OVERDUE FINES ARE 25¢ PER DAY . PER ITEM Return to book drop to remove this checkout from your record. 1 ELECTROPHYSIOLOGICAL STUDIES ON THE TEGUMENT OF SCHISTOSOMA MANSONI By Raymond Hugh Fetterer Jr. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY Department of Zoology 1979 ABSTRACT ELECTROPHYSIOLOGICAL STUDIES ON THE TEGUMENT OF SCHISTOSOMA MANSONI BY Raymond Hugh Fetterer Jr. An electrical potential having a value of -35 mV (Sd = 7.4) was recorded upon penetration of the ventral surface of adult male Schistosoma mansoni with a microelectrode. Histological studies using horseradish peroxidase as a marker injected iontophorectically through the recording electrode indicate that this potential originates in the tegumental epithelium. Recordings from animals with contractile activity showed spontaneous non-overshooting depolarizations with durations of 20 to 100 msec and ampli- tudes between 5 and 15 mV. Slow-wave depolarizations were also observed and were most frequent in animals showing only slight movement. Hyperpolariza- tion of the membrane potential by current injection through the recording electrode caused their amplitude to be decreased indicating the slow-wave depolarizations were not caused by mechanical artifact. The tegumental membrane potential appears to be dependent primarily on the K+ gradient across the surface since a ten fold increase in external K+ caused a 30 mV depolarization in the potential. Altering the external concentra- tion of Cl " or Ca H had little effect on the tegumental potential but lowering the external Na+ concentration to 37 mM caused a 10 mV depolarization. Raymond Hugh Fetterer Jr. An active Na-K pump may also contribute significantly to maintenance of the tegumental potential. The cardiac glycoside ouabain (0.1 mM) rapidly depolarizes the tegumental potential without altering membrane resistance. The potential is also temperature sensitive with exposure of worms to tem- peratures below 30°C causing reversible depolarization. Substitution of LiCl for NaCl also caused a 16 mV depolarization of the tegument. The antiparasitic agent, praziquantel (PZ), has little initial effect on the tegumental membrane potential. A slow depolarization does occur, however, which reaches a significant level about 10 minutes after P2 (1 uM) addition. Carbachol and dopamine are without effect on the tegumental membrane potential. To Ellen ii ACKNOWLEDGMENTS I wish to express my appreciation to Dr. Ralph A. Fax for his guidance, patience and helpful critisism during the preparation of this dissertation. I also thank Drs. D. Nyquist, G. Gebber and R. Hill for their advice and critical reading of the manuscript. Special thanks goes to Dr. James L. Bennett for the encouragement and support he provided during the course of this project. I also wish to thank my colleagues in the laboratory, Connie Bricker, Todd Hickox, Tim Martin and Carla Siefker for their advice and companionship. TABLE OF CONTENTS Page LIST OF TABLES .......................... vi LIST OF FIGURES .......................... vii INTRODUCTION .......................... I Schistosoma mansoni ....................... 1 General Anatomy ....................... I Muscle Systems ....................... 4 The Nervous System ..................... 5 Neurobiology ......................... 6 The Tegument ........................ 7 Function of the Tegument ...... . . . ..... . . . . . 11 OBJECTIVES ............................ 13 METHODS ............................. 14 Source and Maintenance of Animals ............... ll: Recording Media . ....................... 14 Microelectrode Recordings .................... 15 Histological Stuides . . . . ................... l6 Ion Substitution Experiments ........... . ....... 19 Sodium ............................ 20 Lithium ........................... 20 Potassium .......................... 20 Calcium ........................... 20 Chloride ...... . .................... 20 Current Injection Experiments .................. 21 Temperature .......................... 21 Pharmacological Agents ..................... 21 Dopamine and Carbachol ............ . . . . . . . 21 Ouabain . . . . . . ..................... 22 Praziquantel ......................... 22 Statistical Procedures ...................... 25 iv RESULTS . . . ..... Microelectrode Recordings ........... . . ...... Spontaneous Depolarizations ............ . . Histological Studies Biophysical Characteristics of the Tegumental Membrane The Effect of Altered Ion Concentrations . . . ......... Potassium . . ..... SOdium O O O O O O O O O O Lithium C O ........... Calcium. . . . Chloride . . . . . Temperature . . . . ...... . . Ouabain....... Pharmacological Agents Dopamine and Carbachol Praziquantel . DISCUSSION. . . . . Anatomical Origin of the Membrane Potential . . . . . . . Electrical Properties of the Tegumental Membrane . . . . . Ionic Basis of the Membrane Potential. . . . . . . . Na-K Transport System in the Tegumental Membrane Characteristics of Other Transport Epithelial . . . . . . . . Functional Significance of the Tegumental Membrane Potential. Membrane Potential Significance to Transport . . . . . . . . . . . . Correlation with Maintained Tension ............. SUMMARY......... LITERATURE CITED Page 26 26 34 39 #6 46 46 57 6“ 64 64 71 80 99 100 Table LIST OF TABLES Page A comparison of membrane potential recorded ineither FCS/Earle'sor in HBS. . . . . . . . . . . . . . . . 29 Experimental differences used to distinquish an electrogenic Na-K pump . . . . . ..... . ...... 92 Epithelial membrane potential from some vertebrate tissues . . . . . . . . . . . ........... 94 vi LIST OF FIGURES FIGURE 1. 10. ll. 12. 13. 1‘4. 15. 16. 17. Drawing illustrating the general anatomic features of male and female Schistosoma mansoni. . . . . . . . . . . . Diagram of Tegumental structure of Schistosoma Mansoni . . . Diagram showing the preparation used in recording experiments . The structure of praziquantel . . . . . . . . . . . . . . . . . Microelectrode recording of membrane potential from Schistosomamansoni Spontaneous depolarizations recorded with microelectrode . . . Microelectrode recordings of slow-wave depolarizations from twodifferentanimals..................... The effect of altered membrane potential on amplitude of slow-wavepotentials..................... Recording of membrane potential made with HRP electrode. . . Photomicrograph of cross section through S. mansoni . . . . . . Recording of membrane potential made with HRP electrode. . . Electron micrograph showing localization of HRP. ..... . . Electron micrograph of tegument and cytoplasmic connections . Electron micrograph of tegumental cyton . . . . . . . . . . . The effect of current injection on membrane potential ..... Graph of current versus membrane potential . . . . . ..... The effect of altered external concentration of potassium on membrane potential . . . . . . . ..... . . ...... vii Page 10 21+ 28 31 33 36 33 41 43 as as so 52 54 56 LIST OF FIGURES—continued Figure 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. The effect of 100 mM potassium on the time course of the tegumental membrane potential. . . . . . . . . . . . . . The effect of decreasing sodium concentrations on membranepotential The effect of substituting LiCl for NaCl on the tegumental membranepotential The effect of varying external calcium concentration on membranepotential.................... The effect of decreasing concentrations of external chloride on membrane potential . . . . . . . . . . . . . . An example of the effect of temperature on tegumental membranepotential The effect of decreasing temperature on tegumental membranepotential . . . . . . . . . . . . . . . ...... The effect of ouabain on membrane potential . . . . . . . The relationship between the resting membrane potential measured in HBS and the amount of depolarization caused by0.lmMouabain...................... The effect of ouabain on membrane resistance . . . . . . . The dose response of P2 on membrane potential . . . . . . The