The structural and functional development of muscle spindles and their connections in fetal sheep

BRAIN RESEARCH ELSEVIER

Brain Research 642 (1994) 185-198

Research Report

The structural and functional development of muscle spindles and their connections in fetal sheep Sandra Rees a,., John Rawson b, Ilias Nitsos b, Carolyn Brumley b a Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3052, Fic., Australia, b Department of Physiology, Monash University, Clayton 3168, 1.7c.,Australia (Accepted 16 December 1993)

Abstract

In this paper we have studied the structural and functional development of hindlimb muscle receptors and the connections of their afferent fibres in fetal sheep (n = 26) from 67-143 days of gestation (term = 146 days). By recording extracellular discharges in dorsal root ganglia (L7, S1) we have shown that muscle spindle afferents first respond to a ramp-and-hold stretch at mid-gestation ( ~ 75 days). Silver-stained preparations of muscle spindles revealed that afferent fibres are just beginning to form annulospiral windings at this age. It therefore appears that the annulospiral formation is not a necessary requirement for the generation of the response. By 87-92 days some receptors had developed a discharge at resting muscle length. Discharges were generally more robust and easier to elicit and static and dynamic components could be identified in the response to stretch. Although static sensitivity was generally low it was more evident than dynamic sensitivity. By 107-115 days it was possible to clearly distinguish between muscle and tendon afferents and to tentatively classify muscle responses as originating from primary or secondary afferent spindle endings. With increasing gestational age there was a progressive increase in the length and complexity of the spindle innervation in parallel with the maturation of functional activity. Biocytin injections into the dorsal root ganglia revealed afferent projections to the motoneuron pools by 67 days. Silver-staining of muscles showed that innervation of extrafusal fibres was also present by this age. We therefore conclude that the neural pathways necessary for reflex activity involving muscle spindles are present and functional from early in gestation and could contribute to early fetal movements.

Key words: Development; Fetal; Muscle spindle; Structure; Function

1. Introduction

The sequence of events involved in the innervation and structural development of mammalian muscle spindles has been well established in several species [3,18,20,21,25-27]. The corresponding functional development of spindles has received less attention however. Studies in the neonatal kitten [12-14,22,23] have shown that at birth, spindles are functional with several features of the adult response already present. Despite the relative maturity of the response, the innervation of the spindle at this stage is immature without any signs of the characteristic annulospiral arrangement of the afferent endings [22]. This would suggest that the size and shape of the afferent terminals was not the pre-

* Corresponding author. Fax: (61) (3) 347 5219. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 6 5 4 - L

dominant factor in determining the response of the spindle to stretch [22]. Currently, the age at which muscle receptors first begin to function in utero is not known except for the rat where some brief observations have been made on the onset of activity in late gestation [10]. Observations of the human fetus with real-time ultrasound have shown that the first discernible movements are detectable at 7 - 8 weeks of age [7]. Movement of individual limbs occurs at about 9 - 1 0 weeks with more complex movements such as sucking and swallowing evident at 12-14 weeks. Thus although it is clear that muscle contractions occur very early in development, it is not certain when afferent fibres from muscle spindles first become active and possibly contribute to this activity. In order to address this question we have studied the structural and functional maturation of muscle receptors and the connections of their afferent fibres

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in the sheep. In this species, which has a long gestational period (term = 146 days), fetal movements can be observed with ultrasound by about 60 days (A. Ramsden, personal communication) that is well within the first half of gestation. The length of gestation, together with the size and relative robustness of the fetus has made it possible to follow developmental events back into fetal life to define precisely when receptor pathways begin to function. In this study we have shown that muscle spindle afferents from the hind limb are capable of discharging in response to muscle stretch by mid-gestation (~ 75 days). At this stage the innervation of the spindle, as observed in silver-stained preparations, is generally restricted in extent and simple in its arrangement although there is evidence that annulospiral endings are just beginning to develop.

2. Materials and methods 2.1. Electrophysiology Experiments were performed on 26 fetal sheep (Border Leiscester Merino cross) at 67-77 days (d) (n = 3), 87-92 d (n = 2), 97-100 d ( n = 3 ) , 107-115 d ( n = 6 ) , 117-126 d ( n = 5 ) and 127-143 d (n = 7). Both the ewe and fetus were initially anaesthetised with pentothal (20 m g / k g , i.v.) and anaesthesia subsequently maintained with 1.5-2.0% halothane in 50:50 nitrous oxide in oxygen for the duration of the experiment. T e m p e r a t u r e was monitored via a rectal probe and maintained at 37-40°C with the aid of a thermal blanket. Blood glucose and blood gas tensions were measured at regular intervals throughout the experiment, from samples obtained via a catheter in the maternal carotid artery. By adjusting the respiratory rate and tidal volume, p C O 2 was maintained at 35-40 m m H g and blood pH 7.35-7.45. On one occasion (at 138 d) fetal and maternal blood were sampled simultaneously over the 9 h duration of the experiment to determine whether or not the maternal blood gas values were an accurate reflection of the fetal values. As this was found to be the case we were then able to routinely avoid the additional surgery of fetal catheterisation. A low midline incision was made in the ewe, the dorsal surface of the fetus exposed, and the skin surrounding the vertebral column reflected and sutured to the uterine muscle. The spinal cord was stabilised with vertebral clamps, and a laminectomy was performed to expose the L7 and S1 segments of the spinal cord and associated dorsal root ganglia (DRG). Exposed tissues were covered with warm (37°C) paraffin oil contained within pools made by the skin flaps. T h e left hindlimb was lifted out of the uterus to allow for dissection of the tendons of triceps surae, tibialis anterior, quadriceps and flexor digitorum longus. Thread was tied onto the cut end of each of the tendons so that the muscle could be attached to a servo-controlled muscle puller used to apply sinusoidal vibration or rampand-hold stretches to the muscle. Cuff electrodes were placed around the sciatic nerve and the leg was wrapped in cotton wool to avoid heat loss. Glass-insulated tungsten microelectrodes (tip exposure 4 - 1 0 ~ m ) were used for recording extracellular discharges of single neurons in the DRG. Signals were first fed into a unity gain preamplifier and then to a main amplifier with a bandpass of 10 Hz to 10 kHz. For visual inspection and photography, signals were fed to an oscilloscope and also into an audio amplifier. All responses were recorded on magnetic tape for later analysis. On the occasions when it was possible to activate the D R G cell of interest by electrical

