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Home , Extrafusal muscle fiber, Muscle spindle, Sarcoplasm

ScandJ Lab Anim SCI. N0 1, 1996 Val 21

The 0f the rat: Peculiarities of motor innervation and ultrastructure and effect of increased activity.

by Tee! Seene & Maria Umnava University of Tartu, Ulikooli Str, 18 EE2400 Tartu, Estonia

Introduction spindles located in different segments of rat‘s The muscle spindle is an encapsulated stretch- hindlimb. receptor, being located among muscle fascicles, As the structure and innervation of rat’s muscle parallel to extrafusal fibers and associated with spindle has been investigated only in some small pericapsular and blood vessels‘ Skeletal muscles of rats and the data about the ef- The spindle—like shape of multilayered outer fect of muscular activity on intrafusal fibers are capsule is named after this receptor organ. Outer scarce, the present study was undertaken to study capsule cells are similar to perineural epithelial the rat‘s intrafusal muscle fibers and the cells of the sheath (Merrillees, ultrastructure of their motor endings in ultrathin 1960, Shamha & Bourrze 1968, Pallen & Ovalle sections in the biggest hindlimb muscle Quad- 1991 ). riceps femorm (Q.FEMOR) and in typical slow— The most essential data on the structure and func- twitch .mleus (SOL) muscle in the usual condi- tion of muscle spindle are found in cat's spindles tions and increased muscular activity. (Boyd 1962). In recent years investigators have shown that the dynamic and static intrafusal sys- Materials and Methods tems 0f rat’s muscle spindle are less sharply de— Male rats of the Wistar strain, 16-17 weeks old, marcated than in the cat (Walra & Kucera, 1985a, were. maintained on a constant diet SDS-RMI (C) Kucere er al. 1989,1991). At the morphological 3/8 (SDS, Witham, England). Food and water level it reveals sharing of sensory terminals be— were given ad libitum. The rats, six in both group, tween the dynamic bag] and static bag2 intrafusal were housed for 12/12 hrs light/dark period. The fibers; in addition fusimotor may some— rats were assigned to control and exercised. The times coinnervate the dynamic and static exercised animals were running at 35 m/min 5 intrafusal muscle fibers of a rat. days a week for 6 weeks. The running time (Table The comparison of structure and motor innerva— 1) and total work per week was progressively in— tion of three muscles in rat’s hindlimb (Salem; creased. In the first week the total work per week Extensor Digitorum Lmtgus, Lumbricalis) has was ~ 4 500 J and in the sixth week of exercise it showed that the spindles of slow» and fast-twitch was 30 000 .1. Fast-twitch and slow-twitch muscle muscles had similarity, whereas differences were fibers from different skeletal muscles of a rat were observed between the structure of the proximal separated as described earlier (Same & Alev and distal muscles (Kucem et al. 1991 ). These au- 1991). Muscle samples for ultrastructural studies thors expect that the structure of muscle spindles were fixed in 25% glutaraldehyde, postfixed in in different muscles may be related to the muscle 1% osmium tetroxide. dehydrated in graded alco— function, with muscle ability to perform delicate hol and embedded in Epon—Arzddite resin. Ultra- motor tasks. It is believed that the ratio of gamma thin sections were cut from transversely and 1011- and beta axons may increase with the increase of gitudinally oriented muqcle blocks, stained with muscle distance from the . Thus, the uranyl acetate and lead citrate, using 3—5 blocks relative contribution of dynamic and static sys- from each animal. In the red deep part of Q. tems to muscle afferent outflow may differ among FEMOR we succeeded to find the whole muscle

19 Scand, J1 Lab Anim. SCL No. 1. 1996. Vol. 23 spindle which transcended the region of extra- bag2, its diameter is the largest; bagl with lesser fusal muscle fibers" endplates. diameter and two small chain fibers. In juxtaequa— The determination of extrafusa] muscle fibre types torial zone of QPEMOR small end- was performed at the u1trastmcmra1 level by the ings with short terminals (length about 12 im) are number of mitochondria, taking into account the pre— located at the distance of about 3—5 1111 from each vious data on the content of myoglobin and other. Numerous light spherical synaptic vesicles eytoehromes aa3 in the investigated bundles of fibers are concentrated in their , dense—core (Umnova & Seem. 1991, Seem: & Umnova 1992). vesicles can often be arranged among them. Within the terminals mitochondria can also be Results observed. Junctinnal contains few The capillaries are usually located inside the ex— small mitochondria. The bagl intrafusal fiber of ternal layer of the outer capsule of rat's muscle Q.FEMOR has four motor endings of this type. spindles in QFEMOR and SOL Mast cells are The last of them is located at the distance of about often observed in the vicinity of blood vessels in 101m from the others. Its postsynaptic membrane muscle spindles. Rat’s muscle spindle of both forms some finger—like projections, muscle nu- QFEMOR and SOL involves 4 intrafusa] fibers: cleus is present in synaptic sareoplasm. In the ,1 m ,