time course of the effect of P2 on membrane potential . . viii Page 59 61 63 66 68 70 73 75 77 79 82 84 INTRODUCTION Studies of parasites have traditionally involved investigations of bio- chemistry, nutrition, structure and life cycles. Although the surface mem- branes of parasites have been studied it has been only recently that detailed investigations into the ultrastructure of surface membranes and their role in transport and physiological processes have been made. The blood fluke, Schistosoma mansoni, is a medically important trematode parasite and in many ways is one of the more thoroughly studied parasites. Even in S. mansoni there has been no previous attempts to use electrophysio- logical techniques to characterize the tegument and tegumental membrane. This is surprising, since electrophysiological techniques have been successfully applied to the study of membrane transport in vertebrate systems. In light of this gap in our knowledge, I have undertaken studies to elucidate some of the basic properties of the tegumental membrane of _S_. mansoni. Schistosoma mansoni General Anatomy Adult male Schistosoma mansoni are about 1 cm in length and up to 0.2 cm in width and have a wet weight of 0.8 mg. The adult female is longer (1.5 mm) and thinner (0.2 mm) than the male (Fig. 1). Both male and female schisto- somes are cream or white in color. Ventral and anterior suckers are present in both sexes. The body of the male has a groove, the gynechophoral canal, along much of its length. The adult female lies in this groove. 1 Figure 1. Drawing illustrating the general anatomic features of male and female Schistosoma mansoni. AS, anterior sucker; VS, ventral sucker; DS, dorsal surface of the male; GC, gynechophoral canal. Asa Ds—j-l ( L\\L \\ Figure l q The body of adult schistosomes possesses a superficial cortex that covers the parenchyma, the muscles and the major nerve cords. This cortex consist of a tegumental epithelium. Beneath this epithelium lies the outer circular and the inner longitudinal muscle layer (Smith _e_t 511., I969; Senft gt a_l., 1961). The digestive system of the schistosome consists of a cecum with two branches. The branches of the cecum join in the posterior part of the animal to form a single intestine. The cecum and intestine are often brown in appearance due to ingested hemoblobin (Erasmus, 1972). The excretory system of S. mansoni is a protonephridial system. Capillary tubules with terminal flame cells connect with paired protonephridial tubules (Meglitsch, I972). The male reproductive system consist of seven testes located near the anterior end of the body. Ducts from the testes join to form a seminal vesicle that leads to a short duct. This short duct opens at the genital pore which is located just posterior to the ventral sucker. The ovary is situated in the posterior half of the body and from it extends a long uterus that in mature worms is filled with eggs (Noble and Noble, 1976). Muscle Systems In general the structure of schistosome muscle is similar to that of most invertebrate muscle (Lowy and Hansen, 1962). Ultrastructural studies have shown each muscle bundle to consist of an array of thick and thin filaments (Silk and Spence, l969a). The thick filaments branch and there is cross linking between the thin and thick filaments. Nuclei are generally ovoid. The sarcoplasmic reticulum is absent or poorly defined but rough elements are present at scattered intervals. Junctional complexes are present between the outer layers of adjoining sarcoiemmas. At these complexes the adjoining sarco- Iemmas are closely apposed at a distance of only 70 to 90 A. These junctions are variable in length but do not cover the total length of the apposed sarcoiemmas (Silk and Spence, 1969a). The Nervous System With the use of cholinesterase staining the nervous system of S. mansoni is revealed to consist of the central ganglion and a major and minor system of nerve cords running the length of animal lateral to the mid-line (Fripp, l967a). The cords connect at the posterior end of the animal and are joined at intervals throughout their length by transverse connections. Small branches of the cords are directed peripherally and terminate against the longitudinal muscle. An- terior to the ventral sucker the nerve cords meet the central ganglion and the sides of the ganglion are joined by both ventral and dorsal circumesophogeal commissures. A nerve cord projects anteriorly from the central ganglion to innervate the oral sucker while branches of the ventral cord innervate the ventral sucker. Studies of the fine structure of the central ganglion have shown the presence of neuron cell bodies with large nuclei, numerous clusters of ribosomes and elongated mitochondria (Silk and Spence, 196%). These cells give rise to groups of closely packed non-myelinated axons which vary in size and shape. Cell bodies are infrequently encountered in sections through the nerve cords. Synapses between axons were observed in the commissures connecting the two sides of the central ganglion as well as in other parts of the nervous system. Structures resembling neuromuscular junctions were also observed in some areas (Silk and Spence, l969a). No direct innervation of the tegument or the region immediately below the tegument has been observed but there are a number of finger-like projections of underlying parenchyma cells into the dorsal surface of the tegu- 6 ment. Each projection covers a sac of nervous tissue from which a single cilium extends into the tegumentary extension (Morris and Threadgold, 1967). Morris and Threadgold (1977) have suggested this structure is a sensory receptor with a probable function in mechano- or rheo-reception but there is no experimental evidence to support this hypothesis. Neurobiology As is the case for most of the flatworms studied, monoamines have been implicated in the function of the nervous and neuromuscular system of S. mansoni. 5-HT is present in S. mansoni as well as in two other schistosome species (Chou gt a_l., 1972). Chou e_t a_l., (1972) also reported that norepi- nephrine but not dopamine is present in S. mansoni; but a recent study utilizing a more sensitive assay system has shown both dopamine and norepinephrine to be present in all regions of the worm (Gianutos and Bennett, 1977). Although 5- HT is present in S. mansoni in concentrations 10 times that of mammalian brain (Bennett e_t a_l., 1969) synthesis of this amine could not be demonstrated (Bennett and Beuding, 1973). A high affinity uptake system is present, however, and provides a method for the animals to obtain 5-HT from the host's plasma (Bennett and Beuding, 1973). The use of histochemical fluorescence techniques has provided infor- mation about the location of amine containing structures within S. mansoni. 5- HT is present in the head region near the commissures and in the vicinity of nerve trunks as well as throughout the parenchyma cells (Bennett and Beuding, 1971). Catecholamine-containing neurons have been observed in the central ganglion and nerve trunks. Nerve fibers emerging from the trunks also contain catecholamines. Catecholamines are also distributed throughout the paren- chyma (Bennett and Beuding, I971; Machado e_t a_l. 1972). Monoamines may modulate motor activity. Using visual observation to assess the motility, Tomosky e_t a_l., (1974) showed that 5-HT significantly increases contractile activity while depamine and acetylcholine have an inhibi- tory effect. Dopamine causes a lengthening response. Cholinergic blockade stimulates motor activity but this stimulation is abolished by 5-HT antagonists (Tomosky gt 31., 1974). Although visual observation has provided some useful data it has been a less than satisfactory method to measure contractile activity from the musculature of S. mansoni. Visual observation neither gives a quantitative measure of muscle tension nor distinguishes contractions of various types. Recently a method has been developed to obtain direct recordings of tension and contractile