Metabolism of cholesterol in developing rat optic and sciatic nerves

Journal oJ the neurological Sciences Elsevier Publishing Company, A m s t e r d a m - Printed in The Netherlands

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Metabolism of Cholesterol in Developing Rat Optic and Sciatic Nerves D. F. M A T H E S O N M.R.C. Research Group in Applied Neurobioloqy, Institute of Neurology, Queen Square, London, W.( ~! (Great lh'itain ) (Received 7 May, 1971)

INTRODUCTION

Numerous workers, in assaying the rate of myelin synthesis in brain, have repeatedly used the rate of uptake of labelled acetate as an index of lipid metabolism (Bloch 1948: Azarnoff. Curran and Williamson 1957: McMillan. Douglas and Mortensen 1957; Nicholas 1957: Rossiter 1957; Nicholas and Thomas 1958: Grossi. Paoletti and Paoletti 1958: Korey and Orchen 1959: Kabara and Okita 1961 : Hajra and Radin 1963: Pritchard 1963: Smith 19641. Reports on the investigation of lipid metabolism in isolated nerves have been restricted to the metabolism of the whole lipid-soluble fractions, either the chloroform methanol or the ethanol-ether fractions • no reports exist on the isolated myelin lipid component. This communication reports on the findings of the lipid synthetic activity of the isolated cholesterol fraction, as assessed by an in vitro technique, in the developing optic and sciatic nerves in the rat. MATERIAL AND METHODS Animals

Female rats of Porton strain of known ages were used. After weaning, they were fed on a diet of"Chardex'" {up to 15 17 g daily) and water ad libitum. Materials

Tissue culture medium "199" (Glaxo Laboratories, Greenford, Middlesex, EnglandJ was prepared from the concentrated form as previously described (Matheson 1970b}. Sodium[14C] acetate (uniformly labelled) specific activity (32.9 mCi/mmole) was purchased from the Radiochemical Centre. Amersham, Buckinghamshire, England. and was dissolved in distilled water; in most cases 3/~Ci were used for incubation. This work was financially supported by the Medical Research Council.

J. neuroL Sci., 1972. 15 : 77-87

78

D . F . MATHESON

Incubation procedure Rats of accurately known ages were killed by an overdose of ether and the optic and sciatic nerves were quickly excised. Up to 12 rats were used for each determination. The optic nerves were excised from the chiasma to their point of entrance into the optic bulb, whilst the sciatic nerves were taken from as high as possible in the thigh to their point of bifurcation into the popliteal nerves. The nerves were washed in the tissue culture medium "199", cleaned of extraneous tissue, and incubated separately at 37°C in 2.0 ml of"199'" medium which had previously been oxygenated with 95/5°.o oxygen:carbon dioxide and which contained 1.5 llCi/ml [14C]acetate (Matheson 1970b). Incubation was for 3 hr unless otherwise stated.

Extraction of the labelled cholesterol fraction After incubation, the nerves were drained for 3 5 sec on Whatman No. 1 filter paper, weighed to the nearest 0.1 mg, homogenised in a Griffith type tissue grinder in 10 - l 5ml of acetone : ethanol : ether (4 : 4: 1, by vol.). This was then heated to 60° C for at least 10 rain and left standing for a further 18 hr in an atmosphere of nitrogen at room temperature. The suspension was then filtered through fat-free filter paper (Whatman No. 1) and the filtrate retained for analysis. The precipitate was washed in a further 5 ml of the acetone : ethanol : ether mixture and the filtrate was added to the first. To the filtrate was added either a solution of tomatine in a mixture of water : glacial acetic acid :ethanol so that the volume added was 6 times the expected quantity of cholesterol in the sample or 1-2 ml digitonin (0.5~,, in 50~o ethanol). The cholesterol tomatinide was allowed to settle overnight before being separated by centrifugation at 1000 x 9 for 20 min. The precipitate was washed in a further 5-10 ml acetone : ethanol : ether mixture and recentrifuged. The cholesterol separated in this way represented the free cholesterol-containing fraction. To determine the amount of ester cholesterol, the filtrate obtained after the addition of the glycoside was concentrated to 3-4 ml, evaporated at room temperature with nitrogen and hydrolysed by 50% (w/v) methanolic K O H at 50°C for 1 hr. On neutralising the alkali extract with 10 o/acetic acid using phenolphthalein as indicator, the ester sterol was precipitated as the tomatinide as described above.

