Joanne M. Bell

Effects of α-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase, on nucleic acids and proteins in developing rat brain: critical perinatal periods for regional selectivity

Ornithine decarboxylase and its metabolic products, the polyamines, are known to coordinate macromolecule synthesis in developing neural tissues; consequently, inhibition of this enzyme by alpha-difluoromethylornithine interferes with cellular replication and differentiation. We examined the regional selectivity of the effect of alpha-difluoromethylornithine administered either postnatally (days 1-19) or during gestation (days 15-17), in order to determine whether specific phases of maturation are particularly sensitive to polyamine depletion. In the cerebellum, which undergoes major phases of replication and differentiation after birth, postnatal alpha-difluoromethylornithine administration caused a profound and progressive deficit in tissue weight gain as well as in DNA, RNA and protein content. Although regions which develop earlier (cerebral cortex, midbrain + brain stem) also showed adverse effects of postnatal alpha-difluoromethylornithine, the deficits were of much smaller magnitude and were comparable to the effect of the drug on general body growth. Despite these regional differences, inhibition of DNA synthesis ([3H]thymidine incorporation) was similar in cerebellum and in midbrain + brain stem, indicating that the direct impact of alpha-difluoromethylornithine-induced polyamine depletion is exerted in both; DNA synthesis in cerebral cortex was spared relative to the other two regions. These data suggested that the impact of alpha-difluoromethylornithine on development depends, in part, upon the relative degree of maturation of each brain region at the time of drug exposure. In confirmation of this hypothesis, prenatal alpha-difluoromethylornithine given on gestational days 15-17 resulted in loss of the specificity toward cerebellar development and enhancement of effects on cerebral cortex, the region which had displayed the least sensitivity to postnatal alpha-difluoromethylornithine.

Biochemical Determinants of Growth Sparing during Neonatal Nutritional Deprivation or Enhancement: Ornithine Decarboxylase, Polyamines, and Macromolecules in Brain Regions and Heart

ABSTRACT. In order to elucidate the biochemical mechanisms operating to protect the brain from growth retardation in response to nutritional deprivation, comparisons were made of markers of cellular development in brain regions (cerebellum, cerebral cortex, midbrain + brainstem) and in a tissue which is not spared (heart). Nutritional status of neonatal rats was manipulated by increasing or decreasing the litter size beginning at birth, and development of DNA, RNA, and proteins followed throughout the neonatal period. In addition, we assessed the activity and levels of ornithine decarboxylase and its metabolic products, the polyamines, which are known to coordinate macromolecule synthesis in immature tissue and to provide an early index of perturbed development. Cardiac ornithine decarboxylase and polyamines were altered within 48 h of initiating the changes in litter size, and the direction and magnitude of these biochemical effects were predictive of subsequent impairment or enhancement of organ growth and of cellular development. All three brain regions were buffered from growth alterations relative to the heart, but the cerebellum, which undergoes major phases of cell replication later than the other two regions, was somewhat less protected. The spared brain regions also showed evidence of compensatory hypertrophy in nutritional deprivation (increased protein/DNA ratio) which accounts for maintenance of growth in the presence of reduced cell numbers. Thus, brain growth sparing involves specific cellular responses which are dependent on the maturational profile of each brain region.

Undernutrition and overnutrition in the neonatal rat: long-term effects on noradrenergic pathways in brain regions.

ABSTRACT: To determine whether neonatal nutrition influences development of CNS noradrenergic systems, litter sizes were manipulated at birth to produce undernutrition (16-17 pups/litter) or overnutrition (five to six pups) and compared to rats reared in normal litter sizes (10-11 pups). Studies were conducted throughout the preweaning period in which nutrition was manipulated, as well as during postweaning nutritional rehabilitation. Sparing of brain growth occurred, evidenced by much smaller changes in brain region wt than in body wt. Similarly, neonatal malnutrition produced major deficits in norepinephrine levels in peripheral sympathetic pathways, but levels in the brain remained within normal limits. Development of [3H]norepinephrine synaptosomal uptake, a biochemical index for presynaptic terminals, was unimpaired by malnutrition; indeed, higher uptake values were seen than in the control population. Nevertheless, norepinephrine turnover was severely attenuated during nutritional restriction and the effect persisted into adulthood; the deficit was greater in the cerebral cortex than in the cerebellum, despite the fact that cerebellar growth showed less sparing. Development of binding capabilities of noradrenergic receptors, particularly the α2- and β-subtypes, were also adversely affected in cerebral cortex, again suggestive of a deleterious effect on synaptic function. Animals exposed to neonatal overnutrition showed only slight effects on brain region wt or norepinephrine levels, but did display some suppression of [3H]norepinephrine synaptosomal uptake and enhancement of norepinephrine turnover; changes in receptor binding capabilities in the overnourished animals were attributable to the small alterations in brain region wt. These data indicate that neonatal nutrition alters presynaptic and postsynaptic markers of noradrenergic function that remain abnormal even when nutritional rehabilitation occurs.

