557

Journal of Physiology (1991), 432, pp. 557-571 With 6 figures Printed in Great Britain

RESPONSES OF CAT MUSCLE SPINDLES WHICH LACK A DYNAMIC FUSIMOTOR SUPPLY BY M. GIOUX, J. PETIT AND U. PROSKE* From the Laboratoire de Neurophysiologie, College de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France

(Received 29 January 1990) SUMMARY

1. The experiments reported here support the view that some spindles in the peroneus tertius muscle of the anaesthetized cat lack a nuclear bag, intrafusal fibre. 2. The bag1 fibre is characterized by the fact that it is innervated exclusively by dynamic fusimotor axons. A method was devised to test each spindle in peroneus tertius for a dynamic fusimotor innervation. The ventral roots containing the muscle's motor supply were subdivided into five portions, approximately equal in terms of the tension they generated, and each piece was stimulated in turn, repetitively, at fusimotor strength, during ramp stretch of the muscle, to look for a large increase in dynamic response. 3. The method allowed confirmation that the majority of spindles in peroneus tertius had a dynamic fusimotor innervation. However, where the dynamic effect was weak and accompanied by a strong static fusimotor action and extrafusal unloading, it risked being overlooked. 4. The confirmatory test for the presence of a bag1 fibre was whether or not the spindle showed a large increase in dynamic response in the presence of the drug succinyl choline (SCh) injected arterially close to the muscle in which the spindle is located. SCh is known to induce a contracture in the bag1 fibre and therefore mimics tonic dynamic fusimotor stimulation. 5. In five experiments, of a total of forty-two spindles with afferents conducting within the group I range, five examples were encountered where there was no increase in dynamic response, either with ventral root stimulation or perfusion with SCh. It was concluded that these were spindles which lacked a bag1 fibre. 6. Passive stretch of such spindles revealed no feature in the response which allowed them to be distinguished from spindles in which the bag1 fibre was present. This conclusion posed the question, what contribution, if any, does the bag1 fibre make to the stretch response ? 7. It was possible to show that under some conditions the bag1 fibre did contribute to the response to stretch. If the spindle was conditioned by repetitive nerve stimulation, at fusimotor strength, at a length longer than that at which a test stretch was applied, the response to the stretch was delayed, and it began at a lower *

Present address: Department of Physiology, Monash University, Clayton, Victoria, Australia.

MS 8227

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M. GIOUX, J. PETIT AND U. PROSKE

rate than after conditioning stimulation at the test length. The delayed response was attributed to the presence of slack in intrafusal fibres. With the spindle in this state, if a second conditioning stimulus was given to a single dynamic fusimotor fibre, supplying the spindle, the onset of the stretch response was earlier, suggesting that slack had been taken up selectively in the bag, fibre, which was now able to contribute to the stretch response. 8. It is concluded that during large ramp stretches of passive spindles the dynamic response is normally generated in the bag2 fibre. The bag, fibre makes a significant contribution only if it is actively contracting or when the stretch immediately follows a period of dynamic fusimotor stimulation. INTRODUCTION

