The Nerve Ring of the Nematode Caenorhabditis elegans: Sensory Input and Motor Output

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Table of contents   -   Abstract   -    Mat. & Methods   -    Observations   -    Discussion & Acknowledgments

Observations

General description - Detailed structure of the organs in C. elegans - Cephalic musculature - Neuropil

Detailed structure of the organs in C. elegans

In C. elegans there are 16 papillary organs and two amphids. Each of the submedial lips (two subdorsal and two subventral) has three distinct cuticu!ar papillary organs, one (IL) containing two ciliated dendrites in a clearly protruding papilla at the tip and two (MSM and LSM) slightly posterior, each containing one dendrite. These three organs are joined to the CNS by a common papillary nerve (fig 2). The lateral lips have two cuticular papillary organs each, one (IL) which is structurally homologous to the submedial IL organs, the other (VL) placed more posteriorly and slightly ventrally but not structurally homologous with either the LSM or the MSM organ of the submedial lips. In addition, the lateral lips have a cyathiform amphid which contains 12 ciliated dendritic endings. The amphidial and lateral papillary dendrites travel to the CNS together in a common lateral nerve, which is therefore larger than the submedial nerves. The lateral nerve contains, in addition, a single neuron fiber which enters into the oesophagus near the opening of the dorsal oesophageal gland as described in other nematodes (Chitwood and Chitwood, '50).

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Fig. 4 - Schematic section through the sensory organs of a submedial lip. MSM.A, LSM.A cuticular appendages of the MSM and LSM terminations respectively. These appear in micrographs as dense areas, not clearly delimited, and are present in all organs embedded in the cuticle (MSM, LSM, VL, Deirid).

Each of these sensory organs has two accessory processes surrounding its dendries, a feature first described in Ascaris by Goldschmidt ('03). The relationship of these cells to the ciliated dendrites is shown schematically in figure 4 for two papillary organs in a submedial lip. The cap cell process (Goldschmidt's "Geleitzelle") surrounds the cilium only at its distal end and contains a few small, irregularly shaped electron-lucent vesicles. It forms a continuous desmosomal junction with itself, with the pocket cell process, and with neighboring cells of the lip tissue but not with the cilium. The pocket cell process (Goldschmidt's "Stutzzelle") has a larger terminal swelling with what appear to be extensive lamellar inclusions (the "myelin figures" of Yuen ['67]). In some sections these appear closed, but in others they seem to be continuous with the outer cell membrane and protrude into the extracellular space surrounding the cilium (fig 5B). Posteriorly the pocket cell process forms a continuous desmosomal junction with each of the ciliated dendrites at its point of penetration (fig 11C). Consequently the entire ciliary portion of the dendrite distal to the origin of its microtubules is isolated within an extracellular space formed by the two accessory processes. The cell bodies of most of the cap and pocket processes are located posteriorly in the vicinity of the first oesophageal swelling (fig 2). They are joined to their anterior specializations by fine fibers which travel within the papillary nerves and are morphologically indistinguishable from nerve fibers.

In addition to the classically described papillary and amphidial organs the lips of C. elegans contain a number of other endings of processes which also travel in the papillary nerves. All but one of these are nervous, as judged by their posterior projection into the nerve ring, and several contain ciliary elements suggestive of sensory function. However, unlike the papillary and amphidial dendries they are not surrounded by accessory cells and do not terminate at the cuticle Instead they end internally with a variety of shapes, some of them highly convoluted.

