Patterns of Activity and the Effects of Activation of the Fast Conducting System on the Behaviour of Unrestrained Leeches

Several studies have shown that the Fast Conducting System (FCS) in the nerve cord of leeches consists of a chain of interneurones, one in each segmental ganglion, connected to each other by means of electrical synapses and impinged upon by impulses of photic and tactile origin, arising in both segmental and suprasegmental receptors (Laverack, 1969; Gardner-Medwin, Jansen & Taxt, 1973; Mistick, 1974; Frank, Jansen & Rinvik, 1975; Bagnoli, Brunelli & Magni, 1972, 1973; Bagnoli et al. 1974, 1975; Carbonetto & Muller, 1977).

Recently it has been shown that FCS activation by electrical and natural stimuli elicits contraction of the muscle fibres of both the nerve cord sheath and the longitudinal muscles of the body wall, and it has been concluded that one of the possible roles of the FCS is the transegmental generalization of the shortening reflex (Magni & Pellegrino, 1975; Magni & Pellegrino, 1978). Since these results were obtained from experiments performed in isolated preparations under artificial conditions, it is difficult to evaluate their relevance to the integrated behaviour of the animal. The present experiments were aimed at studying the activity and the effects of activation of the FCS in unrestrained behaving leeches, by recording simultaneously the be haviour of the animal and the electrical activity of the nerve cord, monitored by means of a chronically implanted electrode. It will be shown that of all the behaviours we have investigated, only the shortening reflex is preceded by FCS discharge, and that ventilatory movements are reversibly blocked following activation of the FCS by photic or mechanical stimulation of the skin.

The experiments were performed in the March-May period on commercially supplied specimens of Hirudo medicinalis.

The chronic electrode is shown in Fig. 1; it consists of a cuff of Sylgard 184 encapsulating resin (1×1×2 mm), which is pierced by a longitudinal hole 200 μm in diameter. An insulated cable, embedded in the resin block, transverses the cuff at right angle to the hole axis and projects into the hole with a hook-shaped platinum wire 50 μm in diameter. A longitudinal slit permits the hole to be pulled open lengthwise, to insert the nerve cord. On releasing the pull, the elasticity of the material closes the slit almost completely.

Under 8 % ethanol anaesthesia, a median longitudinal cut was made on the dorsal side of one segment across the length of the four annuli that occur between those containing the sensilla, to expose a single interganglionic connective, which was inserted into the hole of the cuff and placed in contact with the platinum hook. The ends of the cuff were sealed with silicon grease, the wound was sutured with silk thread, and the cable emerging from the cuff was secured in place by means of two cross-stitches. Correct placement of the electrode was tested by its ability to record the FCS impulses, identified on the basis of their size, bidirectionality of conduction, and the fact that they were elicited in response to photic and tactile stimuli applied to the skin of every segment (Bagnoli et al. 1973). After a postoperative period of 24 h, the successfully implanted animals were transferred to a glass aquarium (20 × 20 × 50 cm), filled with spring water at room temperature. The chronic electrode and a reference electrode placed in the aquarium water were connected to the input of a differential preamplifier, the output of which was displayed on one beam of a CRO and photographed. The behaviour of the animal was monitored by a ciné camera at 8 or 2 frames/s. The camera provided an output signal for each frame, which was displayed on a second beam of the CRO and was used for synchronizing the electrophysiological with the behavioural records.

The chronically implanted animals display all the behavioural activities which can be observed in intact leeches (Mann, 1962). Throughout the observation time they alternated periods in which they remained motionless with searching, swimming, creeping or ventilation episodes.

The leech can stay motionless in a variety of positions for a considerable period (up to several hours). Its body may be shortened or elongated ; the anterior portion of the body may assume a variety of orientations with respect to the light source; the animal may be attached with the suckers to a vertical wall or to the bottom of the aquarium. However, irrespective of its attitude, no activity of the FCS can be recorded and only a continuous multi-unit, small amplitude activity is recorded by the chronic electrode both in light and in darkness.

In a motionless leech, FCS activation can be induced by at least three kinds of stimuli, namely: (a) a quick change of the level of illumination; (b) a touch applied anywhere on the skin, and (c) a water displacement or a vibration produced by a moving object immersed in the aquarium, but not actually in contact with the animal’s surface.

