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c mice
EXPERIMENTAL
85,223-228
NEUROLOGY
(1984)
RESEARCH
NOTE
Effects of Fasting during Gestation on Brain Development in BALB/c Mice PATRICIA Department
of Health Received
WAINWRIGHT
Studies, October
University
AND MERRILLA of Waterloo,
24, 1983; revision
Waterloo,
received
February
GAGNON’ Ontario
N2L
3G1, Canada
16, 1984
The development of the corpus callosum (CC) was examined in BALB/cCF mice in relation to the effects of a 32-h period of maternal fasting. The treatment was imposed on days 15 and 16 during gestation, which immediately precedes the time when the initial callosal axons cross the midline. The BALB/c strain was used because it is prone to developing a corpus callosum which is either small in size or absent at the midline. A split-litter design was used where fetal brain development was asses& on days 22 and 50 postconception (birth and weaning). The treatment did not increase the incidence of absent CC. On day 22 the midsagittal cross-sectional area of the CC, as well as brain and body weight, were smaller in the experimental group. Covariance analysis showed the effect on brain weight to be related to the overall growth retardation seen in the low body weight. In the control group, CC area and the brain weight were positively correlated, but in the experimental group this was not the case, suggesting the effect on the CC was independent of growth retardation. By day 50 there were no significant differences between the groups on any measure, although the data did show a trend toward a smaller CC in the, fasted animals.
Prior research (12) indicated that protein malnutrition during gestation increases the number of animals with defective corpus callosum (CC) development in BALB/c mice. This strain is prone to deficiency of this forebrain fiber tract where in some animals the commissure is either completely missing at midline or is abnormally small in size (10). Our purpose was to examine Abbreviations: CC-corpus callosum, CA-anterior commissure. ’ The authors thank Mr. W. Zagaja for animal maintenance, Mrs. K. Koenderink for technical assistance, Mrs. P. Sapay for secretarial assistance, and Dr. D. Wahlsten for helpful discussion. The research was supported by a Natural Sciences and Engineering Research Council of Canada grant A7617 to P.W. 223 0014-4886184 $3.00 Copyriat 0 1984 by Academic Press, Inc. All rights of reproduction in any form rmrved.
224
WAINWRIGHT
AND
GAGNON
the effects of an acute period of maternal food deprivation, i.e., fasting, on the growth of the brain and particularly of the CC in these animals. Studies on mice (8, 7) showed that maternal fasting during the 2nd week of gestation has teratogenic effects involving defects of the axial skeleton. Because the cells of origin of the CC arise from the ventricular layer after day 13 postconception (2) and their axons first cross the midline by day 17 ( 11), it was hypothesized that the developing CC would be sensitive to the effects of a period of maternal starvation imposed during that time. Measurements included the cross-sectional area of the anterior commissure (CA) as well as the CC in order to assess the specificity of any effect. Separate groups of animals were tested at birth and at weaning to ascertain whether any effects were permanent. The parents were BALB/cCF mice between 3 and 6 months of age obtained from a breeding colony at the University of Waterloo. Only virgin females were used. They were housed in standard opaque plastic mouse cages with free access to laboratory cubes (Maple Leaf Mills, Toronto) and tap water, and under a 12-h light: 12-h dark cycle at a temperature of 22 f 1 “C. The animals were mated between 1600 and 0900 h. The morning a plug was discovered was designated day 0 and the pregnant female was then housed separately. As it has been shown that the first axons cross the midline by day 17 (1 l), the period of maternal starvation was imposed from 0900 h on day 15 to 1700 h on day 16, i.e., for 32 h immediately prior to the time of crossing, when interruption of growth could be critical in terms of further development. At the initiation of the fast the mouse was weighed and all food removed, but water remained. Thirty-two hours later the mouse was again weighed and the food returned. Controls were handled similarly, except for food deprivation. Animals were checked daily for birth and on day 22 half the pups in each litter were killed. Birth occurred between days 19 