Spinal cord-muscle relations: their role in neuro-muscular development in birds.

In the present study we focused our attention on the role of spinal cord-muscle interactions in the development of muscle and spinal cord cells. Four experimental approaches were used: 1) muscle fiber-spinal cord co-culture; 2) chronic spinal cord stimulation in chick embryos; 3) direct electrical stimulation of the denervated chick muscle; 4) skeletal muscle transplantation in close apposition to the spinal cord in chick embryos. The characteristics of mATPase and energetic metabolism enzyme activities and of myosin isoform expression were used as markers for fiber types in two peculiar muscles, the fast-twitch PLD and the slow-tonic ALD. In vitro, in the absence of neurons, myoblasts can express some characteristics of either slow or fast muscle types according to their origin, while in the presence of neurons, muscle fiber differentiation seems to be related to the spontaneous rhythm delivered by the neurons. The in ovo experiments of chronic spinal cord stimulation demonstrate that the differentiation of the fast and slow muscle features appears to be rhythm dependent. In the chick, direct stimulation of denervated muscles shows that the rhythm of the muscle activity is also involved in the control of muscle properties. In chick embryos developing ALD, the changes induced by modifications of muscle tension demonstrate that this factor also influences muscle development. Other experiments show that muscle back-transplantation can alter the early spinal cord development.

Role of nerve and tension in maturation of posthatching slow-tonic muscle in chicken.

The role of motor innervation and muscle tension in the posthatching maturation of the slow-tonic anterior latissimus dorsi (ALD) muscle of the chicken has been investigated. Modification of the muscle tension was obtained either by maintaining ALD in a shortened state or by stretching, after or without denervation. In denervated as well as in innervated ALD, shortening resulted in atrophy and inhibition of developmental change in muscle fiber population. In contrast, stretch causes hypertrophy, transformation of all 3B fibers, increase in SM2 isomyosin expression, and decrease in Ca2+-activated myosin ATPase in innervated or denervated ALD. On the other hand oxidative activity in ALD fibers was strikingly reduced after denervation even in presence of stretch-induced hypertrophy. This study suggests that a passive stretch can be involved in some, but not all, changes in ALD characteristics occurring after denervation and may be also involved in normal posthatching development of the slow-tonic muscle. Possible clinical implications of these results in relation to treatments for preventing muscle atrophy resulting from immobilization or disuse are suggested.

Change in motor neurone activity modifies the differentiation of a slow muscle in chick embryo.

Slow-tonic anterior latissimus dorsi (ALD) muscle properties were studied following chronic spinal cord stimulation in chick embryo. Stimulation at a fast rhythm was applied from day 7, 8 or 10 of development until the end of embryonic life. When stimulation was applied from day 7 up to day 18 of development, ALD muscle exhibited at day 18 a large decrease in half time to peak of tetanic contraction, a large proportion of fast type II fibres and an increase in fast myosin light chain content as compared to control muscle. When stimulation started at day 8 of development, changes in properties of ALD muscle were reduced when compared to the previous experimental series. Indeed, no fast type II fibres were observed within the muscle, even when stimulation was prolongated until the 20th day of embryonic development. In addition, chronic stimulation at a fast rhythm initiated at day 10 of development did not modify ALD muscle differentiation. The present results indicate that a fast pattern of motor neurone activity can induce some slow-to-fast transformations of ALD muscle fibres. However, after the first week of embryonic life, ALD myotubes appeared refractory to these transformations. The possible mechanisms responsible for the transformation of slow myotubes and for their further loss of plasticity are discussed.

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 alpha-bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.

Developmental changes in myosin isoforms from slow and fast latissimus dorsi muscles in the chicken.

In the course of muscle differentiation, changes in fibre-type population and in myosin composition occur. In this work, the expression of native myosin isoforms in developing fast-twitch (posterior latissimus dorsi; PLD) and slow-tonic (anterior latissimus dorsi; ALD) muscles of the chick was examined using electrophoresis under nondissociating conditions. The major isomyosin of 11-day-old embryonic PLD comigrated with the adult fast myosin FM3. Two additional components indistinguishable from adult fast FM2 and FM1 isomyosins appeared successively during the embryonic development. The relative proportion of these latter isoforms increased with age, and the adult pattern was established by the end of the 1st month after hatching. Between day 11 and day 16 of embryonic development, PLD muscle fibres also contained small amounts of slow isomyosins SM1 and SM2. This synthesis of slow isoforms may be related to the presence of slow fibres within the muscle. At all embryonic and posthatch stages, ALD was composed essentially of slow isomyosins that comigrated with the two slow components SM1 and SM2 identified in adult. Several studies have reported that the SM1:SM2 ratio decreases progressively throughout embryonic and posthatching development, SM2 being predominant in the adult. In contrast, we observed a transient increase in SM1:SM2 ratio at the end of embryonic life. This could reflect a transitional neonatal stage in myosin expression. In addition, the presence in trace amounts of fast isomyosins in developing ALD muscle could be related to the presence of a population of fast fibres within this muscle.

