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Tenotomy decreases sympathetic neuronal survival factors in avian smooth muscle
DevelopmentalBrain Research, 22 (1985) 93-97 Elsevier
93
BRD50263
Tenotomy Decreases Sympathetic Neuronal Survival Factors in Avian Smooth Muscle R. A. RUSH
Centrefor Neuroscienceand Human Physiology Department, The Flinders Universityof South Australia, Bedford Park, S.A. 5042 (Australia) (Accepted March 5th, 1985)
Key words: expansor secundariorum - - dopamine/3-hydroxylase- - trophic regulation - - chicken - - tension
The expansor secundariorum of the chicken wing has a high concentration of survival factor activity for sympathetic neurons. The effect of tenotomy on this activity has been examined in newly hatched and older birds. Survival factor activity was assayed with dissociated embryonic neurons and found to be decreased after tenotomy to low levels in the newly hatched but not the older birds. No change in dopamine fl-hydroxylase concentration was detected, suggesting that tenotomy does not significantly alter impulse activity in the sympathetic innervation. The results are compared with findings after tenotomy in skeletal muscles and contrasted with increased survival factor activity produced by denervation of the expansor secundariorum. INTRODUCTION Current hypotheses of neuronal d e v e l o p m e n t usually include the assumption that target tissues for specific neuronal populations p r o d u c e a survival factor which is secreted, internalized by the terminal processes of innervating neurons and t r a n s p o r t e d retrogradely to the cell s o m a where it exerts effects on cellular metabolism. Recently, this l a b o r a t o r y described the presence of survival factor activity for sympathetic neurons in the avian e x p a n s o r secundariorum 1, a smooth muscle densely innervated by noradrenergic nerves 2. D e n e r v a t i o n of this unique muscle results in an increase in the concentration of survival factor activity suggesting that it is under neuronal control 10. The regulation of this e n d o g e n o u s activity has been investigated further in the studies rep o r t e d below, that examine the influence of tension within the developing muscle. The studies were possible as the presence of a t e n d o n allowed a reduction in tension to be achieved by t e n o t o m y . MATERIALS AND METHODS
Surgery White Leghorn male chickens o b t a i n e d from A n -
derson Chicks ( A d e l a i d e ) were used throughout. Young chickens were no m o r e than 3 days posthatch and older birds were 8 weeks posthatch at the time of tenotomy. T e n o t o m y was p e r f o r m e d under e t h e r anaesthesia and was achieved by section of the tendon at two points 1 - 3 cm a p a r t with the distal cut being within 5 m m of the b o d y of the muscle. The tendons Were examined 5 days later and freed from connective tissue where necessary.
Assay procedures Muscles were r e m o v e d after a further 5 days and homogenized in 10 vols of ice cold Ca 2+-, Mg2+-free physiological buffer, p H 7.4 (ref. 12). D o p a m i n e / 3 hydroxylase ( D B H ) was estimated on individual muscle homogenates; survival factor activity was determined on the supernatants of individual (older birds) or two p o o l e d muscles (young birds) following centrifugation at 18,000 g for 10 rain. D o p a m i n e /3-hydroxylase activity was estimated spectrophotometrically, by the m e t h o d of Nagatsu and UdenfriendS. Results are expressed as nmol of o c t o p a m i n e formed p e r mg protein per h. Protein estimates were p e r f o r m e d according to B r a d f o r d 3 using bovine y-globulin as a standard. Survival activity for sympathetic neurons was as-
Correspondence: R. A. Rush, Centre for Neuroscience and Human Physiology Department, The Flinders University of South Australia, Bedford Park, S.A. 5042, Australia. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
94 sayed by the m e t h o d of Varon and R a i b o r n ~2, with minor modifications as r e p o r t e d earlier 1. Briefly, dissociated sympathetic neurons were p r e p a r e d from l l - d a y - i n c u b a t e d chick embryos by trypsin incubation and trituration. Three thousand cells were plated with muscle extract dilutions ranging from 0.3 to 10%. Assays of t e n o t o m i z e d and contralateral muscles were always p e r f o r m e d simultaneously and after adjusting extracts to the same protein concentrations, using the same batch of neurons. The cells were fixed 24 h later and counted for the presence of neurons and non-neuronal cells. Neurons were counted as neurite-bearing by the presence of processes of at least two cell diameters. It has been shown in this assay p r o c e d u r e that after 24 h of culture, only neurons with neurites will survive the subsequent 24 kJ.
