125 001 THESIU LIBRARY f refit-his: an State University This is to certify that the thesis entitled Characterization of Surface Electrical Activity Recorded from Adult Male Schistosoma Mansoni presented by David Robert Semeyn has been accepted towards fulfillment of the requirements for Masters degree in _Zoolog;L_ r ajor professor Date August 1981 0-7639 OVERDUE FINES: 25¢ per day per ite- ll 1‘ --\\}‘\ t RETURNING LIBRARY MATERIALS: _____________ 3. ”:9 Place in book return to mauve ‘V 4 charge from circulation records CHARACTERIZATION OF SURFACE ELECTRICAL ACTIVITY RECORDED FROM ADULT MALE SCHISTOSOMA MANSONI By David Robert Semeyn A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1981 'J' N G//f§ 97" ABSTRACT CHARACTERIZATION OF SURFACE ELECTRICAL ACTIVITY RECORDED FROM ADULT MALE SCHISTOSOMA MANSONI By David Robert Semeyn Spontaneous electrical activity can be recorded by means of suction electrodes placed on the dorsal surface of adult male Schisto- soma mansoni. This electrical activity is a complex of bi- and tri- phasic potentials which range from smaller amplitude waves (0-40 uV) of high frequency (10-40/sec), to randomly occurring larger amplitude waves (AG-1000+ uV) of lower frequency (O-S/sec). Many of the larger potentials (>100 uV) appear to be propagated. Regional variations of this activity exist, with the posterior region consistently exhibiting potentials greater than 120 uV, while at the anterior region activity greater than 40 uV is only rarely seen. Decreased concentrations of 0a“ (0.00, 0.14, and 0.52 mM) or elimination of Ca2+ plus addition of 4M EGTA, increased concentrations of Mg2+ (3.0, 10.0, and 30.0 5x10“ mM), or addition of 1 mM CoCl2 significantly decreased the level of electrical activity. Drug concentrations of lxlO-BM carbachol, lxlO-6M metrifonate, lxlO-BM dopamine, lxlO-SM pentobarbital, and lxlO-SM antimony tartrate also significantly decreased electrical activity. In contrast, 5-HT (lxlO-7M) significantly increased the level of electrical activity. To Mom and Dad ii ACKNOWLEDGEMENTS I would first like to thank Dr. Ralph A. Fax, for his patience, guidance and helpful criticism during the course of this research. I also thank Dr. Charles Tweedle (Dept. of Anatomy, Michigan State University) and Dr. James L. Bennett (Dept. of Pharmacology and Toxicology, Michigan State University) for serving on my guidance committee. Special thanks goes to Dr. Rick Hult (School of Pharmacy, Ferris State College) for his critical reading of the manuscript and his helpful suggestions. Finally, I wish to thank my colleagues in the laboratory, Connie Bricker, Carla Siefker, Joe Depenbusch, Tim "Bozo" Martin and David "Big Goof" Thompson, for making every day in "Bobby's werld" interest- ing and enjoyable. iii TABLE OF CONTENTS Page DEDICATION' ii ACKNOWLEDGEMENTS iii LIST OF TABLES vi LIST OF FIGURES vii INTRODUCTION 1 General Anatomy 2 The Tegument 2 Musculature 8 The Nervous System 9 Neurobiology ll Comparative Electrophysiology 13 OBJECTIVE 14 MATERIALS AND METHODS 15 Source and Maintenance of Parasitic worms 15 Recording Procedures 15 Ion Substitution Experiments l7 Pharmacological Agents 18 Statistical Procedures 19 RESULTS L 20 Electrical Activity Recorded 20 Characteristics of Spontaneous Electrical Activity--—-— 20 Conduction of Potentials 20 Activity in Sectioned worms 25 Regional Variation of Electrical Activity 25 Effect of Incubation Media on Electrical Activity ------ 28 Ionic Alteration Experiments 30 Calcium 30 Magnesium 30 Cobalt 36 iv TABLE OF CONTENTS (continued) Page Responses to Pharmacological Agents 36 Carbachol 37 Metrifonate , 37 5-HT 44 Dopamine 44 Pentobarbital 51 Antimony tartrate 51 DISCUSSION 59 Possible Sources of Electrical Activity 59 Effects of Ionic Alterations 61 Effect of Pentobarbital 62 Sensitivity of Method a 63 SUMMARY 65 BIBLIOGRAPHY 66 LIST OF TABLES Table Page 1 Regional variation and effect of incubation medium on surface electrical activity 29 2 I Effects of altered ionic concentrations on surface electrical activity recorded from the midbody position at 10 minutes following medium exchange 35 vi Figure 10 11 LIST OF FIGURES Chart recording showing electrical activity recorded simultaneously at two positions from.worms incubated in HBS Spontaneous electrical activity recorded at three different sites on the dorsal surface of worms incu- bated in HBS Amplitude histograms (representing electrical activity recorded at three different sites) from worms incubated in HBS The effects of altered ionic concentrations on surface electrical activity recorded from the midbody position of worms first incubated in HBS Amplitude histograms representing the change from con- trol in frequency of potentials in response to various ionic alterations Amplitude histograms representing the change from con- trol in frequency of potentials in response to various concentrations of carbachol Amplitude histograms representing the change from con- trol in frequency of potentials in response to two con- centrations of metrifonate Chart recordings showing electrical activity recorded from the midbody position of worms incubated in HBS in response to carbachol and metrifonate Amplitude histograms representing the change from con- trol in frequency of potentials in response to various, concentrations of 5—HT vii Page Scanning electron micrograph of male and female Schistosoma mansoni 3 Schematic of tegument and muscle layers in Schistosoma mansoni 5 21 23 26 31 33 38 40 42 45 LIST OF FIGURES (continued) Figure 12 13 14 15 16 Page Amplitude histograms representing the change from con- trol in frequency of potentials in response to various concentrations of dopamine 47 Chart recordings showing electrical activity recorded from the midbody position of worms incubated in HBS in response to 5-HT and dopamine 49 Amplitude histogram representing the change from con- trol in frequency of potentials in response to two con- centrations of pentobarbital 52 Amplitude histogram representing the change from con- trol in frequency of potentials in response to two con- centrations of antimony tartrate Chart recordings showing electrical activity recorded from the midbody position of worms incubated in HBS in response to pentobarbital and antimony tartrate---—---- viii 54 56 :-z=M~:i INTRODUCTION The blood fluke, Schistosoma mansoni, is a digenetic trematode which infects humans during the cercarial stage of its life cycle. Adult schistosomes migrate to and live in the mesenteric and portal venous system. Eggs deposited by the adult female in mesenteric .JL “M: capillaries are responsible for most of the pathology associated with the disease (Nobel and Nobel, 1976). A large portion of the world's population is infected by some form of schistosomiasis, making this trematode a medically important parasite. Several approaches are possible in assessing the effects of experimental treatments on nerve or muscle in Schistosoma mansoni. Biochemical and histochemical studies have provided considerable information about putative neurotransmitters present in schistosomes (Barker st 31., 1966; Bennett 35 EL-: 1969; Bennett and Bueding, 1971; Chou 32 al., 1972; Hillman and Senft, 1975) but give no information about the action of these compounds on nerve or muscle activity. Photoelectric and ultrasonic methods measuring overall movements of schistosomes have also been reported (Brown ggflal., 1973; Hillman and Senft, 1973). These methods also provide little information about activity in the nervous system or about details of locomotory beha- vior. 2 The method described by Fetter 22 31. (1977, 1978), which allows one to record spontaneous electrical activity by means of a suction electrode placed on the surface of the parasite, avoids some of the difficulties described above. This thesis describes studies examining the nature of the electrical activity recorded in this way from the surface of adult male schistosomes. If this activity has its genesis in the nervous system of the parasite, monitoring this activity may provide a sensitive means for examining subtle effects which experi- mental treatments have on the nervous system of this animal, effects which may not be overtly manifested as changes in the motor activity of the animal. General Anatomy Adult male Schistosoma mansoni are approximately 1 cm long, 0.2 cm wide and have a wet weight of 0.8 mg. Adult females are slightly longer (1.5 cm) and much thinner (0.2 mm) than males (Figure 1). Rostrally located ventral and anterior (oral) suckers are present in both male and female. The flat body of the male is curved to form a ventral, longitudinal groove, the gynecophoric canal, within which the adult female normally lies. Paired intestinal ceca converge and fuse at the midpoint of the adult animal, and then continue as a single gut posteriorly (Schmidt and Roberts, 1977). The Tegyment The outer covering of Schistosoma mansoni, called the integument or tegument, is a rough surfaced structure with numberous spines which appears to be an anatomic syncytium of anuclear material (Figure 2) Figure 1. Scanning electron micrograph of male and female Schistosoma mansoni. Female is lying within the gynecophoric canal of the male. OS, oral sucker; VS, ventral sucker; GC, gynecophoric canal; M, male; F, female; P, posterior. Calibration bar: 0.8 mm. Kindly supplied by C.S. Bricker, Michigan State University. Figure l .oomam umaaaaoomuuxo .m0m uxoaaaoo Hmcoauoasfi .00 «mnoaosc .z "souho Houcoaswou .09 “Hosanna oaammHmouzo .00 uoHomsfi Hosanna Ifiwcoa .2: noaomaa umaaoufio .20 unfluuma Hwfiufiumuouca .mzH “undead Amman .Am “magma Hmucossmmu .m muaoazwou man no meanness Hosanna“ .zH mocmupaoa Houno .20 “unmaswou .00H .fiaomcma maomoumfizom ca muozma saunas 0cm unwaswou mo oHumEozom .N ouowfim N shaman 7 (Silk gt E£°’ 1969). This outer anuclear epithelium is in cytoplasmic continuity via channels with tegumental cytons situated beneath the underlying musculature (Silk and Spence, 1969; Fetter ggnal., 1980). These nucleus-containing cytons manufacture and secrete the matrix of the tegument and possess many organelles generally associated with secretory processes (Wilson and Barnes, 1974). Junctional complexes between muscle cells and the membranes of tegumental cytons are also observed (Silk 25 31., 1969). The distal border of the outer tegument is a heptalaminate mem- brane with a thickness of 11 nm while the inner border is a more conventional trilaminate membrane (Hockley EEH§1., 1975). A basement membrane (basal lamina) which lies beneath the inner plasma membrane of the tegumental epithelium, separates the tegumental cytoplasm from a layer of fibrous connective tissue, and an outer circular and inner 1onngitudinal muscle layer. The thickness of the tegumental epithe- lium ranges from 1 to 5 um (Wilson and Barnes, 1977) varying with the contractile state of the parasite and the region in which it is measured, in general being thicker on the dorsal surface. The tegument has been implicated in a number of physiologically important processes. In addition to forming the host-parasite inter- face, carrier systems for the transport of hexoses (Fripp, 1967; Isseroff 35 al,, 1972; Rogers and Bueding, 1975; Uglem and Read, 1975; Cornford and Oldendorf, 1979), amino acids (Senft, 1968; Chappell, 1974; Asch and Read, 197Sa,b; Isseroff 35 31., 1976; Cornford and Oldendorf, 1979) and purine and pyrimidine compounds (Levy and Read, 1975) have been described. The outer surface of the tegumental 8 membrane also contains enzymes aiding in the digestion and absorption of nutrients (Pappas and Read, 1975; Lumsden, 1975). Musculature In general, the musculature of the schistosome resembles that of most invertebrate muscle, appearing to be of the smooth variety with no striations (Lowy and Hansen, 1962)., It is located immediately beneath the basement membrane of the tegument and consists primarily of an outer, circular and an inner, longitudinal muscle layer. Radially oriented muscle fibers are also present but are well de- veloped only in the proximal and distal areas of the acetabulum (Silk and Spence, 1969a). The female musculature is not as well developed as that of the male (Smith 35 31., 1969; Silk and Spence, 1969a). In the adult parasite, the longitudinal muscle layers are most prominent, especially in the dorsal surface (Smith 35 El': 1969). Ultrastructural studies show that the myofibrils consist of arrays of thick (18-40 nm diameter) myofilaments surrounded by a relatively large number (8-14) of thin (5 nm diameter) filaments (Silk and Spence, 1969a). This ratio of thin to thick filaments resembles that normally observed in vertebrate smooth muscle (Perry and Grand, 1979). The thin filaments show considerable branching and cross- 1inking between thick and thin filaments while the thick filaments are arranged in parallel arrays (Silk and Spence, 1969a). The sarco- Plasmic reticulum is poorly developed or absent but rough elements appear at scattered intervals. There is also a lack of tranverse tubules and microtubules which is characteristic of other smooth muscile preparations (Silk and Spence, 1969a). 9 Junctional complexes are observed between muscle cells, with the outer layers of adjoining sarcolemmas being closely apposed at a distance of 7-9 nm. These junctions are of variable length but seldom cover the total length of apposing sarcolemmas. Similar junctional complexes exist between muscle cells and the tegumental cytons (Figure 2) (Silk and Spence, 1969a). The Nervous System The nervous system of Schistosoma mansoni follows the same basic pattern of other trematodes as has been described by Bullock and Horridge (1965). Cholinesterase staining reveals the nervous system to consist of two pairs of anteriorly located central ganglia lying on either side of the esophagus which are joined by circumesophageal commissures. Two major pairs of nerve trunks, dorsal and ventral, extend longitudinally from the ganglia to both the anterior and posterior portions of the parasite (Fripp, 1967b). These nerve cords follow the lateral contours of the animal and are located within the parenchyma. The cords converge at the posterior end of the animal and are connected at intervals throughout their length by numerous dorsal and ventral transverse commissures. Small transverse branches of the cords project peripherally and terminate near the subtegumentary longitudinal musculature. Although no direct innervation of the tegument or the region immediately below the tegument has been ob- served, presumed sensory structures located within the tegument have been described (Morris and Threadgold, 1967; Silk and Spence, 1969b). 10 Anteriorly, a nerve cord projects from the central ganglion to inner- vate the oral sucker, and two branches of the ventral nerve cords innervate the ventral sucker (Fripp, 1967b). The primary study dealing with the ultrastructure of the nervous system of the schistosome was conducted by Silk and Spence (1969b). The central ganglia contain cells with large nuclei and prominent nucleoli. These cells give rise to groups of closely packed non- myelinated axons of variable shape, size and content. Adjacent axo- lemmas are separated from each other and the surrounding musculature and other tissue by a 11-12.5 nm uniform layer of weakly osmiophilic cement substance. A variety of organelles are present within the predominantly electron-lucent axoplasm. Large, dense granules of 100- 160 nm enclosed by a single membrane are found which may be neuro- secretory in nature. These are often present in single rows or small aggregations within nerve processes investing muscle and other cells. These large granules are accompanied by large, ovoid, clear axoplasmic vesicles 50-150 nm in length. Other dense osmiophilic granules 32-90 nm in diameter found in the axoplasm appear to be similar to those containing catecholamines in other neural tissues. This type of granule is often found in association with collections of clear synap- tic vesicles 20-50 nm in diameter. Other osmiophilic granules found within the axoplasm include stellate clusters resembling alpha-glycogen and individual 20 nm granules with the appearance of beta-glycogen. Synapses between axons are observed within the central ganglia and in other regions. These synapses are characterized by accumulations ll of the clear, 20—50 nm diameter type vesicles attached to the pre- synaptic membrane which is separated from the post-synaptic membrane by 9-17 nm.synaptic cleft. Post-synaptic membranes are relatively devoid of cytoplasmic inclusions and appear more thickened and osmio- philic than the pre-synaptic membranes. Neuromuscular junctions are essentially similar to the axo-axonal synapses just described. The junctional cleft is approximately 10 mm wide and is a symmetrically thickened. Post-synaptic elements consist of broadened, osmiophilic sarcolemmas of the adjoining muscle cells. In addition to the clear synaptic vesicles associated with the smaller dense granules, the large osmiophilic neurosecretory granules and their associated clear axoplasmic vesicles are also observed within the sarcoplasm of muscle cells. Neurobiology A number of compounds known to be neurotransmitters in other animal systems have been identified via biochemical and histochemical techniques in the schistosome. The presence and histochemical locali- zation of acetylcholinesterases within schistosomes suggests that acetylcholine may play an important physiological role (Beuding, 1952; Fripp, 1967b). Cholinomimetic agents, e.g. carbachol, have been demonstrated to inhibit spontaneous contractions, decrease tension in the musculature, and to decrease surface electrical activity (Barker gt 31,, 1966; Hillman and Senft, 1973; Fetterer gg_al,, 1977). Car- bachol, metrifonate and eserine have also been reported to block electrically induced muscle excitation in the schistosome (Pax £5 31., 12 1981). These effects have led these authors to the suggestion that acetylcholine may be acting as an inhibitory transmitter in this parasite. Monoamines have also been implicated as neurotransmitters in schistosomes. Fluorescence histochemistry and radioenzymatic assays have been used to confirm the presence of and to localize the cate— cholamines norepinephrine and dopamine within the nervous system of the worm (Bennett and Bueding, 1971; Chou 35 31,, 1972; Gianutsos and Bennett, 1977). These two compounds produce a characteristic leng- thening response of the parasite's musculature, an effect shown pharmacologically to be mediated via a dopamine receptor (Tomosky g; 31,, 1974). Fluorescence histochemistry has also revealed the distribution of 5-HT within the nervous system of the schistosome (Bennett and Bueding, 1971). Although the concentration of S-HT in the schistosome is 10 times that of the mammalian brain (Bennett 35 31., 1969), synthesis of this amine via tryptophan hydroxylase could not be demonstrated (Bennett and Bueding, 1973). It appears that high affinity uptake systems for S-HT as well as norepinephrine are present, which indi- cates that the worm may obtain biogenic amines from the host's plasma (Bennett and Bueding, 1973). Several studies have demonstrated S-HT's ability to increase contractile activity in adult male schistosomes (Barker g£_al,, 1966; Tomosky g£_al,, 1974; Fetterer g£_a1,, 1977). Imipramine and fluoxetine, compounds known to inhibit the active transport system of 5-HT in mammalian CNS neurons, cause a marked decrease in muscle tension and contractile activity in the schistosome 13 (Pax gt $1., 1979). This same study also demonstrated fluoxetine's ability to block the excitatory effects of 5-HT on the musculature. These observations have led to the suggestion that 5-HT may be serving as an excitatory transmitter in this parasite. It should be noted that although these putative neurotransmitters appear most concentrated in neural tissues, their distribution is not limited to the nervous system. ‘Whether or not these substances act directly on the musculature or on structures which innervate the musculature is difficult to determine. It is possible that these substances may have functions in addition to neurotransmission. Dopamine is a good substrate for phenoloxidase (Mansour, 1958) and may be involved with egg production by the female (Seed gt a1., 1978). Comparative Electrophysiology Electrophysiology techniques employing suction electrodes have been used to study the neurobiology of a variety of invertebrate species. A number of preparations, in particular ctenophores (Hor- ridge, 1965), coelenterate hydromedusae CMackie and Pasano, 1968; Josephson and Schwaab, 1979), enteropneusts (Pickens, 1970) and poly- clad flatworms (Koopowitz, 1973, 1975; Stone and Koopowitz, 1976; Keenan gt al., 1979; Koopowitz £5 21,, 1979b) exhibit spontaneous and evoked electrical activity that markedly resembles that recorded from the surface of S, mansoni. Amplitudes, durations and conduction velocities of potentials recorded from these preparations are com- parable to those seen in the adult male schistosome by Fetterer st 31. (1977). OBJECTIVE The present study represents an extension of the work conducted by Fetterer st 21. (1977) in which a method is described which allows spontaneous electrical activity to be recorded by means of a suction electrode placed on the surface of a schistosome. The objectives of this study are l) a more detailed characterization of the surface electrical activity recorded, 2) to explore the possible sources of the activity, and 3) to determine the applicability of this method in assessing the effects of experimental treatments on this parasite. 14 MATERIALS AND METHODS Source and Maintenance of Parasitic WOrms Adult male schistosomes, Schistosoma mansoni, (St. Lucian strain) were isolated 45-60 days post-infection from the mesenteric and portal veins of infected female white mice obtained from the laboratory of Dr. J.L. Bennett, Department of Pharmacology and Toxicology, Michigan State University. Isolated worms were maintained in 1) Hank's balanced salt solution (HBS) (Grand Island Biological, New York, NY), 2) Earle's balanced salt solution (G-ll; Grand Island Biological) containing 50% heat-inactivated horse serum (BBS/HS), or 3) RPMI-l640 (H-18; Grand Island Biological) containing 10% heat-inactivated fetal calf serum (RPMI-l640/FCS). All three media were buffered at pH 7.4 with 0.02 M Hepes (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; Sigma Chemical Co., St. Louis, MO) and contained 100 units/ml penicillin- streptomycin (Grand Island Biological). Parasites were maintained at 35°C until used and all experiments were performed within 8 hours after removal of worms from host animals. Recording Procedures Recordings of surface electrical activity were made by means of suction electrodes. These consisted of polyethylene tubing (PE-20; Clay-Adams, Parsippeny, NJ) pulled to a fine tip (100 um inside 15 16 diameter). A short piece of this tubing (approximately 2 cm) was affixed to a 3/8" 26 gauge hypodermic needle connected to a 1 cc tuberculin syringe barrel containing a silver-silver chloride wire. Prior to recording, electrodes were filled with a sufficient amount of incubation medium to come in contact with the silver wire. A silver- silver chloride wire placed in the recording chamber served as a ground. The electrical signal, recorded differentially, was passed through a preamplifier (Model P-lS, Grass Instrument Co., Quincy, MA; filters set at 0-3 Hz and 0.3 kHz), displayed on an oscilloscope (Model 5113, Tektronix, Inc., Beaverton, OR) and a chart recorder (Physiograph Four, Narco Biosystems Inc., Houston, TX), and then stored on magnetic tape (Model B, Vetter Instrument Co., Rebersburg, PA; recording speed 3 3/4 i.p.s.) for later analysis. In cases where mechanical activity was also monitored, the method described by Fetterer 55 El; (1977, 1978) was used with the recording electrode simultaneously serving as the stationary electrode. The recording chamber consisted of a polyethylene dish containing 2.5 m1 of incubation medium. The temperature of the bathing medium was maintained at 37°C with a thermoelectric heater placed under the recording chamber. A thermistor was used to monitor the temperature of the bathing medium. Surface electrical activity was recorded by means of the suction electrode placed mid-dorsally on the male schistosome either a) at the anterior end above the ventral sucker, b) at the midbody 0.1 mm posterior to the gut bifurcation, or c) at the posterior end 0.25 mm 17 from the tip of the tail., After placement of the electrode, 10 minutes equilibration time was allowed before data collection was begun. All results presented are from adult male schistosomes still paired with females. The recorded electrical activity was quantified by replaying tapes (at 3 3/4 i.p.s.) and counting all negative potentials in excess of 10 uV over 40 second intervals. Potentials less than 10 uV were not counted since they were often indistinguishable from the back; ground electrical noise of the system. The counts were determined by passing the output of the tape recorder through a window discriminator (Model 120, WPI, Inc., Hamden, CN) connected to a digital counter. Starting with the upper limit of the window discriminator set at 10 uV, "above window" counts were determined. The upper limit was then raised in 10 uV increments up to a value of 120 uV, with "above window" counts being made at each setting. In some instances "within window" counts over the same range of 10 0V increments were also determined. From these data, amplitude histograms of the electrical activity were constructed. Ion Substitution Experiments The effects of altered external ion concentrations on surface electrical activity were determined by first monitoring the electrical activity from animals bathed in normal HBS for 10 minutes and then exchanging the HBS for a modified saline containing the altered ion concentration desired. Electrical activity was then monitored for a minimum of 15 minutes with one minute samples of the activity being 18 recorded on tape at 5 minute intervals. Control responses for these experiments were obtained in a separate group of worms in the same manner except that after 10 minutes the HBS in the recording chamber was exchanged for fresh HBS. Electrical activity was then again monitored for a minimum of 15 minutes with one minute samples of activity being recorded on tape at 5 minute intervals. Calcium. Ca2+ concentrations of 0.52, 0.14 and 0.00 mM were tested. These solutions were obtained by altering or eliminating the CaCl2 concentration in HBS. The effect of 0 Ca2+ HBS containing 5x10-4M EGTA was also tested. Maggesium. Mg2+ concentrations of 30.0, 10.0 and 3.0 mM were tested. These solutions were obtained by altering the MgSO4 concen- tration of HBS. Cobalt. A Co2+ concentration of 1 mM was tested. This solution was obtained by adding the appropriate amount of CoCl2 to HBS. Pharmacological Agents Compounds tested were carbachol (Carb) (carbamylcholine chloride), dopamine (DA) (3-hydroxytyramine hydrochloride), pentobarbital (PB) (pentobarbitone sodium), 5-hydroxytryptamine creatinine sulfate (S-HT), all from Sigma Chemical Co., St. Louis, MO; cobalt chloride (CoClz), Mallinckrodt, St. Louis, MO; metrifonate (MT), kindly supplied by Drs. P. Andrews and H. Thomas of the Bayer A.G., and potassium antimony tartrate, kindly supplied by Dr. E. Bueding of the Johns Hopkins University, Baltimore, MD. All compounds were dissolved in double l9 distilled water at the necessary concentrations immediately prior to use. In these experiments, control activity was monitored for 10 minutes prior to drug addition. Twenty-five microliters of the desired solution were then added to the recording chamber to give the final concentration wanted. Electrical activity was then monitored for a minimum of 10 minutes with one minute samples of activity being recorded at 5 minute intervals during the test period. Control re- ' .1. spouses for these studies were obtained from.animals which received twenty-five microliters of distilled water instead of a solution m: containing one of the compounds. Statistical Procedures All values presented in histograms and tables represent the mean ill standard error of the mean (SEM) for a minimum of 6 animals unless otherwise noted. All tests for significance of difference between means were performed using Student's Eftest (non-paired). RESULTS Electrical Activity Recorded Characteristics of Spontaneous Electrical Activity The electrical activity recordable from the adult male schistosome ..r by way of a'suction electrode is a complex of bi- and triphasic poten— tials occurring at a rate of about 60-70/sec with amplitudes ranging :u from greater than 1 mV down to values indistinguishable from the background electrical noise of the system (<10 uV) (Figures 3 and 4). Activity varies from a more or less tonic firing of mixed amplitude potentials in some preparations to more phasic and irregular bursts of potentials in others. In general, smaller potentials have durations of 4 to 20 msec with mean rise times of 7 uV/msec while larger poten- tials have durations ranging from 25 msec to 1.2 sec and rise times which vary from 2.1 uV/msec to 7.3 uV/msec. This electrical activity has its source within the living parasite since exposure of the worm to a high concentration of ethanol abolished all activity within 30 to 60 seconds (Figure 4). Exposure of worms to high temperature (>42°C) or overnight refrigeration also abolished all electrical activity. Conduction of Potentials In several preparations, simultaneous recordings were made by means of two electrodes located at varying distances apart on the 20 21 .oomuo nonuo ecu aw muao>o umaaaam nag: woumaouuoo mum oomuu oco a“ vouuooou mamwuaouom Howuma Ham no: use acme ouoz .mumn an woumofioaw moaau uo>o moomuu soma: no maoauomm woodmaxo mum moonuu mo muom umSoq .moomuu Ham How mama onu ma nofiumunwamu owmuao> .4 ou uofiuouwom as o.~ ucaoa m Boom wouuooou mua>fiuom Hmofiuuooao msoosnuaaafim um maofiuamod zuonwfia Hmmuoo ecu aoum wovuooou hufi>auom Hmofiuuooao u< .mmz :H voumnaodw mauoz aoum maoaufimoa ozu um aamaooamuasaam voouooou >ua>auon Hmofiuuomao wcfisonm wafiouooou uumno .m oudwfim 22 uou m6 m muswfim >300— uon ad. uonn 23 Figure 4. Spontaneous electrical activity recorded at three different sites on the dorsal surface of worms incubated in HBS. At the arrow ethanol was added to the recording chamber (Final concentration of ethanol approximately 502). .24 ANTERIOR t EtOH MIDBODY EtOH CAUDAL Figure 4 IOO UV 2min. 25 dorsal surface of worms incubated in HBS. The small size, frequency and absence of distinguishing characteristics made it impossible to determine whether the smaller amplitude potentials were propagated. However, many of the larger (>100 uV) potentials did appear to be propagated (Figure 3). Conduction of these potentials was almost always from anterior to posterior, occurring a small percentage of the time in the opposite direction. The conduction velocity of these potentials in HBS averaged 40;: 4 cm/sec (N=11) for distances from 0.5 to 3.0 mm.‘ Activity in Sectioned worms In some experiments worms were cut into three equal length pieces. The level and pattern of electrical activity recorded from each of the pieces was indistinguishable from that recorded from the intact animals. From this it appears the activity recorded at any particular region of the worm is independent of adjacent regions and that the activity arises locally and is not generated by some single central pacemaker. REgional Variation of Electrical Activity The exact pattern and level of electrical activity recorded depends on the area on the worm from which the recordings are taken (Figures 4 and 5). At the midbody position potentials ranged in amplitude from less than 10 0V to greater than 120 uV with approxi- mately 13% being greater than 40 uV and 1% being greater than 120 uV. At the anterior position the frequency of low amplitude signals was 26 .mamfiwcm acoquMHv xfim unmoa on you mwofiuoa eaooom 0H uo>o vocfiauouoe mums can some you oom\mua:oo .mcoaufimom wawouoomu uoauoumom A00 wan .avonvfis Amv “Hoauoucm Afiuom Hmofiuuomao wcfiucomoummuv mamuwoumfis ovsuaaaa< .m muzwfim 27 ON 0— m «woman 13.33:..24‘ 8. E 3 t, ’ P ’ ’ O? O— 6 N aaS/Sinnoa 28 only slightly less than at the midbody, but in contrast to the mid- body, signals greater than 40 0V were very rare and no potentials greater than 120 uV were ever recorded. At the posterior, larger potentials were much more prevalent with nearly 33% of the recorded potentials exceeding 40 uV; nearly 6% exceeded 120 uV and in some .instances potentials as large as 1 mV were recorded. By contrast, the apparent frequency of potentials in the 10 to 30 uV range was only about one-half that seen at the midbody or anterior region. This apparent lower frequency of the smaller amplitude potentials at the posterior may be an artifact introduced by our method of counting these potentials. With the presence of a significant number of large potentials with comparatively long time courses, many of the smaller, more rapidly occurring potentials may have been obscured and thus not counted by the window discriminator. Effect of Incubation Media on Electrical Activity HBS, RPMI-l640/FCS and EBS/HS were tested for their effect on the electrical activity recorded from the various positions. The fre- quency and pattern of electrical activity appeared to be affected to only a minor degree by the incubation medium (Table 1). The most notable effect was that the frequency of potentials, both smaller and larger, was greater at the anterior region of worms incubated in EBS/HS. In this medium potentials greater than 100 uV were recorded, while no potentials exceeding 70 uV were recorded from the anterior of worms incubated in the other two media. 29 .mHmaH:m xwm mo asawafia m now 20m H Mucous oSu mm co>Hw mum can swamp oesufiadem woumawfimou onu :H mamwuaouoa mo uom oa\mucooo aw honozwoum mnu usomoueou mo=Hm> o a o o a o o a 0 cm .11 .ON vavoa o .01 vavmo n .Hoo v11 new + HHN umN + 11s n11 + m1 cam + com as + AA mas + mos m=\mmm 01m + mks 01m + «as was + «a nmm + mom on + 1 UN. + «mm onoHIHzmm an H i... a. H won 2 H 2 2 H at N H s m... H .12 mm: >1 onR1a¢ >1 osw1a1 as >1 os.1a< >1 oaw1a1 as >1 os.1a< >1 oswaa1 as assume uoauoumom xvonofiz uoauouq< mufi>fiuo< Hmoauuooam doomuam so sauna: cofiumnaoaH mo uoommm can sowumaum> Hmcowwom H mdm<8 30 Ionic Alteration Experiments In an effort to gain some insight into the nature and origins of the electrical activity recorded, the effects of ionic alterations in the bathing medium on the activity were determined. Quantitative data are presented for the midbody position only but similar responses were seen at the anterior and posterior. Calcium. Decreasing or eliminating Ca2+ from the bathing medium (HBS) decreased the level of electrical activity (Figures 6 and 7, Table 2). ~Reducing the CaCl2 concentration to 0.52 mM from its usual value of 1.40 mM in HBS eliminated all potentials greater than 100 uV within 10 minutes and significantly decreased the frequency of poten- « tials less than 100 uV. Further reduction of the CaCl2 concentration to 0.14 mM eliminated all potentials greater than 60 uV within 10 minutes and decreased the frequency of potentials less than 60 uV by 60%. Complete elimination of CaCl from.the bathing medium.abolished 2 all potentials greater than 50 uV within 10 minutes. Few potentials exceeding 30 uV were recorded and the frequency of potentials less than 50 uV was decreased by half. Complete elimination of CaCl2 from the bathing medium plus addition of EGTA (5x10-4M) further decreased electrical activity to the extent that no potentials greater than 30 uV were recorded and the frequency of potentials less than 30 uV was reduced by 89%. Maggesium. Increasing the concentration of Mg2+ in HBS from its normal value of 1.0 mM to 30.0 mM resulted within 5 minutes in an overall decrease in the level of electrical activity, but after a 31 .28 mm ao>Hw mum mcofiumuucoocoo coH .oomcmno Ixo mmB endows cowumnooafi was scans wsauao mafia usu mmumofiuafi ouooou osu cw xmoun one .mmm ca woumnnoafi umufim maho3 mo aowufimoa muon0fia oSu Bonn emouooou mua>auom Hwofiuuomao oommusm no mcoaumuucooaoo oases wououam mo muoomwo was .0 madman 32 0 ouawfim 18 36+ 33 .>: oNH coco noumouw mamauaouoa mucomoumou aduwoumas sumo mo unmfiu Hum onu no can one .owcmu >1 ONIOH onu cw manquaouon auaz amuwoumfis some mo umoa onu do maaaafiwoa mucus loses“ >2 0H cw mum mean .mcoaunuouam UHGOH mnowum> ou uncommon ca mamfiucouom mo moaoavuum a“ Houuaoo aouw mwcmso osu meducommumou mamumouman monogama< .m madman 34 n ouawfim +N°U 2E— .aou z... :6 .3. 'o 335 /S.anOD V 35 TABLE 2 Effects of Altered Ionic Concentrations on Surface Electrical Activity Recorded from the Midbody Position at 10 Minutes Following Medium Exchange MEDIUM 10 uV40 uV was 648 i 23 93 i 15 0.52 mM 01:: 348 i 28: 16 i 3: 0J4m0a wzimb 5: R 0 Ca2+/No EGTA 344-i-251 1 fig 0 0 012+ with EGTA 84 i 29 0 3 mM Mggi 316 i 30: 21 i 7: 10 mM 1132+ 266 i 301 8 i 41) 30mMMg 322:29 13: 2 1 mM 002+ 254 i 283 1 i 0b a b p<.001; .001: CNH coca Houmoum mamauaouom mucomouaou amuwoumwn some mo uzwau you any so can one .omcmn >2 om Ioa onu cw mHmHquBOQ pugs amuwoumws some «0 umoa new so mafiaaawmn mucoaouocfi >1 0H a“ mum scum .Honomnumo mo mcoaumuucoocoo maoaum> ou uncommou a“ mamfiuaouom mo zocoavouw ca Houucoo aoum omnmzo onu wafiucomoumou mameOumH: ovsufiaaa< .m ounmfih 39 U' FU' FL] 4 ‘0 M Figure 8 -9 lo M [11W Fufuf G 5 IO 45‘ ' '20i .254 DES/SINOODV Figure 9. 40 Amplitude histogram representing the change from control in frequency of potentials in response to two concentrations of metrifonate. Bins are in 10 0V increments beginning on the left of each histogram with potentials in the 10-20 uV range. The bin on the far right of each histogram represents potentials greater than 120 uV. 41 Ll LI 10'6M -51 -101 Umm \thDOU < .r ..I -20« Figure 9 42 .moomuu soon you mama use mum chfiumunaHmU owmuao> use mafia .uonamno woweuooou on» on women mma Aoomuu HoBoHv oumcowwuuoa ZwIOHxH Ho Aoomuu Hommav Hosomnumo z IOHNH .3ouum ecu u< .oumaOMHuuoa can Honomnuno ou uncommon ca mm: aw nouns ocfi mauos mo coauamon anonewa may aouw voouooou hufi>fiuom Hmofiuuooam wsazonm mwaavuoomu uumno .oH eenwum 43 o. 8.11.1 :1; 44 greater than 30 uV were recorded by 20 minutes, and the frequency of potentials less than 30 uV was decreased by 76%. BEBE; The effect of 5-HT on the electrical activity appeared complex (Figures 11 and 13). One effect appears to be an increase in the frequency of potentials greater than 40 uV, an increase detectable at a concentration of 5-HT as low as 1x10-7M. Along with the increased frequency of larger potentials, however, there was a depression in the frequency of potentials counted in the 10 to 30 uV range. This de- pression is detectable even at the lowest concentration of 5-HT tested (lxlO-BM). However, as mentioned previously, the counted frequency of small potentials may be artificially decreased in the presence of many larger potentials. Since 5-HT does increase the frequency of larger potentials, it is not possible to conclude with certainty whether the depression of lower amplitude potentials is real or is simply an artifact of this method of recording them. Dopamine. A concentration of dopamine of lxlO-SM, though it had no observable effect on motility, had a significant depressive effect on electrical activity (Figures 12 and 13). With higher concentra- tions of dopamine, 1x10-6M or greater, the activity corresponded with a decreased mechanical activity and a characteristic lengthening 6M dopamine produced response of the schistosome's musculature. 1x10- its greatest depression of activity within 5 minutes, eliminating all potentials greater than 60 uV and significantly decreasing the number of potentials less than 60 uV. Partial recovery of electrical acti— vity was seen with this concentration, with the frequency of potentials 45 .>1 ONH cmnu Houmouw maoauaouom munomouaou amquumfin some 00 unwau umm onu so can one .owcmu >1 CNIOH ago a“ mamfiuaouoe nu«3.amumOumH£ zoom 00 umoa sea :0 wcficcfiwon muaoaouoafi >1 0H a« mum moan .931m 00 mnofiumuueooaoo msofium> ou uncommou ca mamfiucouod mo mocosvoum ca Houuooo aoum owcmso as» wawucomouaou mameOumas ovsufiama< .11 811111 46 HA ouswum 20— : 111.1 .m p. 6 '7 d) a 335/ smnoa V 47 .>1 ONH cmnu Hounouw mamauaouoa mucomoumou anuwoumac some 00 unwau new onu do can one .owcmu >1 ONIOH onu ca mamauaouod nods amuwoumfis some 00 umoa onu do wnaaawmon mo:mEouoca >1 0H 1H mum mafia .oafiEmeoo mo mcoaumuusooaoo msoaum> ou uncommon ca mamaucou01 mo zoomscoum :« Houuaoo aoum omen:o onu wafiucomouaou manumouma: monuHH1a< .NH 811111 48 NH tnnmam 3Y0— d rfi—l 57 V 335/51N003 oh I 49 .moomuu noon uom.oamm can own meowumunuamo omMuHo> new mafia .uonamno wcwvuooou onu ou woven mmB Aoomuu HoBOHv mafiamaov z Ioaxa no Aoomuu Hommav Hmln z oaxa .3ouum osu u< .oafiamaoo can Balm ou uncommou ma man :« woumnaoafi mauo3 mo clsoaufimoe heonoaa ecu aoum woeuooou >uw>auom Hmoauuooao mafiaonm mwcfiouooou uumno .mH 811111 50 ma spamfim mam mafia .uonamno mcavuooou mnu ou compo mma Amoouu uozoav mumuuHMu zcoafiuam sqloaxa no Amomuu Hoaanv Hmuanumnouamm z IonH .3ouum onu u< .oumuuumu >ooaauam was kuwnumnouaon ou uncommon :« mm: a woumnsocfi mahoB mo :ofiuamoa >vonvfia onu Eoum cmvuoomu mufi>auom Hmofiuuomao waasonm mwaavuooou uumno .oH muawfim 57 .58 N >3 oo— _ oH ouswwm 58 tartrate, and the number of potentials less than 90 uV was somewhat reduced, but not significantly (p>.20). By 20 minutes few potentials greater than 50 uV were recorded and all potentials exceeding 80 uV were eliminated. The frequency of potentials less than 80 uV was reduced by 15% (.05100 uV) appear to be propagated significant distances. Regional variations in the level of electrical activity exist, with the posterior region consistently exhibiting potentials greater than 120 uV, some of which exceeded 1 mV. At the anterior region, activity greater than 40 uV is only rarely seen. Decreased concentrations of Ca2+ (0.00, 0.14 and 0.52 mM) or elimination of Ca2+ plus addition of 5x10-4M EGTA, increased concentrations of Mg2+ (3.0, 10.0 and 30.0 mM), or addition of 1 mM CoCl2 significantly decreased the level of electrical activity. Drug concentrations of lxlO-BM carbachol, 1x10-6M metrifonate, 8M dopamine, lxlO-SM pentobarbital, and 1x10-5M antimony 1x10— tartrate also significantly decreased electrical activity. In contrast, S-HT (lxlO-7M) significantly increased the level of electrical activity. 65 BIBLIOGRAPHY BIBLIOGRAPHY Altura, B.T., P. Turlapaty and B.M. Altura. 1980. Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle. Biochem. Biophys. Acta 595: 309-312. Asch, H.L. and C.P. Read. 1975a. Transtegumental absorption of amino acids by adult male Schistosoma mansoni. J. Parasitol. 61: 378- 379. Asch, H.L. and C.P. Read. 1975b. Membrane transport in Schistosoma mansoni. Transport of amino acids by adult males. Exp. Parasi- tol. 38: 123-135. Barker, L.R., E. Bueding and A.R. Timms. 1966. The possible role of acetylcholine in g, mansoni. Brit. J. Pharmacol. Chemother. 26: 656-665. Bennett, J.L. and E. Bueding. 1971. Localization of biogenic amines in g, mansoni. Comp. Biochem. Physiol. 39A: 859-867. Bennett, J.L., E. Bueding, A.R. Timms and R. Engstrom. 1969. Occur- rence and levels of 5—HT in S, mansoni. Mol. Pharmacol. 5: 542- 545. Bricker, C.S., R.A. Fax and J.L. Bennett. 1981. Microelectrode studies of the tegument and subtegumental compartments of male Schistosoma mansoni. I. Anatomical localization of sources of electrical potentials. J. Parasitol. (Submitted). Brown, M.C., M. Koura, D.R. Bell and H.M. Gilles. 1973. An i§_vitro activity monitor for schistosomes: A preliminary report. Ann. Trop. Med. Parasit. 76: 369-370. Bueding, E. 1952. Acetylcholinesterase activity in S, mansoni. Brit. J. Pharmacol. 7: 563-566. Bullock, T.H. and G.A. Horridge. 1965. Structure and function in the nervous systems of invertebrates, V0. 1. W.H. Freeman and Co., San Francisco. Chappell, L.H. 1974. Methionine uptake by larval and adult Schisto- soma mansoni. Int. J. Parasitol. 4: 361-369. 66 67 Chou, T.C., J.L. Bennett and E. Bueding. 1972. Occurrence and con- centration of biogenic amines in trematodes. J. Parasitol. 58: 1098-1102. Cornford, E.M. and W.H. Oldendorf. 1979. Transintegumental uptake of metabolic substrates in male and female Schistosoma mansoni. J. Parasitol. 65: 357-363. Eckert, R. and H.D. Lux. 1976. A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J. Physiol. (London) 254: 129-151. Fatt, P. and B.L. Ginsborg. 1958. The ionic requirements for the production of action potentials in crustacean muscle fibers. J. Physiol. (London) 142: 516-543. Fetterer, RyH., R.A. Pax and J.L. Bennett. 1977. Schistosoma mansoni: Direct method for simultaneous recording of electrical and motor activity. Exp. Parasitol. 43: 286-294. Fetterer, R.H., R.A. Fax and J.L. Bennett. 1978. Schistosoma mansoni: Physical and chemical factors affecting the mechanical properties of the adult male musculature. Exp. Parasitol. 46: 59-71. Fetterer, R.H., R.A. Fax and J.L. Bennett. 1980. Characterization of the electrical potential from the tegument of adult males. Exp. Parasitol. 49: 353-365. Fripp, P.J. 1967a. The sites of 14C-glucose assimilation in Schisto- soma mansoni and Fasciola hepatica. Comp. Biochem. Physiol. 58: 157—159. Fripp, P.J. 1967b. Histochemical localization of esterase activity in schistosomes. Exp. Parasitol. 21: 380-390. Geduldig, D. and D. Junge. 1968. Sodium and calcium components of action potentials in the Aplysia giant neurone. J. Physiol. (London) 199: 347-365. Gianutsos, G. and J.L. Bennett. 1977. The regional distribution of dopamine and norepinephrine in S, mansoni and F, hepatica. Comp. Biochem. Physiol. 58C: 157-159. Guerrero, S. and W.K. Riker. 1973. (Effects of some divalent cations on sympathetic ganglion function. J. Pharmacol. Exp. Ther. 186: 152-159. Hagiwara, S., H. Hayashi and K. Takahashi. 1969. Calcium and potas- sium currents of the membrane of a barnacle muscle fiber in relation to the calcium spike. J. Physiol. (London) 205: 115- 129. 68 Hagiwara, S. and K. Naka. 1964. The initiation of+§pike potentials in barnacle musle fibers under low internal Ca . J. Gen. Physiol. 48: 141-162. Hagiwara, S. and S. Nakajima. 1966. The difference in Na and Ca spikes as examined by application of tetrodotoxin, procaine and manganese ions. J. Gen. Physiol. 49: 793-806. Hagiwara, S. and K. Takahashi. 1967. Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. Gen. Physiol. 50: 583-601. Harvey, A.M. and F.C. MacIntosh. 1940. Calcium and synaptic trans- mission in a sympathetic ganglion. J. Physiol. (London) 97: 408- 416. Hillman, G.R. and A.W. Senft. 1973. Schistosome motility measure- ments: Responses to drugs. J. Pharmacol. Exp. Ther. 185: 177- 184. Hillman, G.R. and A.W. Senft. 1975. Anticholinergic properties of the antischistosomal drug hycanthone. Am. J. Trop. Med. Hyg. 24: 827—834. . Hockley, D.J., D.J. McClaren, B.J. ward and M.V. Nermut. 1975. A. freeze fracture study of the tegumental membrane of Schistosoma mansoni (Platyhelminthes:Tremetoda). Tissue and Cell 7: 485-496. Horridge, G.A. 1965. Relations between nerves and cilia in Cteno- phores. Am. 2001. 5: 357-375. Hutter, 0.F. and K. Kostial. 1954. Effect of Mg2+ and Ca2+ ions on the release of acetylcholine. J. Physiol. (London) 124: 234-241. Isseroff, H., C.Y. Bonta and M.G. Levy. .1972. Mbnosaccharide absorp- tion by Schistosoma mansoni. 1. Kinetic characteristics. Comp. Biochem. Physiol. 43A: 849-858. Isserhoff, H., J.C. Ertel and M.G. Levy. 1976. Absorption of amino acids by Schistosoma mansoni. Comp. Biochem. Physiol. 54B: 125- 133. Jerelova, 0.M., I.V. Krasts, and B.N. Veprintsev. 1971. The effect of sodium, calcium, and magnesium on the amplitude of the action potential from giant neurons of Limnaea stangalis. Comp. Biochem. Physiol. 40A: 281-293. Josephson, R.K. and W.E. Schwab. 1979. Electrical properties of an excitable epithelium. J. Gen. Physiol. 74: 213-236. 69 Katz, B. and R. Miledi.. 1965. The effect of calcium on acetylcholine release from motor nerve terminals. Proc. Roy. Soc. (London) B161: 496-503. Kerkut, G.A. and D.R. Gardner. 1967. The role of calcium ions in the action potentials of Helix aspersa neurones. Comp. Biochem. Physiol. 20: 147-162. Keenan, L., H. Koopowitz and K. Bernardo. 1979. Primitive nervous systems: Action of amdnergic drugs and blocking agents on acti- vity in the ventral nerve cord of the flatworm Notoplana acticola. J. Neurobiol. 10: 397-407. Koopowitz, H. 1973. Primitive nervous systems: A sensory nerve net in the polyclad flatworm Notoplana acticola. Biol. Bull. 145: Koopowitz, H. 1975. Electrophysiology of the peripheral nerve net in the polyclad flatworm Freemania litoricola. J. Exp. Biol. 62: Koopowitz, H., K. Bernardo and L. Keenan. 1979. Primitive nervous systems: Electrical activity in the ventral nerve cords of the flatworm Notoplana acticola. J. Neurobiol. 10: 367-381. Koopowitz, H., L. Keenan and K. Bernardo. 1979. Primitive nervous systems: Electrophysiology of inhibitory events in flatworm nerve cords. J. Neurobiol. 10: 383-395. Levy, M.G. and C.P. Read. 1975. Purine and pyrimidine transport in Schistosoma mansoni. J. Parasitol. 61: 627-632. Lowy, J. and J. Hansen. 1962. Ultrastructure of invertebrate smooth muscle. Physiol. Rev. Suppl. 5: 34-238. Lumsden, R.D. 1975. Surface ultrastructure and cytochemistry of parasitic helminths. Exp. Parasitol. 37: 267-339. Mackie, G.0. and L.M. Passano. 1968. Epithelial conduction in Hydro- medusae. J. Gen. Physiol. 52: 600-621. Mansour, T.E. 1958. Effect of serotonin on phenol oxidase from the liver fluke Fasciola hepatica and from other sources. Biochem. Biophys. Acta 30: 492-500. Meves, H. 1968. The ionic requirements for the production of action potentials in Helix pomatia neurones. Pflugers Arch. Physiol. 304: 215-241. Morris, C.P. and L.T. Threadgold. _1967. A presumed sensory structure associated with the tegument of Schistosoma mansoni. J. Parasitol. 53: 537-539. 70 Noble, E. and G. Noble. 1976. "Parasitology". Lea and Febiger, Philadelphia, p. 563. Pappas, P.W. and C.P. Read. 1975. Membrane transport in helminth parasites: A review. Exp. Parasitol. 37: 469-530. Pax, R.A., R.H. Fetterer and J.L. Bennett. 1979. Effects of fluoxe- tine and imipramine on male Schistosoma mansoni. Comp. Biochem. Physiol. 64C: 123-127. Pax, R.A., C. Siefker, T. Hickox and J.L. Bennett. 1981. Schistosoma ‘mansoni: Neurotransmitters, longitudinal musculature and effects of electrical stimulation. Parasitol. (In press). Perry, S.V. and B.J. Grand. 1979. Mechanisms of contraction and the specialized protein components of smooth muscle. Brit. Med. Bull. 35: 219-226. Pickens, P.E. 1970. Conduction along the ventral nerve cord of a hemichordate worm. J. Exp. Biol. 53: 515-528. Rogers, S.H. and E. Bueding. 1975. Anatomical localization of glucose uptake by Schistosoma mansoni adults. Int. J. Parasitol. 5: 369- 371. Schmidt, G.D. and L.S. Roberts. 1977. "Foundations of Parasitology". C.V. Mbsby Co., St. Louis, p. 273. ‘ Seed, J.L., M. Boff and J.L. Bennett. 1978. Phenol oxidase activity: Induction in female schistosomes by ig_vitro incubation. J. Parasitol. 64: 283—289. - Silk, M.H. and I. Spence. 1969a. Ultrastructural studies on the blood fluke S, mansoni. 'II. The musculature. S. Afr. J. Med. Silk, M.H. and I. Spence. 1969b. Ultrastructural studies on the blood fluke S, mansoni. III. The nerve and sensory structures. S. Afr. J. Med. 3C1. 34: 93-1040 Silk, M.H., I.A. Spence and J.H. Gear. 1969. Ultrastructural studies on the blood fluke g, mansoni. I. The tegument. S. Afr. J. Med. Sci. 34: 1-10. ' Smith, J.H., E.S. Renolds and F. von Lichtenberg. 1969. The integu— ment of Schistosoma mansoni. Am. J. Trop. Med. Hyg. 18: 28-49. Stanbury, J.B. 1948. The blocking action of Mg2+ ion on sympathetic ganglia. J. Pharmacol. Exp. Ther. 93: 52-62. 71 Standen, N.B. 1975. Ca1cium.and sodium ions as charge carriers in the action potentials of an identified snail neurone. J. Physiol. (London) 249: 241-252. Stone, G.C. and H. Koopowitz. 1976. Primitive nervous systems: Electrophysiology of the pharynx of the polyclad flatworm, Enchiridium punctatum. J. Exp. Biol. 65: 627-642. Thompson, D.P., R.A. Fax and J.L. Bennett. 1981. .Microelectrode studies of the tegument and subtegumental compartments of male Schistosoma mansoni. II. An analysis of electrophysiological properties. J. Parasitol. (Submitted). Tomosky, T.K., J.L. Bennett and E. Bueding. 1974. Tryptaminergic and dopaminergic responses of S, mansoni. J. Pharmacol. Exp. Ther. 190: 260-271. Uglem, G.L. and C.P. Read. 1975. Sugar transport and metabolism in Schistosoma mansoni. J. Parasitol. 61: 390—397. wald, F. 1972. Ionic differences between somatic and axonal action potentials in snail giant neurones. J. Physiol. (London) 220: Werman, R. and H. Grundfest. 1961. Graded and all-or-none electro- genesis in arthropod muscle. 11. The effects of alkali-earth and onium ions on lobster muscle fibers. J. Gen. Physiol. 44: 997-1027. Wilson, R.A. and P.E. Barnes. 1974. An in_vitro investigation of dynamic processes occurring in schistosome tegument, using compounds known to disrupt secretory processes. Parasitol. 68: 239-270. Wilson, R.A. and P.E. Barnes. 1977. The formation and turnover of the membranocalyx of the tegument of S, mansoni. Parasitol. 74: 11-72 0