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Sleep-waking cycle in microencephalic rats induced by prenatal methylazoxymethanol application
Electroencephalography and Clinical Neurophysiology , 1980, 48: 73--79
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© Elsevier/North-Holland Scientific Publishers, Ltd.
SLEEP-WAKING CYCLE IN MICROENCEPHALIC RATS INDUCED BY P R E N A T A L M E T H Y L A Z O X Y M E T H A N O L APPLICATION 1 S. KIYONO, M. SEO and M. SHIBAGAKI
Department of Physiology, Institute for Developmental Research, Aichi Prefectural Colony for the Handicapped, Kasugai, Aichi 480-03 (Japan) (Accepted for publication: April 18, 1979)
Fischer rats injected with 20 mg/kg methylazoxymethanol (MAM) acetate late in pregnancy (15th day of gestation), were found to produce young with considerably reduced cerebral hemispheres (Spatz and Laqueur 1968). The magnitude of the brain deficit was dose dependent, and the forebrain mass was reduced by as much as 60% (Haddad et al. 1972). MAM had a toxic effect on the neuroblast of the rat fetus; the neo- and paleocortices were mainly affected and almost no change was found in the brain stem or cerebellum. Neurons in the neocortex and hippocampus decreased in number (Haddad et al. 1969). Recently, Johnston et al. (1979) reported a virtual absence of neurons in layers II and III, and some disruption of pyramidal cell orientation in the remaining layers. The neocortical cell layer V was lacking due to the destruction of columnar epithelial cells of the periventricular wall in the fetus (Matsutani et al. 1972). Ectopic neurons have been detected in the hippocampus of postnatal h o o d e d rats exposed to MAM during fetal development (Singh 1977). Microencephalic rats performed comparably to controls on several operant tasks (Rabe and Haddad 1972) and conditioned flavor aversion (Woods et al. 1974). On the other hand, MAM-produced microencephalic rats were inferior to controls on several maze
and operant tasks (Haddad et al. 1969; Rabe and Haddad 1972; Matsutani et al. 1973). We have observed a reduction of paradoxical sleep in severely mentally retarded children with various etiologies including microcephalus (Shibagaki et al. 1976), and also a tendency to paradoxical sleep reduction in prenatally X-irradiated microencephalic adult rats (Kiyono et al. 1975a). The present study was carried o u t to reveal sleep-wakefulness characteristics in MAM animals as one of a series of sleep studies in mentally handicapped subjects in our laboratory 2
I This study was supported by a grant from the Ministry of Welfare of Japan for the Study of Pathophysiology of Cerebral Palsy.
2 The article of Sano et al. (1977) was published after completion of Experiment 1 and publication of the preliminary reports (Kiyono et al. 1976a and b).
Methods
Experiment 1 (MAM, 20 mg/kg) The experimental subjects (17 male rats) were offspring of five Sprague--Dawley females, each of which had been injected intraperitoneally with a dose of 20 mg/kg of MAM acetate (diluted to a concentration of 10 mg/ml in 0.9% NaC1) on the 15th day of gestation. The day on which a positive smear was obtained was designated as day 1 of pregnancy. The control rats (15 male) were the young of three females injected with an equivalent volume of diluent. The young, all born within a week, were a b o u t three months old at the start of polygraphic recording.
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Under pentobarbital anesthesia, chronic electrodes were implanted to record EEG, EMG and ocular movements. Following a 5-day recovery period, animals were adapted to a square recording chamber (20 X 25 X 50 cm) with a 12 h Day ( 0 6 : 0 0 - - 1 8 : 0 0 ) -- Night ( 1 8 : 0 0 - - 0 6 : 0 0 ) cycle by a 2 4 h exposure. F o o d and water were given ad libitum. The recording was started at 12:00 at a paper speed of 15 mm/sec, and all animals were gently handled for 1 min just before the recording so that t hey were fully aroused. Each recorded page (20 sec) was scored as either arousal (At), slow wave sleep (SS), or paradoxical sleep (PS) according to the pred o m i n a n t pattern. The sleep-wakefulness parameters studied were as follows: %Ar (ratio of Ar to total recording time), %SS, %PS, PS/TS (ratio of PS to total sleep time), mean PS duration and n u m b e r of PS epochs. Abortive PSs, having durations of less than 40 sec, were included in %PS, but were discarded in the calculation of mean PS duration and the number of PS epochs. After the polygraphic recordings, spontaneous motility was measured using an Animex apparatus (Farad, t y p e S) for 24 h, putting the rat singly in a plastic cage (33 X 38 X 18 cm) with f ood and water ad libitum. At the conclusion of the experiment, rats were killed by an overdose of pentobarbital. Brains were perfused with 10% formalin solution and removed for morphological examination. Comparison of normal and MAMinduced microencephalic data was accomplished by Student's t-test.
Experiment 2 (MAM, 25 mg/kg) The results of Sano et al.'s study (1977) along the same lines as ours suggested that a decrease in PS/TS at the MAM dose of 25 mg/ kg tended to be lower than in controls (0.05 < P < 0.075), and so we replicated their experiment. Experimental procedures were the same as in E x p e r i m e n t 1, except for the MAM dose (25 mg/kg instead of 20 mg/kg) and use of slip-rings to avoid distortion of the leading wires. Polygraphic recordings were
made in 13 control and 13 MAM-treated rats (approximately 3 m ont hs old) for 24 h.
