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L . . . . ; . . ufiwfinfi , . . . . . .4.va...........uw flfiwmmrMMAfifiv LIBRARY Michigan State Tniversity This is to certify that the thesis entitled Neuromuscular Physiology of the Longitudinal Muscle of the EarthWOrm, Lumbrlcus terregigial $189519“ Charles David Drewes has been accepted towards fulfillment of the requirements for _______P__h_-. __D__~_ degree in Zoology Major professor , l Date 53 4b 73 0-7539 ABSTRACT NEUROMUSCULAR PHYSIOLOGY OF THE LONGITUDINAL MUSCLE OF THE EARTHWORM, LUMBRICUS TERRESTRIS LINNAEUS BY Charles David Drewes Nerve—muscle relationships in the longitudinal muscle of the earthworm, Lumbricus terrestris Linnaeus, were examined using an isolated nerve-muscle preparation. Individual segmental nerves were stimulated and mechanical and electrical responses of the longitudinal muscle recorded. In initial experiments a saline was used which is similar in composition to salines used by previous investi— gators. Use of this saline resulted in an increase in the excitability of motor axons, a decrease in external muscle potentials (to less than 100 uV), an increased suscepti- bility of muscle fibers to injury spiking during micro- electrode penetration, and a decrease in muscle resting potentials (to - 36.1 mVi2.5 mV SE). These problems were overcome when a new saline corresponding more closely to the ionic composition of earthworm coelomic fluid and blood was used. The Charles David Drewes composition of this saline was: 77 mM Na+, 4.0 mM K+, 6.0 +, 43 mM C1-, 26 mM SO --, 2.0 mM Tris, mM Ca++, 1.0 mM Mg+ 4 55 mM sucrose, pH 7.4, 167 mOsM. In the new saline external muscle responses were 1 to 5 mV and resting potentials averaged -47.9 mV i 2.5 mV SE. The longitudinal muscle is innervated by a fast and a slow axon from each segmental nerve (SN I and SN II—III). The conduction velocity for the fast axon is 0.37 m/sec and for the slow 0.16 m/sec. Single-pulse stimulation of the fast axon produces large external muscle potentials (1 to 5 mV) and small twitch-like contractions which with repetitive stimulation are antifacilitating. Single-pulse stimulation of the slow axon produces only small external muscle potentials and no measurable muscle contractions. Repetitive stimulation produces large, slowly developing and sustained muscle responses with both the electrical and mechanical responses showing summation and facilitation. The amplitude and time course of such mechanical responses are directly related to the frequency of stimulation within a range of 2 to 20 Hz. No evidence for peripheral inhi- bition was found in any experiment. Individual longitudinal muscle fibers are inner- vated by either the fast or slow axon in a nerve, or by both fast and slow axons. Intracellular responses to segmental nerve stimulation consist of excitatory post- synaptic potentials ranging from 1 to 10 mV. Charles David Drewes The motor field for each pair of segmental nerves covers an area nearly equal to that of a segment but is not confined to one segment. The anterior pair of nerves (SN I) innervates approximately the anterior two-thirds of its segment and a small portion of the segment just anterior to it. The posterior pair of nerves (SN II—III) innervates approximately the posterior two-thirds of its segment and a small portion of the segment just posterior to it. Adjacent nerves (both intrasegmental and inter- segmental) have partially overlapping motor fields. NEUROMUSCULAR PHYSIOLOGY OF THE LONGITUDINAL MUSCLE OF THE EARTHWORM, LUMBRICUS TERRESTRIS LINNAEUS BY Charles David Drewes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1973 ufi’ , 3 . /e ., (i n '3. r. 3' 5% ACKNOWLEDGMENTS '4 'CT' . ‘4 g, -- ;.".'o I wish to express my sincere appreciation to my major professor, Dr. Ralph A. Fax, for his guidance and constructive criticism throughout this investigation. I also thank Dr. R. Neal Band, Dr. Gerard Gebber, and Dr. Rudy Bernard who helpfully served as committee members and consultants; and to Mrs. B. Henderson for her help in eliminating considerable red tape for me. Thanks is also given to my colleagues, Chuck Fourtner, Vin Palese, Ben Cathey, and Fred Divers, who provided friendship and helpful ideas. I would like to express my deepest gratitude to my parents, Mr. and Mrs. Henry Drewes, who have provided immeasurable encouragement and support throughout my graduate studies. ii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O V LIST OF FIGURES. . . . . . . . . . . . . vi INTRODUCTION 0 O O O O O O O 0 O O O O O l Difficulties in Studying Annelid Nerve and Muscle. 1 Annelid Neuromuscular Systems . . . . . . . 4 Polychaeta . . . . . . . . . . . . . 4 Hirudinea . . . . . . . . . . . . . 6 Oligochaeta. . . . . . . . . . . . . 8 OBJECT IVES O O O O O O O O O O O O O O 1 1- MATERIAIJS AND METHODS I O O O O O O O O O 0 13 Source and Maintenance of Animals . . . . . . 13 Dissection. . . . . . . . . . . . . 13 Physiological Salines . . . . . . . . 14 Stimulation and External Electrical Recordings. . l6 Intracellular Electrical Recordings . . . . . 17 Mechanical Recordings . . . . . . . . . . 19 RESULTS 0 O O O O O O O O O O O O O O 2 2 Nerve and Muscle Activity in Pantin's Saline . . 22 External Electrical Activity of Nerve and Muscle. . . . . . . . . 23 Intracellular Electrical Activity of Muscle . . 29 Nerve-Muscle Physiology in the New Saline . .A . 34 Responses to Single Stimuli . . . . . . . 35 External electrical responses . . . . . . 35 Mechanical responses . . . . . . . . . 41 iii Page Responses to Twin Pulses . . . . . . . . . 47 External electrical responses. . . . . . . 47 Mechanical responses. . . . . . . . . . 51 Responses to Repetitive Stimulation . . . . . 54 Correlation of mechanical and electrical responses. . . . . . . . . . . . . 54 Frequency-dependence of slow mechanical responses. . . . . . . . . . . . . 59 Intracellular Recordings . . . . . . . . . 70 Resting and spontaneous activity. . . . . . 70 Excitatory postsynaptic potentials . . . . . 71 Mapping of Motor Fields . . . . . . . . . 77 External electrical recordings . . . . . . 80 Intracellular recordings . . . . . . . . 87 DISCUSSION 0 O O O O O O O O O O 0 O O O 91 Activity of Nerve and Muscle in Various Salines . . 91 Patterns of Innervation . . . . . . . . . . 97 Fast Motor Systems . . . . . . . . . . . 97 Slow Motor Systems . . . . . . . . . . . 101 Inhibition. O O O O O O O O O O O O O 103 Functional Significance of Innervation Patterns . . 104 Fast and Slow Systems . . . . . .' . . . . 104 Motor Fields . . . . . . . . . . . . . 106 Phylogenetic Considerations. . . . . . . . . 108 SUMMARY 0 O O O O O O O O O O O 0 O O O 1 10 LITERATURE CITED . . . . . . . . . . . . . 112 iv LIST OF TABLES Table Page 1. Composition of the two earthworm saline solutions . . . . . . . . . . . . 15 2. Analysis of single-stimulus responses to fast axon stimulation in each segmental nerve. . 46 LIST OF FIGURES Figure 1. Diagram of muscle clamp and strip of body wall muscle . . . . . . . . . . . 2. Decline of external muscle potentials in Pantin's saline. . . . . . . . . . 3. Repetitive activity in nerve and muscle following a single stimulus. . . . . . 4. Longitudinal muscle responses to a single Stimulus O O O O O O O O O O O O 5. Spiking activity in a longitudinal muscle fiber . . . . . . . . . . . . . 6. External electrical responses of the longi- tudinal muscle to single stimuli . . . . 7. Mechanical responses of the longitudinal muscle to single and paired pulses . . . 8. External electrical responses to twin-pulse stimulation of SN I . . . . . . . . 9. Comparison of mechanical responses to single- and paired-pulse stimulation of fast axons O O O O O O O O O O O O O 10. Mechanical and electrical responses to repetitive stimulation of fast and slow axons O O O O O I O O O O O O O 11. Mechanical responses of longitudinal muscle to repetitive stimulation . . . . . . 12. Relationship between onset latency of the slow response and the frequency of stimulation . vi Page 21 25 28 28 33 37 43 49 53 57 62 64 Figure 13. Relationship between the time to 67% of the peak amplitude of slow responses and the stimulus frequency . . . . . . . . . 14. Relationship between the peak tension of slow responses and the stimulus frequency . . 15. Excitatory postsynaptic potentials in longi- tudinal muscle fibers . . . . . . . . 16. Innervation patterns of longitudinal muscle fibers 0 O O O O O O O O O O O O 17. Mapping of external electrical responses of the longitudinal muscle. . . . . . . . . 18. Overlapping motor fields of adjacent segmental nerves 0 O O O O O O O O O O O 0 l9. Excitatory inputs to a muscle fiber from two different segmental nerves . . . . . . vii Page 67 69 73 79 82 86 90 INTRODUCTION There have been numerous investigations of neuro- muscular systems in annelids but most physiological infor- mation concerning annelid neuromuscular systems has been obtained from studies in only two classes, the Polychaeta (polychaetes) and the Hirudinea (leeches). Nerve-muscle relationships in the Oligochaeta are not well understood. In light of this gap in our understanding of annelid neuromuscular systems, I have examined the physiological basis for nerve-muscle relationships in the oligochaete earthworm, Lumbricus terrestris Linnaeus. Difficulties in Studying Annelid Nerve and Muscle Several problems are encountered in any investi- gation of annelid neuromuscular relationships. These problems are related to the general organization of the nervous and muscular systems. The body wall musculature of annelids is typically not organized into distinct bundles of muscle fibers as in many arthropods and chordates. Instead the body muscle is composed of cylindrical sheets of muscle fibers surrounding the body cavity. These muscle sheets extend continuously along the length of the worm and are organized into concentric layers of antagonistic muscle, an outer circular layer and an inner longitudinal layer (Laverack, 1963). As a result of this organization it is difficult to obtain a discrete nerve-muscle preparation. Thus it is not possible to study the functioning of annelid muscle using the same direct approaches used in studying the functioning of muscle composed of distinct bundles of fibers. The structure of individual muscle fibers in annelid muscle leads to technical problems in both histological and physiological studies. The muscle fibers of several annelids, including earthworm longitudinal muscle fibers, are "obliquely striated," that is, there is an angle of 5 to 30° between the striations and the longitudinal axis of the muscle fiber (Hanson, 1957; Heumann and Zebe, 1967; Rosenbluth, 1968; Mill and Knapp, 1970a; Knapp and Mill, 1971). This complication in structure leads to consider- able difficulty in interpreting the over-all structure of muscle fibers. For example no precise determinations of sarcomere lengths have been possible in earthworm muscle. Thus no statements can be made regarding the possibility of uniformity or diversity in muscle fiber structure. From a physiological standpoint the structure of annelid muscle also presents technical problems. For example in earthworm longitudinal muscle the muscle fibers are quite small, making penetration with microelectrodes difficult. Hanson (1957) has described earthworm longi— tudinal muscle fibers as flat and ribbon-shaped, with a thickness of only 2 to 3 u and a width of about 20 u. The length of individual fibers, however, may be as great as 2 to 3 mm. The nervous systems of annelids, particularly those of oligochaetes, are ill-suited to physiological investi- gation. The central nervous system in the earthworm, for example, is surrounded by a thick sheath consisting of muscular and connective tissues. This sheath prevents easy access with microelectrodes to central neurons which are generally less than 50 u in diameter (Staubesand £3 31., 1963; Gunther, 1971). In contrast to the nervous organi- zation in the earthworm, the central nervous system of leeches is relatively simple and well suited to electro- physiological investigation (Coggeshall and Fawcett, 1964). The cell bodies of central neurons are easily identifiable from one preparation to another and are readily accessible to intracellular electrodes (Kuffler and Potter, 1964; Nicholls and Purves, 1970; Stuart, 1969, 1970). Another difficulty in studying annelid neuro- muscular physiology involves the small size of peripheral nerves. In earthworms segmental nerves are about 50 u in diameter and a few tenths of a