Oscar Resnick

The effects of protein malnutrition on the developing central nervous system in the rat

Abstract We have carried out a multi-disciplinary study of the effects of prenatal protein malnutrition on the developing rat brain. These experiments, involving anatomical, physiological, biochemical, and behavioral approaches, have revealed that malnutrition induced prenatally can affect various parameters of brain growth and development. Some of these effects can be reversed depending on when dietary restitutions are carried out. However, if protein malnutrition is maintained during the brain growth spurt or critical growth periods there are many permanent sequelae that cannot be reversed by subsequent restitution of high protein diets. We have reviewed the concept of critical periods of brain growth relative to the various aspects of neural morphogenesis in the rat, that is, the birth of neurons, migration of neurons, differentiation of neurons, and synapse formation. We have also discussed the rapid phases of brain growth in the rat as compared to similar phases in other species as a basis for determining whether the rat model can provide time-tables for brain growth in other species, including man. Different components of the brain, both morphological and chemical, have their own cycles of rapid development so that insults to the brain at particular periods affect particular aspects of brain chemistry and neuronal systems. Development of chemical circuits in the brain, such as the aminergic neurons, and their eventual adequate functioning, depends on development of the neurotransmitters themselves. These latter are markedly affected by protein malnutrition. Major physiological-behavioral states, such as the sleep-waking continuum, are markedly affected by protein malnutrition as are many behaviors. Some of these latter are merely late or retarded in development but others remain permanently altered. By approaching the problem of protein malnutrition from multiple points of view we have been able to pinpoint several brain areas showing the most drastic residua of early protein malnutrition and are beginning, by use of morphometric, electro-ontogenetic, biochemical development and behavioral studies, to define brain locales and basic mechanisms by which these insults produce their effects.

Ontogeny of the levels of biogenic amines in various parts of the brain and in peripheral tissues in normal and protein malnourished rats.

Abstract The ontogenetic development of serotonin, 5-hydroxyindoleacetic acid, and norepinephrine in brain regions and in peripheral tissues was examined in normal and protein malnourished rats from birth to age 300 days. The malnourished rats, which received a diet low in protein starting 5 weeks prior to conception, showed significantly elevated brain and peripheral tissue levels of the biogenic amines and 5-hydroxyindoleacetic acid at birth. This is one of the earliest ages at which protein malnutrition has been reported to affect a major biochemical measure in the brain. In malnourished rats, brain concentrations of serotonin and 5-hydroxydoleacetic acid remained elevated at older ages, up to 300 days, with the largest effects (up to 200% increase) occurring in subtelencephalic brain regions. These changes in brain indole levels probably represent a general metabolic alteration of indoleamine metabolism since elevated indole concentrations were also observed in the heart, lung, and stomach. At most ages the increase in brain norepinephrine levels in malnourished rats was less pronounced than for the indoles. Also, no increase in norepinephrine concentration in the peripheral tissues were observed. With respect to norepinephrine concentrations, the brain appears to be more sensitive to the insult of protein malnutrition than do peripheral tissues. The present results demonstrate that rearing rats on a diet low in protein, but adequate in all other respects, significantly elevates the brain amine content at most ages from birth through 300 days of age.

Seizure susceptibility and brain amine levels following protein malnutrition during development in the rat

Abstract This report examines the change in seizure susceptibility in adult rats which were reared on a diet containing either normal or inadequate levels of protein. A significantly greater percentage of the protein malnourished rats convulsed at low to moderate intensities of electroconvulsive shock (ECS) than normals. Also, seizure duration in the malnourished subjects teneed to be longer than in the normals. Switching adult rats to the opposite diet, i.e. rats reared on normal diets now receive low protein diet and vice versa, had a moderating effect on seizure susceptibility, but full reversal of the effects of protein malnutrition was not achieved. In sum, protein malnutrition during development led to enhanced seizure susceptibility in adulthood, an effect which was only partially ameliorated by restoration of adequate dietary protein levels in adulthood. Prior studies have shown a strong inverse relationship between brain biogenic amine levels and seizure activity. In a second study we therefore investigated whether decreased regional brain levels of norepinephrine and serotonin were produced by chronic protein malnutrition. Suprrisingly, levels of these two amines were elevated in the brains of the protein malnourished rats and, therefore, changes in the levels of these neurochemicals cannot simply account for the increased seizure activity seen in rats reared on inadequate amounts of protein.

