Talk:Paper - The development of nerve-endings in the respiratory muscles of the sheep (1940)

From Embryology



By LILIAN M. DICKSON Department of Anatomy, London School of Medicine for Women

‘Tue recent work of Barcroft et al. (1936), and of Barcroft & Barron (1936, 1937) on the genesis of somatic movement in sheep foetuses included a study of the development of respiratory movement, and by the kindness of Sir Joseph Barcroft the histological examination of respiratory structures was entrusted to the author. The present paper deals with the condition of nerves and nerve-endings in the intercostal muscles and diaphragm of some of the subjects of the Cambridge experiments, and attention has been specially directed to the development of muscle spindles and motor end-plates.

The first object of the investigation was to show what peripheral nervous structures are present in respiratory muscles when respiratory movement is developing, and if possible to throw some light on their function. But the examination of a series of foetal material has also made possible a study of the course of development of muscle spindles and motor end-plates, and has established for one vertebrate species within fairly narrow limits the age at which these structures begin to form.


The material used was provided by Sir Joseph Barcroft from foetuses whose movements had previously been studied by himself and Dr Barron. It consists of the respiratory muscles of twelve sheep foetuses of ages ranging from 40 to 139 days of gestation.

The tissues of nine of the twelve foetuses were fixed in 7% formol and impregnated by Hewer’s (1988) modification of Ranson’s silver method. In addition, Bodian’s (1986) silver method was used for some of the material from two of the nine foetuses. Paraffin sections were cut serially at 7-5 and 10 yp.

The Ranson sections for the most part show good impregnation; Bodian’s method reveals very clearly the cross-striation of the muscle fibres and the axis cylinders but has not picked out nerve-endings, probably because alcohol fixation, which Bodian advises, was not possible in this case. Only those pieces of material which showed good preservation and impregnation by silver were used; and for each foetus at least 100 sections each of diaphragm and intercostal muscle were examined, and in most cases more than double that number. Hence, while recognizing the erratic character of silver impregnation, it is felt that a fair measure of reliance can be placed on negative as well as positive findings in respect of nerve-endings in these specimens. Nerve-endings in the respiratory muscles of the sheep 269

The remaining three foetuses were prepared in the Cambridge laboratories. They were stained by de Castro’s silver method and show excellent impregnation.

Table I. Number and age of specimens. (Gestation period of sheep =157 days)

Age (days) Method of silver impregnation 40 (foetal) de Castro 45


— i


47 Ranson (Hewer’s modification)

58 de Castro

66 Ranson (Hewer’s modification)

80 ” 2” .

84 9 ”

” and Bodian 96 ” ”

100 ” ” and Bodian

111 ” ”

137 ” ”


The muscle fibre (a) Intercostal muscles.

In foetuses 1-3 (40-47 days), muscle fibres are very slender, faint crossstriation is visible, and centrally placed nuclei form swellings on the course of the fibres. Fibrils are faintly visible.

In foetus 5 (66 days) the short oval nuclei just fill the width of the fibre.

In foetus 6 (80 days) the nuclei begin to take up a lateral position and cross-striation is well defined.

In foetus 12 (187 days) the muscle fibre has all the adult characters.

(b) Diaphragm.

In foetuses 1-5, the muscle fibres appear slightly less mature than those of the intercostal muscles of the same foetuses; but in the 80-day and older specimens there is no noticeable difference between the fibres in the two groups of muscles.

Nerve fibres and nerve-endings

(a) In intercostal muscles.

Undifferentiated endings. In foetus 1 (40 days) nerve fibres are plentiful, and many terminate in contact with muscle fibres, but no differentiated endings are present. It seems reasonable to presume that some of these endings are motor, although it is not possible to assign a function with any certainty (Pl. IT, fig. 1).

Sensory endings. In foetuses 2 and 8 (45 and 47 days) formations suggesting very early spindles are present. Since a good photograph of these structures was unobtainable, a camera lucida drawing of one of them, found in the 45-day specimen, is given (Text-fig. 1). 270 - Inhan M. Dickson

Fine branches of a nerve fibre partially enwrap one or more muscle fibres, forming a very simple network. The structure of the muscle fibre was not clearly made out. There is no fusiform enlargement, but the whole fibre is thicker than those in the vicinity, and it may be that more than one is enclosed within the nervous network. There is no surrounding connective tissue reaction.

In foetus 4 (58 days), endings were found similar to those just described but more clearly defined, and a sensory function can be assigned to them with some confidence. One is photographed (Pl. I, fig. 1); as in the younger . specimen, there is no fusiform enlargement of the muscle fibres, no surrounding capsule and no lymphatic space.