Muscle spindles are stretch receptors found in most skeletal muscles. The large afferent fibre, the I a fibre, makes an annulospiral termination, the primary ending, on the equatorial regions of nuclear bag and nuclear chain intrafusal fibres. Group II axons make secondary sensory endings predominantly on nuclear chain fibres. The distinguishing physiological characteristic of the primary sensory ending is that it is dynamically sensitive, responding both to the length change as well as the rate of length change of the muscle. Secondary endings have rather little dynamic sensitivity. Current ideas about the origin of the dynamic sensitivity of the primary ending are based on the mechanical properties of the underlying intrafusal fibres (Matthews, 1972). The equatorial regions of intrafusal fibres show structural specializations, including an accumulation of nuclei and an almost complete lack of myofibrillar material. This is particularly pronounced for the nuclear bag fibres. In nuclear chain fibres the nuclei are surrounded by a wider rim of contractile filaments (Corvaja, Marinozzi & Pompeiano, 1969). During stretch of the spindle, because of the lack of myofibrils, the compliant equatorial regions are extended at the expense of the stiffer polar segments, leading to opening of the spirals of the sensory endings and the initiation of an afferent response. The degree of extension is dependent on the rate of stretch: the higher the rate, the greater the viscous stiffness of the poles and therefore the larger the proportion of the stretch taken up by the equatorial region. It has.generally been assumed that the dynamic response of the spindle during a ramp stretch arises in nuclear bag fibres since these show the greater structural specialization in their equatorial region. The majority of spindles have two nuclear bag fibres, the dynamic bag1 fibre and the static bag2 fibre (Barker, 1974; Boyd, 1981). The two fibres differ in their mechanical properties, the bag2 fibre contracting more rapidly than bag, (Bessou & Pages, 1975; Boyd, 1976). Apart from its slow rate of contraction the important feature of the bag1 fibre is that it is the intrafusal fibre which is innervated by dynamic fusimotor axons. By implication, it has generally been assumed that during stretch of the passive spindle it is predominantly the bag1 fibre which gives rise to the dynamic response. Indeed, such a proposal has been put forward. Observations on isolated spindles suggested that during a ramp stretch the degree of opening and closing of the sensory spirals on the bag1 fibre corresponded closely to the instantaneous rate in the afferent response (Boyd, Gladden & Ward, 1981).

559 SPINDLE DYNAMIC RESPONSE Muscle spindles are sensitive to the drug succinylcholine (SCh) (Granit, Skoglund & Thesleff, 1953). As well as becoming tonically active in the presence of SCh, they typically show a large increase in dynamic response during a stretch. The increase in dynamic sensitivity is thought to be the result of a drug-induced bag, fibre contracture (Rack & Westbury, 1966). It has recently been reported that some muscle spindles of neck muscles do not show an increase in dynamic response in the presence of SCh (Price & Dutia, 1987). This was interpreted as evidence for spindles lacking a bag1 fibre. It is known that in neck muscles many spindles are complexes, 'tandem spindles', in which the bag2 fibre supplies more than one encapsulated region. Each encapsulation is associated with nuclear chain fibres while only the largest capsules also include bag1 fibres (Banks, Barker & Stacey, 1982; Richmond, Bakker, Bakker & Stacey, 1986). Price and Dutia showed that spindles which lacked a large dynamic response in the presence of SCh, and therefore presumably lacked a bag1 fibre, were virtually indistinguishable from other spindles in their passive response to stretch. All of this raises the question, where in the spindle does the dynamic response to passive stretch arise? Is the bag1 fibre involved at all? It has recently been reported that up to 20 % of muscle spindle capsules in the peroneus tertius muscle enclose intrafusal bundles of the b2c configuration, that is, they lack a bag1 fibre (Scott & Young, 1987). Here we report a study in which the sensitivity of spindles of peroneus tertius to SCh was systematically tested in the peroneus tertius muscle. Several spindles were encountered, presumably examples of b2c spindles, which showed no increase in dynamic response in the presence of SCh. These findings have led to a reevaluation of the origin of the dynamic response in all spindles. METHODS

Five experiments were performed on adult cats anaesthetized with pentobarbitone sodium (Pentobarbital, Sanofi 45 mg kg-', I.P.). The peroneus tertius muscle and its tendon were dissected free and the tendon was attached to a servo-regulated muscle stretcher which generated ramp stretches. A strain gauge, placed in series between the tendon and stretcher, allowed the recording of isometric tension. The position of the stretcher was adjusted so that the muscle was held 0 5-1 mm above the minimum in situ length and that the final length did not exceed the muscle's maximum in situ length. The muscle nerve was dissected free over a length of 15-20 mm and mounted on a unipolar recording electrode for the recording of action potentials in afferent fibres. The indifferent electrode was placed in contact with the preparation at some distance from the muscle. After an extensive denervation of the hip, tail and lower hindlimb, functionally single afferent fibres from primary endings of peroneus tertius spindles were isolated in dorsal root filaments. They were identified on the basis of their conduction velocity (above 72 m s-1) and their response to stretch. Each filament containing a single functional afferent was placed on a multipolar electrode, and connected through conventional amplifiers to an instantaneous frequency meter. Five to ten filaments were prepared in each experiment. On several occasions functionally single fusimotor axons were isolated, by dissecting cut ventral roots, until orthodromic stimulation of a root filament elicited in the muscle nerve an all-or-none action potential whose conduction velocity was below 50 m s-1. The effect of stimulating each fusimotor fibre at different rates was observed on the discharge of spindle primary endings during slow muscle stretch (2-5 mm at 3-5 mm s-1) to allow identification of static and dynamic fibres. The search for spindles which lacked a bag, fibre was carried out by combining muscle stretch with stimulation of large pieces of ventral root (Fig. 1). Where no increase in dynamic response could be detected during stimulation of any of the ventral root pieces, the spindle was provisionally identified as lacking a dynamic fusimotor innervation. The confirmatory test was to use SCh.