stimulation of the sciatic nerve via the cuff electrode, the latency between the stimulus artifact and the evoked response in the D R G was determined. Conduction velocity was then calculated by dividing the latency of the response by the length of the conduction path, determined at the end of the experiment. The response properties of sensory neurons were determined by applying ramp-and-hold, and vibratory stimuli. Discharges were identified as originating from muscle receptors rather than Golgi tendon organs if they ceased firing during a muscle twitch evoked by stimulation of the sciatic nerve. We know from an anatomical study [15] that there are distinct primary and secondary endings in muscle spindles in sheep. However, as there is no data about the specific response properties of these different endings in the hindlimb of neonatal or adult sheep, we based our identification on data primarily from the cat [19]. We could not therefore unequivocally designate the present responses as originating from primary or secondary endings and the following classification of stretch receptors must be regarded as tentative. Primary afferent endings were distinguished from secondary endings on the basis of their high dynamic sensitivity to muscle stretch; that is, the firing rate increased concomitantly with an increasing rate of rise of the ramp (dynamic component) and their ability to follow a high-frequency vibratory stimulus. Classification as a spindle secondary afferent fibre was indicated if firing rate was maintained during the hold phase of an applied r a m p (static component) but the unit demonstrated little dynamic sensitivity and no ability to follow a high-frequency vibratory stimulus. It is possible that a similar response pattern could also be obtained from an immature primary ending so clasification of muscle afferent fibres can indeed only be tentative in this study. In addition to firing during a twitch, the identification of Golgi tendon organs was confirmed if the unit failed to follow high-frequency vibration ( > 50 Hz).

2.2. Histology 2.2.1. Biocytin staining of afferents to the spinal cord Prior to the c o m m e n c e m e n t of electrophysiological recordings, Biocytin [16] was injected into the D R G on the opposite side in fetuses at 67, 76 and 92 d. In a fourth sheep the same procedure was carried out on a fetus at 56 d but electrophysiological recordings were not made. A glass micropipette containing 5% Biocytin (Sigma) in 0.05 M Tris buffer (pH 7.4) was inserted into the exposed ganglia (L7 and $1), and the tracer was injected by pressure using a picospritzer (General Valve Corp). Several injections of about 1 ~1 were made in each ganglia to ensure that the ganglia were saturated with tracer. After a post-injection survival time of 8 - 9 h, which corresponded to the duration of the experiment, the fetus was given an overdose of sodium pentobarbitone and perfused as described below. The relevant segments of spinal cord and associated D R G were dissected out and placed in 10% sucrose for 1 - 2 h. Sections (50 p.m) of spinal cord and D R G were cut on a freezing microtome and collected serially in phosphate buffer and placed in 0.3% hydrogen peroxide ( H 2 0 2) in methanol for 20 rain to block any endogenous peroxidase activity. Three, 10 min washes in buffer were then carried out and sections were incubated for 2 h in Avidin-peroxidase (Sigma) diluted 1:5,000 in 0.1 M phosphate buffer and 0.75% Triton X-100. After a thorough washing (5 × 10 min rinses) in phosphate buffer, sections were processed for horseradish peroxidase histochemistry using the cobalt and nickeMntensified diaminobenzidine (DAB) reaction [1]. Sections were washed in buffer and m o u n t e d onto gelatinised slides. Next, they were air dried, counterstained with Neutral red, dehydrated, cleared in histoclear and coverslipped. Sections were viewed and photographed under the light microscope, and selected sections were drawn at 150× using an Olympus projecting microscope. In this study, the innervation of the ventral horn will be described. We will report in detail on the innervation of the dorsal horn in a separate paper.

S. Rees et al. /Brain Research 642 (1994) 185-198

2.2.2. Silver staining of muscle innervation At the conclusion of the experiment, the ewe and fetus were given an overdose of sodium pentobarbitone (130 mg/kg, i.v.) and the fetus perfused through the descending aorta with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The triceps surae, tibialis anterior and quadriceps muscles were removed from the right hindlimb from fetal sheep at 67 d ( n = l ) , 83 d (n = 1), 100 d (n = 2), 115 d (n = 2), 120 d (n= 1), 126 d (n = 1), 134 d (n = 1) and 143 d (n = 2), and processed using the silver method of Barker and Ip [2] to stain the motor and sensory innervation of the spindle, and the motor innervation of extrafusal fibres. Briefly, the muscles were placed in the prescribed fixative for 4-6 d. The tissue was then washed in running water for 24 h, dried and placed in alcohol/ammonia solution for 48 h followed by 5 d in 1.5% silver nitrate (37°C in the dark). Muscles were then placed in reducing solution for 2 d before being rinsed in distilled water and stored in glycerol for at least 14 d. Spindles were then teased from the surrounding extrafusal muscle fibres under a dissecting microscope using fine dissecting needles and forceps. Once isolated, spindles were mounted in glycerol and coverslipped. The muscle spindle preparations were examined under oil immersion (1,250 × ). The diameter of the primary afferent fibre before its first branch point was measured at three internodal sites and the number of branches counted in 4 spindles at each age. Camera lucida drawings (490 x ) were made of representative spindles at each age and the length of the spindles measured on an image analyser. Photographs of the spindles were taken (40×) and photomontages made of complete spindles. Z2.3. Haematoxylin and Eosin staining of muscle fibres The soleus muscle from one fetus (126 d) was removed, cut into 3 equal sections, further fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin. At 300 tzm intervals throughout the length of the muscle, 6 serial sections (10/zm) were cut on a Jung rotary microtome and stained with haematoxylin and eosin. The sections were examined to determine the arrangement of spindles within the muscle and the number of intrafusal fibres within a capsule.

