The Posterior Nervous System of the Nematode Caenorhabditis elegans

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Table of contents   -    Abstract   -     Mat. & Methods   -     Introduction   -     Results   -     Discussion


General anatomy of the posterior nervous system Some of the general anatomical features of the C. elegans hermaphrodite tail are indicated schematically in Figure 1. The tail tapers to a fine tip posteriorly, with small lateral openings in the cuticle for the phasmids, a bilateral pair of sense organs, and a larger ventral opening for the anus (Fig. 1A). Dark-staining rectal cells interrupt the passage from the intestine to the anus. A sphincter muscle cell (Fig. 1A, SI) controls the rectal opening, while single large muscle cells operate to open the anus (depressor ani muscle; Fig. 1A, D) and to squeeze the posterior intestine (stomatointestinal muscle; Fig. 1A. SI). The ventral cord terminates at the preanal ganglion, which contains 13 loosely associated cell bodies (Fig. 1B). Slightly above and to the rear of this ganglion is the small dorsorectal ganglion, whose 3 cells are joined to the preanal ganglion by a pair of circumrectal commissures. Extending posteriorly from the preanal ganglion are a pair of ventrolateral commissures, which pass on either side of the anus to reach 2 bilaterally symmetrical lumbar ganglia, each containing 12 loosely associated neuronal cell bodies. Figure 1B shows a view from the right side, where only the rightward commissures and the right lumbar ganglion are evident. The dorsal cord terminates in the tail by trifurcating to form a pair of circumferential commissures, which enter the lumbar ganglia, plus an additional commissure that extends quite far posteriorly before reversing to extend anteriorly and subventrally to enter the preanal ganglion. Extending posteriorly from the lumbar ganglia are a pair of caudal nerves. A portion of each caudal nerve terminates in the ipsilateral member of the phasmids. The remaining portion of each caudal nerve continues past the phasmid to the extreme tail tip. In addition, fine ventrolateral and dorsolateral nerves extend anteriorly and posteriorly from each lumbar ganglion.

Figure 1

Figure 1. General anatomy of the tail. A, Outline of the hermaphrodite tail viewed from the right side showing the tail musculature in relation to the intestine, anus, and phasmidial opening. Intestinal cells are light gray; rectal gland cells are dark gray. Groups of muscle contractile units are schematized as sets of thin parallel lines. B, View of the nervous system from the right side (left lumbar ganglion, left lateral nerves, and leftward commissures omitted). One pair of "lumbar" neuron somata lie separately, in register with the dorsolateral nerves. The right-hand member of this pair is the unlabeled cell lying just posterior to the 3 DRG cells. Arrows at the bottom of the figure indicate the relative positions of thin sections shown in Figure 3. For abbreviations, see Table 1.

For purposes of reference in the following sections, Figure 2 shows reconstructions of several selected posterior neurons, viewed from the right side, and histograms showing the frequency of synaptic contacts within 3 regions of the tail. Figure 3 shows 3 of the coronal thin sections from which these reconstructions were made. Somata, axons, and dendrites of a limited number of neurons and muscle cells are marked, concentrating on features of the reconstructed cells from Figures 1A and 2. Figure 4 depicts relative cell body positions of all identified neurons from a dorsal aspect, using a symbolism denoting inferred function as well as position; representations are given for both B126 and B136 to show animal-to-animal reproducibility. Figures 5 and 6 show schematic reconstructions of every posterior neuron, viewed from a dorsal aspect. Figure 7 compares the positions of identified axons in both animals in the Iumbar- preanal commissures (Fig. 7A) and the preanal ganglion (8), in coronal sections.

Lumbar ganglion cells: anatomical description and example of serial reconstruction of sensory neurons