activity from the musculature of S. mansoni (Fetterer e_t 31., 1977). In addition to measurement of contractile activity this method also allows for simultaneous recording of electrical activity from the animal's surface. Using this method, Fetterer e_t g” (1977) confirmed that 5—HT at concentrations as low as 10'6M increases contraction rates with a corre- sponding increase in surface electrical activity. This study also demonstrated that the Cholinergic antagonist carbachol (104M) inhibits spontaneous con- tractions and surface electrical activity and decreases the tension of the musculature. The Tegument The tegument of S. mansoni is a syncytium and can be separated into three regions: (I) A superficial anuclear layer (tegumental epithelium), (2) cytOplasmic channels that connect the epithelium to (3), subjacent tegumental cytons (Silk e_t a_l., 1969) (Fig. 2). The outer surface of the tegument is highly invaginated, forming a series of pits or channels projecting as much as 0.5 urn 8 into the cytoplasm of the tegumental epithelium. Though not shown in Figure 2, the inner plasma membrane is thrown into numerous narrow invaginations which project upward into the cytoplasm of the tegumental epithelium. The surface channels and basal invaginations may be separated from each other by as little as 0.25 um (Wilson and Barnes, 1974b). The outer tegumental membrane has a heptalaminate appearance with a total thickness of 110 A while the inner surface is delimited by a more conventional trilaminate membrane (Hockley e_t a_l., 1975). Freeze fracture studies on the outer membrane have shown it to be composed of two trilaminate membranes in intimate contact (Hockley and McClaren, I973; Hockley e_t a_l., 1975; Torpier e_ta_l., 1977; McClaren 3t a_l., 1978). Beneath the inner plasma membrane of the tegumental epithelium are consecutively, a basement membrane, a layer of fibrous connective tissue and the outer circular and inner longitudinal muscles (Morris and Threadgold, 1968). The tegumental cytons lie beneath the muscle fibers and are connected to the tegumental epithelium by cytoplasmic channels of variable length (Smith e_t 31., 1969; Smith and Von Lichtenberg, I974). The thickness of the tegumental epithelium varies with the contractile state of the animal as well as with the region in which it is measured. In general it ranges from 1 to 5 um in thickness (Wilson and Barnes, 1977). Several different types of cytoplasmic inclusions can be observed in the tegumental epithelium. These include mitochondria, multilaminate vesicles, discoid granules and crystalline spines (Smith e_t 31., I969; Silk 3t 31., 1969). The tegumental cytons by contrast have an abundance of cellular organelles which are usually associated with secretory processes (e.g., polysomes and golgi apparatus) (Wilson and Barnes, 19703). Junctional complexes between the membrane of the tegumental cytons and their cyt0plasmic channels on the one Figure 2. Diagram of tegumental structure of S. mansoni. T, Tegumental epithelium; TC, tegumental cyton; N, nucleus of cyton,; CC, cytoplasmic channels; P, pits; OM, outer membrane; 1M, inner membrane; BL, basal lamina; S, spine; LM, longitudinal muscle; Cm, circular muscle; L, lipids; MP, membranous particles; EB, elongated body; IMa, interstititial matrix. 10 Figure 2 11 hand and membrane of adjacent muscle cells on the other have been observed (Silk e_t a_l., 1969). Function of the Tegument The tegument of S. mansoni forms the host-parasite interface and as such has been implicated in some physiologically important processes. The bulk of attention has been given to the tegument's transport function. Even though S. mansoni possesses a gut, the tegument seems to be the site of absorbtion of a number of important solutes. Autoradiographic techniques have been used to show that the tegument is the primary point of entry for glucose (Fripp, l967b). Many amino acids that are utilized by schistosomes are also transported across the tegument (Senft, 1968). An important enzyme that is associated with ion transport, ouabain sensitive Na+-K+-ATPase, is also found in the tegument (G. Cain and J. Oakes, personal communication). The outer tegumental membrane of S. mansoni and many other trema- todes is invested with a superficially positioned coat (the glycocalyx), which varies considerably in morphology and chemistry between different devel- opment stages (Lumsden, 1975). The surface coat is typically inconspicuous, usually present as thin line (Bogitsh and Aldridge, 1967). Histo- and cyto- chemical methods indicate the presence of acid muc0polysacarides in the glycocalyx (Stein and Lumsden, 1973). Although the physiological role of the glycocalyx is unclear, a number of possible functions have been proposed. Clegg (1972) has suggested that the fixed negative charges associated with the glycocaylyx of the schistosome's tegument may aid in adsorbtion of cellular elements from the host and thereby serve to disguise the worm from immuno- logical recognition. 12 The surface of the outer tegumental membrane of S. mansoni has also been shown to contain enzymes which may aid in digestion and absorbtion of nutrients from the host's plasma (Pappas and Read, 1975; Lumsden, 1975; Earnst, 1976). OBJECTIVES Schistosoma mansoni is an important parasitic trematode of humans and the disease caused by this organism has increased in many parts of the world in recent years. Although there has been much research concerning the effect of anti-helminthic drugs on S. mansoni, little is known about the physiological characteristics of the tegumental membrane. Since the tegument forms the host-parasite interface, a knowledge of tegument function is essential to the understanding of the basic physiology of the parasite. I have therefore undertaken a study to characterize electrophyiological properties of the tegument. Preliminary studies have revealed the existence of a previously unreported membrane potential from the tegument. The objectives of this study are to determine the electrical properties of the tegumental membrane and the ionic basis of the membrane potential as well as to investigate the contribution of active transport to the tegumental membrane potential. These studies represent a new approach to the study of the tegument in S. mansoni. It is hoped that the results of these experiments will increase understanding of the basic physiology of flatworm parasites as well as providing information useful to the design and selection of effective antiparasitic drugs. 13 METHODS Source and Maintenance of Animals Female laboratory mice (Mus musculus) infected with Schistosoma mansoni (Puerto Rican strain) were obtained from the laboratory of Dr. J. Bennett, Department of Pharmacology, Michigan State University. Parasites were removed from the portal system of mice 45-55 days post-infection and were maintained in a medium (FCS/Earle's) consisting of a 1:1 mixture of Earle's salt solution (G-ll; Grand Island Biological) and heat inactivated fetal calf serum (FCS) buffered at pH 7.4 with 20 mM Heppes (N-2hydroxymethyl piperazine, Sigma), 100 units/ml pennicilin-streptomyocin (Grand Island Bio- logical) and a final glucose concentration of 0.296. The worms were maintained at 37°C and used for experiments within 12 hours after removal from mice. Recording Media In all recording experiments the standard medium used was Hanks balanced salt solution (HBS). This saline is a desirable medium for recording experiments since it has previously been shown to be an adequate medium in which to record mechanical activity from schistosomes (Fetterer Lani" 1978) and it is a saline with a defined concentration of inorganic ions suitable for performing ion substitution experiments. The final concentrations of con- stituents in HBS are: Na+ 138 mm, K+ 5.9 mM, Ca++ 1.4 mM, Mg++ 0.5 mM, PO 0.5 mM, C1 147, 2 mM, SO 0.5 mM, glucose 5 mM and Heppes 20 mM. 4 4 The pH was adjusted to 7.4 using 6 N NaOH and the osmolality was 300 mOsm. In some experiments recordings made in HBS were compared with recordings made in a standard culture medium and for these experiments FCS/Earle's was used as the medium. 