ELSEVIER Neuroscience Letters 171 (1994) 67 69 NEUROSClENCE LETTERS Blocking actions of picrotoxinin analogues on insect (Periplaneta americana) GABA receptors N.M. Anthony a, C.W. Holyoke Jr. b, D.B. Sattelle ~'* "AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3E J, UK 6E.1. du Pont de Nemours, Stine-Haskell Research Center, Elkton Road, PO Box 30, Newark, DE 19714, USA Received 14 April 1993; Revised version received 3 January 1994; Accepted 18 February 1994 Abstract Five picrotoxinin analogues were examined on GABA-gated chloride channels of an identifiable cockroach (Periplanetaamericana) motor neurone (DO. Substitution of the bridgehead hydroxyl at the C-6 position of the picrotoxinin molecule by a fluorine atom (fluoropicrotoxinin) had little effect, whereas acetylation of the same functional group (picrotoxinin acetate) substantially reduced the effectiveness of the parent compound. Conversion of the terminal isopropenyl group to an acetyl (c~-picrotoxinone) or hydration of the double bond (picrotin) also reduced activity. Dendrobine, a naturally-occurring picrotoxinin-like compound had very little effect on GABA-induced responses at concentrations up to 1.0 x 10 -5 M. The present results suggest that the size and the ability of the bridgehead hydroxyl to undergo hydrogen bond formation and the lipophilic nature of the terminal isopropenyl group profoundly affect the inhibitory actions of the picrotoxinin molecule on insect neuronal GABA-gated chloride channels. Key words'. GABA receptor; Identified neuron; Picrotoxinin; Structure-activity; Insect; Periplaneta americana In insects, ?,-aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the nervous system and on muscle cells (see [10] for review). Recent studies using an identifiable motor neurone, the fast coxal depressor neurone (DO in the metathoracic ganglion of adult cock- roaches [8,11], have indicated that GABA gates a chlo- ride ion channel which is sensitive to the blocking actions of the vertebrate GABA A receptor antagonist picrotox- inin. Here we examine the relative potency of a series of picrotoxinin analogues with the aim of identifying re- gions of the molecule that are critical for blocking insect GABA receptors. The actions of several picrotoxinin analogues have been assessed in [3H]~-dihydropicrotoxinin binding stud- ies on crustacean muscle [6], in housefly toxicity tests [4], in physiological studies using an in situ housefly nervous system preparation [5] and a cockroach neuromuscular [6] preparation. These studies have indicated that the orientation of the terminal isopropenyl group in relation to the bridged bicyclic lactone skeleton and the presence of the bridgehead hydroxyl group are important for in- * Corresponding author. Fax: (44) 223-461954. Elsevier Science Ireland Ltd. SSD1 0304-3940(94)00171-6 secticidal activity. However, no detailed electrophysio- logical studies have been undertaken on picrotoxinin structure-activity relationships. In the present study a series of picrotoxinin analogues, including a novel fluoro-substituted compound, have been tested on the response to GABA of an identifiable insect motor neu- rone. The chemical structures of these analogues are il- lustrated in Fig. 1. The actions of dendrobine, a naturally occurring picrotoxinin-like compound [9] (Fig. 1), have also been investigated. The picrotoxinin derivatives fluoropicrotoxinin, picrotoxinin acetate and ~-picrotoxinone were synthe- sized as described elsewhere [1]. Dendrobine was a gener- ous gift from Dr K Yamada (Nagoya University, Japan). Picrotoxinin and picrotin were purchased from Sigma Chemicals, St. Louis, MO, USA. Adult, male cock- roaches (Periplaneta americana) were used in all experi- ments. Cockroaches were reared at 27-32°C with freely available food and water. A portion of the nerve cord containing the meso- and metathoracic ganglia and at least three of the six abdom- inal ganglia was isolated and mounted in a 0.5 ml volume Perspex experimental chamber. The preparation was