millimeter in length (personal observation). The small size of nerves makes their manipulation difficult and precludes the possibility of physically separating individual nerves into their axonal components. Annelid Neuromuscular Systems Despite inherent difficulties in studying annelid neuromuscular systems, there have been a few meaningful investigations of such systems in two of the annelid classes, the Polychaeta and the Hirudinea. The major contributions to our understanding of annelid neuro- muscular systems are given below for each class of annelids. Polychaeta The thorough description of the nervous system in nereid polychaetes by Smith (1957) has led to several meaningful analyses of the neural control of muscle movements in nereids. One movement which is well under- stood is the longitudinal rapid response of certain nereid worms. Horridge (1959) has described the neural pathway for this response in two polychaetes, Nereis virens and Harmothoé imbricata. In these animals the longitudinal musculature of each lateral half of a segment is inner- vated (via segmental nerve IV) by a large axon arising from an identifiable unipolar cell located in the central nervous system. This motor neuron, after making synaptic contact with a lateral giant fiber in the central nervous system, gives rise to the efferent pathway of the rapid response. Using a nerve-muscle preparation consisting of a short strip of body wall and segmental nerve stumps, the functional characteristics of the efferent pathway of the rapid response were described (Horridge, 1959). The results of this study indicated that the efferent pathway had functional characteristics similar to the so-called "fast" motor systems seen in a variety of invertebrates. In these cases the term "fast" describes the rapid muscle contraction mediated by the "fast" axon (Bullock and Horridge, 1965). Selective stimulation of the fast axon in Nereis, as indicated by a single sharp threshold, resulted in a large external electrical response in the longitudinal muscle (Horridge, 1959). With repetitive stimulation these responses declined rapidly in amplitude. In addition to the fast motor system evidence was given for a slower electrical response apparently resulting in a slow muscle contraction. A similar study of the functional aspects of longitudinal muscle innervation in Nereis by Wilson (1960a) supports the results of Horridge. In addition, this study provides more detail regarding longitudinal muscle innervation, in particular detail regarding the slow electrical and mechanical responses. Wilson showed that low-strength stimulation of segmental nerve IV results in a large and sharp muscle potential with a single threshold. The short onset latency of the response and the susceptibility of the response to fatigue indicate that it is mediated by the fast axon, previously identified by Horridge. A second response, also with a sharp threshold, is seen at higher stimulus strengths. This response is characterized by a smaller amplitude and longer latency than the fast response. This response facilitates with repetitive stimulation suggesting that the response is mediated by a slow axon similar to the slow systems described in other invertebrates. No evidence is given for peripheral inhibition in any of these studies. The occurrence of two functionally distinct motor systems, fast and slow, in polychaetes correlates well with structural analyses of neuromuscular junctions. Dorsett (1963) has identified two morphologically distinct types of nerve terminals on the longitudinal muscle fibers of Nereis. Thus she suggests that the fast and slow responses may be mediated through morphologically distinct nerve endings. Hirudinea Nervous control of movements in leeches has been studied by Wilson (1960a). Stimulation of peripheral nerve stumps produced visible longitudinal muscle con- tractions with a tetanus to twitch ratio of several hundred to one. The external electrical responses from the muscle were graded in amplitude depending on the stimulus strength, thus suggesting numerous motor axons in each nerve. With repetitive stimulation facilitation of the electrical responses was observed, suggesting a slow motor system, similar to that of polychaetes. Recently a much more detailed investigation of motor innervation in leech muscle has been carried out by Stuart (1969, 1970). These studies have involved mapping of motor neurons in the ventral cord as well as mapping of innervated territories in the muscle layers. Stuart has shown that within each lateral half of a segmental ganglion there are six identifiable excitatory motor neurons which directly innervate the longitudinal muscle. The largest of these neurons innervates the entire contralateral half of the longitudinal layer from dorsal to ventral midlines in that segment. The other five motor neurons innervate smaller patches of muscle. The electrical and mechanical responses mediated by all six excitatory axons are charac- terized by summation and facilitation, thus suggesting that there is only a slow system of innervation in the longitudinal muscle. In addition to numerous excitatory inputs, the longitudinal muscle is apparently innervated by two inhi— bitory axons. These results are in contrast to the situation in polychaetes where both fast and slow systems are seen but no inhibition has been found. Oligochaeta In earthw0rms the central nervous system consists of a ventral nerve cord extending the length of the animal. In each segment the ventral nerve cord gives rise to three pairs of segmental nerves which innervate the body wall musculature. All segmental nerves are mixed (sensory and motor) and may be numbered according to their position in the segment, segmental nerve I (SN I) being located anteriorly in each segment and segmental nerves II and III located posteriorly. Segmental nerves II and III (SN II— III) are located so close together they appear as a single nerve, termed the "double nerve." Virtually all information regarding nerve-muscle relationships in oligochaetes is derived from experiments on earthworm longitudinal muscle. Nearly all these experi— ments have had some serious limitation in approach which has prevented a clear understanding of nerve-muscle relationships. The studies of Roberts (1962a,b, 1966) have defined the role of the giant fibers in the reflex pathway of the longitudinal escape response of the earthworm, Lumbricus terrestris. The efferent side of this pathway apparently involves several giant motor neurons whose activity is coupled to giant fiber activity (Gunther, 1972). Though these studies have demonstrated some of the important functional components of the reflex pathway, they do not clearly demonstrate the functional properties of neuromuscular junctions nor do they indicate the over—all pattern of innervation of the longitudinal muscle. To do this a discrete nerve-muscle preparation free from central nervous influence is required. Recently Drewes and Pax (1971) have described a suitable nerve—muscle preparation for the body wall muscle of Lumbricus terrestris. To obtain the preparation the ventral nerve cord was removed leaving only a short strip of muscle along with the segmental nerves which innervate the muscle. Using this preparation a major problem was encountered which precluded clear-cut analysis of nerve- muscle relationships. This difficulty was related to the tendency for repetitive firing of motor fibers following a single stimulus to a segmental nerve. This made analysis of the properties of motor fibers in each nerve difficult. Intracellular recordings from the longitudinal muscle have recently been obtained in two earthworms, Pheretimg communissima and Pheretima hawayana, by Hidaka gt gt. (1969a,b) and Chang (1969), respectively. These studies showed that muscle fibers are characterized by low resting potentials (35 to 36 mV) and by "spontaneous" trains of overshooting muscle spikes. In addition to spiking activity, Hidaka gt gt. (1969c) and Ito gt gt. (1969a,b) have recorded small, spontaneous depolarizing and hyper- polarizing potentials in longitudinal muscle fibers; these are suggested to be excitatory and inhibitory junction potentials, respectively. The frequency of such potentials 10 increased following direct stimulation of the muscle strip. No fixed latencies or sharp thresholds for any of the potentials were indicated and such potentials were often erratic and irregular in shape. Although inhibitory and excitatory inputs to longitudinal muscle are implied by these studies, no clear- cut conclusions can be drawn regarding the number of motor axons present, the distribution of motor axons in the segmental nerves or the over-all pattern of innervation of the longitudinal muscle. Histological investigations, although numerous, have been of little help in clearly establishing the patterns of innervation in earthworm muscle. In some studies two structurally distinct nerve endings have been identified in the longitudinal muscle (Smallwood, 1926; Rosenbluth, 1972). However, in a similar study Mill and Knapp (1970b) found only one morphological type of nerve ending in the longitudinal muscle. Thus neither physio- logical nor structural investigations have established clear—cut evidence for a specific pattern of innervation in earthworm muscle. OBJECT IVES Neuromuscular physiology in earthworms is not well understood, due in part to difficulties in obtaining a suitable nerve—muscle preparation. Further difficulties are indicated by the presence of low muscle resting potentials, spontaneous spiking in muscle fibers, low- amplitude and erratic junction potentials, and repetitive firing following stimulation of motor fibers. The nature of these difficulties suggests abnormal excitability of nerve and muscle membranes brought about by ionic im- balances in the bathing medium. Therefore, in this study I have developed a new earthworm saline with a composition differing considerably from the composition of salines used in previous investi- gations of earthworm muscle physiology, but agreeing well with the ionic composition of earthworm coelomic fluid and blood as determined by Kamemoto gt gt. (1962) and Dietz and Alvarado (1970). Use of the new saline along with a suitable nerve- muscle preparation constitutes a new approach to nerve- muscle physiology in the earthworm. This approach has 11 12 permitted a detailed analysis of the neural control of electrical and mechanical events involved in longitudinal muscle contraction. Information obtained from this investigation provides a basis for comparing earthworm nerve-muscle relationships to those of other annelids. A preliminary report of this work has appeared elsewhere (Drewes and Pax, 1972). MATERIALS AND METHODS Source and Maintenance of Animals Earthworms, Lumbricus terrestris, were used in all experiments. Specimens were either obtained from the Wholesale Bait Company (Hamilton, Ohio) or collected locally. Animals ranged from 0.5 to 1.0 cm in diameter, and from 12 to 18 cm in length. All animals were main- tained in Buss Bed-ding (Buss Mfg. Co., Lanark, Illinois) and stored in styrofoam boxes in 12 to 14° C. Animals could be kept alive for several months under these con- ditions, but most animals were used within two weeks of collection. Dissection Animals were lightly anesthetized by placing them on ice for several minutes before dissection. The animal was pinned dorsal side up into a shallow paraffin tray. In most cases the animal was opened with a dorsal midline incision beginning posterior to the clitellum and extending 10 to 20 segments posteriorly. However, in experiments involving mapping of motor fields it was necessary to make the incision approximately 2 to 3 mm lateral to the dorsal 13 14 midline. The body wall was spread open by cutting the septal connections between the body wall and intestine. The body wall was pinned flat and transverse cuts were made through it at the anterior and posterior boundaries of the incision. It was then possible to completely remove the undissected anterior and posterior regions of the animal along with the intact digestive tract. To allow clear viewing of the ventral nerve cord the ventral blood vessel was dissected free from its connections with the ventral body wall. The entire ventral nerve cord was then removed by severing all segmental nerves close to their central connections with the ventral nerve cord. To permit clear Viewing of the segmental nerve stumps and longitudinal muscle the nephridia in