Neuromuscular junctions in adult and developing fast and slow muscles

Functional changes that occur just before hatching in future fast muscles of the chicken are thought to be influenced by the pattern of innervation. We have compared the neuromuscular junctions of two fast muscles, the posterior latissimus dorsi (PLD) and the pectoralis, which differ in their myosin composition at 18 days in ovo. We have also presented new information on the neuromuscular junctions of the adult fast muscles and an adult slow muscle, the anterior latissimus dorsi (ALD). Both categories of adult muscles were heterogeneous, and there was little difference between endplates of the two fast muscles or between the fast and slow muscles. In contrast, there were significant structural differences between the two fast muscles during embryonic development. In early embryonic muscle fibers, which synthesize embryonic forms of myosin, individual motor endplates were contacted by multiple axon terminals. At 18 days in ovo, the majority of the neuromuscular junctions in the pectoralis continued to be multiterminal, whereas all but one of the terminals had been withdrawn from each endplate in the PLD. This single terminal had a unique form that distinguished it from the embryonic pectoralis and also from the two adult muscles. By 7 days after hatching, the neuromuscular junctions of both muscles had single terminals. They were different from the embryonic terminals, though not necessarily equivalent to adult terminals. The results show that multiple terminals persist at 18 days in ovo in the muscle that continues to express an embryonic myosin, but they have been withdrawn from the muscle that has lost this myosin. It is concluded, from combined data on the two muscles, that maturation of the neuromuscular junction during embryonic and late posthatch development is correlated with transitions in the myosin pattern and in contractile properties.     

The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chick muscles

Summary The differentiation of neuromuscular junctions of multiply innervated, slow, anterior latissimus dorsi (ALD) and focally innervated, fast, posterior latissimus dorsi (PLD) muscles was studied in normal and curarized chick embryos. At 16 days of incubation, fibres of both muscles are contacted by several axon profiles, the number of which falls with age. In 18-day-old embryos individual endplates in ALD are usually contacted by three axon profiles, whereas in PLD, endplates are contacted only by a single large terminal profile. At this time, there is already a significant accumulation of cell organelles in the postsynaptic area.Treatment of embryos with curare during the 7th and 12th day of incubation delays the differentiation of the neuromuscular junction in both muscles. The paralysis dramatically affects the decrease of the number of axon profiles at individual endplates in both muscles. At 16 days the number of axon profiles was greater in embryos treated with curare than in the untreated controls. At 18 days when the number of axon profiles normally decreases, the endplates of both types of curarized muscles have an even greater number of axon profiles than at 16 days. Endplates in curarized PLD had up to 13 and in curarized ALD up to 12 axon profiles. The effects of curare gradually wore off and when the movements of the embryos again became more vigorous, the normal differentiation of neuromuscular junctions continued. At 21 days of incubation many embryos recover from curare and show endplates of normal appearance in both muscles. These results suggest that activity of the muscle is essential for the maturation of the neuromuscular junctions.     

Independent development of contractile properties and myosin light chains in embryonic chick fast and slow muscle

1. The contractile speeds and tetanus/twitch ratios of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles were studied during embryonic development and correlated with the type of myosin light chains present in these muscles as studied by one and two dimensional polyacrylamide gel electrophoresis. 2. At a time when the contractions of PLD were slow, i.e. in 15 day old embryos, the myosin light chains in this muscle were of the fast type. The slow contraction of this muscle may be due to incomplete and slow activation of the contractile elements. The tetanus/twitch ratio of muscles from 15 day old embryos is low and increases sharply with age. This increase could be due to the maturation of the internal membrane system, and occurs at about the same time as the increase in the speed of contraction. 3. ALD muscles contract slowly during all stages of development, although their tetanus/twitch ratio also increases with age. At 13 days they contain a mixture of fast and slow type myosin light chains and with increasing age the proportion of the slow type myosin light chains increases at the expense of the fast type. The slow time course of contraction of ALD is consistent with the presence of slow type myosin light chains. 4. The possibility that the synthesis of the slow type myosin light chains in ALD is induced by early motor activity in chick embryos is discussed.     

Ontogeny of spontaneous antigen-binding cells in developing chick embryos.

Bursal and splenic lymphocytes in developing chick embryos were examined for the number of antigen binding cells (ABC) which could form rosettes with red blood cells from sheep, guinea-pig, rabbit or dinitrophenylated sheep red blood cells (DNP-SRBC). The kinetics of the development of bursal ABC to rabbit red blood cells (RRBC) did not vary significantly with age of embryo examined, and its frequency was constantly high. The number of cells which bind to SRBC, guinea-pig red blood cells (GPRBC) or DNP-SRBC was low in the bursa and increased in an approximately linear fashion with the age of embryo. With the exception of RRBC-ABC, similar results were obtained with splenic ABC. Furthermore, the frequencies of ABC were higher in the spleen than in the bursa. These events suggest the antigen-independent generation of clonal diversity during the developmental stage of chick embryos.     

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.     

Patterns of gene expression in the developing chick thymus.

Gene expression in thymic T cells during late embryogenesis and early growth in chicks was examined using cDNA microarrays. Gene expression patterns were profiled into nine clusters by using self-organizing maps (SOM) clustering analysis. The expression patterns for a set of genes confirmed current information on the development of immune response. Expression of cell surface markers (MHC class I alpha chain, MHC class II associated invariant chain, CD8 beta chain, CD18, and beta2-microglobulin), and genes involved in the innate immune response (NK lysin-like) increased with age, and these patterns were consistent with an increase in the immune responsiveness of the young chick. The expression of cytokine receptor common gamma chain (gammac), death receptor-3 (DR3), and TCR alpha chain increased up to 1 day of age and then decreased. DR3 could play a role in the apoptosis during T-cell maturation, while the differential expression of TCR genes could reflect regulation of the rearrangement of TCR genes and TCR-mediated signal transduction during T cell development. Three genes coding for previously uncharacterized proteins are included in the clusters. These gene expression profiling studies provide background information on the developing chick immune system and provide preliminary functional information on unknown proteins.     

