discussion

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Table of contents   -    Introduction   -    Mat. & Methods   -    Discussion   -    References


(a) How is it made?
(b) Guidance
(c) Polarity
(d) Lineage
(e) Competition
(f) Synaptic specificity
(g) How does it work?

DISCUSSION

The reconstructions which have been described have all been done on the anterior part of the cord. The longest series (the U series) covers just under one half of the probable total cord logic. J. Hodgkin has reconstructed some series that have been cut in the tail, covering the last segment and the beginning of the pre-anal ganglion, and it is possible to identify all five classes of motor neurone in the region on morphological criteria. It is not yet clear, however, whether the same set of interneurones are driving the class A, AS and B motor neurones in the head.

All the connections that have been described between the various classes of cell were allocated on morphological criteria alone. Mark, Marotte & Mart (1972) have suggested that morphologically normal looking synapses may not be functionally active in some instances. Neurones in C. elegans are so limited as to the extent and the diversity of their synaptic input that it seems unlikely that any particular group of synapses from one class of cell to another is 'silent'; however, this remains a possibility. It has been suggested that the vesicles in inhibitory synaptic terminals are more elongated than those from excitatory terminals (Uchinzono 1967). No consistent differences in vesicle morphology could be detected in the various classes of cells in the ventral cord so either none of the synapses described are inhibitory or more probably this criterion is not applicable to C. elegans.

Gap junctions are presumably the sites of electrotonic coupling between cells (Furshpan & Potter 1968), but again their morphological appearance gives little indication as to the electrical characteristics of this coupling .

(a) How is it made?

The structure of the ventral and dorsal cords of C. elegans has been described in some detail. Although this information does not provide unequivocal evidence for any particular way in which the structures might assemble themselves certain features do suggest possible mechanisms that may be used.

(b) Guidance

Neurones always lie in close apposition to hypodermal cells and are enclosed in a 'bag' of basement membrane. Muscle cells are situated on the other side of these membranes with neuromuscular connections apparently being made through them. It seems likely that the system of hypodermal cells with their associated basement membranes could act as a system of 'conduits' which constrain and guide growing nerve processes to grow along well defined tracts. It has been demonstrated that growing neurones will preferentially grow along ordered arrays of collagen filaments (Weiss 1934). The striations which have been seen in basement membranes might similarly provide some directional cues to growing fibres as they are oriented parallel to the fibre bundle. It might also be the case that these 'tram lines' have a polarity so that growing fibres can only follow them in one particular direction.

Growing processes may also take directional cues from existing fibres and follow them along. The observation that fibre bundles maintain their spatial coherence over long distances and that processes from a given class are grouped together is suggestive that this could be happening. Guidance along pre-existing fibres has been suggested by several authors (Harrison 1910; Trujillo-Cenoz & Melamed 1973; Lo Presti, Mecagno & Levinthal 1973).

(c) Polarity

The type A motor neurones have the opposite polarity to the type B motor neurones. The arms giving n.m.js from type A's run forward on both the ventral and dorsal sides. The dendritic arms from the dorsal and ventral members of each class do not run in the same direction however; the dorsal type A and the ventral type B run forward whereas the dorsal type B and the ventral type A run backward (figure 20). One possible explanation of this behaviour is that their axons (the branch that gives n.m.js) have a preferred direction of growth in the cords, being forward in the case of the type A and backward for the type B. This preference could be as a result of the interaction of the growing axon and the basement membrane which it will be assumed has an inherent polarity. The dendritic arms of the motor neurones run in the centre of the cord away from the basement membrane whereas the axons take up a position adjacent to these membranes when they give n.m.js. If one considers the direction of the dendritic branches, the dorsal members of a class can be seen to be inverted relative to the ventral members of the same class. When the dorsal members send out their axons into the motor end plate region next to the basement membrane it could be that they find they are growing in the wrong direction relative to the polarity written in the basement membrane and they then try to turn right round (like a shoot from a seed that is planted upside-down) and in doing so end up between the muscles and hypodermal cells where they are guided round as a commissure to the dorsal side to complete the turn and then can run up in their appropriate directions. Thus the only difference that there may be between dorsal and ventral members of the class A and B is that the dorsal ones are born upside-down relative to the ventral. Van der Loos (1965) has observed a similar behaviour of the pyramidal cells in the rabbit cerebral cortex. Cells which are 'improperly' oriented, i.e. upside-down, send out axons which double back and run in the normal direction whereas the dendrites run in the same direction relative to the cell body which is in the opposite direction to those from normally oriented cells. Axons from Maunthner's neurones have also been shown to have a preferred direction of growth (Hibbard 1965).

