Proteins in cerebrospinal fluid and plasma of fetal rats during development

DEVELOPMENTALBIOLOGY83, 193-200 (1981)

Proteins in Cerebrospinal Fluid and Plasma of Fetal Rats during Development K. M. DZIEGIELEWSKA,C. A. N. EVANS,P. C. W. LAI,* F. L. LORSCHEIDER,*D. H. MALINOWSKA, K. MflLLG,~RD,t AND N. R. SAUNDERS

Department of Physiology and Centre for Neuroscience, University College London, Gower Street, London WCIE6BT, England; *Division of Medical Physiology, Faculty of Medicine, University of Calgary, Alberta, Canada; and tlnstitute of Anatomy A, University of Copenhagen, Copenhagen, Denmark Received May 27, 1980; accepted in revised form August 19, 1980 The concentrations of total protein, albumin, and a-fetoprotein have been measured in the cerebrospinal fluid (csf) and plasma of fetal (12 to 22 days gestation) and neonatal (0 to 10 days postnatal) rats. Total protein concentration in cisternal csf increased from about 140 rag/100 ml at 12 days to reach a peak of over 300 rag/100 ml around the time of birth. In the postnatal period the total protein concentration declined to about 100 rag/100 ml at 10 days; the adult value was 24 _+ 8 rag/100 ml. There was substantially more a-fetoprotein than albumin in csf at 12 days gestation. Both increased in concentration toward the end of gestation; a-fetoprotein reached a plateau of about 100 rag/100 ml at 17-19 days after which it declined markedly to about 5 rag/100 ml at 10 days postnatal; albumin reached a plateau in csf of about 90 rag/100 ml around the time of birth and declined subsequently. Albumin and a-fetoprotein constituted over 50% of the total protein concentration in csf at all fetal ages studied. In plasma these two proteins made up only 35% of the total protein at 17 days gestation but by the time of birth their contribution had doubled. Total protein concentration in plasma increased throughout the developmental period studied as did that of albumin, a-Fetoprotein was at its highest concentration (440 rag/100 ml) at 19 days gestation; it declined markedly in the postnatal period. Other proteins identified in csf and plasma were: transferrin, al-antitrypsin, and IgG. Evidence is discussed which suggests that the high concentration of protein in fetal csf results from specific transfer of plasma protein across the developing choroid plexus rather than from immaturity of the blood-csf barrier. INTRODUCTION

There have been several reports that the concentration of protein in the cerebrospinal fluid (csf) of the fetus is very high compared with that in the adult (see Saunders, 1977; and Dziegielewska et al., 1980a, for references). In all species so far investigated with the exception of the chick embryo (Birge et al., 1974), the csf protein concentration was highest in the earliest stages of development. The csf of the rat fetus has not been investigated although it has been reported that the csf protein concentration in the newborn rat is raised (Amtorp and S~lrensen, 1974). This paper presents results of estimation of total protein, albumin, and afetoprotein (AFP) together with immunological identification of several other proteins in csf and plasma from midgestation until the early postnatal period (term in the rat is 22 days). The results are discussed in relation to mechanisms of blood-csf exchange of plasma proteins during fetal development and its possible significance for brain development. MATERIALS AND METHODS

Female Sprague-Dawley rats were mated at known times and anesthetized 12 to 22 days later using sodium

pentobarbitone (6 mg/100 g body wt) given intraperitoneally. Two- and ten-day-old litters of postnatal rats were also used (anesthetized with ether). Clear csf, free of fetal blood contamination, was obtained by cisternal puncture using 23- to 30-gauge needles; in some fetuses csf was obtained by lateral ventricular puncture. Unless otherwise stated, results for csf refer to cisternal csf. Blood was collected from an umbilical artery or by direct heart puncture into heparinized syringes and then centrifuged. The csf and plasma samples were stored at -18~ Especially for the smaller fetuses it was necessary to pool samples from several fetuses in order to have enough material to make all the protein measurements. Because of sampling difficulties paired samples were not available from the smaller fetuses. Total protein concentrations were estimated using the method of Lowry et al. (1951). The standard was Versatol (William Warner & Co., Ltd., Eastleigh, Hampshire, England) which is a pooled human serum standard. Albumin concentrations were estimated by radial immunodiffusion assay (Mancini et al., 1965). An albumin reference was prepared by repurification of rat serum albumin (Miles) by chromatography on Blue Sepharose (Pharmacia Ltd.); it was standardized against Versatol