14 l5 Microelectrode Recordings Microelectrodes were pulled from 1.5 mm capillary tubing (Omega Dot; Fredrick Haer, Brunswick, Me) with a horizontal electrode puller (Narashige Instruments). Electrodes prepared in this manner were filled with either 2.5 M potassium acetate or 3M potassium chloride and had resistances between 30 and 80 meghoms. The electrodes were connected to a microelectrode preamplifier (WP Instruments, M-4A) and the resulting signal displayed on an osciloscope (Textronix 502A) or chart recorder (Gould Model 220) and stored on an FM tape recorder (Vetter Model B). A silver silver-chloride wire or a KCI agar bridge placed in the preparation bath served as a ground. Current pulses could be delivered through the recording electrode using the circuitry of the pre— amplifier and in such experiments the duration and amplitude of the current pulse were controlled with a stimulator (Grass Instruments, Model S-4). The recording chamber consisted of a 10 ml glass petri dish the bottom of which was lined with Sylgard resin (Dow-Corning, Midland, MI). The tem- perature of solutions placed in the chamber was maintained at 37°C with a thermoelectric heater (Cambion Electronics) placed under the recording chamber. Temperature of solutions in the chamber were monitored with a thermistor. Schistosomes were prepared for microelectrode recording experiments by placing them in the recording chamber containing HBS and 0.0396 pentobarbital (PB) (Sigma). This treatment quickly immobilized the worms. The female was removed from the male and the male was pinned to the sylgard using minuten insect pins placed in the lateral margins of the parasite's body. The animal was pinned so that its dorsal surface was against the bottom of the chamber thus exposing the ventral surface. Care was taken to secure the worm so that the portion of the body being pinned was as flat as possible. A diagram of the 16 preparation is shown in Figure 3. After securing the parasite to the bottom of the recording chamber the HBS-pentobarbital solution was removed, the preparation washed with 3 volumes of HBS and then replaced with normal HBS. Within two to three minutes motility returned but a minimum of 10 minutes of recovery time in HBS was allowed before any experiments were begun. Although motility could be observed in the animal in general, the portion of the worm that was restrained by pins was relatively immobile. Occasionally violet contractions of the musculature could cause the worm's body to tear away from the pins. In such cases the animal was discarded. After a worm was prepared in the above manner a microelectrode was brought near a region of the ventral surface lateral to the gut and medial to the lateral edge of the worm. As the electrode was advanced through the ventral surface, a rapidly occurring potential change was observed. The average baseline value of this potential is referred to as the resting membrane potential. Rapidly occurring fluctuations in the membrane potential were also observed and will be referred to as spontaneous depolarizations. Histological Studies In order to gain an insight into the origin of the membrane potential that is observed upon penetration of the ventral surface of S. mansoni, a marker substance, horseradish peroxidase (HRP; E.C. 1.11.1.7) was injected, in some experiments through the recording electrode. For these experiments a micro- electrode was filled with a solution containing 496 HRP (Sigma Type VI). 0.05 M Tris (tris-hydroxymethylaminomethane hydrochloride, Sigma) and 0.2 or 0.3 M KCl (Snow 31 a_l., I976). The pH of the solution was adjusted to 8.6 with 6 N NaOH. Electrodes filled with this solution had resistances between 80 and 110 megohms. The ventral surface of a worm was pentrated in the manner 17 Figure 3. Diagram showing the preparation used in recording experiments. DS, dorsal surface; VS, ventral surface, G, gut; S, ventral sucker; AS, anterior sucker; E, recording electrode; P, pins. 19 described above except that the animals was placed in HBS containing 0.03% PB in order to prevent movement which could dislodge the electrode during injection of the HRP. After penetration of the ventral surface and observation of the membrane potential, the HRP was ejected from the electrode using positive current of 40 to 80 nA with a microiontophoresis programmer (WP Instruments Model 160) serving as the current source. Three to four current pulses with durations of 1 to 2 seconds were used to inject HRP. After injection of the HRP the membrane potential was again monitored before electrode withdrawal. The animal was then immediately fixed in 496 glutar- aldahyde (in 0.1 M phosphate buffer pH 7.4) while still pinned in the recording chamber. After 5 minutes the animal was removed from the recording chamber and fixation continued for a minimum of three hours at 4°C. After glutaralda- hyde fixation, worms were placed in a solution containing 30 mg% 3,3', diaminobenzidine (Sigma) in 0.1 M phosphate buffer pH 7.4 for 10 minutes, hydrogen peroxide was then added and the reaction allowed to continue for 15 minutes (Granam and Karnovsky, 1966). Some specimens were post-fixed with 1% osmium tetroxide in cacodylate buffer (pH 7.4). The tissue was dehydrated in an alcoholic series and embedded in a low viscosity embedding medium (Spurr, 1969). Thick sections (1-2 um) were cut for light microscopy and stained with 196 toluidine blue. Thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope (Philips Model 300). Ion Substitution Experiments The effects of altered external ion concentrations on membrane potential were determined by measuring the membrane potential from animals bathed in normal HBS and then exchanging the HBS for a modified saline containing the altered ion concentration. After ten minutes the membrane potential was again measured. The preparation was then placed back in HBS and recovery of 20 membrane potential to control values was noted. A minimum of ten imple- ments of the tegument were made first in the control and then altered saline and no more than two concentrations of an ion were used on a single animal. The manner in which the HBS was altered differed for each of the ions studied but in general the methods of Fetterer 3t 31., (1978) were followed. Sodium: Sodium concentrations of 28, 37, 58, 90 and 138 mM were obtained by lowering the NaCl concentration and adding Tris HCL as a substitute. The concentration of other constituents was the same as normal HBS except that Heppes was ommitted and Tris was used as a buffer (pH 7.4). In some experiments glucosamine HC1 (Sigma) was used as a substitute for NaCl instead of Tris HC1. Lithium: In order to determine the effect of Li+ on the membrane potential a modified HBS was used in which LiC1 was substituted for NaCl. Experiments were performed using 138 mM Li+ (complete substitution of LiCl for NaCl) and 69 mM Li+ (replacement of 5096 of NaCl with LiCl). Potassium: Concentrations of 3, 10, 60 and 100 mM were tested. The concentration of potassium in the modified HBS was adjusted by adding or deleting KC1. The chloride concentration was held constant by adding or substracting NaCl. Calcium: Calcium concentrations of 0.01, 0.03, 1, 3, and 10 mM were tested. The appropriate concentration of calcium was obtained by adding or deleting CaClz. When 10 mM Ca++ HBS was made, phosphate and sulphate ions were omitted from HBS to prevent precipitation of Ca“. Chloride: Chloride concentrations of 100, 44 and 17 mM were used. The chloride concentration was altered by decreasing NaCl and replacing Na+ with Na 2504. 