Internal labial papillae. The internal labial papillae are sImilar in all six lips. They consist (fig 5) of the two associated cell processes and, as in all cases in which they have been studied (Yuen, '67; Mc-Laren, '72; DeGrisse et al., 74; 8aldwin and Hlrschmann, '74), two ciliated dendrites of about the same diameter (130 nm), each ending at a slightly different level within the structure. One of these dendrites (ILR) possesses a rootlet four microns in length with a characteristic asymmetrical banding pattern (fig 5D), while the other (ILN) has a rudimentary rootlet observed only in longitudinal sections. The tips of these cilia, which are the most anterior ending of the sensory neurons, are exposed to the exterior through a pore in a cuticular cap which protrudes from the front of the animal at an angle of about 30° from the longitudinal (figs 3B, 5A). No cuticular sheath has been observed surrounding these dendrites. The exact terminal course of the two dendrites, not easily represented schematically (fig 4), consists of a series of reproducible right angle bends just beneath the protrusion. The result is that the lLR fiber ends abutting perpendicular to the ILN fiber at the inner margin of the cuticle and within the pore whereas the ILN fiber proceeds to the end of the channel (fig 5A, inset). At its proximal point of entry into the pocket cell the ILN dendrite possesses a swelling m which there are a number of electron lucent vesicles of about 65 nm diameter. These are the only papillary organs which possess a marked extracellular space into which fine filaments from the pocket cell processes project (fig 5B). This space, though distinct, is small compared to that found surrounding dendrites in insect pressure-sensitive sensilia (e.g., Gnatzy and Schmidt, '71) and occurs from the distal rootlet region of the ILR dendrite to the cuticular opening on the snout.

Lateral submedial papillae. These organs (fig 6) have no cuticular protrusion. The sensory organ consists of a single nonrootleted ciliated fiber (LSM) embedded terminally in the cuticle for a distance of two and one-half microns, plus the two associated cell processes. Compared to the internal labial organ, the lateral submedial has only a very narrow extracellular space between its dendrite and the laminated associated cell. At its terminal end the distal segment swells to a diameter of about 350 nm and contains 10 to 15 diffuse electron dense rodlike structures which are not always discrete but sometimes merge into one another eiher by contact or by interposition of additional dense material. Each of the rods is surrounded by a complement of about five microtubules (figs 6B, 7B). The general appearance of this terminal specialization is similar to the tubular body structure frequently observed in insect mechanoreceptors. As in the amphidial and ILN dendrites, there is a swelling filled with 65 nm diameter vesicles at the entry of this fiber into the proximal part of its pocket cell.

It is interesting in several respects to compare this organ in C. elegans with the homologous organ of Ascaris described by Goldschmidt ('03). First, Goldschmidt found the nerve fiber supplying the organ to be thick relative to the other nerve fibers in the submedial bundles. This is likewise true in C. elegans. Second, the pocket cell of this organ in Ascaris was found not to end in a monopolar cell body between the nerve ring and snout as is the case with other papillary associated cells, but rather to have its cell body at the nerve ring, where it takes on the nature of a glial cell, ensheathing the neuropil of the ring with a thin layer of its cytoplasm and separating it from the surrounding mesodermal tissue. This is also true for the lateral submedial pocket cells in C. elegans (figs 17-19, 24, 28, 29, 33). ThIrd, in his original paper ('03) Goldschmidt was unable to locate cell bodies for the nerve fibers of these organs anterior to the nerve ring. In a later paper (Goldschmidt, '08) he amended this finding and called them his cells 50 in the subventral papillary ganglia, with no explicit mention of them sub- dorsally In C. elegans we have found that the nerve cell bodies of these organs are located just posterior to the nerve ring subdorsally and just anterior to it subventrally (fig 18, LSM). These are the only cell bodies of homologous sensory neurons which do not occupy corresponding positions about the nerve ring in C. elegans.

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Fig. 5 - A. Longitudinal section through the rootleted dendrite of the internal labial organ (ILR). Inset: Longitudinal section showing the extension of the ILN dendrite into the IL pore. B. Perpendicular sectino through the internal labial organ showing the protrusions of the surrounding pocket process into the extracellular space (ES) surrounding the ILN and ILR dendrites. C. Cell bodies of the ILR, VL, and 2-neurons at approximately the maximum diameters of their nuclei, showing the characteristically large nuclear to cytoplasm ratio present in these cells. D. Original and optically averaged micrographs of the ILR rootlet.

Medial submedial papillae Like the lateral submedial organs, these also have no cuticular protrusion from the front of the animal In contrast, they do possess a long striated rootlet with the same ultrastructural banding pattern as observed in the rootleted internal labial dendrite (fig 7). The single ciliated fiber (MSM) ends in an elongated 130 nm diameter termination which is embedded in the cuticle for a distance of about two microns. The terminal specialization of the dendrite is difficult to classify in terms of known invertebrate receptors. That portion embedded in the cuticle has a smooth surface toward the outside of the lip, whereas on the inside it is highly crenated. Within the termination there are long discrete electron dense rods. Four of these extend throughout the embedded ending and are located at the corners of a square. Four others extend for only the proximal half of the ending and are located at the corners of a slightly larger square rotated 45° with respect to the first (figs 6B, 7B). No microtubules have been observed in perpendicular sections of that part of the dendrite embeded in the cuticle.