3. Results 3.1. Electrophysiology 3.1.1. Recording in the dorsal root ganglia E x t r a c e l l u l a r r e c o r d i n g s w e r e m a d e f r o m cells in t h e D R G in fetal s h e e p b e t w e e n t h e ages o f 67 a n d 143 d ( t e r m = 146 d). A s w e d i d n o t d e t e c t any m a r k e d d i f f e r e n c e s in t h e r e s p o n s e s r e c o r d e d f r o m r e c e p t o r s in t h e f o u r m u s c l e s e x a m i n e d in this s t u d y the r e s p o n s e s will b e d e s c r i b e d t o g e t h e r . 6 7 - 7 7 days (n = 3). A t 67 d t h e r e was no s p o n t a n e o u s activity in t h e D R G i n d i c a t i n g t h a t m u s c l e affere n t fibres h a d n o t d e v e l o p e d a d i s c h a r g e at resting m u s c l e length. N e i t h e r could a r e s p o n s e b e e v o k e d by a p p l i e d stretch. I n 75 a n d 77 d fetuses, tonic activity was r e c o r d e d f r o m 3 ceils b u t it was n o t p o s s i b l e to identify t h e m u n e q u i v o c a l l y as m u s c l e a f f e r e n t s as we w e r e u n a b l e to a l t e r t h e i r firing r a t e with m u s c l e stretch. In t h e s e f e t u s e s t h e first r e p r o d u c i b l e responses to s t r e t c h w e r e o b s e r v e d in a few cells (6 units) which d i s c h a r g e d p h a s i c a l l y with a b r i e f b u r s t o f im-

187

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200 ms Fig. 1. Extracellular discharges recorded in the D R G (L7) at 75 d in response to a stretch applied to the tendon of triceps surae. The stretch was applied manually.

pulses ( ~ 20 H z ) (Fig. 1) F o r t e c h n i c a l r e a s o n s we w e r e u n a b l e to a p p l y c o n t r o l l e d stretch to t h e t e n d o n via t h e muscle puller and the muscles were stretched manually. N e v e r t h e l e s s it was c l e a r t h a t t h e s e units res p o n d e d only at the o n s e t o f stretch a n d s h o w e d no s u s t a i n e d activity to muscle l e n g t h e n i n g . T h e r e s p o n s e was also labile a n d d i d n o t always o c c u r with e a c h stretch. It was n o t p o s s i b l e to identify t h e r e c e p t o r type at this age. M a n y p e n e t r a t i o n s w e r e m a d e t h r o u g h t h e L7 a n d S1 ganglia in t h e s e fetuses, a n d it was c l e a r t h a t t h e cells w e r e c a p a b l e o f g e n e r a t i n g a c t i o n p o t e n t i a l s as it was c o m m o n to s e e injury d i s c h a r g e s a n d t h e cells w o u l d also d i s c h a r g e tonically in r e s p o n s e to p r e s s u r e f r o m the m i c r o e l e c t r o d e . T h e r e f o r e , p r e s u m a b l y , m o s t m u s c l e r e c e p t o r s a r e still t o o i m m a t u r e to g e n e r a t e d i s c h a r g e s at resting muscle l e n g t h s o r to r e s p o n d to a n a p p l i e d stretch. 8 7 - 9 2 days (n = 2). D R G cells with a resting disc h a r g e ( 8 - 1 0 Hz) w e r e e n c o u n t e r e d (5 units). T h e s e tonically active units r e s p o n d e d to m u s c l e stretch with an i n c r e a s e d firing r a t e a n d so could b e i d e n t i f i e d as m u s c l e r e c e p t o r s . D i s c h a r g e s o f cells w e r e in g e n e r a l m o r e r o b u s t a n d e a s i e r to elicit in r e s p o n s e to stretch t h a n with t h e e a r l i e s t r e s p o n s e s . In r e s p o n s e to a r a m p - a n d - h o l d s t r e t c h o f 2 m m extension, 6 / 9 units e x h i b i t e d a static c o m p o n e n t in the d i s c h a r g e p a t t e r n (Fig. 2A,B), 2 / 9 a static a n d d y n a m i c c o m p o n e n t (Fig. 2 C , D ) a n d 1 / 9 solely a d y n a m i c c o m p o n e n t . Static sensitivity was g e n e r a l l y low. O f 4 units t e s t e d in m o r e detail, 2 s u s t a i n e d t h e i r d i s c h a r g e r a t e ( ~ 13 H z ) with an i n c r e a s e d r a m p l e n g t h (cf. Fig. 2A,B) a n d in ano t h e r t h e firing r a t e i n c r e a s e d slightly with an i n c r e a s e in the final extension o f the r a m p . In units d i s p l a y i n g b o t h static a n d d y n a m i c sensitivity b o t h c o m p o n e n t s , b u t p a r t i c u l a r l y t h e static, b e g a n to f a t i g u e after 3 a p p l i c a t i o n s o f the stimulus at 0.5 H z (cf Fig. 2C,D). W h e n a v i b r a t o r y stimulus was a p p l i e d to the t e n d o n ( 2 5 - 4 0 Hz), t h e r e c e p t o r c o n t i n u e d to fire (3 units) b u t failed to r e s p o n d to e a c h vibration. T h e a m p l i t u d e o f