Results
MAM, 20 mg/kg All the experimental animals were microencephalic, having lower brain weight than the control animals. Wet weights of various organs were presented in Table I. Cerebral hemispheres in MAM rats decreased by 44.4% in comparison with the controls. Fresh weights of the cerebellum, liver, kidney and spleen as well as the b o d y weight at the time of autopsy showed no difference between the two groups. A set of parameters studied in the sleepwakefulness cycle is summarized in Table II. No items showed a significant difference between the MAM-treated and control groups. It should be particularly not ed that no difference was found in %SS, %PS and mean PS duration. Other parameters such as maximal duration of a single epoch of Ar ( 7 2 ' 0 8 " +_ 26'01" in controls and 1 0 6 ' 5 8 " + 79'52" in MAM group; mean ± S.D.) and PS (55'00" ± 29'38" vs. 49'28" ± 25'48"), and mean latencies for the first SS ( 1 0 ' 2 9 " + 10'06" vs. 9 ' 3 0 " ± 3'10") and PS ( 5 5 ' 0 0 " 5 29'38" vs. 49'28" ± 25'48") also revealed no difference between the two groups in spite of the remarkable reduction in cerebral mass. Each item was further analysed for Day and Night data, and again no difference was detected. Usually 50--60 PS epochs of varying duration occurred over 24 h. Visual inspection of the polygraphic traces, however, suggested frequent spindle bursts during PS in the MAM group. As is well known, spindles oft en appear during the light phase of sleep and are rarely seen during PS in normal rats. To verify this observation, the mean occurrence rate of spindle bursts per PS epoch was calculated for 8 control and 7 MAM animals, in which well developed spindles have been observed. The sample size of PS examined was 465 for the control and
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S L E E P IN M A M - I N D U C E D M I C R O E N C E P H A L I C R A T S TABLE I Wet w e i g h t s o f various organs in n o r m a l and m i c r o e n c e p h a l i c rats (20 mg/kg). Control Cerebral h e m i s p h e r e s Cerebellum Liver Kidney Spleen Body weight
1.42 0.54 19.00 3.49 1.03 459.3
Experimental + 0.08 * + 0.05 -+ 1.77 + 0.41 -+ 0.26 + 50.6
0.79 0.56 19.10 3.29 0.91 433.3
-+ 0.09 -+ 0.07 + 2.09 -+ 0.26 + 0.17 + 39.8
P < 0.001 N.S. N.S. N.S. N.S. N.S.
* Mean -+ S.D. (g).
348 for the MAM group. The results showed that the rate was 0.44 + 0.30 (range: 0.08-1.14) in controls and 1.45 + 1.15 (0.14--4.05) in the MAM group, the difference being significant (P < 0.05). It should be noted, however, that in the MAM group some animals had almost no spindles during PS (e.g., 0.14), and a control rat had a rate as high as 1.14. As for the spontaneous motility, the mean and S.D. in the MAM group over 24 h was 111.8 + 25.6 against 100.0 + 19.3 in controls, taking the mean value obtained from the control group as the reference. The difference was not statistically significant. The corresponding values were 92.3 + 25.6 vs. 100.0 + 25.5 (N.S.) for Day and 120.0 + 30.6 vs. 100.0 + 22.1 ( P < 0.05) for Night. Thus, a significant increase in the spontaneous motility was found only during the Night.
MAM, 25 mg/kg Body weight (427.2 +-28.1 g in controls and 425.2 + 44.6 g in the MAM group) and cerebellar weight (0.51 + 0.05 g vs. 0.48 + 0.06 g) showed no difference, whereas the wet weight of the cerebral hemispheres in MAM rats decreased by 42.3% (1.49 + 0.08 g in controls and 0.86 + 0.12 g in MAM group), the difference being highly significant (P < 0.001). In terms of the severity of MAMinduced microencephaly, a dose dependency was n o t found. Table III shows the results obtained in sleep-wakefulness paramaters. One can see that, in contrast to Experiment 1, %PS, PS/TS and mean number of PS epochs in microencephalic rats decreased significantly in comparison to normal controls in 24 h recordings. Mean PS duration tended to decrease in MAM
T A B L E II S l e e p - w a k e f u l n e s s p a r a m e t e r s in c o n t r o l a n d M A M - t r e a t e d m i c r o e n c e p h a l i c rats (20 mg/kg) ( m e a n + S.D.).
% Ar
% SS
49.1 + 3.0 48.3 -+ 5.0
43.1 -+ 3.6 44.2 -+ 4.2
34.9 + 6.4 31.9 -+ 5.1 62.6 -+ 7.6 64.3 +- 9.4
% PS
PS/TS (%)
Mean PS duration (sec)
Number of PS e p o c h s
8.0 -+ 1.5 7.6 -+ 1.7
15.6 -+ 3.2 14.5 -+ 2.7
113.5 + 9.7 110.1 -+ 12.0
56.9 -+ 10.8 53.2 + 12.5
54.9 -+ 5.2 57.7 -+ 3.7
10.9 + 2.5 10.4 -+ 3.0
16.3 -+ 3.2 15.1 -+ 3.7
121.4 -+ 13.4 119.1 -+ 15.6
36.7 + 8.5 33.5 -+ 10.6
31.6 + 6.9 30.9 -+ 7.7
5.2 -+ 1.7 4.8 -+ 2.4
14.1 -+ 4.3 12.6 -+ 5.4
101.0 -+ 11.0 92.2 -+ 28.4
20.1 -+ 6.4 19.8 + 10.2
24 h Control MAM
Day Control MAM
Night Control MAM
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S. KIYONO ET AL.
TABLE III Sleep-wakefulness parameters in control and MAM-treated microencephalic rats (25 mg/kg) (mean ± S.D.). % Ar
% SS
% PS
46.9± 5.1 49.2-+ 6.3
44.5-+ 4.4 42.5-+ 4.0
35.2± 7.5 34.6± 5.2
53.8± 6.6 53.6± 4.6
11.0-+1.7 9.8-+2.6
58.3-+ 8.8 63.7 ± 13.0
35.4± 6.9 31.3 ± 10.9
6.3-+2.4 5.0 ± 2.6
PS/TS (%)
MeanPS duration ~sec)
Number of PS epochs
24 h
Control MAM
8.6±1.1 7.4L0.8"**
16.3±1.7 127.8-+1 0 . 1 14.8±1.5"* 119.9±11.7"
56.7±9.8 49.2-+6.7"*
16.9-+1.9 14.9-+2.5"
133.4 ± 8.8 126.5±14.2
34.425.9 31.9±6.8
15.4±4.0 13.4 ± 4.5
116.2±15.5 104.0 ± 16.3 *
22.3±7.5 18.8 ± 9.5
Day
Control MAM Nigh t
Control MAM
* P < 0.10, ** P < 0.05, ***P< 0.01. group. As in MAM-20 mg/kg, %At and %SS showed no difference between the two groups. The difference became less conspicuous when the data were subdivided into Day and Night halves. Only PS/TS in Day and mean PS duration in Night t ended to decrease in the MAM group ( P < 0.10). Spindle bursts appeared frequently during PS as in Experim e n t 1. The occurrence of spindles per PS epoch was calculated in 11 c ont r ol (551 PS epochs) and 13 microencephalic rats (572 epochs). The spindle/PS ratio was 0.17 ± 0.11 (range 0.03--0.34) in co n t r ol and 1.18 + 0.64 (0.14-2.60) in MAM animals, the difference being highly significant (P < 0.001). Wet weights of visceral organs and spontaneous motility were n o t measured.