The typical effects of a photic stimulus are shown in Fig. 2. The animal, previously adapted to a low level of illumination (frames 1–6 of Fig. 2 A), was stimulated with a square pulse of bright light about 1 s in duration (frames 7–13 of Fig. 2 A). The onset of the photic stimulus was followed by a short-lasting burst of FCS spikes at a frequency of about 60/3 and a latency of 100–200 ms, as shown in the record of Fig. 2B, in which the numbered signals refer to the frames of Fig. 2 A. The response was transient in nature and no off response was observed; however, in animals adapted to bright light, sudden lowering of the light intensity sometimes elicited a FCS discharge. In either case no clear-cut behavioural correlate of the FCS discharge was apparent.

The FCS response can also be elicited by mechanical stimulation of the skin. The typical response to a mechanical stimulus is shown in Fig. 3. In this experiment the stimulation was performed with a glass rod, which was slowly lowered through the water to touch the animal; contact was made in frame 4 (arrow in Fig. 3 A). The FCS response following the contact between the rod and the skin (Fig. 3B) consists of an irregular burst of action potentials at an initial frequency of about 140/s. This stimulus elicited a clear-cut behavioural response, namely a shortening reflex promptly generalized to the entire body length (frames 6–14 of Fig. 3 A). Apart from the initial burst, the FCS remained silent for all the time taken by the shortening reflex to develop (Fig. 3 B).

A similar response, both behavioural and electrical, can be elicited by a glass rod quickly moved through the water, without actually touching the animal, or by a second leech swimming close to the experimental animal.

The leech exhibits two distinct types of locomotor activities: creeping on a solid substratum and swimming. The mechanisms which underly these behaviours have been analysed in terms of the groups of muscles involved (Gray, Lissman & Pumphrey, 1938; Kristan, Stent & Ort, 1974 a) and the neural control of swimming has been elucidated (Kristan, Stent & Ort, 1974b; Ort, Kristan & Stent, 1974; Kristan & Calabrese, 1976). A third kind of behaviour of the leech, not involving movement of the whole animal, is ventilation (see Mann, 1962). It consists of anti-phasic contractions of dorsal and ventral longitudinal muscles, which appear to be controlled by the same neuronal mechanisms involved in swimming. The only difference between the two kinds of behaviour is that during ventilation movement of the animal is prevented by the attachment of one of the suckers on the substratum.

During creeping, the body of the leech reaches the extreme physiological values of shortening and lengthening; the animal also moves its body with respect to water and places alternatively its suckers and portions of body in contact with the sub-stratum. Throughout the entire cycle of movements that constitutes a ‘step’ (Fig. 4 A), the FCS remains silent (Fig. 4B).

Swimming and ventilation are associated with a characteristic pattern of cord activity, consisting of a series of multi-unit bursts of small amplitude, synchronous with the swimming cycles. This kind of activity has been attributed to the discharge of interganglionic interneurones responsible for the transegmental propagation of the swimming rhythm along the nerve cord (Kristan et al. 1974b). However, FCS activity is never observed during swimming or ventilation.

Photic and mechanical stimuli delivered during creeping or swimming are able to fire the FCS ; however, their effects on these behaviours are unpredictable, due to the difficulty of precisely grading the stimulus intensity and of locking the stimulus to a given phase of the movement. Only a strong mechanical stimulus applied everywhere on the skin during the extension phase of the step is consistently followed by the interruption of creeping and by the initiation of shortening. No obvious effects of this kind have been observed on swimming, which appears to be the least affected of the behaviours we have studied.

By contrast, photic and tactile stimuli delivered during ventilatory movements elicit consistently an interesting response, which consists of a FCS discharge and a reversible blockade of both the ventilatory movements (Kaiser, 1954) and of the associated bursting activity of the cord. These effects are illustrated in Fig. 5, which shows that, as the light intensity was changed, the ventilatory movements displayed by an intact animal (frames 1–16 of Fig. 5) were completely arrested for more than 4 3 (frames 19–27 of Fig. 5). Fig. 6 shows the results of a similar experiment performed on a leech carrying an implanted electrode. The bursting activity, associated with ventilation, was completely suppressed and a FCS discharge was elicited by a step increase in the illumination level. The ventilation-associated bursting activity resumed after several seconds, coincident with the reappearance of the ventilatory movements. A similar effect is elicited by tactile stimulation of the skin.

