Experimental Brain Research
Exp. Brain Res. 31,433-443 (1978)
9 Springer-Verlag1978
Some Observations on the Efferent Innervation of Rat Soleus Muscle Spindles B.L. Andrew, G.C. Leslie and N.J. Part Department of Physiology, The University, Dundee DD 1 4HN, Scotland, U.K.
Summary.In
the small segmental tail muscles of the rat beta fibres provide exclusively the dynamic fusimotor control, while gamma fibres provide exclusively the static fusimor control. The present experiments were made to investigate the fusimotor innervation of spindles in a large muscle of the rat, the soleus, and thus to determine the occurrence and significance of beta innervation in this muscle. Our results have revealed no case of beta innervation in the rat soleus. As a consequence of our experimental method, however, we would not claim that beta innervation does not exist in the soleus, only that it must play an insignificant role relative to that seen in the tail segmental muscles. Investigations of the fusimotor innervation of eight spindles were sufficiently complete to warrant detailed illustration. The number of gamma fibres ranged from two to four. In every case the slowest conducting gamma fibre was dynamic. However, the conduction velocity spectra for the static and dynamic gamma fibres to rat soleus overlap to such an extent that it is impossible to use conduction velocity as the sole guide to functional gamma fibre classification. The pooled results from the eight spindles fully investigated provide a ratio of static to dynamic gamma fibres of approximately 1:1. Other evidence discussed in the paper suggests that in the muscle nerve the ratio is considerably higher. These differences are reconciled if the dynamic gamma fibres branch more profusely and innervate more spindles than do the static gamma fibres. Key words: Rat - Soleus - Fusimotor innervation - Skeletofusimotor innervation An efferent nerve fibre to a mammalian muscle spindle may be either purely fusimotor (gamma) or mixed skeletofusimotor (beta). The importance of the gamma system is firmly established. In the cat the beta system is known to be important in a number of small muscles (Bessou et al., 1965; Ellaway et al., 1971; McWilliam, 1975) and recently has been shown to be present in large
0 0 1 4 - 4 8 1 9 / 7 8 / 0 0 3 1 / 0 4 3 3 / $ 2.20
434
B.L. Andrew et al.
hind limb muscles ( E m o n e t - D d n a n d et al., 1975; E m o n e t - D 6 n a n d an d L a p o r t e , 1975). I n the rat th e small s e g m e n t a l muscles of t h e tail r e c e i v e t h ei r d y n a m i c f u s i m o t o r i n n e r v a t i o n exclusively in the f o r m o f b e t a fibres an d t h ei r static supply exclusively as g a m m a fibres ( A n d r e w and Part, 1974). W e h a v e i n v e s t i g a t e d the f u s i m o t o r n e r v e supply of t h e soleus muscle o f the rat in o r d e r to d e t e r m i n e w h e t h e r t h e f u s i m o t o r supply is a r r a n g e d in a m a n n e r similar to that in t h e tail muscles of t h e rat or in th e m o r e familiar m a n n e r f o u n d in t h e soleus o f t h e cat in w h ic h g a m m a fibres are b o t h static and d y n a m i c in type. E a r l y w o r k on b e t a i n n e r v a t i o n was p e r f o r m e d on small m u scl es b e c a u s e of t h e g r e a t e r t echn i c a l ease of id e n ti f y i n g this t y p e of i n n e r v a t i o n with a s m a l l e r n u m b e r o f c o m b i n a t i o n s of a f f e r e n t a n d e f f e r e n t n e r v e s to t h e muscle. F o r this r e a s o n E m o n e t - D 6 n a n d et al. (1975) in effect m a d e the soleus an d o t h e r large hind limb muscles o f t h e cat into small m u s cl es by cutting b r a n c h e s of t h e i r muscle n e r v e and w o r k i n g with just a small p o r t i o n o f t h e t o t al muscle. T h e soleus muscle o f t h e rat, h o w e v e r , is small e n o u g h to be i n v e s t i g a t e d as a c o m p l e t e muscle. T h e results of t h e s e e x p e r i m e n t s , whilst n o t able to e l i m i n a t e t h e o c c u r r e n c e of b e t a i n n e r v a t i o n in t h e soleus m u s c l e o f th e rat, do strongly suggest t h a t b e t a i n n e r v a t i o n does n o t play the i m p o r t a n t r o l e that it does in t h e tail muscles.
Methods The experiments were carried out on a total of twenty-six female rats, 300450 g body weight. Anaesthesia was induced with trichlorethylene vapour and maintained by intraperitoneal injection of urethane solution, dosage 1.5 mg urethane/g body weight. Supplementary injections were given as necessary. The lumbar spinal roots were exposed by laminectomy and covered with warmed Tyrode solution and paraffin oil. The soleus muscle was dissected free from surrounding tissue, the muscle nerve cleared for the placement of electrodes and all other muscles in the leg denervated. The leg was passed through an aperture in the side of a Perspex bath and a skin flap from the thigh tied round a flange surrounding the hole to form a leak-proof seal (Close, 1967). The bath was filled with paraffin oil floating on Tyrode solution and was maintained at about 35~ C by a coil of circulating warm water. The proximal tendon of the soleus muscle was attached to a rigid support and the distal tendon attached, via a tension transducer, to an electromagnetic puller (Ling-Pye V 47/3) with associated displacement transducer. The nerve fibres to soleus pass through dorsal and ventral roots L4, L5 and L6 with the greatest number of fibres in L5. All these spinal roots were cut near their entry to the cord. Pairs of electrodes, switchable from stimulating to recording, were placed on the appropriate dorsal and ventral roots and on the muscle nerve. Two experimental protocols were performed. In the earlier experiments the ventral root was first divided to give a functionally single gamma fibre (less than 32 m/sec [Andrew and Part, 1972]). This filament was then stimulated at 100 and 200 stimuli/sec both with the muscle held at constant length and with the muscle oscillated sinusoidally through 1 mm at 3 Hz. The dorsal root was then divided until there was obtained a filament containing a single soleus spindle afferent acted upon by the efferent. In the later experiments the dorsal root was the first to be divided to a single spindle primary afferent. The ventral root was then divided into about twenty filaments. Each of these filaments contained between zero and three alpha fibres. If the filament contained more than three alpha fibres it was further divided until three was the maximum number of apha fibres. Because of the off-loading effects of extrafusal muscle fibre contraction, three alpha fibres was considered to be the maximum acceptable in such coarse ventral root filaments. Wherever possible filaments with less
Rat Soleus Muscle Spindles
435
A
Fig. 1. Recordings of alpha and gamma nerve action potentials. Upper trace: An alpha (A) and a gamma (G) action potential recorded in a ventral root filament on supramaximal stimulation of the soleus muscle nerve. L o w e r trace: An alpha (A) and a gamma (G) action potential recorded in the intact soleus muscle nerve on supramaximal stimulation of the above filament. The large amplitude, low frequency deflection is the electromyogram of the motor unit of the alpha fibre. S marks the stimulus artifacts. Both records are the result of averaging thirty-two consecutive records