In foetus 5 (66 days) a clearly defined spindle (Pl. I, fig. 2) is present. It lies within a lymphatic space and consists of a coarse nervous network enclosing several fine muscle fibres. It is not yet “spindle” shaped. In all

Text-fig. 1. Camera lucida drawing of an early sensory formation, probably a muscle spindle, in ‘the intercostal muscle of a 45-day sheep foetus. x 380. A, muscle fibre. B, sensory formation (the thickness of the component branches is slightly exaggerated).

the older foetuses, spindles were found, and by mid-term they are well formed (Pl. I, fig. 3). In the 90-day specimen spindles are long, fusiform, and highly complex in structure. Free interfascicular endings were not found in abundance but are certainly present at 80 days. One is seen in Pl. II, fig. 2. In the absence of any neighbouring blood vessel, its sensory rather than autonomic character may be safely assumed.

Motor endings. As stated above, at 40 days many nerve fibres end in contact with muscle fibres but they do not differ from one another, and so it is impossible to assign to them their different functions.

With the beginning of spindle differentiation at 45 days, it can safely be assumed that many of the still undifferentiated endings are motor, but they undergo surprisingly little change even up to 100 days. The fibre may end singly, or by simple branching, or in a spray, and there are tiny beads at each terminal twig. In the older specimens (80-100 days), the fibres thicken, the Nerve-endings in the respiratory muscles of the sheep 271

beads slightly enlarge and the fibre tends to approach the muscle fibre at right angles to its long axis. This mode of approach is perhaps the most characteristic feature of the motor fibres.

In the 111-day foetus the motor ending is better defined. The terminal “bead” takes the form of an oval plate in which a network is apparent. In one specimen observed, the axis cylinder appeared to branch into two just before reaching the muscle fibre and both branches ended in oval swellings on the same muscle fibre. This seems to be the stage just before true ‘“‘motor

plate” formation. There is as yet no sign of nuclear aggregation into a sole plate (Pl. II, fig. 3).

Text-fig. 2. Camera lucida drawing of a young motor end-plate in the intercostal muscle of a 137-day sheep foetus. x 380. (A microphotograph of the same structure appears on PI. II, fig. 4.) A, muscle fibre with B, sarcolemma torn apparently near junction with neurilemma. C, nerve fibre. D, motor end-plate. E, sole plate.

In the 137-day foetus a true motor plate is present and is figured (Pl. II, fig. 4 and Text-fig. 2). It is far from fully differentiated, but the “sole” has formed and the ending is clearly hypolemmal in position.

In the younger specimens, the relation of the ending to the sarcolemma could not be established with certainty.

(b) In diaphragm.

Sensory and motor endings. In the younger specimens development runs much the same course as in the intercostal muscles with perhaps a slight lag. Foetus 6 (80 days) was the youngest in which a spindle was found but it is difficult to believe that they are not present earlier. At 84 days, fairly complex spindles are present. A 90-day specimen has been photographed (PI. I, fig. 4). As in the intercostal muscles, a true motor end-plate is seen only in foetus 12 (187 days) and is then still in an early stage of development. 272 Tilian M. Dickson

Table II. Motor and sensory nerve-endings in intercostal muscles and diaphragm of foetal sheep (gestation period =157 days)

e Intercostal muscle Foetus in c A ~ Diaphragm no. days Sensory Motor sensory and motor

1 40 Nerve fibres in contact with muscle fibres. Endings Muscle not fully formed not differentiated No endings observed

2 at Formations suggesting Simple beaded endings, Motor as in intercostal

3 47 early spindle not clearly differentiated No sensory formations


4 58 Better defined “‘pre- No material spindle”

5 66 Simple, but definite Endings numerous, single, Motor as in intercostal, spindle spray-like, or show no spindles observed

simple branching 6 80 Free interfascicular end- Fibres tend to approach Well formed spindle

ing formed muscle fibres at right | angles to long axis of muscle 7 84 Well-formed spindle * As in intercostal 8 92 Complex spindle ” ” 9 96 ” ” ” 10 100 ” ” ” ll lll ” Fibres now end in oval ” swellings of plexiform structure 12 137 ” True end-plate, of simple ” form, with well-defined sole plate

The results may be briefly summarized as follows:

Appearances suggestive of early spindle formation are present at 45-47 days; spindles are well defined, though simple at 66 days and well formed by 84 days. Motor end-plates are not present at 111 days; they are present in simple form with true sole plate at 137 days.


