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Studies on choline transport and incorporation into lecithin of synaptosomes of developing rat brain
224
SHORT COMMUNICATIONS
Studies on choline transport and incorporation into lecithin of synaptosomes of developing rat brain The transport processes across the limiting membrane of synaptosomes have been studied in the past by several investigators and are well established for the uptake of cholinell, 19, tryptophan 13, noradrenaline 9, sodium 16 and potassiumS, 12. Previous studies on Na+-K+-stimulated, ouabain-sensitive ATPase 3 and Na ÷ uptake ~ into synaptosomes of the developing rat brain showed the highest level of N a + - K +ATPase activity and maximal accumulation of Na ÷, in the presence of ouabain, to occur during the period between 12-20 days after birth. The present studies on the permeability and incorporation of [Me-a4C]choline into lecithin of synaptosomes of the developing brain were undertaken in the hope of throwing more light on the relationship between changes in permeability and metabolism of the neuronal membrane and the period when functional changes are formed in rat brain. Rats at ages 5 days to adult (older than 80 days) were sacrificed, their cerebra removed and homogenized in 0.25 M sucrose. The synaptosomes were obtained by subfractionating the crude mitochondrial fraction by means of discontinuous densitygradient centrifugation in a sucrose-Ficoll medium as described previously 1. However, in the present work the crude mitochondrial fraction was washed once with 0.25 M sucrose before layering on the gradient. The incubation medium for [14C]choline uptake into synaptosomes was essentially similar to that described by Diamond and Kennedy 11 except that 2 m M Mg 2+ and 100 m M Na + were added to the uptake medium in a final volume of 0.5 ml. After incubation at 37°C for 10 rain the uptake mixture was passed over columns (10 cm × 0.5 cm) filled with Amberlite IRC-50 (Na ÷) cation exchange resin (25-50 mesh). The eluted synaptosomes were homogenized in 9 ml of water and the proteins were determined 17 on an aliquot portion of the homogenate. The remainder was centrifuged at 81,495 × g for 30 min and the radioactivity of the supernatant was measured. The supernatant was lyophilized then analyzed by means of paper chromatography and paper electrophoresis, in 0.2 M acetate buffer, p H 4.5, and in general most of the radioactivity was recovered in choline and much lesser activity was found in phosphorylcholine and acetylcholine. Diamond and Kennedy ~1 showed that after entry into synaptosomes, 60% of the radioactivity could be recovered as choline, 11% as phosphorylcholine, 10% as betaine and less than 5 % as acetylcholine. The specific radioactivity was calculated as counts/min/mg of synaptosomal proteins. Incubations for [Me-14C]choline incorporation into lecithin were carried out aerobically in Erlenmeyer flasks (25 ml capacity) for 2 h with shaking in a water bath shaker at 37°C. In general, synaptosomes, suspended in 1 ml of 0.45 M sucrose and corresponding to 2.5-5 mg protein, were added to an incubation medium which consisted of 33.3 m M potassium phosphate buffer, p H 7.4, 20 m M sodium pyruvate, 1.2 m M sodium malate, 5 m M MgC12 and 2 #Ci of [Me-14C] choline (525 nmoles) in a final volume of 3.0 ml. Lipids were extracted with chloroform-methanol-HC1 (200 : 100 : 1) and lecithin was isolated by means of two-dimensional T L C and its specific radioactivity determined as previously described 4. Choline uptake was investigated in synaptosomes from rats at 3 developmental Brain Research, 31 (1971) 224--228
SHORT COMMUNICATIONS
225
7xlO5 ,
{/) Z t~J 0~
6xlO 3 •
5xlO 3 •
12.
<~ 4xlO5" 0
Z
m
2x103.
