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.

In adult mammals, conversion of fast muscle towards the slow type is obtained by reinnervating the muscle with a slow nerve or by chronic stimulation of the motor nerve at a slow rhythm (for review, see Pette & Vrbovà, 1985). In contrast, slow-twitch soleus exhibits minor alterations when reinnervated by fast motor neurones (Dum et al. 1985; Gordon et al. 1986), or submitted to chronic indirect stimulation at a fast rhythm (Brown et al. 1976). The differential response of fast and slow muscles after such experimental procedures has been attributed to an intrinsic lack of plasticity of slow muscle fibres (Foehring et al. 1987).

In adult birds, when the innervation of the fast-twitch posterior latissimus dorsi (PLD) muscle is supplied by the nerve of the slow-tonic anterior latissimus dorsi (ALD) muscle and vice versa, most muscle characteristics remain unchanged (Koenig & Fardeau, 1973). However, when nerves are exchanged in newly hatched chickens, structural (Bennett & Pettigrew, 1974b), biochemical (Syrovy & Zelenà, 1975) and histochemical features (Cosmos et al. 1979) of fast PLD muscle resemble slow-tonic ALD properties after a few weeks.

Chronic indirect stimulation at a slow rhythm during four weeks also causes a partial fast-to-slow transformation in chicken PLD muscle (Barnard et al. 1986). Nevertheless, even during development, slow-tonic ALD is resistant to control by fast PLD nerve after cross-reinnervation in chicken and slow characteristics still persist in this muscle even months after operation (Hnik et al. 1961’, Jirmanovà et al. 1971; Jirmanovà & Zelenà, 1973). This finding has been interpreted as a result of reinnervation of chicken ALD muscle by slow fibres present in PLD nerve (Hnik et al. 1967). Alternatively, according to Kikuchi et al. (1986), the two muscles may contain inherent characteristics which, in some instances, prevent their complete conversion by a foreign nerve.

However, it is noteworthy that plasticity of a fast muscle is largely increased when the rhythm of nerve activity is altered earlier in the development, i.e. during embryogenesis. Indeed, the changes of fast properties towards the slow type that are obtained after several weeks of chronic indirect stimulation at a low frequency in chicken PLD can be produced in a few days in chick embryo. Spinal cord stimulation at a slow rhythm causes in embryonic PLD a slowing down in speed of contraction (Renaud et al. 1978), prevents the regression of embryonic slow fibres (Renaud et al. 1983) and induces a decrease in fast myosin light chains content (Gardahaut et al. 1985). The question arises whether ALD muscle exhibits a strong resistance to transformation after alteration of motor innervation because its slow properties are not under motor nerve control. Alternatively, it is possible that changes in this muscle can be induced only during early embryonic development.

The aim of this study was to investigate this problem. For this purpose, we choose to analyze the role of the rhythm of motor neurone activity on the differentiation of embryonic slow-tonic ALD muscle. Spinal cord stimulation at a fast rhythm was imposed during early embryogenesis and the effects on contractile properties, histochemical features and myosin light chain pattern of slow ALD muscle were studied.

Experiments were performed on the slow-tonic anterior latissimus dorsi (ALD) muscle of outbred White Leghorn chick embryos.

Chronic spinal cord stimulation

The method for chronic electrical stimulation of the spinal cord has been described elsewhere (Renaud et al. 1978). Briefly, perimedullary electrodes were implanted in chick embryo at day 6 in ovo and the stimulation was initiated at day 7, 8 or 10 according to the experimental series. Pulses of 5 ms duration were delivered at 30Hz frequency in Is bursts. The bursts were repeated every 8 s. Four experimental series were performed: 1) Stimulation from day 7 up to day 18 of embryonic development, 2) from day 8 up to day 18, 3) from day 8 up to day 20 and 4) from day 10 up to day 18. Stimulation efficiency was verified by two methods (Toutant et al. 1980). First, ALD muscles of some experimental embryos were exposed by excision of the skin at day 7, day 8 or day 10 of the embryonic period and their contraction resulting from the stimulations was directly observed. Second, stimulation efficiency was daily ascertained in ovo by observation of the movements of the wings induced by the bursts of stimuli. If necessary, the stimulus voltage was adjusted to maintain effective stimulation. In a preliminary series of experiments, 200 μ1 curare solution (10 μgμl−1) was dropped onto the chorioallantoic membrane of day 7, day 8 or day 10 embryo. In all stages, the curarization of the embryo suppressed the response to electrical pulses which indicates that the contraction of ALD muscle did not result from direct electrical stimulation but was neurally evoked. In control embryos, perimedullary electrodes were implanted but no electrical stimulation was delivered.