(1) The present findings compared with the results of other workers

The finding of well-developed muscle spindles at an age at which the earliest differentiation of motor nerve-endings is hardly discernible is in accordance with the results of other workers in this field. Tello (1917, 1922) finds well-marked muscle spindles in the cat embryo at 30 days and in the human foetus at 6 months, whereas in the 30-day kitten motor end-plates have not begun to form and in the human foetus at term they are still incomplete, although in some muscles, for example the tongue, they approach completion. Hewer (1935), working on human material, finds ‘fairly complete” spindles at 20 (foetal) weeks when the motor nerve-endings are just beginning to differentiate, and makes the further observation that in the human foetus at term the motor nerve-endings in the tongue, diaphragm and intercostal muscles are fully formed, although they are still immature in the Nerve-endings in the respiratory muscles of the sheep 273

muscles of the limbs. Hewer has also examined the neck muscles of some of the foetuses used for the present investigation and obtained the following results which she allows to be quoted, namely that at 80 days primitive spindles are present, but at 187 days there is still no sign of developing motor end-plates. Table III shows the comparative results.

Table III. Age of appearance of nerve-endings in different vertebrate species

Foetal Gestation age period Animal Region days Results days A. Muscle spindles Sheep Respiratory muscles (author) 45 Suggestive sensory formation 157 .” % 66 Definite spindle 2 ” 80 Well-formed spindle Neck muscles (Hewer) 80 Primitive spindle Chick ? (Tello) 14 Well-formed spindle 21 Cat Foot muscles (Hewer) 30 ” ” 60 Human Limb muscles (Hewer) 140 9 280 Leg muscles (Tello) 168 Very complex spindle B. Motor endings Sheep Respiratory muscles (author) 137 Simple motor end-plate with 157 sole plate Neck muscles (Hewer) 137 Not begun to form 157 Human Tongue (Hewer) 200 Motor plates present but un- 280 finished ”» ” 280 Highly differentiated 280 » (Tello) 280 Fairly well-developed with 280 sole plate Intercostals (Hewer) 280 Highly differentiated 280 i (Tello) 280 Slightly less than tongue 280 Forearm (Hewer) 200 Just beginning to differentiate 280 Leg (Hewer) 280 Not nearly completed 280 Leg (M. soleus) (Tello) 280 Beginning 280

These comparisons bring out two points of interest.

(1) In the species examined muscle spindles are present and generally well formed by mid-term, whereas motor end-plates only begin to differentiate after mid-term.

(2) In the sheep at least, both muscle spindles and motor end-plates develop earlier in intercostal and lingual muscles than in the muscles of the limbs, and the early development of motor end-plates in the human tongue and intercostal muscles suggests that a similar age difference may exist in other species. Whether this difference has a functional significance cannot at present be discussed for lack of sufficient data.

(2) Relation of the age of appearance of nerve-endings to length of gestation period and the maturity of the species at term

In comparing the age of development of nerve-endings in different species it is convenient to correlate the histological findings with the length of the gestation period, but in using these results to assess the probable time of appearance of nerve-endings in another species, it may be necessary to take 274 Inlian M. Dickson

into account a further factor, namely the degree of maturity attained by the animal at term. Nerve-endings which are present at mid-term in the sheep, which is post-mature at birth (as compared with the human baby), might well not appear until much later in the rat, for example, which is very immature at birth. In fact, if the findings recorded above in respect of sheep are representative of a wide range of animals, one would not expect to find completed motor end-plates in the rat until well after birth. It is hoped to obtain exact evidence on this point shortly.

The matter is of some interest in view of the vast body of contemporary experimental work, some of it on foetal material, dealing with the release of transmittor substances at the neuro-muscular junction. The presence or absence of a sole plate, amongst other histological factors, might well affect chemical transmission.


Movement Jerky—>sustained —————-—rigidity——— inhibited

Respiration gasping——-~>continuous——~ inhibited Age in days 40 45 50 55 60 65 70 75 80 85 Condition of Nerve fibre Suggestive Well- Definite Wellnerves and in contact pre- defined simple formed nerve-endings with spindle” “pre- spindle spindle in respiratory muscle formation spindle” muscles fibre. (45) (58)

No endings No motor end-plates. Motor fibres end simply or in simple

differentiated branching or sprays with small terminal beads

Text-fig. 3. This record of observed movements was made by Dr D. H. Barron, and is reproduced by his permission. The histological findings were added by the author.

