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We established an in vitro system of central serotonergic neurons by culturing dissociated rat embryonic (El4) brainstem cells to 14 days in vitro and monitored the serotonergic neuronal growth by measuring 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents in the cells with...

We established an in vitro system of central serotonergic neurons by culturing dissociated rat embryonic (El4) brainstem cells to 14 days in vitro and monitored the serotonergic neuronal growth by measuring 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents in the cells with hish performance liquid chromatography with electrochemical detection (HPLC-EC). We studied also tile effects of various drugs on the contents of 5-HT and 5-HIAA, confirming in vivo reports. The 5-HT content (13 ng/mg protein) and 5-HT turnover rate (17 pmol/mg protein/h) at 14 days in vitro were in good agreement with those reported in the adult rat brain. The 5-HT content was more easily depleted with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor than with NSD 1015 (3-hydroxybenzylhydrazine), an aromatic L-amino acid decarboxylase (AADC) inhibitor. Incubation of the cultures with tryptophan or 5-hydroxytryptophan increased the rate of serotonin formation implying that neither tryptophan hydroxylase nor AADC is saturated with its amino acid substrate in this in vitro system . The 5-HT content was depleted by reserpine. The 5-HT and 5-HIAA contents were increased and decreased, respectively, by monoamine oxidase inhibitors. All the above results indicate that the biochemical properties of the central serotonergic neurons in this culture system reflect reliably those of central serotonergic neurons in vivo. We suggest that measuring 5-HT and 5-HIAA contents in the primary cultured dissociated brainstem-cells with HPLC-EC is useful in the study of pharmacology as well as toxicolgy of the central serotonergic neurons.

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