193 0012-1606/81/050193-08502.00/0 Copyright 9 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTAL ]~IOLOGY

using the method of Lowry et al. (1951). Antialbumin was raised in New Zealand White rabbits by serial intracutaneous injection of the repurified rat albumin mixed with Freund's incomplete adjuvant (Harboe and Ingild, 1973). The same immunisation procedure was used to raise anti-fetal rat serum for crossed immunoelectrophoresis of csf and plasma samples (see below). AFP concentrations were estimated by radioimmunoassay (Lai et al., 1976). Crossed immunoelectrophoretic plates of csf and plasma at each age were prepared according to the technique of Laurell (1965). The first dimension was run at 10 V/cm for 60 rain. The second dimension was run at 2 V/cm overnight using an intermediate gel containing 0.25 ml anti-adult rat serum (Dakopatts, Denmark) in 4 ml 1% agarose in barbitone buffer, pH 8.6, and a top gel containing 1.0 ml anti-fetal rat serum in 5 ml 1% agarose. The following specific antisera were used for the identification of individual proteins: anti-human transferrin and al-antitrypsin (Dakopatts); goat antipig a2-macroglobulin (kindly supplied by Dr. G. Kocsis); anti-rat IgG (Miles Laboratories); rabbit anti-rat afetoprotein. Statistical comparisons were made using Student's t test. RESULTS

Quantification of proteins in csf and plasma. The levels of total protein, AFP, and albumin in cisternal csf and plasma at different ages are shown in Table 1 and Fig. 1. In plasma the total protein concentration increased from 1584 _ 109 (SEM) mg/100 ml at 17 days gestation to 2883 + 93 mg/100 ml in the newborn and to 3988 + 72 mg/100 ml by 10 days after birth. The A F P concentration in fetal plasma was about 350 to 450 rag/ 100 ml between Days 17 and 22 but declined rapidly after birth. At 17 days the plasma albumin concentration was about 220 mg/100 ml (Table 1) but thereafter it increased throughout the rest of gestation and in the postnatal period. Albumin and A F P together made up only about 35% of the total protein in plasma at 17 days but by around the time of birth they contributed 6575% of the total protein. The cisternal csf concentration of total protein was 144 _+ 20 mg/100 ml at 12-13 days gestation after which it rose to reach a peak of over 300 mg/100 ml near the time of birth (Table 1, Fig. 1). In the postnatal period the total protein concentration in csf declined considerably by 10 days to 103 + 11 mg/100 ml; the adult value was 24 + 8 mg/100 mi. Total protein concentration in fetal ventricular csf at 15 days gestation was significantly (P < 0.05) higher than comparable cisterhal csf: 198 + 7 (n = 5) mg/100 ml compared with 171 + 6 (n = 12) mg/100 ml. There was substantially more

VOLUME83, 1981

A F P than albumin in csf from early in gestation (1213 days) until Day 20-21 when the concentrations were similar. The albumin in csf remained at around 90 m g / 100 ml until 2 days postnatal whereas the A F P had already begun to decline after 19 days gestation. In the postnatal period both proteins declined but AFP did so more rapidly (Fig. 1). AFP and albumin contributed approximately 55% of the total protein in csf at 12-17 days gestation; between 19 and 21 days the proportion increased to over 60% but from birth until at least 10 days postnatal, A F P and albumin made up less than 50% of the total protein in csf (Fig. 1). It is clear from the above results that although albumin and A F P contributed substantially to the total protein concentration in fetal csf and plasma, they by no means account for all of it, particularly not in early fetal plasma (17 days, Table 1,) and in late gestation and postnatal csf (Table 1, Fig. 1). Identification o f proteins in fetal csf . The protein composition of fetal and neonatal csf and plasma was further investigated using crossed immunoelectrophoresis (Laurell, 1965). This is illustrated in Figs. 2 and 4 for a range of ages. An intermediate gel containing antiadult rat serum was used together with a "top" gel containing anti-fetal rat serum for all fetal and neonatal samples. This allowed a clear distinction between "adult" and "fetal" plasma proteins. Also it improved the detection of some proteins in fetal samples because the anti-adult serum was stronger than the anti-fetal serum (probably because of the poor antigenecity of some fetal proteins (see Chism et al., 1978)). Using both the anti-adult and anti-fetal sera at 13 days gestation the csf could be shown to contain eight distinct peaks and several additional very faint ones (Fig. 2, i). The following proteins were identified by their cross-reactivity with specific antisera: albumin, AFP, transferrin, IgG, and al-antitrypsin. The quantitative changes in albumin and A F P have been described above. Transferrin was one of the largest peaks at 13 days and increased to a maximum by 22 days followed by a decline in the neonatal period (Fig. 2); it was still present in the adult (Fig. 3). al-Antitrypsin was only just detectable at 13 days gestation but also increased to a peak at 22 days followed by a decline in the neonatal period (Fig. 2). IgG was present at all ages examined (Fig. 2). There was one peak with a rather large area in an a~ position (alx in Fig. 2, i) that was prominent a t 12 to 15 days but declined subsequently. Another peak, designated "prealbumin" because of its mobility was clearly visible at 13 days and increased markedly after 15 days to reach a peak at 17 to 19 days after which it declined and became undetectable in the newborn and neonatal period (Fig. 2). It reappeared in the adult (Fig. 3). The other peak which was clearly apparent at 13