than three alpha fibres were used. Along with the alpha fibres in these filaments there were a widely variable number of gamma fibres. Such ventral root filaments were stimulated at 100-200 stimuli/sec and any excitatory action resulting in the primary afferent recorded. If an excitatory action was observed the ventral root filament was split until the fibre, or fibres, responsible for the excitation were obtained in isolation. One of the objects of these experiments was to determine the extent of beta innervation to the rat soleus. Therefore all efferent nerve fibres, both fast and slow, to the soleus were tested for fusimotor action. If stimulation of a fast efferent nerve fibre produced both extrafusal muscle fibre contraction and increased activity in the spindle afferent, two tests were carried out to establish whether the spindle activation was caused by stretch of the spindle brought about by the extrafusal contraction (freakish pull) or by true fusimotor activation of the spindle. The first test involved stimulating the efferent nerve at frequencies up to and beyond the tetanic fusion frequency of the extrafusal motor unit (Bessou et al., 1965). Increase of the spindle response at frequencies beyond that producing the maximum response of the extrafusal motor unit is indicative of fusimotion. The other test required the selective block of the extrafusal neuromuscular junctions with topically applied Flaxedil. The blockage of the extrafusal neuromuscular junctions was accelerated by stimulation of the efferent nerve at 100 shocks sec -1 for half a second once every second and a half (Bessou et al., 1965). The persistence of spindle excitation after extrafusal blockade is another indication of true fusimotor action. The conduction velocity of action potentials in efferent filaments was determined first by stimulation of the muscle nerve and recording the evoked response in the filament (see Fig. 1, upper record). When the filament was stimulated, recordings were taken from the muscle nerve and averaged using a DL 4000 averager (Data Laboratories Ltd.). The lower record in Figure 1 is one such averaged response. The conducted alpha response is clearly seen together with its associated myogram. Superimposed on the e.m.g, is the gamma action potential, corresponding in position with the same gamma potential when first recorded in the filament. This averaged response from the muscle nerve was used routinely to determine that the fibres in the efferent filament were being stimulated supramaximally and that activity in these fibres was reaching the muscle nerve (and passing onward to the muscle itself). Furthermore, this averaging technique ensured that no gamma fibre was being inadvertently stimulated when investigating fibres suspected of being beta-axons. The effects of stimulation of the fusimotor fibres at frequencies of 10-300 shocks per second on the response of the spindle endings to sinusoidal and ramp stretches were recorded on magnetic tape along with records of muscle length and tension. Post-experimentally, the action potential recordings were subjected to instantaneous frequency analysis which was photographed from a C.R.O. screen along with the length and tension records. So that the shock artifacts did not interfere with the recordings a shock-artifact suppressing device (Frederick Haer) was used. The time programming of stimulation of the efferent fibres and the pulling of the muscle was arranged with a Digitimer 4030 (Digitimer Ltd). Wave-forms to operate the muscle puller were produced with a Servomex generator type LF 141.
436
B.L. Andrew et al.
Results
Efferent Innervation of the Rat Soleus If a survey of the efferent innervation of the muscle spindles of the soleus is to be useful, it must be possible to obtain the greater proportion of the efferent nerve fibres in isolation or near isolation in ventral root filaments. The rat soleus has been shown to have about thirty motor units by physiological (Close, 1967; Andrew and Part, 1972) and by histological methods (Zelen~ and Hnlk, 1963; Gutmann and Hanzl~kovfi, 1966). The number of gamma fibres has been estimated histologically as about twenty-two (Zelenfi and Hnfk, 1963). In 10 experiments in the present work, the number of isolated alpha fibres ranged from 20 to 31 but predominantly between 26 and 31. Similarly the number of gamma fibres isolated ranged from 18 to 25. In three other experiments, the numbers of alpha and gamma fibres isolated exceeded noticeably the numbers expected to innervate the soleus. In these cases it was noted however that part of lateral gastrocnemius was innervated by a branch of the soleus muscle nerve. If the number of alpha fibres observed was twenty-six or more and the number of gamma fibres eighteen or more, it was considered that a sufficiently complete survey of the efferent innervation had been carried out.
Beta Innervation A number of fast extrafusal muscle innervating nerve fibres excited also the spindle primary afferent. However in no case, using the techniques given in Methods, were we able to obtain proof positive of the fusimotor origin of this excitation. Presumably the excitation was caused by the extrafusal contraction causing stretch of the spindle. Only those fast efferent fibres which excited the spindle during extrafusal muscle contraction were fully tested with the stimulus frequency and selective block tests; those fast efferents which did not excite the primary afferent were not investigated further. Emonet-D6nand et al. (1975) have examined fast efferent fibres that only fully revealed their beta nature after the off-loading effect of the extrafusal muscle fibres had been removed by selective gallamine block.
Gamma Innervation Two series of experiments were performed on the gamma innervation. In the first of these the gamma fibres were first isolated in ventral root filaments and then their action, static or dynamic, determined by tests on subsequently isolated dorsal root filaments containing either primary or secondary afferents. The second series of experiments involved isolating primary spindle afferents in dorsal root filaments and then isolating in ventral root filaments the fusimotor fibres active on that afferent. The classification of a gamma fibre, as static or dynamic, was made from records such as those shown in Figures 2 and 3. The tests themselves were
Rat Soleus Muscle Spindles
0
ls
437
"
F
. . . .
10
.'g" ]':'.'i:....
,.
,
2OO A
i
75
/%~k,VVVN~J~
i-
Fig. 2. Action of a static gamma fibre. Left hand column of traces: Effects of stimulating a static gamma fibre at the frequencies shown at the left on the response of a spindle primary afferent to 3 Hz sinusoidal stretching of 1 mm peak to peak amplitude. The duration of stimulation is shown by the horizontal bar beneath the records. Right hand column of traces: Effects of stimulating the static gamma fibre at the same frequencies on the response of the same spindle primary afferent to 1 mm amplitude ramp stretch at 10 mm sec-~. The duration of stimulation is shown by the horizontal bar beneath the records. The conduction velocity of the gamma fibre was 18.l m/s -1 and that of the spindle primary 61.3 m/s -1. The right hand vertical calibration bar shows the frequency meter calibration in action potentials per second
d e s i g n e d to o b s e r v e t h e effects of f u s i m o t o r s t i m u l a t i o n ( 1 0 - 2 0 0 s h o c k s / s e c ) on t h e a f f e r e n t d i s c h a r g e s o c c u r r i n g in r e s p o n s e to m u s c l e l e n g t h changes. T h e m u s c l e l e n g t h c h a n g e s w e r e e i t h e r l a r g e a m p l i t u d e (1 ram) 3 H z s i n u s o i d a l stretch o r 1 m m r a m p stretches (at a v e l o c i t y of 10 m m sec-1). O n e of t h e c r i t e r i a u s e d to classify t h e f u s i m o t o r fibres was t h e d y n a m i c i n d e x ( C r o w e a n d M a t t h e w s , 1964), m e a s u r e d f r o m t h e r e c o r d s o f r a m p stretch; s t i m u l a t i o n o f d y n a m i c f u s i m o t o r fibres causes an i n c r e a s e o f t h e i n d e x (see Fig. 3), static f u s i m o t o r s t i m u l a t i o n d e c r e a s e s , o r l e a v e s u n c h a n g e d , the d y n a m i c index. I n o u r e x p e r i m e n t s all t h e static f u s i m o t o r fibres w e r e s e e n to d e c r e a s e the d y n a m i c i n d e x (see Fig. 2).
