Ž . Molecular Brain Research 74 1999 242–246 www.elsevier.comrlocaterbres Short communication Mytilus edulis pedal ganglia express m opiate receptor transcripts exhibiting high sequence identity with human neuronal m1 Patrick Cadet, George B. Stefano ) Neuroscience Research Institute, State UniÕersity of New York, College at Old Westbury, Old Westbury, NY 11568-0210, USA Accepted 14 September 1999 Abstract Previous pharmacological and biochemical evidence suggests that m-subtype opiate receptors are expressed in the mollusk Mytilus Ž . edulis Bivalve , including the organism’s ganglia. In this study, we present molecular evidence of m opiate receptor expression. Using Ž . primers derived from the human neuronal m1 opiate receptor, we used reverse transcription-polymerase chain reaction RT-PCR to detect expression of m transcripts from Mytilus pedal ganglia. Sequence analysis of the RT-PCR products revealed 95% identity with the neuronal human m1 receptor. Furthermore, interleukin-1 and morphine exposure to excised pedal ganglia resulted in up- and down-regulation of the m receptor transcripts, respectively. This study provides molecular evidence that m-type opiate receptors are expressed in molluscan ganglia, suggesting that they first appear in invertebrate organisms and are retained during evolution. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Opiate; Molluscan; Neural; m Opiate receptor transcript; Interleukin 1. Introduction Most of our knowledge of opioid receptors is derived from studies of these receptors in the mammalian nervous system. Opioid receptors have been classified as d, m and k , and heterogeneity has been shown for each of these major subtypes, which differ in anatomic distribution and w x subserve various functions 1,7,9,10,17,25,35 . In earlier studies, our laboratory has documented the presence of high affinity, saturable and naloxone sensitive opioid receptors in Mytilus edulis neural and immune w x tissues 3,16,17,20,22,33 . Opiate binding and pharmaco- logical techniques found in these studies strongly suggest the presence of m and d opiate receptor subtypes. Given this preponderance of pharmacologic and biochemical evi- dence of m opiate receptor expression, specifically in Mytilus pedal ganglia, we were interested in directly ana- lyzing expression of the m opiate receptor expression at the molecular level. ) Corresponding author. Fax: q1-516-876-2727; e-mail: stefanog@surg.som.sunysb.edu, gstefano@li.net 2. Material and methods 2.1. Isolation of total RNA Ž . Tissue samples excised pedal ganglia were homoge- Ž nized in Tri-Reagent Molecular Research Center, Cincin- . nati, OH using a polytron homogenizer. The homogenates were stored at room temperature for 5 min to allow complete dissociation of nucleoprotein. 0.1 ml of 1-bromo- Ž . 3-chloropropane BCP per 1 ml of Tri-Reagent was added to the homogenates. The samples were vortexed vigorously for 15 s and then stored at room temperature for 7 min. After centrifugation of the samples for 15 min at 12,000 = g , the aqueous phase was transferred to a fresh tube. RNA was isopropanol precipitated, washed with 75% ethanol, centrifuged and then air-dried. The pellet was then resus- pended in Rnase-free water. RNA was analyzed spec- trophotometrically and on a 1% agarose gel for purity and concentration. ( ) 2.2. ReÕerse transcription RT -coupled polymerase chain ( ) reaction PCR of total RNA First strand cDNA synthesis was performed using ran- Ž . dom hexamers Gibco BRL, Gaithesburg, MD . A 3 mg of 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 99 00287-9