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Effects of ethanol on postnatal cell acquisition in the rat cerebellum
388
Brain Research, 271 (1983)388-391 Elsevier
Effects of ethanol on postnatal cell acquisition in the rat cerebellum SALVADOR BORGES and PAUL D. LEWIS* Department of Histopathology, Royal PostgraduateMedicalSchool, DucaneRoad, London WI2 0HS (U. K.) (Accepted March 15th, 1983) Key words: ethanol - - brain development- - cerebellum - - neurogenesis - - external granular layer
Cell generation and cell survival were investigated in the cerebellum of young rats exposed to 10% v/v ethanol in drinking water throughout gestation and lactation. At 12 days postpartum, cell cycle parameters m the external granular layer showed no significant change from control values, and the cell acquisition rate was unaffected. However, the external granular layer appeared thicker in ethanol-treated than in control 12-day-oldanimals, and a significant increase in cell death in the internal granular layer was observed. It is suggested that the effect of ethanol on the developingcerebellum may involve reduction of granule cell number consequent to increased granule cell death, and possiblyretarded migration from the proliferating precursor cell pool. It is now well known that prolonged exposure to ethanol during gestation and lactation is correlated with a pattern of abnormal development in the newbornll.15. In 1973, Jones and Smiths called this development disturbance the 'fetal alcohol syndrome'. Since then a body of evidence has accumulated to indicate that the developing nervous system is particularly vulnerable to ethanol exposure 4.5. In the cerebellum14 and hippocampus 1, extensive cellular changes have been attributed to ethanol or ethanol metabolites in maternal blood. It has been shown that neuropathological changes may occur after early postnatal exposure to ethanol 3.7A3. However, in these studies, effects on the nervous system were achieved as a result of high peak alcohol blood levels in the neonates; thus Bauer-Moffett and Altman3 reported levels of 239 mg/dl in animals exposed by ethanol inhalation, whilst Diaz and Samson 7 recorded levels of 279 mg/dl with intragastric administration. Borges and Lewis 5, investigating the effects of alcohol on rats, used the same route of administration through which humans are exposed, giving their animals 10% v/v ethanol in drinking water. The blood levels achieved and the ingested alcohol intake using this method fell within the range associated in humans with fetal alcohol syndrome 11.15.18. In maternal * To whom correspondence should be addressed. 0006-8993/83/$03.00 (~) 1983Elsevier Science Publishers B.V.
blood, peak alcohol levels obtained were 118 mg/dl in mid-gestation and 135 mg/dl during lactation. Using this regime, it was established in a recent study6 that total number of cerebellar cortical granule cells was significantly reduced, resulting in decrease in ratio of granule cells to Purkinje cells by between 9% and 14% in 3 selected lobules of the cerebellum. The pyknotic index in the external granular layer (EGL) appeared significantly increased at 21 days, while mitotic activity was unchanged (see Table III). Although cell death might have contributed to reduction of granule cell precursor numbers, it was suggested that there might also be a disturbance of cell proliferation kinetics in the EGL. One or both of these factors could be responsible for the apparent diminution in cell number in the internal granular layer (IGL). In the present study an attempt has been made to determine the extent to which ethanol may affect cell acquisition in the cerebellum, thereby influencing its development. Pregnant Wistar rats weighing between 150 and 200 g were fed pelleted food (PRD Labsure Animal Foods), and either water or 10% ethanol in drinking water, Food and liquid was administered ad libitum throughout gestation and to 12 days postnatally, and weight gain and caloric and liquid intake of animals were monitoredS.
