Brain Research, 114 (1976) 471479 (© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
471
M O N O A M 1 N E METABOLISM IN T H E D E V E L O P I N G RAT BRAIN A N D EFFECTS OF I O N I Z I N G R A D I A T I O N
D. B. HUDSON, T. VALCANA and P. S. TIMIRAS Department of Physiology-Anatomy, University of California, Berkeley, Calif. 94720 ( U.S.A. )
(Accepted February 20th, 1976)
SUMMARY Neonatal X-radiation induces profound changes in monoamine metabolism in the developing CNS. NE and 5-HT concentrations increase 7 days post-radiation in all CNS regions undergoing rapid axonal growth and proliferation, but not in the region of the cell bodies from which the respective neurotransmitters originate. The increase in NE and 5-HT levels is accompanied by a concomitant increase in the rate of synthesis. While these changes are evident as late as 22 days of age, the monoaminergic systems revert to normal by maturity. It is suggested that these alterations reflect an imbalance in the density of nerve endings to the region where these terminate. These regions are immature at birth and cell proliferation, a process which is affected by X-radiation, is still occurring at the time of exposure.
INTRODUCTION Previous studies in the rat have shown that neonatal X-radiation alters the development of the cholinergic system 28-3o and of GABA 27 in various brain regions. Generally, the changes induced are in the direction of an increase in the concentration of these substances. Our current interest is to determine whether other neurotransmitters are similarly affected by early exposure to X-radiation. Neurotransmitter functions have been ascribed to norepinephrine (NE), serotonin (5-HT) and dopamine (DA), and their intracerebral and subcellular distribution is well known 26. Although various investigations have been conducted on the effects of X-radiation on N E and 5-HT in the central nervous system (CNS), many of the findings reported remain controversial. For example, in the rat, Ershoff and G/d 11 reported no significant change in 5-HT after whole-body X-radiation as compared to untreated, pair-fed controls; whereas Renson and Fisher 23 reported a significant decrease of 5-HT in the hypothalamus after X-radiation. Egafia and Velarde 1° reported that 5-HT increases in various brain areas of the rat following fl-internal (~2p)
472 irradiation of the CNS. The developmental pattern of change of these neurotransmitters in discrete CNS regions has not been extensively studied in terms of levels or in terms of turnover, and we know of no study designed to assess the effects of Xradiation on NE and 5-HT content or turnover at this critical maturational time. On the other hand, the enzymes involved in 5-HT and NE metabolism have been studied, and reportedly increase during the first 3 weeks of postnatal development in the rat 1,5-7,9,,~0. Because the levels of the monoamines differ from one brain region to another, and each has its own timetable of development 17,z5, any study of the effects of X-radiation on their metabolism depends upon the measurement of their turnover with respect to brain area and age. Accordingly, our study was designed to (1) assess the turnover of monoamines in various brain regions, and (2) investigate the effects of neonatal X-radiation on turnover and levels in each region at various developmental ages. in this way, it will be possible to ascertain whether and for how long such changes as do occur are maintained. MATERIALS AND METHODS
Turnover study control We studied the turnover of NE and 5-HT by inhibiting monoamine oxidase and measuring the rate of accumulation o f these substances. Accumulation of catecholamines after inhibition of their catabolism via monoamine oxidase is not a particularly effective manner of assessing turnover since their synthesis is subject to endproduct inhibition 12. Turnover is generally assessed by the fall in monoamine concentration after inhibition of synthesis. However, we found that within the first 90 min of pargyline injection one could assess the rate of synthesis inasmuch as the rates obtained in this manner and the rates obtained by inhibiting synthesis with a-methyltyrosine and p-chlorophenylalanine give similar results 14. A complete analysis should ideally include confirmation by more than one methodological approach. However, because of logistics, this was not possible in the present case. Nine-day-old. male Long-Evans rats were injected mtraperitoneally with an MAO inhibitor, pargyline (75 mg/kg) in isotonic saline, and sacrificed by decapitation 15, 30, 60 and 90 rain lated 2,~1. Control animals received equal volumes o f saline, and one group of controls was sacrificed immediately after saline injection in order to measure zero-time levels: the others were sacrificed on the time schedule described above. The brain was removed, immediately place on ice, and divided into (a) cerebral hemispheres which include the entire neocortex, hippocampus, amygdala, septum and caudate; (b) the cerebellum which was removed by cutting its peduncular connections with brain stem; and the brain stem which was divided into (c) the mesodiencephalon, and (d) the pons-medulla by an oblique cut running from the posterior border of the inferior colliculus on the dorsal surface to the anterior border of the pons on the ventral surface. The levels of NE and 5-HT were determined in individual samples, except in the cerebellum, where two to three samples were combined from the 9-day-old rats. according to the procedures developed by Chang 4 and Maickel et al. is. The tissues were sonified rather than homogenized.
