Neonatal malnutrition: neurochemical, hormonal and behavioral manifestations

Brain Research, 65 (1974) 443-457 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

443

NEONATAL MALNUTRITION: NEUROCHEMICAL, HORMONAL AND BEHAVIORAL MANIFESTATIONS

THOMAS J. SOBOTKA, MICHELLE P. COOK

AND

ROBERT E. BRODIE

Division of Toxicology, Food and Drug Administration, Department of Health, Education, and Welfare, Washington, D.C. 20204 (U.S.A.) (Accepted July 4th, 1973)

SUMMARY

An effective state of malnutrition was imposed on rat pups throughout their postnatal period of development by feeding the lactating dams a 12 ~ casein diet. Controls were fed a 24 ~ casein diet. The malnourished pups exhibited such signs of retarded physical development as reduced body growth and delayed eye opening. At weaning brain weights were reduced and regional brain analysis revealed distortions of chemical composition. These changes included a general reduction of cholesterol concentration, decreased cerebellar DNA content (indicative of cell number), and reduced telencephalic and brain stem protein : DNA ratios (reflecting cell size). The cerebellum also displayed reduced AChE activity suggesting an appreciable effect on its synaptic elements, particularly involving the cholinergic system. Brain stem concentrations of 5-HT and 5-HIAA were increased, implying activation of the central serotonergic neurohumoral system. Possibly related to this, a concomitant increase of adrenal corticosterone was revealed, implicating the additional involvement of the pituitary-adrenal axis in the consequences of postnatal malnutrition. Behaviorally, the previously malnourished weanlings displayed a state of heightened 'emotionality'. Their performance in a 2-way shuttle task was also markedly abnormal. It is proposed that the enhanced activation of the brain stem serotonergic system may, at least partially, underlie the state of heightened 'emotionality', which is the most characteristic behavioral manifestation of perinatal malnutrition.

INTRODUCTION

The vulnerability of the developing brain to changes in its environment is clearly demonstrated by the consequences of dietary inadequacy during the ontogenetic period. Extensive investigations have revealed that perinatal malnutrition results

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in a diversity of long-term deficits and distortions of brain chemical composition and function 15. The changes elicited in general brain chemistry reflect attenuated cell proliferation (particularly in the cerebellum, which undergoes rapid development during the postnatal period of life), reduced cell size, decreased cortical dendritic growth and depressed myelination - - all indicative of retarded brain development. Previous investigators have also reported various modifications of several central neurotransmitter systems following perinatal malnutrition3,23,31, 32. Behaviorally, the most characteristic manifestation of early malnutrition in animals is heightened 'emotional' responsiveness to stressful conditionsS,Z4, 25. Other reported behavioral changes, such as decreased exploratory drive, increased aggressiveness and delayed extinction of a conditioned response, appear to be related to the lower stress thresholdg, ~5. In addition to such 'emotional' disturbances, depressed learning ability has been reported in the literature 9,37. However, there is some question as to whether this poor learning efficiency is due to an actual impaired capacity to learn or is the result of altered motivational or 'emotional' factorsS, 17. One of the more elusive problems of early malnutrition is that of defining those neurochemical or hormonal modifications that might underlie the abnormal states. In an effort to further elucidate such relationships, the present investigation will endeavor to confirm and extend previous findings regarding brain composition, central neurotransmitter systems, hormonal conditions and behavioral manifestations of neonatal protein deficiency. Particular consideration will be given those factors relevant to the reported occurrence of heightened stress responsiveness. MATERIALS AND METHODS