Determination of the spec!fic radioactivity of the cholesterolj~'action A known volume of glacial acetic acid was added to the tomatinide or digitonide cholesterol fraction and the solution heated to 50-60 ° C for 2 3 rain. An aliquot of the acetic acid solution was counted by scintillation counting using NE221 (Nuclear Enterprises Ltd, Edinburgh, Scotland) as the scintillation solvent. The tissue specific activity of the cholesterol fraction was expressed as disint./min per mg wet weight of the nerve.

Estimation of the cholesterol concentration in fi'esh nerves The concentration of cholesterol was estimated either by the method of Searcy, Berquist and Jung (1960) or by a modification of the Liebermann-Burchard reaction. Freshly excised nerves were blotted on W h a t m a n No. 1 filter paper for 3 5 sec J. neurol. Sci., 1972, 15:77 87

CHOLESTEROL IN D E V E L O P I N G RAT O P T I C A N D SCIATIC NERVES

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and weighed to the nearest 0.1 mg. They were then hand-homogenised in approximately 10 ml of ethanol : ether (3 : 1, v/v) solution for up to 20 min per sample in the case of the sciatic nerves (Friede and Hu 1967). The samples were then heated to boiling fo~~ 1 min in a water bath and kept at room temperature for 16--18 hr. The volume of each sample was then made up to 15,0 ml with ethanol, the residue separated by centrifugation at 1000 x 9 for 10 rain, and the supernatant was pipetted off. A known volume of the lipid-soluble fraction from each sample was allowed to evaporate at room temperature under nitrogen, when the method of Searcy et al. was used and 3.0 ml of a saturated solution of ferric sulphate-acetic acid reagent was added together with 0.75 ml concentrated sulphuric acid. The optical density was measured using a Unicam SP549 spectrophotometer at 490 nm with 4.5 silica cuvette.

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Fig. 1. The cholesterol content in optic nerves (A) and sciatic nerves (B) with development. The cholesterol concentration was measured by the method of Searcy et al. 1960 ( 0 ) and that of Kabara et al. I961 ( × and expressed as #g/rag wet weight of nerve. N o ester cholesterol was found in any of the nerve samples by the Liebermann-Burchard method (Kabara et al. 1961). Each point represents the mean of two samples.

The concentration of cholesterol was measuredagainst a standard solution of purified cholesterol (Koch-Light Ltd, Colnbrook, Bucks., England) and the results expressed as #g/mg wet weight of nerve. The Liebermann-Burchard reaction was performed as described by Kabara, McLaughlin and Riegel (1961). .1. neurol. ScL. 1972. 1 5 : 7 7 - 8 7

80

D. F. MATHESON RESULTS

Cholesterol concentration in developing optzc and sciatic nerves Analysis of the cholesterol content in developing rat optic and sciatic nerve showed that there was no difference in the amount of cholesterol precipitated using either tomatine or digitonin as the precipitating agent. During development, the amount of cholesterol in the optic nerve was found to rise gradually from approximately 13 #g/rag at 15 days to reach approximately 25 #g/rag in the adult (of367 days) (Fig. 1) : no ester cholesterol was found at any age investigated. These results support previous observations that throughout this period of development there is not only an increase in the number of myelinated fibres appearing (Matheson 1970a), but also corresponding change in the metabolic activities of the optic nerves (Matheson 1970b). A different pattern of cholesterol accumulation as assessed by the Liebermann Burchard reaction was apparent in the developing sciatic nerve. From 21 40 days there was approximately a 50 "~, rise in the sterol content ; after this age there was little change in the level (Fig. 1). As with the optic nerves, no ester cholesterol was found in any of the samples of the nerves analysed.

Uptake of[ 14C]acetate into the free cholesterol.[?action o(nerve Time. The incorporation of [-l ~C] acetate into the tomatine-precipitated fraction was found to be linear with respect to the time of incubation up to 6 hr in both the adult optic and sciatic nerves (Fig. 2).

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Fig. 2. Relationship between the rate of incorporation of [~4C]acetate into the cholesterol fraction and time of incubation. The optic (@) and sciatic ( × ) nerves from rats of known ages were incubated at 37"C in 2.0 ml of"199" medium containing 1.5/xCi/ml [14C]acetate for varying time intervals. The total incorporation was measured as disint./min per mg of nerve. Each point represents the mean of at least two readings and the bars (I) the range of deviation.

