The influence of collagen gels on the orientation of the polarity of epithelial thyroid cells in culture was studied under four different conditions . (a) Isolated cells cultured on the surface of a collagen gel formed a monolayer. The apical pole was in contact with the culture medium and the basal membrane was attached to the substratum .(b) Isolated cells embedded inside the gel organized within 8 d into follicles . The basal pole was in contact with collagen and the apical pole was oriented towards the interior of the follicular lumen .(c) Cells were first organized into floating vesicles, structures in which the apical surface is in contact with the culture medium, and the vesicles were embedded inside the collagen gel . After 3 d, cell polarity was inverted, the apical pole being oriented towards the cavity encompassed by cells . Vesicles had been transformed into follicles.(d) Monolayers formed on collagen gels as in a were overlaid with a second layer of collagen, which was polymerized in contact with the apical cell surface. A disorganization of the continuous pavement occurred within 24 h; cells attached to the upper layer of collagen and reorganized into follicles in the collagen sandwich within 4-8 d .A similar process occurred when the monolayer was grown on plastic and overlaid with collagen, or grown on collagen and covered with small pieces of glass cover slips. No reorganization was observed between two glass surfaces .In conclusion, first, a basal pole was always formed in the area of contact between the cell membrane and an adhesive surface and, second, the interaction of a preformed apical pole with an adhesive surface was not compatible with the stability of this domain of the plasma membrane . The interaction of the cell membrane with extracellular components having adhesive properties appears to be a determinant factor in the orientation and stabilization of epithelial cell polarity .The formation of a polarized epithelial cell monolayer involves two types of events : (a) the formation of intercellular junctions, the most typical for epithelia being the tight junction (14, 36) ; and (b) cell polarization by an asymmetrical distribution of membrane components between the apical and basolateral domains (8,21) and by a polar distribution of intracellular

Striatal neurons were cultured from the fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

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