Experimental design The animals used for the present report consisted of the dietary control groups from a second, concurrent study initiated to determine any interaction of malnutrition and perinatal exposure to environmental contaminants. Adult pregnant Charles River rats were allowed to litter normally. On the day following delivery (day 1), the litters were culled to 9 or 10 per litter and assigned randomly to either a control (5 litters; 23 male pups and 24 female pups) or experimental group (5 litters; 27 male pups and 21 females). The mothers of the control group were fed a normal protein diet containing 24 ~ casein and the mothers of the experimental (malnourished) group were fed a 1 2 ~ casein, protein-deficient diet. In accordance with the protocol of the second larger study, all of the male pups (in both control and protein-deficient groups) were given daily oral doses of water (0.01 ml/g body weight) from age 2 through 20 days. Daily body weights of the male pups were recorded and the age of eye opening was noted. Upon weaning at 21 days of age, the male offspring (females were discarded) were randomly assigned to either a 'biochemical group' or a 'behavioral group', and housed individually. All rats were maintained on an ad fibitum normal protein diet. At 22 days of age, the 'biochemical group' was sacrificed. The brains, adrenals and thyroids were excised, weighed and frozen for subsequent analysis. The brains

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were divided into the following 3 regions: telencephalon, brain stem and cerebellum. Regional brain analysis included determinations of protein, cholesterol, DNA, acetylcholinesterase (ACHE) activity, butyrylcholinesterase (BuChE) activity, norepinephrine (NE), dopamine (DA), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA). Concentrations of adrenal corticosterone and thyroidal thyroxine were also determined. Behavioral testing of the remaining weanlings commenced at 22 days of age, and included measurements indicative of responsiveness to stressful situations (exploratory behavior, step-out latency and passive avoidance), general motor activity and learning ability (2-way shuttle procedure). The testing procedures took approximately 1 month to complete. Biochemical analyses Protein. Regional brain protein determinations were made according to the method of Lowry et al. 26. Cholesterol. Total cholesterol concentration in each brain part was measured colorimetrically by using the Harleco Cholesterol Kit. DNA. The DNA was extracted with perchloric acid and quantitated by using the diphenylamine reaction as described by Hatcher and Goldstein 21. NE, DA, 5-HT, and 5-HIAA. The method described by Welch and Welch 36 was used to determine brain concentrations of NE, DA, 5-HT and 5-HIAA. Concentrations of these substances in the cerebellum were not measured. AChE and BuChE. Enzymatic activity was measured by using an automated titrimetric procedure tl. To estimate AChE activity, acetyl-/%methylcholine bromide was used as substrate 7. BuChE activity was determined by using a specific substrate, butyrylcholine chloride, in combination with a specific inhibitor for AChE (Burroughs Wellcome, BW 284 C51 dibromide). Corticosterone. Adrenal corticosterone was determined fluorometrically by a modification of the method of Glick et al. 19. Thyroxine. Thyroxine was extracted from thyroid tissue by a column chromatographic separatory procedure and determined colorimetrically according to the Harleco T-4 analytical method. Behavioral procedures To gauge the state of 'emotionality' or stress responsiveness of the weanling rats, the following indices were used: (1) exploratory activity in a novel environment2°; (2) step-out latency into a large chamber4°; and (3) passive avoidance of an aversive stimulus3L Exploratory and general motor activity were measured in a photoactometer. Each rat was placed only once in the actometer. Since the animals were naive to the chamber, the initial 15-min activity was used to represent the degree of exploratory activity. The activity during the second 30 min was taken as a measure of general motor activity. Fear-induced suppression of motor activity (passive avoidance) was measured

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Fig. 1. Growth curves of neonatal male rat pups raised by dams fed control (24 ~ casein) or lowprotein (12 ~ casein) diets during the 3-week postnatal period. in an apparatus, described by Weiss et al. 35, consisting of 2 adjoining chambers, one smaller than the other, separated by a sliding door. Both chambers had a metal grid floor. Each rat was placed in the small compartment facing away from the door. After the door was opened, the latency to enter the large chamber was measured. Immediately after the animal entered the large chamber, the door was closed and a 9 mA/0.2-sec foot-shock was administered. The rat was returned to its home cage and retested on the following day for latency to enter the large (shock) chamber. A maximum latency of 180 sec was allowed. N o shock was given on the retest day. The initial entrance latency (preshoek latency) represents another estimate of exploratory activity in a novel environment. The second entrance time (postshoek latency) is a measure of the aversive reaction displayed in a strongly stressful situation. Learning ability was determined using a 2-way shuttle procedure. The apparatus consisted of 2 adjoining chambers, each 9 in. × 9 in. × 8.5 in. Each trial consisted of 3 sec of conditioning stimuli (light and sound), followed immediately by 3 sec of conditioning stimuli plus the unconditioned stimulus (grid foot-shock). The intertrial interval was 14 sec. Each rat was given a total of 40 trials over 2 days. The mean percent avoidance, escape and no-response scores for the entire 40 trials were calculated. Data on eye opening were analyzed by the g 2 test. All other data were evaluated by the Student's t test.

