Cerebellar DNA Research Articles

Chronic oxytocin treatment during late gestation and lactation impairs development of rat offspring

The nonapeptide oxytocin (OT) plays an important role in timing and course of parturition, and in milk ejection during lactation. Exogenously enhanced OT levels were reported to impair body development of rat offspring at birth and during postnatal stages. In the present study, this effect was further investigated by giving pregnant rats of postcoital day 17 a SC implant that delivers small amounts of OT for 2 months (approximately threefold enhancement of OT levels), and by introducing a crossfostering protocol for the offspring. A slightly reduced body weight of 5 to 7% was again observed in pups born to OT-implanted dams. When reared postnatally by OT-treated mothers, pups lost weight gain (− 7 to −10%). During the weaning period, however, body size caught up with that of control animals. When nursed by an untreated mother, this recovery took place before that period. Growth of control offspring was also hampered when placed with OT-treated mothers, but these pups failed to recover from low body weights which lasted up to at least 70 days of age (−7%). Daily urine production of the pups born of and reared by the OT-treated mothers was reduced at 1 month of age, but this effect was only transient and had disappeared at 70 days of age. Notwithstanding, the recovery of body growth, brain sizes, and cerebellar DNA, i.e., cell content was reduced in the pups born and reared by OT-treated mothers, indicative of a lasting effect on brain development. Such late brain changes were not observed in pups of OT-treated mothers nursed by untreated mothers, and in control pups nursed by both types of mothers. In conclusion, one might say that physiologically enhanced levels of maternal OT can induce subtle alterations in brain development that are potentially indicative for lasting physiological and behavioral changes in the offspring.

Effect of Protein Malnutrition on Glycoprotein, Protein and Lipid Synthesis in the Rat Cerebellum during the Period of Brain Growth Spurt

Female Wistar rats were fed a normal-protein diet (25% casein) or a low-protein diet (8% casein) during pregnancy and lactation. The two diets were isocaloric and contained appropriate amounts of mineral salts and vitamins. Pups from dams submitted to the low-protein diet had a lower body weight than normally fed controls as early as on the day of birth, but a difference in cerebellar weight between the two groups was observed only on the 15th postnatal day. Malnutrition had no effect on cerebellar protein concentration, which increased with age in both groups. The cerebellar DNA concentration was higher at 7 and 15 days of age in normally fed rats than in malnourished rats, whereas at 21 days of age it was higher in the malnourished animals. [U-14C]Leucine and [2-3H]mannose incorporation into proteins and lipid synthesis from acetyl coenzyme A (CoA) derived from [U-14C]leucine markedly decreased with age in the cerebellum of rats fed both diets. [2-3H]Mannose incorporation into cerebellar glycoproteins was greater in malnourished rats during the period of brain growth spurt than in normally fed rats at all ages studied. Prenatal and postnatal protein malnutrition had no effect on [U-14C]leucine incorporation into cerebellar proteins or on cerebellar lipid synthesis from acetyl-CoA derived from [U-14C]leucine during the period of brain growth spurt.

Body and brain growth following continuous perinatal administration of arginine- and lysine-vasopressin to the homozygous brattleboro rat

Using a recently developed controlled-drug-delivery implantation technique, arginine-vasopressin (AVP) or lysine-vasopressin (LVP) was administered to homozygous (HOM) Brattleboro rats throughout pregnancy in order to study the influence of compensation for the deficiency of AVP on body and brain development in their HOM offspring. This mutant is retarded in both body and brain growth from the neonatal period onwards. In one subgroup the LVP-treatment was continued postnatally by means of subcutaneous implantation in the pups. AVP treatment had no growth-stimulating effect either on pup body weight at day one or on postnatal body growth, nor did it affect noticeably the day of eye opening, or a number of brain parameters measured at one month of age. LVP treatment, in contrast, resulted in higher body weights at birth, which could be maintained postnatally if the pups were reared with a Wistar foster-mother. At one month of age body as well as brain weights were still larger in the treated pups. Although cerebellar weight was larger than in untreated Brattleboro pups in this group, cerebellar DNA content or gross morphology, known to be impaired in HOM rats, were not changed. LVP treatment of the pups, as well as maternal AVP-treatment beginning on day 15 of pregnancy, had inhibiting rather than growth-stimulating effects, high-lighting the different effects created by these two peptides at different stages of development.