~E ck
i~103 -
0
5
I0
15 AGE
20
II
ADULT
(doys)
Fig. 1. [Me-14C]Choline uptake into synaptosomes of developing rat brain in absence of added agents (curve A) and in the presence of unlabeled choline (curve B) or hemicholinium-3 (curve C). Curves B and C represent the specific choline uptake. They were obtained by subtracting the choline which leaked into the synaptosomes in the presence of unlabeled choline or hemicholinium from the total choline uptake (curve A). Incubation medium contained about 2 mg of synaptosomes obtained from 5-day, 17-day or adult rats, in sucrose (0.32 M); [Me:4C]choline (0.35/~Ci, equivalent to 0.15 mM); physostigmine sulfate (0.1 mM), 2-mercaptoethanol (2.5 raM); inorganic phosphate (5 mM); potassium (15 mM); glycine buffer (50 mM) of pH 9.0; MgCI~(2 mM) and NaCI (100 mM) in a final volume of 0.5 ml. Each point on the curves represents a different experiment and each experiment was run in duplicates. stages, namely at 5 days of age (prior to active myelination), 17 days of age (during active myelination) and adult (Fig. 1). While the total choline uptake into synaptosomes from 17-day-old rats was only slightly higher than that of the 5-dayold, it was more than twice as high as that of the adult (Fig. 1 A). These differences in choline uptake with brain maturation become more significant when the specific choline uptake 11, which was mostly inhibited by 15 m M unlabeled choline and to a lesser extent by 0.15 m M hemicholinium (Fig. 1 B and C respectively), is plotted against age. A higher concentration of hemicholinium than the 0.15 m M used in the present work is probably required in order to achieve the same inhibitory effect on the choline transport system in synaptosomes, obtained by 15 m M unlabeled choline. However, further work on the differences and similarities between the effect of high concentrations of unlabeled choline and hemicholinium on the specific and nonspecific transport of choline in synaptosomes is needed before any definitive conclusion can be made on the differences obtained in the present work by both pharmacological agents on the choline transport system (Fig. 1 B and C). The specific choline uptake into synaptosomes from the 5-day-old and adult, when reported as percentage of choline uptake in the 17-day-old, is 85 ~ and 3 6 ~ in the presence of unlabeled choline and 5 5 ~ and 49~o in the presence of hemicholinium respectively (Fig. l). These data Brain Research, 31 (1971) 224-228
226
SHORT COMMUNICATIONS
TABLE I In vitro INCORPORATION
OF [ME-14C]CHOLINE INTO LIPIDS OF DEVELOPING RAT BRAIN SYNAPTOSOMES
UNDER UPTAKE CONDITIONS
Incubation conditions are as described under Fig. 1 and text for uptake of [14C]choline. Total lipids were extracted from the lysed synaptosomes after recovery from the Amberlite IRC-50 column. Values represent specific radioactivities for total lipids and are mean for the number of experiments quoted. Each experiment was run in duplicate and the values for the different experiments agreed within 0-7 ~. Additions
Specific radioactivity (counts/min/ktmole PO Age (days)
None Choline (15 raM) Number of experiments
5
17
Adult
319 215 3
460 133 2
397 195 2
suggest that while the carrier-mediated transport of choline increases with brain maturation, the nonspecific transport (see ref. 11) decreases and the sum of both systems (total transport) is highest during the stage of active myelination. Following this rise in the 17-day-old the total transport drops considerably in the adult (Fig. 1 A). In this connection Lajtha 15, working in vivo with the uptake of 36-chloride and thiocyanate by developing brain showed that the rate of uptake by the brain of the two anions decreases with increasing age of the animal. Furthermore Lahiri and Lajtha 14 showed that changes in uptake by brain slices during development are not uniform among the amino acids. Thus these authors observed that the L- and D-isomers of lysine were taken up by slices from newborn brain to a greater extent than by slices from adult brain, while the opposite was true for other amino acids. Under the present uptake conditions, choline is also incorporated into lipids (Table I) with more than 95 ~ of the lipid radioactivity recovered in lecithin, about 4 - 5 ~ in lysolecithin and a negligible amount was found in sphingomyelin. Again maximal incorporation occurred in synaptosomes from the 17-day-old rats. The finding that maximal inhibition (71 ~ ) occurred in the presence of unlabeled choline could be due to the higher uptake of the latter during this period of development as was shown in the permeability studies (Fig. 1B). This observation was confirmed through experiments in which the synaptosomes were incubated for a longer period of time and under conditions of oxidative phosphorylation (Fig. 2). During the period of active myelination, [14C]choline incorporation into lecithin was 12 times as high as that of the adult and almost 3 times that of the 3-day-old rats (Fig. 2). Further studies on [14C]choline incorporation into lecithin showed it to be stimulated by malate plus pyruvate, 1 m M Ca 2+ and inhibited by 1 m M serine. The rapid rise in lecithin labeling correlates well with the changes in choline transport into the synaptosomes during brain growth and development observed in the present studies and is in accord with our previous studies in which the highest rate of N a + uptake 5 and a2Pi incorporation Brain Research, 31 (1971) 224-228
SHORT COMMUNICATIONS
227
~4xlO3~ 12xlO E c:
>, -~. o
IOx107 8xl
1
,~ 4x I011
2xl03l 0~
I0
20
30
40
50
60
I
I-
Adult
Age (doys)
Fig. 2. Effect of age on specific radioactivity of lecithin. Synaptosomes from rat cerebra at different ages and equivalent to 3.8 mg protein were incubated as described in the text. Lecithin was isolated from the lipid extract by means of two-dimensional TLC. The first solvent system consisted of: chloroform-methanol-acetic acid-water (25 : 15:4:2, v/v) and the second of: the upper phase of: nbutanol-pyridine-water (45 : 5 •20, v/v). Lecithin was eluted and its specificradioactivity determined as described previously4. Each point on the curve represents two different experiments and each experiment was run in duplicates. The values for the different experiments agreed within 0-5 %.
