From the Laboratory of NVeuroanatomical Sciences
J. Physiol. (1963), 169, pp. 707-712 With 1 plate and 2 text-figures Printed in Great Britain
707
THE DEVELOPING MOTOR END-PLATE: PHARMACOLOGICAL STUDIES IN THE CHICK EMBRYO
BY D. B. DRACHMAN From the Laboratory of NVeuroanatomical Sciences, National Institute of Neurological Diseases and Blindness, National Institutes of Health, U.S. Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland, U.S.A.
(Received 11 February 1963)
Spontaneous contractions of skeletal muscle can be observed as early as the 4th day of incubation in the embryo of the domestic fowl. It is not yet known whether they arise as the result of impulses initiated in the muscle membrane or transmitted from motor nerves. Nevertheless, from their inception such movements may be blocked by curare (Kuo, 1939) or curare-like agents. These drugs are thought to act at the level of the receptor substance of the neuromuscular junction; it is therefore of interest to examine their pharmacological effects during a period featured by significant changes in the morphology of the junction. In the present study the minimum paralytic dosages (MPDs) of curare, succinylcholine and decamethonium were determined in chick embryos from 7 to 15 days of incubation age. Histochemical staining demonstrated the profound structural changes in the neuromuscular junction which take place within the same period. METHODS Each egg was 'candled' in order to determine the position of the embryo and of a large overlying chorio-allantoic vein. The air sac was punctured, and a relatively large window was made in the shell and shell membrane so as to afford an optimum view of the embryo. The egg was placed in a warmed chamber under a dissecting microscope, brightly illuminated by an adjustable lamp. The chorio-allantoic membrane was kept moist with unbuffered isotonic salt solution. A polyethylene microcatheter was inserted into a large chorio-allantoic vein, according to the method previously described by Drachman & Coulombre (1962). Drug voluimes of 2-5-10 ,lI. were delivered by a micrometer-driven Becton-Dickinson 0-25 ml. syringe, sealed with high-vacuum grease. This technique of intravenous injection has the advantages of permitting accurate dosage directly into the extra-embryonic circulation, and at the same time of presenting a clear view of the undisturbed embryo. The drugs used in these experiments included tubocurarine chloride (Squibb), succinylcholine chloride, and decamethoniulm bromide (Burroughs Weilcome), diluted with isotonic saline solution. After the injection was complete a 5 mini period was allowed to elapse for the drug to take full effect. The embryo was then observed directly through the dissecting microscope for a 5 min test period, during which all discrete muscular movements seen were recorded on a 45 Physiol. 169 Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
708
D. B. DRACHMAN
manual counter. A 'discrete muscular movement' was defined arbitrarily as a single unidirectional displacement of any part of the body, regardless of its amplitude. Skeletal muscle movements were readily distinguished from the slower, swinging contractions of the smooth-muscle component of the amnion. Although this method of quantitating movement relies on subjective observations, the results were consistently reproducible, even when recorded by different observers. The earliest movements can be detected during the 4th day of incubation, and consist of alternate flexion and extension of the neck and trunk. By the 5th or 6th day clear-cut movements of the extremities are present. At first these movements occur in brief bursts separated by 1 min or more of inactivity. The contractions increase in frequency and vigour, and the quiescent periods become correspondingly less prominent, until the end of the second week. The MPDs for curare, succinylcholine and decamethonium were determined for 7-, 9-, 12-, and 15-day embryos by the following procedure: successive (usually two-fold) increments of the drug were administered to embryos of each age until the smallest dose which would eliminate spontaneous movement was found. It was considered that spontaneous movements were eliminated when a group of five embryos showed an average of fewer than 2 movements/5 min observation period. Another group of five embryos which received half the MPD continued to show active spontaneous movement. Throughout these experiments only one dose was given to each chick embryo. In order to correct for the embryo's rapid increase in weight with advancing age, the ratio MPD :g was calculated by dividing the MPD at a given age by the average wet weight of chick embryos of the same age. To obtain the figure for wet weights ten untreated embryos at each age were removed from the egg with the extra-embryonic vessels severed at the body wall, quickly blotted on paper towelling, and weighed on a Sartorius balance. Motor end-plates were stained for cholinesterase according to the modification of the thioacetic acid technique described by Mumenthaler & Engel (1961).