all segments were removed. The final nerve-muscle preparation consisted of a flat sheet of body wall (approximately 15 segments in length) along with the segmental nerve stumps innervating those segments. The entire dissection was completed in 15 to 20 minutes. Physiological Salines Approximately midway through the dissection and for the remainder of an experiment it was necessary to bathe the preparation in a physiological saline solution. Two different salines were used (Table l). Saline I is the physiological saline recommended by Pantin (1946). This saline is very similar in ionic 15 Table 1. Composition of the two earthworm saline solutions. I(mM) II (mM) Na 135.0 77.0 K 2.7 4.0 Co 1.8 6.0 Mg 0.4 1.0 CI 142.0 43.0 504 0.4 26.0 P04 1.0 — Tris — 20 sucrose — 55.0 mOsM 210.0 167.0 pH 7.4 7.4 16 composition to salines used in all previous investigations of earthworm muscle physiology. Its composition, however, does not correspond with the ionic composition of earthworm coelomic fluid or blood (Kamemoto gt gt., 1962; Dietz and Alvarado, 1970). Saline II, a new saline which I have developed, corresponds much more closely to the ionic composition of earthworm coelomic fluid and blood. In this saline the concentrations of Na+, K+, Ca++, and C1- are all nearly identical to those in earthworm coelomic fluid and blood. Since the desired chloride concentration was low it was necessary to add another anion, sulphate, to the medium. An appropriate amount of sucrose was added to obtain an osmotic concentration identical to that reported for earthworm body fluid (Ramsay, 1949). Osmotic concentrations were measured with an Osmette Precision Osmometer (Pre- cision Systems). Throughout all experiments the temper- ature of the preparation was maintained at 12 to 14° C by placing the paraffin tray in an ice bath. Stimulation and External Electrical Recordings For electrical stimulation of individual segmental nerves, a nerve stump was drawn up into the tip of a polyethylene suction electrode. The inner diameter of the electrode tip was 100 to 150 u. Biphasic pulses were delivered to two silver wires, one inside the electrode tip l7 and the other coiled around the outside of the tip. The leads of the stimulating electrode were connected to a Grass Model S4 stimulator through a Grass stimulus iso— lation unit. Unless stated otherwise, Stimulus durations were always 0.2 msec. Electrical activity was recorded using a suction electrode similar to that used for stimulation. Recordings were single-ended against a silver wire ground electrode placed in the bath. Unless otherwise stated, all records were taken from a position approximately 2.0 mm lateral to the segmental nerve being stimulated. The longitudinal co- ordinate of this recording site corresponds to a level midway between the two pairs of setae in the lateral half of each segment. The lateral co-ordinate corresponds to a line through the anterior one-third of the segment when stimulating segmental nerve I (SN I), or through the posterior one-third of the segment when stimulating segmental nerve II-III (SN II-III). Electrical activity was amplified using a Grass P15 AC preamplifier and monitored on a Tektronix 502A oscilloscope. Records were photographed using a Grass C4 oscillosc0pe camera or a Polaroid C27 oscilloscope camera. Intracellular Electrical Recordings Intracellular longitudinal muscle activity was recorded with glass microelectrodes obtained by drawing out Kimax glass tubing (0.8 mm outside diameter) with a 18 Narashige microelectrode puller. The microelectrodes, secured to a glass slide with a rubber band, were submerged in a vacuum flask containing hot 3M KCl solution. A controlled vacuum was applied to the flask causing a gentle boiling. After boiling ten minutes, the flask was sealed with its vacuum and allowed to stand ten minutes. The vacuum was then slowly released. In my experience this procedure was sufficient to fill most of the electrodes. Electrodes were stored in a coplin jar filled with 3M KCl. The microelectrode assembly consisted of the glass microelectrode fitted over a Ag—AgCl wire (no. 34), the microelectrode probe, and the Narashige micromanipulator. The lead from the probe was connected to a W-P Model M-4A electrometer (W-P Instruments, Inc.) and monitored on an oscilloscope. The ground electrode was a KCl-agar bridge con- sisting of an L-shaped glass tube (1/8" outside diameter) filled with a 1% solution of agar in BM KCl. One end of the bridge touched the saline while the other was connected with a Ag-AgCl wire. Using this ground electrode, micro- electrode tip potentials were as 1ow as 2 mV. In all experiments microelectrode resistances ranged from 8 to 20 M9. 19 Mechanical Recordings A somewhat smaller nerve-muscle preparation was required for mechanical recordings. The original muscle strip was cut into two unequal halves by making a longi- tudinal cut approximately 1 mm lateral to the ventral midline. That portion which included the ventral midline was further trimmed down to a length of five or six seg— ments. The remaining preparation, consisting of slightly more than one lateral half of five or six segments, was then used for mechanical recordings. The preparation was attached to a plexiglass muscle clamp (weight = 0.025 g) as shown in Figure 1. One end of the preparation was gripped by the clamp; the other was pinned securely to the tray. The clamp was allowed to pivot freely on an insect pin stuck into the tray. A short thread connected the clamp to a microdisplacement myograph transducer (Linear Core F-SO; Narco Biosystems, Inc.). A resting tension of 0.5 to 1.0 g was applied to the prepa- ration, thus stretching the muscle strip longitudinally to a length about one and a half times the resting length. Muscle contractions were monitored on a Model Four Physio- graph (Narco Biosystems, Inc.). 20 .omuouflcofi mum coeucasswum o>umc on momcommmu maomSE Hmcflcsu neocoH opp can ARV cmmunu 6 mo memos >3 umoscmcmnu m ou cmnomuum we mEmHo one .AmHv mafia pommcfl mo mcomE ha mHomsE may mmflum guess mEmHo ecu ou cmnompum ma mfluum mHomsE mzu no one one .Ava mm>ums Hmucmsmmm Hm5©fl>ficcfl mumadEHum ou poms we Amv ooouuowam mcflumassflum coHBOSm « .maomss Hams moon mo mfluum cam cacao maomsfi mo EMHmMHQ .H musmflm H enamflm RESULTS Nerve and Muscle Activity in Pantin's Saline The experiments which follow were performed using the physiological earthworm saline of Pantin (1946). This saline was suspected of being inappropriate for studying nerve-muscle physiology because its ionic composition differs considerably from that of earthworm coelomic fluid or blood. Several approaches were used to examine this question. First, longitudinal muscle potentials were recorded extracellularly following indirect stimulation. The amplitude of these responses was measured at various times during an experiment. This approach gives some indication of the functional stability of the nerve-muscle preparation while bathed in the saline. Second, electrical activity in segmental nerves was studied following nerve stimulation. By correlating nerve and muscle activity, it was then possible to determine the general level of excitability in motor axons. Finally, the intracellular electrical activity of longitudinal muscle fibers was examined. From these results it is possible to determine the general level of 22 23 excitability of muscle. Using all these approaches one can determine the adequacy or usefulness of Pantin's saline in studies of nerve—muscle relationships in the earthworm. External Electrical Activity of Nerve and Muscle External muscle potentials were examined in eight preparations at various times after the initial exposure to Pantin's saline during dissection. A suction electrode was applied to the surface of the longitudinal muscle approximately 2.0 mm lateral to the segmental nerve being tested. A segmental nerve was stimulated supramaximally with a single stimulus (6.0 V). The results were similar in all preparations and indistinguishable for the two different segmental nerves. Results from a typical experiment are shown in Figure 2. Each point represents the mean amplitude of external muscle potentials recorded from six different segments following stimulation of SN II-III. Each initial recording, taken 20 minutes after exposure to the saline, consisted of a large, single negative potential with a mean amplitude of 2.75 mV : 1.02 mV SD. After another 20 minutes the mean amplitude had decreased to 190 uV t 25 uV SD, or less than one tenth the initial value. The mean amplitude of these potentials continued to decrease until they were almost non-recordable; after one hour the mean amplitude was only 17 uV i 11 uV SD. 24 .mmmcmn ecu oumoflpcfl mmcfla accepnm> .Hofiwcm mco Eoum mucmEmHSmmmE ucmumwmac xflm mo some n mucwmmummu ucflom 30mm .mcflamm map ou musmomxm mo mEHu Houou map pmcflmmm cmpuoam ma HHHIHH 2m mo soapmHsEHum op mmmcommmn maomss cmcnoomn haamcuwuxw mo ocsuHHmEm one .wcflamm m.cflpcmm CH mamflucmpom maomss accumuxm mo mcaaomo .m musmflm 25 O N 'h—r . is n J 0. 0. O. 0. v co N '- (Aw) SSNOdSBH 1VD|813313 TIME IN SALINE (min) Figure 2 26 Correlated closely with the decrement in external muscle potentials was a tendency for motor axons in the segmental nerves to become hyperexcitable, such hyper- excitability being indicated by repetitive firing in nerve and muscle in response to a single stimulus. To record this activity in the segmental nerves a suction recording electrode was placed along side and slightly distal to the stimulating electrode. Thus both stimulating and recording electrodes were in contact with the nerve. Results obtained from each of the two nerves were identical. Figure 3 shows the repetitive activity in SN II-III in response to a single stimulus. The activity consists of a series of spikes of various amplitudes, with the spiking activity gradually decreasing in frequency. Such activity lasted up to several seconds and was often recorded at stimulus intensities only slightly above threshold. However in general the duration of spiking activity in- creased with increases in the stimulus intensity. The repetitive activity in nerve following a single stimulus is at least partly due to repetitive spiking in the motor axons which innervate the longitudinal muscle. This was shown by recording external electrical activity of longitudinal muscle in response to a single nerve stimulus. Each stimulus resulted in a prolonged burst of low- amplitude muscle potentials (Figures 3 and 4). The duration of this burst corresponds with the duration of Figure 3. Figure 4. 27 Repetitive activity in nerve and muscle following a single stimulus. In the top record repetitive spiking activity is recorded from SN II-III in response to a single stimu- lus (8 V). In the bottom record repetitive activity is recorded from the longitudinal muscle following another stimulus to the nerve. Vertical calibration: 0.2 mV. Horizontal calibration: top record, 100 msec; bottom record, 50 msec. Longitudinal muscle responses to a single stimulus. SN II-III is stimulated with a single strong stimulus (20 V, 1.5 msec). A burst of electrical activity in the muscle (lower trace) is sufficient to bring about a large contraction of the muscle (upper trace). Vertical calibration: upper trace, 1.0 9; lower trace, 0.1 mV. Horizontal calibration: 2 sec. 28 Figure 3 Figure 4 29 repetitive nerve activity. With high stimulus strengths the burst of muscle activity lasted several seconds and resulted in a large recordable mechanical response (Figure 4). This mechanical response was often irregular in shape, particularly during the relaxation phase. The amplitude of the contraction was directly related to the amount of bursting activity recorded from the nerve and muscle. Thus there was no clear threshold for the mechani— cal response and there was no stimulus strength which appeared to be supramaximal. These results all suggest that with prolonged exposure of the nerve-muscle preparation to Pantin's saline motor axons in the segmental nerves become hyperexcitable. In this condition nerves fire repetitively following a single stimulus and the muscle responds with a series of external potentials often leading to contraction. These problems make a more detailed analysis of muscle inner- vation nearly impossible. Intracellular Electrical Activity of Muscle The direct effect of Pantin's saline on the longitudinal muscle was studied with intracellular micro- electrodes. Of particular interest was the effect of this