There are, perhaps, two fundamental questions in the field of neurobiology: how neurons
organize themselves during development into specifically interconnected networks, and how
such a network functions. A knowledge of the detailed structure of a nematode's nervous system
does not in itself provide any answers to these questions, but it does at least provide a framework
within which it is possible to pose rather more specific questions.
The development of a nervous system can be divided into three separate phases. The first is the generation of a group of differentiated neurons; the second is the outgrowth and guidance of processes from these neurons and the third is the establishment of connections between processes. The structural data on the nervous system provides information that is most pertinent to the last two phases. This is because the final structure represents the ultimate consequences of the execution of these two processes. We will go on to discuss how these two developmental processes, together with the question of nervous system function, may be further explored in C. elegans.
One of the most striking features of the nervous system of C. elegans is the precision with which
processes are positioned relative to their neighbours within process bundles. Synaptic contacts
are made en passant between adjacent processes; the set of possible synaptic partners that a
neurone may have is therefore limited to the set of processes that are neighbours. Given the
unbranched nature of nematode neurons, this set is usually a relatively small subset of the total
complement of neurons that make up the nervous system. Within this neighbourhood, however,
neurons are fairly highly connected, making connections to nearly half their neighbours on
average (White et al. 1983). Furthermore, there is circumstantial evidence that this level of
connectivity may be independent of neighbourhood, i.e. that a given neuron may make
synaptic connections to more or less the same percentage ofits neighbours no matter what class
they may be (White et al. 1983). Thus process placement must be a major determinant in the
establishment of the patterns of connectivity within the nervous system
of C. elegans.
It seems likely that there may be two aspects of process placement: substrate guidance of pioneering processes to establish process tracts (Berlot & Goodman 1984), and the positioning of processes relative to their neighbours within bundles once process tracts have become established. A distinctive feature of the organization of processes within bundles is the close associations that are seen between specific processes, or between a process and the basal lamina. Such associations are probably the consequence of selective adhesive affinities between the associating entities. Given the probable importance of selective adhesivity in determining connectivity, it is worth considering, within the context of the nematode's nervous system, how such phenomena may be further investigated. Many behavioural mutants have been isolated in C. elegans; it is likely that most of their phenotypes are the consequence of alterations in the nervous system. It is also likely that some of these alterations could take the form of misplaced processes. Up to now, relatively few behavioural mutants have been analysed at the ultrastructural level. This is mainly because of the considerable effort that is required to reconstruct a significant portion of the nervous system from electron micrographs. Recently, staining techniques have been developed that allow the visualization of specific processes or process bundles in whole mounts of C. elegans when viewed with the light microscope. In one of these techniques, sensory process tracts are labelled by dye filling (Hedgecock et al. 1984). In another, processes of certain neuron classes are labelled with monoclonal antibodies and viewed by immunofluorescence in whole mounts (Okamoto & Thomson 1984). Such techniques will facilitate the pre-screening of behavioural mutants for those that have abnormalities in process placement. Selected mutants may then be subjected to a full ultrastructural analysis.
With the dye uptake technique, certain mutants have been found to have abnormal projections from sensory receptors (Hedgecock et al. 1984); such mutants could be candidates for substrate guidance. The defects in these mutants could either be located in the neurons, or in the substrate upon which they grow. It may be possible to distinguish between these two possibilities by means of mosaic analysis (Herman 1984).
Of the mutants that have been analysed by serial section reconstruction, one (unc-30) has been found to have misplaced processes on the VDn and DDn motoneuron classes (J. G. White, S. Brenner & R. Durbin, unpublished observations). The disposition of the processes of the other motoneuron classes in the ventral cord appears normal. It seems possible that such a mutant could be defective in the class-specific expression of an adhesion factor. The molecular analysis of genes that affect process placement may provide a route to an eventual understanding of the function and deployment of region-specific adhesion molecules. Another route to the same end may be taken by directly looking for putative adhesion molecules. Candidate molecules would be expected to be common to a group of processes that are closely associated together. Such a molecule could be sought either directly by using antibodies, or indirectly by looking for species of messenger RNA that show the appropriate neuronal distribution.
Although we have played down the role of synaptic specificity in the generation of the
pattern of connections within the nervous system of C. elegans to a certain extent, it is clear that
there has to be some level ofspecificity. On average, a neuron is presynaptic to about 15%
of its neighbours (unpublished observations). The subset of neighbours that are postsynaptic
to a given neuron is fairly constant from animal to animal, and So is presumably actively
selected. It is likely that synaptogenesis is initiated by a cell-cell recognition event. Such an
event may involve the binding of a surface receptor molecule on one cell to a matching 'label'
molecule on another cell. If all cell classes had single distinguishing label and receptor types,
then the set of synaptic partners of a given cell class could never intersect with that of another.
Such intersections are, in fact, the general rule in the nervous system. Therefore, if such a
label-receptor system is the basis of synaptic specificity, then the labels (and/or receptors) have
to be arranged combinatorially.