Measurement of contraction parameters

ALD muscles from control and stimulated embryos of the same stage were removed after decapitation and immediately placed vertically between two horizontally aligned silversilver chloride electrodes. The chamber contained Krebs-Henseleit solution (pH 7·4) at 28°C gassed with 95 % O2–5 % CO2 mixture. Isometric contraction was recorded using a mecanoelectric transducer (RCA 5734) as described by Khaskiye et al. (1987). The muscle was set to the length at which maximal tension was produced by stimulation (40 Hz frequency, pulses of 2 ms duration). Tetanic contraction was recorded and maximal tetanic tension and tetanic half time to peak ( ttp) were measured. No noticeable change was observed between successive muscle responses elicited by stimulations separated by 10 min rest interval.

Histochemical procedures

After measurement of contractile properties, muscles were immediately frozen in isopentane cooled by liquid nitrogen. Cryostat-cut sections (15 μm thick) were collected on glass slides. Myofibrillar ATPase activity was revealed according to the method described by Guth & Samaha (1970). For each muscle, two serial transverse sections from the largest part of the muscle were used, one with acid preincubation (pH 4·2) and the other with alkaline preincubation (pH 10·4). For each histochemical reaction, transverse sections from experimental and control muscles were collected on the same slides in order to rule out the possibility of artefactual differences resulting from the histochemical procedures. The nomenclature defined by Barnard et al. (1982) was used to classify fibre types in ALD muscle. Quantitative evaluation of fibre population within the muscle was performed by counting the total number of fibres and the number of fibres from different types in the entire transverse section of the largest part of ALD muscle.

Electrophoretic analyses of myosin light chains (MLC)

After revelation of myosin ATPase activity by histochemistry, muscles were selected for MLC analysis. In experimental series in which muscles exhibited an abnormal fibre type profile, ALD that presented less than 20 % modified muscle fibres (type II or intermediate fibres described in Results) when compared to control were discarded. Samples of about 200 mg (wet wt.) of muscles were frozen in liquid nitrogen. A sample required 42 muscles from 18-day-old control embryo, 30 muscles from 20-day-old control embryo. For experimental series, a sample required 30 muscles from embryos stimulated from day 7 up to day 18, 36 muscles from embryos stimulated from day 8 up to day 18, 28 muscles from embryos stimulated from day 8 up to day 20 and 38 muscles from embryos stimulated from day 10 up to day 18. Actomyosin was extracted according to the procedure described elsewhere (Gardahaut et al. 1985). Two-dimensional gel electrophoresis was carried out according to O’Farrell’s method (1975), using ampholines (LKB) at 1·6% (pH5–8) and 0·4% (pH3·5–10) for the first dimension. The second dimension gels were 15 % polyacrylamide-SDS cross-linked with 0-1% bis-acrylamide. Gels were stained with Coomassie Blue, then destained in a methanol/acid acetic/water mixture. Spots of MLC were identified by comigration with purified myosin extracted from adult muscles according to the method of Weeds et al. (1974). The quantitative analysis of MLC was performed by using a Vernon densitometer equipped with an automatic integrator. A low-density wedge (0·2) and a wide-band filter (550 nm) were used in order to obtain optimal sensitivity. Optical density of each MLC detected within the gel was expressed as a percentage of the optical density in all MLC. For each experimental series, one sample was produced allowing 2-to 4-gel electrophoresis depending on protein concentration (n in table expresses the number of gels used in experimental and control series).

Statistical analyses

Statistical analyses were performed by Student’s t-test with a P of 0·05 for tests of significance.