Phasmidial neurons (PHA, PHB). The most obvious posterior sensilla are the 2 phasmids, which open laterally about 40 um posterior to the anus, at a point where the tail's diameter has diminished from about 70 um to about 15 um (Figs. 1, 2). Each phasmid consists of 2 ciliated dendritic endings, one ventral (PHA), and one dorsal (PHB), within an extracellular pocket formed by 2 accessory cells, the socket cell (Phsol) and the sheath cell (Phsh; Fig. 3A). The ciliated endings of PHA and PHB protrude through the sheath cell and appear to be in direct contact with the exterior of the animal. The phasmidial cilia have recognizable basal bodies but no striated rootlets. The ultrastructure of the phasmid has been shown in greater detail by Hall (1977) and by Sulston et al. (1980). These features suggest a chemosensory role for the phasmid and are homologous to those already described for 2 types of putatively chemosensory anterior sensilla, the amphids and the inner labial papillae (Ward et al., 1975; Ware et al., 1975).
     Synaptic endings of PHA and PHB neurons occur only in the preanal ganglion; they contain large clear vesicles (50 ± 10 nm diameter), in contrast to the smaller vesicles (41 ± 9 nm diameter) seen in most other neurons. Figure 2A depicts the soma and processes of PHBR as an example, showing a lateral view. Figure 3 reveals 3 cross-sections from one animal in which PHBR can be seen as a ciliated ending (Fig. 3A), as a soma in the lumbar ganglion (Fig. 3B), and as an axon within the preanal ganglion (too small to be visible in the PAG area in Fig. 3C). The PHBR axon exits its soma and travels ventrally into a commissure within the section depicted in Figure 3B. Several thousand cross-sections were used to assemble detailed reconstructions of 2 animals at various magnifications to confirm this type of detail for every cell type. All of the neuronal and accessory cell somata positions are compared in Figure 4 for 2 animals. Figures 4 and 5 show schematic views from above, demonstrating the bilateral nature of the lumbar ganglia. Note that each PHB soma is located near the dorsal edge of the lumbar ganglion, near the exit of the lumbar-dorsal cord commissure. The positions of the PHB axons are easily seen in Figure 7, which depicts their location within the lumbar-preanal commissures (Fig. 7A) and within the preanal ganglion (8) in both B126 and B136. Note that each neuron is generally represented by only one profile in a given coronal section, as most neurons lack any secondary branching in the tail.

Figure 2. Serial reconstruction of neurons and synapse distribution. A,Neurons PHBR and AVAR shown from the right side. This view was drawn approximately to scale by hand from serial reconstruction data. The approximate dimensions of the extracellular pocket of the phasmid is indicated. Swellings along the axon are not to scale; axon profiles are actually enlarged for clarity. B, Neurons DA9, DVA, and VA12 shown schematically from the right, as above. C, Three histograms referenced to the above drawings illustrate the longitudinal distribution of synaptic contacts within the tail for B136, an animal in which every morphologically identifiable synapse has been cataloged. NMJs onto the defecation muscles are omitted, as they are not well demarked in B136. Numbers of synapses have been summed over intervals of 3 um. The general density of synaptic contacts in the ventral cord is much lower than in the preanal ganglion (White et al., 1976). For abbreviations, see Table 2.

Figure 3. Sample sections from a reconstruction. Three coronal sections demonstrating some of the cell positions schematized in Figures I and 2 are shown. A, Cut at the level of the phasmids. B, Cut at the level of the lumbar and dorsorectal ganglia. C, Cut at the level of the preanal ganglion. In A, both pairs of chemosensory cilia can be seen within the extracellular pocket of the phasmid sheath cell. Arrows at the bottom of B indicate the relative positions of these 3 sections. Scale bars: A. 1 um; B and C, 5 um. For abbreviations, see Tables 1 and 2.

Figure 4. Cell body positions. Schematic views in 2 animals (B126, above; B136, below) of locations of cell bodies of neurons and accessory cells. The view is from a dorsal aspect, with the most dorsal region peeled away to the bottom of each panel. Cell bodies of ALN neurons lie in dorsal positions outside the lumbar ganglia proper. Labels for each soma are placed inside symbols indicating presumed functions; actual soma shapes are generally larger and more space filling. Triangles. sensory neurons; hexagons, interneurons; circles. motoneurons; squares. accessory cells of the phasmids. For abbreviations, see Table 2.

Figure 5. Schematic models of all lumbar ganglion neurons. The arborization of each lumbar neuron is shown, using the same perspective as in Figure 4. Because these cells have no secondary branches, the schematic model is a fair representation of the actual cell shape. The caliber of the neurites has been enlarged for clarity. No attempt is made to show the close approximations of homologous neurites as they traverse the preanal ganglion. The processes of PLML/R and PLNL/R travel in ventrolateral positions, and those of ALNL/R travel in dorsolateral positions. For abbreviations, see Table 2.

