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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