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Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum
EXPERIMENTAL
NEUROLOGY
38, 406-419 (1973)
Effects of Different Schedules of Early Undernutrition Preweaning Growth of the Rat Cerebellum DAVID Laboratory
BARNES
of Developwwatal Purdue
AND JOSEPH ALTMAN
Neurobiology,
Department
University, Lafayette, Received September
on the
i
of Biological
Sciettces,
Indiana 47907 5, 1972
The ad lib. food intake of rats during gestation and lactation was determined and under different schedules experimental animals were fed 75 or 50% of that diet (mild and severe undernutrition). Mild or severe undernutrition during gestation alone had only transient effects on the preweaning weight of the brain or number of cells in the cerebellum. Mild or severe undernutrition during lactation, whether delayed (unfostered) or instant (fostered), had appreciable effects on body and brain growth. Severe lactational undernutrition resulted in a reduction in the number of cells in both the germinal external granular layer and the internal granular layer, the instant group being affected to a greater extent than the delayed group. The effects of combined gestational-lactational undernutrition were comparable to those produced by instant lactational restriction alone in both brain weight and cerebellar cell population.
INTRODUCTION Recent studies have established that dietary deprivation in rodents during suckling irreversibly retards the growth of the brain (9, 12, 15, 16, 20). The effects are most severe in regions such as the cerebellum (6, 8, 11, 13, 14, IS), in which cell proliferation leading to neurogenesis is pronounced at the time of deprivation (1, 2). It was suggested recently that in addition to a general “vulnerable period” (12) of brain development, there may be periods of selective vulnerability of various brain regions and brain constituents depending on the chronology of their maturation (4). In the rat and other altricial species (like the mouse, cat, or dog), the afferent, efferent, relay, and commissural nerve cells (“macroneurons”) come into existence before birth ; the bulk of the *This research program is supported by the National Institute of Mental Health and the U. S. Atomic Energy Commission. The assistance of Kathi Miller, Sharon Evander, Zeynep Kurgun, and Donna Whitehurst is gratefully acknowledged. 406 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserve&
UN~ERN~JTRITION
407
small interneurons (“microneurons”) in some brain regions such as the olfactory bulb, hippocampus, and cerebellum, develop during the suckling period ; a large proportion of neuroglia cells appear during the early postweaning period. Accordingly, dietary deprivation during the gestation period may be expected to interfere with the formation of macroneurons and presumably, as a consequence, with the subsequent organization of the entire brain. Dietary deprivation during the suckling period may inflict less harm on the elements already formed, but would be expected to interfere with the formation of microneurons and thus with the “fine wiring” of the brain. Finally, dietary deprivation during the early postweaning period would prevent the acquisition of a full complement of neuroglia, and thereby disturb the final steps in the maturation of the brain (such as myelination). Of course, the maturation of these three basic constituents of the nervous system is not synchronous throughout the brain nor is it in phase with the three major periods (fetal, infantile, and childhood) of development. A brain structure for which this synchrony holds, such as the cerebellum, could serve as a model for the examination of the differential vulnerability of selected cellular constituents of the nervous system. A variety of techniques have been used to undernourish suckling rats, such as separating the pups for variable periods from the mothers (16), changing litter size (20) or undernourishing the mothers (10). It was shown recently that the latter is a particularly reliable and consistent method of undernutrition (5) and can be used during gestation as well as lactation. In this study the differential effects of gestation and lactation, separately or combined, have been examined, as were the differential effects of “delayed” and “instant” undernutrition during the suckling period. A previous study (5) showed that regardless of whether changing litter size, or direct deprivation of the mothers was used to produce postnatal undernutrition, the effective deprivation of the young is delayed for several days, during which time cell proliferation in the cerebellum was quite brisk. This was attributed to the circumstance that the mothers have ample reserves to satisfy the relatively meager needs of the small neonates. Therefore, a fostering technique was introduced and the effects of delayed deprivation were compared with the effects of instant deprivation through fostering to mothers whose dietary deprivation was started before parturition. The microneurons of the cerebellar cortex (basket, stellate, and granule cells) originate from a subpial proliferative zone, the external granular layer. In the rat this germinal zone increasesrapidly after birth, begins to decline in thickness during the second week, and disappears by 21 days (2). The differentiating granule cells, which constitute the bulk of the
408
BARNES
AND
ALTMAN
cerebellar microneurons, migrate into the internal granular layer. Because of a lag between morphogenic and synaptogenic maturation, synaptogensis is still in progress at 30 days (3). In this study, we determined the gross changes produced during the preweaning period in the growth of the body and brain by different schedules of undernutrition and we also examined their effects on the cell population of the external granular and internal granular layers. In a subsequent study we will describe the effects of these infantile treatments following nutritional rehabilitation during the postweaning periods of adolescence and adulthood. MATERIALS
AND
METHODS
Treatment of Aninzals. Female primiparous Purdue/Wistar albino rats, approximately 3 months of age and weighing 225-275 g were used. They were placed in a cage with adult males in the evening and insemination was established the next morning by the presence of sperm in the vaginal smear or the presence of a vaginal plug. The morning of detection was considered day 0 of pregnancy. The mothers were placed on a controlled diet (75 or 50% of the ad lib. food consumption) from day 0 of gestation, or the day after parturition, or during both gestation and lactation. In order to establish the ad lib. food consumption during these periods, an excess amount of mash food was made available to 50 females. The food mixture consisted of 65% by weight of Wayne Lab Chow in meal form, 32% water, and 3% Mazola corn oil. This was placed in glass receptacles 11.9 cm high held upright in the cage; this method eliminated spillage and prevented the young from gaining access to the food. The average food consumption for each day of gestation and lactation was determined for blocks of 3 days and served as the ad lib. standard for “mild” (75%) and “severe” (50%) undernutrition of the mothers. All the mothers raised a standard litter of eight pups, either their own or pups fostered to them. In the nonfostered procedure all mothers were fed ad lib. during gestation. At parturition the mothers were left either on the ad lib. diet or they were placed on a restricted diet of 75 or 50% for the lactational period of 21 days. The experimental pups in this group were considered to have been subjected to “delayed mild” or “delayed severe” lactational undernutrition on the basis of previous observations (5) that with this procedure the pups are only slightly affected for the first 5 days. In one of the cross-fostered procedures the pups of mothers that were on an ad lib. diet during gestation were transferred to mothers that were kept either on the same diet or on 75 or 500/O diets during both gestation and lactation. The latter two constituted the “instant mild” or “instant severe” undernourished groups, respectively.
