Neurotoxicity of phenytoin administered to newborn mice on developing cerebellum

Neurotoxicologyand Teratology, Vol. 14, pp. 159-165,1992

0892-0362/92$5.00 + .00 Copyright©1992PergamonPress Ltd.

Printedin the U.S.A. All rights reserved.

Neurotoxicity of Phenytoin Administered to Newborn Mice on Developing Cerebellum HIROMITSU OHMORI, l TAKASHI KOBAYASHI AND MINEO YASUDA

Department o f A n a t o m y , Hiroshima University School o f Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan Received 28 J a n u a r y 1992 OHMORI, H., T. KOBAYASHI AND M. YASUDA. Neurotoxicity of phenytoin administered to newborn mice on developing cerebellum. NEUROTOXICOL TERATOL 14(3), 159-165, 1992.--To examine the neurotoxic effects of phenytoin (PHT) on cerebellar development, we administered 50 mg/kg PHT suspended in sesame oil orally to newborn JchlCR mice once a day during postnatal days 2-14 and determined plasma PHT concentrations at designated intervals during the administration period. In the treated group, walking reflex and negative geotaxis were poorly developed on postnatal day 14. Pyknotic cells in the external granular layer (EGL) significantly increased and were prominent in the vermis area compared with controls on postnatal day 14. Plasma PHT levels were 34-36 #g/ml on the 3rd day of PHT treatment and approached a steady-state situation. Total brain weight, size of the cerebellum, and cerebellar weight were significantly reduced in the treated group on postnatal day 56. Accordingly, oral administration of PHT in the neonatal period induced neurotoxic damage on the developing cerebellum. Phenytoin

Mouse cerebellum

Neonatal period

Bioavailability of phenytoin

the plasma concentrations of PHT, and examined the neurotoxic effects on the development of the cerebellum reflexologically, histologically and morphometrically.

PHENYTOIN (PHT) is a commonly prescribed anticonvulsant drug. Chronic P H T administration to epileptic patients may cause cerebellar dysfunction and, in some cases, cerebellar degeneration (4,13,27). P H T is a weakly acidic and slightly soluble drug, thus, various attempts have been made to increase its solubility and improve its bioavailability to attain effective blood concentrations (17,26). In recent years, it has been reported that oral administration of an oily suspension of PHT has resulted in a significant increase of bioavailability in comparison with powder form or aqueous suspension (32,33). Cerebellar neurotoxicity of P H T in mature experimental animals is well established (22,24,35,36). Also, it has been reported that prenatal P H T exposure induces various neurobehavioral teratogenic effects in experimental animals (1,3739). However, developmental neurotoxicity of PHT on the cerebellum in the early postnatal period has not been established (7). Squier et al. (34) reported a human neonate with neocerebellar hypoplasia following intrauterine exposure to anticonvulsant drugs, including PHT. In addition, it has been discovered that the neonatal period of development in the central nervous system (CNS) in mice corresponds to the last trimester in humans (9). Therefore, we administered a sesame oil suspension of P H T orally to newborn mice, determined

METHOD JcI:ICR mice, purchased from Japan CLEA Co., Ltd. (Tokyo), were bred in our laboratory at 22 _+ 2°C with 50 _+ 10°70 humidity. The mice were mated overnight and when a vaginal plug was found the day was designated as gestational day 0. Pregnant mice were housed separately in a plastic cage. We used only mice delivered spontaneously on gestational day 19. The day of birth was designated as postnatal day 0. Pups were marked directly by branding on their body and were marked again by coloring with picric acid on postnatal day 10. At birth, the size of each litter was culled to 10 pups per dam. The pups of each litter, without regard to sex, were divided into treated and control groups with the ratio of about 3 : 2 by use of a table of random numbers. In total, 11 litters were used for morphological and behavioral studies and 9 litters were used for determination of PHT concentrations. A fine powder of PHT with a mean particle size of 17/zm was specially prepared from commercial PHT (Dainihon Pharmaceutical Co., Ltd., Tokyo) by passing it through a fine

1 Requests for reprints should be addressed to Hiromitsu Ohmori, Department of Anatomy, Hiroshima University School of Medicine, Kasumi I-2-3, Minami-ku, Hiroshima 734, Japan. 159

160

OHMORI, KOBAYASHI AND YASUDA I

i

I

I

A

t..a,

r

B

l

D I . . . .

