Competitive displacement binding assay on rat brain sections and using a β-imager: Application to μ-opioid ligands

A new approach of competitive displacement binding assay using brain sections and a β-imager is presented to estimate binding parameters such as affinity and selectivity of new compounds or to characterize receptor families or subtypes of receptors in small brain regions. This method includes a preliminary saturation assay intended to define the optimal concentration of displaceable radio-labeled ligand followed by the determination of displacement constants (IC50 and Ki) in cerebral regions rich in studied receptor. The technique application was demonstrated in seven rat brain structures, using displacement of the selective tritiated μ-opioid ligand [3H]-DAMGO by six opioid ligands: a specific agonist (DAMGO), less specific agonists (morphine, remifentanil), a non-specific antagonist with good affinity for μ receptors (naloxone) and ligands specific of other opioid subtypes (naltrindole, U50.488). Radioactivity counts were collected during 48 h. The assay-validation was performed by measuring intra- and inter-assay variation on determinations and by comparing presently obtained Ki values with data from recognised methodologies. Both prove the accuracy of the proposed method.