409
UNDERNUTRITION
In another cross-fostering procedure the p~tps of mothers that were on a 75 or 50% diet during gestation were transferred to mothers that were on an ad lib. during both gestation and lactation. These pups were considered to have been subjected to mild or severe undernutrition during “gestation alone.” Finally. in a parallel-fostering procedure, the pups of mothers on an ad lib. 75 or 50% diet were transferred to mothers that were on the same diet both during gestation and lactation. The deprived animals constituted the undernourished mild or severe, “combined gestational and lactational” groups. The total number of animals studied in each deprivation procedure and age is summarized in Table 1. All mothers and litters were weighedandfed
Treatment Control 75 % 75y0 7.5% 7.5% 50% 50% 50% 50% Total
Gestation Delayed lactation Instant lactation Gestation and lactation Gestation Delayed lactation Instant lactation Gestation and lactation
Birth
5
10
1.5
20
33 24 18
40 21 23 32 29 37
32 24 23 32 31 32
3.2 2-l 32 31 32 37
32 16 32 32 32 32
169 109 110 128 125 186
-
23 32 26
2-l 30 32
32 32 32
23 32 32
102 126 122
263
261
285
263
1177
10.5
Total
at approximately the same time each morning. The temperature, humidity, and light cycle were carefully controlled. Preparation of the diet was done in a separate room, and the assigned personnel were admitted to the breeding room for a short period only to minimize disturbance of the mothers. Histological Pre)aration. Three or four litters in each nutritional condtion were killed at 0, 5, 10, 15. and 20 days. In most cases the newborn p~ips were killed within 2 hr after birth, from the excess of animals born. Animals were anesthetized with ether and their brains removed in a standardized manner. Wet weight was determined on an analytical balance and the brains were immersed in Bouin’s fluid. After 6 hr, the brains were cut midsagittally and fixed in Rouin’s fixative for 23 hr more. This was
BARNES ANY ALTMAN
410
followed by several changes of 10% neutral formalin for at least 48 hr after which they were dehydrated in a graded series of alcohols and embedded in Paraplast. In a supplementary experiment, the wet and dry weights of the cerebellum were determined at 20 days for mild and severe instantly undernourished animals. Five litters of each of the experimental groups and five control litters were killed as above and the cerebella dissected rapidly. Immediately after weighing, the cerebella were immrsed in liquid nitrogen in small preweighed test tubes. The tubes were placed in a Thermovac freeze-dryer at a pressure of 100 mTorrs for 24 hr at which time the tissue was reweighed and the water content and dry weights determined. Quantitative Evaluation. For quantitative analysis, section were cut at 6 pm from one hemisphere in matched parasagittal sections and stained with Harris’ hematoxylin and eosin. To measure the area, the outline of the
B
1 +-
r
I
FIG. 1. Number of pups delivered, delivered by mothers deprived during
x i z 2 and body gestation.
and brain weights at birth of pups Vertical bars represent 1 S.D.
pyramis (lobe VIII) and the boundaries of the layers were drawn on paper by projection with a Zeiss micro-macro projector magniiied X200 (5-10 days) or x 100 (15-20 days). The boundaries of the layers were traced twice with a Keuffel and Esser compensating polar planimeter and the average of the two readings was used. The cell population of the external granular layer of the pyramis was sampled at 10 days while the cell population in the internal granular layer at 10, 15, and 20 days. All the granule cells lying within a grid (0.0025 mm2) were counted at xl500 magnification with an oil immersion lens. Ten samples were taken from each section. Total granule cells were estimated by multiplying granule cells per square millimeter times the area of the appropriate layer.
411
UNDERPCUTRITTON
RESULTS Gross Changes in Body aud Brain Gestational Undenzz!trition. The number of pups delivered by the ad lib., mildly undernourished, and severely undernourished mothers was nearly identical (Fig. 1A) and there was virtually no infant mortality in any of the groups, showing that in no case was the deprivation excessive. Body (,Fig. 1B) and brain (Fig. 1C) weights at birth were not significantly affected in the mildly undernourished animals, although there was a subsequent transient retardation in body growth at 5-15 days (Table 3). The severely undernourished mothers gave birth to pups whose body weights were significantly (P < .Ol) less than those of the controls (Fig. lB), TABLE SIGNIFICANCE
LEVELS
(t TESTS)
OF
2 DIFFERENCES
IN
BODY
N’EIGHTS
P Values Treatments Control 7.59/, Control 7.5’%,, Control 750/,, Control 75’j& lactation Control SOoj,, Control SO%, Control-SOa/ Control SO%, lactation
Birth
5 Days
10 Days
15 Days
20 Days
gestation lactation lactation gestation
alone delayed instant and
NS -
NS
NS
gestation lactation lactation gestation
alone delayed instant and
NS
-
and this reduction was apparent through day 15 (Table 2). The brain weights were not significantly affected at birth (Fig. 1C) ; however, there was a significant reduction between 5 and 20 days (Table 3). Lactational Undernutrition. From the tenth day onward there was a moderate and significant reduction in the body weights of the mildly under.nourished rats, but there were no differences in magnitude between the delayed and instant lactational groups. The growth of the brain was not significantly affected in the 75% delayed group (Table 3) but there was a small significant reduction in the 75% instant group from the tenth day onward. In the 50% delayed group there was no reduction in body and brain weight at 5 days (Tables 2-3 ; Fig. 2A, B) but a reduction was seen at this age in the 50% instant group. From the tenth day onward body and brain growth was retarded significantly in both groups (Tables 2, 3),
412
BARNES
AND
ALTMAN
P Values Treatment Control 75y0, Control 7.570, Control 7570, Control 750/,, lactation Control SO%‘,, Control SO%, Control SO%, Control SO%, lactation
gestation lactation lactation gestation
alone delayed instant and
gestation lactation lactation gestation
alone delayed instant and
Birth
5 Days
10 Days
15 Days
20 Days
NS
NS NS
NS -
<0.0.5
-
-
-
but the magnitude of retardation was much greater in the instantly undernourished animals (Fig. 2). In the supplementary study the effects of 7.5@O / instant and 50% instant undernutrition were determined on wet whole brain (Fig. 3B) and wet (‘Fig. 3C) and dry (Fig. 3D) cerebellar weights. Brain weight reduction in the 75% instant group was slight (3-4s) but significant (P < 0.001). The wet weight of the whole brain was reduced 14% in the 50% instant animals (P < 0.001); the reduction was of greater magnitude (21%) in the cerebellum (P < 0.001 j. Comparable results were obtained with cerebellar dry weight measurements (Fig. 3D). Gestational and Lactational Usdernzltritior~ Cowzbiwd. The effect of combined mild undernutrition on body and brain growth was minimal and not appreciably different from that during lactation alone. The effects of combined gestational and lactational severe undernutrition were slightly greater at 5 days on both the body and brain (Fig. 2). Interestingly, the additive effect of gestational undernutrition was not pronounced thereafter. &antitative
Histological
Changes
in the Cevebellztm
The histological examination was restricted to the severely undernourished groups (Table 4). Figure 4 shows the effects of various methods of severe undernutrition on the germinal cell population of the external granular layer of the pyramis at clay 10. As expected, the animals undergoing delayed lactation undernutrition showed no deficit at this age in cell population. Although the group undernourished during gestation alone had similar brain weights as the above group (Fig. 2B) this group did show
413
UNDERNUTRITIOIi
NUMBER
OF
CEREBELLA
USED
FOR
QUANTITATIVE
DETERMINATIOKS
10 rkYs
Treatments -__. 50%
HISTOLOGICAL
20 Days
.~-.-Control Delayed, lactation 50% Instant lactation 50% Gestation 507, Gestation and lactation
15 1.5 16 19 6
16 13 20 20 20
some cell reduction (15.2% ; P < 0.01) in the external granular layer. The other two methods of undernutrition (lactation-instant and gestationlactation combined) had a more severe effect of comparable magnitude (30%) on the cell population of the external granular layer.