Figure 7. Coronal sections of the ventrolateral commissures and the preanal ganglion. A, Left and right ventrolateral commissures are shown for B126 (top) and B136 (bottom), as tracings from equivalent regions in the 2 animals. Besides the lumbar neurites, which predominate, there are also 3 neurites from preanal ganglion neurons that are passing toward the dorsal cord via these commissures. B, Sections from the anterior portion of the preanal ganglion in B136 (left) and B126 (right). Because neurons are largely unbranched, most are represented as single profiles. Note that bilateral homologues generally lie quite close to one another. B126 has been rotated 30° clockwise for convenience in presentation. The soma of DD6 is marked by a heavy circle, as are neighboring muscle (M) and hypodermal (H) cells. Magnification is approximately 25,000x. For abbreviations, see Table 2.

Neuron wilh a buried ending (PQR). A third type of accessory cell, the wing cell (Phso2), is more loosely associated with each phasmid, sending abroad, winglike process along much of the phasmid's medial surface. Near the base of the left phasmid, but not the right, the process of Phso2 is invaginated by a different type of ciliated ending, PQR, for which it provides a thin wrapping for some distance posteriorly (Hall, 1977; Sulston et al., 1980). The basal body of the PQR cilium is located near the point of entry into the wing cell, and the distal portion of the ending is markedly spatulate. From its location near the axis of the tail, the PQR ending might be preferentially sensitive to pronounced, deep mechanical deformations.

Neurons with postphasmidial processes (PHC). Posterior to the phasmids, each caudal nerve contains a small process (PHC) that runs in a ventrolateral position for 65 um to the extreme tip of the tail. As the tail tip narrows posteriorly, the 2 processes come to lie adjacent to one another, and for a considerable distance, the fine tail tip consists only of these 2 processes, surrounded by a thin hypodermal layer and the overlying cuticle (Hall, 1977). This fine tip undergoes pronounced deformation, often folding back on itself, when the animal moves backwards. Although the PHC processes are not ciliated, their physical location suggests a possible mechanosensory function. Like the PHA and PHB sensory neurons, the PHC neurons have distinctively larger vesicles at their synapses than do other posterior neurons.

Neurons with microtubule-filled processes (PLM. PVR). From each lumbar ganglion there extends, both anteriorly and posteriorly, a fine ventrolateral nerve consisting of 2 processes. As has been previously described (Chalfie and Thomson, 1979), 1 of these 2 processes (PLM) contains a distinctive array of about 40 microtubules. Each PLM process runs quite near the surface, separated from the cuticle by only a thin layer of hypodermal cytoplasm. The PLM processes are sensory receptors for "light touch" delivered to the posterior portions of the animal (Chalfie and Sulston, 1981; Chalfie et al., 1985).
      One unpaired lumbar neuron, PVR, sometimes has a posterior process running caudally from its soma to a dorsal position in the extreme tail tip (White et al., 1986). In B126, this process contains a small bundle of microtubules, suggesting a sensory function. In B136, no posterior process is found for PVR. Immunochemical staining has revealed that the microtubule bundle in PVR shares the same acetylated alpha-tubulin that is otherwise characteristic of 3 pairs of mechanosensory cells (PLM, AVM, ALM; Siddiqui et al., 1989).

Dorsorectal ganglion cells

DVA, DVB, and DVC each send single processes rostrally through circumrectal commissures into the preanal ganglion and thence into the ventral nerve cord (Figs. 2B and 3B show DVA as an example). The DVB process is greatly enlarged and filled with synaptic vesicles as it crosses the ventral hypodermal ridge to enter the preanal ganglion. Although postsynaptic specializations could not be seen, it seems likely that DVB acts as a defecation motoneuron with synaptic outputs onto arms of the defecation muscles (D, S, SI, VM), which lie along the dorsal surface of the hypodermal ridge. The proximity of the DVB axon and the SI muscle arms can be noted in Figure 3C.

Preanal ganglion cells

Interneurons.Of the 13 neurons with cell bodies in the preanal ganglion, only VA12 has significant synaptic output to neurons within the preanal ganglion itself. VA12 also receives a number of synaptic inputs in the ganglion and thus qualifies as an interneuron. However, VA12 additionally sends a process forward into the ventral nerve cord, which makes a number of NMJs of the type previously described as "class A " (White et al., 1976, 1986). Thus, VA12 acts both as an interneuron and as a motoneuron. PVPL/R and PVT are also intemeurons; most of their synapses are in the ventral cord or the nerve ring (White et al., 1986).

