Cerebellar DNA Content Research Articles

A single peripheral injection of basic fibroblast growth factor (bFGF) stimulates granule cell production and increases cerebellar growth in newborn rats.

The control of neuronal number is critical for coordinating innervation and target organ requirements. Although basic fibroblast growth factor (bFGF) is known to regulate neuron number in the developing embryonic cortex, its potential role during postnatal brain development remains undefined. To address this issue, the cerebellum, a site of postnatal neurogenesis, was used. Previously, we found that a single peripheral injection of bFGF in newborn rats elicited mitosis of neuronal precursors in the external germinal layer (EGL) 8 h after administration. We now define the sustained effects of bFGF treatment on postnatal granule cell production and cerebellar growth. Seventy-two h after a single injection of bFGF (20 ng/g) in newborn rats, the fraction of BrdU-labeled cells in the EGL increased by 46% without altering apoptotic cell number, consistent with enhanced precursor proliferation. Moreover, bFGF increased mitotically labeled cells by 100% and total cell density by 33% in the internal granular layer (IGL), the final destination of the EGL precursors. Because cerebellar volume also increased by 22%, bFGF-induced proliferation enhanced generation of total IGL neurons and increased cerebellar growth. These morphometric measures were corroborated independently by using DNA quantitation: cerebellar DNA content increased 16% after bFGF injection, consistent with increased neuron number. Furthermore, using DNA quantitation as an index, increased total cerebellar cell number elicited by bFGF injection persisted beyond the neurogenetic period, until P35. We conclude that a single postnatal injection of bFGF increases granule neuron number and enhances cerebellar growth following mitotic stimulation.

In vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors

The in vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development have been investigated in transgenic (Tg) mice (IGF-II/I Tg mice) in whom an IGF-II promoter-driven IGF-I transgene is highly expressed in cerebellum. Compared to normal littermates, the brains of IGF-II/I Tg mice exhibited overgrowth beginning from the second week of postnatal life. Among the brain regions examined, cerebellum exhibited the greatest increase in size, such that by 50 days of age cerebellar weight and DNA content were increased by 90% and 143%, respectively, compared to littermate controls. Morphological studies of adult IGF-II/I Tg mice showed that the total number of granule and Purkinje cells was increased by 82% and 20%, respectively, findings consistent with the increased cerebellar DNA content and indicating that the increased cerebellar weight was due in part to an increase in cell number. The thickness of the molecular layer also was increased in IGF-II/I Tg mice. During early postnatal development the number of external granular layer cells, as well as the number of BrdU labeled external granular cells, was increased. These data strongly indicate that IGF-I increases granule cell number by a mechanism that involves the stimulation of granule cell progenitor proliferation. Our findings also indicate that IGF-I influences the growth of Purkinje cells and possibly of other cell types in the cerebellum.

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.

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.

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 < 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.

RELATION OF THYMIDYLATE SYNTHETASE (TS) ACTIVITY TO DNA ACCUMULATION RATE IN DEVELOPING RAT CEREBELLUM: EFFECT OF HYPER- AND HYPOTHYROIDISM

TS, an enzyme of the pathway by which uridylate (an RNA precursor) is converted to thymidylate (a DNA precursor), is elevated in cells undergoing replicative DNA synthesis.We measured TS activity and DNA content in developing cerebellum of normal, thyroxine-treated, and propylthiouracil-treated neonatal rats. An expression for cerebellar DNA accumulation rate (DAR) was derived by differentiation of the equation obtained using the method of least squares to fit a slgmoid curve (y=A/l+be−cc) to cerebellar DNA content versus age.Hyperthyroidism induced by thyroxine significantly increased both TS activity and DAR above control values on days 2-7, and significantly decreased both measurements below control on days 9-15. The coefficient of correlation between TS activity and DAR was 0.96 for control and hyperthyrold pups.Hypothyroidism induced by administration of propylthiouracil (PTU) to nursing dams significantly decreased both TS activity and DAR below control values by age 5 days and significantly increased both measurements above control on days 15-21. The coefficient of correlation between TS activity and DAR was 0.98 for pups of both control and PTU-treated dams.The close correlation between cerebellar TS and DAR in control, hyperthyroid, and hypothyroid animals supports the possibility that TS may be critical in the control of cell replication rate in developing rat cerebellum.