[Innervation, activity rhythm and accumulation of myosin light chains in a fast muscle of chickens]. / Innervation, rythme d'activité et accumulation des chaînes légères de myosine d'un muscle rapide de poulet.

The fast posterior latissimus dorsi muscle (PLD) of the chick ceases to accumulate slow myosin light chains (MLC) during neonatal development. On day 18 of embryonic life slow MLC represented only 2% of total MLC, and LC3F was first detected. In chick embryo, spinal cord stimulation at a slow rhythm modifies PLD differentiation toward the slow type: LC3F did not accumulate and slow MLC increased. In contrast, stimulation at a fast rhythm accelerated LC3F accumulation. PLD denervation on day 2 after hatching inhibited the synthesis of LC3F. Direct stimulation at a fast rhythm led to post-hatching development into normal fast type while a slow rhythm influenced the development of denervated PLD towards the slow type. In innervated PLD, the effect of stimulation at a slow rhythm was less important than in denervated PLD. These results suggest that the rhythm of the neural and/or contractile activity plays an important role in the MLC expression during embryonic and post-natal development of the chicken 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.

Effects of electrical stimulation upon post-hatching development of fibre types in normally innervated fast and slow latissimus dorsii muscles of the chicken.

In chicken, the main characteristic properties of muscle fibre types in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii are acquired during post-hatching development. At day 4 it becomes possible to distinguish between alpha' and beta' fibre types in ALD muscle. At the same time, mATPase staining and NADH-TR activity permit recognition of alpha w and alpha R fibres within PLD muscle. During further development, muscle fibre typology progressively changes towards the adult slow and fast type. Chronic stimulation at a slow rhythm (5 Hz) of PLD prevents the change in relative proportions of alpha R and alpha W fibres within the muscle that occurs in normal post-hatching development and increases the number of beta R fibres. Moreover, oxidative activity is increased in all muscle fibre types following stimulation. In ALD muscle, chronic stimulation at a fast rhythm (40 Hz) results in a decrease in oxidative activity and inhibits the differentiation of alpha' and beta' muscle fibre types. This study demonstrates that in young chicken, the pattern of activity influences the differenciation of fibre types in slow and fast muscles.

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.

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.

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

Latissimus 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 alpha- and beta-tropomyosin subunits. Later, in the slow muscle the ratio beta:alpha decreased and in chicken the amounts of the two components were about the same. In the fast muscle, the alpha-subunit increased and reached 66% in young chicken. In the fast muscle, the alpha-subunit increased and reached 66% in young chicken. In the 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 rhythm was performed from day 10 of embryonic development to day 15 or 16. In both muscles from spinal cord-stimulated embryos, the beta-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.

Influence of neurons on the occurrence of fibre types and myosin light chains in cultured presumptive slow and fast myoblasts.

Myoblasts from 9-day-old quail embryo slow anterior latissimus dorsi (ALD) and fast posterior and latissimus dorsi (PLD) muscles were co-cultured with neurons. The presence of neurons allowed ALD-derived muscle fibres to express characteristic features of a slow muscle (occurrence of alpha' and of beta' fibres and predominance of slow myosin light chains). On the contrary, PLD-derived fibres did not differentiate into normal fast fibres (occurrence of alpha'-like fibres and absence of LC3f). These results are compared with the differentiation of ALD and PLD myoblasts in aneural condition. It is suggested that neurons can modify some phenotypic expression of presumptive slow or fast myoblasts.

Sensitivity of cultured chick embryo heart cells to acetylcholine. Evidence for nicotinic and muscarinic receptors.

