Comparison of the expression, transcription and genomic organization of D 2 dopamine receptors in outbred and inbred strains of rat

Abstract

Three outbred (Sprague-Dawley, Wistar and Long-Evans) and five inbred (Brown-Norway, Buffalo, DA, Fisher and Lewis) strains of rat were used to investigate the extent of genetic variation in the expression and organization of the rat D 2 receptor locus. Radioligand binding studies were performed using 125I-iodobenzamide ([ 125I]IBZM), a high-affinity antagonist for D 2 dopamine receptors, to determine the extent of variation in the expression of D 2 receptors in these strains of rat. A comparison of the affinities ( K d = 0.26–0.38nM) and densities (450–580 fmol/mg of protein) of binding sites for [ 125I]IBZM in the striatum of the eight strains of rat did not reveal statistically significant differences. Solution hybridization using 32P-labeled riboprobes complementary to the coding region of the third intracellular loop of the D 2 receptor was used to investigate the extent of variation in transcription of the long (D 2L) and short (D 2S) isoforms of D 2 receptor mRNA in rat striatal tissue. The level of expression of these two mRNA isoforms was found to be invariant in the strains of rats that were examined. The genomic organization of the D 2 receptor locus for each strain of rat was compared using Southern blot hybridization. Southern blots were hybridized with a DNA probe that codes for the D 2L receptor isoform. Restriction fragment lengths were conserved between each rat strain for genomic DNA digested with BamHI, EcoRI, HindIII, PstI and TaqI. Restriction fragment length polymorphisms (RFLPs) were identified when genomic DNA was digested with XbaI or MspI. The XbaI polymorphism was mapped to within 2 kb of the exon coding for the third intracellular loop of the D 2 receptor. Both RFLPs differentiated Sprague-Dawley and Brown-Norway rats from Wistar, Long-Evans, Buffalo, DA, Lewis and Fisher rats. The RFLPs for the rat D 2 receptor locus provide genetic markers that can be used with classic genetic studies to determine whether strain differences in behavior and/or in the response to pharmacologic intervention are determined by genetic elements linked to the D 2 receptor locus.