and 2 1. Day 22 was chosen for killing in case the experimental treatment affected gestation length. Selection of pups to be killed was made by assigning pups to matched pairs according to weight. Then one member of each pair was randomly selected to be killed on day 22, and the other was maintained to weaning on day 50 postconception. This eliminated the possibility that exists with a small number of animals where random assignment may result in all the smaller (or larger) animals in one group. Handling of the growing pups was restricted to weekly bedding changes. Pups to be killed were weighed before anesthetization with chloroform, followed by cardiac perfusion with 10% buffered Formalin. The rest of the procedure has been described in detail elsewhere (12). Briefly, this consisted
FASTING
AND
BRAIN
DEVELOPMENT
225
of fixation of the brain in Formalin, followed by weighing, embedding in gelatin, and cutting in the sagittal plane at 33 pm. Prior to being weighed the brains were placed on their ventral surface and trimmed by cutting perpendicularly through the brain stem caudal to the cerebellum. The olfactory bulbs were left intact. Sections were stained with hematoxylin and eosin and the cross-sectional area of the CC and CA determined from midsagittal sections. The data were analyzed using a nested analysis of variance where the between-litter variance was used as the error term (1). This was followed by analysis of covariance on individual scores to determine whether or not effects on brain growth were independent of those on correlated variables such as body weight (5). A one-tailed hypothesis test was used and significance was set at a! = 0.05. Fasting of the pregnant mice caused them to lose weight during the 32-h period compared with the weight gain seen in the controls (weight change, experimental = -4.1 g, control = +3.2 g, P < 0.000 1). With one exception, all control animals gave birth on day 19, whereas in the experimental animals gestation length ranged between 19 and 2 1 days [control X = 19.1 days, experimental j; = 20.0 days; Mann-Whitney U = 4.71, P c 0.001 (9)]. An unexpected effect of the treatment was maternal cannibalism of pups in the experimental group. Of the 15 pregnant mice which were fasted, 6 ate all their pups and 4 ate some. This behavior was not seen in any of the control animals. The fetal data are presented in Table 1. There was no evidence that the treatment increased the incidence of absent CC; in fact, no animal in the study showed this defect. There was clear indication of an effect on overall growth on day 22. Body and brain weights, as well as CC area were significantly smaller in the fasted group. The effect on the CA area, although showing a trend, was not statistically significant. As is apparent from the adjusted mean scores, covariance analysis indicated that the effect on brain weight was related to the overall growth retardation seen in the small body weight. The relationship between CC area and brain weight was significantly different in the two groups at birth (experimental r2 = 0.01, P > 0.05, control r2 = 0.60, P < 0.05). This indicates that the effect of the treatment on CC growth was independent of the effect on the overall growth of the brain. By day 50 there was no evidence of any effect, except for a trend toward a smaller CC in the fasted group. These results do not provide any support for the experimental hypothesis that 32 h of maternal fasting between days 15 and 16 of gestation would increase the number of animals with an absent CC as measured on day 22 postconception. This is in contrast to the previous study (12) where a low
226
WAINWRIGHT
AND GAGNON
TABLE
1
Effect of a 32-h Fast during Gestation on Body and Brain Growth in BALB/c Mice” Fasted N = 17 (nine litters) Day 22 postconception (birth) Body weight (g) Brain weight (g) Mean adjusted for body weight Corpus callosum area (mm2) Anterior commissure area (mm2)
1.67 0.128 0.141 0.4265 0.0673
Day 50 postconception (weaning) Body weight (g) Brain weight (g) Corpus callosum area (mm2) Anterior commissure area (mm2)
13.07 0.451 0.8752 0.1300
f + f 2 +
0.063 0.0035 0.0026 0.0275 0.0022
+ 0.378 + 0.007 f 0.0296 Tk0.0035
Control N = 22 (seven litters)
2.19 0.154 0.145 0.5332 0.0753
+ O.lOO** f 0.0046*** f 0.0022 f 0.0325, k 0.0029’
13.74 + 0.371 0.454 + 0.008 0.95 15 + 0.0232 b 0.1256 A 0.0038
’ Data are means + SE; analyzed using between-litter error term. b0.1 > P> 0.05. * P c 0.05. ** P c 0.01. *** P c 0.001 (one-tailed).
