The literature dealing with the peripheral components of the nervous system of the earthworm has been reviewed recently by Stephenson (1930) and Prosser (19346). Hence it is necessary only to mention the principal elements. The sensory endorgans are said to consist of (1) a variety of epidermal cells (“sensory cells”) which have peripheral terminations, are of different shapes, and may be grouped as “sense organs” or may be isolated, (2) free nerve endings, some of which are closely associated with the glandular cells, and (3) intermuscular cells. Certain of the epidermal sensory cells have been shown to be photosensitive; the function of the others is uncertain. Most of them are connected to a subepidermal plexus which appears to be a true network of anastomosing fibres and to contain nerve cells. In each segment three pairs of nerves lead from the central ganglion to nerve rings from which fibres are distributed either by nerve branches or by the peripheral network to the muscles and sensory cells. There is some evidence for and some against conduction of locomotor impulses by the peripheral plexus across segments lacking the nerve cord. Prosser (19346) concluded that the contrary evidence is slightly stronger.
To investigate the nature of some of the peripheral nervous elements in the earthworm, action potentials in the segmental nerves of Lumbricus terrestris were studied by means of an amplifier, loud-speaker and Matthews oscillograph. Small silver hooks, coated with AgCl2, were used for leads, the wires, 1 mm. apart, being supported and insulated by sealing wax.
A worm was pinned to a block of paraffin wax, ventral side up; a ventral incision was made and the nerve cord and segmental nerves exposed for several segments with as little injury to the circulation as possible. The nerves were mounted on the electrodes with the aid of a binocular dissecting microscope. Coelomic fluid continually seeped in around the electrodes. This kept the nerves moist, but it was so abundant that it was necessary to remove it at intervals with filter paper to prevent short-circuiting.
The nerves are small and very fragile, and when a little excess tension or traction is applied, they usually die. They die also if the contents of the intestine come into contact with them. If, however, they are not seriously disturbed they continue to conduct impulses when resting on the wire leads for two or three hours. When cut or crushed at one end they often fail to conduct after a few minutes. Hence most of the observations were made with the nerves in continuity with the nerve cord at one end and the muscles at the other. This has the theoretical disadvantage of allowing muscle action currents to be picked up by the electrodes on the nerve, but numerous control experiments showed that the impulses observed were also present when the nerve was isolated electrically at one end from all surrounding tissue. Further, when a thread, tucked under the musculature at both ends, passed over the electrodes no deflections were observed except occasional very slight deflections when the muscles immediately in contact with the thread were active; these were not at all comparable in size to the nerve impulses. Also no comparable electrical changes were found in a dead nerve with both ends in contact with the body and with active muscle near it. It seems certain, therefore, that few if any muscle action currents were picked up by the leads.
Observations were made in several segments of each worm, usually in the segments anterior to the clitellum. Records have been obtained from approximately 120 worms.
SENSORY IMPULSES IN SEGMENTAL NERVES
Typical records of sensory impulses in a segmental nerve are given in Fig. 1. Afferent impulses have been observed in response to proprioceptive, tactile, chemical, and photic stimulation, the efferent impulses being eliminated by crushing or cutting the nerves centrally.
When a worm was spontaneously active or when tension receptors were stimulated by pushing or pulling on the epithelium with a glass needle, impulses such as those shown in Figs. 1 A and 6 were observed. These were slow, of very large potentials, 30–50 microvolts as recorded. Frequently it was possible to observe rhythmic discharges, presumably in single units. The maximum frequency observed in these rhythmic discharges was 18 per second. These impulses had the same general size and shape whether the stimulated muscle was on the extreme dorsal side, far from the electrodes, or was near the electrodes.
The impulses observed in response to a light touch on the epidermis with a fine glass needle were, as shown in Figs. 1 B and 3, more varied in size, some reaching the potentials of the responses to proprioceptive stimulation. The number of impulses increased with increasing area of stimulation. For example, in one experiment when an area of 0·01 mm.2 was stimulated by a touch, three or four impulses were observed, and when an area twice that in size was touched, four or five impulses were observed in the same nerve. The tactile endings became adapted readily, and the response to a single touch in which numerous endings were stimulated was a brief burst of asynchronous impulses. There were more impulses in such a response in the anterior segments for a given area of stimulation than in the segments in the middle of the body.