Motoneurons. Eight of the 13 neurons of the preanal ganglion are motoneurons, but because most NMJs to ventral body muscles occur anterior to the preanal ganglion, few of these junctions were seen in our reconstructed series. A reconstruction of the dorsal cord in B136 revealed many NMJs for some of these neurons.
      Two neurons, DA8 and DA9, send anterior processes into both the dorsal and the ventral nerve cords (Fig. 2B shows DA9 as an example). These are "class A " motoneurons (White et al., 1976), and in both the dorsal and the ventral nerve cords, their processes run together in the position typical of class A processes. In the dorsal cord, DA9 was found to make a series of NMJs in the reconstructed region of B136. In the same region, DA8 had no NMJs, but its dorsal process extended anteriorly beyond the region of reconstruction. Its expected territory for NMJs should lie just rostral to that of DA9 (White et al., 1976). VA12 is also a class A motoneuron, with NMJs along the ventral cord. VA11 lies rostral to the preanal ganglion proper, outside the region reconstructed, but is listed as part of this set of motoneurons due to its lineal relationship to other preanal neurons (cf. Sulston and Horvitz, 1977). PDA forms NMJs along the dorsal cord.
      Three neurons, VD13, VD12, and DD6, send single processes forward from the preanal ganglion into the ventral cord. Each process has a commissure more anteriorly, which goes to the dorsal cord (White et al., 1986), and in the dorsal cord, we have identified what appear to be the caudal projections from these commissures. VD13 and VD12 receive synaptic input in the dorsal cord and make NMJs in the ventral cord, whereas DD6 receives input ventrally and makes NMJs dorsally. These neurons are thus typical "class D" motoneurons (White et al., 1976), and in the portion of the dorsal cord we have reconstructed, their processes run in the positions typical of class D processes (VDz, VDy, DDz in Table 3).

Neurons of uncertain function. AS11 and PDB would be expected from their lineages (fourth-most anterior daughters of P neuroblasts) to be "class A " motoneurons with a short dorsal process forming NMJs (Sulston, 1976; Sulston and Horvitz, 1977). AS11 differs from other AS motoneurons, which have only a rostral branch in the dorsal cord. AS11 also lacks any NMJs along its dorsal processes (followed to their endpoints in B136). However, both the dorsal and ventral processes of AS11 run in positions typical of other AS motoneurons, and the synaptic input to AS11 in the ventral cord resembles input to other class A motoneurons. Thus, in many respects, AS11 resembles atypical AS motoneuron, but in the absence of observed output, its function remains unclear.
      The posterior process of PDB travels by an unusual ventral route far into the tail tip (more than 10 um posterior to the phasmidial openings) before coursing dorsally and returning anteriorly to enter the emerging dorsal cord (Figs. 1B, 6). The dorsal process travels anteriorly as part of the dorsal nerve cord and terminates within the region we have reconstructed in B136. Neither the ventral processes nor the dorsal process of this cell have synaptic connections that we can observe. A few NMJs may occur in the dorsal cord according to White et al. (1986). The deviation of AS11 and PDB from their expected fates resembles similar deviations observed among the daughters of extremely anterior P neuroblasts (Sulston and Horvitz, 1977). There remains the possibility that PDB, AS11, and other tail neurons might also be involved in synapses that remain morphologically undetected by present methods. Small gap junctions, in particular, are often difficult to identify in conventional thin sections (cf. Hall et al., 1983). They might also be involved in transient synaptic interactions at another developmental stage.

