Molecular cloning studies have defined a family of dopamine D2-like receptors (D2, D3, and D4), which are the products of separate genes. Our previous work has shown that stimulation of dopamine D2-like receptors in cultures of fetal cortical neurons increases the extension and branching of neurites. To determine which D2-like receptors possess morphogenic potentials, a clonal mesencephalic cell line (MN9D) was transfected with D2, D3, or D4 receptor subtypes and treated with quinpirole, an agonist of D2-like receptors, and changes in morphological characteristics were quantitated. Stimulation of D2 receptors increased the number and branching of neurites with little effect on neurite extension; stimulation of D3 and D4 receptors increased the branching and extension of neurites. Similar results were found for primary mesencephalic cultures stimulated with quinpirole. These results suggest that the known D2-like receptors have specific developmental roles in regulating neuronal morphogenesis of dopaminergic pathways.The expression of neurotransmitters prior to the establishment of synaptic circuitry suggests they play a nonsynaptic role in development (1). Recent studies demonstrate that neurotransmitters expressed early are involved in regulating neuronal outgrowth and survival and thus have the potential to control the development of their own synaptogenesis and subsequent adult form. Such neurotrophic roles have been best documented for excitatory amino acids in the developing cortex (2, 3) or hippocampus (4, 5). However, acetylcholine (6), t-aminobutyric acid (7), serotonin (8, 9), and opioids (10) Living cells were photographed after 90-115 hr in culture by using phase-contrast microscopy (Nikon Diaphot) or a digital image-processing system (Image-1; Universal Imaging, Media, PA). Morphological characteristics of individual cells were quantitated at x 1600 by using a computerinterfaced drawing system with a digitizing light pad (Bioquant) as previously described (11,23). Without knowledge of the treatment condition, cells were measured consecutively over two to three dishes. A neurite was defined as a cell process arising from the soma; a neurite branch was defined as a bifurcation of a neurite; neurite length was defined as the distance from the soma to the tip of the longest branch; total neurite extent represented the combined lengths of all neurites and neurite branches per cell (23). Processes shorter than 5 ,um could not be reliably remeasured (23) and were excluded from morphometric analysis. All statistical analy§To whom reprint requests should be addressed.
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