Velocities of conduction of some of the impulses in proprioceptive and tactile responses were calculated by measuring the time to the beginning of the second phase in a diphasic impulse. This method involves a considerable error with electrodes so close together (Hill, 1934), but it is the only one which could be employed with such a short nerve. The measurements give velocities from 30 to 100 mm. per second, the majority falling between 40 and 80 mm. per second. It is difficult to distinguish between two summated impulses and one very slow impulse; similarly there are some very fast impulses in which the beginning of the second phase cannot be detected. Hence there are probably some impulses which fall outside the limits given, but certainly the majority travel at less than 10 cm. per second. These rates are comparable to the rate of 2·5 cm. per second observed by Bovard (1915) in fibres other than giant fibres in the nerve cord.
Responses to HC1, NaOH, and NaCl applied to the epithelium were observed. None was obtained with sucrose. When a drop of a solution of one of the three electrolytes was placed on the epithelium, the impulses observed were, as shown in Fig. 1 C and D, smaller than those in response to proprioceptive and tactile stimulation, the largest being 10–15 microvolts. Occasionally movements of the muscles were induced by the stimulation and the much larger proprioceptive impulses appeared. HC1 N/50 elicited a very slight response, N/20 a good response, and N/10 killed all epidermal sensory endings. NaOH N/20 elicited a very small response, N/10 a good response.
When an earthworm which had been in darkness for at least half an hour was illuminated, impulses in a segmental nerve increased in number, slowly at first and more rapidly after approximately one second illumination. A maximum number was usually reached in two to four seconds. These impulses, as shown in Figs, 1 F and 2, were of very small potential, approximately 5 microvolts as recorded. After several seconds’ illumination the worm started to contract and many large proprioceptive impulses completely obliterated the small ones which constituted the direct response to illumination. To rule out the possibility of stimulation of the nerve by the action of light on the silver chloride the electrodes were shaded; this did not affect the result. The possibility that heat might be involved was ruled out by the fact that holding a hot soldering iron a few millimetres from the worm caused no noticeable response. Furthermore, long periods of dark adaptation were required to obtain the response to light. For example, in one specimen which had been illuminated during other observations for one hour no response was obtained to illumination after fifteen minutes in darkness, a small response after thirty minutes in darkness, and a good response after one hour in darkness. No quantitative study of this response was made, but there was definitely a larger response with illumination of higher than of lower intensities. The nature of this response to illumination is similar to that obtained in the photosensitive caudal ganglion of the crayfish (Prosser, 1934 a).
DISTRIBUTION OF SENSORY FIBRES
When one segmental nerve was on the electrodes, responses were obtained when tactile, proprioceptive, or chemical stimuli were applied to the same segment as that of the nerve and to the segments immediately anterior and posterior to it. Fig. 3 shows responses in one nerve to tactile stimulation in three adjacent segments. The area from which a response could be elicited was strictly homolateral, stopping at the midline dorsally and ventrally. Localised stimulation gave fewer impulses from adjacent segments than from the segment from which the nerve was derived.
Moreover, when the stimulation was confined to very small areas by touching with a fine needle, 0·12 mm. in diameter, there were small areas from which no response was obtained. The typical sensory fields of three segmental nerves are indicated in Fig. 4 as drawn after mapping the sensitive areas. The first segmental nerve received impulses from most of the homolateral epithelium of the same segment, a large part of the segment anterior to it and from small areas of the segment behind. The second segmental nerve received impulses from most parts of the segment innervated and had approximately equal silent areas in the two adjacent segments. The third segmental nerve usually received impulses from most parts of the same segment and the one posterior to it, but from only local areas of the segment ahead. In general, there were more silent spots in the middle than in the ventral and dorsal regions of a segment. Thus there was considerable overlap within a segment as well as from segment to segment. Much variation was observed from segment to segment and in different preparations, but a given sensory field remained constant, at least for several hours. Sometimes the response in one nerve was limited to two segments, sometimes responses were obtained from a small area on a fourth, but usually the three segmental rule applied.
