J. exp. Biol. 136, 333-350 (1988) 333 Printed in Great Britain © The Company of Biologists Limited 1988 UNPAIRED MEDIAN NEURONES IN A LEPIDOPTERAN LARVA (ANTHERAEA PERNYI) II. PERIPHERAL EFFECTS AND PHARMACOLOGY BY S. J. H. BROOKES* Department of Zoology, University of Bristol, UK Accepted 5 January 1988 Summary Two unpaired median cells (MCI and MC2) had a temporal pattern of firing that correlated with phasic muscular activity in preparations of larval Antheraea pernyi, and previous work has indicated that the axons of median cells are associated with nerve trunks innervating blocks of muscle. In spite of this, action potentials in median cells were not found to have any one-for-one effects on either the tension or the electrical activity of somatic muscle fibres. However, bursts of action potentials in MC2 were shown to modulate both tension production and electrophysiological activity of a number of motor units. These effects consisted of an increase in twitch tension, a relaxation of basal resting tension, an increase in relaxation rate following contractions, a hyperpolarization of some muscle fibres and an increase in amplitude of excitatory junction potentials. The relative potency of these different effects varied between fast and slow muscles. All of these effects were mimicked by the application of octopamine and synephrine, and in higher concentrations by a number of other biogenic amines and adrenergic agonists. The possibility that the effects of median cell activity were mediated by the release of endogenous octopamine was supported by the observation that phentolamine (10~5molP1) blocked the effects of both MC2 impulses and the application of exogenous octopamine, whereas propanolol affected neither set of responses. This observation also indicated a pharmacological similarity with a number of other octopamine-sensitive insect tissue preparations. MCI had similar effects to MC2 on the electrical activity of a number of muscles, suggesting that these two cells play a similar role. These observations provide strong evidence for the presence of an identifiable octopaminergic system of neurones, similar to the dorsal unpaired median (DUM) neurones that have been extensively studied in the locust. Introduction The phenomenon of neuromodulatory control of muscular activity is now well- established in invertebrate preparations. Serotonergic neurones in Aplysia and in •Present address: Department of Human Physiology, Flinders Medical Centre, Bedford Park, South Australia 5042. Key words: unpaired median neurones, octopamine, modulation, Lepidoptera. 334 S. J. H. BROOKES leeches have been demonstrated to modify muscle contractions evoked by motoneurone activity (Weiss, Cohen & Kupfermann, 1975, 1978; Mason & Kristan, 1982). In insects, the DUM (dorsal unpaired median) cell system has been extensively studied in locusts and one neurone (DUMETi) has been shown to have neurosecretory-type nerve endings in a skeletal muscle (Hoyle, Dagan, Moberly & Colquhoun, 1974). DUMETi has been shown to modulate the myogenic activity of a specialized group of fibres in the extensor tibialis (Hoyle, 1975; Evans & O'Shea, 1978) and to modulate neuromuscular transmission and tension development (Evans & O'Shea, 1977; O'Shea & Evans, 1979). Suggestions that DUMETi acted through the release of octopamine have been confirmed (Morton & Evans, 1984), and several subtypes of octopamine receptors have been reported to mediate the effects on the muscle and nerve terminals of the extensor tibialis preparation (Evans, 1981). In the lepidopteran Manduca sexta, exogenous octopamine can also modulate neuromuscular transmission in the dorsal longitudinal flight muscles of immature and adult moths, in a manner similar to that reported in locusts (Klaassen & Kammer, 1985). Octopamine is present in the haemolymph of Manduca sexta in physiologically significant quantities (Klaassen & Kammer, 1985; Davenport & Wright, 1986). Octopamine can be synthesized in nervous tissue of Lepidoptera (Maxwell, Tait & Hildebrand, 1978) and can be detected in many parts of the central nervous system (Davenport & Wright, 1986). In spite of this circumstantial evidence for a physiological role for octopamine in Lepidoptera, the source of this biogenic amine in moths and caterpillars is a cause of speculation. The relative importance of circulating octopamine and the possibility of direct octopaminergic innervation of muscles remain in question. The aim of this study was to test the hypothesis that the unpaired median cells (MCI and MC2) were involved in the modulation of muscle activity in a manner similar to that of DUMETi in the locust extensor tibialis. Additionally, the possibility that the peripheral effects might be mediated through the release of octopamine