Cerebellar deoxyribonucleases and DNA in the androgenized female rat.

Postnatal developmental patterns of activity for acid and alkaline deoxyribonuclease (DNase) were determined in the cerebellum of the androgenized female rat. Littermates were injected s.c. with either 1.25 mg testosterone propionate (TP) or vehicle 48 hours after birth. The enzymes and DNA content were measured in the cerebellum at 3, 6, 10, 15, 18, 22, 35, and 60 days of age. Neonatally administered TP affects neither the activities nor the developmental patterns of the DNases through age 60 days. However, the DNA content was significantly lower in the cerebellum of the TP-treated rats at ages 10 and 15 days. These data demonstrate that the TP-induced alterations in cerebellar DNA content are not mediated by acid or alkaline DNase.

Cerebellar DNA polymerase activity in hypothyroid and malnourished rats.

Neonatal hypothyroidism in rats resulted in a decrease in the rate of cerebellar cell division and a delay in the time when cell division ceased. Thus, by 35 days total cell number, as measured by DNA content, was normal. The prolongation of cell division was reflected by an elevation of DNA polymerase activity that persisted beyond the time enzyme activity dropped to adult levels in either normal animals or animals equally retarded in their growth by malnutrition. This increase in polymerase activity was associated with a sevenfold increase in tritiated thymidine incorporation into DNA at 21 days of age. These findings support the view that the effects of hypothyroidism on the timing of cell proliferation in the cerebellum are not mediated solely by the well-known decrease in food intake that occurs in hypothyroidism and that might be expected to result in some degree of malnutrition.

Effect of hyperphenylalaninemia induced during suckling on brain DNA metabolism in rat pups.

We studied DNA metabolism (synthesis and degradation) in brain to investigate the effect of hyperphenylalaninemia induced in rats by treatment with PCPA or alpha MPA plus PHE during suckling (4th-20th days of postnatal age) on cell proliferation and naturally occurring cell death. The incorporation of 14C in DNA as percent of total radioactivity in the tissue, 30 min after administration of [14C]thymidine served as a measure of DNA synthesis in vivo, and the amount of radioactivity recovered in DNA as percent of total 14C in the tissues of 21 day old rats, injected with [14C]thymidine on 2nd day after birth, indicated the turnover (degradation) of DNA. The results showed that the DNA content of cerebellum as well as cerebrum was reduced by treatment with PCPA plus PHE, while treatment with alpha MPA plus PHE had no effect on DNA content in cerebellum but reduced the levels in cerebrum. Treatment with PCPA or alpha MPA plus PHE reduced the synthesis of DNA in cerebrum of 11 day old rats but not in 21 day old rats, and the treatments did not affect DNA synthesis in cerebellum of either 11 or 21 day old rats. The turnover (degradation) of DNA was increased in both cerebellum and cerebrum from rats treated with PCPA plus PHE but alpha MPA plus PHE treatment did not alter the DNA turnover either in cerebellum or in cerebrum. The activity of acid DNase was reduced in both cerebellum and cerebrum from 11 as well as 21 day old rats treated with PCPA plus PHE, but the enzyme activity was not altered in the tissues from rats of both ages treated with alpha MPA plus PHE. The data thus indicate that in rats treated with PCPA plus PHE the reduction in cerebral DNA levels occurs due to reduced synthesis and/or increased turnover (degradation) of DNA but that the reduction in cerebellar DNA may occur only as a result of increased turnover (degradation), and that in rats treated with alpha MPA plus PHE the reduction in cerebral DNA must occur due to reduced synthesis. This suggests that treatment of rats with PCPA plus PHE during suckling inhibits cell proliferation and/or increases naturally occurring cell death in both cerebellum and cerebrum while treatment with alpha MPA plus PHE inhibits only cell proliferation and in cerebrum alone.

