Didier Renaud

Enzymatic differentiation of muscle fibre types in embryonic Latissimus dorsii of the chick: effects of spinal cord stimulation

Abstract Myofibrillar ATPase differentiation in anterior latissimus dorsi (ALD) and posterior latissimus dorsii (PLD) muscles has been studied in chick embryo. At stage day 8, ALD fibres have acid-stable ATPase while the enzymatic activity is acid-labile in PLD fibres. In older stages, PLD exhibits α and β fibres which both have a single spot of acetyl cholinesterase (ACHE) activity. Electrical spinal cord stimulation performed for several days from day 10 of incubation leads to an increase in β fibre-type ratio in PLD, these β fibres exhibit a multiplicity of ACHE spots and are assumed to be multiinnervated.

Spinal cord stimulation in chick embryo: Effects on development of the posterior latissimus dorsi muscle and neuromuscular junctions

Abstract The purpose of this work was to study whether or not stimulation of spinal cord at a critical stage of development in chick embryo would result in a modification of neuromuscular differentiation. The electrodes were implanted anteriorly and posteriorly to the level of motor innervation of the wing. Stimulation was delivered from the 10th day of development, and the posterior latissimus dorsi muscle was studied at Day 14 for cholinesterase activity and innervation or at Day 17 for mechanical activity. In normal development of chick embryo, the posterior latissimus dorsi differentiates into a fast muscle whose fibers have a single end plate. In spinal cord-stimulated embryos, we observed a multiplicity of sites of cholinesterase activity and multi innervation in the posterior latissimus dorsi muscle fibers. Moreover, speeds of contraction and relaxation were slower than in control muscles. These results show that electrically induced activity of motoneurons at a critical stage of embryogenesis modifies the development of the neuromuscular system. Possible explantions of this phenomenon are discussed.

Chronic stimulation of the spinal cord in developing chick embryo causes the differentiation of multiple clusters of acetylcholine receptor in the posterior latissimus dorsi muscle

Abstract Electrodes were implanted around the spinal cord of 7-day-old chick embryos and electric pulses delivered at 0.5-Hz frequency from the 10th to 15th day of incubation. At Day 15, the posterior latissimus dorsi (PLD) muscle, which, in control animals, is focally innervated, was dissected. The number and distribution of AChR clusters revealed by autoradiography after labeling with 125I-α-bungarotoxin was quantitatively studied on isolated muscle fiber fragments and on serial sections of the whole muscle. After chronic stimulation, muscle fibers with multiple AChR clusters were observed. The distribution of the clusters appeared less regular than in the anterior latissimus dorsi muscle which, in control embryos, receives a multiple innervation. The total number of AChR clusters per PLD muscle increased about 1.8 times as a consequence of the stimulation without significant change of the total number of muscle fibers.

Influence of chronic spinal cord stimulation upon differentiation of β muscle fibers in a fast muscle (posterior latissimus dorsi) of the chick embryo

The aim of this work was to study the influence upon differentiation of muscle fiber types of the multiple innervation induced in a fast muscle by chronic spinal cord stimulation. In previous work, we showed that low-frequency stimulation applied to the spinal cord of the chick embryo caused a distributed innervation of muscle fibers in the posterior latissimus dorsi (PLD). In normal development, some beta fibers differentiate within this muscle, the maximal number being attained by 14 to 15 days of embryonic development. Later, the numbers of beta fibers decreased with age. In spinal cord-stimulated embryos the beta muscle fibers within the PLD were stabilized and did not disappear. After the cessation of spinal cord stimulation, the number of beta fibers within the PLD muscle did not decrease. There are possible explanations of the influence of chronic spinal cord stimulation and muscular activity upon formation and persistence of beta fibers within a fast muscle.

Effects of low and high frequency patterns of stimulation on contractile properties, enzyme activities and myosin light chain accumulation in slow and fast denervated muscles of the chicken

The effects of denervation and direct stimulation in fast and slow latissimus dorsii muscles were investigated in chicken. In slow ALD muscle, denervation resulted in an incompleteness of the relaxation, a decrease in MDH and CPK activities and an increase in fast myosin light chains (MLC) accumulation. Direct stimulation at either fast or slow rhythm prevented the effects of denervation on relaxation and CPK activity but was ineffective on MDH activity and fast MLC accumulation. Moreover, direct stimulation of denervated ALD caused rhythm-dependent change in tetanic contraction. In fast PLD muscle, the main changes in muscle properties following denervation were a slowing down of the time course of the twitch and an incompleteness of the relaxation, a decrease in LDH and CPK activities and in LC3F accumulation. Stimulation at a high frequency partly prevented the effects of denervation and resulted in a large accumulation of LC3F, while a low frequency stimulation did not restore the twitch time to peak, increased MDH activity and induced synthesis of slow MLC. This study emphasizes the role of muscle activity and its pattern in some properties of slow and fast chicken muscles following denervation.