Discussion The decrease in wet weight of cerebral hemispheres in the experimental group was 44.4% at a dose o f 20 mg/kg and 42.3% at a dose o f 25 mg/kg, which roughly corresponds with the value (about 40%) r e por t ed originally b y Spatz and Laqueur (1968) and Haddad et al. (1969). The fact that the wet weights of liver, kidney and spleen were normal at 20 mg/kg confirmed t ha t the induced
microencephaly was not a part of general microsplanchnia (Spatz and Laqueur 1968), although Fischer et al. (1972) r e p o r t e d a significant decrease in the weights of liver, kidney, spleen and testis in 35-day-old rats exposed in utero to m e t h y l a z o x y m e t h y l acetate, the dose corresponding to 20 mg/kg of MAM acetate. It is not clear why the results failed to show a dose d e p e n d e n c y in terms of the severity of the microencephaly. The results of Sano et al. (1977) were essentially the same as ours. T hey used 20, 25 and 30 mg/kg of MAM on the 15th day of gestation and found no difference in the sleep-wakefulness cycle in adult microencephalic rats up to the dose of 25 mg/kg, except that the decrease in PS/TS was almost significant in the MAM group. The results of a MAM dose of 30 mg/kg were n o t presented. Since the same authors used fewer animals (5 in each MAM group and 5 controls), we replicated their experiment and confirmed the results of MAM-20 mg/kg administration. In addition, we found t hat PS parameters were significantly decreased compared to the controls. In MAM-treated adult rats (20 mg/kg, i.p., on day 15 of gestation as in the present experiments), an increase of specific concentration of 5-HT in the cerebral hemispheres (Matsutani et al. 1973), an increase o f specific
SLEEP IN MAM-INDUCED MICROENCEPHALIC RATS
concentration of norepinephrine and a decrease in total a m o u n t of norepinephrine and GABA (Johnston et al. 1979) have been reported. The role of 5-HT and norepinephfine in the sleep-wakefulness cycle remains controversial. Nevertheless, normal values were obtained in all sleep parameters in the microencephalic animals at a dose of 20 mg/ kg, and there was a significant decrease in PS items at 25 mg/kg. Thus, the present results suggest that firstly, microencephaly per se is n o t necessarily accompanied b y a change of sleep, with particular reference to a decrease in PS, as in the case of prenatally X-irradiated microencephalic adult rats; secondly, a probable permanent increase in specific concentration and a decrease in the total a m o u n t of such biogenic amines as 5-HT and norepinephrine are also not necessarily accompanied by a change of sleep. As described under Methods, we carried o u t two independent experiments using different doses of MAM. In Tables II and III one can see a considerable difference in the mean PS duration (P < 0.01), whereas the t w o control groups failed to show any statistically significant difference in all other items. Accordingly, we could n o t pool the groups into one, and the comparison was made for each experiment. Mean PS duration was longer in Experiment 2 than in Experiment 1 because of the use of slip-rings in the latter, which would afford more natural conditions in terms of less distortion in leading wires from the animal head. The fact that %PS and mean PS duration were in the normal range at the MAM dose of 20 mg/kg might correspond to earlier findings that mild mental retardates show normal %PS (Fujisawa 1974). On the other hand, it has been generally accepted that the %PS decreases in severe mental retardates including microencephalic cases (Petre-Quadens and Jouvet 1966; Petre-Quadens 1972; Shibagaki et al. 1975), and a positive correlation is found between the learning ability and %PS in various inbred strains of mice (Pagel et al. 1972). We have reported a decreasing ten-
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dency of %PS and mean PS duration in adult rats suffering from icterus gravis neonatorum due to prenatal phenylhydrazine administration (Kiyono et al. 1974a), in microencephalic rats due to prenatal X-irradiation (Kiyono et al. 1975a), and in chronic intoxication due to arsenic trioxide application from birth to weaning (Kiyono et al. 1974b). In the Gunn strains of rats, however, the sleep parameters were normal, although they were genetically hyperbilirubinemic with a remarkable cerebellar hypoplasia (Kiyono 1975b). The fact that several PS items significantly decreased at a dose of 25 mg/kg may correspond to the clinically severe cases of mental retardation, although we did n o t find p r o o f that the greater the MAM dose, the more severe the induced microencephaly. Another finding in the present study was the frequent occurrence of spindle bursts during PS in MAM rats. This was also reported by Sano et al. (1977). Since spindle bursts usually appear in the light phase of sleep in normal animals, one can suppose that PS in MAM-induced microencephalic rats is less deep than PS in controls. It has been observed from a sleep deprivation study in cats that the depth of PS is n o t homogeneous; PS becomes deeper after total sleep deprivation than before it (Kiyono et al. 1965). Supposing that PS in the MAM group is less deep than in controls, one could say that sleep in the MAMtreated group (20 mg/kg) is quantitatively similar to that of controls, b u t qualitatively different from normal sleep. As for the sleep spindles in the mental retardates, there are contradictory findings. Gibbs and Gibbs (1962) observed 'extreme spindles', whereas Petre-Quadens found spindles less frequently than in age-matched normal controls. Recently, we have observed that the lower the developmental quotient the fewer the spindle bursts in mental retardates up to 8 years old (Shibagaki et al. in press). Further analyses are necessary to reach a definite conclusion.
78 Summary Sleep-wakefulness cycle o f a d u l t microencephalic rats due to prenatal a d m i n i s t r a t i o n o f m e t h y l a z o x y m e t h a n o l (MAM) acetate at the dose o f 20 m g / k g on the 15th d a y o f gestation s h o w e d n o difference f r o m the normal c o n t r o l g r o u p , whereas at t h e dose o f 25 m g / k g , %PS and P S / T S decreased significantly. M A M - i n d u c e d m i c r o e n c e p h a l i c rats revealed a significant increase o f spindle bursts during PS, suggesting t h a t their PS is less deep than t h a t o f the controls.