A total of 320 embryos was subjected to experimental manipulation. The rate of mortality between day 6 and the end of embryonic development was similar in both experimental and control series (60%), as a result of surgical trauma. Embryos without obvious abnormalities (e.g. scoliosis) were selected for this study (n = 70 for experimental series and n = 36 for control). The time course of development was slightly delayed in control or stimulated embryos, with regards to gross morphology. However, the growth of ALD muscle was much greater in all experimental series than in normal embryos, as reflected by muscle fibre diameter. Indeed, although this criterion did not significantly differ in 18-day-old implanted and normal embryos (17 ±4μm, n = 7 versus 19 ± 1 μm, n = 7), muscle fibre diameter was greater in ALD of stimulated embryos than in normal muscle, in all experimental series (23±2μm, n = 9 for 7–18 days; 23·5 ± 2 μm for 8–18 days; 23±2μm for 8–20 days; 22 ± 1μm for 10–18 days versus 19±lμm). In addition, the number of muscle fibres was not significantly different in ALD of normal and experimental embryos. At day 18 of embryonic development, this number was 4759 ± 360 (n = 7) in stimulated embryos versus 4408 ± 382 (n = 7) in normal embryos. These values were not noticeably different from the data given by Bourgeois & Toutant (1982).

Characteristics of ALD muscle from control embryo at the end of development

At day 18 of embryonic development, myosin-ATPase activity of most fibres from slow-tonic ALD muscle was characterized by a resistance to both alkali and acid preincubations (Fig. 1B,G). Thus, ALD muscle was nearly entirely composed of type III fibres according to the classification of Barnard et al. (1982). In addition, rare fibres exhibited myosin-ATPase activity that was inhibited after acid preincubation (Fig. 1A,F). These type II fibres (according to the nomenclature of Barnard et al. 1982) were mostly located at the anterior part of the muscle (Fig. 1A,F). In 20-day-old embryo, ALD muscle exhibited a similar histochemical profile to ALD of 18-day-old embryo, composed of type III fibres, except for the presence in some cases of scarce fibres that displayed a weak myosin-ATPase activity after both alkali and acid preincubations (type IIIA fibres according to the nomenclature of Barnard et al. 1982).

Fig. 1.

Staining for myofibrillar ATPase activity in ALD muscle fibres of chick embryo. On the left: alkaline preincubation at pH 10·4. On the right: acid preincubation at pH 4·2. (A,F) Serial cross-sections from 18-day-old control embryo: anterior part of the muscle; Heads of arrows indicate some type If fibres. (B,G) Serial cross-sections from 18-day-old control embryo in another pan of the muscle. In this area, no type II fibres is present. (C,H) Serial cross-sections from 18-day-old stimulated embryo (7 to 18 days). Heads of arrows indicate some type II fibres. (D,I) Serial cross-sections from 18-day-old stimulated embryo (8 to 18 days). Arrows indicate some intermediate fibres. (E,J) Serial cross-sections from 20-day-old stimulated embryo (8 to 20 days). Arrows indicate some intermediate fibres. Scale bar: 50μm for all the photographs.

Fig. 1.

Staining for myofibrillar ATPase activity in ALD muscle fibres of chick embryo. On the left: alkaline preincubation at pH 10·4. On the right: acid preincubation at pH 4·2. (A,F) Serial cross-sections from 18-day-old control embryo: anterior part of the muscle; Heads of arrows indicate some type If fibres. (B,G) Serial cross-sections from 18-day-old control embryo in another pan of the muscle. In this area, no type II fibres is present. (C,H) Serial cross-sections from 18-day-old stimulated embryo (7 to 18 days). Heads of arrows indicate some type II fibres. (D,I) Serial cross-sections from 18-day-old stimulated embryo (8 to 18 days). Arrows indicate some intermediate fibres. (E,J) Serial cross-sections from 20-day-old stimulated embryo (8 to 20 days). Arrows indicate some intermediate fibres. Scale bar: 50μm for all the photographs.

At the end of embryonic development (18- and 20-day-old embryos), fibres from slow-tonic ALD contained a high proportion (about 80%) of slow (LCiS and LC2S) MLC while fast (LC1 and LC2F) MLC content was low (about 20%), (Table 2, Fig. 2).

Table 2.

Light chain composition (mean ± S.E.M.) of myosin from embryonic ALD muscle. Each myosin light chain (MLC) is expressed as a percentage of the total MLC

Light chain composition (mean ± S.E.M.) of myosin from embryonic ALD muscle. Each myosin light chain (MLC) is expressed as a percentage of the total MLC
Light chain composition (mean ± S.E.M.) of myosin from embryonic ALD muscle. Each myosin light chain (MLC) is expressed as a percentage of the total MLC
Fig. 2.

Two-dimensional gel electrophoresis of actomyosin prepared from ALD muscles of 18-day-old embryos.(A) Control muscle; (B) muscle from stimulated embryo (7 to 18 days).