Ventrolateral papillae As in Ascaris, the ventrolateral organ (fig 8) is distinct from either the lateral or medial submedial. The dendrite (VL) is surrounded by cap and pocket cell processes, but its terminal cuticle-embedded portion is much shorter than in either submedial organ and is more nearly spherical (about 300 nm in diameter). A further difference is that there is but a single very electron dense inclusion with about ten microtubules positioned only about its periphery (fig 8B). A similar type of terminal specialization, with a larger number of surrounding microtubules, is observed in the classically described deirids (fig 8C), two cervical sense organs conventionally assumed to be pressure receptors and located laterally within the cuticle, one on each side of the animal, just posterior to the nerve ring.

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Fig. 6 - A. Longitudinal section of the LSM dendritic termination. B. Perpendicular section of the tubular body-like termination (TB) of the LSM dendrite. The more distal portion of the MSM termination is also present at this level. Des, desmosomal seal of the MSM cap cell with itself.

The ventrolateral organ is of particular comparative interest because in Ascaris it was shown by Goldschmidt to consist of two neurons, one as described, surrounded by the usual cap and pocket cell processes, and one just outside of these associated fibers and ending bluntly against, not within, the cuticle. Goldschmidt also stated that in one special case he observed that this latter dendrite appeared swollen into a club-shape beneath the cuticle. We observe just such a club-shaped ending within the lateral lip tissue in a slightly ventrolateral position (figs 3, 9). This dendrite has a normal striated rootlet and cilium. It is unique in that its terminal specialization involves no inclusions, but simply a flaccid swelling. The distal end of this swelling breaks up into a number of finger-like projections arranged in a petal-like formation which indent the cap cell of the lateral internal labial organ of the same lip (figs 8B, 15C,D). A further peculiarity of this neuron is that its cell body is quite distinct from those of the other papillary neurons, both in size (fig 9B) and in its projection into the nerve ring. Structurally, both the nucleus and cell body are large compared with those of the other papillary nucleus, although the cytoplasm is similar in appearance. In projecting into the nerve ring, the nerve fiber does not enter with the others in the characteristic papillary nerve U-turn mentioned above. Whether this dendrite represents a regression in C. elegans of the outer ventrolateral dendrite of a more basic organ, exemplified in Ascaris, or is just another of the dendrites which are not differentiated classically and which end in the lip tissue is an open question.

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Fig. 7 - A. Longitudinal section of the MSM dendritic termination. B. Perpendicular section of the MSM cuticular termination taken slightly posterior to that of figure 6B. Note the presence of eight rods are present at the level of figure 6B, and the anterior end of the MSM pocket cell completely sealed to the surrounding cap cell by a desmosome.

Amphids The two amphids, one on each laleral lip, are larger and more complicated than the papillary organs. Like them, they are composed of a cap and a pocket cell process, although the extracellular space they surround (the amphidial pouch) is larger and in more dIrect contact with the exterior (fig 10). Instead of one or two neuronal dendrites, the amphidial pouch contains eleven. A twelfth ciliated dendrite invades the pockel cell without ever entering the pouch itself. These dendrites are shown in figure 12. Like the papillary dendrites they enter the pouch by proximal penetration of the pocket cell, where they are sealed with a continuous desmosomal junction (fig. 11C). The pouch itself contains an amorphous, slightly electron dense material which surrounds the 11 dendrites contained within it. Vesicles of uniform diameter (about 65 nm) but varying degrees of electron opacity are commonly seen in many of the dendrites just proximal to their entry into the extracellular space and near to the desmosomal junction (figs 10, 11C). Most often these vesicles are tightly clustered either at the membrane or internally. All dendrites are approximately 350 nm in diameter and possess a rudimentary rootlet, seen only in longitudinal sections (fig 10). They possess microtubules in a configuration similar to that described by Hansen and Heumann ('71) in Phormia, basically a 9 x 2 + m arrangement, where m varies from 1 to 6, with the extra tubules interior to the circle of doublets.

