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Organism The nematode used in this work was Caenorhabditis elegans (var. Bristol). It was grown at room temperature on agar petri plates seeded with E. coli as described by Brenner ('74).
Fixation and embedding
Worms were rinsed off a fresh petri plate
and fixed in 0.5% OsO4 in 0.1 M sodium
phosphate, pH 7.4, for one hour at room
temperature. Some specimens were prefixed in glutaraldehyde by rinsing them off
plates with ice-cold 3% glutaraldehyde in
0.1 M sodium phosphate, pH 7.4, and then
cutting them in half to allow penetration of
the fixative. After two hours on ice, worms
were removed from glutaraldehyde by centrifugation, rinsed and then post-fixed in
OsO4 as above.
After fixation, the worms were spread on
a thin layer of 1% agar and any uncut
worms were cut in half or cut nearer the
head if only the tip of the head were going
to be sectioned. The cut worms were covered with a drop of 1% agar, and blocks
of agar containing a single specimen were
removed, dehydrated through a graded series of alcohols to propylene oxide, then
propylene oxide and araldite (CY 212 resin, CIBA, Ltd.) and then into araldite at
room temperature overnight. The following
day they were transferred to fresh araldite
and polymerized in gelatin capsules overnight at 60°C.
Sectioning, staining and electron microscopy Serial sections of approximately 500 Å were cut with diamond knives on an LKB Ultratome III. Most of the series were cut transversely to the long axis of the nematode but several series were cut longitudinally. Ribbons of sections were routinely picked up on formvar-coated 75-mesh copper grids taking care not to align a ribbon with a grid bar. When every section was needed ribbons were collected on formvar-coated slot grids. For analysis of the sensory endings mesh grids were satisfactory. Grids were stained with 5% aqueous uranyl acetate for ten minutes at 60°C and then with lead citrate for five minutes at room temperature according to the procedure of Reynolds. Each section was photographed onto a single plate with an AEI EM 6B or AEI EM 802 electron microscope, except for the region of the nerve ring which was photographed in four parts and the prints assembled into a montage.
Reconstruction
The anterior tip of the head of the nema-
tode contains the specialized endings of
sensory neurons and processes of a number
of associated cells. In this region the processes have distinctive morphological features which can be reconstructed from
about 200 serial sections. Four such series
have been analyzed completely, and many
of the features have been confirmed in less
complete series from about 25 additional
animals. Each series of sections was enlarged and printed in rough
alignment. All
individual cell processes were followed from
print to print and labelled with colored
pens on successive photographs. Small
groups of processes were followed together
to make identification easier and to prevent
accidental jumps.
The ciliated endings of many of the processes in the head suggested that these were
dendrites of sensory neurons. To confirm
this and to determine the axonal projections
of these neurons, every process in the head
was followed in its entirety. This involved
complete reconstruction of a series of 1,600
sections which was carried out as follows.
The processes were traced back from the
tip of the head for 850 sections. They form
six nerve cords each of which was traced
independently. At the same time reconstruction was begun from the other end of
the same series, identifying cell bodies and
their axonal projections. When the two sets
of assignments were joined, symmetrical
cells were compared. Complete concordance
was found. Thus the classification of cells
by their positions and axonal projections
matched the classification of processes by
the similarity of their neuron endings. The
central structures have been confirmed on
an independent series but tracing the nerve
cords has only been done once. Nevertheless, the concordance found gives us
confidence in the assignments made.
The diagrams showing the structures of
the various sensilla and the positions of
cell bodies (figs. 4, 14, 18, 40) were prepared by tracing the outlines of cells in a
sensillum from equally spaced transverse
sections. The longitudinal projection of the
structure was then drawn by hand, Longitudinal series of sections confirmed that interpretations based on transverse sections
were correct.
To reconstruct the shapes of the amphidial cells the transverse outlines of each
cell were traced into a computer directly
from the photographs using a coordinate
digitizer to draw the cell outlines. A computer graphics terminal then displayed
longitudinal projections which could be
rotated to aid visualization. The drawings
of these cells shown in figures 19, 20 and
21 were prepared from print-outs of the
computer display. The details of the system
will be described elsewhere.
Terminology We call all the sense organs in the tip of the nematode's head "sensilla" which cornforms with the definition given by Bullock and Horridge ('65, p. 1608) "...simple types of sense organs involving only a few neurons." In much of the earlier nematode literature, sensilla has been used only for the amphids; other sense organs are designated as "papillae, setae, etc." We also adopt two new terms "sheath" and "socket" cells for the two non-neuronal cells invariably associated with each sensillum. The reasons for this are presented in the discussion. We have followed the nomenclature of DeConinck ('65) in naming the individual sensilla.
Web adaptation, Thomas Boulin, for Wormatlas, 2002