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The effect of neonatal anoxia on brain cholecystokinin-8-like immunoreactivity and monoamine levels of mature rats
Developmental Brain Research, 26 (1986) 285-288 Elsevier
285
BRD 60136
The effect of neonatal anoxia on brain cholecystokinin-8-1ike immunoreactivity and monoamine levels of mature rats HIDEKO YAMAMOTO and TAKESHI KATO
Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227 (Japan) (Accepted November 19th, 1985)
Key words: anoxia - - cholecystokinin - - neonatal - - rat brain - - catecholamine
The effect of neonatal anoxia upon cholecystokinin-8-1ike immunoreactivity (CCK-8-I) concentrations was investigated in different brain areas of mature rats. Anoxia within 24 h after birth resulted in significantly lower CCK-8-I levels in the cortex, nucleus accumbens, amygdala and hypothalamus of 10-week-old rats. In contrast, no change was observed in the monoamine levels of these brain areas. The data suggest that neonatal anoxia selectively affects CCK-containing neurons.
Prolonged hypoxia leads to persistent brain damage. H o w e v e r , not all regions of the brain are equally affected and neuronal alterations especially appeared in some vulnerable areas 5. In m o n k e y s a total asphyxia up to 20 min in the neonatal period induces a highly reproducible pattern of injury involving structures in the brainstem, but the cerebral cortex and basal ganglia are d a m a g e d either very late in the process, or not at all 9. Rats submitted postnatally to anoxia exhibited behavioral changes and a significant increase in the concentration of muscarinic receptors and fl-adrenergic receptors in the h i p p o c a m p u s during their d e v e l o p m e n t 6. N u m e r o u s studies have demonstrated behavioral effects after perinatal oxygen deprivationS. These effects include both learning deficits and changes in emotionality. In general, peripherally administered cholecystokinin (CCK) affects b e h a v i o r - r e l a t e d satiety. W h e n administered i.p. or i.v., C C K also reduces a series of behaviors including exploration, social interactions and long pauses of behavioral inactivity, and exhibits rapid habituation to a novel e n v i r o n m e n t 1. However, it was not d o c u m e n t e d w h e t h e r hypoxia might affect peptide neurons in the brain or not. In the present study, we describe the effect of postnatal
anoxia on the CCK-8-1ike immunoreactivity (CCK8-1) and m o n o a m i n e levels in the brains of m a t u r e rats. Pregnant S p r a g u e - D a w l e y rats were k e p t under regulated conditions, i.e. 2 5 - 2 7 °C, on a 12-h light/ dark cycle (light on at 08.00 h) and were allowed food and water ad libitum. Pups of either sex were exposed to anoxia on the first postnatal day according to Hershkowitz et al. 6. T h e y were placed in a glass chamber with an inlet and outlet for gasses and exposed to 100% nitrogen for 25 min. Control pups received a similar t r e a t m e n t except for the nitrogen exposure. They were d e c a p i t a t e d at the age of 4 or 10 weeks and tissues were dissected on ice and stored at - 7 0 °C until assayed, H o m o g e n i z a t i o n of the tissues was p e r f o r m e d in 250/~1 of ice-cold distilled water using sonication. O n e portion (50/~1) was a d d e d immediately to 100/~1 of 0.3 M perchloric acid for the m e a s u r e m e n t of m o n o a m i n e s according to the method of Kilts et al. 7. A n o t h e r portion (100 /~1) was added to 900/~1 ice-cold m e t h a n o l for the measurement of CCK-8. Both solutions were centrifuged at 1000 g for 30 min at 4 °C. The supernatant of the perchloric acid solution was directly injected into an H P L C - s y s t e m e q u i p p e d with a LC-4B a m p e r o m e t r i c
Correspondence: H. Yamamoto, Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227 (Japan). 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
