Cannabinoid CB1 receptors colocalize with tyrosine hydroxylase in culturedfetal mesencephalic neurons and their activation increases the levels of this enzyme

The incubation of cultured fetal mesencephalic neurons with Delta(9)-tetrah ydrocannabinol (Delta(9)-THC) increased the activity of tyrosine hydroxylas e (TH) and this increase was reversed by SR141716A, a specific antagonist f or cannabinoid CB, receptors. In the present work, we extended these earlie r observations by addressing two objectives. First, we characterized at a m olecular level the presence of CB, receptors in cultured fetal mesencephali c neurons using two strategies: (i) analyzing the presence of CB, receptor gene transcripts by Northern blot, and (ii) measuring [H-3]WIN-55,212-2 bin ding in membrane fractions obtained from these cells, as well as evaluating the potential increase in [S-35]-guanylyl-5'-O-(gamma-thio)-triphosphate ( [35S]GTP gamma S) binding caused by the activation of these receptors with WIN-55,212-2, a synthetic agonist. Northern blot analyses demonstrated the presence of small, bur measurable levels of CB, receptor mRNA in cultured f etal mesencephalic neurons. The presence of these transcripts was accompani ed by the presence of receptor binding protein, as revealed by a small, but specific, [H-3]WIN-55,212-2 binding in membrane fractions obtained from th ese cells. These CB, receptors are coupled to GTP-binding proteins, as the incubation of membrane fractions obtained from these cells with WIN-55.212- 2 slightly, bur significantly increased [S-35]GTP gamma S binding. This fac t indicated the existence, not only of receptor binding, but also of a func tional receptor transduction pathway. As a second objective, we examined th e potential colocalization of CB, receptors and TH in these cells by double -labelling immunocytochemistry. We also determined by Western blotting whet her the previously observed Delta(9)-THC-induced increase in TH activity wa s accompanied by increased TH protein levels. Cultured fetal mesencephalic neurons exhibit diverse cell phenotypes, with CB, receptors localized only on TH-containing neurons. In addition, we found that the incubation of feta l mesencephalic neurons with medium containing Delta(9)-THC increased TH pr otein levels, in concordance with the previously reported increase in TH ac tivity. Collectively, our results support the notion that CB, receptors are present in cultured fetal mesencephalic TH-containing neurons, despite the ir absence in the corresponding neurons in the adult brain. Thus, it is lik ely that the effects of cannabinoids on TH activity are direct. All this da ta strengthen the view that cannabinoid receptors are atypically located du ring brain development and that they might play an important role during th is process, in particular on the phenotypical expression of TH-containing n eurons. (C) 2000 Elsevier Science B.V. All rights reserved.