RESULTS
In the chick embryo the MPD: g for curare, succinylcholine and decamethonium remains essentially constant from the 7th to the 15th day of incubation. Although the uncorrected MPD rises sharply with advancing embryonic age (broken lines in Text-fig. 1), this correlates with the increase in weight of the embryo during development. The solid lines in Text-fig. 1 represent the weight-corrected term, MPDD g, and are essentially straight and parallel to the X-axis. Indeed, the highest MPDJ g differs from the lowest by a factor of only 1-8 for curare, 1-6 for succinylcholine, and 2-4 for decamethonium, which is within the error of the experimental method. The effect of progressively increasing doses of curare on the frequency of spontaneous movement in the 9-day-old chick embryo is illustrated in Text-fig. 2. It is clear that the number of discrete movements declines as the dose is increased. Essentially complete paralysis occurs at the MPD. Six-, 12-, and 15-day embryos gave dose-response curves of similar form with curare. In our histological material the earliest cholinesterase-containing endplates were seen in the paraspinal muscles at 10 days of incubation. They were well developed in this region by 12 days. In the distal muscles of the
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
THE EMBRYONIC NEUROMUSCULAR JUNCTION 709 leg end-plates were first seen at 12-13 days (P1. 1), and were well developed by 14 days. The arrows in Text-fig. 1 bracket the period during which cholinesterase-containing motor end-plates make their appearance in the chick a
3*0 _
/
b
/ 250 2e5 2.0~~~~~~~~~~~~*~
/
2~0
~~~~05-
~~~1~~~5 os/
0.5
J
-~~~~~~~
7
T.5
0
/ 12
9
912
7
15
15
Age (days)
Age (days) C
20
10 /. 0.5/ 0
7
T 9
I
I
r 12
_I15 I
Age (days)
Text-fig. 1. MPD (O --- 0) (a) curare, (b) succinylcholine, (c) decamethonium, in chick embryos from 7 to 15 days of incubation. The weight-corrected ratio MPD: g (@-@) is essentially constant in each graph. Arrows indicate the period during which cholinesterase-containing motor end-plates first appear. 45-2 Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
D. B. DRACHMAN embryo. It is evident that the age range encompassed by the pharmacological experiments extends over the periods before, during, and after cholinesterase becomes demonstrable at the neuromuscular junction. 710
160 140
120 E
100 _
t
E
\
0
,4,
0>60-
C20_ _ 40200 0
1
2
3 4 6 5 Dose of curare (#g)
7
8
Text-fig. 2. Counts of spontaneous movements observed in 9 day chick embryos treated with curare; note the progressive decline in number of movements with increasing dosage. The MPD is seen to be 755,g. Each point represents the mean of 5-10 observations, except for the untreated group which consists of the mean of the control counts made on all chicks which were subsequently treated. S.E. of mean indicated for each group. DISCUSSION
The time at which the earliest anatomical connexion between nerve and muscle occurs in the chick embryo is not yet settled. With silver-impregnation techniques anatomists have found evidence of neuromuscular contact as early as the 4th day of incubation (Visintini & Levi-Montalcini, 1939), or failed to find it until the 7th day (Tello, 1917; Mumenthaler & Engel, 1961). With histochemical methods cholinesterase appears diffusely distributed throughout the myoblasts as early as the 3rd-6th days, and thereafter gradually recedes toward the presumptive end-plate and myotendinous junction regions (Begliomini & Moriconi, 1959; Gerebtzoff, 1959; Mumenthaler & Engel, 1961). In our material the earliest discrete end-plates containing cholinesterase were found on the 10th day in the paraspinal musculature. In the distal muscles of the leg they first appeared on the 12th-13th day, and were well developed by the 14th day. Localized cholinesterase activity represents an early response of the embryo's muscle membrane to innervation (Zelena, 1962). Its appearance was therefore used as a guide to the timing of our pharmacological experiments. Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