A ,'- 60 E:: w z I I--
4o O rr Z ~Z w O
.,...r-
MUSCLE EXTRACT C,~,1100,~,~ ~ , ~
60
.~ 4o
Portions of several control and t e n o t o m i z e d muscles from both the young and older birds were cut from various regions of the tissue and fixed and processed for routine histochemistry. A f t e r wax e m b e d ding and sectioning, 10-/~m sections were cut and stained with haemotoxylin and eosin.
w z ~_ z w 0
sulted in the dramatic loss of nearly 70% of survival activity within the muscle (Fig. 1A). The concentration curve indicates that the m a x i m u m neuronal survival for control muscle was approx. 68% and that 5.6/~g of muscle extract protein is necessary to support 50% of this n u m b e r which is in a g r e e m e n t with our earlier findings1,10. The m a x i m u m n u m b e r of neurons s u p p o r t e d by extract from t e n o t o m i z e d muscle was lower (53%) and 3-fold m o r e protein (17.6/~g) was required for 50% of the cells to produce neurites. This is indicative of a change in the concentration of survival activity within the tenotomized muscle with a possible alteration of the type of activity. However, it is not possible of course to directly
,'f
B
o
Tendons were carefully examined both at 5 days after section and at termination of the experiments. The proximal end of the muscle was generally found to be loosely adhering to proliferating connective tissue but the muscles a p p e a r e d to be free of tension. T e n o t o m y for 10 days in newly hatched chicks re-
/
!.,f
lx
Histology
RESULTS
20
2C
o_
0
2
'4 ' 6 MUSCLE EXTRACT (~l/lOOzul)
8
1'0
Fig. 1. Dose-response curves showing numbers of cells corresponding with increasing concentration of muscle extract from young birds (A) and from older birds (B). Results from control muscles are drawn with solid lines and those from tenotomized muscles with broken lines. The concentration of protein in cell homogenates in A was adjusted to 8 mg/ml and in B to 5.5 mg/ml. All results are expressed as the mean _+ S.E.M., n = 4.
compare half-maxima responses when the maxima are not the same, as different factors may be responsible. In older birds however, there was little or no change in activity within the t e n o t o m i z e d muscle (Fig. 1B). The m a x i m u m neuronal survival was similar to that seen for the young control birds (approx. 65%) and the amount of protein required to produce half-maximum survival was 6.3/~g protein for both tenotomized and control muscles. D B H enzymic activity in the muscle was estimated 11 days after t e n o t o m y in newly hatched birds and 4 and 10 days after t e n o t o m y in older birds. No change in D B H activity was detected in any of the muscles examined, whether expressed as enzyme activity per
95 TABLE I
Effect of tenotomy on DBH activity Using Student's t-test no significant differences were found between control and tenotomized muscles for any of the data shown. T h e n u m b e r of samples for each group was 6; the results from older birds (46 and 66 days old) were analyzed as individual muscles, those from young birds (14-days-old) were analysed as muscle pairs (i.e. from 6 pairs of birds).
Age of birds (days)
Day3-14
(11 days)
Procedure
Muscle wet wt. (mg)
D BH activity nmol octopamine/ mg protein/h
nmol octopamine/ mg wet weight/h
nmol octopamine/ muscle/h
control tenotomized
13.9 + 1.3 12.8 + 1.1
12.4 ___1.6 11.1 + 0.9
1.2 _+ 0.03 1.1 ___0.02
16.5 _ 1.5 15.6 + 1.0
D a y 4 2 - 4 6 (4 days)
control tenotomized
58.3 + 4.1 45.9 + 3.3
9.9 + 0.4 11.3 __+0.6
1.8 _+ 0.04 2.1 _+ 0.04
99.9 + 6.9 91.4 + 5.5
Day 56-66 (10 days)
control tenotomized
51.5 + 7.9 52.2 + 5.4
8.8 -+ 0.2 9.4 + 0.2
1.7 _-+0.04 1.8 + 0.07
95.0 + 15.8 110.1 + 18.5
mg of protein or per mg wet wt. (Table I). The total enzyme content in each muscle was also unchanged as no significant alteration in muscle weight or protein content was apparent after tenotomy (Table
I). These findings are in agreement with the histological findings that indicated that tissue taken from both young and older muscles were unaffected by tenotomy. Fig. 2 shows a section through a control
Fig. 2. Histological sections of normal (A) and l l - d a y tenotomized (B) muscles. Note that in the tenotomized organ there is no atrophy of the s m o o t h muscle and no indication of replacement of fibres with connective tissue. The portions of muscle were taken from a 12-day-old bird, about one third of the way into the organ from the insertion of the tendon. Bars, 20ktm.