286 detector (Bioanalytical Systems, West Lafayette, lN, U.S.A.) using a glassy carbon working electrode. Chromatographic separation was accomplished on a Biophase column (30 cm × 4 mm i.d.) and the mobile phase consisted of a mixture of 0.05 M citrate-phosphate buffer, pH 3.5, and acetonitrile (5%, v/v) containing 1 mM sodium heptanesulfonate. The supernatant of the methanol solution (800 pl) was lyophilized and frozen at - 2 0 °C until assayed. Anti-CCK8-specific antiserum was prepared according to the method of Hashimura et al. 2. This antiserum showed ca. 5% of cross-reactivity with CCK tetrapeptide or CCK-8 non-sulfate and 74.5% with human gastrinI1_17 sulfate. No cross-reaction was observed with substance P and methionine enkephalin up to 10-~ M. The CCK content of the brain areas was measured using a modification of the method of Stjernschantz et al.t~ The detailed procedure will be reported in a separate paper ~3. Protein content was determined using the Bio-Rad protein assay kit (Bio-Rad, Richmond. CA~ U.S.A.). Data were presented as means + S.E.M. and statistical significance of the differences between control and treated animals was determined using Student's two-tailed t-test. The body weights of animals in the anoxia group did not differ significantly from those in the control group at both ages. At 28 days of age, the body weight of rats in the control group was 70.2 + 7.8 g and of those in the anoxia group 65.8 + 7.4 g. At 70 days of age, weights of rats in the control group amounted to 240 + 35 g, and of those in the anoxia group to 215 + 27 g. Fig. 1 presents the effect of neonatal anoxia treatment on the CCK-8-I levels in the mature rat brain. In 28-day-old rats CCK-8-I levels in the anoxia group were significantly decreased in the frontal cortex (44.9%) but not in the hippocampus (123.9%) as compared to controls (data not shown). In 70-day-old rats CCK-8-1 levels in the anoxiatreated group were significantly decreased in the frontal cortex (70%), anterior cingulate cortex (55.6%), frontal parietal cortex motor area (46.1%), somatosensory area (53.3%), nucleus accumbens (45.2%), hypothalamus (54.4%) and amygdala (54.3%), but not in hippocampus (109%) and substantia nigra (98.7%) as compared to the control group.
o
-7-
4
3
2 o
r-n control
6
I
onoxio
I)iiII
Fr.C Ace F'PM F'PS N.Ac Str
Hyp Hip Amg SN Fig. I. Effect of postnatal anoxia upon the concentration in various brain areas of CCK-8-1 of 70-day-old rats. The data represent the means _+ S.E.M. of 7 8 preparations from control animals (~) and animals previously exposed to anoxia for 25 rain (~). Fr.C, frontal cortex: ACC. ant. cingulate cortex: FPM, frontal parietal motor ~rea: FPS, frontal parietal somatosensory area; N.Ac, nucleus accumbens; Str, striatum: Hyp, hypothalamus; Hip, hippocampus; Amg, amygdala: SN, substantia nigra. *, P < 0.05: **. P < 0.0I.
In contrast, at both ages catecholamine and sen)tonin (5-HT) levels in control and anoxia-treated rats did not differ significantly in the majority of brain areas (Tables I and Ill. Although 5-HT levels were decreased significantly in the striatum at 28 days of age, the levels of 5-hydroxyindole acetic ( 5 - H I A A ) acid, a 5-HT metabotite, were not changed in these animals (67.8 + 6.1 pmol/mg protein in the control group vs 57.1 + 3.3 pmol/mg protein in the anoxia TABLE 1
Effect of postnatal anoxia (25 rain) upon the concentrations <~/ the monoamines NE, 5-HT and DA in 28-day-old rats Values are means + S.E.M. for the number of determinations indicated in parentheses. Concentrations of the monoamines are expressed as pmol/mg protein. NE
Frontal cortex control 10.7+0.8 (7) affected 13.3+2.6(7) Hippocampus control 15.6_+1.4(7) affected 13.9+-0.9 (6) Nucleus accumbens control affected Striatum control affected Amygdala control affected
5-HT
DA
24.5+- 1.7 (7) 23.2-+1.2(7) 17.5+-1.2(7) 2(1.3-+1.2 (7) 43.4+-5.{) (6) 45.9-+5.2 (7)
317-+50 353+-33
(6) (7)
28.9-+3.5 (7) 17,2+-1.1 (7)
449_+24 (7) 436_+39 (7)
58.6-+4.7 (7) 62.4+-5.3(6)
61.7_+7.6 (7) 68.1+13.8(6)
287 TABLE II Effect o f postnatal anoxia (25 rain) on rnonoamine concentrations in 70-day-old rats