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Fig. 8 - A. Longitudinal section of the ventrolateral dendritic termination. VL.A, cuticular appendage of the VL dendritic termination. B. Perpendicular section of the VL dendritic termination showing the electron dense specialization (D) surrounded by microtubules. The anterior end of the amphid (AP), beyond the termination of its dendrites, is also seen. C. Perpendicular section through the cuticular termination of the deirid, or cervical sensory organ, showing a similar electron dense specialization surrounded by microtubules. LA, two of the three lateral alae, the third being taken over by the deirid at this level.

One of the most striking features of the amphid, observable only in complete sets of serial sections, is the constancy of its structure from animal to animal. There are three types of dendrite, the same type occurring at a corresponding location in all animals studied, so that a single transverse section which contains all dendrites is sufficient to identify each of them unambiguously.

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Fig. 9 - A. Longitudinal view of the termination of the club-shaped dendrite (C). B. Cell bodies of the C and MSM neurons at approximately the maximum diameters of their nuclei, showing the characteristically larger diameter of the C-nucleus.

Fig. 10 - Longitudinal section through the amphid at its opening to the exterior. The desmosomal junction between the amphidial cap and pocket cell fibers (Des) is shown.

The three dendrites of the first category (1-3 in fig 12) give rise to terminal portions which do not end after the termination of the microtubules, but instead leave the pouch and reinvade the pocket cell. They continue anteriorly as long thin filamentous extensions but still within the pocket cell. Slightly anterior to figure 11A these extensions reach halfway around the snout dorsally and ventrally, approaching their counterparts from the other lateral lip at the medial poles of the animal. Of these dendrites, 1 and 2 form a single filamentous extension each, while fiber 3 branches posterior to its microtubules to form a pair. The eight dendrites of the second category (4-11 in fig 12) end within the pouch at different but characteristic levels shortly after the termination of their microtubules. Two of these (4 and 5), like fiber 3, branch to form two cilia each, whereas the rest are singlets. The one dendrite of the third category (12 in fig 12) contains microtubuiar inclusions like those of the other fibers, but instead of entering the pouch it remains within the pocket cell, sending out on the order of 50 finger-like projections which indent the pocket cell membrane (figs 11A, B, 12). These projections probably correspond to the "microvilli" observed by Baldwin and Hirschmann ('73) in the amphid of M. incognita.

Several features of the amphid emphasize its difference from the papillary organs. First, whereas papillary neuron cell bodies are located anterior to the nerve ring, amphidial cell bodies, which are much larger (fig 13) and contain a distinctive cytoplasm, are positioned behind it in the so-called lateral ganglia, one on either side of the animal as in other nematodes. Secondly, whereas the papillary fibers project into the nerve ring following a direct course well within the mesodermal tissue, the amphidial fibers follow a more circuitous route into the ventral ganglion (fig 2). Output fibers from the amphidial cell bodies of one side join into a bundle at the center of the lateral ganglion and emerge radially, traveling until they reach the interior margin of the cuticle, where they turn 90° and run for about 30° ventrally between the muscle cells and the cuticle (fig 14). When they have nearly reached the ventral line they again turn abruptly and travel radially inward to the ventral ganglion. Third, there is a pronounced difference in the structure of the cytoplasm of the papillary and amphidial pocket cells. Along its course from the nerve ring to the snout the amphidial pockel cell process contains much reticular material granules which are presumably ribosomes, and many Golgi-like structures. The fiber itself has a diameter nearly equal to that of the entire lateral papillary nerve (fig 28A). The cell body of the amphidial pocket cell, located more or less dorsally in the lateral ganglion along with the other amphidial cells (fig 18), shows an even more striking glandular appearance and contains, in addition to the above mentioned inclusions, many non-membrane bound lacunae (fig 13).

