William L. Whitmore

Effects of prenatal nicotine exposure on neuronal development: Selective actions on central and peripheral catecholaminergic pathways

The effects of prenatal nicotine exposure on development of catecholaminergic pathways were examined through measurements of transmitter turnover and levels in both the central and peripheral nervous system. Administration of nicotine (3 mg/kg SC, twice daily) to pregnant rats on gestational days 3 through 20 resulted in growth retardation which did not spare the brain and which did not resolve until after weaning. Nicotine exposure produced an elevation in transmitter turnover in central noradrenergic pathways with a regional selectivity reflecting the timetable of cellular development: the most profound effects were seen in late-developing regions (cerebellum), intermediate effects were found in earlier-developing areas (cerebral cortex) and the least effect was obtained where maturation occurs earliest (midbrain + brainstem). Dopaminergic pathways were much less vulnerable than was the noradrenergic system. Effects on the peripheral sympathetic nervous system also were targeted toward specific neuronal populations: renal, cardiac and adrenal pathways were activated by prenatal nicotine exposure, whereas sympathetic innervation of the lung showed reduced activity. All the peripheral effects appeared only after the second postnatal week. These results indicate that prenatal nicotine exposure produces profound alterations in transmitter disposition which are targeted toward specific neuronal populations and which may depend upon generalized effects on cellular development of specific brain regions. Because altered peripheral nerve activity was seen only after the onset of central control of sympathetic tone, actions on central regulation may play a role in the relatively more profound effects in the autonomic nervous system.

Development of α‐Noradrenergic and Dopaminergic Receptor Systems Depends on Maturation of Their Presynaptic Nerve Terminals in the Rat Brain

Abstract: To study the relationship between ontogeny of rat brain catecholamine nerve terminals and the receptor systems for the catecholamine transmitters, the developmental patterns of synaptosomal uptake mechanisms were compared with those of α‐noradrenergic and dopaminergic receptor‐mediated effects. Uptakes of [3H]dopamine or [3H]norepinephrine into dopaminergic and noradrenergic nerve terminals were low during the 1st week postpartum and increased rapidly during the 2nd week. A similar pattern was obtained for ontogeny of dopaminergic receptor binding sites, as evaluated by [3H]domperidone binding. Stimulation of incorporation of 33Pi into brain phospholipids (elicited by intracisternal injection of dopamine), which is mediated by dopaminergic receptors, was shown to be highly correlated with the maturation of both receptor binding sites and presynaptic nerve terminal uptake. A similar result was seen with norepinephrine, in that the synaptosomal uptake mechanism and norepinephrine‐induced stimulation of 33Pi incorporation into phospholipids, an α‐noradrenergic effect, developed in a parallel fashion. To test the hypothesis that development of the receptor systems is linked to nerve terminal ontogeny, presynaptic nerve terminals were destroyed in neonates by intracisternal administration of 6‐hydroxydopamine. The lesions prevented the maturational increase in the number of dopamine receptor binding sites and produced a defect in development of the dopamine‐ and norepinephrine‐induced stimulation of 33Pi incorporation. The results suggest that ontogeny of both dopaminergic and α‐noradrenergic receptor systems depend upon development of the presynaptic nerve terminals containing the transmitters.

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.

Effects of material methadone administration on ornithine decarboxylase in brain and heart of the offspring: Relationships of enzyme activity to dose and to growth impairment in the rat

Abstract Administration of 5 mg/kg of methadone daily to pregnant and nursing rats produced substantial retardation of body, brain and heart growth in the offspring; alterations in biochemical development also were present in the methadone-exposed pups, as evidenced by delays in the maturational declines of brain and heart ornithine decarboxylase (ODC) activity. Lowering the dose of methadone to levels which did not affect growth or brain ODC, still resulted in pro-found abnormalities in the developmental pattern of heart ODC. These studies indicate that downward adjustment of maternal methadone dosage to a point where birthweights and body and organ growth rates are normal, does not eliminate all of the biochemical alterations associated with the perinatal opiate syndrome.

Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat

Abstract Administration of methodone to pregnant and nursing rats or direct treatment of developing pups with methadone resulted in deficits of development of body weight, brain weight and synaptosomal uptake of 5-hydroxytryptamine, dopamine and norepinephrine. Defective synaptic development was most apparent immediately after birth in pups whose mothers received methadone; while some recovery occurred by 3 weeks of age, there was a subsequent deficit in synaptosomal uptake post-weaning. A similar pattern also was seen in development of synaptic vesicle amine uptake. Direct treatment of neonates with methadone also caused reductions in development of synaptosomal and vesicular uptake mechanisms, but the patterns of alteration were different from those in the maternal treatment group. These studies show that the adverse effect of opiates on general brain growth are accompanied by a slowing of synaptic development of biogenic amine systems.

Rat brain synaptic vesicles: uptake specificities of [3H]norepinephrine and [3H]serotonin in preparations from whole brain and brain regions.

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [3H]norepinephrine and [3H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The Km for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6‐hydroxydopamine, but not of 5,6‐dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat‐ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6‐hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP‐Mg2+ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with Kis similar to those for inhibition of norepinephrine uptake. Whereas 5,6‐dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6‐hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.

Uptake of (−) 3H-norepinephrine by storage vesicles prepared from whole rat brain: Properties of the uptake system and its inhibition by drugs

Abstract Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of (−) 3H-norepinephrine was determined in vitro . Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg2+, and was inhibited in vitro by reserpine. Uptake was linear for 5 min at 30°, but not at 37°. The uptake was saturable and displayed a single Km value of 4 × 10−7 M. Other phenylamines and indoleamines displayed competitive inhibition of norepinephrine uptake; the affinities followed the rank order: reserpine>harmaline>serotonin>epinephrine> dopamine>norepinephrine>metaraminol. Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated with 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotonergic, vesicles. This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake in vitro after administration of reserpine in vivo .

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.

Adrenergic control of DNA synthesis in developing rat brain regions: Effects of intracisternal administration of isoproterenol☆

Catecholamines are hypothesized to control cellular development in the central nervous system. In the current study, isoproterenol administered intracisternally to neonatal rats was found to inhibit DNA synthesis [( 3H]thymidine incorporation) in brain regions. The regional selectivity of effect corresponded to the sequence of cellular maturation, namely midbrain + brainstem greater than cerebral cortex greater than cerebellum, suggesting that the specific linkage of beta-adrenergic receptors to cessation of cell replication occurs during a specific maturational stage.

Effects of neonatal hypoxia on brain development in the rat: immediate and long-term biochemical alterations in discrete regions

To evaluate the sensitivity of immature brain tissue to hypoxic insult, neonatal rats were exposed to 7% O2 for 2 h at critical stages of development (1, 8, 15, 23 days of postnatal age); the immediate and long-term impact of hypoxia was then assessed in cerebellum, cerebral cortex and midbrain through measurement of ornithine decarboxylase (ODC) activity, a biochemical determinant of cellular injury and subsequent maturation, and through measurements of protein synthesis, growth and synaptosomal uptake of norepinephrine (an index of noradrenergic synaptogenesis). In one-day-old rats, hypoxia caused stimulation of protein synthesis and short-term suppression of ODC activity which persisted for several hours after termination of low O2 exposure; over the ensuing days, there was a prolonged elevation of enzyme activity and a subsequent, regionally selective increase in synaptosomal uptake of norepinephrine without changes in brain growth. In contrast, hypoxia in 8-day-old rats produced signs of metabolic injury, with a short-term elevation of ODC throughout the brain and reduced protein synthetic rates, eventual shortfalls in brain regional growth and no net increase in synaptosomal uptake. The effects of hypoxia on brain regional growth in 8-day-old animals appeared to represent an age-specific effect, as low as O2 conditions in older animals did not affect growth (animals made hypoxic at 15 or 23 days), but did produce an eventual reduction in synaptosomal uptake (hypoxia at 15 days). Differences between one-day-old and 8-day-old rats were also apparent in cerebral responses simply to a 2-h separation from the dam under normoxic conditions. These results support the view that cellular development and synaptogenesis are compromised when neonatal brain tissue is exposed to hypoxic conditions, and that there are critical periods of sensitivity in which processes undergoing rapid maturational change are particularly vulnerable.