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Regional changes in monoamine content and uptake of the rat brain during postnatal development
Brain Research, 101 (1976) 305-315
305
(t) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
R E G I O N A L C H A N G E S IN M O N O A M I N E C O N T E N T A N D U P T A K E OF T H E RAT BRAIN D U R I N G P O S T N A T A L D E V E L O P M E N T
Y A S U Y U K I N O M U R A , F U M I K O NAITOH AND TOMIO SEGAWA
Department of Pharmacology, Institute of Pharmaceutical Sciences, Hiroshima University School of Medichte, Kasumi 1-2-3, Hiroshima 734 (Japan) (Accepted June 27th, 1975)
SUMMARY
Regional norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) contents in the developing rat brain were estimated. The rate of increase in N E content was the highest in diencephalon, followed by the lower brain stem, limbic-striatum, neocortex and cerebellum. With postnatal aging, DA concentration increased markedly in limbic-striatum, slightly in the neocortex and negligibly in other regions. In each region except cerebellum, 5-HT content increased gradually but the rate of increase in diencephalon was relatively high. Comparison of the kinetics of high affinity uptake of L-[3H]NE and [aH]5-HT between the neonatal and the adult brain indicated that Km values of L-[3H]NE and [~H]5-HT uptake were 2.9 x 10 v M and 1.7 × 10-7 M respectively in neocortex, diencephalon and lower brain stem and 4.3 × 10-7 M and 2.3 × 10-7 M in limbic-striatum in the neonate as well as in the adult. Vm~ values of both amines uptake differed regionally and the values in the neonate were lower than those in the adult in all regions. Limbicstriatum showed a higher Vm~x value than other regions in uptake of both amines. These results suggested that innervation of monoaminergic neurons in the brain progressed with increasing age, that projections of both NE and 5-HT neurons were relatively high into hypothalamus and limbic-striatum and that DA neuron projections concentrated at striatum. Although the brain, except for limbic-striatum, showed neither regional nor developmental differences in affinity of L-[3H]NE and [3H]5-HT to synaptosomes, the density of nerve terminal of both monoaminergic neurons increased in all regions of the brain during postnatal development. In limbic-striatum, higher Km and Vm~x values of both amines, uptake suggest the existence of both amines' uptake into DA terminal to some extent.
I NTRODUCTION
Several works have been reported on the development of monoaminergic
306 neurons in the animal brain. Karki et a/.lr' found that the brain levels ofnorepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) in the guinea-pig, which display an organized pattern of behavior at birth, were almost as high at birth as in the adult, but that the neonatal rat, whose functional development is poor, had lower concentrations of monoamines in the brain. Although the concentrations of brain NE, dopamine (DA) and 5-HT were low in the neonatal rat brain, they progressively increased to the adult levels `) .~,s.t6,1s,24 and the rate of increase in the concentration of monoamines differed among parts of the developing rat brain jT,e~.e". In addition, the adult brain has revealed regional differences in the kinetics of high affinity uptake of catecholamines into homogenates '~s and 5-HT into brain slices eT. It has been suggested that the ability of tissues to take up [3H]NE was a sensitive and accurate index of the extent of sympathetic innervation r~. Coyle and Axelrod showed 7 that K,, values of [aH]NE uptake to synaptosomes did not change, but V,~x values increased during fetal and postnatal periods. But little has been reported about the development of 5-HT uptake in the brain. The objective of the present study is to investigate in detail regional changes of N E, DA and 5-HT contents during postnatal periods and to compare the kinetics of high affinity uptake of L-[3H]N E and [ZH]5-HT between different regions of the neonatal and the adult brain. MATERIALS AND METHODS
Albino rats of both sexes of a laboratory inbred Wistar strain were used throughout. They were maintained at 23 ~C and kept under normal daylight conditions. The time of birth of the pups was noted to within 12 h. The pups were weaned at 21 days after birth. Preparation of each region of the bra& The areas analyzed were designated as neocortex, limbic system-striatum (limbic-striatum), diencephalon, mesencephalon-pons-medulla (lower brain stem) and cerebellum. Pineal gland and meninges were discarded. Neocortex was the cerebral cortex except hippocampus, amygdala and septum. Limbic-striatum contained olfactory bulb, hippocampus, amygdala, striatum and septum. Diencephalon was separated from remaining brain stem at the anterior border of superior colliculus and the posterior border of optic chiasm. Cerebellum was isolated by cutting its peduncular connection with the brain stem. Lower brain stem was separated from spinal cord by cutting under part of the obex. At various days after birth the rats were sacrificed by decapitation between 10.00 a.m. and 3.00 p.m.. Dissections were performed over ice. The brain samples were washed with the cold saline, weighed and homogenized in acidified n-butanol for amine determination and in 0.32 M sucrose for synaptosomal preparation. Estimation of NE, DA and 5-HT NE and DA were extracted by the method of Maickel et aL 2z and spectro-
307 photofluorimetrically assayed by the modified method of Chang 6. 5-HT was extracted and determined spectrophotofluorimetrically by the method of Maickel et al. 22.