Other anterior endings. In addition to the club-shaped ending described above as part of the ventrolateral organ, the lips of C. elegans contain four additional types of non-cuticular ending from processes contained within the papillary nerves. Three of these, whose cell bodies are labeled 1, 2, and 3 in figure 18, are apparently nervous since they project through the papillary U-turn into the nerve ring. Cells of type 1, in the subdorsal and subventral papillary nerves only, become associated with the MSM papillary organs in a peculiar manner, having a sheet-like ending along the medial surface of the pocket cells of these organs (fig 15A). Cells of type 2 end without specialization next to the oesophagus near the anterior end of its musculature. Cells of type 3, present only subdorsally, possess a ciliary ending with a rudimentary rootlet. Although the 3 fibers travel in the subdorsal bundles for most of the length of the cephalic region, they undergo a right angle turn within the lips, so that their rootlet lies nearly perpendicular to the body axis, and proceed to their neighboring lateral lips. Once there they turn anteriorly again and branch to form a number of long, fine processes which deeply invaginate the cap cell of the lateral internal labial sense organ (figs 15B-D).

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Fig. 11 - Anterior to posterior series of perpendicular sections through the amphidial pouch. A. Anteriormost. Fi: filamentous endings of amphidial neurons 1-3 as described in the text. C. Taken near the proximal entry of some of the amphidial neurons into the pocket cell, where they have a swelling containing clusters of vesicles (V), and the desmosomal junction (Des) between the dendrites and pocket process is present.

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Fig. 12 - Schematic section of the amphidial pouch at a level corresponding to figure 11B. The relative positions of the dendrites are constant from animal to animal, enabling each of them to be identified unambiguously in a single section.

Fig. 13 - Cell bodies of the amphidial pocket cell (A.P) and some amphidial neurons (AN). Comparison with figures 5C and 9B shows the characteristically richer cytoplasm of the amphidial neurons compared with the papillary neurons, their larger nuclear and cell body radii and their smaller nuclear to cytoplasm ratio.

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Fig. 14 - A. Perpendicular section through the ventral third of the animal showing the course of the fibers of the amphidial connective (AC) described in the text. B. Longitudinal section through the ventral quadrant of the worm at a level shown by the arrow in figure 10A where it turns from a circumferential to a radial direction and enters the ventral ganglion. 10C. Higher magnification sagittal view of the amphidial connective beneath the cuticle and a lesser slightly more posterior connective which here is separated off from the larger one. Note that the amphidial connective contains more than 12 fiber profiles, indicating that some lateral ganglion neurons other than amphidial neurons may also be represented.

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Fig. 15 - A. Perpendicular section through the subdorsal lips at a level where the terminal swellings of the 1-fibers expand against the MSM pocket cell. B. Perpendicular section through the lateral and two neighboring submedial lips showing the 3-fiber (outlined) in its course from the subdorsal to the lateral lip, as described in the text. Part of its nearly vertical rootlet (R) is shown. C, D. Lateral lip at a region where the C-fiber and the 3-fiber indent the cap cell fiber of the lateral internal labial sensory organ.

A fourth cell type, not described in Ascaris but which has been found to contribute a fiber to the papillary nerves of C. elegansis labeled as Cell 60 in figure 18. The fiber of this monopolar cell separates off from the rest of the papillary bundle just anterior to the nerve ring and does not enter the U-turn. Posterior to its separation it swells into abroad sheet like expansion which is immediately apposed to the oesophagus and which extends for about 60° circumferentially under nearly all of the nerve ring neuropil (figs 26, 33). The sheets of some of these cells are observed to make what appear to be very broad gap junction contacts with at least one fiber of the nerve ring whose cell body is designated as DM in figure 18. The 60-cell bodies are different from all others observed, being densely packed with small dark granules and non-membrane bound clear areas (fig 16). Anteriorly, the 60-processes end without specialization next to the oesophagus near the anterior end of its musculature.

Figure 16

Fig. 16 - Perpendicular section through one of the 60-cell bodies taken at approximately the maximum diameter of its nucleus showing its peculiar granular, vacuolated cytoplasm. The two anteriorly directed processes of the 3-cell are shown just anterior to the cell body.