Neurons with anterior somata

A number of anterior neurons, whose somata lie rostral to the region reconstructed, send axons into the preanal ganglion via the ventral cord. Processes of 13 neurons have sufficiently distinctive characteristics within the preanal ganglion itself to permit repeated identification in different individuals, and we were able, with varying degrees of certainty, to associate all but one of these with specific identified anterior somata by the following criteria: In the limited portion of the ventral nerve cord we reconstructed anterior to the preanal ganglion, the general structure of the cord is very similar to that described previously for the anterior ventral cord (White et al., 1976). Accordingly, characteristic features such as fiber size, cytoplasmic appearance, and relative position within the cord were used to identify 3 major pairs of cord interneurons (PVC, AVA, AVD), 2 of which had anterior somata (AVA, AVD; Hall, 1977). The patterns of synaptic interaction involving major interneurons in the tail are largely as predicted by homology with the anterior regions of the ventral cord (White et al., 1976, 1986; Hall, 1977). The caudal extension of AVAR into the preanal ganglion is depicted in Figure 2A as an example. In the same fashion, several motoneuron axons could be identified by their relative positions in the caudal region of the motor end plates.
      Our results were compared by White with a series that extends further anterior (White et al., 1986); this comparison confirmed all of our major interneuron identifications. Based on fiber positions and in some cases on limited amounts of specific synaptic input, White could also provide a more tentative identification of 7 other interneuron processes, leaving only 1 process unidentified (J. G. White, personal communication). Figure 6 shows schematic views of ALA, AVHL, and AVFR axons.
      A few of these anterior processes have unique features that are not shown in the figures. The 2 AVH processes run near the dorsal extreme of the ganglion and are distinguished by their relatively small size compared to their neighbors. Another pair, AVFL/R, also run near the dorsal extreme of the ganglion but are larger and have a series of rather marked swellings along their lengths. The AVFR process continues caudally through the preanal ganglion, enters the right circumrectal commissure, then passes over the anal ridge where its swellings occur. AVFL ends in swellings within the preanal ganglion. The AVF processes also have a series of vesicle-filled swellings in the head (White et al., 1986). AVG runs as a flattened profile on the right of the preanal ganglion and usually enters the right circumrectal commissure together with DVA and AVFR. (This feature was not observed in an animal reconstructed by Lois Edgar). AVL was identified in only 1 of 2 animals (B126), where it enters the left circumrectal commissure with DVB and DVC. An unidentified process (Fig. 7B, r) runs near the middle of the preanal ganglion in both B126 and B136; it has few synaptic interactions and no other obvious distinguishing features.
      Single processes running in the 2 lateral hypodermal cords are probably the distal extensions of the ALA neuron, a single cell whose soma lies in the dorsal ganglion in the head. These 2 processes terminate by invaginating into the PVC somata to form a series of synapses. Identification of these processes is still tentative (White et al., 1986; White, personal communication).

Figure 6. Schematic models of all preanal and dorsorectal ganglion neurons. Arborizations are shown using the same conventions as in Figure 5. PVPL is shown with 2 short extra branches. Other preanal neurons lack such extra branches. The route of the PDB commissure is unusual. The lower 2 panels on the right show the posterior arbors of a few neurons with anterior somata. The 2 axons from anterior neuron ALA lie in the lateral nerves. Broken line. indicate commissures that lie outside the reconstructed region. For abbreviations, see Table 2.

Ordering of processes within bundles

There are 5 regions of the tail in which many neuronal fibers course together as a bundle through surrounding tissue; these are the 2 ventrolateral commissures (left and right), the preanal ganglion itself (which is in essence a bundle of parallel fibers), and the posterior dorsal and ventral cords. Within such bundles, a degree of ordering is observed that appears as reproducible as the positioning of cell bodies. Figure 7A illustrates this ordering at 1 level of the left and right ventrolateral commissures, for both B126 and B136; the ordering is most easily appreciated by comparing small groups of fibers (e.g., PHA, PHB, and PVQ) with their bilateral homologues across the midline or with their direct homologues in the second animal. While the ordering is certainly not absolute, it is clearly nonrandom. Within these bundles, there is a loose relationship between cell body position and fiber position; lumbar neurons with the most rostral cell bodies tend to have fibers that lie on the ventral side of the commissure.
      Within the preanal ganglion itself, as shown in Figure 7B, the number of fibers is larger, but a similar sort of ordering persists. Interestingly, homologous fibers from the 2 lumbar ganglia come to lie close to one another in the preanal ganglion, suggesting some sort of homologue affinity or a common response to guiding principles (cf. White et al., 1983). In addition, the ordering is dorsoventrally inverted relative to the ventrolateral commissures; those fibers running most ventrally in the commissures occupy dorsal positions in the preanal ganglion.