[-

I I I

C

. . . . . . . . .

T I

r

I I I

FIG. 1. Measurement of each part of the brain. A) Transverse width of the cerebrum; B) Longitudinal width of the cerebrum; C) Transverse width of the cerebellum; and D) Longitudinal width of the cerebellum.

mesh. P H T was suspended in sesame oil (Maruishi Pharmaceutical Co., Ltd., Osaka) by an ultrasonic homogenizer (Model UR-200P, TOMY SEIKO Co., Ltd., Tokyo) to make a 0.5% P H T suspension. To the pups allotted to the treated group, the sesame oil suspension of P H T was administered orally through a polyethylene tube (0.28 mm inside diameter) by a hypodermic syringe with a 27-gauge needle at a dose volume of 10 ml/kg body weight (corresponding to 50 mg P H T / k g body weight) once a day for the period of postnatal days 2-14. In the control group, sesame oil alone was administered at 10 ml/kg body weight once a day for the period of

postnatal days 2-14. The pups were weighed every day from birth to postnatal day 14, and thereafter, they were weighed weekly. On postnatal days 14 and 21, walking reflex and negative geotaxis were tested according to the method of Fox (10). The pups were weaned on postnatal day 21. For examination of early pathological changes, 3 male pups and 4 female pups were killed with an overdose of diethyl ether on postnatal day 14. The brains were removed and immersed in neutralized 15°70 formalin. The cerebella were dehydrated, embedded in paraffin, and sectioned serially at 10/~m thickness in the sagittal plane and stained with hematoxylin and eosin. At 56 days of age, the remaining animals were killed with an overdose of diethyl ether. The brains were removed and weighed. The size of the cerebrum and the cerebellum was measured using an ocular micrometer under a dissection microscope (Fig. 1). Statistical comparisons of differences between the treated and control groups were made using Student's t test. The plasma concentration of PHT was determined as follows: The mice were anesthetized with diethyl ether and about 100-200 /~1 of blood was collected into heparinized tubes. Each animal was used only once for blood sampling. Plasma PHT levels were determined at designated intervals following oral administration of PHT. The blood was centrifuged at 10,000 rpm for 5 rain with a microcentrifuge to obtain the plasma fraction. An internal standard of 5-(4methylphenyl)-5-phenylhydantoin (Aldrich Chemical Co., Milwaukee, WI) was added to give a concentration of 10 izg/ ml of plasma. A plasma sample, 40-100 #1, was then extracted with 7 ml of ethylacetate by vigorously shaking for 20 min. The extract was evaporated under reduced pressure and reconstituted in methanol. Concentration of PHT was determined by high performance liquid chromatography (HPLC) according to the method of Billings (6) with minor modification. The solvent system was acetonitrile/water/acetic acid (2:3:0.05). The flow rate was 0.9 ml/min. Twenty #1 of the solution were injected on a HPLC column. A column of TSKgel ODS80TMCTR (100 x 4.6 mm) (Toso Manufacturing Co., Ltd., Tokyo) was used. The compounds were detected at 235 nm with UV-8 model II wavelength detector (Toso Manufacturing Co., Ltd., Tokyo). PHT was eluted from the column in 4.0 min. Retention time of the internal standard was 5.8 min. RESULTS

From the third day of PHT treatment onward, all treated pups showed clinical deterioration with anorexia, motor hypoactivity, and motor incoordination, and some showed ataxia. Total mortality in the treated group was 42% in males and 39% in females (Table 1). Mortality in the treated group was significantly higher during the first 7 days of PHT treatment

TABLE 1 MORTALITY

IN THE

PHT-TREATED

ANIMALS

Number of Dying Animals on Postnatal Days

PHT-treated (males) PHT-treated (females)

Total Mortality

2- 7

8 - 14

15 - 21

>21 (days)

13/31 (42o70) 11/28 (39°70)