S. Rees et al. // Brain Research 642 (1994) 185-198

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since they were silent during muscle twitch (Fig. 4A), exhibited dynamic sensitivity to a ramp-and-hold stimulus (Fig. 4B) and followed a vibratory stimulus at 50, 90 (Fig. 4C) and 200 Hz. At 300 Hz (Fig. 4D) the response was phase-locked for approximately 40 ms before it began to fail. One of these units showed no evidence of fatigue after 10 min of intermittent bursts of vibratory stimulation at 50 Hz. These fibres had conduction velocities of 32 and 34 m / s which corresponded to the fastest conducting sciatic nerve fibres at this fetal age [17]. Two units could be tentatively identified as spindle secondaries on the grounds of failing to fire during a twitch and exhibiting strong static but little dynamic

500 ms

Fig. 2. Responses of muscle receptors at 87 d. (A) and (B). T h e unit had no resting discharge but discharged at ~ 13 Hz when a muscle stretch was applied (A). With increasing ramp length the response was maintained (B). (C) and (D). This unit displayed both dynamic and static sensitivity to a ramp-and-hold stimulus (C). Both phases of the response, but particularly the static began to fail after three applications of the stimulus at 0.5 Hz (D).

the evoked discharges was unstable, and often altered during the course of a response. 97-100 days (n = 3). By 97 d, 15/28 stretch-related units had developed a stable tonic discharge (8-10 Hz) at resting muscle length. Analysis of units which discharged only during an applied stretch showed that the static component still predominated but that some receptors also exhibited a marked dynamic responsiveness. 107-115 days (n =6). A resting discharge was recorded from 30/78 units (Fig. 3A-C, Fig. 4A,B). The response to a ramp-and-hold stimulus was analysed in 39 units, 14 with a background discharge and 25 responding only phasically to the applied stretch. Of these 39 units, 17 displayed both dynamic and static responsiveness, 16 static alone and 6 dynamic alone. Fig. 3 illustrates a spontaneously discharging unit that displayed an increase in dynamic sensitivity in response to the increasingly rapid application of the ramp, and static sensitivity in response to increasing muscle extension. The firing rate of the unit increased from a spontaneous discharge of 9 Hz to 29 Hz with a 6.5 mm stretch showing that there was a length dependent component but that this static sensitivity was rather low. This unit, like several others recorded at this age, also exhibited a 'slipper response' [12]. That is, following a muscle stretch applied to a spontaneously firing unit, the unit ceased firing for 250-300 ms before resuming again at the prestretch rate (see also Fig. 5C). Within this age period it was possible to clearly distinguish between muscle and tendon afferents and to tentatively classify the muscle response as that of a primary or secondary afferent spindle ending. Two units could be identified as possible primary endings

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1()0 m s Fig. 3. Responses of a muscle receptor at 107 d. This unit had a resting discharge rate of 9 Hz which increased to 18 Hz with a 2.5 m m stretch (A), 26 Hz with a 6.5 m m stretch (B), and 29 Hz with the same extension but a more rapid rise time of the ramp (C). With these stimulus parameters, the dynamic sensitivity of the unit was also revealed. This unit displayed the characteristics of a 'slipper response', that is it ceased firing for ~ 250 ms after a stretch before resuming at the prestretch rate.

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37 units analysed in detail 19/37 revealed both static and dynamic sensitivity to a ramp-and-hold stretch. In the unit illustrated in Fig. 5A,B, the more rapid application of a ramp (Fig. 5B compared with Fig. 5A) increased the dynamic response. It is notable that during the hold phase of the stretch there was a significant increase in firing frequency as compared to the same extension in the earlier trial. A similar response was also observed at an earlier age (see Fig. 3A-C). This effect could possibly be due to fluctuations in fusimotor activity. We noted that at this age (127-143 d), afferent fibres could be driven to discharge at rates of about 90 Hz during the hold phase of a muscle stretch and well in excess of this during the application of the ramp (Fig. 5C). Several units exhibiting 'slipper responses' were recorded (Fig. 5C). Conduction velocities were recorded for primary afferent fibres at 77 + 2 m / s , secondary fibres at 44 + 1 m / s and for Golgi tendon organ afferents at 68 + 2 m / s .

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1'oom~ Fig. 4. Responses could be identified as originating from muscles spindles or Golgi tendon organs from 107 d. (A-D) Muscle spindles. This unit was silent during a muscle twitch (A), exhibited dynamic sensitivity to a ramp-and-hold stimulus (B), followed a vibratory stimulus at 90 Hz (C), followed at 300 Hz for 40 ms before failing to respond to each vibration (D). Golgi tendon organ. This unit fired during a muscle twitch (E), exhibited no tonic discharge and no dynamic component in response to muscle stretch (F).