_ BRAIN WEIGHT 1.4 -
B
1.2 -
---0 - --o -----A
1 0 FIG.
during
2. Growth gestation,
5
IO TIME-days
Ad Lib (N = 169) Gestation Alone (N=l86) Loctatwn. Delayed lN=135) Loctolo,,. Instant (N*l59) Geshfion - Lactation CN=l701 15
curves of body and brain of rats subjected lactation, or gestation-lactation combined.
2
1
to severe
undernutrition
414
1thRNE.S
AND
AL’rBfAN
The effects of deprivation in the different severely undernourished groups on the cell population of internal granule cells of the pyramis at the time of weaning are summarized in Fig. 5. The animals undernourished during gestation alone showed a slight, nonsignificant reduction in granular nerve cells. In the delayed-lactational group the reduction was greater (14.2%) and still greater in the instant-lactational group (21.8%). The additive effect of combined gestational-lactational undernutrition led to a 26.5% reduction of granule cells at 20 days. DISCUSSION Deprivation During Gestation Alone. Pilot studies not reported in this paper showed that the consequences of undernutrition during gestation and lactation were influenced greatly by the age and weight of the mothers. Deprivation had more deleterious effects in young (2-2.5 months) rats
1
200.
1.4 . 12. 1.0.
FIG. 3. Effects and wet and dry
ll
of severe cerebellar
i or mild instant lactational weights at 20 days.
undernutrition
on the body,
brain,
with little fat reserves (200 g) than in heavier and older animals. Accordingly, females were used only in this experiment that were within the same age and weight range. Similarly, the breeding environment had an appreciable influence ; any disturbance of the undernourished mothers seemed to have an adverse effect on maternal behavior. For this reason, extreme care was taken to maintain optimal breeding conditions and except for the short period of feeding and weighing, the mothers were left undisturbed in a quiet, isolated and spacious breeding room. Perhaps some of these precautions account for the less severe effects obtained in this experiment than were reported earlier by other investigators. Chow and Lee (10) reported that 50% dietary restriction during gestation led to a reduction in the number of pups delivered (McCollum ; we obtained a minimal reduction strain of rats) from 9.3 to 6 (-35%)
N
INTERNAL e
0
GRANULE 02
CELLS z
(X103) 6 2
416
RARNIBAND ALTMAN
3), and that in the 50% group the reduction in males was from 204 to 139 g at 3 months, and from 388 to 291 g at 8 months (see their Table 6), we foulld that the reduction was quite small during illfancy and that by 20 days of age the difference had almost disappeared. In addition to possible differences in breeding conditions, the results of Chow and Lee must be interpreted to be partly due to combined gestational-lactational effects, since they did not foster the newborns to mothers that were not deprived on the preceding days (lactational rehabilitation). Notwithstanding the minimal reduction seen in this study in the birth weight of the pups of the severely undernourished mothers and the absence of a reduction in brain weight, and considering the attempted rehabilitation by fostering the young immediately after birth to normally fed mothers, it was surprising that there was a significant reduction in postnatal brain weight from days 5 to 20. The conclusion that may be drawn is that in spite of the considerable “sparing” manifest during gestation in fetal growth, the nervous system is vulnerable during this period. Interestingly, this retardation is not evident at birth, but becomes apparent during the suckling period. Similar delayed effects were reported previously by Venkatachalam and Ramanathan (19). The effects of gestational undernutrition were also seen in the slight (nonsignificant) reduction of germinal esternal granule cells at 10 days and in the reduction of differentiated granule cells at 20 days. In pilot studies we also attempted to determine the effects on brain growth of more severe gestational undernutrition. However, if mothers were fed only 30% of the ad lib. diet during gestation, there was a very high incidence of deaths among the mothers (SO%)>, of resorption, stillbirth and infantile mortality. Reducing the diet from 50 to 30% appeared to represent a crossing of a threshold similar to that reported previously in comparing 643% protein diet during gestation, which has relatively small effects (19, 22, 23) with a diet of 57’O, which has very serious consequences on reproductive success (7, 17). Deprizlation During Lactation: Delayed or Instant. The effects of depriving mothers during lactation are much more serious than depriving them during gestation. This is presumably due to the selective physiological protection of the embryo and fetus at the expense of the mother during gestation, and the selective behavioral protection of the mother at the expense of the nursing infants during lactation. Thus mild undernutrition during lactation unlike mild undernutrition during gestation had a greater and lasting effect on body and brain development. Because the irreversible effect of lactational undernutrition on the development of the brain is attributed to a reduction of newly forming cells (20) and because the commencement of deprivation at parturition does not have’
UNDERNUTRITION
417
detectable effects for several days on the pups (5) we compared in this study a nonfostered and a fostered method. In the latter case the pups delivered by ad lib. mothers were transferred at birth to mothers who were deprived during gestation. Among the mildly undernourished animals there was no difference (except at 20 days) in the growth of pups of the delayed and instant groups, presumably because even the previously deprived foster mothers could adequately cater for the small needs of the young for the first few days. The effect of mild undernutrition on body growth was small and on brain growth it was significant only in the instantly deprived group. This reduction was also small in cerebellar weight, indicating “sparing” of even this briskly proliferting system by mild undernutrition. In contrast to mild ~mclernutrition, severe lactational undernutrition had appreciable effects on the growth of the brain, and the effects were much more pronounced in the instant than the delayed