21 Current Injection Experiments After penetration of the ventral surface and recording of the membrane potential, current pulses of 0 to 2 nA in amplitude and 100 msec duration were delivered through the recording electrode and the resulting change in membrane potential measured on a storage osciloscope. The membrane resistance was determined from the slope of the current versus membrane potential curve. Resistance measurements were made on animals bathed in either normal HBS or HBS with 0.03% PB. During all current injection experiments care was taken to check the bridge balance and upon indication of bridge imbalance the data were discarded. Temperature The effect of temperature on the tegumental membrane potential was determined in animals bathed in HBS. The temperature of the bathing media was altered by adjusting current flow through the thermoelectric heater. Temperature of the bathing media was monitored continously by a thermistor with output displayed on one channel of a chart recorder (Gould Model 220). The temperature was lowered from an initial temperature of 37°C to 8°C and then rewarmed to 37°C. The membrane potential was measured at 100 sec intervals during the course of the temperature alteration. Pharmacological Agents Dopamine and Carbachol: The putative neurotransmitter dopamine (DA) (3-hydroxytyramine hydrochloride, Sigma) and the Cholinergic agonist carbachol (Carb) (carbamylcholine chloride, Sigma) were dissolved in double distilled water immediately prior to use. The compounds were then diluted to the desired concentration with HBS. Experiments were performed by measuring the membrane potential from an animal in HBS and then exchanging the normal 22 HBS for saline containing the desired concentration of the drug. After ten minutes the membrane potential was remeasured. A minimum of ten impale- ments of the tegument were made in both control and drug containing salines. Only one preparation per drug concentration was used. Ouabain: The cardiac glycoside ouabain (Sigma) was disolved in dime- thylsulfoxide (DMSO) and then diluted to the final concentration with HBS. The time course of the effect of ouabain on membrane potential was determined by measuring the membrane potential from animals bathed in normal saline at 1 minute intervals for 5 minutes and then exchanging the HBS for saline containing 0.1M ouabain. The membrane potential was then measured at 1 minute intervals for 15 minutes. Praziquantel: The antiparasitic compound praziquantel (Gonnert and Andrews, 1977) (Fig. 4) (Bayer Corp.) was dissolved in DMSO and then diluted with HBS to give the desired concentration. Both the dose response of praziquantel (P2) and the time course of the effect on the membrane potential were determined. A dose-response curve was constructed using praziquantel concentrations of 10”, 10'5 M. Control values of membrane potential were determined in HBS and then the HBS exchanged for HBS with the desired PZ concentration and after 10 minutes the membrane potential was remeasured. Only one animal per concentration of PZ was used. Ten measurements of the membrane potential were made first in HBS and then in drug containing HBS. Results from these experiments are expressed as change in membrane potential compared to control. The time course of the P2 effect was determined by measuring control values of membrane potential in HBS and then exchanging the saline for one 6 containing 10' M PZ. Measurements were made at 1 minute intervals for 12 23 Figure 4. The structure of praziquantel. 25 minutes following addition of P2. Between 12 and 12.5 minutes following PZ addition HBS containing P2 was exchanged for HBS containing 100 mM K+ and measurements of membrane potential at one minute intervals was continued for an additional 3 minutes. Statistical Procedures Results from experiments were generally expressed as means with one standard error. Statistical differences between means were determined using a test and lines were fitted to points using linear regression analysis (Steele and Torre, 1960). RESULTS Microelectrode Recordings Resting Membrane Potential: Upon penetration of the ventral surface of an adult male S. mansoni with a microelectrode, a potential difference negative to bath potential is recorded (Fig. 5). Recordings as long as 3 minutes were sometimes possible but this was only in cases in which the worms showed little 'or no contractile activity. In worms displaying any amount of spontaneous contractile activity, such extended periods of recordings were not possible. Pentobarbital (PB) at a concentration of 0.0396 causes relaxation of the worm's musculature and inhibition of spontaneous contractions. When worms were bathed in HBS containing 0.03% PB, continous recordings as long as 5 minutes were possible. Unless otherwise noted all values given below were obtained from animals bath in HBS only. The mean value of membrane potential was -35 mV (SD = 7.4; N = 100) with a range of -20.2 to -56 mV. The standard deviation from a minimum of five measurements of RMP per animal for the 100 animals ranged from 1.8 to 9.4. The adequacy of HBS as a medium in which to record membrane potential was determined. The membrane potential from a group of worms bathed in culture medium (FCS/Earle's) which is known to maintain the i_n vitJ motility of schistosomes for long periods (Fetterer g: a_l., 1978) was compared to the membrane potential from another group of animals bathed in HBS (Table 1). The mean membrane potential for eight animals in FCS/Earle's 26 27 Figure 5. Microelectrode recording of membrane potential from S. mansoni. The sharp vertical drop indicates penetration of the bentral surface. Calibration: Vertical, 10 mV; Horizontal, 2 sec. 28 Figure 5 29 TABLE 1. A COMPARISON OF MEMBRANE POTENTIAL RECORDED IN EITHER FCS/EARLE’S OR HBS. VALUES ARE MEAN MEMBRANE POTENTIAL (2*- STANDARD ERROR) FOR 10 MEASUREMENTS PER ANIMAL. mamazs ”5 4&2rL6 663:33 qaoiLe $01iL9 -%2:1J -m5:12 AOBitl sautfz -%J:1J -m3:1A sasiag -y3:11 5&3iL2 -Q£i22 512:24 ngizs =-ms X=MJ SE=1.9 SE=1.8 30 Figure 6. Spontaneous depolarizations recorded with microelectrode. Top traces: a low-speed chart recording showing spontaneous depolarization (left) and a high-speed recording from another animal (right). Bottom traces: Spontaneous depolarizations superimposed on slow-wave activity. A low- speed recording (right) and the same trace at a high sweep speed (left). Calibration: vertical, 10 mV; Horizontal, 2 sec (right), 80 msec (bottom left) and 600 msec (top left). m OcswE 32 Figure 7. Microelectrode recordings of slow-wave depolarizations from two different animals. Slow speed recordings on top races and high speed recordings on bottom traces. Resting membrane potential is -30 mV (left) and -40 mV (right). Calibration: Vertical, 5 mV (top right), 10 mV (top left); Horizontal, 2 sec (top traces), 80 msec (bottom right), 40 msec (bottom left). 33 .). ) I\,/ I... ,xrllc .../lx.. [IRS/1.x. /Il.. FEEEEEEEEEEE 34 was -34.9 mV (SE = 1.9) while the mean membrane potential for eight animals in HBS was -34.7 mV (SE = 1.8). These means are not significantly different 0.25 (P >0.5) from each other. Spontaneous depolarizations: During recordings of the potential, spon- taneous depolarizations were often observed. One type consisted of relatively short duration (50 to 100 msec), positive going waves with amplitudes of 5 to 10 mV and rise times ranging from 0.2 to 2.5 mV/msec (Fig. 6). Such potentials were usually present in animals which showed visible contractions but were usually not present in those with no observable contractions. The second type of spontaneous depolarization observed consisted of slow- wave depolarizations of relatively long duration (100 to 500 msec), low amplitude (0.5 to 5 mV) and slow rise times (0.1 to 0.3 mV/msec (Fig. 7). They were most often observed in animals showing infrequent spontaneous contrac- tions. Unlike the fast depolarization which were not observed in animals bathed in HBS containing 0.03% PB, the slow potentials were often present in animals in this bathing medium. The nature of the slow-wave potentials was investigated by altering the membrane potential using either hyperpolarizing or depolarizing current pulses (1 nA) injected through the recording electrode. When the membrane potential was made more negative the amplitude of the slow-wave activity increased but when the membrane was partially depolarized, the amplitude of the slow-wave activity decreased or in some cases was absent (Fig. 8). When the membrane potential was returned to normal the amplitude of the slow potentials returned to control values. The alteration of membrane potential had no effect on the frequency of slow potentials. 