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B sensitivity. Conduction velocities of these afferents were 20 and 22 m / s . Further evidence of the developing resistance of the spindle response to fatigue was obtained from a unit which responded with a steady discharge of 15-17 Hz while a ramp-and-hold stimulus was applied for several minutes. Two units fulfilled the criteria for Golgi tendon organs (Figs. 4E,F) since they exhibited no tonic discharge, fired during a muscle twitch with a graded response related to increasing muscle tension (Fig. 4E) and exhibited no dynamic component in response to muscle stretch (Fig. 4F). 117-126 days (n =5). A t this age 2 5 / 5 6 stretch receptor afferents were firing spontaneously at resting muscle length. Of the 33 units analysed in detail, 22 were firing spontaneously, and 11 discharged only during an applied stretch. The majority of these units (25/33) responded with static and dynamic responsiveness, 6 with a static response alone and 2 with a dynamic component alone. Ten units were identified as spindle afferents (3 as possible primary endings) and one, as a Golgi tendon organ. 127-143 days (n = 7). Spontaneous firing (up to 35 Hz) was recorded in 5 1 / 9 1 units (see Fig. 5C). Of the

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200 ms Fig. 5. Muscle spindle responses at 127 d. (A) and (B). In this unit the more rapid application of the ramp increased the dynamic response (B compared with A). Note that during the hold phase of the stretch (B) there was a significant increase in firing frequency for the same extension (2 mm) as in the earlier trial (A). (C). This unit exhibits a spontaneous discharge rate of a 35 Hz, marked dynamic sensitivity and a pronounced 'slipper' effect.

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3.2. Histology 3.2.1. Biocytin tracing of afferents to the spinal cord At 56 d of gestation individual fibres entered the grey matter and penetrated as far as the intermediate zone (lamina VII) of the spinal cord although occasionally a few fibres entered the ventral horn (Fig. 6A). At 67 d fibres had traversed the intermediate zone and were observed to grow along the mid-line of the spinal cord and to curve laterally as they projected towards the ventral region of the medial motoneuron pool (Fig. 6B) and formed varicosities in this region (Fig. 7A,B). A number of fibres also emerged from the intermediate zone and formed varicosities in the ventral region of the lateral motoneuron pool (Figs. 6B, 7A,B). Overall, at this age, it appeared that there was a denser innervation of the medial than of the lateral motoneuron pools. It should be noted that most of the terminal branching and bouton formation occurred after the fibres had reached the vicinity of the motoneuron cell bodies which were closely packed at this age (Fig. 7B). Another important observation was that primary afferents destined to innervate the medial or lateral motoneuron pools left the intermediate zone in separate bundles indicating a possible topographical distribution of primary afferent fibres in the ventral horn (Fig. 6B). A similar arrangement has been observed in the rat [24]. By 76 d the density of innervation in the ventral horn had increased (Fig. 6C). This is illustrated in Fig. 7C where several fibres can be seen innervating neurons in the lateral motor neuron pool compared to the one or two fibres which innervate this group of neurons at 67 d (Figs. 7A,B). Single varicose fibres were observed to make contact with several motoneurons and the number of varicosities apparently terminating on each neuron had increased (Fig. 7D). By 92 d the density of innervation in both the medial and lateral motoneuron pools had increased considerably (Fig. 6D). Axons appeared to be thicker (when compared with earlier ages) and branched extensively within these regions (Fig. 7E,F). Although we could not identify the sources of individual afferents it seems highly probable that those terminating in the motoneuron pools are group Ia fibres as these are the only known primary afferents that project directly to motoneurons. 3.2.2. Silver staining of muscle innervation Muscle spindles from triceps surae, tibialis anterior and quadriceps muscles have been studied from fetal sheep at 67 d (6 spindles), 83 d (8 spindles), 101 d (11 spindles), 107 d (9 spindles), 110-118 d (9 spindles), 127-143 d (10 spindles). We did not detect any specific differences in the pattern of innervation between the spindles from different muscles, although at any age spindles were at slightly different stages of develop-

ment within a particular muscle. It has not been possible to identify the intrafusal fibre types in this study.

67 days. Muscle tissue was fragile at this age and nerve fibres generally stained lightly however it was possible to identify and dissect 6 spindles. In each case, one or more axons could be seen to enter the spindle and divide into 2 major branches from which a fine network of fibres extended with some sidebranches ending in globular heads (Fig. 8A). Some of the spindles showed more complex branching than this, with several loose coils around the intrafusal fibres, but complete annulospiral windings were not observed. Spindles at this age were 0.56 + 0.05 m m in length. 83 days. At this stage several axons innervated the spindles which were now 1.1 + 0.02 m m in length. The thickest of the fibres which was presumably the pri-

56D

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67 D

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it Fig. 6. Drawings of the distribution of primary afferent fibres in the developing fetal sheep spinal cord. At each gestational age the section of cord which contained the greatest number of stained fibres has been selected for presentation. (A) At 56 d fibres have entered the spinal cord and penetrated as far as the intermediate zone with a few fibres projecting into the ventral horn. (B) By 67 d primary afferents converge in the intermediate zone (arrows) before they spread out and extend into the medial (M) and lateral (L) motor neuron pools. With advancing gestational age at 76 d (C) and 92 d (D) the number of primary afferents entering the spinal cord and following the trajectory described above increased. The density of innervation of both the medial and ventral motor neuron pools increases concomitantly. (A-D) Bar = 300/.L DH: Dorsal horn; VH: Ventral horn.

