Early nutrition and the activity and feeding of rats in an artificial environment

Male hooded rats were divided into 3 equal groups: (1) controls were given a complete diet ad libitum at all times; (2) for 8 weeks from the age of 28 days, undernourished (UN) rats received the complete diet in restricted amounts; (3) low protein (LP) rats received a low-protein, high-carbohydrate diet for the same period. From 12 weeks all rats were given the complete diet ad libitum, until tested at 35 weeks. Each rat was then put alone, for 12 days, in a residential “plus-maze” with a central nest box and 4 arms radiating from it; food was supplied at the end of one arm, water in the nest box. On Days 4 to 9 each rat had access to the arms for only 1 hr daily. Entries into the arms, and duration of stay, were recorded automatically. All arms were regularly visited by rats of all classes, even when access was only for 1 hr. Visits per day to the 3 nonfood arms combined were usually higher than visits to the food arm, but duration of stay in the food arm was higher than the total duration in the other 3 arms. LP rats made most visits to the arms and were especially active after the 6 days of restriction; but they were less adaptable than the control and UN rats in adjusting food intake during the period of restricted access. In contrast, the UN rats spent most time in the arms.     

Passive avoidance behavior and response thresholds in adult male rats after early postnatal undernutrition.

Rats were undernourished from birth to 25 days of age. Although severely growth retarded, they had probably received more attention from their mothers than had well fed pups. When the previously undernourished rats were tested as adults in a passive avoidance situation they showed stronger passive avoidance behavior than controls rats. When the rats were retested at a later age in a situation where they were given different intensities of inescapable electric shock, the previously undernourished rats showed more jump responses, and more frequent vocalizations than control rats. Analysis of jump responses revealed that the previously undernourished rats had a lower jump threshold than control rats.     

Locomotor activity and social behavior of old rats after preweaning undernutrition

Rat pups were undernourished from birth to 25 days of age by giving them a nipple-ligated mother and a normal lactating mother for alternate periods of 12 hr/day, thus providing adequate full-time maternal care. Male and female rats undernourished neonatally had greater locomotor activity than controls in old age (16–20 months). Females were more active than males, irrespective of neonatal undernutrition. At the age of 24 months, social behavior was recorded at various times of the day for pairs of male rats (1 previously undernourished and 1 control). With one minor exception, undernourished animals did not differ significantly from controls in the incidence of the various behavioral items that were scored. Neonatal social factors may compensate for possible negative effects of neonatal undernutrition on later social behavior.     

Dominance relationships among previously undernourished and well fed male rats.

Experimental rats were growth retarded by underfeeding their mothers during pregnancy and lactation. All rats were fed ad lib from weaning. Pairs of previously undernourished (PU) and control (C) males were housed together from 30 or 80 days. Social behavior within these groups was observed at about 175 days. PU rats submitted to other PU rats more frequently than indicated by the null hypothesis and C rats allogroomed PU rats less frequently. Some of these groups were divided into pairs comprising one C and one PU rat. In a water-spout competition situation, thirsty PU rats were rather more successful than controls in that they tended to possess the water-spout longer. The rats were then separated and after one week of isolation were reunited in an observation box for 10 min a day on 4 successive days. PU rats allogroomed and walked over or crawled under C rats more frequently. In general, PU rats were the more likely to be the active partners in social encounters, with C rats assuming a passive role.     

Social behaviour of rats following pre- and early postnatal undernutrition

The social behaviour of adult male rats which had been undernourished at some stage before weaning was investigated. Litters were cross-fostered at birth between well fed and underfed dams. Thus, some were undernourished only during gestation, others only during the suckling period and others throughout both periods. After weaning all animals had free access to food and were grouped with two or three littermates. Each adult previously undernourished rat was allocated its own control partner which had been well fed throughout life. Four measures of social interaction were recorded during five 10 min tests. Postnatal undernutrition increased the frequency of allogroom and walk over and pre with postnatal undernutrition had a similar effect. Rearing and mount were unaffected by either treatment. Prenatally growth restricted rats did not differ from controls on any measure. Thus, adult social behaviour was more affected by the postnatal than by the prenatal experience. In another experiment smaller control rats were found not to differ in their social behaviour from larger control rats, indicating that size is not an important factor in these studies.     