96 and tenotomized muscle taken from a single young bird. DISCUSSION The object of the study presented in this communication was to assess the importance of tension within the expansor secundariorum on the concentration of sympathetic survival factor activity. The study was made possible by the presence of a discrete tendon and a high concentration of survival factor in this unique muscle. A role has been established for the innervation in regulating not only the concentration of this activity but also its nature w. Abolition of tension was achieved by tenotomy which dramatically lowered the concentration of survival factor activity during the early posthatch period but not in older birds. Although unlikely, the production of an inhibitory agent by young tenotomized muscle cannot be excluded. It is of interest to note that this change is in marked contrast to the increased concentration that occurs in this muscle as a result of denervation in young birds. This change after denervation may be due to loss of trophic regulation from the nerve but an alternative explanation may be that the increased survival factor activity is due to an increase in tension within the muscle that is also responsible for the considerable hypertrophy that occurs 10. Thus it might reasonably be argued, at least in young birds, that tension alone may be the major factor regulating the metabolic change, although this hypothesis has so far proved difficult to test. The striking difference between the level of survival activity in normal and tenotomized muscles from young, developing birds clearly indicates the importance of tension. Because survival effects were being examined in these experiments and since neuronal activity can influence neuronal growth 6,11~13, it was important to determine enzyme levels in the current experiments as an estimation of impulse traffic carried by the sympathetic fibres. A sensory input from the expansor secundariorum to the autonomic motor fibres may be present 4. DBH levels have been shown to increase in neurons carrying increased impulse traffic and to decrease in decentralized neurons 14. The complete absence of effect of tenotomy on DBH activity suggests that there is little alteration to the impulse traffic in these fibres. It is important to re-
member that the expansor secundariorum is a muscle controlling flight feathers and that young birds (particularly chickens!) have no ability to fly until many weeks of age. Thus it is entirely possible that the impulse activity within these nerves is minimal under normal conditions. No direct comparison of these results with others is possible because of the absence of tendons to most smooth muscles; it is therefore only possible to examine the results from experiments with skeletal muscles, whilst recognising that tenotomy will not necessarily produce similar effects on both muscle types. Most authors agree that the use of tenotomy as a means of abolishing tension within a muscle makes interpretation of results difficult, primarily because of the effects that tenotomy may have on the sensory input to motor neurons. However, in a review of hindlimb muscles, Estavillo et al.5 have concluded that tenotomy probably limits, but does not abolish, impulse activity in sensory and motor nerves. In addition, Lomo 7 has demonstrated that alterations to fibre length and sarcomere number produced by forced, maintained shortening of the rat soleus muscle is independent of neural influences, suggesting that tension within the muscle may be responsible. Perhaps the most relevant work from experiments on immobilized skeletal muscle is that reported by Pittman and Oppenheim 9. These workers demonstrated that immobilization of embryos with neuromuscular blocking agents resulted in an increase in the number of motoneurons in the spinal cord. One explanation for this phenomenon is that during normal development nerve cells compete for a survival factor that is produced by their target tissues in limited supply. Thus as the muscle becomes active on innervation, the survival factor production is lowered resulting in the death of some of the nerves projecting to it. The decreased survival factor activity for sympathetic neurons that we have seen in the expansor secundariorum following tenotomy would therefore seem to contrast with the increased motor neuron survival seen after neuromuscular blockade, although further interpretation will have to await experiments that determine whether the survival factor activity is secreted, whether secretion rate is altered by tenotomy, and if neuromuscular blockade produces the same effect as tenotomy in this muscle. The discovery of survival factor activity for sympa-
97 thetic n e u r o n s in the e x p a n s o r s e c u n d a r i o r u m has
ACKNOWLEDGEMENTS
p r o v i d e d an i m p o r t a n t n e w s o u r c e of a n e r v e g r o w t h factor-like m a t e r i a l f r o m the bird. T h a t its c o n c e n t r a -
This w o r k was s u p p o r t e d by the N a t i o n a l H e a l t h
tion can be r e g u l a t e d by d e n e r v a t i o n and n o w t e n o t o -
and M e d i c a l R e s e a r c h C o u n c i l of A u s t r a l i a . Mrs.