Rdsumd
Cycle veille-sommeil chez des rats rendus microcdphales par administration prg-natale de m~thylazoxymdthanol Les auteurs o n t 6tudi6 le cycle veilles o m m e i l chez des rats adultes d o n t la microc6phalie a 6td p r o v o q u d e par a d m i n i s t r a t i o n de m d t h y l a z o x y m d t h a n o l . A u c u n e diff6rence n ' e s t observde p o u r des doses de 20 m g / k g , mais p o u r des doses de 25 m g / k g une diminution significative du p o u r c e n t a g e de sommeil p a r a d o x a l et du r a p p o r t s o m m e i l p a r a d o x a l sur sommeil t o t a l est constat6e. Les rats microcdphales m o n t r e n t une a u g m e n t a t i o n significative des fuseaux c o r t i c a u x group6s en b o u f f 6 e s p e n d a n t le s o m m e i l p a r a d o x a l , ce qui fait s u p p o s e r que leur sommeil p a r a d o x a l est m o i n s p r o f o n d que celui des a n i m a u x de contrble.
References Fujisawa, K. Nocturnal sleep EEG in olygophrenic children. Clin. Electroenceph., 1974, 16:347--355 (Japanese). Fischer, M.H., Welker, C. and Waisman, H.A. Generalized growth retardation in rats induced by prenatal exposure to methylazoxymethyl acetate. Teratology, 1972, 5: 223--232. Gibbs, E.L. and Gibbs, F.A. Extreme spindles: Correlation of electroencephalographic sleep pattern
S. KIYONO ET AL. with mental retardation. Science, 1962, 138: 1106--1107. Haddad, R.K., Rabe, A., Laqueur, G.L., Spatz, M. and Valsamis, M.P. Intellectual deficit associated with transplacentaUy induced microcephaly in the rat. Science, 1969, 163 : 88--90. Haddad, R.K., Rabe, A. and Dumas, R. Comparison of effects of methylazoxymethanol acetate on brain development in different species. Fed. Proc., 1972, 31: 1520--1523. Haddad, R.K., Rabe, A. and Lawson, W.E. Reversal of conditioned aversion in normal and microencephalic rats. J. Comp. Physiol. Psychol., 1974, 86: 531--534. Johnston, M.V., Grzanna, R. and Coyle, J.T. Methylazoxymethanol treatment of fetal rat results in abnormally dense noradrenergic innervation of neocortex. Science, 1979, 203: 369--371. Kiyono, S., Kawamoto, T., Sakakura, H. and Iwama, K. Effects of sleep deprivation upon the paradoxical phase of sleep in cats. Electroenceph. clin. Neurophysiol., 1965, 19: 34--40. Kiyono, S., Hasui, K. and Takasu, K. Sleep-wakefulness cycle in adult rats suffering from icterus gravis neonatorum. J. Physiol. Soc. Japan, 1974a, 36: 106--107. Kiyono, S., Hasui, K., Takasu, K. and Seo, M. Sleepwakefulness cycle in adult rats applied arsenic trioxide from birth to weaning. J. Physiol. Soc. Japan, 1974b, 36: 233--234. Kiyono, S., Seo, M., Takasu, K., Shoji, R., Takeuchi, I. and Murakami, U. Spontaneous motility, EEG and sleep-wakefulness cycle disturbances in rats suffering from fetal X-irradiation. J. Physiol. Soc. Japan, 1975a, 37: 31--32. Kiyono, S. Sleep studies in Gunn rats. In: M.H. Chase et al~ (Eds.), Sleep Research, Vol. 4. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1975b: 227. Kiyono, S., Seo, M. and Shibagaki, M. Sleep-wakefulness parameters in microencephalic rats due to prenatal MAIMtreatment. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 5. Brain Information Service/ Brain Research Institute, UCLA, Los Angeles, 1976a: 73. Kiyono, S., Seo, M. and Shibagaki, M. Sleep-wakefulness cycle in MAMqnduced microencephalic rats. J. Physiol. Soc. Japan, 1976b, 38:114 (Japanese). Matsutani, T., Tsukada, Y., Matsuyama, H. and Koya, G. Changes in cerebral lysosomal enzymes in rat fetus after methylazoxymethanol application. Neurochemistry, 1972, 11 : 48--51 (Japanese). Matsutani, T., Okada, S., Nagayoshi, M., Tsukada, Y. and Nomura, M. Biochemical and behavioral changes in microencephalic rats due to methylazoxymethanol application. Neurochemistry, 1973, 12:28--31 (Japanese).
SLEEP IN MAM-INDUCED MICROENCEPHALIC RATS Pagel, J., Pegram, V., Vaughn, S., Donaldson, P. and Bridgers, W. The relationship of REM sleep with learning and memory in mice. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 1. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1972: 93. Petre-Quadens, O. Sleep in mental retardation. In: C.D. Clemente et al. (Eds.), Sleep and Maturing Nervous System. Academic Press, New York, 1972: 383--417. Petre-Quadens, O. and Jouvet, M. Paradoxical sleep and dreaming in the mentally retarded. J. Neurol. Sci., 1966, 3: 608--612. Rabe, A. and Haddad, R.K. Methylazoxymethanolinduced microencephaly in rats: behavioral studies. Fed. Proc., 1972, 31: 1536--1539. Sano, K., Kawabuchi, J., Tanaka, T., Masuda, Y. and Matsumoto, J. Polygraphic analysis of sleepwakefulness cycle in microencephalic rats induced by methylazoxymethanol acetate. Arch. Tokushima Univ. (Pedag. Sci.), 1977, 2 6 : 4 1 - - 5 0 (Japanese).
79 Shibagaki, M., Matsuno, Y. and Kiyono, S. Sleep studies in the mental retardates. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 4. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1975: 203. Shibagaki, M., Kiyono, S. and Watanabe, K. Developmental studies on the nocturnal sleep in the mental retardates. Jap. J. EEG EMG, 1976, 4: 192-198 (Japanese). Shibagaki, M., Kiyono, S. and Watanabe, K. Evolution of nocturnal sleep spindles in mentally retarded infants. Jap. J. EEG EMG in press. Singh, S.C. Ectopic neurones in the hippocampus of the postnatal rat exposed to methylazoxymethanol during foetal development. Acta neuropath. (Berl.), 1977, 40: 111--116. Spatz, M. and Laqueur, G.L. Transplacental chemical induction of microencephaly in two strains of rats. Proc. Soc. exp. Biol. Med., 1968, 129: 705--710. Woods, S.C. and Lawson, R. Reversal of conditioned aversion in normal and microencephalic rats. J. comp. physiol. Psychol., 1974, 86: 531--534.