Concentration of[14C]acetate. The total tissue-specific radioactivity (disint./min per mg tissue) of the cholesterol fraction was found to be proportional to the concentration of [l*C]acetate in the incubation medium in both nerves (Fig. 3). For each nerve the line passed through the zero-zero axis. J. neurol. Sci., 1972, 15:77 87

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Fig. 3. Relationship between the total mcorporanon into the free cholesterol fraction and the concentration of the [14C] acetate. The optic I O) and the sciatic [ × ) nerves from rats of known ages were incubated at 37° C for 3 hr in 2.0 ml "'199" medium containing varying concentrations of [14C]acetate. Each point represents the mean value of at least two readings and the bars fI'J represent the range of deviation.

Total radioactivity ql'the jree cholesterol ]faction in relation to aye The total specific radioactivity of the precipitable digitonide or tomatinide fractions was found to fall sharply with development in both the optic and the sciatic nerves (Fig. 4). No difference was observed in the tissue-specific radioactivity using either precipitating agent. In the optic nerve the decline is most marked between the ages of 19 days and approximately 70-80 days : from 80 days onwards the decline is slow. eventually reaching the adult level at 367 days of only 1/30th of the radioactivity at t9 days. A similar pattern change in the uptake of the labelled acetate into the cholesterol fraction also occurred in the developing sciatic nerve. Thus the total specific radioactivity fell from 460 disint./min per mg at 19 days to approximately 1/15th of this value at 180 days (30 disint./min per mg) and reached a final figure of 3 disint. /min per mg in the adult. Relationship between the uptake of [14C]acetate into the JJ'ee cholesterol lraction and the concentration of cholesterol in the nerve During development of both the optic and the sciatic nerves, there was found to bc a decrease in the rate of incorporation of labelled acetate into the free cholesterol fraction concomitant with the increase in the concentration of cholesterol (Fig. 1). The concentration of cholesterol in the optic nerve was shown to be inversely proportional to the rate of uptake into the free cholesterol fraction : for such a relatibnship the correlation coefficient (r) is - 0.88 and the regression equation of y on x is : v=

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Fig. 4. Relationship between the total incorporation of [14C]acetate into the free cholesterol fraction and age in the optic and sciatic nerves. The optic (A) and sciatic (B) nerves of known ages of rats were incubated at 37~C in 2.0 ml "199" medium containing 1.5/~Ci/ml [14C]acetate for 3 hr. Up to 12 nerves were pooled for each reading and the values were the mean of at least two readings. The total incorporation was expressed as disint./min per mg of nerve.

Similarly, for the sciatic nerve, an inverse r e l a t i o n s h i p is also a p p a r e n t for these two p a r a m e t e r s , the c o r r e l a t i o n coefficient being - 0 . 7 7 a n d the regression e q u a t i o n of y on x is: y = - 31.2x + 866 N o difference was o b s e r v e d between the c o n c e n t r a t i o n of cholesterol present in fresh optic or sciatic nerves a n d nerves i n c u b a t e d for 3 hr. J. neurol. Sci., 1972, 15 : 77-87

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The uptake of [14C]acetate into.lree cholesterol.li'action in relation to the nuch'ar population Optic nerves. Calculation of the uptake of the labelled acetate into the free cho.lesterol fraction found in the optic nerve after incubation can be expressed against the nuclear population at different ages during development. These calculations are based on the data shown in Fig. 4A and on the findings of the total wet weight of the nervc and estimations of the nuclear population reported earlier (Matheson t970b). Such an expression, i.e. the total radioactivity of the free cholesterol fraction pef: nucleus (disint./min per nucleus), serves as an index of the rate of myelination in the developing nerve (Majno and Karnovsky 1958) and falls slowly from 19 days of age to reach approximately half this value at 214 days (Fig. 6A). During subsequent devclopment, the index continues to fall reaching 1/10th that found in the youngest animals (of 19 days). It appears, therefore, that myelination continues to an advanced age in the rat, confirming the findings previously described (Matheson 1970a, b). Sciatic nerves. The total rate of incorporation of the [~C]acetate into the free cholesterol fraction expressed per nucleus (disint./min per nucleus) in the sciatic nerve falls sharply during the early stages of development ; with later development the decline is less marked.