389 A group of 12 control and 14 ethanol-exposed rats aged 12 days were given an intraperitoneal injection of 2.5/~Ci/g body weight [3H]thymidine (spot. act. 22 Ci/mmol; Radiochemical Centre, Amersham). At various times between 1 and 30 h after injection of [3H]thymidine, the rats were killed under chloroform anaesthesia by aortic perfusion-fixation with formolacetic acid (1% glacial acetic acid in 10% neutral formalin). After 24 h post-fixation in neutral formalin, the brain was transected horizontally between the inferior and superior colliculi and sagittally through brainstem and cerebellum. Mid-sagittal paraffin sections of the cerebellum cut at 7/~m were mounted on chrome-gelatin slides, and autoradiographs prepared using Kodak AR-10 plates. After exposure for 3 weeks at 4 °C in light-tight boxes containing desiccant, slides were developed for 5 min at 20 °C in Kodak D-19, fixed, and stained with Mayer's haemalum. Duplicate non-autoradiographic sections were stained with haematoxylin and eosin. For measurements of the thickness of the E G L , additional haematoxylin and eosin slides of 12-day animals reared under the same conditions were also used. The E G L of the cerebellum was examined under a 100 x oil-immersion objective. Mitotic and pyknotic indices were derived from counts of 1000 nuclei in haematoxylin- and eosin-stained sections in each of these animals. In autoradiographs, nuclei were scored as labelled if associated with more than 3 silver grains. Labelling indices were derived from counts of 1000 nuclei in animals killed 1 h after injection of [3H]thymidine. Between 50 and 70 mitoses (metaphases and anaphases) were counted in the same areas of E G L of each brain, and percentages of labelled mitoses plotted against time 16. Percentage labelled mitoses data were analyzed according to the method of Barrett2 as modified by Steel and Hanes 17
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-
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.
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4
8
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i
20
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32
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Fig. 1. Percentage of mitoses labelled (PLM) in 12-day rat corebe]Jar external granular layer at different times after injection
of [3H]thymidine. O . . . . . ethanol-treated animals.
O,
control;
~-------O,
in which computer-generated curves, based on independent, lognormally distributed values of G1, S and G 2 are fitted to the experimental findings. Turnover time (the time taken for a renewing tissue in a steadystate to generate a cell number equal to the total population) was calculated as 100 x computed S-phase duration/labelling index 19. The thickness of the E G L was derived from the mean of 9 measurements in each animal taken at representative positions (centrally and halfway to the base of the fissure on either side) in 3 selected lobules of the mid-sagittal cerebellum (lobules II, IV and VIII). Cell cycle parameters derived from curves fitted to percentage labelled mitoses data are given in Table I and are illustrated in Fig. 1. It appears that there is a tendency for S-phase to be longer in the ethanol-exposed animals, but its value is within our laboratory normal range and this is therefore unlikely to be significant. The labelling TABLE II Effect of ethanol on cell acquisition and cell death in the cerebellum of l2-day rats
Values are mean + S.D. n = 11 in each group. TABLE I Effect of ethanol on cell cycle parameters in cerebellar external granular layer at 12 days
Median values (mean + S.D. in brackets). All values in hours.
Cell cycle time Length of S-phase Length of G2-phase Length ofGz-phase
Control
Ethanol
17.3 10.5 (10.7 + 1.9) 2.8(3.1 + 1 . 4 ) 3.1 (4.4 ___4.5)
20.9 12.4(12.7 + 3.0) 3.0(3.2 + 1.1) 4.5 (5.5 + 3.7)
Control
Ethanol
1.63 + 0.19 0.52 + 0.22 20.78 + 3.29 51.5
1.49 + 0.27 0.61 + 0.18 21.24+ 4.13 59.8
46.6
40.0
(1) External granular layer
Mitotic index (%) Pyknotic index (%) Labelling index (%) Turnover time (h) Cell acquisition rate (%/24 h) (2) Internal granular layer
Pyknotic index (%) * P<
0.01.