THE EMBRYONIC NEUROMUSCULAR JUNCTION 711 In the present study we have made quantitative determinations of the sensitivity of the chick embryo to curare, succinyicholine and decamethonium at successive stages of development. These three pharmacological agents are thought to act at the receptor substance of the muscle membrane, although the precise mechanisms of their actions differ (Paton, 1956; del Castillo & Katz, 1957; Taylor, 1959; Zaimis, 1959). A sufficiently wide range of ages was used to include the period during which the most striking alterations in end-plate structure could be demonstrated with cholinesterase staining. The present findings show that the response of receptor substance to pharmacological blocking agents is not altered qualitatively or quantitatively during a period of profound change in the localization of end-plate cholinesterase. This tends to support Miledi's (1962) contention based on observations in normal and denervated muscle that receptors and cholinesterase are separate in location and behaviour. SUMMARY
1. The minimum paralytic dosages (MPDs) of curare, succinylcholine and decamethonium have been determined for chick embryos of 7-15 days of incubation age. After a correction was made for the increase in embryonic weight with advancing age, it was found that the ratio MPD:g for each drug remained constant throughout the ages examined. 2. Histochemical staining revealed that cholinesterase-containing motor end-plates may first be seen between 10 and 13 days of incubation age. 3. The precise site and mechanism of action of curare, succinyleholine and decamethonium have not been established in the embryo, but it is likely that they act at the level of receptor substance. The experimental results suggest that the sensitivity of receptor substance to these drugs remains constant in the chick embryo, while profound changes are taking place in the morphology of the neuromuscular junction. The author acknowledges with gratitude the encouragement and helpful suggestions of Dr A. J. Coulombre, who also participated directly in the early experiments involved in this study. Miss Sonia Steinberg provided technical assistance. REFERENCES
BEGLIOMINI, A. & MoRIcoNI, A. (1959) Sull'Epoca Della Localizzazione Della Colinesterasi Nel Musculo Striato Dell' Embrione Di Pollo. Rev. Biol. 51, 517-531. DEL CASTILO, J. & KATZ, B. (1957). A study of curare action with an electrical micromethod. Proc. Roy. Soc. B, 146, 339-356. DRACHMAN, D. B. & COULOMBRE, A. J. (1962). Method for continuous infusion of fluids into the chorioallantoic circulation of the chick embryo. Science, 138, 144-145. GEREBTZOFF, M. A. (1959). Cholinesterases. pp. 40-43. New York: Pergamon Press.
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
712
D. B. DRACHMAN
Kuo, Z. Y. (1939). Studies in the physiology of the embryonic nervous system. I. Effect of curare on motor activity of the chick embryo. J. exp. Zool. 82, 371-396. MILEDI, R. (1962). Induction of Receptors. In Ciba Foundation Symposium on enzymes and drug action. Ed. MONGAR, J. L. & DE REUCK, A. V. S. pp. 220-238. London: Churchill. MUMENTHALER, M. & ENGEL, W. K. (1961). Cytological localization of cholinesterase in developing chick embryo skeletal muscle. Acta anat. 47, 274-299. PATON, W. D. M. (1956). Mode of action of neuromuscular blocking agents. Brit. J. Anaesth. 28, 470-480. TAYLOR, D. B. (1959). The mechanism of action of muscle relaxants and their antagonists. Anesthesiol. 20, 439-452. TELLO, J. F. (1917). G6nesis de las Terminaciones Nerviosas Motrices v Sensitivas. Trab. Inst. Cajal Invest. biol. 15, 101-199. VIsINTINI, F. & LEvI-MONTALCINI, R. (1939). Relazione tra Differenziazione Strutturale e Funcionale dei Centri e Delle Vie Nervose nell'embrione di Pollo. Schweiz. Arch. Neurol. Psychi at. 43, 381-393, 44, 119-150. ZAIMIS, E. (1959). Mechanisms of neuromuscular blockade. In Curare and Curare-Like Agents. Ed. BOVET, D., BOVET-NI=T, F. & MARINI-BETTOLO, G. B. New York: Elsevier. ZELENA, J. (1962). The Effect of Denervation on Muscle Development. In The Denervated Muscle. Ed. GUTMANN, E. Chap. III. Prague: Czech. Acad. Sci. EXPLANATION OF PLATE Group of motor end-plates in the calf muscles of a 13-day chick embryo; stained for cholinesterase; x 390.