my, e l e v a t e s its status f r o m a n o t h e r unusual source to
Susie M u r d o c h and Miss F i o n a R e n t o n p r o v i d e d ex-
that of a target o r g a n - d e r i v e d c o m p o u n d that m a y be
cellent technical assistance which is greatly a p p r e -
active o n i n n e r v a t i n g s y m p a t h e t i c nerves.
ciated.
REFERENCES
York, 1976, pp. 289-321. 8 Nagatsu, T. and Udenfriend, S., Photometric assay of dopamine-fl-hydroxylase activity in human blood, Clin. Chem., 18 (1972) 980-983. 9 Pittman, R. and Oppenheim, R. W., Cell death of motoneurons in the chick embryo spinal cord. IV. Evidence that a functional neuromuscular interaction is involved in the regulation of naturally occurring cell death and the stabilization of synapses, J. comp. Neurol., 187 (1979) 425-446. 10 Rush, R. A., Abrahamson, I. K., Belford, D. A., Murdoch, S. Y. and Wilson, P. A., Regulation of sympathetic trophic activity in smooth muscle, submitted. 11 Russell, N. J. W., Axonal conduction velocity changes following muscle tenotomy or deafferentiation during development in the rat, J. Physiol. (Lond.), 298 (1980) 347-360. 12 Varon, S. and Raiborn, C., Dissociation, fractionation and culture of chick embryo sympathetic ganglionic cells, J. Neurocytol., 1 (1972) 211-221. 13 Wiesel, T. N. and Hubel, D. H., Effects of visual deprivation on the morphology and physiology of cells in the cat lateral geniculate body, J. Neurophysiol., 26 (1963) 978-993. 14 Zigmond, R. E. and Bowers, C. W., Influence of nerve activity on the macromolecular content of neurons and their effector organs, Ann. Rev. Physiol., 43 (1981)673-687.
1 Belford, D. A. and Rush, R. A., A survival factor for sympathetic neurons from avian smooth muscle, Develop. Brain Res., 6 (1983) 304-308. 2 Bennett, T. and Malmfors, T., The adrenergic nervous system of the domestic fowl, Z. Zellforsch., 106 (1970) 22-50. 3 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 4 Cobb, J, L. S. and Bennett, T., Herbst corpuscles in the smooth muscles in the wings of chicks, Experientia, 26 (1970) 768-769. 5 Estavillo, J., Yellin, H., Sasaki, Y. and Eldred, E., Observations on the expected decrease in proprioceptive discharge and purported advent of non-proprioceptive activity from the chronically tenotomized muscle, Brain Res., 63 (1973) 75-91. 6 Hubel, D. H. and Wiesel, T. N., The period of susceptibility to the physiological effects of unilateral eye closure in kittens, J. Physiol. (Lond.), 206 (1970) 419-436. 7 Lomo, T., The role of activity in the control of membrane and contractile properties of skeletal muscle. In S. Thesleff (Ed.), Motor Innervation of Muscle, Academic Press, New
93
BRD50263
Tenotomy Decreases Sympathetic Neuronal Survival Factors in Avian Smooth Muscle R. A. RUSH
Centrefor Neuroscienceand Human Physiology Department, The Flinders Universityof South Australia, Bedford Park, S.A. 5042 (Australia) (Accepted March 5th, 1985)
Key words: expansor secundariorum - - dopamine/3-hydroxylase- - trophic regulation - - chicken - - tension
The expansor secundariorum of the chicken wing has a high concentration of survival factor activity for sympathetic neurons. The effect of tenotomy on this activity has been examined in newly hatched and older birds. Survival factor activity was assayed with dissociated embryonic neurons and found to be decreased after tenotomy to low levels in the newly hatched but not the older birds. No change in dopamine fl-hydroxylase concentration was detected, suggesting that tenotomy does not significantly alter impulse activity in the sympathetic innervation. The results are compared with findings after tenotomy in skeletal muscles and contrasted with increased survival factor activity produced by denervation of the expansor secundariorum. INTRODUCTION Current hypotheses of neuronal d e v e l o p m e n t usually