S. Rees et al. /Brain Research 642 (1994) 185-198

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Fig. 7. Photomicrographs of primary afferent fibres innervating motor neurons in the ventral horn of the fetal sheep spinal cord. (A) At 67 d fibres enter the ventral horn medially and then sweep laterally to innervate the motor neurons in the ventral region of the medial motor neuron pool. Fibres were also seen to innervate the ventral region of the lateral motor neuron pool. (B) Higher magnification of the lateral and medial motor neuron pools. Individual fibres innervate both the medial and lateral motor neurons separately (large arrows) and the fibres only became varicose once they have reached the vicinity of the motor neurons they are to innervate (small arrows). (C) At 76 d there are increasing numbers of fibres (large arrows) innervating the motor neuron pools, in this case, the lateral extent of the lateral motoneuron pool. (D) Higher magnification of neurons in the lateral motor neuron pool. The number of varicosities innervating a single motor neuron had increased (small arrows) when compared with earlier ages. (E) By 92 d a dense innervation of motor neurons in both the medial and lateral motor neuron pools were observed. (F) Higher magnification of the lateral motor neuron pool. The number of varicose fibres innervating the motor neurons had increased (small arrows). In both (E) and (F) axons (large arrows) had increased in diameter compared to earlier ages. A, C, E, Bar = 85/xm; B, D, F, Bar = 3 5 / z m . L: Lateral motor neuron pool; M: Medial motor neuron pool.

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mary ending, displayed extensive branching usually forming a loose network but it was also possible to detect a few well-formed annulospiral windings (Fig. 8B). The mean diameter of this fibre was 3.0 + 0.02 /xm. One spindle was also observed at a much more advanced stage of development (Fig. 8C). H e r e extensive annulospiral windings were observed on two intrafusal fibres. Short branches which extended from the main fibre frequently ended in a globular head (Fig. 8C). Thinner fibres, possibly secondary endings were observed to enter the spindle and run along the intrafusal fibre with a few branches forming a loose network. Fusimotor innervation was present at this age but the fibres were very fine and did not appear to have formed specialised endings (Fig. 10A).

lar to those illustrated in Fig. 10B for 118 d. Spindles were occasionally seen in tandem (Fig. 9B). On rare occasions 2 pairs of spindles in tandem were located adjacent to each other. The finer secondary fibres could now be distinguished more reliably from the thicker primary terminals. They were mainly arranged in a loose network (Fig. 9B).

101-107 days. The length of the spindle (3.05 + 0.03

110-118 days. The mean diameter of the primary axon was 5.4 + 0.2 /zm. The branches of the axon formed extensive annulospiral endings with small side branches extending from these coils to end in globular expansions (Fig. 9C) as seen earlier. The thinner, secondary endings, formed a loose network that appeared to wrap around several intrafusal fibres. Fusimotor innervation was present with well developed plate endings clearly visible (Fig. 10B). Spindles were 4.0 + 0.3/zm in length.

mm) and the extent of the innervation had increased considerably (Fig. 9A). The primary ending generally bifurcated within the spindle and these fibres then branched extensively forming several annulospiral turns. The mean diameter of the primary fibre was 4.4 + 0.2 /zm, prior to the bifurcation. Fusimotor innervation was now more pronounced with typically 1-3 fibres running along the edge of the intrafusal fibres before terminating in specialised plate endings [5] simi-

127-143 days. At this stage spindles (5.0 _+ 0.5 m m in length) were of a mature form (Fig. 9D). Distinctive, compact annulospiral endings of primary afferent fibres had formed around 2 of the intrafusal fibres by 127 d. The primary afferent fibre was 6.3 _+ 0.13 ~zm in diameter and divided into 2 or 3 branches before entering the spindle. The extent and complexity of the muscle spindle

83D

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Fig. 8. Photomicrographs of silver-stained sensory endings on muscle spindles from tibialis anterior (A) and triceps surae (B) and (C). At 67 d axons enter the spindle and divide into 2 branches. A fine network of fibres extends from the major branches with some side branches ending in globular heads (small arrows) (A). At 83 d axons are beginning to form annulospiral windings (B) although on a few occasions a more advanced annulospiral arrangement was observed (C). In both B and C annulospiral windings occur between the arrowheads. Globular heads are indicated by small arrows in (C) Bar = 50 ~m for A, B and C. P: Primary ending.

S. Rees et al. /Brain Research 642 (1994) 185-198

101 D

A

193

C

115D

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! Fig. 9. Photomicrographs of silver-stained sensory endings on muscle spindles from tibialis anterior and triceps surae at 101 d (A); spindles in tandem at 101 d (B); 115 d (C); 127 d (D). Note the development of the compact annulospiral windings of the primary afferent fibres as gestational age increases. The finer secondary fibres could be seen terminating in a loose network from 101 days onwards (large arrows). Fusimotor fibres can be observed running along the edge of the spindle (small arrow in A and C) In A - D annulospiral endings occur between the arrow heads (A) Bar = 10/zm, (B) Bar = 40/zm, (C) Bar = 20/zm, (D) Bar = 50/~m.

S. Rees et al. / Brain Research 642 (1994) 185-198

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Fig. 10. (A) and (B). Fusimotor innervation of muscle spindles from triceps surae at 83 d (A) and 118 d (B). At 83 d the fibres lacked a specialised ending (arrow). At 118 d the fusimotor fibres branched, terminating in well developed plate endings (arrows) Bar = 5 0 / ~ m (A and B). (C-E). Motor innervation of extrafusal fibres at 83 d (C); 101 d (D); 134 d (E). At 83 d a clearly defined endplate was lacking (arrows) but was evident from 101 d onwards (arrows). (F) Haematoxylin and eosin-stained transverse sections (10 ~ m ) of the soleus muscle at 126 d. Spindles usually occurred singly or in pairs. There were usually three or four but occasionally five intrafusal fibres within a spindle capsule. (C) Bar = 20 ~ m , (D) and (E) Bar = 3 3 / z m , (F) Bar = 60 p.m. EF: Extrafusal fibre; IF: Intrafusal fibre.