F igure 1. Transversal ultrathin section through intracapsulated distal part of quadriceps femoris mucle spindle pole. A. Nerve bundle (l) is conected with outer capsule Of mucle spindle (2), a few intrafusal fibers are present in periaxial space: bagZ (3), bagl (4), chain 1 (5), chain 2 (6), separated by inner capsule. Sixth gamma motor ending (7) is on the bagl fiber. Location of gamma motor nerve (8). Magnifica- tion x 2,500. B. Postsynaptic membrane of the bagl intrafusal fiber forms finger-Iike projections (9) in the Vicinity of gamma terminal (7). Magnification x 20,000.

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Seand. .1. Lab. Anim. SCI. No. 1. 1996. V01. 23

.. «I ~ .. .‘. F igure 2. Transversal ultrathin section through intracaptulated distal part of the quadriceps femoris mucle spindle pole. A. Nerve bundle (1) is separated from the muscle spindle. The bag] (2) and chain 2 (3) intrafusal fibers are located in different compartments. The bag2 (4) and chain 1 (5) fibers are localized in the same compartment. Gamma axon forms motor ending (6) on the bag2 fiber. Magnification x 1,900 B. Postsynaptic membrane of the bag2 fiber forms two short projections (7) in the Vicinity of gamma motor (6). Magnification x 15,000. distal part of intracapsulated region of muscle spindle two other motor endings (5th and 6th) are located on the bagl fiber. They have a larger size, area of synaptic contact, narrower synaptic cleft and more complicated structure of postsynaptic

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Figure 3. Transversa] ultrathin section through the Figure 4. Beta motor axon ending (1) on the bagl gamma motor axon terminal (1) being located on (2) intrafusal fiber in extracapsulated region. the chain 1 intrafusal fiber (2). Mag’nifieation x Lipid inclusions (3), myelinized motor nerve (4). 7,500. Magnification x 9,000.

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Scand. .1. Lab. Anirn. SCI. No. 1. 1996. Vol. 23 zone in comparison With motor endings of of terminal and postsynaptic area. The length of juxtaeqnatorial region. In the postjunctional the third ending is about 40 1111, its postsynaptic sarcoplasm a muscle nucleus, lots of mitochon— membrane forms small protrusions and single fin- dria, polyribosomes, profiles of rough-surfaced ger—Iike projections (Figure 2ab). , elements of the Golgi Only one pole of the chain intrafusal fiber is inner- complex, small secondary lysosomes can be ob— vated in intracapsulated region of QFEMOR mus— served. The surface of the bag1 intrafusal fibers is cle spindle. At the transversal sections of muscle devoid of typical postsynaptic folds, but always spindle, motor axon terminal surrounds about a forms some finger—like projections in the motor half perimeter of the chain intrafusal fiber. axon terminals (Figure lab). The 1ength of the Postsynaptic membrane of the chain fiber does not sixth motor ending is 38 1111. form protrusions and projections, its surface is The bagZ intrafusal fiber in Q.FEMOR is inner- smooth. Large mitochondria, polyribosomes, mus- vated at fewer sites than is the bagl in the same Cle nuclei, secondary lysosomes and other spindle. Three motor endings are observed on the organelles are concentrated in synaptic sareoplasm bag2 fiber.The first of them is at the distance of (Figure 3). The second chain intrafusal fiber is de- about 100 im from the first motor ending on the void of motor innervation. This muscle spindle bag] fiber. It has two terminals (long and short) pole is thinner and shorter than the innervated pat- and lies on the smooth surface of an intrafusal tern, It should be pointed out that in the same fixa— fiber. To some extent the bagZ motor endings look tion conditions all Inithochondria in this similar to those being located in juxtaequatoria] noninnervated chain fiber are swolIen. zone on the bag] fiber, but they have a larger size ln extracapsulated region of muscle spindle in rat’s

Figure 5. A. Ultrathin section of the beta motor end—plate ( l) in the vicinity of extrafusal muscle fiber (2), adjacent to the bagl intrafusal fiber (3). The bagZ (4) intrafusa] fiber and intramuscular nerve bundle (5). Magnification x 2,200. B. Two beta axonal terminals (1). Mitochondria (6) in postsynaptic region of (3). Magnification x 15,000.