General distribution and appearance of synapses

Figure 2C depicts the distribution of synaptic contacts within the posterior region of C. elegans. The number of synapses found in 1 animal are summed over 3-um intervals and are graphed separately for the lumbar ganglia, the dorsal nerve cord, and the preanal ganglion. Synapses occur primarily within the preanal ganglion. Their distribution is quite reproducible in the animals examined by ourselves and by White et al. (1986). Most of the preanal synapses involve overlapping fibers from disparate origins; this region of neuropil is the only zone of overlap between lumbar ganglion sense cells and the fibers from many ventral cord interneurons (note the overlap of PHBR and AVAR axons in Fig. 2A). Few preanal synapses involve the 13 cells whose somata lie within the ganglion.
      As Figure 2C also makes clear, most of the synapses are chemical in nature, particularly within the preanal ganglion proper. There are a few NMJs at the anterior end of the ganglion as the pattern of synapses grades over to that more typical of the ventral nerve cord (White et al., 1976). The zone of NMJs along the dorsal cord extends further caudally.
      The general appearance of some representative preanal ganglion synapses is shown in Figure 8. To accentuate membrane profiles for fiber tracing, the fixation conditions for most specimens involve osmication without prior aldehyde fixation. Some ultrastructural detail has consequently been lost, but that which remains is still adequate for general purposes (Fig. 8A). Figure 8, B and C, shows another animal that did receive a primary aldehyde fixative.
      The occasional gap junctions, as shown in Figure 8A, exhibit extended zones of low curvature in which 2 cells show a close apposition. The outer membrane leaflets at these junctions are separated by a 1-nm gap, and the membranes appear more regular, with slightly increased membrane density along the cytoplasmic surface. The typical gap junction is about 100-200 nm in diameter. These small gap junctions are somewhat more difficult to count or compare from animal to animal. The frequency of gap junctions is substantially lower in the tail than has been found elsewhere in the C. elegans nervous system (White et al., 1976,1986).
      Chemical synapses (Fig. 8A,B) are usually en passant, involving unbranched fibers; neuronal somata adjacent to the preanal ganglion neuropil rarely participate in synaptic connections. The presynaptic process is generally enlarged locally and filled with synaptic vesicles. The individual presynaptic profiles are usually 400-700 nm in diameter, compared to the 100-200 nm diameter in nonsynaptic regions of most processes. Serial section evidence shows that the number of synaptic vesicles at a synapse may vary from 25 to 150 or more. Large synaptic swellings may have more than 1 synaptic output to different postsynaptic processes. In most neurons, the vesicles are round and about 40 nm in diameter; however, some sensory fibers (PHA, PHB, PHC) have vesicles of about 50-nm diameter, which are more irregular in outline. There is always an electron-dense button or rod in the cytoplasm immediately adjacent to the presynaptic membrane. There is little postsynaptic specialization, though a slight increase in membrane density is sometimes evident.
      A surprising feature of the chemical synapses is the high proportion of dyadic contacts, that is, synapses with 1 presynaptic element and 2 postsynaptic ones. The proportion of dyadic synapses is 82% in B126 and 86% in B136; this is a much higher proportion than has been noted elsewhere in the C. elegans nervous system, specifically in the pharynx (Albertson and Thomson, 1976), the ventral cord (White et al., 1976), and the nerve ring (White et al., 1986). In the absence of postsynaptic specializations, the identification of dyadic synapses must rest on the geometrical placement of the apparent participants. The 3 neurons participating in one such punctate dyadic contact are commonly seen to participate together in the same relationship in other adjacent punctate dyads, and rather strict rules govern the occurrence of neuron types together as common postsynaptic partners in a given contact. Interestingly, of the remaining 14- 18% of the chemical synapses that are not dyadic, 2/3 are triads (with 3 postsynaptic partners), and only 1/3 (about 5-6% of the total) are monads. Occasional synapses appear to include hypodermal tissue (nematode epithelium) as one of the "postsynaptic" targets or, rarely, as the only target.
      NMJs are similar to those in the ventral cord and are also mostly (75%) dyadic, with a presynaptic motoneuron simultaneously contacting a muscle arm and another neurite, usually the "dendrite" of a class D motoneuron (Fig. 8B,C; see White et al., 1976). NMJs in C. elegans appear identical morphologically to other chemical synapses, except for the possible intervention of basement membrane between nerve and muscle arms.

Figure 8. Synapse morphologies. Electron micrographs of typical synapses in the tail. In A, no aldehyde was included in the primary fixative (see Materials and Methods). A, Two chemical dyadic synapses (large arrows) and a gap junction (small arrows) in the caudal preanal ganglion in B126. B,A chemical synapse (large arrow) and an NMJ (open arrow; also dyadic) in the rostral preanal ganglion. C, An NMJ (open arrow) in the dorsal nerve cord. For abbreviations, see Tables 1 and 2.