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TABLE I EFFECT OF POSTNATAL MALNUTRITION ON AGE OF EYE OPENING IN RAT PUPS

Dietary group

Control (24~casein) Malnourished (127ooCasein)

N

Percentage of neonates with eyes open at days 12

13

14

15

16

17

22

0

18

46

100

25

0

0

15 42 96 P < 0.025 P < 0.001

100

RESULTS

The experimental diets were given to the lactating dams when the pups were 1 day old. Significant changes in the body weights of the malnourished male pups did not occur until they were 5 days old, a latency of 4 days (Fig. 1). Thereafter, body weight differences between the normal and malnourished groups continued to increase steadily throughout the remainder of the postnatal period. By age 20 days, the deprived pups Weighed only 46 ~o of controls. The protein-deficient diet did not affect the mortality rate of the pups. Another sign of retarded development in the experimental pups was the delay in the age at which eye opening occurred (Table I). The controls had completed this phase of development by 15 days of age, whereas the deprived group did not achieve 100 ~ eye opening until 17 days of age, this delay was statistically significant. Brain composition Brain weight. T h e r e g i o n a l b r a i n w e i g h t s o f the m a l n o u r i s h e d w e a n l i n g s at 22 d a y s o f age were, as e x p e c t e d , significantly l o w e r t h a n t h o s e o f c o n t r o l s ( T a b l e II). TABLE II EFFECT OF POSTNATAL MALNUTRITION ON BODY AND REGIONAL BRAIN WEIGHTS OF 2 2 - D A Y - O L D WEANLING RATS

Values are means i S.E.M. of 15 control and 14 malnourished rats.

Body weight (g) Absolute weights (nag) Telencephalon Cerebellum Brain stern Relative weights (rng/g body weight) Telencephalon Cerebellum Brain stem

Control (24 % casein)

Malnourished (12 % casein)

% Change

56.3 4- 2.0

24.8 4- 1.4

--56 P < 0.001

933 211 386

±34 4-3 4-4

17.5 4- 0.6 3.8 ± 0.1 7.0 4- 0.2

807 155 325

4-11 4-2 4-6

33.6 :J_ 1.4 6.5 4- 0.3 13.5 4- 0.6

--14P<0.005 ---27P<0.001 --16P<0.001 +92 P < 0.001 +69 P < 0.001 4-94 P < 0.001

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IV

EFFECT OF POSTNATAL MALNUTRITION IN RATS ON REGIONAL BRAIN ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE ACTIVITY AT 2 2 DAYS OF AGE V a l u e s a r e m e a n s -4- S . E . M . o b t a i n e d f r o m 6 t o 7 d e t e r m i n a t i o n s .