(d) Lineage

Certain cells in the ventral cord are grouped together in pairs; a dorsal type A neurone is usually next to a dorsal type B, a ventral type A is usually next to a ventral type B and a dorsal type AS is usually next to a ventral type D (figure 24). It seems likely that each of these pairs might be the daughter of a single precursor cell, thus the differentiation of the neurones into their respective classes would be occurring during or after this last division. This has shown to be the case for the ventral A and B pair and the D and AS pair (Sulston 1976). Class A and B neurones have intrinsic polarities which are opposite. The orientations of the two daughter cells (extrinsic polarity) must be in opposite directions so that if it is assumed that the divisions are in the transverse plane then there are two possibilities for the daugher cells: that their intrinsic and extrinsic polarities are aligned, or that they are opposed. In the first case both the axons find their direction of growth in accord with the polarity of the cord and innervate the ventral side, whereas in the second case this will not be so and they will turn and go into the dorsal cord where they will run in their preferred directions innervating the dorsal side.

(e) Competition

The different classes of motor neurones form repeating regions of neuromuscular contacts with fairly sharp points of demarcation where one member of a class ceases to give synapses and its neighbour takes over. These transition regions are not so precisely aligned between the classes (figures 18 and 19) and so it seems unlikely that there are any sort of precisely defined segmental boundaries constraining all neurones of a segment to form their n.m.js in that region. If that were so one might expect that the registration between the classes was as good if not better than that between adjacent members of the same class.

Competition between adjacent neurones has been postulated as a possible mechanism for the establishment of fields of innervation (Cajal 1929; Stirling 1970) and it seems likely that motor neurones within a given class compete with each other for sites in the n.m.j. region. The class D motor neurones and dendrites might be a more extreme example, where not only is the formation of neuromuscular connection inhibited by the presence of a neighbouring type D's but also the growth of the axon. The other classes of motor neurone send their axons well beyond the region where they form n.m.js but these branches seem to be completely non-functional being devoid of any synapses. The recognition of a neighbouring neurone of the same class and consequent partitioning off of territories may still be mediated gap junctions as it seems to be an almost universal rule in C. elegans that neurones of a given class form gap junctions with other members of its class. The formation of transient gap junctions between the growing fibres of the ommatidium and the neuroblasts of the optic lamina has been shown by Lo Presti et al. (1974). Cell contacts that are made via these gap junctions may serve to signal the ommatidial fibres to stop growing and even, as they suggest, to determine the fate of the target neuroblast cells.

(f) Synaptic specificity

It has been shown that the various classes of motor neurones receive their synaptic inputs from specific classes of interneurones, the rules being summarized by the connectivity graph of figure 21. It seems highly likely that some sort of chemical specificity exists and is giving rise to these rules. The dispositions of the cell bodies and dendrites of ventral class A and dorsal class B are identical as far as can be seen yet they receive different synaptic inputs. While it may be possible to postulate that a timing effect is producing these differences it seems far more likely that some sort of cell-cell recognition is taking place. The specificity seems only to exist between classes; neurones are fairly indifferent as to which members of a particular class they form connections. This seems to be determined by the mechanical dispositions of the fibres, connections being formed when two members of the appropriate classes come together. The specificities giving rise to the class connectivity rules are not absolute. Two instances have been cited where the rules break down. These synapses look fairly normal and are indistinguishable from synapses which obey the class rules. Thus it looks as if, once the formation of a synapse is initiated, a morphologically normal synapse is produced even if it is to be the 'wrong' class of cell. It seems therefore, as if the initiation is a probabilistic process, the probabilities varying according to the classes of cells involved.