saline on the resting membrane potentials of individual cells. Initial measurements of resting potentials were made 15 to 20 minutes after exposure to the saline; later 30 records were taken approximately one hour after the exposure. In all these experiments no stimulation of nerve or muscle was given. Initial measurements were made from 37 cells (four animals), the mean resting potential being -44.2 mV i 2.9 mV SE. The majority of these cells were quiescent. After one hour in the saline, however, the mean resting potential had decreased to -36.1 mV i 2.5 mV SE (150 cells, four animals). Of these cells 25% were quiescent, while 75% showed "spontaneous" spiking activity, such as that shown in Figure 5. Generally the spiking activity consisted of a regular series of spikes, ranging in frequency from 0.2/sec to 15/sec, with the frequency usually decreasing with time. The amplitude of these spikes varied from one cell to another, with spikes in some cells overshooting zero potential by as much as 10 to 15 mV. In other cells spikes were as small as 7 mV. These results suggest that exposure to Pantin's saline brings about a gradual depolar- ization in muscle cells which is accompanied by an increased tendency for repetitive spiking. The origin of such spiking appears to be in the mechanical disruption, or penetration of the muscle fibers. This was clearly demonstrated by monitoring the external electrical activity of muscle fibers prior to, during and after penetration with a microelectrode. To do this the 31 suction recording electrode and microelectrode were placed in very close proximity (less than 50 u), so that both electrodes could be used to record activity from the same muscle fiber. Figure 5 shows the intracellular and external electrical activity recorded from a longitudinal muscle fiber. Prior to penetration no external activity is recorded. Immediately after penetration a series of spikes is recorded intracellularly. Simultaneously there occur small external potentials with a time course essentially identical to the time course of intracellular spikes. Once the electrode is withdrawn no spiking activity is recorded externally. These results indicate that the so-called "spontaneous" spikes occur only during penetration, and they may therefore be considered injury spikes rather than true spontaneously occurring spikes. The spiking activity in the longitudinal muscle of the earthworm is similar to that recorded from other annelids (Washizu, 1967; Hidaka gt gt., 1969a,b; Chang, 1969). In these studies, however, such spiking was suggested to be true myogenic activity. In other experiments I have attempted to record postsynaptic muscle potentials in response to segmental nerve stimulation. Several difficulties were encountered in such experiments. One major problem was the presence of injury spikes which made analysis of intracellular junction potentials extremely difficult. Also the irregular 32 .ommE om "Coflumunfiamo HmuCONHnom .>E oa cuoomu Mozoa “>8 H.o .cuoomn Comm: “Cofipmnnflamo Hoofluuw> .Hamo waomsE wCu mo Coflumuumcmm an CGUSCCH mH hpfl>flpom mCflmem pony oCHumoHcCH .C3mucCuH3 mfl moonuomao can we mmoum wufl>fluom mCflxflmm .mconuowao HCHSHHooprCH mCu ou wpflEonum wmoHo CH we COHCB .Aummmsv wcouuowao HCCHmuxm Cm mCflms cmcuoomu omHm we wufl>fluom mfiCB .maumasaaoocuuCH copuooou mwxflmw mo mwflumm m we oumnu cofipmuuoCmm mCHBOHHom .AuosoHv cuoowu umHsHHoomuuCfl mCu mo oCHmemn opp CH umaCm m>flpmmmC wmuma mCu ha pmaamcmflm mH occupomamouofifi mCu Spas coflumupmcmm .uwnflm oHomCE HmcflcspflmcoH m Cs mufl>flpom mCHmem .m onsmflm m muooflm 34 mechanical contractions which often followed nerve stimu- lation made it difficult to maintain an intracellular record for more than a few seconds. Despite such diffi- culties, small depolarizing potentials (less than 1 mV in amplitude) were recorded on rare occasions from a few cells. These cells were normally quiescent in their activity. '- From the preceding results it appears that Pantin's saline has numerous undesirable effects on the nerve— muscle preparation. First, there is a decrease in the amplitude of externally recorded muscle responses to nerve stimulation. Also there is an apparent hyperexcitability of motor axons when exposed to the saline. Finally there is a decrease in the muscle resting potential, which is accompanied by a tendency for repetitive spiking in muscle fibers following penetration with a microelectrode. These undesirable effects all point to the inappropriateness of this saline for studies of nerve-muscle physiology. Nerve-Muscle Physiology in the New Saline The difficulty of analyzing the functional relationships of nerve and muscle bathed in Pantin's saline led to an examination of these relationships in a new physiological saline, whose ionic composition corre- sponded closely to that of earthworm coelomic fluid and blood (see Table 1 for ionic composition of this saline). 35 Using this more appropriate saline it was possible to analyze in detail the functional relationships between earthworm nerve and muscle. This analysis involved the recording of mechanical and electrical muscle responses to stimulation of individual segmental nerves. Responses to Single Stimuli External electrical responses.--External electrical responses of the longitudinal muscle to nerve stimulation were recorded from 12 animals after exposure to the new saline. The suction recording electrode was applied to the surface of the muscle 2.0 mm lateral to the segmental nerve being stimulated. Single stimuli were then delivered to the segmental nerve. In general muscle responses to supramaximal stimuli were large, usually 1 to 5 mV. If tested at 20 minute intervals, these responses remained relatively stable in amplitude for more than one hour of experi- mentation. This is in contrast to the situation in Pantin's saline in which responses declined rapidly in amplitude during the first hour of experimentation (Figure 2). Typical external potentials recorded from the longitudinal muscle are shown in Figure 6. The number of thresholds and the time course of the responses to stimu- lation of SN I were indistinguishable from those of 36 .oomE OH "Coeuonu leamo HouCoNHHom .>E m.o "Coauouneamo Hooepuo> .omnsoo oEeu pco caonmounu pConommec m won noon nomo mane .omCommoH HmeueCe onu mo moH mCHCeHooU onu Co comomEHHoQSm we noens xmom Houoa CCm pCooom m CH mCHuHsmon .conomon we paonmonnu muonm ncooom o Coeumasaepm mo mnpmnonpm nonmen npflz .Caonmounu Hoepenw mnmnm o o>ono posh mnmeonpm msHSEeum Cues coouooou me noon pomonmuonCHm omnma o mommo Ham CH .nonnOCm oCo C0 comomEHquSm Czonw oum momCommoH 03» cnooon nomo CH .HHH IHH 2m mo CoHpmHsEHpm ou momcommon Bonm Amv moomuu Hosea oCo .H Zm mo Coeumaseeum ou momnommou Bonm Amy mooouu Home: one .easfieum onCem ou oaomss HmCHCsuHmCoH one mo momCommou Hooeuuooao HoCHouxm .m ouomem 38 SN II-III. No differences were seen between responses in different segments of the same preparation. The first electrical response of the muscle to gradually increasing stimulus strengths is a single and large negative potential, usually 1 to 5 mV in amplitude. The threshold of this large initial response was sharp and occurred in an all—or-none fashion, thresholds ranging from 1.8 to 5.1 V in different preparations. The mean onset latency for the response to stimu- lation of SN I was 4.8 msec i 0.7 msec SE, with a mean time from stimulus to peak being 8.9 msec i 1.0 msec SE. For SN II-III the mean onset latency was 4.6 msec i 0.8 msec SE, with a mean time from stimulus to peak being 8.7 msec i 1.3 msec SE. The large amplitude and the slow smooth time course of these responses suggests that they are extra- cellular measurements of the postsynaptic potentials from many longitudinal muscle fibers. This idea receives more conclusive support in a later analysis of intracellular muscle responses. In addition, the all-or-none threshold and smooth time course of the responses in all segments suggests that the response is mediated by a single motor axon. The similarity in the time courses of the response mediated by the two nerves suggests that a similar motor axon exists in each segmental nerve. On the basis of the onset latencies of the external responses and the known distances from stimulating to 39 recording electrodes, it is possible to roughly calculate conduction velocities for the proposed axons. For SN I the calculated mean conduction velocity was 0.37 m/sec i 0.04 m/sec SE, and for SN II-III the mean conduction velocity was 0.39 m/sec i 0.04 m/sec SE. These values do not take into consideration possible synaptic delay at the neuro- muscular junction. Assuming a synaptic delay of 1.0 msec estimates for conduction velocities would be approximately 0.5 m/sec. In all segments a second threshold-dependent electrical response of the muscle was also found (Figure 6). This response was seen at stimulus strengths ranging from 0.1 V to several volts above that of the first threshold. The threshold for this second response was also sharp, occurring in all—or-none fashion. Each of these responses consists of a relatively small negative potential superimposed on the declining leg of the large initial peak, thus giving the appearance of a double-peaked potential. The second peak is generally reached 11 to 15 msec after the stimulus. No significant differences were found between the two segmental nerves with respect to the appearance of this second response. The consistently sharp threshold and smooth appearance of this later response suggests the presence of a second, more slowly conducting, motor axon in each of the segmental nerves. For convenience and for reasons made 40 clear later the two proposed axons will hereafter be termed fast and slow axons on the basis of their apparent differ- ences in conduction velocities. An accurate measure of the conduction velocity of the slow axon was difficult, since its threshold was nearly always above that of the fast axon. Due to electrical summation of the two muscle responses, the true time course of the second potential was not clear. However in one instance the threshold for the slow axon was slightly below that of the fast axon, thus allowing isolation and analysis of the muscle response mediated by the slow axon. In this case the electrical response consisted of a smooth negative wave approximately 300 uV in amplitude, or roughly one tenth the amplitude of the faster response. The onset latency of the response was 10 msec with a peak at 14 msec following stimulation. The conduction velocity of the slow axon in this case was 0.16 m/sec, or approximately one half that of the fast axon. The relatively high threshold and slow conduction velocity of this axon suggests that its diameter is smaller than that of the fast axon. In rare instances a third type of muscle response was recorded, occurring 10 to 50 msec later than the re5ponse mediated by the slow axon. The onset latency and amplitude of the response were extremely variable making analysis difficult. The threshold of this response was also variable and usually several volts above that of the slow axon. The erratic nature of this response suggests 41 that it may not represent a third motor axon, but is perhaps a result of double firing of one of the two motor axons, such activity being induced by the relatively high stimulus strength. In summary, an analysis of nerve-muscle relation— ships using external recording techniques and a new saline suggests the presence of a fast and a slow conducting motor axon in each segmental nerve. Responses mediated by each of the axons appear relatively stable following exposure to the new saline. Also there is little or no tendency for repetitive nerve and muscle activity following a single stimulus, indicating relatively normal excitability of motor axons. It therefore appears that the new saline is suitable for use in an electrophysiological investigation of nerve-muscle relationships. Mechanical responses.--Mechanica1 responses of the longitudinal muscle to single, supramaximal stimuli were recorded in eight preparations. Typical responses to stimulation of each of the segmental nerves are shown in Figure 7A,B. ReSponses always consisted of very small twitches barely measurable even at maximum transducer sensitivities. In all cases the responses were clearly visible under the dissection microscope, with contraction being localized in the ventromedial aspect of the longi- tudinal muscle of one or two segments. 