Developmental protein malnutrition: Influences on the central nervous system of the rat ☆

Our group has been carrying out interdisciplinary studies on the effects of prenatal and postnatal protein malnutrition on the developing rat brain. Anatomical, physiological, biochemical and behavioral approaches using the same animal model have revealed that protein malnutrition affects the brain at various levels, i.e., (1) anatomical, as revealed by Golgi findings of deranged dendritic trees on analysis of cortical and subcortical areas; (2) physiological, as revealed by delayed sleep pattern maturation, disturbances in seizure thresholds, slowing of sensory cortico-cortical and thalamocortical evoked potentials, and changed power in hippocampal theta activity; (3) biochemical, as revealed by marked increases in biogenic amines dating from birth, as well as modifications in tryptophan metabolism; and (4) behavioral, as revealed by various changes in responses to different kinds of aversive stimulation. Reversal studies have revealed that many changes are permanent and not amenable to nutritional rehabilitation even at birth, which is before the brain growth spurt in the rat. Our paradigm closely mimicks the human condition of low level, chronic protein undernutrition and thus reveals the underlying disturbances due to malnutrition. The dietary reversal studies are attempts at pin-pointing critical brain growth periods, beyond which recovery of functions is not possible.

The effects of p-chlorophenylalanine, a depletor of brain serotonin, on behavior: II. Retardation of passive avoidance learning

Abstract Rats were tested for passive avoidance learning following injection of p-chlorophenylalanine in Steroid Suspending Vehicle, or the vehicle only. In Exp. 1, the subjects were trained for 20 trials before testing; in Exp. 2, the subjects were trained to a criterion before testing. In both experiments, subjects given p-ClPhe learned more slowly than controls. This supported the hypothesis that p-ClPhe, a specific depletor of brain serotonin, reduces emotionality rather than increases learning ability.

Nucleus raphe dorsalis: A morphometric golgi study in rats of three age groups

Using Rapid Golgi and Nissl techniques, three major cell types: fusiform, multipolar and ovoid-shaped were identified in the nucleus raphe dorsalis of male rats at 30, 90, and 220 days of age. We have described the orientation and dendritic architecture of raphe cells as to type and the relationships of these cells to blood vessels and surrounding structures. For each cell type, the origin of the axon is characteristic. One hundred neurons per age group were measured at their maximal linear extent and the number of spines on the somal surface was counted. Dendritic number, linear extent, diameter and the number of spines along a 50 micron segment near the mid-point of dendritic length in an equal number of primary and secondary dendrites were quantified in each age group. The most striking age-related changes in the multipolar and ovoid-shaped cells were dendritic number, diameter and spine number as well as the number of perisomatic spines. The fusiform cells showed the least age-related changes. In general, the nucleus raphe dorsalis is organized as a reticular nucleus with neurons having few, straight and poorly ramified dendrites.

Overt and hidden forms of chronic malnutrition in the rat and their relevance to man.