DISCUSSION

Digitonin as well as tomatine have been used extensively as precipitating agents for cholesterol in nervous tissue (Kabara 1954; Azarnoff et al. 1957 ; Davison, Dobbing, Morgan and Wright 1958 ; Davison, Morgan, Wadja and Wright 1959 : Davison and Wadja 1959; Kabara et al. 1961), but their relative efficiency regarding cholesterol isolation has not been compared. It was found, however, that no difference existed in either the concentration or the degree of recovery of the labelled cholesterol fractions when isolated by either glycoside. Moreover, although no further purification of the digitonide- or the tomatinide-precipitable fractions was routinely performed, thinlayer chromatographic analysis revealed that only cholesterol was detected (personal observation). The Liebermann-Burchard reaction for the quantitative estimation of the cholesterol from the fraction thus isolated has been severely criticised for its dependence upon temperature, its sensitivity to light and the instability of the measurable endproduct (Kabara 1954; Friede and Hu 1967). These criticisms, however, have been annulled using the method described (Kabara et all 1961). Comparison between the two methods of cholesterol determination in the developing optic nerve showed good agreement (Fig. 1). In this study no esterified cholesterol was found in the optic nerves at any of the ages investigated (14-367 days). Although esterified cholesterol has been found in the immature nervous system (Johnson, McNabb and Rossiter 1948 ; Adams and Davison 1960 ; Clarenberg, Chaikoffand Morris 1963 ; see Kabara 1967), its amount relative to the free cholesterol is always very small. The differential rate of incorporation of the [14C]acetate into the free cholesterol fraction with age also closely parallels the incorporation of the same precursor into J. neurol. Sci.,1972,15:77 87

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Fig. 5. Relationship between concentration and specific radioactivity of the free cholesterol fraction in the optic and sciatic nerves. The cholesterol fraction was determined by the method of Kabara et al. 1961 and the tissue-specific radioactivity obtained from Fig. 4 of rats of the same age. Twice the standard error of estimate is shown on either side of the regression lines. Note the different scales for the total incorporation r,~r the optic nerve (A) and the sciatic nerve (B).

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the lipid-soluble fraction of the optic nerve (Matheson 1970b) and the sciatic nerve (Majno and Karnovsky 1958). Moreover, the rate of uptake in ritro of labelled acetate into the tomatine-precipb table fraction in both nerves is proportional to. the concentration of the cholesterol il-~ the fraction of each nerve (Fig. 5). The significance of this finding has not yet beep~ evaluated. Various factors, however, may be responsible for this negative correlation. among these being the half-life of the free cholesterol at the various ages, the size and activity of the cholesterol pools and the degree of compactness of the myelin lamellac with age. The validity of this negative correlation assumes, however, that the proportion of the radioactivity of the true cholesterol remains constant throughout the age range considered. That the parameter of cholesterol concentration is valid is substantiated by comparing the two methods of cholesterol analysis and has been discussed. Calculation of the rate of myelination can be made from the myelin lipid activity and from the population of the myelin-forming cells, oligodendria and Schwann cells (Majno and Karnovsky 1958, Matheson 1970b). These accounts, however, deal with the rate of incorporation of radioactive acetate into the chloroform-methanol fraction in the sciatic nerve and into the ethanol ether fraction in the optic nerve respectively. Of the various lipids present in myelih, free cholesterol ~s well as sphingomyelin and cerebroside are indicative of myelination. Free cholesterol has the advantage of being easily and specifically extracted and determined. However, its disadvantages as ~:, myelin marker have been discussed elsewhere (Matheson 1971a). In the optic nerve, the radioactivity of the free cholesterol fraction, expressed against the total nuclear population, falls gradually with development (Fig. 6). That this index, i.e. the radioactivity of the free cholesterol fraction per nucleus, is a function of oligodendroglial actlwtx is suggested by Vaughn (19691, who found that in the ral optic nerve the number of oligodendrocytes, and also the ratio of these cells to the total nuclear population, remains constant after approximately 20 days of age In the sciatic nerve, however, this index declines fairly sharply during the initial stages of post-natal development and after about 50-60 days there ts a more gradual rate m the decline. The total nuclear population (from Matheson 1968} was again used m this calculation. Schwann cell nuclei constitute the largest part of the total nuclear population {Abercrombie and Johnson t946" Gamble 1964: Thomas 1969). The validity of this index in assessing the rate of myelination depends on there being no change in the proportion of Schwann cells and. as in the optic nerve, no change in the metabolic activity of the non-myelin bearing cell population. The evidence presented here appears to suggest thai whereas in the optic nerve the rate of myelination changes gradually during development and continues even at an advanced age, in the scmtic nerve the greatest degree of myelination occurs early in life and that. with progressive development, the rate slowly diminishes. The conclusion reached for the optic nerve agrees substantially with the histological and biochemical findings made on the metabolism of the total lipid fractions as previously described (Matheson 1970a. bl.