35 Figure 8. The effect of altered membrane potential on amplitude of slow- wave potentials. Membrane potential (top traces) was altered by injecting hyperpolarizing (left) or depolarizing (right) current (bottom traces) through the recording electrode. Calibration: vertical, 10mV (top) 1 nA (bottom); Horizontal, 2 sec. 37 Figure 9. Recording of membrane potential made with HRP electrode. Injection of HRP from recording electrode was performed between arrows. Calibration: Vertical, 10 mV; Horizontal, 5 sec. 39 Histological Studies The anatomical localization of HRP injected through the recording electrode was undertaken to determine the origin of the membrane potential that is recorded upon penetration of the ventral surface of S. mansoni. These experiments were performed in HBS containing 0.03% PB to reduce movement. Using HRP filled electrodes, the membrane potential recorded from four animals ranged from -32 to -40 mV and resembled potentials that were recorded under similar conditions with KCl filled electrodes. Based on gross inspection, the HRP reaction product was confined to a roughly circular region 75 to 200 um in diameter on the ventral surface of the animal in the area in which the recording electrode was placed. Light microscopic examination of thick sections (1-2 um) gives further information as to localization of the HRP reaction product relative to the internal anatomy of the worm. Examples are shown in Figures 9-14. In an animal in which the membrane potential was -35 mV before and after injection of HRP, (Fig. 9), the optically dense reaction product was observed to be mainly localized in the ventral tegumental epithelium (Fig. 10) but it was also present in the cytoplasmic channels and the tegumental cytons. There was no localization of the reaction product in the muscle layer. Since the HRP reaction product is electron dense (Tweedle, 1978), examination of thin sections through the area of the reaction product gives further detail about the localization. Examination of a preparation following recording of the membrane potential and injection of HRP (Fig. 11) again shows localization of the HRP reaction product in the ventral tegument (Fig. 12). The circular and longitudinal muscle layers that underlie the tegument can be clearly seen in the electron micrographs. The cytoplasmic channels which pass through the muscle layers contain reaction product but no localization of the 40 Figure 10. Photomicrograph of cross section through S. mansoni. The HRP reaction product can be seen in the ventral tegument and cytoplasmic channels. VT, ventral tegument; DT, dorsal tegument; M, Muscle layer. Calibration, 100 uM. 41 Figure 10 42 Figure 11. Recording of membrane potential made with HRP electrode. The two traces are a continuous recording and HRP and HRP injection was performed between the arrows. Calibration: Vertical, 10 mV; Horizontal, 5 sec. 41+ Figure 12. Electron micrograph showing localization of HRP. The HRP reaction product can be seen in the ventral tegument and cytoplasmic connections. VT, ventral tegument; CC, cytoplasmic channels; M, muscle Calibration: 5 uM. 45 Figure 12 46 reaction products seen in the muscle layer itself (Fig. 13). The tegumental cytons are located several microns below the tegument. They and their processes can also be seen to be filled with the reaction product (Fig. 14). Biophysical Characteristics of the Tegumental Membrane The membrane resistance and time constant of the tegumental membrane were measured by injecting current through the recording electrode and noting the resulting change in membrane potential. Experiments were performed on animals bathed in either HBS or HBS containing 0.03% PB. The change in membrane potential observed upon delivery of current pulses appeared to be a linear function of the amount of current injected (Fig. 15). The membrane resistance was determined from the slope of the current-versus-membrane potential curve (Fig. 16). The membrane resistance determined for five animals bathed in normal HBS was 5.2 megohms and for five worms bathed in HBS with 0.03% PB was 4.3 megohms. These means were not significantly different (P>0.2). The membrane time constant was estimated by injecting a -1 nA current pulse and measuring the time required for the resulting perturbation in membrane potential to reach 63% of its maximum value. The mean time constant for three animals bathed in HBS was 0.35 msec and for three animals in HBS containing PB was 0.40 msec. The Effect of Altered Ion Concentrations Potassium: The relationship between external K+ concentration and membrane potential is shown in Figure 17. Clearly there is no simple Nernst relationship but between 30 and 100 mM external K+ there is nearly a linear relationship between membrane potential and K+ concentration. In this range of external K+ concentrations, the approximate relationship is 30 mV per 10- fold change in external K+. Decreasing K+ to levels lower than 47 Figure 13. Electron micrograph of tegument and cytoplasmic connections. VT, ventral tegument; CC, cytoplasmic connections; CM, circular muscle; LM, longitudinal muscle. Calibration: 2 microns. 48 Figure 13 49 Figure 14. Electron micrograph of tegumental Cyton. Electron dense HRP reaction product can be seen in a cyton and its cytoplasmic con- nection. VT, ventral tegument; CY; cyton; CC, cytoplasmic connection. Calibration, 2 microns. 50 Figure 14 51 Figure 15. The effect of current injection on membrane potential. Several superimposed osciloscope traces are shown. The magnitude of the current pulse is shown on the top trace and membrane potential on the botton trace. Calibration: Vertical, 2 nA (top), 10 mV (bottom); Horizontal, 20 msec. 53 Figure 16. Graph of current versus membrane potential. All points are means with one standard error for 5 experiments. Lines were fitted to points using linear regression. AE mV 0-5 Figure 16 1'5 I M o- H 85 0-0-03xPB NP 55 Figure 17. The effect of altered external concentration of potassium on membrane potential. Values are means with one standard error for 6 animals. 56 : Rama AZEV:O_.o:coucoU v. B ._. A. m. 1 m 2 15m ION IO— ( Am) logiuoiod euoiqwow 57 that in HBS (5.9 mM) causes partial depolarization of the membrane so that at 3 mM external KJr the membrane potential is 6 mV less negative than that at 5.9 mM K+. The effect of altered K+ on membrane potential was readily reversed by replacing the altered saline with normal HBS (Fig. 18). Both fast and slow spontaneous depolarizations were present in 10 and 30 mM K+ concentration below 5.9 mM usually resulted in decreased activity. Sodium: The effect of decreasing concentrations of external Na+ on membrane potential is shown in Figure 19. The membrane potential is unchanged by lowering the external Na)r concentration to 90 mM, but concen- trations of Na+ less than this cause partial depolarization. This effect is maximal at an external Na+ concentration of 37 mM. Lowering the Na+ concentration to 24 mM also causes a depolarization, relative to normal Na+ concentration, but it is not Significantly different (0.5>P>0.3) from than observed at 37 mM. Placing animals in the lower Na+ concentrations (24 or 37 mM) usually resulted in a decrease in both fast and slow depolarizations. The effects of external Na+ on membrane potential and spontaneous depolarizations were not readily reversed upon return to normal HBS. Using glucosamine as a Na+ substitute yielded results similar to those obtained when tris was used as a substitute. Lithium: The effect of substituting LiCI for NaCl is shown in Figure 20. Complete substitution of LiCl for NaCl causes a