modification of the motor reflex responses due to ... - Semantic Scholar

J. Exp. BM. (1973), 59, 383-403 tWith 8 text-figures ^Printed in Great Britain

383

MODIFICATION OF THE MOTOR REFLEX RESPONSES DUE TO REPETITION OF THE PERIPHERAL STIMULUS IN THE COCKROACH II. CONDITIONS OF ACTIVATION OF THE MOTONEURONES BY N. F. ZILBER-GACHELIN* AND M. P. CHARTIER University de Paris, Laboratoire de Neurophysiologie comparie, 9, qucd Saint-Bernard, 75005 Paris, France {Received 16 February 1973) INTRODUCTION

The cockroach reacts to an air puff applied to its cerci by a forward escape reflex which decreases or even disappears after a few successive stimuli have been applied (Roeder, 1948). In the preceding paper (Zilber-Gachelin & Chartier, 1973) we have shown that the response is already modified by habituating effects at the first synaptic relay, in the 6th abdominal ganglion (A.G.). The present paper extends this work and studies the effect of the second synaptic relay, the 3rd thoracic ganglion (T.G.), on the abdominal cord to motor nerve transmission. In the final section we study how these properties, combined with those we described in the 6th A.G., can explain the modification of the motor response observed on the isolated reflex loop when repetitive cereal air puffs are delivered. Finally, the bearing of these results on the behaviour of the whole free cockroach is discussed. A brief report of this work has appeared previously (Chartier & Zilber-Gachelin, 1969). MATERIALS AND METHODS

All the experiments were carried out on male Blabera craniifer. The general methods and the materials used have been described previously (Zilber-Gachelin & Chartier, 1973). We shall only point out here some procedures particular to the experiments described in this paper. (1) The thoracic synapses studied have appeared to be particularly sensitive to hypoxia, so that two precautions have been taken: (a) First, the whole animal was used in the experiments. It was fixed on its back to a cork plate by entomological pins through the pronotum and drops of a wax of low melting-point (Adheso sticky wax, S. S. White Company) at a few points along the abdomen. This wax was also used to fix the coxae of the legs away from the middle line, thus exposing the sternal thoracic region under which the proximal levels of the leg nerves are located; but the legs themselves were left free so that their movements could be observed. (b) Secondly, the cuticle was opened only as far as was necessary for access to the • Present address: Laboratoire de Neurobiologie cellulaire, C.N.R.S., 91190 Gif-sur-Yvette, France.

384

N. F. ZILBER-GACHELIN AND M. P. CHARTIER

leg nerves. In this way the ganglion itself was always covered with cuticle; and, tracheae having been preserved, oxygenation remained as good as possible. (2) To study the transmission properties of the synapses of the 3rd T.G. on the motoneurones we stimulated electrically the pre-synaptic fibres running in both connectives in the abdominal cord. Stimulation between the 5th and 6th A.G. is expected to lead to a one-to-one stimulation of the 3rd T.G. since the afferent fibres have been found to run uninterruptedly to the 3rd T.G. from the 6th A.G. (Roeder, 1948; Hess, 1958; Harris & Smyth, 1971) and to follow up to a rate of stimulation of 600/sec (Roeder, 1953). Stimulating closer to the 3rd T.G. will activate other fibres which originate in the higher abdominal ganglia and result in a more mixed input to the 3rd T.G. Ideally, the cereal nerve afferents should be stimulated in order to obtain a pure response. However, in this case the synaptic contribution of the 6th A.G. would be added. All factors considered, it appeared best to isolate the 6th A.G. and to stimulate the stump entering the 5th A.G. The cord was of course left intact for the experiments involving cereal air puff stimulation and stimulation of the cereal nerve. (3) The motor response was recorded on nerve 4 (N 4) (nomenclature of Pringle, 1939) of a posterior leg. This nerve innervates essentially the main extensors of the trochanter, whose contraction is an important element of the escape reflex to an air puff applied to the cerci (Hughes, 1965). N 4 being purely motor (Nijenhuis & Dresden, 1955; Pipa & Cook, 1959), there was no need to cut it distally relative to the electrodes. This allowed the observation of the muscular effects corresponding to the impulses recorded on N 4. When the electromyographic response itself was to be recorded, two small insect pins (no. 015) were inserted directly into the immobilized leg coxa and their distal ends were rigidly secured to the cork plate. (4) We suppressed the possible rostral influences from the cerebral, sub-oesophageal, and 1st and 2nd thoracic ganglia by cutting the cord between the 2nd and the 3rd T.G. We also severed the sensitive and mixed nerves of the 3rd T.G. (nerves 2, 3, 5 and 6) and the nervous filament joining nerves 4 and 5 (cf. Zilber-Gachelin, Paupardin & Chartier, 1973); as a matter of fact we do not know whether its fibres are motor or sensory, but we thus avoided recording in N 4 the eventual potentials coming from nerve 5 which might be sensory. Additionally, the nerves from the abdominal ganglia and finally the abdominal nervous cord itself distal to the stimulation point were severed. In this way a preparation is obtained in which only those pathways directly related to our studies are known to exist. (5) Finally, after the dissection was completed and the electrodes (bipolar choridized silver hooks) were in place under the nervous structures to be studied, desiccation of the preparation was avoided by covering the dissected parts with a thin film of petroleum jelly (Diathera), pure enough to have no effect upon the nervous activity. This jelly had to be applied as a liquid to limit its thickness and thus to avoid blocking conduction in N 4. (6) The devices used to measure the number of spikes of a response or the instantaneous frequency of a discharge have been described in the previous paper (ZilberGachelin & Chartier, 1973). Two differences must be noted. (a) An anti-coincidence circuit was used instead of a threshold circuit. Since, as in all arthropod leg nerves, N 4 contains only a limited number of fibres (ten according to Dresden & Nijenhuis, 1958), the different units could be easily distinguished froni|

Activation of cockroach motoneurones

385

w e another within the general extracellular recording. The response pattern of the different units could thus be studied separately by means of an 'anti-coincidence circuit' which made it possible to select in a complex record the potentials of an amplitude between any two pre-set values. (b) When we stimulated electrically the cereal nerve, the abdominal cord response was better synchronized than after an air-puff stimulation. Because of the summations occurring on the recording the amplitude of the response was then evaluated not by the number of spikes but by the surface of the response; for this purpose it was rectified (double alternation rectification) and then integrated. RESULTS

The final aim of this work and of that described in the preceding paper (ZilberGachelin & Chartier, 1973) was to explain the behaviour of the whole free animal in terms of the properties of the synapses involved in the reflex under study. It may therefore be useful to begin with a short description of this behaviour as we could observe it, so that the bearing on this problem of the results described below can more easily be appreciated. If air puffs are repetitively delivered to the cockroach cerci, the following gross behaviour is observed: (i) an escape reaction is elicited by the first or the first few puffs with (in the latter case) an apparent decrease in the distance covered; (ii) then the animal only takes one or two steps, or makes movements of the legs without moving forward; (iii) finally the animal appears not to react any longer, but careful observation from beneath often makes it possible to see a retraction of the legs under the body or a slight movement of the body and legs (even if they cannot be clearly observed, a small response can always be seen on the electro-myogram recorded from the base of a leg; it becomes weaker and weaker but appears to persist). This third type of reaction will be referred to as a 'startle reaction'. Often no escape reaction at all can be elicited. Finally, if the stimuli are near enough to each other, an initial 'sensitization' (Kandel & Spencer, 1968) can eventually precede this habituation (over 3-4 stimuli). Nerve 4 spontaneous activity; its different types of units The slow system

If the activity of N 4 is recorded in the absence of any stimulation, it can be seen that some units are firing at low frequency (generally 1-20/sec). This spontaneous tonic activity is not accompanied by limb movements but simply by a certain level of tonic contraction; furthermore it sometimes occurs in rhythmic bursts, and a parallel variation of the tonus of the innervated muscles can then be noted. These excitatory fibres probably correspond to the slow system, one of the two types of motor units described initially by Pringle (1939). This slow system is defined by its response to stimulation of the motor fibres. Stimulation by single shocks does not give rise to a muscular contraction but repetitive stimulation induces a slow contraction. The contraction amplitude is directly related to the frequency, up to 100150/sec, where it will reach a plateau. This plateau is about 40 % of that produced by fast axon.