Evidence of reorganization in the hippocampal mossy fiber synapses of adult rats rehabilitated after prolonged undernutrition

We have previously demonstrated that prolonged low-protein diet leads to irreversible cell loss in the hippocampal formation of the adult rat. Because the extent of the resulting hippocampal synaptic alterations is not well characterized, we studied the contacts between mossy fibers and the dendritic excrescences of CA3 pyramidal cells (MF-CA3 synapses) using quantitative methods. Moreover, we investigated whether rehabilitation from undernutrition would influence the morphology of hippocampal synapses. To address these issues, three groups of adult rats were compared: (a) rats fed with a normal diet for 12 months (control rats); (b) rats treated during the same period with low-protein diet (undernourished rats); and (c) rats undernourished for 6 months and then switched to normal diet for 6 months (recovery rats). Timm staining and electron microscopy were employed to estimate the volume of the mossy fiber system and the number and related quantitative features of MF-CA3 synapses. The volume of the suprapyramidal bundle of the mossy fiber system and its total number of synapses were smaller in undernourished rats than in control and recovery animals. These parameters did not differ between the latter two groups. The size of mossy fiber terminals and dendritic excrescences and the surface area of synapses were smaller in undernourished than in control and recovery groups. Conversely, in recovery animals, the volume of the suprapyramidal bundle of the mossy fiber system, the size of mossy fiber terminals and dendritic excrescences, and the total number and surface area of synapses were similar to those of controls. These findings indicate that, following rehabilitation, the pre- and postsynaptic compartments of MF-CA3 synapses undergo structural alterations which compensate for the neuronal loss induced by undernutrition.     

Effects of postweaning undernutrition on exploratory behavior, memory and sensory reactivity in rats: implication of the dopaminergic system

The effects of early undernutrition on behavior and brain biochemistry were examined in rats. At weaning, rats were provided either an ad lib diet (control group) or maintained at 80% of the weight of their control littermates (undernourished group). Three weeks into the diet they were tested in an open field. After 6 weeks of diet, HPLC analyses were conducted on sample brains from each group to assess levels of dopamine and metabolites, respectively dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. At seven weeks of diet, remaining rats were trained in an 8-arm radial maze, and a retention test conducted 72 h after attaining the learning criterion. At fourteen weeks of diet, sensory reactivity was measured by tail-immersion in a water bath maintained at constant temperature 50 +/- 1 degrees C. Undernourished rats exhibited hyperactivity and increased exploratory behavior in the open field, as well as increased sensory reactivity in the tail flick test. In the radial maze, however, undernourished rats did not differ from controls in either learning or retention. Haloperidol (i. p. injection) impaired retention by control but not undernourished animals. HPLC analyses showed an increase in dopamine turnover in the striatum of undernourished rats. Our results suggest that, unlike its effects when induced immediately at birth or in adulthood, undernutrition at weaning does not appear to influence learning and retention but induced an hyperactivity and alterations in striatal DA turnover which was associated with a decrease in responsiveness to i. p. haloperidol injection.     

Lipid peroxidation in developing rat brain during undernutrition

The effect of undernutrition has been studied in relation to lipid peroxidation, both in vitro and in vivo, in the brain of developing rats. Lipid peroxidation in brain homogenates as well as in the mitochondrial fraction of brain was significantly higher in undernourished groups than in controls. This difference was maximal at the 12th postnatal day. Following rehabilitation for two weeks, this increased lipid peroxidation in brains of undernourished rats could no longer be observed. Determination of malonaldehyde levels in vivo did not show an appreciable difference between control and undernourished animals.     

Reaction to omission of reward in an operant conditioning situation in adult rats after undernutrition early in life

Rats were undernourished during the suckling and early post-weaning periods and were then fed a good quality diet ad lib from 43 to 100 days, prior to behavioural testing. Male, previously undernourished and control rats were exposed to a number of operant conditioning tasks involving partial or total omission of a food reward. In the first condition each lever press response was rewarded for five sessions, and then two sessions were given in which food was discontinued. Both groups showed an initial increase in response rate upon omission of reward, but did not behave differently. Following this, subjects were trained for 38 sessions on a fixed-interval 60 sec schedule. Under this schedule no differences were found between the groups in either post-reinforcement pause or response rate. After this, subjects received 10 sessions in which half the rewards at the end of the 60 sec intervals were randomly replaced by brief blackouts. Both groups of subjects paused for longer after food than after blackout but differed in that response rates of previously undernourished rats after both food and blackout increased markedly over sessions whereas those of the controls did not. Finally food was discontinued and both groups showed a rapid drop in response rate, as well as spontaneous recovery effects. The above results, in conjunction with those from other studies indicate that reward omission per se dose not produce higher than normal response rates in previously undernourished rats, but that unpredictable reward omission may do so.     