8 7

3 3

2 1

0 0

NEONATAL PHENYTOIN NEUROTOXICITY

40

=

(Males)

and thereafter decreased, but from postnatal day 21 onward, the remaining treated pups were all alive (Table 1). At termination of PHT administration, the treated pups displayed lesser body weight (mean values: males 8.5 g; females 8.2 g) in comparison with controls (mean value: males 11.5 g; females 11.2 g). Thereafter, the treated pups displayed increased weight gain and recovered to control body weights on postnatal day 21. However, treated pups showed a growth deficiency from postnatal day 28 onward (Fig. 2). At even 56 days of age, the body weight of treated male pups was less than that of controls (Table 2). In reflexologic tests, walking reflex and negative geotaxis were poorly developed in the treated group compared with the control group on postnatal day 14 (Table 3). But, on postnatal day 21, walking reflex and negative geotaxis were acquired by all treated pups. Histologically, pyknotic cells in the EGL significantly increased in the treated group compared with the control group in sagittal sections of the 14-day-old mice cerebella (Fig. 3). Pyknotic cells were also observed in the molecular layer and the internal granular layer in the treated group. In addition, the EGL in the treated group was thicker than that in the control group, indicating a developmental delay. These findings were prominent in the vermis area and suggest that proliferation and migration of the granule cell populations suffered interference. At 56 days of age, total brain weight was reduced in the treated group compared with the control group (Table 2). The size and weight of the cerebella were significantly reduced in the treated group compared with the control group (Table 2). The size of the cerebrum, on the other hand, did not differ between the treated and control groups. Cerebella of the treated group were clearly smaller than those of the control group (Fig. 4). Plasma PHT level was 12.5 ± 0.8 #g/ml at 3 h after dosing on the first day of PHT treatment and did not decline at even 24 h (Fig. 5). Plasma PHT level was 34.9 ± 2.1 #g/ml at 3 h after dosing on the third day of PHT treatment, indicating a tendency toward accumulation (Fig. 5). Thereafter, plasma PHT levels at 3 h after dosing were 34-36 #g/ml on days 3-9 of PHT treatment and approached a steady-state situation (Fig.6). However, the plasma PHT level was 19.7

= Control

*-----* P H T - t r e a t e d

(Females)

161

p

., h " "

Control ....... PHT- treated

.,"

30

,A

:" :'

,A,"" j.-

E o ,.- 20 Control

¢-

Control

?;

(m

-7 /

:,S.!f<~ P H T - t r ea ted (males)

@ II1 7,~;t,"

PHT

treated

(females)

.,*'j

PHT AdministrationJ 0

i

7

i

~

i

i

i

e

|

P

14 21 28 35 42 49 56 Age

in

Days

FIG. 2. Mean body weight of the control and treated groups during postnatal days 2-56. The treated pups were administered orally with 50 mg/kg PHT suspended in sesame oil on postnatal days 2-14.

TABLE 2 EFFECT OF PHENYTOIN (PHT) ON BRAIN DEVELOPMENTOF 56-DAY-OLDMICE Male Control Number of mice examined Body weight (g) Brain weight (rag) Cerebellar weight (mg) Size of the cerebrum (A) Transverse width (mm) (B) Longitudinal width (mm) The size of the cerebellum (C) Transverse width (mm) (D) Longitudinal width (mm) *p < 0.05.

Female PHT-treated

Control

PHT-treated

13 41.6 + 2.4 517.4 :L 19.8 71.6 + 2.6

15 37.2 + 2.0* 438.3 + 16.6" 55.4 + 3.1"

19 32.7 + 1.5 511.0 + 2 4 . 9 70.3 + 3.1

13 31.3 + 2.5 432.4 + 17.1" 53.5 + 6.4*

10.3 + 0.3 8.4 + 0.2

10.1 + 0.2 8.3 + 0.3

10.3 + 0.2 8.4 + 0.1

10.0 :l: 0.3 8.2 + 0.2

8.2 + 0.3 4.5 + 0.2

7.8 + 0.2* 4.0 + 0.1"

8.2 =t= 0.2 4.6 + 0.2

7.9 _+ 0.3* 4.0 + 0.4*

OHMORI, KOBAYASHI AND YASUDA

162

TABLE 3 WALKING REFLEX AND NEGATIVE GEOTAXIS ON POSTNATAL DAY 14 Male

Walking reflex Negative geotaxis

Female

Control*

PHT-Treatedl"

Control

PHT-Treated:[:

100 (%) 100 (%)

73.3 (e/0) 86.7 (%)

100 (e/0) 100 (%)

61.5 (%) 76.9 (%)

*n = 13. in = 15.:~n = 19.