0.24 + 0.30
0.67 _+0.36*
390 TABLE ]II Effects of ethanol on the cerebellum of 12- and 21-day rats
decrease; 1' increase. 12 days (1) Whole cerebellum Cerebellar weight Cerebellar vermal area (2) External granular layer Mitotic index Pyknotic index Area/thickness (3) Internal granular layer Pyknotic index Area
21 days a
NS NS
$* $*
NS NS 1' *
NS 1"* ~.u
t * NS
NS ~ **
* P < 0.05; ** P < 0.01; NS not significant a Data from Borges and Lewis (ref. 6). b At 21 days the EGL in control animals has almost totally involuted, but in ethanol-exposed animals a complete single layer of cells may still be present. index at 1 h after injection of [3H]thymidine, the turnover times, and the cell acquisition rates are shown in Table II. T u r n o v e r time and cell acquisition rate in ethanole x p o s e d material do not a p p e a r to be different from control values. The mitotic index is unchanged (Table II), as was previously n o t e d at 21 days (see Table III). H o w e v e r , the pyknotic index amongst cells of the internal granular layer is significantly increased at 12 days. This i m p a i r e d post-mitotic cell survival would a p p e a r to be a factor related to the changes seen at 21 days, i.e. reduction of cerebellar weight and decrease in the area of the mid-sagittal vermis and I G L (Table III). The E G L thickness in ethanol-exposed rats was increased (47.27 + 2.33/~m) in comparison with controls (45.26 + 2.79 a m ) (S.D., P < 0.05). This increased thickness was also observed in earlier stud-
1 Barnes, D. E. and Walker, D. W., Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus, Develop. Brain Res., 1 (1981) 333-340. 2 Barrett, J. C., A mathematical model of the mitotic cycle and its application to the interpretation of percentage labelled mitoses data, J. Nat. Cancer Inst., 37 (1966) 443--450. 3 Bauer-Moffett, C. and Altman, J., The effect of ethanol chronically administered to preweanling rats on cerebeUar development: a morphological study, Brain Research, 119 (1977) 249-268. 4 Borges, S. and Lewis, P. D., A study of alcohol effects on
ies 9, when m e a s u r e m e n t s of the E G L areas were det e r m i n e d in similar aged animals. Kornguth et al. 9 suggested that the increased E G L thickness in their e t h a n o l - t r e a t e d group m a y have been due to the reduced levels of serum thyroxine recorded in these animals. H o w e v e r , thyroid-deficient rats are known l0 eventually to attain n o r m a l cell numbers in the cerebellum, but only after 5 postnatal weeks due to the persistence of the E G L up to this time, a p h e n o m e non not evident in the present study. T h e results of this study suggest that ethanol may p r o d u c e reduction of final granule cell n u m b e r in the developing c e r e b e l l u m in two ways. The first is by increasing the rate of cell death amongst granule cells, as is seen in thyroid deficiency 10. In this condition there is evidence that cell death is related to reduced opportunities for synaptogenesis. This has not as yet been investigated in e t h a n o l - t r e a t e d animals, and cannot be excluded. H o w e v e r , tentatively it can be suggested that direct neurotoxicity may be involved. The second is an alteration of the kinetics of migration from E G L . R e t e n t i o n of cells that would otherwise migrate out of the E G L is thought to be the cause of expansion of this layer in thyroid hormonetreated ratsl2. A widened E G L was also p r e s e n t here at 12 days. Provided the retained cell fraction did not increase the proliferative pool and ultimately degenerated in situ (note increased E G L cell death at 21 days; Table III), it is possible that this mechanism could also contribute modestly to the final reduction of granule cell n u m b e r seen with ethanol t r e a t m e n t . W e are grateful to Dr. G. G o r d o n Steel for computer analysis of percentage labelled mitoses data. This work was supported by G r a n t 9979 241N from the M e d i c a l Research Council to P . D . L .
5 6 7 8
the developing nervous system, Trends Neurosci., 4 (1981) 13-15. Borges, S. and Lewis, P. D., A study of alcohol effects on the brain during gestation and lactation, Teratology, 25 (1982) 283-289. Borges, S. and Lewis, P. D., The effect of ethanol on the cellular composition of the cerebellum, Neuropath. appl. NeurobioL, 9 (1983) 53--60. Diaz, J. and Samson, H. H., Impaired brain growth in neonatal rats exposed to ethanol, Science, 208 (1980) 751-753. Jones, K. L. and Smith, D. W., Recognition of the fetal alcohol syndrome in early infancy, Lancet, 2 (1973) 999-1001.
