Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Abstract

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Spleen-Cell-Conditioned Medium on [³H]-Choline Uptake by Retinal Cells in vitro Is Mediated by IL-2

“…However, the mechanisms leading to cellular differentiation and response to traumatic or degenerative events are not fully understood. Previous works have suggested that the development, survival and differentiation of retinal cells are regulated by cell-to-cell contacts and by diffusible molecules produced by the retinal microenvironment [7,19,30]. Our results illustrate the role of IL-2, a prototypic immunoregulatory cytokine, in the development of retinal cells, controlling neuronal survival and differentiation of the cholinergic phenotype.…”

“…It was demonstrated that histotypic interactions between retinal cells are essential for the production of cholinergic differentiation-promoting factors. The cholinergic phenotype is lost in monolayer cultures of chick retinal cells and is maintained in aggregate cultures, perhaps because either the appropriate cell contacts were lost on monolayer cultures or because the production of cholinergic-promoting factors are dis- Sholl-Franco/Marques/Paes-de-Carvalho/ de Araujo tinct in these two different cultures [30]. Indeed, treatment of monolayer cultures with conditioned medium from aggregates has been shown to rescue the cholinergic phenotype, as evaluated by ChAT enzyme activity [30].…”

“…The cholinergic phenotype is lost in monolayer cultures of chick retinal cells and is maintained in aggregate cultures, perhaps because either the appropriate cell contacts were lost on monolayer cultures or because the production of cholinergic-promoting factors are dis- Sholl-Franco/Marques/Paes-de-Carvalho/ de Araujo tinct in these two different cultures [30]. Indeed, treatment of monolayer cultures with conditioned medium from aggregates has been shown to rescue the cholinergic phenotype, as evaluated by ChAT enzyme activity [30]. Our data clearly demonstrated that cultures of retinal cells treated with SCM showed a time-dependent increase in [ 3 H]-choline uptake that reaches a high and stable level after 72 h. However, in control cultures, [ 3 H]-choline uptake increases up to 48 h and decreases after this time, possibly suggesting a decrease in the number of cholinergic neurons.…”

Sympathetic neuronal survival induced by retinal trophic factors

“…Conditioned medium (CM) prepared from either rat retina (Araújo and Linden, 1993) or from chicken retina (Ary-Pires et al, 1997) promote the survival of dissociated retinal ganglion cells in culture. In addition, chicken retina CM promotes the differentiation of cholinergic neurons of the retina (de Mello et al, 1990). Skorupa and Klein (1993) showed that a factor produced by mature retinal cells was involved in mAChR gene transcription.…”

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system

“…Thus, all cholinergic markers can be analyzed in this tissue without interference of eventual contaminants from other regions of the nervous system. In addition mixed (glia and neuron) primary cultures of embryonic avian retina develop many neurochemical properties of the intact tissue, including the cholinergic phenotype (De Mello et al . 1990).…”