MULTIPLE FLUORESCENT LIGANDS FOR DOPAMINE-RECEPTORS .2. VISUALIZATION IN NEURAL TISSUES

Selective dopamine receptor ligands, (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high affinity D1 receptor antagonist SCH 23390, the high affinity D2 receptor antagonist N-(p-aminophenethyl)-spiperone or NAPS, and the D2 selective agonist, 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin or PPHT were chemically coupled to the fluorescent compounds, Bodipy, Cascade blue, coumarin, fluorescein, rhodamine, or Texas red. The utility of the 6 fluorescent moieties linked to the 3 dopamine receptor binding ligands for anatomical study of regional and cellular distribution patterns of the two dopaminergic receptor subtypes has been assessed in frozen sections of the rat striatum and compared to our previous report using the rhodamine-labeled antagonists. The regional staining for the two dopaminergic receptor binding sites supports previous work using in vitro receptor autoradiographic analyses; the D1 receptor binding was more robust than that of D2 receptors in the caudate nucleus. The cellular element which most frequently expressed striatal D1 binding sites had a medium-diameter cell body. Medium-sized cells also exhibited fluorescence for the D2 binding site, as did a much larger diameter element; potentially the cholinergic interneuron of the caudate nucleus. The pharmacological specificity for each of the different D1 fluorescent antagonist ligands in the tissues was determined by competition with 100-fold excess of unlabeled SCH 23390 (non-specific binding), spiroperidol (binding selectivity), the stereoactive paired isomers of butaclamol, and the serotonin 5-HT2 receptor antagonist ketanserin. The same criteria were used to assess the different D2 fluorescent agonist and antagonist ligand derivatives. The anatomical efficacy of these novel ligands was determined using selective dichroic filters to stimulate the fluorescent moieties in the optimal excitation wavelength, and the amount of fluorescent dopamine receptor binding was photographically measured and contrasted for each of the newly synthesized fluoroprobes. Using the most pharmacologically specific and anatomically efficient of these novel fluoroprobes, we determined the localization pattern of the D1 and D2 dopamine receptor binding sites in tissues reported to exhibit both subtypes of the receptor. The cellular distribution of the dopamine receptor binding sites was determined concurrently using fluoroprobes in the forebrain, mesencephalon, pituitary, retina, and superior cervical ganglion of the rodent, and bovine adrenal medullary chromaffin cells were examined using the rhodamine-labeled antagonists.