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

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Table of contents  -  Abstract  -   Introduction  -   Materials & Methods  -   Results  -   Discussion  -   References

Material and Methods

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