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The Journal of Physiology, Vol. 169, No. 4
Plate I
D. B. DRACHMAN
(Facing p. 712)
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
707
THE DEVELOPING MOTOR END-PLATE: PHARMACOLOGICAL STUDIES IN THE CHICK EMBRYO
BY D. B. DRACHMAN From the Laboratory of NVeuroanatomical Sciences, National Institute of Neurological Diseases and Blindness, National Institutes of Health, U.S. Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland, U.S.A.
(Received 11 February 1963)
Spontaneous contractions of skeletal muscle can be observed as early as the 4th day of incubation in the embryo of the domestic fowl. It is not yet known whether they arise as the result of impulses initiated in the muscle membrane or transmitted from motor nerves. Nevertheless, from their inception such movements may be blocked by curare (Kuo, 1939) or curare-like agents. These drugs are thought to act at the level of the receptor substance of the neuromuscular junction; it is therefore of interest to examine their pharmacological effects during a period featured by significant changes in the morphology of the junction. In the present study the minimum paralytic dosages (MPDs) of curare, succinylcholine and decamethonium were determined in chick embryos from 7 to 15 days of incubation age. Histochemical staining demonstrated the profound structural changes in the neuromuscular junction which take place within the same period. METHODS Each egg was 'candled' in order to determine the position of the embryo and of a large overlying chorio-allantoic vein. The air sac was punctured, and a relatively large window was made in the shell and shell membrane so as to afford an optimum view of the embryo. The egg was placed in a warmed chamber under a dissecting microscope, brightly illuminated by an adjustable lamp. The chorio-allantoic membrane was kept moist with unbuffered isotonic salt solution. A polyethylene microcatheter was inserted into a large chorio-allantoic vein, according to the method previously described by Drachman & Coulombre (1962). Drug voluimes of 2-5-10 ,lI. were delivered by a micrometer-driven Becton-Dickinson 0-25 ml. syringe, sealed with high-vacuum grease. This technique of intravenous injection has the advantages of permitting accurate dosage directly into the extra-embryonic circulation, and at the same time of presenting a clear view of the undisturbed embryo. The drugs used in these experiments included tubocurarine chloride (Squibb), succinylcholine chloride, and decamethoniulm bromide (Burroughs Weilcome), diluted with isotonic saline solution. After the injection was complete a 5 mini period was allowed to elapse for the drug to take full effect. The embryo was then observed directly through the dissecting microscope for a 5 min test period, during which all discrete muscular movements seen were recorded on a 45 Physiol. 169 Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
708
D. B. DRACHMAN
manual counter. A 'discrete muscular movement' was defined arbitrarily as a single unidirectional displacement of any part of the body, regardless of its amplitude. Skeletal muscle movements were readily distinguished from the slower, swinging contractions of the smooth-muscle component of the amnion. Although this method of quantitating movement relies on subjective observations, the results were consistently reproducible, even when recorded by different observers. The earliest movements can be detected during the 4th day of incubation, and consist of alternate flexion and extension of the neck and trunk. By the 5th or 6th day clear-cut movements of the extremities are present. At first these movements occur in brief bursts separated by 1 min or more of inactivity. The contractions increase in frequency and vigour, and the quiescent periods become correspondingly less prominent, until the end of the second week. The MPDs for curare, succinylcholine and decamethonium were determined for 7-, 9-, 12-, and 15-day embryos by the following procedure: successive (usually two-fold) increments of the drug were administered to embryos of each age until the smallest dose which would eliminate spontaneous movement was found. It was considered that spontaneous movements were eliminated when a group of five embryos showed an average of fewer than 2 movements/5 min observation period. Another group of five embryos which received half the MPD continued to show active spontaneous movement. Throughout these experiments only one dose was given to each chick embryo. In order to correct for the embryo's rapid increase in weight with advancing age, the ratio MPD :g was calculated by dividing the MPD at a given age by the average wet weight of chick embryos of the same age. To obtain the figure for wet weights ten untreated embryos at each age were removed from the egg with the extra-embryonic vessels severed at the body wall, quickly blotted on paper towelling, and weighed on a Sartorius balance. Motor end-plates were stained for cholinesterase according to the modification of the thioacetic acid technique described by Mumenthaler & Engel (1961).
