Based on the general hypothesis that microheterogeneity of dopamine receptors prevents prediction of functional effects simply on the basis of D1 or D2 biochemical characteristics, we propose to study a high affinity, specific D1-like recognition site, not previously characterized, that can mediate profound antidopaminergic behavioral effects. To accomplish this objective, [3H]-SCH23390 has been synthesized, and its chemical and biological identity to the authentic SCH23390 verified. We have demonstrated that there is a persistence of SCH23390 in brain, and prolonged behavioral and biochemical effects, hours after the blood concentration of drug is undetectable, directly opposite to our data with other neuroleptics. Thus, the metabolism of SCH23390 and its time course of distribution (especially in discrete brain regions) will be thoroughly studied. Using classical radioligand methods, the interactions of [3H]-SCH23390 with its binding sites in crude brain membrane fractions will be characterized, utilizing both in vitro and in vivo binding methods, and compared to other ligands which are believed to bind to sites usually considered to be dopamine receptors. The properties of the iodine analog of SCH23390 will be evaluated, and if similar to SCH23390, [125I]-SCH23390 will be used as a very high specific activity tool for receptor studies. Autoradiographic techniques will be used to map the distribution of [125I]-SCH23390 or [3H]-SCH23390 binding sites in brain after administration of the radioligands in vivo, or treatment of brain slices in vitro. Since we have shown that SCH23390 will bind tenaciously to its physiologically important receptor(s), the subcellular localization of the radioactive drug in cellular fractions prepared from brain regions of rats treated in vivo with [125I]-SCH23390 or [3H]-SCH23390 will be determined. The effects of lesioning dopamine pathways or chronic treatment with antipsychotics on SCH23390 recognition sites will also be examined. If these studies are successful, solubilization and purification of the SCH23390 binding sites will be attempted, relying heavily on affinity chromatography to take advantage of the apparent high affinity of these binding sites.
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