Sensitivity of cultured chick embryo heart cells to acetylcholine changes with time in culture. In 24 h cultures, about 25% of the cells exhibit a positive chronotropic response to acetylcholine. This effect is no longer observed after 48 h in culture. Positive and negative chronotropic effects of acetylcholine can be related to the presence of nicotinic and muscarinic receptors evidenced by autoradiography. Some data suggest a possible relationship between the type of sensitivity to acetylcholine and the changes in cell membrane properties occurring in culture.

Developmental change in choline acetyltransferase activity in nerve endings of latissimus dorsii muscles in the chick embryo: influence of chronic spinal cord stimulation.

Choline acetyltransferase (CAT) activity of chick latissimus dorsii muscles was studied during embryonic development and at post-hatching states. CAT activity was always higher in anterior (ALD) than in posterior (PLD) muscles. At embryonic stages, chronic spinal cord stimulation at a low rhythm did not modify CAT activity in ALD nerve endings but caused a transient increase in PLD terminals. This increase in CAT activity seems to be related to an acceleration of neuronal maturation rather than to the occurrence of the multiple innervation that results from the central stimulation.

In vitro differentiation in the absence of nerve of avian myoblasts derived from slow and fast muscle rudiments.

Slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of 9-day-old quail embryos were cultured in vitro without neurons for 1 to 12 weeks. Several differences could be observed between ALD- and PLD-derived cells. PLD cultures proliferated less rapidly than ALD cultures. ALD-derived muscle fibres exhibited wide Z lines, numerous mitochondria, and a poorly developed sarcotubular system, while PLD-derived muscle fibres exhibited narrow Z lines, few mitochondria, and an abundant sarcotubular system. Staining for myofibrillar ATPase revealed that all well-differentiated ALD-derived muscle fibres were of the beta' type, while PLD-derived fibres were of beta and beta R types. These results show that myoblasts from slow and fast muscle rudiments can express in vitro some of the characteristic features of slow and fast muscle fibres, independently of motor innervation.

Influence of chronic spinal cord stimulation upon differentiation of beta 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.

[Effect of a return of motor neurons to spontaneous activity on the multi-innervation of muscular fibers induced in chick embryo by chronic slow-rhythm spinal cord stimulation]. / Effet d'un retour à l'activité spontanée des motoneurones sur la multi-innervation de fibres musculaires provoquée chez l'embryon de poulet par une stimulation médullaire chronique à rythme lent.

The multi-innervation of posterior latissimus dorsi muscle caused in Chick embryo by chronic spinal cord stimulation at low rhythm (0.5 HZ) from the 10th until 14th day of incubation remains but does not increase if the stimulating period is prolonged. If the stimulating period is stopped and the embryo allowed to develop for several more days the number of acetylcholinerase sites on the muscle fibres decreases. This result suggests that the multi-innervation regresses after the cessation of the experimentally-imposed nervous signal.

Histochemical properties of the biventer cervicis muscle of the chick: a relationship between multiple innervation and slow-tonic fibre types.

Chick biventer cervicis muscle fibres have been studied histochemically. Fast-twitch, focally innervated (alpha) fibres represent 70-80% of the total fibres in this muscles. Two histochemical profiles of slow-tonic multi-innervated (beta) fibres have been observed from embryonic life the adult (three-months) stage. These two slow-tonic types differ in the activity of their histochemically demonstrated myofibrillar ATPase after either acid or alkaline preincubation, and after formalin fixation. Both slow-tonic fibre types have a high oxidative metabolism and are PAS-negative. They are referred as to beta 1 and beta 2R fibre types (slow-tonic oxidative) in an expansion of Ashmore's nomenclature, and compared to avian slow-tonic sub-types that have been described in recent reports. beta 1 and beta 2 fibre types exhibit a similar pattern of innervation. Possible explanations of the origin of histochemical heterogeneity in multiple innervated fibres are discussed.

[Effects of chronic medullary stimulation on the total number of sites of acetylcholinesterase activity of the posterior latissimus dorsi muscle of chick embryo]. / Effet de la stimulation médullaire chronique sur le nombre total de sites d'activité acétylcholinestérasique du muscle posterior latissimus dorsi de l'embryon de Poulet.

From incubation day 10 to 15, the spinal cord of Chick embryos are electrically stimulated in ovo at 0.5 Hz at the level of the motor roots innervating the latissimus dorsi muscles. As a consequence, the total number of spots of acetylcholinesterase determined on serial sections of posterior latissimus dorsi muscle increased 2.3 fold. This increase parallels that of acetylcholine receptor clusters, supporting the interpretation that these clusters are of synaptic origin.