391
9 Kornguth, S. E., Rutledge, J. J., Sunderland, E., Siegel, F., Carlson, I., Smollens, J., Juhl, U. and Young, B., Impeded cerebellar development and reduced thyroxine levels associated with fetal alcohol intoxication, Brain Research, 177 (1979) 347-360. 10 Lewis, P. D., Patel, A. J., Johnson, A. L. and Balazs, R., Effects of thyroid deficiency on cell acquisition in the postnatal rat brain: a quantitative histological study, Brain Research, 104 (1976) 49-62. 11 Neugat, R. H., Epidemiol6~ical appraisal of the literature on the fetal alcohol syndrome in humans, Early Human Develop., 5 (1981) 411-429. 12 Patel, A. S., Lewis, P. D., Balazs, R., Lai, M. and Bailey, P., Effects of thyroid hormone on postnatal cell acquisition in the rat brain, Brain Research, 172 (1979) 57-72. 13 Phillips, S. C. and Cragg, B. G., A change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal or adult life, Neuropath. appl. Neurobiol., 8 (1982) 441-454. 14 Phillips, S. C. and Cragg, B. G., A change in susceptibility
15
16 17 18
19
of rat cerebellar Purkinje cells to damage by acetaldehyde during fetal, neonatal and adult life, Neuropath. appl. Neurobiol., 8 (1982) 455-463. Potter, B. J and Hetzel, B. S., Fetal alcohol syndrome. In B. S. Hetzel and R. M. Smith (Eds.), FetalBrain Disorders, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, pp. 297-333. Quastler, H. and Sherman, F. G., Cell population kinetics in the intestinal epithelium of the mouse, Exp. Cell Res., 17 (1959) 420--438. Steel, G. G. and Hanes, S., The technique of labelled mitoses: analysis by automatic curve-fitting, Cell Tiss. Kinet., 4 (1971) 93-105. Streissguth, A. P., Landesman-Dwyer, S., Martin, J. C. and Smith, D. W., Teratogenic effects of alcohol in humans and laboratory animals, Science, 209 (1980) 353-361. Thrasher, J. D., Analysis of renewing epithelial cell populations. In D. M. Prescott (Ed.), Methods in Cell Physiology, Vol. 2, Academic Press, New York, 1966, pp. 323-357.
Brain Research, 271 (1983)388-391 Elsevier
Effects of ethanol on postnatal cell acquisition in the rat cerebellum SALVADOR BORGES and PAUL D. LEWIS* Department of Histopathology, Royal PostgraduateMedicalSchool, DucaneRoad, London WI2 0HS (U. K.) (Accepted March 15th, 1983) Key words: ethanol - - brain development- - cerebellum - - neurogenesis - - external granular layer
Cell generation and cell survival were investigated in the cerebellum of young rats exposed to 10% v/v ethanol in drinking water throughout gestation and lactation. At 12 days postpartum, cell cycle parameters m the external granular layer showed no significant change from control values, and the cell acquisition rate was unaffected. However, the external granular layer appeared thicker in ethanol-treated than in control 12-day-oldanimals, and a significant increase in cell death in the internal granular layer was observed. It is suggested that the effect of ethanol on the developingcerebellum may involve reduction of granule cell number consequent to increased granule cell death, and possiblyretarded migration from the proliferating precursor cell pool. It is now well known that prolonged exposure to ethanol during gestation and lactation is correlated with a pattern of abnormal development in the newbornll.15. In 1973, Jones and Smiths called this development disturbance the 'fetal alcohol syndrome'. Since then a body of evidence has accumulated to indicate that the developing nervous system is particularly vulnerable to ethanol exposure 4.5. In the cerebellum14 and hippocampus 1, extensive cellular changes have been attributed to ethanol or ethanol metabolites in maternal blood. It has been shown that neuropathological changes may occur after early postnatal exposure to ethanol 3.7A3. However, in these studies, effects on the nervous system were achieved as a result of high peak alcohol blood levels in the neonates; thus Bauer-Moffett and Altman3 reported levels of 239 mg/dl in animals exposed by ethanol inhalation, whilst Diaz and Samson 7 recorded levels of 279 mg/dl with intragastric administration. Borges and Lewis 5, investigating the effects of alcohol on rats, used the same route of administration through which humans are exposed, giving their animals 10% v/v ethanol in drinking water. The blood levels achieved and the ingested alcohol intake using this method fell within the range associated in humans with fetal alcohol syndrome 11.15.18. In maternal * To whom correspondence should be addressed. 0006-8993/83/$03.00 (~) 1983Elsevier Science Publishers B.V.
