Maravene Miller

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.

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.

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.

Effects of developmental protein malnutrition on tryptophan utilization in brain and peripheral tissues

Abstract Rats born of mothers fed a low protein diet (8% casein) compared to control rats on a normal diet (25% casein) started 5 weeks prior to mating showed significant decreases in the incorporation of parenterally injected 14C-tryptophan into protein in the brain and peripheral tissues. These effects were observed from day of birth to age 21 days, the oldest age assessed. Also, while different time-dependent patterns of uptake of labelled tryptophan were observed between the two diet groups at birth, this was not pronounced during the subsequent ages examined (Days 5–21). However, significant increases in uptake of 14C-tryptophan into brain and kidney of the 8% casein rats as compared to the 25% casein animals at ages 11 and 21 days may indicate the presence of a more active transport system for tryptophan in the malnourished animals. The significantly lower uptake, incorporation and percent incorporation of the tracer into liver of the 8% casein rats during ontogenetic development may indicate more rapid catabolism of liver proteins to maintain the concentrations of circulating amino acids. Overall, the data indicate that developmental protein malnutrition causes significant alterations in both brain and peripheral utilization of tryptophan.

Effects of growth hormone on brain biogenic amine levels

The effects of IP administered bovine growth hormone (GH) on regional brain serotonin, 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine levels in rats were examined. GH decreased the levels of both monoamines and 5-HIAA in the diencephalon and brainstem while not affecting telencephalic concentrations. In hypophysectomized rats, however GH produced significant elevations of monoamine and 5-HIAA levels in all brain regions. In normal rats the decreases in norepinephrine content produced by GH were correlated with a reduction in the stimulatory action of d-amphetamine on general activity levels. These results demonstrate that GH can affect brain biogenic amines and that these effects have behavioral consequences.

Tryptophan availability: The importance of prepartum and postpartum dietary protein on brain indoleamine metabolism in rats

Abstract Alterations in brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid concentrations occurred in rats as a consequence of dietary protein insufficiencies during prenatal and/or postnatal development. Rats born to dams fed normal (25% casein) or low (8% casein) protein diets starting 5 weeks prior to conception were cross-fostered at birth to dams of the opposite diet. At 21 days of age, their indoleamine metabolism was compared to that of unswitched rats of each diet group. All animals subjected to protein malnutrition during either the prenatal and/or postnatal developmental periods had similar elevations in brain tryptophan, amine, and metabolite concentrations compared to developmentally well-nourished pups. Although the three groups of animals which experienced protein deprivation displayed similar increases in brain indoleamine concentrations, this resulted from different modifications in peripheral metabolic processes controlling free plasma tryptophan availability. For both the prenatal/postnatal and postnatal only protein-deprived pups, lactational malnutrition produced increases in free tryptophan availability for brain metabolism by decreasing a major determinant of this process (the tryptophan to albumin ratio) as a result of the significant lowering of total tryptophan and albumin concentrations in plasma. Animals experiencing protein deficiencies during prepartum existence only, showed increases in free tryptophan availability due to permanent alterations in constituents (total tryptophan, albumin, and nonesterified fatty acid concentrations) affecting the competition between nonesterified fatty acids and tryptophan for binding on albumin that were not reversed by adequate postpartum nutrition. Overall, the present data demonstrate that the nutritional status with respect to the amount of dietary protein during prepartum and postpartum development has an important role in determining subsequent brain tryptophan metabolism.

Protein malnutrition in rats: response of brain amines and behavior to foot shock stress.

Abstract The changes in regional brain levels of serotonin, 5-hydroxyindoleacetic acid, and norepinephrine following an acute shock stress to the feet were examined in adult rats which had been fed a protein-poor diet or a normal diet during the developmental period and adulthood. Regional brain levels of the amines and 5-hydroxyindolacetic acid showed little change in normal rats following up to 90 min of shock, whereas depletions of up to 50% occurred in chronically protein malnourished rats. These neurochemical changes in the chronically malnourished rats were pronounced in the diencephalon and in the midbrain-pons-medulla brain regions. Normal rats which were switched in adulthood to the low protein diet showed minimal decreases in brain amine and 5-hydroxyindoleacetic acid levels following foot shock. This suggests that the effects observed in the chronically malnourished rats reflect a developmental interaction and were not due to the diet being administered at the time of testing. Behavioral measures of reactivity to foot shock, however, did not reveal marked diet related effects. Possible reasons for the differences in the behavioral vs. central neurochemical responses to foot shock are discussed. The results regarding the amine and 5-hydroxyindoleacetic acid levels demonstrate that the brains of chronically protein malnourished rats do not compensate to the same extent as the brains of normal rats (or normal rats switched to a low protein diet at adulthood) for the effects of an acute stress.

Phenylalanine utilization in brain and peripheral tissues during development in normal and protein malnourished rats

Rats born of mothers fed a low protein diet (8% casein) compared to control rats on a normal diet (25% casein) started 5 weeks prior to mating showed significant increases in uptake, incorporation and percent incorporation into protein of 14C-phynylalanine into brain and peripheral tissues. These effects were most pronounced on the day of birth. Also, different patterns of uptake of radioactive phenylalanine were observed between the two diet groups at birth. At ages 5, 11 and 21 days the 8% casein rats showed significant increases only in uptake and incorporation of 14C-phenylalanine into brain tissues as compared to the 25% casein animals with the percent incorporation of total radioactivity into brain protein being the same for both diet groups. For the most part, there were no significant changes in uptake and incorporation of radioactivity in peripheral tissues for the two diet groups on these post-birth days. Overall, the data indicate the possible presence of a brain specific effect whereby preferential utilization of an essential amino acid (phenylalanine) by the central nervous system occurs when rats are fed a low protein diet.