386


The fast system The fast system differs from the slow system by the larger diameter of its nervous fibres and by the fact that their spikes give rise to muscular potentials of a higher amplitude. Moreover, each potential of these fast fibres produces a muscular twitch with a rapid rise time. Such fibres do not discharge in the resting animal, but they can be observed in N 4 during spontaneous and sudden extensions of the leg. They appear then, over an increase in the discharge rate of the slow fibres, as higher-amplitude units which exist only during these movements. On N 4 recordings we have also noticed the presence of another type of fibre. It had a tonic discharge which was inhibited throughout the entire periods of leg movements (flexions as well as extensions). These fibres were perhaps inhibitory. But they were observed only in two preparations and thus could not be systematically studied. Motor responses to single electrical shocks on the abdominal cord

An electrical stimulation of the abdominal cord by a single shock does not generally give rise, regardless of its intensity, to any response of the fast excitatory fibres of N 4, nor to any sudden extension of the trochanter. But a response of a slow excitatory fibre can be noted (Fig. 1, inset); it appears with a latency of about 20 msec and consists of an 'initial burst' of three or four closely clustered spikes (frequency of 100-300/sec), which is followed, if the stimulation is intense enough, by a later response which we called 'after-discharge', the same units firing at a lower frequency (generally 15-50/sec) during, on an average, 200-500 msec. This after-discharge is sometimes difficult to distinguish from the spontaneous activity when the latter occurs as volleys of spikes firing at a similar frequency. At the periphery only a slight 'startle' (as defined above), concomitant with such a nervous response, is observed. This N 4 response will be referred to later as R 1. Motor responses to repeated electrical shocks on the abdominal cord

If the 3rd T.G. synapses are labile in the way described by Roeder (1948), the response will decrease with stimulus repetition. So we shall examine how the R 1 response evolves when abdominal stimulations are repeated. As a matter of fact, several cases are found, according to the repetition rate: (a) The response of the slow fibres does not vary for stimulation frequencies up to 0-5-2/sec, depending on the animal. (b) Above this limit of frequency, and below about 10/sec, the initial burst of the responses still remains unchanged (Figs. 2, 3A); only the initial burst in response to the very first stimulus sometimes has one more spike than any subsequent response (Fig. i i ) . On the other hand, if the response contains an afterdischarge, its duration and firing frequency decrease progressively until they get stabilized at a non-zero plateau (Fig. 1 a). Habituation therefore is present at some level in the thoracic ganglion. Let us emphasize, however, that when the stimulation is too weak to produce an afterdischarge, no habituation can be detected at these frequencies since the initial burst remains constant. (c) Finally, if the stimulations are repeated at a still higher frequency (general!

1


387

10r

n 5

0L

1 5 10 15 Number of stimulations (1/sec) Fig. 1. Modification of the response of a slow excitatory fibre to stimulation of the abdominal cord, through repetition of the stimuli at a rate of i/sec. In the inset is displayed a response of a slow excitatory fibre of the N 4 innervating the left posterior leg; b indicates what we call the 'initial burst' of the response, which is followed by an 'afterdischarge' of three spikes. The first small vertical deflexion at the left of the trace is the stimulation artifact. The graph represents the total number of spikes (a) and the number of spikes of the initial burst (4) of the response to successive electrical stimuli delivered on to the abdominal cord at a frequency of i/sec. Data are averages of 15 similar experiments. While the number of spikes of the afterdischarges decreases, the initial train remains constant; only the first dot is slightly higher, which corresponds to the fact that, in some series, the initial burst had one more spike.

above 10/sec), the fast fibres are activated. This is indicated by a sudden extension of the trochanter and the appearance of large spikes in the N 4 record. This appearance of a fast-fibre discharge (Fig. 3, B-4) marks important variations in the pattern of the slow-fibre responses. The fast-fibre responses are temporally independent of the stimuli and the slow fibres also appear at this time to fire with no fixed temporal relation to the stimuli. Both responses continue to depend on the presence of the stimulus although they show some inertia in that they continue after the cessation of stimulus for varying times (on the order of a few hundreds milliseconds). However, if the stimulations are continued for several seconds, a habituation process sets in d the fast-fibre response disappears, leaving only an R 1 response.

r

25

E X B 59

388


Fig. 2. Non-modification of the initial burst of the response of an N 4 slow excitatory fibre to stimulation of the abdominal cord when the stimulation rate is increased up to 20/sec. The height of each bar represents the number of spikes of the initial burst. Series of eight successive stimulations of the abdominal cord separated by the same time interval (At) were delivered. The value of At for each series is indicated on the lower line (in seconds).

This type of response with activation of die fast fibres had to be subdivided into two categories: (1) The fibres can discharge, during cord stimulation, at a more or less constant frequency (Fig. 3C). Although die spikes have no precise temporal relation with die stimuli, the higher the stimulation rate, die higher will be the firing rate. When die stimulations are stopped, trie frequency of die spikes decreases progressively, die terminal condition being silence for die fast fibres, and die spontaneous resting rate for the slow. (2) At other times the two types of fibres fire in more or less rhythmic bursts of spikes triggered and maintained by the cord stimulations; diese rhythmic discharges continue after die end of die stimuli for a period which can be as long as 1 or 2 min. They can take two different forms: (2 a) Eidier die bursts are separated by periods of total silence of die fast fibres and recur about every second (from 500 msec to 2 sec); tiiis causes at die peripheral level alternations of extension and flexion of die trochanter, which can be compared to walking movements. (2b) Or die fast-fibre bursts are not separated by silent periods but by a continuous low-frequency activity; moreover, the bursts tiien occur at a much higher frequency (about every 100 msec). At die muscular level, one observes, on a background of continuous extension, rhytiimic supplementary extensions, which should perhaps be related to die leg movements during flying. So, in all cases, activation of die fast excitatory fibres requires temporal summation. The latter must on die otiier hand be associated witii a certain spatial summation since, if repetitive stimulations at low intensity are delivered to die cord (and even at a high frequency), only die R 1 response of die slow fibres is obtained. These two factors (temporal and spatial summation) are not independent; increasing spatial summation by augmentation of the intensity or die duration of die stimulus results in a response of die fast fibres for a lower minimum frequency of stimulation. Thus^