560

M. GIOUX, J. PETIT AND U. PROSKE

SCh was injected by means of a centrally directed cannula in the femoral artery of the contralateral hindlimb. The mouth of the cannula lay close to the femoral bifurcation, allowing the injected drug passage into the arterial blood supply on the experimental side. Doses of 50-500 sg kg-' were given, injected manually. Before injection, control responses were recorded from two spindles, one known to have a dynamic fusimotor supply, the other being the tested spindle. Following drug infusion, responses to passive stretch were recorded every 30 s for fifteen successive trials. In practice, at the end of a series most of the effect of a dose of SCh had worn off. During the course of an experiment, all spindles were eventually tested for a response to SCh, even those which were known to have a dynamic fusimotor supply. The ramp stretch used in the experiments employing SCh was of 2-5 mm amplitude at a rate of 3.5 mm s-1. In a second series, involving muscle conditioning, the same size of stretch was used but at a rate of 0-2 mm s-'. Two forms of conditioning were used: a 1 s train of stimuli, at 100 pulses s-1, at fusimotor strength, applied to the muscle nerve 6 s before the test stretch, carried out either at the test length or at a length 2-5 mm longer. When conditioning was at the longer length, the muscle was held there for a further 3 s after stimulation before returning it to the test length. The experiments were carried out under computer control, and all data was stored on flexible discs. RESULTS

Since the histological data had suggested that only one spindle in five was of the b2c type, it was necessary to devise a method which would help in the search for such spindles. The way this was done was to subdivide the ventral roots containing the motor supply to peroneus tertius (L7 and Si) into five portions, approximately equal in terms of the amount of tension they developed in the muscle. Then each portion was stimulated repetitively at 100 pulses s-1, at a strength sufficient to recruit fusimotor fibres, during ramp stretch of the muscle. When the piece of ventral root contained a dynamic fusimotor fibre the afferent response showed a large increase in dynamic response. Since dynamic fusimotor fibres innervate almost exclusively bag, fibres, this was therefore a test for the presence of a bag, fibre. An example is shown in Fig. 1. The response to passive stretch was compared with responses to stimulating each of five filaments, F1-5. Filament 2 probably contained only a-motoneurones and during their stimulation the extrafusal contraction unloaded the stretch response. In filaments 1, 3 and 5 there were static fusimotor fibres as well, since stimulation produced a strong excitatory effect without significantly increasing the dynamic response. The large increase in dynamic response. during stimulation of filament 4 signalled the presence of a dynamic fusimotor fibre. This method was successful in identifying three out of every four spindles with a dynamic fusimotor innervation. Invariably, however, there were several spindles in each preparation which showed no hint of an augmented dynamic response during stimulation of any of the ventral root filaments but which subsequently, in the presence of SCh, showed a response typical of a spindle with a bag, fibre. For one such spindle, after the effects of SCh had worn off, the ventral root filaments were retested to make quite sure that a weak dynamic fusimotor effect had not been overlooked. On that occasion it was confirmed that no increase in dynamic response could be reliably identified. Only when one of the filaments was subdivided into smaller portions, and each piece stimulated separately, did a dynamic effect eventually emerge, the result of stimulation of a dynamic fl-fibre. In other words, the method