We have examined the physiological weight changes seen in rat dams and their offspring as sequelae of either an overt or a hidden form of chronic protein malnutrition. In the overt model, which was produced by feeding dams a very low protein diet (6% casein) starting 5 weeks prior to conception and continued through lactation, the females showed significant weight losses at all ages compared to dams maintained on a normal diet (25% casein). This caused the malnourished 6% dams to have offspring that were categorized as small-for-date at birth in terms of their weight indices and peripheral metabolic profiles. Also, the inadequate milk production of these dams resulted in their pups displaying the almost total failure of growth (greater than 60% decreases in body weights) and peripheral imbalances characteristic of infantile marasmus by day 8 of lactation. Consequently, at all times examined the 6% dams and pups showed most of the typical responses seen in the more severe forms of in utero and lactational malnutrition of mankind. In contrast, the hidden form of malnutrition produced by feeding dams a somewhat higher protein diet (8% casein) throughout the same time periods caused no marked weight losses by these females during their pregnancy compared to the normal dams. Although the 8% pups had the same birth weight indices as the normal offspring, previous data from our group have indicated that the 8% progeny show many metabolic imbalances at birth which are indicators of severe gestational malnutrition in humans. Moreover, while the 8% dams displayed lactational insufficiencies as noted by their pups retarded postnatal growth, nursing of these offspring by 25% dams allowed them to maintain a normal lactational growth curve. However, not only was this cross-fostering unable to rehabilitate most of the prenatally determined biochemical alterations affecting the 8% pups but, additionally, this form of malnutrition will remain undetected if weight indices alone are used as assessors of normalcy. Thus, it appears that the 8% rats may serve as a useful model for the hidden forms of malnutrition in man.

Tryptophan availability: relation to elevated brain serotonin in developmentally protein-malnourished rats.

Abstract Developmental changes in tryptophan, serotonin, and 5-hydroxyindoleacetic acid in many brain regions were examined in normal and protein-malnourished rats from birth to age 30 days. The malnourished rats, whose dams received a diet low in protein starting 5 weeks prior to conception, showed significantly elevated brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid at most ages examined. Brain tryptophan concentrations for both groups of animals showed a positive correlation with their respective unbound plasma tryptophan concentrations. Although the malnourished animals showed lower total plasma tryptophan concentrations than the control group, the amount of free plasma tryptophan available for brain metabolism was significantly higher in the malnourished rats. This was due, in part, to a decrease in the molar ratio of bound tryptophan to albumin in the malnourished animals. In addition, those malnourished rats had lower albumin levels and higher concentrations of nonesterified fatty acids as compared to the normal animals, causing more tryptophan to be available as the free form in plasma. Overall, the present results demonstrate that rearing rats on a diet low in protein but adequate in all other respects significantly elevates brain tryptophan and amine concentrations, probably as a consequence of developmental alterations in plasma tryptophan availability.

Ontogeny of the levels of serotonin in various parts of the brain in severely protein malnourished rats.

We have previously reported that irreversible central and peripheral chemical changes and irreversible changes in spontaneous activity of single neurons in the frontal cortex are seen in adult rats who were born to mothers fed an 8% isocaloric casein diet 5 weeks before mating and during gestation, who were cross-fostered at birth by control (25% casein diet fed) dams and who showed a normal growth curve. Thus, in the rat, a normal growth curve does not necessarily mean a normal development. We now report similar irreversible, albeit larger, changes in small-for-gestational-age (SGA) rats born to mothers fed a 6% isocaloric diet, nursed by 6% casein fed dams and who showed an abnormal growth curve. The regional ontogeny of serotonin, tryptophan and 5-HIAA in the brain and plasma levels of tryptophan, protein, albumin and NEFA are reported. Thus, similar changes were observed in animals with a normal growth curve and in animals with a very markedly decreased growth curve resembling marasmus in humans.

Effects of maternal dietary protein restriction on growth of the brain and body in the rat.

We studied the growth of the brain and body in rats born of dams fed a low-protein (8% casein) diet ad lib beginning 5 weeks prior to mating and continuing throughout gestation and lactation. Control dams were fed an isocaloric 25% casein diet. Litters were culled at birth to 8 pups. At birth, brain weights and body weights of pups of protein restricted dams were similar to those of control pups. During the period of lactation, pups of restricted exhibited severely retarded body growth but only mildly retarded brain growth resulting in an elevated brain/body weight ratio. This relative macrocephaly was maximal at 10-20 days of age, declining completely by 35 days of age. The relative macrocephaly could not be accounted for by increased retention of water in the brain of the malnourished rats. Following weaning, pups were maintained ad lib on the diets fed their mothers. At adulthood brain/body weight ratios were normal in the protein restricted group.