Inhibitory effect of tunicamycin on the adhesivity and aggregation of embryonic brain cells.

Tunicamycin (TM) - (0.1, 1.0 and 10 micrograms/ml) inhibits insignificantly the adhesivity of embryonic mice brain cells during the first 120 min of incubation. The effect is not dose dependent. The concentration of 10 micrograms/ml added at the onset of experiments has a drastic effect during the time period in which cell regeneration, cell movement and formation of aggregates occurs. Up to the 5th day in vitro (DIV), aggregation is completely inhibited and disrupted parts of cells are mostly present in the medium. The concentration of 1 microgram/ml is less effective, and 0.1 microgram/ml is practically without effect. EM analysis shows that tunicamycin (10 micrograms and 1 microgram/ml) diminish the regeneration and integrity of plasmatic membranes, 10 micrograms/ml of tunicamycin destroys cytoplasmatic organelles which is probably the cause of the decline of cellular regeneration and of aggregate formation. Tunicamycin (10 micrograms/ml), if added to the already formed aggregates evokes their disintegration and lower doses (1 microgram/ml) liberated cells from aggregates into the medium.

Ultrastructural and protein synthetic changes in embryonic brain: cells aggregated at 0 degree C.

Cells dissociated from brains of 16 to 18 day-old mice embrya were rotated at 37 degrees C and 0 degree C for 7--8 days. While cells aggregated at 37 degrees C formed compact aggregates, cells aggregated at 0 degree C were found in clusters or were randomly distributed. Cells aggregated in the cold did not differ markedly from the controls in their ultrastructural organisation till the 2--3 day in vitro (DIV). Later, significant structural changes, such as distention of cytoplasmic membranes, destruction of mitochondrial membranes, disappearance of ribosomes, shrinkage of nuclei and disturbance of cytoplasmic membranes were apparent. On the 6--7 DIV, groups of cells were separated by a distance of 100 nm and more, and large parts of their cytoplasm disappeared and outside cell perikarya fragments of membranes appeared forming dense debris. However, even at this period some cells were found which did not show signs of degeneration. Protein synthetic activity in aggregated cells increased linearly at 37 degrees C till 7 DIV, whereas in cells aggregated at 0 degree C an inhibition of about 34% was found at 4 DIV and at 7 DIV the curve of 14C leucine incorporation declined almost to zero. It is thus evident that cells aggregated at 0 degree C maintain an almost normal ultrastructural pattern during the first days of cultivation and only protein synthetic activity is lowered. Cellular membranes, damaged during the dissociation partly regenerated even at 0 degrees C and membraneous contacts were formed between several cells.

Enzyme activity pattern in developing mouse brain in situ in embryonic brain aggregated cells at 37 degrees C and 0 degree C.

In aggregates of nervous tissue, cultivated for 1--7 days at 0 degree C and 37 degrees C, respectively, the activities of seven enzymes of energy liberating metabolism were estimated, in order to evaluate their metabolic "profiles" and changes during cultivation. The enzymes used as markers of different pathways of energy liberation from substrates were: lactate dehydrogenase - LDH - (EC 1.1.1.27), triose-3-phosphate dehydrogenase - TPDH - (EC 1.2.1.12), glycerol-3-phosphate dehydrogenase - GPDH - (EC 1.1.1.8), hexokinase - HK - (EC 2.7.1.1.), malate:NAD dehydrogenase - MDH - (EC 1.1.1.37), citrate synthase - CS - (EC 4.1.3.7), and 3-hydroxyacetyl CoA dehydrogenase - HOADH - (EC 1.1.1.35). During the cultivation, some changes in the metabolic "profiles" were observed. Although some of these changes as well as the differences between the cultivation at 0 degree C and 37 degrees C, were statistically significant, they were not greater than the variations between different samples of any tissue taken at different times. They were not, therefore considered to be of major significance. However, all the aggregates exhibited "profiles" characteristic for the nervous tissue, with relatively very high activity of HK, high activity of MDH and CS (carbohydrate breakdown) and low activity of GPDH and HOADH (lipid catabolism).

Reaggregation of human, chick, and human embryonic brain cells. Factors influencing the formation of a histiotypic unit.