438
B.L. Andrew et al. i
0
ls
10 200
11o
25 !
75
9
200
t
.
~
A
..
,
9
~
,,
^
,
.
.
l
...
. . . . . . . . . . . . .
~
,,,
Fig. 3. Action of a dynamic gamma fibre. Left hand column of traces: Effects of stimulating a dynamic gamma fibre at the frequencies shown at the left on the response of a spindle primary afferent to 3 Hz sinusoidal stretching of 1 mm peak to peak amplitude. The duration of stimulation is shown by the horizontal bar beneath the records. Right hand column of traces: Effects of stimulating the dynamic gamma at the same frequencies on the response of the same spindle primary afferent to 1 mm amplitude ramp stretch at 10 mm sec-1. The duration of stimulation is shown by the horizontal bar beneath the records. The conduction velocity of the gamma fibre was 15.4 ms-1 and that of the spindle primary 64.5 ms-1. The right hand vertical calibration bar shows the frequency meter calibration in action potentials per second
T h e typical r e s p o n s e of a passive s p i n d l e p r i m a r y a f f e r e n t to 3 H z s i n u s o i d a l stretch is to d i s c h a r g e o n l y on t h e e x t e n s i o n p h a s e a n d to b e silent d u r i n g relaxation. D u r i n g high f r e q u e n c y ( 1 0 0 - 2 0 0 H z ) static f u s i m o t o r s t i m u l a t i o n , t h e p r i m a r y e n d i n g is c a u s e d to d i s c h a r g e t h r o u g h o u t t h e w h o l e o f the cycle (see Fig. 2). E v e n at t h e highest f r e q u e n c y (200 H z ) o f stimulation, d y n a m i c g a m m a fibres fail to elicit a c o n t i n u o u s d i s c h a r g e f r o m t h e afferent, t h a t is t h e e n d - o r g a n fails to sustain a d i s c h a r g e during t h e w h o l e of the r e l e a s e p h a s e o f t h e cycle (see
Rat SoleusMuscleSpindles
439
Fig. 3). This is a feature of the dynamic gamma fibre (Lennerstrand and Thoden, 1968). Another feature of dynamic gamma stimulation is that it increases the modulation of the discharge frequency brought about by large amplitude sinusoidal stretching of the muscle. Unfortunately amplitude of modulation is not satisfactorily expressed by instantaneous frequency records but requires the fitting of a sinusoid, to include negative values, to the cycle histogram of the afferent firing throughout the cycle (Hulliger et al., 1977). Thus for the simple purpose of classification of gamma fibres as static or dynamic, lack of discharge during sinusoidal release is probably a prime indicator. In these experiments no difficulty was found in classifying all but one of the fifty-nine gamma fibres isolated; the results from the ramp stretches and sinusoidal tests complemented each other. The one exceptional fibre was classified as dynamic but possessed some static features that would place it in one of the intermediate categories of Emonet-D6nand et al. (1977). Inspection of Figures 2 and 3 will reveal other differences in the consequences of stimulation of static and dynamic gamma fibres. A persistence of the dynamic action for a cycle of sinusoidal stretch after the cessation of dynamic gamma stimulation can be seen whereas the static gamma fibre action ceases rapidly at the end of stimulation. The ramp stretch records with dynamic stimulation shows an adaptation of discharge frequency during the extension period not seen in the static gamma records. This was a feature of the dynamic gamma fibres and indeed is considered by Emonet-D6nand et al. (1977) as the best single distinguishing feature for a dynamic gamma fibre. The results of the first series of experiments are summarized in Fig. 4A. Twenty gamma fibres were isolated. Nineteen of these fibres were static and one was dynamic. The ratio of static to dynamic gamma fibres at nineteen to one, obtained from this first series of experiments differs considerably from that of four to one quoted by Matthews (1972) for cat soleus. This apparent deficiency of dynamic gamma innervation led us to suspect that the dynamic supply to these spindles was from a beta source (Andrew et al., 1976) as in the segmental tail muscles of the rat (Andrew and Part, 1974). It was to investigate this point that the second series of experiments was performed. The testing of efferent fibres on a previously isolated spindle afferent enabled an idea of the total fusimotor innervation of one spindle to be built up. A total of thirty-nine gamma fibres was investigated with this protocol. In contrast to the ratio of nineteen static to one dynamic obtained in the first series of experiments this second series produced a ratio of twenty-one static to eighteen dynamic. Histograms of the conduction velocities of the gamma fibres studied in these two experiments are given in Figure 4. The average conduction velocity of the "afferent first" static gammas was 27.8 m/s -1 _+ 6.8 m/s -I standard deviation and of the dynamic gamma fibres 17.3 m/s -1 + 3.8 m/s -1 standard deviation; these two values are significantly different at the 0.001 level.
Fusimotor Innervation of Individual Muscle Spindles Eight spindles received a complete investigation of their fusimotor innervation subject to the limits of the experimental protocol (see Fig. 5). In each of these
440
B. L~ Andrew et al.
A
::.
9
:.
9 9149149
9 9 9149149
B
99
9
9
1'0
conduction
9
000
2b
0000
9
3'0
00 i
40
velocity
5o
m/see
Fig. 4. Conduction velocities of the isolated gamma fibres. A Conduction velocities of the twenty gamma fibres isolated and then subsequently categorized against spindle afferents. The static gamma fibres are represented by circles and the dynamic gamma fibre by a triangle. B Conduction velocities of the gamma fibres which were categorized against a previously isolated spindle primary afferent, The upper group of triangles shows the dynamic gamma fibres and the lower group of circles shows the static gamma fibres. It is noteworthy that in these experiments there were isolated three static gamma fibres whose conduction velocities exceeded the value of 32 m/sec thought previously (Andrew and Part, 1972) to be the upper limit in the range of gamma fibre conduction velocity
Spindle ~1
9
9
b
-
C
9
d
9
e
9
f
~
g
V
9
9 9
9
9
9
9
9
h
0
~
9
9
9
9
I
I
I
I
I
10
20
30
40
50
conduction
velocity
m/sec
Fig. 5. The gamma innervation of eight soleus muscle spindles. The dynamic gamma fibres are represented by triangles and the static gamma fibres by circles
eight examples the number of efferent nerve fibres investigated in the ventral root filaments was an acceptable fraction of the efferents innervating the soleus (see Results section Efferent Innervation of the Rat Soleus). The most striking feature is that in all eight spindles the slowest conducting gamma fibre was dynamic. Six of the spindles had only one dynamic gamma fibre but two of the
Rat Soleus Muscle Spindles
441
spindles had two. In one of these spindles the two dynamic gamma fibres were the two slowest but in the other there was a static gamma fibre placed between the two dynamic ones. It should also be noted that all the spindles had both a static and a dynamic gamma nerve supply.