group. The greater retardation produced in body growth in the instant group could be attributed to the retardation of weight increment by the fifth day; in the delayed group this was not seen until the tenth day. The same pattern was also seen in the growth of the brain. As expected and in agreement with earlier studies (S, 11, 13, 14, 1S) the reduction in cerebellar weight was greater than in the brain as a whole. Our hypothesis concerning the “delayed” effect of deprivation if the undernutrition of the mother is started at birth was best exemplified by the effect at 10 days on the external granular layer. While there \vas no apparent effect on the “delayed” animals, there was a considerable reduction in this germinal layer in the “instant” animals. This early effect then led presumably to the greater reduction in differentiating granule cells at 20 days in the instant than in the delayed undernourished animals. Dcprizlation Duri)lg Gcstatiofz and Lactatio,l. The combination of severe undernutrition during gestation and lactation retarded body and brain growth considerably although not to the extent expected. Winick (21) concluded that “if an animal is exposed to both prenatal and postnatal malnutrition there is a much greater deficit in total brain cell number by the time of weaning than one would expect from just summing prenatal and postnatal effects alone” (pp. 1510-1511). This is not borne out by the data of this study with the cross-fostering technique which show that instant-mild or severe undernutrition during lactation alone had comparable effects with combined gestational and lactational undernutrition, with the added gestational undernutrition failing to produce a clear cumulative effect. We cannot at present adequately interpret the discrepancy in the magnitude of reduction in the germinal and internal granular layers. That there
418
BARNES
AND
ALTMAN
was no reduction in the external granular layer at 10 days in the delayedlactation group but that a small reduction was apparent by 20 days in the internal granular layer is readily explainable. But it is not clear why the 40% reduction of external granule cells in the instant-lactation and the combined gestation-lactation groups led only to a 21-26s reduction in internal granule cells. The increase and decline in the population of external granule cells is a composite function of the rates of cell proliferation, cell differentiation and cell migration and it remains to be determined which of these is affected by undernutrition and to what extent. REFERENCES J. 1966. Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. J. Comb. Nelbvol. 128: 431-473. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neuroof genesis. III. Dating the time of production and onset of differentiation cerebellar microneurons in rats. J. Colrrp. Nez~rol. 136: 269-294. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granular layer. J. Conzp. Newel. 145: 465-514. ALTMAN, J., G. D. DAS, and K. SUDARSHAN. 1970. The influence of nutrition on neural and behavioral development. I. Critical review of some data on the growth of the body and the brain following dietary deprivation during gestation and lactation. Devel. Psychobiol. 3: 281301. ALTMAN, J., G. D. DAS, K. SUDARSHAN, and J. B. ANDERSON. 1971. The influence of nutrition on neural and behavioral development. II. Growth of body and brain in infant rats using different techniques of undernutrition. Devel. Psychobiol. 4: s-70. ALTMAN, J., and B. MCCRADY. 1972. The influence of nutrition on neural and behavioral development. IV. Effects of infantile undernutrition on the growth of the cerebellum. De&. Psychobiol. 5: 111-122. AMBEGAOKAR, S. D., and K. CHANDRAN. 1959. Physiological effects of low protein diets. I. Studies on nitrogen balance, growth, reproduction and lactation and basal metabolism in rats. Indian J. Med. Res. 4: 539. CHASE, H. P., W. F. B. LINDSLEY, and D. O’BRIEN. 1969. Undernutrition and cerebellar development. Nafzrre (London) 221: 554-55.5. CHEEK, D. B., J. E. GRAYSTONE, and R. D. ROWE. 1969. Hypoxia and malnutrition in newborn rats : Effects on RNA, DNA and protein in tissue. Alxcr. J. Plzysiol. 217 : 642-645. CHOW, B. F., and C. U. LEE. 1964. Effect of dietary restriction of pregnant rats on body weight gain of the offspring. J. Nzttr. 82: 10-18. CULLEY, W. J., and R. 0. LINEBERGER. 1968. Effect of undernutrition on the size and composition of the rat brain. J. N&r. 96: 375-381. DOBBING, J. 1968. Vulnerable periods in developing brain, pp. 287-316. In “Applied Neurochemistry.” A, $I. Davison and J. Dobbing [Eds.]. Davis, Philadelphia, Pennsylvania.
1. ALTMAN,
2. 3. 4.
5.
6. 7.
8.
9. 10. 11. 12.
UNDERNUTRITION
13.
14. 15.
16.
17. 18. 19.
20. 21. 22. 23.
419
J., J. W. HOPEWELL, and A. LYNCH. 1971. Vulnerability of developing brain: VII. Permanent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition. Exp. Nezwol. 32: 439-447. FISH, I., and M. WINICK. 1969. Effect of malnutrition on regional growth of the developing rat brain. Exp. Ncnrol. 25: 536540. GUTHRIE, H. A., and M. L. BROWN. 1968. Effect of severe undernutrition in early life on growth, brain size and composition in adult rats. J. Nutr. 94: 419426. HOWARD, E., and D. M. GRAKOFF. 1968. Effect of neonatal food restriction in mice on brain growth, DNA and cholesterol and on adult delayed response learning. J. Nutr. 95: 111-121. NELSON, M. M., and H. M. EVANS. 1953. Relation of dietary protein levels to reproduction in the rat. J. Nzhtr. 51: 71-84. NEVILLE, H. W., and H. P. CHASE. 1971. Undernutrition and cerebellar development. Exp. Newel. 33: 485-497. VENKATACHALAM, P. S., and K. S. RAMANATHAN. 1964. Effect of protein deficiency during gestation and lactation on body weight and composition of offspring, J. Nutr. 84 : 3&42. WINICK, M., and A. NOBLE. 1966. Cellular response in rats during malnutrition at various ages. J. Nutr. 89: 300-306. WINICK, M. 1970. Nutrition and nerve cell growth. Fed. Proc. 29: 1510-1515. ZAMENHOF, S., E. V. MARTHENS, and F. L. MARGOLIS. 1968. DNA (cell number) and protein in neonatal brain : Alteration by maternal dietary protein restriction. Science 160 : 322-323. ZEMAN, F. J., and E. C. STANBROUGH. 1969. Effect of maternal protein deficiency on cellular development in the fetal rat. J. Nutr. 99: 274-282.