depolarization of the tegumental membrane, so that by ten minutes the membrane is depolarized by 16 mV (0.025_P>0.2) on the membrane potential. The mean peak depolarization was observed at 13°C. Lowering the temperature to 8°C did not cause any additional depolarization (P >0.5). Ouabain The effect of ouabain on membrane potential is shown in Figure 25. The addition of ouabain (0.1 mM) had a marked effect on membrane potential. The mean time for the peak depolarization to occur was 10.0 minutes (SE = 1.0) and the mean time for the depolorization to reach half peak value was 2.8 minutes (SE = 0.04). At 10 minutes after ouabain application the membrane was depolarized by 20.7 mV (0.025 >P<0.05). In doing these experiments it was noted that animals which had a relatively low (less negative) membrane potential were depolarized less by ouabain than animals which had a greater (more negative) membrane potential in HBS. Figure 26 shows the relationship between the resting membrane potential measured in HBS and the mean depolarization caused by ouabain (0.1 mM). A strong correlation (r = 0.99) exist between the resting membrane potential in HBS and the amount of depolarization caused by the ouabain. Membrane resistance was also measured before and after ouabain treat- ment (Fig. 27). The resistance measured in HBS had a mean value of 4.9 megohms (SE = 0.1; Range 4,8 to 5.1; N = 3) while after ouabain (0.1 mM) the mean resistance was 4.98 megohms (SE = 0.3; Range 4.5 5.4). There was no significant difference (P> 0.5) between these two means. 72 Figure 24. The effect of decreasing temperature on tegumental membrane potential. All values are means with one standard error. N = 5. l o ‘7 73 AI- Inueeged eeluquew IO 20 temperature °C Figure 24 74 Figure 25. The effect of ouabain on membrane potential. All values are means with one standard error. N = 8. 75 o 1 nm 23E 835E 25 h 0 m 5 r 4 d q q 0V... can ON... Aug logiueiod éuoiqmaw 76 Figure 26. The relationship between the resting membrane potential measured in HBS and the amount of depolarization caused by 0.1 mM ouabain. Each point represents data from one animal. 77 on. SN 23E DI .l—elee.‘ EUR—Io: mV.. 0V: 3 ON on A. gels-es“ emu-queen ' 78 Figure 27. The effect of ouabain and membrane resistance. All points are means with one standard error. N = 3. Open circles indicate measurements made in ouabain (0.1 mM) and closed circles represent measurements made in HBS. A!“ 79 .12 .10 e f—TS—‘tr—ofr .O .10 .12 Figure 27 80 Pharmacological Agents Dopamine and Carbachol: The effects of dopamine (DA) and carbachol (Carb) on membrane potential were determined. Both compounds were used a a concentration of 104 M since they ad previously been shown to give maximum effects on schistosome's musculature (Fetterer e_t a_l., 1977). Dopamine caused a slight but non-significant (P> 0.5) depolarization (-32.8 mV; Range -21.8 to - 36; SE = 1.5; N = 7 compared to a mean control value of -35.2 mV; Range -27 to 31; SE = 2.0; N = 7). In Carb the mean membrane potential was -35.4 mV (Range -22 to -38; SE = 2.6; compared to a control value of -31.6; Range -21.8 to 38.7; SE = 2.2). Carbachol did not cause a significant change in the membrane potential (P> 0.5). Depolarizations of the fast type were present in worms treated with DA but never observed in worms bathed in Carb. Slow- wave depolarizations, however, were often observed in worms treated with either carb or DA. Praziquantel: The dose-response effect of praziquantel (PZ) on membrane potential after 10 minutes of treatment was determined for concentrations of 10-5, 10'6 and 10’7M (Fig. 28). At a concentration 10"7 -6 M only a slight depolarization (0.8 mV; SE = 1.7) was noted. At 10 M, PZ significantly (0.25< P< 0.05) depolarized the membrane by a mean value of 9.1 mV (SE = 0.7). The depolarization caused by 10'5 M P2 was not Significantly greater than that -6 caused by 10 M PZ. The higher concentrations of PZ completely eliminated any spontaneous depolarizations and caused tonic contracture of the parasite's musculature. At 10'7 M, PZ had no observable effect on spontaneous depolar- izations. The time course of the effect of P2 on membrane potential was also determined (Fig. 29). There is very little effect on membrane potential during the first 2 minutes after PZ addition but by 10 mintes, a significant 81 Figure 28. The dose response of PZ on membrane potential. Each point represents a mean and one standard error. A minimum of five animals per praziquantel concentration were used. 82 mm Semi ‘ [e_elueleelou N.- P2 42 o— N— (A') gnu-ego; eeeaqelew ' 83 Figure 29. The time course of the effect of P2 on membrane potential. All values are means with one standard error. N=3. 84 n— mm 8:5 33...... E: mm: A- Ion-om «us-nu 85 (0.025< P< 0.05) depolarization was noted. The time for the depolarization to reach half its peak value was 7 minutes. When HBS containing PZ was exchanged for HBS with 100 mM K+ an additional rapidly occuring depolariza- tion was observed which reached its maximum within 30 seconds. DISCUSSION Anatomical Origin of the Membrane Potential From my studies it seems clear that the electrical potential I have described originates across the outer tegumental membrane. Horseradish peroxidase (HRP) injected through the recording electrode is confined to the ventral tegumental epithelium, the cytoplasmic connections and the cytons (Fig. 10-14). Since the membrane potential was still recorded after injection of the HRP it seems highly unlikely that the electrode was dislodged from its recording site during HRP injection. Locations other than the ventral tegument for the electrode tip during my recordings seem unlikely for other reasons also. One possibility might be that the electrode tip was located very near but external to the ventral tegument. In such a case, active uptake of the HRP by the tegument might have caused it to be localized in the ventral tegument. It seems unlikely that such a process could be completed in the short diffusion time allowed (3 minutes). Moreover, it has been shown that the ventral tegument has very little ability to take up HRP from the external medium (Smith e_t 31., 1969). Another possibility is that the electrode was actually at a location deeper than the tegument, e.g. in the extra-cellular space or in the muscle and that the HRP was actually removed by the ventral tegument. This also seems unlikely for the reasons cited above. Moreover, in no case was there ever seen even residual amounts of HRP in any structure other than the ventral tegument and its cytons. 86 87 Several previous workers have concluded from ultrastructural evidence that the cytoplasmic channels connect the surface tegumental syncytium with the underlying cytons (Silk e_t a_l., 1969; Smith e_t a_l., I969). The present study Shows that there is a true functional continuity between these structures since a protein (HRP) placed into the tegument quickly moves to adjacent regions of the surface syncytium as well as into the cytoplasmic channels and cytons. Electrical Properties of the Tegumental Membrane Current injection experiments showed the current-voltage relationship to be linear. No evidence of an active membrane response was observed. Therefore, the tegumental membrane appears to be electrically inexcitable. Though spontaneous depolarizations were recorded at times, they were not overshooting. This is consistent with the apparent electrical inexcitability of the tegumental membrane. The total input resistance of the tegumental membrane measured from animals bathed in HBS was 5.2 megohms (Fig. 16). This input resistance cannot be compared to values from other membranes since the area over which the resistance was measured was unknown. However, the values of the total input resistance measured for the tegumental membrane are in the range reported for total input resistance from snail neurons (6.8 megohms) and the goldfish Mauthner cell (4.2 megohms) (Prosser, 1972). The measured time constant of 0.32 ms is in the lower range of time constants reported from a large number of nerve and muscle cells (Prosser, 1972). Since the time constant is equal to the product of the membrane resistance and capacitance (Cm) and C m decreases as the thickness of the membrane increases, a somewhat shorter time constant might be expected since the thickness of the outer tegumental membrane is 110 A (Hockley and McClaren, 1973) compared to 70 to 90 A for nerve or muscle membrane. 