Discussion In this project we have looked for beta innervation in the soleus muscle of the rat. Our experiments have not produced any proven cases of beta innervation. However, because of the limitations of the experimental protocol given above, we would be reluctant to state that beta innervation does not exist in this muscle. Indirect histological evidence for beta innervation in the rat soleus has been reported (Kucera, 1977). Nevertheless our failure to detect beta innervation strongly suggests that beta innervation is not of the great significance that it is in the segmental tail muscles in which the dynamic fusimotor innervation is entirely from beta fibres (Andrew and Part, 1974). Emonet-D6nand et al. (1975) have established the existence of beta innervation in the cat soleus but are not able to state the significance of beta innervation in this muscle. In some muscles of the cat, especially small ones such as abductor digiti quinti medius (McWilliam, 1975) beta innervation is of great significance and indeed in that particular muscle 30% of the fast axons are beta. Other recent work by Emonet-Ddnand and Laporte (1975) has reported that in the large peroneus brevis muscle of the cat 18% of axons supplying extrafusal muscle, supplied also spindles and that 72 % of spindles received beta axons. The gamma innervation of the soleus spindles has several interesting features. The first of these to be considered is the difference in ratio of static to dynamic gamma fibres obtained from the two experimental protocols. When the gamma fibre was isolated first and its function then determined, the ratio of static to dynamic was nineteen to one. In the second series when the afferent was first isolated, the ratio of static to dynamic was twenty-one to eighteen. The likelihood of this difference in ratios arising by chance is slight. Differential branching of static and dynamic gamma fibres provides a possible explanation of these different ratios. If the dynamic gamma fibres branch so as to innervate a greater number of spindles than do the static gamma fibres, then one would expect our two experimental protocols to produce different ratios in much the way that has been seen here. From the second series of experiments we know that each spindle receives approximately the same number of static and dynamic gamma fibres. A greater degree of branching of the dynamic gamma fibres than the statics would mean that the dynamic supply would originate from a smaller total number of parent dynamic gamma axons than parent static axons; hence the high ratio of static to dynamic seen when isolating gamma fibres first. The only fully satisfactory experimental test that could be applied to this proposition would be to isolate and categorize all the gamma fibres to a muscle. This, regrettably, did not prove possible. In one experiment three different dynamic gamma fibres were isolated out of a total gamma innervation of
442
B.L. Andrew et al.
eighteen fibres. This result in itself suggests that differential branching is unlikely to be the full explanation of the different ratios because the static: dynamic ratio must beat most fiveto one in this muscle, as opposed to the ratio of nineteen to one for the first series of experiments. This discrepancy may be accounted for by g a m m a fibres which were isolated in the first series of experiments but which were not successfully cross-matched with a spindle afferent. It may be that a majority of these were dynamic fibres whose action might not have been strong enough to prevent masking of the activated spindle afferents by other active fibres in the dorsal root. The matter of g a m m a fibre conduction velocity has been given an appreciable amount of attention because of the experimental convenience of being able to distinguish the function of nerve fibres by conduction velocity (Matthews, 1972; p. 220). It has not proved possible to m a k e a clear differentiation into static and dynamic g a m m a fibres by conduction velocity. Nevertheless, in cat tibialis posterior (Brown et al., 1965) and rabbit tenuissimus ( E m o n e t - D 6 n a n d et al., 1966) the slowest g a m m a fibres tend to be static. This is not seen in our results from the rat soleus; taking the results from single muscle spindles, the slowest g a m m a fibre is invariably dynamic (Fig. 5). If all the results of these experiments are pooled (Fig. 4), it is apparent that, although the dynamic g a m m a fibres form a slower group than the static g a m m a fibres, the degree of overlap between the two groups is considerable. If we now also bring into consideration the results of the first series of experiments (Fig. 4A), the chance of a given slow g a m m a fibre being dynamic is considerably reduced. Thus, although our results from single spindles show a distinct division of g a m m a fibres by conduction velocity, unfortunately it is probable that this division will be of little practical application when dealing with the whole muscle.
References Andrew, B.L., Part, N.J.: Properties of fast and slow motor units in hind limb and tail muscles of the rat. Quart. J. exp. Physiol. 57, 213-225 (1972) Andrew, B.L., Part, N. J,: The division of control of muscle spindles between fusimotor and mixed skeletofusimotor fibres in a rat caudal muscle. Quart. J. exp. Physiol. 59, 331-349 (1974) Andrew, B.L., Leslie, G.C., Part, N.J.: The ratio of static to dynamic gamma fusimotor fibres innervating the soleus muscle of the rat. J. Physiol. (Lond.) 256, 118-119P (1976) Bessou, P., Emonet-Ddnand, F., Laporte, Y.: Motor fibres innervating extrafusal and intrafusal muscle fibres in the cat. J. Physiol. (Lond.) 180, 649-672 (1965) Brown, M.C., Crowe, A., Manhews, P.B.C.: Observations on the fusimotor fibres of the tibialis posterior muscle of the cat. J. Physiol. (Lond.) 17"/, 140-159 (1965) Close, R.: Properties of motor units in fast and slow skeletal muscles of the rat. J. Physiol. (Lond.) 193, 45-55 (1967) Crowe, A., Matthews, P. B. C.: The effects of stimulation of static and dynamic fusimotor fibres on the response to stretching of the primary endings of muscle spindles. J. Physiol. (Lond.) 174, 109-131 (1964) Ellaway, P., Emonet-D6nand, F., Joffroy, M.: Mise en evidence d'axones squeletto-fusimoteurs (axones 6) dans le muscle premier lombrical superficiel du chat. J. Physiol. (Paris) 63, 617-623 (1971)
Rat Soleus Muscle Spindles
443
Emonet-D6nand, F., Laporte, Y., Pagbs, B.: Fibres fusimotrices statiques et fibres fusimotrices dynamiques chez le lapin. Arch. ital. Biol. 104, 195-213 (1966) Emonet-D6nand, F., Jami, L., Laporte, Y.: Skeleto-fusimotor axons in hind limb muscles of the cat. J. Physiol. (Lond.) 249, 153-166 (1975) Emonet-D6nand, F., Laporte, Y.: Proportion of muscle spindles supplied by skeletofusimotor axons (~-axons) in peroneus brevis muscle of the cat. J. Neurophysiol. 38, 1390-1394 (1975) Emonet-D6nand, F., Laporte, Y., Matthews, P.B.C., Petit, J.: On the subdivision of static and dynamic fusimotor actions on the primary ending of the cat muscle spindle. J. Physiol. (L0nd.) 268, 827-861 (1977) Gutmann, E., Hanzlikovfi, V.: Motor unit in old age. Nature (Lond.) 209, 921-922 (1966) Hulliger, M., Matthews, P. B. C., Noth, J.: Static and dynamic fusimotor stimulation on the response of Ia fibres to low frequency sinusoidal stretching of widely ranging amplitudes. J. Physiol. (Lond.) 267, 811-838 (1977) Kucera, J.: Histochemistry of intrafusal muscle fibres outside the spindle capsule. Amer. J. Anat. 148, 427-432 (1977) Lennerstrand, O., Thoden, U.: Position and velocity sensitivity of muscle spindles in the cat. II. Dynamic fusimotor single fibre activation of primary endings. Acta physiol, scand. 74, 16-29 (1968) Matthews, P. B. C.: Mammalian muscle receptors and their central actions. London: Edward Arnold 1972 MacWilliam, P. N.: The incidence and properties of [3 axons to muscle spindles in the cat hind limb. Quart. J. exp. Physiol. 60, 25-36 (1975) Zelenfi, J., Hnik, P.: Motor and receptor units in the soleus muscle after nerve regeneration in very young rats. Physiol. bohemoslov. 12, 277-289 (1963) Received September 19, 1977
Exp. Brain Res. 31,433-443 (1978)
9 Springer-Verlag1978
Some Observations on the Efferent Innervation of Rat Soleus Muscle Spindles B.L. Andrew, G.C. Leslie and N.J. Part Department of Physiology, The University, Dundee DD 1 4HN, Scotland, U.K.