Values are means + S.E.M. for the number of determinations indicated in parentheses. Concentrations of the monoamines are expressed as pmol/mg protein. NE
Frontal cortex control 30.0_+0.7(5) affected 27.6+1.1(3) Anterior cingulate cortex control 43.0+ 1.5 (6) affected 48.4+2.7 (3) Hippocampus control 36.9+3.4 (6) affected 36.2+2.9 (3)
5-HT
5-HIAA
78.7+4.4 (5) 101+9 (3)
49.7_+5.1 (5) 60.1+9.5(3)
56.7+3.7 (6) 68.4+2.4 (3)
61.5+3.0 (6) 63.3+7.0 (3)
45.3+3.3 (6) 51.2+2.4 (3)
63.1+2.9 (6) 66.2+0.9 (3)
group). N o r e p i n e p h r i n e (NE) and 5-HT levels both increased with age. N e o n a t a l anoxia t r e a t m e n t of the rats left no effect on the catecholamine and 5-HT levels during their d e v e l o p m e n t and maturity. Observations in neonatal and developing animals during hypoxia have d e m o n s t r a t e d a m a r k e d decrease in the activities of tyrosine hydroxylase and tryptophan hydroxylase 3-5. On the other hand, H e d n e r has r e p o r t e d that total anoxia for 20 min did not have any persistent effects on the e n d o g e n o u s levels of the neurotransmitters D A , N E and 5-HT in the whole brain at 28 days of age 5. In our present investigation neonatal anoxia for 25 min did not affect the levels of the neurotransmitters D A , N E and 5H T in the m a j o r i t y of brain areas studied at 28 days or 70 days of age. In addition, the levels of h o m o v a nillic acid ( H V A ) and 5 - H I A A , the metabolites of D A and 5-HT, had not changed, either. A l t h o u g h persistent effects on N E and 5-HT metabolism have
1 Crawley, J.N., Cholecystokinin accelerates the rate of habituation to a novel environment, Pharmacol. Biochem. Behav., 20 (1984) 23-27. 2 Hashimura, E., Shimizu, F., Nishino, T., Imagawa, K., Tateishi, K. and Hamaoka, T., Production of rabbit antibody specific for aminoterminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response, J. Immunol. Meth., 55 (1982) 375-387. 3 Hedner, T. and Lundborg, P., The effect of hypoxia on mono-amine synthesis in brains of developing rats, Biol. Neonate, 31 (1977) 122-126. 4 Hedner, T., Lundborg, P. and Engel, J., The effect of hypoxia on monoamine synthesis in brains of developing rats.
been found in rats exposed to long-lasting asphyxia (45 m i n - 2 h) in infancy t0, the effects of acute hypoxia on the brain levels of the m o n o a m i n e s N E , D A and 5-HT do not a p p e a r to remain for a long time. On the contrary, exposure of neonatal rats to acute anoxia resulted in significantly lower brain CCK-8-I levels at adult age. Especially C C K neurons in the cortex ( F r . C / + A C C + F P M + FPS, see Fig. 1) were m a r k e d l y affected in 70-day-old rats. These findings coincide with the r e p o r t e d m a r k e d loss of cortical neurons after neonatal anoxia ~2. The data suggest that there is a remaining effect of neonatal anoxia on C C K neurons in the cortex, nucleus accumbens and amygdala, but not in the h i p p o c a m p u s and substantia nigra. Similar results were o b t a i n e d with rats at 28 days of age. It is known that the hippocampus is an area of selective vulnerability to ischemia and that, especially in the neonatal period, it is among the first structures to be d a m a g e d 5. Therefore, if it is assumed that C C K neurons in the various brain areas are equally injured during neonatal anoxia, it is p r o b a b l e that there are differences between these brain areas in the rate of recovery of CCK-containing neurons during development. F u r t h e r m o r e it is possible that a hitherto unknown factor could enhance the regeneration of h i p p o c a m p a l C C K neurons. In the present study, neonatal anoxia resulted in a m a r k e d decrease of CCK-8-I levels in the cortex, but not in the hippocampus of m a t u r e rats, whereas there were no changes in D A , N E and 5-HT levels in the brains of these rats. The alterations in CCK-8-I levels may be partially responsible for the behavioral changes in anoxia exposed animals. We thank Dr. J.C. van O e n e for helpful advice in preparing the manuscript.