Activation of cockroach motoneurones Ar=200msec

389

/ = 50msec

50 msec Fig. 3. Patterns of response of N 4 slow and fast excitatory fibres to stimulation of the abdominal cord. (A and B) Recording effected on N 4 of the left posterior leg. Fifty successive stimulations were delivered in each case with a constant inter-stimulus interval (At). The numbers on the left side of the traces indicate the rank of the stimulation in the series. An absence of number indicates an absence of stimulation; the successive traces in this case are also separated by At. For At = 200 msec (A) the response consists of a train of three or four slow-fibre spikes and is not modified by repetition of the stimulus. For A t = 50 msec (B) there appears after the 4th stimulus a fast-fibre discharge, and the pattern of the slow-fibre response is then modified. The inferior trace (C) represents the response of a fast excitatory fibre in another preparation, for an inter-stimulus interval of 25 msec. The stimulation artifacts are indicated by arrows. No slow-fibre response can be seen at this amplification.

for instance in the case illustrated in Fig. 4, for shocks of a duration of 0-4 msec these spikes could be made to appear for a minimum frequency of stimulation of 5/sec, while with shocks 1 msec long they could be obtained for a frequency of 2/sec. Motor responses to successive trains of electrical shocks to the abdominal cord

We have just seen that if single electrical stimulations are applied to the abdominal cord at a high enough frequency they lead to a peripheral motor response similar to kthe escape reaction. Now during natural cereal stimulation by an air puff the recorded 25-3

39°


c 0-8 o 0-6 0-4 0-2

0

1 2 3 4 5 6 Threshold frequency (shocks/sec)

Fig. 4. Conditions of stimulation of the abdominal cord allowing the appearance of a fastfibre response in N 4. The curve represents, for different durations of the electrical shocks applied to the abdominal cord, the corresponding minimum frequency of stimulation allowing the appearance of a response of fa»t fibres in N 4. Too much attention should not be paid to the perfect linearity, for only the values of frequency noted on the X axis have been tested.

response from the abdominal cord is a train of spikes (Zilber-Gachelin & Chartier, 1973), and we know that the corresponding motor response habituates by repetition of the air puffs. To examine whether the thoracic synapses play a role in this habituation, we studied the response of N 4 and of the leg during repetition of trains of electrical shocks applied to the abdominal cord, to simulate the trains seen with cereal stimulation. In order to get closer to the conditions where we delivered air puffs of 1 sec duration to describe the escape reflex habituation (Zilber-Gachelin & Chartier, 1973), the stimulating trains were generally maintained during the same time. The frequency of the shocks inside each train was of 10-200/sec, the trains were separated by constant intervals of 3-10 sec, and 10-15 such trains were delivered in each series. Under these conditions the fast fibres, once activated, continue to fire in spite of the interruptions of a few seconds between the trains. This firing appears, as found previously, either as rhythmic bursts or as a continuous firing of spikes, and the frequency of firing is independent of the repetition rate of both the stimuli within a train and of the trains of stimuli themselves (cf. Fig. 5). If the stimulation parameters are such that a single train does not activate the fast fibres, they can be made to fire by repetition of this train (after 2-6 trains as the case may be). If the trains continue to be repeated at the same rate, this response then increases regularly. This progressive augmentation of reactivity of the ganglion through repetition of the trains has been observed for all the intervals between trains that have been tested (3-10 sec). Such a facilitation probably reflects a very efficient post-tetanic potentiation. However, if the trains are continued the response then decreases progressively and can even disappear completely, showing now the opposite phenomenon of habituation (Fig. 5, 1). The thoracic synapses thus show two antagonistic types of properties: (i) habitua^


5sec

• i1 i Fig. 5. Response of an N 4 excitatory fibre to successive series of trains of electrical shocks applied to the abdominal cord. The traces read from right to left, as indicated by the arrow. Each one represents the analogous curve of the instantaneous frequency of the discharges of an N 4 fast fibre. Successive trains of electrical shocks were applied every 3-2 sec on the abdominal cord; each one is indicated by a horizontal line located under the curves; each train consisted of 0-8 msec shocks delivered at a io/sec rate during 1 sec. Seven series of 15 trains have been delivered with an inter-series interval of 4 min; the responses to the ist, 5th and 7th series are presented. The artifact due to each stimulus being of the same amplitude as the fast-fibre spikes, it was counted by the frequency to voltage converter. The ' response' to, for instance, the first train of the second trace is thus uniquely produced by these artifacts.

tion and (ii) facilitation of transmission through temporal summation and post-tetanic potentiation. According to the parameters of stimulation and largely the intrinsic reactivity of the preparation, the interaction of the two phenomena can be different. There can thus occur an increase of the fast-fibre responses followed by a diminution, as described above. Either habituation shows itself very rapidly, the response disappearing completely after 8 or 9 trains (Fig. 5), or it may begin to decrease only after up to 30 trains have been delivered. In other ganglia a response can be observed with the first train, and a decrease can occur from the second train, without any detectable phase of facilitation. However, in all cases, provided the trains are continued a long enough time, the diminution of the response always finally prevails. The records shown in Fig. 5 represent the activity of a fibre of the fast type, but the activity of the slow fibres increases and decreases simultaneously with that of the fast fibres. The only difference is that the response of the slow fibres reverses to the R 1 type when that of the fast fibres disappears. Moreover, if such series of trains are repeated, with rest intervals of 2-4 min between the series, the decrease in reactivity resulting from repetition of the trains in the preceding series persists, although attenuated, in the following series. Thus, ^ successive series, the response of the fast fibres appears after a greater and greater

392


number of trains, is constituted by a discharge at a lower frequency, and finally appears more and more rapidly (Fig. 5). So, not only does habituation prevail over facilitation within a series of trains, but also it persists longer after the end of the series. The simultaneous action of two antagonistic properties having a different time course can explain this complexity in the change of the response during repetition of trains of stimuli applied to the abdominal cord. Motor responses to repeated activation of a cereal nerve

In this last part of our work we isolated the whole of the reflex loop, from the cerci to the muscles of a posterior leg, and recorded simultaneously the responses of the abdominal cord and of an N 4 to electrical stimulations of a cereal nerve and to air puffs applied to the cerci. We have already found (Zilber-Gachelin & Chartier, 1973) that the cereal nerve response is constant under the stimulation parameters used. On the other hand, for both electrical stimulation of the cereal nerve and air-puff stimulation of the cerci, we find modification of the responses at the abdominal cord and N 4 levels. It thus seems reasonable to assume that a comparison of the responses at these levels can lead to some conclusions about the respective roles of the mechanisms we have described at the two ganglionic levels in the observed behavioural reactions to cereal stimulation. Electrical stimulation of a cereal nerve

If the intensity of stimulation of the cereal nerve by a single shock is increased progressively, there appears first a response on the abdominal cord, in the form of a more and more complex volley of spikes (cf. Zilber-Gachelin & Chartier, 1973, Fig. 2), without any change on N 4. Only for higher intensities of stimulation does N 4 respond. Only the slow fibres respond, in a similar way to that obtained through stimulation of the abdominal cord by single shocks (R 1 response). It occurs as a burst of 3 or 4 spikes which, if the stimulation intensity is sufficient, is followed by an afterdischarge at a lower frequency. But activation of the fast fibres is very difficult to observe using only electrical stimulation. If the cereal nerve stimulations are repeated, the responses of the abdominal cord and of N 4 exhibit parallel behaviour. At a low rate of stimulation such that the abdominal cord response does not habituate (one stimulation every 30 sec for instance), no alteration is seen in the slow-fibre responses to successive stimuli. At increased frequencies a habituation develops and it seems to occur for about the same range of frequencies in both records. Moreover, the time course of this habituation (down to a plateau above the zero level, Fig. 6), and of spontaneous restoration of the responses, appears approximately the same in the two records. At the periphery the usual decreasing but persistent startle reaction is observed. Even if no movement can be seen, a response is detected in the electromyographic recording. Air-puff

cereal stimulation

We then activated the cerealfibresby air puffs applied to the cerci. This stimulation should lead to two results. First, it may produce fast-fibre responses where electrical stimulation did not (see above); and secondly, the extent to which the isolated pre.


