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

The incubation of cultured fetal mesencephalic neurons with Δ9-tetrahydrocannabinol (Δ9-THC) increased the activity of tyrosine hydroxylase (TH) and this increase was reversed by SR141716A, a specific antagonist for cannabinoid CB1 receptors. In the present work, we extended these earlier observations by addressing two objectives. First, we characterized at a molecular level the presence of CB1 receptors in cultured fetal mesencephalic neurons using two strategies: (i) analyzing the presence of CB1 receptor gene transcripts by Northern blot, and (ii) measuring []WIN-55,212-2 binding in membrane fractions obtained from these cells, as well as evaluating the potential increase in []-guanylyl-5′-O-(γ-thio)-triphosphate ([]GTPγS) binding caused by the activation of these receptors with WIN-55,212-2, a synthetic agonist. Northern blot analyses demonstrated the presence of small, but measurable levels of CB1 receptor mRNA in cultured fetal mesencephalic neurons. The presence of these transcripts was accompanied by the presence of receptor binding protein, as revealed by a small, but specific, []WIN-55,212-2 binding in membrane fractions obtained from these cells. These CB1 receptors are coupled to GTP-binding proteins, as the incubation of membrane fractions obtained from these cells with WIN-55,212-2 slightly, but significantly increased []GTPγS binding. This fact indicated the existence, not only of receptor binding, but also of a functional receptor transduction pathway. As a second objective, we examined the potential colocalization of CB1 receptors and TH in these cells by double-labelling immunocytochemistry. We also determined by Western blotting whether the previously observed Δ9-THC-induced increase in TH activity was accompanied by increased TH protein levels. Cultured fetal mesencephalic neurons exhibit diverse cell phenotypes, with CB1 receptors localized only on TH-containing neurons. In addition, we found that the incubation of fetal mesencephalic neurons with medium containing Δ9-THC increased TH protein levels, in concordance with the previously reported increase in TH activity. Collectively, our results support the notion that CB1 receptors are present in cultured fetal mesencephalic TH-containing neurons, despite their absence in the corresponding neurons in the adult brain. Thus, it is likely that the effects of cannabinoids on TH activity are direct. All this data strengthen the view that cannabinoid receptors are atypically located during brain development and that they might play an important role during this process, in particular on the phenotypical expression of TH-containing neurons.