+_ 0 . 8 / ~ g / m l at 3 h after dosing on the twelfth day of P H T treatment. There was no difference in plasma P H T levels between males and females. DISCUSSION Most epileptic w o m e n take anticonvulsant drugs during pregnancy. The teratogenicity o f anticonvulsant drugs is most notable for P H T (15). It has been reported that the incidence of fetal hydantoin syndrome is 1 0 % - 3 0 % o f the infants born to w o m e n taking 100-800 r a g / d a y o f P H T during at least the first trimester of pregnancy or beyond (1,20,21). Five infants

originally described as having fetal hydantoin syndrome were all microcephalic but without severe cerebellar malformations (14). However, three infants have been described with cerebellar malformations following intrauterine exposure to anticonvulsant drugs, including P H T (12,25,34). Chronic P H T administration may cause cerebellar dysfunction and cerebellar degeneration in man (4,13,27). Ghatak et al. (13) reported that long-term P H T therapy with epileptic patients caused organic cerebellar damage with loss of Purkinje cells, a rarefied granular cell layer, and gliosis of the cerebellar cortex. Baler et al. (4) also observed cerebellar atrophy as shown by computed tomography. Chronic exposure to

FIG. 3. Early pathological changes of the vermis in 14-day-old mice treated with 50 mg/kg PHT suspended in sesame oil on postnatal days 2-14. (A,B) The vermis in the control group. (C,D) In the treated group, many pyknotic cells (arrows) were observed in the external granular layer and the external granular layer was thicker than that in the control group. Bars: (A,C) 50 /~m, (B,D) 20 #m.

N E O N A T A L P H E N Y T O I N NEUROTOXICITY

163

FIG. 4. Dorsal view of m o u s e brains at 56 days o f age. Left: n o r m a l brain f r o m a control mouse. Right: small cerebellum from a m o u s e treated with 50 m g / k g P H T suspended in sesame oil.

P H T in mature animals induced dystrophic and swollen axons o f Purkinje ceils and degeneration of granule ceils, but the weight of the cerebellum was not significantly decreased after adult exposure, as it was after neonatal exposure in the present study (22,35,36). There have been many studies on the effects o f X-irradiation (2,3,18) or various chemical agents, e.g., methylazoxymethanol (MAM) (5,19), 5-bromodeoxyuridine (BrdU) (41, 42), cytosine arabinoside (40), and ochratoxin A (11) on cerebellar development in the neonatal period. The neonatal period o f CNS development in mice has been compared to the last trimester in humans (9). In the mouse cerebellum, very active cellular proliferation takes place in the EGL after birth and the microneurons, such as basket cells, stellate cells, and granule cells, are produced in this layer during the first 2

E "~ 40

weeks o f life (30,40). Therefore, the EGL is especially sensitive to X-irradiation, some radiomimetic chemicals, and special viral infections during the neonatal period (2,40). Altman et al. (2) exposed the heads of 3-day-old rats to 200 rad X-irradiation and observed many pyknotic ceils in the EGL at 8 and 12 h after treatment. Fukui et al. (11) also observed many pyknotic cells throughout the EGL at 10 to 12 h after ochratoxin A injection, and moreover, reported that cerebellar weight was significantly reduced and that the number of folia was decreased at 30 days of age. Yamano et al. (40) reported that neonatal administration of cytosine arabinoside caused necrosis of the proliferating ceils in the EGL, that the size and foliation of the cerebella were decreased, and that the width o f the EGL was thicker than that in the control. It has been reported that consecutive injections o f BrdU, during postnatal

40-

O - - - O Day ! of PHT Treatment ~ Day 3 of PHT Treatment

E 30-

0

O

30-

c-

~ 20F"1~ 10-

0

E O

20-

k)

,S

0

1

F-

!