Summary.In
the small segmental tail muscles of the rat beta fibres provide exclusively the dynamic fusimotor control, while gamma fibres provide exclusively the static fusimor control. The present experiments were made to investigate the fusimotor innervation of spindles in a large muscle of the rat, the soleus, and thus to determine the occurrence and significance of beta innervation in this muscle. Our results have revealed no case of beta innervation in the rat soleus. As a consequence of our experimental method, however, we would not claim that beta innervation does not exist in the soleus, only that it must play an insignificant role relative to that seen in the tail segmental muscles. Investigations of the fusimotor innervation of eight spindles were sufficiently complete to warrant detailed illustration. The number of gamma fibres ranged from two to four. In every case the slowest conducting gamma fibre was dynamic. However, the conduction velocity spectra for the static and dynamic gamma fibres to rat soleus overlap to such an extent that it is impossible to use conduction velocity as the sole guide to functional gamma fibre classification. The pooled results from the eight spindles fully investigated provide a ratio of static to dynamic gamma fibres of approximately 1:1. Other evidence discussed in the paper suggests that in the muscle nerve the ratio is considerably higher. These differences are reconciled if the dynamic gamma fibres branch more profusely and innervate more spindles than do the static gamma fibres. Key words: Rat - Soleus - Fusimotor innervation - Skeletofusimotor innervation An efferent nerve fibre to a mammalian muscle spindle may be either purely fusimotor (gamma) or mixed skeletofusimotor (beta). The importance of the gamma system is firmly established. In the cat the beta system is known to be important in a number of small muscles (Bessou et al., 1965; Ellaway et al., 1971; McWilliam, 1975) and recently has been shown to be present in large
0 0 1 4 - 4 8 1 9 / 7 8 / 0 0 3 1 / 0 4 3 3 / $ 2.20
434
B.L. Andrew et al.
hind limb muscles ( E m o n e t - D d n a n d et al., 1975; E m o n e t - D 6 n a n d an d L a p o r t e , 1975). I n the rat th e small s e g m e n t a l muscles of t h e tail r e c e i v e t h ei r d y n a m i c f u s i m o t o r i n n e r v a t i o n exclusively in the f o r m o f b e t a fibres an d t h ei r static supply exclusively as g a m m a fibres ( A n d r e w and Part, 1974). W e h a v e i n v e s t i g a t e d the f u s i m o t o r n e r v e supply of t h e soleus muscle o f the rat in o r d e r to d e t e r m i n e w h e t h e r t h e f u s i m o t o r supply is a r r a n g e d in a m a n n e r similar to that in t h e tail muscles of t h e rat or in th e m o r e familiar m a n n e r f o u n d in t h e soleus o f t h e cat in w h ic h g a m m a fibres are b o t h static and d y n a m i c in type. E a r l y w o r k on b e t a i n n e r v a t i o n was p e r f o r m e d on small m u scl es b e c a u s e of t h e g r e a t e r t echn i c a l ease of id e n ti f y i n g this t y p e of i n n e r v a t i o n with a s m a l l e r n u m b e r o f c o m b i n a t i o n s of a f f e r e n t a n d e f f e r e n t n e r v e s to t h e muscle. F o r this r e a s o n E m o n e t - D 6 n a n d et al. (1975) in effect m a d e the soleus an d o t h e r large hind limb muscles o f t h e cat into small m u s cl es by cutting b r a n c h e s of t h e i r muscle n e r v e and w o r k i n g with just a small p o r t i o n o f t h e t o t al muscle. T h e soleus muscle o f t h e rat, h o w e v e r , is small e n o u g h to be i n v e s t i g a t e d as a c o m p l e t e muscle. T h e results of t h e s e e x p e r i m e n t s , whilst n o t able to e l i m i n a t e t h e o c c u r r e n c e of b e t a i n n e r v a t i o n in t h e soleus m u s c l e o f th e rat, do strongly suggest t h a t b e t a i n n e r v a t i o n does n o t play the i m p o r t a n t r o l e that it does in t h e tail muscles.
Methods The experiments were carried out on a total of twenty-six female rats, 300450 g body weight. Anaesthesia was induced with trichlorethylene vapour and maintained by intraperitoneal injection of urethane solution, dosage 1.5 mg urethane/g body weight. Supplementary injections were given as necessary. The lumbar spinal roots were exposed by laminectomy and covered with warmed Tyrode solution and paraffin oil. The soleus muscle was dissected free from surrounding tissue, the muscle nerve cleared for the placement of electrodes and all other muscles in the leg denervated. The leg was passed through an aperture in the side of a Perspex bath and a skin flap from the thigh tied round a flange surrounding the hole to form a leak-proof seal (Close, 1967). The bath was filled with paraffin oil floating on Tyrode solution and was maintained at about 35~ C by a coil of circulating warm water. The proximal tendon of the soleus muscle was attached to a rigid support and the distal tendon attached, via a tension transducer, to an electromagnetic puller (Ling-Pye V 47/3) with associated displacement transducer. The nerve fibres to soleus pass through dorsal and ventral roots L4, L5 and L6 with the greatest number of fibres in L5. All these spinal roots were cut near their entry to the cord. Pairs of electrodes, switchable from stimulating to recording, were placed on the appropriate dorsal and ventral roots and on the muscle nerve. Two experimental protocols were performed. In the earlier experiments the ventral root was first divided to give a functionally single gamma fibre (less than 32 m/sec [Andrew and Part, 1972]). This filament was then stimulated at 100 and 200 stimuli/sec both with the muscle held at constant length and with the muscle oscillated sinusoidally through 1 mm at 3 Hz. The dorsal root was then divided until there was obtained a filament containing a single soleus spindle afferent acted upon by the efferent. In the later experiments the dorsal root was the first to be divided to a single spindle primary afferent. The ventral root was then divided into about twenty filaments. Each of these filaments contained between zero and three alpha fibres. If the filament contained more than three alpha fibres it was further divided until three was the maximum number of apha fibres. Because of the off-loading effects of extrafusal muscle fibre contraction, three alpha fibres was considered to be the maximum acceptable in such coarse ventral root filaments. Wherever possible filaments with less
Rat Soleus Muscle Spindles
435
A
Fig. 1. Recordings of alpha and gamma nerve action potentials. Upper trace: An alpha (A) and a gamma (G) action potential recorded in a ventral root filament on supramaximal stimulation of the soleus muscle nerve. L o w e r trace: An alpha (A) and a gamma (G) action potential recorded in the intact soleus muscle nerve on supramaximal stimulation of the above filament. The large amplitude, low frequency deflection is the electromyogram of the motor unit of the alpha fibre. S marks the stimulus artifacts. Both records are the result of averaging thirty-two consecutive records