42 .oom m~.o ”CoHuounHHoo HCHCON anom .m mo.o “COHumunHHmo HmoHuuo> .HHHnHH 2m CH Coxo ummm on» Ho CoHpmHCEHum omHsm conHom ans copuooou mH omCOQmoH HmHHEHm o C CH .AoomE mH .Hm>HoHCH mCHCEHumV Csonm mH H 2m CH Coxm Home on» mo COHumHCEHHm omHsmnconHom on omCommoH one 0 CH .omCommou HoHHEHm Hon .nomnoH pmnBoEOm m CH mCHuHsmon HHHIHH zw CH Coxo umom onu on Co>Hm mH mCHCEHum onCHm o m CH .omCommon oxHHunopHsu HHoEm m CH mCHpHCmon H Zm CH Coxm poem onu on Co>Hm mH mCHssHum onCHm m a CH .momHsm conHom CCm onCHm op oHomCE HMCHcCuHmCoH on“ Ho momCommoH HCOHCmnooz .e ousmHm 43 44 An analysis of these responses was difficult because twitches were highly labile, usually becoming too small to measure after one hour of experimentation. A further complication was that twitch responses were subject to rapid and long-lasting fatigue. After only two or three single stimuli to a segmental nerve responses were often completely extinguished, seldom recovering within the duration of the experiment. Consequently all records of the twitch response were taken from previously unstimulated segments and were obtained only during the first hour of experimentation. Experiments were performed to identify which of the proposed axons (fast or Slow) mediates the twitch response described above. To do this mechanical responses were correlated with external electrical responses by simultaneously recording mechanical and electrical activity from the same segment. The threshold for the twitch response always correlated exactly with that of the fast axon; that is, when the stimulus strength was raised just above threshold for the fast axon a single and large negative potential is recorded (Figure 6) along with a small muscle twitch (Figure 7A,B). Raising the stimulus strength above threshold for the slow axon, as indicated by the appearance of a second and later electrical response, contributed no measurable addition to the mechanical response. Thus it 45 appears that the fast axon in each nerve is excitatory and mediates the twitch response of the longitudinal muscle. A quantitative analysis of the time course and amplitude of twitch responses to single stimuli is given in Table 2. Responses to stimulation of the fast axon in SN I were always smaller than those of SN 11-111. In fact, in many segments twitch responses mediated by SN I were too small to be recorded even at maximum transducer sensi- tivities. Stimulation of the fast axon in SN II-III, however, nearly always produced a measurable twitch. The time course of the twitch response to stimu- lation of SN I was nearly identical to that of SN II-III (Table 2). For example the mean onset latency for responses mediated by SN I was 60 msec, as compared to 59 msec for SN II-III. Likewise the times to the peak amplitude of the two responses were nearly identical. From these data it is possible to calculate the excitation—contraction coupling time for the twitch response of the longitudinal muscle. This is done by subtracting the onset latency time for the electrical response of the muscle (about 5 msec) from that of the mechanical response. In the case of SN I a time of 55 msec is obtained; for SN II-III the value is 54 msec. These values represent the time required for excitatory electri- cal events in the longitudinal muscle to bring about a measurable contraction. 46 ZOx< emHoC HopCoEmom nomo CH CoHumHsEHum Coxm umom ou momCommoH mCHCEHumIonCHm mo mHmeHmCdll.N oHnoe 47 Responses to Twin Pulses Stimulation with paired pulses permits a more detailed examination of the characteristics of neuro- muscular junctions than is possible with single stimuli. In particular it is possible to study such phenomena as facilitation and antifacilitation, terms frequently used to describe increases (facilitation) or decreases (anti— facilitation) in the amplitude of successive electrical or mechanical events involved in muscle contraction (Bullock and Horridge, 1965). Thus we may speak of facilitation of a mechanical response when there is an increase in the amplitude of the mechanical responses to successive closely spaced stimuli. Likewise there may be facilitation of electrical responses to successive stimuli. There is however not necessarily a close correlation between electrical and mechanical facilitation since the time courses of the two may differ significantly. External electrical responses.--To study the possi— bility of electrical facilitation of muscle responses the fast and slow axons in the segmental nerves were stimulated using paired pulses. Four preparations were examined, in each preparation the responses to stimulation of SN I being essentially the same as those of SN II-III. Three examples of typical responses to twin-pulse stimulation of SN I are shown in Figure 8. In each record responses to twin pulses just above threshold for the fast Figure 8. 48 External electrical responses to twin-pulse stimulation of SN I. Stimulus intervals are 13 msec (upper), 18 msec (middle), and 27 msec (lower). Each record shows the single-phased muscle responses to fast axon stimulation. In each case the fast axon response to the first stimulus is larger than that to the second. At higher stimulus strengths the slow axon is also stimulated (middle and lower traces), as indi- cated by a second peak superimposed on the fast axon response. The slow axon response to the second stimulus is considerably larger than that to the first, suggesting facilitation. Vertical calibration: 0.5 mV. Horizontal calibration: 10 msec. 50 axon are shown. A large negative potential is recorded in response to the first stimulus, while a much smaller but otherwise similar potential is recorded in response to the second stimulus. In each instance the amplitude of the second response was approximately 60% of the first. This decrement in amplitude of the second response suggests a process of antifacilitation of the response to fast axon stimulation. In contrast to the decrement of responses mediated by the fast axon, responses to slow axon stimulation involve an apparent facilitation. Figure 8 shows two typical examples of this electrical facilitation. In these two cases the stimulus strength, already above threshold for the fast axon, is raised just above threshold for the response mediated by the slow axon. Thus the responses to slow axon stimulation are superimposed on those mediated by the fast axon. Though there is still obvious decrement of the faster response, there is a large increase in the amplitude of the second, slower response. This increase was difficult to quantify but generally the amplitude of the peak appeared to increase two to four times with stimuli spaced 20 to 30 msec apart. These results suggest a facilitation of the response mediated by the slow axon and tend to further substantiate differences in the functioning of the two motor axons. 51 Mechanical responses.-—Although the electrical responses mediated by the fast axon show characteristics of antifacilitation in response to closely spaced stimuli, the mechanical events mediated by the fast axon do not appear to reflect this situation. Instead twitch responses to closely spaced pulses appear to facilitate, facilitation in these cases involving a large increase in the amplitude of the mechanical response to the second stimulus. To examine this mechanical facilitation the fast axon in each nerve was stimulated with paired pulses (15 to 20 msec stimulus interval). Occasionally both fast and slow axons were stimulated, but in these cases there appeared to be no additional contribution to the mechanical response due to slow axon stimulation. Figure 7C,D shows examples of mechanical responses to twin-pulse stimulation of the fast axon in each seg- mental nerve. The individual responses to each stimulus are not apparent because of the close spacing of stimuli. Facilitation is indicated, however, since the amplitude of the response to twin pulses is much more than twice the response to a single stimulus. Figure 9 shows a quantitative comparison of the amplitudes of responses to fast axon stimulation using single and twin pulses. The mean amplitude of the responses to twin-pulse stimulation of SN I was more than three times that of the response to a single stimulus. Likewise the mean response to twin pulses to SN II—III was more than 52 .om H H ouonoCH moCHH HCOHpHo> .o>HoC HmpCosmom nooo CH Coxo ummw onw en CouoHcoE momCommoH HCOHCmnooE Ho ocspHHmEm Coos onu muComonmoH Hon nomm .mCoxm ummm Ho COHHMH ICEHHm omHlecoHHmm CCo IonCHm ou momCommoH HMUHConooE Ho COmHHomEoo .m onsmHm 53 :_I: Zm e. .Zm m oHCmHm :_I__ Zm 405.9%». Hanna 6...??? wwvnuén 4...?th mmmLDm 05:5. --—_—-—-—-——_--- mmmaam 3025 _Zm 5.0. «0.0 mod v0.0 (5) NOISNBJ. )IVBd 54 three times that of the single stimulus response. These results suggest that one important characteristic of the twitch response mediated by the fast axon is the potential for mechanical facilitation. Responses to Repetitive Stimulation Correlation of mechanical and electrical responses. --An examination of muscle responses to repetitive nerve stimulation is particularly valuable in describing the functional characteristics of motor axons in a nerve-muscle preparation. To do this in the earthworm it was necessary to clearly distinguish responses mediated by the slow axon from responses mediated by the fast axon. This is diffi— cult since the thresholds for the two axons are often close. The problem was resolved by simultaneously recording the external electrical and mechanical responses of the longitudinal muscle. Thus it was possible to determine whether one or two axons were stimulated and what type of mechanical response accompanied such stimu- lation. Simultaneous recordings of this type were success- fully made in four preparations with several segments from each preparation being examined. The most suitable range of stimulus frequencies for this study was 5 to 10 Hz. At frequencies higher than 10 Hz thresholds of the two axons became increasingly difficult to separate, and at 55 frequencies below 5 Hz mechanical responses were very small and therefore difficult to analyze. The results from a typical experiment involving stimulation of SN I are shown in Figure 10. In this experiment the fast axon was stimulated at a frequency of 10 Hz and at a stimulus strength just above threshold for the fast axon. The external electrical response to the first stimulus is a large negative potential, identical to the fast axon responses shown in Figure 6. With successive stimuli there is a rapid decrement of electrical responses. At a stimulus frequency of 10 Hz the mean amplitude of the fast axon response to the second stimulus was only 61% of the initial response to the first stimulus (range 50 to 71%, N = 10). By the tenth stimulus the response was only 39% of the initial response (range 31 to 48%, N = 9). With further stimulation responses decreased only slightly and then maintained a stable but low amplitude. A similar pattern of responses was recorded with stimulation of the fast axon in SN II-III. Correlated with these electrical responses to fast axon stimulation, there is a small twitch-like mechanical response which differs only slightly from the twitch response to a single stimulus shown in Figure 7. The particular response shown in Figure 10 is slightly larger and more sustained than responses shown in Figure 7. In most cases, however, the mechanical responses to repetitive stimulation of the fast axon were indistinguishable in 56 .oomE om .cnooou HoBOH “come 0mm .cnooon Homms "CoHpounHHoo HmuCoNHuom .