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Fig. 1. Methods used to identify spindles with a dynamic fusimotor innervation. In each of the six panels the upper trace shows the instantaneous frequency display of a spindle primary ending from the peroneus tertius muscle, the lower trace muscle tension. At the bottom of the figure is shown muscle length. The top left-hand panel ('passive') shows the response of the spindle to passive stretch (2-5 mm at 3-5 mm s-1). Each of the five remaining panels shows the response to stretch during stimulation of a filament of ventral root (F1-5) containing a portion of the muscle's motor supply. Duration of stimulation at 100 pulses s'I is shown by the bar at the bottom of the figure. Filament 2 contained only skeletomotor neurones, filaments 1, 3 and 5, skeletomotor and static fusimotor neurones, while filament 4 included a dynamic fusimotor neurone.

for identifying bag, fibres by means of stimulation of large pieces of ventral root was not entirely reliable. It was our experience that whenever a weak dynamic effect was combined with a strong static action and some extrafusal unloading, the dynamic effect risked being overlooked. It was therefore always necessary to confirm, with SCh, the preliminary classification made with ventral root stimulation In the five experiments a total of forty-two afferents were isolated, identified as coming from muscle spindles, conducting in the range -72-106 m s51. Of these,

M. GIOUX, J. PETIT AND U. PROSKE

562

twenty-six were identified by ventral root stimulation as receiving a dynamic fusimotor innervation. Of the remaining sixteen, ten showed large increases in dynamic responses in the presence of SCh, presumably the result of a bag, contracture. One spindle responded in a manner intermediate between that typical Passive

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is Fig. 2. Responses of two primary endings of peroneus tertius spindles to ramp stretches of 2-5 mm amplitude and rate of 3 5 mm s-1. Length records shown at the bottom of the figure. The four frequency traces show the responses of the two spindles before (passive) and 30 s after the injection of 400 ,ug kg-' SCh into the femoral artery (SCh). The spindle represented in the upper traces was one which had previously been shown to receive a dynamic fusimotor innervation. For the spindle shown on the lower traces no dynamic fusimotor innervation could be detected. Only the spindle shown in the upper traces responded to SCh with a large increase in dynamic response.

of primary and secondary endings. That left five spindles, one from each of three experiments and two from the fourth where there was no sign of an increase in dynamic response, either from ventral root stimulation or in the presence of SCh. Conduction velocities of the five afferents were 83, 88, 90, 96 and 97 m s-1. That is, conduction velocity lay well within the group I range. It ruled out the possibility that we were dealing with spindle secondary endings. An example of such a spindle is shown in Fig. 2. During SCh infusion the practice was always to continuously record the activity of two spindles at a time. Responses to stretch were tested every 30 s following drug administration. The example in Fig. 2 shows the responses to a 2-5 mm stretch at 3-5 mm s-1 before SCh was given and at the peak of its action, 30 s later. Here the dose of SCh was 0 4 mg kg-', sufficient to

563 SPINDLE DYNAMIC RESPONSE evoke a near-maximal response. The spindle shown in the top traces had previously been identified, using ventral root stimulation, as having a dynamic fusimotor supply. In the presence of SCh it showed the large dynamic response characteristic of a bag, contracture. The second spindle (on the right, axonal conduction velocity

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is Fig. 3. Frequency traces (upper four records) and length traces (at the bottom of the figure) for the two spindles shown in Fig. 2, after the effects of SCh had worn off, to 2-5 mm amplitude ramp stretches at two different rates, 1-75 (left) and 7 0 mm s-1 (right). The spindle which had responded with a large dynamic increase in SCh is shown in the upper frequency traces. There is little to distinguish the responses of the two spindles to passive stretch.

97 m s-1) had a control response which was little different from that of the first spindle except for the absence, at this muscle length, of a resting discharge. In the presence of SCh it showed no large dynamic response, even though it had now adopted a high resting rate. In fact the dynamic index (Jansen & Matthews, 1962) actually fell during SCh because of the large increase in static response. All of the five spindles which lacked a large dynamic response in the presence of SCh showed a big increase in resting rate and a reduction in dynamic index. The increased resting rate has previously been interpreted as the result of a bag2 fibre contracture (response phase III, Dutia, 1980). Although the effect of SCh wore off rapidly after each dose, the effect on the dynamic response, if one was present, always lasted long enough to be reliably detected in several test trials. There was no possibility of overlooking such responses. Furthermore, when a spindle was found which lacked a dynamic increase in the presence of SCh it was always tested during repeated doses to confirm the result. Clearly, therefore, spindles could respond quite differently to stretch in the

