The reconstructed nervous systems described in this study were all derived from the nematode Caenorhabditis elegans (var. Bristol); these were cultured on lawns of E. coli grown on agar Petri plates (Brenner 1974).
Worms were rinsed off Petri plates and fixed in 1% osmium tetroxide in 0.1 M sodium phosphate, pH 7.4 for one hour at 20 C. Pre-fixing in glutaraldehyde was not done in this work because, although this method gives better preservation of fine structure, we found that osmium alone gave better contrast to cell membranes, and this facilitated the resolution of process outlines in regions of dense neuropile.
After fixation, the worms were spread on a thin layer of 1% agar and cut in half. The cut worms were covered with a drop of molten 1% agar, and blocks of agar containing a single half worm were cut out. These were dehydrated through a graded series of alcohols to propylene oxide, then to propylene oxide plus 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 60C.
An LKB ultratome III was used with a diamond knife to cut transverse serial sections of approximately 50 nm thickness. Ribbons of sections were generally picked up on Formvar coated 75-mesh copper grids. The sections in the region of the head, where most of the nervous system is situated, were picked up on slot grids, as it was found to be necessary to have every section in this region for successful reconstructions. Grids were stained with a 5% aqueous solution of uranyl acetate for 10 min at 60 C and then with lead citrate for 5 min at room temperature according to the procedure of Reynolds (1963). Sections were photographed on cut film with an AEI 6B or an AEI 802 electron microscope. Most reconstructions were done directly from prints of micrographs of nervous tissue. In the region of the nerve ring, four-way montages were necessary; in other regions, single prints were sufficient. Every section was photographed in the region of the nerve ring and other areas of dense neuropile: photographs of every third section usually sufficed for following process bundles. Some use was made of a computer-aided reconstruction system described by White (1974) and Stevens & White (1979), but most of the reconstructions were done by hand from a total of about 8000 prints.
Small groups of processes were given arbitrary labels, which were written onto the prints with Rotring drafting pens. These labels were carried through all the pictures in which the associated processes were present, and this procedure was repeated until all process profiles were labelled. Processes could then be joined to other processes where branches had occurred, or ultimately be assigned to particular neurons if their cell bodies were within the scope of the reconstruction. When all the labelling was completed, each process was individually followed through every section in which it appeared, and a list was compiled of all the synaptic contacts that it made. In this way all synaptic contacts were recorded twice, once for each member of an interacting pair of processes. This provided a useful check on synapse scoring as any synaptic contact that was only scored once was reappraised.
The reconstructions were done piecemeal with data from five overlapping series; these were designated N2T, N2U, JSH, N2Y and JSE (figure A1, Appendix 1). The structure was found to be sufficiently invariant for equivalent processes and cell bodies to be identified in the region of overlap of two series. The N2T series was the first extended series to be cut in the head; the reconstructions of the head sensilla described by Ward et al. (1975) were based on this series. Although this series extended through the nerve ring and into the ventral cord, mesh grids were used and it was found that the inevitable occasional section loss, through obscuration by grid bars, allowed only a limited reconstruction to be done of these regions. The N2U series was from an old hermaphrodite that gave good quality pictures. It was sectioned on slot grids through the nerve ring and anterior ventral cord and a complete reconstruction of this region was obtained. This series also covered more than half the body length of the animal and enabled the anterior ventral and dorsal cords to be reconstructed. The JSH animal was a fourth stage (L4) larva, which was sectioned on slot grids. A complete reconstruction of the nervous system in the nerve ring and anterior ventral cord was obtained from this animal. This allowed the structure deduced from the N2U series to be validated in these regions, which are the most difficult to reconstruct because they contain dense neuropile with many processes that run close to the plane of sectioning. Few significant differences in structure that could be age-related were seen between the N2U and JSH series. The tail ganglia and some of the posterior ventral and dorsal cord were covered in the JSE reconstruction. The region between the anterior extremity of the JSE series and the posterior extremity of the N2U series has not been reconstructed in a hermaphrodite. A long series that overlapped at both ends, designated N2Y, was obtained from a male animal (Sulston et al. 1980, in which it was referred to as series 4). The motoneurons of the ventral cord and the cells from the posterior lateral ganglion were reconstructed from this animal. The motoneurons (with the exception of the sex-specific VCn class) exhibited essentially the same synaptic behaviour as their anterior counterparts in the hermaphrodite. As there was also no reason to expect any sex-related differences in the cells of the posterior lateral ganglia, these data were incorporated to enable a complete reconstruction of the whole nervous system to be obtained. The structure that is described is a composite that has been derived from all these series except JSH.