,1. neurol. Sci., 1972, 15 : 77--87

86

D. F. MATHESON

SUMMARY

Biochemical analyses were performed on post-natal developing rat optic and sciatic nerves. The cholesterol content was measured and was found to rise markedly in each nerve with age. Using an in vitro technique, [I,~C] acetate incorporation was measured in tomatineor digitonin-precipitated fractions. In both nerves, there was a sharp decline in the tissue-specific activity during post-natal development. An inverse relationship existed in each nerve for the concentration of cholesterol and the rate of uptake of the labelled tomatine- or digitonin-precipitable fraction. The ratio of radioactivity of this fraction to the total nuclear population declines in a characteristic manner in each nerve. The significance of this finding is discussed in relation to myelination.

ACKNOWLEDGEMENTS

I wish to thank Prof. J. B. Cavanagh for all his encouragement and advice and Miss R. Macfarlane for her skilled technical assistance in performing some of the experi= ments. REFERENCES ABERCROMBIE, M. AND M. L. JOHNSON (1946) Quantitative histology of Wallerian degeneration, Part 1 (Nuclear polulation in rabbit sciatic nerve), J. Anat. (Lond.), 80: 37-50. ADAMS, C. W. M. AND A. N. DAVISON (19603 The form in which cholesterol occurs in the adult CNS, J. Neurochem., 5: 293-296. AZARNOFF, D. L., G. g. CURRAN AND W. P. WILLIAMSON (19573 Incorporation of acetate-l-C 14 into cholesterol by human brain tumors, Fed. Proc., 16: 148. BLOCH, K. (1948)Biological synthesis of lipids, Cold Spring Harbor Symp. guam. Biol., 13:29 34. CLARENBERG, R., L. CHAIKOFFAND M. MORRIS (19633 Incorporation of injected cholesterol into myelinating brain of the 17 day old rat, J. Neurochem., 10: 135-142. DAV1SON, A. N. AND M. WADJA (19593 Metabolism of myelin lipids; estimation and separation of brain lipids in the developing rabbit, J. Neurochem., 4: 353-359. DAVISON, A. N., J. DOBB1NG, R. S. MORGAN AND G. PAYLING WRIGHT (1958) The deposition and disposal of (4-C x4) cholesterol in the brain of growing chickens, J. Neurochem., 3:89 94. DAV1SON,A. N., R. S. MORGAN. M. WADJA AND G. PAYLING WRIGHT (1959) Metabolism of myelin lipids ; incorporation of (3-C 14) serine in brain lipids of the developing rabbit and the persistence in the central nervous system, J. Neurochem., 4:360 365. F;'RIEDE,R. L. AND K. H. Hu (1967) Increase in cholesterol along the human optic nerve. J. Neurochem., 4: 307-315. GAMBLE, H. J. (19643 Comparative electron-microscopic observations of the connective tissue of a peripheral nerve and a spinal nerve root in the rat, J. Anat. (Lond.), 98: 17-25. GROSSI, E., P. PAOLETT1AND R. PAOLETTI (1958) An analysis of brain cholesterol and fatty acid biosynthesis, Arch. int. Physiol. Biochem., 66: 564-572. HAJRA, A. AND N. RADIN (19633 lsotopic studies of the biosynthesis of cerebroside fatty acids in rats, J. Lipid Res., 4: 270-278. JOHNSON,A. C., A. R. MCNABB AND R. J. ROSSITER(1948) Lipids of normal brain, Biochem. J., 43:573 577. JOHNSON, A. C., A. R. MCNABB AND R. J. ROSSITER(1949a) Concentration of lipids in the brain of infants and adults, Biochem. J., 44: 494498. JOHNSON, A. C., A. R. MCNABB AND R, J. ROSSITER (1949b) Chemical studies of peripheral nerve during Wallerian degeneration, Part 1 (Lipids), Biochem. J., 45: 500-508. KABARA, J. J. (19543 The light intensity of the Liebermann-Burchard reaction during spectrometric determination of cholesterol. J. Lab. olin. Med., 44: 246-249. J. neurol. Sci., 1972, 15:77 87