473 Turnover study - - X-radiation
Animals were exposed at 2 days of age to a single dose of 500 R whole body Xirradiation from a 180 kV 5-mA X-ray machine with 0.5 mm of Cu and 1.0 mm of Al filters, as described in Maletta and Timiras 19. Monoamine metabolism was investigated at 9, 22 and 64 days of age. X-radiated and sham-radiated controls were injected with pargyline or saline, as described above, and sacrificed 30 rain post-injection. Zero-time levels were determined in saline-injected sham-radiated controls and salineinjected, X-radiated animals. RESULTS Turnover studies in control 9-day-old animals" Serotonin. Unique regional patterns in neurotransmitter levels appeared in
pargyline-treated 9-day-old animals (Fig. 1). The maximum rate of increase of 5-HT occurred within 30 min in the mesodiencephalon and pons-medulla; however, in the pons-medulla, the increase continued throughout the 90-min period studied. A minimal increase was seen in the cerebral hemispheres while in the cerebellum 5-HT levels were lower for 60 min after pargyline than after saline injection, but by 90 min they had reached control levels. 2,8-
Cerebral hemispheres
Cerebellum
Mesodiencepholon
Pons-medullo
I
./
SEROTONIN 2.4-
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./
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08-
0.4-
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i
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NOREPINEPHRINE
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'o 6'0 "o Time
'0 Jo 9'o
~o e" ~o
(minutes)
Fig. 1. Turnover of monoamines (serotonin and norepinephrine) in discrete regions of the rat brain at 9 days of age. Pargyline, a monoamine oxidase inhibitor, at a dose of 75 mg/kg body weight was injected intraperitoneally and the animals were sacrificed after 15, 30, 60, and 90 min. Control animals received saline injections. The zero-time levels of the monoarnines were obtained from animals sacrificed immediately after saline injection. Each point represents the mean of 3-4 determinations. The standard error is of the range given in Tables I and Ii, and is omitted here for clarity.
Radiated
Control
Radiated
Control
Radiated
Control
32
o~ Change**
192 ± 20* (16) 279 ± 23 (18) P < 0.01 *** 63 ~- 14 (8) 123 ± 13 (6) P < 0.01 500 ± 47 (22) 498 ± 56 (18) 954 ± 118 (22) 952 :~ 104 (18) 32
96 ± 25 (5) 533 ± 165 (5) P < 0.05 1075 ~ 96 (14) t065 ~ 81 (I1) 2031 ~- 201 (13) 1877 ± 200 (11)
358 ± (13) 449 ± (10)
925
1077
567
575
410
33
170
160
97
113
114
115
333
52
61
86
558 (8) 611 (9) 584 (9) 708 (9)
± 58
-¢- 57
:~ 56
± 53
133 i 10 (8) 183 ! 30 (10)
302 ± 14 (8) 334 ± 20 (10) 71
50
190 ± 33 (5) 413 4- 42 (5) P < 0.01 1488 ~- 56 (5) 1455 ± 71 (5) 1850 ~ 98 (5) 2001 ± 230 (5)
759 ± (5) 789 ± (5)
30 min
0 time
ng/g/0.5 h
0 time
30 min
22 days o f age
9 days o f age
1293
1266
844
930
230
57
455
457
ng/g/'0.5 h
183
217
138
167
126
43
136
151
°o Change
* n g 5-HT/g tissue ± S.E. for the number of animals shown in parentheses; ** ~0 change of 5-HT levels between time 0 and 30 mm: *** P values indicate significance of difference from control.
Pons-Medulla
Mesodiencephalon
Cerebellum
Control
Cerebral hemispheres
Radiated
Treatment
Region
Effects o f neonatal X-irradiation on the levels and turnover o f serotonin in various regions of' the rat brain at 9 and 22 days o f age
TABLE I
4~ 4~
475
Norepinephrine. Pargyline induced an increase in NE in the cerebral hemispheres within the first 30 min, after which the levels fell to the range of the saline-injected controls. In the mesodiencephalon, a peak was reached within 30 min, remained at this level until one hour post-injection, and by 90 rain the levels compared with those of saline-injected control values. In the pons-medulla, however, levels continued to increase throughout the period studied. In the cerebellum, no appreciable change was detected in the levels of NE following pargyline treatment.