M. GIOUX, J. PETIT AND U. PROSKE

564

presence of SCh. For the two spindles in Fig. 2, after the effects of the drug had worn off, responses to ramp stretch were recompared to confirm that there really was no systematic difference between them. Responses to stretches at two different rates, 1P75 and 7 mm s-I are shown in Fig. 3, the upper traces representing the spindle which had a large dynamic response in the presence of SCh. 10 .~8

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Dynamic index Fig. 4. Histogram showing distribution of values of dynamic index for all forty-two primary endings of spindles during passive stretches of 2-5 mm amplitude at 3-5 mm s-1. Values represented by the filled areas were for spindles which lacked an increase in dynamic response in the presence of the drug SCh.

Although there were minor differences, especially at the higher stretch rate, it was not possible to identify any consistent feature which could be used to distinguish the two spindles. A similar conclusion was reached with the other three spindles which lacked a large dynamic response in the presence of SCh. For each of the forty-two primary endings the dynamic index, that is, the peak response during a stretch minus that half a second later (Jansen & Matthews, 1962), was measured for 2-5 mm stretches at 3-5 mm s-1 and the distribution of values is shown in Fig. 4. Spindles which lacked an enhanced dynamic response in the presence of SCh had dynamic indices during passive stretch which lay within the limits of the range of values. The possibility remained that a weak dynamic effect had been overlooked with ventral root stimulation and that for some reason on this occasion SCh had not been able to show up the presence of the bag, fibre. So for two spindles, after noting the absence of a dynamic response in SCh, when the effects of the drug had worn off, the ventral root supply was re-examined and all fusimotor fibres to these spindles were isolated. A total of five fusimotor fibres could be identified for one spindle and three for the other, each with a clearly static action. There was no sign of a dynamic fusimotor supply to either spindle. One spindle with an afferent fibre conducting at 83 m s-1 was found to have a response intermediate between a truly augmented dynamic response and no dynamic increase in the presence of SCh (category V of Emonet-Denand, Laporte, Matthews & Petit, 1977). No attempt was made to draw conclusions about the intrafusal fibres

SPINDLE DYNAMIC RESPONSE 565 contained within this spindle. At all other times it was possible to classify an afferent unequivocally, as belonging to one or other of the two categories. Here an additional feature during SCh infusions was that each increase in dynamic response was accompanied by a pronounced slow decline in firing at the end of stretch, the 'creep' phase of the response (Boyd et al. 1981). It was always quite easy to decide whether or not creep was present. The data of Figs 3 and 4 suggested that there was no difference in passive stretch response between spindles with and without a bag, fibre. One other way of studying the responses of spindles to passive stretch is by altering the responses using muscle conditioning. If the muscle nerve was stimulated repetitively, at fusimotor strength, at a length several millimetres longer than the test length, and held there for several seconds after stimulation, on returning to the test length the response to a subsequent slow stretch showed a later onset and lower rate of discharge than after conditioning stimulation carried out at the test length (Gregory, Morgan & Proske,

1988). For the two spindles of Fig. 2 the responses to slow stretch (02 mm s-1) following the two forms of conditioning are shown in Fig. 5. The upper pair of traces is from the spindle with a dynamic fusimotor innervation. For each spindle, following conditioning nerve stimulation at a length longer than the test length, the response to the slow stretch began later and at a lower rate than after conditioning at the test length. However, again there were no obvious features which could be used to distinguish the responses of the two spindles. It is interesting that after conditioning stimulation at the test length, following an initial steep increase in firing at stretch onset, which probably corresponded to the 'initial burst' seen with higher stretch rates, there were repeated slow rises and falls in the firing rate. These undulations were quite reproducible and persisted over a range of muscle lengths. They were particularly marked for the spindle shown in the upper traces of Fig. 5. However, there were almost no fluctuations in the responses of both spindles after conditioning stimulation at a length longer than the test length. This kind of behaviour was consistently seen in each experiment. The data compiled up to this point were leading us to the conclusion that whether or not the spindle contained a bag, fibre, as determined by the response to SCh, made little difference to the passive stretch response. This result raised the possibility that the bag1 fibre might not contribute at all to the passive stretch response. However, it is possible to show experimentally that under certain conditions the bag1 fibre does, in fact, make some contribution. The reason why, after conditioning stimulation at the longer-than-test length, the response to stretch was so much smaller (Fig. 5) is that conditioning introduces slack in intrafusal fibres (see Morgan, Prochazka & Proske, 1984). When slack is present there is less strain on the sensory region of intrafusal fibres during the test stretch, producing a weaker response. Since conditioning used muscle nerve stimulation all of the intrafusal fibres would be expected to fall slack. For a spindle with a dynamic fusimotor innervation it is possible to isolate the dynamic axon in a ventral root filament and, following the introduction of slack in all intrafusal fibres, to take up the slack selectively in the fibre innervated by the dynamic axon by means of a second conditioning stimulus.