was investigated. Materials and methods Fifth-instar insect larvae were prepared for neurophysiological recording in the way described previously (Brookes & Weevers, 1988). The entire central nervous system (apart from the brain and the segmental ganglion being studied) was removed to minimize spontaneous muscular activity. The gut and silk glands were removed and the fat body and heart tissue were dissected away from the segment under study (usually segments 4 or 5). Care was taken to avoid disruption of the tracheal supply to the musculature, and a fine jet of air was blown at the spiracle. Standard saline (Weevers, 1966) was oxygenated and flowed at a rate of l-3mlmin~' through the body cavity of the caterpillar. The posterior end of the segment was pinned through the wall into a fixed Sylgard block and the anterior end of the muscle being investigated was attached by an array of fine hooks to a home-built transducer consisting of two Pixie Pharmacology of lepidopteran neurones 335 transducer elements (Endevco, Royston, Herts). Signals from this transducer were passed through a four-pole filter, differentiated by an operational amplifier differentiator (Buchan & Evans, 1980) and recorded on a Racal Store 4 DC tape recorder. The motoneurone innervating the muscle under study was stimulated by a fine pair of silver/silver chloride wires (insulated apart from their tips) that were laid against a single muscle fibre. Careful adjustment of electrode position, polarity and current allowed single motor units to be selectively activated. Muscle activity was monitored intracellularly by a low-resistance floating microelectrode (Woodbury & Brady, 1956). Motoneurone activity was also monitored by recording antidromic impulses in the root of nerve 1 with a silver hook electrode that could be drawn into a paraffin-filled glass sleeve. Median cell activity was controlled either by stimulating the neurone with intracellular depolarizing pulses or by extracellular stimulation of an axon sidebranch at a site remote from the muscle being studied. In the latter case, impulses of the median cell were recorded en passant between the site of stimulation and the muscle under study, using the paraffin hook electrode. This method could be used with confidence since the median cell extracellular impulse had a unique time course and its appearance always correlated with an antidromic impulse recorded when intracellular recording was also used. This preparation had the advantage that the segmental ganglion could be removed thus preventing all spontaneous muscular activity. Where drug applications to the muscle were intended, the entire preparation was tilted at an angle of 10° to the horizontal, to reduce the dead space of saline above the muscle. This meant that the volume of saline surrounding the muscle at any time could not be directly measured, but the changeover time (assessed visually with dye solutions) was of the order of a few seconds only. Solutions of drugs were made up on the day in normal saline. DL-octopamine-HCl, /S-phenylethylamine-HCl, DL-phenylethanolamine, dopamine-HCl, noradrena- line-HCl, adrenaline bitartrate, tyramine-HCl, 5-hydroxytryptamine creatinine sulphate, DL-synephrine-HCl, histamine dihydrochloride, naphazoline-HCl, tola- zoline-HCl, clonidine-HCl and DL-propranolol hydrochloride were all obtained from Sigma Chemical Co. Phentolamine mesylate was obtained from Ciba and chlordimeform was a gift from ICI. Ninety-two preparations were used in the present study; in a few cases recordings were made from several segments in one animal. Results Median cells normally showed a slow rate of spontaneous firing (from 005-0-5impulsess-1) during extended periods of recording from Antheraea pernyi larvae. Occasionally, however, the cell would fire bursts of impulses at much higher frequency, although rarely exceeding 2 spikes s~'. Observation of the animal at such times always revealed strong spontaneous muscular activity. When gross muscular activity was monitored by attaching a transducer to the tail, the 336 S. J. H. BROOKES 50 mV MCI -LU4 Tension -' B ]50mV MCI-I— Tension ]50mV MC1- Tension - 10s D 5mV MCI Tension Fig. 1. Recordings were made from MCI in ganglion 5 of a fifth-instar caterpillar. An uncalibrated transducer attached to the tail hook gave a reflection of the gross longitudinal muscle tone of the animal. It registered an upward deflection when the animal contracted. The nerve cord was intact in this preparation. (A) The rate of firing of the median cell increased with spontaneous jerks and during prolonged muscular activity of the animal. (B) A slow oscillation of tension recorded in an animal that did not appear to involve the phasic longitudinal muscles, was probably due to variations of firing