protein diet increased the number of animals lacking a CC at day 18.5 postconception. This could be an indication that the two different types of malnutrition do in fact have different effects on brain growth. An alternative explanation is that the large number of defective animals in the first study were an indication of developmental delay specific to the CC, and that the structure eventually would have crossed the midline in some of these animals had they been allowed to develop until day 22. A pilot study was initiated to test this, but day 22 postconception is about 2 days after birth, and unfortunately all the maternal animals on a low protein diet cannibalized their pups. Similarly, in the present study, it is possible that the maternal cannibalism in the experimental group introduced a conservative bias to the data by eliminating the more severely affected animals. It is also possible that the critical period for causing callosal deficits through starvation is other than that used in the present study. There was an effect on CC growth at birth, resulting in a smaller crosssectional area in the experimental group which appeared to be independent of the effect on brain weight. At weaning the data showed a trend in the same direction. Both body and brain weight at birth were smaller in the
FASTING
AND
BRAIN
DEVELOPMENT
221
experimental group and there was no evidence of “brain sparing,” i.e., a smaller effect on brain than body weight. The latter observation is somewhat surprising. Glucose is the major substrate utilized for the energy requirements of the fetus and since gluconeogenetic mechanisms in the fetal rodent are limited (4) it is possible that this disturbance in carbohydrate metabolism was responsible for the retarded body growth. It has, however, been demonstrated that during maternal starvation the concentration of ketones in fetal blood is elevated (4). As the perinatal rat brain as well as the fetal human brain are able to utilize these as an energy substrate (4) it might have been expected that the effect on brain weight would be smaller than that on body weight. An alternative explanation is that the effect on brain growth was caused by a decrease in the availability of amino acids to the fetus due to the increase in maternal gluconeogenesis (4). It has been shown that maternal deprivation of essential amino acids decreases fetal cerebral weight, DNA, and protein content (13). It should be noted that in this study the timing of the treatment preceded that of the brain “growth spurt” in rodents (3) when effects of the treatment on growth might have been even greater. The present findings on body weight differ from those in the rat (6) which showed that fetal weight on day 19 was not significantly reduced by fasting adequately nourished mothers between days 17 and 19. This could be accounted for by differences in the timing of the treatment or by differences in response between the two species. In summary, the results of this study showed that maternal fasting during gestation had an effect on body growth and brain weight and CC area as measured at birth, but provide little evidence that the effect on these variables was enduring. REFERENCES 1. ABBEY, H., AND E. HOWARD. 1973. Statistical procedure in developmental studies on species with multiple offspring. Dev. Psychobiol. 6: 329-336. 2. ANGEVINE, J. B. 1970. Critical cellular events in the shaping of neural centres. Pages 6212 in F. 0. SCHMITT, Ed., The Neurosciences Second Study Program. Rockefeller Univ. Press, New York. 3. DUBBING, J., AND J. SANDS. 1979. Comparative aspects of the brain growth spurt. Early Hum.
Dev. 3: 19-83.
4. HAHN, P. 1979. Nutrition and metabolic development in mammals. Pages 1-39 in M. WINICK, Ed. Nutrition: Pre- and Postnatal Development. Vol. 1 in R. B. ALPIN-SLATER AND D. KRITCHEVSKY, Eds., Human Nutrition: A Comprehensive Treatise. Plenum, New York. 5. KLEINBAUM, D. G., AND L. L. KUPPER. 1978. Applied Regression Analysis and Other Multivariate Methods. Duxbury Press, North Scituate, MA. 6. LEDERMAN, S. A., AND P. Rosso. 1981. Effects of fasting during pregnancy on maternal
228
7. 8. 9.
10. 11. 12. 13.
WAINWRIGHT
AND GAGNON
and fetal weight and body composition in well-nourished and undernourished rats. J. Nutr. 111: 1823-1832. MILLER, J. R. 1962. A strain difference in response to the teratogenic effect of maternal fasting in the house mouse. Can. J. Genet. Cytd. 4: 69-78. RUNNER,M. N., AND J. R. MILLER. 1956. Congenital deformity in the mouSe as a consequence of fasting. Anal. Rec. 124: 437-438. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioural Sciences. McGraw-Hill, New York. WAHLSTEN, D. 1974. Heritable aspects of anomalous myelinated fibre tracts in the forebrain of the laboratory mouse. Brain Res. 68: 1-18. WAHLSTEN, D. 198 1. Prenatal schedule of appearance of mouse brain commissures. Dw. Brain Res. 1: 461-473. WAINWRIGHT, P., AND R. STEFANESCU. 1983. Prenatal protein deprivation increases defects of the corpus callosum in BALB/c laboratory mice. Exp. Neurol. 81: 694-702. ZAMENHOF, S., S. M. HALL, L. GRAUEL, E. VAN MARTHENS, AND M. J. DONAHUE. 1974. Deprivation of amino acids and prenatal brain development in rats. J. Nutr. 104: 10021007.