Transmitter biochemistry and histochemistry of the hypoplastic cerebellum in mice after neonatal administration of cytosine arabinoside

Administration of cytosine arabinoside, a DNA polymeraerase inhibitor, to mice (50 mg/kg body weight) on postnatal days 2, 3 and 4 resulted in severe hypoplasia of the cerebellar cortex accompanied by a large decrease in the cerebellar DNA and RNA contents. On postnatal days 10, 20, 30 and 70, concentrations of the putative neurotransmitters, noradrenaline and serotonin, were significantly increased in the affected cerebellum. In contrast, γ-aminobutyric acid concentration was little changed, although a decrease in the content of-γ-aminobutyric acid per cerebellum was observed from postnatal day 20. No changes were found in the concentrations of these transmitter candidates nor in dopamine in the cerebral cortex or the brain stem. Histofluorescence observations revealed abnormally dense noradrenergic fibers in a poorly organized cerebellar cortex. The distribution of these fibers was characterized by a honeycomb pattern in the Purkinje cell and granular layers, and a dense plexus in the lower part of the thin molecular layer. However, the significance of neither the density of the noradrenergic fibers nor their abnormal distribution was apparent from the results of this study.

Cerebellar DNA and tissue water changes in the brain of diabetes insipidus brattleboro rats are already present at birth

Homozygous (HOM) Brattleboro rat newborns, congenitally lacking vasopressin, weighed less than heterozygous controls born from HOM females and reared as 9-pup litters. Whole brain and specific brain regions (cerebral cortex, medulla oblongata and colliculi but not the cerebellum) were also reduced in weight. Decreased tissue water content was observed in the regions affected in weight. In the cerebellum, on the other hand, a 20% lower DNA content was measured, which points to a prenatal origin for the impaired cerebellar neurogenesis which is known to exist postnatally.

Cytotoxic effects of methylnitrosourea on developing brain.

The effect of methylnitrosourea (MNU) on cerebellar and cerebral DNA, RNA, protein, lysosomal enzymes (acid DNase, RNase, phosphatase, and beta-glucuronidase), and 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activities was studied in rats from birth through 12 days of age. Subcutaneous injection of MNU in a dose of 0.625 mmol/kg caused a suppression of increase in weights and content of DNA, RNA, and protein of cerebellum, but no changes in those of the cerebrum or in body weight. Ratios of protein and RNA to DNA were substantially elevated by MNU in the cerebellum but not in the cerebrum. Acid DNase and acid RNase activities of MNU-treated rats were significantly elevated beyond the increase of these activities in controls in the cerebellum, but no change in these activities by MNU was observed in the cerebrum. A slight elevation in acid phosphatase activity was observed in the cerebellum but not in the cerebrum after MNU pretreatment. Beta-glucuronidase and 2',3'-CNPase activities were not changed in the cerebellum or in the cerebrum. These results suggest that in the developing brain, especially in the cerebellum at the mitotic stage, MNU caused cell damage and inhibited cell mitosis.

DNA concentrations in the human cerebellum. Computation from kinetics of deoxyribose extraction in hot acid.

DNA can be measured in mammalian tissues by extracting deoxyribose from unfixed, lyophilized tissue specimens with 0.5 N perchloric acid at 90 degrees C. Deoxyribose concentrations in the extract are determined photometrically by reaction with diphenylamine. Inevitably, some deoxyribose is destroyed during exposure to the hot acid. A computer program has been written which corrects photometric absorbance data for such loss of deoxyribose. When extrapolated to infinite duration of extraction, the corrected absorbances yield a measure of the DNA content of the specimen. This method was used to estimate DNA concentrations in human cerebellar cortex and white matter. The results are discussed in relation to stable carbon isotope ratios of human cerebellar DNA.