Nutritional Influences on Adrenal Chromaffin Cell Development: Comparison with Central Neurons

ABSTRACT: Neurotransmitter systems in the developing brain are generally protected from growth retardation associated with nutritional deprivation. To investigate if such protective mechanisms extend to similar tissues in the peripheral sympathetic system, maturation of the chromaffin cells of the adrenal medulla and development of their centrally derived splanchnic innervation were evaluated in rats whose nutritional status had been altered during the neonatal period by increasing (16–17 pups/litter) or decreasing (five to six pups/litter) the litter size from the standard (11–12 pups/litter). Ontogeny of adrenal catecholamine stores and activities of catecholamine-biosynthetic enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase were monitored, along with activity of choline acetyltransferase, a marker enzyme for the preganglionic neurons innervating the chromaffin cells. Neonatal nutritional deprivation slowed body weight gain and retarded development of the chromaffin cells, as evidenced by subnormal catecholamine stores, tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities. The effects persisted despite the complete recovery of body weights postweaning. The developmental alterations were not caused by overcrowding stress, as plasma corticosterone levels were not elevated in the large litter group. Neonatal nutritional enrichment promoted body weight gain but failed to enhance development of adrenal catecholamines; tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities were elevated only in the preweaning period. In contrast to effects on the chromaffin cells, altered neonatal nutritional status had only minor, transient effects on the development of the centrally derived cholinergic innervation of the adrenal and produced only small changes (<10%) in brain tyrosine hydroxylase activity. These results suggest that, unlike central transmitter systems, maturation of chromaffin cells is adversely affected by neonatal nutritional deprivation; ontogenetic gains may already be close to optimum at normal nutritional status.

Impaired morphological development of the dorsal cochlear nucleus in hamsters treated postnatally with α -difluoromethylornithine

The dorsal cochlear nucleus is a highly organized nucleus in the auditory system in which the ramifications of depletion of specific cell types during development can be studied. Granule cells, small interneurons that are located in all layers of the DCN in the adult hamster, proliferate postnatally and are, therefore, potentially vulnerable to anti-mitotic agents that are administered after birth. The present experiments describe the effects of alpha-difluoromethylornithine, a drug that inhibits proliferation of cerebellar granule cells, on the granule cells in the dorsal cochlear nucleus. As in the cerebellum, the density of granule cells in the dorsal cochlear nucleus is reduced after alpha-difluoromethylornithine treatment. In hamsters treated with alpha-difluoromethylornithine (200 or 500 mg/kg subcutaneously (s.c.), twice daily on postnatal days 4-14), the numerical density of granule cells was reduced in the superficial dorsal cochlear nucleus at 15 days; by 40 days this effect was also apparent in the deep layer, suggesting that cells located superficially that would have migrated into the deep dorsal cochlear nucleus had either failed to develop or did not arrive at their final location. This evidence suggests that the cells normally migrate down from the superficial proliferative zone into the deeper layers. In the drug-treated animals, a layer of mixed granule cells and fusiform cells was thinner than in controls probably due to the reduction in interspersed granule cells since the number of fusiform cells was unaffected. There was also a dose-dependent effect on cell growth; fusiform cells were affected at both doses, while giant cells were only affected at the highest dose. Granule cells form a major input to the fusiform cells and their depletion may account for some of the effects on fusiform cell growth. There could also be additional direct actions of alpha-difluoromethylornithine on this population.

Perinatal dietary supplementation with a soy lecithin preparation: Effects on development of central catecholaminergic neurotransmitter systems☆

Previous work has shown that exposure of developing rats to soy lecithin preparations (SLP) influences macromolecular constituents of immature brain cells and causes abnormal behavioral patterns. To determine if synaptic mechanisms are adversely affected by SLP, we examined the developmental characteristics of noradrenergic and dopaminergic pathways in discrete brain regions. Although transmitter levels were unaffected, the utilization rates of both catecholamines were profoundly disturbed in an age-dependent, regionally-selective manner. Utilization tended to be subnormal in the preweanling stage, but demonstrated a postweaning elevation in cerebellum and midbrain + brainstem. Enhanced utilization persisted in the latter region only, and cerebral cortex actually showed a lowered utilization rate in adulthood (60 days of age). Transmitter uptake capabilities were also affected by developmental exposure to SLP, as was tyrosine hydroxylase activity. The patterns of effects on these two variables indicated that the altered transmitter utilization rate probably reflected a change in impulse activity in the affected neuron populations, with promotion of activity in the midbrain + brainstem and reduced activity in the cerebral cortex. These data indicate that dietary supplementation with SLP throughout perinatal development alters synaptic characteristics in a manner consistent with disturbances in neural function.