Effects of X-radiation Serotonin. In the cerebral hemispheres and cerebellum of 9-day-old X-radiated rats, 5-HT levels were higher than in controls, and the higher levels persisted at 22 days of age (Table l). The turnover of 5-HT subsequent to pargyline injection was not markedly different in control and X-radiated animals except in the cerebellum, where it was markedly higher than in controls. This increase in the rate of accumulation in the cerebellum exists whether the results are expressed as ng/g tissue/0.5 h or as ng/ cerebellum/0.5 h. Norepinephrine. NE levels were elevated by radiation in all brain regions at 9 days, markedly so in the cerebellum and cerebral hemispheres (Table ll) and the elevation persisted at 22 days. In all regions, except the cerebellum, turnover at 9 days was only slightly higher in the X-radiated group than in the control group. In the cerebellum of the control there was no apparent turnover of NE, whereas in the radiated cerebellum NE turnover was significantly higher than in any other region studied, control or radiated. Furthermore, between 9 and 22 days of age, NE turnover in the cerebellum increased 10-fold in control animals and declined in the radiated, so that at 22 days no significant difference in turnover was observed between control and radiated animals. DISCUSSION Low NE levels and corresponding low MAO activity reflect the low rate of catecholamine synthesis in the immature CNS TM. In addition, the levels of 5-HT and NE and their accumulation after MAO inhibition, differ with the brain area (Fig. 1), being highest in the pons-medulla and mesodiencephalon, regions which contain the cell bodies of these specific neuronal systems at and which are known to mature early. The increased rate of synthesis of neurotransmitters with age and its dependence upon the region and the transmitter considered, is illustrated by two examples: the turnover of NE generally increases in all brain regions from 9 to 22 days of age, with the highest rate of increase in the cerebellum where a 10-fold increase in the rate of NE accumulation is associated with a decrease in actual levels. The developmental pattern of 5-HT represents another example. The high levels of 5-HT in the mesodiencephalon and pons-medulla correspond to the well-known raphe nuclei which extend up through the mesencephalon~ ; although 5-HT levels do not increase appreci-
Radiated
Control
Radiated
Control
Radiated
Control
126" 4- 4 (21) 149 4- 9 (17) P -< 0.02*** 227 4- 14 (12) 343 ± 42 (9) P < 0.02 222 i 12 (22) 257 i 10 (17) P < 0.05 483 ± 31 (22) 501 4- 34 (19)
0 time
9 days o f age
240 i 14 (5) 684 ± 107 (4) P < 0.01 365 ± 22 (I 3) 456 ± 30 (10) P -< 0.05 720 4- 33 (14) 757 ± 43 (11)
193 ± 4 (13) 2325_ 20 (lO)
30 rain
256
237
199
143
341
t3
83
67
ng/g/0.5 h
57
49
77
64
99
6
56
53
177 4- 1 (11) 259 4- 14 (10) P .< 0.001 375 4- 22 (8) 480 4- 33 (9) P < 0.02 630 4- 36 (9) 744 4- 39 (9) P -~. 0.05
169 4- 6 (8) 180± 9 (8)
% Change** 0 time
1084 3_ 8 (5) 1138 3_ 46 (5)
276 4- 10 (5) 313 ~ 5 (5) P • 0.02 294 4- 10 (6) 405 4- 14 (6) P < 0.001 675 ~_ 26 (5) 830 i 74 (5)
30 rain
22 days o f age
394
454
350
300
146
117
133
107
ng/g/0.5 h
53
72
73
80
56
66
74
63
% Change
*ng NE/g tissue ± S.E. for the number of animals shown in parentheses; ** °i, change of NE levels between time 0 and 30 rain ; *** P values indicate significance of difference from control.
Pons-Medulla
Mesodiencephalon
Cerebellum
Control
Cerebral hemispheres
Radiated
Treatment
Region
Effects o f neonatal X-irradiation on the levels and turnover o f norepinephrine in various regions o f the rat brain at 9 and 22 days o/'age
TABLE II
4~ --0
477 ably in these two regions with age, 5-HT rates of turnover increase markedly. Neonatal X-radiation markedly increases the levels of NE and 5-HT in several brain areas - - changes similar to those observed previously in other neurotransmitters, such as ACh and GABA, under the same experimental conditions z7,28,33. In general, the high levels of monoamines are accompanied by an increase in their rates of synthesis. Thus, in the cerebellum, both levels and turnover rates are higher in the irradiated animals than in controls (Tables I and I1). In the cerebral hemispheres, the 5-HT levels are high, but not the rate of turnover; it is possible that the high levels of 5-HT induce a negative feedback on the rate of synthesis initiated at a time prior to the age studied. The high levels of NE in the X-radiated 9-day-old cerebellum are also accompanied by a more rapid turnover which declines by 22 days. However, no significantly higher turnover is evident in the other regions with the exception of the cerebral hemispheres where, although the levels remain high, the turnover in the Xradiated group decreases, particularly at longer time intervals post-radiation (22 days). This may also be a consequence of a negative feedback mechanism, similar to that proposed for 5-HT. The mode of action of X-radiation on the metabolism of the biogenic amines studied is difficult to interpret. Neonatal X-radiation markedly influences cell number and cell growth in the developing brain ~0, the alterations observed being more marked in the less developed regions, i.e., cerebellum and cerebral hemispheres (where cell division and growth were active at the time of the exposure) than in the more mature regions, i.e., mesodiencephalon and pons-medulla. The lack of significant change in 5HT content and metabolism in the pons-medulla is of interest because this region has already matured in terms of neuronal proliferation at the time of irradiation and the number of neurons would therefore not be affected; however, serotonergic pathways extend from this region to the cerebral hemispheres where 5-HT metabolism is affected by X-radiation. One might speculate, therefore, that the effects of X-radiation in this region reflect: (a) an imbalance in the density of these nerve endings due to a decrease in other cell types in the area2; (b) an abnormal metabolism due to imbalances in other neurotransmitters 27,28 ; or, (c) a direct effect of X-radiation on storage through abnormalities induced in membrane properties of the nerve endings, thereby affecting local amine distributionS, 22. The greater vulnerability of the developing CNS to radiation and the transitory nature (except in the cerebellum) of the alterations reported, indicate that X-radiation interferes with developmental aspects of monoamine metabolism such as cell population of the receptor area and/or branching of axonal terminals of the particular transmitter neuron. This is supported by the following observations: (a) when animals are X-radiated with high levels of radiation in adulthood, a time when neuronal density and neuronal proliferation are already established, no changes are found in monoaminergic neuronsS, 32, or cholinergic neurons29; and (b) when other conditions of abnormal cell growth and proliferation are studied such as those caused by genetic mutation (e.g., the Reeler mouse, in which cerebellar growth is significantly stunted, to a degree comparable to that shown after irradiation), an increase in neurotransmitter levels is similarly observed13,15.