Effect of naloxone and D-Met 2-Pro 5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met 2-Pro 5-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of 3H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of 3H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met 2-Pro 5-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.

Increased cerebellar thymidine kinase and DNA during early postnatal development of diethylstilbestrol-treated rats

Thymidine kinase activity and DNA content were determined in the cerebellum after the administration of diethylstilbestrol (DES) to neonatal female rats. Rats were injected s.c. with either 30 μg DES or vehicle 48 h after birth. DNA and thymidine kinase were measured in the cerebellum at 3, 7, 10, 18, 21, 26, and 60 days of age. Thymidine kinase activity and DNA content in the DES-treated littermates were significantly increased between ages 3 to 21 days and ages 3 to 26 days, respectively. The data suggest diethylstilbestrol-induced relationships between thymidine kinase activity and DNA synthesis during the period of rapid cerebellar development.

Effect of prenatal phenytoin administration on postnatal development of the rat: a behavioral teratology study.

Adult pregnant Wistar rats were treated with phenytoin (100 Mg/kg) orally from day 7 to 19 of pregnancy, and a control group was pair-fed during the whole treatment period. Within 24 hours after parturition , the offspring were culled to six to eight per litter and reared by fostering or cross-fostering. The physical and behavioral development of the offspring was observe up to 90 days of age. There was a reduced survival of the offspring and a reduction in body weight which persisted to the end of the experiment, though both of these effects could be reduced by cross-fostering. Certain neurological defects were also seen in the prenatal phenytoin group. For example, there was a delay of up to 15 days in the development of the dynamic righting reflex, a decrease in ability of offspring to stay on a rotating rod, and a decrease in ability to walk along elevated parallel rods. There seemed also to be some loss of cliff avoidance. However, there was no change in the development of crawling and walking activities at 9-21 days of age, and no important changes were observed in a head dipping test or in a conditioned avoidance test at 26-34 days. There was a significant decrease in brain weight of the treated group at age 3 days which remained significantly lower than the controls even at 90 days, but no change in the brain/body weight ratio. There was no difference in cerebellar DNA content.

The effect of late prenatal and/or early postnatal zinc deficiency on the development and some biochemical aspects of the cerebellum and hippocampus in rats

In rats, late prenatal and/or early postnatal zinc deficiency results in behavioural anomalies in adult animals, but not in overt dysmorphogenesis of the central nervous system. Cerebellar and hippocampal development occurs mainly in the first three weeks postnatally and zinc accumulates specifically in the mossy fibres of the hippocampus during this period. In the present investigation, rat pups were suckled by dams fed a zinc-deficient (<0.5 mg/kg) diet either from day 19 of pregnancy or from parturition. Control animals were restricted-fed the same diet supplemented with 100 mg zinc/kg. Studies were performed on pups either on day 18 postpartum in the case of animals fed the experimental diets from parturition, or on day 20 for pups which received treatment from day 19 of gestation. Cerebellar and hippocampal weights were lower in pups suckling from zinc-deficient dams but zinc levels were not affected in either organ, although histological evidence suggested less zinc in the hippocampal mossy fibres. Incorporation of H-thymidine into cerebellar and hippocampal DNA was not affected by maternal zinc status, nor was the activity of the zinc metalloenzyme alkaline phosphatase. The activity of the myelin-marker enzyme 2′, 3′-cyclic nucleotide 3′-phosphohydrolase was substantially lower in both regions of the brain in zinc deprived pups, especially in the hippocampus. Activity of the zinc metalloenzyme L-glutamic acid dehydrogenase was also diminished in both tissues from 20-day-old pups and in the hippocampi of 18-day-old animals. The data suggest that cerebellar and hippocampal DNA synthesis is not seriously affected by late prenatal and/or early postnatal zinc depletion, but that the activities of two enzymes associated with neural function are. The possibility is raised that these defects may be associated with the behavioural changes observed in rats subjected to zinc impoverishment during the period of maximal cerebellar and the hippocampal development.

