IMMUNOHISTOCHEMICAL STUDIES ON PHOSPHORYLATION OF TYROSINE-HYDROXYLASE IN CENTRAL CATECHOLAMINE NEURONS USING SITE-SPECIFIC AND PHOSPHORYLATION STATE-SPECIFIC ANTIBODIES
Zq. Xu et al., IMMUNOHISTOCHEMICAL STUDIES ON PHOSPHORYLATION OF TYROSINE-HYDROXYLASE IN CENTRAL CATECHOLAMINE NEURONS USING SITE-SPECIFIC AND PHOSPHORYLATION STATE-SPECIFIC ANTIBODIES, Neuroscience, 82(3), 1998, pp. 727-738
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.