393

100%

Ar=O-5sec

50%

100%,-

50% L

Fig. 6. Parallel habituation of the abdominal cord responses and of those of an N 4 slow fibre to stimulation of a cereal nerve. The curves represent the amplitude of abdominal responses (Abd.) and of the responses of an N 4 slow excitatory fibre to stimulation of an ipsilateral cereal nerve as a function of the number of stimuli delivered. The inter-stimulus interval, At, was o-s sec. The amplitude of the abdominal responses was measured by their surface and that of the N 4 responses by the number of potentials of the responses (initial burst and afterdischarge). These amplitudes are expressed as a percent of the amplitude of the first response.

paration resembles the intact animal may thus be more easily assessed (cf. Discussion). The air puffs for this part of the study were delivered in the conditions established in the first study (Zilber-Gachelin & Chartier, 1973). They were delivered at regular intervals which varied from 5 sec to 2 min. In these conditions the stimulus can induce a complex motor response of both the slow-type and the fast-type motor axons, very similar to that obtained by repetitive electrical stimulation of the abdominal cord, and which will be referred to as R 3. The slow-fibre response (Fig. 7, trace E8) can be divided into three parts: (a) It always begins with a response of the R 1 type. (b) Then the firing rate can remain at the mean rate of the 'afterdischarge' of R i, thus simply increasing the duration of R i, or it can undergo a further increase, which occurs either after the frequency has decreased down to the resting level (Fig. 7) or before. This is the very variable part of the response pattern, the slow fibres often showing several irregular oscillations of their firing rate. (c) Finally, the firing rate increases abruptly to a much higher value (175-250/sec instead of 8-50/sec); from then on the activity consists of a train of spike bursts occurring fairly regularly (around every 1-3 sec); the mean maximum firing frequency in each burst is in the range just mentioned, but with a tendency first to increase then to decrease slightly, until this particular pattern of firing suddenly stops, sometimes after 1 or 2 bursts at a much lower firing rate (maximum of 50-90/sec); but this last part of the response is much more irregular, somewhat as at the beginning of the response. The rhythmic train response has a duration of about 14 sec (13-16 but never more nor less), whatever the frequency of the air puffs and their duration; even if the interval between puffs is smaller than 14 sec, the arrival of air puffs in the middle

394


62-5 sp/sec EF

125 sp/sec

250 sp/sec

AM.

Pi

5sec

Fig. 7. Responses of a slow excitatory fibre and of a fast excitatory fibre of N 4 to air puffs applied to the cerci. The different analogous curves represent the instantaneous frequency of the discharges (i) of a fast excitatory fibre ( £ » of N 4 of the left posterior leg, (ii) of a slow excitatory fibre (Es) of this nerve, and (iii) of the abdominal cord (Abd.), in response to cereal air puffs (Jh and pj. The two stimuli fa and p, are separated by 20 sec. On the right side are the calibrations of the curves (number of spikes per second for the vertical deflexion indicated). The smallest spikes of the abdominal recording were not counted by the frequency to voltage converter.

of the response does not change its pattern or duration in any way; if an air puff is delivered just at the end of the usual response, it will not give any response nor increase the duration of R 3. Thus there seems to exist some kind of threshold; if it is crossed, the train type of response will always develop, independently of other air-puff stimuli, as a practically all-or-none response. When an R 3 response occurs, the fast fibres are always activated (Fig. 7, trace EF). They fire, as do the slow ones, in bursts which recur at the same rhythm as the former. Two differences must nevertheless be noted. First, the fast-fibre activity always appears after the beginning of the slow-fibre train response and often ends before it, so that its mean duration is shorter (around 11 sec). Secondly, the maximum firing rate of the spikes within the bursts is smaller, around 50/sec. This R 3 response leads to large rhythmic extensions of the trochanter. When the air puffs are repeated, R 3 can disappear as early as the second stimulus, while the corresponding abdominal response simply decreases slightly. An example is shown in Fig. 7, where the interval between stimuli was 20 sec. If this interval is larger (e.g. 1-2 min), several R 3 responses will probably be obtained. In this case the complete disappearance of R 3 is usually preceded by a drop out of one or two R 3 responses. This latter behaviour may be likened to that of a graded turn-off of an all-or-none process. The stimuli which lead to a failure of R 3 and those whicM


395

Ffollow the last R 3 response produce a response of the R 1 type, but where the firing rate is generally higher and where there often occur several small irregular bursts of spikes in the slow fibres (Fig. 7, p2), with sometimes a few spikes in the fast fibres. This type of response will be referred to as R 2. At the periphery some small movements of the leg can be observed. When the stimuli are repeated, the fast-fibre spikes disappear altogether and the response gets stabilized to a small R 1. A small but persistent startle is then observed. At the same time, the abdominal response decreases down to a plateau, as described previously (Zilber-Gachelin & Chartier, 1973). The problem of the abdominal determinism of R 3 will be discussed below. Let us only note here that the abdominal decrease is not perfectly regular, and the abdominal response to a late trial may be of an amplitude comparable to that of the first stimulus of the series; nevertheless no R 3 response will reappear. This implies that an overriding phenomenon establishes itself with successive stimuli. It is apparent that this phenomenon resides in the habituability of some synaptic junctions in the thoracic ganglion. During the R 3 response spikes of very low amplitude could be recorded on the abdominal cord. Since such responses have never been observed on isolated abdomen preparations and since these units fired during spontaneous agitation, they probably correspond to activation of descending fibres and were not considered to be part of the abdominal cord response in these analyses. In many preparations no R 3 response was obtained at all, even to the first air puff, but only R 1, just as described for the cereal nerve stimulation, with the same peripheral startle reaction. An additional observation was made in the case of air-puff stimulation. In several cases, if the repetition rate of the stimulus was around 5-10/sec, an apparent sensitization of the response occurred; after 1-4 stimuli leading to R1, an R 3 response appeared, in spite of the continuing decrease of the abdominal cord response. This is very similar to the observations made when identical trains of electrical stimuli were repeated on the abdominal cord and led to the appearance of a fast-fibre response (cf. Fig. 5, trace 7). The sensitization observed is thus due to the properties described at the thoracic level. As in the latter case, the response is not temporally related in a precise way to the stimulus; it can appear up to 3-5 sec after the beginning of the air puff. Of course, if the stimulations are continued R 3 disappears, the sensitization being overshadowed by habituation taking place both at the abdominal and thoracic levels.

