Gloria L. Pleger

Chapter 16 Isolation of Micro- and Macronuclei of Tetrahymena pyriformis

Publisher Summary This chapter discusses the isolation of micro- and macronuclei of Tetrahymena Pyriformis. With the exception of a few amicronucleate or partially amicronucleate strains of Tetrahymenaa each cell contains two types of nuclei, a smaller micronucleus and a larger macronucleus. Although these two nuclei are formed from daughter nuclei of a single mitotic division (the second postzygotic division) during conjugation, they differ in most of their structural and functional properties in vegetative cells. Micronuclei divide mitotically prior to cell division, undergo DNA synthesis early in the cell cycle, do not contain nucleoli or heterogeneous ribonucleoprotein particles, and are genetically inactive. Macronuclei divide amitotically when the cell divides, synthesize DNA in the middle third of the cell cycle, have large numbers of RNA-containing inclusions, and are responsible for the genetic activity which controls the phenotype of the vegetative cell. Micro- and macronuclei thus have a common origin during conjugation and reside in the same cytoplasm during vegetative growth, yet differ strikingly in structure and function. Therefore, it is suggested that these two nuclei may be used as a model system to explore the mechanisms whereby the same (or closely related) genetic information is maintained in different structural and functional states in eukaryotic cells.

GABA-stimulated chloride uptake and enhancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development.

The present study was undertaken to evaluate the ability of diazepam (DZ) to elicit a response in the brains of developing animals. gamma-Aminobutyric acid (GABA)-stimulated 36Cl- uptake in the presence and absence of DZ was measured in synaptoneurosomal preparations from whole brain of fetal rats at 20 and 21 days gestation and from cerebral cortex of rats at 7, 14, 21, 28 and 60-90 days postnatal age. The ability of GABA to stimulate 36Cl- uptake in a concentration-dependent manner was evident from gestational day 20. The EC50 for GABA stimulation but not the maximum stimulation increased significantly from day 20 to day 21 of gestation. Postnatally, only moderate changes in the EC50 were evident, but the maximum 36Cl- transported increased significantly from 7 to 14 days and remained stable thereafter. DZ enhanced GABA-mediated 36Cl- influx from 20 days gestation, and this enhancement was seen as a decrease in the EC50 for GABA stimulation. However, DZ also significantly increased maximum GABA-stimulated 36Cl- transport in synaptoneurosomal preparations at 21 days gestation and at 7 days postnatal age, a response to DZ not seen at older ages. DZ had a less robust effect on GABA-mediated 36Cl- transport at 28 days postnatal age than at any other age, a result consistent with functional observations of decreased responsivity to DZ in late juvenile, early adolescent rats. The benzodiazepine antagonist Ro 15-1788 prevented the effect of DA on GABA-stimulated 36Cl- uptake in tissue from 21-day fetuses and from 60- to 90-day-old adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered stressor-induced changes in GABAA receptor function in the cerebral cortex of adult rats exposed in utero to diazepam

Prenatal administration of the anxiolytic drug diazepam (DZP), 2.5 mg/kg) to the pregnant rat over gestational days 14-20 altered function and stressor-induced responsiveness of the GABAA receptor in the cerebral cortex of exposed animals as adults. In Experiment 1, the impact of 15 min of restraint on chloride-facilitated benzodiazepine binding was evaluated in male and female rats at 70-90 days of age. Early exposure to DZP led to an enhanced potency of chloride on binding in both males and females. In Experiment 2, GABA stimulation of 36chloride uptake was measured in male rats at 35 or 70 days of age following 10 min of forced swimming at ambient temperature. In control animals, stressor-induced changes in receptor function were not evident until 70 days, and in DZP-exposed rats the stressor had no effect on receptor function at either age. These changes in GABAA receptor responsiveness induced by early exposure to DZP may underlie the disrupted behavioral responses to environmental challenge that have been previously reported.

Importance of hypothalamic function to stressor-induced responsiveness of the GABAA receptor in the cerebral cortex: a non-corticosterone influence

Catecholamine terminals in the paraventricular nucleus (PVN) of the hypothalamus of 60-day-old rats were destroyed by the stereotaxic injection of 6-hydroxydopamine into the PVN (6-OHDA; 9 micrograms/1.5 microliters bilaterally), and the rats were tested 2 weeks later. Lesions led to a 70% reduction of norepinephrine in the hypothalamus and a loss of dopamine-beta-hydroxylase immunoreactivity in the PVN. Furthermore, 6-OHDA lesions in the hypothalamus disrupted stressor-induced (15 min of restraint) changes in GABAA receptor function in the cerebral cortex (assessed by measuring chloride-facilitated benzodiazepine binding) but did not alter stressor-induced increases in plasma corticosterone levels. Additionally, the lesion did not change the responsiveness of the GABAA receptor to the corticosterone metabolite, allotetrahydrodeoxycorticosterone. These results indicate that stressor-induced changes in cortical GABAA receptor function are not driven by the stressor-induced release of corticosterone. A separate group of animals were tested for behavioral responses to challenge, and while 6-OHDA-induced lesions did not alter total scores in the test of environment-specific social interaction, the lesions did induce a change in composition of the behavior. Lesioned animals demonstrated increased physical (vigorous contact) interactions, similar to behavior previously observed in younger rats. The results of the behavioral study support a role for the GABAA receptor in the cerebral cortex in mediating appropriate behavioral responses to challenge in the adult rat. Thus, a hypothalamic lesion that prevented challenge-induced changes in GABAA receptor function in the cortex (with no change in the corticosterone response to the stressor) also led to altered behavioral responses to challenge.