It has been shown that repeated presentations of the stimuli leads to the reversible disappearance of the FCS response (Laverack, 1969; Bagnoli et al. 1973; Mistick, 1974). In this condition the same stimulus fails to produce the blockade of ventilatory movements, as illustrated in Fig. 7. In this experiment the rhythmic cord bursts associated with ventilation were not evident; therefore, ventilatory movements were monitored by recording the water displacements caused by the animal’s activity. The first of a series of mechanical (Fig. 7 A) and photic (Fig. 7C) stimuli elicited a FCS discharge, followed by a reversible arrest of ventilation. After a series of repeated presentations of the same stimuli at the same intensity the response of the FCS to both stimuli disappeared and ventilation was completely unaffected (Fig. 7B, D). This effect is fully reversible, FCS response and blockade of ventilation reappearing some minutes after the stimulations are interrupted.

The disappearance through repeated presentations of the FCS response to one modality of stimulation does not prevent afferent impulses of a different modality from firing the FCS (Laverack, 1969). Similarly, FCS discharge and blockade of ventilation, which had vanished following repeated presentations of a photic stimulus, could again be obtained upon mechanical stimulation of the skin (not illustrated).

Our observation that in the resting animal, irrespective of its body attitude, the FCS does not discharge is consistent with the phasic nature of the system, suggested by other investigations (Laverack, 1969; Gardner-Medwin et al. 1973; Mistick, 1974; Frank et al. 1975; Bagnoli et al. 1973, 1974, 1975), which showed that it responds only to sudden changes in stimulus intensity.

The fact that photic and weak tactile stimuli are capable of firing the FCS in the motionless animal shows that the absence of FCS activity observed in these conditions is not due to a lowering of its excitability, but simply to the lack of an adequate synaptic input. Moreover, the finding that a tactile stimulus, as weak as that caused by a small water vibration, which is adequate to activate T cells (Nicholls & Baylor, 1968), elicits a FCS burst is consistent with the demonstrated projection of the T cells onto the FCS neurone (Bagnoli et al. 1975).

The role of the FCS in the transegmental generalization of the shortening reflex, suggested by Smith & Page (1974) on the basis of the data of Gardner-Medwin et al. (1973) and supported by the investigations of Magni & Pellegrino (1978), is consistent with our finding that in the intact, motionless animal a FCS discharge elicited by a tactile stimulus is followed by a generalized body shortening. The lack of an observable shortening following activation of the FCS by a diffuse photic stimulus stands in apparent contrast to the observation of Gee (1912), who reported shortening in response to photic stimulation. The reasons for this discrepancy are difficult to explain on the basis of the present evidence. It may be tentatively suggested that in our conditions the shortening induced by a photic stimulus is too small to be detected, possibly on account of the low frequency of the photically induced FCS response (see Figs. 2 and 3 ; see also Bagnoli et al. 1973).

A surprising outcome of our investigations is that the FCS is completely silent not only in a motionless animal, but also during active movements. The lack of discharge of the FCS during the whole step-cycle of creeping, in which the physiological extremes of body length are attained, appears incompatible with the suggestion advanced by Smith & Page (1974) for Haemopis that the FCS is primarily activated by postulated stretch receptors of the nerve cord sheath. Certainly, however, apart from the hypothetical cord stretch receptors, a wealth of exteroceptive afferent impulses, which have been shown to activate the FCS, must be elicited during all the kinds of active behaviours we have studied. In particular, in both swimming and creeping the intensity of the stimuli impinging upon skin mechanoreceptors is quite comparable to (and likely to be greater than) that capable of activating the FCS in a motionless leech. The problem therefore arises of the mechanisms by which reafferent excitatory impulses (Von Holst & Mittelstaedt, 1950) are prevented from firing the FCS during active movements, i.e. whether this is due to a reduction of the afferent barrage on the FCS or to a lowering of its own excitability, or both.