into phospholipids 6 of synaptosomes was observed during the period between the 12th and 20th day post partum. It is possible that the findings of several investigators 2,7,1°,2° on the rise of lipid synthesis during the period of active myelination could be partly due to an increase in the activities of the transport systems of their respective precursors, followed by an increase in the permeability of the neuronal membranes to the various lipid precursors. This could result in an increase in the incorporation of the latter into their respective phospholipids. However, the rise in phospholipid synthesis during this period of development could also be due to an increase in the activities o f the enzymes involved in their biosynthesis. Thus Aeberhard et al.7 showed maximal elongation of fatty acids in both microsomes and mitochondria from rat brain to occur around 15 days after birth, and Maker and Hauser Is working with rat brain slices showed the biosynthesis of gangliosides and cerebrosides, by using [U-14C]glucose as precursor, to occur at a maximum rate at about 11-12 days after birth. Thus the rise in lipid synthesis, both in vivo and in vitro, during the period when functional changes are formed could be due to an increase in the transport systems for their precursors as well as in the enzymes involved in their biosynthesis. The authors wish to thank Gerald Elwinger and Robert Frothingham for technical assistance. Brain Research, 31 (1971) 224-228
228
SHORT COMMUNICATIONS T h e s e studies
were supported
by R e s e a r c h
Grant
NS-07876-04
from
the
National Institute of Neurological Diseases and Stroke.
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Ga. 30902 (U.S.A.)
ATA A. ABDEL-LATIF JACK P. SMITH
1 ABDEL-LATIF,A. A., A simple method for isolation of nerve-ending particles from rat brain, Biochim. biophys. Acta ( Amst.), 121 (1966) 403-406. 2 ABDEL-LATIE,A. A., AND ABOOD, L. G., Incorporation of a2p into phospholipids and phosphoproteins of cytoplasmic fractions of developing rat brain, J. Neurochem., 12 (1965) 157-166. 3 ABDEL-LATIF,A. A., BRODY,J., ANDRAMAHI,H., Studies on Na+-K+-ATPase of the nerve endings and appearance of electrical activity in developing rat brain, J. Neurochem., 14 0967) 1133-1141. 4 ABDEL-LATIF, A. A., AND SMITH, J. P., In vivo incorporation of choline, glycerol and orthophosphate into lecithin and other phospholipids of subcellular fractions of rat cerebrum, Biochim. biophys. Acta (Amst.), 218 (1970) 134-140. 5 ABDEL-LATIF,A. A., YAMAGUCHI,M., SMITH, J., AND YAMAGUCHI,T., Studies on the effect of ouabain on sodium and phosphate uptake into nerve endings of developing rat brain, Life Sci., 7 (1968) 1325-1338. 6 ABDEL-LATIF,A. A., YAMAGUCHI,T., YAMAGUCHI,M., AND CHANG, F., Studies on [a2P]orthophosphate incorporation into nucleotides, phospholipids and phosphoproteins of isolated nerve endings from developing rat brain, Brain Research, 10 (1968) 307-321. 7 AEaERHARD, E., GRIPPO, J., AND MENKES, J. H., Fatty acid synthesis in the developing brain, Pediat. Res., 3 (1969) 590-596. 8 BRADFORD, H. F., Respiration in vitro of synaptosomes from mammalian cerebral cortex, J. Neurochem., 16 (1969) 675-684. 9 COLBURN, R. W., GOODWIN, E. K., MURPHY, D. L., BUNNEY, W. E., JR., AND DAVIS, J. M., Quantitative studies of norepinephrine uptake by synaptosomes, Biochem. Pharmacol., 17 (1968) 957-964. 10 DAVISON,A. N., AND DOBBING,J., The developing brain. In A. N. DAVISONANDJ. DOBmNG(Eds.), Applied Neurochemistry, Davis, Philadelphia, 1968, pp. 253-286. 11 DIAMOND, I., AND KENNEDY, E. P., Carrier-mediated transport of choline into synaptic nerve endings, J. biol. Chem., 244 (1969) 3258-3263. 12 ESCUETA,A. V., AND APELL, S. H., Biochemical studies of synapses in vitro. II. Potassium transport, Biochemistry, 8 (1969) 725-733. 13 GRAHAME-SMITH,D. G., AND PARE1TT, A. G., Tryptophan transport across the synaptosomal membrane, J. Neurochem., 17 (1970) 1339-1353. 14 LAmRI, S., AND LAJTHA, A., Cerebral amino acid transport in vitro. I, J. Neurochem., 11 (1964) 77-86. 