566

M. GIOUX, J. PETIT AND U. PROSKE

The experiment is illustrated in Fig. 6. The conditioning-test sequence is shown, diagrammatically, at the top. The response shown in trace A was obtained following conditioning stimulation at the test length, stimulating the muscle nerve at fusimotor strength. Here therefore any pre-existing slack had been removed. A single

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12.5 mm 2s Fig. 5. Responses of the same two spindles as shown in Figs 2 and 3, but during slow stretch (2-5 mm amplitude at 0-2 mm s-1) (length trace at the bottom of the figure). Upper pair of traces from the spindle with a demonstrated dynamic fusimotor innervation. For each spindle responses to two successive stretches have been compared, but following different forms of muscle conditioning. In each pair the lower frequency trace represents the stretch response after conditioning stimulation of the muscle nerve (100 pulses s-1 at fusimotor strength for 1 s at length 2-5 mm longer than that at the onset of the stretch. After stimulation the muscle was held for a further 3 s at the longer length before returning to the test length and applying the stretch. The upper frequency traces in each pair of records represents the response after conditioning nerve stimulation at the test length. Although the two forms of conditioning produce quite different responses to stretch from both spindles there is no obvious feature that distinguishes one spindle from the other.

conditioning tetanus only was given (tetanus 1). In B the same conditioning tetanus was given, but with the muscle held at the longer-than-test length (tetanus 2). This time all intrafusal fibres would be expected to lie slack at stretch onset. In C and D, a tetanus at 2 was given but this was followed by a second period of stimulation indicated at 3. Here for trace C the entire motor supply to peroneus tertius (L7 plus Si ventral roots) was stimulated, minus the dynamic fusimotor fibre, while for trace D the dynamic fusimotor fibre alone was stimulated.

567 SPINDLE DYNAMIC RESPONSE For the response shown in C the second period of stimulation led to slack being removed in all intrafusal fibres except that innervated by the dynamic axon. Here the response to stretch was little different from that in A. In D slack was taken up only in the fibre innervated by the dynamic axon. The fact that the response to

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Fig. 6. Experiment to demonstrate that the intrafusal fibre innervated by the dynamic fusimotor axon, the bag, fibre, may contribute to the stretch response. At the top of the figure the sequence of conditioning stimulation and test stretch is shown schematically. The four frequency traces are all responses to slow stretch of the same spindle. Length traces at the bottom of the figure. In A, the response to stretch follows conditioning nerve stimulation at 1 only. The other three traces, B, C and D, are after conditioning stimulation at 2. In C and D a second conditioning tetanic stimulus was given. For trace C, the ventral root (VR) supply to peroneus tertius was stimulated at 3, but minus an identified dynamic fusimotor axon to that spindle. In D only the dynamic axon (y) was stimulated at 3. Since the response in D was different from that in B, it shows that conditioning dynamic fusimotor stimulation can modify the stretch response.

stretch in trace D differed significantly from that in trace B strongly suggests that selective removal of slack from the bag1 intrafusal fibre can modify the response to stretch. In other words, the earlier onset of the response to stretch in D, compared with B, must be taken as representing a contribution to the stretch response by the bag, fibre. DISCUSSION

