PubMed Health⌕ Search

Biomedical subjects

A Cestelli

Publications and source records attributed to A Cestelli.

At least 37 records · Page 2Linked to original sources

Neuronal cell cultures: a tool for investigations in developmental neurobiology.

The aim of this review is to describe environmental requirements for survival of neuronal cells in culture, and secondly to survey the complex interplay between hormones, neurotrophic factors, transport- and extracellular matrix- proteins, which characterize the developmental program of differentiating neurons. An overall reconsideration of the literature in this vast field is above the limits of the present paper; since progress and refinement in the techniques of neuronal cell cultures have paralleled the advancement in Developmental Neurobiology, we will run instead through the main steps which form the conceptual framework of neuronal cell cultures.

Animals↗

Developmental changes of neuron-specific enolase mRNA in primary cultures of rat neurons.

1. The level of mRNAs for neuron-specific enolase (NSE) and nonneuronal enolase (NNE) was studied in developing rat brain and in pure neuronal cultures of corresponding ages treated or not treated with triiodothyronine (T3). 2. In brain cortices both messages are already detectable at the earliest age (embryonal day 16; E16). During development the mRNA for NNE remains at a steady level, with a transient decline at postnatal day 5 (P5). 3. On the other hand, NSE mRNA follows a biphasic curve: the signal increases threefold from E-16 to P0 and threefold from P5 to P18, with a plateau between P0 and P5. 4. In neuronal cultures the NNE message is present at a constant level until day 10 and declines sharply thereafter, while in T3-treated cultures it reaches a minimum beforehand. 5. The NSE mRNA, on the other hand, increases continuously throughout the whole culture life span, and a slightly higher level is observed in T3-treated cells during the first ten days.

Animals↗

The relative proportion of H1(0) and A24 is reversed in oligodendrocytes during rat brain development.

1. The histone complement of oligodendrocyte chromatin at different stages of brain development was studied after acid extraction of nuclei. 2. HCl-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1(0) with differentiation. 4. The increase in H1(0) is accompanied by a concomitant decrease in the total amount of the ubiquitinated form of histone H2A (A24).

Aging↗

Cellular mechanism of action of thyroid hormones.

It has emerged in the last decade that the molecular mechanism of action of thyroid hormones resembles that of steroids; thyroid hormones indeed exert their effects mainly by directly regulating gene expression, on association with specific chromatin-bound receptors. Of the two thyroid hormones, thyroxine (T4) appears to be a sort of prohormone, whereas triiodothyronine (T3) seems to be the active form; in this respect, T4-deiodination, which occurs at the level of the target tissues, may be crucial in the local homeostasis of T3. Moreover, many cellular compartments, other than the nucleus, can bind thyroid hormone, and at least some of these further sites might play some role in modulating T3 supply to the nucleus. The binding of the T3-receptor complex to chromatin is likely to regulate the structural organization of specific genes and, in some instances, of the chromatin as a whole.

Animals↗

Triiodothyronine-induced shortening of chromatin repeat length in neurons cultured in a chemically defined medium.

At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from approximately 200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one.

Animals↗

Inhibition of glial proliferation in vitro by serum from patients with multiple sclerosis.

Primary cell cultures from fetal rat CNS have been employed to evaluate the effects caused by the addition of serum from patients affected by multiple sclerosis (MS). MS-serum supplemented media caused a decrease in [3H]-thymidine incorporation into the cultures, thus indicating an inhibitory effect on proliferating glial cells. Sera from patients in remission stage of the disease showed an inhibitory effect not significatively lower than those from patients in acute stage. These results suggest that glial cells may be a target of circulating factors present in MS.

Adult↗

Rat CNS neurons are not yet programmed to shorten their chromatin repeat length at the end of fetal neurogenesis.

Neurons from rat fetal cerebral hemispheres were grown in a synthetic medium (Maat medium), as previously described, for different periods of time. The repeat length of their chromatin was determined by micrococcal nuclease digestion and compared with that of neurons isolated from postnatal rat brain of corresponding ages. In contrast to the in vivo situation, we found that neurons, dissociated at the 16th gestational day and cultured in vitro, did not undergo the shortening of their chromatin repeat, thus indicating that, at the end of their mitotic cycles, they are not yet programmed to this event.

Aging↗

Formulation of a novel synthetic medium for selectively culturing rat CNS neurons.

Dissociated cells from rat fetal cerebral hemispheres were grown in surface adhering culture using a novel synthetic medium (Maat medium) and compared with those grown either in the presence of serum or in the chemically defined medium described by Bottenstein and Sato. The addition of various compound combinations allowed us to lower insulin concentration to almost physiological levels. Maat medium improved the purity and longevity of neuronal cultures. The purity of neuronal cultures grown in different media was checked both by immunofluorescence and by the analysis of [3H]thymidine incorporation.

Animals↗

A low repeat length in oligodendrocyte chromatin.

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."

Age Factors↗

Rat CNS cell culture. Enhancement of neuronal survival and delay of glial proliferation by serum from patients with multiple sclerosis. A morphological study.

The addition of serum from multiple sclerosis (MS) patients to the culture medium of dissociated cells from cerebral hemispheres of rat embryos caused a delay in glial proliferation and an enhancement of neuronal survival. Sera from normal individuals and patients with other neurological diseases failed to show this effect. These morphological observations are interpreted as the outcome of inhibition of in vitro gliogenesis.

Animals↗

Selective culture of rat CNS neurons in a synthetic medium.

A Chemically Defined Medium is described which facilitates the survival of neurons in primary cultures of rat cerebral hemispheres. More than 90% of all cells were identified as neurons using neurofilament as a marker in an immunocytochemical assay. In contrast, serum-supplemented medium, by stimulating nonneuronal cell proliferation, hinders the survival of neurons in culture.

Animals↗

The use of liposomes as acceptors for the assay of lipid glycosyltransferases from rat brain.

Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first time as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose: ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1 : 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucostyltransferase. For the assay UDP-galactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing alpha-hydroxy fatty acids, in a molar ratio of 6 : 0.57 : 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200--500 A. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesulfonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography.

Animals↗