88 The fast depolarizations (Fig. 6) are the most prevalent when movements of the animal are present. Any treatment which eliminates movement also eliminates these depolarizations. For this reason the information currently available does not allow elimination of the possibility that these fast depolari» zations are simple mechanical artifact due to bending of the microelectrode tip. Relatively low amplitude slow-wave depolarizations are also often re- corded from the ventral tegument (Fig. 7). These potentials, in contrast to the fast potentials, are definitely not caused by mechanical artifact Since altera- tion of the membrane potential by injecting hyperpolarizing current causes an increase in their amplitude while depolarizing current decreases their amplitude (Fig. 8). There are several possible sources for these spontaneous depolarizations. As mentioned above the fast potentials could result simply from mechanical artifact. Anatomical evidence indicates that there are junctions between the adjacent membranes of the muscle, cytons and cytoplasmic channels (Silk e_t 31., 1969). These junctions are reported to be up to 400mu in length but do not cover the full length of the apposed membranes. From this it appears there may be electrical coupling between the muscles and the tegument. Thus on the basis of the information currently available the spontaneous depolarizations that are recorded could represent elecrotonic spread of electrical activity from muscle into the tegument. A third possibility is that they represent activity located completely in the tegument. Ionic Basis of the Membrane Potential The tegumental potential appears to be primarily a K” potential since alteration of the external K+ concentration has a major effect on it. A 30 mV change in membrane potential per 10 fold change in K+ concentration is noted 89 at external K+ concentrations between 30 and 100 mM (Fig. 17). Although significant, this change is much less than the approximatley 60 mV change predicted by the Nernst equation. Potassium concentrations lower than normal (5.9 mM) cause a depolarization of the tegumental membrane (Fig. 17). This result is quite different from the hyperpolarization that is predicted by the Nernst equation. The deviation from the value predicted by the Nernst equation when the tegumental membrane is depolarized by K+ concentrations between 30 and 100 mM could be caused by the presence of other permeable ion species such as Na+ or Cl'. Since the membrane potential is altered only slightly by decreasing concentrations of Cl', it seems unlikely that in this membrane there is a high CI' permeability relative to K+ permeability as is observed in some muscle cells (Brading and Caldwell, 1974). If the membrane were significantly permeable to Na+ and there were no active transport of Na+ occurring, one would expect a hyperpolarization of the membrane upon reducing external Na+ concentration. Decreasing the Na+ concentration in the external medium from 57 to 37 mM however causes the tegumental membrane to be depolarized by approximatley 10 mV. Na-K Transport System in the Tegumental Membrane Both the effects of altered K+ and Na+ could be explained if a Na-K transport system (pump) were present and necessary for at least partial maintenance of the membrane potential. If such a pump were present one would expect lowered Na+ concentration to depolarize the membrane due to an inactivation of the pump (Thomas, 1972; Casteels e_t _a_l.,l972). Lowered external K+ concentration would also be expected to inactivate such a pump and depolarize the membrane (Thomas, 1972). Several other lines of evidence 90 support the possibility that a Na—K transport system plays a significant role in the maintenance of the tegumental membrane potential. The cardiac glycoside ouabain is a specific inhibitor of active Na-K transport (Glynn, 1964). If a Na-K transport system is present in the tegumental membrane, then inhibition of the pump by ouabain should also lead to a depolarization of the membrane. Ouabain (0.1 mM) causes a rapid depolarization of the tegument (Fig. 25). No significant change in membrane resistance was observed so it appears ouabain is not acting by activating a potential dependent membrane conductance (Fig. 27). The amount of depolari- zation of the tegumental membrane noted upon addition of ouabain ranged from 15 to 29 mV. This range of responses to ouabain would be explained if the contribution of the active transport system to the membrane potential is greater in some animals than in others. This would explain my observation that animals with greater (more negative) membrane potentials are depolarized to a greater extent by the addition of ouabain (Fig. 26). The effect of temperature on the tegumental potential also indicates a Na-K transport system may be present. Though passive properties of the membrane potential that are due to ion diffusion potentials are affected by temperature, one would expect that over the range of temperatures I tested such effects would be quite small. The magnitude by which the diffusion potential can be expected to be altered by temperature can be estimated by the Nernst equation: E = RT/F in (ion)out/(ion) in. The extent of the effect due to decreasing the temperature from 37 to 8°C is given by Tl/Tz: 281 K0/310 K0 = 0.91. This change in temperature would result in an approximately 10% decrease in membrane potential. For a resting membrane potential of -40 mV, the effect of altering the passive properties of the membrane would result in a depolarization of at most 4 mV. This is much less than the 20 mV change in the tegumental membrane potential that I observed upon lowering the temperature 91 from 37 to 80C (Fig. 23). Thus, it would seem that some active temperature- sensitive process, e.g. a Na-K pump, is responsible for generation of at least a portion of the tegumental potential. The fact that substitution of Li+ for Na+ causes depolarization also supports the possibility that a Na-K transport system exists in the tegumental membrane. Lithium ions have a permeability similar to Na+ but Li+ is not transported by the Na-K pump (Diamond and Wright, 1969; Skou, 1964). In a membrane with a Na-K pump substitution of Li+ for Na+ would result in accumulation of Li+ in the cell. Since Li+ cannot substitute for Na+ ions in activating the pump, the pump is inhibited. All of the above lines of evidence strongly suggest that an active Na-K pump is present in the tegumental membrane. Whether this pump is electro- genic, i.e. has a coupling ratio greater than one, is another question. Thomas (1972) has listed several criteria that can be used to distinquish between an electrogenic and an electrically neutral pump. A comparison of Thomas's criteria with my experimental observations on the tegumental membrane is shown in table 2. An assumption underlying the experimental criteria shown in Table 2 is that these treatments cause an instantaneous inhibition of the Na-K pump without altering the internal concentration of either Na+ or K+ (Thomas, 1972). However, as. Thomas (1972) points out any treatment which inhibits the Na-K pump will result in a loss of some internal K+ with a resulting depolarization of the membrane. This effect is particularly important in small cells since the internal ion concentration of small cells could change quite rapidly upon inhibition of the Na-K pump. The ventral tegumental epithelium of S. mansoni is thin (1-3 um thick) with a relatively large surface area compared to its volume. 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