Patterns of troponin T expression in mammalian fast,slow and promiscuous muscle fibres

Summary The distribution of troponin T(TnT) species in typed single muscle fibres was analysed using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and a monoclonal antibody specific for fast TnT. Fibres taken from erector spinae (Es), plantaris (Plt), diaphragm (Dia) and soleus (Sol) muscles of adult rabbits were pretyped as fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), slow-twitch-oxidative (SO) or promiscuous (P) using a combination of histochemical staining and PAGE. Although none of the four size classes of TnT was either muscle or fibre type specific, their pattern of expression differed in each muscle and between the fibre types. FG fibres expressed TnT_1f or TnT_2f as predominant species, depending on the muscle; TnT_3f and TnT_4f were minor components. In contrast, all size classes of TnT were expressed in varying proportions in FOG fibres from Es and Plt, while those from Dia resembled FG fibres, expressing TnT_1f as their major species. P fibres from Es, Plt, and Sol exhibited a distinctive pattern of fast TnT expression, TnT_3f being the predominant species. Dia differed from the other muscles as TnT_1f was the dominant fast TnT species in its P fibres as it is in the Dia fast fibres. Quantitative analysis of one- and two-dimensional gels revealed that the P fibres could be divided into two classes, those that exhibited discoordinate expression of fast and slow TnTs, myosin light chains and myosin heavy chains and those in which their expression was coordinate. In addition low levels of TnT_4f were detected in SO fibres and of slow TnT in fast fibres.     

Cross-reinnervation changes the expression patterns of the monocarboxylate transporters 1 and 4: An experimental study in slow and fast rat skeletal muscle

The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.     

大鼠比目鱼肌肌纤维总数、横截面积及其内慢肌纤维和快肌纤维横截面积与年龄相关的变化

目的 探讨SD大鼠比目鱼肌的形态学特征随年龄增长的变化情况。方法 4、18、25和30月龄雄性SD大鼠比目鱼肌各5块,通过HE染色、琥珀酸脱氢酶（SDH）活性检测和免疫组织化学方法,观测比目鱼肌纤维形态、数目、肌肉横截面积及其内慢肌纤维和快肌纤维分布特征、横截面积与年龄相关的变化。 结果 比目鱼肌内肌纤维形状随年龄增长变得不规则,并伴以肌纤维劈裂和簇聚。30月龄较4月龄大鼠的比目鱼肌纤维总数减少（P＜0.05）,而且其与其余年龄组大鼠的肌肉横截面积比较亦明显减小（P＜0.01）。仅4月龄和25月龄大鼠比目鱼肌内慢肌纤维横截面积相比无差别（P＞0.05）,而快肌纤维横截面积则表现为18月龄和25月龄比较无差异（P＞0.05）。结论 30月龄SD大鼠比目鱼肌纤维总数、肌肉横截面积及其内慢肌纤维和快肌纤维横截面积与其他年龄组大鼠相比差异显著,提示在衰老后期骨骼肌功能可能会发生明显变化。     

Differences in ammonia and adenylate metabolism in contracting fast and slow muscle.

The time course of AMP deamination and IMP reamination was studied during in situ stimulation and subsequent recovery in fast-twitch (gastrocnemius) and slow-twitch (soleus) muscles of pentobarbital-anesthetized rats. Muscles were stimulated tetanically at rates that initially produced comparable decreases (40%) in tension development. In fast muscle, progressive decreases in total adenine nucleotide (TAN) contents of up to 50% were balanced by equivalent increases in IMP contents. Ammonia concentration initially increased in a 1:1 stoichiometry with changes in IMP and TAN. During recovery following stimulation, IMP removal matched, but NH3 removal exceeded the rate of TAN resynthesis. In contrast, TAN content in slow muscle was only slightly decreased (10%) during stimulation and there were no increases in IMP or NH3. Stimulation of the soleus following ligation of the blood supply did not increase TAN depletion. In both fast and slow muscle, changes in glutamate, aspartate, and alanine could be accounted for by transamination. These results illustrate a fundamental difference in adenylate metabolism during intense muscle stimulation between fast- and slow-twitch mammalian muscle.     

Developmental changes of glycogen enzymes in fast and slow muscles of the rat.

Glycogen synthase (GS), Branching enzyme (BE), Glycogen Phosphorylase (GPho) and the Debranching enzyme (DE) activities were studied in the white extensor digitorum longus (ELD) and in the red soleus (S) muscles of rats during the developmental period from birth to the young adulthood. During the first days of postnatal life all enzyme activities similarly increase in both ELD and in S. BE and DE seem to rise faster and reach the adult levels earlier than GS and GPho. Significant differences can be seen between ELD and S by age 14--21 days: glycogenolytic enzymes rise higher in the ELD, GS activity is similar in both muscles whereas BE activity is higher in S. All these develop changes are completed in 25 days. Thereafter, only GPho activity in the ELD continues to slowly increase.     

Acetylcholine synthesis in nerve endings to slow and fast muscles of developing chicks: Effect of muscle activity

The activity of the acetylcholine-synthesizing enzyme choline acetyltransferase was estimated in nerve terminals to fast, focally-innervated and slow, multiply-innervated muscles of chick embryos. Terminals in fast muscles contain considerably more choline acetyltransferase than terminals in slow muscles, and this could be related to factors determining the different patterns of innervation of the muscles. Neuromuscular blockade by curare increases the choline acetyltransferase content of both types of muscle, suggesting that more nerve terminals survive in inactive muscles.