Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex

Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.     

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.     

Changes in metabolic activity of neurons in the anterior hypothalamus nuclei during hyperthermia, fever, and hypothermia

Differently directed changes in metabolic activity of anterior hypothalamic nuclei's neurons in rats during hyperthermia, fever, and hypothermia were revealed with histochemical methods. During hyperthermia, the activity of energy metabolism enzymes increased as well as RNA content in the neurons of supraoptic, paraventricular and median preoptic anterior hypothalamic nuclei. This is shown by an increase in the metabolic activity of neurons of these nuclei. Metabolic activity in neurons of median preoptic nuclei decreased and was not changed considerably in neurons of supraoptic and paraventricular nuclei during endotoxin-induced fever. The development of hypothermia was characterised by a decrease in metabolic activity of neurons of supraoptic, paraventricular and medium preoptic nuclei. It is supposed that differently directed metabolic activity changes in neurons of anterior hypothalamic nuclei during hyperthermia are connected with the mechanisms of body temperature regulation (median preoptic nuclei) and neurosecretory processes (supraoptic and paraventricular nuclei).     

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.     

A quantitative comparison of the hominoid thalamus: II. Limbic nuclei anterior principalis and lateralis dorsalis

Structures in the limbic system are commonly thought to be similar in form and function in all mammalian brains. In the study reported here, two thalamic limbic nuclei, N. anterior principles and N. lateralis dorsalis, were compared among a group of extant of extant hominoids. The nuclear volumes, neuronal densities, number of neurons per nucleus, and volumes of neuronal perikarya were measured. Humans have much larger nuclei but the nuclei constitute a similar proportion of the whole thalamus as found in the other hominoids. Whereas the human limbic nuclei were observed to have a decrease in the densities of nerve cells compared with those of the other hominoids, this difference is less than that found in most other thalamic nuclei. Consequently the estimated number of neurons is much higher for humans. The total number of neurons best separates the human limbic nuclei from those of the other hominoids. This preliminary study suggests that during hominid evolution neurons were preferentially added to the limbic nuclei of the thalamus.     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Effect of neonatal androgenization on the septal neurons of the brain in female rats in early postnatal ontogeny

Newborn female rats were androgenized, and the reaction of neurons of brain septum on excessive quantity of exogenous androgens, introduced during so-called "crucial" period of formation of centers of gonadotropic regulation of sexual cycles, has been studied in 3, 5, 7, 10, 20, 30, and 60 days old animals. Morphometry of brain septum cell nuclei revealed that most neuron nuclei shrink after androgenization. Monoamine content was significantly increased in septum nuclei of experimental animals. Neonatal androgenization led to the increased capacity of septal complex neurons to bind 3H-estradiol and to the decreased 3H-testosterone binding. The data obtained suggest that the brain septum neurons of female rats depend on sex steroids, particularly during "crucial" period of development.     

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.     

Differential genotoxicity of ethyl methanesulphonate,N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays

The purpose of this study was to determine if mutagen-induced DNA damage is correlated with the frequency of induced recombination events. The alkylating agents ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU), and the plant growth regulator and herbicide maleic hydrazide (MH) were compared in tobacco seedlings for their ability to induce DNA damage measured by the Comet assay, and recombination activity measured by the GUS gene reactivation assay, and by the somatic twin sectors assay. While EMS and ENU induced a dose-dependent increase in DNA damage in leaf nuclei, MH had no significant effect. By contrast, MH induced a 6-fold higher frequency of homologous recombination as expressed by the GUS assay and a 2.8-fold higher frequency of somatic twin sectors than after EMS treatments.     

Flow cytometric analysis of the cell-cycle distribution of spleen lymphocytes isolated from Fischer 344 rats exposed to ethyl nitrosourea

Current studies in our laboratory are designed to determine the frequency of genotoxic responses induced in lymphocytes isolated from Fischer 344 rats. To evaluate the effect of a model compound, N-ethyl-N-nitrosourea (ENU), on the cell-cycle distribution of spleen lymphocytes, 8-week old, female Fischer 344 rats were injected i.p. with ENU and sacrificed 1, 2, 4, and 6 weeks afterexposure. Four replicate cultures per dose per exposure period were established and cells were cultured for 66 hr. Colcernid, an agent which blocks cells in mitosis and induces an accumulation of cells in the G₂ + M peak, was added to two of the four cultures as a positive control. After a 3 hr incubation, the cells were harvested, the nuclei stained with propidium iodide, and the DNA content of the individual nuclei was quantified by flow cytometry. As expected, exposure to Colcemid resulted in an accumulation of cells in the G₂ + M phase of the cell cycle, which was accompanied by a decrease in the Go + GI population. The increase in the G₂ + M population was significant (p < 0.05) in cultures of lymphocytes assayed at 4 and 6 weeks after exposure. The eflect of increasing ENU concentratiorl was an increase in the percentage of Sphase cells (p =0.05) and a decrease (p < 0.02) in the percentage of G₀ + G₁ cells. This finding was observed only in those lymphocytes isolated 1 week after exposure. These findings indicate that flow cytometric analysis of the distribution of cells within the cell-cycle may provide insight into the eflects of toxicant exposure on mamnzalian cells.Abbreviations BRdU bromodeoxyuridine - ENU N-ethyl-N-nitrosourea - FCM flow cytometry - PHA phytohemagglutinin - PI propidium iodide     

大白鼠中缝核一氧化氮合酶阳性神经元的组织化学观察

中脑和脑桥部中缝核被认为与睡眠有直接和间接关系的重要脑结构。本文用一氧化氮合酶（ＮＯＳ）组织化学结合荧光组织化学方法证实在中缝核群中,ＮＯＳ阳性神经元主要定位于这两个脑部的中缝核内,ＮＯＳ产生的ＮＯ能使脑血管扩张,参与脑血流的调节。提示这二个脑部中缝核内的ＮＯＳ阳性神经元可能作为多种因素之一,参于睡眠状态下基本脑血流的维持     

Otolith and semicircular canal inputs to single vestibular neurons in cats.