DOBBING,
NEUROLOGY
38, 406-419 (1973)
Effects of Different Schedules of Early Undernutrition Preweaning Growth of the Rat Cerebellum DAVID Laboratory
BARNES
of Developwwatal Purdue
AND JOSEPH ALTMAN
Neurobiology,
Department
University, Lafayette, Received September
on the
i
of Biological
Sciettces,
Indiana 47907 5, 1972
The ad lib. food intake of rats during gestation and lactation was determined and under different schedules experimental animals were fed 75 or 50% of that diet (mild and severe undernutrition). Mild or severe undernutrition during gestation alone had only transient effects on the preweaning weight of the brain or number of cells in the cerebellum. Mild or severe undernutrition during lactation, whether delayed (unfostered) or instant (fostered), had appreciable effects on body and brain growth. Severe lactational undernutrition resulted in a reduction in the number of cells in both the germinal external granular layer and the internal granular layer, the instant group being affected to a greater extent than the delayed group. The effects of combined gestational-lactational undernutrition were comparable to those produced by instant lactational restriction alone in both brain weight and cerebellar cell population.
INTRODUCTION Recent studies have established that dietary deprivation in rodents during suckling irreversibly retards the growth of the brain (9, 12, 15, 16, 20). The effects are most severe in regions such as the cerebellum (6, 8, 11, 13, 14, IS), in which cell proliferation leading to neurogenesis is pronounced at the time of deprivation (1, 2). It was suggested recently that in addition to a general “vulnerable period” (12) of brain development, there may be periods of selective vulnerability of various brain regions and brain constituents depending on the chronology of their maturation (4). In the rat and other altricial species (like the mouse, cat, or dog), the afferent, efferent, relay, and commissural nerve cells (“macroneurons”) come into existence before birth ; the bulk of the *This research program is supported by the National Institute of Mental Health and the U. S. Atomic Energy Commission. The assistance of Kathi Miller, Sharon Evander, Zeynep Kurgun, and Donna Whitehurst is gratefully acknowledged. 406 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserve&
UN~ERN~JTRITION
407
small interneurons (“microneurons”) in some brain regions such as the olfactory bulb, hippocampus, and cerebellum, develop during the suckling period ; a large proportion of neuroglia cells appear during the early postweaning period. Accordingly, dietary deprivation during the gestation period may be expected to interfere with the formation of macroneurons and presumably, as a consequence, with the subsequent organization of the entire brain. Dietary deprivation during the suckling period may inflict less harm on the elements already formed, but would be expected to interfere with the formation of microneurons and thus with the “fine wiring” of the brain. Finally, dietary deprivation during the early postweaning period would prevent the acquisition of a full complement of neuroglia, and thereby disturb the final steps in the maturation of the brain (such as myelination). Of course, the maturation of these three basic constituents of the nervous system is not synchronous throughout the brain nor is it in phase with the three major periods (fetal, infantile, and childhood) of development. A brain structure for which this synchrony holds, such as the cerebellum, could serve as a model for the examination of the differential vulnerability of selected cellular constituents of the nervous system. A variety of techniques have been used to undernourish suckling rats, such as separating the pups for variable periods from the mothers (16), changing litter size (20) or undernourishing the mothers (10). It was shown recently that the latter is a particularly reliable and consistent method of undernutrition (5) and can be used during gestation as well as lactation. In this study the differential effects of gestation and lactation, separately or combined, have been examined, as were the differential effects of “delayed” and “instant” undernutrition during the suckling period. A previous study (5) showed that regardless of whether changing litter size, or direct deprivation of the mothers was used to produce postnatal undernutrition, the effective deprivation of the young is delayed for several days, during which time cell proliferation in the cerebellum was quite brisk. This was attributed to the circumstance that the mothers have ample reserves to satisfy the relatively meager needs of the small neonates. Therefore, a fostering technique was introduced and the effects of delayed deprivation were compared with the effects of instant deprivation through fostering to mothers whose dietary deprivation was started before parturition. The microneurons of the cerebellar cortex (basket, stellate, and granule cells) originate from a subpial proliferative zone, the external granular layer. In the rat this germinal zone increasesrapidly after birth, begins to decline in thickness during the second week, and disappears by 21 days (2). The differentiating granule cells, which constitute the bulk of the
408
BARNES
AND
ALTMAN
cerebellar microneurons, migrate into the internal granular layer. Because of a lag between morphogenic and synaptogenic maturation, synaptogensis is still in progress at 30 days (3). In this study, we determined the gross changes produced during the preweaning period in the growth of the body and brain by different schedules of undernutrition and we also examined their effects on the cell population of the external granular and internal granular layers. In a subsequent study we will describe the effects of these infantile treatments following nutritional rehabilitation during the postweaning periods of adolescence and adulthood. MATERIALS
AND
METHODS
Treatment of Aninzals. Female primiparous Purdue/Wistar albino rats, approximately 3 months of age and weighing 225-275 g were used. They were placed in a cage with adult males in the evening and insemination was established the next morning by the presence of sperm in the vaginal smear or the presence of a vaginal plug. The morning of detection was considered day 0 of pregnancy. The mothers were placed on a controlled diet (75 or 50% of the ad lib. food consumption) from day 0 of gestation, or the day after parturition, or during both gestation and lactation. In order to establish the ad lib. food consumption during these periods, an excess amount of mash food was made available to 50 females. The food mixture consisted of 65% by weight of Wayne Lab Chow in meal form, 32% water, and 3% Mazola corn oil. This was placed in glass receptacles 11.9 cm high held upright in the cage; this method eliminated spillage and prevented the young from gaining access to the food. The average food consumption for each day of gestation and lactation was determined for blocks of 3 days and served as the ad lib. standard for “mild” (75%) and “severe” (50%) undernutrition of the mothers. All the mothers raised a standard litter of eight pups, either their own or pups fostered to them. In the nonfostered procedure all mothers were fed ad lib. during gestation. At parturition the mothers were left either on the ad lib. diet or they were placed on a restricted diet of 75 or 50% for the lactational period of 21 days. The experimental pups in this group were considered to have been subjected to “delayed mild” or “delayed severe” lactational undernutrition on the basis of previous observations (5) that with this procedure the pups are only slightly affected for the first 5 days. In one of the cross-fostered procedures the pups of mothers that were on an ad lib. diet during gestation were transferred to mothers that were kept either on the same diet or on 75 or 500/O diets during both gestation and lactation. The latter two constituted the “instant mild” or “instant severe” undernourished groups, respectively.