15 LAJTHA, A., The development of the blood-brain barrier, J. Neurochem., 1 (1957) 216-227. 16 LING, C.-M., AND ABDEL-LATIF,A. A., Studies on sodium transport in rat brain nerve-ending particles, J. Neurochem., 15 (1968)721-729. 17 LOWRY,O. H., ROSESROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 MAKER, H. S., AND HAUSER,G., Incorporation of glucose carbon into gangliosides and cerebrosides by slices of developing rat brain, J. Neurochem., 14 (1967) 457J,64. 19 MARCHBANKS,R. M., The uptake of [14C]choline into synaptosomes in vitro, Biochem. J., 110 (1968) 533-541. 20 SPERRY,W. M., The biochemistry of the brain during early development. In K. A. C. ELLIOTT,I. H. PAGE AND J. H. QUASTEL(Eds.), Neurochemistry, Thomas, Springfield, I11., 1962, pp. 55-84. (Accepted May 14th, 1971)
Brain Research, 31 (1971) 224-228
SHORT COMMUNICATIONS
Studies on choline transport and incorporation into lecithin of synaptosomes of developing rat brain The transport processes across the limiting membrane of synaptosomes have been studied in the past by several investigators and are well established for the uptake of cholinell, 19, tryptophan 13, noradrenaline 9, sodium 16 and potassiumS, 12. Previous studies on Na+-K+-stimulated, ouabain-sensitive ATPase 3 and Na ÷ uptake ~ into synaptosomes of the developing rat brain showed the highest level of N a + - K +ATPase activity and maximal accumulation of Na ÷, in the presence of ouabain, to occur during the period between 12-20 days after birth. The present studies on the permeability and incorporation of [Me-a4C]choline into lecithin of synaptosomes of the developing brain were undertaken in the hope of throwing more light on the relationship between changes in permeability and metabolism of the neuronal membrane and the period when functional changes are formed in rat brain. Rats at ages 5 days to adult (older than 80 days) were sacrificed, their cerebra removed and homogenized in 0.25 M sucrose. The synaptosomes were obtained by subfractionating the crude mitochondrial fraction by means of discontinuous densitygradient centrifugation in a sucrose-Ficoll medium as described previously 1. However, in the present work the crude mitochondrial fraction was washed once with 0.25 M sucrose before layering on the gradient. The incubation medium for [14C]choline uptake into synaptosomes was essentially similar to that described by Diamond and Kennedy 11 except that 2 m M Mg 2+ and 100 m M Na + were added to the uptake medium in a final volume of 0.5 ml. After incubation at 37°C for 10 rain the uptake mixture was passed over columns (10 cm × 0.5 cm) filled with Amberlite IRC-50 (Na ÷) cation exchange resin (25-50 mesh). The eluted synaptosomes were homogenized in 9 ml of water and the proteins were determined 17 on an aliquot portion of the homogenate. The remainder was centrifuged at 81,495 × g for 30 min and the radioactivity of the supernatant was measured. The supernatant was lyophilized then analyzed by means of paper chromatography and paper electrophoresis, in 0.2 M acetate buffer, p H 4.5, and in general most of the radioactivity was recovered in choline and much lesser activity was found in phosphorylcholine and acetylcholine. Diamond and Kennedy ~1 showed that after entry into synaptosomes, 60% of the radioactivity could be recovered as choline, 11% as phosphorylcholine, 10% as betaine and less than 5 % as acetylcholine. The specific radioactivity was calculated as counts/min/mg of synaptosomal proteins. Incubations for [Me-14C]choline incorporation into lecithin were carried out aerobically in Erlenmeyer flasks (25 ml capacity) for 2 h with shaking in a water bath shaker at 37°C. In general, synaptosomes, suspended in 1 ml of 0.45 M sucrose and corresponding to 2.5-5 mg protein, were added to an incubation medium which consisted of 33.3 m M potassium phosphate buffer, p H 7.4, 20 m M sodium pyruvate, 1.2 m M sodium malate, 5 m M MgC12 and 2 #Ci of [Me-14C] choline (525 nmoles) in a final volume of 3.0 ml. Lipids were extracted with chloroform-methanol-HC1 (200 : 100 : 1) and lecithin was isolated by means of two-dimensional T L C and its specific radioactivity determined as previously described 4. Choline uptake was investigated in synaptosomes from rats at 3 developmental Brain Research, 31 (1971) 224--228
