Monolayer cultures of perikarya isolated from postnatal rat cerebellum.

Viable cerebellar perikarya of mixed cell type obtained from 7--9-day-old rats were maintained in monolayer cutlure for up to 12 days. During this time extensive neurite formation and outgrowth occurred. The large majority of the cells developing in culture were tentatively identified as granule neurons. This identification was based on the large number isolated from the starting tissue, and the cells' general morphological features in culture such as perikaryal and nuclear size, the bipolar nature of neurite extension, and their migratory behaviour.     

Monolayer cultures of normal adult rat adrenocortical cells: Steroidogenic responses to nucleotides,bacterial toxins and antimicrotubular agents

Summary Monolayer cultures of normal rat adrenocortical cells were treated with agents which stimulate steroidogenesis by Y-1 adrenal tumour cells. Choleragen was active, whereas cyclic nucleotides other than cyclic AMP, bacterial endotoxins and antimicrotubular agents were inactive.This work was supported by the Medical Research Council of Great Britain (grant No. 970/656/B). I am grateful to MissR. Magee for technical assistance and Prof.A. M. Neville for continued support.     

Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes

Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (4% O2) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO2 (4% O2). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies. The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.     

Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes

Summary Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (5% O₂) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO₂ (4% O₂). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies.The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.     

Monolayer cultures of normal adult rat adrenocortical cells: steroidogenic responses to nucleotides, bacterial toxins and antimicrotubular agents.

Monolayer cultures of normal rat adrenocortical cells were treated with agents which stimulate steroidogenesis by Y-1 adrenal tumour cells. Choleragen was active, whereas cyclic nucleotides other than cyclic AMP, bacterial endotoxins and antimicrotubular agents were inactive.     

Preparation of isolated cells from rat heart

Summary Suspensions of isolated cells from rat heart were prepared and the data for viability and yield are given. Glucose uptake by the cells was mediated by a carrier system.The technical assistance of Mr.E. T. Potter, Mr.K. D. Patel and Mr.M. Griffin is gratefully acknowledged.     

Cilia in stellate neurons of the rat cerebellum

Summary Cilia in stellate neurons of the normal rat cerebellum are described. 8 cilia were observed in a total of 60 cells studied. An 8+1 pattern was found throughout their length. Furthermore, no arms, spokes or other accessory structures necessary for ciliary motion were seen. These findings make it possible to suggest that these cilia are probably without function and are related to the epithelial origin of these cells.This work was granted by INIC: Centro de Morfologia Experimental (MbP₁).Acknowledgments. The authors thank M.A. Rodrigues, M. M. Pacheco, M. C.Pinho and L. B. Nunes for technical assistance.     

An improved technique of preparing primary cultures of isolated cells from adult frog kidney

Zusammenfassung Eine verbesserte Technik zur Gewinnung der ersten Einzelschichtbildungen von Froschnierenzellen wurde entwickelt. Die Zellen werden in neuem auflösendem Nährboden isoliert und wachsen auf modifiziertem Eagle-Nährboden mit 13% Kalbsserum. Nach 10 Tagen zeigt sich stark aktives mitotisches Zellwachstum in der Kultur.     

Ethanol influence on insulin secretion from isolated rat islets

This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.     

Ethanol influence on insulin secretion from isolated rat islets

Summary This study was done to delineate the role of - and -adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an -adrenergic blocker, phentolamine, or a -adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.Supported by the U.S. Veterans Administration     

Immunohistological investigations of N-Acetylserotonin in the rat cerebellum after parachlorophenylalanine treatment

Summary The amount of N-acetylserotonin (NAS) in the granule layer of the rat cerebellum was investigated using immunohistologic double antibody technique. After 5 days of treatment with parachlorophenylalanine (PCPA) an increase of NAS was observed. The possibility of a differential effect of PCPA on serotonin synthesis in the neurons and the nerve terminals is discussed.     

Immunoelectrophoresis of soluble proteins isolated from cellular fractions of regenerating rat liver

Riassunto Gli autori hanno studiato, mediante elettroforesi su agar ed immunoelettroforesi, il comportamento delle proteine solubili isolate dalle frazioni citoplasmatica e mitocondriale del fegato rigenerante di ratto. Sono state rilevate alcune modificazioni delle proprietà immunochimiche di taluni componenti proteici.     

The postnatal evolution of muscular twitches in the developing rat

Summary The reported study concerns the evolution of muscular twitches during the 21 postnatal days of the normal rat pups. The data indicate an age-dependent progression of these twitches in different body regions.Acknowledgment. We thank Dr K. Chaudhary for the revision of the english text and Mrs D. Huot-Blais for her secretarial assistance.     

Neuritic plaque-like structures in the rat cerebellum following prolonged alcohol consumpotion

Summary Primitive neuritic plaques were observed in the inner third of the molecular layer of the cerebellar cortex of rats following chronic alcohol consumption. Neurites were indentified as dystrophic parallel fiber bouton. Amyloid material dispersed among neurites was not clearly recognized, dystrophic some fibrils were frequently seen among them. Astrocytic processes were noted in the periphery of the plaque. Microglial reaction, however, was non-existent. The rarity of these lesions in the rat cerebellum and their probable relation to long periods of alcohol consumption is discussed.This work was granted by I.N.I.C., project MbPl, and Stiftung Volkswagenwerk, project I/37424.     

Immunohistological investigation of N-acetylserotonin in the rat cerebellum after parachlorophenylalanine treatment.

The amount of N-acetylserotonin (NAS) in the granule layer of the rat cerebellum was investigated using immunolohistologic double antibody technique. After 5 days of treatment with parachlorophenylalanine (PCPA) an increase of NAS was observed. The possibility of a differential effect of PCPA on serotonin synthesis in the neurons and the nerve terminals is discussed.