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Effects of early undernutrition on individual cerebellar lobes in male and female rats
202
Brain Research, 157 (1978) 202-205 ~ Elsevier/North-Holland Biomedical Pres~
Effects of early undernutrition on individual cerebellar lobes in male and female rats
TIMOTHY CHARLES JORDAN and KENNETH FRANK HOWELLS Neurobiology Laboratory, Biology Department, O~[brd Polytechnic, Headington, Oxford 03(30BP (Great Britain)
(Accepted July 13th, 1978)
Recent studies have demonstrated the vulnerability of the brain to early undernutrition 6, with the cerebellum being particularly susceptible during its 'growth spurt', where significant reductions have been found in weight and cell number2,Z,L Histological studies have shown reductions in vermis cross-sectional area', but it is not clear precisely where these deficits lie. Proportionately greater decreases have been separately reported for the granular layer ~, molecular layer 4, or individual neurons within these layers 13. In these studies, the areas within the cerebellum chosen for analysis were from limited regions and selected in a rather arbitrary manner, where at most portions of two lobes were used. These apparent inconsistencies suggest that selective differences may occur within the cerebellum. Investigations on male and female animals have also implicated an additional differential response to early undernutrition s. Other studies have either used male animalsl,~, 11 or not reported the sex of the animals4,13. It was therefore decides to investigate the effects of early (pre- and perinatal) undernutrition on the individual lobes within the cerebellum in male and female rats. Pregnant Wistar rats were given 50 % of their normal food supply (as defined by the controls' food intake during these periods) during pregnancy and lactation, water being available ad libitum. At birth, control and experimental litters were standardized to 8. After weaning, all animals received an ad libitum diet. They were sacrificed at age 36 weeks. After perfusion with 10 % formalin and determination of total brain weight, the cerebella were weighed and processed for histological examination. Sagittal sections of 8 # m from the vermis were projected on to drawing paper, and outlines drawn of the vermis and its component layers. The individual lobes of the cerebellum (using the notation of Larsell 9) were delineated, cut out and weighed, lit was not found feasible to determine the white matter content of individual lobes, so the values determined for lobe areas refer to the molecular and granular layers only. The results were analyzed for significance using the Student's t-test. The undernutrition regimen caused significant (P < 0.001) decreases in body weight (20 ~ ) in both male and female rats. Significant (P < 0.01) reductions in whole brain (10 ~), and cerebellum (13 ~o) weight were also found. In terms of vermis cross-
203
371
Experimental • Granularlayer O Molecularlayer
]~
MALES
X
Control
rnm?
3
.vln
• Granularlayer ] [] Molecularlayer FEMALES
I -2
"~ Control
mm.2 "3
Figs. 1 and 2. Effects of malnutrition on lobe cross-sectional areas of the cerebella. The standard deviation bars given (n = 4) are for total lobe areas.
204 sectional areas decreases (P -< 0.01) were larger in male (31 "/~/oJthan female ,120v"~/,,, animals. The results for individual lobes are given in Figs. 1 and 2 for male and female animals respectively. Taking the molecular and granular layers together, there was only a small effect of undernutrition on lobe I (which was not statistically significant) and no effects on lobe II. In lobes Ill, IV, V and VI, the decreases seen were only significant in male animals. In both males and females, significant deficits were found (P < 0.001) in lobes VII-X. i f the molecular and granular layers are considered separately, small fluctuations in the extent of involvement can be seen. With the female animals, a greater deficit in the molecular layers of the lobes was found. This was particularly apparent in lobe Vi, where the molecular layer showed a significant decrease (P <~ 0.01), although the total lobe area deficit was not significant. Similarly, the decrease in lobe VIII in females was largely due to the molecular layer. The undernutrition regimen was found to cause significant and permanent reductions in body, brain and cerebellum weight and vermis cross-sectional area. Within the cerebellum, these deficits were restricted to certain lobes, and a differential response was shown by male and female rats, females being less severely affected. These differences may partially account for discrepancies shown by the results of earlier studies. With sample sites taken from either side of the fissura prima (between lobes V and VI), Closet a15 found greater reductions in the molecular (24 ¢~i) than the granular (16 o~) layer. No mention was made of the sex of the animals used. From our results, the males showed no such difference in lobe V, and in lobe V1 only a slightly greater deficit in the molecular layer was seen. If male and female animals are taken together, the significant decrease in the molecular layer oftobe VI shown by the female animals brings our findings into line with those of Clos et al. 4. West and Kemper ~3 reported no proportionately greater decrease in either the molecular or the granular layer. Unfortunately, since neither the site of sampling nor the sex of the animals was given, comparisons with the present results are not feasible. Dobbing el al. '~found a proportionately greater deficit in the granular layer of lobe VI in male animals. Our results are at variance with this finding, although precise comparisons may not be possible due to the different method used by these workers to effect malnutrition (increasing litter size as opposed to restricting food intake). It is known that fostering large litters does have significant effects on maternal contact and caring time v, which in turn can produce marked effects on brain growth 1°,12. In such cases this may cause different effects in the cerebellum. The results obtained clearly demonstrate that further work on the histological and cytological effects of undernutrition on the cerebellum must take into account the differential effects shown in the present study of selective lobe involvement and male :female differences. Work is currently in progress investigating the cell populations of the lobes, together with an analysis of the developmental sequelae of these changes.