Cell Body Locations

The neuron cell bodies of one of four completely sectioned nerve rings are shown in three oblique schematic views of the same animal in figure 17. This figure encompasses the region of the nerve ring from the anterior-most neurons through the beginning of the smaller ventral ganglion, some cells of which are present in the posterior regions of the figure. All of those cells which have anteriorly directed processes are identified by the nerve bundles of which they are a part. Figure 18 shows the cell body locations in the nerve ring and anterior part of the ventral ganglion of two animals in a diagram representing the worms cut along their ventral midlines and then flattened out into a plane. In each case, identification of a cell body was made by following its peripheral process to its terminal specialization on the lips in complete series of electron micrographs. In the figure, neurons of the papillary bundles are identified both by the bundle of which they are a part and their sensory nature. One of the noteworthy features is the high degree of bilateral symmetry in cell body position. Comparison of different animals has shown that these cells are distributed in a similar manner in all cases studied.

Figure 17

Fig. 17 - Perspective drawing, three views of the same animal, of the nerve ring cell bodies from the most anterior to the beginning of the ventral ganglion. Fibers of the nerve ring are located in the region about the oesophagus where cell bodies are absent. Cross-hatch: subdorsal papillary cells; lines: lateral papillary cells; dots: subventral papillary cells; black: LSM pocket cell nuclei.

Figure 18

Fig. 18 - Diagrams representing two worms as if cut along their ventral midlines and flattened into a plane, anterior end up, for the same region of the CNS represented in figure 17. The shaded area indicates the region of the neuropil of the nerve ring, above, and the anterior portion of the ventral ganglion, below, which are continuous. Since the nerve ring is inclined at a slight angle to the body axis, corresponding points around it lie on an arc (dotted lines) rather than on a straight horizontal line. Small circles represent cell bodies which have not been characterized. Black circles represent nuclei of the LSM pocket cell. A: amphidial neurons; AP: nuclei of the amphidial pocket cell; VM: two characteristic neurons on the dorsal and ventral midlines, respectively; Oe: cell sending a fiber to the anterior end of the oesophagus by way of the lateral papillary nerve bundle.

In marked contrast to the observations on Ascaris (Goldschmidt '08), we find only a very limited aggregation of cell bodies into the classical ganglia in C. elegans. Rather, they seem to form a loosely packed continuum of cell bodies which are occasionally separated from the surrounding hypodermal cells by thin glial processes of the LSM pocket cells, but more often are in direct contact with them.The ventral and lateral ganglia are differentiated by dividing the animal into four imaginary quadrants, two medial and two lateral, delineating their boundaries by the four symmetrical cephalic muscle bundle entries (see discussion of cephalic musculature). The lateral ganglia are taken to be composed of those cell bodies, including the amphidial cells, in the lateral quadrants, and the ventral ganglion of those cell bodies located in the ventral quadrant and clustered about the ventral posterior extension of the nerve ring neuropil. The absence of the commonly called papillary ganglia is easily observed in figure 18, where the neurons contributing to the papillary nerves are seen to be present both anterior and posterior to the nerve ring neuropil.

The correspondence in position around the ring of cells having homologous endings in the snout is apparent, with the exception, as mentioned, of the LSM nerve cells, which have their cell bodies anterior to the ring subventrally and posterior subdorsally. Of particular interest is that the nerve cell bodies of the VL organs are nowhere near the corresponding position of either of the two submedial organ neurons with which they might be homologous on the basis of classical studies, namely, the LSM and MSM. The cell bodies of the rootleted club-shaped dendrites (C) are located in a position laterally corresponding to the MSM neurons, but as has been described they are so distinct from the other papillary neurons, both in structure nad in nerve ring projection that they can not be described as being homologous with them.

The subdorsal LSM and 3-neurons and the 60-cells are unusual in structure. First, whereas all other cells contributing to the papillary bundle are anterior to the ring, these are posterior. Secondly, all other neurons are bipolar with one anteriorly and one posteriorly directed process. In contrast, the subdorsal LSM neurons are monopolar with a branch point at the U-turn, one branch traveling to the corresponding lip, the other entering the ring just posterior to the U-turn. The 60-cells are monopolar. The subdorsal 3- neurons are bipolar, but with both processes directed anteriorly, one going to the snout, the other to the nerve ring just posterior to the subdorsal U-turn. This difference has been found consistently with but a single exception. On one side of one animal, the 3-cells was found to be monopolar with one branch point, the two fibers coming from it duplicating the courses of the two normally unbranched fibers.


Web adaptation, Thomas Boulin, for Wormatlas, 2002