(g) How does it work?

The small size of C. elegans presents severe technical problems for the investigation of the physiological and electrophysiological properties of the muscle cells; however Ascaris muscles have been fairly extensively investigated, notably by del Castillo and his co-workers. This work has been reviewed by De Bell (1965). Little is known, at the moment, of the physiology of C. elegans muscle but the close homologies which exist between both the structure of the muscles of the two animals and the nervous system innervating them leads one to suspect that the two may also be similar in their physiological properties.

Isolated preparations of Ascaris body muscles exhibit endogenous myogenic spike activity. This activity can be modulated by the application of acetylcholine which increases the spike frequency and y-amino-butyric acid (GABA) which decreases it. It is likely that these neurotransmitters are used by the various classes of motor neurone; the acetylcholine ones being excitatory and the GABA containing ones inhibitory. The spike activity in adjacent cells is strongly correlated with little or no latency suggesting that the muscle cells are coupled together electrically. This coupling is probably mediated by gap junctions such as those that are seen between muscle cells in C. elegans. Crofton (1966) has suggested on the basis of these observations that the waves of muscle contraction that travel down the body when the animal is moving forwards or backwards, are propagated myogenically, the muscles acting as their own proprioreceptors and the nervous system serving only to modulate this activity.

The synaptic circuitry associated with the various classes of motor neurone innervating the body muscles gives a certain amount of support for these ideas. None of the neurones described in the ventral cord have any structures that would suggest that they may be mechanosensory. If the nervous system were being used for wave propogation one might suppose that such cells would be necessary in order to provide feedback to the neural circuit driving the muscles of information on posture. Motor neurones of class A, B and AS are driven via their respective sets of interneurones from the central nervous system (c.n.s.) i.e. the nerve ring. Each member of a given class receives the same synaptic input regardless of its anterior/posterior position in the cord. It seems unlikely that these are used for the propagation of waves but rather that they are used for the global on/off control of wave generation and propagation by the c.n.s. Classes A and B are the only neurones present in both the dorsal and ventral sides and so one is probably excitatory and the other inhibitory. AS motor neurones are only present on the dorsal side and could be used to set up a certain posture, such as the deep ventral bend that usually accompanies the transition from backward to forward motion (R. Freedman, unpublished observations).

Class D motor neurones are not driven by the c.n.s. but rather from the other classes of motor neurone. They receive their synaptic input on the opposite side from the regions where they give n.m.js suggesting that they may form a system of reciprocal inhibition to ensure that the front and the back work antagonistically. It is probable that they are only used when the muscle system is first switched on and act to set the initial phases of the muscle oscillators.

There are fewer class C motor neurones than other types and in the hermaphrodite they predominantly innervate the vulva. They are more numerous in the male (Sulston 1976) and probably receive their synaptic input from the ganglia in the tail which are much larger and more complex than in the hermaphrodite. It is possible that they are involved in male copulatory behaviour and hermaphrodite egg laying.

The animals move forward by propagating sinusoidal waves backward along the body and, conversely, move backward by propagating forward directed waves. The end of the body, where the waves are progressing from, seems more active and vigorous than the other end which often seems to be passively following, as though there is a gradient of muscle activity which may be produced by a similar gradient of motor neurone activity. If the muscle cells have endogenous myogenic activity and are stretched activated, as are Ascaris muscles, then the muscle cells along the body will behave as a set of coupled oscillators. If there is a gradient of synaptic input along the body, the muscles with the highest excitation will oscillate with the highest amplitude and frequency and entrain all the more distal muscle oscillators causing waves to propagate away from the point of highest excitation. Thus, for forward movement there might be an anterior/posterior gradient of excitation and for backward movement this gradient would be reversed.


Web adaptation, Chris Crocker, for Wormatlas, 2008