Fig. 2.

Two-dimensional gel electrophoresis of actomyosin prepared from ALD muscles of 18-day-old embryos.(A) Control muscle; (B) muscle from stimulated embryo (7 to 18 days).

Effects of early (7 to 18 and 8 to 18 days) embryonic spinal cord stimulation at a fast rhythm on ALD muscle

When spinal cord stimulation at 30 Hz frequency was delivered from day 7 up to day 18 of embryonic development, ALD muscle exhibited a 50% decrease in ttp of tetanic contraction when compared to control (Table 1). This change in contractile properties of slow muscle was less important when stimulation was initiated one day later during development. Indeed, ttp of tetanic contraction decreased by 30 % in ALD of embryos stimulated from day 8 up to day 18 of development when compared to control muscle (Table 1).

Table 1.

Parameters of tetanic contraction of embryonic ALD muscle

Parameters of tetanic contraction of embryonic ALD muscle
Parameters of tetanic contraction of embryonic ALD muscle

Revelation of myosin ATPase activity showed a modified fibre type profile in ALD muscle of stimulated embryos. When spinal cord stimulation at a fast rhythm started at day 7 of development, ALD of 18-day-old embryos contained a great proportion of fibres in which this enzymic activity was completely inhibited after acid preincubation (type II fibres according to Barnard et al. 1982), (Fig. 1C,H). These fibres represented about 30% (1410 ± 173) of the total muscle fibre population (4759 ±360). In contrast, ALD muscle of embryos stimulated from day 8 up to day 18 of development was characterized by the occurrence of fibres in which myosin-ATPase activity was not completely inhibited after acid preincubation (Fig. 1D,I). Indeed, after this treatment, enzymic activity revealed that 30% (1503 ± 164) of the total number of ALD muscle fibres (4759 ± 360) were lightly stained. These acid-labile fibres could be classified as ‘intermediate’ fibres.

MLC pattern was also modified in ALD muscle of embryos submitted to spinal cord stimulation. Stimulation at 30 Hz frequency delivered from day 7 up to day 18 of embryonic development causes a 90 % increase in fast MLC content when compared to control muscle (38% versus 20%), (Table 2, Fig. 2). However, when spinal cord stimulation was initiated at day 8 of develop-ment, synthesis of fast MLC in slow ALD of 18-day-old stimulated embryos was only increased by 45 % when compared to control (29% versus 20%), (Table 2, Fig. 2). In both experimental series, changes affected phosphorylated myosin light chains (LC2S and LC2F) while alkali myosin light chains (LCiS and LQF) were not modified (Table 2).

Effects of the duration of the spinal cord stimulation period on ALD muscle

In order to determine whether the differences between ALD muscles of embryos stimulated from day 7 up to day 18 and embryos stimulated from day 8 up to day 18 could be attributed to the differences between durations of stimulation periods, we analyzed the effects of spinal cord stimulation applied from day 8 until the end of the embryonic period.

When spinal cord stimulation at 30 Hz frequency was applied from day 8 up to day 20 of incubation, ttp of tetanic contraction was decreased (about 40 %) in ALD muscle when compared to control (Table 1). In addition, an important proportion (about 45%) of acid labile ‘intermediate’ fibres was observed within ALD muscle (Fig. 1E,J). However, type II fibres were never found in ALD of this experimental series. On the other hand, fast MLC content was markedly increased when compared to control (Table 2, Fig. 2). Nevertheless, when the stimulation was initiated at embryonic day 8, the proportion of fast MLC in ALD muscle was no higher after twelve days of stimulation (26 %) than after ten days (29%). It is noteworthy that a stimulation applied from day 7 up to day 18 of development resulted in a higher amount of fast MLC (38 %) in ALD muscle.

Effects of late (10 to 18 days) embryonic spinal cord stimulation at a fast rhythm on ALD muscle

When spinal cord stimulation at 30 Hz frequency was initiated at day 10 of embryonic development, properties of ALD muscle did not differ between 18-day-old stimulated and control embryos. Indeed, parameters of tetanic contraction (Table 1), myosin-ATPase activity of muscle fibres and MLC content (Table 2) were not modified by spinal cord stimulation in this experimental series.

