IMMUNOHISTOCHEMICAL STUDIES ON PHOSPHORYLATION OF TYROSINE-HYDROXYLASE IN CENTRAL CATECHOLAMINE NEURONS USING SITE-SPECIFIC AND PHOSPHORYLATION STATE-SPECIFIC ANTIBODIES

Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, wer e applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanth ine, IBMX) and on forskolin on formalin-perfused rat brains. Four anti sera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyros ine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse mono clonal antibody to (iv) native tyrosine hydroxylase. In the in vitro s tudies THS40p-like immunoreactivity was not observed unless slices wer e treated with IBMX-forskolin after which a dense fibre network was fo und in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Altho ugh these cells were pan-tyrosine hydroxylase-positive, several of the m were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclo nal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the serine 40-phosphorylated form of tyrosine hydroxylase. Treated sli ces did not show any THS40p-immunoreactive cell bodies in the dopamine rgic All cell group and only a few, weakly fluorescent neurons were ob served in locus coeruleus. However, a sparse fibre plexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the pe rfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 are a, and in noradrenergic locus coeruleus neurons, whereas THS19p-like i mmunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum a nd the tyrosine hydroxylase-monoclonal antibody. In forebrain areas kn own to be innervated by mesencephalic dopamine neurons, no THS40p-posi tive fibres were observed, whereas THS19p-immunoreactive fibres were f ound in subregions of. the striatum, olfactory tubercle and nucleus ac cumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggest that antibodies directed against phosphorylaled forms of tyro sine hydroxylase can be used to evaluate the state of tyrosine hydroxy lase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo.