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Y Michigan 5mm University This is to certify that the thesis entitled QWch flag on Biophysical We of Tegument and Subtegumental Compartments in Schistosoma mansoni presented by p 5 David Paul Thomffpon has been accepted towards fulfillment of the requirements for __M££er_3_ degree in _§§lenc.e__ 0-7639 OVERDUE FINES: 25¢ per W per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulatlon records BIOPHYSICAL CHARACTERIZATION OF TEGUMENT AND SUBTEGUMENTAL COMPARTMENTS IN SCHISTOSOMA MANSONI By David Paul Thompson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1981 ABSTRACT BIOPHYSICAL CHARACTERIZATION OF THE TEGUMENT AND SUBTEGUMENTAL COMPARTMENTS IN SCHISTOSOMA MANSONI BY David Paul Thompson Standard intracellular microelectrode techniques were used to determine the electrical properties of the tegument and subtegumental regions in male Schisto- soma mansoni. Three distinct compartments of electrical potential were observed. Resting potentials recorded in these compartments were -45.9 i 2.5 mV (Eteg), }. *3 42.0 t 1.1 mV (E2), and -4.7 t 0.3 mV (E3). Input resistance was measured in ,9 we each compartment and found to be 4.5 MOhms (tegument), 9.2 MOhms (£2), and 3.5 (:5 MOhms (E3). Time constants for the tegument, E2, and E3 compartments were 0.24 t. 0.01 msec, 0.25 :t 0.01 msec, and 0.13 1- 0.01 msec, respectively. Multiple electrode experiments revealed that the tegument and E2 compart- ment are electrical syncytia with similar current spreading capabilities. Low resistance pathways appear to connect the tegument and E2 region, as electrotonic signals initiated in either of those compartments experience only a 15-25% reduction upon passing into the other. Injecting large (> 200 nA) depolarizing current pulses into the tegument or Eg compartment often elicited active membrane responses. These responses were not actively propagated. The addition of a non-diffusible solute to the recording medium immobilized the parasites and greatly reduced the current spreading capacity of both the tegument and E2 syncytia. Active responses could not be evoked in schistosomes exposed to this treatment. To Mom and Dad ii ACKNOWLEDGEMENTS I would first like to thank Dr. Ralph A. Pax for giving a liberal artist his first chance in science, and for providing a great deal of guidance during the course of this research. A I also thank Dr. Frank Blatt (Dept. of Physics, Michigan State University) and Dr. Charles Tweedle (Dept. of Anatomy, Michigan State University) for their suggestions and for serving on my guidance committee. Dr. James Bennett (Dept. of Pharmacology/Toxicology, Michigan State University) deserves a special thanks for masterminding the schistosome project and for providing support. I would also like to thank my colleagues and friends in the laboratory, C. M. Siefker, T. C. Martin, and D. R. Semeyn for helping to make every day enjoyable, and C. M. Bricker who, along with R. H. Fetterer, provided the foundations upon which my investigations were based. Finally, thanks to 'I‘rygve Aaby (Dept. of Biophysics, Michigan State University) for lending his technical expertise on many occasions. iii TABLE OF CONTENTS Page LISTOFTABLES OOOOOOOOOOOOOOOOOO0.0.00.0...OOOOOOOOOOOOOOOOOOO Vi LISTOFFIGURES .0O0..0.0...00...OOOOOOOOOOOOOOOOOOO0.0.0000... Vii INTRODUCTION.OOOOOOOIOOOOOOOOOO000......OOOOOOOOOOOOOOOOOOOOO 1 Schistosoma mansoni 1 GeneralAnatomy 2 The Tegument 2 Muscle System 6 Parenchymaand ExtracellularSpaces......................... 10 Comparative Electrophysiology 17 Specific Objectives......................................... 20 MATERIALSANDMETHODS............... ...... .. ....... 21 SourceandMaintenanceofAnimals 21 RecordingMedia.............................................. 21 Preparationfor ElectricalRecording 21 StatisticalProcedures......................................... 22 ElectricalRecordings.......................................... 23 Compartmentalization of Electrical Potentials................. 24 Current/VoltageRelations 27 TimeConstants............................................ 29 SpaceConstants........................................... 30 TissueCouplingRatios...................................... 31 RecordingsinHyperosmoticSolution 31 RESULTSOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOO 00...... ........ O 32 Single Electrode Experiments 32 Compartmentalization of Electrical Potential.................. 32 Current/VoltageRelations 34 Multiple Electrode Experiments................................. 39 Current Spread Though Electrical Compartments 39 RiseTimesforInjectedCurrents............................. 44 Intercompartmental Coupling Ratios.......................... 44 ActiveMembraneResponses.................. ..... 49 Effects of Hyperosmotic Solutions..... ............. ...... 56 iv DISCUSSION 0.0.0.0....00....OOOOOOO-OOOOOOOOOOOOOOOOOOOOOOOOOOOO Morphological Substrates of Electrical Potentials... . . .. . . . .. . . . . .. Passive Current Spreading Properties Current/Voltage Relations and Time Constants.................... Intercompartmental Electrical Coupling.......................... Active Membrane Responses.................................... SUMMARYOOOOOOOOO0.0.0.0.0...00.0.00...OOOOOOOOOOOOOOOOOOOOOOO LITERATURECITED. ..... 0......0.0.0.0.000000000000000000000IOO 56 65 65 72 79 81 83 85 LIST OF TABLES Table Page 1. Summary of data obtained from single electrode tests . . . ..... . . . . 38 2. Summary of data obtained from multiple electrode tests . . . . ..... 64 vi LIST OF FIGURES FIGURE Page 1. Scanning electron micrograph of paired male and female SChiStosomes OOOOOOOOOOOOOOOOOOO0..00.000000...OOOOOOOOOOOOO 4 2. Transmission electron micrograph of HRP labelled tegument and associatedstructures 8 3. Transmission electron micrograph of HRP labelled circular andlongitUdinal mUSCIe 00......OOOOOOOOOOOOOOOOOOOOIOO0...... 12 4. Transmission electron micrograph of HRP injected into extracellular spaces around muscle bundles . . . . . . . . . . . . . . . . . . . . . 14 5. Schematic oftegument and musclelayers 16 6. Microelectrode recording of three distinct zones of electricalpotentiaIOOOOOOOOOOOOOOOOO...0.0.0.0...0.0.0.000... 26 7. Graph of current-versus-membrane potential measured in each of the electricalcompartments........................... 36 8. Depiction of the apparatus used during recordings for currentspreadOO...OOOOOOOOOOOOOOOOO00.000.000.00.0.00.00... 41 9. Graph of current decay in each of the electrical compartments COO...I0.0.0.0000...OIOOOOOOOOOO0.0.0.0.000... 43 10. Graph of the time course of electrotonic potentials spreading through the tegument and E2 compartment . ..... . . . . . . 46 11. Depiction of the apparatus used during recordings for tissue couplingOO0.000000IOOOOOOOOOOOIOOOOOOOOOOOOO... ....... 48 12. Microelectrode recordings of active membrane responses eIiCitedinthetegument OIOOOOOOOOO...OOOOOOOOOOOIOOOOOOOOOOO 51 13. Microelectrode recordings of active membrane responses eIiCitedintheEzcompartment OOOOOOOOOOOOOOOOOIOOOOOI0...... 53 14. Microelectrode recordings of active membrane reponses recorded simultaneously in injected and non-injected electrical compartments 55 vii 15. 16. 17. 18. Graph of current-versus-membrane potential measured in the tegument and E2 compartment of schistosomes bathed inahyperosmOtic salutiODOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.000 Graph of the time course of electrotonic potentials spreading through the tegument of E2 compartment of schistosomes bathed in a hyperosmotic solution . . . . . . . . . . . . . . . Graph of current decay in the tegument and E2 compartment of schistosomes bathed in a hyperosmotic solution . . . . . . . . . . . . . . . . . Semilogarithmic plot expressing current decay as a function ofinterelectrode separation viii 59 61 63 68 INTRODUCTION Schistosoma mansoni is a trematode Platyhelminth capable of infecting humans during one stage of its polymorphic life-cycle. Free-swimming cercariae deposited in fresh water penetrate the skin of humans and eventually migrate to the liver where they mature. Adult schistosomes infiltrate the mesenteric veins and capillaries where they desposit eggs that penetrate the intestinal wall and are expelled in feces. Most of the pathology associated with the disease stems from hydrodynamic disruptions of the mesenteric venous system (Noble and Noble, 1976). It has been estimated that one of every twelve peOple in the world is infected by some form of schistosomiasis, making it second only to malaria in occurence among infectious diseases. Currently available antihelminthics are either marginally effective or too expensive to be made available in adequate quantities. Although many features concerning schistosome anatomy, biochemistry, and pharmacology have been well documented, the principles governing the neuro- muscular system are poorly understood. While methods for monitoring locomotion (Brown e_t g, 1973; Hillman and Senft, 1973), and muscle contraction (Fetterer _e_t a_l., 1977) are making quantifiable assessments of experimental treatments possible, they leave many questions concerning the underlying physiological mechanisms unanswered. In particular, they cannot answer questions regarding events that must be occurring at the membrane level. Analysis of these processes is best achieved by microelectrode techniques that have been only recently introduced to the study of schistosomes. At this time, Fetterer's e_t a_l. (1980) characterization of the tegumental potential, and Bricker's g a_l. (1981) identification of an electrical compartment that appears to represent muscle bundles are the only reported studies of this kind. Therefore, the primary objective of the present study is to extend the work of those investigators in order to achieve a more thorough understanding of the biophysical prOperties of the tegument and muscle membranes in .S_. mansoni. It is hoped that this information will make possible increasingly rational approaches to the clinical control of this disease. General anatomy Adult male S_. mansoni are approximately 0.8 cm long, 0.2 cm wide, and weigh 0.8 mg. The ventral body of male worms has a groove (gynecOphoric canal) along most of its length within which the female lies. Females are slightly longer than males, but only about one-tenth as wide. They leave the gynecOphoric canal only to penetrate the narrowest capillaries possible while laying their eggs. Both sexes possess rostrally located anterior and ventral suckers that are presumably involved in locomotion and/or stabilization (Figure 1). The gross morphology of schistosomes is relatively simple. The tegument and underlying muscle bundles form a shell within which intestinal cecae, protone- phridia, and gonads are suspended in a parenchymal matrix composed largely of lipid and glycogen particles and extracellular space. The nervous system is composed of two paired circumesophageal ganglia within which all nerve nuclei are located, and four lateral nerve trunks. From these trunks extend smaller fibers that synapse with muscle bundles, and may innervate putative sensory receptors located within the tegument (Silk and Spence, 1969b; Morris and Threadgold, 1967). The Tegument Adult male S. mansoni possess an outer covering, the tegument, that appears to be an anatomic syncytium of anuclear material (Silk e_t a_l., 1969). It is bound superficially by a 110 X basement membrane (Hockey _e_t a_l., 1975) which separates it from a layer of fibrous connective tissue that lies above the muscle layers (Morris and Threadgold, 1968). The tegument ranges in thickness from 1-5 microns Figure 1. Scanning electron micrograph of male and female _S_. 