The biggest problem that was encountered in the course of the reconstruction work was the location of errors. Errors were generally made in one of three ways. (1) The most prevalent was human error, which would occur when following long featureless process bundles and which typically resulted in switches in process labels. (2) Many processes run close to the plane of sectioning in the vicinity of the nerve ring, with the result that the membranes of these processes would often be cut obliquely and give indistinct images. This made process identification very difficult in such situations, leading to the second most prevalent source of errors. (3) Similar errors of process identification also occurred in regions of poor image quality caused by dirt on sections or loss of sections on grid bars although, surprisingly, this was the least prevalent source of errors.
Errors generally manifested themselves by the appearance of an improbable structure, such as a process that was joined to more than one cell body or conversely not joined to any at all. Much of the nervous system was found to be bilaterally symmetrical; some of the sensory receptors in the head have higher levels of symmetry. Any deviations that were seen from expected symmetries were considered suspect. Errors were located either by exhaustive searching of every section that contained the process that was in question, or by looking at the reconstructions for discontinuities in synaptic behaviour, and then closely checking the regions of the process where the discontinuities occurred. In this way a complete, self-consistent structure was built up. The structures of the major regions of neuropile have been validated by separate reconstructions; the JSH series in the case of the nerve ring and the N2S series in the case of the ventral cord (White et al. 1976). Hall has undertaken an independent reconstruction of the tail ganglia; the structure that he describes is essentially the same as the structure that we describe here (Hall 1977).
We are reasonably confident that the structure that we present is substantially correct and gives a reasonable picture of the organization of the nervous system in a typical C. elegans hermaphrodite. It is likely that in the elaboration of a structure of this complexity that a few small errors might have crept in, but we feel that these may be quite limited because of the amount of cross-checking that was done. A few minor ambiguities still exist, however, which would require a considerable effort to clear up. These are described in Appendix 2.
We have adopted a uniform system of nomenclature for naming the neurons and associated cells of C. elegans. Unfortunately it was not practicable to make such a system compatible with the various nomenclatures that have been used up till now. Appendix 3 lists the equivalences between these systems and the one used in this study. Neurons are given arbitrary names consisting of three upper case letters. The last letter can alternatively be a number of up to two digits. Additional symmetry descriptors are added to the name in the cases of groups of cells that are in the same class and related to each other by simple geometric symmetries. These descriptors are D or V (dorsal or ventral) and L or R (left or right). A group of cells with six-fold symmetry, such as IL1, has as its members: IL1DL, IL1DR, IL1L, IL1R, IL1VL and IL1VR. The members of the classes of motoneuron in the ventral cord do not have these symmetrical relations with each other. In these cases, the third digit of the class name is a numeral, which represents the anterior or posterior location of the neuron relative to its fellow class members; for example, VA3 is the third VA motoneuron. The use of the three-letter name without descriptors implies all members of the class if there is more than one. For the motoneurons, a lower case n is used in the third digit position to represent the generic name for all class members (for example, VAn). A slight modification of this system is used to describe the associated cells of sensilla, i.e. the sheath and socket cells. A sheath cell is designated by 'sh' and a socket cell 'so'. Thus in the case of the right sub-dorsal cephalic sensillum, the neuron is referred to as CEPDR, the sheath cell as CEPshDR and the socket cell as CEPsoDR.
Web adaptation, Thomas Boulin, for Wormatlas, 2001, 2002