478 The altered n e u r o t r a n s m i t t e r metabolism m a y underlie the f u n c t i o n a l a b n o r m a l ities z4 o f the C N S i n d u c e d by n e o n a t a l X - r a d i a t i o n , specifically the higher overall excitability observed in developing a n i m a l s exposed to n e o n a t a l X - r a d i a t i o n 3:3. The total response of the C N S is the algebraic sum o f the excitatory a n d inhibitory i n p u t s of the various b r a i n regions, a n d the overall alterations i n d u c e d by X-radiation would reflect the relative i m p a i r m e n t of the various n e u r o t r a n s m i t t e r s in these regions. ACKNOWLEDGEMENTS We t h a n k Ms. C. Miller for her technical assistance. We also t h a n k A b b o t t L a b o r a t o r i e s for their generous gift o f Pargyline. This work was s u p p o r t e d by A,E.C. C o n t r a c t No. AT(04-3)-34, Project 82.
REFERENCES 1 Agrawal, H. C., Glisson, S. M. and Himwich, W. A., Changes in monoamines of rat brain during postnatal ontogeny, Biochim. biophys, acta (Amst.), 130 (1966) 511-513. 2 Airman, J., Anderson, W. T. and Wright, K. H., Selected destruction of precursors of microneurons of the cerebellar cortex with fractionated low-dose X-rays, Exp. NeuroL, 17 (1967) 481497. 3 And6n, N. E., Dahlstr6m, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Scand., 67 (1966) 313-326. 4 Chang, C. C., A sensitive method for spectrofluorometric assay of catecholamines, Int. J. Neuropharmacol., 3 (1964) 643-649. 5 Coyle, J. T. and Axelrod, J., Development of the uptake and storage of L-(3H) norepinephrine in the rat brain, J. Neurochem., 18 (1971) 2061-2075. 6 Coyle, J. T. and Axelrod, J., Dopamine-fl-hydroxylase in the rat brain: developmental characteristics, J. Neurochem., 19 (1972) 449-459. 7 Coyle, J. T. and Axelrod, J., Tyrosine hydroxylase in rat brain: developmental characteristics, J. Neurochem., 19 (1972) 1117-1126. 8 Dahlstr6m, A., H~iggendal, J. and Rosengren, B., The effect of roentgen irradiation on monoamine containing neurons in the rat brain, Acta Radiol., 12 (1973) 191-200. 9 Deguchi, T. and Barchas, J., Regional distribution and developmental change of tryptophan hydroxylase activity in rat brain, J. Neurochem., 19 (1972) 927-929. 10 Egafla, E. and Velarde, M. I., Effects of E-internal (p32) irradiation on the rat 5-HT content of CNS levels, Experientia (Basel), 23 (1967) 526-527. 11 Ershoff, B. H. and G,'il, E. M., Effects of radiation on tissue serotonin levels in the rat. Proc. Soc. exp. Biol. (N. Y.J, 108 (1961) 160-162. 12 Everett, G. M., Wiegand, R. G. and Rinaldi, F. U., Pharmacologic studies of some nonhydrazine MAO inhibitors, Ann. N. Y. Acad. Sci., 107 (1963) 1068-1080. 13 Hamburgh, M., Analysis of the postnatal developmental effects of 'reeler', a neurological mutation in mice. A study in developmental genetics, Develop. BioL, 8 (1963) 165-185. 14 Hudson, D. B., Merrill, B. J. and Sands, L. A., Effects of prenatal and postnatal nicotine administration on biochemical asl~ts of brain development. In A. Vernadakis and N. Weiner (Eds.), Drugs and the Developing Brain, Plenum Press, New York, 1974, pp. 243-256. 15 Hudson, D., Valcana, T., Bean, G. and Timiras, P. S., Glutamic acid: a strong candidate as the neurotransmitter of the cereheltar granule cell, Neurochem. Res., (1976) in press. 16 Kulkarni, A. S. and Shideman, F. E., Catecholamine accumulation in the brains of infant and adult rats after monoamine oxidase inhibition, Europ. J. PharmacoL, 3 (1968) 269-271. 17 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.
