Brain Research, 97 (1975) 163-166 163 (~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands Tryptophan hydroxylase in the rat brain M. J. BROWNSTE1N, M. PALKOV1TS, J. M. SAAVEDRA AND J. S. KIZER Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.) attd (M.P.) Semmelweis Medical University, Tiizult6-t~ 58 (Hungary) (Accepted June 25th, 1975) Tryptophan hydroxylase (TH) (E.C. 1.14.16.4) is present in the brain and spinal cord and catalyzes the conversion of tryptophan to 5-hydroxytryptophan (5-HTP). The latter compound is decarboxylated by 1-aromatic amino acid decarboxylase to yield serotonin which is thought to function as a neurotransmitter. The distribution of TH among nuclei of the brain should reflect the distribution of serotonergic nerve cells and their processes. Furthermore, since TH is the rate-limiting enzyme in the biosynthesis of serotonin, it should be useful to study the effects of pharmacological, physiological, and surgical manipulations on its activity. As a prelude to such studies the distribution of TH among nuclei of the rat brain has been determined. Nuclei and regions of the hypothalamus 7, hindbrain 6 and limbic system s were removed from frozen sections of the brain with small hollow needles as previously described ~. Pellets from similar areas of two brains were pooled and homogenized in 20 #1 of ice cold 0.05 M Hepes buffer, pH 7.4, containing 0.002 M dithiothreitol. The buffer should be prepared immediately prior to use for best results. Two micro- liters of the homogenate were removed for protein determination according to the method of Lowry et al. 4; bovine serum albumin served as the standard. The remainder of the homogenate was centrifuged at 30,000 x g for 20 min at 2 °C. Ten microliters of the supernatant were then assayed for TH by means of a sensitive radiometric method 3. In the first stage of this assay, 5-HTP is generated from tryptophan. Then the 5-HTP is enzymatically converted into radiolabeled melatonin. The amount of melatonin produced is proportional to the amount of 5-HTP which is made initially. The method is quite sensitive; TH activity can be determined in as little as 50 Fg of brain stem. The levels of TH (ng of 5-HTP/mg protein/h) in areas of the brain appear in Table I. There is a 43-fold difference between the highest concentration measured and the lowest one. The raphe nuclei which contain large numbers of serotonergic cell bodies 1,2, are especially rich in TH. The enzyme is also concentrated in regions of the brain through which dense bundles of serotonergic axons travel. Notable among these is the medial forebrain bundle. Fairly high levels of serotonin have been found in individual nuclei of the hypothalamus 1° and limbic system 9. These nuclei all have substantial amounts of