Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons.

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.