L- [aH] NE and [3H] 5-HT uptake Uptake procedure of L-[~H]NE or [3H]5-HT into synaptosomes was carried out by the modified method of Nomura and Segawa 25. Each area of the brain (cerebellum was not examined in the uptake experiment) was minced and homogenized in 9 vol. of ice cold 0.32 M sucrose bubbled with 95 ~/o 02-5 ~ CO2 gas for 10 rain using Teflon homogenizer. The homogenate was centrifuged at 900 × g for 10 rain to make a sediment of the cell debris and nucleus. The supernatant fluid was then centrifuged at 11,500 × g for 20 min to yield the P2 fraction in sediment. Pz fractions were suspended in Krebs-Ringer solution (pH 7.2) containing 2 × 10-~ M pheniprazine (a monoamine oxidase inhibitor) and 0 . 2 ~ ascorbic acid to give a final concentration of 0.4-0.8 mg protein/ml. The suspension was divided into 1.8 ml units each of which was transferred into 10 ml polyethylene tubes. After pre-incubation for l0 rain at 37 °C, 0.2 ml of the various concentrations of L-[3H]NE (8.8 Ci/ mmole, Radiochemical Centre, Amersham) or [aH]5-HT (0.5 Ci/mmole, Radiochemical Centre, Amersham) were added and a further incubation was carried out at 37 °C for 5 rain for L-[3H]NE and 2 min for [3H]5-HT. At the end of the incubation period, 6 ml of the cold saline were added to the reaction mixture and centrifuged at 11,500 × g for 20 min at 4 °C and the supernatant fluid was decanted. The pellet of synaptosomes was resuspended in 8 ml of the cold saline. After re-centrifugation at 11,500 × g for 20 min, a 0.1 ml portion of 1 M NaOH solution was added to the pellet, left overnight and neutralized with 0.1 ml of 1 M HCI solution. Radioactivity was determined in 8 ml of Bray's solution by a Packard Tri-Carb liquid scintillation counter. Counting efficiency was 2 2 - 2 5 ~ . Preliminary experiments indicated that after 2 min or 5 rain incubation, above 90 ~o of the radioactivity in the pellet of synaptosomes was unchanged monoamines. The extent of active uptake was expressed as the difference between the values at 37 °C and at 0 °C. Estimation of protein was carried out by the Folin reagent method of Lowry et al. 21. RESULTS
Changes of the brain weight The weights of each region of the brain progressively increased during postnatal period and the rate of increase during 14 days after birth was higher than that of the following days (Fig. 1). Changes of NE content Fig. 2 shows the changes of NE content in each region at days 0.5, 3, 7, 14, 30 and 60 after birth. NE levels at the neonatal brain were lower than the adult levels in all regions. Although N E content gradually increased, a different rate of increase was found in each area. N E in the diencephalon showed marked increase and in the adult its content was approximately 6 times that of the neonatal level. In the lower brain
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stem the rate of increase during 14 days after birth was highest, and the content in the adult was about 4 times that of the neonatal value. In comparison with the diencephalon and lower brain stem, the rates of increase of NE concentration in neocortex and limbic-striatum were relatively low and that of the cerebellum was negligible.
Changes of DA content With progressive age, DA content in the limbic-striatum markedly increased and at the age of 30 days its concentration arrived at the adult level (Fig. 3). A
309
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Fig. 2. Concentrations of norepinephrine in each region of the rat brain during postnatal development. Each point represents an average with S.E.M. of 3-12 determinations.
gradual increase was found in neocortex but not in diencephalon, lower brain stem and cerebellum.
Changes of 5-HT content Fig. 4 shows a progressive and slight increase of 5-HT concentration in all regions. The postnatal increase of 5-HT level in diencephalon was higher than those in other areas and in the adult 5-HT content was approximately 2.5 times that of the neonatal value.
Difference of L-f3H] NE uptake between the neonatal and the adult brain The P2 fractions of each region prepared from the neonatal and the adult brain were incubated at both 37 °C and 0 °C for 5 rain with L-[3H]NE in concentrations varying from 0.102 × 10 -v M to 0.51 × 10 -7 M; 4-8 experiments were performed at each concentration of L-[3H]NE. Results of active uptake to each concentration were expressed graphically as double reciprocal plots (Fig. 5), from which kinetic constants were obtained. Differences in Km values were not found in the neocortex, diencephalon or lower brain stem in the adult as well as in the neonate and its value was 2.9 × 10 -7 M. Limbic-striatum had a higher Km value (4.3 × 10 -7 M) in the
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Fig. 4. Concentrations of 5-hydroxytryptamine in each region of the rat brain during postnatal development. Each point represents an average with S.E.M. of 3-10 determinations.
Jo
311
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Fig. 5. Double reciprocal plot of the initial active uptake of L-[aH]norepinephrine into P2 fractions prepared from different regions of the neonatal and the adult rat brain. P2 fractions were incubated for 5 min with L-[ZH]norepinephrine in concentrations varying from 0.102 × 10 7 M to 0.51 /. 10 7 M. Active uptake is expressed as 10 -a nmole of L-[3H]norepinephrine taken up per mg protein of brain in 5 rain. Each point represents an average obtained from 4-8 determinations.