II. Different length of exposure, Biol. Neonate, 32 (1977) 229-236. 5 Hedner, T., Central monoamine metabolism and neonatal oxygen deprivation. An experimental study in the rat brain, Acta Physiol. Scand., Suppl. 460 (1978) 1-34. 6 Hershkowitz, M., Grimm, V.E. and Speiser, Z., The effects of postnatal anoxia on behaviour and on the muscarinic and fl-adrenergic receptors in the hippocampus of the developing rat, Dev. Brain Res., 7 (1983) 147-155. 7 Kilts, C.D., Breese, G.R. and Mailman, R.B., Simultaneous quantification of dopamine, 5-hydroxytryptamine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography
288 with electrochemical detection, J. Chromatogr., 225 (1981) 347-357. 8 Meier, G.W., Hypoxia. In E. Furchtgott (Ed.), Pharmacological and biophysical agents and behavior. Academic Press, New York, 1971, pp. 99-142. 9 Myers, R.E., Two patterns of perinatal brain damage and their conditions of occurrence, Am. J. Obstet. Gynecol., 112 (1972) 246-276. 10 Simon, N. and Volicer, L., Neonatal asphyxia in the rat: greater vulnerability of males and persistent effects on brain monoamine synthesis, J. Neurochem., 26 (1976)
893-900. l l Stjernschantz, J., Gregerson, D., Bausher, L. and Sears, M., Enzyme-linked immunosorbent assay of substance P: a study in the eye, J, Neurochem., 38 (t982) 1323-1328. 12 Towbin, A., Organic causes of minimal brain dysfunction: perinatal origin of minimal cerebral lesions, JAMA. 217 (1971) 1207-1214. 13 Yamamoto, H. and Kato, T., Enzyme immunoassay for cholecystokinin octapeptide sulfate and its application, J. Neurochem., in print.
285
BRD 60136
The effect of neonatal anoxia on brain cholecystokinin-8-1ike immunoreactivity and monoamine levels of mature rats HIDEKO YAMAMOTO and TAKESHI KATO
Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227 (Japan) (Accepted November 19th, 1985)
Key words: anoxia - - cholecystokinin - - neonatal - - rat brain - - catecholamine
The effect of neonatal anoxia upon cholecystokinin-8-1ike immunoreactivity (CCK-8-I) concentrations was investigated in different brain areas of mature rats. Anoxia within 24 h after birth resulted in significantly lower CCK-8-I levels in the cortex, nucleus accumbens, amygdala and hypothalamus of 10-week-old rats. In contrast, no change was observed in the monoamine levels of these brain areas. The data suggest that neonatal anoxia selectively affects CCK-containing neurons.
