Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies

• Published in: Neuroscience 1998-02, Vol.82 (3), p.727-738
• Main Authors: XU, Z.-Q, LEW, J. Y, HARADA, K, AMAN, K, GOLDSTEIN, M, DEUTCH, A, HAYCOCK, J. W, HÖKFELT, T
• Format: Article
• Language: English
• Subjects: 1-Methyl-3-isobutylxanthine - pharmacology; Animals; Binding Sites, Antibody; Biochemistry and metabolism; Biological and medical sciences; Catecholamines - metabolism; Central nervous system; Dopamine - physiology; Enzyme Inhibitors - pharmacology; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Immunohistochemistry; Male; Medicin och hälsovetenskap; Neurons - enzymology; Neurons - metabolism; Norepinephrine - physiology; Phosphorylation; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase - antagonists & inhibitors; Tyrosine 3-Monooxygenase - metabolism; Vertebrates: nervous system and sense organs
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(97)00189-9

Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and on forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX-forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal 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 slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre plexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggests that antibodies directed against phosphor