Coordination of cell development by the ornithine decarboxylase (ODC)/polyamine pathway as an underlying mechanism in developmental neurotoxic events

Publisher Summary This chapter discusses the ornithine decarboxylase (ODC)/polyamine pathway as a potential underlying biochemical mechanism of functional teratogenesis. Through study of its direct inhibition, this pathway is now known to regulate nucleic acid and protein synthesis in developing tissues, and thus may act as the common target for neurobehavioral, synaptogenic, and morphological as well as cellular deficits in response to a diverse spectrum of developmental insults. The appearance of alterations in behavioral function caused by a teratogen must have, as its basis, a primary action on cellular mechanisms. Teratological changes confined to behavioral (or biochemical) function can be as damaging as obvious gross skeletal or morphological abnormalities: subtle deficits may go unnoticed at birth and lead to a cascade of alterations in neurobehavioral functioning as the animal matures that make it progressively difficult to determine underlying primary alterations induced by the teratogen. The selectivity of behavioral effects of α-difluoromethylornithine (DFMO) exposure for these periods paralleled the regional selectivity generated by the biochemical and anatomical timetable of maturation of each brain region.

Perinatal dietary exposure to soy lecithin: Altered sensitivity to central cholinergic stimulation☆

The effects of perinatal exposure to soy lecithin preparation (SLP) on the development of cholinergic responses in the rat brain were examined by assessing the ability of intracisternally administered carbachol to stimulate 33Pi incorporation into phospholipids in vivo, an effect of carbachol mediated by muscarinic cholinergic receptors. Maternal intake of SLP produced a suppression of the cholinergic response in the offspring, an effect which was specific in that basal (unstimulated) incorporation rates were not reduced (in fact, they eventually became elevated), nor was the response to another neurotransmitter (dopamine) compromised. The effect occurred early in the preweanling stage, a period in which SLP exposure also enhances development of cholinergic nerve terminals. These results suggest that SLP exposure has a major effect on cholinergic synaptic development and reactivity, followed by secondary changes in other neurotransmitter pathways and by more generalized effects on basal membrane phospholipid turnover.

Neonatal Nutritional Deprivation or Enhancement: The Cardiac-Sympathetic Axis and its Role in Cardiac Growth and Stress Responses

ABSTRACT: To determine the mechanisms by which neuronal input influences cardiac growth during altered neonatal nutritional status, rats were reared in small, standard, or large litter sizes and the adrenergically mediated stimulation of cardiac ornithine decarboxylase was determined; ornithine decarboxylase provides a mechanistic link connecting adrenergic input to cardiac growth. Nutritionally deprived pups showed impaired development of sympathetic reflex stimulation as shown by the attenuation of the cardiac ornithine decarboxylase response to hydralazine-induced hypotension throughout the preweanling period. The subnormal reactivity to hydralazine reflected a defect in neurotransmission, as a full response was obtained with direct β-receptor stimulation (isoproterenol). Nevertheless, cardiac hypertrophy in response to repeated isoproterenol administration was markedly suppressed in nutritionally deprived animals, suggesting that the β-receptor/ornithine decarboxylase pathway had become uncoupled from growth. Because maturation of neural connections to peripheral tissues causes a loss of hypoxia tolerance, nutritional status also influenced the ability of neonatal rats to survive hypoxia. These data indicate that cardiac growth suppression or enhancement caused by nutritional manipulations may be mediated, in part, through alterations in the development of neuronal input to the tissue, and that similar factors influence survival during hypoxic stress.

Postnatal nutritional status influences development of cardiac adrenergic receptor binding sites

Nutritional status in neonatal rats was manipulated by altering the litter size to produce overnourished (5-6 pups/litter) and undernourished animals (16-17 pups/litter) for comparison with standard nutritive status (10-11 pups/litter). Nutritionally deprived pups showed impaired body and cardiac growth and a slowing of development of cardiac membrane binding sites for alpha 1- and beta-receptor ligands. Overnourishment enhanced growth and receptor development. In all cases, restitution of receptor binding characteristics to normal occurred at the beginning of nutritional rehabilitation (i.e., at weaning), well before the return of body and organ weights. These results thus suggest that the ontogenetic pattern of receptor binding sites is dependent upon nutritional intake rather than on weight gains per se. Receptor deficits caused by neonatal malnutrition probably contribute to reduced responsiveness to adrenergic stimulation.