title: Noninvasive nuclear imaging enables the in vivo quantification of striatal dopamine receptor expression and raclopride affinity in mice.
abstract: The increasing use of genetically engineered mice as animal models of human disease in biomedical research, latest advances in imaging technologies, and development of novel, highly specific radiolabeled biomarkers provide great potential to study receptor expression and gene function in vivo in mice. (11)C-raclopride is a widely used PET tracer to measure striatal D(2) receptor binding and was used to test the feasibility of the multiple-ligand-concentration receptor assay for D(2) receptor quantification.
  
  
  METHODS
  Mice underwent a total of 4 scans with decreasing specific activities from 141 to 0.4 GBq/μmol, corresponding to (11)C-raclopride injected doses of 2.4 to 1,274 nmol/kg, using either a standard bolus injection protocol (n = 12) or a bolus-plus-constant infusion protocol to attain true equilibrium conditions (n = 7). Receptor occupancy was plotted as a function of raclopride dose, and D(2) receptor density and raclopride affinity were calculated using linear and nonlinear regression analysis, respectively. In addition, we used ex vivo autoradiography, a more spatially accurate imaging technology, to validate the in vivo PET measurements, and we performed test-retest experiments to determine the reproducibility and reliability of the PET-derived measures.
  
  
  RESULTS
  The receptor occupancy curves showed that an injected tracer dose of 4.5 nmol/kg induces approximately 10% receptor occupancy, whereas 1% receptor occupancy will be achieved at tracer doses of approximately 0.45 nmol/kg. Using the bolus injection protocol and nonlinear regression analysis, we determined that the average D(2) receptor density was 9.6 ± 1.1 pmol/mL, and the apparent raclopride affinity was 5.0 ± 0.6 pmol/mL. These values agreed well with those obtained at true equilibrium conditions. In contrast, linear Scatchard analysis did not lead to the expected linear relationship because nonsaturable binding was observed at high raclopride concentrations, and thus, it seems to be unsuitable for quantitative (11)C-raclopride analysis in mice.
  
  
  CONCLUSIONS
  Our data showed that the tracer mass, if higher than 4 nmol/kg, can strongly affect binding parameter estimations and must be considered when performing kinetic analysis, specifically in mice. We also demonstrated that the in vivo determination of D(2) receptor density and raclopride affinity is feasible in mice using multiple-injection protocols and nonlinear regression analysis.
authors.affiliation: Department of Preclinical Imaging and Radiopharmacy, Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, Eberhard-Karls University of Tuebingen, Tuebingen, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Fischer
authors.forename: Vesna
authors.initials: V
authors.lastname: Sossi
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.forename: Martin
authors.initials: M
authors.lastname: Thunemann
authors.forename: Florian C
authors.initials: FC
authors.lastname: Maier
authors.forename: Martin S
authors.initials: MS
authors.lastname: Judenhofer
authors.forename: Julia G
authors.initials: JG
authors.lastname: Mannheim
authors.forename: Gerald
authors.initials: G
authors.lastname: Reischl
authors.forename: Bernd J
authors.initials: BJ
authors.lastname: Pichler
mesh_terms: D000818:Animals; D001345:Autoradiography; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008297:Male; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D017072:Neostriatum / Q000378:metabolism*; D011485:Protein Binding; D020891:Raclopride / Q000378:metabolism*; D011954:Receptors, Dopamine / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D011954:Receptors, Dopamine; D020891:Raclopride