SHORT COMMUNICATIONS
225
7xlO5 ,
{/) Z t~J 0~
6xlO 3 •
5xlO 3 •
12.
<~ 4xlO5" 0
Z
m
2x103.
~E ck
i~103 -
0
5
I0
15 AGE
20
II
ADULT
(doys)
Fig. 1. [Me-14C]Choline uptake into synaptosomes of developing rat brain in absence of added agents (curve A) and in the presence of unlabeled choline (curve B) or hemicholinium-3 (curve C). Curves B and C represent the specific choline uptake. They were obtained by subtracting the choline which leaked into the synaptosomes in the presence of unlabeled choline or hemicholinium from the total choline uptake (curve A). Incubation medium contained about 2 mg of synaptosomes obtained from 5-day, 17-day or adult rats, in sucrose (0.32 M); [Me:4C]choline (0.35/~Ci, equivalent to 0.15 mM); physostigmine sulfate (0.1 mM), 2-mercaptoethanol (2.5 raM); inorganic phosphate (5 mM); potassium (15 mM); glycine buffer (50 mM) of pH 9.0; MgCI~(2 mM) and NaCI (100 mM) in a final volume of 0.5 ml. Each point on the curves represents a different experiment and each experiment was run in duplicates. stages, namely at 5 days of age (prior to active myelination), 17 days of age (during active myelination) and adult (Fig. 1). While the total choline uptake into synaptosomes from 17-day-old rats was only slightly higher than that of the 5-dayold, it was more than twice as high as that of the adult (Fig. 1 A). These differences in choline uptake with brain maturation become more significant when the specific choline uptake 11, which was mostly inhibited by 15 m M unlabeled choline and to a lesser extent by 0.15 m M hemicholinium (Fig. 1 B and C respectively), is plotted against age. A higher concentration of hemicholinium than the 0.15 m M used in the present work is probably required in order to achieve the same inhibitory effect on the choline transport system in synaptosomes, obtained by 15 m M unlabeled choline. However, further work on the differences and similarities between the effect of high concentrations of unlabeled choline and hemicholinium on the specific and nonspecific transport of choline in synaptosomes is needed before any definitive conclusion can be made on the differences obtained in the present work by both pharmacological agents on the choline transport system (Fig. 1 B and C). The specific choline uptake into synaptosomes from the 5-day-old and adult, when reported as percentage of choline uptake in the 17-day-old, is 85 ~ and 3 6 ~ in the presence of unlabeled choline and 5 5 ~ and 49~o in the presence of hemicholinium respectively (Fig. l). These data Brain Research, 31 (1971) 224-228
226
SHORT COMMUNICATIONS
TABLE I In vitro INCORPORATION
OF [ME-14C]CHOLINE INTO LIPIDS OF DEVELOPING RAT BRAIN SYNAPTOSOMES
UNDER UPTAKE CONDITIONS
Incubation conditions are as described under Fig. 1 and text for uptake of [14C]choline. Total lipids were extracted from the lysed synaptosomes after recovery from the Amberlite IRC-50 column. Values represent specific radioactivities for total lipids and are mean for the number of experiments quoted. Each experiment was run in duplicate and the values for the different experiments agreed within 0-7 ~. Additions
Specific radioactivity (counts/min/ktmole PO Age (days)
None Choline (15 raM) Number of experiments
5
17
Adult
319 215 3
460 133 2
397 195 2
suggest that while the carrier-mediated transport of choline increases with brain maturation, the nonspecific transport (see ref. 11) decreases and the sum of both systems (total transport) is highest during the stage of active myelination. Following this rise in the 17-day-old the total transport drops considerably in the adult (Fig. 1 A). In this connection Lajtha 15, working in vivo with the uptake of 36-chloride and thiocyanate by developing brain showed that the rate of uptake by the brain of the two anions decreases with increasing age of the animal. Furthermore Lahiri and Lajtha 14 showed that changes in uptake by brain slices during