AChE (#moles MeCh/g/min) Telencephalon Cerebellum Brain stern BuChE (#moles BuCh/g/min) Telencephalon Cerebellum Brain stem

Control (24 % ease±n)

Malnourished (12 % casein)

% Change

8.01 -4- 0.64 6.85 -4- 0.24 7.33 -4-0.67

6.75 -4- 0.36 4.73 -4-0.32 6.61 -4- 0.41

--16 --31 --10

NS P < 0.001 NS

10.73 -4- 1.02 7.90 -4- 0.64 9.57 -4- 0.71

8.44 ~ 0.67 7.32 -4-0.63 9.40 -4- 0.20

--21 -- 7 -- 2

NS NS NS

The cerebellum appeared to be most affected; weights o f the telencephalon and brain stem were reduced by 14 and 1 6 ~ , respectively, while the weight of the cerebellum was 27 ~ below the control value. In terms of relative brain weight (brain : body weight ratio), the ratios of all 3 brain regions of the malnourished weanlings were significantly greater than those of controls, and reflect the greater reduction in body weight (--56 ~ ) than in brain weight. Cholesterol, protein and DNA (Table III). Cholesterol concentrations, which serve to index the extent of lipid accumulation, were significantly reduced in all 3 brain regions of the malnourished weanlings. Protein concentration was, however, unaffected. The D N A changes were found to exhibit regional differences. In the experimental animals, the telencephalic and brain stem regions had significantly greater concentrations of D N A per unit weight of tissue than did those of controls; the total D N A content per brain part, however, remained unchanged. The opposite occurred in the cerebellum; its D N A concentration was unchanged, while the total D N A content was reduced by 30 ~o of control. Similarly, regional differences were also found with regard to protein : D N A ratios. The telencephalon and brain stem of malnourished rats exhibited reduced protein: D N A ratios, whereas the cerebellar ratio was unaffected. AChE and BuChE activities. Postnatal malnutrition appears to exert a significant depressant effect on cerebellar AChE activity of the 22-day-old weanlings (Table IV). The reduced enzyme activities in the other 2 brain regions were not statistically significant. No statistically significant regional changes in BuChE activity were revealed. 5-HT, 5-HIAA, NE and DA. The primary neurohumoral effect of postnatal malnutrition appeared to involve the serotonergic system. Brain stem concentrations of 5-HT ( + 40 ~ ) and its primary metabolite, 5-HIAA ( + 102 ~), were both significantly increased above control values in the 22-day-old weanlings (Table V). No other statistically significant neurohumoral changes were found.

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EFFECTS IN BRAIN

TABLE VII EFFECT OF POSTNATAL MALNUTRITION ON INDICES OF EMOTIONALITY IN THE WEANLING RAT

Values are means -4- S.E.M. from 7 control and 11 malnourished rats.

Exploratory activity* Step-out latency (see) Preshock Postshock

Control (24 °/o casein)

Malnourished (12 % casein)

% Change

171.8 i 16

123.6 -4- 14

--28 P < 0.01

44.7 -4- 14 107.0 -4- 34

124.1 ± 16 180.0 -4- 00

+178 P < 0.005 +68 P < 0.05

* Five-rain averages for first 15 min in photoactometer.

Hormonal state. The absolute weight of the adrenal glands in the 22-day-old malnourished weanlings was significantly below that of controls (Table VI). While this fact alone might imply attenuated adrenal activity, quite the opposite appeared to occur. This is shown by the fact that not only was relative adrenal weight (adrenal : body ratio) significantly greater than control, but the adrenal concentration of corticosterone was also markedly greater (155 ~ ) than control. Similarly, the thyroids of the malnourished animals weighed less than those of controls but their relative weights were significantly greater. However, in contrast to the adrenal gland, the thyroidal concentration of its hormone, thyroxine, was not significantly different from the control value. Behavioral manifestations

Examination of the parameters used to index 'emotionality' (Table VII) reveals a state of heightened responsiveness to stress in the weanling rats previously malnourished during their postnatal period of development. In response to the mild stress of a novel environment, the malnourished offspring demonstrated a 28 ~ decrease in total exploratory activity in the photoactometer, as well as a 178 ~ increase in preshock latency to enter a large chamber. In the more intensely stressing situation involving passive avoidance of a 'shock chamber,' the malnourished rats displayed a marked increase in fear-induced suppression of m o t o r activity as evidenced by their significantly longer postshock latency to enter the 'shock chamber' than that of the TABLE VIII EFFECT OF EARLY MALNUTRITION ON SHUTTLE-BOX PERFORMANCE OF 25-DAY-OLD WEANLING RATS