When a longitudinal (horizontal) cut was made through the epidermis to and sometimes through the circular muscles, all responses to stimulation in the area dorsal to the cut were lost. If such a cut did not traverse an entire segment the response dorsal to it was lost and that of the same level in the intact part was weakened. There was no effect on the responses from similar dorsal levels of the adjacent segments. A dorsoventral (vertical) cut weakened but did not abolish the response on either side. These results indicate that fibres from the sensory endings pass both longitudinally and vertically in a segment, but that the majority run vertically toward the segmental nerves.
When a drop of 0·01 per cent, solution of nicotine was injected beneath the epithelium there was slight initial stimulation, but after one or two minutes all responses to stimulation in the region around the injected spot disappeared. When injected just beneath the epithelium the proprioceptive responses often remained, while the tactile and chemical responses were lost. Strychnine 0·05 per cent, had no effect on the sensory field. It did, however, stimulate the subepidermal cells to discharge.
DISTRIBUTION OF MOTOR FIBRES
When the connection of the segmental nerve with the ganglion was left intact and afferent impulses were eliminated by cutting or crushing the nerve peripherally, efferent impulses were observed passing out from the ganglion. These increased as motor responses were induced by stimulation in distant regions. Such impulses are shown in Fig. 5. They were somewhat smaller than the proprioceptive responses but of slightly larger potentials than the impulses in response to chemical stimulation.
The study of the distribution of these motor impulses in the segmental nerves proved to be more difficult than the analysis of the sensory field. An attempt was made to record action potentials in the muscles with silver needles inserted in the muscles, and some potentials, similar in size and slightly longer in duration than the large nerve impulses, were observed. These were not obtained consistently, however, partly because all the muscle fibres of a segment do not appear to contract each time a peristaltic wave passes and partly because of very considerable base line irregularities. The results obtained suggested that there was some contraction in a segment lacking its nerves if adjacent to segments with nerves intact but not if the adjacent segments were also denervated.
A better test was provided by a record of the proprioceptive impulses set up in the muscle receptors. The results are shown in Fig. 6. As a peristaltic wave passed, fewer proprioceptive impulses arose in muscles of a segment when the nerves of adjacent segments were cut than when they were intact. No impulses whatever appeared when the segment in which impulses were recorded was separated from an intact region by two segments in which the nerves were cut. This indicated that some motor fibres overlap one but not two segments on either side of the segment of origin.
When the nerves of one segment were stimulated electrically and those of the adjacent segments were cut, good contractions were observed in the same segment, less contraction of the segment ahead, and very little of the segment behind. The evidence, therefore, with respect to the motor field indicates that there is a three segmental overlap as in the sensory field, but that the effect on adjacent segments is much less than on the segment in which the particular nerve arises.
Evidence concerning conduction of impulses by the peripheral system was obtained by removing the nerve cord for different lengths. The operated segments were pinned firmly to eliminate the effects of tension and traction which Friedlander (1894) and Garrey and Moore (1915),et al. have shown to be so important in locomotion. A marked diminution in efficiency of conduction of a peristaltic wave was observed in specimens in which the cord was severed when compared with the controls in which only the muscles were cut. This confirms many previous observations (see Prosser (1934 b) for references). When one ganglion was removed or the nerves and cord cut the contraction waves passed even less frequently. When one or more additional ganglia were removed these segments did not contract and no wave passed unless the worm were free enough to pull on the intact segments beyond and thus set up a contraction wave. Moreover, when a crystal of salt was placed on segments in which the segmental nerves had been cut, no general responses were obtained such as occurred when intact segments were stimulated. It appears, therefore, that the peripheral plexus is incapable of conducting impulses through more than one segment.
FUNCTION OF THE SUBEPIDERMAL PLEXUS
The study of action potentials in the segmental nerves of the earthworm gives information which could not be obtained by other methods concerning conduction in the peripheral elements of the nervous system. The evidence presented indicates that the impulses in responses to proprioceptive stimulation are of large potential, the impulses in response to chemical stimulation are of less and to photic stimulation of still less potential. The responses to tactile stimuli are more mixed than the others. Apparently these different types of response travel in different sets of fibres, and if a non-polarised nerve net is involved in the sensory mechanism, there must be a separate net for each of these types of response. This seems unlikely.