DISCUSSION

In the present study, in order to unravel the respective roles of each relay of the escape reflex to a cereal air puff, we had to suppress, as thoroughly as possible, interfering influences. The drawback of this procedure can be of course the difficulty in relating the observed results to what is happening in the intact free animal. In this last condition we did not study quantitatively the behavioural effects of successive air puffs - movements of the different joints of a leg, co-ordination of the legs, etc. But the gross behaviour has been described (cf. beginning of the Results section). It seems reasonable to assume that (i) the large rhythmic movements due to fast-fibre activation (R 3) observed in the case of the isolated reflex loop are associated

396


with the escape response; (ii) R 2 corresponds to the habituated forward reaction;" (iii) the observed startle reactions due only to activation of the slow fibres (R 1) are associated with the startle reactions in the intact animal. The gross behaviour thus appears to be the same in the isolated and in the intact preparation. Moreover, Ewing & Manning (1966) recorded in muscles of whole free-walking cockroaches the same kind of pattern of slow and fast units that we have seen in N 4 for the reflex loop. The general model which will be given for the isolated loop would thus be valid in the whole free animal, i.e. the major components of this biologically important plastic behaviour would be explicable in terms of relatively simple processes at the neuronal level. The different influences interfering with the reflex in normal conditions would modulate the response (cf. below) but not change its general trend of variation with repetition of air puffs applied to the cerci. Let us now examine how this paper and the previous one (Zilber-Gachelin & Chartier, 1973) can explain the variations of the reflex motor output of the isolated reflex loop due to repetition of the reflexogenic stimulus in terms of the dynamic properties of the two central relays involved in this reflex. (a) We described in the preceding paper (Zilber-Gachelin & Chartier, 1973) a habituation of the abdominal cord responses down to a non-zero plateau after passage of the impulses through the 6th A.G. (b) In the first parts of the present paper we described in the same way the dynamic properties of the 3rd T.G. Let us first note that this study has been carried out with electrical stimulations of the abdominal cord, which risked activation of abdominal fibres not involved in the escape reflex. This risk was reduced to a minimum by stimulating the connectives between the 6th and the 5th A.G. Moreover, the properties displayed through abdominal stimulation are consistent with the rest of this study. In particular, in the last part of this paper we also studied N 4 responses to cereal stimulation and the different types of motor responses were similar in both cases. So it seems that the mode of stimulation used has been relatively selective, probably because the fibres involved in the reflex are essentially located in the dorsal part of the abdominal connectives, near to the stimulating electrodes. Our results suggest that, at the level of the 3rd T.G., the abdominal cord afferents are projected through two main types of synaptic pathways on to the motoneurones: (i) One on to the slow excitatory fibres which always respond to over-threshold abdominal stimulation (in spite of a diminution, their responses never disappear completely through repetition of the stimuli); they are responsible for tonus, small movements and startle reactions. (ii) One on to the fast excitatoryfibres,activation of which we showed to result from an important temporal and spatial summation and to lead to sudden and large movements of the leg. Activity of the fast excitatory fibres is always accompanied by an i ncrease of that of the slow excitatory fibres. Furthermore, both types of synapses show, during repetitive stimulation, antagonistic properties of facilitation and habituation. (c) Finally, the anatomical and functional link between the two central relays (6th A.G. and 3rd T.G.) was preserved and the whole reflex loop studied with cereal stimulation. No precise correlations could be expected to be found between the two recordings since they were made with external electrodes, so that the abdominal recording probably did not include the activity of all the fibres involved in the real response or, t


397

) on the contrary, could include activity from fibres not involved in the reflex under study; in particular Dagan & Parnas (1970) claimed that the giant fibres do not synapse with the motoneurones of the legs. But the question remains open since (i) these authors only considered the largest giants, (ii) collaterals of the giant axons have been shown in the thoracic ganglia (Farley & Milburn, 1969; Harris & Smyth, 1971) which might well synapse with the motoneurones (Farley & Milburn, 1969), and (iii) it is quite possible that activity in these giants produces a d.c. shift in the thoracic neuropil which can lead to activation of the motoneurones (Hoyle, 1970). However that may be, two groups of relevant results have been obtained. (c 1) If only the R 1 response responsible for the startle reaction is obtained on N 4, a gross parallelism has been shown between the changes of R 1 and of the abdominal response due to repetition of the stimuli. This parallelism is striking enough to suggest that a major role is played by the habituation properties of the 6th A.G. in the startle decrease and its persistence through repetition of cereal air puffs. This is consistent with our finding that R 1 is not very sensitive to repetition of electrical stimuli on the abdominal cord (cf. also Schlue, 1967). (c 2) In the cases where the R 3 response which includes activation of the fast fibres of the motor nerve is elicited, this rhythmic complex response disappears very rapidly if the air puffs are repeated, even at low frequency (e.g. i/min), whereas the abdominal response persists. Only a response of the slow fibres, R 2 then R 1, is left on N 4. Since it has been observed that the fast-fibre response appears only if a stiff!cient temporal summation is obtained at the 3rd T.G. by repetitive stimulation, we can assume likewise that its disappearance is related, in addition to the thoracic habituability, to the observed decrease, even small, in the impulse frequency of the abdominal response. Our results thus, contrary to those of Roeder (1948, 1959), stress the important role of the 6th A.G. in habituation of the peripheral response. Since, on the one hand, the thoracic synapses have been shown to be habituable (cf. also Hoyle, 1970) and, on the other hand, their output is very dependent on the frequency of the abdominal input, it is clear that the overall habituation effect of repetitive air puffs will be highly reinforced on the motor nerve relative to the habituation observed in the 6th A.G. In the case of reflexes involving many interneurones, such effects could explain a massive peripheral habituation (cf. Thorpe, 1956) relative to that observed at the level of each relay. (c 3) If the first air puff produces an R 1 response, and if this stimulus is repeated about every 7 or 10 sec, the abdominal response shows the usual habituation but R 3 may appear, before fading away again, after a few puffs. This sensitization (cf. also Roberts, 1966) is probably due to the post-tetanic potentiation which we have shown to exist at the thoracic level and which is overshadowed by habituation after a few puffs. It of course appears more easily for these short inter-stimulus intervals, but, for still shorter ones, habituation which develops quicker (cf. Zilber-Gachelin & Chartier, 1973) prevails over potentiation from the beginning of the series of puffs, so that no sensitization can be observed. The different conclusions we have come to can be set into an operational model (Fig. 8, A 1): the maximum frequency of the abdominal discharge (Abd. /max) should exceed a threshold critical value (cf.) in order to produce a fast-fibre R 3 response. Moreover, the appearance on N 4 of a fast-fibre response with repetition

398

N. F. ZILBER-GACHELIN AND M. P. CHARTIER 1 C^t Puff)

A r

2

R-2

(«th Puff) " l

c.f.

0

D

"lj

40 sec

20

40 sec

1}

fTr\Tk

E

c.f.