Dietary group

Control (24 ~ casein) Malnourished (12~ casein)

N

7 11

% Response (average response for 40 trials) Avoidance

Escape

No response

26 3

48 20

26 77

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T.J. SOBOTKAet al.

controls. These effects were shown not to be due to a general reduction in motor activity, since both groups displayed similar general motor activity scores during the second 30-min period in the photoactometer (56.3 ± 15 for controls and 56.4 4- 9 for experimentals). The performance of the malnourished rats in the shuttle-box task was markedly abnormal in comparison to the controls (Table VIII). Since the deprived animals displayed an obvious debility in effecting even an escape from the aversive (shock) stimulus, little can be said concerning their capability to learn the avoidance component of this task. DISCUSSION

A protein-restricted diet was fed to lactating dams, thereby imposing an effective state of nutritional deprivation on the neonate rats during the 3-week postnatal nursing period. Attenuated body growth became evident 4 days after the experimental diets were given to the mothers. This latency period presumably reflects the maternal nutritional reserve capacity. Upon depletion of this reserve, neonatal body weight reductions occurred and remained significantly below controls throughout the experimental period. At the time of weaning, deprived pups had attained only 44 ~ of control body weight. The delay in age of eye opening further evidenced obvious developmental abnormalities. The ontogeny of these and other physical features and natural reflexes are known to be affected by perinatal malnutrition3L At weaning, the chemical composition of all 3 brain regions was found to be affected by the postnatal malnutrition. However, there did occur a differential effect, depending on the particular brain region, as reported by previous investigators6, a0. Such regional sensitivity is based on the fact that, following embryonic differentiation, the maturation of the brain is not synchronous throughout the nervous system5. Proliferation of the neuronal cell population of the forebrain and brain stem occurs primarily prenatally in the rodent. Postnatally, these cells undergo growth and maturation, including such changes as development and migration of neuronal processes (axons and dendrites), synaptogenesis and myelination. The cerebellum, in contrast, undergoes only moderate cell formation prenatally (involving primarily Purkinje cell formation) but very brisk cell proliferation postnatally. Subsequently, growth and maturation of cerebellar neuronal processes occur. In consonance with such differential development, nutritional deprivation, sustained during the postnatal period only, has relatively little effect on telencephalic or brain stem cell number (normal DNA content) but does appear to reduce the cell size (reduced protein/DNA) and to influence subsequent neuronal maturation, as described below. In contrast, the rapidly developing cerebellum encountered an appreciable reduction in cell population (reduced DNA content by 30 ~) but little change in cell size. Two components of nerve cell maturation, namely, myelination and synaptogenesis, both of which are of considerable importance to the functional development of the central nervous system, also appear to be affected by neonatal malnutrition. Significant reductions in cholesterol concentration of all 3 brain regions suggest