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© Elsevier/North-Holland Scientific Publishers, Ltd.
SLEEP-WAKING CYCLE IN MICROENCEPHALIC RATS INDUCED BY P R E N A T A L M E T H Y L A Z O X Y M E T H A N O L APPLICATION 1 S. KIYONO, M. SEO and M. SHIBAGAKI
Department of Physiology, Institute for Developmental Research, Aichi Prefectural Colony for the Handicapped, Kasugai, Aichi 480-03 (Japan) (Accepted for publication: April 18, 1979)
Fischer rats injected with 20 mg/kg methylazoxymethanol (MAM) acetate late in pregnancy (15th day of gestation), were found to produce young with considerably reduced cerebral hemispheres (Spatz and Laqueur 1968). The magnitude of the brain deficit was dose dependent, and the forebrain mass was reduced by as much as 60% (Haddad et al. 1972). MAM had a toxic effect on the neuroblast of the rat fetus; the neo- and paleocortices were mainly affected and almost no change was found in the brain stem or cerebellum. Neurons in the neocortex and hippocampus decreased in number (Haddad et al. 1969). Recently, Johnston et al. (1979) reported a virtual absence of neurons in layers II and III, and some disruption of pyramidal cell orientation in the remaining layers. The neocortical cell layer V was lacking due to the destruction of columnar epithelial cells of the periventricular wall in the fetus (Matsutani et al. 1972). Ectopic neurons have been detected in the hippocampus of postnatal h o o d e d rats exposed to MAM during fetal development (Singh 1977). Microencephalic rats performed comparably to controls on several operant tasks (Rabe and Haddad 1972) and conditioned flavor aversion (Woods et al. 1974). On the other hand, MAM-produced microencephalic rats were inferior to controls on several maze
and operant tasks (Haddad et al. 1969; Rabe and Haddad 1972; Matsutani et al. 1973). We have observed a reduction of paradoxical sleep in severely mentally retarded children with various etiologies including microcephalus (Shibagaki et al. 1976), and also a tendency to paradoxical sleep reduction in prenatally X-irradiated microencephalic adult rats (Kiyono et al. 1975a). The present study was carried o u t to reveal sleep-wakefulness characteristics in MAM animals as one of a series of sleep studies in mentally handicapped subjects in our laboratory 2
I This study was supported by a grant from the Ministry of Welfare of Japan for the Study of Pathophysiology of Cerebral Palsy.
2 The article of Sano et al. (1977) was published after completion of Experiment 1 and publication of the preliminary reports (Kiyono et al. 1976a and b).
Methods
Experiment 1 (MAM, 20 mg/kg) The experimental subjects (17 male rats) were offspring of five Sprague--Dawley females, each of which had been injected intraperitoneally with a dose of 20 mg/kg of MAM acetate (diluted to a concentration of 10 mg/ml in 0.9% NaC1) on the 15th day of gestation. The day on which a positive smear was obtained was designated as day 1 of pregnancy. The control rats (15 male) were the young of three females injected with an equivalent volume of diluent. The young, all born within a week, were a b o u t three months old at the start of polygraphic recording.
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S. KIYONO ET AL.
Under pentobarbital anesthesia, chronic electrodes were implanted to record EEG, EMG and ocular movements. Following a 5-day recovery period, animals were adapted to a square recording chamber (20 X 25 X 50 cm) with a 12 h Day ( 0 6 : 0 0 - - 1 8 : 0 0 ) -- Night ( 1 8 : 0 0 - - 0 6 : 0 0 ) cycle by a 2 4 h exposure. F o o d and water were given ad libitum. The recording was started at 12:00 at a paper speed of 15 mm/sec, and all animals were gently handled for 1 min just before the recording so that t hey were fully aroused. Each recorded page (20 sec) was scored as either arousal (At), slow wave sleep (SS), or paradoxical sleep (PS) according to the pred o m i n a n t pattern. The sleep-wakefulness parameters studied were as follows: %Ar (ratio of Ar to total recording time), %SS, %PS, PS/TS (ratio of PS to total sleep time), mean PS duration and n u m b e r of PS epochs. Abortive PSs, having durations of less than 40 sec, were included in %PS, but were discarded in the calculation of mean PS duration and the number of PS epochs. After the polygraphic recordings, spontaneous motility was measured using an Animex apparatus (Farad, t y p e S) for 24 h, putting the rat singly in a plastic cage (33 X 38 X 18 cm) with f ood and water ad libitum. At the conclusion of the experiment, rats were killed by an overdose of pentobarbital. Brains were perfused with 10% formalin solution and removed for morphological examination. Comparison of normal and MAMinduced microencephalic data was accomplished by Student's t-test.
Experiment 2 (MAM, 25 mg/kg) The results of Sano et al.'s study (1977) along the same lines as ours suggested that a decrease in PS/TS at the MAM dose of 25 mg/ kg tended to be lower than in controls (0.05 < P < 0.075), and so we replicated their experiment. Experimental procedures were the same as in E x p e r i m e n t 1, except for the MAM dose (25 mg/kg instead of 20 mg/kg) and use of slip-rings to avoid distortion of the leading wires. Polygraphic recordings were
made in 13 control and 13 MAM-treated rats (approximately 3 m ont hs old) for 24 h.