205 1 Baird, A., Widdowson, E. M. and Cowley, J. J., Effects of calorie and protein deficiencies early in life on the subsequent learning ability of rats, Brit. J. Nutr., 25 (1971) 391403. 2 Barnes, D. and Altman, J., Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum, Exp. Neurol., 38 (1973) 406 419. 3 Chase, H. P., Lindsley, W. and O'Brien, D., Undernutrition and cerebellar development, Nature (Lond.), 221 (1969) 554-555. 4 Clos, J., Favre, C., Selme-Matrat, M. and Legrand, J., Effects of undernutrition on cell formation in the rat brain and specially on cellular composition of the cerebellum, Brain Research, 123 (1977) 13 26. 5 Dobbing, J., Hopewell, J. W. and Lynch, A., Vulnerability of developing brain: VII. Permanent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition, Exp. Neurol., 32 (1971) 439 447. 6 Dobbing, J. and Smart, J. L., Vulnerability of developing brain and behaviour, Brit. reed. Bull., 30 (1974) 164-168. 7 Francova, S., Effects of protein-calorie malnutrition on the development of social behaviour in rats, Develop. Psychobiol., 6 (1973) 35 43. 8 Howells, K. F., Jordan, T. C. and Pigott, S. M., Effects of pre- and perinatal food intake on the rat cerebellum, J. PhysioL (Lond.), 272 (1977) 6-7P. 9 Larsell, O., The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat, J. comp. Neurol., 97 (1952) 281-356. 10 Levine, S., The effects of differential infantile stimulation on emotionality at weaning, Canad. J. Psycho/., 13 (1959) 243-247. I I Lynch, A., Smart, J. L. and Dobbing, J., Motor coordination and cerebellar size in adult rats undernourished in early life, Brain Research, 83 (1975) 249-259. 12 Smart, J. L. and Preece, J., Maternal behaviour of undernourished mother rats, Animal Behav., 21 (1973) 613-619. 13 West, C. D. and Kemper, T., The effect of a low protein diet on the anatomical development of the rat brain, Brain Research, 107 (1976) 221-237.
Brain Research, 157 (1978) 202-205 ~ Elsevier/North-Holland Biomedical Pres~
Effects of early undernutrition on individual cerebellar lobes in male and female rats
TIMOTHY CHARLES JORDAN and KENNETH FRANK HOWELLS Neurobiology Laboratory, Biology Department, O~[brd Polytechnic, Headington, Oxford 03(30BP (Great Britain)
(Accepted July 13th, 1978)
Recent studies have demonstrated the vulnerability of the brain to early undernutrition 6, with the cerebellum being particularly susceptible during its 'growth spurt', where significant reductions have been found in weight and cell number2,Z,L Histological studies have shown reductions in vermis cross-sectional area', but it is not clear precisely where these deficits lie. Proportionately greater decreases have been separately reported for the granular layer ~, molecular layer 4, or individual neurons within these layers 13. In these studies, the areas within the cerebellum chosen for analysis were from limited regions and selected in a rather arbitrary manner, where at most portions of two lobes were used. These apparent inconsistencies suggest that selective differences may occur within the cerebellum. Investigations on male and female animals have also implicated an additional differential response to early undernutrition s. Other studies have either used male animalsl,~, 11 or not reported the sex of the animals4,13. It was therefore decides to investigate the effects of early (pre- and perinatal) undernutrition on the individual lobes within the cerebellum in male and female rats. Pregnant Wistar rats were given 50 % of their normal food supply (as defined by the controls' food intake during these periods) during pregnancy and lactation, water being available ad libitum. At birth, control and experimental litters were standardized to 8. After weaning, all animals received an ad libitum diet. They were sacrificed at age 36 weeks. After perfusion with 10 % formalin and determination of total brain weight, the cerebella were weighed and processed for histological examination. Sagittal sections of 8 # m from the vermis were projected on to drawing paper, and outlines drawn of the vermis and its component layers. The individual lobes of the cerebellum (using the notation of Larsell 9) were delineated, cut out and weighed, lit was not found feasible to determine the white matter content of individual lobes, so the values determined for lobe areas refer to the molecular and granular layers only. The results were analyzed for significance using the Student's t-test. The undernutrition regimen caused significant (P < 0.001) decreases in body weight (20 ~ ) in both male and female rats. Significant (P < 0.01) reductions in whole brain (10 ~), and cerebellum (13 ~o) weight were also found. In terms of vermis cross-
203
371
Experimental • Granularlayer O Molecularlayer
]~
MALES
X
Control
rnm?