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an interference with the process of myelination throughout the brain. Similar findings have been reported by previous investigators14,16. This defective myelination has been postulated to be the result of insufficient differentiation and retarded migration of neuroglial elements within the brain6,10. If this suggestion is true, then it seems somewhat surprising that none of the brain regions displayed a statistically significant change in BuChE activity, said to be localized to a great extent in neuroglial cells22. Adlard and Dobbing 3 reported similar lack of effect on whole brain BuChE activity. There is, however, a possible explanation as to why no changes in BuChE activity were found. If the migration of neuroglial cells is impaired, then they might be expected to be concentrated closer to the sites of their postnatal origin, that is, the secondary germinal matrices, such as the subependymal layer of the cerebrum or the cerebellar subpial, external granular layer5. Relatively high levels of BuChE activity would also be expected to accompany the higher concentrations of neuroglial cells. Enzyme analysis using whole brain or relatively large brain regions (as in the present experiment) may detect no differences between malnourished and control animals, whereas more discrete regional analysis may reveal very significant differences in enzyme activity. Investigations have established the fact that a high proportion of the brain's AChE activity is located in the synaptosomal subceUular fraction, which contains pre- and postsynaptic neuronal elements~,13. Consequently, AChE measurements may be related to the metabolic activity of the cholinergic neurotransmitter system and/or to the quantity or maturity of at least a certain population of synaptic complexes2,L In view of this, the significant 31 700reduction of cerebeUar AChE activity in malnourished offspring implies an appreciable effect on the synaptic elements within this brain region, particularly involving the cholinergic system. Adlard and Dobbing 3, however, reported a small but non-significant reduction in cerebellar AChE activity. The apparent marked vulnerability of the cerebellum to early malnutrition would seem to be more consistent with our finding, which suggests attenuated synaptogenesis. In contrast, we found no statistically significant reductions in either telencephalic or brain stem AChE activity in the 22-day-old malnourished weanling. Similar negative results were reported by Sereni e t al. 3~ who also employed dietary deprivation only during the postnatal period. However, they did find that AChE activity was reduced in younger pups (14 days and younger) but that it returned to control levels by 21 days of age, indicating that the rate of AChE development, and presumably synaptogenesis, is retarded by under-nutrition. Adlard and Dobbing 2,3 have reported similar conclusions. In their investigations, however, forebrain and brain stern enzyme concentrations were still depressed at 21 days of age in contrast to our results and those of Sereni e t al. 31. This longer period of enzyme depression may be related to the fact that Adlard and Dobbing imposed nutritional deprivation, both pre- and postnatally, thereby inducing a comparatively more severe condition. In a more recent article these authors 4 reported a rebound accumulation of AChE activity in homogenates and subcellular fractions of brain regions as the perinatally malnourished pups reached adulthood. The previously malnourished rat had higher AChE concentrations than adult controls. Gambetti e t al. is similarly reported in-

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creased AChE activity in synaptosomal fractions of cerebral cortex but this increase was found only in weanling rats previously malnourished. This synaptosomal increase in enzymatic activity of early deprived weanling rat brain may represent the initial subcellular stage of recovery and rebound which subsequently becomes evident in the whole homogenate in the adult, as was reported by Adlard and Dobbing 4. The latter authors offered as possible explanations for this rebound phenomenon altered enzyme kinetics, a general increase in enzyme concentration (possibly reflecting a compensatory change) or a 'sparing' of nerve endings. Resolution of this problem must await further information. The available information does show that during development the process of brain synaptogenesis, at least involving a select population of cholinergic nerve endings, is offset by malnutrition, particularly in the cerebellum. The involvement of neurohumoral systems, other than the cholinergic, in early malnutrition has been indicated by previous reportsZ3,31,3L Pre- and postnatal malnutrition results in decreases of brain NE and DA in weanling rats z3,32. However, the interpretation of these changes is made uncertain by the conflicting reports of increased32 and decreasedz3 tyrosine hydroxylase activity, which is the rate-limiting step in catecholamine metabolism. Restricting the time of malnourishment to the postnatal period only reportedly decreases DA but not NE 3z. Sereni et al. 31 did report reduced NE levels in the young neonates but no alteration in weanling rats. In our experiments we also found no evidence of decreased NE, nor could we confirm the previous report of decreased DA in either the telencephalon or brain stem of the weanling rat following postnatal malnutrition. We did, however, find a significant change of the serotonergic system, particularly in the brain stem region, consisting of increased 5-HT, as well as its primary metabolite, 5-HIAA. These changes were interpreted as indicative of an activation of the brain serotonergic system. To date, the only available information pertaining to serotonin and early malnutrition is a report by Sereni et al. 31. Measuring whole brain serotonin, they found decreased levels in neonates up to 8 days of age but no changes thereafter in older pups. Our finding of increased brain serotonergic activation offers a plausible neurochemical explanation for the behavioral state following perinatal malnutrition. As mentioned previously, the most characteristic behavioral manifestation of early malnutrition in animals is, reportedly, heightened emotional responsiveness to stressful conditionsS,24,25. This was confirmed in the present experiment. The nutritionally deprived weanlings exhibited decreased exploration in a novel environment, increased latency to leave a start box to enter a strange chamber, and facilitation of passive avoidance responding. These behavioral changes manifest behavioral suppression and reveal an exaggerated state of fear responding or pronounced anxiety2°,35,4°. Serotonergic activation has been implicated as a primary component mediating behavioral suppression in response to conditioned fearas and in the induction of pronounced states of anxiety by yohimbinezs. More recently, Wise et al. ~9 have suggested that minor tranquilizers exert their anxiety-reducing effects by a reduction of serotonin turnover in the brain, the greatest effects being concentrated in the midand hindbrain regions. In view of this suggested relationship between serotonin and