The principal finding of these experiments is that in peroneus tertius, as in neck muscles (Price & Dutia, 1987), there are some spindles which in the presence of SCh do not show a heightened dynamic response. All of the currently available evidence suggests that the dynamic response during SCh infusion is due to a bag1 contracture. From direct observations of isolated muscle spindles, Gladden (1976) concluded that

M. GIOUX, J. PETIT AND U. PROSKE bag, fibres had the lowest contraction threshold to acetylcholine added to the bathing fluid. These fibres also showed back-slippage or 'creep' after stretch of the spindle or after nerve stimulation. The only afferent response which corresponds in its time course with that of creep is the decline at the end of a ramp stretch during dynamic fusimotor stimulation (Boyd et al. 1981). It could be argued that during SCh infusion an occasional spindle in the muscle, perhaps because of a poor local circulation, received less than the dose required for a bag1 contracture and therefore did not show a heightened dynamic response. Such an interpretation seems unlikely for the five spindles in this study because in the presence of the drug they all showed quite dramatic increases in their firing rates, suggestive of bag2 fibre contractures. It would therefore have to be postulated that for these spindles there was some kind of specific diffusion barrier restricting access to the bag1 fibre. That seems very unlikely. Furthermore, on the occasion on which an attempt was made to isolate the full complement of fusimotor fibres supplying two of these spindles, no dynamic fusimotor fibre was found. In another recent experiment Price & Dutia (1990) were able to locate individual spindles in the tenuissimus muscle while recording from their afferent fibres. They then tested each spindle with a topical application of SCh. On one occasion a response was observed of a strong biasing action without an accompanying increase in dynamic response. Subsequent histological analysis showed that this spindle contained only one bag fibre. Single bag spindles are almost invariably b2c spindles (Kucera, 1982). From their larger sample of responses of b2c spindles from neck muscles Price & Dutia (1987) concluded that with respect to afferent conduction velocity, discharge variability and vibration sensitivity, b2c spindles lie in a position intermediate between that of b1b2c spindles and spindle secondary endings. Our own observations, while from a much more limited sample, suggest that there is no single detectable feature, at least in the response to passive ramp stretch, which allows the two kinds of spindles to be distinguished. It is probably for that reason that Banks, Ellaway & Scott (1980) were not able to find any evidence of two populations of primary endings in peroneus brevis and tertius. Thus the important conclusion is reached that the typical response to ramp stretch of the primary ending of the passive spindle does not require the presence of a bag1 fibre. Similarly, Price & Dutia (1990) concluded '...the bag1 fibre is not the major determinant of dynamic responsiveness in the passive spindle'. That conclusion led us to carry out the experiment illustrated in Fig. 6. By contracting the muscle at a muscle length longer than the test length and then returning it to the test length it was possible to introduce slack in intrafusal fibres (Morgan et al. 1984). We don't know if the same amount of slack develops in all of the fibres, but it is reasonable to assume that that is so. Following the development of slack, the response to a slow stretch began later and at a lower rate than in the absence of slack (Figs 5 and 6). However, if after the intrafusal fibres had been made slack only the dynamic fusimotor fibre was stimulated, the subsequent response to stretch was always consistently different. We concluded that taking up the slack, selectively, in the bag1 fibre by dynamic fusimotor stimulation led to a modification of the stretch response. Indeed, the same 568