£L

3

'

~

~

10-

9 ~ 2'4

Time (hr)

FIG. 5. Representative plasma P H T levels in mice at various time intervals after dosing on the first a n d the third day of P H T treatment. Each point represents the m e a n + SD o f four mice ( P H T was administered o n postnatal days 2-4).

Days of PHT Treatment

FIG. 6. Representative plasma P H T levels at 3 h after dosing from the first to the twelfth day o f P H T treatment. Each point represents the m e a n + SD o f four mice ( P H T was administered on postnatal days 2-14).

164

OHMORI, KOBAYASHI AND YASUDA

days 2, 3, and 4, caused marked cerebellar hypoplasia, necrosis of the cells of the E G L , and delayed dissolution o f the E G L in the developing cerebellum (41). Similar results were obtained by Bejar et al. (5) in the developing cerebella o f mice injected with M A M . A b n o r m a l behavior induced by M A M or X-irradiation has been also reported (3,19). In the present study, pyknotic cells in the E G L , wide E G L , and reduction in cerebellar weight were observed and retardation of m o t o r and behavioral development were seen. The agreement of these findings for many agents with the present P H T results suggests that the effects of neonatal P H T administration are more related to a disruption of development than to the toxicity o f the drug in adults. The plasma P H T levels in our study, with the range o f 3436 /zg/ml on the third day o f P H T treatment, were significantly higher than 10-20/~g/ml which is considered the therapeutic range in humans. These plasma levels correspond to the toxic range in humans. Therefore, it is no wonder that cerebellar and behavioral effects of P H T were seen. The plasma levels decreased to less than 20 # g / m l on the twelfth day of P H T treatment. This may be related to the postnatal development of the activity o f the cytochrome P-450-containing enzyme system in the mouse (23,28). It has been reported that undernutrition reduces brain weight and cerebellar weight (8,16). Because the treated pups exhibited anorexia, the effects

may be due to malnourishment of the pups in addition to P H T ' s direct effects. The treated pups showed a growth deficit after weaning. It has been reported that this kind of late growth deficiency is seen with prenatal P H T exposure (38) and is characteristic of other teratogens producing endocrine failure (29, 31). Therefore, the growth effects of P H T may be related to the damage to the growth-controlling systems of the hypothalamic-pituitary axis. In conclusion, our results indicate that developing cerebellar tissue early in life is susceptible to the neurotoxic effects o f P H T and that oral administration of P H T interferes with development o f the cerebellum. Hence, during pregnancy, epileptic w o m e n should be carefully given P H T in moderate doses while monitoring plasma P H T levels properly. Children exposed prenatally to P H T should be closely examined for neurological abnormalities, including cerebellar malformations. ACKNOWLEDGEMENTS We thank Dr. Y. Higashi and Professor N. Yata, Institute o f Pharmaceutical Sciences, Hiroshima University School of Medicine, for determination of plasma P H T concentrations. The technical assistance of H. Maki and H. Ishihara, Department of A n a t o m y , Hiroshima University School o f Medicine is also gratefully acknowledged.