General pattern of synaptic interactions

Table 3 lists all the synaptic interactions observed in the 2 animals examined. Chemical synapses are the predominant class. Multiple separate punctate contacts involving the same cells are common; each such contact is counted separately. Synaptic interactions involving unidentified processes number less than 10% of the total interactions. NMJs are also listed, including those in the dorsal cord.
      The few synapses outside the preanal ganglion occur in the dorsal cord or within the lumbar ganglia or their associated commissures. In the lumbar ganglion, reproducible chemical synapses are formed by a pair of lateral fibers (perhaps ALA) to the PVC interneurons. These synapses are monadic and occur in specialized depressions invaginating the surface of the PVC somata. Reproducible gap junctions in the ventrolateral commissures occur between processes of the PLM sensory neurons and the LUA interneurons. Nonreproducible (i.e., single-instance) lumbar gap junctions were also observed between PLM processes and several other cells (Table 3, gap junctions), suggesting a particularly strong tendency of PLM cells to form gap junctions within the lumbar ganglia.
      Within the preanal ganglion, few gap junctions were observed reproducibly (i.e., in both animals). Many of the cases listed in Table 3 involved a bilaterally homologous sensory cell pair. In the animal examined by White et al. (1986), a somewhat larger number of gap junctions were identified, including most of those described here. Of the additional gap junctions they noted, many are between other bilateral homologues. Others are between nonhomologues that are also involved in chemical synapses with each other, as well as some additional PLM contacts in the ventrolateral commissures.

Reproducibility of interactions. In the listings in Table 3, bilateral homologues are clearly involved in similar patterns of chemical synaptic interaction. All contacts involving either the right or left member of each cell pair have been grouped together in order to emphasize this point. The number of synapses seen between most 3-cell groups are not highly repeated in a given animal or when comparing B126 to B136. In fact, many specific dyadic interactions are seen only once in one animal and not at all in the other. However, when one sums all of the contacts between cell pairs disregarding dyadic partners the correspondence is much better (Table 4; Hall, 1977), and when one combines the data from bilateral homologues, the correspondence is very high (Table 5; see Discussion).
      Each neuron synapses onto a restricted set of cells. For most presynaptic neurons, the list of postsynaptic targets is rather small, and certain pairs of postsynaptic targets are clearly preferred (see Discussion). A few presynaptic neurons do not appear to focus on preferred pairs, but choose a wider assortment of targets (e.g., PHA and DVB) without many repeated contacts to the same targets. PHB synaptic outputs are more typical. Note, in Table 3, the number of different permutations by which PHBL/R neurons contact PVCL/R and AVAL/R simultaneously, and the relatively large number of these contacts in each animal. There is a preponderance of ipsilateral contacts among the PHB->PVC+AVA synapses, and PVCL receives many more contacts than PVCR. This ipsilateral bias seems to stem mostly from an incomplete mixing of left and right lumbar homologues as they enter the preanal neuropil; more rostral portions of the preanal neuropil show more frequent contralateral contacts in many synapse classes.
      A few neurons are contacted only infrequently by PHB neurons. By comparing data from several animals, it is possible to determine that the less common synaptic targets are, in fact, quite variable and may be contacted once or twice in some animals, but not in others. This fact is more clearly noted by ignoring dyadic relationships, and reclassifying all synaptic contacts onto a 2-way table. Table 4 shows a subset of our data from Table 3, displaying the number of synaptic contacts between 26 neurons in the tail, where a dyadic synapse made by PHBL-> PVCL + VA12 is shown as 2 contacts: one from PHBL-> PVCL and another from PHBL-> VA12. For ease of presentation, Table 4 shows only about half of the possible synapse combinations, which require a 40 x 40 matrix to include all cells present in the preanal neuropil. However, Table 4 is arranged to include almost all common synapse types; most of the other boxes in the full 40 x 40 matrix are empty. Table 4 also includes the same type of data obtained from an animal reconstructed by White et al. (1986), using numbers compiled from their illustrations. Some synapses are very highly conserved, especially PHBL-> PVCL, which is the most highly repeated contact in each animal. As discussed below, we designate synapses that are repeated 6 times or more in the 3 animals combined as the normal targets (lumping all contacts involving bilateral homologues or homologous motoneurons). Thus, the PHB-> PVC boxes are marked by a heavy outline, as are the less common PHB-> PHB boxes. The latter are exclusively contralateral as there are no self-synapses, whereas the former are more often ipsilateral. There are scattered synaptic contacts shown in Table 4 that do not fall within the heavily outlined boxes; these are considered "infrequent synapses," and below, we discuss the possibility that they represent developmental "noise". When the entire data set is examined on a 2-way table, the number of infrequent synapses may total about 20% of all contacts, depending on how they are defined.