include the assumption that target tissues for specific neuronal populations p r o d u c e a survival factor which is secreted, internalized by the terminal processes of innervating neurons and t r a n s p o r t e d retrogradely to the cell s o m a where it exerts effects on cellular metabolism. Recently, this l a b o r a t o r y described the presence of survival factor activity for sympathetic neurons in the avian e x p a n s o r secundariorum 1, a smooth muscle densely innervated by noradrenergic nerves 2. D e n e r v a t i o n of this unique muscle results in an increase in the concentration of survival factor activity suggesting that it is under neuronal control 10. The regulation of this e n d o g e n o u s activity has been investigated further in the studies rep o r t e d below, that examine the influence of tension within the developing muscle. The studies were possible as the presence of a t e n d o n allowed a reduction in tension to be achieved by t e n o t o m y . MATERIALS AND METHODS
Surgery White Leghorn male chickens o b t a i n e d from A n -
derson Chicks ( A d e l a i d e ) were used throughout. Young chickens were no m o r e than 3 days posthatch and older birds were 8 weeks posthatch at the time of tenotomy. T e n o t o m y was p e r f o r m e d under e t h e r anaesthesia and was achieved by section of the tendon at two points 1 - 3 cm a p a r t with the distal cut being within 5 m m of the b o d y of the muscle. The tendons Were examined 5 days later and freed from connective tissue where necessary.
Assay procedures Muscles were r e m o v e d after a further 5 days and homogenized in 10 vols of ice cold Ca 2+-, Mg2+-free physiological buffer, p H 7.4 (ref. 12). D o p a m i n e / 3 hydroxylase ( D B H ) was estimated on individual muscle homogenates; survival factor activity was determined on the supernatants of individual (older birds) or two p o o l e d muscles (young birds) following centrifugation at 18,000 g for 10 rain. D o p a m i n e /3-hydroxylase activity was estimated spectrophotometrically, by the m e t h o d of Nagatsu and UdenfriendS. Results are expressed as nmol of o c t o p a m i n e formed p e r mg protein per h. Protein estimates were p e r f o r m e d according to B r a d f o r d 3 using bovine y-globulin as a standard. Survival activity for sympathetic neurons was as-
Correspondence: R. A. Rush, Centre for Neuroscience and Human Physiology Department, The Flinders University of South Australia, Bedford Park, S.A. 5042, Australia. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
94 sayed by the m e t h o d of Varon and R a i b o r n ~2, with minor modifications as r e p o r t e d earlier 1. Briefly, dissociated sympathetic neurons were p r e p a r e d from l l - d a y - i n c u b a t e d chick embryos by trypsin incubation and trituration. Three thousand cells were plated with muscle extract dilutions ranging from 0.3 to 10%. Assays of t e n o t o m i z e d and contralateral muscles were always p e r f o r m e d simultaneously and after adjusting extracts to the same protein concentrations, using the same batch of neurons. The cells were fixed 24 h later and counted for the presence of neurons and non-neuronal cells. Neurons were counted as neurite-bearing by the presence of processes of at least two cell diameters. It has been shown in this assay p r o c e d u r e that after 24 h of culture, only neurons with neurites will survive the subsequent 24 kJ.
A ,'- 60 E:: w z I I--
4o O rr Z ~Z w O
.,...r-
MUSCLE EXTRACT C,~,1100,~,~ ~ , ~
60
.~ 4o
Portions of several control and t e n o t o m i z e d muscles from both the young and older birds were cut from various regions of the tissue and fixed and processed for routine histochemistry. A f t e r wax e m b e d ding and sectioning, 10-/~m sections were cut and stained with haemotoxylin and eosin.