>E m.o .pnooon HosoH um mo.o lcuooon nomad "CoHumHnHHmo HCUHuHo> .HHCEHum o>Hmmouosm nuHB oCCHHHdEo CH momooHOCH noHnB omCommou HMOHHuooHo onu CH noon HouoH .UCooom o CCm .CmoumHm COHmCou mCHmoHo>oU eHonw lomHoH m CH mCHuHCmoH .Coxm Bon onu now cHonwonnu o>ono umsn comHoH mH nmeoHum msH iCEHum onu m #4 .mHmHHCouom oHomsE CH uCoEouoop CHmmH pCo omCommoH oxHHunouHsu HHmEm m CH mCHpHCmoH lCoxo ummH onu How UHonmoan o>ono nmsH eomHmn mH Cumcounm msHsanm one a on .Nm OH pm H 2m Ho eoHumH ICEHHm on omCommoH CH Csonm one Apnooou nosoHv momCOQon HCOHHuooHo HCCHoon UCo Acuooou Hommsv momCommon HMUHCmnooz .mCoxm 30Hm CCo umom Ho CoHuoHCEHHm o>HuHuomoH on momCommoH HooHHuooHo UCo HmoHCmnooz .OH museHC OH OHJU e .m J .E m "coflumnnaamo HMOfluum> .coflumNHumHommp mo mommnm pocflumfio 03» we» >3 ©mumoa©cfl mm .AGOHnm>Hwacfl Hmspv mcoxm 30am Usm ummm anon hp ©0um>umccfl mum 0 ca muwnflw maomsz .d ca mmogu cmnu mmusoo QEHu Hm3on cam hocmpma ummco HmmcoH m o>ms m ca mamauswpom we» muoz .soxm 30Hm we» we cwum>uwccfl mum m CH mmonn paw .coxm ummm map maco wa Umum>uwcsH mum 4 CH mumnfiw OHomsz .HHHIHH Zm on Haseflum mamcflm Op wmsommmu cfl csonm mum muwnflm mHomsa ucmHUMMHU xflm Eoum mwuoomu MOHSHHUOMNHGH .muwnflm maomSE Hmcflpsuflmcoa CH mamflpcwuom oeummcmmumom >u0pmufloxm .mH museum 74 mediated by SN I was 6.0 msec i 0.8 msec SD. The potential developed rapidly, reaching a peak in a mean time of 1.6 t 0.6 msec SD following the onset of the potential. Similar responses were recorded with stimulation of SN II-III, the mean onset latency being 5.6 msec i 0.8 msec SD and the time to the peak being 1.4 msec i 0.7 msec SD. The amplitude of these potentials was quite variable from one cell to another, and much variation was also seen within a cell if single stimuli were delivered every few seconds. The mean amplitude of responses mediated by SN I was 2.3 mV (range 1 to 6 mV) and that for SN II-III was 2.7 mV (range 1 to 10 mV). The threshold for this first type of response was determined by gradually decreasing the stimulus strength and observing responses to single stimuli. In all cases the threshold for this response was sharp and coincided exactly with the threshold of the external electrical response mediated by the fast axon (Figure 6). Thus these muscle fibers appeared to be innervated by the fast excitatory axon. The conduction velocity for the fast axon, as calculated from intracellular records, was 0.32 m/sec (N = 38 for SN I, N = 28 for SN II-III). This compares quite favorably with the values based on extracellular recordings (0.37 m/sec). A second type of postsynaptic potential was recorded in about 10% of all muscle fibers. This response 75 consisted of a single, smooth depolarization which occurred much later and had a slower time course than the response mediated by the fast axon (Figure 158). For stimulation of SN I the mean onset latency of this response was 10.3 msec i 1.2 msec SD and the mean time from the onset of the potential to its peak was 2.3 msec i 0.5 msec SD. Similar responses were obtained with stimulation of SN II-III, the mean onset latency being 10.2 msec i 1.1 msec SD and the mean time to the peak being 3.6 msec i 1.0 msec SD. The amplitude of these potentials was variable from one cell to another. The mean amplitude of the response to stimulation of SN I was 2.3 mV (range 1 to 3 mV), but the responses to stimulation of SN II-III were somewhat larger, the mean amplitude being 3.9 mV (range 2 to 9 mV). Thresholds for these responses were determined by gradually lowering the stimulus strength. In every case the threshold occurred in a sharp, all-or-none fashion and coincided exactly with the threshold of external electrical responses mediated by the slow axon (Figure 6). Thus these muscle fibers appear to be innervated solely by the slow excitatory axon. Conduction velocities for the slow axon, as calcu- lated using measurements from intracellular records, were 0.17 m/sec for SN I and 0.18 m/sec for SN II-III, about one-half the velocity for the fast axon. These values are quite comparable to the corresponding values based on 76 extracellular recordings (0.16 m/sec). All these calcu- lations of conduction velocities do not take into consider- ation the possibility of synaptic delay. Thus true conduction velocities for the fast and slow axons are probably slightly greater than those which are given. A third type of response was recorded from about 20% of all muscle fibers. This response differed from the other two in that it consisted of two distinct phases of depolarization (Figure 15C). The first phase of the response was a rapid and smooth depolarization closely resembling the fast axon response shown in Figure 15A. The second phase was a slower and later depolarization closely resembling the slow axon response shown in Figure 15B. The occurrence of this two-phased response suggested that it is composed of two different excitatory postsynaptic potentials superimposed on one another. This idea was substantiated by determining the thresholds for the potential. In nearly all cases thresholds for the two phases of the response were clearly separable, and these thresholds coincided exactly with those of extracellular responses mediated by the fast and slow axon. Thus these muscle fibers appear to receive dual excitatory innervation, that is, innervation from both fast and slow axons. In the remaining 30% of all muscle fibers no measurable postsynaptic responses to nerve stimulation 77 were recorded. These fibers did not appear to be damaged since resting potentials in these fibers were not signifi- cantly different from resting potentials in innervated fibers. These results suggest that such muscle fibers were not innervated by the nerve being tested and perhaps are innervated by another segmental nerve. In summary, an analysis of intracellular responses to segmental nerve stimulation provides further evidence for the presence of two functionally different excitatory axons in each segmental nerve (SN I and SN II-III). Some muscle fibers appear to be innervated by only one or the other of these axons but a significant percentage of muscle fibers are innervated by both of these axons, thus indi- cating dual excitatory innervation. A quantitative analysis of the pattern of innervation of longitudinal fibers is given in Figure 16. These figures represent results obtained from only one specific region of the longitudinal muscle, and thus may not reflect the pattern of innervation for the entire muscle layer. Mapping of Motor Fields Prosser (1935) first studied motor fields in earthworm longitudinal muscle by viewing the spatial limits of muscle contractions in response to nerve stimu- lation. Based on his Observations he suggested that the motor fields for one segmental nerve may extend into 78 .m>umc Hmucmemmm may Scum usage muoumuwoxm ucmummmm on pm>flmomn mumnflm waomse mEOm .ACOHum>umccH Hmspv mcoxm 3Oam tam ummm anon an pmum>uwccH was mmmucmoumm ummHmH umszmeom m can .OCOHm coxm wuoumufloxm 30am mnu an pmum>uwccfl mmz mnmnflm mHomsa mo mmwucmoumm HHmEm m .coEEOO umoE mmz coxm muoumufloxm ummm on» an coflum> Imogen mm>nmc nuon nom .muwnflw OHOmDE om mo meEmm m mucwmmummn nmmum roam .HHHIHH zm on Hmumumfl uwnuo may cam H 2m ou Hmumuma coeumasmom mco .mnwnww OHomsE mo mcofiumHsmom 03¢ cw coflumHszum w>umc HmucmEmmm Op mwmcommwn maomse umHsHHmomuucH mcHHmEMm an pmcfimuno mums mpHSmmn one .mnmnflm OHOmSE Hmcflpsuflocoa mo mcumuumm coeum>nmccH .mH musmflm ___I__ _¢mw 0V SH museum $838k! 50 % IhOm 304m ._.m .qz> lmcflapflfi HmmHOp .Aza .Aummmsv mmnaa msqflano map an pmumoapcfl mcflmn mmHHQ>O mo mmum men .Hmnuocm mco mmaum>o mpcmEmmm mcflnonnmflmc ca mm>umc unconnpm “Om mpamflm on» Omam .Aumzoav mmcfla msqflano man an pODMOHch mcflmn awaum>o mo mmum wen .nmnuocm mco mmanm>o ucmEmmm QEMm onu ca mm>nmc “snowmpm How mpamew Use .mamapcmpom OHOmSE accumuxm mcwmmma an pmcfienmump mums .mmcfla pmuuop an powwowpcfl .waomsfi HmcflpsuamcoH may now mpamflm uouoE mo mmflumpcson one .pnoo m>umc Hwnucm> may Eoum mmflnm noflnz mm>umc Hmucwfimmm men can HOMMH maomsfi chflpsuflchH on» mcfisonm ammo Gama we Euo3nuumm on» no Hams moon one .mw>nmc Hmucwfimmm accommpm mo mpamflm Houoe mcflmmmaum>o .mH musmflm 86 ms musmflm A \ I I k I I _ N a .......................... mm m ............................ F. 2m §§§ NI... .. z... I.“ ......................... um. N M N .......................... M. m... w §S§S§%fli z. w, .......................... I...” IN \ Huh 420 \Iullm f MID mm} 87 of the fields of adjacent nerves. Such overlap is both intrasegmental and intersegmental. The apparent overlap of motor fields of adjacent segmental nerves may be explained in two ways. First, there may be coincidental and overlapping excitatory innervation of muscle fibers from different segmental nerves. This explanation seems likely from a physical standpoint, since Hanson (1957) has shown that longitudinal muscle fibers may be two or three times longer than a single segment. An alternative explanation is that muscle fibers are not really innervated by two different nerves, but instead branches of motor axons from one nerve inter- digitate with branches of adjacent nerves, thus giving only the appearance of overlapping motor fields. Intracellular recordings.--In the following experi- ments I have used intracellular recordings to study the possibility of coincidental and overlapping innervation of muscle fibers by different segmental nerves. To study the possibility of overlapping innervation between segmental nerves in the same segment (intrasegmental overlap), recordings were made approximately midway between the septal boundaries of a segment. To study overlap between seg- mental nerves of different segments (intersegmental over- lap), recordings were made near the septal boundary between the adjacent segmental nerves. Segmental nerves 88 were stimulated individually using supramaximal stimulus intensities and a low frequency of stimulation (5 Hz). Four preparations were examined with similar results obtained from each preparation. In each prepa- ration muscle fibers were commonly found which received innervation from both SN I and SN II-III in the same segment as shown in Figure 19. The response to stimu- lation of SN I at 5 Hz (upper trace) consists of a series of excitatory postsynaptic potentials ranging from 1 to 4 mV in amplitude. A slight summation of the potentials is apparent. The response of the same muscle fiber to stimulation of SN II-III (lower trace) consists of a similar series of excitatory postsynaptic potentials ranging from 2 to 5 mV in amplitude. In addition to summation of the potentials there is also a slight facili- tation. Similar results were obtained when testing for intersegmental overlap of motor fields; that is, excitatory postsynaptic potentials were recorded from muscle fibers following stimulation of either of two adjacent segmental nerves in two different segments. These experiments lend further support to the idea of overlap Of motor fields for adjacent segmental nerves. 89 .omw m.o "COHHMHQHHmo HmucoNHnom .>E m "coHumunHHmo HMOHuHm> .ucmfimmm mEmm may cH mm>umc Hmucmammm ucouwmep 039 an HOQHH mHomSE HmcHtsuHmcoH m Ho COHum>HmccH muoumuHoxw pwmmmSm muHsmmu one .mHmHchuom mcHumEESm mo moHHom m 2H mpHSme ome Nu m pm HHHIHH Zm mo coHumHDEHuw m>Hqumme pucomu HoBOH map CH .mHmHucmuom UHHQMQMm Tumom mcHumEESm mo wwHHmm m :H mHHSmmH um m um H Zm Ho coHumHsEHum puoomn Homes mgp cH .HcmEmmm mEmm on» CH mm>nmc Hmucwfimwm mo coHumH IsEHum O>HuHummon HmEmeEmHQSm mcH3oHHOw HOQHH mHomSE HmcH©5pHmcoH mEmm may cH >HH>Huom HMHDHHwomuusH 30cm mpnoowu npom .mw>nwc HmpcmEmwm ucwummme oBu Eoum HmnHm mHowsE m on musmcH muoumuHoxm .aH mnsmHm mH mucm DISCUSSION Activity of Nerve and Muscle in Various Salines The functional properties of the neuromuscular systems of animals are frequently studied by using isolated nerve-muscle preparations and a suitable physiological saline solution. In general suitable salines are obtained by determining the concentrations of major ions in an animal's body fluids and duplicating these concentrations in the form of a saline solution. Any alteration in the concentrations of major ions often leads to malfunctioning or rapid deterioration of the preparation (Lockwood, 1961). In the case of the earthworm, analyses of the ionic composition of body fluids have been carried out only recently. From these analyses it is obvious that the ionic composition of earthworm body fluids does not coincide with the ionic composition of commonly used earthworm salines, such as that of Pantin (1946). There are considerable differences in the concentrations of major ions, such as + ++ Na , Ca , and Cl-. It is these differences in ionic composition which may result in the observed deterioration 91 92 and malfunctioning of earthworm nerve-muscle preparations bathed in Pantin's saline. Although the present investigation has not clearly established which ions are most critical in maintaining the normal functioning of earthworm nerve and muscle, there is reason to believe that calcium ions may be particularly important in accounting for the abnormal and normal functioning observed in the two salines. It is known, for example, that the concentration of external calcium determines the excitability, or polari- zability, of nervous tissue (Brink, 1954). This effect has been demonstrated in both vertebrate and invertebrate nerve preparations. The excitability of frog nerve, for example, increases in a low-calcium saline and at concentrations less than 0.3 mM alpha fibers in the sciatic nerve may become spontaneously active (Brink 35 21., 1946). In lobster axons similar increases in excitability and spontaneous discharging are observed in calcium-free solutions (Adelman, 1956; Adelman and Adams, 1959). Also Rathmayer (1969) using a calcium-free saline observed prolonged repetitive firing in spider motor axons following a single nerve stimulus. Such repetitive firing suggests that a lack of calcium brings about hyperexcitability of motor axons. The general influence of calcium on nerve excita- bility may also explain the hyperexcitability and repetitive firing of earthworm motor axons bathed in Pantin's saline. 