479 18 Maickel, R. P., Cox, R. H. Jr., Saillant, J. and Miller, F. P., A method for the determination of serotonin and norepinephrine in discrete areas of the brain, Int. J. Neuropharmacol., 7 (1968) 275-281. 19 Maletta, G. J. and Timiras, P. S., Acetyl- and btttyryl-cholinesterase activity of selected brain areas in developing rats after neonatal X-irradiation, J. Neurochem., 13 (1966) 75-84. 20 Nachmias, V. T., Amine oxidase and 5-hydroxytryptamine in developing rat brain, J. Neurochem., 6 (1960) 99-104. 21 Neff, N. H. and Costa, E., Application of steady-state kinetics to the study of catecholamine turnover after monoamine oxidase inhibition or reserpine administration, J. Pharmaeol. exp. Ther., 160 (1968) 4 0 4 7 . 22 Palaid, D. J. and Supek, Z., Liberation of brain 5-hydroxytryptamine and noradrenaline by X-ray treatment in the new-born and adult rat, J. Neurochem., 13 (1966) 705-709. 23 Renson, J. et Fisher, P., Lib6ration de 5-hydroxytryptamine par la rayonnement X, Arch. int. Physiol., 67 (1959) 142-144. 24 Siggins, G. R., Hofl'er, B. J., Oliver, A. P. and Bloom, F. E., Activation of a central noradrenergic projection to cerebellum, Nature (Lond.), 233 (1971) 481483. 25 Timiras, P. S., Vernadakis, A. and Sherwood, N. M., Development and plasticity of the nervous system. In N. S. Assali (Ed.), Biology of Gestation, Vol. H, Academic Press, New York, 1968, pp. 261 319. 26 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand., 367, Suppl. (1971) 1-122. 27 Valcana, T., Hudson, D. and Timiras, P. S., Effects of X-radiation on amino acid content in the developing rat cerebellum, J. Neurochem., 19 (1972) 2229-2232. 28 Valcana, T., Liao, C. and Timiras, P. S., Effects of X-radiation on the development of the cholinergic system of the rat brain. II. Investigation of alterations in acetylcholine content, Environ. Physiol. Biochem., 4 (1974) 58 63. 29 Valcana, T. and Timiras, P. S., Effects of X-radiation on the development of the cholinergic system of the rat brain. I. Study of alterations in choline acetyltransferase and acetylcholinesterase activity and acetylcholinesterase synthesis, Environ. Physiol, Biochem., 4 (1974) 47 57. 30 Valcana, T., Vernadakis, A. and Timiras, P. S., Effects of neonatal X-radiation on choline acetyltransferase activity in various areas of the developing central nervous system. In M. R. Sikov and D. D. Mahlum (Eds.), Radiation Biology of the Fetal and Juvenile Mammal, U.S. Atomic Energy Commission, Washington, 1969, pp. 887-898. 31 Valzelli, L. and Garattini, S., Biogenic amines in discrete brain areas after treatment with monoamine oxidase inhibitors, J. Neurochem., 15 (1968) 259-261. 32 Van Woert, M. H. and Korb, T., Effect of whole-body X-irradiation on tyrosine hydroxylase and catecholamine levels, Life Sci., 9 (I) (1970) 227-232. 33 Vernadakis, A. and Timiras, P. S., Effects of whole-body X-irradiation on electroshock seizure responses in developing rats, Amer. J. Physiol., 205 (1963) 177 1 80.
471
M O N O A M 1 N E METABOLISM IN T H E D E V E L O P I N G RAT BRAIN A N D EFFECTS OF I O N I Z I N G R A D I A T I O N
D. B. HUDSON, T. VALCANA and P. S. TIMIRAS Department of Physiology-Anatomy, University of California, Berkeley, Calif. 94720 ( U.S.A. )
(Accepted February 20th, 1976)
SUMMARY Neonatal X-radiation induces profound changes in monoamine metabolism in the developing CNS. NE and 5-HT concentrations increase 7 days post-radiation in all CNS regions undergoing rapid axonal growth and proliferation, but not in the region of the cell bodies from which the respective neurotransmitters originate. The increase in NE and 5-HT levels is accompanied by a concomitant increase in the rate of synthesis. While these changes are evident as late as 22 days of age, the monoaminergic systems revert to normal by maturity. It is suggested that these alterations reflect an imbalance in the density of nerve endings to the region where these terminate. These regions are immature at birth and cell proliferation, a process which is affected by X-radiation, is still occurring at the time of exposure.