REFERENCES 1. Adams, J.; Vorhees, C. V.; Middaugh, L. D. Developmental neurotoxicity of anticonvulsants: Human and animal evidence on phenytoin. Neurotoxicol. Teratol. 12:203-214; 1990. 2. Altman, J.,; Anderson, W. J.; Wright, K. A. Reconstitution of the external granular layer of the cerebellar cortex in infant rats after low-level X-irradiation. Anat. Rec. 163:453-472; 1969. 3. Altman, J.; Anderson, W. J.; Strop, M. Retardation of cerebellar and motor development by focal X-irradiation during infancy. Physiol. Behav. 7:143-150; 1971. 4. Baier, W. K.; Beck, U.; Doose, H. Cerebellar atrophy following diphenylhydantoin intoxication. Neuropediatrics 15:76-81; 1984. 5. Bejar, A.; Roujansky, P.; De Barry, J.; Gombos, G. Different effect of methylazoxymethanol on mouse cerebellar development depending on the age of injection. Exp. Brain Res. 57:279-285; 1985. 6. Billings, R. E. Decreased hepatic 5,10-methylenetetrahydrofolate reductase activity in mice after chronic phenytoin treatment. Mol. Pharmacol. 25:459-466; 1984. 7. Blank, N. K.; Nishimura, R. N.; Seil, F. J. Phenytoin neurotoxicity in developing mouse cerebellum in tissue culture. J. Neurol. Sci. 55:91-97; 1982. 8. De Guglielmone, A. E. R.; Soto, A. M.; Duvilanski, B. H. Neonatal undernutrition and RNA synthesis in developing rat brain. J. Neurochem. 22:529-533; 1974. 9. Dobbing, J.; Sands, J. Quantitative growth and development of human brain. Arch. Dis. Child. 48:757-767; 1973. 10. Fox, W. M. Reflex-ontogeny and behavioural development of the mouse. Anim. Behav. 13:234-241; 1965. 11. Fukui, Y.; Hoshino, K.; Kameyama, Y. Developmental abnormalities of mouse cerebellum induced by intracisternal injection of ochratoxin A in neonatal period. Exp. Neurol. 98:54-66; 1987. 12. Gadisseux, J. F.; Rodriguez, J.; Lyon, G. Pontoneocerebellar hypoplasia--A probable consequence of prenatal destruction of the pontine nuclei and a possible role of phenytoin intoxication. Clin. Neuropathol. 3:160-167; 1984. 13. Ghatak, N. R.; Santoso, R. A.; McKinney, W. M. Cerebellar degeneration following long-term phenytoin therapy. Neurology 26:818-820; 1976. 14. Hanson, J. W.; Smith, D. W. The fetal hydantoin syndrome. J. Pediatr. 87:285-290; 1975.

15. Hanson, J. W. Teratogen update: Fetal hydantoin syndrome. Teratology 33:349-353; 1986. 16. Hillman, D. E.; Chen, S. Vulnerability of cerebellar development in malnutrition. I. Quantitation of layer volume and neuron numbers. Neuroscience 6:1249-1262; 1981. 17. Hodges, S.; Forsythe, W. I.; Gillies, D.; Remington, H.; Cawood, A. Bioavailability and dissolution of three phenytoin preparations for children. Dev. Med. Child Neurol. 28:708-712; 1986. 18. Inouye, M.; Kameyama, Y. Cell death in the developing rat cerebellum following X-irradiation of 3 to 100 rad: A quantitative study. J. Radiat. Res. (Tokyo) 24:259-269; 1983. 19. Kabat, K.; Buterbaugh, G. G.; Eccles, C. U. Methylazoxymethanol as a developmental model of neurotoxicity. Neurobehav. Toxicol. Terotol. 7:519-525; 1985. 20. Kelly, T. E. Teratogenicity of anticonvulsant drugs. I: Review of the literature. Am. J. Med. Genet. 19:413-434; 1984. 21. Kelly, T. E.; Edwards, P.; Rein, M.; Miller, J.; Dreifuss, F. Teratogenicity of anticonvulsant drugs. II: A prospective study. Am. J. Med. Genet. 19:435-443; 1984. 22. Kiefer, R.; Knoth, R.; Anagnostopoulos, J.; Volk, B. Cerebellar injury due to phenytoin. Identification and evolution of Purkinje cell axonal swellings in deep cerebellar nuclei of mice. Acta Neuropathol. (Berl) 77:289-298; 1989. 23. Klinger, W.; Mueller, D.; Kleeberg, U. Induction and its dependence on development. In: Estabrook, R. W.; Lindenlaub, E., eds. The induction of drug metabolism. Stuttgart: F. K. Schattauer Verlag; 1978:517-544. 24. Lettmann, E.; Rune, G. M.; Korfsmeiner, K. H. Neurotoxicity of diphenylhydantoin (DPH) in the cerebellum of mongolian gerbils.- An enzyme histochemical study-J. Environ. Pathol. Toxicol. Oncol. 7(4):17-26; 1987. 25. Mallow, D. W.; Herrick, M. K.; Gathman, G. Fetal exposure to anticonvulsant drugs-Detailed pathological study of a case. Arch. Pathol. Lab. Med. 104:215-218; 1980. 26. Manson, J. I.; Beard, S. M.; Magarey, A.; Pollard, A. G.; O'Reilly, W. J. Bioavailability of phenytoin from various pharmaceutical preparations in children. Med. J. Aust. 2:590-592; 1975.