development are not uniform among the amino acids. Thus these authors observed that the L- and D-isomers of lysine were taken up by slices from newborn brain to a greater extent than by slices from adult brain, while the opposite was true for other amino acids. Under the present uptake conditions, choline is also incorporated into lipids (Table I) with more than 95 ~ of the lipid radioactivity recovered in lecithin, about 4 - 5 ~ in lysolecithin and a negligible amount was found in sphingomyelin. Again maximal incorporation occurred in synaptosomes from the 17-day-old rats. The finding that maximal inhibition (71 ~ ) occurred in the presence of unlabeled choline could be due to the higher uptake of the latter during this period of development as was shown in the permeability studies (Fig. 1B). This observation was confirmed through experiments in which the synaptosomes were incubated for a longer period of time and under conditions of oxidative phosphorylation (Fig. 2). During the period of active myelination, [14C]choline incorporation into lecithin was 12 times as high as that of the adult and almost 3 times that of the 3-day-old rats (Fig. 2). Further studies on [14C]choline incorporation into lecithin showed it to be stimulated by malate plus pyruvate, 1 m M Ca 2+ and inhibited by 1 m M serine. The rapid rise in lecithin labeling correlates well with the changes in choline transport into the synaptosomes during brain growth and development observed in the present studies and is in accord with our previous studies in which the highest rate of N a + uptake 5 and a2Pi incorporation Brain Research, 31 (1971) 224-228
SHORT COMMUNICATIONS
227
~4xlO3~ 12xlO E c:
>, -~. o
IOx107 8xl
1
,~ 4x I011
2xl03l 0~
I0
20
30
40
50
60
I
I-
Adult
Age (doys)
Fig. 2. Effect of age on specific radioactivity of lecithin. Synaptosomes from rat cerebra at different ages and equivalent to 3.8 mg protein were incubated as described in the text. Lecithin was isolated from the lipid extract by means of two-dimensional TLC. The first solvent system consisted of: chloroform-methanol-acetic acid-water (25 : 15:4:2, v/v) and the second of: the upper phase of: nbutanol-pyridine-water (45 : 5 •20, v/v). Lecithin was eluted and its specificradioactivity determined as described previously4. Each point on the curve represents two different experiments and each experiment was run in duplicates. The values for the different experiments agreed within 0-5 %.
into phospholipids 6 of synaptosomes was observed during the period between the 12th and 20th day post partum. It is possible that the findings of several investigators 2,7,1°,2° on the rise of lipid synthesis during the period of active myelination could be partly due to an increase in the activities of the transport systems of their respective precursors, followed by an increase in the permeability of the neuronal membranes to the various lipid precursors. This could result in an increase in the incorporation of the latter into their respective phospholipids. However, the rise in phospholipid synthesis during this period of development could also be due to an increase in the activities o f the enzymes involved in their biosynthesis. Thus Aeberhard et al.7 showed maximal elongation of fatty acids in both microsomes and mitochondria from rat brain to occur around 15 days after birth, and Maker and Hauser Is working with rat brain slices showed the biosynthesis of gangliosides and cerebrosides, by using [U-14C]glucose as precursor, to occur at a maximum rate at about 11-12 days after birth. Thus the rise in lipid synthesis, both in vivo and in vitro, during the period when functional changes are formed could be due to an increase in the transport systems for their precursors as well as in the enzymes involved in their biosynthesis. The authors wish to thank Gerald Elwinger and Robert Frothingham for technical assistance. Brain Research, 31 (1971) 224-228
228
SHORT COMMUNICATIONS T h e s e studies
were supported
by R e s e a r c h
Grant
NS-07876-04
from
the
National Institute of Neurological Diseases and Stroke.
