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Dopamine (DA)-producing neurons in the ventral midbrain are generated from a specified neuronal lineage and form selective axonal pathways that mediate multiple CNS functions. Expression of the gene encoding tyrosine hydroxylase (TH), which is a key enzyme of catecholamine biosynthesis, is regulated during the development of midbrain DA neurons. In the present study, we report the developmental regulation and cell type specificity of TH gene promoter in the ventral midbrain by using a green fluorescent protein (GFP) reporter system. Transgenic mice were generated that express GFP in the majority of midbrain DA neurons under the control of the 9-kb upstream region of the rat TH gene. At an early embryonic stage, GFP expression was induced in the developing DA neurons, and the expression was then markedly down-regulated at later embryonic stages. However, the expression was reactivated and approached the adult levels during early postnatal development. These developmental changes in GFP expression patterns suggest the presence of multistep regulatory mechanisms for TH gene expression during DA neuron development. The TH promoter appears to possess transcriptional elements at least necessary for the induction of TH expression at the early embryonic stage and its reactivation during the post-natal development.

The aim of this study was to elucidate electrophysiologically the actions of dopamine and SKF38393, a D1-like dopamine receptor agonist, on the membrane excitability of striatal large aspiny neurons (cholinergic interneurons). Whole-cell and perforated patch-clamp recordings were made of striatal cholinergic neurons in rat brain slice preparations. Bath application of dopamine (1-100 microM) evoked a depolarization/inward current with an increase, a decrease, or no change in membrane conductance in a dose-dependent manner. This effect was antagonized by SCH23390, a D1-like dopamine receptor antagonist. The current-voltage relationships of the dopamine-induced current determined in 23 cells suggested two conductances. In 10 cells the current reversed at -94 mV, approximately equal to the K+ equilibrium potential (EK); in three cells the I-V curves remained parallel, whereas in 10 cells the current reversed at -42 mV, which suggested an involvement of a cation permeable channel. Change in external K+ concentration shifted the reversal potential as expected for Ek in low Na+ solution. The current observed in 2 mM Ba2+-containing solution reversed at -28 mV. These actions of dopamine were mimicked by application of SKF38393 (1-50 microM) or forskolin (10 microM), an adenylyl cyclase activator, and were blocked by SCH23390 (10 microM) or SQ22536 (300 microM), an inhibitor of adenylyl cyclase. These data indicate, first, that dopamine depolarizes the striatal large aspiny neurons by a D1-mediated suppression of resting K+ conductance and an opening of a nonselective cation channel and, second, that both mechanisms are mediated by an adenylyl cyclase-dependent pathway.

Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/ AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation.The RET proto-oncogene encodes a receptor tyrosine kinase the ligands of which are members of the glial cell line-derived neurotrophic factor (GDNF) 1 protein family, including GDNF, neurturin, artemin, and persephin (1, 2). These neurotrophic factors signal through multisubunit receptor complexes consisting of RET and glycosylphosphatidylinositol-anchored coreceptor called GDNF family receptor ␣1-4(GFR␣1-4). It turned out that the GDNF/RET signaling plays an important role in survival or differentiation of various neurons as well as kidney organogenesis (3-6). In addition, RET mutations are responsible for development of several human diseases such as papillary thyroid carcinoma, multiple endocrine neoplasia types 2A and 2B, and Hirschsprung's disease (1, 2).RET can activate a variety of intracellular signaling pathways, including RAS/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, and phospholipase C ␥ pathways (1, 2). As is the case for other receptor tyrosine kinases, phosphorylated tyrosine residues in RET represent docking sites for several adaptor and effector molecules. For example, tyrosines at codons 905, 1015, 1062, and 1096 were identified as docking sites for Grb7/Grb10, phospholipase C ␥, Shc/Enigma/Frs2/IRS-1/Dok, and Grb2, respectively (7-17). In particular, phosphorylation of tyrosine 1062 is crucial for activation of major intracellular signaling pathways, including the RAS/ERK, PI3K/AKT, JNK, p38 MAPK, and ERK5 pathways (15, 18 -21). RET can also activate Rho family GTPases, including Rho, Rac, and Cdc42 that are involved in reorganization of the actin ...

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