409
UNDERNUTRITION
In another cross-fostering procedure the p~tps of mothers that were on a 75 or 50% diet during gestation were transferred to mothers that were on an ad lib. during both gestation and lactation. These pups were considered to have been subjected to mild or severe undernutrition during “gestation alone.” Finally. in a parallel-fostering procedure, the pups of mothers on an ad lib. 75 or 50% diet were transferred to mothers that were on the same diet both during gestation and lactation. The deprived animals constituted the undernourished mild or severe, “combined gestational and lactational” groups. The total number of animals studied in each deprivation procedure and age is summarized in Table 1. All mothers and litters were weighedandfed
Treatment Control 75 % 75y0 7.5% 7.5% 50% 50% 50% 50% Total
Gestation Delayed lactation Instant lactation Gestation and lactation Gestation Delayed lactation Instant lactation Gestation and lactation
Birth
5
10
1.5
20
33 24 18
40 21 23 32 29 37
32 24 23 32 31 32
3.2 2-l 32 31 32 37
32 16 32 32 32 32
169 109 110 128 125 186
-
23 32 26
2-l 30 32
32 32 32
23 32 32
102 126 122
263
261
285
263
1177
10.5
Total
at approximately the same time each morning. The temperature, humidity, and light cycle were carefully controlled. Preparation of the diet was done in a separate room, and the assigned personnel were admitted to the breeding room for a short period only to minimize disturbance of the mothers. Histological Pre)aration. Three or four litters in each nutritional condtion were killed at 0, 5, 10, 15. and 20 days. In most cases the newborn p~ips were killed within 2 hr after birth, from the excess of animals born. Animals were anesthetized with ether and their brains removed in a standardized manner. Wet weight was determined on an analytical balance and the brains were immersed in Bouin’s fluid. After 6 hr, the brains were cut midsagittally and fixed in Rouin’s fixative for 23 hr more. This was
BARNES ANY ALTMAN
410
followed by several changes of 10% neutral formalin for at least 48 hr after which they were dehydrated in a graded series of alcohols and embedded in Paraplast. In a supplementary experiment, the wet and dry weights of the cerebellum were determined at 20 days for mild and severe instantly undernourished animals. Five litters of each of the experimental groups and five control litters were killed as above and the cerebella dissected rapidly. Immediately after weighing, the cerebella were immrsed in liquid nitrogen in small preweighed test tubes. The tubes were placed in a Thermovac freeze-dryer at a pressure of 100 mTorrs for 24 hr at which time the tissue was reweighed and the water content and dry weights determined. Quantitative Evaluation. For quantitative analysis, section were cut at 6 pm from one hemisphere in matched parasagittal sections and stained with Harris’ hematoxylin and eosin. To measure the area, the outline of the
B
1 +-
r
I
FIG. 1. Number of pups delivered, delivered by mothers deprived during
x i z 2 and body gestation.
and brain weights at birth of pups Vertical bars represent 1 S.D.
pyramis (lobe VIII) and the boundaries of the layers were drawn on paper by projection with a Zeiss micro-macro projector magniiied X200 (5-10 days) or x 100 (15-20 days). The boundaries of the layers were traced twice with a Keuffel and Esser compensating polar planimeter and the average of the two readings was used. The cell population of the external granular layer of the pyramis was sampled at 10 days while the cell population in the internal granular layer at 10, 15, and 20 days. All the granule cells lying within a grid (0.0025 mm2) were counted at xl500 magnification with an oil immersion lens. Ten samples were taken from each section. Total granule cells were estimated by multiplying granule cells per square millimeter times the area of the appropriate layer.
411
UNDERPCUTRITTON
RESULTS Gross Changes in Body aud Brain Gestational Undenzz!trition. The number of pups delivered by the ad lib., mildly undernourished, and severely undernourished mothers was nearly identical (Fig. 1A) and there was virtually no infant mortality in any of the groups, showing that in no case was the deprivation excessive. Body (,Fig. 1B) and brain (Fig. 1C) weights at birth were not significantly affected in the mildly undernourished animals, although there was a subsequent transient retardation in body growth at 5-15 days (Table 3). The severely undernourished mothers gave birth to pups whose body weights were significantly (P < .Ol) less than those of the controls (Fig. lB), TABLE SIGNIFICANCE
LEVELS
(t TESTS)
OF
2 DIFFERENCES
IN
BODY
N’EIGHTS
P Values Treatments Control 7.59/, Control 7.5’%,, Control 750/,, Control 75’j& lactation Control SOoj,, Control SO%, Control-SOa/ Control SO%, lactation
Birth
5 Days
10 Days
15 Days
20 Days
gestation lactation lactation gestation
alone delayed instant and
NS -
NS
NS
gestation lactation lactation gestation
alone delayed instant and
NS
-
and this reduction was apparent through day 15 (Table 2). The brain weights were not significantly affected at birth (Fig. 1C) ; however, there was a significant reduction between 5 and 20 days (Table 3). Lactational Undernutrition. From the tenth day onward there was a moderate and significant reduction in the body weights of the mildly under.nourished rats, but there were no differences in magnitude between the delayed and instant lactational groups. The growth of the brain was not significantly affected in the 75% delayed group (Table 3) but there was a small significant reduction in the 75% instant group from the tenth day onward. In the 50% delayed group there was no reduction in body and brain weight at 5 days (Tables 2-3 ; Fig. 2A, B) but a reduction was seen at this age in the 50% instant group. From the tenth day onward body and brain growth was retarded significantly in both groups (Tables 2, 3),
412
BARNES
AND
ALTMAN
P Values Treatment Control 75y0, Control 7.570, Control 7570, Control 750/,, lactation Control SO%‘,, Control SO%, Control SO%, Control SO%, lactation
gestation lactation lactation gestation
alone delayed instant and
gestation lactation lactation gestation
alone delayed instant and
Birth
5 Days
10 Days
15 Days
20 Days
NS
NS NS
NS -
<0.0.5
-
-
-
but the magnitude of retardation was much greater in the instantly undernourished animals (Fig. 2). In the supplementary study the effects of 7.5@O / instant and 50% instant undernutrition were determined on wet whole brain (Fig. 3B) and wet (‘Fig. 3C) and dry (Fig. 3D) cerebellar weights. Brain weight reduction in the 75% instant group was slight (3-4s) but significant (P < 0.001). The wet weight of the whole brain was reduced 14% in the 50% instant animals (P < 0.001); the reduction was of greater magnitude (21%) in the cerebellum (P < 0.001 j. Comparable results were obtained with cerebellar dry weight measurements (Fig. 3D). Gestational and Lactational Usdernzltritior~ Cowzbiwd. The effect of combined mild undernutrition on body and brain growth was minimal and not appreciably different from that during lactation alone. The effects of combined gestational and lactational severe undernutrition were slightly greater at 5 days on both the body and brain (Fig. 2). Interestingly, the additive effect of gestational undernutrition was not pronounced thereafter. &antitative
Histological
Changes
in the Cevebellztm
The histological examination was restricted to the severely undernourished groups (Table 4). Figure 4 shows the effects of various methods of severe undernutrition on the germinal cell population of the external granular layer of the pyramis at clay 10. As expected, the animals undergoing delayed lactation undernutrition showed no deficit at this age in cell population. Although the group undernourished during gestation alone had similar brain weights as the above group (Fig. 2B) this group did show
413
UNDERNUTRITIOIi
NUMBER
OF
CEREBELLA
USED
FOR
QUANTITATIVE
DETERMINATIOKS
10 rkYs
Treatments -__. 50%
HISTOLOGICAL
20 Days
.~-.-Control Delayed, lactation 50% Instant lactation 50% Gestation 507, Gestation and lactation
15 1.5 16 19 6
16 13 20 20 20
some cell reduction (15.2% ; P < 0.01) in the external granular layer. The other two methods of undernutrition (lactation-instant and gestationlactation combined) had a more severe effect of comparable magnitude (30%) on the cell population of the external granular layer.