3
.vln
• Granularlayer ] [] Molecularlayer FEMALES
I -2
"~ Control
mm.2 "3
Figs. 1 and 2. Effects of malnutrition on lobe cross-sectional areas of the cerebella. The standard deviation bars given (n = 4) are for total lobe areas.
204 sectional areas decreases (P -< 0.01) were larger in male (31 "/~/oJthan female ,120v"~/,,, animals. The results for individual lobes are given in Figs. 1 and 2 for male and female animals respectively. Taking the molecular and granular layers together, there was only a small effect of undernutrition on lobe I (which was not statistically significant) and no effects on lobe II. In lobes Ill, IV, V and VI, the decreases seen were only significant in male animals. In both males and females, significant deficits were found (P < 0.001) in lobes VII-X. i f the molecular and granular layers are considered separately, small fluctuations in the extent of involvement can be seen. With the female animals, a greater deficit in the molecular layers of the lobes was found. This was particularly apparent in lobe Vi, where the molecular layer showed a significant decrease (P <~ 0.01), although the total lobe area deficit was not significant. Similarly, the decrease in lobe VIII in females was largely due to the molecular layer. The undernutrition regimen was found to cause significant and permanent reductions in body, brain and cerebellum weight and vermis cross-sectional area. Within the cerebellum, these deficits were restricted to certain lobes, and a differential response was shown by male and female rats, females being less severely affected. These differences may partially account for discrepancies shown by the results of earlier studies. With sample sites taken from either side of the fissura prima (between lobes V and VI), Closet a15 found greater reductions in the molecular (24 ¢~i) than the granular (16 o~) layer. No mention was made of the sex of the animals used. From our results, the males showed no such difference in lobe V, and in lobe V1 only a slightly greater deficit in the molecular layer was seen. If male and female animals are taken together, the significant decrease in the molecular layer oftobe VI shown by the female animals brings our findings into line with those of Clos et al. 4. West and Kemper ~3 reported no proportionately greater decrease in either the molecular or the granular layer. Unfortunately, since neither the site of sampling nor the sex of the animals was given, comparisons with the present results are not feasible. Dobbing el al. '~found a proportionately greater deficit in the granular layer of lobe VI in male animals. Our results are at variance with this finding, although precise comparisons may not be possible due to the different method used by these workers to effect malnutrition (increasing litter size as opposed to restricting food intake). It is known that fostering large litters does have significant effects on maternal contact and caring time v, which in turn can produce marked effects on brain growth 1°,12. In such cases this may cause different effects in the cerebellum. The results obtained clearly demonstrate that further work on the histological and cytological effects of undernutrition on the cerebellum must take into account the differential effects shown in the present study of selective lobe involvement and male :female differences. Work is currently in progress investigating the cell populations of the lobes, together with an analysis of the developmental sequelae of these changes.
205 1 Baird, A., Widdowson, E. M. and Cowley, J. J., Effects of calorie and protein deficiencies early in life on the subsequent learning ability of rats, Brit. J. Nutr., 25 (1971) 391403. 2 Barnes, D. and Altman, J., Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum, Exp. Neurol., 38 (1973) 406 419. 3 Chase, H. P., Lindsley, W. and O'Brien, D., Undernutrition and cerebellar development, Nature (Lond.), 221 (1969) 554-555. 4 Clos, J., Favre, C., Selme-Matrat, M. and Legrand, J., Effects of undernutrition on cell formation in the rat brain and specially on cellular composition of the cerebellum, Brain Research, 123 (1977) 13 26. 5 Dobbing, J., Hopewell, J. W. and Lynch, A., Vulnerability of developing brain: VII. Permanent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition, Exp. Neurol., 32 (1971) 439 447. 6 Dobbing, J. and Smart, J. L., Vulnerability of developing brain and behaviour, Brit. reed. Bull., 30 (1974) 164-168. 7 Francova, S., Effects of protein-calorie malnutrition on the development of social behaviour in rats, Develop. Psychobiol., 6 (1973) 35 43. 8 Howells, K. F., Jordan, T. C. and Pigott, S. M., Effects of pre- and perinatal food intake on the rat cerebellum, J. PhysioL (Lond.), 272 (1977) 6-7P. 9 Larsell, O., The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat, J. comp. Neurol., 97 (1952) 281-356. 10 Levine, S., The effects of differential infantile stimulation on emotionality at weaning, Canad. J. Psycho/., 13 (1959) 243-247. I I Lynch, A., Smart, J. L. and Dobbing, J., Motor coordination and cerebellar size in adult rats undernourished in early life, Brain Research, 83 (1975) 249-259. 12 Smart, J. L. and Preece, J., Maternal behaviour of undernourished mother rats, Animal Behav., 21 (1973) 613-619. 13 West, C. D. and Kemper, T., The effect of a low protein diet on the anatomical development of the rat brain, Brain Research, 107 (1976) 221-237.