BRIEF NOTES

195

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FIG. l. Concentration of total protein, a - f e t o p r o t e i n and a l b u m i n in csf of fetal and p o s t n a t a l rats. Abscissa= age in days from conception (left side) or b i r t h (right side). Ordinate: p r o t e i n concentration in mg/lO0 ml. Mean -+ SE of mean.

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days (aox, Fig. 2) also increased markedly up to 22 days and then declined in the neonatal period. Its immunoelectrophoretic arc was very wide and rather fuzzy; the latter appearance is characteristic of glycoproteins. From this and its mobility it is suggested that it may be al-acid glycoprotein. Many other peaks which were indistinct or not apparent at 15 days appeared in csf over the last few days of gestation and generally increased in area up to 22 days gestation and subsequently declined in the neonatal period. One peak showed an unusual fluctuation (/~xin Fig. 2). At 13 days and even at 15 days (not shown) it was only very faint; it showed a marked increase in area by 17 days (Fig. 2, ii) followed by a decline such that it was barely detectable at 22 days. In the newborn csf it increased again with a further considerable increase by 2 days postnatal but was not detectable by 10 days postnatal. Although the total protein concentration in ventricular csf at 15 days gestation was significantly higher than in cisternal csf (see previous section) there was no marked difference in the crossed immunoelectrophoretic plates for csf from the two sites. Identification of proteins in fetal plasma. We were unsuccessful in obtaining blood samples uncontaminated by tissue or amniotic fluid earlier than at 17 days gestation. The immunoelectrophoretic pattern of plasma at this stage of development is shown in Fig. 4 (i). Subsequent changes in plasma proteins are shown in Fig. 4. a2-Macroglobulin was identified in plasma (Fig. 4, iii)

196

DEVELOPMENTAL BIOLOGY

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VOLUME83, 1981

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igG FIG. 2. Crossed immunoelectrophoretic plates of csf at different fetal and postnatal ages in the rat. The age in days from conception or birth is indicated on each plate. Undiluted csf (5 ~l) was used. The intermediate gel contained 0.25 ml anti-adult serum (in 4 ml of gel). The top gel contained 1.0 ml anti-fetal serum (in 5 ml of gel). a, Albumin; a, a-fetoprotein; pa, prealbumin; t, transferrin; IgG, immunoglobulin G; alat, al-antitrypsin.

BRIEF NOTES

197

FIG. 3. Crossed immunoelectrophoretic plates of csf and plasma of adult rat. The csf (5 ~1) was undiluted. The plasma (5 ~l) was diluted 20 times. The gel contained 0.25 ml anti-adult serum in 8 ml of gel.