w z ~_ z w 0
sulted in the dramatic loss of nearly 70% of survival activity within the muscle (Fig. 1A). The concentration curve indicates that the m a x i m u m neuronal survival for control muscle was approx. 68% and that 5.6/~g of muscle extract protein is necessary to support 50% of this n u m b e r which is in a g r e e m e n t with our earlier findings1,10. The m a x i m u m n u m b e r of neurons s u p p o r t e d by extract from t e n o t o m i z e d muscle was lower (53%) and 3-fold m o r e protein (17.6/~g) was required for 50% of the cells to produce neurites. This is indicative of a change in the concentration of survival activity within the tenotomized muscle with a possible alteration of the type of activity. However, it is not possible of course to directly
,'f
B
o
Tendons were carefully examined both at 5 days after section and at termination of the experiments. The proximal end of the muscle was generally found to be loosely adhering to proliferating connective tissue but the muscles a p p e a r e d to be free of tension. T e n o t o m y for 10 days in newly hatched chicks re-
/
!.,f
lx
Histology
RESULTS
20
2C
o_
0
2
'4 ' 6 MUSCLE EXTRACT (~l/lOOzul)
8
1'0
Fig. 1. Dose-response curves showing numbers of cells corresponding with increasing concentration of muscle extract from young birds (A) and from older birds (B). Results from control muscles are drawn with solid lines and those from tenotomized muscles with broken lines. The concentration of protein in cell homogenates in A was adjusted to 8 mg/ml and in B to 5.5 mg/ml. All results are expressed as the mean _+ S.E.M., n = 4.
compare half-maxima responses when the maxima are not the same, as different factors may be responsible. In older birds however, there was little or no change in activity within the t e n o t o m i z e d muscle (Fig. 1B). The m a x i m u m neuronal survival was similar to that seen for the young control birds (approx. 65%) and the amount of protein required to produce half-maximum survival was 6.3/~g protein for both tenotomized and control muscles. D B H enzymic activity in the muscle was estimated 11 days after t e n o t o m y in newly hatched birds and 4 and 10 days after t e n o t o m y in older birds. No change in D B H activity was detected in any of the muscles examined, whether expressed as enzyme activity per
95 TABLE I
Effect of tenotomy on DBH activity Using Student's t-test no significant differences were found between control and tenotomized muscles for any of the data shown. T h e n u m b e r of samples for each group was 6; the results from older birds (46 and 66 days old) were analyzed as individual muscles, those from young birds (14-days-old) were analysed as muscle pairs (i.e. from 6 pairs of birds).
Age of birds (days)
Day3-14
(11 days)
Procedure
Muscle wet wt. (mg)
D BH activity nmol octopamine/ mg protein/h
nmol octopamine/ mg wet weight/h
nmol octopamine/ muscle/h
control tenotomized
13.9 + 1.3 12.8 + 1.1
12.4 ___1.6 11.1 + 0.9
1.2 _+ 0.03 1.1 ___0.02
16.5 _ 1.5 15.6 + 1.0
D a y 4 2 - 4 6 (4 days)
control tenotomized
58.3 + 4.1 45.9 + 3.3
9.9 + 0.4 11.3 __+0.6
1.8 _+ 0.04 2.1 _+ 0.04
99.9 + 6.9 91.4 + 5.5
Day 56-66 (10 days)
control tenotomized
51.5 + 7.9 52.2 + 5.4
8.8 -+ 0.2 9.4 + 0.2
1.7 _-+0.04 1.8 + 0.07
95.0 + 15.8 110.1 + 18.5
mg of protein or per mg wet wt. (Table I). The total enzyme content in each muscle was also unchanged as no significant alteration in muscle weight or protein content was apparent after tenotomy (Table
I). These findings are in agreement with the histological findings that indicated that tissue taken from both young and older muscles were unaffected by tenotomy. Fig. 2 shows a section through a control
Fig. 2. Histological sections of normal (A) and l l - d a y tenotomized (B) muscles. Note that in the tenotomized organ there is no atrophy of the s m o o t h muscle and no indication of replacement of fibres with connective tissue. The portions of muscle were taken from a 12-day-old bird, about one third of the way into the organ from the insertion of the tendon. Bars, 20ktm.