TABLE 1 COMPARISON OF Wmax VALUES FOR THE INITIAL ACTIVE UPTAKE OF L-[aH]NOREP1NEPHR1NE AND
[3H]5-
HYDROXYTRYPTAMINE INTO SYNAPTOSOMES IN DIFFERENT REGIONS BETWEEN TIlE NEONATAL AND THE A DU L T RAT BRAIN
Vm~x values for initial active uptake of L-[aH]NE and [3H]5-HT were obtained from the double reciprocal plots in Fig. 5 (L-[ZH]NE) and Fig. 6 ([aH]5-HT). Vn~x value is expressed as 10 a nmole/ mg protein/5 rain for L-[ZH]NE and 10 3 nmole/mg protein/2 rain for [aH]5-HT.
Region
Neocortex Limbic-striatum Diencephalon Lower brain stem
L-13H JN E uptake
[ aH 15-HT uptake
Neonate
Adult
Neonate
Adult
0.71 3.70 1.92 1.05
0.95 6.25 2.22 1.28
4.35 8.00 6.25 6.67
5,26 12,50 7.15 8.06
t
312 adult and the neonate. In contrast, the Vma,, value of L-[ZH]NE uptake was different in all regions. In the adult brain, the highest value was shown in the limbic-striatum followed by the diencephalon, lower brain stem and neocortex (Table I). In the neonate, all regions of the brain had lower Vmax values than those of the adult, The limbic-striatum showed the highest increase of Vmax value during development.
Difference of/3H/5-HT uptake between the neonatal and the adult brain The kinetics of [ZH]5-HT uptake was analyzed by the same procedure as in L-[ZH]NE uptake. P,, fractions suspended in Krebs-Ringer solution (pH 7.2) were incubated at both 37 'C and 0 ~C for 2 rain with [aH]5-HT in concentrations varying from 0.18 > 10-7 M to 0.9 < 10 7 M. Fig. 6 shows the double reciprocal plots of these results. Although K,,,. values of [ZH]5-HT uptake did not ditfer regionally in both the neonate and the adult brain (Kn,~ : 1.7 ::< 10 7 M), that of the limbicstriatum was somewhat higher than others (K,, 2.3 >; 10 -~ M). Vm~x values were different not only among each region but also between the neonatal and the adult brain (Table !). In the adult brain the limbic-striatum had the highest V,:,~ values followed by the lower brain stem, diencephalon and neocortex. The brain,
25
[3H]5-HTNeocortexUptake l Diencephalon )Km : 1.7 X 10-7 M Lowerbrain stem| Limbic-striatum, Km: 2.3 XI0"? M
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Fig. 6. Double reciprocal plot of the initial active uptake of [aH]5-hydroxytryptarnine into P2 fractions prepared from different regions of the neonatal and the adult rat brain. P2 fractions were incubated for 2 min with [3H]5-hydroxytryptarnine in concentrations varying from 0118 x 10-7 M to 0.9 x IO-? M. Active uptake is expressed as 10 2 nrnole of [3H]5-hydroxytryptarnine taken up per rng protein of brain in 2 rnin. Each point represents an average obtained from 4-8 determinations.
313 however, showed lower Vmax values in the neonate than in the adult in all regions, especially the limbic-striatum (Table I). DISCUSSION
A high rate of increase in the brain weight was found during 14 days after birth and thereafter the rate of increase became low (Fig. 1). The developmental stage of the rat brain, 4-15 days after birth, has been called the 'critical period '23. Morphological observation has indicated a marked growth of axons and dendrites from neurons and active myelin formation at this stage. Furthermore, the brain at this stage showed a high content of nucleic acid and a marked increase of various enzyme activitiesg, 13. N E content, being lower at birth, was lower in the rostral part than in the caudal part (Fig. 2) and this finding corresponded with the result of Loizou 19. A progressive increase of NE content was found in all regions of which the highest increase was in the diencephalon, while the lower brain stem had the highest rate of increase of NE level during 14 days after birth. At this 'critical period' NE may be synthesized actively in the cell body of NE neurons located in the locus coeruleusl0,11. From the result that in the adult lower brain stem there was a relatively higher NE content but a lower Vmax value, it was presumed that the lower brain stem had NE originating from axons and cell bodies. Although in the neocortex, diencephalon and lower brain stem, Km values did not differ regionally or developmentally, Vmax values were different among these regions and the neonatal values were lower than those of the adult (Fig. 5, Table I). These results suggested that the affinity of L[aH]NE to nerve terminals did not differ among regions and ages but that the density of nerve terminals differs in each region and increases with age. In the limbic-striatum the Km value, which was equal in both the neonate and the adult, was higher than those of other regions (Table I), suggesting the existence of a non-specific uptake in the limbic-striatum. In addition the limbic-striatum revealed the highest value of Vmax in both the neonate and the adult. In spite of this higher Vmax value in the limbicstriatum than those of other regions, it was shown to be. lower in endogenous NE content than in the diencephalon and lower brain stem. Snyder and Coyle have documented z8 that the striatal region in the adult brain had not only a higher Km value but also a higher Vmax value of I.-[aH]NE uptake and that striatal uptake of [3H]DA was inhibited competitively by L-NE. The existence of this avid DA uptake mechanism in the striatum explains the present phenomena of a higher Kin, higher Vmax and a lower content of NE in the limbic-striatum. Although neonatal DA concentration was not different among each region, its rate of increase was marked in the limbic-striatum (Fig. 3). In the neocortex, but not in the diencephalon, lower brain stem and cerebellum, DA content slightly increased, indicating that the projection of DA neurons from the substantia nigra concentrated into the striatum and, more or less, a projection occurred into the neocortex with progressing age. It has been observed that the adult striatum showed the highest Vmax value of [3H]DA uptake 28.
