Autoradiographic localization of opioid binding sites combined with immunogold detection of Leu-enkephalin, crustacean hyperglycaemic hormone and moult inhibiting hormone at the electron microscopic level in the sinus gland of the shore crab, Carcinus maenas.

Double labelling experiments were performed on the same tissue section at the electron microscopic level, in order to show the involvement of the opioid leucine-enkephalin (Leu-enk) in the modulation of crustacean hyperglycaemic hormone (CHH) mobilization. Both neuropeptides were stored in distinct axon terminals of the sinus gland of Carcinus maenas. A post-embedding immunogold cytochemical technique for Leu-enk, CHH and the CHH neurohormone related moult inhibiting hormone (MIH) was combined with a scintillator intensified autoradiographic method to demonstrate binding of the opioid antagonist [3H] naloxone. Ultrathin sections were successively incubated with antisera against Leu-enk, CHH or MIH, and the corresponding colloidal gold labelled antisera, followed by autoradiographic processing. At the ultrastructural level [3H] naloxone binding sites were easily recognized by their silver tracks after development. Opioid binding sites for [3H] naloxone were visualized only at membranes of CHH-containing axon terminals. These results provide morphological evidence for direct enkephalinergic control of CHH containing neurons in the sinus gland of C. maenas and are furthermore the first autoradiographic demonstration of opioid binding sites in the nervous system of invertebrates.