The compensatory role of food-motivation in the maze learning performance of lactationally undernourished rats.

To test the hypothesis that the motivational effects of neonatal undernutrition might conceal the detrimental effects on learning, we tested previously undernourished and normally nourished Sprague-Dawley rats on learning of a novel maze pattern under either latent learning (nonappetitive) or food-motivated conditions. Under the nonappetitive conditions, the previously undernourished rats learned significantly less than the normal controls, but when motivated for food, the undernourished rats performed as well as the controls. When learning performance measures are sensitive to motivation, differential motivation between undernourished and normal subjects must be controlled or eliminated.     

Undernutrition in early life: lasting effects on activity and social behavior of male and female rats.

The growth-retarded offspring of mother rats undernourished throughout pregnancy and lactation were fed ad libitum from weaning. Males were tested at 100 days of age, and ovariectomized females at 150 days. After 2 weeks of isolation, pairs of males consisting of 1 control (C) and 1 previously undernourished (PU) rat were observed for 10 min on each of 9 consecutive days. Similarly paired and treated female rats were tested a total of 8 times, the first 3 tests being separated from the last 5 by a 10-day interval. The PU rats, whether male or female, performed more rearing responses and PU male rats ambulated more in the social situation than C rats. The PU males also performed more of the social responses allogroom, walk over, and mount than C males. Similarly, PU females performed more allogroom and crawl under than C females. In general, PU rats were more active than C rats and more socially responsive.     

Developmental delays in offspring of rats undernourished or zinc deprived during lactation

Offspring of rats who were zinc or calorie deprived during lactation were administered a battery of reflex and motor tests from postnatal Day 4 to Day 21. Compared to offspring of ad lib-fed control rats, both zinc deprived and undernourished offspring exhibited developmental delays in reflexes which appeared after the first postnatal week (auditory startle, air righting, and rope descent). As the deficiencies continued the delays appeared to be more pronounced. The zinc deficiency did not add to the deficits associated with calorie restriction alone because there were no significant differences between the zinc deficient and undernourished pups on any of the measures except eye opening. When rehabilitated offspring were tested at 45 and 60 days of age for motor deficits there were no significant impairments resulting from preweaning dietary conditions. However, the growth retardation of zinc deprived and undernourished rats persisted long after dietary rehabilitation was implemented.     

Mesial prefrontal cortical lesions and timidity in rats. II. Reactivity to novel stimuli

An earlier set of experiments suggested that mesial prefrontal cortical (MFC) lesions in rats enhanced timidity. It was uncertain whether this increased timidity was a general phenomenon, or was restricted to fear of bright, open spaces. The experiments reported here measured behavioral reactivity to a variety of stimuli, under situations where light/dark differences were minimized. It was found that MFC rats were slowed in leaving an open field to enter a small box. In the open field, MFC subjects showed signs of enhanced reactivity, but only when the field was novel and the subjects unhandled. When allowed to choose between four alleys containing varying stimuli, brain-damaged rats avoided novel objects and complex stimuli, but spent more time than controls in contact with other rats in the apparatus. In a test of food neophobia, MFC subjects were not neophobic in a familiar test environment, but did avoid the experimenter more than controls. Finally, duration of barbiturate anesthesia was shortened by MFC lesions, but only under conditions of high novelty. It is concluded that MFC lesions produce a timidity which is not restricted to photophobia.     