314 The pattern of regional localization of 5-HT content in the neonate wab the same as in the adult and the increase of its content was shown to be slight in aii regions, of which the diencephalon had a relatively high rate of increase (Fig. 4). It is suggested that in the prenatal period 5-HT neurons to some extent project from the midbrain raphe nucleus into each area and that postnatal innervation is less than that of NE or DA neurons. The kinetics of [:~H]5-HT uptake (Fig. 6) indicated neither developmental nor regional differences in affinity of [aH]5-HT to synaptosomes in the neocortex, diencephaton and lower brain stem. In these regions, Vmax values revealed differences among each part and developmental stage (Table I) as well as in L-[3H]NE uptake (Table 1). Although the limbic-striatum showed a lower 5-HT content (Fig. 4), it had the highest Vmax value of [aH]5-HT uptake, it also had a higher K m value than other regions, suggesting that [aH]5-HT may enter the D A terminal. In this respect it was speculated that 5-HT, especially at a higher concentration in medium~ may be taken up by striatum z7"9. The diencephalon had a higher 5-HT concentration but not a higher Vmax value, the reason |i~r which is not clearly understood. A higher content of 5-HT in the lower brain stern at both stages suggests that the 5-HT estimated, originated, to a certain extent, from cell bodies and axons. As there were no developmental differences of Km values in both L-[:~H]NE and [aH]5-HT uptake, it was presumed that the character of both L-[3H]NE and [aH]5-HT uptake was not essentially modified at the developmental stage. In contrast, the regional and developmental differences of Vrnax value indicated that NE and 5-HT neurons innervated each region differently, in this case there is a need for more accurate discussion to consider a monoamine concentration together, although the Vmax value of the uptake is a sensitive index of innervation of monoaminergic neuron. Quantitative electron microscopic findings have revealed postnatal development of synaptic contacts 1,a0. With terminal formation during the postnatal period, synaptic contacts also progressed and physiological functions of monoaminergic neurons increased.
REFERENCES 1 AGHAJANIAN, G. K., AND BLOOM, F. E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Research, 6 (1967) 716-727. 2 AGRAWAL,H. C., GLISSON,S. N., AND HIMWICH,W. A., Changes in monoamines of rat brain during postnatal ontogeny, Biochim. Biophys. Acta (Amst.), 130 (1966) 511-513. 3 BAKER, P. C., AND QUAY, W. B., 5-Hydroxytryptamine metabolism in early embryogenesis and the development of brain and retinal tissues. A Review, Brain Research, 12 (1969) 273-295. 4 BENNETT,O. S., ANDGIARMAN,N. J., Schedule of appearance of 5-hydroxytryptamine (serotonin) and associated enzymes in the developing rat brain, J. Neurochem., 12 (1965) 911-918. 5 BREESE,G. R., AND TRALOR,T. D., Developmental characteristics of brain catecholamines and tyrosine hydroxylase in the rat: effects of 6-hydroxydopamine, Brit. J. Pharamacol., 44 (1972) 210-222. 6 CHANG,C. C., A sensitive method for spectrophotofluorometric assay of catecholamines, Int. J. Neuropharmacol., 3 (1964) 643-649. 7 COYLE, J. T., ANDAXELROD,J., Development of the uptake and storage of L-[aH]norepinephrine in the rat brain, J. Neurochem., 18 (1971) 2061-2075.