Influence of spinal cord stimulation upon myosin light chain and tropomyosin subunit expression in a fast muscle (posterior latissimus dorsi) of the chick embryo

SummaryLatissimus dorsi muscles of the chick consist of a slow (ALD) and a fast (PLD) muscle. The influence of chronic spinal cord stimulation in the chick embryo upon the expression of myosin light chains and tropomyosin subunits was investigated. Early in development the two muscles exhibited the same ratio of α- and β-tropomyosin subunits. Later, in the slow muscle the ratio β:α decreased and in chicken the amounts of the two components were about the same. In the fast muscle, the α-subunit increased and reached 66% in young chicken. In the early stages of embryonic development, both muscles accumulated slow and fast light chains. However, in ALD the amount of slow light chains was greater than that of fast light chains and the reverse was observed in PLD muscle. Later during development, the slow components decreased in PLD while the fast components increased; the reverse was observed in ALD muscle. The fast myosin LC3f has been detected in 18-day-old embryonic PLD.Chronic spinal cord stimulation at a low rhytm was performed from day 10 of embryonic development to day 15 or 16. In both muscles from spinal cord-stimulated embryos, the β-tropomyosin subunit was lower than in control embryos. In ALD, the pattern of light chains was unaffected by chronic stimulation while in PLD muscle the slow and fast components were modified. In particular the ratio LCs:LCf was increased in spinal cord-stimulated embryos with regard to controls.

Effects of spinal cord stimulation on the differentiation of posterior latissimus dorsi nerve in the chick embryo

Abstract Spinal cord stimulation was carried out during embryonic development of the chick from the 10th day until 13.5, 14.5, or 15.5 days of embryonic life. The effects of this superimposed neuronal activity upon the cytodifferentiation of the nerve entering the posterior latissimus dorsi (PLD) muscle were studied by comparative analysis of electron micrographs of PLD nerves from control and spinal cord-stimulated embryos. For each embryonic stage studied, electrical stimulation of the spinal cord appeared to accelerate the growth of axons, to enhance Schwann cell mitosis, and the enclosure of a single axon in a Schwann cell, i.e., the process of myelination. These results underline the role of neuronal activity in the maturation of a developing nerve.

Effects of denervation and direct electrical stimulation upon the post-hatching differentiation of posterior latissimus dorsi muscle in chicken

The influences of denervation and of direct electrical stimulation of denervated muscle upon the post-hatching differentiation of fibre types in the fast avian muscle posterior latissimus dorsi have been investigated. Denervation inhibits the normal decrease in number of muscle fibres exhibiting acid-stable myofibrillar ATPase activity and leads to weak oxidative activity in all the fibres. Direct stimulation at a low rhythm of denervated muscle induces the normal decrease of fibres exhibiting acid-stable myofibrillar ATPase but does not allow the occurrence of normal oxidative activity pattern. The results emphasize the role of muscular activity upon the differentiation of fibre types in a developing muscle.

Effect of spinal cord stimulation on the metabolism of developing latissimus dorsii muscles in chick embryo

Influence of chronic spinal cord stimulation upon some characteristic enzyme activities of energy metabolism was investigated in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii muscles of the chick embryo. During embryonic life, oxidative metabolism (as evaluated by the activity of malate dehydrogenase (MDH] represents the main energetic pathway in both slow and fast muscles. At the end of embryonic life, an increase in anaerobic (as evaluated by the activity of lactate dehydrogenase (LDH] and creatine phosphokinase (CPK) activities occurs in PLD muscle. Chronic spinal cord stimulation at a low frequency was performed from the 10th day to the 16th day of embryonic development. In ALD, the enzyme activities were unaffected, while in PLD a concomitant decrease in LDH and CPK activities was observed.

Nicotinic and muscarinic responses of embryonic chick ventricular myocardium to acetylcholine: chronotropic and electrophysiological effects

Sensitivity of 7‐day‐old chick embryo ventricular heart fragments to acetylcholine was investigated. Low doses mainly produced a positive chronotropic effect, whereas high doses of acetylcholine provoked a decrease in the heart beat rhythm. The positive chronotropic effect of acetylcholine was related to the presence of nicotinic receptors that were evidenced within ventricular myocardium by autoradiography. Membrane potential recording showed that acetylcholine hyperpolarizes the diastolic membrane potential when the drug had a negative chronotropic effect. This effect of acetylcholine on the membrane potential was not observed when the drug had a positive chronotropic effect. In many cases, the diastolic membrane potential exhibited spontaneous small depolarizing potentials. Their amplitude was low and their frequency was irregular. These potentials were suppressed by treatment with α‐bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.