A microassay for simultaneous measurement of in vivo rates of tryptophan hydroxylation and levels of serotonin in discrete brain nuclei

Abstract A sensitive procedure for simultaneous determination of in vivo rates of tryptophan hydroxylation and levels of serotonin (5-HT) in discrete rat brain nuclei is described. Rates of tryptophan hydroxylation are estimated by 5-hydroxytryptophan (5-HTP) accumulation following l -aromatic amino acid decarboxylase inhibition by R04-4602/1. 5-HTP is separated from 5-HT by liquid cation exchange after which both 5-HTP and 5-HT are measured by a sensitive radioenzymatic assay. Following decarboxylase inhibition 5-HTP accumulates over 30 min in 6 brain nuclei examined, with negligible levels of 5-HTP being measured in the absence of decarboxylase inhibition. 5-HT levels do not change significantly up to 45 min after decarboxylase inhibition. Comparison of rates of tryptophan hydroxylation determined in 12 different microdissected rat brain areas reveals a greater rate of 5-HT biosynthetic activity in raphe nuclei containing 5-HT cell bodies than in nuclei containing 5-HT terminals. Pretreatment with para -chlorophenylalanine markedly reduces both 5-HTP accumulation and 5-HT levels in the nucleus raphe dorsalis. With this procedure quantities as little as 10 pg of both 5-HTP and 5-HT can be measured, allowing estimation of in vivo serotonin biosynthesis in microgram quantities of brain tissue.