Results
MAM, 20 mg/kg All the experimental animals were microencephalic, having lower brain weight than the control animals. Wet weights of various organs were presented in Table I. Cerebral hemispheres in MAM rats decreased by 44.4% in comparison with the controls. Fresh weights of the cerebellum, liver, kidney and spleen as well as the b o d y weight at the time of autopsy showed no difference between the two groups. A set of parameters studied in the sleepwakefulness cycle is summarized in Table II. No items showed a significant difference between the MAM-treated and control groups. It should be particularly not ed that no difference was found in %SS, %PS and mean PS duration. Other parameters such as maximal duration of a single epoch of Ar ( 7 2 ' 0 8 " +_ 26'01" in controls and 1 0 6 ' 5 8 " + 79'52" in MAM group; mean ± S.D.) and PS (55'00" ± 29'38" vs. 49'28" ± 25'48"), and mean latencies for the first SS ( 1 0 ' 2 9 " + 10'06" vs. 9 ' 3 0 " ± 3'10") and PS ( 5 5 ' 0 0 " 5 29'38" vs. 49'28" ± 25'48") also revealed no difference between the two groups in spite of the remarkable reduction in cerebral mass. Each item was further analysed for Day and Night data, and again no difference was detected. Usually 50--60 PS epochs of varying duration occurred over 24 h. Visual inspection of the polygraphic traces, however, suggested frequent spindle bursts during PS in the MAM group. As is well known, spindles oft en appear during the light phase of sleep and are rarely seen during PS in normal rats. To verify this observation, the mean occurrence rate of spindle bursts per PS epoch was calculated for 8 control and 7 MAM animals, in which well developed spindles have been observed. The sample size of PS examined was 465 for the control and
75
S L E E P IN M A M - I N D U C E D M I C R O E N C E P H A L I C R A T S TABLE I Wet w e i g h t s o f various organs in n o r m a l and m i c r o e n c e p h a l i c rats (20 mg/kg). Control Cerebral h e m i s p h e r e s Cerebellum Liver Kidney Spleen Body weight
1.42 0.54 19.00 3.49 1.03 459.3
Experimental + 0.08 * + 0.05 -+ 1.77 + 0.41 -+ 0.26 + 50.6
0.79 0.56 19.10 3.29 0.91 433.3
-+ 0.09 -+ 0.07 + 2.09 -+ 0.26 + 0.17 + 39.8
P < 0.001 N.S. N.S. N.S. N.S. N.S.
* Mean -+ S.D. (g).
348 for the MAM group. The results showed that the rate was 0.44 + 0.30 (range: 0.08-1.14) in controls and 1.45 + 1.15 (0.14--4.05) in the MAM group, the difference being significant (P < 0.05). It should be noted, however, that in the MAM group some animals had almost no spindles during PS (e.g., 0.14), and a control rat had a rate as high as 1.14. As for the spontaneous motility, the mean and S.D. in the MAM group over 24 h was 111.8 + 25.6 against 100.0 + 19.3 in controls, taking the mean value obtained from the control group as the reference. The difference was not statistically significant. The corresponding values were 92.3 + 25.6 vs. 100.0 + 25.5 (N.S.) for Day and 120.0 + 30.6 vs. 100.0 + 22.1 ( P < 0.05) for Night. Thus, a significant increase in the spontaneous motility was found only during the Night.
MAM, 25 mg/kg Body weight (427.2 +-28.1 g in controls and 425.2 + 44.6 g in the MAM group) and cerebellar weight (0.51 + 0.05 g vs. 0.48 + 0.06 g) showed no difference, whereas the wet weight of the cerebral hemispheres in MAM rats decreased by 42.3% (1.49 + 0.08 g in controls and 0.86 + 0.12 g in MAM group), the difference being highly significant (P < 0.001). In terms of the severity of MAMinduced microencephaly, a dose dependency was n o t found. Table III shows the results obtained in sleep-wakefulness paramaters. One can see that, in contrast to Experiment 1, %PS, PS/TS and mean number of PS epochs in microencephalic rats decreased significantly in comparison to normal controls in 24 h recordings. Mean PS duration tended to decrease in MAM
T A B L E II S l e e p - w a k e f u l n e s s p a r a m e t e r s in c o n t r o l a n d M A M - t r e a t e d m i c r o e n c e p h a l i c rats (20 mg/kg) ( m e a n + S.D.).
% Ar
% SS
49.1 + 3.0 48.3 -+ 5.0
43.1 -+ 3.6 44.2 -+ 4.2
34.9 + 6.4 31.9 -+ 5.1 62.6 -+ 7.6 64.3 +- 9.4
% PS
PS/TS (%)
Mean PS duration (sec)
Number of PS e p o c h s
8.0 -+ 1.5 7.6 -+ 1.7
15.6 -+ 3.2 14.5 -+ 2.7
113.5 + 9.7 110.1 -+ 12.0
56.9 -+ 10.8 53.2 + 12.5
54.9 -+ 5.2 57.7 -+ 3.7
10.9 + 2.5 10.4 -+ 3.0
16.3 -+ 3.2 15.1 -+ 3.7
121.4 -+ 13.4 119.1 -+ 15.6
36.7 + 8.5 33.5 -+ 10.6
31.6 + 6.9 30.9 -+ 7.7
5.2 -+ 1.7 4.8 -+ 2.4
14.1 -+ 4.3 12.6 -+ 5.4
101.0 -+ 11.0 92.2 -+ 28.4
20.1 -+ 6.4 19.8 + 10.2
24 h Control MAM
Day Control MAM
Night Control MAM
76
S. KIYONO ET AL.
TABLE III Sleep-wakefulness parameters in control and MAM-treated microencephalic rats (25 mg/kg) (mean ± S.D.). % Ar
% SS
% PS
46.9± 5.1 49.2-+ 6.3
44.5-+ 4.4 42.5-+ 4.0
35.2± 7.5 34.6± 5.2
53.8± 6.6 53.6± 4.6
11.0-+1.7 9.8-+2.6
58.3-+ 8.8 63.7 ± 13.0
35.4± 6.9 31.3 ± 10.9
6.3-+2.4 5.0 ± 2.6
PS/TS (%)
MeanPS duration ~sec)
Number of PS epochs
24 h
Control MAM
8.6±1.1 7.4L0.8"**
16.3±1.7 127.8-+1 0 . 1 14.8±1.5"* 119.9±11.7"
56.7±9.8 49.2-+6.7"*
16.9-+1.9 14.9-+2.5"
133.4 ± 8.8 126.5±14.2
34.425.9 31.9±6.8
15.4±4.0 13.4 ± 4.5
116.2±15.5 104.0 ± 16.3 *
22.3±7.5 18.8 ± 9.5
Day
Control MAM Nigh t
Control MAM
* P < 0.10, ** P < 0.05, ***P< 0.01. group. As in MAM-20 mg/kg, %At and %SS showed no difference between the two groups. The difference became less conspicuous when the data were subdivided into Day and Night halves. Only PS/TS in Day and mean PS duration in Night t ended to decrease in the MAM group ( P < 0.10). Spindle bursts appeared frequently during PS as in Experim e n t 1. The occurrence of spindles per PS epoch was calculated in 11 c ont r ol (551 PS epochs) and 13 microencephalic rats (572 epochs). The spindle/PS ratio was 0.17 ± 0.11 (range 0.03--0.34) in co n t r ol and 1.18 + 0.64 (0.14-2.60) in MAM animals, the difference being highly significant (P < 0.001). Wet weights of visceral organs and spontaneous motility were n o t measured.