20 D

LJ

0 7 14 21 28 35 42 sec Fig. 8. Operational model intended to explain the modification of the motor reflex response of N 4, due to repetition of cereal air puffs. The two parts of each diagram have the aame .XT-axis. The upper part represents the thoracic central excitatory state (Th.c.e.8.), and the lower part the maximum frequency of the abdominal response (Abd. / , „ ) as a function of time. The type of response obtained on N 4 is indicated above the diagrams. E, Threshold of Th.c.e.s. above which an R 3 response is obtained, cf., Critical frequency of Abd. fmxx under which no R 3 response can be obtained to a single puff. (A) Assumptions of the model. An air puff is delivered at the level of each arrow. The larger the increase in Th.c.e.s. due to a puff, the longer it lasts (Zilber-Gachelin, 1966, 1970). (A 1) Responses to the first puff of an experiment; the larger the abdominal response, the larger the increase in Th.ae.s. (Abd. f,^) 1 > c.f.: R 3 is obtained. (Abd. /„,„) 2 = c.f.: R 2 is obtained. (Abd. /„*,) 3 < c.f.: R 1 is obtained. (A 2) Responses to the nth puff (n> 1) in the same animal; due to habituation in the 3rd T.G. the same abdominal response as in A 1 produces a smaller increase in Th.c.e.s., so that: (Abd. / „ „ ) 1 > c.f. leads to an R 2 response, and (Abd. / J 3 to an R I response whose amplitude is smaller than in the previous case. (B) Habituation when the first response is R 3; R 3 disappears here from the second puff. (C) Same inter-stimulus interval (20 sec) as in (B) but the first response is R 1; R 1 progressively decreases to a non-zero level (as does the abdominal response). (D) Same response to the first air puff as in (C), but the inter-stimulus interval is smaller (7 sec). The successive puffs are thus delivered before the Th.c.e.s. has returned to its resting level, and a sensitization effect is produced: R 3 appears to the 3rd puff. This practically all-ornone response lasts 14 sec, so that the 4th and the 5th puffs have apparently no peripheral effect.


399

P>f the stimuli, provided that tne frequency of stimulation is high enough, seems to suggest that each stimulus leads, within the thoracic ganglion, to the build-up of some 'central excitatory state' (c.e.s.) which dissipates with time. The higher Abd. /max, the higher the increase in c.e.s. in the thoracic ganglion. A fast-fibre response would be elicited when this c.e.s. exceeds a threshold E; for a value around E, R 2 will be obtained, and, for a subthreshold value, R 1. The amplitude of R 1 would reflect that of the increase in c.e.s. Moreover, the habituability of the thoracic synapses is taken into account in the model where, for a given amplitude of the abdominal response, a stimulus of the rank n > 1 leads to a smaller increase in c.e.s. than the first stimulus (Fig. 8, A 2). Finally, in a series of cereal stimulations the R 3 response does not suddenly and definitively disappear, but shows a few reappearances to the stimuli following its first disappearance; this could be accounted for in the model by small variations in the level of E, all the more as the spontaneous activity recorded on N 4 showed also irregular variations, with temporary increases of activity. It should be noted that this model is purely operational and does not prejudge the actual mechanism of this build-up of c.e.s.: it could very well be a disinhibition, since, in thoracic interneurones of a resting cockroach, many spontaneous i.p.s.p.'s have been observed which were suppressed by cereal air-puff stimulation (Bentley, 1969). However that may be, this simple model can explain all the different observations above (cf. Fig. 8B, C, D) and, besides, fits very well with the results we obtained on the variation of the motor response through application of different peripheral stimuli (Zilber-Gachelin, 1966, 1967, 1970; Zilber-Gachelin & Chartier, 1969). No experiments were carried out to examine the possibility of habituation effects at the neuro-muscular junctions (cf. Bruner & Kennedy, 1970), but the properties we observed in the central synapses seem to be sufficient to explain the peripheral effects. The motor response with activation of the fast units has appeared to us as being partially independent of the abdominal impulses which trigger it; the timing of the spike discharge is in no way related to that of the abdominal stimuli, and the response can last a long time after the end of the stimulations. This type of response, which implies the existence of a neural pattern generator in the 3rd T.G., appears to be characteristic of insect thoracic neurones. It has been described for instance by Luco (1963), who called it 'natural response', by Svidersky (1965), and by Wilson (1965a, 1966a, 1967), who called it 'tonic reflex'. It has been claimed (Hughes, 1965) that air puffs applied to the cerci induce a simple retraction of the legs, the rhythmic movements being dependent on a feedback loop from the periphery. Since in our experiments all the sensory fibres were cut, which excluded any possible peripheral feedback, our results suggest that, on the contrary, the rhythmical movements evoked by the peripheral stimulations are precoded in the 3rd T.G. That central oscillators might well control insect muscles during stereotyped behaviour has been postulated for a long time (e.g. von Hoist, 1948; Hughes, 1958; Huber, 1962; Hoyle, 1964; Wilson, 19656, 1966 a, b; Wendler, 1966; Wilson, 1967). The central patterning of motoneuronal rhythmic activity has been even demonstrated for insect flight (Wilson, 1961, 19656; Wyman, 1965, 1966; Wilson, 1968) and insect ventilation (Miller, 1965; Mill & Hughes, 1966; Miller, 1966; Farley, Case & Roeder, 967). But the problem of the control of rhythmic walking leg movements is still

400


debated. The co-ordination of the six legs is probably largely dependent on proprioQ ceptive feedback (e.g. von Hoist, 1935; Hughes, 1957; Delcomyn, 1971), but the question remains open for the rhythmical activity of each leg. The work of Usherwood and his associates (Runion & Usherwood, 1968; Usherwood, Runion & Campbell, 1968; Usherwood & Runion, 1970) stresses the considerable importance of sensory information in determining the activity of the leg motoneurones during walking, while the work of Pearson & Bergman (1969) and Pearson & lies (1970) indicates a central patterning. It is quite possible that the mechanism is different according to the species; the former work was done on the locust and the latter on the cockroach. Moreover, Bentley (1969) could never, in the cricket, obtain rhythmical bursts of activity in the motoneurones in response to cereal air puffs, while we saw it very easily in the cockroach. As we pointed out at the beginning of the discussion, and as discussed by Pearson (1972), this rhythmic activity in the de-afferented preparation is probably the basic pattern of walking. A central locomotor rhythm generator thus appears to exist in the cockroach metathoracic ganglion. In the whole animal the feed-backs from the legs, which are known to exist (e.g. Pringle, 1940; Wilson, 1965 c; Delcomyn, 1971), would modulate the stereotyped centrally generated pattern we described (cf. Wendler, 1961, 1964, 1966, for the stick insect). The existence of centrally determined programs modulated by peripheral feedbacks might well appear to be fairly common in arthropods (cf. e.g. Eisner & Huber, 1969). Let us finally emphasize that, from our work, it is clear that the existence of a habituation phenomenon does not necessitate the presence of the cephalic ganglia of the animal. Some experiments in which the nerve cord was left intact between the 3rd T.G. and the cerebral ganglia (unpublished results) allowed us to see that habituation of the N 4 responses had apparently identical characteristics to those obtained after section of the nerve cord. Although it cannot be denied that the anterior centres modulate the reactivity of the animal (Weiant, 1958; Roeder, 1959; Huber, 1965; Hughes, 1965; Rowell, 1970), they seem to have little influence on the habituation of the reflex we have studied. The same appears to be true in nereid polychaetes (Evans, 1969). Moreover, we have shown (Zilber-Gachelin, 1970; Zilber-Gachelin & Chartier, 1969) that peripheral influences from different sensory modalities affect the peripheral response by some sensory-motor integration taking place inside the thoracic ganglia, also independently of the head. In particular, dishabituation of the escape reflex, which does not exist in the 6th A.G. (Zilber-Gachelin & Chartier, 1973), would take place at the thoracic level (Zilber-Gachelin, 1970). To our knowledge it is the first time that habituation of a reflex response is shown to involve two successive habituable synapses with dishabituation by an extra stimulus taking place at only one of them. SUMMARY