than three alpha fibres were used. Along with the alpha fibres in these filaments there were a widely variable number of gamma fibres. Such ventral root filaments were stimulated at 100-200 stimuli/sec and any excitatory action resulting in the primary afferent recorded. If an excitatory action was observed the ventral root filament was split until the fibre, or fibres, responsible for the excitation were obtained in isolation. One of the objects of these experiments was to determine the extent of beta innervation to the rat soleus. Therefore all efferent nerve fibres, both fast and slow, to the soleus were tested for fusimotor action. If stimulation of a fast efferent nerve fibre produced both extrafusal muscle fibre contraction and increased activity in the spindle afferent, two tests were carried out to establish whether the spindle activation was caused by stretch of the spindle brought about by the extrafusal contraction (freakish pull) or by true fusimotor activation of the spindle. The first test involved stimulating the efferent nerve at frequencies up to and beyond the tetanic fusion frequency of the extrafusal motor unit (Bessou et al., 1965). Increase of the spindle response at frequencies beyond that producing the maximum response of the extrafusal motor unit is indicative of fusimotion. The other test required the selective block of the extrafusal neuromuscular junctions with topically applied Flaxedil. The blockage of the extrafusal neuromuscular junctions was accelerated by stimulation of the efferent nerve at 100 shocks sec -1 for half a second once every second and a half (Bessou et al., 1965). The persistence of spindle excitation after extrafusal blockade is another indication of true fusimotor action. The conduction velocity of action potentials in efferent filaments was determined first by stimulation of the muscle nerve and recording the evoked response in the filament (see Fig. 1, upper record). When the filament was stimulated, recordings were taken from the muscle nerve and averaged using a DL 4000 averager (Data Laboratories Ltd.). The lower record in Figure 1 is one such averaged response. The conducted alpha response is clearly seen together with its associated myogram. Superimposed on the e.m.g, is the gamma action potential, corresponding in position with the same gamma potential when first recorded in the filament. This averaged response from the muscle nerve was used routinely to determine that the fibres in the efferent filament were being stimulated supramaximally and that activity in these fibres was reaching the muscle nerve (and passing onward to the muscle itself). Furthermore, this averaging technique ensured that no gamma fibre was being inadvertently stimulated when investigating fibres suspected of being beta-axons. The effects of stimulation of the fusimotor fibres at frequencies of 10-300 shocks per second on the response of the spindle endings to sinusoidal and ramp stretches were recorded on magnetic tape along with records of muscle length and tension. Post-experimentally, the action potential recordings were subjected to instantaneous frequency analysis which was photographed from a C.R.O. screen along with the length and tension records. So that the shock artifacts did not interfere with the recordings a shock-artifact suppressing device (Frederick Haer) was used. The time programming of stimulation of the efferent fibres and the pulling of the muscle was arranged with a Digitimer 4030 (Digitimer Ltd). Wave-forms to operate the muscle puller were produced with a Servomex generator type LF 141.
436
B.L. Andrew et al.
Results
Efferent Innervation of the Rat Soleus If a survey of the efferent innervation of the muscle spindles of the soleus is to be useful, it must be possible to obtain the greater proportion of the efferent nerve fibres in isolation or near isolation in ventral root filaments. The rat soleus has been shown to have about thirty motor units by physiological (Close, 1967; Andrew and Part, 1972) and by histological methods (Zelen~ and Hnlk, 1963; Gutmann and Hanzl~kovfi, 1966). The number of gamma fibres has been estimated histologically as about twenty-two (Zelenfi and Hnfk, 1963). In 10 experiments in the present work, the number of isolated alpha fibres ranged from 20 to 31 but predominantly between 26 and 31. Similarly the number of gamma fibres isolated ranged from 18 to 25. In three other experiments, the numbers of alpha and gamma fibres isolated exceeded noticeably the numbers expected to innervate the soleus. In these cases it was noted however that part of lateral gastrocnemius was innervated by a branch of the soleus muscle nerve. If the number of alpha fibres observed was twenty-six or more and the number of gamma fibres eighteen or more, it was considered that a sufficiently complete survey of the efferent innervation had been carried out.
Beta Innervation A number of fast extrafusal muscle innervating nerve fibres excited also the spindle primary afferent. However in no case, using the techniques given in Methods, were we able to obtain proof positive of the fusimotor origin of this excitation. Presumably the excitation was caused by the extrafusal contraction causing stretch of the spindle. Only those fast efferent fibres which excited the spindle during extrafusal muscle contraction were fully tested with the stimulus frequency and selective block tests; those fast efferents which did not excite the primary afferent were not investigated further. Emonet-D6nand et al. (1975) have examined fast efferent fibres that only fully revealed their beta nature after the off-loading effect of the extrafusal muscle fibres had been removed by selective gallamine block.
Gamma Innervation Two series of experiments were performed on the gamma innervation. In the first of these the gamma fibres were first isolated in ventral root filaments and then their action, static or dynamic, determined by tests on subsequently isolated dorsal root filaments containing either primary or secondary afferents. The second series of experiments involved isolating primary spindle afferents in dorsal root filaments and then isolating in ventral root filaments the fusimotor fibres active on that afferent. The classification of a gamma fibre, as static or dynamic, was made from records such as those shown in Figures 2 and 3. The tests themselves were
Rat Soleus Muscle Spindles
0
ls
437
"
F
. . . .
10
.'g" ]':'.'i:....
,.
,
2OO A
i
75
/%~k,VVVN~J~
i-
Fig. 2. Action of a static gamma fibre. Left hand column of traces: Effects of stimulating a static gamma fibre at the frequencies shown at the left on the response of a spindle primary afferent to 3 Hz sinusoidal stretching of 1 mm peak to peak amplitude. The duration of stimulation is shown by the horizontal bar beneath the records. Right hand column of traces: Effects of stimulating the static gamma fibre at the same frequencies on the response of the same spindle primary afferent to 1 mm amplitude ramp stretch at 10 mm sec-~. The duration of stimulation is shown by the horizontal bar beneath the records. The conduction velocity of the gamma fibre was 18.l m/s -1 and that of the spindle primary 61.3 m/s -1. The right hand vertical calibration bar shows the frequency meter calibration in action potentials per second
d e s i g n e d to o b s e r v e t h e effects of f u s i m o t o r s t i m u l a t i o n ( 1 0 - 2 0 0 s h o c k s / s e c ) on t h e a f f e r e n t d i s c h a r g e s o c c u r r i n g in r e s p o n s e to m u s c l e l e n g t h changes. T h e m u s c l e l e n g t h c h a n g e s w e r e e i t h e r l a r g e a m p l i t u d e (1 ram) 3 H z s i n u s o i d a l stretch o r 1 m m r a m p stretches (at a v e l o c i t y of 10 m m sec-1). O n e of t h e c r i t e r i a u s e d to classify t h e f u s i m o t o r fibres was t h e d y n a m i c i n d e x ( C r o w e a n d M a t t h e w s , 1964), m e a s u r e d f r o m t h e r e c o r d s o f r a m p stretch; s t i m u l a t i o n o f d y n a m i c f u s i m o t o r fibres causes an i n c r e a s e o f t h e i n d e x (see Fig. 3), static f u s i m o t o r s t i m u l a t i o n d e c r e a s e s , o r l e a v e s u n c h a n g e d , the d y n a m i c index. I n o u r e x p e r i m e n t s all t h e static f u s i m o t o r fibres w e r e s e e n to d e c r e a s e the d y n a m i c i n d e x (see Fig. 2).