It is probably not reasonable to assume that the pattern of connections seen between processes in a particular neighbourhood is solely the consequence of the intrinsic specificities of the neurons involved. There are suggestions that interactions between synapses may act to modify certain patterns of synaptic connection that might otherwise form as a consequence of specific neuron-neuron recognition. There are slight differences in connectivity between the dorsal and ventral members of the classes SMB, SAA, OLQ and RMD. These differences are manifested as reciprocal substitutions of gap junctions for chemical synapses and chemical synapses for gap junctions. This behaviour may suggest that there are interactions between these types of connection in these circumstances, and that these interactions result in a mutual exclusivity of chemical synapses and gap junctions.
We have used the criteria of morphology and connectivity to define the 118 classes ofneuron that have been described. Given that a particular neuron can only select synaptic partners from its neighbourhood, it is probable that there are classes that we have defined that have the same intrinsic synaptic potential; in other words, if placed in the same neighbourhood they would select the same subset of neighbours as synaptic partners. Therefore, the number of classes that we have defined (118) is almost certainly an overestimate of the number of neuron types that are intrinsically different in their specificities. It is strongly suspected, on the basis of morphology, that AQR and PQR are members ora single class, as are ALM and PLM, ALN and PLN, and AVM and PVM. It is probable that there are other class equivalences that are not so obvious, particularly among the interneurons, which often do not have distinguishably different morphologies. It may be possible to identify such 'superclasses' by a neighbourhood analysis. If the neighbourhoods from two classes are compared and common neighbours are identified, then it is possible that the two classes may be members of a superclass, if the pattern of synaptic connections made to the common neighbours is the same in each case. By considering all pairwise combinations of classes, and then reiterating the process considering all members of putative superclasses as equivalent, it may be possible to arrive at a logically consistent set of superclasses. These superclasses will define groups of cells that have intrinsically identical synaptic specificities. Such an endeavour may notjust be an idle intellectual exercise, as a knowledge of such 'supergroups' could facilitate the identification of mutants that have altered labels or receptors. Such mutations would be expected to have pleiotropic consequences, affecting all the members of a supergroup. Thus mutants that affect connectivity of all the members of a particular supergroup are candidates for mutants with altered labels and/or receptors. An analysis of such mutants may provide a possible route towards an understanding of the molecular basis of synaptic specificity.
The relative simplicity of the structure of the nervous system of C. elegans provides a challenge
to determine how it functions. The main disadvantage of this nervous system from the point
of view of functional studies is that the small size of the component neurons precludes the use
of electrophysiological recording techniques. Such techniques can, however, be used with
Ascaris. There are considerable homologies between the ventral cord motoneurons of Ascaris
and C. elegans (Stretton et al. 1978); more recently, similar homologies have been seen in the
interneurons of the retro-vesicular ganglion (Donmoyer, Angstadt and Stretton, personal
communication). The neurotransmitter dopamine has been shown to be present in the same
classes ofcells in the two animals (Sulston et al. 1975). It seems likely that such structural and
biochemical similarities may indicate an underlying functional similarity, justifying the
extrapolation of data obtained from one animal to the other. Electrophysiological studies on
homologous cells in Ascaris suggest that the DAn, DBn, and ASn motoneurons of C. elegans are excitatory, whereas the DDn and VDn motoneurons are inhibitory (Johnson & Stretton 1980).
Further work may yield information about the role of the interneurons of the ventral cord in
activating the motoneurons.
The functional aspects of the nervous system of C. elegans may be studied directly by
characterizing the behavioural consequences of specific lesions in the nervous system. Lesions
may be produced by laser microsurgery (Sulston & White 1980), a technique that is capable
of removing any cell or small group of cells within the nervous system. As an alternative, use
may be made of lesions produced as a consequence of mutations. For example, one mutant,
unc-30, specifically affects the organization of the VDn and DDn motoneurons in the ventral
cord, leaving the other motoneuron classes relatively unaffected (J. G. White, S. Brenner &
R. Durbin, unpublished observations). This mutant is uncoordinated in forward and backward
locomotion. When stimulated by a tap on the head, instead of backing away, these animals
shorten by simultaneously activating both their ventral and their dorsal muscles. This
behaviour is what one would predict if cross-inhibition between the dorsal and ventral sides
were lacking. This observation reinforces the suggestion, originally made on morphological
criteria, that the VDn and DDn classes function as cross-inhibitors.
The combined techniques of laser microsurgery, mutants and tests for drug responsiveness
have been used to produce detailed models for the function of the circuitry associated with the
touch response (Chalfie et al. 1984), and the circuitry that controls egg-laying (Horvitz et al.
1984). Other areas of the nervous system should be equally amenable to such methodologies,
particularly the chemosensory system. This system is particularly attractive, as the chemotactic
response has been characterized (Ward 1973; Dusenbery 1974) and many mutants that are
defective in chemotaxis ha ve been isolated (Dusenbery et al.
1975; Lewis & Hodgkin 1977).
We would like to thank our colleagues who, over the years, have offered advice and encouragement for this work. We would particularly like to mention Donna Albertson, Martin Chalfie, Richard Durbin, Edward Hedgecock, Robert Horvitz and John Sulston for the many stimulating discussions that we have had together, and also Donna Albertson, Leon Nawrocki and John Sulston for reading and commenting on the manuscript.
Web adaptation, Thomas Boulin, for Wormatlas, 2001, 2002