blood, peak alcohol levels obtained were 118 mg/dl in mid-gestation and 135 mg/dl during lactation. Using this regime, it was established in a recent study6 that total number of cerebellar cortical granule cells was significantly reduced, resulting in decrease in ratio of granule cells to Purkinje cells by between 9% and 14% in 3 selected lobules of the cerebellum. The pyknotic index in the external granular layer (EGL) appeared significantly increased at 21 days, while mitotic activity was unchanged (see Table III). Although cell death might have contributed to reduction of granule cell precursor numbers, it was suggested that there might also be a disturbance of cell proliferation kinetics in the EGL. One or both of these factors could be responsible for the apparent diminution in cell number in the internal granular layer (IGL). In the present study an attempt has been made to determine the extent to which ethanol may affect cell acquisition in the cerebellum, thereby influencing its development. Pregnant Wistar rats weighing between 150 and 200 g were fed pelleted food (PRD Labsure Animal Foods), and either water or 10% ethanol in drinking water, Food and liquid was administered ad libitum throughout gestation and to 12 days postnatally, and weight gain and caloric and liquid intake of animals were monitoredS.
389 A group of 12 control and 14 ethanol-exposed rats aged 12 days were given an intraperitoneal injection of 2.5/~Ci/g body weight [3H]thymidine (spot. act. 22 Ci/mmol; Radiochemical Centre, Amersham). At various times between 1 and 30 h after injection of [3H]thymidine, the rats were killed under chloroform anaesthesia by aortic perfusion-fixation with formolacetic acid (1% glacial acetic acid in 10% neutral formalin). After 24 h post-fixation in neutral formalin, the brain was transected horizontally between the inferior and superior colliculi and sagittally through brainstem and cerebellum. Mid-sagittal paraffin sections of the cerebellum cut at 7/~m were mounted on chrome-gelatin slides, and autoradiographs prepared using Kodak AR-10 plates. After exposure for 3 weeks at 4 °C in light-tight boxes containing desiccant, slides were developed for 5 min at 20 °C in Kodak D-19, fixed, and stained with Mayer's haemalum. Duplicate non-autoradiographic sections were stained with haematoxylin and eosin. For measurements of the thickness of the E G L , additional haematoxylin and eosin slides of 12-day animals reared under the same conditions were also used. The E G L of the cerebellum was examined under a 100 x oil-immersion objective. Mitotic and pyknotic indices were derived from counts of 1000 nuclei in haematoxylin- and eosin-stained sections in each of these animals. In autoradiographs, nuclei were scored as labelled if associated with more than 3 silver grains. Labelling indices were derived from counts of 1000 nuclei in animals killed 1 h after injection of [3H]thymidine. Between 50 and 70 mitoses (metaphases and anaphases) were counted in the same areas of E G L of each brain, and percentages of labelled mitoses plotted against time 16. Percentage labelled mitoses data were analyzed according to the method of Barrett2 as modified by Steel and Hanes 17
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'..,\ 6o-
~
-
/
-
,o/ 40-
;...---,
i
•
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.
/°
o
,,.
.
/
°
i
i
i
4
8
12
i6
l
I
i
!
i
20
24
28
32
hours
Fig. 1. Percentage of mitoses labelled (PLM) in 12-day rat corebe]Jar external granular layer at different times after injection
of [3H]thymidine. O . . . . . ethanol-treated animals.