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Ga. 30902 (U.S.A.)
ATA A. ABDEL-LATIF JACK P. SMITH
1 ABDEL-LATIF,A. A., A simple method for isolation of nerve-ending particles from rat brain, Biochim. biophys. Acta ( Amst.), 121 (1966) 403-406. 2 ABDEL-LATIE,A. A., AND ABOOD, L. G., Incorporation of a2p into phospholipids and phosphoproteins of cytoplasmic fractions of developing rat brain, J. Neurochem., 12 (1965) 157-166. 3 ABDEL-LATIF,A. A., BRODY,J., ANDRAMAHI,H., Studies on Na+-K+-ATPase of the nerve endings and appearance of electrical activity in developing rat brain, J. Neurochem., 14 0967) 1133-1141. 4 ABDEL-LATIF, A. A., AND SMITH, J. P., In vivo incorporation of choline, glycerol and orthophosphate into lecithin and other phospholipids of subcellular fractions of rat cerebrum, Biochim. biophys. Acta (Amst.), 218 (1970) 134-140. 5 ABDEL-LATIF,A. A., YAMAGUCHI,M., SMITH, J., AND YAMAGUCHI,T., Studies on the effect of ouabain on sodium and phosphate uptake into nerve endings of developing rat brain, Life Sci., 7 (1968) 1325-1338. 6 ABDEL-LATIF,A. A., YAMAGUCHI,T., YAMAGUCHI,M., AND CHANG, F., Studies on [a2P]orthophosphate incorporation into nucleotides, phospholipids and phosphoproteins of isolated nerve endings from developing rat brain, Brain Research, 10 (1968) 307-321. 7 AEaERHARD, E., GRIPPO, J., AND MENKES, J. H., Fatty acid synthesis in the developing brain, Pediat. Res., 3 (1969) 590-596. 8 BRADFORD, H. F., Respiration in vitro of synaptosomes from mammalian cerebral cortex, J. Neurochem., 16 (1969) 675-684. 9 COLBURN, R. W., GOODWIN, E. K., MURPHY, D. L., BUNNEY, W. E., JR., AND DAVIS, J. M., Quantitative studies of norepinephrine uptake by synaptosomes, Biochem. Pharmacol., 17 (1968) 957-964. 10 DAVISON,A. N., AND DOBBING,J., The developing brain. In A. N. DAVISONANDJ. DOBmNG(Eds.), Applied Neurochemistry, Davis, Philadelphia, 1968, pp. 253-286. 11 DIAMOND, I., AND KENNEDY, E. P., Carrier-mediated transport of choline into synaptic nerve endings, J. biol. Chem., 244 (1969) 3258-3263. 12 ESCUETA,A. V., AND APELL, S. H., Biochemical studies of synapses in vitro. II. Potassium transport, Biochemistry, 8 (1969) 725-733. 13 GRAHAME-SMITH,D. G., AND PARE1TT, A. G., Tryptophan transport across the synaptosomal membrane, J. Neurochem., 17 (1970) 1339-1353. 14 LAmRI, S., AND LAJTHA, A., Cerebral amino acid transport in vitro. I, J. Neurochem., 11 (1964) 77-86. 15 LAJTHA, A., The development of the blood-brain barrier, J. Neurochem., 1 (1957) 216-227. 16 LING, C.-M., AND ABDEL-LATIF,A. A., Studies on sodium transport in rat brain nerve-ending particles, J. Neurochem., 15 (1968)721-729. 17 LOWRY,O. H., ROSESROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 MAKER, H. S., AND HAUSER,G., Incorporation of glucose carbon into gangliosides and cerebrosides by slices of developing rat brain, J. Neurochem., 14 (1967) 457J,64. 19 MARCHBANKS,R. M., The uptake of [14C]choline into synaptosomes in vitro, Biochem. J., 110 (1968) 533-541. 20 SPERRY,W. M., The biochemistry of the brain during early development. In K. A. C. ELLIOTT,I. H. PAGE AND J. H. QUASTEL(Eds.), Neurochemistry, Thomas, Springfield, I11., 1962, pp. 55-84. (Accepted May 14th, 1971)
Brain Research, 31 (1971) 224-228