The Posterior Nervous System of the Nematode Caenorhabditis elegans

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

Material and methods

Fixalion and embedding. Caenorhabdilis elegans (var. Bristol, strain N2) were grown monoxenically at 20°C in Petri plates containing nematode growth medium (NGM) agar preseeded with Escherichia coli strain OP50 (Brenner, 1974). Synchronous populations (initiated during a 45-min hatching period) were grown at 20°C for 50 hr, at which point they had just begun to lay eggs (Byerly et al., 1976). Subsequent fixation and embedding steps were as described by Ware et al. (1975). Briefly, animals were rinsed off plates and washed several times in S medium (Brenner, 1974), anesthetized for 15 min in 0.5% 1-phenoxy-2-propanol in 0.1 M cacodylate buffer (pH, 7.4), and fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer (pH, 7.4) for 1-4 hr at 23°C. The tails were then cut off with an X-acto blade and stained for 45 min in 1% uranyl acetate, 0.05 M maleate buffer (pH, 6.1). After dehydration through a graded series of ethanol and acetone, the cut pieces were embedded in Epon-Araldite.
      The lack of an aldehyde fixation resulted in a rather washed-out appearance, which aided the serial reconstruction without preventing the identification of synaptic contacts. However, for some of the micrographs shown in Figure 8, aldehyde-fixed tissues were used to allow a more representative view of the cytoplasmic features of chemical synapses.

Sectioning and reconstruction. For microtomy, individual tail pieces were cut out of a flat mold and mounted on support blocks with epoxy cement. Serial sections were cut perpendicular to the body axis with a diamond knife on a Sorvall MT2B ultramicrotome at a thickness of 50-60 nm. Sections were collected in strips on slot grids (15-30 sections per grid). Continuity was very good, as the loss of sections was generally 3% or less. Typical series were 1000-2000 sections long.
      Sectional series were used to derive general information from 8 adult animals (Hall, 1977). All of the general features described, down to the level of cell numbers and positions, have been confirmed in 4 or more animals. More detailed information, including the identification of cell types by fiber tracing, were principally derived from 2 long series, B126 and B136, which are essentially complete for all of the posterior ganglia, for their associated commissures, and for the included synapses. In addition, many of the reported morphological findings were confirmed or extended by Lois Edgar in an independently reconstructed series (L. Edgar, personal communication) and by White et al. (1986) in a reconstructed series. Repeated reexamination of our series and those of White et al. were required to settle a few difficult points, particularly where several fibers pass together through tortuous commissures. These comparisons served to double-check the identities and features of posterior cells, but more importantly, they allowed many anteriorly projecting processes to be identified as distal parts of already described anterior cells (White et al., 1986). There are several minor changes in axon identifications compared to those previously reported (Hall, 1977).
      The reconstruction of long fiber tracts (500-800 sections) was facilitated by serial-section cinematography, using methods and equipment designed by R. Ware (Levinthal and Ware, 1972; Ware et al., 1975). The ganglia were reconstructed from high-magnification prints of every section; this was required to catalog every synapse and to trace difficult commissures. Microscopy was done on Zeiss EM9 and Philips 300 electron microscopes.

Nomenclature. We have adopted the cell-naming system of White et al. (1986). Bilateral pairs of tail neurons are referred to together by a 3-letter name and separately by the same name followed by L or R to indicate the left or right member of the pair. Ventral cord motoneurons have 2-letter names, followed by a number that corresponds to their place in order along the ventral cord (see Table 1 for a list of abbreviations and Table 2 for a summary of cell types and nomenclature).
Our characterizations of neurons as motoneurons, sensory neurons, or interneurons were based only on our anatomical findings, except for the PLM sensory neurons, where reported studies using laser microbeam ablation and mutants have provided direct functional evidence for the mediation of a touch response (Chalfie and Sulston, 1981; Chalfie et al., 1985). Among all other neurons, those with reproducible synaptic outputs onto muscle cells were called motoneurons, those with apparent sensory specializations were called sensory neurons, and all others were called interneurons, provided that they involved both pre- and post- synaptically somewhere in the animal (not necessarily in the tail). One limitation of this scheme is that we may have misclassified sensory neurons whose sensory specializations were not obvious. A few neurons have properties combining those of 2 described classes; such instances are dealt with specifically in the text.
      Among the ascribed interneurons, we have designated as "major" and "minor" those with many and few synaptic contacts, respectively. However, because the physiological strength of the synapses cannot be determined with present methodology, these designations do not rule out the possibility that "minor" interneurons, with only infrequent synaptic interactions, may nonetheless make significant contributions to the animals behavior.

Enumeralion of synapses. For the cataloging of synapses, 2 entire sectional series described in this paper (B126 and B136) were used. In each series, occasional sections were missing, and occasional sections were relatively poor in quality, but there were no serious gaps in either series. A primary list of synapses was made for each animal, noting the position of each synapse by section number. Chemical synapses included in this list showed presynaptic specializations for 3-10 successive sections (an electron-dense button or rod along the cytoplasmic face of the presynaptic membrane and increased membrane density, accompanied by a cluster of vesicles). A secondary list of possible chemical synapses was made where 2 or 3 sections adjoining missing or damaged sections displayed indications of a presynaptic specialization. The pattern of interactions in each secondary list was not noticeably different from that in the primary lists; hence, the 2 lists were combined in each case. For B126 and for B136, the combined lists contained 159 and 144 synapses, respectively. These lists include every morphologically identifiable posterior synapse in the corresponding animal, exclusive of neuromuscular junctions (NMJs) in the dorsal cord. Synaptic contacts in the dorsal nerve cord were enumerated separately in an essentially complele series from B136.
      Gap junctions (electrical synapses) tend to be rather small and variable, depending upon section angle. Thus, their identification is more subjective and difficult to codify. It is less certain that we have enumerated all gap junctions in the posterior nervous system in either B126 or B136.

Web adaptation, Thomas Boulin, for Wormatlas, 2002