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fear responding, our results indicate that enhanced activation of the brain stem serotonergic system in the weanling rat may, at least partially, underlie the state of heightened emotionality following early nutritional deprivation. Besides the increased serotonin metabolism, the malnourished weanlings also displayed a marked (155 %) increase of adrenal corticosterone. This is not totally unexpected in view of the apparently low threshold to stress of the malnourished neonates. That the adrenal and serotonin effects may be connected is consistent with reports of an important relationship between brain serotonin and the pituitaryadrenal system12,27, 29. However, the exact nature of this relationship is presently a matter of controversy. The increased adrenal corticosterone, together with the increased relative adrenal and thyroid weights in malnourished offspring, does suggest enhanced pituitary activity. Consequently, previous reports of growth hormone deficiency in malnourished neonates 34 should not be interpreted to signify pan-hypopituitarism but rather may indicate a more specific endocrinologic dysfunction. In addition to the emotional lability, the malnourished weanlings also exhibited a marked performance deficit in the 2-way shuttle box. The animals were incapable even of escaping effectively from the aversive stimulus. Since the rats demonstrated no abnormality in general m o t o r activity, it is tempting to speculate that this inability to escape may stem from the exaggerated fear-induced immobility exhibited by the malnourished weanlings. However, other explanations are possible 17. In summary, abnormalities in physical development, structural brain chemistry and subsequent behavioral characteristics were demonstrated in confirmation of previous reports emphasizing the extent to which basic brain functions can be altered following postnatal nutritional deprivation. In addition, the brain serotonergic neurohumoral system was also found to be altered. This alteration was suggested to underlie the exaggerated responsiveness to stress, a characteristic sequel of early malnutrition. Altered adrenal activity was also demonstrated and may be involved with the serotonergic-behavioral interaction.

REFERENCES 1 ABDEL-LATIF,A., SMITH, J., AND ELLINGTON, E., Subcellular distribution of sodium-potassium adenosine triphosphatase, acetylcholine and acetylcholinesterase in developing rat brain, Brain Research, 18 (1970) 441-450. 2 ADLARD, B. P., AND DOBBING, J., Vulnerability of developing brain. III. Development of four enzymes in the brain of normal and undernourished ra~, Brain Research, 28 (1971) 97-107. 3 ADLARD, B. P., AND DOBBING,J., Vulnerability of developing brain. V. Effects of total and postnatal undernutrition on regional brain enzyme activities in three-week-old rats, Pediat. Res., 6, (1972) 38-42. 4 ADLARD,B. P., AND DOBBING,J., Vulnerability of developing brain. VIII. Regional acetylcholinesteraseactivity in the brains of adult rats undernourished in early life, Brit. J. Nutr., 28 (1972) 139-143. 5 ALTMAN, J., Postnatal neurogenesis and the problem of neural plasticity. In A. LAJTHA(Ed.), Developmental Neurobiology, Thomas, Springfield, Ill., 1970, pp. 197-237. 6 ALTMAN, J., Nutritional deprivation of neural development. In M. STERMAN,D. McGINTYAND A. ADINOLFI(Eds.), Brain Development and Behavior, Academic Press, New York, 1971, pp. 359-368.

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