1. Aggregation of embryo human, mouse, and chick brain cells was studied. The optimum age interval of donors from different species was determined. 2. The significance of different dissociation procedures (mild trypsinisation followed by sieving, trypsinisation + DNA digestion, mechanical dissociation in 1 or 2 steps, and Ca2+ chelation by EGTA) for the rate of aggregation was estimated. A significant reduction of aggregation was observed after one step mechanical dissociation. Nonspecific adhesion of cells on DNA molecules was found only during the first stages of aggregation. 3. The curve of aggregation kinetics follows the curve of floculation kinetics. 90% free cells disappear from the medium after 2 h of aggregation and a large number of microaggregates are formed which condense after 20 to 24 h into compact aggregates. The time course of aggregation was similar for all cells dissociated by different means. Small differences in the rate of aggregation, caused by dissociation procedures, were apparent only during the first stages of aggregation. 4. The histiotypic unit formed by aggregation of human, mouse, and chick embryo brain cells exhibits some common and some specific features. During aggregation a multiple structural reconstruction takes place and a limited number of cells are exchanged or sorted out from aggregates into the medium. 5. The structural organisation of aggregates from differently dissociated cells differs in several aspects. This indicates that membrane surface structures are influenced differently by dissociation and behave differently during distinct stages of aggregation.

Adhesion of dissociated mouse embryonic brain cells. A new method of quantitative evaluation.

The adhesion of mouse embryonic brain cells was measured in a rotating chamber. A method is proposed for quantitative evaluation of adhesion kinetics. Dissociated cells were incubated in a planparallel chamber and pictures were taken between time 0-120 min. Film negatives were evaluated by computer--controlled scanning. Ten thousand individual data area obtained from one frame and 1,000 levels of absorbency are distinguished. A method is described which allows the discrimination of area, density and the shape of adhering cells. The influence of the dissociation procedure on cellular adhesion was studied. Short trypsinisation (0.025% trypsin for 5 min) followed by sieving was most favourable for adhesion. Mechanical sieving and dissociation with EGTA (Ca2+ chelator) gave less satisfactory results. Significantly diminished adhesion was observed after prolonged trypsinisation. If cells were incubated in media lacking Ca2+, adhesion was significantly inhibited. The kinetics of adhesion follows the curve of flocculation kinetics independently of the dissociation procedure and composition of the medium.

Structure and ultrastructure of cultivated glial cells from Corpus callosum.

1. Explants and dissociated cells from Corpus callosum (c. c.) of rats and rabbits were cultivated in Petri dishes and Rose chambers. 2. Different types of glial cells were found in the cultivated Corpus callosum (c. c.) explanted from 12 days old rats: a) adendritic glial cells, typical for migrating oligodendroglial cells, b)-migrating large, nondifferentiated astrocytes with pronounced phagocytosing activity, c) macro- and microglial cells which differentiated during cultivation. 3. The population of differentiated glial cells is mostly composed of oligodendroglia, less of astrocytes and microglial cells are rare. 4. Differentiation of dissociated cells from c. c. in homogenous and mixed population was studied. The appearance of first processes of macroglial cells is postponed to 6 to 10 days of cultivation. No substantial difference was observed between homogenous and mixed population. A higher incidence of macrophages was observed in the later. 5. Glial cells differentiate surrounded by degenerated nerve fibers and myelin, exhibiting phagocytoses and cleaning reaction.

Differentiation of corpus callosum glial cells and factors influencing their maturation in vitro.

1. Explants of Corpus callosum (c. c.) from 12-day-old rats were cultivated under different experimental conditions. 2. Migration and differentiation is activated by the presence of neighbouring explants, toward which glial cells predominantly migrate. Glial cells migrate if closely adhering to the supporting collagen and the process of differentiation is enhanced by presence of underlying cell layers. 3. Migratory activity of glial cells decreases and is delayed with age of donors. Migrating cells have a similar appearance as in cultures from 12 days old donors. The presence of immature types of glial cells in c. c. of adult animals was proved. 4. Glucose was found to be an adequate metabolical substrate, utilisation of glucose being lower than in cultivated neurons. In the absence of glucose or serum in the medium, neither migration nor differentiation of glial cells was observed. 5. The addition of embryonal extract and embryonic brain extract enhanced only initial stages of cell migration and differentiation.