Sex-specific effects of in utero manipulation of GABAA receptors on pre- and postnatal expression of BDNF in rats

Exposure to diazepam (DZ) during the last week of in utero development in rats induces neurobehavioral effects that do not become apparent in exposed animals until young adult ages. Some of the effects are sex specific. This study evaluated the hypothesis that late gestational exposure to DZ, a positive modulator of GABA(A) receptors, affects the developmental appearance of brain-derived neurotrophic factor (BDNF), an effect that could be linked to the later consequences of the exposure. Pregnant Long-Evans rats were injected with DZ (2.5 mg/kg) over gestation days 14-20, and their male and female offspring were evaluated for levels of BDNF mRNA and protein in the cerebral cortex and hypothalamus at fetal day 20 and at postnatal ages spanning birth to young adulthood. The effects of the exposure were sex and region specific. At fetal day 20 the expression of BDNF was reduced by about 20% in the hypothalamus of males only. The early exposure affected postnatal expression of BDNF in the hypothalamus only modestly, influencing the age-related profile in both sexes. Postnatal development of BDNF in the cerebral cortex was significantly affected by the in utero exposure in males only with mRNA levels lower in the exposed group and protein levels higher during juvenile ages. At adulthood, both levels were lower in DZ-exposed males. GABA serves a role as a trophic factor during early development, and these results suggest that manipulation of GABA(A) receptors during early development could interact with the developmental action of other trophic factors thereby leading to altered neural organization and later neurobehavioral dysfunction.

NEUROSTEROID ACTION AT THE GABAA RECEPTOR IN FETAL RAT FOREBRAIN

In utero exposure to diazepam (DZ), a positive modulator of the GABAA (gamma-aminobutyric acid type A) receptor exerts profound effects on the offspring that become most apparent after the maturation of the brain during puberty and that are often sex specific, suggesting that the early exposure might have interfered with organizing actions of sex steroids. In addition to genomic actions, many reduced steroids interact directly with membrane receptors, including the GABAA receptor. In the present study, the effect of in vitro exposure to neurosteroids on GABA-stimulated 36chloride uptake in synaptoneurosomes from adult cerebral cortex or fetal forebrain (gestation day 20) was examined. The initial study examined the effects of incubation with DZ (10 microM) and the neuroactive steroid, 3 alpha,5 beta-THP (500 nM), alone and in combination. In adult tissue, the presence of either drug alone decreased the EC50 for GABA stimulation, and incubation with both drugs had an additive effect. In fetal tissue, while both compounds decreased the EC50, an additive effect was apparent only when comparing the combined exposure to 3 alpha,5 beta-THP alone. DZ alone reduced the EC50 as much as both drugs together. In the second study, the effect of in vitro exposure to androsterone (2.5 microM) was evaluated in male and female fetal tissue separately as well as in the adult. Androsterone enhanced the sensitivity to GABA in all groups but also reduced the efficacy of GABA in fetal tissue, irrespective of gender. While neurosteroids and DZ elicited similar responses in fetal and adult tissue, the study identified a greater vulnerability of fetal GABAA receptors to modulatory compounds.

Effect of Prenatal Exposure to Diazepam on Brain GABAA Receptor mRNA Levels in Rats Examined at Late Fetal or Adult Ages

This study tested the hypotheses that in utero exposure to diazepam (DZ): (1) exerts long-lasting effects on GABAA receptor function by altering GABAA receptor subunit mRNA levels in specific brain regions of adult animals and/or (2) alters GABAA subunit mRNA expression in exposed fetuses. We assayed levels of mRNAs encoding several of the most predominant GABAA receptor subunits as well as cyclophilin mRNA. Analysis of mRNA levels in the cortex in adults showed that only γ2S mRNA levels varied significantly with prenatal drug exposure, an effect unrelated to DZ action to the GABAA receptor. Analysis in fetuses indicated that mRNA levels varied as a function of both fetal sex and fetal drug environment. Irrespective of sex, DZ exposure increased both α1 and cyclophilin mRNAs in fetal brainstem whereas the mRNA levels of γ2S were increased and decreased, respectively, in the telencephalon and hypothalamus of DZ-exposed fetuses.

Region-, age-, and sex-specific effects of fetal diazepam exposure on the postnatal development of neurosteroids

Fetal exposure to diazepam (DZ), a positive modulator of GABA(A) receptors and an agonist at mitochondrial benzodiazine receptors, induces long-term neural and behavioral effects. This study evaluated whether the early manipulation influenced the normal development of brain levels of neurosteroids or altered steroid action at GABA(A) receptors. Pregnant dams were injected over gestation days 14 through 20 with DZ (2.5 mg/kg) or the vehicle. Male and female offspring were analyzed at five postnatal ages. The levels of progesterone (P), dihydroprogesterone (DHP), 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), testosterone (T), dihydrotestosterone, and 5alpha-androstan-3alpha,17beta diol were measured in the cerebral cortex and diencephalon. The results indicated that development of brain steroid levels and the impact of fetal DZ exposure were region- and sex-specific. Age-related changes in brain steroids did not mirror associated changes in circulating P and T. Age regulated the levels of all 3 progestins in the cerebral cortex, and fetal DZ exposure interacted with the development of P and DHP. The development of 3alpha,5alpha-THP in the cortex was markedly influenced by sex, with levels in males decreasing over postnatal development whereas they increased over postpubertal development in females. An adolescent surge in T levels was observed in male cortex and fetal DZ exposure prevented that surge. Steroid levels in the diencephalon were altered by age mainly in females, and DZ exposure had little effect in this region. The data support region-specific regulation of brain steroid synthesis. Only in the cerebral cortex are relevant mechanisms readily modifiable by fetal DZ exposure. However, neither sex nor fetal DZ exposure altered the response of GABA(A) receptors in adult cortex to neurosteroid.