In the absence of direct evidence, any conclusion on this point must necessarily Remain tentative. Concerning swimming and ventilation, however, a reasonable hypothesis could be advanced, on the basis of the known synaptic connexions between some of the neurones in the leech segmental ganglion. The large longitudinal motor neurones (L cells; Stuart, 1970) receive a synaptic input by means of electrical rectifying synapses from both the tactile mechanoreceptive neurones (T cells, Nicholls & Baylor, 1968) and the FCS neurone, as recently suggested (Magni & Pellegrino, 1978). During swimming or ventilation the L motor neurones are tonically inhibited (hyperpolarized) through the alternating action of four groups of motor neurones controlling the dorsal and ventral longitudinal muscles, by means of both electrical and chemical synapses (see Fig. 20 in Ort et al. 1974). It is conceivable that hyperpolarization of the L motor neurone could spread back across the rectifying electrical synapses to both T and FCS neurones and hence prevent the response of the FCS to a tactile stimulus, both by interfering negatively with the intraganglionic conduction of afferent impulses responsible for the activation of the FCS neurone and by decreasing the excitability of the FCS neurone itself. The latter mechanism is supported by the finding that the electrical excitability of the FCS is decreased by hyperpolarization of the L motor neurone (Magni & Pellegrino, unpublished). According to this hypothesis, the control of the reafferent barrage during swimming and ventilation would be brought about through the operation of an electrical synapse at both presynaptic and postsynaptic levels. Investigations aimed at its direct verification are presently being performed.

We can provide no plausible explanation of the FCS silence observed during creeping, because of the lack of detailed knowledge of the neural mechanisms involved in this kind of behaviour: the problem must be deferred until more information is available.

We have observed that if the FCS is made to discharge during the extension phase of creeping or during ventilation, the ongoing activity is interrupted and a shortening reflex may be initiated, whereas swimming appears not to be consistently affected. For the reason outlined above it is not possible to discuss the neural mechanism responsible for the arrest of creeping; our attention will therefore be focused on ventilation and swimming, the neural bases of which are more completely understood.

Concerning ventilation, our finding that its blockade by tactile and photic stimulation is invariably associated with a FCS discharge and disappears as soon as the FCS is adapted to these stimuli suggests that the FCS discharge is instrumental in producing the arrest of ventilation. Laverack’s conclusion (1969) that the adaptation is modality-specific is confirmed by our observation.

It has been demonstrated that the FCS neurone drives the L motor neurones at the level of each ganglion, thereby contributing to the initiation and promoting the intersegmental generalization of the shortening reflex (Magni & Pellegrino, 1978). It could be proposed, therefore, that excitation of the FCS produces the arrest of ventilation through the activation of the L motor neurones, which in turn cause the synchronization, and hence the suppression of the antiphasic discharge, of dorsal and ventral excitors in each ganglion (Ort et al. 1974). The synaptic connexions shown by Ort et al. (1974) between L motor neurones and dorsal and ventral excitors and inhibitors, namely that the L motor neurone is coupled to the excitors by means of an electrical rectifying synapse and receives an inhibitory chemical synapse from the inhibitors, are quite consistent with this proposed mechanism.

According to this interpretation, the inefficacy of photic and mild mechanical stimuli to block swimming movements in the intact freely moving animal could be attributed to a higher level of ‘oscillatory drive’ witnessed by the higher beating frequency and larger amplitude of the oscillation; this would result in a higher hyperpolarization of the L motor neurone and hence a more powerful cut-down of the efficacy of a given afferent impulse barrage. Obviously this hypothesis can only be validated by the demonstration that the same neural mechanisms underly both swimming and ventilation; this appears however, a likely possibility, in view of the fact that frequency and amplitude of the swimming movements can vary over a wide range (Kristan et al. 1974a).

It is therefore possible that ventilation and fast swimming occupy the extremes of an ‘oscillatory drive’ continuum (of unknown nature), the intensity of which determines frequency and amplitude of the movements on one hand, and the degree of the control of reafferent inputs on the other. It would follow that an afferent impulse barrage, produced by an external stimulus (exafferent input; von Holst & Mittel-staedt, 1950) is more likely to block the ongoing behaviour when it occurs at the lower end of the continuum (ventilation) than at its upper end (swimming); a prediction which is amenable to experimental test.