RESULTS
In the chick embryo the MPD: g for curare, succinylcholine and decamethonium remains essentially constant from the 7th to the 15th day of incubation. Although the uncorrected MPD rises sharply with advancing embryonic age (broken lines in Text-fig. 1), this correlates with the increase in weight of the embryo during development. The solid lines in Text-fig. 1 represent the weight-corrected term, MPDD g, and are essentially straight and parallel to the X-axis. Indeed, the highest MPDJ g differs from the lowest by a factor of only 1-8 for curare, 1-6 for succinylcholine, and 2-4 for decamethonium, which is within the error of the experimental method. The effect of progressively increasing doses of curare on the frequency of spontaneous movement in the 9-day-old chick embryo is illustrated in Text-fig. 2. It is clear that the number of discrete movements declines as the dose is increased. Essentially complete paralysis occurs at the MPD. Six-, 12-, and 15-day embryos gave dose-response curves of similar form with curare. In our histological material the earliest cholinesterase-containing endplates were seen in the paraspinal muscles at 10 days of incubation. They were well developed in this region by 12 days. In the distal muscles of the
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
THE EMBRYONIC NEUROMUSCULAR JUNCTION 709 leg end-plates were first seen at 12-13 days (P1. 1), and were well developed by 14 days. The arrows in Text-fig. 1 bracket the period during which cholinesterase-containing motor end-plates make their appearance in the chick a
3*0 _
/
b
/ 250 2e5 2.0~~~~~~~~~~~~*~
/
2~0
~~~~05-
~~~1~~~5 os/
0.5
J
-~~~~~~~
7
T.5
0
/ 12
9
912
7
15
15
Age (days)
Age (days) C
20
10 /. 0.5/ 0
7
T 9
I
I
r 12
_I15 I
Age (days)
Text-fig. 1. MPD (O --- 0) (a) curare, (b) succinylcholine, (c) decamethonium, in chick embryos from 7 to 15 days of incubation. The weight-corrected ratio MPD: g (@-@) is essentially constant in each graph. Arrows indicate the period during which cholinesterase-containing motor end-plates first appear. 45-2 Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
D. B. DRACHMAN embryo. It is evident that the age range encompassed by the pharmacological experiments extends over the periods before, during, and after cholinesterase becomes demonstrable at the neuromuscular junction. 710
160 140
120 E
100 _
t
E
\
0
,4,
0>60-
C20_ _ 40200 0
1
2
3 4 6 5 Dose of curare (#g)
7
8
Text-fig. 2. Counts of spontaneous movements observed in 9 day chick embryos treated with curare; note the progressive decline in number of movements with increasing dosage. The MPD is seen to be 755,g. Each point represents the mean of 5-10 observations, except for the untreated group which consists of the mean of the control counts made on all chicks which were subsequently treated. S.E. of mean indicated for each group. DISCUSSION
The time at which the earliest anatomical connexion between nerve and muscle occurs in the chick embryo is not yet settled. With silver-impregnation techniques anatomists have found evidence of neuromuscular contact as early as the 4th day of incubation (Visintini & Levi-Montalcini, 1939), or failed to find it until the 7th day (Tello, 1917; Mumenthaler & Engel, 1961). With histochemical methods cholinesterase appears diffusely distributed throughout the myoblasts as early as the 3rd-6th days, and thereafter gradually recedes toward the presumptive end-plate and myotendinous junction regions (Begliomini & Moriconi, 1959; Gerebtzoff, 1959; Mumenthaler & Engel, 1961). In our material the earliest discrete end-plates containing cholinesterase were found on the 10th day in the paraspinal musculature. In the distal muscles of the leg they first appeared on the 12th-13th day, and were well developed by the 14th day. Localized cholinesterase activity represents an early response of the embryo's muscle membrane to innervation (Zelena, 1962). Its appearance was therefore used as a guide to the timing of our pharmacological experiments. Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