S. Rees et al. / Brain Research 642 (1994) 185-198

innervation at 67, 83, 101, 115 and 127 d can be appreciated in the camera lucida drawings of the spindles (Fig. 11) which have all been drawn at the same magnification.

3.2.3. Innervation of extrafusal muscle fibres Motor innervation of extrafusal fibres was evident at 67 d in the silver-stained preparations but a clearly defined endplate region did not appear to have developed. By 83 d (Fig. 10C) the motor fibre bundles had a characteristic appearance with a central branch giving off several smaller side branches perpendicular to the main branch. Distinctive end plate specialisations could be detected at 101 d (Fig. 10D). The end plate regions increased in size with increasing gestational age (134 d) (Fig. 10E) and motor fibres appeared to increase in diameter, although measurements were not made.

3.2.4. Light histology of soleus muscle Examination of transverse sections of the soleus muscle at 126 d revealed that spindles usually occurred singly or in pairs. On rare occasions up to four spindles were located adjacent to each other (Fig. 10F). There were usually three or four but occasionally five intrafusal fibres within a spindle capsule (Fig. 10F).

4. Discussion

In this study we have described the prenatal structural and functional development of muscle receptors and their afferent connections, from the earliest age at which activity can be evoked (75 d) to just prior to term when the response is seemingly mature (143 d). This report appears to be the first detailed description of the ontogeny of muscle receptor function during gestation. It has been reported briefly, that muscle afferents in the fetal rat discharge in response to a small change in limb position from embryonic day 17 onwards (term = 22 d) [10], but there has not been an extensive developmental study in the fetal rat. Muscle spindles in the sheep at 75 d are small (~ 0.5 mm in length) and their afferent fibres are just beginning to form the annulospiral windings, characteristic of the mature spindle. Since it was possible to evoke activity in spindle afferents with an applied stretch at this age, it appears that the annulospiral formation is not a necessary requirement for the generation of a response. The spindle, at this age however, does not appear to be capable of generating a consistent tonic response at resting muscle length, nor does it respond to stretch with a sustained train of discharges. Within two weeks of the onset of activity (that is by about 87 d) responses to stretch were easier to elicit. Receptors were beginning to develop a tonic discharge at resting muscle length and the stretch-evoked re-

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sponse displayed clear static and dynamic sensitivities with the static component predominating. Spindles were still relatively immature but annulospiral windings were beginning to form, and in a few spindles were well developed. These results, in general, support the findings of Patak et al. [22] who reported that afferent responses demonstrating several features of the adult response are present well before an annulospiral structure has developed. Contrary to their results, however, we found that static sensitivity, while low, consistently appeared earlier in development than did dynamic sensitivity. During reinnervation of muscle spindles following nerve regeneration it has been shown that here, too, both static and dynamic responses can be obtained from spindles lacking a completely reformed annulospiral ending [4]. Spontaneous and evoked, activity became more stable with increasing gestational age (up to 143 d). The frequency of the tonic discharge increased from 10 Hz at 87 d to 35 Hz at 127 d and both static and dynamic sensitivities became more pronounced. Parallel to this functional development was a progressive increase in the length and complexity of the spindle innervation~ There were however, no specific structural changes which could be correlated with particular aspects of functional development. By 127 d the spindle appeared mature with the primary afferent fibre ending in a compact annulospiral arrangement. Secondary endings were never observed to form the flower spray endings, described for other species [3], but always ended in loose axonal networks encircling the intrafusal fibres. Fusimotor innervation of intrafusal fibres was evident from 83 d although specialised endings did not form until about 101 d. For technical reasons we were unable to determine when fusimotor innervation first became functional and influenced the spindle response. There were however occasional fluctuations in the static spindle responses after about 107 d which could have been due to changes in background fusimotor activity. Conduction velocity of all muscle receptor afferents increased markedly during the last four weeks of gestation. This is not unexpected in a precocious animal like the sheep which needs to have well developed motor control immediately after birth. The only previous report of muscle spindle development in the sheep [8] suggested that very immature innervation of muscle spindles was present as early as 47 d in intercostal muscles. The illustrations of this innervation suggest that it is at a much earlier stage of development than we describe at 67 d for the hindlimb muscles. It is, in any case, to be expected that innervation of the intercostal muscles develops earlier than that of the hindlimb as there is a rostro-caudal gradient of development in the spinal cord. The biocytin injections in the DRG revealed afferent projections to the motoneuron pools by 67 d, which

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was just before muscle receptors had started to respond to stretch. The possibility therefore exists for even the earliest muscle receptor activity to influence the motoneurons, and presumably other target neurons in the spinal cord. Furthermore there was evidence of innervation of extrafusal muscle fibres by 67 d. Thus the neural pathways required for reflex activity involving muscle spindles are present from early in gestation. At present one can only speculate on the possible functional role of muscle spindle activity in utero but it is possible that activation of the receptors during gestation might be important for some aspect of neural development. In the developing mammalian visual system it has been clearly demonstrated that appropriate functional activity is a necessary requirement for normal development [11]. The requirements might be somewhat different, however, for the development of synaptic connections between afferent fibres and spinal neurons. It has been shown that patterned neuronal activity at least in birds, is not essential for the specification of pathways in the stretch reflex [9]. Rather, it has been suggested that pre- and post-synaptic neurons have a distinctive molecular phenotype at the time of synaptogenesis and that this forms the basis for the formation of the correct pattern of synaptic connections. Whether this also applies to mammals has yet to be determined. In any event neuronal activity could play an important role in the reinforcement of pathways once they have formed. In conclusion, we have shown that the timing of the structural and functional development of muscle receptors and their afferent connections is closely associated with both the innervation of extrafusal muscle fibres and the appearance of early fetal movements. Obviously, caution must be exercised in extrapolating these findings to other animals because the time at which a given function develops may vary relative to birth. However, it has been generally found that the sequence in which functions develop tends to be preserved across species. It is therefore possible that the close correspondence found here between the onset of activity in muscles and their receptors represents a general feature of motor system development.

