IMPROVED QUANTITATIVE AUTORADIOGRAPHY OF RAT-BRAIN DELTA-OPIOID BINDING-SITES USING [H-3] DSTBULET, A NEW HIGHLY POTENT AND SELECTIVE LINEAR ENKEPHALIN ANALOG

The binding properties of [3H]DSTBULET, a new potent and specific ligand for δ-opioid binding sites, were characterized using slices from rat brain striatum. Specific [3H]DSTBULET binding reached steady-state within 60 min at 20°C, was saturable and reversible and the ligand appeared to interact with a single class of binding sites, characterized as δ by competition experiments. Scatchard analysis of the saturation isotherm plots yielded a KD value of 5.73 nM. At this concentration the specific binding was 94% of the total binding. Unlabeled DSTBULET had a low affinity (K1 = 422 nM) for μ binding sites labeled by [3H]DAGO and at 5 nM [3H]DSTBULET interacted with a maximum of 1.2% of total μ sites. [3H]DSTBULET was therefore used for a detailed quantitative radioautographic analysis of the distribution of rat brain δ-opioid binding sites in order to clarify some inconsistencies in the relative densities of these sites previously reported using either various non specific ligands or [3H]DPDPE, a cyclic enkephalin endowed with high δ selectivity but relatively low affinity. In addition to the claustrum, basolateral and posteromedial cortical amygdaloid nuclei, nucleus accumbens ahd caudate putamen, regions well-known to contain very high levels of δ sites, a [3H]DSTBULET labeling was also found in the intermediodorsal, rhomboid and mediodorsal (lat.) thalamic nuclei, cortex frontoparietal (layers II + III and V + VI), ventroposterior (parvocellular) and reuniens thalamic nuclei, and anterior olfactory nucleus (posterior part). In the thalamus, the mediodorsal, central medial and centrolateral nuclei and the posterior thalamic nucleus group were moderately labeled. The superior colliculus (superficial gray layer), the interpeduncular nucleus, the stria terminalis and the nucleus solitary tractus were moderately labeled. Lower labeling was found in the medial habenula, ventromedial, laterodorsal and ventroposterior (median) thalamic nuclei, dentate gyrus and the pyramidal cell layer of the hippocampus. In the globus pallidus, ventromedial hypothalamic nucleus, ventroposterior (lateralis) thalamic nucleus, substantia nigra, ventral tegmental area, periaqueductal gray matter and dorsal raphe the labeling was scarce but significant. At all levels in the spinal cord, a low labeling was found, restricted to the substantia gelatinosa. Interestingly δ-opioid receptors are highly concentrated in the more recently developed areas of the brain (cortex, neostriatum), where they could be involved in modulation of integrational processes mainly through regulation of aminergic (especially dopamine) pathways. Therefore, owing to its high affinity and good selectivity, [3H]DSTBULET seems to be the most appropriate radiolabeled probe currently available to investigate the δ-opioid receptors and to quantify these binding sites by radioautography with a high sensitivity and accuracy. Accordingly, labeling was found in several areas (ventral tegmental area, substantia nigra, nucleus tractus solitarius) where a mismatch between the site of action of δ-opioids and the presence of δ sites has been previously hypothesized. © 1990.