315 8 COYLE, J. T., AND HENRY, D., Catecholamines in fetal and newborn rat brain, J. Neurochem., 21 0973) 61-67. 9 DAHL, D. R., AND SAMSON, F. E., JR., Metabolism of rat brain mitochondria during postnatal development, Amer. J. Physiol., 196 (1959) 470-472. 10 DAHLSTR~3M,A., AND IZUXE,K., A method for the demonstration of monoamine containing nerve fibres in the central nervous system, Acta physiol, scand., 60 (1964) 293-294. 11 DAHLSTR6M, A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, seand., 62, Suppl. 232 (1964) 1-55. 12 HENN, F. A., AND HAMBERGER, A., Glial cell function: uptake of transmitter, Proc. nat. Acad. Sei. (Wash.), 68 (1971) 2686-2690. 13 HIMWICH, W. A., Biochemical and neurophysiological development of the brain in the neonatal period, Int. Rev. Neurobiol., 4 (1962) 117-158. 14 lVERSEN, L. L., GLOWINSKI, J., AND AXELROD, J., The physiologic disposition and metabolism of norepinephrine in immunosympathectomized animals, J. Pharmacol. exp. Ther., 151 (1966) 273-284. 15 KARKI, N., KUNTZMAN, R., AND BROD~E,B. B., Storage, synthesis and metabolism of monoamines in the developing brain, J. Neurochem., 9 (1962) 53-58. 16 KATO, R., Serotonin content of rat brain in relation to sex and age, J. Neurochem., 5 (1960) 202. 17 KELLER, H. H., BARTHOUNI, G., AND PLETCHER, A., Spontaneous and drug-induced changes of cerebral dopamine turnover during postnatal development of rats, Brain Research, 64 (1973) 371-378. 18 KULKARNI, A. S., AND SHIDEMAN, E. E., Sensitivities of the brains of infant and adult rats to the catecholamine-depleting actions of reserpine and tetrabenazine, J. Pharmaeol. exp. Ther., 153 (1966) 428-433. 19 LoIzou, L. A., The postnatal ontogeny of monoamine-containing neurons in the central nervous system of the albino rat, Brain Research, 40 (1972) 395-418. 20 Lo~zou, L. A., AND SALT, P., Regional changes in monoamines of the rat brain during postnatal development, Brain Research, 20 (1970) 467-470. 21 LOWRY, O. H., ROSENBROUGH, J. N., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 22 MMCKEL, R. G., COx, R. H., JR., SA1LLANT,J. AND M1LLER, F. P., A method for the determination of serotonin and norepinephrine in discrete areas of rat brain, Int. J. Neuropharmacol., 7 (1968) 275-281. 23 MCILWMN, H., Biochemistry o f the Central Nervous System, Churchill, London, 1965, 270 pp. 24 NACHMIAS,V. T., Amine oxidase and 5-hydroxytryptamine in developing rat brain, J. Neurochem., 6 (1960) 99-104. 25 NOMURA, Y., AND SEGAWA, T., Influences of colchicine and vinblastine on the uptake of 5-hydroxytryptamine and norepinephrine by rat brain synaptosomes and small vesicle fractions, J. Neurochem., 24 (1975) 1257-1259. 26 PORtliER, W., AND HEELER, A., Regional development of catecholamine biosynthesis in rat brain, J. Neurochem., 19 (1972) 1917-1930. 27 SHASKAN,E. G., AND SNYDER, S. H., Kinetics of serotonin accumulation into slices from rat brain : relationship to catecholamine uptake, J. Pharmacol. exp. Ther., 175 (1970) 404-418. 28 SNYDER, S. H., AND COYLE, J. T., Regional differences in 3H-norepinephrine and 3H-dopamine uptake into rat brain homogenates, J. Pharmacol. exp. Ther., 165 (1969) 78-86. 29 SNYDER, S. H., SHASKAN, E. G., AND KUHAR, J., Serotonin uptake systems in brain tissue. In J. BARCHAS AND E. USDIN (Ed.), Serotonin and Behavior, Academic Press, New York, 1973, pp. 97-108. 30 WOODWARD, D. J., HOFFER, B. J., SIGGINS, G. R., AND BLOOM, F. E., The ontogenic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar Purkinje cells, Brain Research, 34 (1971) 73-97.
305
(t) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
R E G I O N A L C H A N G E S IN M O N O A M I N E C O N T E N T A N D U P T A K E OF T H E RAT BRAIN D U R I N G P O S T N A T A L D E V E L O P M E N T
Y A S U Y U K I N O M U R A , F U M I K O NAITOH AND TOMIO SEGAWA
Department of Pharmacology, Institute of Pharmaceutical Sciences, Hiroshima University School of Medichte, Kasumi 1-2-3, Hiroshima 734 (Japan) (Accepted June 27th, 1975)
SUMMARY
Regional norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) contents in the developing rat brain were estimated. The rate of increase in N E content was the highest in diencephalon, followed by the lower brain stem, limbic-striatum, neocortex and cerebellum. With postnatal aging, DA concentration increased markedly in limbic-striatum, slightly in the neocortex and negligibly in other regions. In each region except cerebellum, 5-HT content increased gradually but the rate of increase in diencephalon was relatively high. Comparison of the kinetics of high affinity uptake of L-[3H]NE and [aH]5-HT between the neonatal and the adult brain indicated that Km values of L-[3H]NE and [~H]5-HT uptake were 2.9 x 10 v M and 1.7 × 10-7 M respectively in neocortex, diencephalon and lower brain stem and 4.3 × 10-7 M and 2.3 × 10-7 M in limbic-striatum in the neonate as well as in the adult. Vm~ values of both amines uptake differed regionally and the values in the neonate were lower than those in the adult in all regions. Limbicstriatum showed a higher Vm~x value than other regions in uptake of both amines. These results suggested that innervation of monoaminergic neurons in the brain progressed with increasing age, that projections of both NE and 5-HT neurons were relatively high into hypothalamus and limbic-striatum and that DA neuron projections concentrated at striatum. Although the brain, except for limbic-striatum, showed neither regional nor developmental differences in affinity of L-[3H]NE and [3H]5-HT to synaptosomes, the density of nerve terminal of both monoaminergic neurons increased in all regions of the brain during postnatal development. In limbic-striatum, higher Km and Vm~x values of both amines, uptake suggest the existence of both amines' uptake into DA terminal to some extent.