Our results show that chronic spinal cord stimulation at a fast rhythm performed from day 7 in chick embryo modifies the development of ALD muscle. The changes comprise a speeding up of tetanic contraction, a great proportion of fast type II fibres and a large increase in fast MLC content. Thus, a fast rhythm of motor neurone activity can produce a change of this embryonic slow muscle towards the fast type.

Nerves first enter the muscle mass from which the ALD forms at about 4 days of incubation (Bennett et al. 1983). At day 12 of embryonic development, nearly all acetylcholine receptor clusters are contacted by nerve terminal profiles (Smith & Slater, 1983). Although the frequency of activation of slow and fast motor units has not been analyzed at early stages of development, O’Donovan (1984) has observed distinct phasic or tonic activation patterns in embryonic motor neurones at day 12 of development. Thus, embryonic chick motor neurones may have different frequencies of firing and could provide differential signals for fibre type differentiation. Indeed, our results show that embryonic myofiber transformation towards the fast type can be induced by a fast pattern of motor neurone activity.

It has been suggested that fibre type specialization during early development is intrinsically determined. Primary myotubes exhibit differences in myosin-ATPase histochemistry (Butler & Cosmos, 1981; McLennan, 1983a,b) and myosin isoform types (Miller & Stockdale, 1986; Stockdale & Miller, 1987; Crow, 1987) as early as day 5 of embryonic development. These characteristics can differentiate without innervation (Butler et al. 1982; Phillips & Bennett, 1984; Sohal & Sickles, 1986; Phillips et al. 1986) or in the absence of neuromuscular transmission (Sohal & Sickles, 1986; Crow & Stockdale, 1986). However, it has been shown that innervation is necessary to maintain the production of secondary myotubes which differentiate later during embryonic development (Harris, 1981; McLennan, 1983c; Crow & Stockdale, 1986; Ross et al. 1987). In embryonic ALD muscle, this generation of myotubes contains both fast and slow myosin isoforms and contributes to the differentiation of type II fibres observed in adult (Schafer et al. 1987; Miller & Stockdale, 1987; Grove & Thornell, 1988). Consequently, when spinal cord stimulation at a fast rhythm is applied to 7-day-old embryos, it could be hypothesized that fast motor neurone activity induces a transformation of secondary myotubes from future type III fibres to future type II fibres. In this case, the frequency pattern of nerve activity would be one important factor that controls the differentiation of secondary myotubes towards fast or slow types during embryogenesis.

In order to know whether a foreign nerve was capable of inducing a change in muscle fibre types, heterotopic innervation of brachial and hindlimb muscles has been done (Khaskiye et al. 1980; Laing & Lamb, 1983; Butler et al. 1986; Vogel & Landmesser, 1987). Thus, Butler et al. (1986) have shown that when slow-tonic ALD muscle is allowed to be innervated by thoracic motor neurones, a compatible union initially exists between the foreign nerve and the muscle, but a subsequent loss of intramuscular nerve branches occurs, resulting in an atrophy of the muscle as early as day 9 of incubation. However, when brachial neural tube is replaced by a lumbosacral segment, normal differentiation of ALD and PLD muscles has been maintained until day 18 of embryonic development (Khaskiye et al. 1980). These experiments clearly show that, although motor neurones arising from different spinal cord segments do not seem to exhibit the same capability to establish durable contacts with muscle fibres, they never induce modifications in muscle fibre types. Cauwenbergs et al. (1986) have determined that during development, wing and leg motility in chick embryo does not exceed 32 movements per min. Based on the fact that, from day 6 to day 16 of the embryonic period, limb movements in the chick are neurogenic in origin and correlated with bursts of activity in developing spinal cord (Provine, 1972,1973), it could be hypothesized that chronic spinal cord stimulation at 30 Hz frequency (1 s bursts every 8 s) would increase motor neurone activity in brachial spinal cord and cause fibre-type changes in ALD muscle. This would not occur in experiments of heterotopic innervation.

However, it is noteworthy that experimental change in parameters of muscle innervation other than its rhythm of activity can modify slow muscle differentiation. Gauthier et al. (1984, 1987) have demonstrated that a change in embryonic pool of motor neurones caused by an inhibition of neuronal cell death after curarization results in a change in the differentiation of ALD muscle in which numerous fibres present low myosin-ATPase activity after acid preincubation. It has been suggested by the authors that curarization of the embryo results in a survival of ‘inappropriate’ motor neurones that normally disappear during cell death period and it can be hypothesized that these ‘foreign’ motor neurones have a rhythm of activity different from motor neurones normally innervating slow ALD muscle.

