[³H]4‐(dimethylamino)‐N‐(4‐(4‐(2‐methoxyphenyl)piperazin‐1‐yl) butyl)benzamide: A selective radioligand for dopamine D₃ receptors. II. Quantitative analysis of dopamine D₃ and D₂ receptor density ratio in the caudate‐putamen

Abstract

4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-benzamide (WC-10), a N-phenyl piperazine analog, displays high affinity and moderate selectivity for dopamine D3 receptors versus dopamine D2 receptors (Chu et al. [2005] Bioorg Med Chem 13:77–87). In this study, WC-10 was radiolabeled with tritium (specific activity = 80 Ci/mmol), and quantitative autoradiography studies were conducted using rhesus monkey and Sprague-Dawley rat brain sections. Kd values for the binding of [3H]WC-10 to D3 receptors obtained from quantitative autoradiography with rhesus monkey and rat brain sections are in agreement with Kd values obtained from cloned human and rat receptors (Xu et al. [2009] Synapse 63:717-728). The D2 selective antagonist [3H]raclopride binds with 11-fold higher affinity to human HEK D2L (Kd = 1.6 nM) than HEK D3 (Kd = 18 nM) receptors; [3H]raclopride binds to rat Sf9 rD2L receptors with a Kd of 6.79 nM, a value that is 4-fold lower than binding to human HEK D2L receptors and 2.5-fold higher than binding to rat Sf9 rD3 receptors. In vitro quantitative autoradiography studies with [3H]WC-10 and [3H]raclopride were conducted on adult rat and rhesus monkey brain sections. A mathematical model for calculating the absolute densities of dopamine D2 and D3 receptors based on the in vitro receptor binding data of [3H]WC-10 and [3H]raclopride was developed.

Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats

“…1, 2g). Quantitative analysis was performed with the program Beta-Vision Plus (BioSpace, France), and the absolute densities of dopamine D 2 and D 3 receptors using the D 3 -preferring radioligand [3H] WC-10 and the D 2 /D 3 ligand, [ 3 H]raclopride were calculated as previously described [14, 17]. …”

“…The apparent binding density for each receptor-bound radioligand was calculated using the specific activity of the radioligand expressed as fmol/mg tissue, as previously described [14]. The experimenter was blinded to all conditions during the analysis.…”

“…DJ-1 and Pink1 knockout rats demonstrate significant motor deficit and age-dependent neuronal loss in SNpc, consistent with human disease. In this study of post-mortem rat brains, we investigated the expression of an array of genes associated with PD and determined the absolute densities of dopamine D 2 and D 3 receptors using the D 3 receptor-specific ligand [ 3 H] WC-10 and the D 2 /D 3 ligand [ 3 H]raclopride [14]. We also measured the densities of dopamine D 1 receptor, DAT and VMAT2 in the striatum of DJ-1 and Pink1 knockout rats by quantitative autoradiography.…”

PET imaging for attention deficit preclinical drug testing in neurofibromatosis-1 mice

“…20-micron coronal sections were generated from flash-frozen brains and processed for autoradiography as previously described (Xu et al, 2009; Xu et al, 2010). Sections were incubated for 60 min at room temperature with [ 3 H]-SCH23390 (D1 receptors), [ 3 H]-raclopride (D2 receptors), [ 3 H]-WC-10 (D3 receptors), [ 3 H]-DTBZ (VMAT2), or [ 3 H]-Win 35428 (DAT) (Frey et al, 1997; Savasta et al, 1986; Xu et al, 2010).…”

“…Sections were incubated for 60 min at room temperature with [ 3 H]-SCH23390 (D1 receptors), [ 3 H]-raclopride (D2 receptors), [ 3 H]-WC-10 (D3 receptors), [ 3 H]-DTBZ (VMAT2), or [ 3 H]-Win 35428 (DAT) (Frey et al, 1997; Savasta et al, 1986; Xu et al, 2010). Non-specific binding was determined following the addition of 1μM (+)-butaclamol, 1μM eticlopride, 1μM tetrabenazine, or 1μM nomifensine, respectively.…”