INTRODUCTION Previous studies in the rat have shown that neonatal X-radiation alters the development of the cholinergic system 28-3o and of GABA 27 in various brain regions. Generally, the changes induced are in the direction of an increase in the concentration of these substances. Our current interest is to determine whether other neurotransmitters are similarly affected by early exposure to X-radiation. Neurotransmitter functions have been ascribed to norepinephrine (NE), serotonin (5-HT) and dopamine (DA), and their intracerebral and subcellular distribution is well known 26. Although various investigations have been conducted on the effects of X-radiation on N E and 5-HT in the central nervous system (CNS), many of the findings reported remain controversial. For example, in the rat, Ershoff and G/d 11 reported no significant change in 5-HT after whole-body X-radiation as compared to untreated, pair-fed controls; whereas Renson and Fisher 23 reported a significant decrease of 5-HT in the hypothalamus after X-radiation. Egafia and Velarde 1° reported that 5-HT increases in various brain areas of the rat following fl-internal (~2p)
472 irradiation of the CNS. The developmental pattern of change of these neurotransmitters in discrete CNS regions has not been extensively studied in terms of levels or in terms of turnover, and we know of no study designed to assess the effects of Xradiation on NE and 5-HT content or turnover at this critical maturational time. On the other hand, the enzymes involved in 5-HT and NE metabolism have been studied, and reportedly increase during the first 3 weeks of postnatal development in the rat 1,5-7,9,,~0. Because the levels of the monoamines differ from one brain region to another, and each has its own timetable of development 17,z5, any study of the effects of X-radiation on their metabolism depends upon the measurement of their turnover with respect to brain area and age. Accordingly, our study was designed to (1) assess the turnover of monoamines in various brain regions, and (2) investigate the effects of neonatal X-radiation on turnover and levels in each region at various developmental ages. in this way, it will be possible to ascertain whether and for how long such changes as do occur are maintained. MATERIALS AND METHODS
Turnover study control We studied the turnover of NE and 5-HT by inhibiting monoamine oxidase and measuring the rate of accumulation o f these substances. Accumulation of catecholamines after inhibition of their catabolism via monoamine oxidase is not a particularly effective manner of assessing turnover since their synthesis is subject to endproduct inhibition 12. Turnover is generally assessed by the fall in monoamine concentration after inhibition of synthesis. However, we found that within the first 90 min of pargyline injection one could assess the rate of synthesis inasmuch as the rates obtained in this manner and the rates obtained by inhibiting synthesis with a-methyltyrosine and p-chlorophenylalanine give similar results 14. A complete analysis should ideally include confirmation by more than one methodological approach. However, because of logistics, this was not possible in the present case. Nine-day-old. male Long-Evans rats were injected mtraperitoneally with an MAO inhibitor, pargyline (75 mg/kg) in isotonic saline, and sacrificed by decapitation 15, 30, 60 and 90 rain lated 2,~1. Control animals received equal volumes o f saline, and one group of controls was sacrificed immediately after saline injection in order to measure zero-time levels: the others were sacrificed on the time schedule described above. The brain was removed, immediately place on ice, and divided into (a) cerebral hemispheres which include the entire neocortex, hippocampus, amygdala, septum and caudate; (b) the cerebellum which was removed by cutting its peduncular connections with brain stem; and the brain stem which was divided into (c) the mesodiencephalon, and (d) the pons-medulla by an oblique cut running from the posterior border of the inferior colliculus on the dorsal surface to the anterior border of the pons on the ventral surface. The levels of NE and 5-HT were determined in individual samples, except in the cerebellum, where two to three samples were combined from the 9-day-old rats. according to the procedures developed by Chang 4 and Maickel et al. is. The tissues were sonified rather than homogenized.
473 Turnover study - - X-radiation
Animals were exposed at 2 days of age to a single dose of 500 R whole body Xirradiation from a 180 kV 5-mA X-ray machine with 0.5 mm of Cu and 1.0 mm of Al filters, as described in Maletta and Timiras 19. Monoamine metabolism was investigated at 9, 22 and 64 days of age. X-radiated and sham-radiated controls were injected with pargyline or saline, as described above, and sacrificed 30 rain post-injection. Zero-time levels were determined in saline-injected sham-radiated controls and salineinjected, X-radiated animals. RESULTS Turnover studies in control 9-day-old animals" Serotonin. Unique regional patterns in neurotransmitter levels appeared in
pargyline-treated 9-day-old animals (Fig. 1). The maximum rate of increase of 5-HT occurred within 30 min in the mesodiencephalon and pons-medulla; however, in the pons-medulla, the increase continued throughout the 90-min period studied. A minimal increase was seen in the cerebral hemispheres while in the cerebellum 5-HT levels were lower for 60 min after pargyline than after saline injection, but by 90 min they had reached control levels. 2,8-
Cerebral hemispheres
Cerebellum
Mesodiencepholon
Pons-medullo
I
./
SEROTONIN 2.4-
2.O-
I So,ne,oi-- 1 Pargyline inj.--~
./
I
/
le~
1.2-
/\/"
08-
0.4-
I
i
I
I
I
I
I
I
i
l
NOREPINEPHRINE
08-
i
/-
I
/d
/
)/
06-
0.4-
,/.
02-
Jo "o ~o
/j~......o~.
'o 6'0 "o Time
'0 Jo 9'o
~o e" ~o
(minutes)
Fig. 1. Turnover of monoamines (serotonin and norepinephrine) in discrete regions of the rat brain at 9 days of age. Pargyline, a monoamine oxidase inhibitor, at a dose of 75 mg/kg body weight was injected intraperitoneally and the animals were sacrificed after 15, 30, 60, and 90 min. Control animals received saline injections. The zero-time levels of the monoarnines were obtained from animals sacrificed immediately after saline injection. Each point represents the mean of 3-4 determinations. The standard error is of the range given in Tables I and Ii, and is omitted here for clarity.