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F igure 6. A. Destruction of (1) in the central part of intrafusal fiber of soleus muscle, isolated mitochondria (2) and single T—tubules (3) can be seen in sarcoplasm. Magnification x 20,000. B. Motor ending on the chain intrafusal fiber of soleus muscle. (1), synaptic vesicles (2), (3) and postsynaptic folds (4). Magnification x 27,000.

Q.FEMOR only one motor ending can be observed. deep, narrow junetional folds which are separated It is located on the bagl intrafusal fiber at the dis« from myofibrils by some muscle nuclei, polyribo- tance of 100 im from the outer capsule of spindle somcs, elements of granular reticulum, aggre- and is indented into its surface. The length of this gations of mitochondria. Extracapsulatcd region of ending is about 21 1111. Axon terminal has short the bag2 and the chain intrafusal fibers of this spin- nonmyclinizcd segment. Postsynaptic membrane dle pole has no motor innervation. forms finger-like projections in the proximal part In the polar region of muscle spindles of exercised of motor endings and short wide junetional folds and control rats’ satellite cells occur under the along the side surfaces of motor axon terminal. At basal membrane of intrafusal fibers in both types the bottom of axon terminal, postsynaptie mem- of muscles (Q.FEMOR and SOL). 12 satellite brane of the bag] fiber is parallel to presynaptie cells (5 - in the bag2, 4 - in the bagl, 3 — in the membrane and does not form junetional folds. chain intrafusal fibers) are being located in the Muscle nucleus, large mitochondria, polyriboso- Q.FEMOR spindle pole within the distance of mes, vesicles, lysosome-like bodies, partly resor- 240-3001111. Large secondary lysosomelike bod- beri light lipid inclusions occur in synaptic sarco- ies were observed in the sarcoplasm of the chain plasm (Figure 4). Extrafusal motor endplate also fibers and in of some satellite cells. has short nonrnyelinized preterminal segment. After muscle activity there are no conspicuous de— Motor endplates are found on 6 extrafusal muscle structive changes in lhe myofibrils of QFEMOR fibers neighbouring the bagl intrafusal muscle intrafusal fibers. However, lysosome-h'kc bodies are fiber bundles. Motor endplates 0f extrafusal mus— always prersent in perinuelear and synaptic sarco- cle fibers have (1-5) small oval axon terminals, in- plasm. At the same time the destruction of myofibrils dented into muscle fibers (Figure 5). Postsynaptie in the peripheral, central and perinuelear parts of membrane of extrafusal fibers form a lot of regular, three types of intrafusal fibers occurs in SOL. 23 Scand. J. Lab. Anim. SCI. No. l. 1990. Vol. 23