96 and tenotomized muscle taken from a single young bird. DISCUSSION The object of the study presented in this communication was to assess the importance of tension within the expansor secundariorum on the concentration of sympathetic survival factor activity. The study was made possible by the presence of a discrete tendon and a high concentration of survival factor in this unique muscle. A role has been established for the innervation in regulating not only the concentration of this activity but also its nature w. Abolition of tension was achieved by tenotomy which dramatically lowered the concentration of survival factor activity during the early posthatch period but not in older birds. Although unlikely, the production of an inhibitory agent by young tenotomized muscle cannot be excluded. It is of interest to note that this change is in marked contrast to the increased concentration that occurs in this muscle as a result of denervation in young birds. This change after denervation may be due to loss of trophic regulation from the nerve but an alternative explanation may be that the increased survival factor activity is due to an increase in tension within the muscle that is also responsible for the considerable hypertrophy that occurs 10. Thus it might reasonably be argued, at least in young birds, that tension alone may be the major factor regulating the metabolic change, although this hypothesis has so far proved difficult to test. The striking difference between the level of survival activity in normal and tenotomized muscles from young, developing birds clearly indicates the importance of tension. Because survival effects were being examined in these experiments and since neuronal activity can influence neuronal growth 6,11~13, it was important to determine enzyme levels in the current experiments as an estimation of impulse traffic carried by the sympathetic fibres. A sensory input from the expansor secundariorum to the autonomic motor fibres may be present 4. DBH levels have been shown to increase in neurons carrying increased impulse traffic and to decrease in decentralized neurons 14. The complete absence of effect of tenotomy on DBH activity suggests that there is little alteration to the impulse traffic in these fibres. It is important to re-
member that the expansor secundariorum is a muscle controlling flight feathers and that young birds (particularly chickens!) have no ability to fly until many weeks of age. Thus it is entirely possible that the impulse activity within these nerves is minimal under normal conditions. No direct comparison of these results with others is possible because of the absence of tendons to most smooth muscles; it is therefore only possible to examine the results from experiments with skeletal muscles, whilst recognising that tenotomy will not necessarily produce similar effects on both muscle types. Most authors agree that the use of tenotomy as a means of abolishing tension within a muscle makes interpretation of results difficult, primarily because of the effects that tenotomy may have on the sensory input to motor neurons. However, in a review of hindlimb muscles, Estavillo et al.5 have concluded that tenotomy probably limits, but does not abolish, impulse activity in sensory and motor nerves. In addition, Lomo 7 has demonstrated that alterations to fibre length and sarcomere number produced by forced, maintained shortening of the rat soleus muscle is independent of neural influences, suggesting that tension within the muscle may be responsible. Perhaps the most relevant work from experiments on immobilized skeletal muscle is that reported by Pittman and Oppenheim 9. These workers demonstrated that immobilization of embryos with neuromuscular blocking agents resulted in an increase in the number of motoneurons in the spinal cord. One explanation for this phenomenon is that during normal development nerve cells compete for a survival factor that is produced by their target tissues in limited supply. Thus as the muscle becomes active on innervation, the survival factor production is lowered resulting in the death of some of the nerves projecting to it. The decreased survival factor activity for sympathetic neurons that we have seen in the expansor secundariorum following tenotomy would therefore seem to contrast with the increased motor neuron survival seen after neuromuscular blockade, although further interpretation will have to await experiments that determine whether the survival factor activity is secreted, whether secretion rate is altered by tenotomy, and if neuromuscular blockade produces the same effect as tenotomy in this muscle. The discovery of survival factor activity for sympa-
97 thetic n e u r o n s in the e x p a n s o r s e c u n d a r i o r u m has
ACKNOWLEDGEMENTS
p r o v i d e d an i m p o r t a n t n e w s o u r c e of a n e r v e g r o w t h factor-like m a t e r i a l f r o m the bird. T h a t its c o n c e n t r a -
This w o r k was s u p p o r t e d by the N a t i o n a l H e a l t h
tion can be r e g u l a t e d by d e n e r v a t i o n and n o w t e n o t o -
and M e d i c a l R e s e a r c h C o u n c i l of A u s t r a l i a . Mrs.
my, e l e v a t e s its status f r o m a n o t h e r unusual source to
Susie M u r d o c h and Miss F i o n a R e n t o n p r o v i d e d ex-
that of a target o r g a n - d e r i v e d c o m p o u n d that m a y be
cellent technical assistance which is greatly a p p r e -
active o n i n n e r v a t i n g s y m p a t h e t i c nerves.
ciated.
REFERENCES
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