_ BRAIN WEIGHT 1.4 -
B
1.2 -
---0 - --o -----A
1 0 FIG.
during
2. Growth gestation,
5
IO TIME-days
Ad Lib (N = 169) Gestation Alone (N=l86) Loctatwn. Delayed lN=135) Loctolo,,. Instant (N*l59) Geshfion - Lactation CN=l701 15
curves of body and brain of rats subjected lactation, or gestation-lactation combined.
2
1
to severe
undernutrition
414
1thRNE.S
AND
AL’rBfAN
The effects of deprivation in the different severely undernourished groups on the cell population of internal granule cells of the pyramis at the time of weaning are summarized in Fig. 5. The animals undernourished during gestation alone showed a slight, nonsignificant reduction in granular nerve cells. In the delayed-lactational group the reduction was greater (14.2%) and still greater in the instant-lactational group (21.8%). The additive effect of combined gestational-lactational undernutrition led to a 26.5% reduction of granule cells at 20 days. DISCUSSION Deprivation During Gestation Alone. Pilot studies not reported in this paper showed that the consequences of undernutrition during gestation and lactation were influenced greatly by the age and weight of the mothers. Deprivation had more deleterious effects in young (2-2.5 months) rats
1
200.
1.4 . 12. 1.0.
FIG. 3. Effects and wet and dry
ll
of severe cerebellar
i or mild instant lactational weights at 20 days.
undernutrition
on the body,
brain,
with little fat reserves (200 g) than in heavier and older animals. Accordingly, females were used only in this experiment that were within the same age and weight range. Similarly, the breeding environment had an appreciable influence ; any disturbance of the undernourished mothers seemed to have an adverse effect on maternal behavior. For this reason, extreme care was taken to maintain optimal breeding conditions and except for the short period of feeding and weighing, the mothers were left undisturbed in a quiet, isolated and spacious breeding room. Perhaps some of these precautions account for the less severe effects obtained in this experiment than were reported earlier by other investigators. Chow and Lee (10) reported that 50% dietary restriction during gestation led to a reduction in the number of pups delivered (McCollum ; we obtained a minimal reduction strain of rats) from 9.3 to 6 (-35%)
N
INTERNAL e
0
GRANULE 02
CELLS z
(X103) 6 2
416
RARNIBAND ALTMAN
3), and that in the 50% group the reduction in males was from 204 to 139 g at 3 months, and from 388 to 291 g at 8 months (see their Table 6), we foulld that the reduction was quite small during illfancy and that by 20 days of age the difference had almost disappeared. In addition to possible differences in breeding conditions, the results of Chow and Lee must be interpreted to be partly due to combined gestational-lactational effects, since they did not foster the newborns to mothers that were not deprived on the preceding days (lactational rehabilitation). Notwithstanding the minimal reduction seen in this study in the birth weight of the pups of the severely undernourished mothers and the absence of a reduction in brain weight, and considering the attempted rehabilitation by fostering the young immediately after birth to normally fed mothers, it was surprising that there was a significant reduction in postnatal brain weight from days 5 to 20. The conclusion that may be drawn is that in spite of the considerable “sparing” manifest during gestation in fetal growth, the nervous system is vulnerable during this period. Interestingly, this retardation is not evident at birth, but becomes apparent during the suckling period. Similar delayed effects were reported previously by Venkatachalam and Ramanathan (19). The effects of gestational undernutrition were also seen in the slight (nonsignificant) reduction of germinal esternal granule cells at 10 days and in the reduction of differentiated granule cells at 20 days. In pilot studies we also attempted to determine the effects on brain growth of more severe gestational undernutrition. However, if mothers were fed only 30% of the ad lib. diet during gestation, there was a very high incidence of deaths among the mothers (SO%)>, of resorption, stillbirth and infantile mortality. Reducing the diet from 50 to 30% appeared to represent a crossing of a threshold similar to that reported previously in comparing 643% protein diet during gestation, which has relatively small effects (19, 22, 23) with a diet of 57’O, which has very serious consequences on reproductive success (7, 17). Deprizlation During Lactation: Delayed or Instant. The effects of depriving mothers during lactation are much more serious than depriving them during gestation. This is presumably due to the selective physiological protection of the embryo and fetus at the expense of the mother during gestation, and the selective behavioral protection of the mother at the expense of the nursing infants during lactation. Thus mild undernutrition during lactation unlike mild undernutrition during gestation had a greater and lasting effect on body and brain development. Because the irreversible effect of lactational undernutrition on the development of the brain is attributed to a reduction of newly forming cells (20) and because the commencement of deprivation at parturition does not have’
UNDERNUTRITION
417
detectable effects for several days on the pups (5) we compared in this study a nonfostered and a fostered method. In the latter case the pups delivered by ad lib. mothers were transferred at birth to mothers who were deprived during gestation. Among the mildly undernourished animals there was no difference (except at 20 days) in the growth of pups of the delayed and instant groups, presumably because even the previously deprived foster mothers could adequately cater for the small needs of the young for the first few days. The effect of mild undernutrition on body growth was small and on brain growth it was significant only in the instantly deprived group. This reduction was also small in cerebellar weight, indicating “sparing” of even this briskly proliferting system by mild undernutrition. In contrast to mild ~mclernutrition, severe lactational undernutrition had appreciable effects on the growth of the brain, and the effects were much more pronounced in the instant than the delayed