Plasticity of ALD muscle fibres decreases as embryonic development proceeds. When spinal cord stimulation of the chick embryo was initiated at day 8, the speeding up of tetanic contraction in ALD was lower than when stimulation started one day before. In addition, we failed to detect fast type II fibres in muscles of this experimental series. Indeed, ALD of embryos stimulated at day 8 always exhibited ‘intermediate’ acid-labile fibres, even when stimulation was prolongated until day 20 of development. On the other hand, ALD muscles of both experimental series (8–18 days and 8–20 days) exhibited a lower content in fast MLC than ALD of embryos in which stimulation was initiated, at day 7. It is noteworthy that, in all experimental series, spinal cord stimulation only changes phosphorylated MLC in ALD muscle. It has been already shown that, in fast muscles, indirect stimulation first modifies phosphorylated MLC (Brown et al. 1983; Gardahaut et al. 1985).

Finally, when spinal cord stimulation at a fast rhythm started at day 10 of incubation, no change was observed at day 18 in contractile properties, histochemical profile and MLC components of ALD muscle. Consequently, our results show that the ability of ALD myotubes to transform into fast and slow types is restricted to a precise period of embryogenesis, i.e. between day 7 and day 10. It could be argued that the lack of effect observed when stimulation (bursts of 30 Hz frequency) started at day 10 of embryonic period could be related to the lower efficiency of this pattern of stimulation as development proceeds. However, this explanation is unlikely because different patterns (10 Hz, 30 Hz and 40 Hz) were applied from day 10 up to day 18 of embryonic development and failed to modify contractile properties, fibre types and MLC pattern of ALD muscle (unpublished data).

In conclusion, our results show that change in the rhythm of motor neurone activity can modify the differentiation of ALD muscle during embryogenesis, but only when it is initiated during early muscle differentiation, i.e. before day 10. The factors responsible for this loss of plasticity of ALD myotubes remain to be determined. It might be hypothesized that the establishment of a particular pattern of innervation of ALD muscle fibres is involved in this phenomenon. Indeed, the distributed innervation characteristic for type III fibres is already established in this muscle at day 9 of embryonic development, although the distance between synaptic sites increases towards the mature value until hatching period (Bennett & Pettigrew, 1974a; Phillips et al. 1985). Alternatively, the capability of secondary myotubes to differentiate into future type III or type II fibres could be restricted to the period of early maturation of these fibres. Further investigations are required to clarify the role of these different factors.

We would like to express our sincere thanks to Dr T. Rouaud for his valuable help throughout this study. This work was supported by a grant from Association Française contre les Myopathies and by Centre National de la Recherche Scientifique (URA n° 1340).

Les propriétés du muscle lent anterior latissimus dorsi (ALD) ont été étudiées après stimulation de la moelle épinière chez l’embryon de poulet. Une stimulation chronique à rythme rapide est délivrée à partir du 7ème, 8ème ou 10ème jour du développement jusque vers la fin de la période embryonnaire. Quand la stimulation est appliquée du 7ème au 18ème jour du développement, le muscle présente une forte diminution du demi-temps de contraction tétanique maximum, une quantité importante de fibres rapides de type II et une augmentation du contenu en chaÎnes légères rapides de la myosine. Quand la stimulation à rythme rapide débute au 8ème jour du développement, les changements des propriétés du muscle sont moins importants que ceux qui sont observés lorsque la stimulation débute au 7ème jour. La stimulation délivrée à partir du 8ème jour n’induit pas l’apparition de fibres rapides de type II même si cette stimulation est prolongée jusqu’au 20ème jour du développement. De plus, la stimulation centrale chronique à rythme rapide ne modifie pas le développement de l’ALD lorsqu’elle est appliquée à partir du 10ème jour de la vie embryonnaire. Les résultats obtenus montrent qu’il est possible, dans une certaine mesure, d’orienter la différenciation de l’ALD vers le type musculaire rapide en imposant aux motoneurones une activité à fréquence rapide à un stade précoce du développement embryonnaire. Toutefois, cette plasticité de l’ALD disparaÎt rapidement. Les mécanismes pouvant rendre compte de la transformation des myotubes lents et de la perte ultérieure de leur plasticité sont discutés.

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