85,223-228
NEUROLOGY
(1984)
RESEARCH
NOTE
Effects of Fasting during Gestation on Brain Development in BALB/c Mice PATRICIA Department
of Health Received
WAINWRIGHT
Studies, October
University
AND MERRILLA of Waterloo,
24, 1983; revision
Waterloo,
received
February
GAGNON’ Ontario
N2L
3G1, Canada
16, 1984
The development of the corpus callosum (CC) was examined in BALB/cCF mice in relation to the effects of a 32-h period of maternal fasting. The treatment was imposed on days 15 and 16 during gestation, which immediately precedes the time when the initial callosal axons cross the midline. The BALB/c strain was used because it is prone to developing a corpus callosum which is either small in size or absent at the midline. A split-litter design was used where fetal brain development was asses& on days 22 and 50 postconception (birth and weaning). The treatment did not increase the incidence of absent CC. On day 22 the midsagittal cross-sectional area of the CC, as well as brain and body weight, were smaller in the experimental group. Covariance analysis showed the effect on brain weight to be related to the overall growth retardation seen in the low body weight. In the control group, CC area and the brain weight were positively correlated, but in the experimental group this was not the case, suggesting the effect on the CC was independent of growth retardation. By day 50 there were no significant differences between the groups on any measure, although the data did show a trend toward a smaller CC in the, fasted animals.
Prior research (12) indicated that protein malnutrition during gestation increases the number of animals with defective corpus callosum (CC) development in BALB/c mice. This strain is prone to deficiency of this forebrain fiber tract where in some animals the commissure is either completely missing at midline or is abnormally small in size (10). Our purpose was to examine Abbreviations: CC-corpus callosum, CA-anterior commissure. ’ The authors thank Mr. W. Zagaja for animal maintenance, Mrs. K. Koenderink for technical assistance, Mrs. P. Sapay for secretarial assistance, and Dr. D. Wahlsten for helpful discussion. The research was supported by a Natural Sciences and Engineering Research Council of Canada grant A7617 to P.W. 223 0014-4886184 $3.00 Copyriat 0 1984 by Academic Press, Inc. All rights of reproduction in any form rmrved.
224
WAINWRIGHT
AND
GAGNON
the effects of an acute period of maternal food deprivation, i.e., fasting, on the growth of the brain and particularly of the CC in these animals. Studies on mice (8, 7) showed that maternal fasting during the 2nd week of gestation has teratogenic effects involving defects of the axial skeleton. Because the cells of origin of the CC arise from the ventricular layer after day 13 postconception (2) and their axons first cross the midline by day 17 ( 11), it was hypothesized that the developing CC would be sensitive to the effects of a period of maternal starvation imposed during that time. Measurements included the cross-sectional area of the anterior commissure (CA) as well as the CC in order to assess the specificity of any effect. Separate groups of animals were tested at birth and at weaning to ascertain whether any effects were permanent. The parents were BALB/cCF mice between 3 and 6 months of age obtained from a breeding colony at the University of Waterloo. Only virgin females were used. They were housed in standard opaque plastic mouse cages with free access to laboratory cubes (Maple Leaf Mills, Toronto) and tap water, and under a 12-h light: 12-h dark cycle at a temperature of 22 f 1 “C. The animals were mated between 1600 and 0900 h. The morning a plug was discovered was designated day 0 and the pregnant female was then housed separately. As it has been shown that the first axons cross the midline by day 17 (1 l), the period of maternal starvation was imposed from 0900 h on day 15 to 1700 h on day 16, i.e., for 32 h immediately prior to the time of crossing, when interruption of growth could be critical in terms of further development. At the initiation of the fast the mouse was weighed and all food removed, but water remained. Thirty-two hours later the mouse was again weighed and the food returned. Controls were handled similarly, except for food deprivation. Animals were checked daily for birth and on day 22 half the pups in each litter were killed. Birth occurred between days 19 and 2 1. Day 22 was chosen for killing