Vulnerability of developing brain: IV. Passive avoidance behavior in young rats following maternal undernutrition

An investigation was carried out of the effect of undernourishing rats during much of gestation and throughout the whole suckling period on their behavior in a passive avoidance situation. All young were fed ad libitum from 21 days of age. Passive avoidance tests were done at 30 and 31 days in a two-compartment box. On the first test normal rats tended to cross between compartments more promptly than previously underfed rats. Cross-over latency was always greater on the second test than on the first. There were no significant differences in test 2 latency or in the ratio, test 2 latency/test 1 latency, between sexes, between normal and previously undernourished rats, between groups with 2-min and 24-hr interest intervals, or between master and yoked control rats. The last finding indicates that the apparent passive avoidance response may have been either nonspecific ‘freezing’ behavior following unpleasant stimulation, or inhibition specifically of movement, due to its association with shock.     

The supraoptic nucleus in hypothyroid and undernourished rats: an experimental morphometric study

The supraoptic nuclei of both male and female 30-day-old rats rendered hypothyroid by daily subcutaneous injection of propylthiouracil were studied and the results were compared with age- and sex-matched rats fed ad libitum (control rats) and with undernourished rats. Morphometric methods were used to evaluate the volume of the supraoptic nucleus and the areal and numerical densities of its neurons. These parameters allowed us to estimate the total number of neurons of this nucleus. In addition, the mean cross-sectioned area and mean nuclear diameter of the same neurons were also evaluated. The volume of the supraoptic nucleus was reduced both in hypothyroid and undernourished animals when compared with normal controls. The areal and numerical densities of neurons from the former groups were increased and the volume density of the neuropil was reduced. As a consequence, the total number of neurons was found to be identical in all groups studied. Furthermore, the mean nuclear diameters and cross-sectioned areas of the supraoptic neurons were similar in all groups. The volumetric differences and the increased packing found were always more marked for hypothyroid than for undernourished rats. Differences were not detected between male and female groups. The present results support the view that the increased cell packing in hypothyroid animals depends upon a reduction in the neuropil of the nucleus, as has been described under similar conditions in other central nervous system areas displaying identical patterns of neurogenesis. In addition, it was found that the effects of undernourishment cannot be discriminated from those dependent on neonatal hypothyroidism.     

Early-life growth hormone treatment to offspring of undernourished mothers alters metabolic parameters in primary adipocytes in adulthood

Abstract

Maternal undernutrition (UN) is associated with the development of obesity and metabolic complications in adult offspring. This study investigated the impact of preweaning growth hormone (GH) treatment on adipocyte functionality in adult male offspring. Sprague-Dawley rats were assigned either standard (C) or undernourished (UN) diet (50% ad libitum) throughout gestation. Postnatal day 3–21, male C/UN pups received either saline (CS, UNS) or GH (2.5?µg/g/d; CGH, UNGH) by subcutaneous injection. Primary adipocytes were isolated following the collagenase digestion of adipose tissue. Primary adipocytes from UN offspring had significantly increased the secretion of pro-inflammatory cytokines accompanied by increased cytokine/cytokine receptor expression. This correlated with increased TLR4/NF-κB signaling. While increased inflammatory potential was not observed in adipocytes derived from UNGH offspring, there was a clear alteration in the expression of genes relating to carbohydrate and lipid metabolism along with nutrient transporters. Overall, preweaning GH treatment alters detrimental patterns of development, which predispose UN offspring to obesity and insulin resistance.     

Effects of early life undernutrition in artificially-reared rats. 2. Subsequent behaviour

Four groups of rat pups were reared: mother-reared control (well-fed) and undernourished (MRC, MRU) and artificially-reared control and undernourished (ARC, ARU). AR rats were fed expressed rats' milk (days 5-7), mixtures of rats' milk and milk substitute (days 8-16), and milk substitute only (days 17-20) by intermittent infusion via a gastric cannula. Undernutrition, imposed from 5-25 days, was effected initially by underfeeding the mother (MRU) or by infusing restricted quantities of milk (ARU). Weaning was at 21 days and undernutrition from 21-25 days was by restricting the supply of solid food. All rats were fed ad lib from 25 days. From 18 weeks, male rats were subjected to 3 behavioural tests: open field, social behaviour (encounters between rats of like treatment group) and bar-pressing for food (CRF, FR5, VI 1', VI 2' and Extinction). Previously undernourished rats responded at higher rates on FR5, received fewer rewards on VI 2' and displayed more aggression during social interaction. These findings may reflect direct effects of nutrition on brain growth. In the open field and social situations ARU were generally less active than ARC animals, whereas MRU and MRC rats differed little. These results are discussed in terms of MRU pups being maternally "buffered" against the worst effects of undernutrition and ARC rats displaying characteristics of "early stimulated" animals.     