5. Acknowledgements We are grateful for the excellent technical assistance of Mrs. Jane Ng and Ms. Sarah Spencer in the preparation, drawing and photography of the muscle

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spindles and of Mr. Jun Ling Liu in the initial electrophysiological experiments. We thank Dr. Uwe Proske for valuable comments during the course of this work, The project was supported by a grant from the National Health and Medical Research Council of Australia.

6. References [1] Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. [2] Barker, D and Ip, M.C., A silver method for demonstrating the innervation of mammalian muscle in teased preparations, J. Physiol., 69 (1963) 73P-74P. [3] Barker, D. and Milburn, A., Development and regeneration of mammalian muscle spindles, Sci. Prog. Oxf., 69 (1984) 45-64. [4] Barker, D., Scott, J.J.A. and Stacey, M.J., Reinnervation and recovery of cat muscle receptors after long term denervation, Exp. Neurol., 94 (1986) 184-202. [5] Barker, D., Stacey, M.J. and Adal, M.N., Fusimotor innervation in the cat, Phil. Trans. Roy. Soc. (Lond), 258 (1970) 315-346. [6] Browne, J.S., The response of de-efferented muscle spindle in sheep extraocular muscles to stretch and vibration, J. Physiol., 242 (1974) 60P. [7] De Vries, J.P.P., Visser, G.H.A. and Prechtl, H.F.F., The emergence of fetal behaviour I. Qualitative aspects, Early Human Dev., 7 (1982) 301-322. [8] Dickson, LM., The development of nerve-endings in the respiratory muscles of the sheep, J. Anat., 74 (1940) 268-278. [9] Frank, E. and Wenner, P., Environmental specification of neuronal connectivity, Neuron, 10 (1993) 779-785. [10] Fitzgerald, M., Spontaneous and evoked activity of fetal primary afferents in vivo, Nature, 326 (1987) 603-605. [11] Goodman, C. and Shatz, C., Developmental mechanisms that generate precise patterns of neuronal connectivity, Cell, Vol. 72, Neuron, Vol. 10, 1993, Suppl. 77-98. [12] Gregory, J.E. and Proske, U., Responses of muscle receptors in the kitten, J. Physiol., 366 (1985) 27-45. [13] Gregory, J.E. and Proske, U., The response of muscle spindles in the kitten to stretch and vibration, Exp. Brain Res., 73 (1988) 606-614. [14] Gregory, J.E. and Proske, U., Extrafusal and intrafusal motor units in the kitten, Int. J. Develop. Neurosci., 9 (1991) 101-109. [15] Harker, D.W., The structure and innervation of sheep superior rectus and levator palpebrae extraocular muscles. II. Muscle spindles, Invest. Opthalmol., 11 (1972) 970-979. [16] King, M.A., Louis, P.M., Hunter, B.E. and Walker, D.W., Biocytin: a versatile anterograde neuroanatomical tract-tracer alternative, Brain Res., 497 (1989) 361-367. [17] Loke, J.P., Harding, R. and Proske, U., Conduction velocity in peripheral nerve of fetal, newborn and adult sheep, Neurosci. Lett., 71 (1986) 317-322. [18] Maeda, N., Osawa, K., Masuda, T., Hakeda, Y. and Kumegawa, M., Postnatal development of annulospiral endings of Ia Fibres in muscle spindles of mice, Acta Anat., 124 (1985) 42-46. [19] Matthews, P.B.C., Mammalian muscle receptors and their central actions, Williams and Wilkins, Baltimore, 1972, pp. 140-194.

Fig. 11. Camera lucida drawings of the entire sensory innervation of the equatorial region of muscle spindles at 67 (A), 83 (B), 101 (C), 115 (D) and 127 d (E). These drawings were made to show the increase in the extent and complexity of the innervation with increasing gestational age. The solid lines represent the fibres on the upper surface of the intrafusal fibre and the unfilled lines, at the back of the fibres. The outlines of the individual intrafusal fibres have been omitted for clarity. Bar = 100/zm for all drawings.

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[20] Milburn, A., The early development of muscle spindles in the rat, J. Cell Sci., 12 (1973) 175-195. [21] Milburn, A., Stages in development of cat muscle spindles, J. Embryol. Exp. Morph. Anat., 82 (1984) 177-216. [22] Patak, A., Proske, U., Turner, H. and Gregory, J.E., Development of sensory innervation of muscle spindles in the kitten, Int. J. Develop. Neurosci., 10 (1992) 81-92. [23] Skoglund, S., The activity of muscle receptors in the kitten, Acta Physiol. Scand., 50 (1960) 203-221. [24] Snider, W.D., Zhang, L., Yusoof, S., Gorukanti, N. and Tsering, C., Interactions between dorsal root axons and their target

motor neurons in developing mammalian spinal cord, J. Neurosci., 12 (1992) 3497-3508. [25] Sutton, A.C., On the development of the neuro-muscular spindle in the extrinsic eye muscles of the pig, Am. J. Anat., 18 (1915) 117-145. [26] Zelena, J., The morphogenetic influence of innervation on the ontogenetic development of muscle spindles, J. Embryol. Exp. Morphol., 5 (1957) 283-292. [27] Zelena, J., The role of sensory innervation in the development of mechanoreceptors, Prog. Brain Res., 43 (1976) 59-64.