438
B.L. Andrew et al. i
0
ls
10 200
11o
25 !
75
9
200
t
.
~
A
..
,
9
~
,,
^
,
.
.
l
...
. . . . . . . . . . . . .
~
,,,
Fig. 3. Action of a dynamic gamma fibre. Left hand column of traces: Effects of stimulating a dynamic gamma fibre at the frequencies shown at the left on the response of a spindle primary afferent to 3 Hz sinusoidal stretching of 1 mm peak to peak amplitude. The duration of stimulation is shown by the horizontal bar beneath the records. Right hand column of traces: Effects of stimulating the dynamic gamma at the same frequencies on the response of the same spindle primary afferent to 1 mm amplitude ramp stretch at 10 mm sec-1. The duration of stimulation is shown by the horizontal bar beneath the records. The conduction velocity of the gamma fibre was 15.4 ms-1 and that of the spindle primary 64.5 ms-1. The right hand vertical calibration bar shows the frequency meter calibration in action potentials per second
T h e typical r e s p o n s e of a passive s p i n d l e p r i m a r y a f f e r e n t to 3 H z s i n u s o i d a l stretch is to d i s c h a r g e o n l y on t h e e x t e n s i o n p h a s e a n d to b e silent d u r i n g relaxation. D u r i n g high f r e q u e n c y ( 1 0 0 - 2 0 0 H z ) static f u s i m o t o r s t i m u l a t i o n , t h e p r i m a r y e n d i n g is c a u s e d to d i s c h a r g e t h r o u g h o u t t h e w h o l e o f the cycle (see Fig. 2). E v e n at t h e highest f r e q u e n c y (200 H z ) o f stimulation, d y n a m i c g a m m a fibres fail to elicit a c o n t i n u o u s d i s c h a r g e f r o m t h e afferent, t h a t is t h e e n d - o r g a n fails to sustain a d i s c h a r g e during t h e w h o l e of the r e l e a s e p h a s e o f t h e cycle (see
Rat SoleusMuscleSpindles
439
Fig. 3). This is a feature of the dynamic gamma fibre (Lennerstrand and Thoden, 1968). Another feature of dynamic gamma stimulation is that it increases the modulation of the discharge frequency brought about by large amplitude sinusoidal stretching of the muscle. Unfortunately amplitude of modulation is not satisfactorily expressed by instantaneous frequency records but requires the fitting of a sinusoid, to include negative values, to the cycle histogram of the afferent firing throughout the cycle (Hulliger et al., 1977). Thus for the simple purpose of classification of gamma fibres as static or dynamic, lack of discharge during sinusoidal release is probably a prime indicator. In these experiments no difficulty was found in classifying all but one of the fifty-nine gamma fibres isolated; the results from the ramp stretches and sinusoidal tests complemented each other. The one exceptional fibre was classified as dynamic but possessed some static features that would place it in one of the intermediate categories of Emonet-D6nand et al. (1977). Inspection of Figures 2 and 3 will reveal other differences in the consequences of stimulation of static and dynamic gamma fibres. A persistence of the dynamic action for a cycle of sinusoidal stretch after the cessation of dynamic gamma stimulation can be seen whereas the static gamma fibre action ceases rapidly at the end of stimulation. The ramp stretch records with dynamic stimulation shows an adaptation of discharge frequency during the extension period not seen in the static gamma records. This was a feature of the dynamic gamma fibres and indeed is considered by Emonet-D6nand et al. (1977) as the best single distinguishing feature for a dynamic gamma fibre. The results of the first series of experiments are summarized in Fig. 4A. Twenty gamma fibres were isolated. Nineteen of these fibres were static and one was dynamic. The ratio of static to dynamic gamma fibres at nineteen to one, obtained from this first series of experiments differs considerably from that of four to one quoted by Matthews (1972) for cat soleus. This apparent deficiency of dynamic gamma innervation led us to suspect that the dynamic supply to these spindles was from a beta source (Andrew et al., 1976) as in the segmental tail muscles of the rat (Andrew and Part, 1974). It was to investigate this point that the second series of experiments was performed. The testing of efferent fibres on a previously isolated spindle afferent enabled an idea of the total fusimotor innervation of one spindle to be built up. A total of thirty-nine gamma fibres was investigated with this protocol. In contrast to the ratio of nineteen static to one dynamic obtained in the first series of experiments this second series produced a ratio of twenty-one static to eighteen dynamic. Histograms of the conduction velocities of the gamma fibres studied in these two experiments are given in Figure 4. The average conduction velocity of the "afferent first" static gammas was 27.8 m/s -1 _+ 6.8 m/s -I standard deviation and of the dynamic gamma fibres 17.3 m/s -1 + 3.8 m/s -1 standard deviation; these two values are significantly different at the 0.001 level.
Fusimotor Innervation of Individual Muscle Spindles Eight spindles received a complete investigation of their fusimotor innervation subject to the limits of the experimental protocol (see Fig. 5). In each of these
440
B. L~ Andrew et al.
A
::.
9
:.
9 9149149
9 9 9149149
B
99
9
9
1'0
conduction
9
000
2b
0000
9
3'0
00 i
40
velocity
5o
m/see
Fig. 4. Conduction velocities of the isolated gamma fibres. A Conduction velocities of the twenty gamma fibres isolated and then subsequently categorized against spindle afferents. The static gamma fibres are represented by circles and the dynamic gamma fibre by a triangle. B Conduction velocities of the gamma fibres which were categorized against a previously isolated spindle primary afferent, The upper group of triangles shows the dynamic gamma fibres and the lower group of circles shows the static gamma fibres. It is noteworthy that in these experiments there were isolated three static gamma fibres whose conduction velocities exceeded the value of 32 m/sec thought previously (Andrew and Part, 1972) to be the upper limit in the range of gamma fibre conduction velocity
Spindle ~1
9
9
b
-
C
9
d
9
e
9
f
~
g
V
9
9 9
9
9
9
9
9
h
0
~
9
9
9
9
I
I
I
I
I
10
20
30
40
50
conduction
velocity
m/sec
Fig. 5. The gamma innervation of eight soleus muscle spindles. The dynamic gamma fibres are represented by triangles and the static gamma fibres by circles
eight examples the number of efferent nerve fibres investigated in the ventral root filaments was an acceptable fraction of the efferents innervating the soleus (see Results section Efferent Innervation of the Rat Soleus). The most striking feature is that in all eight spindles the slowest conducting gamma fibre was dynamic. Six of the spindles had only one dynamic gamma fibre but two of the
Rat Soleus Muscle Spindles
441
spindles had two. In one of these spindles the two dynamic gamma fibres were the two slowest but in the other there was a static gamma fibre placed between the two dynamic ones. It should also be noted that all the spindles had both a static and a dynamic gamma nerve supply.
