66 Brain Research. 531 (1990) 66--71 Elsevier BRES 15955 Evidence for an enkephalinergic system in the nervous system of the pond snail, Lymnaea stagnalis Michael K. Leung 1, Harry H. Boer 2, Jan van Minnen 2, Jonathan Lundy 1 and George B. Stefano 1 1Departments of Chemistry and BiologicalSciences, State Universityof New York, Collegeat Old Westbury, Old Westbury, NY (U.S.A.) and 2Biological Laboratory, Free University,Amsterdam (The Netherlands) (Accepted 24 April 1990) Key words: Enkephalin; Pond snail; [3H]Etorphine; Lymnaea stagnalis Evidence for the presence of an enkephalinergic system in the ganglia of the pond snail, Lymnaea stagnalis, has been obtained with 3 experimental approaches. Scatchard analysis with [3H]etorphine reveals a monophasic high-affinity opiate binding site (Ka 2.3 nM) which is naloxone-sensitive. Immunocytochemical localization of Met- and Leu-enkephalin-like substances as well as a-MSH- and ACTH-Iike materials was demonstrated within specific populations of neurons. Substances with Met- and Leu-enkephalin and Met-enkephalin sulfoxide RIA reactivities were detected also in HPLC fractions corresponding to the retention times of authentic enkephalin standards. Together, the results provide strong evidence for the presence of enkephalinergic mechanisms in the nervous system of Lymnaea stagnalis. Additionally, the report provides indirect evidence for the existence of a macromolecular opioid precursor. This enkephalinergic system shows striking similarities to opioid mechanisms found in vertebrates and bespeaks a common evolutionary origin. INTRODUCTION Since the discovery of endogenous opiate-like sub- stances in animal tissues many studies have been per- formed which illustrate the importance of these neuro- peptides in mechanisms that transcend the original nociceptive/analgesic concept. Their importance is fur- ther demonstrated by their discovery in invertebrates 1°. These findings support the concept of 'ancestral protein molecules' and 'analogy between neurosecretory systems of higher invertebrates and vertebrates '13'14. Our current broadly based understanding of the nature of endogenous opioid systems strongly suggests that the origin of this important signal system lies in primitive organisms. Immunocytochemical and pharmacologial techniques have been the main tools used in the study of compar- ative opioid biology 16. The results of investigations with these procedures have demonstrated the presence of opioid-like substances and mechanisms in a number of invertebrates 11. However, as these are indirect method- ologies, more concrete proof for the presence of such signal molecules in a particular organism requires evi- dence from more than one experimental approach. In the case of substance P, for example, various investigators have found immunoreactive substance P-like materials in invertebrate neural tissues. Yet, attempts to isolate substance P in the neural tissues of Mytilus edulis, Leucophaea rnaderae, and Helix aspersa by biochemical methods were unsuccessful s. Definitive proof of the presence of opioid substances in invertebrates has been provided in Mytilus edulis 9't7. Convincing HPLC evi- dence for the presence of opioid substances in Carcinus maenas has also been reported recently 3'4. Given the great wealth of neurobiological information concerning the pond snail, Lymnaea stagnalis 1'2, it was of interest to determine if its neural tissues contain opioid material. The present report is based on binding analysis, immunocytochemistry and HPLC coupled with radioim- munoassay (RIA) to demonstrate the occurrence of such an opioid system in Lymnaea stagnalis. METHODS Binding analysis Tissue preparation. Lymnaea stagnalis employed in this study were mature snails (shell height 25-30 mm) reared under laboratory conditions. For binding studies, the esophageal ganglionic complex was dissected on ice, and pooled tissue from 240 animals was homogenized in 50 vols. of 0.32 M sucrose, pH 7.4 at 4 *C, by use of a Brinkmann polytron (30 s, setting No. 5). The crude homogenate was processed as described in detail elsewhere 7. Binding assay. Binding analyses were carried out by the method Correspondence: M.K. Leung, Department of Chemistry, SUNY/ColIege at Old Westbury, P.O. Box 210, Old Westbury, NY 11568-0210, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)