Researchers studied the convergence of the vertical posterior semicircular canal (PC), saccular nerves (SAC), utricular nerves (UT), and horizontal semicircular canal nerves (HC) on single vestibular neurons. The vestibular neurons were categorized by their innervating targets. Vestibular neurons were classified as vestibulospinal proper neurons (VS), vestibulo-ocular proper neurons (VO), vestibulo-oculo-spinal neurons sending axon collaterals to the extraocular motoneuron pools and spinal cord (VOS), and vestibular nucleus neurons without axons to the oculomotor nuclei or the spinal cord (V). Results indicate that the percentage of convergence of VS neurons was higher that that of neurons sending axons to the oculomotor nuclei (VO and VOS). They conclude that the convergence of canal and otolith inputs likely contributes mainly to vestibulospinal reflexes by sending inputs to the neck and other muscles during head inclination, which creates the combined stimuli of angular and linear acceleration.     

Administration of ethylnitrosourea to neonate hamsters increases growth and frequency of SV40-induced fibrosarcomas

The in vivo interaction between the chemical carcinogen ethylnitrosourea (ENU) and the oncogenic simian virus 40 (SV40) was studied. Inbred newborn Syrian golden hamsters were injected subcutaneously with SV40 (5 x 10(6) plaque-forming units), ENU (0.5% solution, 125 or 25 mg/kg body wt), or equal mixtures of the two. Animals that received SV40 and ENU developed more tumors (100% vs 52%) within a shorter latent period (10 weeks vs 18 weeks) than animals that received SV40 alone. Animals given SV40 and ENU showed increased mortality and increased metastatic tumors (54.2% vs 30.8%) compared with those given SV40 alone. The SV40 and ENU group also exhibited multiple (greater than 10 nodules) pulmonary metastases (33.3% vs 7.7%) and metastases in multiple organs (12.5% vs 0%) compared with animals injected with SV40 alone. No difference in primary tumor size, histology, and SV40 T-antigen content was detected between SV40- and SV40/ENU-induced tumors. Four weeks after SV40 or SV40 plus ENU treatment, animals were challenged intradermally with 2.7 x 10(6) SV40-transformed hamster cells. Five weeks after challenge, 89.5% of the animals treated with SV40 and ENU and 45.4% of animals treated with SV40 developed tumors at the challenge site. Newborn animals given SV40 and ENU developed larger tumors at the challenge site (P less than 0.002) than newborns treated with SV40 alone. Thus, administration of ENU to hamsters during the neonatal stage of development produced a long-lasting systemic effect that enhanced tumor development by transplanted SV40-transformed hamster cells.     

Sociality and oxytocin and vasopressin in the brain of male and female dominant and subordinate mandarin voles

The dominant–subordinate hierarchy in animals often needs to be established via agonistic encounters and consequently affects reproduction and survival. Differences in brain neuropeptides and sociality among dominant and subordinate males and females remain poorly understood. Here we explore neuropeptide levels and sociality during agonistic encounter tests in mandarin voles. We found that dominant mandarin voles engaged in higher levels of approaching, investigating, self-grooming and exploring behavior than subordinates. Dominant males habituated better to a stimulus vole than dominant females. Dominant males displayed significantly less oxytocin-immunoreactive neurons in the paraventricular nuclei and more vasopressin-immunoreactive neurons in the paraventricular nuclei, supraoptic nuclei, and the lateral and anterior hypothalamus than subordinates. Dominant females displayed significantly more vasopressin-immunoreactive neurons in the lateral hypothalamus and anterior hypothalamus than subordinates. Sex differences were found in the level of oxytocin and vasopressin. These results indicate that distinct parameters related to central nervous oxytocin and vasopressin are associated with behaviors during agonistic encounters in a sex-specific manner in mandarin voles.     

The chromatin repeat length of brain cortex and cerebellar neurons changes concomitant with terminal differentiation.

Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis. The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages. Administration of [3H]thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually. This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length. In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30. Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes. No changes were detected in liver nuclei during the same period. Non-astrocytic glia cells of the adult cortex had 200 bp repeats.     

Age-related changes in nitroxidergic neurons in some nuclei of rat medulla oblongata

The paper presents a comparative study of NO neurons in the solitary tract nucleus, giant-cell, and lateral reticular nuclei in rats at 4, 7, 10, 14, 30, 45, and 60 days old and 3, 6, 12, 18, 24 months old. We determine the active quantitative and qualitative changes that occur in NO-positive neurons in the studied nuclei during the course of postnatal development. A low level of enzyme activity is observed on the first day; it reaches a peak level around the first-third month, then slowly declines. The size and number of nitroxidergic neurons increases, while the relative cell density decreases until the third month of life. We reveal local differences in the ontogenetic development of NO neurons in the studied nuclei. Solitary tract neurons have the highest rate of development, while NO neurons of old animals undergo early and extreme changes as compared to other studied nuclei of rat medulla oblongata.