their cell bodies and dendrites (Blagburn, Beadle & Sattelle, 1985; Blagburn &. Sattelle, 1987c). The aim of this study was to investigate the sensitivity of insect.
J. exp. Biol. 137, 603-607 (1988) Printed in Great Britain © The Company of Biologists Limited 1988
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SHORT COMMUNICATION LOPHOTOXIN BLOCKS SYNAPTIC ACETYLCHOLINE RECEPTORS IN THE COCKROACH PERIPLANETA AMERICANA BY JONATHAN M. BLAGBURN* AND DAVID B. SATTELLE AFRC Unit of Insect Neufophysiology and Pharmacology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK Accepted 11 February 1988 The actions of lophotoxin on synaptic acetylcholine receptors were investigated, using the cholinergic synapse between the lateral filiform hair sensory neurone and giant interneurone 3 in the first-instar cockroach Periplaneta americana. Lophotoxin (1-Oxl0~ 6 moll~ 1 ) blocked transmission without affecting the passive or active membrane properties of the pre- and postsynaptic neurones, and without affecting non-cholinergic synaptic inputs. Micromolar concentrations of lophotoxin also blocked the depolarizing response of giant interneurone 3 to carbamylcholine, when applied ionophoretically to its dendritic branches. The toxin shifted , the dose-response curve to the right by two orders of magnitude, suggesting that it was acting as a competitive antagonist. Lophotoxin is a diterpene lactone isolated from gorgonian corals of the genus Lophogorgia (Fenical et al. 1981). It has recently been shown to block nicotinic neuromuscular transmission in vertebrates at micromolar concentrations (Culver & Jacobs, 1981; Langdon & Jacobs, 1983; Atchison, Narahashi & Vogel, 1984), and binding studies have shown that it blocks transmission by binding to the acetylcholine recognition sites of muscle nicotinic receptors (Culver, Fenical & Taylor, 1984). Recently, lophotoxin has emerged as a selective, high-affinity antagonist at neuronal nicotinic receptors, blocking transmission in autonomic ganglia of the chick and rat at concentrations in the range 1-Oxl0~6moll~1 to 3-2xlO~:>moll~1 (Sorenson, Culver & Chiappinelli, 1987). However, the toxin has not been tested on invertebrate nervous tissues. There is considerable evidence that cereal afferent, giant interneurone synapses of the cockroach Periplaneta americana are cholinergic (Sattelle, 1985), and the nicotinic antagonist a'-bungarotoxin blocks transmission at nanomolar concentrations (Sattelle et al. 1983). The first-instar terminal abdominal ganglion is a useful preparation for studies of axonal and synaptic physiology since the * Permanent address: Institute of Neurobiology, 201 Boulevard del Valle, Old San Juan, Puerto Rico 00901. Key words: acetylcholine, nicotinic receptor, synapse, insect, lophotoxin.
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J. M. BLAGBURN AND D. B. SATTELLE
presynaptic filiform hair sensory neurones and postsynaptic giant interneurones are individually identifiable. The filiform hair sensory neurone, giant interneurone synapses in the first-instar cockroach exhibit nicotinic properties (Blagburn & Sattelle, 1987c) and giant interneurones bear nicotinic acetylcholine receptors on their cell bodies and dendrites (Blagburn, Beadle & Sattelle, 1985; Blagburn & Sattelle, 1987c). The aim of this study was to investigate the sensitivity of insect synaptic acetylcholine receptors to lophotoxin. Recently hatched first-instar cockroach nymphs (Periplaneta americana) were dissected in saline of the following composition (in mmoll" 1 ): NaCl, 150; KC1, 3; CaCl2, 5; MgCl2, 1; NaOH, 2; TES, 5; trehalose, 5; sucrose, 50; pH7-2. The methods for dissection of the terminal ganglion preparation, for intracellular recording from the lateral filiform hair sensory neurone (LFHSN) and giant interneurone 3 (GI3), and for ionophoretic application of cholinergic agonists have all been described in detail elsewhere (Blagburn et al. 1985; Blagburn & Sattelle, 1987a,b). Membrane input resistance was monitored by measuring the voltage change due to the injection of a — 0-5 nA current pulse. The microelectrode resistance was subtracted electronically. Lophotoxin was dissolved in dimethyl sulphoxide to give a 5-0xl0~ 2 moll~ 1 stock solution. Samples of this were added to saline to give the final concentration. The maximum concentration of dimethyl sulphoxide present was 0-02% (v/v). In control experiments 1% dimethylsulphoxide had no significant effects on the properties of either LFHSN or GI3. The dose-response curve for carbamylcholine application is representative of two similar experiments. The resting potential of GI 3 was, as reported previously, about —75 to —80 mV, compared with - 5 0 to - 6 0 mV for the LFHSN axon (Blagburn & Sattelle, 1987c). The action potentials in LFHSN elicited monosynaptic excitatory postsynaptic potentials (EPSPs) in GI3 with a mean amplitude of 6-7mV (Fig. 1A; Blagburn & Sattelle, 1987a). Ionophoretic application of the cholinergic agonist carbamylcholine (CCh) to the small dendritic field situated on the neurite of GI 3 resulted in depolarizing responses which were recorded from the cell body (Fig. 1C). It has previously been shown that these depolarizing cholinergic responses are not abolished by a high Mg 2+ : Ca 2+ ratio, and hence are due to acetylcholine receptors on GI3 itself. It has also been shown that these cholinergic responses are of the nicotinic type (Blagburn & Sattelle, 1987c). Lophotoxin at a concentration of 1-Oxl0~ 7 moir l produced no discernible effect on EPSPs or CCh responses (two experiments). At 1-Oxl0~ 6 moir' lophotoxin, however, EPSPs were totally abolished in 10±2min (mean±s.D., N=3) (Fig. IB), and at l-OxlO^moll" 1 they were abolished in 8±l-4min (N = 2). Occasional depolarizing synaptic potentials remained in GI 3, which were not synchronized with LFHSN spikes (Fig. IB). These may have been due to noncholinergic synaptic input from other neurones. There were no effects on the resting potential and input resistance of GI3 and LFHSN, and the sensory axo spikes were unaffected. Washing the preparation in normal saline for 60min di not reverse the synaptic block produced by lophotoxin. Following a 10-min
Actions of lophotoxin
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