Prolonged hypoxia leads to persistent brain damage. H o w e v e r , not all regions of the brain are equally affected and neuronal alterations especially appeared in some vulnerable areas 5. In m o n k e y s a total asphyxia up to 20 min in the neonatal period induces a highly reproducible pattern of injury involving structures in the brainstem, but the cerebral cortex and basal ganglia are d a m a g e d either very late in the process, or not at all 9. Rats submitted postnatally to anoxia exhibited behavioral changes and a significant increase in the concentration of muscarinic receptors and fl-adrenergic receptors in the h i p p o c a m p u s during their d e v e l o p m e n t 6. N u m e r o u s studies have demonstrated behavioral effects after perinatal oxygen deprivationS. These effects include both learning deficits and changes in emotionality. In general, peripherally administered cholecystokinin (CCK) affects b e h a v i o r - r e l a t e d satiety. W h e n administered i.p. or i.v., C C K also reduces a series of behaviors including exploration, social interactions and long pauses of behavioral inactivity, and exhibits rapid habituation to a novel e n v i r o n m e n t 1. However, it was not d o c u m e n t e d w h e t h e r hypoxia might affect peptide neurons in the brain or not. In the present study, we describe the effect of postnatal
anoxia on the CCK-8-1ike immunoreactivity (CCK8-1) and m o n o a m i n e levels in the brains of m a t u r e rats. Pregnant S p r a g u e - D a w l e y rats were k e p t under regulated conditions, i.e. 2 5 - 2 7 °C, on a 12-h light/ dark cycle (light on at 08.00 h) and were allowed food and water ad libitum. Pups of either sex were exposed to anoxia on the first postnatal day according to Hershkowitz et al. 6. T h e y were placed in a glass chamber with an inlet and outlet for gasses and exposed to 100% nitrogen for 25 min. Control pups received a similar t r e a t m e n t except for the nitrogen exposure. They were d e c a p i t a t e d at the age of 4 or 10 weeks and tissues were dissected on ice and stored at - 7 0 °C until assayed, H o m o g e n i z a t i o n of the tissues was p e r f o r m e d in 250/~1 of ice-cold distilled water using sonication. O n e portion (50/~1) was a d d e d immediately to 100/~1 of 0.3 M perchloric acid for the m e a s u r e m e n t of m o n o a m i n e s according to the method of Kilts et al. 7. A n o t h e r portion (100 /~1) was added to 900/~1 ice-cold m e t h a n o l for the measurement of CCK-8. Both solutions were centrifuged at 1000 g for 30 min at 4 °C. The supernatant of the perchloric acid solution was directly injected into an H P L C - s y s t e m e q u i p p e d with a LC-4B a m p e r o m e t r i c
Correspondence: H. Yamamoto, Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227 (Japan). 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
286 detector (Bioanalytical Systems, West Lafayette, lN, U.S.A.) using a glassy carbon working electrode. Chromatographic separation was accomplished on a Biophase column (30 cm × 4 mm i.d.) and the mobile phase consisted of a mixture of 0.05 M citrate-phosphate buffer, pH 3.5, and acetonitrile (5%, v/v) containing 1 mM sodium heptanesulfonate. The supernatant of the methanol solution (800 pl) was lyophilized and frozen at - 2 0 °C until assayed. Anti-CCK8-specific antiserum was prepared according to the method of Hashimura et al. 2. This antiserum showed ca. 5% of cross-reactivity with CCK tetrapeptide or CCK-8 non-sulfate and 74.5% with human gastrinI1_17 sulfate. No cross-reaction was observed with substance P and methionine enkephalin up to 10-~ M. The CCK content of the brain areas was measured using a modification of the method of Stjernschantz et al.t~ The detailed procedure will be reported in a separate paper ~3. Protein content was determined using the Bio-Rad protein assay kit (Bio-Rad, Richmond. CA~ U.S.A.). Data were presented as means + S.E.M. and statistical significance of the differences between control and treated animals was determined using Student's two-tailed t-test. The body weights of animals in the anoxia group did not differ significantly from those in the control group at both ages. At 28 days of age, the body weight of rats in the control group was 70.2 + 7.8 g and of those in the anoxia group 65.8 + 7.4 g. At 70 days of age, weights of rats in the control group amounted to 240 + 35 g, and of those in the anoxia group to 215 + 27 g. Fig. 1 presents the effect of neonatal anoxia treatment on the CCK-8-I levels in the mature rat brain. In 28-day-old rats CCK-8-I levels in the anoxia group were significantly decreased in the frontal cortex (44.9%) but not in the hippocampus (123.9%) as compared to controls (data not shown). In 70-day-old rats CCK-8-1 levels in the anoxiatreated group were significantly decreased in the frontal cortex (70%), anterior cingulate cortex (55.6%), frontal parietal cortex motor area (46.1%), somatosensory area (53.3%), nucleus accumbens (45.2%), hypothalamus (54.4%) and amygdala (54.3%), but not in hippocampus (109%) and substantia nigra (98.7%) as compared to the control group.