1. The synaptic transfer properties within the 3rd thoracic ganglion (T.G.) from the abdominal cord axons to the motoneurones has been studied in the cockroach. This ganglion was completely de-afferented except for motor nerve 4, whose links with the muscles of the posterior legs were left intact. 2. The response of one of these nerves to electrical stimulation of the abdominal cord involves activation of 2 types of units:


401

(a) Slow excitatory fibres which have a tonic discharge and respond to each lowthreshold abdominal cord stimulation by a transient increase of this firing rate; these units are responsible for muscular tonus and for low amplitude movements (startle reactions). (b) Fast excitatory fibres, which have no tonic discharge and require for their activation higher intensity and frequency of stimulation, i.e. an important temporal and spatial summation. They are responsible for larger and more rapid movements. They fire without any precise chronological relation with the stimuli, often in bursts which continue after the end of the stimulations and cause sudden rhythmic movements. 3. During repetition of the stimuli, the two types of synaptic pathways show both habituation and facilitation through temporal summation and post-tetanic potentiation. These two phenomena persist after the end of the stimulations and have long (minutes) but different time courses. Moreover, habituation always prevails over facilitation if stimulations are continued during a sufficient time. These antagonistic properties existing at the same time might explain the complex way in which the motor responses develop with the application of repetitive trains of stimuli to the cord. 4. The role of these properties in the changes of the reflex motor responses to successive air puffs applied to the cerci has been studied. These properties appear to be responsible for the sensitization of the responses which can be sometimes observed. They lead, in conjunction with the habituation properties of the 6th abdominal ganglion (A.G.), to the disappearance of the escape reflex involving firing of both fast and slow fibres. Finally, they seem to have a minor role in habituation of the startle reactions (involving firing of only the slow fibres) which would be mainly due to the 6th A.G. habituability. We are greatly indebted to Dr A. Mallart for his invaluable help and his thoughtful comments during preparation of the manuscript, and to Dr R. Kado for his so careful and critical reading of this manuscript. Thanks are also due to Dr J. Bruner for very useful discussions and to Mrs R. Zilber for translation of Svidersky's paper. REFERENCES BENTLEY, D. R. (1969). Intracellular activity in cricket neurons during the generation of behaviour patterns. J. Insect Physiol. is, 677-99. BRUNER, J. & KENNEDY, D. (1970). Habituation, occurrence at a neuromuscular junction. Science, N. Y. 169, 92. CHARTIER, M. P. & ZILBKR-GACHELIN, N. F. (1969). Indications sur le r61e des ganglions thoraciques dans 1'habituation, chez la Blatte, du reflexe de fuite a une bouffee d'air sur les cerques. J. Physiol., Paris 61, 242. DAGAN, D. & PARNAS, I. (1970). Giant fibre and "mull fibre pathways involved in the evasive response of the cockroach, Periplantta americana. J. exp. Biol. 53, 313-324. DELCOMYN, F. (1971). The effect of limb amputation on locomotion in the cockroach, Periplaneta americana. J. exp. Biol. 54, 453-69. DRESDEN, D. & NIJENHUIS, E. D. (1958). Fibre analysis of the nerves of the second thoracic leg in Periplaneta americana. Proc. K. ned. Akad. Wet. (C) 61, 213-23. ELSNER, N. & HOBER, F. (1969). Die Organisation des Werbegesanges der Heuschrecke Gomphocerippus rufus in Abhfingigkeit von zentralen und periphere Bedingungen. Z. vergl. Physiol. 65, 380-423. EVANS, S. M. (1969). Habituation of the withdrawal response in nereid polychaetes. 2. Rates of habituation in intact and decerebrate worms. Biol. Bull. mar. biol. Lab., Woods Hole 137, 105-17. EWING, A- W. & MANNING, A. (1966). Some aspects of the efferent control of walking in three cockroach species. J. Insect Physiol. ia, 1115-18.

402


FAKLBY, R. D., CASE, J. F. & ROEDER, K. D. (1967). Pacemaker of tracheal ventilation in the cockroacW

Periplaneta americana. J. Insect Pkytiol. 13, 1713-28. FARLEY, R. D. & MILBURN, N. S. (1969). Structure and function of the giant fibre system in the cockroach, Periplaneta americana. J. Insect Physiol, 15, 457—76.