Discussion In this project we have looked for beta innervation in the soleus muscle of the rat. Our experiments have not produced any proven cases of beta innervation. However, because of the limitations of the experimental protocol given above, we would be reluctant to state that beta innervation does not exist in this muscle. Indirect histological evidence for beta innervation in the rat soleus has been reported (Kucera, 1977). Nevertheless our failure to detect beta innervation strongly suggests that beta innervation is not of the great significance that it is in the segmental tail muscles in which the dynamic fusimotor innervation is entirely from beta fibres (Andrew and Part, 1974). Emonet-D6nand et al. (1975) have established the existence of beta innervation in the cat soleus but are not able to state the significance of beta innervation in this muscle. In some muscles of the cat, especially small ones such as abductor digiti quinti medius (McWilliam, 1975) beta innervation is of great significance and indeed in that particular muscle 30% of the fast axons are beta. Other recent work by Emonet-Ddnand and Laporte (1975) has reported that in the large peroneus brevis muscle of the cat 18% of axons supplying extrafusal muscle, supplied also spindles and that 72 % of spindles received beta axons. The gamma innervation of the soleus spindles has several interesting features. The first of these to be considered is the difference in ratio of static to dynamic gamma fibres obtained from the two experimental protocols. When the gamma fibre was isolated first and its function then determined, the ratio of static to dynamic was nineteen to one. In the second series when the afferent was first isolated, the ratio of static to dynamic was twenty-one to eighteen. The likelihood of this difference in ratios arising by chance is slight. Differential branching of static and dynamic gamma fibres provides a possible explanation of these different ratios. If the dynamic gamma fibres branch so as to innervate a greater number of spindles than do the static gamma fibres, then one would expect our two experimental protocols to produce different ratios in much the way that has been seen here. From the second series of experiments we know that each spindle receives approximately the same number of static and dynamic gamma fibres. A greater degree of branching of the dynamic gamma fibres than the statics would mean that the dynamic supply would originate from a smaller total number of parent dynamic gamma axons than parent static axons; hence the high ratio of static to dynamic seen when isolating gamma fibres first. The only fully satisfactory experimental test that could be applied to this proposition would be to isolate and categorize all the gamma fibres to a muscle. This, regrettably, did not prove possible. In one experiment three different dynamic gamma fibres were isolated out of a total gamma innervation of
442
B.L. Andrew et al.
eighteen fibres. This result in itself suggests that differential branching is unlikely to be the full explanation of the different ratios because the static: dynamic ratio must beat most fiveto one in this muscle, as opposed to the ratio of nineteen to one for the first series of experiments. This discrepancy may be accounted for by g a m m a fibres which were isolated in the first series of experiments but which were not successfully cross-matched with a spindle afferent. It may be that a majority of these were dynamic fibres whose action might not have been strong enough to prevent masking of the activated spindle afferents by other active fibres in the dorsal root. The matter of g a m m a fibre conduction velocity has been given an appreciable amount of attention because of the experimental convenience of being able to distinguish the function of nerve fibres by conduction velocity (Matthews, 1972; p. 220). It has not proved possible to m a k e a clear differentiation into static and dynamic g a m m a fibres by conduction velocity. Nevertheless, in cat tibialis posterior (Brown et al., 1965) and rabbit tenuissimus ( E m o n e t - D 6 n a n d et al., 1966) the slowest g a m m a fibres tend to be static. This is not seen in our results from the rat soleus; taking the results from single muscle spindles, the slowest g a m m a fibre is invariably dynamic (Fig. 5). If all the results of these experiments are pooled (Fig. 4), it is apparent that, although the dynamic g a m m a fibres form a slower group than the static g a m m a fibres, the degree of overlap between the two groups is considerable. If we now also bring into consideration the results of the first series of experiments (Fig. 4A), the chance of a given slow g a m m a fibre being dynamic is considerably reduced. Thus, although our results from single spindles show a distinct division of g a m m a fibres by conduction velocity, unfortunately it is probable that this division will be of little practical application when dealing with the whole muscle.
References Andrew, B.L., Part, N.J.: Properties of fast and slow motor units in hind limb and tail muscles of the rat. Quart. J. exp. Physiol. 57, 213-225 (1972) Andrew, B.L., Part, N. J,: The division of control of muscle spindles between fusimotor and mixed skeletofusimotor fibres in a rat caudal muscle. Quart. J. exp. Physiol. 59, 331-349 (1974) Andrew, B.L., Leslie, G.C., Part, N.J.: The ratio of static to dynamic gamma fusimotor fibres innervating the soleus muscle of the rat. J. Physiol. (Lond.) 256, 118-119P (1976) Bessou, P., Emonet-Ddnand, F., Laporte, Y.: Motor fibres innervating extrafusal and intrafusal muscle fibres in the cat. J. Physiol. (Lond.) 180, 649-672 (1965) Brown, M.C., Crowe, A., Manhews, P.B.C.: Observations on the fusimotor fibres of the tibialis posterior muscle of the cat. J. Physiol. (Lond.) 17"/, 140-159 (1965) Close, R.: Properties of motor units in fast and slow skeletal muscles of the rat. J. Physiol. (Lond.) 193, 45-55 (1967) Crowe, A., Matthews, P. B. C.: The effects of stimulation of static and dynamic fusimotor fibres on the response to stretching of the primary endings of muscle spindles. J. Physiol. (Lond.) 174, 109-131 (1964) Ellaway, P., Emonet-D6nand, F., Joffroy, M.: Mise en evidence d'axones squeletto-fusimoteurs (axones 6) dans le muscle premier lombrical superficiel du chat. J. Physiol. (Paris) 63, 617-623 (1971)
Rat Soleus Muscle Spindles
443
Emonet-D6nand, F., Laporte, Y., Pagbs, B.: Fibres fusimotrices statiques et fibres fusimotrices dynamiques chez le lapin. Arch. ital. Biol. 104, 195-213 (1966) Emonet-D6nand, F., Jami, L., Laporte, Y.: Skeleto-fusimotor axons in hind limb muscles of the cat. J. Physiol. (Lond.) 249, 153-166 (1975) Emonet-D6nand, F., Laporte, Y.: Proportion of muscle spindles supplied by skeletofusimotor axons (~-axons) in peroneus brevis muscle of the cat. J. Neurophysiol. 38, 1390-1394 (1975) Emonet-D6nand, F., Laporte, Y., Matthews, P.B.C., Petit, J.: On the subdivision of static and dynamic fusimotor actions on the primary ending of the cat muscle spindle. J. Physiol. (L0nd.) 268, 827-861 (1977) Gutmann, E., Hanzlikovfi, V.: Motor unit in old age. Nature (Lond.) 209, 921-922 (1966) Hulliger, M., Matthews, P. B. C., Noth, J.: Static and dynamic fusimotor stimulation on the response of Ia fibres to low frequency sinusoidal stretching of widely ranging amplitudes. J. Physiol. (Lond.) 267, 811-838 (1977) Kucera, J.: Histochemistry of intrafusal muscle fibres outside the spindle capsule. Amer. J. Anat. 148, 427-432 (1977) Lennerstrand, O., Thoden, U.: Position and velocity sensitivity of muscle spindles in the cat. II. Dynamic fusimotor single fibre activation of primary endings. Acta physiol, scand. 74, 16-29 (1968) Matthews, P. B. C.: Mammalian muscle receptors and their central actions. London: Edward Arnold 1972 MacWilliam, P. N.: The incidence and properties of [3 axons to muscle spindles in the cat hind limb. Quart. J. exp. Physiol. 60, 25-36 (1975) Zelenfi, J., Hnik, P.: Motor and receptor units in the soleus muscle after nerve regeneration in very young rats. Physiol. bohemoslov. 12, 277-289 (1963) Received September 19, 1977