by its cross-reactivity with anti-pig 0/2-macroglobulin. Clearly even by 17 days gestation there were many more proteins present in plasma than in csf; most of them showed an increase in peak area and distinctness of their precipitation lines with increasing gestational age. Most of the changes described in csf between 17 and 22 days gestation were paralleled by similar changes in plasma. Notable among these was the increasing prominence of transferrin and al-antitrypsin. The initial increase in "prealbumin" followed by its decline by 22 days and disappearance in the neonatal period was similar in csf and plasma. The ~x protein showed a pattern of change which was generally different from that in csf. Thus its peak area was approximately similar during the period 17 to 22 days although in csf it increased on the day of birth. Unlike csf, in plasma Bx had declined by 2 days postnatal but was still prominent at 10 days. The wide aox peak increased considerably to reach a maximum at 20 days gestation. In both csf and plasma this peak appears to be made up of two confluent components of different mobility. In csf the faster component was the more prominent whereas in plasma it was the slower component which was larger. DISCUSSION

The results presented in this paper show that the concentration of total protein in csf of the fetal rat is high as has been demonstrated for several other species (see Saunders, 1977; Dziegielewska et al., 1980a). In the other mammalian species the total protein concentration was highest at the earliest stage of gestation investigated. However, in the rat, there was a peak concentration of protein in csf at the time of birth, the

concentration increasing during the last few days of gestation and then declining after birth (Fig. 1, Table 1). The rat fetus was also strikingly different from the fetuses of other species in that there was a very large number of protein peaks in both csf and plasma toward the end of gestation. During the days before birth the protein peaks increased rapidly both in number and in area. The plasma concentration of AFP was maximum at 19-20 days gestation but declined rapidly in the postnatal period, as previously described by Lai et al. (1976). In most species a low level of AFP is reached well before the time of birth (see Gitlin and Boesman, 1967; Adinolfi and Haddad, 1977; Invarsson et al., 1978; Lai et al., 1978, Smith et al., 1979; Dziegielewska et al., 1980a,b). The only other species so far reported to have a rather high concentration of AFP in fetal plasma up until the time of birth is the rabbit (Branch, 1972). The relatively low concentration of albumin in fetal plasma especially at the earliest age (17 days) investigated does not appear .to have been reported before in the rat. Clearly AFP and albumin are important plasma proteins in the fetal rat. However, it is unclear which other proteins are quantitatively significant in fetal rat plasma especially around 17 days gestation when AFP and albumin together contribute only 35% of the total protein (Table 1). Most protein in plasma showed a rapid and progressive increase in peak area over the last few days of gestation. One of those in the 0/2 region presumably corresponds to the fetal rat Ra2-globulin reported by Gitlin and Boesman (1967) which they described as increasing from 13 days to a peak at the time of birth with a subsequent decline in the postnatal period. One protein showed a marked increase in concentration (as

198

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DEVELOPMENTAL BIOLOGY

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FIG. 4. Crossed immunoelectrophoretic plates of plasma at different fetal and postnatal ages in the rat. The age in days from conception or b i r t h is indicated on each plate. Plasma (1,1) was used. The intermediate gel contained 0.25 ml of anti-adult serum (in 4 ml of gel). The top gel contained 1.0 ml of anti-fetal serum (in 5 ml of gel). Symbols as in Fig. 2. ~2m, a2-Macroglobulin.