Interrnyofibrillar space increases simultaneously. (Walru & Kucem 1985 ab, Kucem er al 1991). They Lyosome—like bodies and big vacuoleilike structures believe that motor innervation has influence upon are formed in the sarcoplasm of SOL intrafusal intrafusal fiber size, oxidative capacity and develop fibers (Figure 6a). In case of muscular activity no ment of sarcotubular system (Walro & Kucem prominent lesions in SOL axon terminals can be 1985a). It is also necessary to note that under the found, but postsynaptic membrane of chain fiber same fixation conditions in noninnervated chain forms short postjunetional folds with unfused basal fiber, mitochondria are swollen. It may be suggested membranes in synaptic clefts (Figure 6b). that chain fibers are in different metabolic state or differ in their biochemical properties (Walro & Discussion Kucem 1988, Kucera & Walro 1987), but it may be In order to study changes in the biggest muscle of possible as well, that this Chain fiber pole lacks mo- the rat’s hindlirnb Q.FEMOR, the ultrastructure of tor innervation. intrafusal muscle fibers and their motor endings All motor endings, being located in intraeapsulated under increased muscular activity, it is essential to area of Q.FEMOR muscle spindle on three types of find out the peculiarities of these structures in the intrafusal fibers occupy superficial position in rela usual cage conditions. tion to fiber surface. They are not indented into Studies of serial ultrathin sections of Q.FEMOR intrafusal fibers and have a small postsynaptic area. muscle spindle pole have revealed 10 motor end- Taking this factor into consideration we suggest ings in intracapsulated region on all intrafusal that motor endings being located in intracapsulated fibers. It is known that the factors which affect region of Q.FEMOR muscle spindle are formed by diversity of motor endings may be the following: gamma motor axons. type, function of motor axon (dynamic or In addition it is essential to compare the ultrastructure static), afferent innervation of intrafusal fibers of muscle spindles and nerve innervation of fast- (Walm & Kucem 1985a). The bagl intrafusal twiteh and slow-twitch extrafusal muscle fibres, fiber has most of the siles of motor innervation (in The satellite cells being located under the basal our case - 6 endings). It is supposed that the small membrane of intrafusal fibers may be the source of size and short length of axon terminals being lo- the formation of new fibers after neonatal de- cated on the bagl fiber in juxtaequatorial region is efferentation of rat’s hindlirnb (chcna & Saukup explained by suppressive action of muscle spindle 1993) and under autografting all the small skeletal sensory region (Walro & Kucera 1985a). muscles (Soukup et al 1990, Umnova ez at 1994). Ultrastructure of motor endings becoming more Extracapsulated region of the bag] intrafusal fiber complicated with the ierease of the distance from and neighbouring extrafusal muscle fibers are equator of muscle spindle confirms this supposi- coinnervated by beta axon. Motor ending, located tion. Undulating contour and finger-like projec- on the bag1 in extraeapsulated region of the spin- tions, formed by the bagl postsynaptic membrane dle pole, is more indented into the intrafusal fiber may probably be used for identification of the in comparison with the superficial bag] fiber in rat’s Q.FEMOR. Postsynaptie mem- formed by the gamma axons in intracapsulated re— brane of the cat’s bagl fiber has the same property gion on the same fiber (Umrtova & Seene 1995). (Boyd & Gladden 1985). Noninyelinized preterrninal segments of motor On the hag2 intrafusal fiber there are three tnotor axon, forming endings on the bag1 fiber in endings being located in the intracapsulated re- extraeapsulated region and on the neighbouring gion. The postsynaptic membrane of the bag2 extrafusal muscle fibers, are short. This feature is fiber has smoother contour than the bagl fiber. also characteristic of the beta axon endings The motor ending being located on the chain fiber (Kucem 1985). The length of the motor ending has the largest size. One chain intrafusal fiber lack- may be considered as a sign of skeletofusimotor ing motor innervation on this pole is thinner and innervation of fibers. Length of the motor ending, shorter than the innervated chain fiber. Some inves- located in extraeapsulated region on the bagl tigators have observed noninnervated intrafusal fiber is 2 times shorter than the length of gamma fibers in other small muscles of rat’s hindliinb motor endings on the same fiber in intracapsula

24 Scand. J. Lab. Anim. SCI. No, 1. 1996. Vol. 23

ted region of this pole. It is similar to average intrafusal fiber is devoid of motor innervation, length of beta axon terminals in muscles of the and polyneuronal innervation of the dynamic other mammalian species (Boyd & Gladden 1985, bag] intrafusal fiber with gamma and beta motor Kumra 1985). As the bagI fiber is innervated by axons of appropriate motoneurons. Reduced mo- the gamma and beta axons, has well developed tor static component and polyneuronal innerva- myofibrillar apparatus (at least in extracapsulated tion strengthen the influences of the dynamic region) and lipid drops, which are subjected to intrafusal fiber in muscle spindle of Q. FEMOR resorption, we suggest that the bagl fiber may be muscle. Ability of the bagl plasma membrane to able to contract. The diversity of motor endings form finger-like projections in the vicinity of mo- located on the bagl intrafusal fiber reflects its po— tor axona] terminals establishes more close lyneural innervation. This fact probably causes synaptic contact and quick transmission of infor- the morphological and histochemical heterogeni- mation concerning changes of muscle length of ty of the bag] muscle fiber in its different regions Q.FEMOR muscle to the CNS. The bagl (Walra & Kucera 1985a, Kuccra & Walro I988, intrafusal fiber is coinnervated by the beta axon Khan & Suukup 1988). It is suggested that the lo- together with oxidative type of extrafusal muscle cation of beta-innervated muscle spindles in mo— fiber. tor region of , where muscle eon- Comparison of the effect of prolonged muscular tractions originate, probably helps to receive im- activity on the ultrastructure of the muscle spin- mediate afferent response. dles in Q.FEMOR and SOL shows that in SOL In the studied region of Q.FEMOR we registered intrafusal muscle fibres indicate some signs of at least 6 red oxidative type extrafusal muscle destruction of myofibrils and other cell organel- fibers coinnervated together with bagl intrafusal les. Intrafusal muscle fibers being located in the fiber and forming one . In usual condi- SOL muscle adapt themselves to increased activi— tions of skeletal muscle function the intrafusal ty with a mechanism of response reaction similar muscle fibers very weakly participate in the proc- to that of extrafusal fibers, although the changes ess of contraction (Kucera et czl. 1991). Consider- in intrafusal muscle fibers are considerably smal- ing some sings of destruction in myofihrils and ler than those in extrafusal fibers. other organelles of intrafusal fibers we may sug- gest that SOL is probably subjected to the major- References ity of workload under prolonged muscular activ- Boyd 1: The structure and innervation of the nu» ity as predominantly slow-twitch skeletal muscle. clear ba g muscle fiber system and the nuclear Summing up, it can be concluded that intrafusal chain muscle fiber system in manunalian mus- muscle fibers being located in the region of slow cle spindles. Phil Trans Roy Soc Lond (Biol). oxidative muscle fibers of SOL adapt themselves 1962, 245, 81-136. to increased activity probably by using a mecha— Boyd I & M Gladden: Review. In: The Muscle nism of response reaction similar to that of Spindle (Boyd LA. and Gladden M.H. eds). extrafusal fibers, although the changes in Macmillan, London, 1985, 3-22. intrafusal muscle fibers are considerably smaller Khan M & T Snukup: Histochemieal heterogene» than in extrafusal fibers, ity of intrafusal muscle fibers in slow and fast skeletal muscle of the rat. Histochemical I. Acknowledgments 1988, 20, 52-60. This study was supperted by the [SF grant LLP 101). K mam J: Distribution of skeletofusimotor axons in lumbrical muscles of the monkey. Anat and Summary Embryol. 1985, 173, 95-104. The ultrastructures of intrafusal muscle fibers and Kucem J & J Walm: The effect of neonatal motor endings in the rat’s Quadrieeps femoris deafferentation or deefferentation on the (Q.FEMOR) have some peculiarities. They are inimunocytochemistry of muscle spindles in expressed by a deficit of static component in 1110- the rat. Histochemistry. 1988. 90, 151—160. tor innervation, as one static nuclear chain

