Quantification of D1B(D5) receptors in dopamine D1A receptor-deficient mice

The unavailability of selective D1A(D1) or D1B(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 D1A null mutants. Coronal brain sections were prepared from the caudal portion of the prefrontal cortex of homozygous or heterozygous D1A 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 D1B(D5) receptors in hippocampus, and also shows that the loss of functional D1A 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).