NEONATAL

PHENYTOIN

NEUROTOXICITY

27. McLain, L. W.; Martin, J. T.; Allen, J. H. Cerebellar degeneration due to chronic phenytoin therapy. Ann. Neurol. 7:18-23; 1980. 28. Pelkonen, O. Prenatal and neonatal development of drug and carcinogen metabolism. In: Estabrook, R. W.; Lindenlaub, E., eds., The induction of drug metabolism. Stuttgart: F. K. Schattauer Verlag; 1978:507-516. 29. Pizzi, W. J.; Barnhardt, J. E. Effects of monosodium glutamate on somatic development, obesity and activity in the mouse. Pharmacol. Biochem. Behav. 5:551-557; 1976. 30. Rodier, P. M. Chronology of neuron development: Animal studies and their clinical implications. Dev. Med. Child Neurol. 22: 525-545; 1980. 31. Rodier, P. M.; Kates, B.; White, W. A.; Muhs, A. Effects of prenatal exposure to methylazoxymethanol (MAM) on brain weight, hypothalamic cell number, pituitary structure, and postnatal growth in the rat. Teratology 43:241-251; 1991. 32. Shinkuma, D.; Hamaguchi, T.; Yamanaka, Y.; Mizuno, N.; Yata, N. Influence of vehicle on gastrointestinal absorption of phenytoin in rats. Chem. Pharm. Bull. (Tokyo) 33:4981-4988; 1985. 33. Shinkuma, D.; Hamaguchi, T.; Yamanaka, Y.; Mizuno, N.; Yata, N. Comparison of the effects of sesame oil and oleic acid as suspension vehicles on gastrointestinal absorption of phenytoin in rats. Chem. Pharm. Bull. (Tokyo) 33:4989-4994; 1985. 34. Squier, W.; Hope, P. L.; Lindenbaum, R. H. Neocerebellar hypoplasia in a neonate following intra-uterine exposure to anticonvulsants. Dev. Med. Child Neurol. 32:737-742; 1990.

165 35. Volk, B.; Kirchg~issner, N. Damage of Purkinje cell axons following chronic phenytoin administration: An animal model of distal axonopathy. Acta Neuropathol. (Bed) 67:67-74; 1985. 36. Volk, B.; Kirchg~issner, N.; Detmar, N. Degeneration of granule cells following chronic phenytoin administration: An electron microscopic investigation of the mouse cerebellum. Exp. Neurol. 91:60-70; 1986. 37. Vorhees, C. V.; Minck, D. R.; Berry, H. K. Anticonvulsants and brain development. Prog. Brain Res. 73:229-244; 1988. 38. Vorhees, C. V.; Rindler, J. M.; Minck, D. R. Effects of exposure period and nutrition on the developmental neurotoxicity of anticonvulsants in rats: Short and long-term effects. Neurotoxicology l 1:273-284; 1990. 39. Weisenburger, W. P.; Minck, D. R.; Acuff, K. D.; Vorhees, C. V. Dose-response effects of prenatal phenytoin exposure in rats: Effects on early locomotion, maze learning, and memory as a function of phenytoin-induced circling behavior. Neurotoxicol. Teratol. 12:145-152; 1990. 40. Yamano, T.; Shimada, M.; Abe, Y.; Ohta., S; Ohno, M. Destruction of external granular layer and subsequent cerebellar abnormalities. Acta Neuropathol. (Bed) 59:41-47; 1983. 41. Yu, W. H. A. The effect of 5-bromodeoxyuridine on the postnatal development of the rat cerebellum: Morphologic and radioautographic studies. Am. J. Anat. 150:89-108; 1977. 42. Yu, W. H. A. Restitution of bromodeoxyuridine-induced changes in cerebellar development. Neuropathol. Appl. Neurobiol. 5:4148; 1979.