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Kucera J, J Walro & J Reichler: The role of nerve and Soukup J,‘ FPedrom & L-E Thomell: Rat intrafu- muscle factors in the development of rat muscle sal fibers regenerated after autotransplantation spindles. Am] Anat. 1989, 186, 144-160. of the EDL. Muscles do not express their typi- Kucera J, J Walro & J Reichler: Neural organiza- cal heavy chain isoenzimes in the ab- tion of spindles in three hindlimb muscles of sence of sensory innervation. Muscle and M0- the rat. Am J Anat. 1991, 190, 74-88. tility. 1990, 2, 1054110. Merillees N: The fine structure of muscle spindles Umnova M d”: T Seene: The effect of increased in the lumbrical muscles of the rat. J Biophys functional load on the activation of satellite Bioehem Cytol. 1960, 7, 725—742. cells in the skeletal muscle of adult rats. Int J Ovalle W: Motor nerve terminal on rat intrafusal Sports Med. 1991, 12, 501-504, muscle fibers a correlated light and electron mi- Umnova M & ’I‘Seene: Ultrastructure of intrafusal croscopic study. J Anat. 1972, 111, 239-252. motor endings in rat muscle spindle (in Rus- Patten R & W Ovalle: Muscle spindle uluastructurc sian). Doklady Akademii Nauk. 1995, 34], revealed by conventional and high resolution 263-269. scanning electron microscopy. Anat Rec. 1991. Umnova M, J Ja‘rva & T Seene: Muscle spindle 230, 183—198. ultrastructurc in rat autografted skeletal muscle Seene T & K Alev: Effect of muscular activity on (mextensor digitorum longus) (in Russian). the turnover rate of and myosin heavy Doklady Akademii Nauk. 1995, 338, 545-550. and light chains in different types of skeletal Walro J & J Kucera: Motor innervation of muscle. Int J Sports Med. 1991, 12, 204-207. intrafusal fibers in rat muscle spindles: Incom— Seene T & M Umnova: Relations between the plete separation of dynamic and static sys- changes in the turnover rate of contractile pro- tems. Am J Anat. 1985, 173, 5568. teins, activation of satellite cells and ultra- Walro J & J Kucera: Rat muscle spindles deficient structural response of neuromuscular junc— in elements of the static gamma system. tions in the fast oxidative—glyeolytie muscle Neurosci Lett. 1985. 59. 303—307. fibers in endurance trained rats. Basic and Ap— Zelena J & T Soukup: Increase in the number of plied Myology. 1992, 2, 39-46. intrafusal muscle fibers in rat muscles after Shantha T & G Bourna: The perineurial epithe- neonatal motor denervation. Neuroscience. lium — a new concept. In: The structure and 1993, 52, 207-218. function of the (Ed.: G.Bourne). Academic Press, New York. 1968, I, 379-459.

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