O,
control;
~-------O,
in which computer-generated curves, based on independent, lognormally distributed values of G1, S and G 2 are fitted to the experimental findings. Turnover time (the time taken for a renewing tissue in a steadystate to generate a cell number equal to the total population) was calculated as 100 x computed S-phase duration/labelling index 19. The thickness of the E G L was derived from the mean of 9 measurements in each animal taken at representative positions (centrally and halfway to the base of the fissure on either side) in 3 selected lobules of the mid-sagittal cerebellum (lobules II, IV and VIII). Cell cycle parameters derived from curves fitted to percentage labelled mitoses data are given in Table I and are illustrated in Fig. 1. It appears that there is a tendency for S-phase to be longer in the ethanol-exposed animals, but its value is within our laboratory normal range and this is therefore unlikely to be significant. The labelling TABLE II Effect of ethanol on cell acquisition and cell death in the cerebellum of l2-day rats
Values are mean + S.D. n = 11 in each group. TABLE I Effect of ethanol on cell cycle parameters in cerebellar external granular layer at 12 days
Median values (mean + S.D. in brackets). All values in hours.
Cell cycle time Length of S-phase Length of G2-phase Length ofGz-phase
Control
Ethanol
17.3 10.5 (10.7 + 1.9) 2.8(3.1 + 1 . 4 ) 3.1 (4.4 ___4.5)
20.9 12.4(12.7 + 3.0) 3.0(3.2 + 1.1) 4.5 (5.5 + 3.7)
Control
Ethanol
1.63 + 0.19 0.52 + 0.22 20.78 + 3.29 51.5
1.49 + 0.27 0.61 + 0.18 21.24+ 4.13 59.8
46.6
40.0
(1) External granular layer
Mitotic index (%) Pyknotic index (%) Labelling index (%) Turnover time (h) Cell acquisition rate (%/24 h) (2) Internal granular layer
Pyknotic index (%) * P<
0.01.
0.24 + 0.30
0.67 _+0.36*
390 TABLE ]II Effects of ethanol on the cerebellum of 12- and 21-day rats
decrease; 1' increase. 12 days (1) Whole cerebellum Cerebellar weight Cerebellar vermal area (2) External granular layer Mitotic index Pyknotic index Area/thickness (3) Internal granular layer Pyknotic index Area
21 days a
NS NS
$* $*
NS NS 1' *
NS 1"* ~.u
t * NS
NS ~ **
* P < 0.05; ** P < 0.01; NS not significant a Data from Borges and Lewis (ref. 6). b At 21 days the EGL in control animals has almost totally involuted, but in ethanol-exposed animals a complete single layer of cells may still be present. index at 1 h after injection of [3H]thymidine, the turnover times, and the cell acquisition rates are shown in Table II. T u r n o v e r time and cell acquisition rate in ethanole x p o s e d material do not a p p e a r to be different from control values. The mitotic index is unchanged (Table II), as was previously n o t e d at 21 days (see Table III). H o w e v e r , the pyknotic index amongst cells of the internal granular layer is significantly increased at 12 days. This i m p a i r e d post-mitotic cell survival would a p p e a r to be a factor related to the changes seen at 21 days, i.e. reduction of cerebellar weight and decrease in the area of the mid-sagittal vermis and I G L (Table III). The E G L thickness in ethanol-exposed rats was increased (47.27 + 2.33/~m) in comparison with controls (45.26 + 2.79 a m ) (S.D., P < 0.05). This increased thickness was also observed in earlier stud-
1 Barnes, D. E. and Walker, D. W., Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus, Develop. Brain Res., 1 (1981) 333-340. 2 Barrett, J. C., A mathematical model of the mitotic cycle and its application to the interpretation of percentage labelled mitoses data, J. Nat. Cancer Inst., 37 (1966) 443--450. 3 Bauer-Moffett, C. and Altman, J., The effect of ethanol chronically administered to preweanling rats on cerebeUar development: a morphological study, Brain Research, 119 (1977) 249-268. 4 Borges, S. and Lewis, P. D., A study of alcohol effects on
ies 9, when m e a s u r e m e n t s of the E G L areas were det e r m i n e d in similar aged animals. Kornguth et al. 9 suggested that the increased E G L thickness in their e t h a n o l - t r e a t e d group m a y have been due to the reduced levels of serum thyroxine recorded in these animals. H o w e v e r , thyroid-deficient rats are known l0 eventually to attain n o r m a l cell numbers in the cerebellum, but only after 5 postnatal weeks due to the persistence of the E G L up to this time, a p h e n o m e non not evident in the present study. T h e results of this study suggest that ethanol may p r o d u c e reduction of final granule cell n u m b e r in the developing c e r e b e l l u m in two ways. The first is by increasing the rate of cell death amongst granule cells, as is seen in thyroid deficiency 10. In this condition there is evidence that cell death is related to reduced opportunities for synaptogenesis. This has not as yet been investigated in e t h a n o l - t r e a t e d animals, and cannot be excluded. H o w e v e r , tentatively it can be suggested that direct neurotoxicity may be involved. The second is an alteration of the kinetics of migration from E G L . R e t e n t i o n of cells that would otherwise migrate out of the E G L is thought to be the cause of expansion of this layer in thyroid hormonetreated ratsl2. A widened E G L was also p r e s e n t here at 12 days. Provided the retained cell fraction did not increase the proliferative pool and ultimately degenerated in situ (note increased E G L cell death at 21 days; Table III), it is possible that this mechanism could also contribute modestly to the final reduction of granule cell n u m b e r seen with ethanol t r e a t m e n t . W e are grateful to Dr. G. G o r d o n Steel for computer analysis of percentage labelled mitoses data. This work was supported by G r a n t 9979 241N from the M e d i c a l Research Council to P . D . L .
