Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes

Abstract

We present data on the effect of elevated concentrations of K + ions (25 mM) and polylysine (PLL) coating of the substratum on the in vitro survival and behaviour of cells derived from 8-day-old rat cerebellum. The cells were grown in Eagle's basal medium in the presence of 10% foetal calf serum and cytosine arabinoside (10 μM), as a mitotic inhibitor. The most conspicuous effect of the high potassium was to facilitate the relatively long survival of the nerve cells, whereas PLL influenced the nerve cell attachment and thereby the size of the aggregates formed in the cultures. When cells were grown in high [K +] on PLL-coated dishes (standard conditions) over 70% of the plated cells survived beyond 7 DIV, and about 95% of the cells were small interneurones, tentatively identified as predominantly granule cells. The most numerous non-neuronal cells were glial fibrillary acid protein (GFA) positive astrocytes. The beneficial effect of high potassium on nerve cell survival was most prominent after 7 DIV, when it is known that transmission-associated neurochemical functions are just becoming detectable under the standard conditions. Initially (at 3 DIV) under all the tested conditions, and throughout the experimental period under the standard conditions, the dominant type of GFA-positive cells was the process bearing ‘differentiated’ astrocyte. When the conditions resulted in a great decrease in nerve cell numbers, on the other hand, flat astroblast-like cells became the most abundant cells in this class. Neurones grown on polylysine in the presence of 25 mM potassium extended neurites as early as 6 h after plating, and with longer culture times, an extensive network of fibres of neuronal origin was generated. Neurites did not seem to follow the processes of GFA-positive astrocytes in the cultures. Although there was a limited tendency for neuronal cell bodies to be positioned around astrocytes at 1 DIV, this became less marked with time, and no preferential association between astrocytes and neurones could be detected in the cultures later than 2 DIV.