J. Insect Physiol. Vol. 35, No. 12, pp. 943-947, 1989 Printed in Great Britain. All rights reserved 0022-1910/89 $3.00 + 0.00 Copyright 0 1989Pergamon Press plc zyxwvutsrqp SEROTONERGIC NEUROHAEMAL TISSUE IN zyxwvutsrqponmlkjihgfedcbaZYXW RHODNIUS PROLIXUS: SYNTHESIS, RELEASE AND UPTAKE OF SEROTONIN IAN ORCHARD Department of Zoology, University of Toronto, 25 Harbord St, Toronto, Ontario, Canada M5S 1Al zyxwvutsrqponm (Received 21 June 1989) Abstract-The abdominal nerves of Rhodnius prolixus possess a serotonergic neurohaemal area. The uptake of [3H] serotonin and [3H] tryptophan into these abdominal nerves has been studied. There is a sodium-sensitive, high-affinity uptake mechanism for serotonin which has an apparent X, of 0.22 PM and a V,, of 585 fmol/lO min. The loaded [3H] serotonin, identified by HPLC, is released in a calcium-depen- dent manner by high-potassium saline. [‘HI tryptophan is also loaded, and about 1.4% of this is converted into serotonin over a 3 h incubation. The synthesised [3H] serotonin is also released by high-potassium in a calcium-dependent manner. Key W ord Index: HPLC; serotonin, tryptophan, calcium-dependent release, high-affinity uptake, insect neurohaemal INTRODUCTION The vertebrate nervous system contains a re-uptake mechanism for biogenic amines (see Iversen, 1967) and a similar mechanism appears to occur in the invertebrates. Thus, a high-affinity uptake system has been demonstrated for octopamine in nervous tissue of insects and leeches (Evans, 1978; Webb and Orchard, 1981; Carlson and Evans, 1986) and for serotonin in neurosecretory terminals in crustacea (Livingstone et af., 1981). In RhodniusproZiwus it has been shown that radiolabel is accumulated by the mesothoracic ganglionic mass and associated abdom- inal nerves when they are incubated in vitro in [3H] serotonin, and that radiolabel is released from the abdominal nerves of such loaded preparations in response to depolarising stimuli (Flanagan and Berlind, 1984). These abdominal nerves are covered in a neurohaemal plexus of serotonin-like immuno- reactive processes (Flanagan, 1984; Lange et al., 1988; Orchard et al., 1988) which is derived from five dorsal unpaired median neurones of the mesothoracic ganglionic mass (Orchard et al., 1989). Recently it was shown that serotonin is present in and released from these abdominal nerves (Lange et al., 1988, 1989). Clearly the loading of radiolabel shown by Flanagan and Berlind (1984) may be due to a high- affinity uptake of [ 3H] serotonin into the serotonergic neurohaemal areas, in a similar manner to that shown in lobsters (Livingstone et al., 1981). Further support for this suggestion comes from work with 5,7-dihy- droxytryptamine, a neurotoxic analogue of serotonin, which is apparently loaded into serotonergic neurones by the high-affinity uptake system (see Baumgarten et al., 1978, 1982; Berge et al., 1985). 5,7_dihydroxytryptamine has a profound effect upon the serotonergic neurohaemal areas of Rhodnius (Orchard et al., 1989) implying a loading into these terminals. In this article the uptake of [ 3Hj serotonin and [‘H] tryptophan into nerves containing the serotonergic system has been examined. There is a sodium-sensi- tive, high-affinity uptake mechanism for serotonin present in the abdominal nerves. Furthermore these areas are capable of releasing the loaded [3H] sero- tonin in a calcium-dependent manner following depo- larising stimuli. In addition to loading [ ‘H’j serotonin this system is capable of loading [ 3H] tryptophan, and about 1.4% of this tryptophan is converted into serotonin, which is also releasable in a calcium- dependent manner. The work lays the foundation for future studies on the regulation of, and influence of pharmacological agents upon, synthesis, release and uptake of sero- tonin in an insect serotonergic neurohaemal complex. MATERIALS AND METHODS Fifth-instar larvae of Rhodnius prolixus Stil, obtained from a long-established colony at the University of Toronto, were used throughout the present study. The bugs were maintained at 25°C in high relative humidity and were used, unfed, within 20-30 days of emergence as fifth instars. The mesothoracic ganglionic mass attached to stretches (approx. 3 mm) of abdominal nerves were dissected and incubated individually in saline (Lange et al., 1988) containing either 5(1,2-3H-N) hydroxy- tryptamine creatinine sulphate, 30 Ci/mmol (DuPont New England Nuclear Corp.) or (3H-G) L-tryp- tophan, 5.8 Ci/mmol (Amersham) in a manner similar to Flanagan and Berlind (1984). Briefly, to determine the uptake of radiolabel, tissues were washed for 2 min in saline following incubation, solubilized overnight in 1 ml of NCS tissue solubilizer (Amersham) and dissolved in 10 ml Econofluor (DuPont New England Nuclear Corporation). Radio- activity was estimated at a counting efficiency of 21% 943