(8) Relation of muscle-spindles and motor end-plates to respiratory movements

The results of Barcroft & Barron’s extensive studies of the genesis of somatic movement in sheep foetuses have been published (1936), 1987), and the reader is referred to those papers. One set of observations will be considered here. They are summarized in the accompanying chart (Text-fig. 3) Nerve-endings in the respiratory muscles of the sheep 275

made by Dr D. H. Barron, and published here by his permission, with the addition of the author’s histological findings. Barcroft & Barron found that movement at different ages shows certain well-defined characters. At first jerky, it gradually becomes sustained, and in older specimens the phenomena, first of rigidity and later of inhibition, are observed. Sustained movement is established by the 48th day. Further, it was found that these changes in muscular behaviour are associated with the forward extension of pathways and the development of inhibition centres in the brain stem (diagram, Textfig. 8). These observations refer to movements as a whole, including those of the trunk, head and limbs. Coming to the special case of respiratory movements the further observations were made:

(1) The earliest involvement of the respiratory musculature was observed in a 88-day foetus and was part of a mass movement of the body. The separation of respiratory from general body movement develops gradually between the 38th and 49th days by the dropping out of the movements of the head and limbs.

(2) The respiratory movement is a rhythmical one. The rhythm is at first jerky and of short duration; it gradually becomes more sustained; and by the 50th day respiratory movements are almost continuous and are of the sustained or tonic type (1987).

(8) The development of a tonic element in the rythmic movement depends on the development of central inhibitory mechanisms (1937).

Turning to the histological findings, we find that:

(1) At 40 days, ie. 2 days after the earliest exhibition of respiratory movement, no specialized nerve-endings are present.

(2) By 50 days, when the change from “‘jerk” to sustained movement is fairly well established, a specialized sensory ending, the “‘pre-spindle” of 45 days, has made its appearance.

(3) The establishment of sustained movement and the manifestation of inhibition take place long before the earliest development of a motor end-plate.

Rhythmic respiratory discharges from the central nervous system depend upon the excitability of the brain which is heightened by the arrival of sensory stimuli—indeed it is an open question whether without such sensory stimuli the excitability would not be below the linal level. One of the obvious sensory avenues is from the nerve endings in the respiratory muscles themselves. The histological findings show that undifferentiated endings, possibly in contact with the muscle fibre, are able to subserve jerky rhythm. Whether for sustained rhythm a more complex type of receptor. organ is essential cannot be said with certainty from the available data, but-it is at least suggestive that the beginning of spindle formation and the’ development: of sustained movement should so closely coincide.

Anatomy LXXxIV 18 276 Iihan M. Dickson


1. The muscle fibre and nerve-endings in the intercostal muscles and diaphragm of 12 sheep foetuses are described.

2. The formation of the muscle spindle is found to begin at about the 45th day of gestation and to be well advanced by mid-term (about 80 days).

8. Motor end-plates are not found at 111 days and are present in simple form at 187 days.

4. The findings (i) are compared with those of other workers in this field, and (ii) are considered in relation to observed movements.


I wish to express my gratitude to Sir Joseph Barcroft for providing the material on which my results are based.

My thanks are also due to Dr D. H. Barron for allowing me to use his diagram and chart, to Dr E. E. Hewer for communicating her unpublished findings, and to Prof. M. F. Lucas Keene for her helpful criticism and advice at many stages of the work.

The micro-photographs were made by Mr Willmott of the British Postgraduate School.


Barcrort, J. & Barron, D. H. (1936). J. Physiol. 88, 56.

(1937). J. Physiol. 91, 329.

Barcrort, J., Barron, D. H. & WinbLE, W. F. (1936). J. Physiol. 87, 73. Bopian, D. (1936). Anat. Rec. p. 89.

Hewer, E. E. (1933). J. Anat., Lond., 67, 350.

—— (1935). J. Anat., Lond., 69, 369.

TELLO, J. F. (1917). Trab. Lab. Invest. Biol. Univ. Madrid, 15, fasc. 2. —— (1922). Z. ges. Anat. 1. Z. Anat. EntwGesch. 64, 348.


PriatE I Figs. 1-4. Microphotographs of developing muscle spindles in the respiratory muscles of sheep foetuses. Fig. 1. 58-day foetus, intercostal muscle. x 750. Fig. 2. 66-day foetus, intercostal muscle. x 450. Fig. 3. 84-day foetus, intercostal muscle. x 450. Fig. 4. 90-day foetus, diaphragm. x 300.

Puate II Figs. 1-4. Microphotographs of developing nerve-endings in intercostal muscles of sheep foetuses. Fig. 1. Undifferentiated nerve-ending in a 45-day foetus. x 335. Fig. 2. Free interfascicular sensory ending in an 80-day foetus. x 335. Fig. 3. Motor ending in a 111-day foetus. x 750. Fig. 4. Young motor end-plate with sole plate (note direction of arrow) in a 137-day foetus. x 450. Journal of Anatomy, Vol. LX XIV, Part 2 Plate I

. Fig. 4.


Fig. 3.