Nucleic acids and protein in brains and cerebella of rats transplacentally exposed to ethylnitrosourea

Female rats were injected (IP) with either 15, 60, 100, or 150 mg/kg N-ethyl-N-nitrosourea (ENU) on gestation day 18. The neurochemical development of the whole brain and the cerebellum was studied in 21- and 60-day old offspring; pups from mothers subjected to 150 mg/kg were examined only at 21 days. Brain weights were significantly reduced in all ENU-exposed groups at 21 days, and the 60 mg/kg group at 60 days. Whole brain DNA and protein concentrations of most ENU groups were elevated at 21 days and RNA content and RNA/DNA ratios were subnormal. Deficits in whole brain DNA content were observed in all ENU groups on day 60. Cerebellar weight was reduced in the 60 and 150 mg/kg groups on day 21 and in the 100 mg/Kg group on day 60. At 21 days, reductions in cerebellar DNA and RNA concentrations and contents were often recorded in several ENU groups. Cerebellar DNA concentration and content of the 60 and 100 mg/kg groups remained subnormal at 60 days of age. In general, a dose-response effect was evident in the number and magnitude of neurochemical alterations produced by ENU. These data suggest that transplacental exposure to ENU, a potent neurotropic carcinogen, affects the biochemical maturation of the brain.

Effects of neonatal estrogenization on thymidine kinase activity and DNA content in the cerebellum of the rat.

Cerebellar thymidine kinase activity and DNA content were determined following the administration of 17 beta-estradiol benzoate (EB) to neonatal rats during the sexually critical period of brain development. Male and female littermates were injected s.c. 24 and 72 h after birth with either 500 microgram EB or vehicle. At 3, 4, 6, 7, 8, 10, 12 and 15 days of age cerebellar thymidine kinase activity and DNA content were measured. A sexual dimorphism in thymidine kinase activity occurred in control rats between 6 and 15 days of age, i.e., enzyme activity was significantly higher in the cerebellum of the female rats. Thymidine kinase activity in both male and female EB-treated rats was (1) significantly greater than that of their littermate controls at 3 and 4 days of age, (2) the same as that for their controls at 6 days of age, (3) significantly lower than control levels between 7 and 12 days of age and, (4) the same as their controls by day 15. Alterations in cerebellar DNA content were similar to those described for the enzyme. These data demonstrate the occurrence of age-dependent estrogen-induced relationship(s) between thymidine kinase activity and DNA synthesis in the cerebellum of the rat during early postnatal development.

The effects of sex and neonatal androgenization on thymidine kinase activity and DNA content in the cerebellum of the rat

The effects of neonatally administered testosterone propionate (TP) were determined on the activity of thymidine kinase and on the content of DNA in the cerebellum of the rat during early postnatal development. Forty-eight hiours after birth, female and male pups were injected s.c. with either 1.5 mg TP or an equivalent volume of vehicle. Cerebellar thymidine kinase activity and DNA content were measured at 3, 4, 6, 7, 8, 10, 12, 15, 18 and 21 days of age. A sexual dimorphism in thymidine kinase activity was found for control rats between 6 and 18 days of age, i.e. the activity of the enzyme was significantly higher in the cerebellum of the female controls. The activity of thymidine kinase in the cerebellum of TP-treated male rats was significantly greater than that of the male controls between 4 and 21 days of age. The activity of thymidine kinase in the cerebellum of TP-treated female pups was significantly lower than that of the female controls between 6 and 12 days of age. Enzyme activity at age 15 days was the same for TP-treated and control female rats. However, thymidine kinase activity in the cerebellum at TP-treated female ratswas significantly increased above that of the controls at 18 and 21 days of age. Thus, the response of the enzyme following the injection of TP 48 h after birth was sex-dependent between 4 and 15 days of age. Alterations in cerebellar DNA content in the TP-treated male and female rats closely paralleled the changes described for thymidine kinase. These data suggest sex-dependent transactions between the animal and the injection of TP as well as androgen-dependent relationships between thymidine kinase and DNA synthesis in the cerebellum. Significant changes in the Michaelis-Menten constant with increasing age also suggest that the developing cerebellum contains more than one form of the enzyme.