I NTRODUCTION
Several works have been reported on the development of monoaminergic
306 neurons in the animal brain. Karki et a/.lr' found that the brain levels ofnorepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) in the guinea-pig, which display an organized pattern of behavior at birth, were almost as high at birth as in the adult, but that the neonatal rat, whose functional development is poor, had lower concentrations of monoamines in the brain. Although the concentrations of brain NE, dopamine (DA) and 5-HT were low in the neonatal rat brain, they progressively increased to the adult levels `) .~,s.t6,1s,24 and the rate of increase in the concentration of monoamines differed among parts of the developing rat brain jT,e~.e". In addition, the adult brain has revealed regional differences in the kinetics of high affinity uptake of catecholamines into homogenates '~s and 5-HT into brain slices eT. It has been suggested that the ability of tissues to take up [3H]NE was a sensitive and accurate index of the extent of sympathetic innervation r~. Coyle and Axelrod showed 7 that K,, values of [aH]NE uptake to synaptosomes did not change, but V,~x values increased during fetal and postnatal periods. But little has been reported about the development of 5-HT uptake in the brain. The objective of the present study is to investigate in detail regional changes of N E, DA and 5-HT contents during postnatal periods and to compare the kinetics of high affinity uptake of L-[3H]N E and [ZH]5-HT between different regions of the neonatal and the adult brain. MATERIALS AND METHODS
Albino rats of both sexes of a laboratory inbred Wistar strain were used throughout. They were maintained at 23 ~C and kept under normal daylight conditions. The time of birth of the pups was noted to within 12 h. The pups were weaned at 21 days after birth. Preparation of each region of the bra& The areas analyzed were designated as neocortex, limbic system-striatum (limbic-striatum), diencephalon, mesencephalon-pons-medulla (lower brain stem) and cerebellum. Pineal gland and meninges were discarded. Neocortex was the cerebral cortex except hippocampus, amygdala and septum. Limbic-striatum contained olfactory bulb, hippocampus, amygdala, striatum and septum. Diencephalon was separated from remaining brain stem at the anterior border of superior colliculus and the posterior border of optic chiasm. Cerebellum was isolated by cutting its peduncular connection with the brain stem. Lower brain stem was separated from spinal cord by cutting under part of the obex. At various days after birth the rats were sacrificed by decapitation between 10.00 a.m. and 3.00 p.m.. Dissections were performed over ice. The brain samples were washed with the cold saline, weighed and homogenized in acidified n-butanol for amine determination and in 0.32 M sucrose for synaptosomal preparation. Estimation of NE, DA and 5-HT NE and DA were extracted by the method of Maickel et aL 2z and spectro-
307 photofluorimetrically assayed by the modified method of Chang 6. 5-HT was extracted and determined spectrophotofluorimetrically by the method of Maickel et al. 22.
L- [aH] NE and [3H] 5-HT uptake Uptake procedure of L-[~H]NE or [3H]5-HT into synaptosomes was carried out by the modified method of Nomura and Segawa 25. Each area of the brain (cerebellum was not examined in the uptake experiment) was minced and homogenized in 9 vol. of ice cold 0.32 M sucrose bubbled with 95 ~/o 02-5 ~ CO2 gas for 10 rain using Teflon homogenizer. The homogenate was centrifuged at 900 × g for 10 rain to make a sediment of the cell debris and nucleus. The supernatant fluid was then centrifuged at 11,500 × g for 20 min to yield the P2 fraction in sediment. Pz fractions were suspended in Krebs-Ringer solution (pH 7.2) containing 2 × 10-~ M pheniprazine (a monoamine oxidase inhibitor) and 0 . 2 ~ ascorbic acid to give a final concentration of 0.4-0.8 mg protein/ml. The suspension was divided into 1.8 ml units each of which was transferred into 10 ml polyethylene tubes. After pre-incubation for l0 rain at 37 °C, 0.2 ml of the various concentrations of L-[3H]NE (8.8 Ci/ mmole, Radiochemical Centre, Amersham) or [aH]5-HT (0.5 Ci/mmole, Radiochemical Centre, Amersham) were added and a further incubation was carried out at 37 °C for 5 rain for L-[3H]NE and 2 min for [3H]5-HT. At the end of the incubation period, 6 ml of the cold saline were added to the reaction mixture and centrifuged at 11,500 × g for 20 min at 4 °C and the supernatant fluid was decanted. The pellet of synaptosomes was resuspended in 8 ml of the cold saline. After re-centrifugation at 11,500 × g for 20 min, a 0.1 ml portion of 1 M NaOH solution was added to the pellet, left overnight and neutralized with 0.1 ml of 1 M HCI solution. Radioactivity was determined in 8 ml of Bray's solution by a Packard Tri-Carb liquid scintillation counter. Counting efficiency was 2 2 - 2 5 ~ . Preliminary experiments indicated that after 2 min or 5 rain incubation, above 90 ~o of the radioactivity in the pellet of synaptosomes was unchanged monoamines. The extent of active uptake was expressed as the difference between the values at 37 °C and at 0 °C. Estimation of protein was carried out by the Folin reagent method of Lowry et al. 21. RESULTS
Changes of the brain weight The weights of each region of the brain progressively increased during postnatal period and the rate of increase during 14 days after birth was higher than that of the following days (Fig. 1). Changes of NE content Fig. 2 shows the changes of NE content in each region at days 0.5, 3, 7, 14, 30 and 60 after birth. NE levels at the neonatal brain were lower than the adult levels in all regions. Although N E content gradually increased, a different rate of increase was found in each area. N E in the diencephalon showed marked increase and in the adult its content was approximately 6 times that of the neonatal level. In the lower brain
308
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stem the rate of increase during 14 days after birth was highest, and the content in the adult was about 4 times that of the neonatal value. In comparison with the diencephalon and lower brain stem, the rates of increase of NE concentration in neocortex and limbic-striatum were relatively low and that of the cerebellum was negligible.