Partner relationships in dyadic synapses. The majority of the dyadic chemical synapses in the preanal ganglion occur between a subset of 21 neurons. The data in Table 5 is derived from that in Table 3 by combining the results from B126 and B136 and by combining the interactions of bilateral homologues. Table 5 lists the possible pairs of postsynaptic partners in a set of 2-dimensional arrays, one array for each important presynaptic neuron class. In each case, the dyadic contacts for a class of presynaptic neurons are listed singly, each position in the array corresponding to the 2 possible types of postsynaptic neurons involved (i.e., as combinatorial pairs of postsynaptic partners).
      Each array in Table 5 includes, as its diagonal, positions for cases in which the 2 postsynaptic partners are homologues. Examination of Table 5 reveals that these diagonals are virtually empty, indicating that bilateral homologues are contacted together very rarely. This is true despite the fact, as shown in Table 3, that it is extremely common for a given presynaptic neuron to contact both members of a bilaterally homologous pair, overall. This can occur, of course, only if the 2 bilaterally homologous cells are virtually always contacted separately, in different punctate contacts, as is indeed the case. This observation suggests that there is a proscription against bilateral homologue involvement at the single contact level, but that this proscription does not apply globally when contacts are summed.
      While the array diagonals are surprisingly empty, each array is characterized by the grouping of observed contacts into a few specific positions, each representing the joint occurrences of a postsynaptic neuron of one specific type with another of a different specific type. The importance of these groupings is supported by examination of the minority of triadic synapses (cf. Table 3); about 75% of these can be considered as modified versions of one or another of the common dyadic classes, in the sense that the first 2 postsynaptic neurons are as in a common dyadic class, and the third is a bilateral homologue of 1 of the first 2. We believe, as discussed below, that these dyadic (and triadic) groupings are functionally important for the divergence of information into a limited number of alternative routes for processing.

Wiring diagram. As the foregoing section makes clear, in the desired wiring diagram, the predominant synapses can be accurately represented as double-headed arrows, going from one presynaptic cell type to 2 different postsynaptic ones. In Figure 9, this symbolism is used, and the thickness of the arrows is used to indicate the frequency of the corresponding synapse type, as judged by the number of separate punctate contacts (though true physiological synapse strength obviously cannot be inferred from this data).
      Certain features, probably of functional importance, stand out in the wiring diagram of Figure 9. First, it is striking how predominant is one class of synapses, that from the PHB sensory cells onto the 2 major interneuron types AVA and PVC. It is noteworthy that the interaction pattern of the PHB cells is quite different from that of the PHA cells, even though these 2 types of sensory cells have adjacent ciliated dendrites in the phasmidial pocket. Both phasmidial neuron types PHA and PHB exhibit what appear in the representation to be self-synapses, but are in fact synapses from one bilateral homologue to the other. There is a strong tendency towards convergence of several sensory neuron outputs onto 3 major interneuron classes, AVA, PVC and AVD; these are major interneurons of the ventral cord and the nerve ring. Frequently, the 2 postsynaptic cells types jointly contacted in a given type of synapse (e.g., AVA and PVC) make direct contact with one another in other synapses; interestingly, such contacts are usually unidirectional (e.g., AVA is presynaptic to PVC but not vice versa). There are few instances of reciprocal synapse formation; in our data, they only involve the major interneuron AVA and the loeal interneuron LUA. [The data in White et al. (1986) include a few reciprocal contacts between AVA and PVC and between LUA and AVD.] Interestingly, and as a corollary of the previous point, there are few dyadic synapses in which AVA and LUA are jointly contacted (none in our data, 4 instances in White's data); indeed, the only significant dyadic input to LUA is from AVA. Finally, for each presynaptic cell type having several different outputs, there is a very strong tendency for the several outputs to share one postsynaptic partner in common, usually either AVA or PVC.
      In the discussion, these features serve as the focus of an attempt to understand both the function and the development of this synaptic circuitry.

Figure 9. Wiring diagram for the tail. Double-headed arrows indicate dyadic chemical synapses. The width of individual lines is proportional to the relative frequency of occurrence of synaptic contacts. Excluded from the diagram, but probably of functional significance, are the following frequent chemical synapse classes: PHA->AVH, PHC->DVA, ALA->PVC, and DVB->DVC. Many of the interneurons have many additional synaptic interactions either in the ventral nerve cord or in the nerve ring. Triangles, sensory neurons; hexagons, interneurons; circles, motoneurons; -|, gap junctions. See Results, Wiring diagram, for format; see Table 2 for abbreviations.

Web adaptation, Thomas Boulin, for Wormatlas, 2002