Radiated
Control
Radiated
Control
Radiated
Control
32
o~ Change**
192 ± 20* (16) 279 ± 23 (18) P < 0.01 *** 63 ~- 14 (8) 123 ± 13 (6) P < 0.01 500 ± 47 (22) 498 ± 56 (18) 954 ± 118 (22) 952 :~ 104 (18) 32
96 ± 25 (5) 533 ± 165 (5) P < 0.05 1075 ~ 96 (14) t065 ~ 81 (I1) 2031 ~- 201 (13) 1877 ± 200 (11)
358 ± (13) 449 ± (10)
925
1077
567
575
410
33
170
160
97
113
114
115
333
52
61
86
558 (8) 611 (9) 584 (9) 708 (9)
± 58
-¢- 57
:~ 56
± 53
133 i 10 (8) 183 ! 30 (10)
302 ± 14 (8) 334 ± 20 (10) 71
50
190 ± 33 (5) 413 4- 42 (5) P < 0.01 1488 ~- 56 (5) 1455 ± 71 (5) 1850 ~ 98 (5) 2001 ± 230 (5)
759 ± (5) 789 ± (5)
30 min
0 time
ng/g/0.5 h
0 time
30 min
22 days o f age
9 days o f age
1293
1266
844
930
230
57
455
457
ng/g/'0.5 h
183
217
138
167
126
43
136
151
°o Change
* n g 5-HT/g tissue ± S.E. for the number of animals shown in parentheses; ** ~0 change of 5-HT levels between time 0 and 30 mm: *** P values indicate significance of difference from control.
Pons-Medulla
Mesodiencephalon
Cerebellum
Control
Cerebral hemispheres
Radiated
Treatment
Region
Effects o f neonatal X-irradiation on the levels and turnover o f serotonin in various regions of' the rat brain at 9 and 22 days o f age
TABLE I
4~ 4~
475
Norepinephrine. Pargyline induced an increase in NE in the cerebral hemispheres within the first 30 min, after which the levels fell to the range of the saline-injected controls. In the mesodiencephalon, a peak was reached within 30 min, remained at this level until one hour post-injection, and by 90 rain the levels compared with those of saline-injected control values. In the pons-medulla, however, levels continued to increase throughout the period studied. In the cerebellum, no appreciable change was detected in the levels of NE following pargyline treatment.
Effects of X-radiation Serotonin. In the cerebral hemispheres and cerebellum of 9-day-old X-radiated rats, 5-HT levels were higher than in controls, and the higher levels persisted at 22 days of age (Table l). The turnover of 5-HT subsequent to pargyline injection was not markedly different in control and X-radiated animals except in the cerebellum, where it was markedly higher than in controls. This increase in the rate of accumulation in the cerebellum exists whether the results are expressed as ng/g tissue/0.5 h or as ng/ cerebellum/0.5 h. Norepinephrine. NE levels were elevated by radiation in all brain regions at 9 days, markedly so in the cerebellum and cerebral hemispheres (Table ll) and the elevation persisted at 22 days. In all regions, except the cerebellum, turnover at 9 days was only slightly higher in the X-radiated group than in the control group. In the cerebellum of the control there was no apparent turnover of NE, whereas in the radiated cerebellum NE turnover was significantly higher than in any other region studied, control or radiated. Furthermore, between 9 and 22 days of age, NE turnover in the cerebellum increased 10-fold in control animals and declined in the radiated, so that at 22 days no significant difference in turnover was observed between control and radiated animals. DISCUSSION Low NE levels and corresponding low MAO activity reflect the low rate of catecholamine synthesis in the immature CNS TM. In addition, the levels of 5-HT and NE and their accumulation after MAO inhibition, differ with the brain area (Fig. 1), being highest in the pons-medulla and mesodiencephalon, regions which contain the cell bodies of these specific neuronal systems at and which are known to mature early. The increased rate of synthesis of neurotransmitters with age and its dependence upon the region and the transmitter considered, is illustrated by two examples: the turnover of NE generally increases in all brain regions from 9 to 22 days of age, with the highest rate of increase in the cerebellum where a 10-fold increase in the rate of NE accumulation is associated with a decrease in actual levels. The developmental pattern of 5-HT represents another example. The high levels of 5-HT in the mesodiencephalon and pons-medulla correspond to the well-known raphe nuclei which extend up through the mesencephalon~ ; although 5-HT levels do not increase appreci-
Radiated
Control
Radiated
Control
Radiated
Control
126" 4- 4 (21) 149 4- 9 (17) P -< 0.02*** 227 4- 14 (12) 343 ± 42 (9) P < 0.02 222 i 12 (22) 257 i 10 (17) P < 0.05 483 ± 31 (22) 501 4- 34 (19)
0 time
9 days o f age
240 i 14 (5) 684 ± 107 (4) P < 0.01 365 ± 22 (I 3) 456 ± 30 (10) P -< 0.05 720 4- 33 (14) 757 ± 43 (11)
193 ± 4 (13) 2325_ 20 (lO)
30 rain
256
237
199
143
341
t3
83
67
ng/g/0.5 h
57
49
77
64
99
6
56
53
177 4- 1 (11) 259 4- 14 (10) P .< 0.001 375 4- 22 (8) 480 4- 33 (9) P < 0.02 630 4- 36 (9) 744 4- 39 (9) P -~. 0.05
169 4- 6 (8) 180± 9 (8)
% Change** 0 time
1084 3_ 8 (5) 1138 3_ 46 (5)
276 4- 10 (5) 313 ~ 5 (5) P • 0.02 294 4- 10 (6) 405 4- 14 (6) P < 0.001 675 ~_ 26 (5) 830 i 74 (5)
30 rain
22 days o f age
394
454
350
300
146
117
133
107
ng/g/0.5 h
53
72
73
80
56
66
74
63
% Change
*ng NE/g tissue ± S.E. for the number of animals shown in parentheses; ** °i, change of NE levels between time 0 and 30 rain ; *** P values indicate significance of difference from control.
