAMS, C. W. M., HALLPIKE, J. F., and BAYLISS, 0. B. (1971). J. Neurochem. 18, 14791483. 2. ADAMS, D. H., and OSBORNE, J. (1973). Neurobiology 3, 91-112. 3. BANIK, N. L., and DAVISON, A. H. (1974). Biothem. J. 143, 39-45. 4. BIGNAMI, A., and DAHL, D. (1975).Develop. Biol. 44, 204-209.
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