CHOLESTEROL IN DEVELOPING RAT OPTIC AN[) SCIATIC NERVES

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KABARA. J. J. (1967) Brain cholesterol: the effect of chemical and physical agents, Adr. Lipid Res., 3 279--387. KABARA, J. J. AND G. OKn'A (1961) Brain cholesterol: biosynthesis with selected precursors m tit,..~i Neurochem., 7:298 304. KABARA, J. J., J. T. McLAUGHLIN AND C. RIEGEL (1961) Quantitative microdetermination of cholestero~ using tomatine as the precipitating agent, Anal. Chem., 33 : 305 -307. KOREV, S. R. AND M. ORCHEN (1959) Plasmalagens of the nervous system. Deposition in developing rat brain and incorporation of C I"~ isotope from acetate and palmitate into unsaturated ether chain. Arc/i Biochem., 83 :' 381--389. MCMILLAN, P. J., G. W. DOUGLAS AND R. A. MORTENSEN (1957) Incorporation of acetate-l-C 1~ and pyruvic-2-C ~4 into brain cholesterol in the intact rat, Proc. Soc. exp. Biol. ~N. Y.), 96:738 740. MAJNO, G. AND M. L. KARNOVSKY(1958) A biochemical and morphological study of myelination and demyelination, Part 1 (Lipid biosynthesis in vitro by normal nervous tissue), J. exp. Med, 107: 476-495. MANDEL, P., R. BIETH AND R. STOLE (1949) La r6partition des divers fraction s lipidiques dans le cerveaux de l'embryon de poulet durant la seconde partie de l'incubation, C. R. Soc. Biol. (Paris), 143 : 1224-1226. MATHESON,D. F. (1968) Stud)' of the Amino-acid Incorporation into Peripheral Nerve, Doctoral thesis, University of London. MATHESON, D. F. (1970a) Some quantitative aspects of myelination of the optic nerve in rat, Brain Research, 24:257 269. MATHESON, D. F. (1970b) Some aspects of lipid and protein metabolism in developing rat optic nerves_ Brain Research, 24: 271-283. MATHESON, D. F. (1971) Evidence in support of centripedal gradient in myelination in rat optic nerve~, Exp. Neurol., 32: 195-205. NICHOLAS, H. J. (1957) Biosynthesis of cholesterol in the central nervous system, l~?d. Proc., 16:324 3 2 5 NICHOLAS, H. J. AND B. THOMAS (1958) Biosynthesis of cholesterol and fatty acids in the adult rat brain. Fed. Proc., 17 : 450. PRITCHARO, E. (1963) The tbrmation of phospholipids from labelled C ~4 precursors in developing rat brain in ritro, J. Neurochem., 10:495 502. ROSSITER, R. (1957) Lipid metabolism. In: D. RICHTER tEd.), Metabolism of the Nervou,~ System, Pergamon, London, pp. 355 379. SEARCY, R. L., L. M. BERQUISTAND R. C. JUNG (1960) Rapid ultramicro-estimation of serum total cholesterol, J. Lipid Bes., 1 : 349-351. SMt'~H, M . E . (1964) Lipid biosynthesis in the central nervous system in experimental allergic encephalomyelitis, J. Neurochem., 11 : 29-37. SPERRY, W. M. (1955) Lipids of the brain during early development. In: H. WAELSCH ted.), Biochemlstrl of the Developin 9 Nervous System, Pergamonl London, pp. 261 267. THOMAS, P. K. (1969) The connective tissue of peripheral nerve: an electron microscopic study, J. Anat (Lond.), 97: 35-44. VAUGHN, J. E. (1969) An electron microscopic analysis of gliogenesis in rat optic nerves, Z Zet(fim~ch., 94:293-324.

J. neurol. Sci., 1972, 15:77-87