Changes of DA content With progressive age, DA content in the limbic-striatum markedly increased and at the age of 30 days its concentration arrived at the adult level (Fig. 3). A
309
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gradual increase was found in neocortex but not in diencephalon, lower brain stem and cerebellum.
Changes of 5-HT content Fig. 4 shows a progressive and slight increase of 5-HT concentration in all regions. The postnatal increase of 5-HT level in diencephalon was higher than those in other areas and in the adult 5-HT content was approximately 2.5 times that of the neonatal value.
Difference of L-f3H] NE uptake between the neonatal and the adult brain The P2 fractions of each region prepared from the neonatal and the adult brain were incubated at both 37 °C and 0 °C for 5 rain with L-[3H]NE in concentrations varying from 0.102 × 10 -v M to 0.51 × 10 -7 M; 4-8 experiments were performed at each concentration of L-[3H]NE. Results of active uptake to each concentration were expressed graphically as double reciprocal plots (Fig. 5), from which kinetic constants were obtained. Differences in Km values were not found in the neocortex, diencephalon or lower brain stem in the adult as well as in the neonate and its value was 2.9 × 10 -7 M. Limbic-striatum had a higher Km value (4.3 × 10 -7 M) in the
310
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Jo
311
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Neocortex-Neonate L-[SH]NE uptake
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Fig. 5. Double reciprocal plot of the initial active uptake of L-[aH]norepinephrine into P2 fractions prepared from different regions of the neonatal and the adult rat brain. P2 fractions were incubated for 5 min with L-[ZH]norepinephrine in concentrations varying from 0.102 × 10 7 M to 0.51 /. 10 7 M. Active uptake is expressed as 10 -a nmole of L-[3H]norepinephrine taken up per mg protein of brain in 5 rain. Each point represents an average obtained from 4-8 determinations.
TABLE 1 COMPARISON OF Wmax VALUES FOR THE INITIAL ACTIVE UPTAKE OF L-[aH]NOREP1NEPHR1NE AND
[3H]5-
HYDROXYTRYPTAMINE INTO SYNAPTOSOMES IN DIFFERENT REGIONS BETWEEN TIlE NEONATAL AND THE A DU L T RAT BRAIN
Vm~x values for initial active uptake of L-[aH]NE and [3H]5-HT were obtained from the double reciprocal plots in Fig. 5 (L-[ZH]NE) and Fig. 6 ([aH]5-HT). Vn~x value is expressed as 10 a nmole/ mg protein/5 rain for L-[ZH]NE and 10 3 nmole/mg protein/2 rain for [aH]5-HT.
Region
Neocortex Limbic-striatum Diencephalon Lower brain stem
L-13H JN E uptake
[ aH 15-HT uptake
Neonate
Adult
Neonate
Adult
0.71 3.70 1.92 1.05
0.95 6.25 2.22 1.28
4.35 8.00 6.25 6.67
5,26 12,50 7.15 8.06
t
312 adult and the neonate. In contrast, the Vma,, value of L-[ZH]NE uptake was different in all regions. In the adult brain, the highest value was shown in the limbic-striatum followed by the diencephalon, lower brain stem and neocortex (Table I). In the neonate, all regions of the brain had lower Vmax values than those of the adult, The limbic-striatum showed the highest increase of Vmax value during development.
Difference of/3H/5-HT uptake between the neonatal and the adult brain The kinetics of [ZH]5-HT uptake was analyzed by the same procedure as in L-[ZH]NE uptake. P,, fractions suspended in Krebs-Ringer solution (pH 7.2) were incubated at both 37 'C and 0 ~C for 2 rain with [aH]5-HT in concentrations varying from 0.18 > 10-7 M to 0.9 < 10 7 M. Fig. 6 shows the double reciprocal plots of these results. Although K,,,. values of [ZH]5-HT uptake did not ditfer regionally in both the neonate and the adult brain (Kn,~ : 1.7 ::< 10 7 M), that of the limbicstriatum was somewhat higher than others (K,, 2.3 >; 10 -~ M). Vm~x values were different not only among each region but also between the neonatal and the adult brain (Table !). In the adult brain the limbic-striatum had the highest V,:,~ values followed by the lower brain stem, diencephalon and neocortex. The brain,
25
[3H]5-HTNeocortexUptake l Diencephalon )Km : 1.7 X 10-7 M Lowerbrain stem| Limbic-striatum, Km: 2.3 XI0"? M
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Fig. 6. Double reciprocal plot of the initial active uptake of [aH]5-hydroxytryptarnine into P2 fractions prepared from different regions of the neonatal and the adult rat brain. P2 fractions were incubated for 2 min with [3H]5-hydroxytryptarnine in concentrations varying from 0118 x 10-7 M to 0.9 x IO-? M. Active uptake is expressed as 10 2 nrnole of [3H]5-hydroxytryptarnine taken up per rng protein of brain in 2 rnin. Each point represents an average obtained from 4-8 determinations.