Graft-versus-host disease impairs cerebellar growth.

Graft-versus-host disease impairs cerebellar growth.

Perinatal methadone exposure and brain development: a biochemical study.

Abstract— The neurochemical effect of maternally administered methadone (5 mg/kg, DL‐methadone‐HCI) on the brain (including the olfactory bulbs, cerebellum, and brain stem) and cerebellum of offspring exposed during gestation and/or lactation was studied in 10‐, 21‐, and 60‐day old rats. Brain weights were significantly reduced in all methadone‐exposed groups at 10 days of age, while only those rats subjected to methadone during gestation or lactation had deficits in brain weights at day 21; no differences were found at 60 days. Brain DNA content was significantly reduced in all opiate‐exposed offspring at every age examined, but RNA/DNA and protein/DNA ratios were only consistently increased in rats of the gestation group. Cerebellar weight was reduced at 10 days in the gestation‐lactation pups, at 21 days in rats of the gestation and lactation groups, and at 60 days in animals of the gestation and gestation‐lactation groups. Cerebellar DNA content was significantly decreased in pups of the gestation group at every age investigated, but only reduced at 21 days in the lactation group and at 60 days in the gestation‐lactation group. Rats in the lactation group had the greatest number of alterations in terms of RNA and protein, with the most noticeable being decreases in mean cellular RNA content on days 21 and 60 and a reduction in the mean cellular protein content on day 60. These data suggest that prenatal and/or postnatal methadone treatment affects the biochemical maturation of the central nervous system; deficits in neurons and/or glia, as well as a reduction in myelination, might be reflected in these changes.

Correlation of DNA accumulation rate with thymidylate synthetase activity in developing rat cerebellum: effect of hypothyroidism.

Experimental hypothyroidism in the neonatal rat is known to result in a delay in cerebellar DNA biosynthesis. Because of the known high correlation between rate of DNA biosynthesis and thymidylate synthetase (TS) activity in neonatal hyperthyroidism in the rat, we studied this correlation under conditions of neonatal hypothyroidism. The equation for the least-squares logistic curve describing cerebellar DNA content in neonatal rats as a function of postnatal age (day of birth is day 0), was differentiated to give an expression for the instantaneous rate of cerebellar DNA accumulation in units of nanograms per hour per milligram wet cerebellum. The rate of accumulation of cerebellar DNA in hypothyroid neonatal rats from litters of propylthiouracil-treated dams was significantly (p &lt; 0.05) decreased below control values at 3, 5, and 9 days of age, and significantly elevated at 15 and 21 days of age; the maximum decrease (78% of control) occurred on day 5, and the maximum elevation (266% of control) occurred on day 21. The developmental pattern of cerebellar TS activity in units of picomoles per hour per milligram wet cerebellum, closely paralleled the pattern for DNA accumulation rate in cerebella of both normal and hypothyroid neonatal rats, with cerebellar TS activity in hypothyroid pups significantly decreased below normal on postnatal days, 5 and 9, and significantly elevated on days 15 and 21. During this early developmental time period, a linear correlation of the rates of cerebellar DNA accumulation with the values of cerebellar TS activity gave correlation coefficients of 0.98 and 0.95 for normal and hypothyroid animals, respectively. These data thus support results from our previous study of thyroxine-induced hyperthyroidism, further suggesting that TS may be critical for maintenance of replicative DNA synthesis in the developing rat cerebellum.