The serotonin signalling system is involved in many cortical brain functions. Its effective extracellular concentrations are controlled by release and uptake systems. In this proposal we plan to study further the high affinity, fluoxetine-sensitive serotonin uptake system we have previously described in primary astrocyte cultures. We will now focus on its development as a function of the age of the animals from which the cultures are derived (E16-P6), to see if we can get an indication when this property is developmentally established in situ. We will also further examine the influence of serum and different defined factors, since we have found that fetal bovine serum increases serotonin uptake in astrocyte cultures. We will attempt to identify the factor or class of factors in serum responsible. We will also examine the effects of co-culture with neurons to see if neuronal influences can stimulate the serotonin uptake system. These studies should give us some clue as to the mechanisms of induction of serotonin uptake. To find evidence that fluoxetine-sensitive serotonin uptake is present in mature astrocytes in situ we will evaluate uptake in astrocytes prepared by density gradient centrifugation or tissue-print techniques, and do further studies to localize uptake sites in situ after intraventricular injection of [3H] serotonin. Uptake will be measured using [3H] serotonin for astrocytes isolated by the density gradient method, and autoradiography to study uptake both in these cells and cells obtained by the tissue-print method. Autoradiography enables uptake to be studied at the single cell level and will be coupled with immuno-staining. We will see if fluoxetine- sensitive serotonin uptake occurs in type 1 and type 2 astrocytes in culture and will use other markers for oligodendrocytes, neurons and microglia to specifically identify all the cells that may take up serotonin in mixed cultures. We will extend our studies to evaluate the presence of serotonin receptors in cultured and bulk-isolated astrocytes as a function of the same conditions used to study serotonin uptake. There are only a few very preliminary reports on the presence of serotonin receptors in primary astrocyte cultures and because of the importance of these receptor systems in the brain and the numerous receptor systems already convincingly demonstrated on astrocytes in culture, the existence of any of the multiple subtypes of serotonin receptors needs to be critically evaluated. We propose that some of the variability seen in the other studies could be due to effects of different growth conditions. These studies should enable us to say when uptake systems develop in astrocytes, what influences this development and whether and how serotonin receptors are localized in astrocytes. From a broader viewpoint they should provide information needed to critically evaluate what role astrocytes may play in the all-important CNS serotonin signalling system.
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