In situ examination of tyrosine hydroxylase activity in the rat locus coeruleus using (3 ',5 ')-[H-3(2)]-alpha-fluoromethyl-tyrosine as substrate of the enzyme

Tyrosine hydroxylase (TH) activity can be modified by changes in the specif ic activity of the enzyme (SA(TH)) or in the levels of active enzyme. We de veloped a methodology making it possible to measure with excellent anatomic al resolution TH enzymatic activity and TH protein quantity by quantitative autoradiography and immunoautoradiography, respectively, from adjacent sec tions taken at serial intervals along the longitudinal extent of a same bra in. SA(TH) was estimated by the slope of linear regressions established bet ween TH activity and TH quantity measured at each anatomical plane. To eval uate TH activity, we used (3',5')-[(3)Ha(2)]-(D, L)-alpha-fluoromethyltyros ine [H-3(2)]-MFMT, which is transformed by TH to [H-3]-MFM-dopa, a potent a nd irreversible substrate for aromatic amino acid decarboxylase. We found t hat the SA(TH) in the cell body area of the LC (PKA) was 48% lower than tha t evaluated in the surrounding pericoerulean neuropil (PCN). In the PCN, 22 % only of TH level exhibited a level of enzymatic activity above threshold. We also examined how SA(TH) was distributed in the LC 15 min and 3 days af ter RU 24722 treatment, a potent phasic and tonic activator of TH enzyme in noradrenergic neurons. Two distinct mechanisms have been observed: the sho rt-term effect was due to an increase in the SA(TH) in the PKA only, while the long-term effect was mainly caused by an increase in the number of acti ve TH proteins in the PCN. These results suggest that the fine regulation o f TH activity which occurs in the different compartments of LC neurons may be critical in the functions involving the LC. (C) 2000 Wiley-Liss, Inc.