o
-7-
4
3
2 o
r-n control
6
I
onoxio
I)iiII
Fr.C Ace F'PM F'PS N.Ac Str
Hyp Hip Amg SN Fig. I. Effect of postnatal anoxia upon the concentration in various brain areas of CCK-8-1 of 70-day-old rats. The data represent the means _+ S.E.M. of 7 8 preparations from control animals (~) and animals previously exposed to anoxia for 25 rain (~). Fr.C, frontal cortex: ACC. ant. cingulate cortex: FPM, frontal parietal motor ~rea: FPS, frontal parietal somatosensory area; N.Ac, nucleus accumbens; Str, striatum: Hyp, hypothalamus; Hip, hippocampus; Amg, amygdala: SN, substantia nigra. *, P < 0.05: **. P < 0.0I.
In contrast, at both ages catecholamine and sen)tonin (5-HT) levels in control and anoxia-treated rats did not differ significantly in the majority of brain areas (Tables I and Ill. Although 5-HT levels were decreased significantly in the striatum at 28 days of age, the levels of 5-hydroxyindole acetic ( 5 - H I A A ) acid, a 5-HT metabotite, were not changed in these animals (67.8 + 6.1 pmol/mg protein in the control group vs 57.1 + 3.3 pmol/mg protein in the anoxia TABLE 1
Effect of postnatal anoxia (25 rain) upon the concentrations <~/ the monoamines NE, 5-HT and DA in 28-day-old rats Values are means + S.E.M. for the number of determinations indicated in parentheses. Concentrations of the monoamines are expressed as pmol/mg protein. NE
Frontal cortex control 10.7+0.8 (7) affected 13.3+2.6(7) Hippocampus control 15.6_+1.4(7) affected 13.9+-0.9 (6) Nucleus accumbens control affected Striatum control affected Amygdala control affected
5-HT
DA
24.5+- 1.7 (7) 23.2-+1.2(7) 17.5+-1.2(7) 2(1.3-+1.2 (7) 43.4+-5.{) (6) 45.9-+5.2 (7)
317-+50 353+-33
(6) (7)
28.9-+3.5 (7) 17,2+-1.1 (7)
449_+24 (7) 436_+39 (7)
58.6-+4.7 (7) 62.4+-5.3(6)
61.7_+7.6 (7) 68.1+13.8(6)
287 TABLE II Effect o f postnatal anoxia (25 rain) on rnonoamine concentrations in 70-day-old rats
Values are means + S.E.M. for the number of determinations indicated in parentheses. Concentrations of the monoamines are expressed as pmol/mg protein. NE
Frontal cortex control 30.0_+0.7(5) affected 27.6+1.1(3) Anterior cingulate cortex control 43.0+ 1.5 (6) affected 48.4+2.7 (3) Hippocampus control 36.9+3.4 (6) affected 36.2+2.9 (3)
5-HT
5-HIAA
78.7+4.4 (5) 101+9 (3)
49.7_+5.1 (5) 60.1+9.5(3)
56.7+3.7 (6) 68.4+2.4 (3)
61.5+3.0 (6) 63.3+7.0 (3)
45.3+3.3 (6) 51.2+2.4 (3)
63.1+2.9 (6) 66.2+0.9 (3)
group). N o r e p i n e p h r i n e (NE) and 5-HT levels both increased with age. N e o n a t a l anoxia t r e a t m e n t of the rats left no effect on the catecholamine and 5-HT levels during their d e v e l o p m e n t and maturity. Observations in neonatal and developing animals during hypoxia have d e m o n s t r a t e d a m a r k e d decrease in the activities of tyrosine hydroxylase and tryptophan hydroxylase 3-5. On the other hand, H e d n e r has r e p o r t e d that total anoxia for 20 min did not have any persistent effects on the e n d o g e n o u s levels of the neurotransmitters D A , N E and 5-HT in the whole brain at 28 days of age 5. In our present investigation neonatal anoxia for 25 min did not affect the levels of the neurotransmitters D A , N E and 5H T in the m a j o r i t y of brain areas studied at 28 days or 70 days of age. In addition, the levels of h o m o v a nillic acid ( H V A ) and 5 - H I A A , the metabolites of D A and 5-HT, had not changed, either. A l t h o u g h persistent effects on N E and 5-HT metabolism have
1 Crawley, J.N., Cholecystokinin accelerates the rate of habituation to a novel environment, Pharmacol. Biochem. Behav., 20 (1984) 23-27. 2 Hashimura, E., Shimizu, F., Nishino, T., Imagawa, K., Tateishi, K. and Hamaoka, T., Production of rabbit antibody specific for aminoterminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response, J. Immunol. Meth., 55 (1982) 375-387. 3 Hedner, T. and Lundborg, P., The effect of hypoxia on mono-amine synthesis in brains of developing rats, Biol. Neonate, 31 (1977) 122-126. 4 Hedner, T., Lundborg, P. and Engel, J., The effect of hypoxia on monoamine synthesis in brains of developing rats.