Further, the evidence that there is a fixed but irregular sensory field for each segmental nerve, usually on a three segmental plan, points to the conclusion that these responses pass from the sensory endings, not by a continuous nerve net or through a synaptic system, but by discrete branching fibres. This conclusion is supported by the following facts (i) conduction of responses to the segmental nerves is prevented by a horizontal and weakened by a vertical cut through the subepidermal plexus, (ii) responses can be blocked locally by nicotine, (iii) strychnine does not alter the sensory field, and (iv) the number of impulses increases with the area of stimulation. Certainly the impulses do not pass round the ends of a cut as they do in a coelenterate net. Although there may be fewer afferent impulses in a nerve when the adjacent segments are stimulated than when the epithelium of its own segment is stimulated, these impulses are of the same size and there is no evidence of the “decremental conduction” which Jordan (1929) states to be a fundamental property of nerve nets. In fact, the distribution of sensory fibres resembles that in the skin of a vertebrate, for example the frog (Adrian, Cattell, and Hoagland, 1931).
Similarly, it appears that motor fibres may extend for one segment on either side of the one in which they arise but that this is less extensive than the sensory overlap. These conclusions support those of Garrey and Moore (1915) and Bovard (1918) but not those of Hess (1925 b) and Janzen (1931). They are in line with the observation of Coonfield (1932) that epithelial secretion fails to occur beyond a stimulated region when the ganglia are removed. It appears likely, therefore, that sensory, motor, and secretory impulses are not conducted in a continuous peripheral nerve net. These results give no information concerning the direct connections from epidermal sensory cells to muscles through the subepidermal plexus as indicated by Hess (1925 a) or concerning the polarisation of these elements as maintained by Shensa and Barrows (1932).
The subepidermal plexus does, however, appear to play an important function as a region of branching of fibres. Langdon (1895) stated that there are, on the average, about 1000 sense organs per segment and that each of these contains approximately thirty sensory cells. There are in addition the free nerve endings, photoreceptors, and isolated sensory cells of the special type described by Smallwood (1926). Smallwood said that there are approximately 1000 longitudinal muscle cells and 500 circular muscle cells per segment. There are, on the other hand, only twenty to thirty fibres in each of the six nerves per segment. These fibres are nonmedullated and are 3–10 miera in diameter.1 Further, the number of impulses observed in response to stimulation of a given area is less than the number of sensory cells in that area according to Langdon’s figures. There must be, therefore, as Smallwood (1926) suggested, considerable branching of the fibres in the segmental nerves to the muscles, and numerous sensory endings must be connected with one fibre in each segmental nerve. The intersegmental overlap indicates an even greater degree of complexity in the distribution of the fibres peripherally.
In the segmental nerves of Lumbricus large repetitive impulses were observed in response to stimulation of proprioceptors, smaller impulses in response to epithelial stimulation by HC1, NaOH, and NaCl and still smaller ones in response to photic stimulation. The response to illumination was a gradual increase in impulses to a maximum; it was then usually interrupted by big proprioceptive impulses initiated by the muscular response. In response to tactile stimulation impulses of a range of amplitudes, rapidly adapting, and increasing in number with increasing area stimulated were observed. The rates of conduction of the majority of the tactile and proprioceptive impulses lie between 4 and 8 cm. per second.
In one segmental nerve responses were obtained to homolateral stimulation of that segment and of the two adjacent segments. Exploration of this sensory field with a fine needle showed local areas from which no response was obtained. A horizontal cut through the epithelium and subepidermal plexus abolished responses dorsal to the cut; a vertical cut weakened them on either side.
Responses were abolished locally by nicotine. Strychnine had no effect on the sensory field.
Efferent impulses, somewhat smaller than the impulses in response to proprioceptive stimulation, passed out from the ganglion in the segmental nerves. Few proprioceptive impulses in a contraction wave arose in a segment after the nerves of the adjacent segment toward the approaching wave were cut, and none when the nerves of the second segment removed were also cut.
Stimulation of one segmental nerve induced contraction of that segment and weak contraction of the two adjacent segments. Peristaltic waves were often conducted but greatly impeded after one ganglion was removed and were not conducted after two or more ganglia were removed.
It is concluded that sensory and motor impulses are conducted in separate branching fibres rather than in a continuous nerve net.
It is a pleasure to acknowledge my indebtedness to Prof. E. D. Adrian for many helpful suggestions and criticisms throughout this investigation.
I am greatly indebted to Mr John Z. Young, Department of Zoology, Oxford, for the loan of the slides from which these measurements were made.