Pons-Medulla
Mesodiencephalon
Cerebellum
Control
Cerebral hemispheres
Radiated
Treatment
Region
Effects o f neonatal X-irradiation on the levels and turnover o f norepinephrine in various regions o f the rat brain at 9 and 22 days o/'age
TABLE II
4~ --0
477 ably in these two regions with age, 5-HT rates of turnover increase markedly. Neonatal X-radiation markedly increases the levels of NE and 5-HT in several brain areas - - changes similar to those observed previously in other neurotransmitters, such as ACh and GABA, under the same experimental conditions z7,28,33. In general, the high levels of monoamines are accompanied by an increase in their rates of synthesis. Thus, in the cerebellum, both levels and turnover rates are higher in the irradiated animals than in controls (Tables I and I1). In the cerebral hemispheres, the 5-HT levels are high, but not the rate of turnover; it is possible that the high levels of 5-HT induce a negative feedback on the rate of synthesis initiated at a time prior to the age studied. The high levels of NE in the X-radiated 9-day-old cerebellum are also accompanied by a more rapid turnover which declines by 22 days. However, no significantly higher turnover is evident in the other regions with the exception of the cerebral hemispheres where, although the levels remain high, the turnover in the Xradiated group decreases, particularly at longer time intervals post-radiation (22 days). This may also be a consequence of a negative feedback mechanism, similar to that proposed for 5-HT. The mode of action of X-radiation on the metabolism of the biogenic amines studied is difficult to interpret. Neonatal X-radiation markedly influences cell number and cell growth in the developing brain ~0, the alterations observed being more marked in the less developed regions, i.e., cerebellum and cerebral hemispheres (where cell division and growth were active at the time of the exposure) than in the more mature regions, i.e., mesodiencephalon and pons-medulla. The lack of significant change in 5HT content and metabolism in the pons-medulla is of interest because this region has already matured in terms of neuronal proliferation at the time of irradiation and the number of neurons would therefore not be affected; however, serotonergic pathways extend from this region to the cerebral hemispheres where 5-HT metabolism is affected by X-radiation. One might speculate, therefore, that the effects of X-radiation in this region reflect: (a) an imbalance in the density of these nerve endings due to a decrease in other cell types in the area2; (b) an abnormal metabolism due to imbalances in other neurotransmitters 27,28 ; or, (c) a direct effect of X-radiation on storage through abnormalities induced in membrane properties of the nerve endings, thereby affecting local amine distributionS, 22. The greater vulnerability of the developing CNS to radiation and the transitory nature (except in the cerebellum) of the alterations reported, indicate that X-radiation interferes with developmental aspects of monoamine metabolism such as cell population of the receptor area and/or branching of axonal terminals of the particular transmitter neuron. This is supported by the following observations: (a) when animals are X-radiated with high levels of radiation in adulthood, a time when neuronal density and neuronal proliferation are already established, no changes are found in monoaminergic neuronsS, 32, or cholinergic neurons29; and (b) when other conditions of abnormal cell growth and proliferation are studied such as those caused by genetic mutation (e.g., the Reeler mouse, in which cerebellar growth is significantly stunted, to a degree comparable to that shown after irradiation), an increase in neurotransmitter levels is similarly observed13,15.
478 The altered n e u r o t r a n s m i t t e r metabolism m a y underlie the f u n c t i o n a l a b n o r m a l ities z4 o f the C N S i n d u c e d by n e o n a t a l X - r a d i a t i o n , specifically the higher overall excitability observed in developing a n i m a l s exposed to n e o n a t a l X - r a d i a t i o n 3:3. The total response of the C N S is the algebraic sum o f the excitatory a n d inhibitory i n p u t s of the various b r a i n regions, a n d the overall alterations i n d u c e d by X-radiation would reflect the relative i m p a i r m e n t of the various n e u r o t r a n s m i t t e r s in these regions. ACKNOWLEDGEMENTS We t h a n k Ms. C. Miller for her technical assistance. We also t h a n k A b b o t t L a b o r a t o r i e s for their generous gift o f Pargyline. This work was s u p p o r t e d by A,E.C. C o n t r a c t No. AT(04-3)-34, Project 82.
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