Effect of thyroid deficiency on cell acquisition in the postnatal rat brain: A quantitative histological study

The mechanisms underlying transient reduction of cell number in the developing cerebellum of thyroid-deficient rats have been studied by quantitative histtological methods. Thyroid deficiency has no significant effect on the generation cyccle of dividing cells in either the subependymal layer of the lateral ventricular walls or the external granular layer of the cerebellum: the length of the cell cycle and the duration of the different phases of the cycle, including the DNA synthesis time appears to be normal. However, the external granular layer of the cerebellum contains fewer cells than in control at 12 days. Pyknotic nuclei are prominent in the granule cell layer of the hypothyroid cerebellum at this age. These amount to an estimated loss of about 1% of the total cerebellar cell population in 24 h. It is suggested that death of granule cells is for the most part a consequence of reduced Purkinje cell dendritic arborization, with failure of parallel fibre/Purkinje cell synaptogeneiis. In the second postnatal week, granule cell death and reduced numbers of cells in the germinal zone can account to a great extent for the observed shortfall in cerebellar DNA content. The eventual attainment of normal cell numbers in the cerebellum of hypothyroid rats is related to a persistent external granular layer in the fourth and fifth postnatal weeks.

Effect of thyroxine on DNA synthesis and thymidine kinase activity during cerebellar development

Body growth, cerebellar growth, cerebellar DNA content, and cerebellar thymidine kinase activity were studied in rats made hyperthyroid from birth, and littermate controls. Hyperthyroidism caused the following. (1) Significantly decreased body weight in treated animals aged 3 days and older. (2) A significant decrease in cerebellar wet weight at age 12 days and later in treated animals. (3) A significant increase in cerebellar DNA by the age of 2 days in treate animals, continuing through the age of 6 days. By the age of 12 days, cerebellar DNA content was significantly decreased below control values in treated animals. (4) An induction of cerebellar thymidine kinase activity in treated animals by age 1 day continuing through age 5 days, with maximum elevation being 137% of control values at age 4 days. By age 9 days, cerebellar thymidine kinase activity in treated animals had fallen to levels significantly below control values and activity was minimal at age 15 days in comparison to disappearance of activity around age 18 days in cerebella of control animals. (5) DNA synthesis is thus accelerated in cerebella of thyroxine treated animals aged 2 to 6 days and subsequently total cerebellar DNA fails to reach normal values. Thymidine kinase activity in cerebella of treated animals is induced by age 1 day, prior to the increase of cerebellar DNA content in treated animals, and subsequently falls below control values by age 9 days, prior to a significant decrease in DNA below control values. This apparent shift to the left in the developmental spectrum of cerebellar thymidine kinase activity compared to the effects of thyroxine on DNA synthesis is discussed in the light of what is known about the effects of hormones on enzyme systems and possible mechanisms of action of thyroid hormones. The conclusions are drawn that thyroxine appears to induce thymidine kinase activity in early cerebellar development and appears to be related to premature decrease of enzyme activity. In addition, our findings support the possibility that thymidine kinase may be an essential regulatory enzyme in DNA biosynthesis, and that the effects on thymidine kinase and DNA biosynthesis of thyroxine in developing cerebellum may be secondary to a modulation of gene activity by the hormone.

Neonatal malnutrition: neurochemical, hormonal and behavioral manifestations

An effective state of malnutrition was imposed on rat pups throughout their postnatal period of development by feeding the lactating dams a 12% casein diet. Controls were fed a 24% casein diet. The malnourished pups exhibited such signs of retarded physical development as reduced body growth and delayed eye opening. At weaning brain weights were reduced and regional brain analysis revealed distortions of chemical composition. These changes included a general reduction of cholesterol concentration, decreased cerebellar DNA content (indicative of cell number), and reduced telencephalic and brain stem protein: DNA ratios (reflecting cell size). The cerebellum also displayed reduced AChE activity suggesting an appreciable effect on its synaptic elements, particularly involving the cholinergic system. Brain stem concentrations of 5-HT and 5-HIAA were increased, implying activation of the central serotonergic neurohumoral system. Possibly related to this, a concomitant increase of adrenal corticosterone was revealed, implicating the additional involvement of the pituitary-adrenal axis in the consequences of postnatal malnutrition. Behaviorally, the previously malnourished weanlings displayed a state of heightened ‘emotionality’. Their performance in a 2-way shuttle task was also markedly abnormal. It is proposed that the enhanced activation of the brain stem serotonergic system may, at least partially, underlie the state of heightened ‘emotionality’, which is the most characteristic behavioral manifestation of perinatal malnutrition.