5 6 7 8
the developing nervous system, Trends Neurosci., 4 (1981) 13-15. Borges, S. and Lewis, P. D., A study of alcohol effects on the brain during gestation and lactation, Teratology, 25 (1982) 283-289. Borges, S. and Lewis, P. D., The effect of ethanol on the cellular composition of the cerebellum, Neuropath. appl. NeurobioL, 9 (1983) 53--60. Diaz, J. and Samson, H. H., Impaired brain growth in neonatal rats exposed to ethanol, Science, 208 (1980) 751-753. Jones, K. L. and Smith, D. W., Recognition of the fetal alcohol syndrome in early infancy, Lancet, 2 (1973) 999-1001.
391
9 Kornguth, S. E., Rutledge, J. J., Sunderland, E., Siegel, F., Carlson, I., Smollens, J., Juhl, U. and Young, B., Impeded cerebellar development and reduced thyroxine levels associated with fetal alcohol intoxication, Brain Research, 177 (1979) 347-360. 10 Lewis, P. D., Patel, A. J., Johnson, A. L. and Balazs, R., Effects of thyroid deficiency on cell acquisition in the postnatal rat brain: a quantitative histological study, Brain Research, 104 (1976) 49-62. 11 Neugat, R. H., Epidemiol6~ical appraisal of the literature on the fetal alcohol syndrome in humans, Early Human Develop., 5 (1981) 411-429. 12 Patel, A. S., Lewis, P. D., Balazs, R., Lai, M. and Bailey, P., Effects of thyroid hormone on postnatal cell acquisition in the rat brain, Brain Research, 172 (1979) 57-72. 13 Phillips, S. C. and Cragg, B. G., A change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal or adult life, Neuropath. appl. Neurobiol., 8 (1982) 441-454. 14 Phillips, S. C. and Cragg, B. G., A change in susceptibility
15
16 17 18
19
of rat cerebellar Purkinje cells to damage by acetaldehyde during fetal, neonatal and adult life, Neuropath. appl. Neurobiol., 8 (1982) 455-463. Potter, B. J and Hetzel, B. S., Fetal alcohol syndrome. In B. S. Hetzel and R. M. Smith (Eds.), FetalBrain Disorders, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, pp. 297-333. Quastler, H. and Sherman, F. G., Cell population kinetics in the intestinal epithelium of the mouse, Exp. Cell Res., 17 (1959) 420--438. Steel, G. G. and Hanes, S., The technique of labelled mitoses: analysis by automatic curve-fitting, Cell Tiss. Kinet., 4 (1971) 93-105. Streissguth, A. P., Landesman-Dwyer, S., Martin, J. C. and Smith, D. W., Teratogenic effects of alcohol in humans and laboratory animals, Science, 209 (1980) 353-361. Thrasher, J. D., Analysis of renewing epithelial cell populations. In D. M. Prescott (Ed.), Methods in Cell Physiology, Vol. 2, Academic Press, New York, 1966, pp. 323-357.