Punishment of runway behaviour in rats with fornical lesions

The performance of rats with fornical lesions was compared to that of operated controls in a runway punishment situation. A number of different performance measures were recorded including start box latency, runway time, and goal box time. In Experiment 1, the runway and goal box were visible from the start box whereas in Experiment 2 neither was visible from the start box. In both experiments the postshock start box latencies of the operated control rats were significantly longer than those of the rats with fornical lesions. In general, rats with fornical lesions hesitated in the presence of stimuli that had been paired with shock while control rats hesitated more in the presence of stimuli that preceded those that had been paired with shock. These results are not easily dealt with by any general inhibitory theory of hippocampal function. The most likely alternative was provided by a spatial, or similar, theory of hippocampal function.     

Fetal undernutrition induces overexpression of CRH mRNA and CRH protein in hypothalamus and increases CRH and corticosterone in plasma during postnatal life in the rat

Prenatal undernutrition induces a variety of cardiovascular alterations in mammals when adults, including hypertension and hypercortisolism, which are thought to be caused by decreased glucocorticoid feedback control of the hypothalamus–pituitary–adrenal (HPA) axis programmed during fetal life. Hypothalamic CRH seems to be involved in blood pressure elevation of spontaneously hypertensive rats and in primary hypertension of humans, but the influence of prenatal undernutrition on CRH expression has deserved little attention. Here, we studied the expression of both CRH mRNA and CRH protein in the hypothalamus of neonatal and juvenile offspring of rats undernourished during fetal life, as well as the plasma levels of CRH and corticosterone. Prenatal undernutrition of pups was induced by submitting pregnant rats to diet restriction (10 g daily of 21% protein standard laboratory diet). Pups born from dams with free access to the standard laboratory diet served as controls. At day 2 of postnatal age, undernourished pups showed lower body and brain weights, but higher plasma CRH and corticosterone than normal pups. At day 40 of age, brain weight was significantly decreased in the undernourished rats, while plasma corticosterone, plasma CRH and systolic pressure were significantly increased in these animals. At days 2 and 40 of postnatal age, increased CRH mRNA expression and CRH concentration were found in the hypothalamus of undernourished rats. Results indicate that, in the rat, prenatal undernutrition led to fetal programming of CRH overexpression, a neuropeptide serving as activating signal to the HPA axis and/or to extrahypothalamic brain regions concerned with cardiovascular regulation.     

Trimethoprim-sulfamethoxazole increase micronuclei formation in peripheral blood from weanling well-nourished and malnourished rats

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is a widely used drug. In spite of this, there are few reports on its genotoxicity, and the results are controversial. Severe malnutrition is a complex condition that increases the susceptibility to infections. Consequently, drugs are extensively used in malnutrition cases. Experimental animal models have been widely used to study the effects of malnutrition. Neonatal rats were experimentally malnourished (UN) during lactation. The UN rats weighed 51.1% less than the well-nourished (WN) controls and had lower concentrations of serum protein and blood lipids. The micronucleus (MN) assay is useful for detecting chromosome damage induced by nutritional deficiencies. In vivo rodent MN assays have been widely used to screen genotoxic agents. In this study, we have evaluated the frequency of spontaneous and TMP-SMX-induced micronuclei in the peripheral blood of weanling (21 days of age) rats using a flow cytometric analysis technique. The spontaneous frequency of micronucleated reticulocytes (MN-RETs) was 2.7 times greater in the UN rats than in the WN rats. In rats that were not treated with TMP-SMX, the percentage of reticulocytes was significantly lower (41.1%) in the UN rats than the WN controls. A therapeutic dose of TMP-SMX (80 mg/kg (TMP), 400 mg/kg (SMX) for 48 hr) increased MN-RETs in the WN and in the UN rats. The data demonstrate the genotoxic effect of this drug. The results indicate that severe protein-calorie restriction and drug treatment enhance DNA damage in rat peripheral blood reticulocytes, potentially increasing the risk of negative effects on health.