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, 0.6 mm. Kindly supplied by C. S. Bricker, Dept. of Zoology, Michigan State University. Figure 1 (Wilson and Barnes, 1977), and is generally about twice as thick at the dorsal surface as it is ventrally. The matrix of the tegument is manufactured and secreted by cells termed "cytons" which lie beneath the muscle layers (Silk g _a_l., 1969). These cells possess a large number: of organelles including those generally associated with secretory processes (Wilson and Barnes, 1974), and are connected to the tegumental surface by cytoplasmic channels (Silk e_t 21., 1969; Fetterer e_t a_l., 1980). The tegument itslef also contains some inclusions such as mitochondria, multilaminate vesicles and crystalline spines (Silk gt _a_l., 1969). The tegument of _S_. mansoni has been implicated in several physiological processes. In addition to providing a barrier that is highly resistant to the host's degradative enzymes, it also appears to be an important site of entry for glucose (Fripp, 1967) and several amino acids (Senft, 1968). A thin layer of material (the glycocalyx) coats the outer surface of the tegument (Bogdish and Aldridge, 1967). It is partially composed of acid mucOpolysaccharides (Stein and Lumsden, 197 3), and may contain enzymes involved in the absorbtion and digestion of nutrients (Lumsden, 1975; Pappas and Read, 1975; Ernst, 1977). There is also anatomical evidence suggesting that innervated sensory structures are located in the tegument that may function to detect the direction of flow of the surrounding medium (Morris and Threadgold, 1967). Recent work conducted by Fetterer gt a_l. (1980) has demonstrated that a well defined tegumental potential (Eteg) of about -35mV exists that can be altered by changing physical and/or chemical qualities of the parasite's environment. Eleva- ted concentrations of external K+, reduced Ca“, cold, and ouabain all induce depolarization of the tegument, indicating that some form of active Na-K transport must be involved in maintaining this potential (Fetterer e_t _a_l., 1980). Fetterer e_t a_l. (1980) employed horseradish peroxidase (HRP) as a marker for determining the location of the recording electrode while gathering these data. During all penetrations in which large potentials were recorded, HRP iontophoreti- cally injected was subsequently found to occupy only those compartments directly associated with the tegument (e.g., cytons, cytOplasmic channels, and the outer tegument itself) (Figure 2). Recordings made in the ventral tegument of unanesthetized parasites revealed spontaneous depolarizations that ranged from 3 to 15 mV in amplitude with 20 to 100 msec durations. Most of this activity was abolished by anesthesia (Fetterer gt a_l., 1980). Other evidence suggesting that the tegument may be actively prepagating electrical activity has been reported in surface electrical studies (Fetterer e_t $1., 1977; Semeyn _et 3%, 1981). Alternatively, Fetterer e_t g. (1977, 1980) suggest that these transients may be volume conducted responses of underlying muscle elements, as they are generally correlated with mechanical contractions of the parasite. A possible morphological substrate for electrotonic coupling of the tegument and muscle has been observed in electron micrographs. Plasma membranes of the integumentary cells appear to form junctional complexes with neighboring muscle cells that closely resemble those connecting most smooth muscle cells (Silk and Spence, 1969b). Muscle System The muscle system of _S; mansoni is primarily located immediately beneath the basement membrane of the tegument, where it is arranged in an outer circular and an inner longitudinal fashion (Silk and Spence, 1969b). All muscle appears to be of the smooth variety with no striations, and in that sense it resembles most invertebrate muscle (Lowy and Hansen, 1962). Thick (180—400 8 diameter) myofilaments are surrounded by 8-14 thin (50 8 diameter) filaments, a ratio very near that observed in vertebrate smooth muscle preparations (Perry and Grand, 1979). The thick filaments are arranged in parallel, while thin filaments exhibit a Figure 2. Transmission electron micrograph of HRP injected tegument and related structures. DT, dorsal tegument; TC, tegumental cyton; CC, cytoplasmic channel; DVM, dorsal-ventral muscle; CM, circular muscle; LM, longitudinal muscle. Kindly supplied by CS Bricker, Dept. of Zoology, Michigan State University. Figure 2 great deal of branching and cross-linking with other thin as well as thick filaments. Ovoid nuclei are normally separate from and deeper than the fiber bundles, and are connected to them by cytOplasmic processes (Silk and Spence, 1969b). The sarcoplasmic reticulum in schistosomes is poorly deve10ped, and appears only at scattered intervals. Mitochondria appear in sac-like distensions of the sarcoplasm along myofibril bundles. Lipid globules as well as a and B glycogen particles are distributed throughout the muscle cells (Silk and Spence, 1969b). Junctional complexes, which are of particular importance to this investigation, are observed between the outer layers of adjoining sarcolem mas. These junctions separate apposing cell membranes by about 7-9 nm, and are up to 400 um long. They appear to be similar to those at the interface between cyton cells and neighboring muscle bundles (Silk and Spence), 1969b). Motility is presumably the most important function of the schistosome's muscle system. Hillman and Senft (1973) as well as Brown _e__t_ a_l. (1973) have developed techniques designed to indirectly measure schistosome motility 'm v_i_t£o using either photoelectric cells or ultrasound. By these methods they have been able to quantify whole body movements in response to various treatments, and thereby gain some understanding of pharmacological effects on schistosome musculature or nerves. Fetterer e_t a_l. (1977) have deve10ped a technique for monitoring mechanical activity directly, using suction electrodes in circuit with a force transducer. This method is, in some cases, as much as 100x more sensitive than previously reported ones. In addition to serving as a sensitive monitor for the effects of various pharmacological agents, this method has demonstrated that the contractile proper- ties of the schistosome musculature are much like those reported for other invertebrate as well as vertebrate muscle preparations. For example, elevated K+ (Fetterer fl a_l., 1977) and hyperosmotic sucrose solutions (Pax _e_t a_l., 1981) both induce paralysis. 10 While Fetterer's it a_l. (1977) method for monitoring muscle activity repre- sents a vast improvement over previously reported techniques, it does not provide the cellular-level information required for determining the membrane electrical events mediating muscle activity. Bricker e_t a_l. (1981), using HRP as a marker, have recently identified a second zone of electrical potential as being endemic to muscle tissue (Figure 3). Resting potentials recorded intramuscularly are generally about one-half as great as those recorded in overlying tegumental regions. Treatments affecting Eteg are always observed to exert a similar effect on muscle potentials simultaneously (Brickler e_t a_l., 1981). Because it has not been firmly established that all recordings from the second electrical compartment represent intramuscular potentials, I will refer to that zone as E2 in the present study. Parenchyma and Extracellular Spaces For the present study, we classify as parenchyma all tissue lying beneath the tegument that is not a component of the tegumental or muscle systems. This includes the cecum and intestine, excretory and reproductive elements, as well as a great deal of extracellular space and fat particles. It is important to note that many transverse muscle fibers do in fact extend across the internal compartment of the parasite (Silk and Spence, 1969b). While distinct microelectrode recordings from individual organs or nerves within the schistosome have not been obtained, Bricker g a_l. (1981) have success- fully recorded from submuscular extracellular regions as evidenced by injections of HRP (Figure 4). This compartment of electrical potential has been labelled as E3 by Bricker gt a_l. (1981), and I will use that terminology to designate this potential. Resting potentials recorded in the parenchyma generally range from -3 mV to -9 mV. The anatomical correlates of the three distinct compartments of electrical potential are depicted in Figure 5. 11 Figure 3. A. Circular muscle injected with HRP. Calibration: 8 microns. B. Longitudinal muscle injected with HRP. Calibration: 8 microns. The electrical potential recorded during these injections was classified as E2 by Bricker et al. (1981). Kindly supplied by C. S. Bricker, Dept. of Zoology, Michigan State University. 12 Figure 3 13 Figure 4. Extracellular spaces injected with HRP. The electrical potential recorded during these injections was classified as E3 by Bricker g al. (1981). DT, dorsal tegument; BL, basal lamina; LM, longitudinal muscle. Calibration: 6 microns. Kindly supplied by C. S. Bricker, Dept. of Zoology, Michigan State University. 14 Figure 4 15 Figure 5. Schematic of tegument and muscle layers in S. mansoni. T, tegument; OM, outer membrane of tegument; BL, basal lamina; IM,'ihtern§I membrane of the tegument; S, spine; MP membranous particles; EB, elongated bodies; IMa, intersti- tial matrix; CM, circular muscle; LM, longitudinal muscle; TC, tegumental cyton; N, nucleus; CC, cytoplasmic channel; JP, juntional process; L, lipid granule; ECS, extracellular space. 16 3k ° ’/IM . u"'lor(""'li’ lMc7I MAG—'2: — _— Figure 5 17 Comparative Electrophysiology Several features of the schistosome's morphology and physiology are shared by other organisms that have been more thoroughly tested by electrophysiological methods. In particular, the schistosome appears to exhibit some characteristics common to: (a) large neurons, (b) epithelial sheets and (c) strips of vertebrate smooth muscle. These similarities point to the possibility that some elements of the electrical models deve10ped for those preparations may also be applicable to the schistosome. The roughly cylindrical shape and syncytial nature of its membrane-bound tegument impart anatomical qualities to _S_. mansoni that approximate those observed in some nerve preparations. There are numerous examples in the literature of nerve preparations for which electrical parameters have been determined using single and double microelectrode methods. Araki and Otani (1955) recorded the magnitude and time course of potential changes developed across the membranes of toad motoneurons during the application of a rectangular current pulse through a bridge-balanced intracellular microelectrode. Frank and Fourtes (1966) adopted this technique for analysis of cat motoneurons, and from the data derived membrane input resistances in the 2-3 MOhm range, and time constants of 0.27 msec. These values are similar to those obtained by Fetterer e_t a_l. (1980) who used similar techniques while recording from the ventral tegument of unanesthetized schistosomes. Length constants of 0.17 mm to 0.48 mm have been reported for cat motoneurons (Coombs gt 311., 1959); these also are consistent with those observed during a preliminary study of ventral (0.20 mm) and dorsal (0.35 mm) tegument in schistosomes (Fetterer e_t _a_l., 1980b). Evidence that the tegument of schistosomes may represent an excitable epithelium is provided by Fetterer e_t 31. from surface electrical recordings using suction electrodes (1977) as well as from microelectrode studies (1980). Pinned 18 flat to the recording chamber floor, anesthetized schistosomes present a structural model closely resembling a two dimensional epithelial sheet similar to that found in several other phyla of animals. Ctenophores (Horridge, 1965) and coelenterate hydromedusae (Mackie and Pasano, 1968; Josephson and Schwab, 1979) exhibit epithelial conduction that markedly resembles that recorded from the surface of schistosomes. Unlike the schistosome's tegument, the nerve-free exumbrellar epithelium of the hydromedusae is a multicelluar syncytium, but gap junctions connecting adjoining cells presumably provide low resistance pathways for current dissemination (Hand and Gobel, 1972; Josephson and Schwab, 1979), so conditions for conduction in these preparations may approximate one another. The elevation of external concentrations of Mg“- to levels that would normally suppress nervous activity does not affect epithelial conduction in hydromedusae (Mackie and Pasano, 1968). This response is also observed in similarly treated schistosomes during recordings from the tegument using suction electrodes (Semeyn gt g1” 1981). Epithelial cells of salivary glands in snails (Kater g 93., 1977), insects (Wiener g g” 1964; Ginsborg gt gl_., 1973), and mice (Kater gt gl_., 197 9) all exhibit electrical coupling, presumably by way of gap junctions. In these preparations, I electrotonic signals can be recorded over relatively large distances, and in some cases evoked action potentials can spread from cell to cell (Kater 53 gl_., 1977). Our preliminary data suggest that these conditions may exist in the schistosome tegument as well (Fetterer gt gl_., 1980b). Other epithelial sheet preparations examined by multiple electrode methods include newt gut (Shiba, 1970), toad urinary bladder, and the gallbladder of Necturus (Reuss and Firm, 1974, 197 5). The latter two examples both exhibit tissue space constants of about 0.4 mm, which is nearly equal to the value obtained in preliminary recordings from the tegument of § mansoni (Fetterer gt 9, 1980b). In most epithelial sheet preparations the tissue segments analyzed are 1-4 cells thick, 19 while other dimensions are assumed to be essentially infinite. Current injected intracellularly at a point source within the sheet does not decay exponentially, but follows a Bessel function-like shape that is initially very steep (Shiba, 1970; Caveney, 1974; Reuss and Finn, 1975). This was also a feature of the response observed in preliminary recordings from the schistosome's tegument (Fetterer g_t g” 1980b). This deviation from classic cable theory prediction is thought to be due to two-dimensional current flow to surrounding areas (Woodbury and Grill, 1961), presumably made possible by low resistance junctional complexes between cells. The abundance of muscle tissue beneath the tegument in schistosomes suggests a comparison with the thin tissue strips often used for analysis of electrical properties in smooth muscle preparations. Responses to stimulation delivered intracellularly with microelectrodes or externally using large plates have been analyzed to determine the cable properties of numerous muscle preparations. Tomita (1966) recording from intracellularly stimulated guinea-pig vas deferens observed linear I/E relations for both hyperpolarizing and depolarizing current pulses. Input resistances ranged from 11-30 MOhms, time constants from 1.5-3.0 msec, and currents decayed rapidly, so the space constant was only about 0.1 mm. Hirst and Neild (1977) used intracellular stimulating and recording techniques in guinea-pig arterioles, and derived input resistances of 2 MOhms to 6 MOhms, and space constants of 1.1 mm to 1.6 mm. Time constants obtained from intracellular current injection of frog atrial tissue were as short as 0.2 msec (Rougier e_t gt, 1968), while those recorded in Tunicate heart cells were 0.4 msec (Kriebel, 1968). These values are all quite close to those recorded from the ventral tegument of adult schistosomes using similar techniques (Fetterer gt 9, 1980, 1980b). Experi- ments performed on the same preparations using field stimulation resulted in values that were 1-2 orders of magnitude greater than those observed during intracellular stimulation (Tomita, 1966; Abe and Tomita, 1968). 20 Specific Objectives Schistosoma mansoni is a medically important endoparasite for which elec- trophysiological analysis is extremely incomplete. Some valuable information regarding several electrical properties of the ventral tegument has been reported (Fetterer _e_t _a_l., 1980, 1980b), but these studies raise nearly as many questions as they answer. The present study represents a logical extension of the work conducted by Fetterer gt a_l. (1980, 1980b). It will attempt to answer the following questions: (1) To what extent, if at all, are the tegument and muscle compartments in .S_. mansoni (as identified by Fetterer e_t a_l., 1980, and Bricker gt a_l., 1981) electrical syncytia? (2) What are the intracellularly injected I/E relations for tegument and muscle? (i.e., Do membrane potential dependent conductance changes occur in response to depolarizing or hyperpolarizing current pulses?) (3) What are the time factors associated with electrotonic potentials recorded at the site of current injection, and if measurable, those spreading through the tissue compartments? (4) Are electrical events transmitted from the tegument to the muscle (and/or vice-versa)? MATERIALS AND METHODS Source and Maintenance of Animals Adult male Schistosoma mansoni (Puerto Rican strain) were obtained from portal and mesenteric veins of female laboratory mice (Mg musculus) 50-60 days after an induced cercarial infection. The parasites were maintained at 35-37°C in a medium consisting of a 1:1 mixture of Earle's salt solution (G-ll, GIBCO, New York, New York) and heat inactivated horse serum (HS, GIBCO) with 20 mM Hepes (N-2 hydroxymethyl piperazine, Sigma, St. Louis, Mo.), 100 units/ml pennicilin- streptomyocin (GIBCO), and a final glucose concentration of 0.296. The medium was adjusted to a pH of 7.4 with 6N NaOH. All experiments were performed within eight hours after parasites were removed from the host. Recordtgg Media All experiments were performed in media consisting of the HS/Earle's mixture to which was added either carbachol (1 x 10‘4, Sigma), sodium pentobar- bital (Na-PB, 50 mg%, Sigma) or sufficient quantities of sucrose to raise the osmotic pressure of the bath by 200 mOsm. These paralytics were necessary to ensure that electrode displacement would not disrupt the electrophysiological monitors conducted. Preparation for Electrical Recordings Before placing parasites into the recording chamber, 5 ml of the standard recording medium (HS/Earle's) was added along with an appropriate quantity of 21 22 either carbachol or Na-PB. Contents of the bath were thoroughly mixed with a pipette. When the desired bath temperature was reached, a parasite was added and 10-15 minutes were allowed for the drug to exert its full effect. Occasionally, longer periods of exposure to the paralytic were required for complete immobiliza- tion, but this seemed nearly always to be associated with worm pairs that were not completely separated. In every case, females were removed from the gynecOphoric canal and discarded prior to testing. Male schistosomes were secured to the chamber floor with two insect pins inserted at the extremities in a manner that exposed the entire dorsal surface, thus rendering it accessible to electrode penetration from above. The parasites were handled as gently as possible to avoid stretching or compression of their body wall, as both appeared to result in significant reductions in resting potentials of the tissue compartments. While insertion of the pins resulted in some morphological damage, data were always collected near central regions of the parasite to reduce any possible adverse effects, such as current shunting, that the pins may have caused. Statistical Procedures Except where noted, at least 6 parasites were tested under each paralytic for the parameters analyzed. Test points for each animal represent the mean of at least 3 penetrations. Most results are expressed as means with one standard error. Standard two-tailed t-tests were used to determine statistical differences, and lines were fitted to points by linear regression analysis (McCall, 1975). 23 Electrical Recordingg All experiments employed microelectrodes pulled on a horizontal micro- electrode puller (Narishige Instruments, Tokyo, Japan) from either 1.5 mm 'kwick- fill' capillary tubing (WPI, New Haven, Conn.) or 1.4 mm 'microstar' tubing (Radnoti, Monrovia, Ca1if.). Electrodes were filled with 3.0 M KC1 and had resistances of 2-15 MOhms. Fluid within the recording chamber was grounded by a 3.0 M KCl agar bridge constructed from 5 mm glass tubing and Ag-AgCl wire. The recording chamber consisted of a 10 ml plastic dish, the base of which was lined with a 5 mm layer of Sylgard resin (Dow Corning, Midland, Mich.). The chamber was partially submerged in a 50 ml petri dish into which a series of heating coils were added. Current to the coils was supplied by the same 6V battery connected to the light source, and was adjustable so that fluids within the chamber could be maintained at 35-370C. Visual inspection of the preparation was maintained at all times through a microscope (Wild Instruments M5A, Switzerland). Single electrode recordings used an electrical circuit described by Araki and Otani (1955). A microelectrode was mounted in a dc bridge circuit so that it could be used simultaneously to pass current and record voltage changes. A square wave stimulator (Grass S4, Quincy, Mass.) and a stimulus isolation unit (Grass SIU-4B) were connected across the bridge and supplied constant current pulses. The ends of the bridge were connected to a preamplifier (WPI M707) whose outputs led to differential amplifiers on a dual beam oscilloscope (Tektronix 5113, Beaverton, Oregon). Channel 1 recorded resting potential and voltage changes in response to currents that were monitored on channel 2. The amount of current delivered through the electrode was controlled by the stimulator. The bridge was balanced extracellularly by passing 5 nA rectangular current pulses of both polarities through the electrode and adjusting the variable bridge 24 resistance until there was no dc deflection on channel 1. At balance, the voltage step recorded by the electrode represented the change in transmembrane potential due to the current pulses. Multiple electrode recordings that measured the decay of electrotonic potentials over distance did not monitor voltage changes generated at the injecting electrode, therefore steps were not taken to balance the circuit. These recordings used large (100-400 nA) current pulses that were supplied by an iontophoretic programmer (WPI 160). Currents of this magnitude exceed the limitations of the bridge circuit, which was therefore temporarily interrupted. During these re- cordings, tests were always conducted to ensure that inter-electrode coupling did not occur. The following is a list of methods employed for determining the biophysical characteristics of the tegument and muscle compartments of adult male schisto- somes: Comfirtmentalization of Electrical Potentials A single microelectrode was lowered into the bath, and advanced by a micromani- pulator (Narishige Instruments) until it just penetrated the dorsal tegument of the preparation at a point just lateral to the gut, and approximately midway along the length of the body. Penetration of the tegument was signaled by an abrupt downward deflection of the trace on the oscilloscope to a steady resting potential that ranged from 30-60 mV negative to bath (Figure 6). This most superficial electrical potential was identified in studies conducted by Fetterer gt a_l. (1980) as the potential developed across the outer tegumental membrane. This potential will be referred to as Eteg- The electrode was then advanced further, into an electrical compartment immediately beneath the tegument. Bricker gt a_l. (1981) have presented evidence suggesting that this potential represents fibers of circular and longitudinal muscle. 25 Figure 6. Microelectrode recording of three distinct zones of electrical potential in an adult male S_. manso___n_i. Eteg: electrode tip within the tegument (Fetterer e_t al., 1980); E2 and E3, most evidence suggests that the electrode tip during these recordings lies within muscle fibers (E2) and extracellular spaces (E3) (Bricker e_t g” 1981). 27 Electrode penetration of that compartment appears on the oscilloscope as a relatively abrupt upswing from the highly polarized tegumental zone, generally to a resting potential of about one-half of that observed within the tegument of the same animal (i.e., to between 15-30 mV negative) (Figure 6). This potential will be referred to as E2. Resting potentials observed within this zone were not as stable as those of the tegument, and sporadic oscillations or a gradual upward drift of the signal was common. This tendency often led to complications that confounded attempts to derive some of the other biophysical parameters, but obtaining muscle resting potentials did not require a sustained recording. Normally, a relatively steady signal plateau could be maintained for up to several seconds. Advancement of the electrode tip to regions deeper than the muscle compartment resulted in a gradual diminution of the negative electrical potential, eventually to levels that approached and occasionally exceeded 0 mV. Evidence obtained during Bricker's HRP marker studies on muscle suggests that this zone represents either submuscular parenchyma or extracellular spaces. Bricker e_t _at. (1981) have designated the compartment as E3. For the present investigation, I have classified data collected from this compartment according to that designa- tion. During attempts to record steady resting potentials from the tegument and particularly the E2 compartment, brief episodes of apparent slow and fast wave transient electrical acitivity often occurred. These responses were observed and recorded, but the rarity of their appearance and their tendency to spontaneously disappear rendered any type of controlled analysis or modification impossible. Current/Voltage Relations Because of the preparation's size and unusual geometry, it was not possible to spatially isolate the morphological substrate of any of the electrical potential 28 compartments. This made it impossible to determine unequivocally the resistance characteristics of the membranes, and underscored the desirability of utilizing alternative techniques. The present investigation employed single and sometimes multiple microelectrode techniques for the determination of current/voltage relations. The input resistance (Rin) of each electrical compartment was obtained by using a bridge circuit, so that a single electrode could be used for both current injection and voltage change measurements (Araki and Otani, 1955). By injecting 500 msec current pulses of alternate polarity, in sequential increments of 1-4 nA into each electrical compartment, current-versus-membrane potential curves were obtained. Rin is given by the lepes of these curves, which are derived by linear regression analysis. Generally, microelectrodes possessing low resistances (2-3 MOhms) proved to be best suited for this test. After being lowered into the recording chamber as near to the preparation without making contact as was possible, current pulses were delivered through the electrode, and the bridge circuit balanced. The tegument was then penetrated, and current pulses were delivered. Voltage changes were recorded in response to currents delivered to that compartment as well as the muscle and parenchyma. Upon completion of each sequence of 1-4 nA current pulses, the microelectrode was withdrawn from the parasite and a 4 nA current of each polarity was once again delivered into the bath to ensure that the impedance of the electrode had not been altered. Data obtained from trials in which electrode impedance changes did occur were discarded. Impedance changes of this kind occurred approximately one- third of the time. In the above procedure, no efforts were made to rebalance the electrode after penetrating the tissue. Pressure on the electrode tip during tissue impale- ment can alter the resistance of the electrode, which can in turn significantly 29 affect voltage changes observed in response to injected currents (Coombs t g” 1959). Since typical membrane time constants are on the order of milliseconds, some investigators suggest that it is permissible to re—adjust the bridge-balance once inside a membrane-bound structure, and thereby cancel any steep initial edge of a voltage signal that does not have a relatively slow (i.e., at least a millisecond) time factor associated with it (Kater _e_t gl_., 1977). This maneuver is thought to be justified according to the principle that the capacitance of a membrane will be charged prior to the deve10pment of any ion fluxes across the membrane. This discharge generally requires at least a millisecond. Therefore, any rapid step in voltage with essentially no time constant probably represents bridge imbalance (Coombs _et gt, 1959). Because of these considerations, the above experiments were repeated using 20 nA currents, and data were recorded only after the bridge balance had been re- adjusted after penetration of the tissue. The larger current was used in an attempt to improve signal to noise ratios. Only the tegument of parasites immobilized with carbachol or by the addition of 200 MOsm sucrose were tested in this manner. The instability of E2 and E3, together with the small portion of the voltage signal with a slow time course resulted in poor signal to noise ratios, and made interpretation of responses in those compartments extremely difficult. Time Constants Single electrode tests using extracellularly bridge-balanced electrodes were conducted concurrently with Rin measurements to determine the time required for voltage signals in response to 3 nA depolarizing current pulses to reach 1 - l/e (or 6396) of their maximum value. A value for this parameter was also obtained from the signals generated during Rin tests for which intracellularly bridge-balanced electrodes were used. In the latter case, these times were of course much longer, due to the fact that the initial steep element of the voltage 30 signal was zeroed out, and only that portion with a slow rise time was considered. Because of the poor signal to noise ratio and high-gain amplifications required for this analysis, the tegument was the only compartment investigated by this procedure. A less direct method for deriving a time constant was also employed. In this test, separate current injecting and recording electrodes were used. The electrode deployment was similar to that used for determination of current/voltage relations over distance. The time required to reach one-half of the maximum amplitude of electrotonic potentials generated in the tegument and muscle was measured at points 0.1, 0.3, and 0.5 mm distal from the site of current injection. Only hyperpolarizing current pulses of 300 nA were used in this test to avoid any complications of interpretation that rectification (or perhaps, electrogenesis) might have introduced. The value of this parameter as an indicator of the time constant is manifested in a cable theory expression developed by Hodgkin and Rushton (1946), who pointed out that the time factor would increase linearly with distance along a cable. The slope (m) of these times when expressed over distance is found to equal the membrane time constant divided by twice the space constant (m=Tau/ 2 A). Space Constants Multiple electrode tests for obtaining information on membrane resistance features and time constants indicated that the longitudinal spread of current within the tegument and muscle was substantial enough to measure at relatively large inter- electrode separations. Separate current injecting and recording electrodes were used to determine the extent to which induced electrotonic potentials spread through the three electrical compartments. During this test, the injecting electrode was inserted into the tegument, muscle, or parenchyma just lateral to the gut, and about midway along the length of the body. A 31 second electrode was inserted into the same compartment at a point 0.1 mm caudal from the injecting electrode, and maintained at this position to serve as the first recording electrode. A third electrode was inserted into the same compartment, but was maneuvered successively to points 0.3, 0.5, and 1.0 mm away from the injection site. Depolarizing and hyperpolarizing current pulses of 200 nA and 500 msec duration were delivered via the iontOphoretic programmer. The first recording electrode served as a monitor to verify (by their magnitude) that signals were in fact being successfully delivered into the compartment of interest, while simultaneously providing data to be used in the space constant determinations. Tissue Coupling Ratios The electrode deployment used to determine tissue coupling ratios is depicted in Figure 11. Current pulses of -200 nA were delivered for 500 msec at one pint in the tegument or muscle, and resulting potential changes were monitored simultaneously in that compartment as well as in one of the two remaining compartments at points equidistant from the current source (0.3 mm). Results were expressed as a ratio of the voltage signal generated in the non-injected compartment (V2) over that recorded from the compartment being injected (V1). Recordingg in Hyperosmotic Solution Recordings similar to those made in schistosomes immobilized in carbachol or Na- PB were also made in parasites immobilized in a solution of HS/Earle's made hyperosmotic by the addition of 200 mOsm sucrose. This raised the osmotic pressure of the recording medium to approximately 500 mOsm. Sucrose is a non- diffusible solute, and therefore draws H20 out of the preparation. This is turn results in cell shrinkage, which is believed to disrupt intercellular gap junctions in cardiac and smooth muscle syncytia (Tomita, 1966). RESULTS Single Electrode Experiments Compartmentalization of Electrical Potential Microelectrode penetrations of .52. mansoni indicate that there exist three discrete compartments of electrical potential (Figure 6). The first of these encountered upon impalement of the parasite's surface has been demonstrated by Fetterer gt g_l. (1980) to be the tegument. Horseradish peroxidase iontOphoretically injected into that compartment was confined exclusively within the tegument and its underlying cyton cell origin (Figure 2). The electrical potential measured in the ventral tegument of unanesthetized parasites bathed in a 1:1 mixture of fetal calf's serum (FCS) and Earle's saline was -34.9 t 1.9 mV (Fetterer gt a_l., 1980). In the present investigation, the PCS constituent of the recording medium was replaced by horse serum (HS), and all recordings were made in the dorsal tegument and underlying tissue layers rather than in the ventral tegument. The mean resting tegumental potential (Eteg) of unanesthetized parasites measured under these conditions was -42.8 :t 1.6 mV. Because of the continuous movement exhibited by unanesthetized worms, microelectrode recordings from the 1-4 microns thick tegumental compartment, as well as other regions, were extremely difficult to maintain for extended periods. For this reason, most of the tests were performed while animals were immobilized with either carbachol (1 x 10‘4M), or sodium pentobarbital (Na-PB, 50 mg%). These drugs, when administered in the above concentrations, inhibited essentially all of the mobility normally exhibited by the parasites, and made prolonged recordings possible. Fetterer's gt a_l. (1980) data suggest that use of 30 mg% Na-PB does not significantly affect resting potential or input resistance 32 33 (Rin) of the tegument. My data are consistent with these findings. Neither carbachol nor Na-PB significantly altered the resting potential of the dorsal tegument. The mean resting potential recorded in parasites immobilized with carbachol was -45.9 i 2.5 mV with a range of -35.3 mV to -49.0 mV. These results are statistically similar to those obtained from parasites immobilized with Na-PB, for which the mean was -40.6 t 2.7 mV, with a range of -34.3 mV to -52.7 mV. A second electrical potential compartment is encountered at once as the microelectrode is advanced through the basement membrane of the tegument. Penetration of this compartment is signaled by the appearance of a steady resting potential that is about one-half of that recorded in the tegument (Figure 6). Bricker gt _a_l. (1981) have demonstrated that this second zone of electrical potential probably originates from fibers of circular or longitudinal muscle that lie immediately beneath the tegument (Figure 3). The mean resting potential of this compartment measured in unanesthetized schistosomes was -20.2 :t 1.0 mV, and values ranged from -17.0 mV to -24.0 mV. Similar muscle recordings in parasites immobilized in carbachol or Na-PB yielded statistically similar results of -22.0 t 1.1 mV (range = -14.0 mV to -32.0 mV) and -21.3 i 1.3 mV (range = -17.0 mV to - 24.3 mV), respectively. These values are similar to those reported by Bricker g_t a_l. (1981). Generally, steady E2 potential recordings were more difficult to maintain than ones from the tegument, even in well anesthetized parasites. After resting within the E2 compartment for a variable period of time (from 3-12 seconds), the electrode usually drifted into a third, less polarized region (Figure 6). It was presumed that this potential represents the parenchymal or extracellular space compartment identified by Bricker _e_t gt. (1981), and classified as E3. The mean resting potential of the E3 compartment in carbachol anesthetized parasites was - 4.7 i 0.3 mV, with a range of -3.0 mV to -7.0 mV. Recordings from the E3 34 compartment of Na-PB anesthetized parasites yielded a value of -3.3 i 0.7 mV and ranged from -0.7 mV to -5.7 mV. These values are consistent with those reported by Bricker e_t gt. (1981). Current/Voltage Relations Through the use of a bridge circuit by which current could be injected through a single recording electrode, I versus E curves were obtained for each of the compartments, and from these, input resistances (Rim) were calculated. Data were obtained by injecting 500 msec depolarizing and hyperpolarizing current pulses into each compartment while simultaneously recording the resulting voltage changes at the electrode tip. Rin values obtained for the dorsal tegument of parasites immobilized with carbachol and Na-PB were 4.5 MOhms and 3.5 MOhms, respec- tively (Figure 7). The difference is not significant (P>.05). These results are Similar to those obtained by Fetterer gt g. (1980) from the ventral tegument of unanesthetized schistosomes (5.2 MOhms), and worms anesthetized in a medium containing a lower concentration (30 mg96) of Na-PB (4.3 MOhms) (Table 1). Recordings from the E2 compartment revealed that the Rin there is nearly twice that recorded in the tegument. Parasites immobilized with carbachol exhibited an E2 input resistance of 9.2 MOhms, while the Na-PB group showed a statistically similar value of 7.3 MOhms (P>.05). Input resistance was also determined for the E3 compartment, but only in parasites immobilized with carbachol. Rin for this compartment was 3.5 MOhms, a value not significantly different from that recorded in the tegument of carbachol treated schistosomes (P>.05). Single electrode tests for the determination of time constants yielded values that were shorter than most of those previously reported for epithelial or smooth muscle membranes (Tomita, 1975; Prosser, 1972). Depolarizing current pulses of 3 nA were delivered to the tegument or muscle while voltage responses were 35 Figure 7. Graph of current-versus-membrane potential measured in each of the electrical compartments in parasites immobilized with 1 x 10"4 carbachol. All points are means with one standard error for 6 parasites. Lines were fitted to points using linear regression. 36 1(nA) O - TEGUMEN T 0'52 0'53 50" L -25 p -50 I- lfigne7 37 analyzed to determine the time required for them to reach 1 - l/e (or 6396) of their maximum value. The tegumental time constant, as measured in this manner, was about 0.25 msec when recorded from schistosomes immobilized in either carbachol or Na-PB (Table 1). The time constants for E2 membranes were essentially identical to this value (P>.05). The values listed above for Rin and Tau were derived from the total potential change recorded in response to currents injected from extracellularly bridge- balanced electrodes. Upon penetration of the parasite, no adjustments were made to compensate for possible electrode impedance alterations that may have been incurred by contact with the tissue (Coombs g g” 1959). Therefore, the steep initial and terminal phases of the voltage responses may have been due in part to increased electrode impedances. For this reason, Rin measurements were made from the tegument using microelectrodes that were re-balanced intracellularly by eliminating the steep initial phase of the voltage perturbation. Eliminating the steep initial phase of the electrotonic potential induced by a 20 nA hyperpolarizing current pulse left only a small fraction of the original signal. Voltage changes that occurred during that portion of the trace characterized by a slow time course ranged from 0.7 mV to 2.0 mV in magnitude, with a mean value of 1.0 t 0.3 mV. This translates into an Rin of approximately 50 KOhms, or about 196 of the value derived from the data obtained from bridge circuits not re-balanced intracellularly. Time constant determinations made using intracellularly bridge-balanced electrodes yielded values larger by over an order of magnitude than those listed above for tests involving bridges not re-balanced. Under these conditions they averaged 4.5 i 0.2 msec, and ranged from 4.3 msec to 4.6 msec. This test was performed while recording from the tegument only of carbachol immobilized parasites. The small voltage changes and poor signal to noise ratios made similar recordings in the E2 or E3 compartments impossible. 38 TABLE I. Biophysical properties of various compartments of male _S_. mansoni using single electrodes. 1x10'4 Carbachol 50 mg% Na-PB +200 mM Sucrose E (mv) tegument -45.9:2.5 -40.6:2.7 -38.6:3.8 compartment 2 -22.0:1.1 -21.3:1.3 -19.7:1.8 compartment 3 -4.710.3 -3.4:0.7 -2.8j-_0.4 Rin (MOth) tegument (e) 4 . 6:1 . 1 3 . 5:0 . 7 6 . 3:1 . 5 tegument (i) .052:.002 N.D. .053:.002 compartment 2 (e) 10.3:1.8 7.3:1.3 11.5:1.8 compartment 3 (e) 3.5:0.8 N.D. N.D. Tau (msec) tegument (e) .24:0.01 0.29:0.02 0.24:0.01 tegument (i) 4.5:0.2 N.D. 5.2:0.3 compartment 2 (e) 0.25: .01 0.28:0.03 0.23:0.01 compartment 3 (e) 0.13:0.01 _ N.D. N.D. e = bridge balanced extracellularly i = bridge balanced intracellularly (these measurements made in tegument only) ND = Parameter not determined 39 Multiple Electrode Studies Current Spread ThrougElectrical Compartments To circumvent some of the problems associated with single electrode techniques (Frank and Fourtes, 1956), membrane electrical properties were also examined using separate current injecting and recording electrodes. The extent of current spread within each compartment was measured by injecting 200 nA hyperpolarizing current pulses and recording the resulting electrotonic potentials at specific distances from the point of injection within the same tissue compartment. From these data, I determined the distance over which the voltage change decayed by a factor of 1 -1/e (Figure 8). I have termed this distance A' in recognition that it does not represent a true space constant (Hodgkin and Rushton, 1946). The X's obtained in this manner were relatively short, and at the lower range of values reported for the space constant in other preparations (Prosser, 1972). The tegument and E2 compartments of parasites immobilized with carbachol had a 1' of 0.55 mm and 0.69 mm, respectively (Figure 9). For Na-PB treated schistosomes, )3 was 0.32 mm (tegument) and 0.36 mm (E2). Recordings were also made in the E3 compartment of six parasites immobilized with carbachol, but 1' was difficult to determine by the same method in that compartment, since the voltage change in response to 200 nA current, even at 0.1 mm, was only about one- third as great as that recorded in the tegument and muscle (Figure 9). From the results of this analysis it is apparent that some form of low resistance pathways within the electrical compartments exist that make longitudi- nal current flow possible. Fetterer gt g. (1980) have demonstrated that the tegument is an anatomical syncytium. Current would be expected to flow through that compartment providing the tegumental membrance is of sufficient resistance. 40 Figure 8. Depiction of the recording apparatus employed during recordings for current spread. At right, some representative oscilloscope traces at points used during the test. 41 llO mV 200 msec Figure 8 42 Figure 9. Graph of current decay in the electrical compartments of S. mansoni immobilized with 1 x 10‘4 carbachol. All points are means with one stan'aafi error for 6 parasites. 43 IOOIAfl 1m 1 [.811 G 2 3 m OM. _ m 110 on to M... .. Iool. ...\.\ K . /- V. I...\ .\ .m. /- /V... Figure 9 44 Currents would also be expected to flow relatively unimpeded through extracellular spaces which appear from marker studies to be continuous within the parasite (Bricker e_t g, 1981). Current spread through the E2 compartment over distances much greater than the length of individual muscle fibers, however, implies that some form of low resistance pathways must interconnect adjoining muscle fibers, if that compartment does in fact represent muscle tissue. Rise Times for Injected Currents The rise times of voltage changes induced when separate injecting and recording electrodes were used appear to increase linearly with distance through both tegumental and E2 syncytia. If the time for an induced voltage change to reach one-half of its steady-state value is plotted agianst the distance between the current injecting and recording electrode, a straight line is obtained (Figure 10). The slope of this line (m) is equal to the membrane time constant divided by twice the space constant (Hodgkin and Rushton, 1946). Slapes derived in this way from tegumental recordings in nine carbachol anesthetized parasites yielded a value of 75.0 E2 recordings revealed a statistically similar value of 66.3 (P>.05). These values coincide well with similarly analyzed smooth muscle preparations (Tomita, Intercompartmental Coupling Ratios In order to determine the degree of electrical coupling between tegument, muscle, and the E3 compartments, hyperpolarizing current pulses of 500 msec and 200 nA were injected into one compartment while the resulting electrotonic potentials were recorded simultaneously in that compartment as well as one of the remaining two. All recordings were made at sites 0.3 mm distal from the injection (Figure 11). This test is somewhat similar to that previously used to determine the degree 45 Figure 10. Graph of the time required for electrotonic potentials spreading through the tegument and E2 compartment to reach one-half of their steady-state amplitudes expressed over inter-electrode separation. All points are means with one standard error for 6 parasites immobilized with 1 x 10"4 carbachol. The slope of these lines (m) is related to the membrane time and length constants according to the equation: m = Tau/ 2 (Lambda). This parameter assumes a value of 75.0 (tegument), and 66.3 (E2). These values are statistically similar (P>.95). 46 O -TEGUMENT O '52 60' «93:; 2:: .qu .33.“...- 0.5 0.1 (mm) Figure 10 47 Figure 11. Depiction of recording apparatus employed during recordings for tissue coupling. Right: representative oscilloscope traces obtained during tegument:E2 recordings. Lower: representative traces obtained while injecting in E2 and recording in tegument (left), or E3 (right). 49 of intercellular coupling in various nerve and epithelial preparations (Kao, 1967; Lowenstein and Karma, 1967; Kater gt g" 1977; M.V.L. Bennett _e_t gl_., 1978). In those tests, current is injected into one cell while voltage changes are simul- taneously recorded in that cell as well as an adjoining cell that appears to be connected by way of a close apposition or "gap junction." The coupling ratio is then given by the ratio: the potential developed in the adjoining cell (V2) divided by the potential developed in the injected cell (V1). By using the method outlined above, and expressing the magnitude of potentials recorded in the non-injected compartment over those recorded in the injected compartment (i.e., V2/V1), tissue coupling ratios were obtained that describe the spread of current among each of the three compartments (Table 2). With a coupling ratio of 0.80, it appears there exist low resistance pathways allowing current to pass in both directions between tegument and E2. There appears to be little interconnection between the E3 compartment and the tegument or E2, as the coupling ratio here was only about 0.15. Active Membrane Responses Active membrane responses often appeared at the recording electrode when depolarizing currents of 200 nA or greater were delivered into the tegument or E2 compartment (Figures 12-14). Such responses were never elicited when the current injecting electrode was located within extracellular spaces. They were generally superimposed on electrotonic signals that were at least 10 mV in magnitude at a point 0.1 mm away from the injection site. The "spikes" usually occurred after the electrotonic potential had achieved a relatively steady plateau, and they were often preceeded by a drop in membrane resistance, as evidenced by the transient reduction in signal magnitude just prior to spike initiation. 50 Figure 12. Oscilloscope traces of responses evoked by injecting depolarizin current pulses into the dorsal tegument of _S_ mansoni immobilized with 1 x 10 carbachol, and 50 mg% Na-PB. All injections and recordings made in dorsal tegument. Upper: aborted spike preceeding large amplitude spike in response to a 200 nA depolarizing current pulse. Inter-electrode separation is 0.15 mm. Calibra- tion: vertical, 5 mV; horizontal, 200 msec. Middle: two overshooting spikes in response to 400 nA current pulse in a Na-PB anesthetized schistosome. Inter- electrode separation is 0.2 mm. Et is -48 mV. Overshoots to +26 mV and +28 mV. Calibration: vertical, 20 mV; horizontal, 200 msec. Lower: spikes and aborted spikes recorded at 0.1 mm (lower), and 0.3 mm (upper) in a schistosome immobilized with 1 x 10’4 carbachol. Eteg is -46 mV. Overshoots to +10 mV. Calibration: vertical, 10 mV; horizontal, 1 sec. ~ l I” .1 UN 52 Figure 13. Oscilloscope traces of responses evoked by injecting depolarizulg current pulses into the E2 compartment of S. mansoni immobilized with 1 x 10 carbachol. All injections and recordings made 1n the dorsal subtegumental (E2) compartment. Upper: series of graded spikes recorded at 0.1 mm and 0. 3 mm away from the injection site in response to 200 nA depolarizing current pulse. Calibration: vertical, 20 mV; horizontal, 100 msec. Middle: single spike recorded at 0.1 mm in response to 300 nA depolarizing current pulse. E2 is -21 mV (upper), and -22 mV (lower). Calibration: vertical, 20 mV; horizontal, 200 msec. Figure 13 54 Figure 14. Representative oscilloscope traces of active responses evoked by injecting depolarizing current pulses into the dorsal tegument or muscle layer of S. mansoni while simultaneously recording in both syncytia at points equidistant (0:3 mm) from the injection site. Upper: single spike recorded in tegument (upper, Eteg = -43 mV) and E2 (lower, E2 = -28 mV) in response to intra-tegumental (Eteg = -46 mV) injection of +300 nA. Calibration: vertical, 20 mV; horizontal, 100 msec. Middle: graded responses in muscle (upper, E2 = -26 mV) and tegument (lower, Rt -48 mV) upon injection of +400 nA current into the E2 compartment. Cah ration: vertical, 10 mV, horizontal, 100 msec. Lower: series of graded responses upon injection of +300 nA into the tegument. Responses recorded in tegument (upper, Ete = -44 mV) and the E2 compartment (lower, E2 = -25 mV). Calibration: vertical, mV, horizontal, 100 msec. 56 The spikes were of variable amplitude and duration. Some were observed to overshoot by as much as 28 mV (Figure 12). Overshooting depolarizations had rise times of up to 3 V/sec, and durations of 10-40 msec. Such overshooting spikes were never recorded at distances greater than 0.15 mm from the injection site. On some occasions, 2-6 spikes of approximately equal magnitude and time course would develop consecutively during an electrotonic depolarization (Figures 12 and 13). Clusters of graded potentials were even more common (Figures 13 and 14). These responses were characterized by a continuum of amplitudes, durations, and waveforms. While spikes were most readily observed in recordings made very near the injection site, they were also recorded on many occasions at points as far away as 1.0 mm (Figure 14). These responses were, however, always reduced in magnitude from those recorded nearer the injection. Usually, large active responses recorded at 0.1 mm could also be monitored at a point further away, but in a reduced form. In such cases, the decrement in response amplitude over distance was approxi- mately equal to that predicted on the basis of the passive current spreading characteristics and the tegument of E2 compartment. On several occasions, spikes were recorded from the tegument in response to current injected into the E2 compartment, and vice-versa (Figure 14). Responses recorded in the non-injected compartment appeared to be identical to those observed in the compartment being stimulated. Effects of Hyperosmotic Solutions My experiments with separate current injecting and voltage recording electrodes reveal that the E2 compartment is a functional syncytium, and that there also exists an electrical coupling between tegument and E2. Anatomical studies have shown the presence of apparent gap junctions between muscle fibers as well as between tegument and muscle (Silk and Spence, 1969b). .- -._:} I. .. 57 Tomita (1966) has shown that gap junctions in other animals are disrupted by exposure to hyperosmotic solutions. To test if hyperosmotic media disrupt the apparent low resistance pathways in schistosomes, the animals were exposed to HS/Earle's solution to which was added sufficient sucrose to raise the osmotic pressure by 200 mOsm (i.e., to a toal concentration of approximately 500 mOsm). The tissue current spreading properties were then re-examined. Table 2 summarizes the results of these experiments. Resting membrane potentials and single electrode time constants were not significantly altered by exposure to the hyperosmotic medium (P>.05). Rin exhibited a 5396 increase in response to the sucrose in the tegument, and a 2696 increase in E2 (Figure 15, Table 1). Multiple electrode tests showed that the time required for electrotonic potentials to reach one-half of their steady-state amplitude as a function of inter- electrode separation yielded a straight line. The slope of this line (m) was 1.5 times greater in the tegument and 1.7 times greater in the muscle than those obtained during recordings form parasites immobilized with carbachol (Figure 16). Since m=Tau/2 A, the results suggest that either the time constant had increased, or else the space constant had decreased. The hyperosmotic medium drastically reduced current spread within both the tegument and E2 compartment. Electrotonic potentials measured at 0.1 mm in response to 200 nA hyperpolarizing current pulses were reduced from 17.5 mV to 4.5 mV in Eg, a 74% drop. In the tegument, the potentials did not decrease so drastically. They dropped from 21.0 mV to 9.5 mV, a 53% drop (Figure 17). The coupling ratio between the tegument and E2 was also reduced, from 0.71 to 0.47. During these tests, no evoked active responses were recorded, nor were spon- taneous depolarizations apparent at any time. 58 Figure 15. Graph of current-versus-membrane potential measured in the tegument and E2 compartment of _S_. mansoni bathed in an HS/Earle's medium made hyperosmotic by the addition of 265 mOsm sucrose. The slopes of these lines are greater than those obtained during similar recordings made in schistosomes immobilized with 1 x 10'4 carbachol or 50 mg% Na-PB, indicating that membrane input resistance had increased upon exposure to the non-diffusible hyperosmotic solution. 59 O - TEGUMENT 0'52 ‘ 50 - -25 - -50 )- Figure 15 60 Figure 16. Graph of the time required for electrotonic potentials spreading through the tegument and E2 compartments to reach one-half of their steady-state amplitudes expressed over inter-electrode separation. Solid lines represent data from schistosomes immobilized with hyperosmotic sucrose solution. Dotted lines represent similar recordings made in schistosomes immobilized in 1 x 10 carbachol (Figure 10). All points represent means with one standard error for 6 parasites. 61 O-TEGUMENT «co-Ev OE: owes—9:0 =2Toco 0.3 0.5 X (mm) 0.1 Figure 16 62 Figure 17. Graph of current decay in the tegument and E2 compartments of S. mansoni immobilized by the hyperosmotic sucrose solution. Note the large drop '13 current spread in both syncytia, which is most pronounced in the E2 compartment. All points are means with one standard error for 6 parasites. 63 _ I-TEGUMENT l' 0'52 L. )- . ¢\‘ * ¢\\ - 1. <>/°—; 4 L / L 011.53.015.1..110 (mm) Figure 17 64 TABLE II. Biophysical properties of various compartments of male g mansoni using multiple electrodes. 1x10"4 Carbachol 50 mg96 Na-PB +200 mM Sucrose Lambda' (mm) tegument 0.55 0.32 C. . compartment 2 0.69 0.36 C.D. compartment 3 C.D. C.D. C.D. m (msec/mm) tegument 75.0 .D. 108.0 compartment 2 66.3 N.D. 110.5 compartment 3 N.D. .D. N.D. Coupling Ratios (no units) tegument: compartment 2 0.71:0.05 0.83:0.05 0.47:0.11 compartment 2: tegument 0.83:0.03 0.79:0.06 0.55:0.02 tegument: compartment 3 0.16:0.03 N.D. C.D. compartment 2: compartment 3 0.14:0.03 C.D. N.D. ND = Parameter not determined CD = cannot determine accurately DISCUSSION Morphological Substrates of Electrical Compartments Electrical recordings obtained during this investigation were identified as being from tegument, E2, or extracellular space according to the conventions established by Fetterer gt g_l_. (1980) and Bricker gt gl_. (1981). These classifications are based on depth of penetration and relative electrical potentials. The first potential encountered upon impalement of the parasite is the tegumental potential (Eteg)- The values I recorded from the dorsal tegument of unanesthetized schistosomes (-42.8 mV) were approximately 8 mV more negative than those recorded from the ventral tegument (-34.9 mV) by Fetterer gt g_l. (1980). This difference may be accounted for on the basis of alterations in the recording medium, or method of immobilization, that is, number and placement of pins. The Eteg recorded is within the range of those reported for various epithelial preparations, such as mouse salivary gland cells (-50mV) (Kater _e_t g, 1978), Amphinuma small intestine mucosal cells (-41 mV) (White, 1976), and Necturus small intestine mucosal cells (-35 mV) (Doherty gt gl_., 197 9). Most evidence suggests that the second potential encountered upon penetra- tion of the schistosome is endemic to the muscle layer (Bricker gt gl_., 1981). My data indicate the E2 potentials are approximately one-half as great as those recorded in the tegument. This finding is consistent with that reported by Bricker e_t g1. (1981). Most vertebrate and invertebrate muscle preparations exhibit larger resting potentials than those recorded in the schistosome. The -20 mV to -29 mV values I recorded from the E2 compartment were significantly lower than those reported for vertebrate smooth muscle preparations, which range from -37 mV in guinea-pig gall bladder (Creed and Kuriyama, 1971) and urinary bladder (Creed, 1979) to -65 mV in rat uterus (Mekata, 1971). However, the muscle resting 65 66 potentials I recorded are similar to those observed in some invertebrate muscle. Intramuscular recordings from the nematode Ascaris yield values ranging from -30 mV to -40 mV (del Castillo gt Q” 1964; Rozhkova _e_t g” 1969). On the basis of HRP injections through microelectrodes, (Bricker e_t gl_., 1981) concluded that the third electrical potential encountered, E3, has its origin in extracellular spaces in the animal. The low value I record for this potential is similar to that reported by Bricker gt gl_. (1981). Similar low negative transcuta- neous potentials are recorded in the gills of the crustacean Callinectes. Mantel (1967) has demonstrated that this potential is due to the active uptake of Cl‘ from the medium Passive Current Spreading Properties of the Electrical Compartments The spread of current signals within the parasite's tegument indicates that this compartment is an electrical syncytium. This confirms earlier electron microscope studies which show that the tegument appears to be an anatomical syncytium (Silk .e_t gl_., 1969), and the studies of Fetterer e_t g. (1980) which show it to have no barriers hindering HRP diffusion. The two-dimensional nature of the schistosome's tegument would allow current injected from a point source to spread in all directions within the space of the syncytium. This would result in a rapid reduction in current density with distance away from the injection site (Jack gt g_l., 1975). While current does decay rapidly in comparison with most nerve preparations, the tegumental membranes do provide enough resistance to make quantifiable measurements of the electrotonic signals possible over distances of up to 1.5 mm. The pattern of current spread in the tegument is well described by a Bessel function (Shiba, 1970). Decay is initially very steep, then begins to flatten-out and becomes exponential about 0.1 mm away from the injection site (Figure 18). This 67 Figure 18. Semilogarithmic plot expressing current decay as a function of inter- electrode separation in the tegument and E2 compartments of _S_. mansoni immobil- ized with 1 x 10'4 carbachol. Note the approximately exponential rate of current decay recorded in both compartments beginning at 0.1 mm distal from the injection site. All points are means with one standard error for 6 parasites. 68 4 40 p? 30 . o-TEGUMENT 0-52 l 20 - o l I o O I 10 - I . i 9 E 1.. I <5 " O i 0.1 0.3 0.5 1.0 X (mm) Figure 18 69 decay characteristic is a common feature of other epithelial syncytia as well, including newt gut (Shiba, 1970) and insect salivary glands (Caveney, 1974). The method I used for quantifying current spread (1') probably yielded artifi- cially high values, as the steepest portion of the decay curve (that area between the injection site and 0.1 mm) was excluded from consideration. This convention was necessary, however, in order to make quantifiable analyses of current decay rates possible. Because currents decay so rapidly in the immediate vicinity of the injecting electrode, N determinations using the injection point as a reference instead of the more distal location where decay becomes exponential would have resulted in extremely low values that could not have been compared across treatments. While the A's of 0.32 mm to 0.55 mm derived for the tegument of _S_._ mansoni are small compared to the length constants reported for most nerve and muscle preparations (Prosser, 1972; Creed, 1980), they do represent a significant portion of the parasite's total body length, which seldom exceeds 6 mm. In preparations in which current flow is not restricted to one plane, voltage signals characteristically decay rapidly (Jack _e_t gl_., 1975). Josephson and Schwab (1979) have measured the length constant in the exumbrellar epithelium of hydromedusae and found it to be 1.3 mm. This preparation, like the tegument of _S_._ mansoni, represents a two- dimensional syncytium. The muscle system of _S_. mansoni, unlike the tegument, does not appear to be an open anatomical syncytium. Electron micrographs show definite cell boundaries between muscle fibers (Silk and Spence, 1969b), and Bricker gt _a_l. (1981) have shown that HRP injected intramuscularly is confined to the injected fiber. In spite of the limitations imposed on the diffusion of large molecular weight substances through the muscle compartment, multiple electrode tests reveal that ionic current does spread over relatively large distances (N: 0.69 mm in carbochol immobilized 70 parasites). I conclude from this that muscle fibers in the schistosome are probably coupled to each other by low resistance pathways. In vertebrate cardiac and smooth muscle tissue, individual muscle fibers are not electrically isolated, but connected to neighboring fibers by low resistance pathways. Most evidence suggests that these connections occur at sites where adjoining fibers closely appose one another to form electrotonic of "gap" junctions (Barr _e_t g_l., 1965). Gap junctions exhibit hexagonal arrays of membrane particles in the junctional plane that presumably form thin channels through which ions and some metabolites can pass from one cell to the next (McNutt and Weinstein, 1970). Gap junctions between adjacent muscle fibers in the schistosome have been described (Silk and Spence, 1979b). They may represent the morphological substrate of the low resistance pathways I describe. The presence of gap junctions could explain the mechanism whereby peris- taltic-like mechanical waves spread from one end of the schistosome to the other (Fetterer gt .a_l., 197 7 ). Electrical coupling of muscle fibers by way of gap junctions would alleviate the necessity for synchronous neural activation of each fiber prior to contraction. Muscle activation in schistosomes could be myogenic, and depend on differential membrane conductance changes in pacemaker cells that lead to rhythmically occurring contractions in those cells and electrically connected neighboring cells (Marshall, gt gt, 1978). Fax gt gt. (1981), on the basis of conducted responses to electrical stimulation, have provided additional evidence which suggests that contraction in schistosomes may be myogenic. Gap junctions are disrupted by exposure to solutions made hyperosmotic by the addition of sucrose, apparently because of cell shrinkage that accompanies water loss (Tomita, 1966). In all preparations exhibiting gap junctions that have been tested to date, disruption of contacts is always associated with a loss of intercellular electrical coupling (M.V.L. Bennett, 1974).. 71 When I exposed schistosomes to solutions made hyperosmotic by the addition of 200 mOsm sucrose they were immediately immobilized, but tegument and E2 resting potentials were not altered. This suggests that the resistance of non- junctional membranes remained the same. Total Rin of both syncytia increased slightly. This rise in Rin upon the addition of sucrose is consistent with previously reported effects on visceral smooth muscle (Nagai and Prosser, 1963). Current spread in the E2 compartment was reduced from 0.69 mm (in carbachol) to less than 0.2 mm. In the tegument, the reduction in current spread was significant, although not as great as that observed in E2. The tegument of §._ mansoni is an open syncytium (Silk gt 9, 1969; Fetterer gt gt, 1980), so the decrease in current spread within this compartment is probably due entirely to longitudinal resistance increases in response to shrinkage. In the E2 compartment, the decrement in current spread is much more pronounced. One explanation for this could be that, in addition to cell shrinkage, there is significant disruption of gap junctions between muscle cells. Analysis of electrotonic potentials initiated in the E2 compartment of schistosomes immobilized with carbachol or Na-PB indicate that current spread in this tissue layer is similar to that occurring in the tegument. This suggests that the sum of resistive barriers encountered by currents injected into either compart- ment are approximately the same. Given the relative locations and internal morphologies of the two compartments, currents spreading through the muscle should encounter more resistance to radial flow because of the overlying tegument, as well as to longitudinal flow, because of the additional fiber and bundle membranes. These factors would exert opposite effects on longitudinal current spread, with (the former enhancing it and' the latter impeding it. The net result appears to be that the different effects are balanced, so that the tegument and muscle have current carrying capabilities that are nearly identical. This model is 72 consistent with Rin measurements indicating that injected currents encounter more resistance in muscle than in tegument. It should be noted that the presence of gap junctions or other ionic channels may not be a prerequisite for multicullular preparations to exhibit cable-like behavior. If intercellular clefts are narrow enough to allow capacitive coupling, then low resistance junctions may not be necessary (Daniel and Sarna, 1978). Determining the precise role that either ohmic or capacitive coupling serve independently to impart syncytial qualities to the muscle compartment in schisto- somes would be difficult, as treatments disrupting one form would likely have a similar effect on the other. Measurements of current spread through the E3 compartment indiCate that decay occurs more rapidly there than in the tegument or muscle. This may be explained on the basis that current injected into extracellular spaces would not be radially confined to a narrow layer of efflux pathways as that injected into the electrical syncytia. The larger volume field available for current flow would tend to dilute current signals more rapidly. It is also possible that a large portion of current injected into the extracellular space escapes the worm by passing through the gut. Current/Voltage Relations and Time Constants Current/voltage recordings obtained by a single electrode bridge-balanced circuit indicate that both tegument and muscle membranes respond to inward and outward current in a linear fashion. This corroborates previous observations made from ventral tegumental recordings in unanesthetized schistosomes (Fetterer e_t gt, 1980). Because conductance changes were observed during subsequent multiple electrode studies that used larger currents, their absence in single electrode tests for Rin and Tau may have been due to the electrical geometry of the preparation. 73 Several investigators have noted that responses in electrically coupled systems (e.g., smooth muscle preparations) to intracellular stimulation are characterized by (a) linear I/E relations, (b) short time constants, (c) short space constants, and (d) the absence or rare appearance of spiking phenomena (Tomita, 1967; Kater gt g" 1977). All of these features accurately describe the responses to intracellular stimulation of the tegument and muscle compartments of §._ mansoni. A number of studies have addressed the question of why current behaves differently when injected into an electrically coupled multi-cellular system from that injected into a nerve or other single celled preparation. Tomita (1966) used intracellular stimulation to depolarize smooth muscle cells of guinea-pig taenia coli. Action potentials were initiated in very few of the cells, and the spatial decay of electrotonic signals was so sharp that they could not be monitored over distances greater than 0.1 mm. Tomita hypothesized that current spread was occurring in three dimensions through interconnections between the muscle cells (probably gap junctions). Under these conditions, the membrane area available for current efflux increases with the cube of the distance away from the current source, rather than linearly as is the case for preparations that are one-dimen- sional. Because of this, membrane near the injection site is shunted by low resistance pathways to adjoining fibers, so the impaled area does not become sufficiently depolarized for spike initiation to occur. Even when normal threshold potentials are achieved at the electrode tip, spikes may not be evoked in the coupled system, because outward current from adjoining membranes can re-charge the membrane capacity as rapidly as it is depolarized (Tomita, 1966). My data suggest that the electrical geometry of _S_. mansoni resembles multidimentional smooth muscle preparations in several respects. The high degree of membrane folding characterizing the tegument, and the multicellular nature of the underlying muscle syncytium both provide the morphological basis for this \‘_ _, 74 condition. Gap junctions that interconnect muscle bundle sarcolemmas (Silk and Spence, 1969b) and perhaps even the tegument and muscle syncytia (Silk e_t gt, 1969) may provide the morphological substrate for this electrical coupling. If coupling within and/or between the two compartments is extensive, then small intracellular depolarizing currents may not be sufficient to depolarize enough membrane for the initiation of action potentials. Jack _e_t_ gt (1975) consider the general situation where multidimensional current flow from a point source is possible. Here, the voltage change over distance can be defined according to the equation: V = C 1n (2 lg/r), where C is a function of the stimulus parameters, 2.2 is the space constant, and r is the distance between the current source and voltage monitor. From this equation it is apparent that using a single electrode bridge circuit will result in enormous voltage changes at the electrode tip, even when only small currents are injected, because under these conditions r approaches zero (Jack gt 9, 197 5). This response is precisely what is observed. To circumvent some of the problems associated with single electrode methodology, other investigators have adopted techniques that use multiple elec- trodes and/or field stimulation (Tomita, 1966; Kater e_t g, 1977). The major disadvantage of these methocb is that it is impossible to determine the precise amount of current entering the cells and causing the voltage changes that are being monitored. In the present study, both single and multiple electrode methods were used whenever possible to analyze the tegument and muscle membrane properties. In spite of possible complications that may have arisen because of the apparent multidimensional nature of the preparation, single electrode Rm values obtained from the dorsal tegument are within the range of those observed in several preparations, including goldfish Mauthner cells (2 MOhms), lamprey touch and pressure cells (5.4 MOhms and 6.3 MOhms), and snail neurons (6.8 MOhms) 75 (Prosser, 1972). The 4.5 MOhms and 3.5 MOhms recorded in carbachol and Na-PB immobilized parasites, respectively, are also similar to those obtained from unanesthetized schistosomes (Fetterer, gt gt, 1980). Recordings from E2 indicate that Rin there is approximately twice as great as that observed in the tegument for each anesthetic used. The recorded values of 9.2 MOhms in carbachol, and 7.3 MOhms in Na-PB are comparable to those reported for several smooth muscle preparations, including guinea-pig vas deferens (11-3-MOhms) (Tomita, 1967), dog arterioles (2-6 MOhms) (Hirst and Neild, 1977), and rabbit anococcygeus (18 MOhms) (Gillespie, gt gt, 1974). In light of the significantly lower resting membrane potential recorded in the E2 compartment as compared with the tegument, it is surprising that the Rim exhibited by the membranes of the E2 compartment are larger. At least two factors could account for this observation. First, voltage signals injected into the E2 compartment are probably affected by resistive barriers presented by the overlying tegument as well as the muscle membranes. This would be a series effect that would tend to increase Rin values measured in the E2 compartment. Second, muscle fiber limiting membranes may confine currents injected intra- muscularly, thereby restricting the amount of tissue across which voltage changes spread. This too would result in higher Rm values. It is not possible at the present time to isolate tegument from muscle, or individual muscle fibers to examine these possibilities more thoroughly. Recordings from E3 indicate that resistance to current injection in extra- cellular regions is similar to that seen in the tegument. These results are difficult to interpret, however, because of the enormous difference in size of the two compartments. It is important to note that while Rin measurements can be used to obtain reliable information concerning other membrane properties in preparations charac- 76 terized by electrically isolated cells, their value in an electrically coupled system is questionable. The Rin of an electrically isolated cell can be used to determine the membrane resistance (Rm = Rin x surface area), an accurate measure of which is essential for determination of all of the cable parameters. The Rin of an epithelial or smooth muscle cell is influenced somewhat by membrane resistance, but much more so by the coupling resistance between it and its neighboring cells (Holman and Neild, 1979). Therefore, the accuracy with which Rm can be used to predict Rm depends a great deal upon the extent to which cells within the tissue are electrically coupled. For most smooth muscle preparations, intercellular coupling is so extensive that a 75% reduction in Rm may be accompanies by only a 5% drop in Rin (Holman and Neild, 1979). Because voltage signals both deve10p and decay rapidly in a multidimensional syncytium (Tomita, 1967), the observed time constants are considerably shorter than many of those reported for various nerve cells (Prosser, 1972), or multi- dimensional preparations for which electrotonic signals were initiated by field stimulation (Tomita, 1967; Holman and Neild, 1979). They are, however, consistent with values obtained for some nerve and muscle preparations injected intracellu- larly. These include cat spinal motoneurons (0.27 msec) (Frank and Fourtes, 1956), goldfish Mauthner cells (0.39 msec) (Furshpan and Furukawa, 1962), and tunicate heart cells (0.4 msec) (Kriebel, 1968). The 0.23 msec to 0.25 msec time constants recorded in the dorsal tegument and muscle are slightly lower than those reported by Fetterer _e_t a_l. (1980). This difference may be due, in part, to anatomical variations between dorsal and ventral aspects of the parasite (Silk gt gt, 1969). In addition to possible complications in single electrode recordings that may have arisen because of multidimensional current flow, Rin and Tau measurements could have been affected by electrode imbalances induced by tissue impalement. Since biological membranes of electrically isolated cells normally