Discussion The decrease in wet weight of cerebral hemispheres in the experimental group was 44.4% at a dose o f 20 mg/kg and 42.3% at a dose o f 25 mg/kg, which roughly corresponds with the value (about 40%) r e por t ed originally b y Spatz and Laqueur (1968) and Haddad et al. (1969). The fact that the wet weights of liver, kidney and spleen were normal at 20 mg/kg confirmed t ha t the induced
microencephaly was not a part of general microsplanchnia (Spatz and Laqueur 1968), although Fischer et al. (1972) r e p o r t e d a significant decrease in the weights of liver, kidney, spleen and testis in 35-day-old rats exposed in utero to m e t h y l a z o x y m e t h y l acetate, the dose corresponding to 20 mg/kg of MAM acetate. It is not clear why the results failed to show a dose d e p e n d e n c y in terms of the severity of the microencephaly. The results of Sano et al. (1977) were essentially the same as ours. T hey used 20, 25 and 30 mg/kg of MAM on the 15th day of gestation and found no difference in the sleep-wakefulness cycle in adult microencephalic rats up to the dose of 25 mg/kg, except that the decrease in PS/TS was almost significant in the MAM group. The results of a MAM dose of 30 mg/kg were n o t presented. Since the same authors used fewer animals (5 in each MAM group and 5 controls), we replicated their experiment and confirmed the results of MAM-20 mg/kg administration. In addition, we found t hat PS parameters were significantly decreased compared to the controls. In MAM-treated adult rats (20 mg/kg, i.p., on day 15 of gestation as in the present experiments), an increase of specific concentration of 5-HT in the cerebral hemispheres (Matsutani et al. 1973), an increase o f specific
SLEEP IN MAM-INDUCED MICROENCEPHALIC RATS
concentration of norepinephrine and a decrease in total a m o u n t of norepinephrine and GABA (Johnston et al. 1979) have been reported. The role of 5-HT and norepinephfine in the sleep-wakefulness cycle remains controversial. Nevertheless, normal values were obtained in all sleep parameters in the microencephalic animals at a dose of 20 mg/ kg, and there was a significant decrease in PS items at 25 mg/kg. Thus, the present results suggest that firstly, microencephaly per se is n o t necessarily accompanied b y a change of sleep, with particular reference to a decrease in PS, as in the case of prenatally X-irradiated microencephalic adult rats; secondly, a probable permanent increase in specific concentration and a decrease in the total a m o u n t of such biogenic amines as 5-HT and norepinephrine are also not necessarily accompanied by a change of sleep. As described under Methods, we carried o u t two independent experiments using different doses of MAM. In Tables II and III one can see a considerable difference in the mean PS duration (P < 0.01), whereas the t w o control groups failed to show any statistically significant difference in all other items. Accordingly, we could n o t pool the groups into one, and the comparison was made for each experiment. Mean PS duration was longer in Experiment 2 than in Experiment 1 because of the use of slip-rings in the latter, which would afford more natural conditions in terms of less distortion in leading wires from the animal head. The fact that %PS and mean PS duration were in the normal range at the MAM dose of 20 mg/kg might correspond to earlier findings that mild mental retardates show normal %PS (Fujisawa 1974). On the other hand, it has been generally accepted that the %PS decreases in severe mental retardates including microencephalic cases (Petre-Quadens and Jouvet 1966; Petre-Quadens 1972; Shibagaki et al. 1975), and a positive correlation is found between the learning ability and %PS in various inbred strains of mice (Pagel et al. 1972). We have reported a decreasing ten-
77
dency of %PS and mean PS duration in adult rats suffering from icterus gravis neonatorum due to prenatal phenylhydrazine administration (Kiyono et al. 1974a), in microencephalic rats due to prenatal X-irradiation (Kiyono et al. 1975a), and in chronic intoxication due to arsenic trioxide application from birth to weaning (Kiyono et al. 1974b). In the Gunn strains of rats, however, the sleep parameters were normal, although they were genetically hyperbilirubinemic with a remarkable cerebellar hypoplasia (Kiyono 1975b). The fact that several PS items significantly decreased at a dose of 25 mg/kg may correspond to the clinically severe cases of mental retardation, although we did n o t find p r o o f that the greater the MAM dose, the more severe the induced microencephaly. Another finding in the present study was the frequent occurrence of spindle bursts during PS in MAM rats. This was also reported by Sano et al. (1977). Since spindle bursts usually appear in the light phase of sleep in normal animals, one can suppose that PS in MAM-induced microencephalic rats is less deep than PS in controls. It has been observed from a sleep deprivation study in cats that the depth of PS is n o t homogeneous; PS becomes deeper after total sleep deprivation than before it (Kiyono et al. 1965). Supposing that PS in the MAM group is less deep than in controls, one could say that sleep in the MAMtreated group (20 mg/kg) is quantitatively similar to that of controls, b u t qualitatively different from normal sleep. As for the sleep spindles in the mental retardates, there are contradictory findings. Gibbs and Gibbs (1962) observed 'extreme spindles', whereas Petre-Quadens found spindles less frequently than in age-matched normal controls. Recently, we have observed that the lower the developmental quotient the fewer the spindle bursts in mental retardates up to 8 years old (Shibagaki et al. in press). Further analyses are necessary to reach a definite conclusion.
78 Summary Sleep-wakefulness cycle o f a d u l t microencephalic rats due to prenatal a d m i n i s t r a t i o n o f m e t h y l a z o x y m e t h a n o l (MAM) acetate at the dose o f 20 m g / k g on the 15th d a y o f gestation s h o w e d n o difference f r o m the normal c o n t r o l g r o u p , whereas at t h e dose o f 25 m g / k g , %PS and P S / T S decreased significantly. M A M - i n d u c e d m i c r o e n c e p h a l i c rats revealed a significant increase o f spindle bursts during PS, suggesting t h a t their PS is less deep than t h a t o f the controls.