BRIEF NOTES

199

judged by the area of its peak in comparable crossed plasma proteins have been demonstrated. It is thought immunoelectrophoretic plates) just after birth com- that developing neurons acquire their intracellular pared with just before birth (/~x in Fig. 4). plasma proteins from csf during their early differenThe rat fetus appears to be similar to the sheep fetus tiation which takes place in the neuroepithelial cell in that the csf/plasma ratios for individual proteins in layer which lines the ventricular system (cf. Sidman both species fall throughout gestation (cf. Table 1 and and Rakic, 1973). However, the significance of these Dziegielewska et al., 1980a). But in the sheep fetus the proteins for neuronal differentiation and development total protein concentration in csf also falls throughout remains to be determined. gestation from at least as early as 35 days, whereas in the rat there is a rise in csf total protein during the REFERENCES last few days before birth. It is clear from Fig. 1 that AFP is not contributing to this final rise in csf protein ADINOLFI, M., and HADDAD, S. A. (1977). Levels of plasma proteins in the rat fetus since the AFP concentration in csf acin human and rat foetal c.s.f, and the development of the bloode.s.f, barrier. Neuropaediatrie 8, 345-353. tually falls at this stage of development (Table 1). AI~rORP, O. (1976). Transfer of II~5-albuminfrom blood into brain and The high concentration of protein in fetal csf has cerebrospinal fluid in newborn and juvenile rats. Acta Physiol. usually been attributed to immaturity of the bloodS~and. 97, 399-406. brain and blood-csf barriers to protein (e.g., Birge et AMTORP,0., and SORENSEN,S. C. (1974). The ontogenetic development al., 1974; Amtorp and S~lrensen, 1974; Adinolfi and Hadof concentration differences for protein and ions between plasma and cerebrospinal fluid in rabbits and rats. J. Physiol. 243, 387-400. dad, 1977; Ramey and Birge, 1979) although a reduced BENNO, R. H., and WILLIAMS,T. H. (1978). Evidence for intracellular sink effect due to a lower csf secretion rate in the imlocalization of alpha-fetoprotein in the developing rat brain. Brain mature brain has also been suggested to contribute Res. 142, 182-186. (Amtorp, 1976). However, the barrier to protein appears BIRGE, W. J., ROSE, A. D., HAYWOOD,J. R., and DOOLIN,P. F. (1974). to be well formed in fetal and newborn rats since maDevelopment of the blood-cerebrospinal fluid barrier to proteins and differentiation of cerebrospinal fluid in the chick embryo. Deture tight junctions have been observed between cerevelop. Biol. 41, 245-254. bral endothelial cells (Caley and Maxwell, 1970; M~lllBRANCH,W. R. (1972). The ontogeny of alpha-foetoprotein in the foeg~rd, Madsen, and Saunders, unpublished) and between tal and neonatal rabbit, and its experimental induction in adult choroid plexus epithelial cells (Tennyson, 1975). There rabbits. Int. J. Cancer 10, 451-457. is also direct evidence against immaturity of the blood- CALEY, W. D., and MAXWELL,D. S. (1970). Development of the blood vessels and extracellular spaces during postnatal maturation of rat brain barrier to fluorescein-labeled albumin in fetal cerebral cortex. J. Comp. Neurol. 138, 31-48. rats as early as 15 days gestation (Olsson et al., 1968). CHISM, S. E., BURTON,R. C., and WARNER,N. L. (1978). ImmunogeThis appears to conflict with the report of Amtorp nicity of oncofetal proteins: A review. Clin. Immunol. Immuno(1976) of an increased penetration of labeled human pa~hol. 11, 346-373. albumin from blood into brain of newborn compared DZIEGIELEWSKA,K. M., EVANS, C. A. N., FOSSAN, G., LORSCHEIDER, F. L., MALINOWSKA,D. H., M~LLGARD, K., REYNOLDS, M. L., with older rats. However, Amtorp did not attempt to SAUNDERS, N. R., and WILKINSON,S. (1980a). Proteins in csf and take account of contamination of brain samples by plasma of fetal sheep during development. J. Physiol. 300, 441-455. blood and csf; also his experiments were of much longer DZIEGIELEWSKA,K. M., KOCSIS, G., and SAUNDERS,N. R. (1980b). duration than those of Olsson et al. (1968). It seems Identification of fetuin and other proteins in cerebrospinal fluid and more likely that much of the high concentration of plasma of fetal pigs during development. Camp. Biochem. Physiol. B 66, 535-541. plasma protein in fetal csf can be accounted for by specific transfer across the choroid plexus as has been de- DZIEGIELEWSKA,K. M., EVANS,C. A. N., MALINOWSKA,D. H., M~LLG~.RD, K., REYNOLDS, M. L., and SAUNDERS,N. R. (1980c). Bloodscribed in the sheep fetus (Dziegielewska et al. 1980c). c.s.f, transfer of plasma proteins during fetal development in the A low turnover of csf may also be a contributory factor sheep. J. Physiol. 300, 457-465. (Woodbury et al., 1974) and this could explain the sus- DZIEOIELEWSKA,K. M., EVANS,C. A. N., MALINOWSKA,D. H., M@LLGARD, K., REYNOLDS,M. L., and SAUNDERS,N. R. (1980d). Distritained increase in one protein in csf (Fig. 2) at 2 days bution of plasma proteins in developing brain. J. Physiol., in press. postpartum when in plasma this peak had declined conGITLIN,D., and BOESMAN,M. (1967). Fetus-specific serum proteins in siderably (Fig. 4). several mammals and their relation to human ~-fetoprotein. Camp. Immunohistochemical studies have demonstrated inBiochem. Physiol. 21, 327-336. tracellular AFP (Benno and Williams, 1978; Trojan and HARBOE, N., and INGILD,A. (1973). Immunization, isolation of immunoglobulins,estimation of antibody titre. In "A Manual of QuanUriel, 1979) and albumin (Benno, personal communititative Immunoelectrophoresis," (N. H. Axelsen, J. Kroll, and B. cation; Trojan and Uriel, 1979) in fetal rat brain. SimWeeke, eds.), Chap. 23, pp. 161-164. Universitet sforlaget, Oslo. ilar observations have been made in human fetal brain INVARSSON, B. I., CARLSSON,R. N. K., and KARLSSON,B. W. (1978). (Mdllg~rd et al., 1979) and in sheep fetal brain (DzieSynthesis of a-fetoprotein, albumin and fetal serum protein in neogielewska et al., 1980d); in these species additional natal pigs. Biol. Neonate 34, 259-268.