THE EMBRYONIC NEUROMUSCULAR JUNCTION 711 In the present study we have made quantitative determinations of the sensitivity of the chick embryo to curare, succinyicholine and decamethonium at successive stages of development. These three pharmacological agents are thought to act at the receptor substance of the muscle membrane, although the precise mechanisms of their actions differ (Paton, 1956; del Castillo & Katz, 1957; Taylor, 1959; Zaimis, 1959). A sufficiently wide range of ages was used to include the period during which the most striking alterations in end-plate structure could be demonstrated with cholinesterase staining. The present findings show that the response of receptor substance to pharmacological blocking agents is not altered qualitatively or quantitatively during a period of profound change in the localization of end-plate cholinesterase. This tends to support Miledi's (1962) contention based on observations in normal and denervated muscle that receptors and cholinesterase are separate in location and behaviour. SUMMARY
1. The minimum paralytic dosages (MPDs) of curare, succinylcholine and decamethonium have been determined for chick embryos of 7-15 days of incubation age. After a correction was made for the increase in embryonic weight with advancing age, it was found that the ratio MPD:g for each drug remained constant throughout the ages examined. 2. Histochemical staining revealed that cholinesterase-containing motor end-plates may first be seen between 10 and 13 days of incubation age. 3. The precise site and mechanism of action of curare, succinyleholine and decamethonium have not been established in the embryo, but it is likely that they act at the level of receptor substance. The experimental results suggest that the sensitivity of receptor substance to these drugs remains constant in the chick embryo, while profound changes are taking place in the morphology of the neuromuscular junction. The author acknowledges with gratitude the encouragement and helpful suggestions of Dr A. J. Coulombre, who also participated directly in the early experiments involved in this study. Miss Sonia Steinberg provided technical assistance. REFERENCES
BEGLIOMINI, A. & MoRIcoNI, A. (1959) Sull'Epoca Della Localizzazione Della Colinesterasi Nel Musculo Striato Dell' Embrione Di Pollo. Rev. Biol. 51, 517-531. DEL CASTILO, J. & KATZ, B. (1957). A study of curare action with an electrical micromethod. Proc. Roy. Soc. B, 146, 339-356. DRACHMAN, D. B. & COULOMBRE, A. J. (1962). Method for continuous infusion of fluids into the chorioallantoic circulation of the chick embryo. Science, 138, 144-145. GEREBTZOFF, M. A. (1959). Cholinesterases. pp. 40-43. New York: Pergamon Press.
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
712
D. B. DRACHMAN
Kuo, Z. Y. (1939). Studies in the physiology of the embryonic nervous system. I. Effect of curare on motor activity of the chick embryo. J. exp. Zool. 82, 371-396. MILEDI, R. (1962). Induction of Receptors. In Ciba Foundation Symposium on enzymes and drug action. Ed. MONGAR, J. L. & DE REUCK, A. V. S. pp. 220-238. London: Churchill. MUMENTHALER, M. & ENGEL, W. K. (1961). Cytological localization of cholinesterase in developing chick embryo skeletal muscle. Acta anat. 47, 274-299. PATON, W. D. M. (1956). Mode of action of neuromuscular blocking agents. Brit. J. Anaesth. 28, 470-480. TAYLOR, D. B. (1959). The mechanism of action of muscle relaxants and their antagonists. Anesthesiol. 20, 439-452. TELLO, J. F. (1917). G6nesis de las Terminaciones Nerviosas Motrices v Sensitivas. Trab. Inst. Cajal Invest. biol. 15, 101-199. VIsINTINI, F. & LEvI-MONTALCINI, R. (1939). Relazione tra Differenziazione Strutturale e Funcionale dei Centri e Delle Vie Nervose nell'embrione di Pollo. Schweiz. Arch. Neurol. Psychi at. 43, 381-393, 44, 119-150. ZAIMIS, E. (1959). Mechanisms of neuromuscular blockade. In Curare and Curare-Like Agents. Ed. BOVET, D., BOVET-NI=T, F. & MARINI-BETTOLO, G. B. New York: Elsevier. ZELENA, J. (1962). The Effect of Denervation on Muscle Development. In The Denervated Muscle. Ed. GUTMANN, E. Chap. III. Prague: Czech. Acad. Sci. EXPLANATION OF PLATE Group of motor end-plates in the calf muscles of a 13-day chick embryo; stained for cholinesterase; x 390.
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
The Journal of Physiology, Vol. 169, No. 4
Plate I
D. B. DRACHMAN
(Facing p. 712)
Downloaded from J Physiol (jp.physoc.org) by guest on July 28, 2011
