Activity-Dependent Phosphorylation of Tyrosine Hydroxylase in Dopaminergic Neurons of the Rat Retina

Abstract

We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABA A and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves.

Neurochemical characterization of tyrosine hydroxylase-immunoreactive interneurons in the developing rat cerebral cortex

“…Because TH activity is regulated by phosphorylation, we examined whether the TH in cortical neurons was phosphorylated by using an antibody against TH phosphorylated at Ser19 (TH phosphoserine19), one of three serine residues, including Ser31 and Ser40, whose phosphorylation is regulated physiologically (Xu et al, 1998;Lew et al, 1999;Witkovsky et al, 2004;Bobrovskaya et al, 2004). Brains from P16 to P40 rats were examined: P16 (n=3 brains), P18 (n=2), P20 (n=1), P24 (n=2), P34 (n=3) and P40 (n=1).…”

“…Additionally, in the developing rat, labeling of the cortical TH-IR neurons was insensitive to 6-hydroxydopamine toxicity, and no endogenous catecholamines were detected in cortical somata (Berger et al, 1985). To address whether or not TH in cortical neurons is active, we examined the phosphorylation of TH at Ser19 because a variety of physiological stimuli induce TH phosphorylation at this residue and at Ser31 and/or Ser40 (Lew et al, 1999;Bobrovskaya et al, 2004;Witkovsky et al, 2004). The majority of cortical interneurons identified by the phosphorylation-independent TH antibody also contained TH phosphorylated at Ser19 in the postnatal and adult rat.…”

“…No immunoreactivity for active caspase 3 was observed in cortical TH cells. Because phosphorylation regulates TH enzyme activity (Xu et al, 1998;Lew et al, 1999;Witkovsky et al, 2004;Bobrovskaya et al, 2004), we assessed TH activity and the possibility of L-3,4-dihydroxyphenylalanine (L-DOPA) production in these cells by immunostaining for one of the phosphorylated forms of TH. The majority of cortical TH-IR cells were colabeled by an antibody against phosphorylated TH.…”

A Specific Survival Response in Dopamine Neurons at Most Risk in Parkinson's Disease

“…More than three coverslips from more than two preparations were used for each experimental group. Activated DA neurons were visualized by phospho-Ser-31 TH (Witkovsky et al, 2004). For analyses of cleaved-caspase-3, phosphoSer-31 TH and phospho-extracellular signal-regulated kinase 1 ⁄2 (Erk1/2) numbers per coverslip (10 images taken with 10ϫ lens were combined) were compared.…”

A New Perspective for Parkinson’s Disease: Circadian Rhythm

“…Many promising initiations have extended light treatment to a wider disease spectrum [106]. Dopamine is a chemical messenger for light adaptation [64], and exposure to light could increase retinal dopamine activity [107]. There is a sign that, circadian light entrainment is increasingly applied to PD patients attempting to reset the biologic clock.…”