exhibit Rin's of 77 at least an order of magnitude less than that of the microelectrode, the total potential change observed upon current delivery may be dominated by properties of the electrode (Coombs e_t g, 1959). During this investigation, bridge-balancing was achieved prior to impalement of the parasite. On those occasions that imbalances were observed upon withdrawal, data were discarded. This procedure is consistent with previous investigators who have either balanced electrodes prior to penetration only (Reuss and Finn, 1974), or subtracted post-withdrawal imbalances from intracellularly recorded signals (Coombs gt gt, 1959). It did not, however, compensate for possible imbalances due to tissue/electrode interactions present during intracellular recording but not present when the electrode was in the bath (Frank and Fourtes, 1956; Coombs gt gt, 1959). Such imbalances, if present, would tend to increase potential changs associated with the passage of current, thereby resulting in artificially high values for Rm. Because of the above considerations, Rin measurements were also made in the tegument using electrodes that were rebalanced intracellularly to eliminate the initial steep phase of the voltage signals. These tests yielded an Bin value of approximately 50 KOhms, a value nearly two orders of magnitude less than that obtained using bridges balanced extracellularly only. The time required for these signals to reach 1 - l/e of Vmax was 4.5 t 0.2 msec, or about 20X the value obtained in measurements using bridges balanced extracellularly only. It has been suggested that this procedure is justified on the basis that membrane time constants are of the order of milliseconds, and any initial edge of a current step will involve the membrane's capacitance and the resistance of the electrode only. Responses characterized by Tau's of much less than a millisecond, such as those obtained using electrodes balanced extracellularly only, probably reflect electrode imbalances caused by electrode tip/tissue interactions (Frank and Fourtes, 1956). 78 Several lines of evidence already mentioned suggest that the schistosome tegument and muscle (or E2) are multidimensional syncytia. If this is the case, then the linear I/ E relations and rapid potential changes obtained from single electrode recordings probably are not artifacts resulting from electrode impedance changes, but reflect an important property of the parasite, that is, the presence of multidimensional current-efflux pathways. By using the same multiple electrode technique by which 1' was determined, electrotonic signals were also analyzed to determine the time required to reach one-half of the stead-state amplitude. For each paralytic, these time factors expressed over distance yielded a straight line. This is a common feature of preparations exhibiting cable-like characteristics. Hodgkin and Rushton (1946) demonstrated in nerve preparations that the slope of this line can be used to determine the space or time constants if one of the two parameters could be obtained experimentally. The equation describing this relationship is m (the slope of the half-times of electrotonic signals) = Tau/ 2 x. We were unable to confidently insert either Tau or X into this expression for the purpose of obtaining the other, but it is noteworthy that the time course of electrotonic potentials recorded in the tegument and E2 compartment of schistosomes is similar to those reported for some smooth muscle preparations, such as guinea-pig taenia coli (Abe and Tomita, 1967) and rabbit artery (Mekata, 1971). It is also noteworthy that the slope of this parameter increased significantly upon the addition of sucrose to the recording medium. This increase implies that either Tau increases, or else the length constant decreases. One would expect an increase in internal resistance to occur as cells shrink in response to hyperosmotic media. Since A = rm/ri, this would result in a decrease in the length constant. This corroborates data I have already presented showing that current spread is drastically reduced by exposure to hyperosmotic media. 79 Intercompartmental Electrical Coupling Bricker e_t gt (1981) have demonstrated that treatments altering the resting potential of the tegument cause a simultaneous shift in the muscle potential as well. The data I have presented in this thesis give one reason why this may be so. Simultaneous recordings from the tegument and E2 during current delivery to the opposite compartment indicate that a large percentage of electrotonic responses initiated in one compartment spread into the other. Tissue coupling ratios obtained by this method in carbachol and Na-PB immobilized parasites indicate that 7 0-8596 of an electrotonic signal initiated in one compartment will spread into the other. This indicates that either a low resistance pathway connects the two syncytia, or else their apposing membranes are close enough to allow an extremely tight capacitive coupling, or both. In most reported cases of electrical coupling between epithelial or muscle cells, the structure believed to be responsible for the connection is the gap junction. Junctional complexes have been observed along the tegument/muscle interface in schistosomes (Silk gt _a_l., 1969b), but they have not been adequately examined to draw definitive conclusions regarding their untra-fine structure. They do appear to resemble gap junctions found in various epithelial (Lowenstein, 1966) and smooth muscle preparations (Barr, 1963). In particular, the junctional complexes in schistosomes closely resemble those connecting epidermis and gastro- dermis in the coelenterate Hydra. They are similar in size and shape, the intercellular space separating apposing membranes is similar, and both connect membranes of different tissues (Hand and Gobel, 1972). If the tegument and muscle compartments in S_ mansoni are electrically coupled, it will be important to determine just how essential this coupling is to the parasite. In schistosomes anesthetized with putative inhibitory neurotransmitters, surface electrical recordings reveal that some activity persists long after most 80 mechanical activity has ceased (Semeyn _et a" 1981). These "spikes" could represent volume conducted nerve activity that has not been eliminated by the drugs, or volume conducted myogenic transients in individual muscle fibers, the contractions of which are not sufficient to cause recordable changes in body tension. A more likely alternative, however, is that these electrical impulses actually develop across and are conducted along the other tegumental membrane (Fetterer gt al., 1977). This type of epithelial conduction has been observed in Hydra, where, as is also the case in _S_._ mansoni, the activity persists in the presence of elevated Mg”, suggesting that it is not neuronally mediated (Mackie and Passano, 1968; Keenan, gtgl_., 1979). If the electrical impulses recorded from the tegument are an inherent feature of that compartment, then it would seem reasonable to speculate that the tegument is serving in some communication capacity, possibly to mediate activity within the underlying muscle compartment. Gap junctions could provide the pathway for this communcation. If, on the other hand, activity recorded in the tegument represents volume conducted muscle activity, then tegumental recor- dings could provide an accessible means by which to monitor the electrical activity of the parasite's effector system. While some reduction (20-35%) in intercompartmental electrical coupling is observed when the parasites are exposed to hyperosmotic media, a relatively large amount (50%) of the current continues to pass into the non-injected syncytium. This could indicate that some low resistance pathways between the two syncytia are not affected by the treatment, or that tegument and muscle are capacitively coupled, and this coupling is more resistant to alterations in the medium. 81 Active Membrance Responses With single electrode current injection, spike initiation was not observed. As mentioned previously, this is probably due to the multidimensional nature of both the tegument and muscle compartments. With multiple electrode systems, however, active responses were frequently evoked by passing large (200-500 nA) depolarizing currents into the tegument or muscle compartments. Their membrane potential dependance, rapid rates of rise, short durations, and tendency to occasionally overshoot zero potential all point to the likelihood that they do in fact represent active membrane responses. Although some apparently all-or-none spikes were recorded, most of the evoked potentials were graded, and their magnitudes were approximately a function of the length constant for tegument and/or muscle. Overshooting spikes were never recorded over distances greater than 0.1 mm distal from the injection site. This suggests that active responses are not propagated over large distances. It must be pointed out, however, that all of these studies were done on carbachol or Na-PB immobilized parasites. These drugs may have had a significant influence on some of the characteristics of these potentials. It is also possible that regenerative action potentials are not a normal feature of schistosome muscle or tegument. There are numerous smooth muscle preparations in which spike generation cannot be induced under any circumstances, including rat anococcygeus (Creed, 1975) and guinea-pig gall bladder (Creed and Kuriyama, 1971). My results indicate that active responses may be initiated across tegument and/or muscle membranes of schistosomes when large enough currents are deli- vered intracellularly to bring a sufficient amount of membrane to threshold. Because the safety factor is relatively low, as indicated by the short space constants, it might be expected that active responses will not be propagated great distances. These features of excitation appear to characterize several other 82 preparations as well, including mouse vas deferens (Furness and Burnstock, 1969), and crayfish somatic musculature (Wiersma, 1961). Because of these considerations, the recording of active responses to intracellular stimulation in schistosomes probably depends most heavily upon the relative locations of the stimulating and recording electrodes. Several investi- gators have suggested that the functional unit of smooth muscle is the muscle bundle (Nagai and Prosser, 1963; Tomita, 1966). Normally, depolarizing currents must simultaneously affect a majority of fibers within a bundle in order for active responses to be initiated. This generally requires the use of large extracellular electrodes (Tomita, 1967). Therefore, recording spikes in intracellularly stimulated schistosomes may be achieved most often when both electrodes are embedded in the same muscle bundle, tightly coupled neighboring muscle bundles, or perhaps, the tegument above the injected muscle bundle. Smaller spikes recorded distally may represent electrotonic spread of active responses intiated at the injection site. While the large depolarizations are an interesting phenomenon, it is quite possible that they do not occur naturally within the parasite. From a pharma- cological perspective, however, they do represent a potentially valuable source of information, as the ionic dependencies of these transients may be similar to those mediating the smaller depolarizations that do appear to be physiologically signifi- cant. It should be noted, however, that spike generation is not always an essential component of smooth muscle contracture (Sakamato and Kuriyama, 1970; Creed, 197 9; Keatinge, 1979).. Further work is obviously needed to determine the physiological importance of active membrane responses to the parasite, as well as their ionic composition. SUMMARY Three distinct compartments of electrical potential were observed upon penetration of _S_._ mansoni immobilized with 1 x 10‘4 carbachol. The resting potentials recorded in these compartments were similar to those reported by Fetterer gt at (1980), and Bricker gt gt (1981) in parasites immobilized with 30 mg% Na-PB. Input resistance of the tegument and E3 compartment were found to be approximately one-half as great as that recorded in the E2 compartment. Current injected into the tegument or E2 compartment was observed to spread through those compartments, demonstrating that both are electrical syncytia. The pattern of current decay recorded is similar to that observed in epithelial sheet preparations. That is, the slope of decay is initially very steep, then decreases and becomes exponential at about 0.1 mm distal from the injection site. Injecting large (> 200 nA) depolarizing current pulses into the tegument or E2 compartment often elicited active membrane responses in those compart- ments. Multiple spikes often occurred, as did overshooting responses. 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