354
Brain Research, 99 (1975) 354 358 ,(~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Consequences of tenotomy on the evolution of multiinnervation in developing rat soleus muscle
PIERRE B E N O I T
AND
JEAN-PIERRE CHANGEUX
Neurobiologie Moldculaire, Ddpartement de Biologie Mol(cutaire, lnstitut Pasteur, 75015 Paris (France)
(Accepted August 12th, 1975)
There exists good experimental evidence6, la that the functional organization of the vertebrate nervous system is under stringent genetic control, although the number o f its identified neuronal circuits and synaptic contacts appears many orders of magnitude larger than the number of genes. In an attempt to solve this paradox, it was suggested that, among other mechanisms, the state of activity o f the developing neuronal network regulates the phenotypic expression of the genes and therefore contributes to the specification of the neuronal network within a low-cost genetic envelope2, 3. In particular, it was assumed that (a) at early stages of development, functional synaptic contacts are present in a larger number than at late stages, (b) a selective stabilization of synapses takes place during maturation and (c) the state o f activity o f a given postsynaptic soma initially controls, in a retrograde manner, the selective stabilization of its afferent synapses. Because o f its simplicity, the neuromuscular junction of vertebrates constitutes a convenient preparation to test the theory. With this system, it is known that (a) at the earliest stages o f development, the contacts established between motor nerves and muscle fibers are functional7,tl, t4, (b) in chick embryo, the chronic block of the neuromuscular synapses by highly specific neurotoxins interfere with their developmerit 4,5 and (c) in chick and rat, the developing muscle fiber receives from different motoneurons several functional nerve terminals which converge at well-defined synaptic spots1,10; in the adult muscle, a single nerve terminal persists per synapse. The aim of the experiments reported in this communication is to study the eventual effect of the activity of developing rat soleus muscle on the evolution of its innervation. Experiments were carried out on soleus muscle from 1- to 3-week-old rats (Sprague-Dawley). The muscle and the corresponding branch of the sciatic nerve were dissected from ether anesthetized animals and kept at room temperature under a continuous flow of oxygenated rat Ringer's solution 9. To facilitate recording of synaptic potentials, the calcium concentration in the physiological solution was raised to 5 m M and 0.5-1.0 × 10 -6 M D-tubocurarine was added to the bath to avoid muscle contraction. The nerve was carefully sucked into a fine micropipette and
355
Fig. l. Example of end-plate potentials obtained by intracellular recording from a curarized soleus muscle of an 8-day-old rat. As the intensity of the stimulus applied to the nerve decreases, the amplitude of the response varies in a stepwise manner. Vertical bar, 2 mV; horizontal bar, 5 msec.
s t i m u l a t e d by short electric pulses o f variable intensity, a p p l i e d between the inside o f the pipette a n d the bath. The p o s t s y n a p t i c p o t e n t i a l s o f individual muscle fibers were r e c o r d e d intracellularly with s t a n d a r d microelectrodes, d i s p l a y e d o n a storage oscilloscope a n d p h o t o g r a p h e d . Fig. 1 shows a typical recording o b t a i n e d with the n e u r o m u s c u l a r j u n c t i o n o f an 8 - d a y - o l d rat. As the strength o f the stimulus progressively increases, the a m p l i t u d e o f the r e c o r d e d e n d - p l a t e p o t e n t i a l increases in a d i s c o n t i n u o u s m a n n e r , t h r o u g h successive steps (3 in this p a r t i c u l a r experiment) instead o f a single step as it does in the a d u l t muscle fiber. Similar observations, r e p o r t e d by R e d f e r n 10 a n d by Bennett a n d Pettigrew 1, reveal a m u l t i i n n e r v a t i o n o f i n d i v i d u a l muscle fibers by several 3
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Brain Research, 99 (1975) 354 358 ,(~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Consequences of tenotomy on the evolution of multiinnervation in developing rat soleus muscle
PIERRE B E N O I T
AND
JEAN-PIERRE CHANGEUX
Neurobiologie Moldculaire, Ddpartement de Biologie Mol(cutaire, lnstitut Pasteur, 75015 Paris (France)
(Accepted August 12th, 1975)
There exists good experimental evidence6, la that the functional organization of the vertebrate nervous system is under stringent genetic control, although the number o f its identified neuronal circuits and synaptic contacts appears many orders of magnitude larger than the number of genes. In an attempt to solve this paradox, it was suggested that, among other mechanisms, the state of activity o f the developing neuronal network regulates the phenotypic expression of the genes and therefore contributes to the specification of the neuronal network within a low-cost genetic envelope2, 3. In particular, it was assumed that (a) at early stages of development, functional synaptic contacts are present in a larger number than at late stages, (b) a selective stabilization of synapses takes place during maturation and (c) the state o f activity o f a given postsynaptic soma initially controls, in a retrograde manner, the selective stabilization of its afferent synapses. Because o f its simplicity, the neuromuscular junction of vertebrates constitutes a convenient preparation to test the theory. With this system, it is known that (a) at the earliest stages o f development, the contacts established between motor nerves and muscle fibers are functional7,tl, t4, (b) in chick embryo, the chronic block of the neuromuscular synapses by highly specific neurotoxins interfere with their developmerit 4,5 and (c) in chick and rat, the developing muscle fiber receives from different motoneurons several functional nerve terminals which converge at well-defined synaptic spots1,10; in the adult muscle, a single nerve terminal persists per synapse. The aim of the experiments reported in this communication is to study the eventual effect of the activity of developing rat soleus muscle on the evolution of its innervation. Experiments were carried out on soleus muscle from 1- to 3-week-old rats (Sprague-Dawley). The muscle and the corresponding branch of the sciatic nerve were dissected from ether anesthetized animals and kept at room temperature under a continuous flow of oxygenated rat Ringer's solution 9. To facilitate recording of synaptic potentials, the calcium concentration in the physiological solution was raised to 5 m M and 0.5-1.0 × 10 -6 M D-tubocurarine was added to the bath to avoid muscle contraction. The nerve was carefully sucked into a fine micropipette and
355
Fig. l. Example of end-plate potentials obtained by intracellular recording from a curarized soleus muscle of an 8-day-old rat. As the intensity of the stimulus applied to the nerve decreases, the amplitude of the response varies in a stepwise manner. Vertical bar, 2 mV; horizontal bar, 5 msec.
s t i m u l a t e d by short electric pulses o f variable intensity, a p p l i e d between the inside o f the pipette a n d the bath. The p o s t s y n a p t i c p o t e n t i a l s o f individual muscle fibers were r e c o r d e d intracellularly with s t a n d a r d microelectrodes, d i s p l a y e d o n a storage oscilloscope a n d p h o t o g r a p h e d . Fig. 1 shows a typical recording o b t a i n e d with the n e u r o m u s c u l a r j u n c t i o n o f an 8 - d a y - o l d rat. As the strength o f the stimulus progressively increases, the a m p l i t u d e o f the r e c o r d e d e n d - p l a t e p o t e n t i a l increases in a d i s c o n t i n u o u s m a n n e r , t h r o u g h successive steps (3 in this p a r t i c u l a r experiment) instead o f a single step as it does in the a d u l t muscle fiber. Similar observations, r e p o r t e d by R e d f e r n 10 a n d by Bennett a n d Pettigrew 1, reveal a m u l t i i n n e r v a t i o n o f i n d i v i d u a l muscle fibers by several 3
-6 c o
~C2 0 •
0
0
0
0 0 o
E
•
8
o o
•
0
-Q--O----O----
g o