313 however, showed lower Vmax values in the neonate than in the adult in all regions, especially the limbic-striatum (Table I). DISCUSSION
A high rate of increase in the brain weight was found during 14 days after birth and thereafter the rate of increase became low (Fig. 1). The developmental stage of the rat brain, 4-15 days after birth, has been called the 'critical period '23. Morphological observation has indicated a marked growth of axons and dendrites from neurons and active myelin formation at this stage. Furthermore, the brain at this stage showed a high content of nucleic acid and a marked increase of various enzyme activitiesg, 13. N E content, being lower at birth, was lower in the rostral part than in the caudal part (Fig. 2) and this finding corresponded with the result of Loizou 19. A progressive increase of NE content was found in all regions of which the highest increase was in the diencephalon, while the lower brain stem had the highest rate of increase of NE level during 14 days after birth. At this 'critical period' NE may be synthesized actively in the cell body of NE neurons located in the locus coeruleusl0,11. From the result that in the adult lower brain stem there was a relatively higher NE content but a lower Vmax value, it was presumed that the lower brain stem had NE originating from axons and cell bodies. Although in the neocortex, diencephalon and lower brain stem, Km values did not differ regionally or developmentally, Vmax values were different among these regions and the neonatal values were lower than those of the adult (Fig. 5, Table I). These results suggested that the affinity of L[aH]NE to nerve terminals did not differ among regions and ages but that the density of nerve terminals differs in each region and increases with age. In the limbic-striatum the Km value, which was equal in both the neonate and the adult, was higher than those of other regions (Table I), suggesting the existence of a non-specific uptake in the limbic-striatum. In addition the limbic-striatum revealed the highest value of Vmax in both the neonate and the adult. In spite of this higher Vmax value in the limbicstriatum than those of other regions, it was shown to be. lower in endogenous NE content than in the diencephalon and lower brain stem. Snyder and Coyle have documented z8 that the striatal region in the adult brain had not only a higher Km value but also a higher Vmax value of I.-[aH]NE uptake and that striatal uptake of [3H]DA was inhibited competitively by L-NE. The existence of this avid DA uptake mechanism in the striatum explains the present phenomena of a higher Kin, higher Vmax and a lower content of NE in the limbic-striatum. Although neonatal DA concentration was not different among each region, its rate of increase was marked in the limbic-striatum (Fig. 3). In the neocortex, but not in the diencephalon, lower brain stem and cerebellum, DA content slightly increased, indicating that the projection of DA neurons from the substantia nigra concentrated into the striatum and, more or less, a projection occurred into the neocortex with progressing age. It has been observed that the adult striatum showed the highest Vmax value of [3H]DA uptake 28.
314 The pattern of regional localization of 5-HT content in the neonate wab the same as in the adult and the increase of its content was shown to be slight in aii regions, of which the diencephalon had a relatively high rate of increase (Fig. 4). It is suggested that in the prenatal period 5-HT neurons to some extent project from the midbrain raphe nucleus into each area and that postnatal innervation is less than that of NE or DA neurons. The kinetics of [:~H]5-HT uptake (Fig. 6) indicated neither developmental nor regional differences in affinity of [aH]5-HT to synaptosomes in the neocortex, diencephaton and lower brain stem. In these regions, Vmax values revealed differences among each part and developmental stage (Table I) as well as in L-[3H]NE uptake (Table 1). Although the limbic-striatum showed a lower 5-HT content (Fig. 4), it had the highest Vmax value of [aH]5-HT uptake, it also had a higher K m value than other regions, suggesting that [aH]5-HT may enter the D A terminal. In this respect it was speculated that 5-HT, especially at a higher concentration in medium~ may be taken up by striatum z7"9. The diencephalon had a higher 5-HT concentration but not a higher Vmax value, the reason |i~r which is not clearly understood. A higher content of 5-HT in the lower brain stern at both stages suggests that the 5-HT estimated, originated, to a certain extent, from cell bodies and axons. As there were no developmental differences of Km values in both L-[:~H]NE and [aH]5-HT uptake, it was presumed that the character of both L-[3H]NE and [aH]5-HT uptake was not essentially modified at the developmental stage. In contrast, the regional and developmental differences of Vrnax value indicated that NE and 5-HT neurons innervated each region differently, in this case there is a need for more accurate discussion to consider a monoamine concentration together, although the Vmax value of the uptake is a sensitive index of innervation of monoaminergic neuron. Quantitative electron microscopic findings have revealed postnatal development of synaptic contacts 1,a0. With terminal formation during the postnatal period, synaptic contacts also progressed and physiological functions of monoaminergic neurons increased.
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