93 Such repetitive firing was suggested by Drewes and Pax (1971) and has been clearly substantiated in the present study. The use of an appropriate saline with a higher calcium concentration appears to restore the normal excitability of motor axons. There is evidence that calcium is not only critical in the functioning of nerve, but also may play an important role in the functioning of muscle. For example, Kuffler (1944) found a potential difference between two ends of frog sartorius muscle, one end bathed in calcium-free Ringer's solution and the other in normal Ringer's solution. The end of muscle in the calcium-free bath was 8 mV negative with respect to the other end. This difference was inter- preted as a depolarization of muscle fibers induced by low calcium. Accompanying this was a tendency for spontaneous activity in muscle fibers, thus suggesting an increase in excitability of muscle membrane. More substantial evidence for such an effect has been shown by Kobayashi (1972) in molluscan muscle. Using the radular protractor muscle of Rapana thomasiana, Kobayashi showed that removal Of external calcium brings about depolarization and spontaneous firing of muscle fibers. In earthworms there appears to be a similar dependence of the muscle membrane potential on the con- centration of external calcium as shown by Hidaka EE.E$' (1969a) and Chang (1969). In these studies the longitudinal 94 muscle of two species of the earthworm, Pheretima, were used along with an earthworm saline which is essentially identical to that of Pantin (1946). Their values for resting potentials of longitudinal muscle fibers are nearly identical to those I have measured in Lumbricus longi- tudinal muscle bathed in Pantin's saline. The membrane potential in the longitudinal muscle of Pheretima is dependent upon concentrations of calcium, sodium and potassium (Hidaka gt $1., 1969a; Chang, 1969). Slight increases in external calcium, for example, cause a hyperpolarization of muscle fibers, while slight decreases cause a depolarization. Depolarization is also brought about by increases in external potassium or sodium. Using the figures of Hidaka and Chang, which show the dependence of the membrane potential on each ionic species, it is possible to roughly predict the membrane potential if ion concentrations were changed to those in the new saline I have developed. In the case of each ionic species a hyperpolarization would be predicted, changes of 8 to 12 mV being predicted following the appropriate changes in calcium and sodium. These predictions coincide well with the mean resting potential I have measured in Lumbricus longitudinal muscle using the new earthworm saline. The relatively large resting potential of muscle fibers bathed in the new saline may be the basis for the decreased tendency for injury spiking seen in the new saline. A greater resting potential, controlled in part 95 by the relatively high calcium concentration, could produce increased membrane stability. Thus any disruption of the muscle membrane, such as penetration with a microelectrode, would be less likely to cause repetitive spiking due to injury. In addition to controlling the excitability of nerve and muscle membranes, calcium also plays a role in controlling events in synaptic transmission, particularly the release of chemical transmitter (Brink, 1954). Using the frog nerve-muscle preparation Kuffler (1944) observed significant reduction in externally recorded end-plate potentials following exposure to low-calcium saline. This was attributed either to decreased transmitter output or to decreased sensitivity of the postsynaptic membrane to the transmitter. The study of Del Castillo and Stark (1952), however, clearly showed the effect of calcium on the end-plate potential was due primarily to decreased transmitter output. A similar dependence of transmitter output on calcium ions has been demonstrated in invertebrate muscle preparations (Bracho and Orkland, 1970; Ortiz and Bracho, 1972). Using a crayfish nerve-muscle preparation it was shown that increases in external calcium resulted in an increase in the average number of quanta of excitatory transmitter released and in an increase in the amplitude of excitatory postsynaptic potentials. Accompanying these 96 changes was an increase in the effective resistance of the muscle membrane, but this change in resistance could only account in small part for the observed increase in the amplitude of postsynaptic potentials (Ortiz and Bracho, 1972). In annelid muscle there appears to be a similar relationship between external calcium concentrations and transmitter output. Stuart (1970) reported a large in- crease in the amplitude of excitatory postsynaptic potentials in leech muscle following increases in external calcium. In the present study I have shown that the size of excitatory postsynaptic potentials in earthworm muscle is greatly reduced when exposed to Pantin's saline. It may be that the relatively low calcium concentration in the saline results in a decrease in excitatory transmitter output, and thus a decrease in the size of excitatory potentials. In summary, the results of the present study indicate the importance of using an appropriate earthworm saline in studying nerve-muscle relationships in the earthworm. It has been shown that an earthworm saline commonly used in previous investigations of earthworm neuromuscular systems is inappropriate for such studies. This suggests that previous investigations of earthworm muscle physiology are subject to re-interpretation. 97 Patterns of Innervation In earthworm longitudinal muscle there appear to be two functionally distinct excitatory systems, a fast system mediating a rapid twitch-like response and a slow system mediating a more slowly developing and sustained response. Each of these responses appears to be mediated by one excitatory axon of a single type, a fast axon mediating the rapid response and a slow axon mediating the sustained response. Each segmental nerve, SN I or SN II-III, contains only one fast and one slow axon which innervate the longitudinal muscle. In many ways this functional differentiation of motor axons resembles that seen in other annelids as well as other invertebrate phyla. Fast Motor Systems The fast system of earthworms resembles that seen in other annelids, particularly nereid polychaetes. Horridge (1959) and Wilson (1960a) have shown a fast response in the longitudinal muscle of Nereis, the response being mediated by a single axon in SN IV. Stimulation of this axon results in a large external muscle potential which shows characteristics of antifacilitation. Thus the properties of the polychaete fast system are similar to those I have described for the earthworm fast system. A major difference is that in the earthworm each side of a segment is innervated by two fast axons rather than one as in Nereis. 98 The fast motor system of earthworms is also comparable to the fast systems in other invertebrates. For example, Prosser and Melton (1954) have demonstrated a fast response in the proboscis retractor muscle of sipunculid worms. This response had a single low threshold, indicating innervation by a single axon, and showed rapid antifacilitation. In certain molluscs, fast systems have also been found. Wilson (1960b) demonstrated a fast antifacilitating response to stellar nerve stimulation in the mantle muscle of cephalopods, Loligo and Octopus. Such responses had little or no refractory period indicating local potentials rather than spiking activity in the muscle. In Loligo the fast mechanical responses summated considerably with repetitive stimulation, but in Octopus no summation was evident indicating a tetanus to twitch ratio of about one to one. Fast motor systems have been extensively studied in arthropods, particularly in the insects and crustaceans (c.f. Usherwood, 1967, and Atwood, 1967, for reviews). In these groups motor systems have reached a high degree of specialization and diversity in both structure and function. This diversity is reflected in the wide range of mechanical and electrical responses obtained with fast axon stimu- lation. For example in the claw closer muscle of the rock lobster, Panulirus, mechanical responses to fast axon 99 stimulation show considerable summation and facilitation, appreciable contraction occurring only at frequencies of stimulation above 10 Hz. In contrast, the claw closer of the crayfish gives strong twitch responses to single stimuli, but repetitive stimulation causes rapid fatigue (Hoyle and Wiersma, 1958). Likewise there is considerable variation in the types of intracellular electrical responses to fast axon stimulation. In some cases electrical responses to fast axon stimulation, although producing a large twitch, consist of graded excitatory postsynaptic potentials as in the claw closer of the crayfish (Hoyle and Wiersma, 1958). In others, such as the grasshopper metathoracic extensor tibiae, responses consist of overshooting spikes (Cerf gt 21., 1959). In some arthropod muscles differentiation of motor axons is such that classification into fast and slow types is difficult. For example in the main flexor muscle of the rock lobster the properties of several motor axons appear to be intermediate between those of fast and slow axons (Hoyle and Wiersma, 1958). The fast system of earthworm longitudinal muscle differs from the fast systems seen in most arthropods. Generally fast systems in arthropods are relatively stable signs of fatigue occurring only after prolonged repetitive stimulation. Also in some arthropods fast responses to repetitive stimulation may summate and facilitate, reaching 100 a large and distinct tension plateau (tetanus). In contrast the fast response of earthworm longitudinal muscle is highly labile, complete mechanical fatigue of the response appearing after only a few stimuli. This rapid fatigue is accompanied by a significant decline in the amplitude of external muscle potentials. The weak mechanical responses to fast axon stimu- lation which I have obtained in these experiments suggest that relatively few muscle fibers produce such responses. Visual observations also indicate these responses are localized appearing to involve only muscle fibers in the ventrolateral region of the segment. However experiments involving intracellular recordings and mapping of motor fields suggest that many muscle fibers in a segment are innervated by the fast axon. Therefore many muscle fibers, though innervated by the fast axon, do not contribute to the mechanical response. It is possible, however, that such fibers respond mechanically to several closely spaced stimuli in the fast axon or to simultaneous stimulation of fast axons in two segmental nerves innervating the same muscle fibers. Alternatively the lack of responsiveness to a single stimulus may indicate that fast responses were partially fatigued due to dissection of the animal. From the results it is not clear what type of intracellular response is required to initiate the twitch response. Such a response in a muscle fiber could be initiated by either a large postsynaptic potential or by 101 an active membrane response (spike). Furthermore it is possible that the level of depolarization necessary for contraction may vary from one cell to another, a situation seen in many arthropods (Atwood, 1967). Slow Motor Systems The slow motor system in earthworm longitudinal muscle closely resembles the slow systems found in other annelids. In nereid polychaetes, for example, Wilson (1960a) has demonstrated a slow electrical response of the longitudinal muscle following stimulation of SN IV. This response is smaller than the fast response, occurs after a longer latency, and shows summating and facilitating characteristics with repetitive stimulation. These characteristics are essentially identical to the electrical characteristics of the slow muscle responses I have identified in the earthworm. The slow system of the earthworm is also somewhat similar to that of the leech longitudinal muscle. In the leech responses to stimulation of slow axons consist of summating and facilitating excitatory postsynaptic po- tentials which give rise to slow and summating mechanical contractions (Stuart, 1970). Similar slow systems are seen in the muscle of other invertebrate phyla such as the sipunculid proboscis retractor muscle (Prosser and Melton, 1954; Prosser and Sperelakis, 1959) and molluscan mantle muscle (Wilson, 102 1960b). In these instances slow responses are character— ized by facilitating electrical responses and slowly developing mechanical responses to repetitive nerve stimu- lation. Slow motor systems are also common in arthropods, having been extensively studied in crustaceans and insects. In these groups specialization and diversity of motor systems is so extensive that clear distinctions between fast and slow systems are not always apparent. Neverthe— less, it is possible to make a few generalizations regarding the functioning of arthropod slow systems. In general, slow responses of the muscle are mediated by one or a few slow axons. Intracellular responses to slow axon stimu- lation usually consist of small excitatory postsynaptic potentials, often a few millivolts or less in amplitude. These potentials summate and facilitate with repetitive stimulation, the resulting depolarization giving rise to a slowly developing mechanical response. The amplitude of this response is dependent on the frequency of stimulation, frequencies up to 100 Hz or more being required for a maximal response in some muscles (Hoyle and Wiersma, 1958). The slow responses of earthworm muscle are similar to those of arthropods. In the earthworm longitudinal muscle, as in many arthropod muscles, the slow response is mediated by only a few axons. Also the amplitude of the slow response is clearly dependent on the frequency of 103 stimulation, frequencies as low as 2 Hz giving responses which are just measurable and frequencies of 20 to 50 Hz giving maximal responses. Inhibition No evidence for peripheral inhibition in the longitudinal muscle of the earthworm could be found. In 'nO cases were threshold-dependent decreases in mechanical or electrical responses of the longitudinal muscle observed. Thus the innervation pattern of earthworm muscle appears similar to that in nereid polychaetes in which fast and slow, but not inhibitory, axons have been demonstrated (Wilson, 1960a). This lack of inhibition is in contrast to the apparent presence of peripheral inhibitory systems in other annelids. For example Hidaka 3E.al. (1969c) and Ito gt_al. (1969a) have recorded spontaneous inhibitory postsynaptic potentials from longitudinal muscle fibers in the earth- worm, Pheretima communissima. Also Stuart (1969, 1970) has clearly demonstrated postsynaptic inhibition in the longi- tudinal muscle of the leech. The results of the present study, though giving no support for inhibition, do not exclude the possibility that peripheral inhibition exists in the longitudinal muscle. It is possible, for example, that inhibitory axons exist in the segmental nerves but thresholds of these axons are not clearly separable from the thresholds of excitatory 104 axons. In this situation stimulation of a segmental nerve might simultaneously excite both inhibitory and excitatory axons, with the net result still being a depolarization in the muscle. An alternative possibility is that a pre- synaptic inhibitory mechanism is involved. With such a mechanism no inhibitory postsynaptic potentials would be seen. Functional Significance of Innervation Patterns Fast and Slow Systems In the earthworm there are two well-known and obvious locomotor activities involving the longitudinal muscle. The first is the rapid escape response character- istic of many oligochaetes and polychaetes. The response is known to consist of a powerful multi-segmental and twitch-like contraction of the longitudinal muscle. The central components of the reflex pathway of the rapid response are the giant fibers (Stough, 1930; Rushton, 1945, 1946; Roberts, 1962a). In the earthworm, giant fibers make functional contact with several motor neurons in each segment and at least some Of these motor neurons appear to innervate the longitudinal muscle (Gunther, 1972). It is possible that the axons arising from these motor neurons correspond to the fast axons I have identified in each segmental nerve. Also it is possible that the activation 105 of these fast axons by the rapidly conducting giant fibers may result in a rapid and synchronous shortening of the animal. Another obvious locomotor activity in earthworms is the slow peristaltic movements of the worm observed during crawling or burrowing. These movements actually involve successive retrograde waves of circular and longitudinal muscle contractions (Gray and Lissman, 1938; Chapman, 1950). In any one segment there is a reciprocal relation- ship between the timing of circular and longitudinal muscle contractions (Seymour, 1969, 1970). Events occurring in the central nervous system during peristaltic contractions are not well understood. Roberts (1967) has identified a slow conducting pathway in the ventral nerve cord which seems to be multisynaptic in nature and which may represent the central pathway for slow peristaltic movements of the animal. The slow and sustained longitudinal muscle con- tractions seen during peristaltic movements are comparable to the contractions I have recorded in response to slow axon stimulation. Perhaps the slow axons may be important in mediating these locomotor movements. If this were the case, then sequential bursts of activity in the slow axons of successive segmental nerves could bring about the wave- like contraction of the worm. 106 Motor Fields The body wall musculature of the earthworm is not organized into distinct segmental bundles of muscle fibers, but instead is organized into continuous layers or sheets of muscle. Because of this particular organization it is often useful to describe patterns of muscle innervation in terms of motor fields or territories. The mapping of motor fields for individual segmental nerves permits a better understanding of the functional significance of these nerve elements in locomotion. Motor fields have been described in only two anne— lids, the leech (Stuart, 1970) and the earthworm (present study). In the leech Stuart described the motor fields for excitatory axons innervating the longitudinal muscle. To do this individual excitatory motor neurons in the seg- mental ganglia were stimulated using intracellular electrodes. The regions of longitudinal muscle which contracted during this stimulation were observed. The innervation of the contracting region was confirmed by intracellular records of muscle fiber activity. Stuart found that the motor fields for each excitatory axon in a segment were different. One ex- citatory axon, arising from the "large longitudinal motoneurone,‘ innervates the entire lateral half of a segment. Since there is electrotonic coupling between the two "large motoneurones" in each segment, Stuart suggests that their axons function to bring about a uniform 107 shortening of the animal. Such a response, she maintains, may occur when the animal withdraws from a noxious stimulus. In contrast the motor fields of the other five excitatory axons innervating the longitudinal muscle are much smaller. These fields subdivide the body wall into small strips or patches of muscle. She suggests that contraction of any one of these strips would cause a bending of the body. The co-ordinated action of these small motor fields may account for the complex bending and twisting movements of the animal. The motor fields in the earthworm longitudinal muscle are somewhat different than those of the leech. In the earthworm motor fields are large, fields of both fast and slow axons extending from dorsal to ventral midlines. The arrangement of these motor fields would probably not permit a wide variety of twisting and bending movements, such as one sees in the leech. However this arrangement would appear to be well-suited to locomotor activites requiring uniform and symmetrical contractions of the segmental musculature. Examples of such movements would be the peristaltic forward locomotion of the worm or the rapid escape response. The only prerequisite for such responses is the synchronous output of right and left sides of the animal. The absence of output from either side would provide the possibility of turning movements. 108 Phylogenetic Considerations An important trend in the phylogeny of invertebrate nervous systems has been the concentration or centralization of diffuse and unspecialized nervous systems, such as nerve nets or nerve plexuses, into distinct and specialized central nervous systems. This centralization has been accomplished by a decrease in the autonomy of the peripheral nervous system. The first significant degree of bilateral cephalization in nervous systems is seen in the free- living flatworms, which possess a distinct central nervous system and a complicated submuscular nerve plexus (Hyman, 1951). Arthropods on the other hand represent one of the highest degrees of centralization in invertebrates, as indicated by the replacement of peripheral nerve plexuses with sensory and motor nerve tracts. Phylogenetically the annelids would appear to fill a position somewhere between the flatworms and arthropods, and thus might be expected to have nervous features common to each group. This idea is supported by several lines of evidence. Most annelids possess nervous elements reminiscent of primitive as well as advanced nervous systems. For example, in addition to a central nervous system with distinct nerve tracts, many annelids possess a complex subepidermal nerve plexus (Stephenson, 1930). On the basis of anatomical investigations the nerve plexus has been considered to be a continuous nerve net (Hess, l925a,b; 109 Smallwood, 1926), although physiological evidence does not support this idea (Janzen, 1931). A possible role of the plexus in peripheral reflex responses of muscle to photic stimulation is suggested by Prosser (1946). In the present investigation an explanation of nerve-muscle relationships is given which requires no motor pathways other than direct ones from motor axons to muscle. In this respect the earthworm neuromuscular system is similar to that of arthropods. The finding that relatively few motor axons innervate each segmental block of earthworm muscle is also a situation found in many arthropods. A further similarity is indicated by the functional differ- entiation of annelid and arthropod motor systems into fast and slow types. S UMMARY The neuromuscular physiology of earthworm longi- tudinal muscle has been examined using a new physiological saline and a suitable nerve-muscle preparation. Stimulation of individual segmental nerves gives two functionally different responses of the longi- tudinal muscle: a rapid twitch-like contraction to a single stimulus, this response being mediated by a fast excitatory axon; and a slowly developing and sustained response to repetitive stimulation, this response being mediated by a slow excitatory axon . The longitudinal musculature of a segment is innervated by relatively few axons, a fast and a slow axon being present in segmental nerve I and in the double nerve, segmental nerve II-III. Electrical and mechanical responses to stimulation of the fast axon show characteristics of anti— facilitation, while responses to slow axon stimu- lation show summation and facilitation. 110 111 Individual longitudinal muscle fibers are inner- vated by one or both excitatory axons in a seg- mental nerve, thus indicating the presence of dual motor innervation. The motor fields of adjacent segmental nerves overlap one another, such overlap occurring between adjacent nerves in the same segment or between adjacent nerves of two neighboring segments. LITERATURE CITED LITERATURE CITED Adelman, W. J. 1956. The effect of external calcium and magnesium depletion on single nerve fibers. J. Gen. Physiol. 22:753-772. Adelman, W. J., and J. Adams. 1959. Effects of calcium lack on action potential of motor axons of the lobster limb. J. Gen. Physiol. 42:655-664. Atwood, H. L. 1967. Crustacean neuromuscular mechanisms. Am. Zool. 1:527-551. Bracho, H., and R. K. Orkland. 1970. 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