in case the experimental treatment affected gestation length. Selection of pups to be killed was made by assigning pups to matched pairs according to weight. Then one member of each pair was randomly selected to be killed on day 22, and the other was maintained to weaning on day 50 postconception. This eliminated the possibility that exists with a small number of animals where random assignment may result in all the smaller (or larger) animals in one group. Handling of the growing pups was restricted to weekly bedding changes. Pups to be killed were weighed before anesthetization with chloroform, followed by cardiac perfusion with 10% buffered Formalin. The rest of the procedure has been described in detail elsewhere (12). Briefly, this consisted
FASTING
AND
BRAIN
DEVELOPMENT
225
of fixation of the brain in Formalin, followed by weighing, embedding in gelatin, and cutting in the sagittal plane at 33 pm. Prior to being weighed the brains were placed on their ventral surface and trimmed by cutting perpendicularly through the brain stem caudal to the cerebellum. The olfactory bulbs were left intact. Sections were stained with hematoxylin and eosin and the cross-sectional area of the CC and CA determined from midsagittal sections. The data were analyzed using a nested analysis of variance where the between-litter variance was used as the error term (1). This was followed by analysis of covariance on individual scores to determine whether or not effects on brain growth were independent of those on correlated variables such as body weight (5). A one-tailed hypothesis test was used and significance was set at a! = 0.05. Fasting of the pregnant mice caused them to lose weight during the 32-h period compared with the weight gain seen in the controls (weight change, experimental = -4.1 g, control = +3.2 g, P < 0.000 1). With one exception, all control animals gave birth on day 19, whereas in the experimental animals gestation length ranged between 19 and 2 1 days [control X = 19.1 days, experimental j; = 20.0 days; Mann-Whitney U = 4.71, P c 0.001 (9)]. An unexpected effect of the treatment was maternal cannibalism of pups in the experimental group. Of the 15 pregnant mice which were fasted, 6 ate all their pups and 4 ate some. This behavior was not seen in any of the control animals. The fetal data are presented in Table 1. There was no evidence that the treatment increased the incidence of absent CC; in fact, no animal in the study showed this defect. There was clear indication of an effect on overall growth on day 22. Body and brain weights, as well as CC area were significantly smaller in the fasted group. The effect on the CA area, although showing a trend, was not statistically significant. As is apparent from the adjusted mean scores, covariance analysis indicated that the effect on brain weight was related to the overall growth retardation seen in the small body weight. The relationship between CC area and brain weight was significantly different in the two groups at birth (experimental r2 = 0.01, P > 0.05, control r2 = 0.60, P < 0.05). This indicates that the effect of the treatment on CC growth was independent of the effect on the overall growth of the brain. By day 50 there was no evidence of any effect, except for a trend toward a smaller CC in the fasted group. These results do not provide any support for the experimental hypothesis that 32 h of maternal fasting between days 15 and 16 of gestation would increase the number of animals with an absent CC as measured on day 22 postconception. This is in contrast to the previous study (12) where a low
226
WAINWRIGHT
AND GAGNON
TABLE
1
Effect of a 32-h Fast during Gestation on Body and Brain Growth in BALB/c Mice” Fasted N = 17 (nine litters) Day 22 postconception (birth) Body weight (g) Brain weight (g) Mean adjusted for body weight Corpus callosum area (mm2) Anterior commissure area (mm2)
1.67 0.128 0.141 0.4265 0.0673
Day 50 postconception (weaning) Body weight (g) Brain weight (g) Corpus callosum area (mm2) Anterior commissure area (mm2)
13.07 0.451 0.8752 0.1300
f + f 2 +
0.063 0.0035 0.0026 0.0275 0.0022
+ 0.378 + 0.007 f 0.0296 Tk0.0035
Control N = 22 (seven litters)
2.19 0.154 0.145 0.5332 0.0753
+ O.lOO** f 0.0046*** f 0.0022 f 0.0325, k 0.0029’
13.74 + 0.371 0.454 + 0.008 0.95 15 + 0.0232 b 0.1256 A 0.0038
’ Data are means + SE; analyzed using between-litter error term. b0.1 > P> 0.05. * P c 0.05. ** P c 0.01. *** P c 0.001 (one-tailed).