group. The greater retardation produced in body growth in the instant group could be attributed to the retardation of weight increment by the fifth day; in the delayed group this was not seen until the tenth day. The same pattern was also seen in the growth of the brain. As expected and in agreement with earlier studies (S, 11, 13, 14, 1S) the reduction in cerebellar weight was greater than in the brain as a whole. Our hypothesis concerning the “delayed” effect of deprivation if the undernutrition of the mother is started at birth was best exemplified by the effect at 10 days on the external granular layer. While there \vas no apparent effect on the “delayed” animals, there was a considerable reduction in this germinal layer in the “instant” animals. This early effect then led presumably to the greater reduction in differentiating granule cells at 20 days in the instant than in the delayed undernourished animals. Dcprizlation Duri)lg Gcstatiofz and Lactatio,l. The combination of severe undernutrition during gestation and lactation retarded body and brain growth considerably although not to the extent expected. Winick (21) concluded that “if an animal is exposed to both prenatal and postnatal malnutrition there is a much greater deficit in total brain cell number by the time of weaning than one would expect from just summing prenatal and postnatal effects alone” (pp. 1510-1511). This is not borne out by the data of this study with the cross-fostering technique which show that instant-mild or severe undernutrition during lactation alone had comparable effects with combined gestational and lactational undernutrition, with the added gestational undernutrition failing to produce a clear cumulative effect. We cannot at present adequately interpret the discrepancy in the magnitude of reduction in the germinal and internal granular layers. That there
418
BARNES
AND
ALTMAN
was no reduction in the external granular layer at 10 days in the delayedlactation group but that a small reduction was apparent by 20 days in the internal granular layer is readily explainable. But it is not clear why the 40% reduction of external granule cells in the instant-lactation and the combined gestation-lactation groups led only to a 21-26s reduction in internal granule cells. The increase and decline in the population of external granule cells is a composite function of the rates of cell proliferation, cell differentiation and cell migration and it remains to be determined which of these is affected by undernutrition and to what extent. REFERENCES J. 1966. Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. J. Comb. Nelbvol. 128: 431-473. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neuroof genesis. III. Dating the time of production and onset of differentiation cerebellar microneurons in rats. J. Colrrp. Nez~rol. 136: 269-294. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granular layer. J. Conzp. Newel. 145: 465-514. ALTMAN, J., G. D. DAS, and K. SUDARSHAN. 1970. The influence of nutrition on neural and behavioral development. I. Critical review of some data on the growth of the body and the brain following dietary deprivation during gestation and lactation. Devel. Psychobiol. 3: 281301. ALTMAN, J., G. D. DAS, K. SUDARSHAN, and J. B. ANDERSON. 1971. The influence of nutrition on neural and behavioral development. II. Growth of body and brain in infant rats using different techniques of undernutrition. Devel. Psychobiol. 4: s-70. ALTMAN, J., and B. MCCRADY. 1972. The influence of nutrition on neural and behavioral development. IV. Effects of infantile undernutrition on the growth of the cerebellum. De&. Psychobiol. 5: 111-122. AMBEGAOKAR, S. D., and K. CHANDRAN. 1959. Physiological effects of low protein diets. I. Studies on nitrogen balance, growth, reproduction and lactation and basal metabolism in rats. Indian J. Med. Res. 4: 539. CHASE, H. P., W. F. B. LINDSLEY, and D. O’BRIEN. 1969. Undernutrition and cerebellar development. Nafzrre (London) 221: 554-55.5. CHEEK, D. B., J. E. GRAYSTONE, and R. D. ROWE. 1969. Hypoxia and malnutrition in newborn rats : Effects on RNA, DNA and protein in tissue. Alxcr. J. Plzysiol. 217 : 642-645. CHOW, B. F., and C. U. LEE. 1964. Effect of dietary restriction of pregnant rats on body weight gain of the offspring. J. Nzttr. 82: 10-18. CULLEY, W. J., and R. 0. LINEBERGER. 1968. Effect of undernutrition on the size and composition of the rat brain. J. N&r. 96: 375-381. DOBBING, J. 1968. Vulnerable periods in developing brain, pp. 287-316. In “Applied Neurochemistry.” A, $I. Davison and J. Dobbing [Eds.]. Davis, Philadelphia, Pennsylvania.
1. ALTMAN,
2. 3. 4.
5.
6. 7.
8.
9. 10. 11. 12.
UNDERNUTRITION
13.
14. 15.
16.
17. 18. 19.
20. 21. 22. 23.
419
J., J. W. HOPEWELL, and A. LYNCH. 1971. Vulnerability of developing brain: VII. Permanent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition. Exp. Nezwol. 32: 439-447. FISH, I., and M. WINICK. 1969. Effect of malnutrition on regional growth of the developing rat brain. Exp. Ncnrol. 25: 536540. GUTHRIE, H. A., and M. L. BROWN. 1968. Effect of severe undernutrition in early life on growth, brain size and composition in adult rats. J. Nutr. 94: 419426. HOWARD, E., and D. M. GRAKOFF. 1968. Effect of neonatal food restriction in mice on brain growth, DNA and cholesterol and on adult delayed response learning. J. Nutr. 95: 111-121. NELSON, M. M., and H. M. EVANS. 1953. Relation of dietary protein levels to reproduction in the rat. J. Nzhtr. 51: 71-84. NEVILLE, H. W., and H. P. CHASE. 1971. Undernutrition and cerebellar development. Exp. Newel. 33: 485-497. VENKATACHALAM, P. S., and K. S. RAMANATHAN. 1964. Effect of protein deficiency during gestation and lactation on body weight and composition of offspring, J. Nutr. 84 : 3&42. WINICK, M., and A. NOBLE. 1966. Cellular response in rats during malnutrition at various ages. J. Nutr. 89: 300-306. WINICK, M. 1970. Nutrition and nerve cell growth. Fed. Proc. 29: 1510-1515. ZAMENHOF, S., E. V. MARTHENS, and F. L. MARGOLIS. 1968. DNA (cell number) and protein in neonatal brain : Alteration by maternal dietary protein restriction. Science 160 : 322-323. ZEMAN, F. J., and E. C. STANBROUGH. 1969. Effect of maternal protein deficiency on cellular development in the fetal rat. J. Nutr. 99: 274-282.
DOBBING,