been found in rats exposed to long-lasting asphyxia (45 m i n - 2 h) in infancy t0, the effects of acute hypoxia on the brain levels of the m o n o a m i n e s N E , D A and 5-HT do not a p p e a r to remain for a long time. On the contrary, exposure of neonatal rats to acute anoxia resulted in significantly lower brain CCK-8-I levels at adult age. Especially C C K neurons in the cortex ( F r . C / + A C C + F P M + FPS, see Fig. 1) were m a r k e d l y affected in 70-day-old rats. These findings coincide with the r e p o r t e d m a r k e d loss of cortical neurons after neonatal anoxia ~2. The data suggest that there is a remaining effect of neonatal anoxia on C C K neurons in the cortex, nucleus accumbens and amygdala, but not in the h i p p o c a m p u s and substantia nigra. Similar results were o b t a i n e d with rats at 28 days of age. It is known that the hippocampus is an area of selective vulnerability to ischemia and that, especially in the neonatal period, it is among the first structures to be d a m a g e d 5. Therefore, if it is assumed that C C K neurons in the various brain areas are equally injured during neonatal anoxia, it is p r o b a b l e that there are differences between these brain areas in the rate of recovery of CCK-containing neurons during development. F u r t h e r m o r e it is possible that a hitherto unknown factor could enhance the regeneration of h i p p o c a m p a l C C K neurons. In the present study, neonatal anoxia resulted in a m a r k e d decrease of CCK-8-I levels in the cortex, but not in the hippocampus of m a t u r e rats, whereas there were no changes in D A , N E and 5-HT levels in the brains of these rats. The alterations in CCK-8-I levels may be partially responsible for the behavioral changes in anoxia exposed animals. We thank Dr. J.C. van O e n e for helpful advice in preparing the manuscript.
II. Different length of exposure, Biol. Neonate, 32 (1977) 229-236. 5 Hedner, T., Central monoamine metabolism and neonatal oxygen deprivation. An experimental study in the rat brain, Acta Physiol. Scand., Suppl. 460 (1978) 1-34. 6 Hershkowitz, M., Grimm, V.E. and Speiser, Z., The effects of postnatal anoxia on behaviour and on the muscarinic and fl-adrenergic receptors in the hippocampus of the developing rat, Dev. Brain Res., 7 (1983) 147-155. 7 Kilts, C.D., Breese, G.R. and Mailman, R.B., Simultaneous quantification of dopamine, 5-hydroxytryptamine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography
288 with electrochemical detection, J. Chromatogr., 225 (1981) 347-357. 8 Meier, G.W., Hypoxia. In E. Furchtgott (Ed.), Pharmacological and biophysical agents and behavior. Academic Press, New York, 1971, pp. 99-142. 9 Myers, R.E., Two patterns of perinatal brain damage and their conditions of occurrence, Am. J. Obstet. Gynecol., 112 (1972) 246-276. 10 Simon, N. and Volicer, L., Neonatal asphyxia in the rat: greater vulnerability of males and persistent effects on brain monoamine synthesis, J. Neurochem., 26 (1976)
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