569 SPINDLE DYNAMIC RESPONSE conclusion can be reached from the experiments of others involving conditioning dynamic fusimotor stimulation (see, for example, Emonet-Denand, Hunt & Laporte, 1985, Fig. 5). In other words, under conditions which are most likely to favour a stretch response from the bag1 fibre, it being taut while all other intrafusal fibres lie slack, a component of the stretch response can be attributed to bag1. It could be argued that the stretch response always arises in an intrafusal fibre other than the bag1 fibre. Taking up the slack in bag1 could, as a result of mechanical linkages, lead to transmission of the shortening movement to adjacent intrafusal fibres, removing some of their slack as well, and therefore lead to a modification of the stretch response. However, this is a more complicated explanation, and therefore seems less likely. In their analysis of the spindle composition of peroneus tertius Scott & Young (1987) found that 67 % of spindle capsules came from tandem spindles, that is, spindles with multiple encapsulations served by separate primary afferents and joined by a common intrafusal fibre, the bag2 fibre (Banks et al. 1982). Thirty-one per cent of tandem spindle capsules were of the b2c kind. In other words, the 21 % of all spindle capsules in peroneus tertius with no bag1 fibre all came from tandem spindles. Accepting this conclusion, it is of interest that the intrafusal contracture induced by SCh in the bag1 fibre at one end of a tandem spindle appears to have no effect on the dynamic response to stretch of an afferent supplying a b2c capsule at the other end. Some comment is required on the possible origin of the fluctuations in frequency seen during slow stretch (Fig. 5). These fluctuations are not due to any obvious friction between the muscle and adjacent tissue, nor are they restricted to spindles from any one part of the muscle. Similar fluctuations have also been observed for spindles in the soleus muscle (J. E. Gregory, D. L. Morgan & U. Proske, unpublished observations). The fluctuations seem to be particularly pronounced after conditioning of the spindle at the test length, that is during slow stretch after all of the intrafusal fibres have been rendered taut by removal of any pre-existing slack. Furthermore they are almost absent after conditioning at a length longer than the test length, that is, in the presence of slack in intrafusal fibres. Our interpretation of these observations is that stretch of a taut intrafusal bundle leads to afferent activity arising from sensory terminals on several intrafusal fibres. As the stretch proceeds and one of these fibres is stretched beyond a yield point, the fibre exhibits some backslippage leading to a transient fall in the discharge. There are several yield points for different intrafusal fibres. We postulate that stretch of a slack intrafusal bundle leads to activity generated in only one intrafusal fibre, the bag2 fibre, and there are therefore no multiple yield points. The above conclusions mean that the current picture of the internal workings of the mammalian muscle spindle must undergo important revisions. During passive muscle stretch, especially if the stretch is repeated, as might occur during repetitive limb movements, spindles will be in a state which corresponds to that in Figs 5 and 6 where all intrafusal fibres lie slack. Under these conditions the source of the afferent activity during stretch is likely to be the sensory terminals on the bag2 fibre. The bag1 fibre will only begin to make a contribution if there is an intervening period of dynamic fusimotor stimulation. The real influence of the bag1 fibre will only emerge

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when it is undergoing stretch while actively contracting. The resulting large increase in afferent response is likely to be due to the fact that most of the stretch is taken up by the sensory region of the bag, fibre since stiffness of polar segments will be high. Extension of the sensory region of bag2 when it is contracting is likely to be less because the bag2 fibre contracts more rapidly than bag1. The viscous stiffness during slow stretch of the actively contracting parts of the intrafusal fibres depends on their contraction speed (Brown, 1971). One consequence of assigning the origin of the stretch response to bag2 is that current hypotheses about stretch activation of intrafusal fibres (stretch-excitation, Laporte, Emonet-Denand & Hunt, 1985) will have to be revised. If stretch excitation occurs, and it contributes to the generation of the dynamic response of the passive spindle, then it must arise largely in bag2. In other words, if there is stretch excitation of bag1 this must be too small to make a significant contribution to the stretch response. To conclude, we have confirmed for peroneus tertius the findings of Price & Dutia (1987, 1990) that in the presence of SCh some spindles, presumably those lacking a bag1 fibre, do not show an increase in dynamic response. The conclusion is reached that in the passive spindle much of the stretch response is generated in the bag2 fibre, although a contribution from bag1 may occur under certain conditions. The emerging picture of the spindle is that of a receptor with two muscle fibres, the nuclear bag fibres, concerned with generation of dynamic responses. When these fibres are actively contracting during muscle stretch, one, the bag1 fibre, will tend to increase the dynamic response, while the other, the bag2 fibre, will tend to reduce it. What perfusion with SCh tells us is that when both are contracting together, the influence of the bag1 fibre contraction prevails. Future evaluations of the role of muscle spindles in motor control will have to take into account this kind of facility. The suggestion to try the effects of SCh on peroneus tertius spindles came from Dr J. J. Scott. We would like to thank D. Couton for technical assistance, Y. Laporte and J. J. Scott for help with the manuscript. REFERENCES

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