Quantification of D_1B (D₅) receptors in dopamine D_1A receptor-deficient mice

The unavailability of selective D_1A (D₁) or D_1B (D₅) dopamine receptor ligands has prevented the direct localization of binding sites for these receptors. Thus, receptor autoradiography with long exposure times was used to detect minor D₁-like binding sites in the brains of D_1A null mutants. Coronal brain sections were prepared from the caudal portion of the prefrontal cortex of homozygous or heterozygous D_1A knockout mice or wildtype mice, and labeled with the D₁ receptor antagonist [³H]-SCH23390. Slides were dried, and apposed to film with polymer-calibrated standards for 90 days to allow visualization of any low abundance binding sites. No binding was detected in most regions of homozygote (-/-) mouse brains that have high densities of D₁ binding in wildtype mice (e.g., the striatum, nucleus accumbens, olfactory tubercles or amygdala). Conversely, small, but detectable amounts of D₁-binding were measured in the hippocampus, albeit with a density less than the lowest standard (ca. 20 fmol/mg). Saturation binding of [³H]-SCH23390 in hippocampal homogenates from homozygous mice confirmed a B_max of 12.3 fmol/mg protein with a K_D of 0.57 nM. The current work demonstrates directly the presence of D_1B (D₅) receptors in hippocampus, and also shows that the loss of functional D_1A gene products almost completely eliminates detectable D₁-binding sites in striatum, as well as in some regions (e.g., the amygdala) where a non-adenylyl cyclase coupled D₁ receptor has been reported. This indicates that these non-adenylyl cyclase coupled D₁-like receptors represent alternate signaling pathways rather than novel gene products(s).