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LAI, P. C. W., FORRESTER,P. I., HANCOCK,R. L., HAY, D. M., and LORSCHEIDER,F. L. (1976). Rat alphafetoprotein: Isolation, radioimmunoassay and fetal-maternal distribution during pregnancy. J. Reprod, Fert. 48, 1-8. LAI, P. C. W., MEARS, G. J., VAN PETrie, G. R., HAY, D. M., and LORSCHEIDER, F. L. (1978). Fetal-maternal distribution of ovine alpha-fetoprotein. Amer. J. PhysioL 235, E27-E31. LAURELL, J. C. (1965). Antigen-antibody crossed electrophoresis. Anal. B/ochem, 10, 358-361. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL,R. J. (1951). Protein measurement with the Folin phenol reagent. J. B/o/. C h e ~ 193, 265-275. MANCINI, G., CARBONARA,A. 0., and HEREMAN8, J. F. (1965). Immunochemical quantification of antigens by single radial immunodiffusion. Int. J. lmmunochem, 2, 235-254. MOLLG~,RD, K., JACOBSEN,M., JACOBSEN,G. K., CLAUSEN,P. P., and SAUNDERS,N. R. (1979). Immunohistechemical evidence for an intra-cellular localization of plasma proteins in human foetal choroid plexus and brain. Neurosci. Left. 14, 85-90. OLSSEN, Y., KLATZO,F., SOURANDER,P., and STEINWM,L, O. (1968). Blood-brain barrier to albumin in embryonic newborn and adult rats. Acta NeuropathoL 10, 117-122. RAMEY,B. A., and BIRGE,W. J. (1979). Development of cerebrospinal

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fluid and the blood-cerebrospinal fluid barrier in rabbits. Deve/op. B/o/. 68, 292-298. SAUNDERS,N. R. (1977). Ontogeny of the blood brain barrier. Exp. Eye Re~ 25 (Suppl.), 523-550. SIDMAN,R. L., and RAKIC,P. (1973). Neuronal migration, with special reference to the developing human brain: A review. Brain Re~ 62, 1-35. SMITH, K.M., LAI, P. C. W., ROBERTSON,H.A., CHURCH, R.B., and LORSCHEIDER, F.L. (1979). Distribution of alpha-l-fetoprotein in fetal plasma, allantoic fluid, amniotic fluid and maternal plasma of cows. J. Reprod. Fert. 57, 235-238. TENNYSON, V. (1975). Ultrastructural characteristics of the talencephalic and myelencephalic choroid plexus in fetus of man and rabbit, and a comparison with the adult choroid plexus in rabbit. In "The Choroid Plexus in Health and Disease" (M. G. Netsky and S. Shuangshoti, eds.), pp. 36-71. John Wright & Sons, Bristol. TROJAN, J., and URIEL, J. (1979). Localisation intracellulaire de l'alphafoetoprot6in et de la s6rumalbumine dans le syst~me nerveux central du Rat au cours du developpement foetal et postnatal. C. R. Acad, Sci. Paris 289, Serie D, 1157-1160. WOODBURY, D. M., JOHANSON,C., and BR~NSTED, M. (1974). Maturation of the blood-brain and blood-cerebrospinal fluid barriers and transport systems. In "Narcotics and Hypothalamus" (E. Zimmerman and R. George, eds.), pp. 225-247. Raven Press, New York.