Rdsumd
Cycle veille-sommeil chez des rats rendus microcdphales par administration prg-natale de m~thylazoxymdthanol Les auteurs o n t 6tudi6 le cycle veilles o m m e i l chez des rats adultes d o n t la microc6phalie a 6td p r o v o q u d e par a d m i n i s t r a t i o n de m d t h y l a z o x y m d t h a n o l . A u c u n e diff6rence n ' e s t observde p o u r des doses de 20 m g / k g , mais p o u r des doses de 25 m g / k g une diminution significative du p o u r c e n t a g e de sommeil p a r a d o x a l et du r a p p o r t s o m m e i l p a r a d o x a l sur sommeil t o t a l est constat6e. Les rats microcdphales m o n t r e n t une a u g m e n t a t i o n significative des fuseaux c o r t i c a u x group6s en b o u f f 6 e s p e n d a n t le s o m m e i l p a r a d o x a l , ce qui fait s u p p o s e r que leur sommeil p a r a d o x a l est m o i n s p r o f o n d que celui des a n i m a u x de contrble.
References Fujisawa, K. Nocturnal sleep EEG in olygophrenic children. Clin. Electroenceph., 1974, 16:347--355 (Japanese). Fischer, M.H., Welker, C. and Waisman, H.A. Generalized growth retardation in rats induced by prenatal exposure to methylazoxymethyl acetate. Teratology, 1972, 5: 223--232. Gibbs, E.L. and Gibbs, F.A. Extreme spindles: Correlation of electroencephalographic sleep pattern
S. KIYONO ET AL. with mental retardation. Science, 1962, 138: 1106--1107. Haddad, R.K., Rabe, A., Laqueur, G.L., Spatz, M. and Valsamis, M.P. Intellectual deficit associated with transplacentaUy induced microcephaly in the rat. Science, 1969, 163 : 88--90. Haddad, R.K., Rabe, A. and Dumas, R. Comparison of effects of methylazoxymethanol acetate on brain development in different species. Fed. Proc., 1972, 31: 1520--1523. Haddad, R.K., Rabe, A. and Lawson, W.E. Reversal of conditioned aversion in normal and microencephalic rats. J. Comp. Physiol. Psychol., 1974, 86: 531--534. Johnston, M.V., Grzanna, R. and Coyle, J.T. Methylazoxymethanol treatment of fetal rat results in abnormally dense noradrenergic innervation of neocortex. Science, 1979, 203: 369--371. Kiyono, S., Kawamoto, T., Sakakura, H. and Iwama, K. Effects of sleep deprivation upon the paradoxical phase of sleep in cats. Electroenceph. clin. Neurophysiol., 1965, 19: 34--40. Kiyono, S., Hasui, K. and Takasu, K. Sleep-wakefulness cycle in adult rats suffering from icterus gravis neonatorum. J. Physiol. Soc. Japan, 1974a, 36: 106--107. Kiyono, S., Hasui, K., Takasu, K. and Seo, M. Sleepwakefulness cycle in adult rats applied arsenic trioxide from birth to weaning. J. Physiol. Soc. Japan, 1974b, 36: 233--234. Kiyono, S., Seo, M., Takasu, K., Shoji, R., Takeuchi, I. and Murakami, U. Spontaneous motility, EEG and sleep-wakefulness cycle disturbances in rats suffering from fetal X-irradiation. J. Physiol. Soc. Japan, 1975a, 37: 31--32. Kiyono, S. Sleep studies in Gunn rats. In: M.H. Chase et al~ (Eds.), Sleep Research, Vol. 4. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1975b: 227. Kiyono, S., Seo, M. and Shibagaki, M. Sleep-wakefulness parameters in microencephalic rats due to prenatal MAIMtreatment. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 5. Brain Information Service/ Brain Research Institute, UCLA, Los Angeles, 1976a: 73. Kiyono, S., Seo, M. and Shibagaki, M. Sleep-wakefulness cycle in MAMqnduced microencephalic rats. J. Physiol. Soc. Japan, 1976b, 38:114 (Japanese). Matsutani, T., Tsukada, Y., Matsuyama, H. and Koya, G. Changes in cerebral lysosomal enzymes in rat fetus after methylazoxymethanol application. Neurochemistry, 1972, 11 : 48--51 (Japanese). Matsutani, T., Okada, S., Nagayoshi, M., Tsukada, Y. and Nomura, M. Biochemical and behavioral changes in microencephalic rats due to methylazoxymethanol application. Neurochemistry, 1973, 12:28--31 (Japanese).
SLEEP IN MAM-INDUCED MICROENCEPHALIC RATS Pagel, J., Pegram, V., Vaughn, S., Donaldson, P. and Bridgers, W. The relationship of REM sleep with learning and memory in mice. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 1. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1972: 93. Petre-Quadens, O. Sleep in mental retardation. In: C.D. Clemente et al. (Eds.), Sleep and Maturing Nervous System. Academic Press, New York, 1972: 383--417. Petre-Quadens, O. and Jouvet, M. Paradoxical sleep and dreaming in the mentally retarded. J. Neurol. Sci., 1966, 3: 608--612. Rabe, A. and Haddad, R.K. Methylazoxymethanolinduced microencephaly in rats: behavioral studies. Fed. Proc., 1972, 31: 1536--1539. Sano, K., Kawabuchi, J., Tanaka, T., Masuda, Y. and Matsumoto, J. Polygraphic analysis of sleepwakefulness cycle in microencephalic rats induced by methylazoxymethanol acetate. Arch. Tokushima Univ. (Pedag. Sci.), 1977, 2 6 : 4 1 - - 5 0 (Japanese).
79 Shibagaki, M., Matsuno, Y. and Kiyono, S. Sleep studies in the mental retardates. In: M.H. Chase et al. (Eds.), Sleep Research, Vol. 4. Brain Information Service/Brain Research Institute, UCLA, Los Angeles, 1975: 203. Shibagaki, M., Kiyono, S. and Watanabe, K. Developmental studies on the nocturnal sleep in the mental retardates. Jap. J. EEG EMG, 1976, 4: 192-198 (Japanese). Shibagaki, M., Kiyono, S. and Watanabe, K. Evolution of nocturnal sleep spindles in mentally retarded infants. Jap. J. EEG EMG in press. Singh, S.C. Ectopic neurones in the hippocampus of the postnatal rat exposed to methylazoxymethanol during foetal development. Acta neuropath. (Berl.), 1977, 40: 111--116. Spatz, M. and Laqueur, G.L. Transplacental chemical induction of microencephaly in two strains of rats. Proc. Soc. exp. Biol. Med., 1968, 129: 705--710. Woods, S.C. and Lawson, R. Reversal of conditioned aversion in normal and microencephalic rats. J. comp. physiol. Psychol., 1974, 86: 531--534.