protein diet increased the number of animals lacking a CC at day 18.5 postconception. This could be an indication that the two different types of malnutrition do in fact have different effects on brain growth. An alternative explanation is that the large number of defective animals in the first study were an indication of developmental delay specific to the CC, and that the structure eventually would have crossed the midline in some of these animals had they been allowed to develop until day 22. A pilot study was initiated to test this, but day 22 postconception is about 2 days after birth, and unfortunately all the maternal animals on a low protein diet cannibalized their pups. Similarly, in the present study, it is possible that the maternal cannibalism in the experimental group introduced a conservative bias to the data by eliminating the more severely affected animals. It is also possible that the critical period for causing callosal deficits through starvation is other than that used in the present study. There was an effect on CC growth at birth, resulting in a smaller crosssectional area in the experimental group which appeared to be independent of the effect on brain weight. At weaning the data showed a trend in the same direction. Both body and brain weight at birth were smaller in the
FASTING
AND
BRAIN
DEVELOPMENT
221
experimental group and there was no evidence of “brain sparing,” i.e., a smaller effect on brain than body weight. The latter observation is somewhat surprising. Glucose is the major substrate utilized for the energy requirements of the fetus and since gluconeogenetic mechanisms in the fetal rodent are limited (4) it is possible that this disturbance in carbohydrate metabolism was responsible for the retarded body growth. It has, however, been demonstrated that during maternal starvation the concentration of ketones in fetal blood is elevated (4). As the perinatal rat brain as well as the fetal human brain are able to utilize these as an energy substrate (4) it might have been expected that the effect on brain weight would be smaller than that on body weight. An alternative explanation is that the effect on brain growth was caused by a decrease in the availability of amino acids to the fetus due to the increase in maternal gluconeogenesis (4). It has been shown that maternal deprivation of essential amino acids decreases fetal cerebral weight, DNA, and protein content (13). It should be noted that in this study the timing of the treatment preceded that of the brain “growth spurt” in rodents (3) when effects of the treatment on growth might have been even greater. The present findings on body weight differ from those in the rat (6) which showed that fetal weight on day 19 was not significantly reduced by fasting adequately nourished mothers between days 17 and 19. This could be accounted for by differences in the timing of the treatment or by differences in response between the two species. In summary, the results of this study showed that maternal fasting during gestation had an effect on body growth and brain weight and CC area as measured at birth, but provide little evidence that the effect on these variables was enduring. REFERENCES 1. ABBEY, H., AND E. HOWARD. 1973. Statistical procedure in developmental studies on species with multiple offspring. Dev. Psychobiol. 6: 329-336. 2. ANGEVINE, J. B. 1970. Critical cellular events in the shaping of neural centres. Pages 6212 in F. 0. SCHMITT, Ed., The Neurosciences Second Study Program. Rockefeller Univ. Press, New York. 3. DUBBING, J., AND J. SANDS. 1979. Comparative aspects of the brain growth spurt. Early Hum.
Dev. 3: 19-83.
4. HAHN, P. 1979. Nutrition and metabolic development in mammals. Pages 1-39 in M. WINICK, Ed. Nutrition: Pre- and Postnatal Development. Vol. 1 in R. B. ALPIN-SLATER AND D. KRITCHEVSKY, Eds., Human Nutrition: A Comprehensive Treatise. Plenum, New York. 5. KLEINBAUM, D. G., AND L. L. KUPPER. 1978. Applied Regression Analysis and Other Multivariate Methods. Duxbury Press, North Scituate, MA. 6. LEDERMAN, S. A., AND P. Rosso. 1981. Effects of fasting during pregnancy on maternal
228
7. 8. 9.
10. 11. 12. 13.
WAINWRIGHT
AND GAGNON
and fetal weight and body composition in well-nourished and undernourished rats. J. Nutr. 111: 1823-1832. MILLER, J. R. 1962. A strain difference in response to the teratogenic effect of maternal fasting in the house mouse. Can. J. Genet. Cytd. 4: 69-78. RUNNER,M. N., AND J. R. MILLER. 1956. Congenital deformity in the mouSe as a consequence of fasting. Anal. Rec. 124: 437-438. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioural Sciences. McGraw-Hill, New York. WAHLSTEN, D. 1974. Heritable aspects of anomalous myelinated fibre tracts in the forebrain of the laboratory mouse. Brain Res. 68: 1-18. WAHLSTEN, D. 198 1. Prenatal schedule of appearance of mouse brain commissures. Dw. Brain Res. 1: 461-473. WAINWRIGHT, P., AND R. STEFANESCU. 1983. Prenatal protein deprivation increases defects of the corpus callosum in BALB/c laboratory mice. Exp. Neurol. 81: 694-702. ZAMENHOF, S., S. M. HALL, L. GRAUEL, E. VAN MARTHENS, AND M. J. DONAHUE. 1974. Deprivation of amino acids and prenatal brain development in rats. J. Nutr. 104: 10021007.