HARRIS, C. L. & SMYTH, T. JR. (1971). Structural details of cockroach giant axons revealed by injected dye. Comp. Biochem. Physiol. 40A, 295-303. HESS, A. (1958). Experimental anatomical studies of pathways in the severed central nerve cord of the cockroach. J. Morph. 103, 479-501. VON HOLST, E. (1935). Die Koordination der Bewegung bei den Arthropoden in Abhlngigkeit von zentralen und peripheren Bedigungen. Biol. Rev. 10, 234—61. VON HOLST, E. (1948). Von der Mathematik der nervOsen Ordnungaleistung. Experientia 4, 374-81. HOYLE, G. (1964). Exploration of neuronal mechanisms underlying behavior in insects. In Neural Theory and Modeling (ed. R. F. Reiss), pp. 346-76. Stanford University Press. HOYLB, G. (1970). Cellular mechanisms underlying behavior. Neuroethology. Adv. Insect Physiol. 7. 349-444HOBER, F. (1962). Central nervous control of sound production in crickets and some speculations on its evolution. Evolution 16, 429-42. HOBER, F. (1965). Brain-controlled instinctive behaviour in Orthopterans. In The Physiology of the Insect Central Nervous System (ed. J. E. Treherne and J. W. L. Beament), pp. 233-46. London: Academic Press. HUGHES, G. M. (1957). The co-ordination of insect movements. II. The effect of limb amputation and the cutting of commissures in the cockroach (Blatta orientalis). J. exp. Biol. 34, 306—33. HUGHES, G. M. (1958). The co-ordination of insect movements. III. Swimming in Dytiscus, Hydrophilus and a dragonfly nymph. J. exp. Biol. 35, 567-83. HUGHES, G. M. (1965). Neuronal pathways in the insect central nervous system. In The Physiology of the Insect Central Nervous System (ed. J. E. Treherne and J. W. L. Beament), pp. 79-112. London: Academic Press. KANDEL, E. R. & SPENCER, W. A. (1968). Cellular neurophysiological approaches in the study of learning. Physiol. Rev. 48, 65-134. Luco, J. V. (1963). Plasticity and the natural response of a nervous organization. In Perspectives in Biology (ed. C. F. Cori, V. G. Foglia, L. F. Leloir and E. Ochoa), pp. 335-60. Amsterdam: Elsevier. MILL, P. J. & HUGHES, G. M. (1966). The nervous control of ventilation in dragonfly larvae. J. exp. Biol. 44, 297-316. MILLER, P. L. (1965). The central nervous control of respiratory movements. In The Physiology of the Insect Central Nervous System (ed. J. E. Treherne and J. W. L. Beament), pp. 141-55. London: Academic Press. MILLER, P. L. (1966). The regulation of breathing in insects. Adv. Insect Physiol. 3, 279-344. NIJENHUIS, E. D. & DRESDEN, D. (1955). On the topographical anatomy of the nervous system of the mesothoracic leg of the american cockroach (Periplaneta americana), I. Proc. K. ned. Akad. Wet. (C) 58, 121-30. PEARSON, K. G. (1972). Central programming and reflex control of walking in the cockroach. J. exp. Biol. 56, 173-93PEARSON, K. G. & BERGMAN, S. J. (1969). Common inhibitory motoneurones in insects. J. exp. Biol. 50, 445-71PEARSON, K. G. & ILES, J. F. (1970). Discharge patterns of coxal levator and depressor motoneurones of the cockroach, Periplaneta americana. J. exp. Biol. 53, 139-65. PIPA, R. L. & COOK, E. F. (1959). Studies on the hexapod nervous system. I. The peripheral distribution of the thoracic nerves of the adult cockroach, Periplaneta americana. Ann. entomol. Soc. Am. 52, 695-710. PRTNGLE, J. W. S. (1939). The motor mechanism of the insect leg. J. exp. Biol. 16, 220-31. PRINGLE, J. W. S. (1940). The reflex mechanism of the insect leg. J. exp. Biol. 17, 8-17. ROBERTS, M. B. V. (1966). Facilitation in the rapid response of the earthworm Lumbricus terrains L. J. exp. Biol. 45, 141-50. ROEDER, K. D. (1948). Organization of the ascending giant fibre system in the cockroach (Periplaneta americana). J. exp. Zool. 108, 243-62. ROEDER, K. D. (1953). Electrical activity in nerves and ganglia. In Insect Physiology (ed. K. D. Roeder), pp. 423-62. New York: John Wiley. ROEDER, K. D. (1959). A physiological approach to the relation between prey and predator. Smithson. misc. Collns 137, 287-306. ROWELL, C. H. F. (1970). Incremental and decremental processes in the insect central nervous system. In Short-term Changes in Neural Activity and Behaviour (ed. G. Horn and R. A. Hinde), pp. 237—80. Cambridge University Press. RUNION, H. I. & USHERWOOD, P. N. R. (1968). Tarsal receptore and leg reflexes in the locust, J. exp. Biol. 49, 421-36.

Activation of cockroach tnotoneurones

403

W. R. (1967). ErregungsUbertragung von Riesen&sem auf Motoneurone bei der Schabe Periplaneta americana L. Experientia 33, 38. SVIDERSKY, V. L. (1965). Activity des neurones isolea du ganglion thoracique du criquet asiatique (Locusta rmgratoria). (In Russian.) Dokl. Akad. Nausk SSSR 164, 1204-7. THORPE, W. H. (1956). Learning and Instinct in Animals. London: Methuen. USHERWOOD, P. N. R. & RUNION, H. I. (1970). Analysis of the mechanical responses of metathoracic extensor tibiae muscles of free-walking locusts. J. exp. Biol. 53, 39-58. USHBRWOOD, P. N. R., RUNION, H. I. & CAMPBELL, J. I. (1968). Structure and physiology of a chordotonal organ in the locust leg. J. exp. Biol. 48, 305-23. WBIANT, E. A. (1958). Control of spontaneous efferent activity in certain efferent nerve fibres from the metathoracic ganglion of the cockroach, Proc. 10th int. Congr. Ent. (1956) a, 81-2. WENDLER, G. (1961). Die Regelung der Korperhaltung bei Stabheuschrecken (Carausius 7/iorosus). Naturwissenschaften 48, 667-77. WENDLER, G. (1964). Laufen und Stehen der Stabheuschrecke Carausius morosus: Sinnesborstenfelder in den Beingelenken als Glieder von Regelkreisen. Z. vergl. Physiol. 48, 108-250. WENDLER, G. (1966). The co-ordination of walking movements in Arthropods. In Nervous and Hormonal Mechanisms of Integration. Symp. Soc. exp. Biol. 30, 220-49. Cambridge University Press. WILSON, D. M. (1961). The central nervous control of flight in a locust. J. exp. Biol. 38, 471-90. WILSON, D. M. (1965a). The nervous co-ordination of insect locomotion. In The Physiology of the Insect Central Nervous System (ed. J. E. Treherne and J. W. L. Beament), pp. 125-40. London and New York: Academic Press. WLLSON, D. M. (19656). Motor output patterns during random and rhythmic stimulation of locust thoracic ganglia. Biophys. J. 5, 121-43. WILSON, D. M. (1965c). Proprioceptive leg reflexes in cockroaches. J. exp. Biol. 43, 397-409. WILSON, D. M. (1966a). Insect walking. Ann. Rev. Entomol. 11, 103—22. WILSON, D. M. (19666). Central nervous mechanisms for the generation of rhythmic behaviour in Arthropods. In Nervous and Hormonal Mechanisms of Integration. Symp. Soc. exp. Biol. 30, 199-228. Cambridge University Press. WILSON, D. M. (1967). An approach to the problem of control of rhythmic behaviour. In Invertebrate Nervous Systems (ed. C. A. G. Wiersma), pp. 219—29. Chicago University Press. WILSON, D. M. (1968). The nervous control of insect flight and related behaviour. Adv. Insect Physiol. 5. 280-338. WYMAN, R. J. (1965). Probabilistic characterization of simultaneous nerve impulse sequences controlling dipteran flight. Biophys. J. 5, 447-71. WYMAN, R. J. (1966). Multistable firing patterns among several neurons. J. Neurophysiol. 39, 807-33. ZILBER-GACHELIN, N. F. (1966). Experiences de sensibilisation chez la Blatte. J. Physiol., Paris 58, 276. ZtLBER-GACHELiN, N. F. (1967). Sensibilisation et desensibilisation chez la Blatte. J. Physiol., Paris 59. 309ZILBER-GACHELIN, N. F. (1970). Etude comportementale et electrophysiologique de quelques facteurs responsables de la variability des reponses motrices reflexes de la Blatte. Thesis, University of Paris. ZILBER-GACHELIN, N. F. & CHARTIER, M. P. (1969). Niveau synaptique de la 'desensibilisation' par appui des pattes chez la Blatte (Blabera craniifer). J. Physiol., Paris 61, 434. ZILBER-GACHELIN, N. F. & CHARTTER, M. P. (1973). Modification of the motor reflex responses due to repetition of the peripheral stimulus in the cockroach. I. Habituation at the level of an isolated abdominal ganglion. J. exp. Biol. 59, 359—81. ZILBER-GACHELIN, N. F., PAUPARDIN, D. & CHARTIER, M. P. (1973). Functional and anatomical aspects of leg innervation in the giant cockroach Blabera craniifer as compared with that in the American cockroach Periplaneta americana. Comp. Biochtm. Physiol. 45A (in the Press).

26

E x B 59